WO2025259992A1 - 5'-modified monomers, oligonucleotides and double-stranded rnas - Google Patents

5'-modified monomers, oligonucleotides and double-stranded rnas

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Publication number
WO2025259992A1
WO2025259992A1 PCT/US2025/033557 US2025033557W WO2025259992A1 WO 2025259992 A1 WO2025259992 A1 WO 2025259992A1 US 2025033557 W US2025033557 W US 2025033557W WO 2025259992 A1 WO2025259992 A1 WO 2025259992A1
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WO
WIPO (PCT)
Prior art keywords
alkyl
compound
oligonucleotide
branched
alkenyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/033557
Other languages
French (fr)
Inventor
Juan SALINAS
Dhrubajyoti Datta
Masaaki Nakata
Joe PANARESE
Jayaprakash K. Nair
Patrick Miller
Mark K. SCHLEGEL
Muthiah Manoharan
Punit Seth
Vasant Jadhav
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Alnylam Pharmaceuticals Inc
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Alnylam Pharmaceuticals Inc
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Application filed by Alnylam Pharmaceuticals Inc filed Critical Alnylam Pharmaceuticals Inc
Publication of WO2025259992A1 publication Critical patent/WO2025259992A1/en
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar, optionally, M is in the south conformation (i.e., C2’-endo); n is an integer selected from 1 - 3; n 5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n 6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n 7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n 8 is an integer selected from 1 - 3 (e
  • Q is not wherein * is the bond to the phosphorous atom.
  • Q is not wherein * is the bond to the phosphorous atom.
  • the compound is not of the formula, .
  • Some compounds of Formula (I) can be of Formula (Ia), For example, some compounds of Formula (I) can be of formula, wherein: Q 1 is -O-, -S-, or -N(R N )-, and R N is hydrogen, methyl, C 1- 3 alkoxy, or C 1-3 acyl.
  • Some compounds of Formula (I) can be of formula, . [0011] Some other compounds of Formula (I) can be of formula, . [0012] Yet some other compounds of Formula (I) can be of formula, , [0013] Still some other compounds of Formula (I) can be of formula, [0014] Some compounds of Formula (I) can be of formula, . [0015] Some compounds of Formula (I) can be of formula, wherein R NQ is hydrogen). [0016] Some other compounds of Formula (I) can be of formula (Ib), [0017] Some other compounds of Formula (I) can be of the structure: .
  • some compounds of Formula (I) can be of the structure: [0018] Yet some other compounds of Formula (I) can be of the structure: For some compounds of Formula (I) can be of the structure: [0019] In some embodiments, the compound of Formula (II) is of Formula (IIa), (IIb), (IIc) or (IId): [0020] Yet some other compounds of Formula (II) can be formula, . [0021] Still some other compounds of Formula (II) can be of formula, . [0022] Some compounds of Formula (III) can be of formula (IIIa), , wherein n is 1, 2 or 3. [0023] In some embodiments Q is where * is the bond to the phosphorous atom. For example Q is .
  • a compound of Formula (IV) is a compound is of Formula (V), where B, X, R P , Q 4 , R 2’ and R 3 ’ are as defined in Formula (IV).
  • the compound is of Formula (V), wherein R 3’ is -OR 30 and R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2 ’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2- (methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N- methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, N-alkenyl
  • the compound is of Formula (V), wherein R 2’ is -OR 30 and R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is -OR 30 and R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 3 ’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,
  • the compound of Formula (IV) is a compound is of Formula (VI): or a salt thereof, wherein: X A is O, S, SO 2 , CH 2 , NHR S’ or N(CO)R S’ , wherein R S’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; Y A is O, S, SO 2 , CH 2 , NHR S’ or N(CO)R S’ , wherein R S’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and R 3’ is hydrogen, halogen, or -OR 30 , wherein: R 30 is hydrogen, a hydroxy protecting group,
  • Y A is O, and the compound is of Form or a salt thereof, where B, X, X A , R P , a 4 nd Q are as defined in Formula (VI).
  • X A is O, and the compound is of Formula (VIII), or a s A P 4 alt thereof, where B, X, Y , R , and Q are as defined in Formula (VI).
  • X A and Y A are O, and the compound is of compound of Formula (IX), or a salt thereof, where B, X, R P , and Q 4 are as defined in Formula (VI).
  • the compound is of Formula (IX), or a salt thereof, wherein: B is an optionally modified nucleobase (e.g., uracil); Q 4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q 4 are optionally and independently replaced with -C(O)-, -S(O) 2 -,
  • the compound of formula (IV) is a compound of Formula (X), or a salt thereof, wherein: X A is O, S, SO 2 , CH 2 , NHR S’ or N(CO)R S’ , wherein R S’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and B, X, R P , R 2’ , R 3’ and Q 4 are as defined in Formula (IV). [0031] In some compounds of Formula (X), X A is O. In some other compounds of Formula (X), X A is S.
  • R 3’ is hydrogen, halogen, or -OR 30
  • R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-
  • R 2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2- methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo- 3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched
  • R 2’ is hydrogen, halogen, or -OR 30
  • R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-
  • R 3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2- methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo- 3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched
  • the compound of formula (IV) is a compound of Formula (XI), or a salt thereof, wherein: X A is O, S SO 2 CH 2 , NHR S’ or N(CO)R S’ , wherein R S’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and B, X, R P , R 2’ , R 3 and Q 4 are as defined in Formula (IV). [0035] In some compounds of Formula (XI), X A is O. In some other compounds of Formula (XI), X A is S.
  • R 3’ is hydrogen, halogen, or -OR 30
  • R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-
  • R 2’ is hydrogen, halogen, or -OR 30
  • R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-
  • the compound of Formula (IV) is of formulae (XII)-(XIV): wherein: X A is O, S SO 2 CH 2 , NHR S’ or N(CO)R S’ , wherein R S’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R A is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; and B, X, R P , R 2’ , R 3 and Q 4 are as defined in Formula (IV).
  • X A is O. In some other compounds of formulae (XII)-(XIV), X A is S.
  • R 3’ is hydrogen, halogen, or -OR 30 , and R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is halogen (e.g., F), alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-
  • R 2’ is F, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2- oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched O-alkenyl,
  • a compound of Formula (XV) has the structure: .
  • n 5 is 2 or 3, e.g., n 5 is 2.
  • a compound of Formula (XVI) has the structure: In some compounds of Formula (XVI), n 6 i 6 s 1 or 2, e.g., n is 1.
  • a compound of Formula (XVII) has the structure: [0045] In some embodiments, a compound of Formula (XVII) has the structure: [0046] In some compounds of Formula (XVII), R 4’ is methyl, ethyl or propyl, e.g., R 4’ is methyl. In some compounds of Formula (XVII), n 7 is 1 or 2, e.g., n 7 is 1. In some compounds of Formula (XVII), X A is O. In some other compounds of Formula (XVII), X A is S. In some compounds of Formula (XVII), n 7 is 1, R 4’ is methyl, and X A is O.
  • a compound of Formula (XX) has the structure: In some c 8 ompounds of Formla (XX), n is 1. In some other compounds of Formula (XX), n 8 is 2. [0048] In some embodiments, a compound of Formula (XXI) has the structure: [0049] In some embodiments, a compound of Formula (XXI) has the structure: [0050] In some compounds of Formula (XXI), R 4’ is methyl, ethyl or propyl, e.g., R 4’ is methyl. In some compounds of Formula (XXI), n 9 is 1. In some compounds of Formula (XXI), n 9 is 2.
  • X A is O. In some other compounds of Formula (XXI), X A is S. In some compounds of Formula (XXI), n 9 is 1, R 4’ is methyl, and X A is O. In some other compounds of Formula (XXI) n 9 is 2, R 4’ is methyl, and X A is O. [0051] In some compounds of Formula (XXI), R 4’ is hydrogen and n 9 is 2. In some other compounds of Formula (XXI), R 4’ is hydrogen and n 9 is 1. In some other compounds of Formula (XXI), R 4’ is hydrogen, X A is O, and n 9 is 2.
  • R 4’ is hydrogen, X A is O and n 9 is 1. In some other compounds of Formula (XXI), R 4’ is hydrogen, X A is S, and n 9 is 2. In some other compounds of Formula (XXI), R 4’ is hydrogen, X A is S and n 9 is 1. Some compounds of Formula (XXI), R 4’ is hydrogen, X A is O, n 9 is 2, and the compounds have the structure: [0052] In some embodiments, a compound of Formula (XVIII) has the structure: [0053] In some compounds of Formula (XVIII), Q 5 is *-NHCH 2 -, where * is the bond to the S(O 2 )R PS group.
  • Q 5 is ethylene. In yet some other compounds of Formula (XVIII), Q 5 is ethenylene. In some compounds of Formula (XVIII), Q 5 is propylene. In some compounds of Formula (XVIII), X A is O. In some other compounds of Formula (XVIII), X A is S. In some compounds of Formula (XVIII), X A is O and Q 5 is methylene, ethylene, ethenylene, or propylene. [0054] In some compounds of Formula (XVIII), X A is O, Q 5 is methylene and the compounds are of the structure: .
  • X A is O, Q 5 is ethylene and the compounds are of the structure: .
  • X A is O, Q 5 is propylene. and the compounds are of the structure: .
  • a compound of Formula (XIX) has the structure: In some other embodiments, a compound of Formula (XIX) has the structure: .
  • R 5X is methyl.
  • X A is O.
  • X A is S.
  • y is 0.
  • y is 1. In some compounds of Formula (XIX), X A is O and R 5X is methyl. In some compounds of Formula (XIX), X A is O, R 5X is methyl, and y 0. In some compounds of Formula (XIX), X A is O, R 5X is methyl, and y is 1. [0058] In some embodiments, a compound of Formula (XXII) has the structure: . In some embodiments, n 10 is 0, 12 or 3 (e.g., 1). [0059] In some embodiments, a compound of Formula (XXIII) has the structure: .
  • R 3’ is hydrogen, halogen, or -OR 30
  • R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkyl,
  • R 3’ is -OR 30
  • R 30 is a reactive phosphorous group (e.g., a phosphoramidite).
  • R 2’ is hydrogen, halogen, or -OR 30
  • R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkyl,
  • R 2’ is hydrogen, halogen (e.g., F), O-alkyl (e.g., -OMe ,-OEt, or -O-2-methoxyethyl), or -OR 30 , and R 30 is hydrogen, a hydroxy protecting group.
  • Q 4 is where * is the bond to the phosphorous atom.
  • Q 4 is where * is the bond to the phosphorous atom.
  • Q 4 is
  • Q 4 is , where * is the bond to the phosphorous atom.
  • Q 4 is where * is the bond to the phosphorous atom.
  • Q 4 is where * is the bond to the phosphorous atom.
  • Q 4 is pref 4 erably Q is where * is the bond to the phosphorous atom.
  • Q 4 is .
  • Q is where * is the bond to the phosphorous atom.
  • Q is where * is the bond to the phosphorous atom.
  • Q is where * is the bond to the phosphorous atom.
  • X is O. In some other embodiments, X is S.
  • A is -C(*)(H)-. In some embodiments, A is -CH 2 C(*)(H)-. In yet some other embodiments, A is -C(*)(H)CH 2 -.
  • E is a bond.
  • each R P is independently -OR O , -SR S , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S .
  • at least one R P is -OR O , e.g., each R P is independently -OR O .
  • Each R O can be independently hydrogen, C 1-6 alkyl (e.g., C 1-3 alkyl), or a hydroxyl protecting group.
  • each R O can be independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, or a hydroxyl protecting group.
  • each R O is hydrogen, methyl, or ethyl.
  • each R O is a hydroxyl protecting group, such as pivaloyloxymethyl ((CH 3 ) 3 CC(O)OCH 2 -, POM).
  • at least one R P is -SR S , e.g., each R P is independently -SR S .
  • Each R S can be independently hydrogen, C 1-3 alkyl, or a thiol protecting group.
  • each R S can be independently hydrogen, methyl, ethyl, propyl, isopropyl, or a thiol protecting group.
  • each R S is hydrogen, methyl or ethyl.
  • each R S is a thiol protecting group.
  • at least one R P is -N(R N ) 2 , e.g., each R P is independently -N(R N ) 2 .
  • Each R N can be independently hydrogen, C 1-3 alkyl, or an amine protecting group.
  • each R N can be independently hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, each R N is hydrogen, methyl or ethyl. In some embodiments, at least one R N is an amine protecting group. [0076] In some compounds of formulae I-XXVII and XIX-XXIII, at least one R P is - N(R N )S(O) 2 R 2S , e.g., each R P is independently -N(R N )S(O) 2 R 2S . R N can be hydrogen, C 1-3 alkyl, or an amine protecting group.
  • R N can be hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, R N is hydrogen, methyl or ethyl. In some embodiments, R N is an amine protecting group.
  • R 2S can be methyl, ethyl, propyl, or isopropyl. In some embodiments, R 2S is methyl or ethyl, e.g., R 2S is methyl. In some embodiments, R 2S is a C 3- 6 cycloalkyl (e.g., cyclopropyl or cyclobutyl).
  • R N is hydrogen, methyl, ethyl, propyl, or isopropyl
  • R 2S is methyl, ethyl, propyl, or isopropyl.
  • R N is an amine protecting group
  • R 2S is methyl, ethyl, propyl, or isopropyl.
  • one R P is -OR O
  • the other R P is -SR S , -N(R N ) 2 , or - N(R N )S(O) 2 R 2S .
  • one R P is -OR O
  • the other R P is -SR S .
  • one R P is -OR O , and the other R P is -N(R N ) 2 . In yet another example, one R P is -OR O , and the other R P is N(R N )S(O) 2 R 2S . [0078] In some other embodiments, one R P is -SR S , and the other R P is -N(R N ) 2 , or - N(R N )S(O) 2 R 2S . For example, one R P is -SR S , and the other R P is -N(R N ) 2 . In another example, one R P is -SR S , and the other R P is N(R N )S(O) 2 R 2S .
  • R PS is -OR O , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S .
  • R PS is -OR O .
  • Each R O can be independently hydrogen, C 1-6 alkyl (e.g., C 1- 3 alkyl), or a hydroxyl protecting group.
  • each R O can be independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, or a hydroxyl protecting group.
  • each R O is hydrogen, methyl, or ethyl.
  • each R O is a hydroxyl protecting group, such as pivaloyloxymethyl ((CH 3 ) 3 CC(O)OCH 2 -, POM).
  • R PS is OH.
  • R PS is -N(R N ) 2 .
  • Each R N can be independently hydrogen, C 1-3 alkyl, or an amine protecting group.
  • each R N can be independently hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, each R N is hydrogen, methyl or ethyl. In some embodiments, at least one R N is an amine protecting group.
  • R PS is -N(R N )S(O) 2 R 2S .
  • R N can be hydrogen, C 1-3 alkyl, or an amine protecting group.
  • R N can be hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, R N is hydrogen, methyl or ethyl.
  • R N is an amine protecting group.
  • R 2S can be methyl, ethyl, propyl, or isopropyl.
  • R 2S is methyl or ethyl, e.g., R 2S is methyl.
  • R 2S is a C 3-6 cycloalkyl (e.g., cyclopropyl or cyclobutyl).
  • R N is hydrogen, methyl, ethyl, propyl, or isopropyl
  • R 2S is methyl, ethyl, propyl, or isopropyl.
  • R N is an amine protecting group
  • R 2S is methyl, ethyl, propyl, or isopropyl.
  • R PS is -N(R N )P(O)(OR O )(R PC ) (e.g., - N(H)P(O)(OR O )(R PC ).
  • R P is -OR O and R PC is C 1-3 alkyl (e.g., methyl), wherein R O is hydrogen, C 1-6 alkyl (e.g., C 1-3 alkyl), or a hydroxyl protecting group.
  • R P is -OR O and R PC is C 1-3 alkyl (e.g,. methyl), wherein R O is hydrogen or C 1-3 alkyl. In some embodiments, R P is methoxy or ethoxy and R PC is methyl. In some embodiments, R P is -OH and R PC is methyl.
  • R 3’ is -OR 30 .
  • R 3’ is -OR 30
  • R 30 is a reactive phosphorous group.
  • R 3’ is -OR 30
  • R 30 is a reactive phosphorous group selected from phosphoramidite, H-phosphonate, alkyl-phosphonate, and phosphate triester, optionally R 30 is a phosphoramidite.
  • R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )N(R P2 ) 2 , - P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, -P(S)(OR P1 )H, - P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P3 is an optionally substituted C 1 -C 30 alkyl, optionally substituted C 2
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • R 3’ is - OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph.
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2 , and where R P1 is –CH 2 CH 2 CN.
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • each R P2 is independently methyl, ethyl, propyl, or isopropyl.
  • R 3’ is -OR 30
  • R 30 is - P(OR P1 )N(R P2 ) 2 , and where each R P2 is isopropyl.
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph
  • each R P2 is independently methyl, ethyl, propyl, or isopropyl
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2 , and where R P1 is – CH 2 CH 2 CN, and each R P2 is isopropyl.
  • R 3’ is -OR 30
  • R 30 is a phosphoramidite group such as 3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or 3'-[(ß-thiobenzoylethyl)-(1-pyrrolidinyl)]- thiophosphoramidite).
  • R 3’ is -OR 30
  • R 30 is hydrogen or a hydroxyl protecting group (e.g., a silyl based hydroxyl protecting group).
  • Some exemplary hydroxyl protecting group for R 30 of R 3’ include, but are not limited to, t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionally, the hydroxyl protecting group is TBDMS.
  • TBDMS t-butyldimethylsilyl
  • TMS trimethylsilyl
  • TES triethylsilyl
  • TIPS triisopropy
  • R 3’ is -OR 30
  • R 30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 30 is a bond to an oligonucleotide.
  • R 3’ can be connected to the 5’- hydroxyl of the nucleoside or nucleotide or the 5’-hydroxyl at the 5’-terminal of the oligonucleotide.
  • the internucleotide linkage between the compound or nucleoside of formulae I-XXIII and the nucleoside, nucleotide, or oligonucleotide it is linked to can be an unmodified internucleotide linkage (i.e., phosphodiester) or a modified internucleotide linkage (e.g., phosphorothioate, MMI or imidp, preferably the modified internucleotide linkage is phosphorothioate). Exemplary modified internucleotide linkages are described herein below. [0092] In some embodiments, R 3’ is hydrogen or halogen (e.g., F, Br, Cl or I).
  • R 3’ is H or F.
  • R 3’ is -OR 20 , where R 20 is optionally substituted C 1-6 alkyl.
  • R 3’ is -OR 20 , where R 20 is methyl, ethyl, or propyl.
  • R 3’ is -OR 20 , where R 20 is methyl.
  • R 3’ is -OR 20 , where R 20 is optionally substituted C 2-6 alkenyl.
  • R 3’ is -OR 20 , where R 20 is vinyl or allyl.
  • R 3’ is -OR 20 , where R 20 is optionally substituted C 2-6 alkynyl.
  • R 3’ is -OR 20 , where R 20 is acetylenyl, propargyl, or 5-hexyn-1-yl.
  • R 3’ is -OR 20 , where R 20 is C 1-6 alkoxyC 1- 6 alkyl.
  • R 3’ is -OR 20 , where R 20 2-methoxyethyl.
  • R 3’ is -OR 30 and R 2’ is hydrogen, halogen, or -OR 20 .
  • R 3’ is - OR 30 and R 2’ is hydrogen, halogen, or -OR 20 , and where R 30 is a reactive phosphorous group, hydroxyl protecting group, a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is hydrogen or halogen (e.g., F, Br, Cl or I).
  • R 2’ is H or F.
  • R 2’ is -OR 20 , where R 20 is optionally substituted C 1-6 alkyl.
  • R 20 is C 1-6 alkyl substituted with one, two, or three substituents selected independently from the group consisting of halogen, -OR 22, -N(R 22 ) 2 , -SR 22 , - C(O)OR 22 , -C(O)N(R 22 ) 2 , wherein R 22 is hydrogen or C 1-3 alkyl (e.g., 2,2,2-trifluoroethyl, 1,3- dimethoxyprop-2-yl).
  • R 20 is C 1-6 alkyl substituted with one or two substituents selected independently from the group consisting of halogen, -OR 22, -N(R 22 ) 2 , -SR 22 , -C(O)OR 22 , - C(O)N(R 22 ) 2 , wherein R 22 is hydrogen or C 1-3 alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxyprop- 2-yl).
  • R 2’ is -OR 20 , where R 20 is methyl, ethyl, or propyl. In some embodiments, R 2’ is -OR 20 , where R 20 is methyl.
  • R 2’ is -OR 20 , where R 20 is C 1- 6 alkoxyC 1-6 alkyl.
  • R 2’ is -OR 20 , where R 20 is 2-methoxyethyl.
  • R 2’ is -OR 20 , where R 20 is N-(C 1- 6 alkyl)aminocarbonylC 1-6 alkyl.
  • R 2’ is -OR 20 , where R 20 is 2-(N-methylamino)-2- oxoethyl or 3-oxo-3-(N-methylamino)prop-1-yl.
  • R 2’ is -OR 30
  • R 30 is hydrogen or a hydroxyl protecting group (e.g., a silyl based hydroxyl protecting group).
  • a hydroxyl protecting group for R 30 of R 2’ include, but are not limited to, t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionally, the hydroxyl protecting group is TBD
  • R 2’ is halogen, (e.g., F), alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2- (methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N- methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O
  • R 2’ is -OR 30 .
  • R 2’ is -OR 30
  • R 30 is a reactive phosphorous group.
  • Exemplary reactive phosphorous groups are described herein below and include, but are not limited to, phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries.
  • R 2’ is -OR 30
  • R 30 is a reactive phosphorous group selected from phosphoramidite, H-phosphonate, alkyl-phosphonate, and phosphate triester, optionally R 30 is a phosphoramidite.
  • R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )N(R P2 ) 2 , - P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, -P(S)(OR P1 )H, - P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P3 is an optionally substituted C 1 -C 30 alkyl, optionally substituted C 2
  • R 2’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • R 2’ is - OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph.
  • R 2’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2 , and where R P1 is –CH 2 CH 2 CN.
  • R 2’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • each R P2 is independently methyl, ethyl, propyl, or isopropyl.
  • R 2’ is -OR 30
  • R 30 is - P(OR P1 )N(R P2 ) 2 , and where each R P2 is isopropyl.
  • R 2’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • R P1 is C 1-6 alkyl substituted with cyano or -SC(O)Ph
  • each R P2 is independently methyl, ethyl, propyl, or isopropyl
  • R 2’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2 , and where R P1 is – CH 2 CH 2 CN, and each R P2 is isopropyl.
  • R 2’ is -OR 30
  • R 30 is a phosphoramidite group such as 2’-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or 2’-[(ß-thiobenzoylethyl)-(1-pyrrolidinyl)]- thiophosphoramidite).
  • R 2’ is -OR 30
  • R 30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 30 is a bond to an oligonucleotide.
  • R 30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide
  • R 2’ can be connected to the 5’- hydroxyl of the nucleoside or nucleotide or the 5’-hydroxyl at the 5’-terminal of the oligonucleotide.
  • the internucleotide linkage between the compound or nucleoside of formulae I-XXIII and the nucleoside, nucleotide, or oligonucleotide it is linked to can be an unmodified internucleotide linkage (i.e., phosphodiester) or a modified internucleotide linkage (e.g., phosphorothioate, MMI or imidp, preferably the modified internucleotide linkage is phosphorothioate). Exemplary modified internucleotide linkages are described herein below. [00110] In compounds of formulae I-XXIII, B is an optionally modified natural or non-natural nucleobase.
  • B is an optionally modified natural or non-natural nucleobase.
  • B is uracil, adenine, cytosine, 5-methylcytosine, guanine, or thymine (i.e., 5- methyluracil).
  • B is a modified or protected nucleobase.
  • B is a protected nucleobase comprising at least one amine or hydroxyl protecting group.
  • B is adenine, cytosine, 5-methylcytosine, or guanine comprising at least one amine protecting group. Exemplary modified, unmodified natural and non-natural nucleobase are described herein below.
  • X is O; each R P is -OR O ; R 2’ is hydrogen, F, or -OR 20 , where R 20 is hydrogen, optionally substituted C 1-6 alkyl (e.g., methyl, ethyl or propyl), such asC 1-6 alkoxyC 1-6 alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C 1- 6 alkyl)aminocarbonylC 1-6 alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl); and R 3’ is -OR 30 , where R 30 is reactive phosphorous group (e.g., a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester).
  • C 1-6 alkyl e.g., methyl, ethyl or propyl
  • R 3 is
  • the reactive phosphorous group is: -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 (e.g., - P(OR P1 )N(R P2 ) 2 , -P(SR P
  • X is O; each R P is -OR O ; R 2’ is hydrogen, F, or -OR 20 , where R 20 is hydrogen, optionally substituted C 1-6 alkyl (e.g., methyl, ethyl or propyl, preferably methyl), such as C 1-6 alkoxyC 1-6 alkyl (e.g., methoxy, 2-methoxyethyl) or N- (C 1-6 alkyl)aminocarbonylC 1-6 alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl) ; and R 3’ is -OR 30 , where R 30 is -P(OR P1 )N(R P2 ) 2 , and where R P1 is – CH 2 CH 2 CN, and each R P2 is is is is
  • X is O; R PS is -OR O ; R 2’ is hydrogen, F, or - OR 20 , where R 20 is hydrogen, optionally substituted C 1-6 alkyl (e.g., methyl, ethyl or propyl), such as C 1-6 alkoxyC 1-6 alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C 1-6 alkyl)aminocarbonylC 1-6 alkyl (e.g.
  • R 3’ is -OR 30 , where R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
  • X is O; R PS is -OR O ; R 2’ is hydrogen, F, or - OR 20 , where R 20 is hydrogen, optionally substituted C 1-6 alkyl (e.g., methyl, ethyl or propyl), such asC 1-6 alkoxyC 1-6 alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C 1-6 alkyl)aminocarbonylC 1-6 alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N-methylamino)prop-1-yl); and R 3’ is -OR 30 , where R 30 is reactive phosphorous group (e.g., a phosphoramidite, H-phosphonate, alkyl- phosphonate, or phosphate triester).
  • C 1-6 alkyl e.g., methyl, ethyl or propyl
  • R 3 is
  • the reactive phosphorous group is: -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , - P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, -P(S)(OR P1 )H, -P(O)(SR P1 )H, - P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 (e.g., -P(OR P1 )N(R P2 ) 2 , -P(SR P
  • X is O; R PS is -OR O ; R 2’ is hydrogen, F, or - OR 20 , where R 20 is hydrogen, optionally substituted C 1-6 alkyl (e.g., methyl, ethyl or propyl, preferably methyl), such as C 1-6 alkoxyC 1-6 alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C 1- 6 alkyl)aminocarbonylC 1-6 alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl) ; and R 3’ is -OR 30 , where R 30 is -P(OR P1 )N(R P2 ) 2 , and where R P1 is – CH 2 CH 2 CN, and each R P2 is isopropyl.
  • C 1-6 alkyl e.g., methyl, eth
  • X is O;
  • R PS is -OR O , where each R O is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl;
  • R 2’ is hydrogen, F, or -OR 20 , where R 20 is hydrogen, methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl;
  • R 3’ is R 30 , where R 30 is - P(OR P1 )N(R P2 ) 2 , where R P1 is –CH 2 CH 2 CN, and each R P2 is isopropyl.
  • the compound is of the formula, or a salt thereof, wherein: Q is wherein: * is the bond to the phosphorous atom; Q 1 is -O-, -S-, or -N(R NQ )-, wherein R NQ is hydrogen, methyl, C 1-3 alkoxy, or C 1-3 acyl; B is an optionally modified nucleobase (e.g., uracil); X is O or S; each R P is independently -OR O , -SR S , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; each R S is independently hydrogen, C 1-3 alkyl, or a thiol protecting group; each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group; and R 2S is C 1-3 alkyl; one of R 2
  • R 20 is hydrogen, hydroxyl protecting group, optionally substituted C 1-6 alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C 2-6 alkenyl, or optionally substituted C 2- 6 alkynyl (e.g., propargyl); and the other of R 2’ and R 3’ is -OR 30 , wherein:
  • R 3’ is -OR 30 .
  • the compound is of formula, . or a salt thereof, wherein: R 3’ is -OR 30 , wherein: R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl,
  • R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • R O is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • R 20 is hydrogen.
  • R 20 is a hydroxyl protecting group (e.g., TBS, TMS).
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, and each R P is - OR O .
  • the compound is of formula,
  • R 3’ is -OR 30 , wherein: R 30 is -P(OR P1 )N(R P2 ) 2 , wherein: each R P1 is optionally substituted C 1-6 alkyl, (e.g., -CH 2 CH 2 CN); each R P2 is independently optionally substituted C 1-6 alkyl (e.g., isopropyl); each R P is -OR O , wherein: each R O is independently C 1-6 alkyl, or a hydroxyl protecting group; X is O; n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, or cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cycl
  • B is uracil or thymine.
  • R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • R O is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • R 20 is hydrogen.
  • R 20 is a hydroxyl protecting group (e.g., TBS, TMS).
  • the compound is of the formula, or a salt thereof, wherein: Q 4 is where * is the bond to the phosphorous atom, and Q 1 is -O-, -S-, or -N(R NQ )-, wherein: R NQ is hydrogen, methyl, C 1-3 alkoxy, or C 1-3 acyl; M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionally modified nucleobase (e.g., uracil); X is O or S; X A is O or S; Y A is O or
  • R 3’ is -OR 30 , wherein: R 30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 3’ is -OR 30 .
  • the compound is of formula,
  • M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar;
  • R 3’ is -OR 30 , wherein: R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide;
  • B is an optionally modified nucleobase (e.g., uracil);
  • Q 4 is propylene, propenylene, propynylene, methylcyclopropyl,
  • M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar;
  • R 3’ is -OR 30 , wherein: R 30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; each R P and R PS is -OR O , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; y is 0 or
  • R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • R O is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • R 20 is hydrogen.
  • R 20 is a hydroxyl protecting group (e.g., TBS, TMS).
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, and each R P is - OR O .
  • the compound is of formula, ,
  • M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar;
  • R 3’ is -OR 30 , wherein: R 30 is -P(OR P1 )N(R P2 ) 2 , wherein: each R P1 is optionally substituted C 1-6 alkyl, (e.g., -CH 2 CH 2 CN); each R P2 is independently optionally substituted C 1-6 alkyl (e.g., isopropyl); each R P and R PS is -OR O ,wherein: each R O is independently C 1-6 alkyl,
  • the compound is of formulae IV-XIV wherein: Q 4 is , where * is the bond to the phosphorous atom; X is O; each R P is independently -OR O or is -N(R N ) 2 , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; and each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group R 3’ is -OR 30 , wherein: R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
  • the compound is of formulae IV-XIV, wherein: Q 4 is , where * is the bond to the phosphorous atom; X is O; each R P is independently -OR O or is -N(R N ) 2 , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; and each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group R 3’ is -OR 30 , wherein: R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
  • the compound is of formulae IV-XIV, wherein: Q 4 is , where* is the bond to the phosphorous atom; X is O; each R P is independently -OR O or is -N(R N ) 2 , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; and each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group R 3’ is -OR 30 , wherein: R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
  • the compound is of formulae IV-XIV, wherein: Q 4 is where* is the bond to the phosphorous atom.
  • each R P is independently -OR O or is -N(R N ) 2 , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; and each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group R 3’ is -OR 30 , wherein: R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group [00139]
  • the compound is of formulae IV-XIV, wherein: Q 4 is where * is the bond to the phosphorous atom; X is O; each R P is independently -OR O or is -N(R N ) 2 , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; and each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group R 3’ is -OR 30 , wherein: R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
  • the compound is of formulae IV-XIV, wherein: Q 4 is , , , , , , where * is the bond to the phosphorous atom;
  • X is O;
  • each R P is independently -OR O or is -N(R N ) 2 , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; and each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group
  • R 3’ is -OR 30 , wherein: R 30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
  • R 3’ is -OR 30 , X is O, and each R P is -OR O .
  • R 3’ is -OR 30 , X is O, each R P is -OR O , and each R O independently is C 1-6 alkyl (e.g., methyl or ethyl).
  • R 3’ is -OR 30 , X is O, each R P is -OR O , and each R O independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • R 3’ is -OR 30 , R 30 is - P(OR P1 )N(R P2 ) 2 , X is O, and one R P is -OR O and the other R P is C 1-3 alkyl.
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, one R P is -OR O , and each R O independently is C 1-6 alkyl (e.g., methyl or ethyl), and the other R P is C 1-3 alkyl.
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • one R P is -OR O
  • each R O independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl)
  • the other R P is C 1-3 alkyl (e.g., methyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • X is O
  • each R P is -OR O .
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • X is O
  • each R P is -OR O
  • each R O independently is C 1-6 alkyl (e.g., methyl or ethyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • X is O
  • each R P is -OR O
  • each R O independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • R 3’ is -OR 30 , R 30 is - P(OR P1 )N(R P2 ) 2 , X is O, and each R P is -OR O .
  • R 3’ is - OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, each R P is -OR O , and each R O independently is C 1-6 alkyl (e.g., methyl or ethyl).
  • R 3’ is -OR 30
  • R 30 is - P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O
  • each R O independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O .
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O
  • each R O independently is C 1-6 alkyl (e.g., methyl or ethyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O
  • each R O independently is a hydroxyl protecting group (e.g.
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • one R P is -OR O and the other R P is C 1-3 alkyl.
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • one R P is -OR O
  • each R O independently is C 1-6 alkyl (e.g., methyl or ethyl)
  • the other R P is C 1-3 alkyl.
  • R 3’ is - OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • one R P is -OR O
  • each R O independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl)
  • the other R P is C 1-3 alkyl (e.g., methyl).
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • R PS is -OR O .
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, R PS is -OR O , and R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, R PS is -OR O , and R P is C 1-3 alkyl (e.g., methyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • X is O
  • R PS is -OR O
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • X is O
  • R PS is -OR O
  • R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • X is O
  • R PS is -OR O
  • R O is a hydroxyl protecting group
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, and R PS is -OR O .
  • R 3’ is -OR 30 , R 30 is -P(OR P1 )N(R P2 ) 2 , X is O, R PS is -OR O , and R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • R PS is -OR O
  • R O is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O .
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O
  • R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • B is uracil or thymidine
  • R 3’ is -OR 30
  • R 30 is -P(OR P1 )N(R P2 ) 2
  • X is O
  • each R P is -OR O
  • each R O is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • R 3’ is -OR 30 and R 30 is a bond to an oligonucleotide, e.g., R 3’ is wherein: Y is O or S (e.g., S) and represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA).
  • Y is O or S (e.g., S) and represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA).
  • the oxygen atom that is illustrated linking the 5’-end of the oligonucleotide to the phosphorous atom is the 5’-oxygen of the 5’-terminal nucleoside of the oligonucleotide.
  • the compound is of formula,
  • R 3 is wherein: Y is O or S; represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA); n is an integer selected from 1 - 3; M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethy
  • Y is S. In other embodiments, Y is O.
  • oligonucleotides may be an RNA, a DNA, a single-stranded RNA, such as an antisense oligonucleotide (ASO), the antisense strand of a double-stranded RNA (such as an siRNA), and oligonucleotide derivatives such as phosphorodiamidate morpholino oligomers (PMOs).
  • ASO antisense oligonucleotide
  • siRNA siRNA
  • PMOs phosphorodiamidate morpholino oligomers
  • X is O
  • each R P is -OR PO
  • R 3’ is -OR 30
  • R 30 is a bond to an oligonucleotide
  • R 3’ is , wherein: Y is O or S (e.g., S) and represents the remainder of an oligonucleotide (e.g., the antisense strand of a double- stranded RNA).
  • the compound is of formula,
  • R 3 is wherein: Y is O or S; represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA); each R P is -OR O , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; X is O; X A is O; Y A is O; n is an integer selected from 1 - 3; M is a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionally modified nucleobase
  • B is uracil or thymine.
  • R O is C 1-6 alkyl (e.g., methyl or ethyl).
  • R O is a hydroxyl protecting group (e.g. pivaloyloxymethyl).
  • Y is S. In other embodiments, Y is O.
  • the compound is selected from the group of compound shown in Table A: Table A: Some exemplary compounds
  • an oligonucleotide with a 5’-terminal phosphate mimic comprising the structure: wherein: * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose); A is -C(*)(H)-, -CH 2 C(*)(H)-, or -C(*)(H)CH 2 -, wherein * is the bond to E; E is a bond or -CH 2 -; Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-
  • an oligonucleotide with a 5’-terminal phosphate mimic comprising the structure: wherein: X is O or S; Q 4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl; each R P is independently -OR O , -SR S , -N(R N ) 2 , or -N(R N )S
  • the sugar moiety of the nucleotide comprising the 5’-terminal phosphate mimic can comprise a 5- or 6- membered ring.
  • the sugar moiety of the nucleotide comprising the above modification can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof).
  • a furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified de
  • the 5’-terminal phosphate mimic can be attached to any atom, e.g., any carbon atom of the sugar moiety.
  • the 5’-terminal phosphate mimic e.g., can replace a -CH 2 OH group or a -OH group (e.g., a -CH 2 OH group) on the sugar moiety of a 5’-terminal nucleotide of the oligonucleotide.
  • the 5’-terminal phosphate mimic e.g., replaces the 4’-CH2OH group on the furanose ring (e.g.
  • ribofuranose arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribofuranose
  • the 5’-CH 2 OH group on the pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • the 5’-terminal nucleotide of the oligonucleotide e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose,
  • the sugar moiety of the nucleotide comprising the , modification i.e., the nucleotide at 5’-end of the oligonucleotide (5’-terminal nucleotide) can comprise a 5- or 6- membered ring.
  • the sugar moiety of the nucleotide comprising the above modification can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof).
  • a furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified de
  • the , modification replaces carbon atom in the ring portion of the sugar moiety of a 5’-terrminal nucleotide of the oligonucleotide.
  • the , modification replaces the 4’-C atom in the furanose (e.g.
  • ribofuranose arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribofuranose
  • the 5’-C atom in the pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • the oligonucleotide comprises at its 5’-end a compound of formulae I-IV described herein.
  • X is O.
  • X is O
  • each R P is OR O .
  • X is O
  • each R P is OH.
  • X is O
  • each R P is ethoxy.
  • X is O
  • each R P is OH.
  • X is O, and each R P is OR O wherein R O is pivaloyloxymethyl.
  • the 5’-terminal nucleotide of the oligonucleotide is of the structure: , , , or a salt thereof, wherein: n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two
  • the 5’-terminal nucleotide of the oligonucleotide has the structure: .
  • the 5’-terminal nucleotide of the oligonucleotide can be of formula, , wherein: Q 1 is -O-, -S-, or -N(R N )-, and R N is hydrogen, methyl, C 1- 3 alkoxy, or C 1-3 acyl.
  • the 5’-terminal nucleotide of the oligonucleotide is of formula, [00163] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00164] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, . [00165] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, .
  • the 5’-terminal nucleotide of the oligonucleotide is of formula, [00167] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00168] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, e.g., of formula .
  • the ‘5-terminal nucleotide of the oligonucleotide is of formula, e.g., of formula [00170] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00171] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, . [00172] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, .
  • the 5’-terminal nucleotide of the oligonucleotide is of formula, , wherein n is 1, 2 or 3.
  • the 5’-terminal nucleotide of the oligonucleotide has the structure: .
  • the 5’-terminal nucleotide of the oligonucleotide is of formula:
  • the 5’-terminal nucleotde is of formula: .
  • the 5’-terminal nucleotde is of formula: .
  • Q 4 is ethylene or ethenylene , where * is the bond to the phosphorous atom).
  • the 5’-terminal nucleotide of the oligonucleotide has the structure:
  • the 5’-terminal nucleotide of the oligonucleotide is of formula: .
  • the 5’-terminal nucleotde is of formula: In some other embodiments, the the 5’-terminal nucleotde is of formula: .
  • Q 4 is ethylene or ethenylene (e.g., preferably where * is the bond to the phosphorous atom).
  • the 5’-terminal nucleotide of the oligonucleotide has the structure:
  • the 5’-terminal nucleotide of the oligonucleotide is of formula:
  • the 5’-terminal nucleotde is of formula:
  • the the 5’-terminal nucleotde is of formula:
  • Q is ethylene or ethenylene (e.g., pref erably , where * is the bond to the phosphorous atom).
  • the 5’-terminal nucleotide of the oligonucleotide has the structure:
  • the 5’-terminal nucleotide of the oligonucleotide is of formula:
  • the 5’-terminal nucleotde is of formula:
  • the 5’-terminal nucleotde is of formula:
  • Q is ethylene or ethenylene (e.g., preferably where * is the bond to the phosphorous atom).
  • the 5’-terminal nucleotide of the oligonucleotide has the structure: , , , , , ,
  • the 5’-terminal nucleotide of the oligonucleotide is of the structure: each as defined in Table 14 (below), where s at the end of the abbreviation indicates the internucleoside linkage between the 5’-terminal nucleoside and the subsequent nucleoside is a phosphorothioate internucleoside linkage, and the absence of “s” indicates that the internucleoside linkage between the 5’-terminal nucleoside and the subsequent nucleoside is a phosophodiester (e.g., a phosphate internucleotide linkage).
  • the oligonucleotide comprises at least three nucleotides.
  • the oligonucleotide comprises from 5 to 100, e.g., from 10 to 50 nucleotides.
  • the oligonucleotide comprises from 15 to 40 nucleotides.
  • the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length.
  • the oligonucleotide is 19, 20, 21, 22, or 23 nucleotides in length.
  • the oligonucleotide described herein can comprise at least one nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications). Exemplary nucleic acid modifications are described herein below, and include, but are not limited to nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and combinations thereof. [00182] In some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-OMe nucleotides.
  • the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermally destabilizing modification of the duplex.
  • the oligonucleotide comprises a thermally destabilizing modification at at least one of position 4, 5, 6, 7, or 8, counting from the 5’-end of the oligonucleotide, where the compound of formulae I-XXIII is at position 1 from the 5’-end of the oligonucleotide; optionally, the thermally destabilizing modification is located at position 6, 7, or 8, counting from the 5’-end of the oligonucleotide, preferably the thermally destabilizing modification is located at position 7, counting from the 5’- end of the oligonucleotide.
  • the oligonucleotide comprises least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-F nucleotides.
  • the oligonucleotide comprises 2, 3, 4, 5, or 62’-F nucleotides, optionally, the oligonucleotide comprises 3, 4, 5 or 6 2’-F nucleotides.
  • the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of formulae I-XXIII is at position 1 from the 5’-end of the oligonucleotide.
  • the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 14 and 16, counting from the 5’-end of the oligonucleotide
  • the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 9, 14 and 16, preferably, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the oligonucleotide.
  • each 2’-F nucleotide is an independently selected nucleotide.
  • the oligonucleotide can also comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide comprises 2, 3, 4, 5, 6, or 7 2’-deoxy nucleotides, optionally, the oligonucleotide comprises 3, 4, 5 or 62’-deoxy nucleotides.
  • the oligonucleotide can comprise a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of formulae I-XXIII is at position 1 from the 5’-end of the oligonucleotide.
  • the oligonucleotide comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of oligonucleotide.
  • the oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, counting from the 5’-end of oligonucleotide.
  • the oligonucleotide comprises a 2’- deoxy nucleotide at least at positions 2, 5, 7, and 12, counting from the 5’-end of oligonucleotide.
  • thew oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’-end of the oligonucleotide. It is noted that when more than one 2’-dexy nucleotide is present in the oligonucleotide, each 2’-deoxy nucleotide is an independently selected nucleotide.
  • One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleobases in the oligonucleotide can be non-natural or modified nucleobases.
  • the oligonucleotide can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified or protected nucleobases.
  • the internucleotide linkages in the oligonucleotide can be independently unmodified (e.g., phosphodiester) or modified (e.g., phosphorothioate).
  • the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleoside linkages.
  • the oligonucleotide comprises at least one (e.g., 1, 2, 4, or 5) modified internucleoside linkages (e.g., phosphorothioate) at the first 1-5 positions at one or both ends of the oligonucleotide.
  • the oligonucleotide comprises a modified oligonucleotide linkage (e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 5’-end of the oligonucleotide; and the oligonucleotide comprises a modified oligonucleotide linkage (e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide.
  • a modified oligonucleotide linkage e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide.
  • the oligonucleotide is covalently linked to a support, e.g., a solid support.
  • a double-stranded RNA dsRNA
  • the sense strand is substantially complementary to the antisense strand
  • one of the sense or the antisense strand is an oligonucleotide described herein, i.e., an oligonucleotide with a 5’-terminal modification comprising the structure: wherein: * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose); A is -C(*)(H)-, -CH 2 C(*)(H)-, or -C(*)(H)CH 2 , wherein * is the bond to E; E is a bond
  • the modification replaces a CH 2 OH group on the sugar moiety of the 5’-terrminal nucleotide of the sense or antisense strand.
  • the modification replaces the 4’-CH 2 OH group on the furanose ring (e.g.
  • ribofuranose arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose
  • the 5’- CH 2 OH group on the pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • the 5’-terminal nucleotide of the sense or antisense strand e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose,
  • the modification replaces carbon atom in the sugar moiety of the 5’-terminal nucleotide of the sense or antisense strand.
  • the modification replaces the 4’-C atom in the furanose (e.g.
  • ribofuranose arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose
  • pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • the sense or antisense strand comprises at its 5’-end a compound of formulae I-XXIII described herein.
  • X is O.
  • X is O
  • each R P is OR O .
  • X is O
  • each R P is OH.
  • X is O
  • each R P is ethoxy.
  • X is O
  • each R P is OH.
  • X is O, and each R P is OR O wherein R O is pivaloyloxymethyl.
  • the 5’-terminal nucleotide of one of the sense or antisense strand is of the structure: , , , , , , where one of R 2’ and R 3’ is -OR 30 , and R 30 is a bond to the rest of the sense or antisense strand, provided that R 3’ is -OR 30 in formulae , , .
  • the antisense strand comprises the above 5’-terminal modification.
  • the method comprises administering to the subject either: (i) a double- stranded RNA described herein, where the antisense strand is substantially complementary to a target gene; or (ii) an oligonucleotide described herein, where the oligonucleotide is substantially complementary to a target gene.
  • a pharmaceutical composition comprising an oligonucleotide or dsRNA molecule described herein alone or in combination with a pharmaceutically acceptable carrier or excipient.
  • a cell comprising an oligonucleotide or dsRNA molecule described herein.
  • a gene silencing kit comprising an oligonucleotide or dsRNA molecule described herein.
  • a method for silencing a target gene, in a cell is also provided herein.
  • the method comprises a step of introducing: (i) a dsRNA molecule described herein into the cell, where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene.
  • a method for inhibiting or reducing the expression of a target gene in a subject is provided herein.
  • the method comprises administering to the subject: (i) a dsRNA molecule described herein, where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene.
  • a dsRNA molecule described herein where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene.
  • FIG. 1 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140883, AD-2140885, AD- 2140887, AD-2140888, and AD-64958 (parent) in mice.
  • FIG. 2 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140884, AD-2140882, AD- 2140886, AD-2261036, and AD-64958 (parent) in mice.
  • FIG. 2 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140884, AD-2140882, AD- 2140886, AD-2261036, and AD-64958 (parent) in mice.
  • FIG. 3 shows mTTR protein levels at different timepoints after administration of a single dose (0.5 mg/kg) of exemplary duplexes AD-286913, AD-2633769, AD-2633771, AD- 2633772, AD-2633774, AD-2633775, AD-2633777, and AD-64958 (parent) in mice.
  • FIG. 4 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140883, AD-2140885, AD- 2140887, AD-2140888, and AD-64958 (parent) in mice.
  • FIG. 4 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140883, AD-2140885, AD- 2140887, AD-2140888, and AD-64958 (parent) in mice.
  • FIG. 5 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2680450, AD-2680452, AD- 2680454, AD-2680455, AD-2680456, and AD-64958 (parent) in mice.
  • FIG. 6 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-401824, AD-2919280, AD- 2919281, AD-2919282, AD-2919283, AD-2919284, AD-2919285, AD-2919286, AD-2919288, and AD-2919289 targeting SOD1 mRNA in mice.
  • FIG. 7 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-401824, AD-401825, AD- 2919282, AD-2919289, AD-3116172 and AD-3116181 targeting SOD1 mRNA in mice.
  • FIG. 8 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-1271086, AD-3367267, and AD- 3367269 targeting SOD1 mRNA in mice.
  • FIG. 8 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-1271086, AD-3367267, and AD- 3367269 targeting SOD1 mRNA in mice.
  • FIG. 9 shows sAPP ⁇ protein levels at different timepoints after intrathecal administration of exemplary duplexes AD-960499, AD-454844, and AD-2905746 in non-human primate.
  • FIG. 10 shows APP mRNA remaining in various organs at day 91 after intrathecal administration of exemplary duplexes AD-960499, AD-454844, and AD-2905746 in non-human primate.
  • FIG. 11 shows MAP2 mRNA remaining in various organs at day 91 after intrathecal administration of exemplary duplexes AD-476454, AD-2912412, and AD-2912413 in non-human primate.
  • FIG. 10 shows APP mRNA remaining in various organs at day 91 after intrathecal administration of exemplary duplexes AD-476454, AD-2912412, and AD-2912413 in non-human primate.
  • FIG. 12 shows APP protein levels after intrathecal administration of exemplary duplexes AD-454844, AD-3175047, and AD-3216841 in non-human primate.
  • FIG.14 shows Configurational and conformational features of LNA, ⁇ -L-LNA, and 5 ⁇ -VP-functionalized LNA and ⁇ -L-LNA studied.
  • FIG.15B shows siRNAs with LNA or ⁇ -L-LNA at the 5' terminus of the antisense strands result in diminished silencing of gene expression in cultured cells or mice.
  • FIG.16A shows the 5'-VP- ⁇ -L-LNA modification results in a more active siRNA than the 5'-VP-LNA modification.
  • Ttr mRNA remaining in primary mouse hepatocytes cultured with the indicated siRNAs at the indicated concentrations under free uptake conditions. mRNAs were quantified by RT-qPCR, and averages ⁇ standard deviations are plotted (n 3).
  • FIG.16B shows the 5'-VP- ⁇ -L-LNA modification results in a more active siRNA than the 5'-VP-LNA modification.
  • FIG.17A shows 5'-VP- ⁇ -L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-Ome, models of antisense strands modified with 5'-VP-2’-Ome.
  • FIG.17B shows 5'-VP- ⁇ -L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-Ome, models of antisense strands modified with 5'-VP- ⁇ -L-LNA.
  • FIG.17C shows 5'-VP- ⁇ -L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-Ome, models of antisense strands modified with 5'-VP-LNA lodged at the Ago2 MID domain binding site.
  • FIG.17D shows 5'-VP- ⁇ -L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-OMe. Overlay of 5'-VP- ⁇ -L-LNA (golden) and 5'-VP-LNA (violet) bound to the Ago2 MID domain.
  • FIG.19 shows RNAi-mediated gene silencing is more efficient when the antisense strand of the siRNA is modified at 5' end with a 5'-vinyl-phosphonate carrying nucleotide that adopts a C3’-exo (South) conformation than a C3′-endo (North) pucker.
  • FIG.20A shows models of 5 ⁇ -terminal guide strand nucleotides lodged at the MID domain of RISC Ago2: LNA with a 5 ⁇ -phosphate.
  • 20B shows models of 5 ⁇ -terminal guide strand nucleotides lodged at the MID domain of RISC Ago2: Overlay of the LNA model shown in FIG.20A, LNA with an E-VP moiety, and E-VP-RNA. Carbon atoms of AS1 residues are colored in magenta, purple and green for P- LNA (5 ⁇ -phosphate LNA), E-VP-LNA and E-VP-RNA, respectively, and the AS1 and AS2 phosphorus atoms of RNA are highlighted in black. The phosphorus position of P-LNA virtually matches the position of the E-VP-RNA phosphorus (FIG.
  • FIG. 20B depicts some exemplary compounds of Formula (V).
  • FIG. 22 depicts a synthetic scheme for synthesis of compounds of Formula (V).
  • Reference (ref) for compound 1 is Ref: Marquez et. al. J. Chem. Soc. Perkins Trans.1: Org. Bioorg. Chem.1997, 1073-1078.
  • FIG.23 depicts some exemplary compounds of Formula (VI).
  • FIG.24 depicts some exemplary compounds of Formula (VII).
  • FIG.25 depicts some exemplary compounds of Formula (VIII).
  • FIG.26 depicts some exemplary compounds of Formula (X).
  • FIG.27 depicts some exemplary compounds of Formula (XI).
  • FIG. 28 depicts some exemplary compounds of Formulae (XII)-(XIV). Reference, Damha et. al. J. Am. Chem. Soc.2017, 139, 14542-14555. [00235] FIG.
  • FIG. 29 depicts % mouse SOD1 (mSOD1) mRNA remaining relative to aCSF in the brain (right hemisphere).
  • FIG. 30 depicts % mSOD1 mRNA remaining relative to aCSF/GADPH.in the brain (right hemisphere), liver and heart.
  • DETAILED DESCRIPTION [00237] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
  • the use of the singular includes the plural unless specifically stated otherwise.
  • the use of “or” means “and/or” unless stated otherwise.
  • the ring M can be a monocyclic or bicyclic ring (such as C 3-8 cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), or a 5- or 6- membered sugar.
  • ring M is a cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl).
  • ring M is a heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl).
  • ring M is a sugar.
  • the sugar moiety can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof).
  • a furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof
  • a pyranose
  • phosphate moiety, B, R 2’ and R 3’ can be attached to any atom, e.g., any carbon atom of the ring M, e.g., of the sugar moiety.
  • ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R 2 is attached to C2 or is absent, R 3’ is attached to C3, and the phosphate moiety is attached to C4 of the furanose.
  • furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof
  • ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R 2 is absent, R 3’ is attached to C2, and the phosphate moiety is attached to C3 of the furanose.
  • furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof
  • B is attached to C1
  • R 2 is absent
  • R 3’ is attached to C2
  • the phosphate moiety is attached to C3 of the furanose.
  • ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R 2 is absent, R 3’ is attached to C4, and the phosphate moiety is attached to C3 of the furanose.
  • furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof
  • B is attached to C1
  • R 2 is absent
  • R 3’ is attached to C4
  • the phosphate moiety is attached to C3 of the furanose.
  • ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R 2 is absent, and both of R 3’ and the phosphate moiety are attached to C3 of the furanose. It is noted that R 2’ , R 3’ and the phosphate moiety can replace the hydroxyl group present on the carbon of the furanose the R 2’ , R 3’ and the phosphate moiety are attached to.
  • furanose e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof
  • B is attached to C1
  • R 2
  • phosphate moiety When the phosphate moiety is attached to C4 of the furanose, it can replace the -CH 2 OH group at the C4 of the ribose.
  • the attachment of the B can be in the alpha or beta configuration.
  • the attachment of the R 2’ , R 3’ and the phosphate moiety independently can be in the R or S configuration.
  • ring M is cyclopentane.
  • ring M is cyclopentane
  • B is attached to C1
  • R 2 is attached to C2 or is absent
  • R 3’ is attached to C3
  • the phosphate moiety is attached to C4 of the cyclopentane.
  • ring M is cyclopentane and B is attached to C1, R 2 is absent, R 3’ is attached to C2, and the phosphate moiety is attached to C3 of the cyclopentane.
  • ring M is cyclopentane, and B is attached to C1, R 2 is absent, R 3’ is attached to C4, and the phosphate moiety is attached to C3 of the cyclopentane.
  • ring M is cyclopentane, and B is attached to C1, R 2 is absent, and both of R 3’ and the phosphate moiety are attached to C3 of the cyclopentyl.
  • carbon of the cyclopentane to which the B is attached is denotated as C1 and numbering proceeds in a clockwise fashion. It is noted that attachment of the B, R 2’ , R 3’ and the phosphate moiety independently can be in the R or S configuration.
  • ring M is a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1 of the pyranose, R 2’ is absent, R 3 ’ is attached to C4 and the phosphate moiety is attached to C5 of the pyranose.
  • pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof
  • B is attached to C1 of the pyranose
  • R 2’ is absent
  • R 2’ , R 3’ and the phosphate moiety can replace the hydroxyl group present on the carbon of the pyranose the R 2’ , R 3’ and the phosphate moiety are attached to.
  • the attachment of the B can be in the alpha or beta configuration.
  • the attachment of the R 2’ , R 3’ and the phosphate moiety independently can be in the R or S configuration.
  • DNA typically favors the C2'-endo sugar pucker (also known as the South conformer). This conformation is associated with the B-form of DNA, which is the canonical double helix structure. In C2'-endo, the C2' carbon atom is positioned above the plane of the sugar ring.
  • ring M is in the south conformation (i.e., C2’- endo).
  • RNA typically favors the C3'-endo sugar pucker (also known as the North conformer). This conformation is associated with the A-form of RNA, which is a more compact helix.
  • C3'-endo the C3' carbon atom is positioned above the plane of the sugar ring.
  • ring M is in the north conformation (i.e., C3’-endo).
  • R 3’ [00246]
  • R 3’ can be hydrogen, halogen, -OR 20 , or -OR 30 .
  • R 3’ is -OR 30 , where R 30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 3’ is -OR 30 and R 30 is hydrogen or hydroxyl protecting group.
  • R 3’ is -OR 30 and R 30 is a reactive phosphorus group.
  • R 30 is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester.
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is C 1-6 alkyl, optionally substituted with 1, 2,
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is C 1-6 alkyl, optionally
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is C 1-6 alkyl, optionally substitute
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is 2-cyanoethyl (-
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is 2-cyanoethyl (-
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is 2-cyanoethyl (-
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , where: RP1 is 2- cyanoethyl (-CH 2 CH 2 CN); each R P2 is independently isopropyl.
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is C 1-6 alkyl, optionally substitute
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is C 1-6 alkyl, optionally substitute
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is 2-cyanoethyl (-
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is 2-cyanoethyl (-
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: RP1 and one of R P2 taken together with the
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: RP1 and one of R P2 taken together with the atom
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: RP1 and one of R P2 taken together with the
  • R 3’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: RP1 and one of R P2 taken together with the
  • R 3’ is -OR 30 and R 30 is a bond to a nucleoside or a nucleotide, or an oligonucleotide.
  • R 30 is a bond to a nucleoside, a nucleotide, or an oligonucleotide
  • the internucleotide linkage between compound of formulae I-IV and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester) internucleotide linkage or a modified (e.g., phosphorothioate) internucleotide linkage.
  • R 3’ is -OR 30 and R 30 is linked to 5’-position of a nucleoside, nucleotide, or oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage (e.g., or a modified (e.g., phosphorothioate) internucleotide linkage.
  • R 30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide.
  • R 30 is linked to the 5’- terminal (e.g., 5’-OH) of the oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage. In some other embodiments, R 30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide by a modified (e.g., phosphorothioate) internucleotide linkage. [00265] In some embodiments, R 3’ is -OR 30 and R 30 is a hydroxyl protecting group.
  • R 3’ is -OR 30 and R 30 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,
  • R 3’ is -OR 30 and R 30 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R 30 is TBDMS.
  • R 3’ is hydrogen or halogen.
  • R 3 is H or F.
  • R 3’ is -OR 20 , where R 20 is hydrogen, hydroxyl protecting group, optionally substituted C 1-6 alkyl, such as C 1-6 alkoxyC 1-6 alkyl (e.g., 2-methoxyethyl) or N-(C 1- 6 alkyl)aminocarbonylC 1-6 alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl).
  • R 3’ is -OR 20 , and R 20 is hydrogen or hydroxyl protecting group.
  • R 3’ is - OR 20 , and R 20 is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, optionally, R 20 is methyl.
  • R 3’ is -OR 20 , and R 20 is C 1-6 alkoxyC 1-6 alkyl.
  • R 3’ is -OR 20 , and R 20 is 2-methoxyethyl.
  • R 3’ is -OR 20 and R 20 is a hydroxyl protecting group.
  • R 3’ is -OR 20 and R 20 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,
  • R 3’ is -OR 20 and R 20 is TBDMS (or TBS), TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R 20 is TBDMS (or TBS).
  • R 3 ’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O
  • R 2’ can be hydrogen, halogen, -OR 20 , or -OR 30 .
  • R 2’ is hydrogen or halogen.
  • R 2’ is H or F.
  • R 2’ is -OR 20 , where R 20 is hydrogen, hydroxyl protecting group, optionally substituted C 1-6 alkyl, such as C 1-6 alkoxyC 1-6 alkyl (e.g., 2-methoxyethyl) or N-(C 1- 6 alkyl)aminocarbonylC 1-6 alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl).
  • R 2’ is -OR 20 , and R 20 is hydrogen or hydroxyl protecting group.
  • R 2’ is -OR 20 , where R 20 is an optionally substituted C 1-6 alkyl.
  • R 2’ is -OR 20
  • R 20 is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, optionally, R 20 is methyl.
  • R 2’ is -OR 20
  • R 2’ is -OR 20
  • R 20 is 2-methoxyethyl.
  • R 2’ is -OR 20 and R 20 is a hydroxyl protecting group.
  • R 2’ is -OR 20 and R 20 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4
  • R 2’ is -OR 20 and R 20 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R 20 is TBDMS.
  • R 2’ is -OR 30 , where R 30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.
  • R 2’ is -OR 30 and R 30 is hydrogen or hydroxyl protecting group.
  • R 2’ is -OR 30 and R 30 is a reactive phosphorus group.
  • R 30 is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester.
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is C 1-6 alkyl, optionally substituted with 1, 2,
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each RP1 is C 1-6 alkyl, optionally
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is C 1-6 alkyl, optionally
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is 2-cyanoethyl (-
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is 2-cyanoethyl (-
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is 2-cyanoethyl (-
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , where: RP1 is 2- cyanoethyl (-CH 2 CH 2 CN); each R P2 is independently isopropyl.
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is C 1-6 alkyl, optionally
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is C 1-6 alkyl, optionally substitute
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is 2-cyanoethyl (-
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: each R P1 is 2-cyanoethyl (-
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: R P1 and one of R P2 taken together with the
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: R P1 and one of R P2 taken together with the
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: R P1 and one of R P2 taken together with the
  • R 2’ is -OR 30 and R 30 is -P(OR P1 )N(R P2 ) 2 , -P(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )H, - P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 , where: R P1 and one of R P2 taken together with the
  • R 2’ is -OR 30 and R 30 is a bond to a nucleoside or a nucleotide, or an oligonucleotide.
  • R 30 is a bond to a nucleoside, a nucleotide, or an oligonucleotide
  • the internucleotide linkage between compound of formulae I-II and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester) internucleotide linkage or a modified (e.g., phosphorothioate) internucleotide linkage.
  • R 2’ is -OR 30 and R 30 is linked to 5’-position of a nucleoside, nucleotide, or oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage (e.g., or a modified (e.g., phosphorothioate) internucleotide linkage.
  • R 30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide.
  • R 30 is linked to the 5’- terminal (e.g., 5’-OH) of the oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage. In some other embodiments, R 30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide by a modified (e.g., phosphorothioate) internucleotide linkage. [00299] In some embodiments, R 2’ is -OR 30 and R 30 is a hydroxyl protecting group.
  • R 2’ is -OR 30 and R 30 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,
  • R 2’ is -OR 30 and R 30 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R 30 is TBDMS.
  • R 2 ’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2- methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo- 3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branche
  • each R P is independently -OR O , -SR S , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S .
  • at least one R P is -OR O , e.g., both R P are independently - OR O .
  • each R O can be independently hydrogen, C 1-3 alkyl, or a hydroxyl protecting group.
  • at least one R P is -OR O , e.g., both R P are independently - OR O and each R O is independently hydrogen or C 1-6 alkyl (e.g., C 1-3 alkyl).
  • At least one R P is -OR O , e.g., both R P are independently -OR O and each R O is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl.
  • at least one R P is -OR O , e.g., both R P are independently -OR O and each R O is hydrogen.
  • at least one R P is -OR O , e.g., both R P are independently -OR O and each R O is methyl.
  • at least one R P is -OR O , e.g., both R P are independently -OR O and each R O is ethyl.
  • At least one R P is -OR O , e.g., both R P are independently -OR O and each R O is tert-butyl.
  • at least one R P is -OR O , e.g., both R P are independently -OR O and each R O is independently a hydroxyl protecting group.
  • At least one R P is -OR O , e.g., both R P are independently -OR O and each R O is independently a hydroxyl protecting group selected from the group consisting of pivaloyloxymethyl (POM), BOC or Boc, MOM, MTM, t- butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t-butoxymethyl, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, SEMOR, THP, 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, MTHP, 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl-S,S-dioxide, CTMP, 1,4-dioxan- 2-yl, tetrahydrofuranyl, tetrahydr
  • At least one R P is -OR O , e.g., both R P are independently -OR O and each R O is independently pivaloyloxymethyl (POM).
  • at least one R P is -SR S , e.g., both R P are independently -SR S .
  • each R S can be independently hydrogen, C 1-3 alkyl, or a thiol protecting group.
  • at least one R P is -SR S , e.g., both R P are independently -SR S and each R S is independently hydrogen or C 1-3 alkyl.
  • At least one R P is -SR S , e.g., both R P are independently -SR S and each R S is hydrogen, methyl, ethyl, or propyl.
  • at least one R P is -SR S , e.g., both R P are independently -SR S and each R S is hydrogen.
  • at least one R P is -SR S , e.g., both R P are independently -SR S and each R S is methyl.
  • at least one R P is -SR S , e.g., both R P are independently -SR S and each R S is ethyl.
  • At least one R P is -SR S , e.g., both R P are independently -SR S and each R S is independently a thiol protecting group. In some embodiments, at least one R P is -SR S , e.g., both R P are independently -SR S and each R S is independently pivaloyloxymethyl. [00305] In some embodiments, at least one R P is -N(R N ) 2 , e.g., both R N are independently - N(R N ) 2 S . When R P is -N(R N ) 2 , each R N can be independently hydrogen, C 1-3 alkyl, or a thiol protecting group.
  • At least one R P is -N(R N ) 2 , e.g., both R P are independently -N(R N ) 2 and each R N is independently hydrogen or C 1-3 alkyl.
  • at least one R P is - N(R N ) 2 , e.g., both R P are independently -N(R N ) 2 and each R N is hydrogen, methyl, ethyl, or propyl.
  • at least one R P is -N(R N ) 2 , e.g., both R P are independently -N(R N ) 2 and each R N is hydrogen.
  • At least one R P is -N(R N ) 2 , e.g., both R P are independently -N(R N ) 2 and each R N is methyl. In yet another example, at least one R P is -N(R N ) 2 , e.g., both R P are independently -N(R N ) 2 and each R N is ethyl. In some embodiments, at least one R P is -N(R N ) 2 , e.g., both R P are independently -N(R N ) 2 and each R N is independently an amine protecting group.
  • At least one R P is -N(R N )S(O) 2 R 2S , e.g., each R P is independently -N(R N )S(O) 2 R 2S .
  • at least one R P is -N(R N )S(O) 2 R 2S , e.g., both R P are independently - N(R N )S(O) 2 R 2S , and each R N is independently hydrogen, methyl, ethyl, propyl, or isopropyl, and each R 2S is independently methyl, ethyl, propyl, or isopropyl.
  • At least one R P is -N(R N )S(O) 2 R 2S , e.g., both R P are independently -N(R N )S(O) 2 R 2S , and each R N is independently an amine protecting group, and each R 2S is independently methyl, ethyl, propyl, or isopropyl.
  • B nucleobase
  • B is an optionally modified nucleobase. It is noted that the nucleobase can be a natural or non-natural nucleobase.
  • non-natural nucleobase means a nucleobase other than adenine, guanine, cytosine, uracil, or thymine.
  • exemplary non-natural nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropy
  • purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, content of all which is incorporated herein by reference.
  • the non-natural nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8- (hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(is
  • a non-natural nucleobase is a modified nucleobase, i.e., the nucleobase comprises a nucleobase modification described herein, e.g., the nucleobase is a substituted or modified analog of any of the natural nucleobases.
  • nucleobase modifications include, but not limited to: C-5 pyrimidine with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities, N 2 - and N 6 - with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities of purines, G-clamps, guanidinium G-clamps, and pseudouridine known in the art.
  • the non-natural nucleobase is a universal nucleobase.
  • a universal nucleobase is any modified or unmodified natural or non-natural nucleobase that can base pair with all of adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide comprising the universal nucleobase.
  • Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza- 7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6- methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4- methylinolyl, 4,6-dimethylindolyl, phenyl, nap
  • the natural or non-natural nucleobase is a protected nucleobase.
  • a “protected nucleobase” refers to a nucleobase comprising a nitrogen protecting group, and/or an oxygen protecting group, and/or a sulfur protecting group.
  • the nucleobase is a pyrimidine modified at the C4 position.
  • the nucleobase is a pyrimidine modified at the C5 position.
  • the nucleobase is a purine modified at the N2 position.
  • the nucleobase is a purine modified at the N6 position.
  • the nucleobase is a purine modified at the C6 position.
  • the nucleobase is a N-7 deaza purine, optionally modified at the N7 position.
  • the nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.
  • the nucleobase is uracil, adenine, guanine, or cytosine, optionally each independently comprising a hydroxyl, or amine protecting group.
  • the nucleobase is selected from the group consisting of: .
  • Double-stranded RNA [00316] The skilled person is well aware that double-stranded RNAs comprising a duplex structure of between 19 and 24, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (RNAi). However, others have found that shorter or longer double-stranded oligonucleotides can be effective as well. Accordingly, in some embodiments, a longer double-stranded oligonucleotide described herein is capable of inducing RNA interference. Stated another way, the longer double-stranded oligonucleotides described herein can mediate RNA interference.
  • RNAi RNA interference
  • RNAi refers to the ability to inhibit or reduce the expression of a target nucleic acid, e.g., a target RNA such as a mRNA in a sequence specific manner.
  • dsRNA double-stranded RNA
  • A is -C(*)(H)-, -CH 2 C(*)(H)-, or -C(*)(H)CH 2 -, wherein * is the bond to E; E is a bond or -CH 2 -;
  • the modification replaces a CH 2 OH group on the sugar moiety of the 5’-terminal nucleotide of the sense or antisense strand.
  • the modification replaces the 4’-CH 2 OH group on the furanose ring (e.g.
  • ribofuranose arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose
  • the 5’- CH 2 OH group on the pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • the 5’-terminal nucleotide of the sense or antisense strand e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose,
  • the modification replaces carbon atom in the sugar moiety of the 5’-terrminal nucleotide of the sense or antisense strand.
  • the modification replaces the 4’-C atom in the furanose (e.g.
  • ribofuranose arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose
  • pyranose e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose
  • the sense or antisense strand comprises at its 5’-end a compound of formulae I-XXI described herein.
  • X is O.
  • X is O
  • each R P is OR O .
  • X is O
  • each R P is OH.
  • X is O
  • each R P is ethoxy.
  • X is O
  • each R P is OH.
  • X is O
  • each R P is OR O wherein R O is pivaloyloxymethyl.
  • the 5’-terminal nucleotide of one of the sense and antisense strand is of the structure: , , , , , , , , , ,
  • n is an integer selected from 1 - 3;
  • A is -C(*)(H)-, -CH 2 C(*)(H)-, or -C(*)(H)CH 2 -, wherein * is the bond to E;
  • E is a bond or -CH 2 -;
  • B is an optionally modified nucleobase (e.g., uracil);
  • Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl,
  • the antisense strand of the dsRNA is an oligonucleotide described herein.
  • the term “antisense strand” refers to an oligonucleotide that is substantially or 100% (e.g., exactly) complementary to a target nucleic acid of interest.
  • an antisense strand can be complementary, in whole or in part, to target nucleic acid of interest, such as a messenger RNA, an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding.
  • each strand of the dsRNA can range from 12-40 nucleotides in length.
  • each strand independently can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 27-35 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19- 23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, 21-23 nucleotides in length, 25-35 nucleotides in length, 26-35 nucleotides in length, 27-34 nucleotides in length, 28- 32 nucleotides in length or 29-31 nucleotides in length.
  • the sense and antisense strands can be equal length or unequal length. In some embodiments, the antisense strand is longer, e.g., by 1, 2, 3, 4, or 5 nucleotides than the sense strand.
  • each of the sense and antisense strand is independently 15, 16, 17, 28, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • each of the sense and antisense strand is independently 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, each strand is independently 19, 20, 21, 22 or 23 nucleotides in length.
  • one strand is 18, 19, 20, 21 or 22 nucleotides in length and the other strand (e.g., the antisense strand) is 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense and antisense strands of the dsRNA molecule are complementary to each other and can hybridize to each other to form a double-stranded or duplex region. Accordingly, the dsRNA molecule has a double-stranded or duplex region.
  • the duplex region double-stranded region can be 17-25 nucleotide base pairs in length.
  • the dsRNA can have a duplex region of 17-24 nucleotide pairs in length.
  • the dsRNA has a duplex region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs in length. In some embodiments, the dsRNA has a duplex region of 19, 20, 21 or 22 nucleotide base pairs in length.
  • the dsRNA molecule can have one or more overhang regions (i.e., single-stranded region) and/or capping groups of dsRNA molecule at the 3’-end, or 5’-end or both ends of a strand. Without limitations, the overhang can be 1-3 nucleotides, e.g., 1, 2 or 3 nucleotides in length.
  • the overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.
  • the overhang can form a mismatch with the sequence being targeted or it can be complementary to the sequence being targeted or can be other sequence.
  • the sense and antisense strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. Without limitations the overhang can be present at the 3’-end of only one of the strands or both strands.
  • the dsRNA molecule comprises a single overhang.
  • the dsRNA molecule has a single overhang and the overhang is no more than one, two or three nucleotides in length.
  • the overhang is 2 nucleotides in length.
  • the overhang is present at the 3’-end of a strand (e.g., the antisense strand).
  • the dsRNA comprises a two-nucleotide overhang at the 3’-end of a strand (e.g., the antisense strand).
  • the overhang is present at the 3’-end of the antisense strand.
  • the antisense comprises a 1 or 2 nucleotide overhang at its 3’-end.
  • the dsRNA can also have a blunt end.
  • one end of the dsRNA is a blunt end and the other end has an overhang.
  • the blunt end can be located at the 5’- end of the antisense strand (or the 3’-end of the sense strand) or vice versa.
  • the antisense strand of the dsRNA has a nucleotide overhang at the 3’-end, and the 5’-end is blunt.
  • the asymmetric blunt end at the 5’-end of the antisense strand and 3’-end overhang of the antisense strand favor the guide strand loading into RISC process.
  • the dsRNA has a 2-nucleotide overhang on the 3’-end of the antisense strand and a blunt end at the 5’-end of the antisense strand.
  • the dsRNA molecule has two blunt ends, i.e., at both ends of the dsRNA.
  • the two strands of the dsRNA are of the same length.
  • the antisense strand is of length 18 to 25 nucleotides.
  • the antisense strand is 21-25, 19-25, 19-21 or 21-23 nucleotides in length.
  • the antisense strand is 23 nucleotides in length.
  • the sense strand can be, in some embodiments, 18-25 nucleotides in length.
  • the sense strand is 21-25, 19-25, 19-21 or 21-23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. [00332] In some embodiments, sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
  • Nucleic acid modifications [00333] The longer double-stranded and single-stranded oligonucleotides described herein can comprise one or more nucleic acid modifications. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates (e.g., ligands), and any combinations thereof.
  • nucleic acid modification(s) can be present in any position of longer double-stranded and single-stranded oligonucleotides.
  • a nucleic acid modification(s) can be present in only one strand or both strands of a dsRNA.
  • only the antisense strand comprises at least one, e.g., two, three, four, five or more nucleic acid modifications.
  • only the sense strand comprises at least one, e.g., two, three, four, five or more nucleic acid modifications.
  • both strands independently comprise at least one, e.g., two, three, four, five or more nucleic acid modifications.
  • Embodiments of the various aspects described herein recite specific position(s) on a strand, counting from an end of a strand.
  • the counting of the position is from the first nucleotide at the specified end.
  • the counting of the position can be from the first nucleotide at the specified end of the strand, or the first base-paired nucleotide in the strand at the specified end.
  • the dsRNA comprises a thermally destabilizing modification.
  • a thermally destabilizing modification is meant modification that result in a dsRNA having a lower overall melting temperature (Tm), preferably a Tm with one, two, three or four degrees lower, than the Tm of the dsRNA without having such a modification.
  • thermally destabilizing modifications are described herein below, and can include, but are not limited to, abasic modifications; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy (i.e., 2’-H) modification, acyclic nucleotide (e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA)), threose nucleic acid (TNA), a nucleotide linked by through its 2’-position (i.e., by its 2’-OH group to 5’-position of the subsequent nucleotide (a 2’-5’ RNA modification)); a Hyp-spacer modification; modified internucleotide linkages that decrease the thermal stability of dsRNA duplexes; or nucleobases with impaired W-C H-bonding to complementary base on the opposite strand.
  • 2’-deoxy (i.e., 2’-H) modification e.g.,
  • the dsRNA comprises at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more independently selected thermally destabilizing modifications.
  • the thermally destabilizing modification can be present at any position of the dsRNA. Further, the thermally destabilizing modifications all can be present in one strand or both strands of the dsRNA. In some embodiments, only the antisense strand comprises at least one, e.g., two, three, four or more thermally destabilizing modifications.
  • only the sense strand comprises at least one, e.g., two, three, four or more thermally destabilizing modifications.
  • both the sense and the antisense strands comprise at least one, e.g., two, three, four or more thermally destabilizing modifications.
  • the thermally destabilizing modification can occur on any nucleotide of the sense strand or antisense strand.
  • the thermally destabilizing modification can occur on every nucleotide on the sense strand and/or antisense strand; each thermally destabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise thermally destabilizing modifications in an alternating pattern.
  • the alternating pattern of the thermally destabilizing modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the thermally destabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the thermally destabilizing modifications on the antisense strand.
  • thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, or preferably at position 4, 5, 6, 7, or 8, counting from the 5’-end of the antisense strand. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5 or 9 from the 5’-end of the antisense strand. In some other embodiments, the thermally destabilizing modification is located at position 6, 7 or 8 from the 5’-end of the antisense strand. In some particular embodiments, the thermally destabilizing modification is located at position 7 from the 5’-end of the antisense strand. [00339] In some embodiments, only the antisense strand comprises a thermally destabilizing modification.
  • only the antisense strand comprises a thermally destabilizing modification and said thermally destabilizing modification is located at position 4, 5, 6, 7, or 8, counting from the 5’-end of the antisense strand, preferably the thermally destabilizing modification is located at position 5, 6, 7, or 8; more preferably the thermally destabilizing modification is located at position 6, 7, or 8.
  • only the antisense strand comprises a thermally destabilizing modification and the thermally destabilizing modification is located at position 7 of the antisense strand, counting from the 5’-end of the antisense strand.
  • a thermally destabilizing modification can be located at one of position 2, 3, 4, 5, 6, 7, 8 or 9, or preferably at position 4, 5, 6, 7, or 8, counting from the 5’- end of the longer-ssNA. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5 or 9 from the 5’-end of the longer-ssNA. In some other embodiments, the thermally destabilizing modification is located at position 6, 7 or 8 from the 5’-end of the longer- ssNA. In some particular embodiments, the thermally destabilizing modification is located at position 7 from the 5’-end of the longer-ssNA.
  • dsRNA comprises a thermally destabilizing modification.
  • a thermally stabilizing modification is meant modification that result in a dsRNA having a higher overall melting temperature (Tm), preferably a Tm with one, two, three or four degrees higher, than the Tm of the dsRNA without having such a modification.
  • Tm overall melting temperature
  • Exemplary thermally destabilizing modifications are described herein below, and can include, but are not limited to, 2’-fluoro nucleotides (2’-F modifications), bridged nucleic acid (BNA), e.g., locked nucleic acid (LNA), and cyclohexene nucleic acid (CeNA).
  • the thermally stabilizing modification is a 2’-fluoro nucleotide.
  • thermally stabilizing modification are described herein below. Additional exemplary abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011/133876 and WO2019222479, contents of both of which are incorporated herein by reference in their entireties.
  • dsRNA can comprise at least two, e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more the thermally stabilizing (e.g., 2’-F) modifications.
  • the thermally stabilizing (e.g., 2’-F) modifications all can be present in one strand or both strands of a dsRNA.
  • the sense strand comprises at least one, e.g., two, three, four or more thermally stabilizing (e.g., 2’-F) modifications.
  • the antisense strand comprises at least one, e.g., two, three, four or more thermally stabilizing (e.g., 2’-F) modifications.
  • both the sense and the antisense strands comprise at least one, e.g., two, three, four or more thermally stabilizing (e.g., 2’- F) modifications.
  • the thermally stabilizing (e.g., 2’-F) modification can occur on any nucleotide of the sense strand or antisense strand.
  • the thermally stabilizing (e.g., 2’-F) modification can occur on every nucleotide on the sense strand and/or antisense strand; each thermally stabilizing (e.g., 2’-F) modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise thermally stabilizing (e.g., 2’-F) modifications in an alternating pattern.
  • the alternating pattern of the thermally stabilizing (e.g., 2’-F) modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the thermally stabilizing (e.g., 2’-F) modifications on the sense strand can have a shift relative to the alternating pattern of the thermally stabilizing (e.g., 2’-F) modifications on the antisense strand.
  • the sense strand of the dsRNA comprises at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more thermally stabilizing (e.g., 2’-F) modifications.
  • the sense strand comprises two, three, four, or five thermally stabilizing (e.g., 2’-F) modifications.
  • the sense strand comprises three or four thermally stabilizing (e.g., 2’-F) modifications.
  • a thermally stabilizing (e.g., 2’-F) modification in the sense strand can be present at any positions.
  • the sense strand comprises at least three thermally stabilizing (e.g., 2’-F) modifications.
  • the sense comprises thermally stabilizing (e.g., 2’-F) modification at least at positions 7, 10 and 11, counting from the 5’-end of the sense strand.
  • the sense strand comprises at least four thermally stabilizing (e.g., 2’-F) modifications.
  • the sense comprises thermally stabilizing (e.g., 2’-F) modification at least at positions 7, 9, 10 and 11, counting from the 5’-end of the sense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5’-end of the antisense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5’-end of the antisense strand.
  • the sense strand comprises a block of two, three or four thermally stabilizing (e.g., 2’-F) modification.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11, counting from the 5’-end of the sense strand
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11 from the 5’-end, counting from the 5’-end of the sense strand
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11, counting from the 5’-end of the sense strand
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the antisense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand.
  • the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’- end of the antisense strand.
  • the sense strand does not comprise a thermally stabilizing (e.g., 2’-F) modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.
  • the antisense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more thermally stabilizing (e.g., 2’-F) modifications.
  • the antisense strand comprises two, three, four, five or six thermally stabilizing (e.g., 2’-F) modifications.
  • a thermally stabilizing (e.g., 2’-F) modification in the antisense strand can be present at any position.
  • the antisense strand comprises at least three thermally stabilizing (e.g., 2’-F) modifications.
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense comprises at least four thermally stabilizing (e.g., 2’-F) modifications.
  • the antisense comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense strand comprises at least five thermally stabilizing (e.g., 2’-F) modifications.
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense strand comprises at least six thermally stabilizing (e.g., 2’-F) modifications.
  • the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense strand comprises at least one thermally stabilizing (e.g., 2’-F) modification adjacent to a stabilizing destabilizing modification.
  • the thermally stabilizing (e.g., 2’-F) modification can be the nucleotide at the 5’-end or the 3’-end of the thermally destabilizing modification, i.e., at position -1 or +1 from the position of the thermally destabilizing modification.
  • the antisense strand comprises a thermally stabilizing (e.g., 2’-F) modification at each of the 5’-end and the 3’-end of the thermally destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the antisense strand comprises at least two stabilizing modifications at the 3’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • the sense strand does not comprise a thermally stabilizing (e.g., 2’-F) modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.
  • the dsRNA described herein can comprise at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more 2’-OMe nucleotides.
  • the 2’- OMe nucleotides all can be present in one strand or both strands of a dsRNA.
  • both the sense and the antisense strands comprise at least one 2’-OMe nucleotide.
  • the 2’-OMe modification can occur on any nucleotide of the sense strand or antisense strand.
  • the 2’-OMe modification can occur on every nucleotide on the sense strand and/or antisense strand; each 2’-OMe modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise 2’-OMe modifications in an alternating pattern.
  • the alternating pattern of the 2’-OMe modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the 2’-OMe modifications on the sense strand can have a shift relative to the alternating pattern of the 2’-OMe modifications on the antisense strand.
  • the antisense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or more 2’-OMe modifications.
  • a 2’-OMe modification in the antisense strand can be present at any position.
  • each nucleotide, except for any other specified modification (e.g., thermally destabilizing modification(s), thermally stabilizing modification(s), and/or 2’-deoxy (2’-H) modification(s)) of the antisense strand is independently a 2’-O-methyl nucleotide.
  • the sense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more 2’-OMe modifications.
  • a 2’-OMe modification in the sense strand can be present at any positions.
  • each nucleotide, except for any other specified modification (e.g., thermally stabilizing modification(s), lipophilic modification(s), inverted nucleotide(s), thermally destabilizing modification(s), and/or 2’-deoxy (2’-H) modification(s)) of the sense strand is independently a 2’-O-methyl nucleotide.
  • 2’-deoxy (2’-H) nucleotides [00169]
  • the dsRNA described herein can comprise a 2’-deoxy, i.e., 2’-H nucleotides.
  • the longer double-stranded and single-stranded oligonucleotides described herein can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) 2’-deoxy nucleotides.
  • a 2’-deoxy nucleotide can be present in any position of the sense or antisense strand. Further, 2’-deoxy nucleotides all can be present in one strand or both strands of the dsRNA.
  • sense strand comprises 1, 2, 3, 4, 5 or more 2’-deoxy nucleotides.
  • the sense strand comprises a 2’-deoxy nucleotide at any one of positions 7, 9 and 11, counting from the 5’-end of the sense strand.
  • the sense strand comprises a 2’-deoxy nucleotide at least at position 9, counting from the 5’-end of the strand.
  • the sense strand comprises a 2’-deoxy nucleotide at least at positions 7 and 9, counting from the 5’-end of the strand.
  • the sense strand comprises a 2’- deoxy nucleotide at least at positions 9 and 11, counting from the 5’-end of the strand.
  • antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8 or more 2’-deoxy nucleotides.
  • the antisense strand comprises a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of the strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, counting from the 5’-end of the strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, and 12, counting from the 5’-end of the strand. In another non-limiting example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’- end of the strand.
  • Lipophilic modifications [00173] In some embodiments, the dsRNA described herein can comprise a lipophilic modification.
  • the longer double-stranded and single-stranded oligonucleotides described herein can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) lipophilic modifications.
  • Exemplary lipophilic modifications include nucleotides modified with a lipophilic group, e.g., nucleotides comprising a lipophilic group (e.g., an C 10 -C 30 alkyl, or a C 10 -C 30 alkenyl group, such as a C 16 alkyl, a C 16 alkenyl, a C 18 alkyl, a C 18 alkenyl, a C 20 alkyl, a C 20 alkenyl, a C 22 alkyl, a C 22 alkenyl, a C 24 alkyl, a C 24 alkenyl; C 15 alkyl, a C 15 alkenyl, a C 17 alkyl, a C 17 alkenyl, a C 19 alkyl, a C 19 alkenyl, a C 21 alkyl, a C 21 alkenyl, a C 23 alkyl, or a C 23 alkenyl) at their 2’-position.
  • Some exemplary lipophilic nucleotides include, but are not limited to, 2’-O-hexadecyl-modified nucleotide (Nhd), 2’-O-docosanyl-modified nucleotide (Nda), 2’-O-(omega-hydroxy-hexadecyl)- modified nucleotide (NhdOH), and 2’-O-(omega-hydroxy-docosanyl)-modified nucleotide (NdaOH).
  • a lipophilic modification can be present in any position of the sense or antisense strand. Further, lipophilic modifications all can be present in one strand or both strands of a dsRNA.
  • only the sense strand comprises a lipophilic modification.
  • the sense strand comprises a lipophilic modification at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 or 18, counting from the 5’-end of the sense strand.
  • the sense strand comprises a lipophilic modification at any one of positions 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 or 18, counting from the 5’-end of the sense strand.
  • each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl, 2’-fluoro, 2’-deoxy, LNA, HNA, CeNA, 2’- methoxyethyl, 2’-O-allyl, or 2’-C-allyl, 2’-deoxy, or.
  • the strands can contain more than one modification.
  • each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl or 2’-fluoro. It is to be understood that these modifications are in addition to any other specified modification (e.g., at least one thermally destabilizing modification of the duplex present in the antisense strand) of dsRNA molecule.
  • the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2’- O-methyl or 2’-deoxy.
  • each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2 ⁇ -deoxy-2’-fluoro nucleotide, 2’-O-N-methylacetamido (2’-O-NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O-aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide.
  • each residue of the sense strand and antisense strand is independently modified with 2’- O-methyl nucleotide, 2’-deoxy nucleotide or 2 ⁇ -deoxy-2’-fluoro nucleotide.
  • these modifications are in addition to any thermally destabilizing modification of the duplex present in the antisense strand.
  • the antisense strand comprises at least one thermally destabilizing modification
  • the remaining nucleotides are independently a 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2 ⁇ -deoxy-2’-fluoro nucleotide, 2’-O-N-methylacetamido (2’-O- NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O- aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide.
  • the antisense strand comprises a thermally destabilizing modification and the remaining nucleotides are independently a 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2 ⁇ -deoxy-2’-fluoro nucleotide.
  • the antisense strand comprises: (i) a thermally destabilizing modification at position 5, 6, 7, or 8, counting from the 5’-end of the antisense strand; (ii) at least two, e.g., 3, 4, 5 or 62’- fluoro nucleotides; and (iii) the remaining nucleotides are independently a 2’-O-methyl nucleotide or 2’-deoxy nucleotide.
  • each nucleotide of the sense strand is independently 2’-O- methyl nucleotide, 2’-deoxy nucleotide, 2 ⁇ -deoxy-2’-fluoro (2’-F) nucleotide, 2’-O-N- methylacetamido (2’-O-NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O-aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide.
  • each nucleotide of the sense strand is independently 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2 ⁇ - deoxy-2’-fluoro nucleotide.
  • the sense strand comprises at least two, e.g., 3, 4, 5 or 6 2’-fluoro nucleotides, and the remaining nucleotides are independently a 2’-O-methyl nucleotide or 2’-deoxy nucleotide.
  • At least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisense strand of a dsRNA can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex.
  • the nucleotide at the 1 position within the duplex region from the 5’-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT.
  • the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair.
  • the first base pair within the duplex region from the 5’- end of the antisense strand is an A:U base pair.
  • the sense strand or the antisense strand can comprise the adenosine (A) nucleotide.
  • Modified internucleotide linkages [00180]
  • the dsRNA described herein can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more modified internucleoside linkages.
  • internucleoside linkage refers to a covalent linkage between adjacent nucleosides.
  • Exemplary modified internucleoside linkage include, but are not limited to, phosphodietetrs, phosphorothioates (R, S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH 2 -N(CH 3 )-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino (—CH 2 -N(CH 3 )-O—CH 2 -), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H) 2 -O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH 2 -N(CH 3
  • a modified internucleotide linkage can occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the internucleotide linkage modification can occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern.
  • the alternating pattern of the internucleotide linkage modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand can have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.
  • the dsRNA comprises the modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage(s) in the overhang region.
  • the overhang region comprises two nucleotides having modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between the two nucleotides.
  • modified internucleoside e.g., phosphorothioate or methylphosphonate internucleotide
  • Internucleotide linkage modifications can also be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region.
  • the overhang nucleotides can be linked through modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage, and optionally, there can be additional modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide.
  • modified internucleoside e.g., phosphorothioate or methylphosphonate internucleotide
  • additional modified internucleoside e.g., phosphorothioate or methylphosphonate internucleotide
  • internucleoside e.g., phosphorothioate or methylphosphonate internucleotide
  • linkage linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide.
  • these terminal three nucleotides can be at the 3’-end of the antisense strand.
  • the indicated position refers to the internucleotide linkage that links the nucleotide at said position with the nucleotide one position down stream from said position.
  • an internucleotide linkage at position N means it is between nucleotides N and N+1.
  • an internucleotide linkage at position 1, counting from the 5’-end means the linker is between the nucleotides at positions 1 and 2, counting from the 5’- end.
  • the sense strand comprises one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the sense strand, and one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the sense strand.
  • modified internucleoside e.g., phosphorothioate or methylphosphonate internucleotide
  • the sense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5’-end of the sense strand
  • sense strand further comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3’-end of the sense strand.
  • the antisense strand comprises one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the antisense strand, and one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the antisense strand.
  • modified internucleoside e.g., phosphorothioate or methylphosphonate internucleotide
  • the antisense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5’-end of the antisense strand
  • the antisense strand further comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3’-end of the antisense strand.
  • the sense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2 counting from the 5’-end of the sense strand, a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2, counting from the 3’-end of the sense strand; and the antisense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2 counting from the 5’-end of the antisense strand, a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2, counting from the 3’-end of the antisense strand.
  • a modified internucleoside e.g., phosphorothi
  • the nucleotides in the overhang region of the dsRNA molecule can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-Fluoro, 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl-5-methyluridine, 2’-O- methoxyethyladenosine, 2’-O-methoxyethyl-5-methylcytidine, GNA (glycol nucleic acid), SNA (serinol nucleic acid), TNA (threose nucleic acid), and any combinations thereof.
  • 2’-sugar modified such as, 2’-Fluoro, 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl-5-methyluridine, 2’-O- methoxyethyladenosine, 2’-O-methoxyethyl-5
  • TT can be an overhang sequence for either end on either strand.
  • the 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule can be phosphorylated.
  • the overhang region contains two nucleotides having a phosphorothioate internucleotide linkage between the two nucleotides, where the two nucleotides in the overhang region can be the same or different.
  • 5’-modifications [00188] The 5’-end of a strand of the dsRNA lacking the 5’-terminal phosphate mimic, can also be modified.
  • Exemplary modifications for the 5’-end include, but are not limited a 5’-morpholino nucleotide (e.g., a nucleotide where the 5’-OH group is replaced with a morpholino group), a 5’- dimethylamino nucleotide (e.g., a nucleotide where the 5’-OH group is replaced with a dimthylamino group, a 5’-deoxy nucleotide, an inverted nucleotide (i.e., a nucleotide linked via a 5’->5’ linkage to the rest of the strand), an inverted abasic nucleotide (e.g., an abasic nucleotide linked by a 5’->5’ linkage), or an inverted abasic locked nucleic acid modification (i.e., an LNA lacking a nucleobase and linked by a 5’->5’ linkage) at the 5’-end.
  • the sense strand of the dsRNA comprises a 5’-morpholino nucleotide, a 5’-dimethylamino nucleotide, a 5’-deoxy nucleotide, an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end.
  • the sense strand comprises an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end.
  • the sense strand of the dsRNA comprises an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 3’-end.
  • the sense strand comprises a ligand at its 3’-end.
  • Ligands [00191]
  • the oligonucleotide can comprise a ligand.
  • a ligand can modify one or more properties of the attached molecule (e.g., the dsRNA described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake (cell targeting), charge and clearance.
  • the ligand is a targeting ligand.
  • targeting ligand refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment.
  • Some exemplary targeting ligands include, but are not limited to, antibodies, antigen binding fragments of antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferring receptor ligands (e.g., transferrin), biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, lipoprotein receptor ligands (e.g., LDL and HDL).
  • receptor ligands e.g., transferrin
  • biotin e.g., serotonin receptor ligands
  • PSMA endothelin
  • GCPII endothelin
  • somatostatin e.g., lipoprotein receptor ligands (e.g., LDL and HDL).
  • Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins.
  • the term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.
  • the ligand is a CNS tissue targeting ligand.
  • CNS tissue targeting ligand can be a lipophilic group that conjugated to an internal or terminal position within an oligonucleotide. Exemplary lipophilic ligands are described below.
  • Certain receptors are known to be present on the surface of CNS cells that may be utilized in order to achieve delivery of an oligonucleotide, such as to a neuronal cell, a glial cell, a microglial cell, an oligodendrocytic cell, an ependymal cell, astrocytic cell, a unipolar cell, a bipolar cell, a multipolar cell, a psuedounipolar cell, a pyramidal cell, a basket cell, a stellate cell, a purkinje cell, a betz cell, an amacrine cell, a granule cell, an ovoid cell, a medium aspiny neuronal cell, and/or a large aspiny neuronal cell.
  • an oligonucleotide such as to a neuronal cell, a glial cell, a microglial cell, an oligodendrocytic cell, an ependymal cell, as
  • CNS tissues such as a forebrain tissue, a midbrain tissue, a hindbrain tissue, a diencephalon tissue, a telencephalon tissue, a myelencepphalon tissue, a metencephalon tissue, a mesencephalon tissue, a prosencephalon tissue, a rhombencephalon tissue, a cortices tissue, a frontal lobe tissue, a parietal lobe tissue, a temporal lobe tissue, an occipital lobe tissue, cerebral tissue, a tissue from the thalamus, a tissue from the hypothalamus, a tissue from the tectum, a tissue from the tegmentum, a tissue from the cerebellum, a tissue from the pons, a tissue from the medulla, a tissue from the amygdala, a tissue from the hippocampus, a basal ganglia tissue, a tissue from the
  • the ligand is an ocular tissue targeting ligand.
  • ocular tissue targeting ligand can be a lipophilic group that conjugated to an internal or terminal position within an oligonucleotide. Exemplary lipophilic ligands are described below.
  • Certain receptors are known to be present on the surface of ocular cells that may be utilized in order to achieve delivery of an oligonucleotide, such as to an optic nerve cell, a trabecular meshwork cell, a Schlemm’s canal cell, a juxtacanalicular tissue cell, a ciliary muscle cell, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell.
  • an optic nerve cell such as to an optic nerve cell, a trabecular meshwork cell, a Schlemm’s canal cell, a juxtacanalicular tissue cell, a ciliary muscle cell, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell.
  • ocular tissues such as a retinal blood vessel , episcleral veins or choroid tissue, including a choroid vessel, cornea, pupil, sclera, conjunctiva, optic nerve, iris, lens, aqueous humor, macula, optic disk, retina, ciliary muscle, vitreous humor, vitreous body, choroid, fovea, ciliary body, blood vessels, muscles (lateral rectus muscle, medial rectus muscle, ciliary muscle), ligaments (suspensory ligaments), anterior chamber, posterior chamber, limbal rings, and fovia.
  • the ligand is an asialoglycoprotein receptor (ASGPR) ligand.
  • ASGPR asialoglycoprotein receptor
  • an ASGPR ligand is meant a ligand that binds the ASGPR.
  • the ASGPR ligand comprises one or more (e.g., 1, 2, 3 or more) GalNAc or GalNAc derivatives attached through a bivalent or trivalent branched linker.
  • An exemplary ASGPR ligand is:
  • n 0 -10 (e.g., 1 or 4);
  • a loop forming oligonucleotide where 3 or 4 consecutive nucleosides are modified with GalNAc containing ligands such as, for example, the loop containing oligonucleotide, (5’-gcagcc(G*A*A*A*)ggcugc3’ ; SEQ ID NO: 113), where each lower case base is 2’-O-Methyl substituted and each G* and A* is substituted at the 2’-O position with .
  • a ligand can be linked at any position of either strands of the dsRNA.
  • the ligand can be at the 5’-end, 3’-end or at an internal position of a strand, e.g., sense or antisense strand of the dsRNA.
  • the sense strand comprises the ligand, i.e., the ligand is conjugated to the sense strand.
  • the ligand is conjugated to the 3′ end of the sense strand.
  • the sense strand or antisense strand comprises a lipophilic moiety (e.g., a in vivo delivery enhancing moiety) and a the targeting moiety.
  • the lipophilic moiety and the targeting moiety are independently present within: (a) an internally-modified nucleosides such as, (i) ( ii) (b) a modified internucleotide linkage such as, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(RL1); or (c) a 5’-terminal modification such as (i) -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is wherein Q2 is a bond, C(O), S(O) 2 , or -P(Y’)(OH)-O-; or (ii) -P(Y)(OH)O-R L3 or -C(O)N(H)R L3 , wherein Y is O or S; or (iii) -R L3 , -C(O)R L3 , wherein Y is O or S;
  • R 2 ’ or R 3 ’groups include, but are not limited to, hydrogen, halogen (e.g., 2’-fluoro), hydroxy, 2’-O-alkyl (e.g., 2’-OMethyl), 2’-O-methoxyalkyl (e.g., 2’-O- methoxymethyl, 2’-O-methoxyethyl, or 2’-O-2-methoxypropanyl) modification, 2’-O-allyl modification, 2’-C-allyl modification, 2'-O-N-methylacetamido (2'-O-NMA, i.e.
  • halogen e.g., 2’-fluoro
  • hydroxy hydroxy
  • 2’-O-alkyl e.g., 2’-OMethyl
  • 2’-O-methoxyalkyl e.g., 2’-O- methoxymethyl, 2’-O-methoxyethyl, or 2
  • R 2 ’ or R 3 ’ may be H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O- dimethylaminoethoxyethyl, or O-aminopropyl.
  • R L1 , R L2 and R L3 are each a group containing a lipophilic moiety, such as a C 10-26 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C 12-26 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C 12-24 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C 14-24 saturated or unsaturated hydrocarbon chain.
  • R L1 , R L2 and R L3 are each a group containing a C 14-18 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a C 16 saturated or unsaturated hydrocarbon chain. In one embodiment, R L1 , R L2 and R L3 are each a group containing a saturated or unsaturated C 22 -hydrocarbon chain. [00201] In other embodiments R L1 , R L2 and R L3 are each a group containing a lipophilic moiety, such as a lipophilic vitamin or steroid, including, but not limited to, Vitamin E, Vitamin A (retinol, retinoic acid), and cholesterol. [00202] In one embodiment, when R L3 comprises a lipophilic moiety, then R L3 can be selected from the group consisting of:
  • integer m is 0-10 (e.g., 0; or 1-10 or 1-8; or 0-6; or 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); W is C 1 -C 4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl); R and R’ are each independently H or C 1 -C 4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, or t-butyl); G is G 1 or a saturated or unsaturated C 10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C 21 hydrocarbon chain e., G together with the carbonyl to which it is attached may form
  • R L3 include, but are not limited to the following structures:
  • R L3 include, but are not limited to,
  • R L1 can be selected from the group consisting of -G 1 and -S(O) 2 G 1 .
  • Examples of R L1 include, but are not limited to, the following structures: Further examples of R L1 include, but are not limited to, the following structures: [00204]
  • R L2 is -C(O)R L3 , wherein R L3 is according to any of the preceding embodiments thereof.
  • R L2 can be selected from the group consisting of wherein integer m is 0-8 (for instance, m is 0; or m is 1-8; or m is 0-6; or m is 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); R and R’ are each independently H or an alkyl group such as a C 1 -C 4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl); G is G 1 or a saturated or unsaturated C 10-26 saturated or unsaturated hydrocarbon chain (e.g., a C 21 hydrocarbon chain, i.e., G together with the carbonyl to which it is attached may form a group with 22 carbons); for instance, G may be a linear or branched C
  • R L2 is a nucleobase modified with a G or G 1 group, wherein G and G 1 are as defined above (e.g., a pyrimidine nucleobase modified at the 5’-position with a group comprising G or G 1 ).
  • B 1 include, but are not limited to, , wherein t is selected from 0 – 20 (e.g., 1-12, or 1-10, or 3-12, or 3-10).
  • in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: wherein: B is an optionally modified nucleobase; G 3 is a saturated or unsaturated C 1-20 hydrocarbon group (e.g., C 1-6 alkylene; C 2-6 alkylene; or hexylene);; L K is a linking group such as -O-, -N(H)-, -S-, -S-S-, -C(O)O-, OC(O)-, -C(O)N(H)-, - N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O) 2 -, -S(O) 2 O-, -S(O) 2 N(H)-, -P(O)(OH)O-, - OP(O)(OH)-, -P(S)(OH)O-, - OP(O)
  • L K contains a carbonyl attached to G 2 (e.g., (-N(H)C(O)- or -OC(O)- ), then G 2 is a C 21 hydrocarbon group; and when L K does not contain a carbonyl attached to G 2 , then G 2 is a C 22 hydrocarbon group.
  • R G is hydrogen.
  • R G is OH, In one embodiment, R G is COOH. In another embodiment, R G is CONH 2 . In one embodiment, R G is amino.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • a modified internucleotide linkage is shown with substituent atoms fully described at the phosphorous atom, e.g., where C’ is the 2’-carbon or 3’-carbon atom of a ribose ring, it is understood that the oxygen having the broken bond is the 5'-oxygen of the subsequent nucleotide.
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: ,wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as .
  • n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as .
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), and R G is hydrogen, hydroxy, amino, -COOH, or - C(O)NH 2, such as [00212]
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), and R G is hydrogen, hydroxy, amino, -COOH, or - C(O)NH2, such as [00213]
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1-10, 1-8, 1-6
  • n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is a C 22 alkyl chain. In one embodiment, G is a C 16 alkyl chain [00214] In some embodiments, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C 22 hydrocarbon chain, optionally substituted with one or two groups selected from the group consisting of halogen, -OR G , -SR G , - N(R G ) 2 , -C(O)OR G , -OC(O)R G , -C(O)N(R G ) 2 , -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(
  • n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is C 10 -C 22 alkyl chain (e.g., a C 14 -C 24 alkyl chain, C 16 -C 22 alkyl chain, or a C 16 alkyl chain, or a C 22 alkyl chain). [00215] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: .
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as [00217] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula
  • the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(R L1 ), wherein R L1 is -G 1 or S(O) 2 -G 1 , each as defined above, wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(O)(X)O-, wherein X is -N(H)(R L1 ), wherein R L1 is (such as wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the in vivo delivery enhancing moiety is present within a [00221] a modified internucleotide linkage of the form, (such as wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the preceding nucleotide contains a 2’- fluoro modification.
  • the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification.
  • the in vivo delivery enhancing moiety is present within a [00223] a modified internucleotide linkage of the form (such as wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the preceding nucleotide contains a 2’- fluoro modification. In certain embodiments, the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification.
  • the in vivo delivery enhancing moiety is present within a [00225] a modified internucleotide linkage of the form, -OP(Y)(X)O-, whereinY is O or S and X is wherein G 1 is defined above, such as wherein n is selected from 7- 23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) or [00226]
  • in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a direct bond or through a carrier or linker.
  • in vivo delivery enhancing moiety is conjugated to the 3’-end of the sense or antisense strand via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of the sense or antisense strand via a direct bond or through a carrier or linker. [00227] In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula wherein X is O or S (e.g., S); and R L3 is according to any of the preceding embodiments there.
  • R L3 can be wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • R L3 can be wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • m can be 2-5, or 1, or 2, or 3, or 4 or 5.
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula -R L3 , wherein R L3 is according to any of the preceding embodiments there.
  • R L3 can be wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • R L3 can be such as wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23), and R G is hydrogen, hydroxy, amino, - COOH, or -C(O)NH 2.
  • m can be 2-5, or 1, or 2, or 3, or 4 or 5.
  • R L3 can be , wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • m can be 2-5, or 1, or 2, or 3, or 4 or 5.
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula: , or a salt thereof, wherein X is O or S (e.g., S); L is a divalent linking group (e.g., C1-20 alkyl or C 1-10 alkyl-S-S-C 1-10 alkyl).
  • in vivo delivery enhancing moiety is conjugated to the 5’- end of one of the sense and antisense strands and is of the formula (such as or a salt thereof, wherein q is selected from 0 – 18 (e.g., 1-11 or 1-8, or 3-11, or 3-8) and X is O or S (e.g., S).
  • R ligand is selected from , wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21); and a, b, and c are independently selected from 1- 22, provided that the sum of a + b + c is selected from 2 to 22.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula [00233] [00234] wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21). In one embodiment, n is 13. In another embodiment, n is 19.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula: wherein m is selected from 1-10; n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); E is -C(O)N(H)-(CH 2 ) p -*, -N(H)C(O)-(CH 2 ) p -*, -C(O)O-(CH 2 ) p -*, -OC(O)-(CH 2 ) p -*, - OP(Y)(OH)O-(CH 2 ) p -*, -O-(CH 2 ) p -, -N(H)-(CH 2 ) p , -S-(CH 2 ) p -, -N(H)-(CH 2 )
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula wherein m is selected from 1-10 (e.g., 3-6, or 3); R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH 2 , and n is selected from 7- 23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23).
  • m can be 3-6 and R G is hydrogen; or m can be 3-6 and R G is COOH.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and the 5’-terminal nucleotide is of the formula: where Lipo in R is one of:
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is:
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is L2 wherein R selected from: L ipo2 wherein R is
  • in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: [00240] In one embodiment, in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is , wherein R L2 selected from:
  • in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a carrier or linker, and the carrier or linker is an inverted abasic nucleotide, such as an inverted abasic deoxyribonucleotide or an inverted abasic ribonucleotide, each connected to the remainder of the oligonucleotide via a phosphodiester (PO) or phosphorothioate (PS) linkage.
  • PO phosphodiester
  • PS phosphorothioate
  • Examples include, but are not limited to, , wherein Q 2 is a bond, C(O), S(O) 2 , or -P(Y’)(OH)-O-, Y and Y’ are independently O or S; and R L3 is as defined above.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide and is of the formula or a salt thereof, wherein each X is independently O or S (e.g., each is S); R ligand is selected from the groups listed in Table R-1; and L is a divalent linking group (e.g., C 1-20 alkyl or C 1-10 alkyl-S-S-C 1-10 alkyl).
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula or a salt thereof, wherein each X is independently O or S (e.g., each is S) and R ligand is selected from wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula or a salt thereof, wherein each X is independently O or S (e.g., each is S) and Rligand is selected from wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).and L is a divalent linking group (e.g., C 1-20 alkyl or C 1-10 alkyl-S-S-C 1-10 alkyl.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula or a salt thereof, wherein each X is O or S (e.g., each is S) and Rligand is selected from ,,wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).
  • R L1 , R L2 , and R L3 are each a group containing at least one targeting moiety, such as an integrin receptor ligand (e.g., avB3, avB5, or avB6-targeting ligand), a chemokine receptor ligand, a transferring receptor ligand (e.g., transferrin), a serotonin receptor ligand, an Asialoglycoprotein receptor-targeting ligand, a lipoprotein receptor ligand (e.g., LRP1- targeting ligand), etc.
  • the targeting moiety can be selected from:
  • each R is independently C 1-10 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl); and R La is hydrogen, C 1-10 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t- butyl, isobutyl, butyl, or hexyl), C 3-8 cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl).
  • R La is hydrogen, C 1-10 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t- butyl, isobutyl, butyl, or hexyl), C 3-8 cycloalkyl, 3-8 membered heterocyclyl,
  • cyclooctyne BCN, or DBCO
  • a tetrazine with a terminal alkyne or cycloalkyne e.g. cyclooctyne
  • a thiol and maleimide with or without hydrolysis of the product.
  • L and L’ are independently one of: (a) -L 1 -[G-L 2 ] q -G-L 3 -* wherein q is 0 or an integer selected from 1-10; (b) -L 1 -G-L 2 -G-L 3 -*; (c) -L 1 -G-L 3 -*; (d) -G-L 3 -*; (e) -L 1 -G-*; or (f) -G-*.
  • L 1 is selected from one of the groups: (a) a bond, C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH), wherein and R N is hydrogen or C 1-6 alkyl; (b) a bond, C(O), P(O)(OH), or P(S)(OH); (c) a bond; (d) C(O); (e) P(O)(OH); or (f) P(S)(OH); each L 2 and L 3 is independently selected from one of the groups: (a) -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, - N(R N )C(O)O-, -N(R N )C(O)O-, -N(R N )C
  • q is 0, 1, 2, 3, 4, or 5. In another embodiment of L or L’, q is 0, 1, 2, 3, or 4. In another embodiment of L or L’, q is 0, 1, 2, or 3. In another embodiment of L or L’, q is 0, 1, or 2. In another embodiment of L or L’, q is 1, 2, 3, 4, or 5, In another embodiment of L or L’, q is 1, 2, 3, or 4. In another embodiment of L or L’, q is 1, 2, or 3. In another embodiment of L or L’, q is 1 or 2. In another embodiment of L or L’, q is 4. In another embodiment of L or L’, q is 3. In another embodiment of L or L’, q is 2.
  • L is one of: (a) wherein k i 1 N s an integer from 1 to 10; L is bond, C(O), C(S), C(NR ), S(O) 2 , P(O)(OH), or P(S)(OH) (e.g., L 1 is a bond, C(O), P(O)(OH), or P(S)(OH)); and R N is hydrogen or C 1-6 alkyl; (b) wherein k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7; (c) wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3); (d) wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3
  • L’ is : (a)*-G-L 1 -, wherein L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); and G is C 1- 10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); and G is C 1- 10 alkyl; (b)*-G-[L 2 -G] q -L 1 -, wherein L 1 is a bond, CH 2 , C(O), S(O) 2 , P(O)(OH), or P(S)(OH); each L 2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -
  • R L and R L1 that comprise a targeting ligand include, but are not limited to, [00254] Additional examples of R L1 and R L3 that comprise a targeting ligand include, but are not limited to, [00255] Examples of R L2 that comprise a targeting ligand include, but are not limited to, [00256] Additional examples of R L2 that comprise a targeting ligand include, but are not limited to, [00257]
  • each R X is an integrin-receptor targeting ligand such as, [00258]
  • R L1 or R L3 is .
  • R L2 is [00260]
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is:
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: [00261]
  • multiple targeting ligands may be connected to a branched multivalent linker.
  • R L1 , R L2 and R L3 can comprise a branched linking group ( ⁇ ) capable of supporting multiple targeting ligands (e.g., at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3).
  • the mutiple targeting moieties can be connected through an R L , R L1 , and R L2 of the form, (R X -L-ZZ-) z - ⁇ -T- , wherein each R X is a targeting moiety; z is at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3, T is -L’-T’-**, wherein ** is the bond to ⁇ , and T’ is O, S, N(H), C(O), S(O) 2 , C(O)N(H), N(H)C(O), OC(O), OC(O), -P(O)(OH)-, -P(S)(OH)-, -OP(O)(OH)-, -OP(S)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, -OP(O)(OH)O-, or -OP(S)
  • T is selected from the following, wherein ** is the bond to ⁇ : (a) -C(O)-X 1 -L 5 -X 2 -C(O)-**, wherein X 1 and X 2 are each independently C 1-10 alkyl; or C 2-10 alkyl; or C 4-10 alkyl; or C 6-10 alkyl; or C 2-8 alkyl; or C 2-6 alkyl; or C 2-4 alkyl; (b) -C(O)-C 2-20 alkyl-C(O)-**, such as -C(O)-C 2-12 alkyl-C(O)-**, (c) -C(O)-C 6-20 alkyl-C(O)-**, such as -C(O)-C 6-12 alkyl-C(O)-**, (d) -C(O)-C 10 alkyl-C(O)-** and (e) -C(O)-CH 2 CH 2
  • T is selected from the following, ** is the bond to ⁇ : (f) -N(H)C(O)-C 2-20 alkyl-C(O)-**, (g) - N(H)C(O)-C 6-20 alkyl-C(O)-**, such as - N(H)C(O)-C 6-12 alkyl-C(O)-**, (h) - N(H)C(O)-C 10 alkyl-C(O)-**, (i) -C(O)-C 2-20 alkyl-C(O)N(H)-**, (j) -C(O)-C 6-20 alkyl-C(O)N(H)-**, (k) -C(O)-C 6-12 alkyl-C(O)N(H)-**, (l) -C(O)-C 10 alkyl-C(O)N(H)-**, (m) -N(H)C
  • T is selected from the following, ** is the bond to ⁇ : (a) -N(H)C(O)-X 3 -ZZ-X 4 -C(O)-**, (b) -C(O)-X 3 -ZZ-X 4 -C(O)N(H)-**, (c) N(H)C(O)-X 3 -ZZ- X 4 -C(O)N(H)-**, wherein X 3 and X4 are independently C 2-12 alkyl; or C 4-10 alkyl; or C 6-10 alkyl; or C 4-8 alkyl; and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, -OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct).
  • T is selected from the following (w) -L 6 -[G 5 -O] q5 -G 5 -L 4 -**, wherein L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C 1- 6 alkyl; each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); each G 5 is independently C 1-10 alkyl; (x) -C(O)-[CH 2 CH 2 -O] q5 -G 5 -L 4 -**, L 4 is -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )
  • L 4 is -A 1 -B 1 or -B 1 -A 1 -, wherein each A 1 is independently -O- or -N(H)-, and each B 1 is independently C(O), G 5 is C 1-10 alkyl (e.g., C 2-10 alkyl or C 2-6 alkyl); and (z) -C(O)-[CH 2 CH 2 -O] q5 - C 2-10 alkyl-C(O)N(H)-**; wherein in each of the preceding ** is the bond to ⁇ , and q5, when present, is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4.)
  • Examples of branched linking group ( ⁇ ) include, but are not limited to, wherein the broken bond is the bond to L’.
  • R L and R L1 that comprise a branched linker to a targeting ligand include, but are not limited to,
  • branched R L1 and R L3 include,
  • branched R 12 examples include,
  • targeting moiety is bonded to the 3’-oxygen of the 3’- terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: selected from 1 - 10 (e.g., r is 7); and each R is:
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is:
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: [00273] In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: selected from 1 - 10 (e.g., r is 7); and each R is: wherein R x is a targeting ligand.
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: ligand.
  • each R x is an integrin-receptor targeting ligand such as,
  • each R x is an ASGPR ligand, such as
  • L and/or L’ may be a bond when ZZ is formed by reaction of a functional group within the ligand or oligonucleotide.
  • ZZ be formed by reaction of: a thiol (SH) group within one or more cysteine (C) residues; or an amino (NH 2 ) group within one or more lysine (K) residues; or a primary amide group (CONH 2 ) within one or more asparagine (N) or glutamine (Q) residues; or an azide group (N 3 ) within one or more modified amino acid residues (e.g., an azide within an 6-azidolysine residue; or a carboxylic acid group within one or more aspartic acid or glutamic acid residues.
  • the dsRNA agent comprises a double-stranded region formed between the sense and antisense strands and optionally one or two single-stranded non-loop overhang, and wherein the one or more lipophilic moieties are conjugated to either the doublestranded region or the non-loop overhang.
  • the dsRNA agent does not contain a loop (e.g., stem loop) region.
  • the dsRNA agent contains a loop (e.g., stem loop) region, and the one or more lipophilic moieties are not conjugated to the loop (e.g., stem loop) region.
  • the dsRNA agent comprises a sense strand of 10 to 53 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand.
  • the sense strand may be 10 to 49, 12 to 49, 12 to 45, 12 to 42, 12 to 40, 15 to 49, 15 to 45, 15 to 42, 15 to 40, 15 to 38, or 15 to 36 nucleotides in length.
  • the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length.
  • the region of complementarity to the target sequence is at least 19 contiguous nucleotides in length.
  • the sense strand comprises at its 3'-end a stem-loop set forth as: S 1 -L-S 2 , in which S 1 is complementary to S 2 , and in which L forms a loop between S 1 and S 2 .
  • the first 17 to 25 nucleotides counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 11 to 28 counting from 5’ end of the sense strand forms a 3'-end a stem-loop set forth as: S 1 -L-S 2 .
  • the length of the stem loop S 1 -L-S 2 is 11 to 28, 13 to 26, or 15 to 24 nucleotides in length. In one embodiment, the stem loop S 1 -L-S 2 is 16 nucleotides in length. In some embodiments, the stem loop S 1 -L-S 2 comprises a sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 105).
  • L is at least 3, 4, or 5 nucleotides in length. In some embodiments, L comprises a sequence of GAAA.
  • the sense strand is 36 nucleotides in length, the first 20 nucleotide counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 16 nucleotides forms a stem loop S 1 -L-S 2 .
  • the 16-nucleotide stem loop S 1 -L-S 2 has the sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 105), wherein L is GAAA.
  • the one or more lipophilic moieties are conjugated to a nonterminal position of the sense strand.
  • the one or more lipophilic moieties are conjugated to one or more nucleotides of the stem loop S 1 -L-S 2 . In some embodiments, the one or more lipophilic moieties are conjugated to one or more nucleotides of the loop L.
  • S 1 and S 2 are complementary and contain 4-10 nucelotides, e.g., S 1 and S 2 each contain 6 complementary nucelotides.
  • S 1 and S 2 are complementary and contain 4-10 nucelotides and L is GAAA, e.g., Si and S2 each contain 6 complementary nucelotides and L is GAAA.
  • the one or more lipophilic moieties containing one or more saturated or unsaturated C 22 hydrocarbon chains are conjugated to one or more internal positions on at least one strand of the dsRNA agent.
  • in vivo delivery enhancing moiety is connected in series with another targeting moiety, as described herein.
  • a sense or antisense strand can contain a series modification at the 3 ’-end or 5 ’-end of the oligonucleotide, such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety.
  • the series modification is of the form, wherein Q is selected from wherein R L2 is according to any preceding embodiment, wherein one of the broken bonds connects to a 5 ’-oxygen of a nucleoside or a 3’- oxygen of a nucleoside and the other connects to a 5 ’-terminal or 3 ’-terminal modification as described herein.
  • a sense or antisense strand can contain a series modification of the form, wherein Q is selected from wherein
  • R L2 is according to any preceding embodiment, each Y is independently O or S; one of the broken bonds connects to a 5 ’ -oxygen of a nucleoside or a 3 ’ -oxygen of a nucleoside and the other connects any of the 5’-terminal modifications described above or 3’-terminal modifications described above.
  • a sense or antisense strand can contain a series modification of the the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; each Y is independently O or S; one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety) and the other comprises a second ligand moiety (e.g., a targeting moiety).
  • each Y is independently O or S
  • one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety) and the other comprises a second ligand moiety (e.g., a targeting moiety).
  • a sense or antisense strand can contain a series modification of the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; each Y is independently O or S; one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of R L or R L2 above) and the other comprises a second ligand moiety (e.g., a targeting moiety).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety, such as any of R L or R L2 above
  • a second ligand moiety e.g., a targeting moiety
  • an oligonucleotide may have a series modification at the 5 ’-end of the formula:
  • n is selected from 7-23 (e.g.,11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each
  • Y is independently O or S and R 510 and R 520 are wherein each Y is independently O or S.
  • a sense or antisense strand can contain a series modification of the form, wherein the broken bond connects to the 3’-oxygen of a 3’-terminal nucleoside; each Y is independently O or S; one of R 31 and R 32 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of R L2 above) and the other comprises a second ligand moiety (e.g., a targeting ligand).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety, such as any of R L2 above
  • a second ligand moiety e.g., a targeting ligand
  • an oligonucleotide may have a series modification at the 3 ’-end of the formula: wherein n is selected from
  • each Y is independently O or S and R 310 and R 320 are indepedently O or S.
  • [00296] in another embodiment, can contain a single modification at the 5’-end, the 3’-end or at an internal position that contains both the in vivo delivery enhancing moiety and the targeting moiety.
  • single modification is bonded to the 3 ’-oxygen of the 3 ’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is wherein R L2 and R L3 comprises the in vivo delivery enhancing moiety (e.g., according to any in vivo delivery enhancing moiety embodiment of R L2 or R L3 above); R TG comprises the targeting moiety (e.g., R TG is according to Formula (X) (below), wherein R 5 is -L-ZZ-L’-, where L, ZZ, and L’ are defined for Formula (X) or an embodiment thereof;
  • E is -C(O)N(H)-(CH 2 ) p -*, -N(H)C(O)-(CH 2 ) p -*, -C(O)O-(CH 2 ) p -*, -OC(O)-(CH 2 ) p -*, - OP(Y)(OH)O-(CH 2 ) p -*, -O-(CH 2 ) p -, -N(H)-(CH 2 ) p , -S-(CH 2 ) p -, -N(H)-O-(CH 2 ) p -, -O-N(H)-(CH 2 ) p -, N(H)N(H)-(CH 2 ) p -, or -S-S-(CH 2 ) p -*, -Ph-(CH 2 ) p -, -OPh-(CH 2 )
  • E 1 is -O-, -S-, or -N(H)-;
  • T is a bond or -L 6 -G 1 -[L 5 -G 1 ] q1 -L 4 -**, wherein ** is the bond to E; ql is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L 4 , L 5 , and L 6 are independently a bond, -A’-B’-A 1 - or ZZ 1 ; ZZ 1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne);
  • a reactive pair e.g., a reaction between an azide and an alkyne or a cycloalkyne
  • each G 1 is independently -D’-E’-F 1 -, wherein D 1 , E 1 , and F 1 are independently a bond, Ci- loalkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups;
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-;
  • each B 1 is independently a bond, C(O), C(S), C(NR N1 ), S(O), S(O) 2 , P(O)(OH),
  • each R N1 is independently hydrogen or C 1-6 alkyl, or two R N1 within an -A’-B’-A 1 - group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl.
  • the single modification is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is wherein R D is as defined above.
  • R D is In one embodiment of the 3’- or 5 ’-modification, R D is for example, G 1 can be C 1-10 alkyl. In one embodiment of the 3’- or 5 ’-modification, R D is wherein n is selected from 7-
  • R G is hydrogen, hydroxy, amino, -COOH, or - C(O)NH 2 ; and G 1 is C 1-10 alkyl.
  • R D is wherein n is selected from 7-23 (e.g., 11-23, or 11 -
  • R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH 2 ; G 1 is C 1-10 alkyl; and R TG is -(N)H)-(CH 2 ) q -N(H)-R Lig , wherein q is selected from 1 - 20 (e.g., 2-20 or 2-18 or 2-16 or 2-14, or 2-12, or 2-10; or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) and R Lig is
  • R D is R L3 . In one embodiment of the 3 or 5 ’-modification, R D is In one embodiment of the 3 ’- or
  • R D is for example, G 1 can be C 2-20 alkyl. In one embodiment of the 3 ’- or 5 ’-modification, wherein n is selected from 7-23
  • R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH 2 ;
  • G 1 is C 2-20 alkyl.
  • R D is wherein n is selected from 7-23 (e.g., 11-23, or 11 - 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23);
  • R G is hydrogen, hydroxy, amino, -COOH, or -C(O)NH 2 ;
  • G 1 is C 2 -2oalkyl, and -C(O)-R TG is , , and the broken bond is the bond between R TG and the nitrogen.
  • a sense or antisense strand can contain two different ligand modifications, one at the 3'-end of the strand and the other at the 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety.
  • a sense or antisense strand can contain two different ligand modifications, one is an internal modified nuceloside or modified internucleotide linkage of the strand and the other at the 3 ’-end or 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety.
  • the modified nuceloside represented by L1 can be located at a position in the strand selected from positions 2 to n-1, where the strand contains n nucleotides (e.g., n-1 is 20 when n is 21).
  • segment (1) of the strand contains 5 nucleotides and segment (2) contains the remainder of the nucleotides within the strand.
  • “Position” herein when referring to a modified nucleotide, nucleoside, or internucleotide linkage is counted from the 5 ’-end of the strand, for example, position 6 includes the 6th nucleotide from the 5 ’-end of the strand and the 6th internucleotide linkage counting from the 5 ’-end of the strand.
  • L1 comprises the in vivo delivery enhancing moiety and a sense or antisense strand can be represented by one of: wherein B is an optionally modified nucleobase (e.g., A, C, G, U, or T); R 13 or R L1 comprises the in vivo delivery enhancing moiety, L2 comprises the targeting moiety.
  • B is an optionally modified nucleobase (e.g., A, C, G, U, or T)
  • R 13 or R L1 comprises the in vivo delivery enhancing moiety
  • L2 comprises the targeting moiety.
  • R L is selected from the group consisting of: wherein n is selected from 7-23 (e.g., 11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 13; and in another embodiment, n is 19.
  • R L1 is selected from the group consisting of: and , wherein n is selected from 7-23 (e.g., 11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 7; and in another embodiment, n is 10.
  • R L is selected from the group consisting of: embodiment, R L1 is selected from the group consisting of:
  • the sense strand of the dsNA has one of the following modification patterns: Table B: Exemplary sense strand motifs
  • n is a 2’-O-methyl-modified nucleotide
  • (dN) is a 2’-deoxy-nucleotide
  • Nf is a 2’-fluoro-modified nucleotide (e.g., 2 ’-deoxy-2’ -fluoro modified nucleotide); and the sense strand optionally comprises either:
  • each (I) is an inverted nucleotide (e.g., an inverted abasic nucleotide, such as an inverted abasic ribonucleotide, such as an inverted abasic deoxyribonucleotide);
  • (L1) and (L2) are independently hydrogen or a group comprising a ligand , wherein the ligand is selected from:
  • a lipophilic group examples include a group comprising an C 10 -C 30 alkyl, or a C 10 -C 30 alkenyl group, e.g., a C 10 alkyl, C 10 alkenyl, C 12 alkyl, C 12 alkenyl, C 14 alkyl, C 14 alkenyl, C 15 alkyl, C 15 alkenyl, C 16 alkyl, a C 16 alkenyl, a C 18 alkyl, a C 18 alkenyl, a C 20 alkyl, a C 20 alkenyl, a C 22 alkyl, a C 22 alkenyl, a C 24 alkyl, a C 24 alkenyl; C 15 alkyl, a C 15 alkenyl, a C 17 alkyl, a C 17 alkenyl, a C 19 alkyl, a C 19 alkenyl, a C 21 alkyl, a C 21 alkenyl, a C 21 alken
  • a cell-receptor targeting ligand such as a group comprising an Asialoglycoprotein receptor-targeting (ASGPR) ligand, an integrin- receptor targeting ligand (e.g., avB3, avB5, or avB6-targeting ligand), a lipoprotein receptor-targeting ligand (e.g., LRP1 -targeting ligand); or
  • ASGPR Asialoglycoprotein receptor-targeting
  • an integrin- receptor targeting ligand e.g., avB3, avB5, or avB6-targeting ligand
  • a lipoprotein receptor-targeting ligand e.g., LRP1 -targeting ligand
  • precursor functional group where the precursor functional group is suitable for post-synthetic functionalization with a ligand (e.g. as descirbed in (i) or (ii)) containing or conjugated to a complementary reactive functional group;
  • precursor functional groups include, but are not limited to, amino, carboxy, primary amido (-C(O)NH 2 ), N- succinamido, azido (-N 3 ), mercapto (-SH), active esters (e.g., an N- hydroxysuccinimde ester (NHS ester) or a pentafluorophenyl ester),
  • cyclooctynyl e.g., bicyclo[6.1.0]nonynyl (BCN) or dibenzocyclooctynyl (DBCO)
  • BCN bicyclo[6.1.0]nonynyl
  • DBCO dibenzocyclooctynyl
  • trans-cyclooctenyl 2-methylsulfonylpyrimidinyl, 4-vinylpyridinyl, and protected forms thereof; or
  • nucleotide comprising a ligand modification, as described above, that replaces the nucleotide decribed above;
  • lipophile modified nucleotides include, (Nhd) - 2’-O-hexadecyl-modified nucleotide; (Nda) - a 2’-O-docosanyl-modified nucleotide; (NhdOH) - 2’-O-(omega-hydroxy-hexadecyl)-modified nucleotide); or (NdaOH) - a 2’-O-(omega-hydroxy-docosanyl)-modified nucleotide.
  • each of the nucleotides are connected in series (i.e., in a 3 ’->5’ manner) via optionally modified internucleotide linkages.
  • each of the nucleotides are connected by phosphodiester or phosphorothioate internucleotide linkages.
  • the nucleotide at one of the internal positions is substituted for a lipophile modified nucleotide.
  • the lipophile modified nucleotide is substituted at one of positions 4-8 or 12-18 of the sense strand, counting from the 5 ’end.
  • the lipophile modified nucleotide is substituted at one of positions 4-8 or 12-18 of the sense strand (e.g., one of position 5, 6, 7, 8, 12, 13, 14, 15, 16, or 17; or at position 5; or at position 6; or at position 7; or at position 8; or at position 12; or at position 13; or at position 14; or at position 15; or at position 16; or at position 17.)
  • the lipophile modified nucleotide is substituted at one of positions 3-6 or 10- 16 of the sense strand (e.g., one of position 3, 4, 5, 6, 10, 11, 12, 13, 14, 15 or 16); or at position 3; or at position 4; or at position 5; or at position 6; or at position 10; or at position 11 ; or at position 12; or at position 13; or at position 14; or at position 15; or at position 16.)
  • Examples of lipophile modified nucleotides include
  • the preceding sense strand examples comprise 5’-(L1)- attached to the 5 ’-terminal nucleotide (e.g., through the 5’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L1 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • L1 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • the preceding sense strand examples comprise -(L2)-3', attached to the 3 ’-terminal nucleotide (e.g., through the 3’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • the nucleotide at one of the internal positions is substituted for a lipophile modified nucleotide and the sense strand comprises 5’-(L1)- attached to the 5’-terminal nucleotide (e.g., through the 5’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L1 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • L1 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L1 is a cell-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L1 is a integrin-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the LI is an avB6 integrin-receptor targeting ligand.
  • the nucleotide at one of the internal positions is substituted for a lipophile modified nucleotide and the sense strand comprises -(L2)-3', attached to the 3’-terminal nucleotide (e.g., through the 3’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
  • the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L2 is a cell-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L2 is a integrin-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L2 is an avB6 integrin-receptor targeting ligand.
  • the preceding sense strand examples comprise 5’-(L1)- attached to the 5 ’-terminal nucleotide (e.g., through the 5’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, and -(L2)-3', attached to the 3 ’-terminal nucleotide (e.g., through the 3’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein one of L1 and L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand; and the other of L1 and L2 is a lipophilic ligand
  • the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the cell-receptor targeting ligand is an avB6 integrin-receptor targeting ligand.
  • the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester linkage linkage s, counting from the 5 ’-end of the oligonucleotide.
  • the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester linkages, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in m nucleotides in length where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in m nucleotides in length where the nucleotide in m nucleotides in length, the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage, and the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 21 and 22 are connected by a phosphorothioate internucleotide linkage; and positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in 21 nucleotides in length For a nucleotide in 21 nucleotides in length, the nucleotides at positions 19 and 20 are connected by a phosphorothioate internucleotide linkage; and positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • each (inv) attached to a 5 ’-terminal nucleotide is connected via a phosphorothioate linkage (5’ -5’).
  • each (inv) attached to a 3 ’-terminal nucleotide is connected via a phosphorothioate linkage (3’ -3’).
  • each (inv) attached to a 5 ’-terminal nucleotide is connected via a phosphorothioate linkage (5 ’-5’), and each (inv) attached to a 3 ’-terminal nucleotide is connected via a phosphorothioate linkage (3’ -3’).
  • the sense strand is according to any one of S1-S35 wherein the nucleotide at any one of positions 4-8 or 13-18 counting from the 5’-end of the strand, is replaced with a nucleotide that is substituted with a ligand group (e.g., a lipophilic group).
  • a ligand group e.g., a lipophilic group
  • the sense strand of the dsNA has one of the following modification patterns:
  • n is a 2’-O-methyl-modified nucleotide
  • (dN) is a 2 ’-deoxy-nucleotide
  • Nf is a 2’-fluoro-modified nucleotide
  • (L) is a ligand-modified nucleotide, e.g., comprising a lipophilic group (e.g., C 16 or C 22 modification), a cell-receptor targeting ligand, such as an Asialoglycoprotein receptor-targeting (ASGPR) ligand, an integrin-receptor targeting ligand, or a lipoprotein receptor-targeting ligand ; and s is a phosphorothioate internucleotide linkage,
  • a lipophilic group e.g., C 16 or C 22 modification
  • a cell-receptor targeting ligand such as an Asialoglycoprotein receptor-targeting (ASGPR) ligand, an integrin-receptor targeting ligand, or a lipoprotein receptor-targeting ligand
  • s is a phosphorothioate internucleotide linkage
  • each (s) is a phosphorothioate or a phosphodiester internucleotide linkage (e.g., in certain embodiments, each (s) is a phosphorothioate); each (J) is independently an optional linking group (such as an inverted nucleotide (I) (e.g., an inverted abasic nucleotide, such as an inverted abasic ribonucleotide, such as an inverted abasic deoxyribonucleotide));
  • I inverted nucleotide
  • I inverted abasic nucleotide
  • inverted abasic ribonucleotide such as an inverted abasic deoxyribonucleotide
  • (L1) and (L2) are independently hydrogen or a group comprising a ligand
  • the sense strand optionally comprises a 5 ’-morpholino nucleotide, a 5’- dimethylamino nucleotide, a 5 ’-deoxy nucleotide, an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5 ’-end.
  • the antisense strand of the dsNA has one of the following modification patterns:
  • n is a 2’-O-methyl-modified nucleotide
  • s is a phosphorothioate internucleotide linkage (3’- 5’)
  • (dN) is a 2’-deoxy-nucleotide
  • Nf is a 2’-fluoro-modified nucleotide
  • * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose);
  • A is -C(*)(H)-, -CH 2 C(*)(H)-, or -C(*)(H)CH 2 , wherein * is the bond to E;
  • E is a bond or -CH 2 -;
  • Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3 -methylcyclobutyl or cyclobutylmethyl;
  • each R P is independently -OR O , -SR s , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; each R s is independently hydrogen, C 1-3 alkyl, or a thiol protecting group; each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group; and
  • R 2S is C 1-3 alkyl, and where the preceding structure replaces the 4’-CH 2 OH group on the ribose ring of the 5 ’-terminal nucleotide; or
  • n 1, 2 or 3;
  • each R P is independently -OR O , -SR s , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; each R s is independently hydrogen, C 1-3 alkyl, or a thiol protecting group; each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group; and
  • R 2S is C 1-3 alkyl; or wherein:
  • each R P is independently -OR O , -SR s , -N(R N ) 2 , or -N(R N )S(O) 2 R 2S , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; each R s is independently hydrogen, C 1-3 alkyl, or a thiol protecting group; each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group; and
  • R 2S is C 1-3 alkyl
  • X A is O or S
  • Y A is O or S
  • Q 4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropyhnethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl; or
  • each R P is independently -OR O , -N(R N )2, or -N(R N )S(O)2R 2S , wherein: each R O is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; each R N is independently hydrogen, C 1-3 alkyl, or an amine protecting group; and
  • R 2S is C 1-3 alkyl
  • Q 5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3 -methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q 5 are optionally and independently replaced with -C(O)-, -S(O) 2 -, -O-, -S-, or -N(R NQ )-, wherein R NQ is hydrogen, methyl,
  • R 2’ and R 3’ are as defined for formulae I-XXIII.
  • each of the nucleotides are connected in series (i.e., in a 3 ’->5’ manner) via phosphodiester or phosphorothioate internucleotide linkages.
  • G thermally destabilizing modification
  • Ngn a glycol nucleic acid, S-isomer
  • N2p a 2'-phosphate nucleotide (i.e., a 3’-RNA connected by 3’-5’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively);
  • MM a nucleobase mismatch to the sense strand
  • the nucleotide position 5, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the nucleotide position 6, counting from the 5’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the nucleotide position 7, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the nucleotide position 8, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the antisense strand may be selected from any one of AS40 - AS58 as recited in Table E:
  • the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5 ’-end of the oligonucleotide.
  • the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
  • nucleotide in m nucleotides in length where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in m nucleotides in length where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide and the 3 ’-terminal nucleoside is linked to a ligand by a phosphorothioate linkage.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide, and the nucleoside at position 23 is linked to a ligand via a phosphorothioate linkage; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide, and the nucleoside at position 21 is linked to a ligand via a phosphorothioate linkage.
  • nucleotide in m nucleotides in length where the nucleotide in m nucleotides in length, the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage, and the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 21 and 22 are connected by a phosphorothioate internucleotide linkage; and positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. [00342] In certain embodiments, where the nucleotide in m nucleotides in length:
  • nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage
  • nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 1 and 2; 2 and 3; 21 and 22; and 22 and 23 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
  • nucleotide in 21 nucleotides in length the nucleotides at positions 1 and 2; 2 and 3; 19 and 20; and 20 and 21 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5 ’-end of the oligonucleotide.
  • nucleotide in m nucleotides in length where the nucleotide in m nucleotides in length:
  • nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage
  • nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
  • nucleotide in 23 nucleotides in length the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 22 and 23 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
  • nucleotide in 21 nucleotides in length the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 20 and 21 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5 ’-end of the oligonucleotide.
  • the antisense strand of the dsNA has one of the following modification patterns in Table F.
  • the nucleotide at one of positions 5-8, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G), such as :
  • N2p a 2'-phosphate nucleotide (i.e., a 3’-RNA connected by 3’-5’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively);
  • MM a nucleobase mismatch to the sense strand (and the nucleotide is optionally modified by 2 ’-fluoro or 2’-OMethyl);
  • the nucleotide position 5, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the nucleotide position 6, counting from the 5’- end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the nucleotide position 7, counting from the 5’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the nucleotide position 8, counting from the 5’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
  • the antisense strand may be selected from any one of AS136 - AS171 as recited in Table G.
  • each of sense strand SI through SI 23 can be hybridized or duplexed with any one of antisense strands AS 1 through AS 171.
  • the longer double-stranded oligonucleotide product described herein comprises any one of the sense strands SI through S123hybridized/duplexed with any one of the antisense strands AS1 through AS171.
  • the sense strand is selected from an embodiment having 21 nucleotides and the antisense strand is selected from an embodiment having 23 nucleotides.
  • the sense strand is selected from an embodiment having 21 nucleotides and the antisense strand is selected from an embodiment having 21 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 19 nucleotides and the antisense strand is selected from an embodiment having 21 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 19 nucleotides and the antisense strand is selected from an embodiment having 19 nucleotides.
  • the longer single-stranded oligonucleotide described herein is any one of the sense strands S1 through S123 or any one of the antisense strands AS1 through AS171.
  • the dsRNA or oligonucleotide described herein can be formulated in compositions.
  • the dsRNA or oligonucleotide described herein can be formulated into pharmaceutical compositions for therapeutic use.
  • a pharmaceutical composition comprising a therapeuticallyeffective amount of one or more of the dsRNA or oligonucleotide described herein, taken alone, or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
  • compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.
  • terapéuticaally effective amount means that amount of a compound, material, or composition comprising a conjugate described herein which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
  • compositions for use with the methods described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • a dsRNA or oligonucleotide described herein can be formulated for administration by, for example, by intravenous, oral, aerosol, or topical route.
  • the compositions can be formulated for intralesional, intratumoral, intraperitoneal, subcutaneous, intramuscular, or intravenous injection, infusion, liposome-mediated delivery, topical, intrathecal, gingival pocket, per rectum, intrabronchial, nasal, transmucosal, intestinal, oral, ocular, or otic delivery.
  • dsRNA described herein can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank’s solution or Ringer’s solution.
  • the dsRNA can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • the dsRNA or oligonucleotide can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the dsRNAs or oligonucleotides described herein can be formulated with one or more lipids for delivery.
  • the dsRNAs or oligonucleotides described herein can be formulated in lipid particles.
  • lipid particle refers to a vesicle formed by one or more lipid components. Lipid particles are typically used as carriers for nucleic acid delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API).
  • lipid particle compositions for such delivery are composed of ionizable or cationic lipids, phospholipids (especially compounds having a phosphatidylcholine group), cholesterol, and a polyethylene glycol (PEG) lipid; however, these compositions may also include other lipids.
  • the ionizable lipid is typically employed to condense the nucleic acid cargo at low pH and to drive membrane association and fusogenicity.
  • the phospholipid is typically employed to enhance fusogenicity.
  • the cholesterol is typically employed to provide membrane integrity.
  • the PEG-lipid is typically employed to provide steric stabilization.
  • the sum composition of lipids typically dictates the surface characteristics and thus the protein (opsonization) content in biological systems thus driving biodistribution and cell uptake properties.
  • a lipid particle can be a lipid nanoparticle (LNP).
  • a lipid particle described herein comprises an ionizable lipid.
  • ionizable lipid refers to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
  • ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower.
  • ionizable lipids have a pK a of the protonatable group in the range of about 4 to about 7. Ionizable lipids are also referred to as cationic lipids herein.
  • the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3) .
  • the ionizable lipid is the lipid ATX-002 ..
  • the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-l-amin.e.
  • the ionizable lipid is Compound 6 or Compound 22 described in WO2015/199952, content of which is incorporated herein by reference in its entirety.
  • ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid particle.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically employed to enhance fusogenicity.
  • non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C 10 -C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids suitable for use in the lipid particles include nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isoprop
  • the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some preferred embodiments, the non-cationic lipid is DPSC.
  • the non-cationic lipid can comprise 0-30% (mol) of the total lipid present in the lipid particle.
  • the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid particle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1.
  • the lipid particle can further comprise a conjugated lipid molecule.
  • conjugated lipid refers to a lipid molecule conjugated with a non-lipid molecule, such as a PEG, polyoxazoline, polyamide, or polymer (e.g., cationic polymer). Generally, these lipids are used to inhibit aggregation of lipid particles and/or provide steric stabilization.
  • conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
  • Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O- (2',3'-di(tetradecanoyloxy)propyl- 1 -O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S- DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)- 1,2- distearoyl-sn-
  • DAG
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol ( 1 -[8'-(Cholest-5-en-3 [beta]-oxy)carboxamido-3',6'- dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4- Ditetradecoxylbenzyl- [omega] -methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol
  • the PEG-lipid can be PEG-DMG, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] .
  • the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid particle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid particle.
  • the lipid particle can further comprise a component, such as a sterol, to provide membrane integrity.
  • a sterol that can be used in the lipid particle is cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5 ⁇ -cholestanol, 5 ⁇ -coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5 ⁇ - cholestane, cholestenone, 5 ⁇ -cholestanone, 5 ⁇ -cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
  • the component providing membrane integrity such as a sterol
  • the lipid particle comprises: an ionizable lipid; a non-cationic lipid; a conjugated lipid that inhibits aggregation of particles; and a sterol.
  • Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed.
  • the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non- cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition.
  • the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic-lipid by mole or by total weight of the composition.
  • the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition.
  • the composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition.
  • the composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition.
  • the formulation may also be a lipid particle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition;
  • the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.
  • the lipid particle comprises: an ionizable lipid in an amount from about 20 mol % to about 90 mol % of the total lipid present in the particle; a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipid present in the particle; a conjugated lipid that inhibits aggregation of particles in an amount from about 0.5 mol % to about 20 mol % of the total lipid present in the particle; and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipid present in the particle.
  • the lipid particle comprises ionizable lipid / non-cationic-lipid / sterol / conjugated lipid at a molar ratio of 50:10:38.5:1.5.
  • the total lipid to nucleic acid (mass or weight) ratio is from about 10: 1 to about 30: 1.
  • the amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.
  • dsRNA and/or oligonucleotide described herein may be formulated for administration in any convenient way for use in medicine, e.g., human or veterinary medicine, by analogy with other pharmaceuticals.
  • dsRNA and/or oligonucleotide described herein or a pharmaceutical composition comprising same can be administered to a subject using different routes of delivery.
  • routes of delivery include, but are not limited to intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
  • the dsRNA and/or oligonucleotide described herein can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be parenteral, topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), or oral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
  • the route and site of administration may be chosen to enhance targeting.
  • Lung cells might be targeted by administering the dsRNA and/or oligonucleotide described herein in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the dsRNA and/or oligonucleotide described herein and mechanically introducing the dsRNA and/or oligonucleotide described herein.
  • a method of administering an dsRNA and/or oligonucleotide described herein, to a subject e.g., a human subject.
  • the present invention relates to an dsRNA and/or oligonucleotide described herein for use in inhibiting expression of a target gene in a subject.
  • the method or the medical use includes administering a unit dose of the dsRNA and/or oligonucleotide described herein.
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target gene.
  • the unit dose for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application
  • the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days.
  • the unit dose is not administered with a frequency (e.g., not a regular frequency).
  • the unit dose may be administered a single time.
  • the effective dose is administered with other traditional therapeutic modalities.
  • dsRNA and/or oligonucleotide described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
  • the administration of the dsRNA and/or oligonucleotide composition described herein is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the medication can be provided in measured doses or in a dispenser which delivers a metered dose.
  • the dsRNA and/or oligonucleotide described herein is administrated via subcutaneous or intravenous administration. [00388] In some embodiments, the dsRNA and/or oligonucleotide described herein is administrated via intrathecal administration.
  • the dsRNA and/or oligonucleotide described herein is administrated via intravitreal administration.
  • the disclosure also provides a cell comprising a compound, dsRNA or oligonucleotide described herein described herein.
  • the term “cell” refers to a single cell as well as to a population of (i.e., more than one) cells.
  • a cell can be a prokaryotic or eukaryotic cell.
  • Exemplary cells include, but are not limited to, bacterial cells, yeast cells, plant cell, animal (including insect) or human cells.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell. It is noted a cell can be in vivo, in vitro or ex vivo.
  • a dsRNA or oligonucleotide described herein can be provided in a kit, e.g., as a component of a kit.
  • the kit includes (a) a dsRNA or oligonucleotide described herein, and optionally (b) informational material.
  • the informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and/or the use of a dsRNA or oligonucleotide described herein for the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the dsRNAs or oligonucleotides, their molecular weight, concentration, date of expiration, batch, or production site information, and so forth. In some embodiments, the informational material relates to using dsRNA or oligonucleotide to treat, prevent, or diagnosis of disorders and conditions.
  • the informational material can include instructions to administer the dsRNA or oligonucleotide in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein).
  • the informational material can include instructions to administer the dsRNA or oligonucleotide to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition needing treatment.
  • the informational material of the kits is not limited in its form.
  • the informational material e.g., instructions
  • the dsRNA or oligonucleotide can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the dsRNA or oligonucleotide be substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • the dsRNA or oligonucleotide is provided as a dried form, reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer, can optionally be provided in the kit.
  • the kit can include one or more containers for the components of the kit.
  • the kit contains separate containers, dividers, or compartments for the different components of the kit.
  • the dsRNA or oligonucleotide can be contained in a bottle, vial, or syringe, and the informational material can be contained association with the container.
  • the separate elements of the kit are contained within a single, undivided container.
  • the dsRNA or oligonucleotide is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more-unit dosage forms of the dsRNA or oligonucleotide.
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the dsRNA or oligonucleotide.
  • the containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
  • the kit optionally includes a device suitable for administration of the dsRNA or oligonucleotide, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.
  • the device is an implantable device that dispenses metered doses of the dsRNA or oligonucleotide.
  • the disclosure also features a method of providing a kit, e.g., by combining components described herein.
  • the kit can further comprise additional components and/or reagents for practicing the methods described herein using the dsRNA or oligonucleotide described herein.
  • aspects of the disclosure also relate to methods for inhibiting the expression of a target gene in a subject.
  • the method comprises administering to the subject in an amount sufficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the antisense strand is substantially complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is substantially complementary to a target gene.
  • the disclosure further relates to a use of dsRNA and/or oligonucleotide for inhibiting expression of a target gene in a target cell.
  • the disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell in vitro.
  • Another aspect the invention relates to a method of modulating the expression of a target gene in a cell, comprising administering to said cell a dsRNA and/or oligonucleotide described herein. It is noted that administering to the cell can be in vitro or in-vivo. Methods for administering a compound to a cell are well known and available to one of skill in the art. As used herein, administering the compound to the cell means contacting the cell with the compound so that the compound is taken up by the cell.
  • the cell can be contacted with the dsRNA/oligonucleotide in a cell culture e.g., in vitro or ex vivo, or the compound can be administrated to a subject, e.g., in vivo.
  • a cell culture e.g., in vitro or ex vivo
  • the compound can be administrated to a subject, e.g., in vivo.
  • the term “contacting” or “contact” as used herein in connection with contacting a cell includes subjecting the cells to an appropriate culture media, which comprises a dsRNA and/or oligonucleotide described herein.
  • “contacting” or “contact” includes administering the dsRNA and/or oligonucleotide described herein, e.g., in a pharmaceutical composition to a subject via an appropriate administration route such that the compound contacts the cell in vivo.
  • said administering to the cell can include subjecting the cell to an appropriate culture media which comprises the dsRNA/oligonucleotide.
  • said administering to the cell includes administering the dsRNA/oligonucleotide to a subject via an appropriate administration route such that the dsRNA/oligonucleotide is administered to the cell in vivo.
  • the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP
  • the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1(YKL-4O), RPS25, a2-AR, and GSK3a.
  • the target gene is selected from the group consisting of myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYSI), glycogen synthase 1
  • the target gene is selected from the group consisting of myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYSI),
  • the target gene is selected from the group consisting of adrenoceptor beta 1 (ADRBl); calcium voltage-gated channel subunit alphal C (CACNA1C); calcium voltage-gated channel subunit alphal G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type l(AGTRl); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban
  • the target gene is selected from the group consisting of MUC5B, TSLP, IL33, ALOX15, AGER(RAGE), MUC5AC, and STAT6.
  • the target gene is selected from the group consisting of Delta 4- Desaturase, Sphingolipid 1 (DEGS1); leptin; folliculin (FLCN); Zinc Finger Protein 423 (ZFP423); Cyclin Dependent Kinase 6 (CDK6); Regulatory Associated Protein Of MTOR Complex 1 (RPTOR); Mechanistic Target Of Rapamycin Kinase, (mTOR); Forkhead Box Pl (FOXP1); Phosphodiesterase 3B (PDE3B); and Activin A Receptor Type 1C (ACVR1C).
  • the target gene is selected from the group consisting of TTR for hATTR (CNS, ocular and systemic), myocilin (MYOC), Ras homolog family member A (RhoA), SSB (small RNA binding exonuclease protection factor La), optineurin, Carbonic Anhydrase 2 (CA2), Rho associated coiled-coil containing protein kinase 1 (ROCK1), Rho associated coiled- coil containing protein kinase 2 (ROCK2), Angiopoietin-Like 7 (ANGPTL7), and cytochrome P450 1B1 (CYP1B1).
  • CNS ocular and systemic
  • MYOC myocilin
  • RhoA Ras homolog family member A
  • SSB small RNA binding exonuclease protection factor La
  • optineurin Carbonic Anhydrase 2 (CA2), Rho associated coiled-coil containing protein kinase 1 (ROCK1)
  • the target gene is selected from the group consisting of PPARG, ADIPOQ, CD36, LPL, ADAMTS9, RASD1, GYS2, CAT, DPYS, MLXIPL, VEGFA, HLA- DQA1, LIPA, CTSC, FCGR2A, GBE1, SH2B3, CTSK, CDKN2B, ELN, ARG1, HHEX, TCF7L2, CYP2A6, ALDH2, ACADS, GLYCTK, LDLR, HAL, ACER3, SLC7A7.
  • PTPN22 CDKN1C, LEPR, SNAI2, PGM1, IGF2BP2, TTPA, ATP7B, ASPA, ADRB3, MAN2B1, RCAN1, PIGL, TBX1, LMNB1, FBP1, ETFA, LMNA, LAT2, PRKAG2, SELENBP1, TKT, PCSK1, PSAP, NDN, ACY1, SATB2, CYP21A2, POMC, CDC73, CTSH, CFTR, CTSA, G6PD, EXT1, EXT2, CPT1A, SEMA5A, WFS1, KIT, ACAT1, GGCX, FKBP6, PPARGC1B, DGCR6, HMGCS2, PEPD, WRN, LCAT, KLF13, SLC16A2, DHCR7, ITPR3, CLDN4, FZD9, SLC30A2, APOA5, HADHA, CDKAL1, PTPN2, LIPC, CD226, PON1, MCCC1, EIF2AK3, GYG1,
  • Embodiments of the various aspects described herein include a reactive phosphorus group.
  • reactive phosphorus groups are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages.
  • Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P in or P v valence state including, but not limited to, phosphoramidite, H- phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries.
  • Reactive phosphorous group in the form of phosphoramidites (P III chemistry) as reactive phosphites are a preferred reactive phosphorous group for solid phase oligonucleotide synthesis.
  • the intermediate phosphite compounds are subsequently oxidized to the Pv state using known methods to yield phosphodiester or phosphorothioate internucleoside linkages.
  • the reactive phosphorous group is -P(OR P1 )N(R P2 ) 2 , - P(SR P1 )N(R P2 ) 2 , -P(O)(OR P1 )N(R P2 ) 2 , -P(S)(OR P1 )N(R P2 ) 2 , -P(R P3 )N(R P2 ) 2 , -P(O)(SR P1 )N(R P2 ) 2 , - P(O)(OR P1 )H, -P(S)(OR P1 )H, -P(O)(SR P1 )H, -P(O)(OR P1 )R P3 , -P(S)(OR P1 )R P3 , or -P(O)(SR P1 )R P3 .
  • R P1 is an optionally substituted C 1-6 alkyl.
  • each R P2 is independently optionally substituted C 1-6 alkyl.
  • each R P2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl. It is noted that when two or more R P2 groups are present in the reactive phosphorous group, they can be same or different. Thus, in some none-limiting examples, when two or more R P2 groups are present, the R P2 groups are different. In some other non-limiting examples, when two or more R P2 groups are present, the R P2 groups are same. In some embodiments, each R P2 is isopropyl.
  • both R P2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl.
  • R P1 and one of R P2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl.
  • each R P3 is independently optionally substituted C 1-3 oalkyl substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl, or optionally substituted C 2 -C 30 alkynyl (e.g., optionally substituted C 1 -C 10 alkyl, optionally substituted C 2 -C 10 alkenyl, or optionally substitutedC 2 -C 10 alkynyl).
  • R P3 is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which can be optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -Cealkoxy.
  • the reactive phosphorous group is -P(OR P1 )(N(R P2 ) 2 ).
  • the reactive phosphorous group is -P(OR P1 )(N(R P2 ) 2 ), where R P1 is 2-cyanoethyl (- CH 2 CH 2 CN) and each R P2 is isopropyl.
  • Hydroxyl protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley
  • Exemplary hydroxyl protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boe), methoxymethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacohnethyl (GUM), t-butoxymethyl, pivaloyloxymethyl (POM), acetyloxymethyl, (AM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3 -bromotetrahydropyranyl, t
  • hydroxyl protecting group is benzyl, benzoyl, 2,6- dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).
  • DMT 4,4'-dimethoxytrityl
  • Pixyl 9-phenylxanthine-9-yl
  • MOX 9-(p-methoxyphenyl)xanthine-9-yl
  • the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4 '-dimethoxytrityl. In some embodiments, hydroxyl protecting group is pivaloyloxymethyl.
  • protected hydroxyl and “protected hydroxyl” as used herein mean a group of the formula -OR Pro , wherein R Pro is an oxygen protecting group as defined herein.
  • amine protecting group also referred to as an amino protecting group herein.
  • Amine protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • Additional exemplary amine protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N - phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N- 2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-l,3,5-triazacyclohexan-2-
  • Sulfur protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • the practice of the present invention can employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.
  • the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • RNA e.g., mRNA
  • mRNA e.g., a transcript of a gene that encodes a protein
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein
  • target gene e.g., a target gene
  • RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNAs, and viral RNAs, can also be targeted.
  • RNAi refers to the ability to silence, in a sequence specific manner, a target gene, e.g., mRNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
  • the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.
  • Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • Other conditions such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • the term “substantially complementary”, with respect to a nucleotide sequence in relation to a reference nucleotide sequence means a nucleotide sequence having a percentage of identity between the substantially complementary nucleotide sequence and the exact complementary sequence of said reference of at least at least 80%. e.g., at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (i.e., exactly complementary).
  • identity is assessed over a length of at least 15, e.g., at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides.
  • off-target and the phrase “off-target effects” refer to any instance in which an effector molecule against a given target causes an unintended affect by interacting either directly or indirectly with another target sequence, a DNA sequence or a cellular protein or other moiety.
  • an “off-target effect” may occur when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of an siRNA.
  • the absence of a given treatment or agent can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant increase in such level.
  • a “terminal” of a strand refers to position 1, counting from the nearest end of the strand.
  • a 5’-terminal refers to position 1, counting from the 5’-end of the strand.
  • a 3 ’-terminal refers to position 1, counting from the 3 ’-end of the strand.
  • a “terminal region” of a strand refers to positions 1-4, e.g., positions 1, 2, 3, and 4, counting from the nearest end of the strand.
  • a 5 ’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 5 ’-end of the strand.
  • a 3’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 3’-end of the strand.
  • a 5 ’-terminal region for the antisense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the antisense strand.
  • a preferred 5 ’-terminal region for the antisense strand is positions 1 , 2 and 3 counting from the 5 ’ -end of the antisense strand.
  • a 3 ’ -terminal region for the antisense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand.
  • a preferred 3 ’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 3’- end of the antisense strand.
  • a 5 ’-terminal region for the sense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the sense strand.
  • a preferred 5 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 5 ’-end of the sense strand.
  • a 3 ’-terminal region for the sense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand.
  • a preferred 3 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 3 ’-end of the sense strand.
  • a “central region” of a strand refers to positions 5-17, e.g., positions 6- 16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5 ’-end of the strand.
  • the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand.
  • a preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5’-end of the sense strand.
  • a more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5 ’-end of the sense strand.
  • a preferred central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 15 16 and 17, counting from 5’-end of the antisense strand.
  • a more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5’- end of the antisense strand.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g. animal or a plant).
  • ex vivo refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).
  • in vivo refers to events that occur within an organism (e.g. animal, plant, and/or microbe).
  • the term "subject" or "patient” refers to any organism to which a composition disclosed herein can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “patient” and “subject” are used interchangeably herein.
  • a subject can be male or female.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders.
  • compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment. Alternatively, a subject can also be one who has not been previously diagnosed.
  • a “subject in need” of testing for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • the subject is human.
  • the subject is an experimental animal or animal substitute as a disease model.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice.
  • the term subject is further intended to include transgenic species.
  • the subject can be of European ancestry.
  • the subject can be of African American ancestry.
  • the subject can be of Asian ancestry.
  • parenteral administration refers to administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein. [00459] As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • a dosage unit refers to a form in which a pharmaceutical agent is provided.
  • a dosage unit is a vial comprising lyophilized antisense oligonucleotide.
  • a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
  • treat By the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) it is meant that the severity of the subject’s condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • prevent refers to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
  • the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present invention.
  • a glycolic nucleic acid is wherein B is a modified or unmodified nucleobase, and * is R, S, or racemic.
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1 ’, C2’, C3’, C4’ or 04’) are independently or in combination absent from the nucleotide.
  • bonds between the ribose carbons e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’
  • bonds between the ribose carbons e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4
  • B is a modified or unmodified nucleobase
  • R 1 and R 2 independently are H, halogen, OR 3 , or alkyl
  • R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.
  • Unlocked nucleic acid (UNA) modification encompasses monomers with bonds between C1’-C4’ being removed (i.e. the covalent carbon-oxygen-carbon bond between the Cl’ and C4’ carbons).
  • bonds between C1’-C4’ being removed
  • the C2’-C3’ bond i.e. the covalent carbon-carbon bond between the C2’ and C3’ carbons
  • the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety).
  • the acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.
  • UNA has the structure: where B is a modified or unmodified nucleobase; R, R’, R”, R’” and R” are independently H, OH, CH 3 , CH 2 CH 3 , O- alkyl, NH 2 , NHMe or NMei; and each * is independently R, S, or racemic.
  • the UNA modification is where B is a modified or unmodified nucleobase and R is H, OH or O-alkyl.
  • Modified unlocked nucleic acid (mUNA) modification include, but are not limited to the following: wherein Base is a modified or unmodified nucleobase, and * is R, S, or racemic.
  • the UNA modification is selected from the group consisting of:
  • abasic modification refers to a nucleotide or analog thereof that does not have a nucleobase.
  • Some exemplary abasic modifications include, but are not limited to, the following:
  • R is H, Me, Et or OMe; R’ is H, Me, Et or OMe; R” is H, Me, Et or OMe; and * represents either R, S or racemic.
  • the thermally destabilizing modification is selected from the group consisting of: wherein B is a modified or unmodified nucleobase, and * is R, S, or racemic.
  • B is a modified or unmodified nucleobase
  • * is R, S, or racemic.
  • the 2 ’-5 ’ RNA is where Base is a modified or unmodifed nucleobase.
  • TNA is a nucleotide comprising a threose sugar instead of a ribose sugar, where its 3 ’-position is linked to the 3’-positoon of the nucleotide upstream of it, and its 2’-psotion is linked to the 5’-positoon of the nucleotide downstream of it.
  • TNA has the structure
  • TNA modifications include, but are not limited to, the following: where B is a modified or unmodified nucleobase.
  • a Hyp-spacer modification comprises a hydroxyprolinol monomer, e.g., where R is H or a modified or unmodified nucleobase.
  • Additional exemplary sugar modifications include, but are not limited to the following: where B is a modified or unmodified nucleobase, and R is H or C 1 - Cealkyl (e.g., methyl or ethyl),
  • nucleotides with impaired W-C H-bonding to complementary base on opposing strand include, but are not limited to, nucleotides comprising a nucleobase independently selected from the following:
  • Exemplary non-canonical bases with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand include, but are not limited to, inosine, nebularine, 2-aminopurine, 2,4-difhiorotoluene, 5 -nitroindole, 3-nitropyrrole, 4-fluoro-6- methylbenimidazole and 4-methylbenzimidazole.
  • Exemplary ⁇ -nucleotides include, but are not limited to, B is a modified or unmodified nucleobase, and R is H, OH, OCH 3 , F, NH2, NHMe, NMe 2 or O-alkyl.
  • Exemplary phosphate modifications known to decrease the thermal stability of dsNA duplexes compared to natural phosphodiester linkages include, but are not limited to, the following: where the alkyl for the R group can be a C 1 -C 6 alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.
  • bridged nucleic acid includes, but is not limited to, nucleotides that comprise a five-membered or six-membered bridged structure with a fixed 3'-endo confirmation, also known as the north confirmation.
  • the bridged structure connects the 2'-arbon (e.g., 2’-oxygen) of the ribose sugar to the 4' carbon of the ribose sugar.
  • Various different bridge structures are possible containing carbon, oxygen, nitrogen, and hydrogen atoms.
  • the BNA is locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • aliphatic means a saturated or unsaturated and straight, branched, and/or cyclic hydrocarbon having the defined number of carbon atom. Examples include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, and cycloalkylalkynyl, having the defined number of carbon atoms.
  • alkyl refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms.
  • alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl.
  • “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl.
  • Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.
  • a “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH2 group to an NH group or an O group).
  • the term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof.
  • the heteroatom(s) is placed at any interior position of the heteroalkyl group.
  • up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3
  • alkenyl refers to an alkyl group containing at least one carbon-carbon double bond.
  • the alkenyl group can be optionally substituted with one or more “alkyl group substituents.”
  • Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-l-yl andheptadec-8,ll-dien-l-yl.
  • alkynyl refers to an alkyl group containing a carbon-carbon triple bond.
  • the alkynyl group can be optionally substituted with one or more “alkyl group substituents.”
  • exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl.
  • Useful alkynyl groups include the lower alkynyl groups.
  • cycloalkyl refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and/or alkylene.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl.
  • Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • Heterocyclyl refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively).
  • Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system.
  • 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent.
  • exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4- morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3- dioxanyl, 1,4-dioxanyland the like.
  • Aryl refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms.
  • the aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and — NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl.
  • Heteroaryl refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • O, N, or S e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzisothi
  • halogen refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen radioisotope or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen-substituted moiety or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.
  • haloalkyl refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different.
  • fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.
  • Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g.
  • halosubstituted (C1-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).
  • amino means -NH2.
  • alkylamino means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl).
  • dialkylamino means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -N(alkyl)(alkyl).
  • alkylamino includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.”
  • arylamino means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and — N(aryl)2.
  • heteroarylamino means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen.
  • NHheteroaryl and — N(heteroaryl)2.
  • two substituents together with the nitrogen can also form a ring.
  • the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like.
  • Exemplary alkylamino includes, but is not limited to, NH(Cl-ClOalkyl), such as — NHCH3, — NHCH2CH3, — NHCH2CH2CH3, and — NHCH(CH3)2.
  • Exemplary dialkylamino includes, but is not limited to, — N(Cl-C10alkyl)2, such as N(CH3)2, — N(CH2CH3)2, — N(CH2CH2CH3)2, and — N(CH(CH3)2)2.
  • aminoalkyl means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms ( — N — ) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl.
  • an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.
  • hydroxyl and “hydroxyl” mean the radical — OH.
  • alkoxyl refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O- alkenyl, and -O-alkynyl.
  • Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
  • the alkoxy and aroxy groups can be substituted as described above for alkyl.
  • Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n- propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O- hexyl, O- cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.
  • carbonyl means the radical — C(O) — . It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.
  • carboxy means the radical — C(O)O — . It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., carboxyl group.
  • cyano means the radical — CN.
  • nitro means the radical — NO2.
  • heteroatom refers to an atom that is not a carbon atom.
  • heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens.
  • alkylthio and thioalkoxy refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur.
  • the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • alkylthio also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups.
  • sulfinyl means the radical — SO — . It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.
  • sulfonyl means the radical — SO2 — . It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.
  • thiocarbonyl means the radical — C(S) — . It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.
  • acyl refers to an alkyl-CO — group, wherein alkyl is as previously described.
  • exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms.
  • Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
  • Aroyl means an aryl-CO — group, wherein aryl is as previously described.
  • Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.
  • Arylthio refers to an aryl-S — group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.
  • Aralkyl refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
  • Aralkyloxy refers to an aralkyl-0 — group, wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxy group is benzyloxy.
  • Aralkylthio refers to an aralkyl-S — group, wherein the aralkyl group is as previously described.
  • An exemplary aralkylthio group is benzylthio.
  • Alkoxycarbonyl refers to an alkyl-0 — CO — group.
  • exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-0 — CO — group.
  • exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-0 — CO — group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an H2N — CO — group.
  • Alkylcarbamoyl refers to a R'RN — CO — group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.
  • Dialkylcarbamoyl refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.
  • “Acyloxy” refers to an acyl-0 — group, wherein acyl is as previously described.
  • “Acylamino” refers to an acyl-NH — group, wherein acyl is as previously described.
  • “Aroylamino” refers to an aroyl-NH — group, wherein aroyl is as previously described.
  • substituted means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified.
  • substituted refers to a group “substituted” on a substituted group at any atom of the substituted group.
  • Suitable substituents include, without limitation, halogen, hydroxyl, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxylalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.
  • two substituents, together with the carbons to which they are attached to can form a ring.
  • an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.
  • any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2 or 3 substituents selected independently from the group consisting of halogen, -OR 22 ’ -N(R 22 ) 2 , -SR 22 , -C(O)OR 22 , -C(O)N(R 22 ) 2 , wherein R 22 is hydrogen or C 1-3 alkyl (e.g., 2,2,2-trifhioroethyl, l,3-dimethoxyprop-2-yl).
  • any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with one or two substituents selected independently from the group consisting of halogen, -OR 22, -N(R 22 ) 2 , -SR 22 , -C(O)OR 22 , -C(O)N(R 22 ) 2 , wherein R 22 is hydrogen or C 1-3 alkyl (e.g., 2,2,2-trifhioroethyl, 1 ,3-dimethoxyprop-2-yl).
  • An “isocyanato” group refers to a NCO group.
  • a “thiocyanato” group refers to a CNS group.
  • An “isothiocyanate” group refers to a NCS group.
  • Compound 16 Compound 15 (600 mg, 0.781 mmol) was dissolved in 80% aqueous AcOH and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-10% MeOH in ethyl acetate) to obtain compound 16 as a white form (332 mg, 91%).
  • Compound 17 A solution of 16 (350 mg, 0.752 mmol) and DIPEA (0.393 mL, 2.26 mL) in CH 2 CI 2 was added dropwise 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.185 mL, 0.827 mmol) at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction was quenched with saturated NaHCO 3 (aq.) and the reaction mixture was stirred for 10 min. The organic layer was washed with saturated NaHCO 3 (aq.), water and brine and dried with Na 2 SO 4 and concentrated under vacuum.
  • Compound 20 A solution of p-toluenesulfonic acid monohydrate (2.99 g, 15.74 mmol) in MeOH/DCM (500 mL) was added to a solution of compound 19 (7.9 g, 12.10 mmol) in a 1:1 mixture of MeOH/DCM (500 mL) at 0°C. . After 30 min at 0°C, solid NaHCO3 was added until the orange color of the solution disappeared. The resulting mixture was filtered, the filtrate was combined with silica gel, and the volatiles were evaporated to dryness. The residue was purified by ISCO automated column (120g) using 0-10% MeOH in DCM as eluant to give compound 20 (2.4 g, 56%).
  • Compound 27 A solution of compound 26 1 (5 g, 12.4 mmol) in pyridine (100 mL) was added 4,4 '-dimethoxytrityl chloride (4.21 g, 12.4 mmol) and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate and saturated aqueous NaHCO 3 . The organic layer was washed with water and brine and dried over Na 2 SO 4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (45-100% ethyl acetate in hexane) to obtain compound 27 (5.8 g, 66%) as a white form.
  • Compound 29 Compound 28 (1.5 g, 2.34 mmol), imidazole (191 mg, 2.81 mmol), triphenylphosphine (1.84 g, 7.02 mmol) and I2 (653 mg, 2.57 mmol) was dissolved in THF (15 mL). The mixture was stirred and heated in a microwave reactor at 150 °C for 6 h. The reaction was cooled to 0 °C and then quenched by addition of saturated NaHCO 3 (aq.) and saturated Na 2 SiO 3 (aq.). The reaction mixture was diluted with ethyl acetate.
  • Compound 34 Compound 27 (500 mg, 0.709 mmol), imidazole (145 mg, 2.13 mmol), triphenylphosphine (223 mg, 0.851 mmol) and h (198 mg, 0.780 mmol) was dissolved in THF (5 mL). The mixture was stirred and heated in a microwave reactor at 100 °C for 2 h. The reaction was cooled to room temperature and quenched by addition of saturated NaHCO 3 (aq.) and saturated Na 2 SiO 3 (aq.) and diluted with ethyl acetate. The organic layer was washed with saturated NaHCO 3 (aq.), water and brine and dried with Na 2 SO 4 and concentrated under vacuum.
  • Compound 42 Note: Two batches in parallel (10.0 g + 80.0 g). Add compound 41 (80.0 g, 158 mmol, 1.00 eq) and uracil (23.1 g, 206 mmol, 1.30 eq) into ACN (800 mL). Add BSA (129 g, 634 mmol, 156 mL, 4.00 eq) into the mixture at 20 °C. The solution in to an ice bath, slowly add TMSOTf (56.3 g, 253 mmol, 45.8 mL, 1.60 eq) at 0 °C -5 °C. Stir at 85 °C for 3 hrs.
  • TMSOTf 56.3 g, 253 mmol, 45.8 mL, 1.60 eq
  • Compound 43 Note: Four batches in parallel. (5.00 g +45.0 g x 3). Add compound 42 (45.0 g, 80.8 mmol, 1.00 eq) in MeOH (450 mL) and CHCI 3 (90 mL). Add MeONa (4.37 g, 80.8 mmol, 1.00 eq) into the mixture. Stir at 30 °C for 12 hrs. Concentrate under reduced pressure. Triturate it with EtOAc (300 mL) at 25 °C for 30 min and collect filter cake. Repeat the operation above again. Triturate it with ACN (300 mL) at 25 °C for 30 min and collect filter cake. Filter the reaction solution and collect the cake. Concentrate under reduced pressure.
  • Compound 45 Note: Four batches in parallel. (5.00 g + 25.0 g + 20.0 g x 2). Add compound 44 (20.0 g, 41.0 mmol, 1.00 eq) andDMAP (9.99 g, 81.7 mmol, 1.99 eq) into ACN (400 mL) at 15-20 °C. Add PhOCSCl (7.80 g, 45.2 mmol, 6.25 mL, 1.10 eq) into the mixture at 15-20 °C. Stir at 15-20 °C (external temperature) for 12 hrs.
  • Compound 46 Note: Four batches in parallel. (5.00 g + 30.0 g + 20.0 g x 2). Add compound 45 (20.0 g, 32.1 mmol, 1.00 eq) into toluene (300 mL) at 20 °C (reactor 1). Add AIBN (1.05 g, 6.42 mmol, 0.2 eq) into toluene (300 mL) at 20 °C (reactor 2). Add Bu 3 SnH (74.7 g, 256 mmol, 68.0 mL, 8.00 eq) into the reaction mixture of AIBN (1.05 g, 6.42 mmol, 0.20 eq) and Tol. (300 mL) at 15-20 °C.
  • Compound 49 Note: Three batches in parallel. (5.00 g x 3). A mixture of compound 48 (5.00 g, 9.42 mmol, 1.00 eq) in dry ACN (20 mL) was cooled to 0-5 °C and degassed and purged with Ar for 3 times, and NaH (1.70 g, 42.4 mmol, 60.0% purity, 4.50 eq) was added at 0 °C, the mixture was stirred at 18 °C for 4 hrs under Ar atmosphere.
  • Compound 50 Add compound 49 (12.0 g, 17.6 mmol, 1.0 eq) into DCM (84.0 mL) at 20 °C. Add dodecane- 1 -thiol (5.35 g, 26.4 mmol, 6.33 mL, 1.50 eq) at 20 °C. Add TFA (3.02 g, 26.4 mmol, 1.96 mL, 1.50 eq) at 20 °C. Stir at 20 °C for 2 hrs. Add H 2 O (50 mL) to the mixture.
  • Compound 53 Note: Three batches in parallel. (150 g x 3). Add Compound 52 (150 g, 438 mmol, 1.00 eq) in Ca(OH) 2 (0.37 M, 1500 mL, 1.26 eq) at 15°C. The suspension was degassed under vacuum and purged with N 2 (15Psi) several times. Stir at 90°C for 12 hrs. Combine with three batches. Filter and collect filter liquor. (Remove excess insoluble impurities). Used to the next. Obtain Compound 53 (900 g, crude) as brown solid.
  • Compound 58B The suspension of uracil (8.73 g, 77.8 mmol, 1.20 eq), HMDS (560 mL), (NH 4 ) 2 SO 4 (750 mg, 5.68 mmol, 424 ⁇ L, 8.75e-2 eq) (catalytic amount) at 20°C. Under a nitrogen atmosphere for 4 h at 135°C. Used to the next. Remove the excess HMDS under high vacuum. Obtain Compound 58B (16.6 g, crude) as white solid.
  • Compound 59 Note: Three batches in parallel. (10.0 g x2 +14.0 g). The suspension of Compound 58B (16.6 g, 64.7 mmol, 1.00 eq) in ACN (200 mL) at 20°C. Add a solution of the Compound 58 (14.0 g, 64.7 mmol, 1.00 eq) in dry ACN (200 mL), and add TMSOTf (15.8 g, 71.2 mmol, 12.8 mL, 1.10 eq) at 20°C. Stir resulting reaction mixture for 1 h at 20°C.
  • Compound 60 Note: Three batches in parallel. (40.0 g +10.0 g +13.0 g). Add Compound 59 (40.0 g, 149 mmol, 1.00 eq) in AcOH (960 mL) and H 2 O (240 mL) at 20°C. Stir at 80°C for 6 hrs. The solvent was concentrated and evaporated with toluene (100 mL) twice. The solvent was concentrated and evaporated with Py. (200 mL) eight times. Obtain Compound 60(crude) (92.0 g) as yellow oil.
  • Compound 62 Note: Five batches in parallel. (5.00 g x 5). A mixture of Compound 61 (5.00 g, 9.42 mmol, 1.00 eq) in dry ACN (25 mL) was cooled to 0-5 °C and degassed and purged with Ar for 3 times, and NaH (1.70 g, 42.4 mmol, 60.0% purity, 4.50 eq) was added at 0 °C, the mixture was stirred at 20 °C for 2 h under Ar atmosphere.

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Abstract

The technology described herein relates to 5'-modified nucleosides, nucleotides, oligonucleotides and double-stranded RNAs, e.g., siRNAs, and kits comprising them and methods of their use for inhibiting target genes.

Description

5’-MODIFIED MONOMERS, OLIGONUCLEOTIDES AND DOUBLE-STRANDED RNAS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No.63/660,129 filed June 14, 2024, and U.S. Provisional Application No.63/705,390 filed October 9, 2024, the contents of all of which are incorporated herein by reference in their entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 12, 2025, is named “ALN-525_ST_26.xml” and is 1,538,503 bytes in size. TECHNICAL FIELD [0003] The technology described herein relates generally to 5’-modified nucleosides, nucleotides, oligonucleotides and double-stranded RNAs, e.g., siRNAs, compositions and kits comprising them and methods of their use for inhibiting target genes. BACKGROUND [0004] There remains a need in the art for oligonucleotides and siRNAs having improved activity and/or pharmacodynamics. The present disclosure addresses some of these needs. SUMMARY [0005] In one aspect, provided herein is a compound of formulae I-XXI,
or a salt thereof, wherein: M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar, optionally, M is in the south conformation (i.e., C2’-endo); n is an integer selected from 1 - 3; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1 or 2); n9 is an integer selected from 1 - 3 (e.g., 1 or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); y is 0 or 1; A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2-, wherein * is the bond to E; E is a bond or -CH2-; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2- methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2- propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; X is O or S; XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; YA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC), wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl; RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; R4’ is C1-6alkyl (e.g., methyl),r C1-6alkoxy (e.g., methoxy), hydrogen; R5X is H or C1-6alkyl (e.g., methyl); one of R2’and R3’is hydrogen, halogen, -OR20, alkyl, branched alkyl, aminoC1- 6alkyl(e.g., branched aminoC1-6alkyl), C2-6alkenyl, C2-6alkynyl, C1-6alkyl ester, C1-6alkylthio (e.g., branched C1-6alkylthio), C1-6alkylamino (e.g., branched N- C1- 6alkylamino), C2-6alkenylthio (e.g., branched C2-6alkenylthio), N- C2- 6alkenylamino (e.g., branched N- C2-6alkenylamino), C2-6alkylthioester, N-C1- 6alkylcarbamyl, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted alkyl, (e.g., optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl, 2-[N,N- dimethyl)aminooxy]ethyl, or 3-oxo-3-(N-methylamino)prop-1-yl)), optionally substituted branched alkyl, optionally substituted alkenyl (e.g., optionally substituted C2-6alkenyl,) or optionally substituted alkynyl, (e.g., optionally substituted C2-6alkynyl (e.g., propargyl); and R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amin, and the other of R2’and R3’is -OR30, wherein R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [0006] In some embodiments, Q is not wherein * is the bond to the phosphorous atom. [0007] In some embodiments, when X is O or S, and each Rp is ORO, wherein each RO is hydrogen or an oxygen protecting group, then Q is not wherein * is the bond to the phosphorous atom. [0008] In some embodiments, the compound is not of the formula, . [0009] Some compounds of Formula (I) can be of Formula (Ia), For example, some compounds of Formula (I) can be of formula, wherein: Q1 is -O-, -S-, or -N(RN)-, and RN is hydrogen, methyl, C1- 3alkoxy, or C1-3acyl. [0010] Some compounds of Formula (I) can be of formula, . [0011] Some other compounds of Formula (I) can be of formula, . [0012] Yet some other compounds of Formula (I) can be of formula, , [0013] Still some other compounds of Formula (I) can be of formula, [0014] Some compounds of Formula (I) can be of formula, . [0015] Some compounds of Formula (I) can be of formula, wherein RNQ is hydrogen). [0016] Some other compounds of Formula (I) can be of formula (Ib), [0017] Some other compounds of Formula (I) can be of the structure: . For example, some compounds of Formula (I) can be of the structure: [0018] Yet some other compounds of Formula (I) can be of the structure: For some compounds of Formula (I) can be of the structure: [0019] In some embodiments, the compound of Formula (II) is of Formula (IIa), (IIb), (IIc) or (IId): [0020] Yet some other compounds of Formula (II) can be formula, . [0021] Still some other compounds of Formula (II) can be of formula, . [0022] Some compounds of Formula (III) can be of formula (IIIa), , wherein n is 1, 2 or 3. [0023] In some embodiments Q is where * is the bond to the phosphorous atom. For example Q is . [0024] In some embodiments, a compound of Formula (IV) is a compound is of Formula (V), where B, X, RP, Q4, R2’ and R3’ are as defined in Formula (IV). For example, the compound is of Formula (V), wherein R3’ is -OR30 and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further aspects of these embodiments, R2’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2- (methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N- methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O- alkyl ester, S-alkyl ester, N-alkyl ester, 1 2 wherein R and R independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0025] In some other embodiments, the compound is of Formula (V), wherein R2’ is -OR30 and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further aspects of these embodiments, R3’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0026] In some embodiments, the compound of Formula (IV) is a compound is of Formula (VI): or a salt thereof, wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; YA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide, and B, X, RP, and Q4 are as defined in Formula (IV). [0027] In some compounds of Formula (VI), YA is O, and the compound is of Form or a salt thereof, where B, X, XA, RP, a 4 nd Q are as defined in Formula (VI). [0028] In some compounds of Formula (VI), XA is O, and the compound is of Formula (VIII), or a s A P 4 alt thereof, where B, X, Y , R , and Q are as defined in Formula (VI). [0029] In some compounds of Formula (VI), XA and YA are O, and the compound is of compound of Formula (IX), or a salt thereof, where B, X, RP, and Q4 are as defined in Formula (VI). For example, the compound is of Formula (IX), or a salt thereof, wherein: B is an optionally modified nucleobase (e.g., uracil); Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [0030] In some embodiments, the compound of formula (IV) is a compound of Formula (X), or a salt thereof, wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and B, X, RP, R2’, R3’ and Q4 are as defined in Formula (IV). [0031] In some compounds of Formula (X), XA is O. In some other compounds of Formula (X), XA is S. [0032] In some compounds of Formula (X), R3’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. For example, R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2- methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo- 3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S- alkenyl, O-alkyl ester, S-alkyl ester, or N-alkyl ester. [0033] In some compounds of Formula (X), R2’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. For example, R3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2- methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo- 3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S- alkenyl, O-alkyl ester, S-alkyl ester, or N-alkyl ester. [0034] In some embodiments, the compound of formula (IV) is a compound of Formula (XI), or a salt thereof, wherein: XA is O, S SO2CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and B, X, RP, R2’, R3 and Q4 are as defined in Formula (IV). [0035] In some compounds of Formula (XI), XA is O. In some other compounds of Formula (XI), XA is S. [0036] In some compounds of Formula (XI), R3’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0037] In some compounds of Formula (XI), R2’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0038] In some embodiments, the compound of Formula (IV) is of formulae (XII)-(XIV): wherein: XA is O, S SO2CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; and B, X, RP, R2’, R3 and Q4 are as defined in Formula (IV). [0039] In some compounds of formulae (XII)-(XIV), XA is O. In some other compounds of formulae (XII)-(XIV), XA is S. [0040] In some compounds of Formula (XI), R3’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R2’ is halogen (e.g., F), alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, or N-alkyl ester. [0041] In some compounds of formulae (XII)-(XIV), R2’ is F, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2- oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N- alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, or wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0042] In some embodiments, a compound of Formula (XV) has the structure: . In some compounds of Formla (XV), n5 is 2 or 3, e.g., n5 is 2. [0043] In some embodiments, a compound of Formula (XVI) has the structure: In some compounds of Formula (XVI), n6 i 6 s 1 or 2, e.g., n is 1. [0044] In some embodiments, a compound of Formula (XVII) has the structure: [0045] In some embodiments, a compound of Formula (XVII) has the structure: [0046] In some compounds of Formula (XVII), R4’ is methyl, ethyl or propyl, e.g., R4’ is methyl. In some compounds of Formula (XVII), n7 is 1 or 2, e.g., n7 is 1. In some compounds of Formula (XVII), XA is O. In some other compounds of Formula (XVII), XA is S. In some compounds of Formula (XVII), n7 is 1, R4’ is methyl, and XA is O. [0047] In some embodiments, a compound of Formula (XX) has the structure: In some c 8 ompounds of Formla (XX), n is 1. In some other compounds of Formula (XX), n8 is 2. [0048] In some embodiments, a compound of Formula (XXI) has the structure: [0049] In some embodiments, a compound of Formula (XXI) has the structure: [0050] In some compounds of Formula (XXI), R4’ is methyl, ethyl or propyl, e.g., R4’ is methyl. In some compounds of Formula (XXI), n9 is 1. In some compounds of Formula (XXI), n9 is 2. In some compounds of Formula (XXI), XA is O. In some other compounds of Formula (XXI), XA is S. In some compounds of Formula (XXI), n9 is 1, R4’ is methyl, and XA is O. In some other compounds of Formula (XXI) n9 is 2, R4’ is methyl, and XA is O. [0051] In some compounds of Formula (XXI), R4’ is hydrogen and n9 is 2. In some other compounds of Formula (XXI), R4’ is hydrogen and n9 is 1. In some other compounds of Formula (XXI), R4’ is hydrogen, XA is O, and n9 is 2. In some other compounds of Formula (XXI), R4’ is hydrogen, XA is O and n9 is 1. In some other compounds of Formula (XXI), R4’ is hydrogen, XA is S, and n9 is 2. In some other compounds of Formula (XXI), R4’ is hydrogen, XA is S and n9 is 1. Some compounds of Formula (XXI), R4’ is hydrogen, XA is O, n9 is 2, and the compounds have the structure: [0052] In some embodiments, a compound of Formula (XVIII) has the structure: [0053] In some compounds of Formula (XVIII), Q5 is *-NHCH2-, where * is the bond to the S(O2)RPS group. In In some other compounds of Formula (XVIII), Q5 is ethylene. In yet some other compounds of Formula (XVIII), Q5 is ethenylene. In some compounds of Formula (XVIII), Q5 is propylene. In some compounds of Formula (XVIII), XA is O. In some other compounds of Formula (XVIII), XA is S. In some compounds of Formula (XVIII), XA is O and Q5 is methylene, ethylene, ethenylene, or propylene. [0054] In some compounds of Formula (XVIII), XA is O, Q5 is methylene and the compounds are of the structure: . [0055] In some compounds of Formula (XVIII), XA is O, Q5 is ethylene and the compounds are of the structure: . [0056] In some compounds of Formula (XVIII), XA is O, Q5 is propylene. and the compounds are of the structure: . [0057] In some embodiments, a compound of Formula (XIX) has the structure: In some other embodiments, a compound of Formula (XIX) has the structure: . In some compounds of Formula (XIX), R5X is methyl. In some compounds of Formula (XIX), XA is O. In some other compounds of Formula (XIX), XA is S. In some compounds of Formula (XIX), y is 0. In some compounds of Formula (XIX) y is 1. In some compounds of Formula (XIX), XA is O and R5X is methyl. In some compounds of Formula (XIX), XA is O, R5X is methyl, and y 0. In some compounds of Formula (XIX), XA is O, R5X is methyl, and y is 1. [0058] In some embodiments, a compound of Formula (XXII) has the structure: . In some embodiments, n10 is 0, 12 or 3 (e.g., 1). [0059] In some embodiments, a compound of Formula (XXIII) has the structure: . [0060] In some compounds of formulae (XV)-(XXIII), R3’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0061] In some compounds of formulae (XV)-(XXIII), R3’is -OR30, and R30 is a reactive phosphorous group (e.g., a phosphoramidite). [0062] In some compounds of formulae (XV)-(XXI), R2’is hydrogen, halogen, or -OR30, and R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. In some further embodiments of these compounds, R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [0063] In some compounds of formulae (XV)-(XXI), R2’is hydrogen, halogen (e.g., F), O-alkyl (e.g., -OMe ,-OEt, or -O-2-methoxyethyl), or -OR30, and R30 is hydrogen, a hydroxy protecting group. [0064] In some embodiments, Q4 is where * is the bond to the phosphorous atom. For example, Q4 is where * is the bond to the phosphorous atom. In some embodiments, Q4 is
For example, Q4 is , where * is the bond to the phosphorous atom. [0065] In some compounds of formulae (IV)-(XIV), Q4 is where * is the bond to the phosphorous atom. For example, in some compounds of formulae (IV)-(XIV), Q4 is where * is the bond to the phosphorous atom. In some preferred embodiments of compounds of formulae (IV)-(XIV), . [0066] In some compounds of Formula (XII), Q4 is where * is the bond to the phosphorous atom. For example, in some compounds of Formula (XII), Q4 is pref 4 erably Q is where * is the bond to the phosphorous atom. In some preferred compounds of Formula (XII), Q4 is . [0067] In some embodiments, Q is where * is the bond to the phosphorous atom. [0068] In some embodiments, Q is where * is the bond to the phosphorous atom. [0069] In some embodiments, Q is where * is the bond to the phosphorous atom [0070] In some compounds of formulae I-XXIII, X is O. In some other embodiments, X is S. [0071] In some embodiments, A is -C(*)(H)-. In some embodiments, A is -CH2C(*)(H)-. In yet some other embodiments, A is -C(*)(H)CH2-. [0072] In some embodiments, E is a bond. In some other embodiments, E is -CH2-. [0073] In compounds of formulae I-XXVII and XIX-XXIII, each RP is independently -ORO, - SRS, -N(RN)2, or -N(RN)S(O)2R2S. In some embodiments, at least one RP is -ORO, e.g., each RP is independently -ORO. Each RO can be independently hydrogen, C1-6alkyl (e.g., C1-3alkyl), or a hydroxyl protecting group. For example, each RO can be independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, or a hydroxyl protecting group. In some embodiments, each RO is hydrogen, methyl, or ethyl. In some embodiments, each RO is a hydroxyl protecting group, such as pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM). [0074] In some compounds of formulae I-XXVII and XIX-XXIII, at least one RP is -SRS, e.g., each RP is independently -SRS. Each RS can be independently hydrogen, C1-3alkyl, or a thiol protecting group. For example, each RS can be independently hydrogen, methyl, ethyl, propyl, isopropyl, or a thiol protecting group. In some embodiments, each RS is hydrogen, methyl or ethyl. In some embodiments, each RS is a thiol protecting group. [0075] In some compounds of formulae I-XXVII and XIX-XXIII, at least one RP is -N(RN)2, e.g., each RP is independently -N(RN)2. Each RN can be independently hydrogen, C1-3alkyl, or an amine protecting group. For example, each RN can be independently hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, each RN is hydrogen, methyl or ethyl. In some embodiments, at least one RN is an amine protecting group. [0076] In some compounds of formulae I-XXVII and XIX-XXIII, at least one RP is - N(RN)S(O)2R2S, e.g., each RP is independently -N(RN)S(O)2R2S. RN can be hydrogen, C1-3alkyl, or an amine protecting group. For example, RN can be hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, RN is hydrogen, methyl or ethyl. In some embodiments, RN is an amine protecting group. R2S can be methyl, ethyl, propyl, or isopropyl. In some embodiments, R2S is methyl or ethyl, e.g., R2S is methyl. In some embodiments, R2S is a C3- 6cycloalkyl (e.g., cyclopropyl or cyclobutyl). In some compounds, RN is hydrogen, methyl, ethyl, propyl, or isopropyl, and R2S is methyl, ethyl, propyl, or isopropyl. In some compounds, RN is an amine protecting group, and R2S is methyl, ethyl, propyl, or isopropyl. [0077] In some embodiments, one RP is -ORO, and the other RP is -SRS, -N(RN)2, or - N(RN)S(O)2R2S. For example, one RP is -ORO, and the other RP is -SRS. In another example, one RP is -ORO, and the other RP is -N(RN)2. In yet another example, one RP is -ORO, and the other RP is N(RN)S(O)2R2S. [0078] In some other embodiments, one RP is -SRS, and the other RP is -N(RN)2, or - N(RN)S(O)2R2S. For example, one RP is -SRS, and the other RP is -N(RN)2. In another example, one RP is -SRS, and the other RP is N(RN)S(O)2R2S. [0079] In yet some other embodiments, one RP is -N(RN)2, and the other RP is -N(RN)S(O)2R2S. [0080] In some compounds of Formula (XVIII), RPS is -ORO, -N(RN)2, or -N(RN)S(O)2R2S. In some embodiments, RPS is -ORO. Each RO can be independently hydrogen, C1-6alkyl (e.g., C1- 3alkyl), or a hydroxyl protecting group. For example, each RO can be independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, or a hydroxyl protecting group. In some embodiments, each RO is hydrogen, methyl, or ethyl. In some embodiments, each RO is a hydroxyl protecting group, such as pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM). In some compounds of Formula (XVIII), RPS is OH. [0081] In some compounds of Formula (XVIII), RPS is -N(RN)2. Each RN can be independently hydrogen, C1-3alkyl, or an amine protecting group. For example, each RN can be independently hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, each RN is hydrogen, methyl or ethyl. In some embodiments, at least one RN is an amine protecting group. [0082] In some compounds of Formula (XVIII), RPS is -N(RN)S(O)2R2S. RN can be hydrogen, C1-3alkyl, or an amine protecting group. For example, RN can be hydrogen, methyl, ethyl, propyl, isopropyl, or an amine protecting group. In some embodiments, RN is hydrogen, methyl or ethyl. In some embodiments, RN is an amine protecting group. R2S can be methyl, ethyl, propyl, or isopropyl. In some embodiments, R2S is methyl or ethyl, e.g., R2S is methyl. In some embodiments, R2S is a C3-6cycloalkyl (e.g., cyclopropyl or cyclobutyl). In some compounds, RN is hydrogen, methyl, ethyl, propyl, or isopropyl, and R2S is methyl, ethyl, propyl, or isopropyl. In some compounds, RN is an amine protecting group, and R2S is methyl, ethyl, propyl, or isopropyl. [0083] In some compounds of Formula (XVIII), RPS is -N(RN)P(O)(ORO)(RPC) (e.g., - N(H)P(O)(ORO)(RPC). For example, RP is -ORO and RPC is C1-3alkyl (e.g., methyl), wherein RO is hydrogen, C1-6alkyl (e.g., C1-3alkyl), or a hydroxyl protecting group. In some embodiments, RP is -ORO and RPC is C1-3alkyl (e.g,. methyl), wherein RO is hydrogen or C1-3alkyl. In some embodiments, RP is methoxy or ethoxy and RPC is methyl. In some embodiments, RP is -OH and RPC is methyl. In some compounds of Formula (XVIII), RPS is -N(H)P(O)(OH)(CH3) [0084] In some compounds of Formula (XVIII), RPS is -N=P(ORO)2(RPC). For example, each RP is -ORO and RPC is C1-3alkyl (e.g,. methyl), wherein RO is hydrogen, C1-6alkyl (e.g., C1-3alkyl), or a hydroxyl protecting group. In some embodiments, each RP is -ORO and RPC is C1-3alkyl (e.g,. methyl), wherein RO is hydrogen or C1-3alkyl. In some embodiments, each RP is methoxy or ethoxy and RPC is methyl. In some embodiments, each RP is -OH and RPC is methyl. [0085] In some compounds described herein, R3’ is -OR30. For example, R3’ is -OR30, and R30 is a reactive phosphorous group. Exemplary reactive phosphorous groups are described herein below and include, but are not limited to, phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. Thus, in some embodiments, R3’ is -OR30, and R30 is a reactive phosphorous group selected from phosphoramidite, H-phosphonate, alkyl-phosphonate, and phosphate triester, optionally R30 is a phosphoramidite. Accordingly, in some compounds, R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, - P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, - P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP3 is an optionally substituted C1-C30alkyl, optionally substituted C2- C30alkenyl, or optionally substituted C2-C30alkynyl (e.g., optionally substituted C1-C10alkyl, optionally substituted C2-C10alkenyl, or optionally substituted C2- C10alkynyl); each RP1 is independently an optionally substituted C1-6alkyl; and each RP2 is independently optionally substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, such as isopropyl) or both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl. [0086] In some embodiments, R3’ is -OR30, and R30 is -P(ORP1)N(RP2)2. For example, R3’ is - OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is C1-6alkyl substituted with cyano or -SC(O)Ph. In some embodiments, R3’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is –CH2CH2CN. [0087] In some embodiments, R3’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R3’ is -OR30, and R30 is - P(ORP1)N(RP2)2, and where each RP2 is isopropyl. [0088] In some preferred embodiments, R3’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is C1-6alkyl substituted with cyano or -SC(O)Ph, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R3’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is – CH2CH2CN, and each RP2 is isopropyl. [0089] In some embodiments, R3’ is -OR30, and R30 is a phosphoramidite group such as 3'-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or 3'-[(ß-thiobenzoylethyl)-(1-pyrrolidinyl)]- thiophosphoramidite). [0090] In another example, R3’ is -OR30, and R30 is hydrogen or a hydroxyl protecting group (e.g., a silyl based hydroxyl protecting group). Some exemplary hydroxyl protecting group for R30 of R3’ include, but are not limited to, t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionally, the hydroxyl protecting group is TBDMS. [0091] In yet another example, R3’ is -OR30, and R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R30 is a bond to an oligonucleotide. When R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide, R3’ can be connected to the 5’- hydroxyl of the nucleoside or nucleotide or the 5’-hydroxyl at the 5’-terminal of the oligonucleotide. It is noted that the internucleotide linkage between the compound or nucleoside of formulae I-XXIII and the nucleoside, nucleotide, or oligonucleotide it is linked to can be an unmodified internucleotide linkage (i.e., phosphodiester) or a modified internucleotide linkage (e.g., phosphorothioate, MMI or imidp, preferably the modified internucleotide linkage is phosphorothioate). Exemplary modified internucleotide linkages are described herein below. [0092] In some embodiments, R3’ is hydrogen or halogen (e.g., F, Br, Cl or I). For example, R3’ is H or F. [0093] In some compounds of formulae I-XXIII, R3’ is -OR20, where R20 is optionally substituted C1-6alkyl. For example, R3’ is -OR20, where R20 is methyl, ethyl, or propyl. In some embodiments, R3’ is -OR20, where R20 is methyl. [0094] In some compounds of formulae I-XXIII, R3’ is -OR20, where R20 is optionally substituted C2-6alkenyl. For example, R3’ is -OR20, where R20 is vinyl or allyl. In some compounds of formulae I-IV, R3’ is -OR20, where R20 is optionally substituted C2-6alkynyl. For example, R3’ is -OR20, where R20 is acetylenyl, propargyl, or 5-hexyn-1-yl. [0095] In some compounds of formulae I-XXIII, R3’ is -OR20, where R20 is C1-6alkoxyC1- 6alkyl. For example, R3’ is -OR20, where R202-methoxyethyl. [0096] Preferably, R3’ is -OR30 and R2’ is hydrogen, halogen, or -OR20. For example, R3’ is - OR30 and R2’ is hydrogen, halogen, or -OR20, and where R30 is a reactive phosphorous group, hydroxyl protecting group, a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [0097] In some embodiments, R2’ is hydrogen or halogen (e.g., F, Br, Cl or I). For example, R2’ is H or F. [0098] In some compounds of formulae I-V and IX-XXI, R2’ is -OR20, where R20 is optionally substituted C1-6alkyl. In one example, R20 is C1-6alkyl substituted with one, two, or three substituents selected independently from the group consisting of halogen, -OR22, -N(R22)2, -SR22, - C(O)OR22, -C(O)N(R22)2, wherein R22 is hydrogen or C1-3alkyl (e.g., 2,2,2-trifluoroethyl, 1,3- dimethoxyprop-2-yl). In another example, R20 is C1-6alkyl substituted with one or two substituents selected independently from the group consisting of halogen, -OR22, -N(R22)2, -SR22, -C(O)OR22, - C(O)N(R22)2, wherein R22 is hydrogen or C1-3alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxyprop- 2-yl). [0099] In another example, R2’ is -OR20, where R20 is methyl, ethyl, or propyl. In some embodiments, R2’ is -OR20, where R20 is methyl. [00100] In some compounds of formulae I-V and IX-XXI, R2’ is -OR20, where R20 is C1- 6alkoxyC1-6alkyl. For example, R2’ is -OR20, where R20 is 2-methoxyethyl. [00101] In some compounds of formulae I-V and IX-XXI, R2’ is -OR20, where R20 is N-(C1- 6alkyl)aminocarbonylC1-6alkyl. For example, R2’ is -OR20, where R20 is 2-(N-methylamino)-2- oxoethyl or 3-oxo-3-(N-methylamino)prop-1-yl. [00102] In another example, R2’ is -OR30, and R30 is hydrogen or a hydroxyl protecting group (e.g., a silyl based hydroxyl protecting group). Some exemplary hydroxyl protecting group for R30 of R2’ include, but are not limited to, t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionally, the hydroxyl protecting group is TBDMS. [00103] In some compounds of formulae I-V and IX-XXI, R2’ is halogen, (e.g., F), alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2- (methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo-3-(N- methylamino)prop-1-yl)),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O- alkyl ester, S-alkyl ester, N-alkyl ester, 1 2 wherein R and R independently are alkyl, branched alkyl, alkyl ester or alkyl amine. [00104] In some compounds described herein, R2’ is -OR30. For example, R2’ is -OR30, and R30 is a reactive phosphorous group. Exemplary reactive phosphorous groups are described herein below and include, but are not limited to, phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. Thus, in some embodiments, R2’ is -OR30, and R30 is a reactive phosphorous group selected from phosphoramidite, H-phosphonate, alkyl-phosphonate, and phosphate triester, optionally R30 is a phosphoramidite. Accordingly, in some compounds, R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, - P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, - P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP3 is an optionally substituted C1-C30alkyl, optionally substituted C2-C30alkenyl, or optionally substituted C2-C30alkynyl (e.g., optionally substituted C1-C10alkyl, optionally substituted C2-C10alkenyl, or optionally substituted C2-C10alkynyl); each RP1 is independently an optionally substituted C1-6alkyl; and each RP2 is independently optionally substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, such as isopropyl) or both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl. [00105] In some embodiments, R2’ is -OR30, and R30 is -P(ORP1)N(RP2)2. For example, R2’ is - OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is C1-6alkyl substituted with cyano or -SC(O)Ph. In some embodiments, R2’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is –CH2CH2CN. [00106] In some embodiments, R2’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R2’ is -OR30, and R30 is - P(ORP1)N(RP2)2, and where each RP2 is isopropyl. [00107] In some preferred embodiments, R2’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is C1-6alkyl substituted with cyano or -SC(O)Ph, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R2’ is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is – CH2CH2CN, and each RP2 is isopropyl. [00108] In some embodiments, R2’ is -OR30, and R30 is a phosphoramidite group such as 2’-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite or 2’-[(ß-thiobenzoylethyl)-(1-pyrrolidinyl)]- thiophosphoramidite). [00109] In yet another example, R2’ is -OR30, and R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R30 is a bond to an oligonucleotide. When R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide, R2’ can be connected to the 5’- hydroxyl of the nucleoside or nucleotide or the 5’-hydroxyl at the 5’-terminal of the oligonucleotide. It is noted that the internucleotide linkage between the compound or nucleoside of formulae I-XXIII and the nucleoside, nucleotide, or oligonucleotide it is linked to can be an unmodified internucleotide linkage (i.e., phosphodiester) or a modified internucleotide linkage (e.g., phosphorothioate, MMI or imidp, preferably the modified internucleotide linkage is phosphorothioate). Exemplary modified internucleotide linkages are described herein below. [00110] In compounds of formulae I-XXIII, B is an optionally modified natural or non-natural nucleobase. For example, B is an optionally modified natural or non-natural nucleobase. For example, B is uracil, adenine, cytosine, 5-methylcytosine, guanine, or thymine (i.e., 5- methyluracil). In some embodiments, B is a modified or protected nucleobase. For example, B is a protected nucleobase comprising at least one amine or hydroxyl protecting group. In some embodiments, B is adenine, cytosine, 5-methylcytosine, or guanine comprising at least one amine protecting group. Exemplary modified, unmodified natural and non-natural nucleobase are described herein below. [00111] In some compounds of formulae I-XVII and XIX-XXIII, X is O; each RP is -ORO; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, optionally substituted C1-6alkyl (e.g., methyl, ethyl or propyl), such as C1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1- 6alkyl)aminocarbonylC1-6alkyl (e.g. 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl); and R3’ is -OR30, where R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00112] In some compounds of formulae I-XVII and XIX-XXIII, X is O; each RP is -ORO; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, optionally substituted C1-6alkyl (e.g., methyl, ethyl or propyl), such asC1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1- 6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl); and R3’ is -OR30, where R30 is reactive phosphorous group (e.g., a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester). In some further embodiments of this, the reactive phosphorous group is: -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3 (e.g., - P(ORP1)N(RP2)2), wherein: each RP3 is an optionally substituted C1-C30alkyl, optionally substituted C2-C30alkenyl, or optionally substituted C2-C30alkynyl (e.g., optionally substituted C1-C10alkyl, optionally substituted C2-C10alkenyl, or optionally substituted C2-C10alkynyl); each RP1 is independently an optionally substituted C1-6alkyl (e.g., C1-6alkyl substituted with cyano or - SC(O)Ph, such as 2-cyanoethyl); and each RP2 is independently optionally substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, preferably isopropyl), or both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl. [00113] In some compounds of formulae I-XVII and XIX-XXIII, X is O; each RP is -ORO; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, optionally substituted C1-6alkyl (e.g., methyl, ethyl or propyl, preferably methyl), such as C1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N- (C1-6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl) ; and R3’ is -OR30, where R30 is -P(ORP1)N(RP2)2, and where RP1 is – CH2CH2CN, and each RP2 is isopropyl. [00114] In some preferred embodiments, X is O; each RP is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl; and R3’ is R30, where R30 is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. [00115] In some compounds of Formula (XVIII), X is O; RPS is -ORO; R2’ is hydrogen, F, or - OR20, where R20 is hydrogen, optionally substituted C1-6alkyl (e.g., methyl, ethyl or propyl), such as C1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1-6alkyl)aminocarbonylC1-6alkyl (e.g. 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N-methylamino)prop-1-yl); and R3’ is -OR30, where R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00116] In some compounds of Formula (XVIII), X is O; RPS is -ORO; R2’ is hydrogen, F, or - OR20, where R20 is hydrogen, optionally substituted C1-6alkyl (e.g., methyl, ethyl or propyl), such asC1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1-6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N-methylamino)prop-1-yl); and R3’ is -OR30, where R30 is reactive phosphorous group (e.g., a phosphoramidite, H-phosphonate, alkyl- phosphonate, or phosphate triester). In some further embodiments of this, the reactive phosphorous group is: -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, - P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3 (e.g., -P(ORP1)N(RP2)2), wherein: each RP3 is an optionally substituted C1-C30alkyl, optionally substituted C2-C30alkenyl, or optionally substituted C2-C30alkynyl (e.g., optionally substituted C1-C10alkyl, optionally substituted C2- C10alkenyl, or optionally substituted C2-C10alkynyl); each RP1 is independently an optionally substituted C1-6alkyl (e.g., C1-6alkyl substituted with cyano or -SC(O)Ph, such as 2-cyanoethyl); and each RP2 is independently optionally substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, preferably isopropyl), or both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl. [00117] In some compounds of Formula (XVIII), X is O; RPS is -ORO; R2’ is hydrogen, F, or - OR20, where R20 is hydrogen, optionally substituted C1-6alkyl (e.g., methyl, ethyl or propyl, preferably methyl), such as C1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1- 6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl) ; and R3’ is -OR30, where R30 is -P(ORP1)N(RP2)2, and where RP1 is – CH2CH2CN, and each RP2 is isopropyl. [00118] In some preferred compounds of Formula (XVIII), X is O; RPS is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl; and R3’ is R30, where R30 is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. [00119] In some embodiments, the compound is of the formula, or a salt thereof, wherein: Q is wherein: * is the bond to the phosphorous atom; Q1 is -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl; B is an optionally modified nucleobase (e.g., uracil); X is O or S; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; one of R2’and R3’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2- 6alkynyl (e.g., propargyl); and the other of R2’and R3’ is -OR30, wherein: R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [00120] In some embodiments, the compound is of the formula, or a salt thereof, wherein: Q is
wherein: * is the bond to the phosphorous atom; B is an optionally modified nucleobase (e.g., uracil); X is O; each RP is independently -ORO, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; one of R2’and R3’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2- 6alkynyl (e.g., propargyl); and the other of R2’and R3’ is -OR30, wherein: R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [00121] In some embodiments, R3’ is -OR30. For example, the compound is of formula, . or a salt thereof, wherein: R3’ is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. [00122] In some embodiments, R3’ is -OR30, X is O, and each RP is -ORO. For example, the compound is of formula,
or a salt thereof, wherein: R3’ is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; each RP is -ORO, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; X is O; n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. [00123] In certain embodiments of the above compounds, RO is C1-6 alkyl (e.g., methyl or ethyl). In certain embodiments, RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). In certain embodiments, R20 is hydrogen. In certain embodiments, R20 is a hydroxyl protecting group (e.g., TBS, TMS). [00124] In some embodiments, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and each RP is - ORO. For example, the compound is of formula,
or a salt thereof, wherein: R3’ is -OR30, wherein: R30 is -P(ORP1)N(RP2)2, wherein: each RP1 is optionally substituted C1-6alkyl, (e.g., -CH2CH2CN); each RP2 is independently optionally substituted C1-6alkyl (e.g., isopropyl); each RP is -ORO, wherein: each RO is independently C1-6alkyl, or a hydroxyl protecting group; X is O; n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, or cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. [00125] In certain embodiments of the above compounds, B is uracil or thymine. In certain embodiments, RO is C1-6 alkyl (e.g., methyl or ethyl). In certain embodiments, RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). In certain embodiments, R20 is hydrogen. In certain embodiments, R20 is a hydroxyl protecting group (e.g., TBS, TMS). [00126] In some embodiments, the compound is of the formula, or a salt thereof, wherein: Q4 is where * is the bond to the phosphorous atom, and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl; M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionally modified nucleobase (e.g., uracil); X is O or S; XA is O or S; YA is O or S; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; R3’ is -OR30, wherein: R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [00127] In some embodiments, the compound is of the formula, or a salt thereof, wherein:
, , ,
, where * is the bond to the phosphorous atom; B is an optionally modified nucleobase (e.g., uracil); X is O; XA is O; YA is O; each RP is independently -ORO, , wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; R3’ is -OR30, wherein: R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. [00128] In some embodiments, R3’ is -OR30. For example, the compound is of formula,
,
, or a salt thereof, wherein: M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; R3’ is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; B is an optionally modified nucleobase (e.g., uracil); Q4 is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2- propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1 or 2); n9 is an integer selected from 1 - 3 (e.g., 1 or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); R4’ is C1-6alkyl (e.g., methyl), C1-6alkoxy (e.g., methoxy), or hydrogen; R5X is H or C1-6alkyl (e.g., methyl); X is O or S; XA is O or S; YA is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; RPS isC1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC), wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl; and R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3- (N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl). [00129] In some embodiments, R3’ is -OR30, X is O, and each RP is -ORO. For example, the compound is of formula,
, , or a salt thereof, wherein: M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; R3’ is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; each RP and RPS is -ORO, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1, or 2); n9 is an integer selected from 1 - 3 (e.g., 1, or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); R4’ is C1-6alkyl (e.g., methyl), C1-6alkoxy (e.g., methoxy), or hydrogen; R5X is H or C1-6alkyl (e.g., methyl); X is O; XA is O; YA is O; RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2- methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; and Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2- propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3- (N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl). [00130] In certain embodiments of the above compounds, RO is C1-6 alkyl (e.g., methyl or ethyl). In certain embodiments, RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). In certain embodiments, R20 is hydrogen. In certain embodiments, R20 is a hydroxyl protecting group (e.g., TBS, TMS). [00131] In some embodiments, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and each RP is - ORO. For example, the compound is of formula, ,
,
, or a salt thereof, wherein: M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; R3’ is -OR30, wherein: R30 is -P(ORP1)N(RP2)2, wherein: each RP1 is optionally substituted C1-6alkyl, (e.g., -CH2CH2CN); each RP2 is independently optionally substituted C1-6alkyl (e.g., isopropyl); each RP and RPS is -ORO,wherein: each RO is independently C1-6alkyl, or a hydroxyl protecting group; y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1, or 2); n9 is an integer selected from 1 - 3 (e.g., 1, or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); R4’ is C1-6alkyl (e.g., methyl), C1-6alkoxy (e.g., methoxy), or hydrogen; R5X is H or C1-6alkyl (e.g., methyl); X is O; XA is O; YA is O; RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; B is an optionally modified nucleobase (e.g., uracil); Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2- methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2- propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; and R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3- (N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl). [00132] In certain embodiments of the above compounds, B is uracil or thymine. In certain embodiments, RO is C1-6 alkyl (e.g., methyl or ethyl). In certain embodiments, RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). In certain embodiments, R20 is hydrogen. In certain embodiments, R20 is a hydroxyl protecting group (e.g., TBS, TMS). [00133] In some embodiments, the compound is of formulae IV-XIV, wherein: Q4 is where * is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00134] In some embodiments, the compound is of formulae IV-XIV wherein:
where * is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00135] In some embodiments, the compound is of formulae IV-XIV wherein: Q4 is , where * is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00136] In some embodiments, the compound is of formulae IV-XIV, wherein: Q4 is , where * is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00137] In some embodiments, the compound is of formulae IV-XIV, wherein: Q4 is , where* is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00138] In some embodiments, the compound is of formulae IV-XIV, wherein: Q4 is where* is the bond to the phosphorous atom. X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group [00139] In some embodiments, the compound is of formulae IV-XIV, wherein: Q4 is where * is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00140] In some embodiments, the compound is of formulae IV-XIV, wherein: Q4 is , , , , , where * is the bond to the phosphorous atom; X is O; each RP is independently -ORO or is -N(RN)2, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each RN is independently hydrogen, C1-3alkyl, or an amine protecting group R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. [00141] In some compounds of formulae IV-XVII and XIX-XIII, R3’ is -OR30, X is O, and each RP is -ORO. In some compounds of Formula (IV), R3’ is -OR30, X is O, each RP is -ORO, and each RO independently is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of Formula (IV), R3’ is -OR30, X is O, each RP is -ORO, and each RO independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00142] In some compounds of formulae IV-VII and XIX-XXIII, R3’ is -OR30, R30 is - P(ORP1)N(RP2)2, X is O, and one RP is -ORO and the other RP is C1-3alkyl. In some compounds of formulae IV-XXIII, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, one RP is -ORO, and each RO independently is C1-6 alkyl (e.g., methyl or ethyl), and the other RP is C1-3alkyl. In some compounds of formulae IV-XXIII, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, one RP is -ORO, and each RO independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl), and the other RP is C1-3alkyl (e.g., methyl). [00143] In some compounds of formulae IV-XVII and XIX-XXIII, B is uracil or thymidine, R3’ is -OR30, X is O, and each RP is -ORO. In some compounds of formulae IV-XXIII, B is uracil or thymidine, R3’ is -OR30, X is O, each RP is -ORO, and each RO independently is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of formulae IV-XXIII, B is uracil or thymidine, R3’ is -OR30, X is O, each RP is -ORO, and each RO independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00144] In certain compounds of formulae IV-XVII and XIX-XXIIII, R3’ is -OR30, R30 is - P(ORP1)N(RP2)2, X is O, and each RP is -ORO. In some compounds of formulae IV-XXIII, R3’ is - OR30, R30 is -P(ORP1)N(RP2)2, X is O, each RP is -ORO, and each RO independently is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of formulae IV-XXIII, R3’ is -OR30, R30 is - P(ORP1)N(RP2)2, X is O, each RP is -ORO, and each RO independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00145] In some compounds of formulae IV-XVII and XIX-XXIII, B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and each RP is -ORO. In some compounds of formulae IV-XXIII, B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, each RP is -ORO, and each RO independently is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of formulae IV-XXIII, B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, each RP is -ORO, and each RO independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00146] In some compounds of formulae IV-XVII and XIX-XXIII, B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and one RP is -ORO and the other RP is C1-3alkyl. In some compounds of formulae IV-XXIII, B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, one RP is -ORO, and each RO independently is C1-6 alkyl (e.g., methyl or ethyl), and the other RP is C1-3alkyl. In some compounds of formulae IV-XXIII, B is uracil or thymidine, R3’ is - OR30, R30 is -P(ORP1)N(RP2)2, X is O, one RP is -ORO, and each RO independently is a hydroxyl protecting group (e.g. pivaloyloxymethyl), and the other RP is C1-3alkyl (e.g., methyl). [00147] In some compounds of Formula (XVIII), R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and RPS is -ORO. In some compounds of Formula (XVIII), R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, RPS is -ORO, and RO is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of Formula (XVIII), R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, RPS is -ORO, and RP is C1-3alkyl (e.g., methyl). [00148] In some compounds of Formula (XVIII), B is uracil or thymidine, R3’ is -OR30, X is O, and RPS is -ORO. In some compounds of Formula (XVIII), B is uracil or thymidine, R3’ is -OR30, X is O, RPS is -ORO, and RO is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of Formula (XVIII), B is uracil or thymidine, R3’ is -OR30, X is O, RPS is -ORO, and RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00149] In certain compounds of Formula (XVIII), R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and RPS is -ORO. In some compounds of Formula (XVIII), R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, RPS is -ORO, and RO is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of Formula (XVIII), R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, RPS is -ORO, and RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00150] In some compounds of formulae IV-XXI, B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, and each RP is -ORO. In some compounds of Formula (IV), B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, each RP is -ORO, and RO is C1-6 alkyl (e.g., methyl or ethyl). In some compounds of Formula (IV), B is uracil or thymidine, R3’ is -OR30, R30 is -P(ORP1)N(RP2)2, X is O, each RP is -ORO, and each RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). [00151] In some embodiments, R3’ is -OR30 and R30 is a bond to an oligonucleotide, e.g., R3’ is wherein: Y is O or S (e.g., S) and represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA). In the preceding, the oxygen atom that is illustrated linking the 5’-end of the oligonucleotide to the phosphorous atom is the 5’-oxygen of the 5’-terminal nucleoside of the oligonucleotide. For example, the compound is of formula,
, , , or a salt thereof, wherein: R3’ is wherein: Y is O or S; represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA); n is an integer selected from 1 - 3; M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; Q4 is ethylene, ethenylene (e.g., preferably ), where * is the bond to the phosphorous atom), propylene, propenylene (e.g., or propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1, or 2); n9 is an integer selected from 1 - 3 (e.g., 1, or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); R4’ is C1-6alkyl (e.g., methyl), C1-6alkoxy (e.g., methoxy), or hydrogen; R5X is H or C1-6alkyl (e.g., methyl); Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC); wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl; X is O or S; XA is O or S; YA is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3- (N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. [00152] In some embodiments, Y is S. In other embodiments, Y is O. [00153] Herein, is used to represent an oligonucleotide; such oligonucleotides may be an RNA, a DNA, a single-stranded RNA, such as an antisense oligonucleotide (ASO), the antisense strand of a double-stranded RNA (such as an siRNA), and oligonucleotide derivatives such as phosphorodiamidate morpholino oligomers (PMOs). [00154] In some embodiments, X is O, each RP is -ORPO, and R3’ is -OR30, where R30 is a bond to an oligonucleotide, e.g., R3’ is , wherein: Y is O or S (e.g., S) and represents the remainder of an oligonucleotide (e.g., the antisense strand of a double- stranded RNA). For example, the compound is of formula,
, or a salt thereof, wherein: R3’ is wherein: Y is O or S; represents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA); each RP is -ORO, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; X is O; XA is O; YA is O; n is an integer selected from 1 - 3; M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; Q4 is ethylene, ethenylene (e.g., or, , preferably , where * is the bond to the phosphorous atom), propylene, propenylene, or propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1, or 2); n9 is an integer selected from 1 - 3 (e.g., 1, or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); R4’ is C1-6alkyl (e.g., methyl), C1-6alkoxy (e.g., methoxy), or hydrogen; R5X is H or C1-6alkyl (e.g., methyl); Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2- propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC); wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl; R2’ is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. [00155] In certain embodiments, B is uracil or thymine. In certain embodiments, RO is C1-6 alkyl (e.g., methyl or ethyl). In certain embodiments, RO is a hydroxyl protecting group (e.g. pivaloyloxymethyl). In some embodiments, Y is S. In other embodiments, Y is O. [00156] In some embodiments, the compound is selected from the group of compound shown in Table A: Table A: Some exemplary compounds
[00166] In another aspect, provided herein is an oligonucleotide with a 5’-terminal phosphate mimic comprising the structure: wherein: * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose); A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2-, wherein * is the bond to E; E is a bond or -CH2-; Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl; X is O or S; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl. [00167] In another aspect, provided herein is an oligonucleotide with a 5’-terminal phosphate mimic comprising the structure: wherein: X is O or S; Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2- methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; and RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S,-N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC); wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl. [00157] It is noted that the sugar moiety of the nucleotide comprising the 5’-terminal phosphate mimic (e.g. i.e., the nucleotide at 5’-end of the oligonucleotide (5’-terminal nucleotide) can comprise a 5- or 6- membered ring. For example, the sugar moiety of the nucleotide comprising the above modification can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof). It is noted that the 5’-terminal phosphate mimic can be attached to any atom, e.g., any carbon atom of the sugar moiety. The 5’-terminal phosphate mimic (e.g., can replace a -CH2OH group or a -OH group (e.g., a -CH2OH group) on the sugar moiety of a 5’-terminal nucleotide of the oligonucleotide. For example, the 5’-terminal phosphate mimic (e.g., replaces the 4’-CH2OH group on the furanose ring (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribofuranose) or the 5’-CH2OH group on the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5’-terminal nucleotide of the oligonucleotide. [00158] Similarly, the sugar moiety of the nucleotide comprising the , modification, i.e., the nucleotide at 5’-end of the oligonucleotide (5’-terminal nucleotide) can comprise a 5- or 6- membered ring. For example, the sugar moiety of the nucleotide comprising the above modification can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof). Generally, the , modification replaces carbon atom in the ring portion of the sugar moiety of a 5’-terrminal nucleotide of the oligonucleotide. For example, the , modification replaces the 4’-C atom in the furanose (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribofuranose) or the 5’-C atom in the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) ring of the 5’- terminal nucleotide of the oligonucleotide. [00159] In some embodiments, the oligonucleotide comprises at its 5’-end a compound of formulae I-IV described herein. In certain embodiments, X is O. In certain embodiments, X is O, and each RP is ORO. In certain embodiments, X is O, and each RP is OH. In certain embodiments, X is O, and each RP is ethoxy. In certain embodiments, X is O, and each RP is OH. In certain embodiments, X is O, and each RP is ORO wherein RO is pivaloyloxymethyl. [00160] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of the structure: , , , or a salt thereof, wherein: n is an integer selected from 1 - 3; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; Q4 is ethylene, ethenylene (e.g., or, , preferably where * is the bond to the phosphorous atom), propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; X is O or S; XA is O or S; YA is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; one of R2’and R3’is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3- (N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); the other of R2’and R3’ is -OR30, wherein: R30 is a bond to the rest of the oligonucleotide; and RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester. [00161] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: . For example, the 5’-terminal nucleotide of the oligonucleotide can be of formula, ,, wherein: Q1 is -O-, -S-, or -N(RN)-, and RN is hydrogen, methyl, C1- 3alkoxy, or C1-3acyl. [00162] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00163] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00164] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, . [00165] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, . [00166] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00167] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00168] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, e.g., of formula . [00169] In some embodiments, the ‘5-terminal nucleotide of the oligonucleotide is of formula, e.g., of formula [00170] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, [00171] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, . [00172] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, . [00173] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of formula, , wherein n is 1, 2 or 3. [00174] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: . For example, the 5’-terminal nucleotide of the oligonucleotide is of formula: In some embodiments, the 5’-terminal nucleotde is of formula: . In some other embodiments, the the 5’-terminal nucleotde is of formula: . In some aspects of these embodiments, Q4 is ethylene or ethenylene , where * is the bond to the phosphorous atom). [00175] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: For example, the 5’-terminal nucleotide of the oligonucleotide is of formula: . In some embodiments, the 5’-terminal nucleotde is of formula: In some other embodiments, the the 5’-terminal nucleotde is of formula: . In some aspects of these embodiments, Q4 is ethylene or ethenylene (e.g., preferably where * is the bond to the phosphorous atom). [00176] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: For example, the 5’-terminal nucleotide of the oligonucleotide is of formula: In some embodiments, the 5’-terminal nucleotde is of formula: . In some other embodiments, the the 5’-terminal nucleotde is of formula: In so 4 me aspects of these embodiments, Q is ethylene or ethenylene (e.g., pref erably , where * is the bond to the phosphorous atom). [00177] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: For example, the 5’-terminal nucleotide of the oligonucleotide is of formula: In some embodiments, the 5’-terminal nucleotde is of formula: In some other embodiments, the the 5’-terminal nucleotde is of formula: In s 4 ome aspects of these embodiments, Q is ethylene or ethenylene (e.g., preferably where * is the bond to the phosphorous atom). [00178] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: , , , ,
[00179] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide is of the structure: each as defined in Table 14 (below), where s at the end of the abbreviation indicates the internucleoside linkage between the 5’-terminal nucleoside and the subsequent nucleoside is a phosphorothioate internucleoside linkage, and the absence of “s” indicates that the internucleoside linkage between the 5’-terminal nucleoside and the subsequent nucleoside is a phosophodiester (e.g., a phosphate internucleotide linkage). [00180] Generally, the oligonucleotide comprises at least three nucleotides. For example, the oligonucleotide comprises from 5 to 100, e.g., from 10 to 50 nucleotides. In some embodiments, the oligonucleotide comprises from 15 to 40 nucleotides. For example, the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. For example, the oligonucleotide is 19, 20, 21, 22, or 23 nucleotides in length. It is noted that the compound of formulae I-XXIII counts as one nucleotide. [00181] The oligonucleotide described herein can comprise at least one nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications). Exemplary nucleic acid modifications are described herein below, and include, but are not limited to nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and combinations thereof. [00182] In some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-OMe nucleotides. [00183] In some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermally destabilizing modification of the duplex. For example, the oligonucleotide comprises a thermally destabilizing modification at at least one of position 4, 5, 6, 7, or 8, counting from the 5’-end of the oligonucleotide, where the compound of formulae I-XXIII is at position 1 from the 5’-end of the oligonucleotide; optionally, the thermally destabilizing modification is located at position 6, 7, or 8, counting from the 5’-end of the oligonucleotide, preferably the thermally destabilizing modification is located at position 7, counting from the 5’- end of the oligonucleotide. [00184] In some embodiments, the oligonucleotide comprises least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-F nucleotides. For example, the oligonucleotide comprises 2, 3, 4, 5, or 62’-F nucleotides, optionally, the oligonucleotide comprises 3, 4, 5 or 6 2’-F nucleotides. In some embodiments, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of formulae I-XXIII is at position 1 from the 5’-end of the oligonucleotide. For example, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 14 and 16, counting from the 5’-end of the oligonucleotide, optionally, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 9, 14 and 16, preferably, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the oligonucleotide. It is noted that when more than one 2’-F nucleotide is present in the oligonucleotide, each 2’-F nucleotide is an independently selected nucleotide. [00185] The oligonucleotide can also comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-deoxy (2’-H) nucleotides. For example, the oligonucleotide comprises 2, 3, 4, 5, 6, or 7 2’-deoxy nucleotides, optionally, the oligonucleotide comprises 3, 4, 5 or 62’-deoxy nucleotides. The oligonucleotide can comprise a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of formulae I-XXIII is at position 1 from the 5’-end of the oligonucleotide. For example, the oligonucleotide comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of oligonucleotide. In some embodiments, the oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, counting from the 5’-end of oligonucleotide. For example, the oligonucleotide comprises a 2’- deoxy nucleotide at least at positions 2, 5, 7, and 12, counting from the 5’-end of oligonucleotide. For example, thew oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’-end of the oligonucleotide. It is noted that when more than one 2’-dexy nucleotide is present in the oligonucleotide, each 2’-deoxy nucleotide is an independently selected nucleotide. [00186] One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleobases in the oligonucleotide can be non-natural or modified nucleobases. For example, the oligonucleotide can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified or protected nucleobases. [00187] The internucleotide linkages in the oligonucleotide can be independently unmodified (e.g., phosphodiester) or modified (e.g., phosphorothioate). Thus, in some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleoside linkages. Generally, the oligonucleotide comprises at least one (e.g., 1, 2, 4, or 5) modified internucleoside linkages (e.g., phosphorothioate) at the first 1-5 positions at one or both ends of the oligonucleotide. For example, the oligonucleotide comprises a modified oligonucleotide linkage (e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 5’-end of the oligonucleotide; and the oligonucleotide comprises a modified oligonucleotide linkage (e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide. [00188] In some embodiments, the oligonucleotide is covalently linked to a support, e.g., a solid support. [00189] In yet another aspect, provided herein is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand is substantially complementary to the antisense strand, and wherein one of the sense or the antisense strand is an oligonucleotide described herein, i.e., an oligonucleotide with a 5’-terminal modification comprising the structure: wherein: * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose); A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2, wherein * is the bond to E; E is a bond or -CH2-; Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, X is O or S; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl. [00190] Generally, the modification replaces a CH2OH group on the sugar moiety of the 5’-terrminal nucleotide of the sense or antisense strand. For example, the modification replaces the 4’-CH2OH group on the furanose ring (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose) or the 5’- CH2OH group on the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5’-terminal nucleotide of the sense or antisense strand. [00191] Similarly, the modification replaces carbon atom in the sugar moiety of the 5’-terminal nucleotide of the sense or antisense strand. For example, the modification replaces the 4’-C atom in the furanose (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose) ring or the 5’-C atom in the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) ring of the 5’-terminal nucleotide of the sense or antisense strand. [00192] In other words, the sense or antisense strand comprises at its 5’-end a compound of formulae I-XXIII described herein. In certain embodiments, X is O. In certain embodiments, X is O, and each RP is ORO. In certain embodiments, X is O, and each RP is OH. In certain embodiments, X is O, and each RP is ethoxy. In certain embodiments, X is O, and each RP is OH. In certain embodiments, X is O, and each RP is ORO wherein RO is pivaloyloxymethyl. [00193] For example, the 5’-terminal nucleotide of one of the sense or antisense strand is of the structure: , , , , , , where one of R2’and R3’ is -OR30, and R30 is a bond to the rest of the sense or antisense strand, provided that R3’ is -OR30 in formulae , , . [00194] Preferably, the antisense strand comprises the above 5’-terminal modification. [00195] In still another aspect, provided herein is a method for reducing the expression of a target gene in a subject. The method comprises administering to the subject either: (i) a double- stranded RNA described herein, where the antisense strand is substantially complementary to a target gene; or (ii) an oligonucleotide described herein, where the oligonucleotide is substantially complementary to a target gene. [00196] In another aspect, provided herein is a pharmaceutical composition comprising an oligonucleotide or dsRNA molecule described herein alone or in combination with a pharmaceutically acceptable carrier or excipient. [00197] In yet another aspect, provided herein is a cell comprising an oligonucleotide or dsRNA molecule described herein. [00198] In still another aspect, provided herein is a gene silencing kit comprising an oligonucleotide or dsRNA molecule described herein. [00199] Also, provided herein is a method for silencing a target gene, in a cell. The method comprises a step of introducing: (i) a dsRNA molecule described herein into the cell, where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene. [00200] In another aspect, provided herein is a method for inhibiting or reducing the expression of a target gene in a subject. The method comprises administering to the subject: (i) a dsRNA molecule described herein, where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide comprises a nucleotide sequence substantially complementary to a nucleotide sequence of the target gene. BRIEF DESCRIPTION OF THE DRAWINGS [00201] FIG. 1 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140883, AD-2140885, AD- 2140887, AD-2140888, and AD-64958 (parent) in mice. [00202] FIG. 2 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140884, AD-2140882, AD- 2140886, AD-2261036, and AD-64958 (parent) in mice. [00203] FIG. 3 shows mTTR protein levels at different timepoints after administration of a single dose (0.5 mg/kg) of exemplary duplexes AD-286913, AD-2633769, AD-2633771, AD- 2633772, AD-2633774, AD-2633775, AD-2633777, and AD-64958 (parent) in mice. [00204] FIG. 4 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2140883, AD-2140885, AD- 2140887, AD-2140888, and AD-64958 (parent) in mice. [00205] FIG. 5 shows mTTR protein levels at different timepoints after administration of a single dose (0.3 mg/kg) of exemplary duplexes AD-286913, AD-2680450, AD-2680452, AD- 2680454, AD-2680455, AD-2680456, and AD-64958 (parent) in mice. [00206] FIG. 6 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-401824, AD-2919280, AD- 2919281, AD-2919282, AD-2919283, AD-2919284, AD-2919285, AD-2919286, AD-2919288, and AD-2919289 targeting SOD1 mRNA in mice. [00207] FIG. 7 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-401824, AD-401825, AD- 2919282, AD-2919289, AD-3116172 and AD-3116181 targeting SOD1 mRNA in mice. [00208] FIG. 8 shows mSOD1 mRNA remaining in brain (right hemisphere) after intracerebroventricular administration of exemplary duplexes AD-1271086, AD-3367267, and AD- 3367269 targeting SOD1 mRNA in mice. [00209] FIG. 9 shows sAPPα protein levels at different timepoints after intrathecal administration of exemplary duplexes AD-960499, AD-454844, and AD-2905746 in non-human primate. [00210] FIG. 10 shows APP mRNA remaining in various organs at day 91 after intrathecal administration of exemplary duplexes AD-960499, AD-454844, and AD-2905746 in non-human primate. [00211] FIG. 11 shows MAP2 mRNA remaining in various organs at day 91 after intrathecal administration of exemplary duplexes AD-476454, AD-2912412, and AD-2912413 in non-human primate. [00212] FIG. 12 shows APP protein levels after intrathecal administration of exemplary duplexes AD-454844, AD-3175047, and AD-3216841 in non-human primate. [00213] FIG 13 shows 5ʹ-(E)-VP modified nucleotides previously tested in the context of siRNAs (I-VI) and conformational equilibrium between C2′-endo (South) and C3′-endo (North) sugars. R = 2’-modification. [00214] FIG.14shows Configurational and conformational features of LNA, α-L-LNA, and 5ʹ-VP-functionalized LNA and α-L-LNA studied. [00215] FIG.15A shows siRNAs with LNA or α-L-LNA at the 5' terminus of the antisense strands result in diminished silencing of gene expression in cultured cells or mice. Ttr mRNA remaining in primary mouse hepatocytes cultured with the indicated siRNAs at the indicated concentrations under free uptake conditions. mRNAs were quantified by RT-qPCR, and averages ± standard deviations are plotted (n=3). normalized to pre-dose levels in individual animals (n=3). [00216] FIG.15B shows siRNAs with LNA or α-L-LNA at the 5' terminus of the antisense strands result in diminished silencing of gene expression in cultured cells or mice. Ttr protein amounts in serum at indicated days after mice were dosed subcutaneously with 1 mg/kg indicated siRNA. Plotted are averages ± standard deviations [00217] FIG.16A shows the 5'-VP-α-L-LNA modification results in a more active siRNA than the 5'-VP-LNA modification. Ttr mRNA remaining in primary mouse hepatocytes cultured with the indicated siRNAs at the indicated concentrations under free uptake conditions. mRNAs were quantified by RT-qPCR, and averages ± standard deviations are plotted (n=3). [00218] FIG.16B shows the 5'-VP-α-L-LNA modification results in a more active siRNA than the 5'-VP-LNA modification. Ttr protein amounts in serum at indicated days after mice were dosed subcutaneously with 0.4 mg/kg indicated siRNA. Plotted are averages ± standard deviations normalized to pre-dose levels in individual animals (n=3). [00219] FIG.17A shows 5'-VP-α-L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-Ome, models of antisense strands modified with 5'-VP-2’-Ome. [00220] FIG.17B shows 5'-VP-α-L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-Ome, models of antisense strands modified with 5'-VP-α-L-LNA. [00221] FIG.17C shows 5'-VP-α-L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-Ome, models of antisense strands modified with 5'-VP-LNA lodged at the Ago2 MID domain binding site. [00222] FIG.17D shows 5'-VP-α-L-LNA makes interactions with the Ago2 MID domain that are similar to those of the antisense strand modified with 5'-VP-OMe. Overlay of 5'-VP-α-L-LNA (golden) and 5'-VP-LNA (violet) bound to the Ago2 MID domain. [00223] FIG.18 shows TTR protein amounts in serum at indicated days after mice were dosed subcutaneously with 1 mg/kg indicated siRNA. Plotted are averages ± standard deviations normalized to pre-dose levels in individual animals (n=3). [00224] FIG.19 shows RNAi-mediated gene silencing is more efficient when the antisense strand of the siRNA is modified at 5' end with a 5'-vinyl-phosphonate carrying nucleotide that adopts a C3’-exo (South) conformation than a C3′-endo (North) pucker. [00225] FIG.20A shows models of 5ʹ-terminal guide strand nucleotides lodged at the MID domain of RISC Ago2: LNA with a 5ʹ-phosphate. [00226] FIG. 20B shows models of 5ʹ-terminal guide strand nucleotides lodged at the MID domain of RISC Ago2: Overlay of the LNA model shown in FIG.20A, LNA with an E-VP moiety, and E-VP-RNA. Carbon atoms of AS1 residues are colored in magenta, purple and green for P- LNA (5ʹ-phosphate LNA), E-VP-LNA and E-VP-RNA, respectively, and the AS1 and AS2 phosphorus atoms of RNA are highlighted in black. The phosphorus position of P-LNA virtually matches the position of the E-VP-RNA phosphorus (FIG. 20B), but the b torsion angle of the former nucleotide is in a gauche conformation to do so (FIG. 20A). Combining LNA with an E- VP moiety at AS1 results in a shift by the phosphorus of almost 1 Å relative to E-VP-RNA in addition to a shift by the entire LNA nucleoside (FIG.20B). [00227] FIG.21 depicts some exemplary compounds of Formula (V). [00228] FIG. 22 depicts a synthetic scheme for synthesis of compounds of Formula (V). Reference (ref) for compound 1 is Ref: Marquez et. al. J. Chem. Soc. Perkins Trans.1: Org. Bioorg. Chem.1997, 1073-1078. [00229] FIG.23 depicts some exemplary compounds of Formula (VI). [00230] FIG.24 depicts some exemplary compounds of Formula (VII). [00231] FIG.25 depicts some exemplary compounds of Formula (VIII). [00232] FIG.26 depicts some exemplary compounds of Formula (X). [00233] FIG.27 depicts some exemplary compounds of Formula (XI). [00234] FIG. 28 depicts some exemplary compounds of Formulae (XII)-(XIV). Reference, Damha et. al. J. Am. Chem. Soc.2017, 139, 14542-14555. [00235] FIG. 29 depicts % mouse SOD1 (mSOD1) mRNA remaining relative to aCSF in the brain (right hemisphere). [00236] FIG. 30 depicts % mSOD1 mRNA remaining relative to aCSF/GADPH.in the brain (right hemisphere), liver and heart. DETAILED DESCRIPTION [00237] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise. [00238] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. M ring [00239] Compounds of Formula (IV) have the structure: . In compounds of Formula (IV) the ring M can be a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl), or a 5- or 6- membered sugar. [00240] In some embodiments, ring M is a cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl). IN some embodiments, ring M is a heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrothienyl).. [00241] In some embodiments, ring M is a sugar. The sugar moiety can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof). It is noted that phosphate moiety, B, R2’ and R3’ can be attached to any atom, e.g., any carbon atom of the ring M, e.g., of the sugar moiety. [00242] In some embodiments, ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R2 is attached to C2 or is absent, R3’ is attached to C3, and the phosphate moiety is attached to C4 of the furanose. In some embodiments, ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R2 is absent, R3’ is attached to C2, and the phosphate moiety is attached to C3 of the furanose. In some embodiments, ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R2 is absent, R3’ is attached to C4, and the phosphate moiety is attached to C3 of the furanose. In some embodiments, ring M is a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1, R2 is absent, and both of R3’ and the phosphate moiety are attached to C3 of the furanose. It is noted that R2’, R3’ and the phosphate moiety can replace the hydroxyl group present on the carbon of the furanose the R2’, R3’ and the phosphate moiety are attached to. When the phosphate moiety is attached to C4 of the furanose, it can replace the -CH2OH group at the C4 of the ribose. The attachment of the B can be in the alpha or beta configuration. The attachment of the R2’, R3’ and the phosphate moiety independently can be in the R or S configuration. [00243] In some embodiments, ring M is cyclopentane. In some embodiments, ring M is cyclopentane, and B is attached to C1, R2 is attached to C2 or is absent, R3’ is attached to C3, and the phosphate moiety is attached to C4 of the cyclopentane. In some embodiments, ring M is cyclopentane and B is attached to C1, R2 is absent, R3’ is attached to C2, and the phosphate moiety is attached to C3 of the cyclopentane. In some embodiments, ring M is cyclopentane, and B is attached to C1, R2 is absent, R3’ is attached to C4, and the phosphate moiety is attached to C3 of the cyclopentane. In some embodiments, ring M is cyclopentane, and B is attached to C1, R2 is absent, and both of R3’ and the phosphate moiety are attached to C3 of the cyclopentyl. It is noted that carbon of the cyclopentane to which the B is attached is denotated as C1 and numbering proceeds in a clockwise fashion. It is noted that attachment of the B, R2’, R3’ and the phosphate moiety independently can be in the R or S configuration. [00244] In some embodiments, ring M is a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), and B is attached to C1 of the pyranose, R2’ is absent, R3’ is attached to C4 and the phosphate moiety is attached to C5 of the pyranose. It is noted that R2’, R3’ and the phosphate moiety can replace the hydroxyl group present on the carbon of the pyranose the R2’, R3’ and the phosphate moiety are attached to. The attachment of the B can be in the alpha or beta configuration. The attachment of the R2’, R3’ and the phosphate moiety independently can be in the R or S configuration. [00245] DNA typically favors the C2'-endo sugar pucker (also known as the South conformer). This conformation is associated with the B-form of DNA, which is the canonical double helix structure. In C2'-endo, the C2' carbon atom is positioned above the plane of the sugar ring. Accordingly, in some embodiments, ring M is in the south conformation (i.e., C2’- endo). On the other hand, RNA typically favors the C3'-endo sugar pucker (also known as the North conformer). This conformation is associated with the A-form of RNA, which is a more compact helix. In C3'-endo, the C3' carbon atom is positioned above the plane of the sugar ring. Accordingly, in some other embodiments, ring M is in the north conformation (i.e., C3’-endo). R3’ [00246] In the various aspects described herein, R3’ can be hydrogen, halogen, -OR20, or -OR30. [00247] In some embodiments, R3’ is -OR30, where R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R3’ is -OR30 and R30 is hydrogen or hydroxyl protecting group. [00248] In some embodiments, R3’ is -OR30 and R30 is a reactive phosphorus group. For example, R30 is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1- C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2— C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m— (CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; each RP2 is independently C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1- C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1- C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1- C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2— [CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6, optionally each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, preferably each RP2 is isopropyl; or both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; and each RP3 is independently C1-30alkyl, C2-C30alkenyl, or C2-C30alkynyl, each optionally independently substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1- C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1- C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2— C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m— (CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6, optionally each RP3 is independently methyl, ethyl, propyl, isopropyl, n- butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00249] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; each RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [00250] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00251] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [00252] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently methyl, ethyl, propyl, isopropyl, n- butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00253] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently isopropyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00254] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, where: RP1 is 2- cyanoethyl (-CH2CH2CN); each RP2 is independently isopropyl. [00255] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently optionally substituted C1-6alkyl. [00256] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00257] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently optionally substituted C1-6alkyl. [00258] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00259] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [00260] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently optionally substituted C1-6alkyl. [00261] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1- C6alkoxy. [00262] In some embodiments, R3’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00263] In some embodiments, R3’ is -OR30 and R30 is a bond to a nucleoside or a nucleotide, or an oligonucleotide. When R30 is a bond to a nucleoside, a nucleotide, or an oligonucleotide, the internucleotide linkage between compound of formulae I-IV and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester) internucleotide linkage or a modified (e.g., phosphorothioate) internucleotide linkage. [00264] In some embodiments, R3’ is -OR30 and R30 is linked to 5’-position of a nucleoside, nucleotide, or oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage (e.g., or a modified (e.g., phosphorothioate) internucleotide linkage. For example, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide. In some embodiments, R30 is linked to the 5’- terminal (e.g., 5’-OH) of the oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage. In some other embodiments, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide by a modified (e.g., phosphorothioate) internucleotide linkage. [00265] In some embodiments, R3’is -OR30 and R30 is a hydroxyl protecting group. For example, R3’is -OR30 and R30 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′- bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate. [00266] In some embodiments, R3’is -OR30 and R30 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R30 is TBDMS. [00267] In some embodiments, R3’ is hydrogen or halogen. For example, R3 is H or F. [00268] In some embodiments, R3’ is -OR20, where R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, such as C1-6alkoxyC1-6alkyl (e.g., 2-methoxyethyl) or N-(C1- 6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl). For example, R3’ is -OR20, and R20 is hydrogen or hydroxyl protecting group. [00269] In some embodiments, R3’ is -OR20, and R20 is C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-4)alkyl, SO2NH(C1-4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1- 4)alkyl]2, C(O)NH2, C(O)NH(C1-6alkyl), C(O)N(C1-6alkyl)2, COOH, COO(C1-6alkyl) (e.g., COOMe), C2-6acyl (e.g., acetyl), (C1-8)alkyl, O(C1-8)alkyl (i.e., C1-8alkoxy), O(C1-8)haloalkyl, (C2-8)alkenyl, (C2-8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R3’ is - OR20, and R20 is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, optionally, R20 is methyl. [00270] In some embodiments, R3’ is -OR20, and R20 is C1-6alkoxyC1-6alkyl. For example, R3’ is -OR20, and R20 is 2-methoxyethyl. [00271] In some embodiments, R3’is -OR20 and R20 is a hydroxyl protecting group. For example, R3’is -OR20 and R20 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′- bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate. [00272] In some embodiments, R3’is -OR20 and R20 is TBDMS (or TBS), TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R20 is TBDMS (or TBS). [00273] In some embodiments, R3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O- alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. R2’ [00274] In the various aspects described herein, R2’ can be hydrogen, halogen, -OR20, or -OR30. [00275] In some embodiments, R2’ is hydrogen or halogen. For example, R2’ is H or F. [00276] In some embodiments, R2’ is -OR20, where R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, such as C1-6alkoxyC1-6alkyl (e.g., 2-methoxyethyl) or N-(C1- 6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl or 3-oxo-3-(N- methylamino)prop-1-yl). For example, R2’ is -OR20, and R20 is hydrogen or hydroxyl protecting group. In other examples, R2’ is -OR20, where R20 is an optionally substituted C1-6alkyl. [00277] In some embodiments, R2’ is -OR20, and R20 is C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1- C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)- C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m— (CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R2’ is -OR20, and R20 is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, optionally, R20 is methyl. [00278] In some embodiments, R2’ is -OR20, and R20 is C1-6alkoxyC1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1- C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2— [CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R2’ is -OR20, and R20 is 2-methoxyethyl. [00279] In some embodiments, R2’is -OR20 and R20 is a hydroxyl protecting group. For example, R2’is -OR20 and R20 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′- bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate. [00280] In some embodiments, R2’is -OR20 and R20 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R20 is TBDMS. [00281] In some embodiments, R2’ is -OR30, where R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R2’ is -OR30 and R30 is hydrogen or hydroxyl protecting group. [00282] In some embodiments, R2’ is -OR30 and R30 is a reactive phosphorus group. For example, R30 is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1- C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2— C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m— (CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; each RP2 is independently C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1- C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1- C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1- C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2— [CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6, optionally each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, preferably each RP2 is isopropyl; or both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; and each RP3 is independently C1-30alkyl, C2-C30alkenyl, or C2-C30alkynyl (e.g.,C1-10alkyl, C2- C10alkenyl, or C2-C10alkynyl , each optionally independently substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m— (CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6, optionally each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00283] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; each RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [00284] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00285] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [00286] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00287] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently isopropyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00288] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, where: RP1 is 2- cyanoethyl (-CH2CH2CN); each RP2 is independently isopropyl. [00289] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently optionally substituted C1-6alkyl. [00290] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionally substituted with a CN or –SC(O)Ph; both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00291] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently optionally substituted C1-6alkyl. [00292] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00293] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [00294] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently optionally substituted C1-6alkyl. [00295] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1- C6alkoxy. [00296] In some embodiments, R2’ is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, - P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. [00297] In some embodiments, R2’ is -OR30 and R30 is a bond to a nucleoside or a nucleotide, or an oligonucleotide. When R30 is a bond to a nucleoside, a nucleotide, or an oligonucleotide, the internucleotide linkage between compound of formulae I-II and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester) internucleotide linkage or a modified (e.g., phosphorothioate) internucleotide linkage. [00298] In some embodiments, R2’ is -OR30 and R30 is linked to 5’-position of a nucleoside, nucleotide, or oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage (e.g., or a modified (e.g., phosphorothioate) internucleotide linkage. For example, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide. In some embodiments, R30 is linked to the 5’- terminal (e.g., 5’-OH) of the oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage. In some other embodiments, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide by a modified (e.g., phosphorothioate) internucleotide linkage. [00299] In some embodiments, R2’is -OR30 and R30 is a hydroxyl protecting group. For example, R2’is -OR30 and R30 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′- bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate. [00300] In some embodiments, R2’is -OR30 and R30 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionally, R30 is TBDMS. [00301] R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkenyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2- methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl) or 3-oxo- 3-(N-methylamino)prop-1-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S- alkyl, O-alkenyl, S-alkenyl, N-alkenyl, branched O-alkenyl, branched N-alkenyl, branched S- alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , , , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. RP [00302] In various aspects described herein, each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S. In some embodiments, at least one RP is -ORO, e.g., both RP are independently - ORO. When RP is -ORO, each RO can be independently hydrogen, C1-3alkyl, or a hydroxyl protecting group. In some embodiments, at least one RP is -ORO, e.g., both RP are independently - ORO and each RO is independently hydrogen or C1-6alkyl (e.g., C1-3 alkyl). For example, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl. For example, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is hydrogen. In another example, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is methyl. In yet another example, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is ethyl. In yet another example, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is tert-butyl. [00303] In some embodiments, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is independently a hydroxyl protecting group. For example, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is independently a hydroxyl protecting group selected from the group consisting of pivaloyloxymethyl (POM), BOC or Boc, MOM, MTM, t- butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t-butoxymethyl, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, SEMOR, THP, 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, MTHP, 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl-S,S-dioxide, CTMP, 1,4-dioxan- 2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7- methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1- methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4- dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3- methyl-2- picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α- naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4- methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl-S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, TBMPS, formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate, preferably the protecting group is POM. Thus, in some embodiments, at least one RP is -ORO, e.g., both RP are independently -ORO and each RO is independently pivaloyloxymethyl (POM). [00304] In some embodiments, at least one RP is -SRS, e.g., both RP are independently -SRS. When RP is - SRS, each RS can be independently hydrogen, C1-3alkyl, or a thiol protecting group. In some embodiments, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is independently hydrogen or C1-3alkyl. For example, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is hydrogen, methyl, ethyl, or propyl. For example, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is hydrogen. In another example, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is methyl. In yet another example, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is ethyl. In some embodiments, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is independently a thiol protecting group. In some embodiments, at least one RP is -SRS, e.g., both RP are independently -SRS and each RS is independently pivaloyloxymethyl. [00305] In some embodiments, at least one RP is -N(RN)2, e.g., both RN are independently - N(RN)2 S. When RP is -N(RN)2, each RN can be independently hydrogen, C1-3alkyl, or a thiol protecting group. In some embodiments, at least one RP is -N(RN)2, e.g., both RP are independently -N(RN)2 and each RN is independently hydrogen or C1-3alkyl. For example, at least one RP is - N(RN)2, e.g., both RP are independently -N(RN)2 and each RN is hydrogen, methyl, ethyl, or propyl. For example, at least one RP is -N(RN)2, e.g., both RP are independently -N(RN)2 and each RN is hydrogen. In another example, at least one RP is -N(RN)2, e.g., both RP are independently -N(RN)2 and each RN is methyl. In yet another example, at least one RP is -N(RN)2, e.g., both RP are independently -N(RN)2 and each RN is ethyl. In some embodiments, at least one RP is -N(RN)2, e.g., both RP are independently -N(RN)2 and each RN is independently an amine protecting group. [00306] In some embodiments, at least one RP is -N(RN)S(O)2R2S, e.g., each RP is independently -N(RN)S(O)2R2S. For example, at least one RP is -N(RN)S(O)2R2S, e.g., both RP are independently - N(RN)S(O)2R2S, and each RN is independently hydrogen, methyl, ethyl, propyl, or isopropyl, and each R2S is independently methyl, ethyl, propyl, or isopropyl. In some embodiments, at least one RP is -N(RN)S(O)2R2S, e.g., both RP are independently -N(RN)S(O)2R2S, and each RN is independently an amine protecting group, and each R2S is independently methyl, ethyl, propyl, or isopropyl. B (nucleobase) [00307] In some embodiments of the various aspects described herein, B is an optionally modified nucleobase. It is noted that the nucleobase can be a natural or non-natural nucleobase. By a “non-natural nucleobase” means a nucleobase other than adenine, guanine, cytosine, uracil, or thymine. Exemplary non-natural nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5- alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6- diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5- nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5- methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5- methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2- methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, content of all which is incorporated herein by reference. [00308] In some embodiments, the non-natural nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8- (hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6, N6-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6- (alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8- (hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5- (alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)- 2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5- (allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3- diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4- (dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2- (thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)- 4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)- 4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)- 2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin- 1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2- (thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7- substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxyl)-1,3-(diaza)-2- (oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7- (aminoalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxyl)-1-(aza)-2- (thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenoxazin-1- yl, 7-(guanidiniumalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl- hydroxyl)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxyl)-1-(aza)-2-(thio)- 3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3- (methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7- (aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5- (trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6- (methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N2-substituted purines, N6-substituted purines, O6-substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof. [00309] In some embodiments, a non-natural nucleobase is a modified nucleobase, i.e., the nucleobase comprises a nucleobase modification described herein, e.g., the nucleobase is a substituted or modified analog of any of the natural nucleobases. Examples of the nucleobase modifications include, but not limited to: C-5 pyrimidine with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities, N2- and N6- with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities of purines, G-clamps, guanidinium G-clamps, and pseudouridine known in the art. [00310] In some embodiments of any one of the aspects, the non-natural nucleobase is a universal nucleobase. As used herein, a universal nucleobase is any modified or unmodified natural or non-natural nucleobase that can base pair with all of adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide comprising the universal nucleobase. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza- 7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6- methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4- methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof. [00311] In some embodiments, the natural or non-natural nucleobase is a protected nucleobase. As used herein, a “protected nucleobase” refers to a nucleobase comprising a nitrogen protecting group, and/or an oxygen protecting group, and/or a sulfur protecting group. [00312] For example, the nucleobase is a pyrimidine modified at the C4 position. In another non-limiting example, the nucleobase is a pyrimidine modified at the C5 position. [00313] In some embodiments, the nucleobase is a purine modified at the N2 position. In some embodiments, the nucleobase is a purine modified at the N6 position. For example, the nucleobase is a purine modified at the C6 position. In some non-limiting examples, the nucleobase is a N-7 deaza purine, optionally modified at the N7 position. [00314] In some embodiments, the nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil. For example, the nucleobase is uracil, adenine, guanine, or cytosine, optionally each independently comprising a hydroxyl, or amine protecting group. [00315] In some embodiments, the nucleobase is selected from the group consisting of: . Double-stranded RNA [00316] The skilled person is well aware that double-stranded RNAs comprising a duplex structure of between 19 and 24, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (RNAi). However, others have found that shorter or longer double-stranded oligonucleotides can be effective as well. Accordingly, in some embodiments, a longer double-stranded oligonucleotide described herein is capable of inducing RNA interference. Stated another way, the longer double-stranded oligonucleotides described herein can mediate RNA interference. As used herein, the phrase “mediates RNAi” refers to the ability to inhibit or reduce the expression of a target nucleic acid, e.g., a target RNA such as a mRNA in a sequence specific manner. [00317] Accordingly, in another aspect provided herein is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand substantially or 100% (e.g., exactly) complementary to the sense strand, and wherein one of the sense and antisense strand has a 5’- terminal modification comprising the structure: wherein: * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose); A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2-, wherein * is the bond to E; E is a bond or -CH2-; Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl; X is O or S; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl. [00318] Generally, the modification replaces a CH2OH group on the sugar moiety of the 5’-terminal nucleotide of the sense or antisense strand. For example, the modification replaces the 4’-CH2OH group on the furanose ring (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose) or the 5’- CH2OH group on the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5’-terminal nucleotide of the sense or antisense strand. [00319] Similarly, the modification replaces carbon atom in the sugar moiety of the 5’-terrminal nucleotide of the sense or antisense strand. For example, the modification replaces the 4’-C atom in the furanose (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribsofuranose) ring or the 5’-C atom in the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, allopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) ring of the 5’-terminal nucleotide of the sense or antisense strand. [00320] Thus, in some embodiments, the sense or antisense strand comprises at its 5’-end a compound of formulae I-XXI described herein. In certain embodiments, X is O. In certain embodiments, X is O, and each RP is ORO. In certain embodiments, X is O, and each RP is OH. In certain embodiments, X is O, and each RP is ethoxy. In certain embodiments, X is O, and each RP is OH. In certain embodiments, X is O, and each RP is ORO wherein RO is pivaloyloxymethyl. [00321] In some embodiments, the 5’-terminal nucleotide of one of the sense and antisense strand is of the structure: , , , , , ,
or a salt thereof, wherein: n is an integer selected from 1 - 3; A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2-, wherein * is the bond to E; E is a bond or -CH2-; B is an optionally modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionally replaced with -N=; Q4 is ethylene, ethenylene (e.g., or, preferably , where * is the bond to the phosphorous atom), propylene, propenylene, or propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionally replaced with -N=; y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n8 is an integer selected from 1 - 3 (e.g., 1, or 2); n9 is an integer selected from 1 - 3 (e.g., 1, or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2- methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=; and RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC); wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl; X is O or S; XA is O or S; YA is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; one of R2’and R3’is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionally substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3- (N-methylamino)prop-1-yl), optionally substituted C2-6alkenyl, or optionally substituted C2-6alkynyl (e.g., propargyl); and RA is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amin, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl) or alkyl ester; the other of R2’and R3’ is -OR30, wherein: R30 is a bond to the rest of the sense or antisense strand, provided that R3’ is -OR30 in formulae . [00322] Preferably, the antisense strand of the dsRNA is an oligonucleotide described herein. [00323] As used herein, the term “antisense strand” refers to an oligonucleotide that is substantially or 100% (e.g., exactly) complementary to a target nucleic acid of interest. For example, an antisense strand can be complementary, in whole or in part, to target nucleic acid of interest, such as a messenger RNA, an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding. [00324] It is noted that each strand of the dsRNA can range from 12-40 nucleotides in length. For example, each strand independently can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 27-35 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19- 23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, 21-23 nucleotides in length, 25-35 nucleotides in length, 26-35 nucleotides in length, 27-34 nucleotides in length, 28- 32 nucleotides in length or 29-31 nucleotides in length. Without limitations, the sense and antisense strands can be equal length or unequal length. In some embodiments, the antisense strand is longer, e.g., by 1, 2, 3, 4, or 5 nucleotides than the sense strand. [00325] In some embodiments, each of the sense and antisense strand is independently 15, 16, 17, 28, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. For, example, each of the sense and antisense strand is independently 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, each strand is independently 19, 20, 21, 22 or 23 nucleotides in length. In some embodiments, one strand (e.g., the sense strand) is 18, 19, 20, 21 or 22 nucleotides in length and the other strand (e.g., the antisense strand) is 21, 22, 23, 24 or 25 nucleotides in length. [00326] The sense and antisense strands of the dsRNA molecule are complementary to each other and can hybridize to each other to form a double-stranded or duplex region. Accordingly, the dsRNA molecule has a double-stranded or duplex region. The duplex region (double-stranded region) can be 17-25 nucleotide base pairs in length. For example, the dsRNA can have a duplex region of 17-24 nucleotide pairs in length. In some embodiments, the dsRNA has a duplex region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs in length. In some embodiments, the dsRNA has a duplex region of 19, 20, 21 or 22 nucleotide base pairs in length. [00327] The dsRNA molecule can have one or more overhang regions (i.e., single-stranded region) and/or capping groups of dsRNA molecule at the 3’-end, or 5’-end or both ends of a strand. Without limitations, the overhang can be 1-3 nucleotides, e.g., 1, 2 or 3 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the sequence being targeted or it can be complementary to the sequence being targeted or can be other sequence. The sense and antisense strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. Without limitations the overhang can be present at the 3’-end of only one of the strands or both strands. [00328] In some embodiments, the dsRNA molecule comprises a single overhang. For example, the dsRNA molecule has a single overhang and the overhang is no more than one, two or three nucleotides in length. Preferably, the overhang is 2 nucleotides in length. In some embodiments, the overhang is present at the 3’-end of a strand (e.g., the antisense strand). In some embodiments, the dsRNA comprises a two-nucleotide overhang at the 3’-end of a strand (e.g., the antisense strand). For example, the overhang is present at the 3’-end of the antisense strand. For example, the antisense comprises a 1 or 2 nucleotide overhang at its 3’-end. [00329] The dsRNA can also have a blunt end. For example, one end of the dsRNA is a blunt end and the other end has an overhang. Without limitations, the blunt end can be located at the 5’- end of the antisense strand (or the 3’-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3’-end, and the 5’-end is blunt. While not bound by theory, the asymmetric blunt end at the 5’-end of the antisense strand and 3’-end overhang of the antisense strand favor the guide strand loading into RISC process. In some embodiments, the dsRNA has a 2-nucleotide overhang on the 3’-end of the antisense strand and a blunt end at the 5’-end of the antisense strand. [00330] In some other embodiments, the dsRNA molecule has two blunt ends, i.e., at both ends of the dsRNA. For example, the two strands of the dsRNA are of the same length. In some embodiments, the antisense strand is of length 18 to 25 nucleotides. In some embodiments, the antisense strand is 21-25, 19-25, 19-21 or 21-23 nucleotides in length. In some particular embodiments, the antisense strand is 23 nucleotides in length. [00331] Similar to the antisense strand, the sense strand can be, in some embodiments, 18-25 nucleotides in length. In some embodiments, the sense strand is 21-25, 19-25, 19-21 or 21-23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. [00332] In some embodiments, sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. Nucleic acid modifications [00333] The longer double-stranded and single-stranded oligonucleotides described herein can comprise one or more nucleic acid modifications. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates (e.g., ligands), and any combinations thereof. It is noted that a nucleic acid modification(s) can be present in any position of longer double-stranded and single-stranded oligonucleotides. A nucleic acid modification(s) can be present in only one strand or both strands of a dsRNA. In some embodiments, only the antisense strand comprises at least one, e.g., two, three, four, five or more nucleic acid modifications. In some embodiments, only the sense strand comprises at least one, e.g., two, three, four, five or more nucleic acid modifications. In some embodiments, both strands independently comprise at least one, e.g., two, three, four, five or more nucleic acid modifications. [00334] Embodiments of the various aspects described herein recite specific position(s) on a strand, counting from an end of a strand. When the strand is single stranded, e.g., a longer-stranded oligonucleotide, the counting of the position is from the first nucleotide at the specified end. When the strand is part of a double-stranded molecule, e.g., a longer double-stranded oligonucleotide, the counting of the position can be from the first nucleotide at the specified end of the strand, or the first base-paired nucleotide in the strand at the specified end. Preferably, counting of the position is from the first nucleotide at the specified end of the strand. Thermally destabilizing modifications [00335] In some embodiments of any one of the aspects described herein, the dsRNA comprises a thermally destabilizing modification. By a “thermally destabilizing modification” is meant modification that result in a dsRNA having a lower overall melting temperature (Tm), preferably a Tm with one, two, three or four degrees lower, than the Tm of the dsRNA without having such a modification. Exemplary thermally destabilizing modifications are described herein below, and can include, but are not limited to, abasic modifications; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy (i.e., 2’-H) modification, acyclic nucleotide (e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA)), threose nucleic acid (TNA), a nucleotide linked by through its 2’-position (i.e., by its 2’-OH group to 5’-position of the subsequent nucleotide (a 2’-5’ RNA modification)); a Hyp-spacer modification; modified internucleotide linkages that decrease the thermal stability of dsRNA duplexes; or nucleobases with impaired W-C H-bonding to complementary base on the opposite strand. [00336] In some embodiments, the dsRNA comprises at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more independently selected thermally destabilizing modifications. The thermally destabilizing modification can be present at any position of the dsRNA. Further, the thermally destabilizing modifications all can be present in one strand or both strands of the dsRNA. In some embodiments, only the antisense strand comprises at least one, e.g., two, three, four or more thermally destabilizing modifications. In some embodiments, only the sense strand comprises at least one, e.g., two, three, four or more thermally destabilizing modifications. In some embodiments, both the sense and the antisense strands comprise at least one, e.g., two, three, four or more thermally destabilizing modifications. [00337] The thermally destabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the thermally destabilizing modification can occur on every nucleotide on the sense strand and/or antisense strand; each thermally destabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise thermally destabilizing modifications in an alternating pattern. The alternating pattern of the thermally destabilizing modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the thermally destabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the thermally destabilizing modifications on the antisense strand. [00338] In some embodiments, thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, or preferably at position 4, 5, 6, 7, or 8, counting from the 5’-end of the antisense strand. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5 or 9 from the 5’-end of the antisense strand. In some other embodiments, the thermally destabilizing modification is located at position 6, 7 or 8 from the 5’-end of the antisense strand. In some particular embodiments, the thermally destabilizing modification is located at position 7 from the 5’-end of the antisense strand. [00339] In some embodiments, only the antisense strand comprises a thermally destabilizing modification. For example, only the antisense strand comprises a thermally destabilizing modification and said thermally destabilizing modification is located at position 4, 5, 6, 7, or 8, counting from the 5’-end of the antisense strand, preferably the thermally destabilizing modification is located at position 5, 6, 7, or 8; more preferably the thermally destabilizing modification is located at position 6, 7, or 8. In some embodiments, only the antisense strand comprises a thermally destabilizing modification and the thermally destabilizing modification is located at position 7 of the antisense strand, counting from the 5’-end of the antisense strand. [00340] Similar to the antisense strand, a thermally destabilizing modification can be located at one of position 2, 3, 4, 5, 6, 7, 8 or 9, or preferably at position 4, 5, 6, 7, or 8, counting from the 5’- end of the longer-ssNA. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5 or 9 from the 5’-end of the longer-ssNA. In some other embodiments, the thermally destabilizing modification is located at position 6, 7 or 8 from the 5’-end of the longer- ssNA. In some particular embodiments, the thermally destabilizing modification is located at position 7 from the 5’-end of the longer-ssNA. Thermally stabilizing modifications [00341] In some embodiments, dsRNA comprises a thermally destabilizing modification. By a “thermally stabilizing modification” is meant modification that result in a dsRNA having a higher overall melting temperature (Tm), preferably a Tm with one, two, three or four degrees higher, than the Tm of the dsRNA without having such a modification. Exemplary thermally destabilizing modifications are described herein below, and can include, but are not limited to, 2’-fluoro nucleotides (2’-F modifications), bridged nucleic acid (BNA), e.g., locked nucleic acid (LNA), and cyclohexene nucleic acid (CeNA). In some preferred embodiments, the thermally stabilizing modification is a 2’-fluoro nucleotide. Exemplary, thermally stabilizing modification are described herein below. Additional exemplary abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011/133876 and WO2019222479, contents of both of which are incorporated herein by reference in their entireties. [00342] In some embodiments, dsRNA can comprise at least two, e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more the thermally stabilizing (e.g., 2’-F) modifications. Without limitations, the thermally stabilizing (e.g., 2’-F) modifications all can be present in one strand or both strands of a dsRNA. In some embodiments, the sense strand comprises at least one, e.g., two, three, four or more thermally stabilizing (e.g., 2’-F) modifications. In some embodiments, the antisense strand comprises at least one, e.g., two, three, four or more thermally stabilizing (e.g., 2’-F) modifications. In some embodiments, both the sense and the antisense strands comprise at least one, e.g., two, three, four or more thermally stabilizing (e.g., 2’- F) modifications. The thermally stabilizing (e.g., 2’-F) modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the thermally stabilizing (e.g., 2’-F) modification can occur on every nucleotide on the sense strand and/or antisense strand; each thermally stabilizing (e.g., 2’-F) modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise thermally stabilizing (e.g., 2’-F) modifications in an alternating pattern. The alternating pattern of the thermally stabilizing (e.g., 2’-F) modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the thermally stabilizing (e.g., 2’-F) modifications on the sense strand can have a shift relative to the alternating pattern of the thermally stabilizing (e.g., 2’-F) modifications on the antisense strand. [00343] In some embodiments, the sense strand of the dsRNA comprises at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more thermally stabilizing (e.g., 2’-F) modifications. In some embodiments, the sense strand comprises two, three, four, or five thermally stabilizing (e.g., 2’-F) modifications. For example, the sense strand comprises three or four thermally stabilizing (e.g., 2’-F) modifications. Without limitations, a thermally stabilizing (e.g., 2’-F) modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises at least three thermally stabilizing (e.g., 2’-F) modifications. For example, the sense comprises thermally stabilizing (e.g., 2’-F) modification at least at positions 7, 10 and 11, counting from the 5’-end of the sense strand. In some other embodiments, the sense strand comprises at least four thermally stabilizing (e.g., 2’-F) modifications. For example, the sense comprises thermally stabilizing (e.g., 2’-F) modification at least at positions 7, 9, 10 and 11, counting from the 5’-end of the sense strand. [00344] In some embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some other embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four thermally stabilizing (e.g., 2’-F) modification. [00345] In some embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11 from the 5’-end, counting from the 5’-end of the sense strand, and the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand. In yet some other embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the antisense strand. [00346] In some embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand. In yet some other embodiments, the sense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’- end of the antisense strand. [00347] In some embodiments, the sense strand does not comprise a thermally stabilizing (e.g., 2’-F) modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand. [00348] The antisense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more thermally stabilizing (e.g., 2’-F) modifications. In some embodiments, the antisense strand comprises two, three, four, five or six thermally stabilizing (e.g., 2’-F) modifications. Without limitations, a thermally stabilizing (e.g., 2’-F) modification in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises at least three thermally stabilizing (e.g., 2’-F) modifications. For example, the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the antisense comprises at least four thermally stabilizing (e.g., 2’-F) modifications. For example, the antisense comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 14 and 16, counting from the 5’-end of the antisense strand. In some further embodiments, the antisense strand comprises at least five thermally stabilizing (e.g., 2’-F) modifications. For example, the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand. In still some further embodiments, the antisense strand comprises at least six thermally stabilizing (e.g., 2’-F) modifications. For example, the antisense strand comprises thermally stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the antisense strand. [00349] In some embodiments, the antisense strand comprises at least one thermally stabilizing (e.g., 2’-F) modification adjacent to a stabilizing destabilizing modification. For example, the thermally stabilizing (e.g., 2’-F) modification can be the nucleotide at the 5’-end or the 3’-end of the thermally destabilizing modification, i.e., at position -1 or +1 from the position of the thermally destabilizing modification. In some embodiments, the antisense strand comprises a thermally stabilizing (e.g., 2’-F) modification at each of the 5’-end and the 3’-end of the thermally destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification. [00350] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification. [00351] In some embodiments, the sense strand does not comprise a thermally stabilizing (e.g., 2’-F) modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand. 2’-OMe nucleotides [00166] In some embodiments, the dsRNA described herein can comprise at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more 2’-OMe nucleotides. Without limitations, the 2’- OMe nucleotides all can be present in one strand or both strands of a dsRNA. In some embodiments, both the sense and the antisense strands comprise at least one 2’-OMe nucleotide. The 2’-OMe modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2’-OMe modification can occur on every nucleotide on the sense strand and/or antisense strand; each 2’-OMe modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise 2’-OMe modifications in an alternating pattern. The alternating pattern of the 2’-OMe modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the 2’-OMe modifications on the sense strand can have a shift relative to the alternating pattern of the 2’-OMe modifications on the antisense strand. [00167] The antisense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or more 2’-OMe modifications. Without limitations, a 2’-OMe modification in the antisense strand can be present at any position. In some embodiments, each nucleotide, except for any other specified modification (e.g., thermally destabilizing modification(s), thermally stabilizing modification(s), and/or 2’-deoxy (2’-H) modification(s)) of the antisense strand is independently a 2’-O-methyl nucleotide. [00168] Like the antisense strand, the sense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more 2’-OMe modifications. Without limitations, a 2’-OMe modification in the sense strand can be present at any positions. In some embodiments, each nucleotide, except for any other specified modification (e.g., thermally stabilizing modification(s), lipophilic modification(s), inverted nucleotide(s), thermally destabilizing modification(s), and/or 2’-deoxy (2’-H) modification(s)) of the sense strand is independently a 2’-O-methyl nucleotide. 2’-deoxy (2’-H) nucleotides [00169] In some embodiments, the dsRNA described herein can comprise a 2’-deoxy, i.e., 2’-H nucleotides. For example, the longer double-stranded and single-stranded oligonucleotides described herein can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) 2’-deoxy nucleotides. [00170] A 2’-deoxy nucleotide can be present in any position of the sense or antisense strand. Further, 2’-deoxy nucleotides all can be present in one strand or both strands of the dsRNA. [00171] In some embodiments, sense strand comprises 1, 2, 3, 4, 5 or more 2’-deoxy nucleotides. For example, the sense strand comprises a 2’-deoxy nucleotide at any one of positions 7, 9 and 11, counting from the 5’-end of the sense strand. In some embodiments, the sense strand comprises a 2’-deoxy nucleotide at least at position 9, counting from the 5’-end of the strand. For example, the sense strand comprises a 2’-deoxy nucleotide at least at positions 7 and 9, counting from the 5’-end of the strand. In another non-limiting example, the sense strand comprises a 2’- deoxy nucleotide at least at positions 9 and 11, counting from the 5’-end of the strand. [00172] In some embodiments, antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8 or more 2’-deoxy nucleotides. For example, the antisense strand comprises a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the antisense strand. In some embodiments, the antisense strand comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of the strand. For example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, counting from the 5’-end of the strand. In another non-limiting example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, and 12, counting from the 5’-end of the strand. In another non-limiting example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’- end of the strand. Lipophilic modifications [00173] In some embodiments, the dsRNA described herein can comprise a lipophilic modification. For example, the longer double-stranded and single-stranded oligonucleotides described herein can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) lipophilic modifications. Exemplary lipophilic modifications include nucleotides modified with a lipophilic group, e.g., nucleotides comprising a lipophilic group (e.g., an C10-C30 alkyl, or a C10-C30 alkenyl group, such as a C16 alkyl, a C16 alkenyl, a C18 alkyl, a C18 alkenyl, a C20alkyl, a C20 alkenyl, a C22 alkyl, a C22 alkenyl, a C24 alkyl, a C24 alkenyl; C15 alkyl, a C15 alkenyl, a C17 alkyl, a C17 alkenyl, a C19alkyl, a C19 alkenyl, a C21 alkyl, a C21 alkenyl, a C23 alkyl, or a C23 alkenyl) at their 2’-position. Some exemplary lipophilic nucleotides include, but are not limited to, 2’-O-hexadecyl-modified nucleotide (Nhd), 2’-O-docosanyl-modified nucleotide (Nda), 2’-O-(omega-hydroxy-hexadecyl)- modified nucleotide (NhdOH), and 2’-O-(omega-hydroxy-docosanyl)-modified nucleotide (NdaOH). [00174] A lipophilic modification can be present in any position of the sense or antisense strand. Further, lipophilic modifications all can be present in one strand or both strands of a dsRNA. In some embodiments, only the sense strand comprises a lipophilic modification. For example, the sense strand comprises a lipophilic modification at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 or 18, counting from the 5’-end of the sense strand. In some embodiments, the sense strand comprises a lipophilic modification at any one of positions 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 or 18, counting from the 5’-end of the sense strand. [00175] In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl, 2’-fluoro, 2’-deoxy, LNA, HNA, CeNA, 2’- methoxyethyl, 2’-O-allyl, or 2’-C-allyl, 2’-deoxy, or. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl or 2’-fluoro. It is to be understood that these modifications are in addition to any other specified modification (e.g., at least one thermally destabilizing modification of the duplex present in the antisense strand) of dsRNA molecule. [00176] In some embodiments, at least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2’-deoxy, 2’-O-methyl or 2’-fluoro modifications, thermally destabilizing modifications. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2’- O-methyl or 2’-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide, 2’-O-N-methylacetamido (2’-O-NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O-aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide. For example, each residue of the sense strand and antisense strand is independently modified with 2’- O-methyl nucleotide, 2’-deoxy nucleotide or 2´-deoxy-2’-fluoro nucleotide. Again, it is to be understood that these modifications are in addition to any thermally destabilizing modification of the duplex present in the antisense strand. [00177] In some embodiments, the antisense strand comprises at least one thermally destabilizing modification, and the remaining nucleotides are independently a 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide, 2’-O-N-methylacetamido (2’-O- NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O- aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide. For example, the antisense strand comprises a thermally destabilizing modification and the remaining nucleotides are independently a 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide. In some embodiments, the antisense strand comprises: (i) a thermally destabilizing modification at position 5, 6, 7, or 8, counting from the 5’-end of the antisense strand; (ii) at least two, e.g., 3, 4, 5 or 62’- fluoro nucleotides; and (iii) the remaining nucleotides are independently a 2’-O-methyl nucleotide or 2’-deoxy nucleotide. [00178] In some embodiments, each nucleotide of the sense strand is independently 2’-O- methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro (2’-F) nucleotide, 2’-O-N- methylacetamido (2’-O-NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O-aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide. For example, each nucleotide of the sense strand is independently 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´- deoxy-2’-fluoro nucleotide. In some embodiments, the sense strand comprises at least two, e.g., 3, 4, 5 or 6 2’-fluoro nucleotides, and the remaining nucleotides are independently a 2’-O-methyl nucleotide or 2’-deoxy nucleotide. [00179] In some embodiments, at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisense strand of a dsRNA can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex. In some embodiments, the nucleotide at the 1 position within the duplex region from the 5’-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5’- end of the antisense strand is an A:U base pair. It is noted that either the sense strand or the antisense strand can comprise the adenosine (A) nucleotide. Modified internucleotide linkages [00180] The dsRNA described herein can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more modified internucleoside linkages. As used herein, “internucleoside linkage” refers to a covalent linkage between adjacent nucleosides. Exemplary modified internucleoside linkage include, but are not limited to, phosphodietetrs, phosphorothioates (R, S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2-O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)- C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’), C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)-O-5', 3'- NHP(O)(OCH3)-O-5’), imidophosphoramidate (“imidp”), 2’->5’ internucleoside linkages, 2’->3’ internucleoside linkages, 3’->3’ internucleoside linkages, and 5’->5’ internucleoside linkages, optionally the modified internucleoside linkage is phosphorothioate, methylphosphonate, imidp or MMI, more preferably the modified internucleoside linkage is phosphorothioate (PS). [00181] A modified internucleotide linkage can occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification can occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand can have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. [00182] In some embodiments, the dsRNA comprises the modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage(s) in the overhang region. For example, the overhang region comprises two nucleotides having modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between the two nucleotides. Internucleotide linkage modifications can also be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides can be linked through modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage, and optionally, there can be additional modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides can be at the 3’-end of the antisense strand. [00183] With respect to position of an internucleotide linkage, the indicated position refers to the internucleotide linkage that links the nucleotide at said position with the nucleotide one position down stream from said position. In other word, an internucleotide linkage at position N means it is between nucleotides N and N+1. Thus, an internucleotide linkage at position 1, counting from the 5’-end, means the linker is between the nucleotides at positions 1 and 2, counting from the 5’- end. [00184] In some embodiments, the sense strand comprises one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the sense strand, and one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the sense strand. For example, the sense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5’-end of the sense strand, and sense strand further comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3’-end of the sense strand. [00185] In some embodiments, the antisense strand comprises one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the antisense strand, and one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the antisense strand. For example, the antisense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5’-end of the antisense strand, and the antisense strand further comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3’-end of the antisense strand. [00186] In some embodiments, the sense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2 counting from the 5’-end of the sense strand, a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2, counting from the 3’-end of the sense strand; and the antisense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2 counting from the 5’-end of the antisense strand, a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2, counting from the 3’-end of the antisense strand. Overhang modifications [00187] The nucleotides in the overhang region of the dsRNA molecule can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-Fluoro, 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl-5-methyluridine, 2’-O- methoxyethyladenosine, 2’-O-methoxyethyl-5-methylcytidine, GNA (glycol nucleic acid), SNA (serinol nucleic acid), TNA (threose nucleic acid), and any combinations thereof. For example, TT (or UU) can be an overhang sequence for either end on either strand. The 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule can be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate internucleotide linkage between the two nucleotides, where the two nucleotides in the overhang region can be the same or different. 5’-modifications [00188] The 5’-end of a strand of the dsRNA lacking the 5’-terminal phosphate mimic, can also be modified. Exemplary modifications for the 5’-end include, but are not limited a 5’-morpholino nucleotide (e.g., a nucleotide where the 5’-OH group is replaced with a morpholino group), a 5’- dimethylamino nucleotide (e.g., a nucleotide where the 5’-OH group is replaced with a dimthylamino group, a 5’-deoxy nucleotide, an inverted nucleotide (i.e., a nucleotide linked via a 5’->5’ linkage to the rest of the strand), an inverted abasic nucleotide (e.g., an abasic nucleotide linked by a 5’->5’ linkage), or an inverted abasic locked nucleic acid modification (i.e., an LNA lacking a nucleobase and linked by a 5’->5’ linkage) at the 5’-end. [00189] In some embodiments, the sense strand of the dsRNA comprises a 5’-morpholino nucleotide, a 5’-dimethylamino nucleotide, a 5’-deoxy nucleotide, an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end. For example, the sense strand comprises an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end. For example, the sense strand of the dsRNA comprises an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 3’-end. [00190] In some embodiments, the sense strand comprises a ligand at its 3’-end. Ligands [00191] In some embodiments, the oligonucleotide can comprise a ligand. Without wishing to be bound by a theory, a ligand can modify one or more properties of the attached molecule (e.g., the dsRNA described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake (cell targeting), charge and clearance. [00192] In some embodiments, the ligand is a targeting ligand. As used herein the term “targeting ligand” refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigen binding fragments of antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferring receptor ligands (e.g., transferrin), biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, lipoprotein receptor ligands (e.g., LDL and HDL). [00193] Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins. The term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule. [00194] In some embodiments, the ligand is a CNS tissue targeting ligand. For example, CNS tissue targeting ligand can be a lipophilic group that conjugated to an internal or terminal position within an oligonucleotide. Exemplary lipophilic ligands are described below. Certain receptors are known to be present on the surface of CNS cells that may be utilized in order to achieve delivery of an oligonucleotide, such as to a neuronal cell, a glial cell, a microglial cell, an oligodendrocytic cell, an ependymal cell, astrocytic cell, a unipolar cell, a bipolar cell, a multipolar cell, a psuedounipolar cell, a pyramidal cell, a basket cell, a stellate cell, a purkinje cell, a betz cell, an amacrine cell, a granule cell, an ovoid cell, a medium aspiny neuronal cell, and/or a large aspiny neuronal cell. Such can be present within CNS tissues, such as a forebrain tissue, a midbrain tissue, a hindbrain tissue, a diencephalon tissue, a telencephalon tissue, a myelencepphalon tissue, a metencephalon tissue, a mesencephalon tissue, a prosencephalon tissue, a rhombencephalon tissue, a cortices tissue, a frontal lobe tissue, a parietal lobe tissue, a temporal lobe tissue, an occipital lobe tissue, cerebral tissue, a tissue from the thalamus, a tissue from the hypothalamus, a tissue from the tectum, a tissue from the tegmentum, a tissue from the cerebellum, a tissue from the pons, a tissue from the medulla, a tissue from the amygdala, a tissue from the hippocampus, a basal ganglia tissue, a tissue from the corpus callosum, a tissue from the pituitary gland, a tissue from the ventral horn, a tissue from the dorsal horn and a white matter tissue. [00195] In some embodiments, the ligand is an ocular tissue targeting ligand. For example, ocular tissue targeting ligand can be a lipophilic group that conjugated to an internal or terminal position within an oligonucleotide. Exemplary lipophilic ligands are described below. Certain receptors are known to be present on the surface of ocular cells that may be utilized in order to achieve delivery of an oligonucleotide, such as to an optic nerve cell, a trabecular meshwork cell, a Schlemm’s canal cell, a juxtacanalicular tissue cell, a ciliary muscle cell, a retinal cell, an astrocyte, a pericyte, a Müller cell, a ganglion cell, an endothelial cell, a photoreceptor cell. Such can be present within ocular tissues, such as a retinal blood vessel , episcleral veins or choroid tissue, including a choroid vessel, cornea, pupil, sclera, conjunctiva, optic nerve, iris, lens, aqueous humor, macula, optic disk, retina, ciliary muscle, vitreous humor, vitreous body, choroid, fovea, ciliary body, blood vessels, muscles (lateral rectus muscle, medial rectus muscle, ciliary muscle), ligaments (suspensory ligaments), anterior chamber, posterior chamber, limbal rings, and fovia. [00196] In some embodiments, the ligand is an asialoglycoprotein receptor (ASGPR) ligand. By an ASGPR ligand is meant a ligand that binds the ASGPR. In some embodiments, the ASGPR ligand comprises one or more (e.g., 1, 2, 3 or more) GalNAc or GalNAc derivatives attached through a bivalent or trivalent branched linker. An exemplary ASGPR ligand is:
where n = 0 -10 (e.g., 1 or 4); or
, or a loop forming oligonucleotide where 3 or 4 consecutive nucleosides are modified with GalNAc containing ligands; such as, for example, the loop containing oligonucleotide, (5’-gcagcc(G*A*A*A*)ggcugc3’ ; SEQ ID NO: 113), where each lower case base is 2’-O-Methyl substituted and each G* and A* is substituted at the 2’-O position with . [00197] It is noted that a ligand can be linked at any position of either strands of the dsRNA. For example, the ligand can be at the 5’-end, 3’-end or at an internal position of a strand, e.g., sense or antisense strand of the dsRNA. [00198] In some embodiments, the sense strand comprises the ligand, i.e., the ligand is conjugated to the sense strand. For example, the ligand is conjugated to the 3′ end of the sense strand. In some embodiments, the sense strand or antisense strand comprises a lipophilic moiety (e.g., a in vivo delivery enhancing moiety) and a the targeting moiety. [00199] In some embodiments, the lipophilic moiety and the targeting moiety are independently present within: (a) an internally-modified nucleosides such as, (i) (ii) (b) a modified internucleotide linkage such as, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(RL1); or (c) a 5’-terminal modification such as (i) -P(Y)(OH)-R5, wherein Y is O or S and R5 is wherein Q2 is a bond, C(O), S(O)2, or -P(Y’)(OH)-O-; or (ii) -P(Y)(OH)O-RL3 or -C(O)N(H)RL3, wherein Y is O or S; or (iii) -RL3, -C(O)RL3, -C(O)N(H)RL3, -S(O)2RL3, -S(O)2N(H)RL3; or (d) a 3’-terminal modification such as (i) -P(Y)(OH)-R3, wherein Y is O or S; and R3 is or (ii) -RL3, -C(O)RL3, -C(O)N(H)RL3, -S(O)2RL3, -S(O)2N(H)RL3; wherein: B is an optionally modified nucleobase; B1 is a nucleobase modified with a lipophilic moiety or a targeting moiety (e.g., a pyrimidine nucleobase modified at the 5-position); RL3, RL1, and RL2 are each a group containing a lipophilic moiety or a targeting moiety; R2’ or R3’ may be any functional group that is an acceptable 2’-modification for a ribose sugar. Examples of suitable R2’ or R3’groups include, but are not limited to, hydrogen, halogen (e.g., 2’-fluoro), hydroxy, 2’-O-alkyl (e.g., 2’-OMethyl), 2’-O-methoxyalkyl (e.g., 2’-O- methoxymethyl, 2’-O-methoxyethyl, or 2’-O-2-methoxypropanyl) modification, 2’-O-allyl modification, 2’-C-allyl modification, 2'-O-N-methylacetamido (2'-O-NMA, i.e. -OCH2C(O)N(H)Me) modification, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) modification, 2'-O-aminopropyl (2'-O-AP) modification, or 2'-ara-F modification. For instance, R2’ or R3’ may be H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O- dimethylaminoethoxyethyl, or O-aminopropyl. [00200] In one embodiment, RL1, RL2 and RL3 are each a group containing a lipophilic moiety, such as a C10-26 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C12-26 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C12-24 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C14-24 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C14-18 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a C16 saturated or unsaturated hydrocarbon chain. In one embodiment, RL1, RL2 and RL3 are each a group containing a saturated or unsaturated C22-hydrocarbon chain. [00201] In other embodiments RL1, RL2 and RL3 are each a group containing a lipophilic moiety, such as a lipophilic vitamin or steroid, including, but not limited to, Vitamin E, Vitamin A (retinol, retinoic acid), and cholesterol. [00202] In one embodiment, when RL3 comprises a lipophilic moiety, then RL3 can be selected from the group consisting of:
wherein integer m is 0-10 (e.g., 0; or 1-10 or 1-8; or 0-6; or 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); W is C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl); R and R’ are each independently H or C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, or t-butyl); G is G1 or a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C21 hydrocarbon chain e., G together with the carbonyl to which it is attached may form a group with 22 carbons) (for instance, G may be a linear or branched C21 alkyl group), wherein G is optionally substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, - N(RG)2, -C(O)ORG, -OC(O)RG, -C(O)N(RG) 2, -N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl (for instance, G is optionally substituted with a -ORG, -C(O)ORG, or -N(RG)C(O)RG); and G1 is a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g., a C22 hydrocarbon chain,for instance, G1 may be a linear or branched C22 alkyl group), wherein G1 is optionally substituted with one or two groups selected from the group consisting of halogen, - ORG1, -SRG1, -N(RG1)2, -C(O)ORG1, -OC(O)RG1, -C(O)N(RG1)2, -N(RG1)C(O)RG1, - N(RG1)C(O)ORG1, -N(RG1)SO2(RG1), or -SO2N(RG1)2, wherein each RG1 is independently hydrogen or C1-C6 alkyl (for instance, G1 is optionally substituted with a -ORG1, -C(O)ORG1, or - N(RG1)C(O)RG1). [00203] Examples of RL3 include, but are not limited to,
Other examples of RL3 include, but are not limited to the following structures:
Further examples of RL3 include, but are not limited to,
In another embodiment, RL1 can be selected from the group consisting of -G1 and -S(O)2G1. Examples of RL1 include, but are not limited to, the following structures: Further examples of RL1 include, but are not limited to, the following structures: [00204] In another embodiment, RL2 is -C(O)RL3, wherein RL3 is according to any of the preceding embodiments thereof. In another embodiment, RL2 can be selected from the group consisting of wherein integer m is 0-8 (for instance, m is 0; or m is 1-8; or m is 0-6; or m is 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); R and R’ are each independently H or an alkyl group such as a C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl); G is G1 or a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g., a C21 hydrocarbon chain, i.e., G together with the carbonyl to which it is attached may form a group with 22 carbons); for instance, G may be a linear or branched C21 alkyl group), wherein G is optionally substituted with one or two groups selected from the group consisting of halogen, - ORG, -SRG, -N(RG)2, -C(O)ORG, -OC(O)RG, -C(O)N(RG) 2, -N(RG)C(O)RG, -N(RG)C(O)ORG, - N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl (for instance, G is optionally substituted with a -ORG, -C(O)ORG, or -N(RG)C(O)RG); and G1 is a saturated or unsaturated C10-26 saturated or unsaturated hydrocarbon chain (e.g.,a C22 hydrocarbon chain,(for instance, G1 may be a linear or branched C22 alkyl group), wherein G1 is optionally substituted with one or two groups selected from the group consisting of halogen, - ORG1, -SRG1, -N(RG1)2, -C(O)ORG1, -OC(O)RG1, -C(O)N(RG1)2, -N(RG1)C(O)RG1, - N(RG1)C(O)ORG1, -N(RG1)SO2(RG1), or -SO2N(RG1)2, wherein each RG1 is independently hydrogen or C1-C6 alkyl (for instance, G1 is optionally substituted with a -ORG1, -C(O)ORG1, or - N(RG1)C(O)RG1). Additional examples of RL2 include, but are not limited to the following structures: Further examples of RL2 include, but are not limited to the following structures: [00205] In some embodiments, B1 is a nucleobase modified with a G or G1 group, wherein G and G1 are as defined above (e.g., a pyrimidine nucleobase modified at the 5’-position with a group comprising G or G1). Examples of B1 include, but are not limited to, , wherein t is selected from 0 – 20 (e.g., 1-12, or 1-10, or 3-12, or 3-10). [00206] In some embodiments, in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: wherein: B is an optionally modified nucleobase; G3 is a saturated or unsaturated C1-20 hydrocarbon group (e.g., C1-6 alkylene; C2-6 alkylene; or hexylene);; LK is a linking group such as -O-, -N(H)-, -S-, -S-S-, -C(O)O-, OC(O)-, -C(O)N(H)-, - N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O)2-, -S(O)2O-, -S(O)2N(H)-, -P(O)(OH)O-, - OP(O)(OH)-, -P(S)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O-, -OP(S)(OH)O-, G2 is a saturated or unsaturated C10-26 hydrocarbon group (e.g., a C14-C24 hydrocarbon group, a C16-C22 hydrocarbon group, or a C21-C22 hydrocarbon group; and RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2. [00207] For example, when LK contains a carbonyl attached to G2 (e.g., (-N(H)C(O)- or -OC(O)- ), then G2 is a C21 hydrocarbon group; and when LK does not contain a carbonyl attached to G2, then G2 is a C22 hydrocarbon group. In one embodiment, RG is hydrogen. In another embodiment, RG is OH, In one embodiment, RG is COOH. In another embodiment, RG is CONH2. In one embodiment, RG is amino. [00208] In the above structures for the lipophilic monomers, the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. Further, in the preceding and throughout the present application, where a modified internucleotide linkage is shown with substituent atoms fully described at the phosphorous atom, e.g., where C’ is the 2’-carbon or 3’-carbon atom of a ribose ring, it is understood that the oxygen having the broken bond is the 5'-oxygen of the subsequent nucleotide. [00209] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: ,wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as . [00210] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as . [00211] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), and RG is hydrogen, hydroxy, amino, -COOH, or - C(O)NH2, such as [00212] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), and RG is hydrogen, hydroxy, amino, -COOH, or - C(O)NH2, such as [00213] In some embodiments, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C10-C22 hydrocarbon chain (e.g., a C16-C22 alkyl chain, or a C16 alkyl chain, or a C22 alkyl chain)., optionally substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, -N(RG)2, -C(O)ORG, - OC(O)RG, -C(O)N(RG)2, -N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or -SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl; and nucleobase B is a modified or unmodified nucleobase. In one embodiment, n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is a C22 alkyl chain. In one embodiment, G is a C16 alkyl chain [00214] In some embodiments, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C22 hydrocarbon chain, optionally substituted with one or two groups selected from the group consisting of halogen, -ORG, -SRG, - N(RG)2, -C(O)ORG, -OC(O)RG, -C(O)N(RG) 2, -N(RG)C(O)RG, -N(RG)C(O)ORG, -N(RG)SO2(RG), or - SO2N(RG)2, wherein each RG is independently hydrogen or C1-C6 alkyl; and nucleobase B is a modified or unmodified nucleobase. In one embodiment, n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is C10-C22 alkyl chain (e.g., a C14-C24 alkyl chain, C16-C22 alkyl chain, or a C16 alkyl chain, or a C22 alkyl chain). [00215] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: . [00216] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula , wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21), such as [00217] In one embodiment, the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula
[00218] In some embodiments, the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(RL1), wherein RL1 is -G1 or S(O)2-G1, each as defined above, wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. [00219] In some embodiments, the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(O)(X)O-, wherein X is -N(H)(RL1), wherein RL1 is (such as wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. [00220] In some embodiments, the in vivo delivery enhancing moiety is present within a [00221] a modified internucleotide linkage of the form, (such as wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. In certain embodiments, the preceding nucleotide contains a 2’- fluoro modification. In certain embodiments, the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification. [00222] In some embodiments, the in vivo delivery enhancing moiety is present within a [00223] a modified internucleotide linkage of the form (such as wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) and the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic. In certain embodiments, the preceding nucleotide contains a 2’- fluoro modification. In certain embodiments, the preceding nucleotide contains a 2’-O-methyl modification. In certain embodiments, the preceding nucleotide contains a 2’-H modification. [00224] In some embodiments, the in vivo delivery enhancing moiety is present within a [00225] a modified internucleotide linkage of the form, -OP(Y)(X)O-, whereinY is O or S and X is wherein G1 is defined above, such as wherein n is selected from 7- 23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21) or [00226] In some embodiments, in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 3’-end of the sense or antisense strand via a direct bond or through a carrier or linker. In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of the sense or antisense strand via a direct bond or through a carrier or linker. [00227] In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula wherein X is O or S (e.g., S); and RL3 is according to any of the preceding embodiments there. For example, RL3 can be wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). In another example, RL3 can be wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). For example, m can be 2-5, or 1, or 2, or 3, or 4 or 5. [00228] In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula -RL3, wherein RL3 is according to any of the preceding embodiments there. For example, RL3 can be wherein n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). In another example, RL3 can be such as wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23), and RG is hydrogen, hydroxy, amino, - COOH, or -C(O)NH2.. For example, m can be 2-5, or 1, or 2, or 3, or 4 or 5. [00229] [00230] In another example, RL3 can be , wherein m is selected from 1-6, and n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). For example, m can be 2-5, or 1, or 2, or 3, or 4 or 5. [00231] In some embodiments, in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the sense and antisense strands (e.g., sense strand) and is of the formula: , or a salt thereof, wherein X is O or S (e.g., S); L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl). In one embodiment, in vivo delivery enhancing moiety is conjugated to the 5’- end of one of the sense and antisense strands and is of the formula (such as or a salt thereof, wherein q is selected from 0 – 18 (e.g., 1-11 or 1-8, or 3-11, or 3-8) and X is O or S (e.g., S). In these embodiments, Rligand is selected from , wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21); and a, b, and c are independently selected from 1- 22, provided that the sum of a + b + c is selected from 2 to 22. [00232] In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula [00233] [00234] wherein n is selected from 7-23 (e.g.,11 – 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21). In one embodiment, n is 13. In another embodiment, n is 19. [00235] In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula: wherein m is selected from 1-10; n is selected from 7-23 (e.g., 11-23, or 11 – 21, or 13-21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); E is -C(O)N(H)-(CH2)p-*, -N(H)C(O)-(CH2)p-*, -C(O)O-(CH2)p-*, -OC(O)-(CH2)p-*, - OP(Y)(OH)O-(CH2)p-*, -O-(CH2)p-, -N(H)-(CH2)p, -S-(CH2)p-, -N(H)-O-(CH2)p-, -O-N(H)-(CH2)p-, N(H)N(H)-(CH2)p-, or -S-S-(CH2)p-*, -Ph-(CH2)p-, -OPh-(CH2)p-, or -ZZ1-(CH2)p-; wherein * is the bond to the alpha-amino acid carbon, Ph is phenyl, Y is =O or =S, p is selected from 1 – 6; RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; and ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne). [00236] In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula wherein m is selected from 1-10 (e.g., 3-6, or 3); RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2, and n is selected from 7- 23 (e.g., 11-23, or 11 – 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23). For example, m can be 3-6 and RG is hydrogen; or m can be 3-6 and RG is COOH. [00237] In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and the 5’-terminal nucleotide is of the formula: where Lipo in R is one of:
[00238] In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is L2 wherein R selected from: Lipo2 wherein R is
[00239] In one embodiment, in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: [00240] In one embodiment, in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is , wherein RL2 selected from:
[00241] In some embodiments, in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’-end of one of the sense and antisense strands via a carrier or linker, and the carrier or linker is an inverted abasic nucleotide, such as an inverted abasic deoxyribonucleotide or an inverted abasic ribonucleotide, each connected to the remainder of the oligonucleotide via a phosphodiester (PO) or phosphorothioate (PS) linkage. Examples include, but are not limited to, , wherein Q2 is a bond, C(O), S(O)2, or -P(Y’)(OH)-O-, Y and Y’ are independently O or S; and RL3 is as defined above. [00242] In some embodiments, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide and is of the formula or a salt thereof, wherein each X is independently O or S (e.g., each is S); Rligand is selected from the groups listed in Table R-1; and L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl). [00243] For example, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula or a salt thereof, wherein each X is independently O or S (e.g., each is S) and Rligand is selected from wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21). [00244] In some embodiments, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula or a salt thereof, wherein each X is independently O or S (e.g., each is S) and Rligand is selected from wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21).and L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl. [00245] In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula or a salt thereof, wherein each X is O or S (e.g., each is S) and Rligand is selected from ,,wherein n is 7-23 (e.g.,11 – 21, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21). [00246] In another embodiment, RL1, RL2, and RL3 are each a group containing at least one targeting moiety, such as an integrin receptor ligand (e.g., avB3, avB5, or avB6-targeting ligand), a chemokine receptor ligand, a transferring receptor ligand (e.g., transferrin), a serotonin receptor ligand, an Asialoglycoprotein receptor-targeting ligand, a lipoprotein receptor ligand (e.g., LRP1- targeting ligand), etc. In one embodiment, the targeting moiety can be selected from:
[00247] In one embodiment, the broken bond is connected to a group of the formula, **-L-ZZ- L’- , wherein ** represents the bond to the targeting ligand, ZZ is a bridging group that may be formed, for example, by reaction of two functional groups, and can be selected from the group consisting of, -C(H)=N-, -C(H)=N-N(H)-, -C(H)=N- N(H)C(O)-, -C(H)=N-N(R)-, -C(H)=N-N(R)C(O)-, -C(H)=N-O-, -C(O)N(H)-, -C(O)N(H)-N(H)- , -C(O)N(H)-N(R)-, -C(O)N(R)-, -C(O)O-, -C(O)S-, -C(O)N(H)-, -C(O)N(H)-N(H)-, -C(O)N(H)- N(R)-, -C(O)N(R)-, -C(S)N(H)-, -C(S)N(R)-, -C(S)O-, -C(S)S-, -C(S)N(H)-, -C(S)N(R)-, - N(H)C(O)N(H)-, -N(H)C(O)N(R)-, -N(H)C(O)O-, -N(H)C(O)S-, -N(H)C(S)N(H)-, - N(H)C(S)N(R)-, -N(H)C(S)O-, -N(H)C(S)S-, -S-S-, -CH2-S-, -CH2-O-, -CH2-N(H)-, -CH=CH-, and a click adduct, for example, comprising a group selected from the following structures:
wherein each R is independently C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, butyl, or hexyl); and RLa is hydrogen, C1-10alkyl (e.g., methyl, ethyl, propyl, isopropyl, t- butyl, isobutyl, butyl, or hexyl), C3-8cycloalkyl, 3-8 membered heterocyclyl, aryl (e.g., phenyl), or heteroaryl (e.g., 2-pyridyl). [00248] “Click adduct” herein included those adducts formed by a copper(I)-catalyzed azide- alkyne cycloaddition reaction, a strain-promoted azide-alkyne cycloaddition, a strain-promoted azide-trans-cycloalkene cycloaddition, and a thiol-maleimide Michael-addition reaction including, for example, (i) an azide with a terminal alkyne or cycloalkyne (e.g. cyclooctyne, BCN, or DBCO); (ii) a tetrazine with a terminal alkyne or cycloalkyne (e.g. cyclooctyne); (iii) a thiol and maleimide (with or without hydrolysis of the product). [00249] In some embodiments, L and L’ are independently one of: (a) -L1-[G-L2]q-G-L3-* wherein q is 0 or an integer selected from 1-10; (b) -L1-G-L2-G-L3-*; (c) -L1-G-L3-*; (d) -G-L3-*; (e) -L1-G-*; or (f) -G-*. wherein in each of (a) -(f), * represent the bond to ZZ; L1 is selected from one of the groups: (a) a bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH), wherein and RN is hydrogen or C1-6alkyl; (b) a bond, C(O), P(O)(OH), or P(S)(OH); (c) a bond; (d) C(O); (e) P(O)(OH); or (f) P(S)(OH); each L2 and L3 is independently selected from one of the groups: (a) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, - N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and - N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (b) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-,- N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (c) -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)N(RN)-, -N(RN)C(O)O-, -N(RN)C(O)N(RN)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (d) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; or (e) -C(O)N(RN)-, -N(RN)C(O)-, -O-, and -N(RN)-, wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently selected from one of the groups: (a) C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or (b) C1-10alkyl, optionally substituted with 1, 2, or 3 R groups (e.g., 1 or 2 R groups; or 1 R group); or wherein each R group is independently selected from the group consisting of R’, C1-6alkyl, C1- 6haloalkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, heterocyclyl, aryl, heteroaryl, C3- 8cycloalkylC1-6alkyl, heterocyclylC1-6alkyl, aryl C1-6alkyl, heteroarylC1-6alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(Rb)2, -O(Ra), -S(R0), -C(O)OR0, C(O)R0, -C(O)N(R0)2, -C(NR0)OR0, -C(NR0)R0, -C(NR0)N(R0)2, -C(S)OR0, -C(S)R0, -C( S)N(R0)2, -S(O)2R0, -S(O)2OR0, -S(O)2N(R0)2, -N(R0)C(O)OR0, -N(R0)C(O)R0, -N(R0)C( O)N(R0)2, -N(R0)S(O)2R0, -N(R0)S(O)2OR0, -N(R0)S(O)2N(R0)2, -OC(O)OR0, -OC(O)R0, -OC(O)N(R0)2, -OS(O)2R0, -OS(O)2OR0, -OS(O)2N(R0)2, or -SC(O)R0, wherein each R0 is independently hydrogen or C1-6alkyl; each Ra is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; and each Rb is independently hydrogen, C1-6alkyl, or a nitrogen protecting group. [00250] In one embodiment of L or L’, q is 0, 1, 2, 3, 4, or 5. In another embodiment of L or L’, q is 0, 1, 2, 3, or 4. In another embodiment of L or L’, q is 0, 1, 2, or 3. In another embodiment of L or L’, q is 0, 1, or 2. In another embodiment of L or L’, q is 1, 2, 3, 4, or 5, In another embodiment of L or L’, q is 1, 2, 3, or 4. In another embodiment of L or L’, q is 1, 2, or 3. In another embodiment of L or L’, q is 1 or 2. In another embodiment of L or L’, q is 4. In another embodiment of L or L’, q is 3. In another embodiment of L or L’, q is 2. [00251] In some embodiments, L is one of: (a) wherein k i 1 N s an integer from 1 to 10; L is bond, C(O), C(S), C(NR ), S(O)2, P(O)(OH), or P(S)(OH) (e.g., L1 is a bond, C(O), P(O)(OH), or P(S)(OH)); and RN is hydrogen or C1-6alkyl; (b) wherein k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7; (c) wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3); (d) wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an integer from 4 to 6); (e) wherein and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6); (f) wherein s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); or and wherein in each of the preceding embodiments of L, * represents the bond to ZZ. [00252] In some embodiments, L’ is : (a)*-G-L1-, wherein L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1- 10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1- 10alkyl; (b)*-G-[L2-G]q-L1-, wherein L1 is a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); each L2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(RN)-, wherein RN is hydrogen or C1-6alkyl each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl; (c) -[G-L2]q-G-L3-*, wherein each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; or each L2 is independently C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O wherein each RN is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (d) *-L3-G-L1-, wherein L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L1 is -OP(O)(OH)O- or -OP(S)(OH)O-; and G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups (e.g., 1 or no R groups); (e) -L1-[G-L2]q-G-*, wherein L1 is a bond or -B-A-; each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl or C2- 10alkenyl, each of which is optionally substituted with 1 or 2 R groups; (f) -[G-L2]q-G-*, wherein each L2 is independently C(O)(NRN) , N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O wherein each RN is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; (g) -C2-30alkyl-*, such as -C5-20alkyl-* or -C10-20alkyl-*; (h) -C1-10alkyl-*, optionally substituted with 1 or 2 R groups; (i) -C(O)-C2-30alkyl-*, such as -C(O)-C5-20alkyl-* or -C(O)-C10-20alkyl-*; wherein in each of (a) - (i), * is the bond to ZZ, and q, when present, is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3; or 0, 1, 2, or 3; or 0, 1, or 2; or 0 or 1; or 0; or 1; or 2). [00253] Examples of RL and RL1 that comprise a targeting ligand include, but are not limited to, [00254] Additional examples of RL1 and RL3 that comprise a targeting ligand include, but are not limited to, [00255] Examples of RL2 that comprise a targeting ligand include, but are not limited to, [00256] Additional examples of RL2 that comprise a targeting ligand include, but are not limited to, [00257] In one embodiment of any of the preceding structures comprising a targeting moiety, each RX is an integrin-receptor targeting ligand such as, [00258] In one embodiment, RL1 or RL3 is . [00259] In one embodiment, RL2 is [00260] In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is:
In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: [00261] In another embodiment, multiple targeting ligands may be connected to a branched multivalent linker. [00262] In certain embodiments, RL1, RL2 and RL3 can comprise a branched linking group (Δ) capable of supporting multiple targeting ligands (e.g., at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3). For example, in one embodiment, the mutiple targeting moieties can be connected through an RL, RL1, and RL2 of the form, (RX-L-ZZ-)z-Δ-T- , wherein each RX is a targeting moiety; z is at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3, T is -L’-T’-**, wherein ** is the bond to Δ, and T’ is O, S, N(H), C(O), S(O)2, C(O)N(H), N(H)C(O), OC(O), OC(O), -P(O)(OH)-, -P(S)(OH)-, -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, -P(S)(OH)O-, -OP(O)(OH)O-, or -OP(S)(OH)O-; and L , L’, and ZZ are each as described above, and each * represents the bond to the targeting ligand. [00263] In some embodiments, T is selected from the following, wherein ** is the bond to Δ: (a) -C(O)-X1-L5-X2-C(O)-**, wherein X1 and X2 are each independently C1-10alkyl; or C2-10alkyl; or C4-10alkyl; or C6-10alkyl; or C2-8alkyl; or C2-6alkyl; or C2-4alkyl; (b) -C(O)-C2-20alkyl-C(O)-**, such as -C(O)-C2-12alkyl-C(O)-**, (c) -C(O)-C6-20alkyl-C(O)-**, such as -C(O)-C6-12alkyl-C(O)-**, (d) -C(O)-C10alkyl-C(O)-** and (e) -C(O)-CH2CH2-C(O)-**, wherein each L5 is a bond, ZZ, or - A1-B1-A1- (e.g., a bond, -B1-A1- or -A1-B1-; or a bond; or ZZ), wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is independently hydrogen or C1-6alkyl; each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, - OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct). [00264] In some embodiments, T is selected from the following, ** is the bond to Δ: (f) -N(H)C(O)-C2-20alkyl-C(O)-**, (g) - N(H)C(O)-C6-20alkyl-C(O)-**, such as - N(H)C(O)-C6-12alkyl-C(O)-**, (h) - N(H)C(O)-C10alkyl-C(O)-**, (i) -C(O)-C2-20alkyl-C(O)N(H)-**, (j) -C(O)-C6-20alkyl-C(O)N(H)-**, (k) -C(O)-C6-12alkyl-C(O)N(H)-**, (l) -C(O)-C10alkyl-C(O)N(H)-**, (m) -N(H)C(O)-C2-20alkyl-C(O)N(H)-**, (n) - N(H)C(O)-C6-20alkyl-C(O)N(H)-**, (o) - N(H)C(O)-C6-12alkyl-C(O)N(H)-**, and (p) - N(H)C(O)-C10alkyl-C(O)N(H)-**. (q) In some embodiments, T is selected from the following, ** is the bond to Δ: (a) -N(H)C(O)-X3-ZZ-X4-C(O)-**, (b) -C(O)-X3-ZZ-X4-C(O)N(H)-**, (c) N(H)C(O)-X3-ZZ- X4-C(O)N(H)-**, wherein X3 and X4 are independently C2-12alkyl; or C4-10alkyl; or C6-10alkyl; or C4-8alkyl; and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, -OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct). [00265] In some embodiments, , T is selected from the following (w) -L6-[G5-O]q5-G5-L4-**, wherein L4 and L6 are independently -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1- 6alkyl; each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G5 is independently C1-10alkyl; (x) -C(O)-[CH2CH2-O]q5-G5-L4-**, L4 is -A1-B1-A1-, wherein each A1 is independently a bond, -O-, -S-, or -N(RN1)-, wherein RN1 is hydrogen or C1-6alkyl; each B1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); G5 is C1-10alkyl. (y) -C(O)-[CH2CH2-O]q5-G5-L4-**, L4 is -A1-B1 or -B1-A1-, wherein each A1 is independently -O- or -N(H)-, and each B1 is independently C(O), G5 is C1-10alkyl (e.g., C2-10alkyl or C2-6alkyl); and (z) -C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-**; wherein in each of the preceding ** is the bond to Δ, and q5, when present, is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4.) Examples of branched linking group (Δ) include, but are not limited to, wherein the broken bond is the bond to L’. [00266] Examples of -T*-Δ- include, but are not limited to,
[00267] Examples of RL and RL1 that comprise a branched linker to a targeting ligand include, but are not limited to,
[00268] Examples of branched RL1 and RL3 include,
[00269] Examples of branched R12 include,
202
[00270] In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’- terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: selected from 1 - 10 (e.g., r is 7); and each R is:
[00271] In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is:
[00272] In some embodiments, targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is: [00273] In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: selected from 1 - 10 (e.g., r is 7); and each R is: wherein Rxis a targeting ligand.
[00274] In some embodiments, targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is: ligand.
[00275] In one embodiment of any of the preceding structures comprising a targeting moiety, each Rxis an integrin-receptor targeting ligand such as,
[00276] In another embodiment of any of the preceding structures comprising a targeting moiety, each Rx is an ASGPR ligand, such as
[00277] In certain embodiments, L and/or L’ may be a bond when ZZ is formed by reaction of a functional group within the ligand or oligonucleotide. For example, when the ligand is an antibody or an antigen binding fragment thereof, then ZZ be formed by reaction of: a thiol (SH) group within one or more cysteine (C) residues; or an amino (NH2) group within one or more lysine (K) residues; or a primary amide group (CONH2) within one or more asparagine (N) or glutamine (Q) residues; or an azide group (N3) within one or more modified amino acid residues (e.g., an azide within an 6-azidolysine residue; or a carboxylic acid group within one or more aspartic acid or glutamic acid residues.
[00278] In some embodiments, the dsRNA agent comprises a double-stranded region formed between the sense and antisense strands and optionally one or two single-stranded non-loop overhang, and wherein the one or more lipophilic moieties are conjugated to either the doublestranded region or the non-loop overhang. In some embodiments, the dsRNA agent does not contain a loop (e.g., stem loop) region. In some embodiments, the dsRNA agent contains a loop (e.g., stem loop) region, and the one or more lipophilic moieties are not conjugated to the loop (e.g., stem loop) region.
[00279] In some embodiments, the dsRNA agent comprises a sense strand of 10 to 53 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand. For instance, the sense strand may be 10 to 49, 12 to 49, 12 to 45, 12 to 42, 12 to 40, 15 to 49, 15 to 45, 15 to 42, 15 to 40, 15 to 38, or 15 to 36 nucleotides in length. In some embodiments, the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length. In some embodiments, the region of complementarity to the target sequence is at least 19 contiguous nucleotides in length.
[00280] In some embodiments, the sense strand comprises at its 3'-end a stem-loop set forth as: S1-L-S2, in which S1 is complementary to S2, and in which L forms a loop between S1 and S2.
[00281] In some embodiments, the first 17 to 25 nucleotides counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 11 to 28 counting from 5’ end of the sense strand forms a 3'-end a stem-loop set forth as: S1-L-S2.
[00282] In some embodiments, the length of the stem loop S1-L-S2 is 11 to 28, 13 to 26, or 15 to 24 nucleotides in length. In one embodiment, the stem loop S1-L-S2 is 16 nucleotides in length. In some embodiments, the stem loop S1-L-S2 comprises a sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 105).
[00283] In some embodiments, L is at least 3, 4, or 5 nucleotides in length. In some embodiments, L comprises a sequence of GAAA.
[00284] In some embodiments, the sense strand is 36 nucleotides in length, the first 20 nucleotide counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 16 nucleotides forms a stem loop S1-L-S2. In one embodiment, the 16-nucleotide stem loop S1-L-S2 has the sequence of GCAGCCGAAAGGCUGC (SEQ ID NO: 105), wherein L is GAAA.
[00285] In some embodiments, the one or more lipophilic moieties are conjugated to a nonterminal position of the sense strand.
[00286] In some embodiments, the one or more lipophilic moieties are conjugated to one or more nucleotides of the stem loop S1-L-S2. In some embodiments, the one or more lipophilic moieties are conjugated to one or more nucleotides of the loop L.
[00287] In some embodiments, S1 and S2 are complementary and contain 4-10 nucelotides, e.g., S1 and S2 each contain 6 complementary nucelotides.
[00288] In some embodiments, S1 and S2 are complementary and contain 4-10 nucelotides and L is GAAA, e.g., Si and S2 each contain 6 complementary nucelotides and L is GAAA.
[00289] In some embodiments, the one or more lipophilic moieties containing one or more saturated or unsaturated C22 hydrocarbon chains are conjugated to one or more internal positions on at least one strand of the dsRNA agent.
Dual Conjugation
[00290] In another embodiment, in vivo delivery enhancing moiety is connected in series with another targeting moiety, as described herein. For example, a sense or antisense strand can contain a series modification at the 3 ’-end or 5 ’-end of the oligonucleotide, such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety.
[00291] In one embodiment, the series modification is of the form, wherein Q is selected from wherein RL2 is according to any preceding embodiment, wherein one of the broken bonds connects to a 5 ’-oxygen of a nucleoside or a 3’- oxygen of a nucleoside and the other connects to a 5 ’-terminal or 3 ’-terminal modification as described herein. [00292] In another example, a sense or antisense strand can contain a series modification of the form, wherein Q is selected from wherein
RL2 is according to any preceding embodiment, each Y is independently O or S; one of the broken bonds connects to a 5 ’ -oxygen of a nucleoside or a 3 ’ -oxygen of a nucleoside and the other connects any of the 5’-terminal modifications described above or 3’-terminal modifications described above.
[00293] In one embodiment, a sense or antisense strand can contain a series modification of the the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; each Y is independently O or S; one of R51 and R52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety) and the other comprises a second ligand moiety (e.g., a targeting moiety).
[00294]
[00295] In another embodiment, a sense or antisense strand can contain a series modification of the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; each Y is independently O or S; one of R51 and R52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of RL or RL2 above) and the other comprises a second ligand moiety (e.g., a targeting moiety).
For example an oligonucleotide may have a series modification at the 5 ’-end of the formula:
wherein n is selected from 7-23 (e.g.,11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each
Y is independently O or S and R510 and R520 are wherein each Y is independently O or S.
In another embodiment, a sense or antisense strand can contain a series modification of the form, wherein the broken bond connects to the 3’-oxygen of a 3’-terminal nucleoside; each Y is independently O or S; one of R31 and R32 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of RL2 above) and the other comprises a second ligand moiety (e.g., a targeting ligand).
For example an oligonucleotide may have a series modification at the 3 ’-end of the formula: wherein n is selected from
7-23 (e.g.,11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); and each Y is independently O or S and R310 and R320 are indepedently O or S.
[00296] In another embodiment, can contain a single modification at the 5’-end, the 3’-end or at an internal position that contains both the in vivo delivery enhancing moiety and the targeting moiety.
[00297] In one embodiment, single modification is bonded to the 3 ’-oxygen of the 3 ’-terminal nucleotide, and is of the formula -P(Y)(OH)-R3, wherein Y is O or S and R3 is wherein RL2 and RL3 comprises the in vivo delivery enhancing moiety (e.g., according to any in vivo delivery enhancing moiety embodiment of RL2 or RL3 above); RTG comprises the targeting moiety (e.g., RTG is according to Formula (X) (below), wherein R5 is -L-ZZ-L’-, where L, ZZ, and L’ are defined for Formula (X) or an embodiment thereof;
[00298] E is -C(O)N(H)-(CH2)p-*, -N(H)C(O)-(CH2)p-*, -C(O)O-(CH2)p-*, -OC(O)-(CH2)p-*, - OP(Y)(OH)O-(CH2)p-*, -O-(CH2)p-, -N(H)-(CH2)p, -S-(CH2)p-, -N(H)-O-(CH2)p-, -O-N(H)-(CH2)p-, N(H)N(H)-(CH2)p-, or -S-S-(CH2)p-*, -Ph-(CH2)p-, -OPh-(CH2)p-, or -ZZ’-(CH2)p-; wherein * is the bond to the alpha-amino acid carbon, Ph is phenyl, Y is =0 or =S, p is selected from 1 - 6; RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; and ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne);
[00299] E1 is -O-, -S-, or -N(H)-;
[00300] T is a bond or -L6-G1-[L5-G1]q1-L4-**, wherein ** is the bond to E; ql is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L4, L5, and L6 are independently a bond, -A’-B’-A1- or ZZ1; ZZ1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne);
[00301] each G1 is independently -D’-E’-F1-, wherein D1, E1, and F1 are independently a bond, Ci- loalkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups;
[00302] each A1 is independently a bond, -O-, -S-, or -N(RN1)-;
[00303] each B1 is independently a bond, C(O), C(S), C(NRN1), S(O), S(O)2, P(O)(OH),
P(S)(OH), or P(S)(SH);
[00304] each RN1 is independently hydrogen or C1-6alkyl, or two RN1 within an -A’-B’-A1- group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl.
[00305] In one embodiment, the single modification is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula -P(Y)(OH)-R5, wherein Y is O or S and R5 is wherein RD is as defined above.
[00306] In one embodiment of the 3’- or 5 ’-modification, RD is In one embodiment of the 3’- or 5 ’-modification, RD is for example, G1 can be C1-10alkyl. In one embodiment of the 3’- or 5 ’-modification, RD is wherein n is selected from 7-
23 (e.g., 11-23, or 11 - 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23); and RG is hydrogen, hydroxy, amino, -COOH, or - C(O)NH2; and G1 is C1-10alkyl. In one embodiment of the 3’- or 5 ’-modification, RD is wherein n is selected from 7-23 (e.g., 11-23, or 11 -
21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23); RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; G1 is C1-10alkyl; and RTG is -(N)H)-(CH2)q-N(H)-RLig, wherein q is selected from 1 - 20 (e.g., 2-20 or 2-18 or 2-16 or 2-14, or 2-12, or 2-10; or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) and RLig is
[00307] In one embodiment of the 3’- or 5 ’-modification, RD is RL3 . In one embodiment of the 3 or 5 ’-modification, RD is In one embodiment of the 3 ’- or
5 ’-modification, RD is for example, G1 can be C2-20alkyl. In one embodiment of the 3 ’- or 5 ’-modification, wherein n is selected from 7-23
(e.g., 11-23, or 11 - 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23); RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2;
G1 is C2-20alkyl.
[00308] In one embodiment of the 3’- or 5 ’-modification, RD is wherein n is selected from 7-23 (e.g., 11-23, or 11 - 21, or 13-21, or is 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23); RG is hydrogen, hydroxy, amino, -COOH, or -C(O)NH2; G1 is C2-2oalkyl, and -C(O)-RTG is , , and the broken bond is the bond between RTG and the nitrogen.
[00310] In another embodiment, a sense or antisense strand can contain two different ligand modifications, one at the 3'-end of the strand and the other at the 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety.
[00311] In another embodiment, a sense or antisense strand can contain two different ligand modifications, one is an internal modified nuceloside or modified internucleotide linkage of the strand and the other at the 3 ’-end or 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises the targeting moiety. The modified nuceloside represented by L1 can be located at a position in the strand selected from positions 2 to n-1, where the strand contains n nucleotides (e.g., n-1 is 20 when n is 21). For example, when L1 is at position 6, then segment (1) of the strand contains 5 nucleotides and segment (2) contains the remainder of the nucleotides within the strand. “Position” herein, when referring to a modified nucleotide, nucleoside, or internucleotide linkage is counted from the 5 ’-end of the strand, for example, position 6 includes the 6th nucleotide from the 5 ’-end of the strand and the 6th internucleotide linkage counting from the 5 ’-end of the strand.
[00312] In one embodiment, L1 comprises the in vivo delivery enhancing moiety and a sense or antisense strand can be represented by one of: wherein B is an optionally modified nucleobase (e.g., A, C, G, U, or T); R13 or RL1 comprises the in vivo delivery enhancing moiety, L2 comprises the targeting moiety.
[00313] In one embodiment, RL is selected from the group consisting of: wherein n is selected from 7-23 (e.g., 11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 13; and in another embodiment, n is 19.
[00314] In one embodiment, RL1 is selected from the group consisting of: and , wherein n is selected from 7-23 (e.g., 11 - 21, or 11, 12, 13, 14 ,15, 16, 17, 18, 19, 20, or 21); for example, in one embodiment, n is 7; and in another embodiment, n is 10.
[00315] In another embodiment, RL is selected from the group consisting of: embodiment, RL1 is selected from the group consisting of:
Exemplary sense strands
[00316] In some embodiments, the sense strand of the dsNA has one of the following modification patterns: Table B: Exemplary sense strand motifs
wherein n is a 2’-O-methyl-modified nucleotide;
(dN) is a 2’-deoxy-nucleotide;
Nf is a 2’-fluoro-modified nucleotide (e.g., 2 ’-deoxy-2’ -fluoro modified nucleotide); and the sense strand optionally comprises either:
(a) a 3 ’-terminal or 5 ’-terminal modification selected from:
(i) 5’-(L1)- attached to the 5’-terminal nucleotide (e.g., through the 5’-O of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage;
(ii) -(L2)-3', attached to the 3’-terminal nucleotide (e.g., through the 3’-O of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage;
(iii) 5’-(L1)(I)- attached to the 5 ’-terminal nucleotide (5 ’-5’), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage; or
(iv) -(I)(L2)-3', attached to the 3’-terminal nucleotide (3’-3’), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage; wherein each (I) is an inverted nucleotide (e.g., an inverted abasic nucleotide, such as an inverted abasic ribonucleotide, such as an inverted abasic deoxyribonucleotide);
(L1) and (L2) are independently hydrogen or a group comprising a ligand , wherein the ligand is selected from:
(i) a lipophilic group; examples include a group comprising an C10-C30 alkyl, or a C10-C30 alkenyl group, e.g., a C10 alkyl, C10 alkenyl, C12 alkyl, C12 alkenyl, C14 alkyl, C14 alkenyl, C15 alkyl, C15 alkenyl, C16 alkyl, a C16 alkenyl, a C18 alkyl, a C18 alkenyl, a C20alkyl, a C20 alkenyl, a C22 alkyl, a C22 alkenyl, a C24 alkyl, a C24 alkenyl; C15 alkyl, a C15 alkenyl, a C17 alkyl, a C17 alkenyl, a C19alkyl, a C19 alkenyl, a C21 alkyl, a C21 alkenyl, a C23 alkyl, or a C23 alkenyl group; examples include, but are not limited to, a hexadecyl group, a docosanyl group, an omega-hydroxy-hexadecyl group, and an omega-hydroxy-docosanyl group; or
(ii) a cell-receptor targeting ligand, such as a group comprising an Asialoglycoprotein receptor-targeting (ASGPR) ligand, an integrin- receptor targeting ligand (e.g., avB3, avB5, or avB6-targeting ligand), a lipoprotein receptor-targeting ligand (e.g., LRP1 -targeting ligand); or
(iii) a precursor functional group, where the precursor functional group is suitable for post-synthetic functionalization with a ligand (e.g. as descirbed in (i) or (ii)) containing or conjugated to a complementary reactive functional group; examples of precursor functional groups include, but are not limited to, amino, carboxy, primary amido (-C(O)NH2), N- succinamido, azido (-N3), mercapto (-SH), active esters (e.g., an N- hydroxysuccinimde ester (NHS ester) or a pentafluorophenyl ester),
1.2.4.5-tetrazinyl (e.g., 3-methyl-l,2,4,5-tetrazinyl, 3-(pyridin-2-yl)-
1.2.4.5-tetrazinyl, or 3-(pyrimidin-2-yl)-l,2,4,5-tetrazinyl-); cyclooctynyl (e.g., bicyclo[6.1.0]nonynyl (BCN) or dibenzocyclooctynyl (DBCO)), trans-cyclooctenyl, 2-methylsulfonylpyrimidinyl, 4-vinylpyridinyl, and protected forms thereof; or
(b) a nucleotide comprising a ligand modification, as described above, that replaces the nucleotide decribed above; examples of lipophile modified nucleotides include, (Nhd) - 2’-O-hexadecyl-modified nucleotide; (Nda) - a 2’-O-docosanyl-modified nucleotide; (NhdOH) - 2’-O-(omega-hydroxy-hexadecyl)-modified nucleotide); or (NdaOH) - a 2’-O-(omega-hydroxy-docosanyl)-modified nucleotide.
[00317] In each of the preceding sense strands, each of the nucleotides are connected in series (i.e., in a 3 ’->5’ manner) via optionally modified internucleotide linkages. For example, each of the nucleotides are connected by phosphodiester or phosphorothioate internucleotide linkages.
[00318]
[00319] In certain embodiments of the preceding sense strand examples, the nucleotide at one of the internal positions is substituted for a lipophile modified nucleotide. For example, the lipophile modified nucleotide is substituted at one of positions 4-8 or 12-18 of the sense strand, counting from the 5 ’end. In particular examples, when the sense strand is 21 nucleotides in lengthe, the lipophile modified nucleotide is substituted at one of positions 4-8 or 12-18 of the sense strand (e.g., one of position 5, 6, 7, 8, 12, 13, 14, 15, 16, or 17; or at position 5; or at position 6; or at position 7; or at position 8; or at position 12; or at position 13; or at position 14; or at position 15; or at position 16; or at position 17.) In other particular examples, when the sense strand is 19 nucleotides in length, the lipophile modified nucleotide is substituted at one of positions 3-6 or 10- 16 of the sense strand (e.g., one of position 3, 4, 5, 6, 10, 11, 12, 13, 14, 15 or 16); or at position 3; or at position 4; or at position 5; or at position 6; or at position 10; or at position 11 ; or at position 12; or at position 13; or at position 14; or at position 15; or at position 16.) Examples of lipophile modified nucleotides include, (Nhd) - 2’-O-hexadecyl-modified nucleotide; (Nda) - a 2’-O- docosanyl-modified nucleotide; (NhdOH) - 2’-O-(omega-hydroxy-hexadecyl)-modified nucleotide); or (NdaOH) - a 2’-O-(omega-hydroxy-docosanyl)-modified nucleotide.
[00320] In certain embodiments, the preceding sense strand examples comprise 5’-(L1)- attached to the 5 ’-terminal nucleotide (e.g., through the 5’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L1 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
[00321] In certain embodiments, the preceding sense strand examples comprise -(L2)-3', attached to the 3 ’-terminal nucleotide (e.g., through the 3’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand.
[00322] In certain embodiments of the preceding sense strand examples, the nucleotide at one of the internal positions is substituted for a lipophile modified nucleotide and the sense strand comprises 5’-(L1)- attached to the 5’-terminal nucleotide (e.g., through the 5’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L1 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L1 is a cell-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L1 is a integrin-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the LI is an avB6 integrin-receptor targeting ligand.
[00323] In certain embodiments of the preceding sense strand examples, the nucleotide at one of the internal positions is substituted for a lipophile modified nucleotide and the sense strand comprises -(L2)-3', attached to the 3’-terminal nucleotide (e.g., through the 3’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L2 is a cell-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L2 is a integrin-receptor targeting ligand. In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the L2 is an avB6 integrin-receptor targeting ligand.
[00324] In certain embodiments, the preceding sense strand examples comprise 5’-(L1)- attached to the 5 ’-terminal nucleotide (e.g., through the 5’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, and -(L2)-3', attached to the 3 ’-terminal nucleotide (e.g., through the 3’-0 of the terminal nucleoside), optionally via a divalent linking group, such as a phosphodiester or phosphorothioate linkage, wherein one of L1 and L2 is a cell-receptor targeting ligand, such as a transferrin-targeting ligand, a lipoprotein targeting ligand, an ASGPR-targeting ligand, an integrin-receptor targeting ligand, or a lipophilic ligand; and the other of L1 and L2 is a lipophilic group (e.g., an in vivo delivery enhancing moiety). In one embodiment, the lipophile modified nucleotide is an in vivo delivery enhancing moiety and the cell-receptor targeting ligand is an avB6 integrin-receptor targeting ligand.In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester linkage linkage s, counting from the 5 ’-end of the oligonucleotide.
[00325] In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester linkages, counting from the 5 ’-end of the oligonucleotide.
[00326] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
[00327] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage, and the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 21 and 22 are connected by a phosphorothioate internucleotide linkage; and positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. For a nucleotide in 21 nucleotides in length, the nucleotides at positions 19 and 20 are connected by a phosphorothioate internucleotide linkage; and positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
[00328] In other embodiments, each (inv) attached to a 5 ’-terminal nucleotide is connected via a phosphorothioate linkage (5’ -5’).
[00329] In other embodiments, each (inv) attached to a 3 ’-terminal nucleotide is connected via a phosphorothioate linkage (3’ -3’).
[00330] In certain other embodiments, each (inv) attached to a 5 ’-terminal nucleotide is connected via a phosphorothioate linkage (5 ’-5’), and each (inv) attached to a 3 ’-terminal nucleotide is connected via a phosphorothioate linkage (3’ -3’).
[00331] In certain embodiments, the sense strand is according to any one of S1-S35 wherein the nucleotide at any one of positions 4-8 or 13-18 counting from the 5’-end of the strand, is replaced with a nucleotide that is substituted with a ligand group (e.g., a lipophilic group).
[00332] For example, in certain embodiments, the sense strand of the dsNA has one of the following modification patterns:
Table C: Additional exemplary sense strand motifs
wherein n is a 2’-O-methyl-modified nucleotide;
(dN) is a 2 ’-deoxy-nucleotide;
Nf is a 2’-fluoro-modified nucleotide;
(L) is a ligand-modified nucleotide, e.g., comprising a lipophilic group (e.g., C16 or C22 modification), a cell-receptor targeting ligand, such as an Asialoglycoprotein receptor-targeting (ASGPR) ligand, an integrin-receptor targeting ligand, or a lipoprotein receptor-targeting ligand ; and s is a phosphorothioate internucleotide linkage,
(s) is a phosphorothioate or a phosphodiester internucleotide linkage (e.g., in certain embodiments, each (s) is a phosphorothioate); each (J) is independently an optional linking group (such as an inverted nucleotide (I) (e.g., an inverted abasic nucleotide, such as an inverted abasic ribonucleotide, such as an inverted abasic deoxyribonucleotide));
(L1) and (L2) are independently hydrogen or a group comprising a ligand, and the sense strand optionally comprises a 5 ’-morpholino nucleotide, a 5’- dimethylamino nucleotide, a 5 ’-deoxy nucleotide, an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5 ’-end.
[00333] In some embodiments, the antisense strand of the dsNA has one of the following modification patterns:
Table D: Exemplary antisense strand motifs
wherein: n is a 2’-O-methyl-modified nucleotide; s is a phosphorothioate internucleotide linkage (3’- 5’);
(dN) is a 2’-deoxy-nucleotide;
Nf is a 2’-fluoro-modified nucleotide;
Z is wherein:
* is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose);
A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2, wherein * is the bond to E;
E is a bond or -CH2-;
Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3 -methylcyclobutyl or cyclobutylmethyl;
X is O; each RP is independently -ORO, -SRs, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each Rs is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and
R2S is C1-3alkyl, and where the preceding structure replaces the 4’-CH2OH group on the ribose ring of the 5 ’-terminal nucleotide; or
Z and monomer (n) at position 1 together are , wherein: n is 1, 2 or 3;
X is O or S; each RP is independently -ORO, -SRs, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each Rs is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and
R2S is C1-3alkyl; or wherein:
X is O or S; each RP is independently -ORO, -SRs, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each Rs is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and
R2S is C1-3alkyl; and
XA is O or S;
YA is O or S; and
Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropyhnethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl; or
Z and monomer (n) at position 1 together are wherein:
X is O or S; each RP is independently -ORO, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and
R2S is C1-3alkyl; and
XA is O or S; YA is O or S; and y is 0 or 1; n5 is an integer selected from 2 - 5 (e.g., 2, 3, or 4, preferably 2); n6 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1); n7 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably l);n8 is an integer selected from 1 - 3 (e.g., 1, or 2); n9 is an integer selected from 1 - 3 (e.g., 1, or 2); n10 is an integer selected from 1 - 3 (e.g., 1 or 2; preferably 1);
Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2- cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3 -methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionally and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionally replaced with -N=;
RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC), wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; RPC is C1-6alkyl (e.g., C1-6alkyl or methyl); and R2S is C1-3alkyl; and
R2’ and R3’ are as defined for formulae I-XXIII.
[00334] In each of the preceding antisense strands, each of the nucleotides are connected in series (i.e., in a 3 ’->5’ manner) via phosphodiester or phosphorothioate internucleotide linkages. [00335] In one embodiment of any one of AS1-AS39, the nucleotide at one of positions 5-8, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G), such as:
(Ngn) - a glycol nucleic acid, S-isomer; (N2p) - a 2'-phosphate nucleotide (i.e., a 3’-RNA connected by 3’-5’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively);
(Tna) - a threose nucleotide (connected by 3’-3’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively);
(MM) a nucleobase mismatch to the sense strand; or
(Nul) an unlocked nucleic acid.
[00336] In one embodiment of any one of AS1-AS39, the nucleotide position 5, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G). In one embodiment of any one of AS1-AS39, the nucleotide position 6, counting from the 5’-end of the antisense strand is replaced with a thermally destabilizing modification (G). In one embodiment of any one of AS1-AS39, the nucleotide position 7, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G). In one embodiment of any one of AS1- AS39, the nucleotide position 8, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
[00337] For example, the antisense strand may be selected from any one of AS40 - AS58 as recited in Table E:
Table E.
[00338] In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5 ’-end of the oligonucleotide. In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
[00339] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide.
[00340] In other embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide and the 3 ’-terminal nucleoside is linked to a ligand by a phosphorothioate linkage. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide, and the nucleoside at position 23 is linked to a ligand via a phosphorothioate linkage; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide, and the nucleoside at position 21 is linked to a ligand via a phosphorothioate linkage.
[00341] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage, and the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 21 and 22 are connected by a phosphorothioate internucleotide linkage; and positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5 ’-end of the oligonucleotide. [00342] In certain embodiments, where the nucleotide in m nucleotides in length:
(a) the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage;
(b) the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage;
(c) the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage; and
(d) the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
[00343] That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 21 and 22; and 22 and 23 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide. And, for a nucleotide in 21 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 19 and 20; and 20 and 21 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5 ’-end of the oligonucleotide.
[00344] In certain embodiments, where the nucleotide in m nucleotides in length:
(a) the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage;
(b) the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage;
(c) the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and
(d) the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide.
[00345] That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 22 and 23 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’- end of the oligonucleotide. And, for a nucleotide in 21 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 20 and 21 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5 ’-end of the oligonucleotide.
[00346] For example, in some embodiments, the antisense strand of the dsNA has one of the following modification patterns in Table F.
Table F: Additional exemplary antisense motifs
[00347] In one embodiment of any one of AS58-AS135, the nucleotide at one of positions 5-8, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G), such as :
(Ngn) - a glycol nucleic acid, S-isomer;
(N2p) - a 2'-phosphate nucleotide (i.e., a 3’-RNA connected by 3’-5’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively);
(Tna) - a threose nucleotide (connected by 3’-3’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively);
(MM) a nucleobase mismatch to the sense strand (and the nucleotide is optionally modified by 2 ’-fluoro or 2’-OMethyl); or
(Nul) an unlocked nucleic acid.
[00348] In one embodiment of any one of AS58-AS135,, the nucleotide position 5, counting from the 5 ’-end of the antisense strand is replaced with a thermally destabilizing modification (G). In one embodiment of any one of AS58-AS135,, the nucleotide position 6, counting from the 5’- end of the antisense strand is replaced with a thermally destabilizing modification (G). In one embodiment of any one of AS58-AS135,, the nucleotide position 7, counting from the 5’-end of the antisense strand is replaced with a thermally destabilizing modification (G). In one embodiment of any one of AS58-AS135,, the nucleotide position 8, counting from the 5’-end of the antisense strand is replaced with a thermally destabilizing modification (G).
[00349] For example, the antisense strand may be selected from any one of AS136 - AS171 as recited in Table G.
Table G.
[00350] In a further embodiment of each of the preceding exemplary sense and antisense strands, each of sense strand SI through SI 23 can be hybridized or duplexed with any one of antisense strands AS 1 through AS 171. In other words, the longer double-stranded oligonucleotide product described herein comprises any one of the sense strands SI through S123hybridized/duplexed with any one of the antisense strands AS1 through AS171. In certain embodiments, the sense strand is selected from an embodiment having 21 nucleotides and the antisense strand is selected from an embodiment having 23 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 21 nucleotides and the antisense strand is selected from an embodiment having 21 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 19 nucleotides and the antisense strand is selected from an embodiment having 21 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 19 nucleotides and the antisense strand is selected from an embodiment having 19 nucleotides.
[00351] In some embodiments, the longer single-stranded oligonucleotide described herein is any one of the sense strands S1 through S123 or any one of the antisense strands AS1 through AS171.
Compositions
[00352] The dsRNA or oligonucleotide described herein can be formulated in compositions. For example, the dsRNA or oligonucleotide described herein can be formulated into pharmaceutical compositions for therapeutic use. Accordingly, in another aspect, provided herein is a pharmaceutical composition comprising a therapeuticallyeffective amount of one or more of the dsRNA or oligonucleotide described herein, taken alone, or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
[00353] The pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous. [00354] The phrase “therapeutically effective amount” as used herein means that amount of a compound, material, or composition comprising a conjugate described herein which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
[00355] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[00356] As used herein, a “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
[00357] Pharmaceutical compositions for use with the methods described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, a dsRNA or oligonucleotide described herein can be formulated for administration by, for example, by intravenous, oral, aerosol, or topical route. The compositions can be formulated for intralesional, intratumoral, intraperitoneal, subcutaneous, intramuscular, or intravenous injection, infusion, liposome-mediated delivery, topical, intrathecal, gingival pocket, per rectum, intrabronchial, nasal, transmucosal, intestinal, oral, ocular, or otic delivery.
[00358] Techniques and formulations generally can be found in Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intrathecal, intravenous, intraperitoneal, and subcutaneous. For injection, a dsRNA described herein can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank’s solution or Ringer’s solution. In addition, the dsRNA can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
[00359] The dsRNA or oligonucleotide can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
LNPand liposome formulations
[00360] The dsRNAs or oligonucleotides described herein can be formulated with one or more lipids for delivery. For example, the dsRNAs or oligonucleotides described herein can be formulated in lipid particles. As used herein, the term “lipid particle” refers to a vesicle formed by one or more lipid components. Lipid particles are typically used as carriers for nucleic acid delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Generally, lipid particle compositions for such delivery are composed of ionizable or cationic lipids, phospholipids (especially compounds having a phosphatidylcholine group), cholesterol, and a polyethylene glycol (PEG) lipid; however, these compositions may also include other lipids. The ionizable lipid is typically employed to condense the nucleic acid cargo at low pH and to drive membrane association and fusogenicity. The phospholipid is typically employed to enhance fusogenicity. The cholesterol is typically employed to provide membrane integrity. The PEG-lipid is typically employed to provide steric stabilization. The sum composition of lipids typically dictates the surface characteristics and thus the protein (opsonization) content in biological systems thus driving biodistribution and cell uptake properties. A lipid particle can be a lipid nanoparticle (LNP).
[00361] In some embodiments, a lipid particle described herein comprises an ionizable lipid. As used herein, the term “ionizable lipid” refers to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Typically, ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. Ionizable lipids are also referred to as cationic lipids herein.
[00362] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3) . In some embodiments, the ionizable lipid is the lipid ATX-002 .. In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-l-amin.e. In some embodiments, the ionizable lipid is Compound 6 or Compound 22 described in WO2015/199952, content of which is incorporated herein by reference in its entirety.
[00363] Without limitations, ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid particle.
[00364] As used herein, the term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid. Accordingly, the non-cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1- trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
[00365] Other examples of non-cationic lipids suitable for use in the lipid particles include nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like. [00366] In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some preferred embodiments, the non-cationic lipid is DPSC.
[00367] tThe non-cationic lipid can comprise 0-30% (mol) of the total lipid present in the lipid particle. For example, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid particle. In various embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1.
[00368] In some embodiments, the lipid particle can further comprise a conjugated lipid molecule. As used herein, the term “conjugated lipid” refers to a lipid molecule conjugated with a non-lipid molecule, such as a PEG, polyoxazoline, polyamide, or polymer (e.g., cationic polymer). Generally, these lipids are used to inhibit aggregation of lipid particles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
[00369] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O- (2',3'-di(tetradecanoyloxy)propyl- 1 -O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S- DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)- 1,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof.
[00370] The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol ( 1 -[8'-(Cholest-5-en-3 [beta]-oxy)carboxamido-3',6'- dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4- Ditetradecoxylbenzyl- [omega] -methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG-lipid can be PEG-DMG, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] . [00371] The PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid particle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid particle.
[00372] In some embodiments, the lipid particle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid particle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5α- cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
[00373] The component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) of the total lipid present in the lipid particle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid particle.
[00374] In one embodiment, the the lipid particle comprises: an ionizable lipid; a non-cationic lipid; a conjugated lipid that inhibits aggregation of particles; and a sterol. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non- cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid particle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.
[00375] In one embodiment, the the lipid particle comprises: an ionizable lipid in an amount from about 20 mol % to about 90 mol % of the total lipid present in the particle; a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipid present in the particle; a conjugated lipid that inhibits aggregation of particles in an amount from about 0.5 mol % to about 20 mol % of the total lipid present in the particle; and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipid present in the particle. In some embodiments, the lipid particle comprises ionizable lipid / non-cationic-lipid / sterol / conjugated lipid at a molar ratio of 50:10:38.5:1.5.
[00376] In one embodiment, the total lipid to nucleic acid (mass or weight) ratio is from about 10: 1 to about 30: 1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.
Administering to a subject
[00377] The dsRNA and/or oligonucleotide described herein may be formulated for administration in any convenient way for use in medicine, e.g., human or veterinary medicine, by analogy with other pharmaceuticals.
[00378] The dsRNA and/or oligonucleotide described herein or a pharmaceutical composition comprising same can be administered to a subject using different routes of delivery. Exemplary routes of delivery include, but are not limited to intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
[00379] The dsRNA and/or oligonucleotide described herein can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be parenteral, topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), or oral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
[00380] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the dsRNA and/or oligonucleotide described herein in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the dsRNA and/or oligonucleotide described herein and mechanically introducing the dsRNA and/or oligonucleotide described herein.
[00381] In one aspect, provided herein is a method of administering an dsRNA and/or oligonucleotide described herein, to a subject (e.g., a human subject). In another aspect, the present invention relates to an dsRNA and/or oligonucleotide described herein for use in inhibiting expression of a target gene in a subject. The method or the medical use includes administering a unit dose of the dsRNA and/or oligonucleotide described herein.
[00382] The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target gene. The unit dose, for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application
[00383] In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.
[00384] In some embodiments, the effective dose is administered with other traditional therapeutic modalities.
[00385] The dsRNA and/or oligonucleotide described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
[00386] In some embodiments, the administration of the dsRNA and/or oligonucleotide composition described herein is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose.
[00387] In some embodiments, the dsRNA and/or oligonucleotide described herein is administrated via subcutaneous or intravenous administration. [00388] In some embodiments, the dsRNA and/or oligonucleotide described herein is administrated via intrathecal administration.
[00389] In some embodiments, the dsRNA and/or oligonucleotide described herein is administrated via intravitreal administration.
Cells
[00390] The disclosure also provides a cell comprising a compound, dsRNA or oligonucleotide described herein described herein. As used herein, the term “cell” refers to a single cell as well as to a population of (i.e., more than one) cells. A cell can be a prokaryotic or eukaryotic cell. Exemplary cells include, but are not limited to, bacterial cells, yeast cells, plant cell, animal (including insect) or human cells. In some embodiments, the cell is a eukaryotic cell. For example, the cell is a mammalian cell. It is noted a cell can be in vivo, in vitro or ex vivo.
Kits
[00391] A dsRNA or oligonucleotide described herein can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) a dsRNA or oligonucleotide described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and/or the use of a dsRNA or oligonucleotide described herein for the methods described herein. The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the dsRNAs or oligonucleotides, their molecular weight, concentration, date of expiration, batch, or production site information, and so forth. In some embodiments, the informational material relates to using dsRNA or oligonucleotide to treat, prevent, or diagnosis of disorders and conditions.
[00392] In some embodiments, the informational material can include instructions to administer the dsRNA or oligonucleotide in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer the dsRNA or oligonucleotide to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition needing treatment.
[00393] The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but can also be in other formats, such as computer readable material.
[00394] Components of the kit, e.g., the dsRNA or oligonucleotide can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the dsRNA or oligonucleotide be substantially pure and/or sterile. When the dsRNA or oligonucleotide is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When the dsRNA or oligonucleotide is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
[00395] The kit can include one or more containers for the components of the kit. In some embodiments, the kit contains separate containers, dividers, or compartments for the different components of the kit. For example, the dsRNA or oligonucleotide can be contained in a bottle, vial, or syringe, and the informational material can be contained association with the container. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the dsRNA or oligonucleotide is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more-unit dosage forms of the dsRNA or oligonucleotide. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the dsRNA or oligonucleotide. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
[00396] The kit optionally includes a device suitable for administration of the dsRNA or oligonucleotide, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses metered doses of the dsRNA or oligonucleotide. The disclosure also features a method of providing a kit, e.g., by combining components described herein.
[00397] In some embodiments, the kit can further comprise additional components and/or reagents for practicing the methods described herein using the dsRNA or oligonucleotide described herein.
Methods of inhibiting expression of a target gene
[00398] Aspects of the disclosure also relate to methods for inhibiting the expression of a target gene in a subject. The method comprises administering to the subject in an amount sufficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the antisense strand is substantially complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is substantially complementary to a target gene.
[00399] The disclosure further relates to a use of dsRNA and/or oligonucleotide for inhibiting expression of a target gene in a target cell. The disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell in vitro.
[00400] Another aspect the invention relates to a method of modulating the expression of a target gene in a cell, comprising administering to said cell a dsRNA and/or oligonucleotide described herein. It is noted that administering to the cell can be in vitro or in-vivo. Methods for administering a compound to a cell are well known and available to one of skill in the art. As used herein, administering the compound to the cell means contacting the cell with the compound so that the compound is taken up by the cell. Generally, the cell can be contacted with the dsRNA/oligonucleotide in a cell culture e.g., in vitro or ex vivo, or the compound can be administrated to a subject, e.g., in vivo. The term “contacting” or “contact” as used herein in connection with contacting a cell includes subjecting the cells to an appropriate culture media, which comprises a dsRNA and/or oligonucleotide described herein. Where the cell is in vivo, “contacting” or “contact” includes administering the dsRNA and/or oligonucleotide described herein, e.g., in a pharmaceutical composition to a subject via an appropriate administration route such that the compound contacts the cell in vivo. For example, when the cell is in vitro, said administering to the cell can include subjecting the cell to an appropriate culture media which comprises the dsRNA/oligonucleotide. Where the cell is in vivo, said administering to the cell includes administering the dsRNA/oligonucleotide to a subject via an appropriate administration route such that the dsRNA/oligonucleotide is administered to the cell in vivo.
[00401] In some embodiments, the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIPl) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.
[00402] In other embodiments, the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1(YKL-4O), RPS25, a2-AR, and GSK3a. [00403] In one embodiment, the target gene is selected from the group consisting of myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYSI), survival of motor neuron 1 (SMN1), alpha-glucosidase (GAA); adrenoceptor beta 1 (ADRBl); calcium voltage-gated channel subunit alphal C (CACNA1C); calcium voltage-gated channel subunit alphal G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type l(AGTRl); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/cahnodulin dependent protein kinase II delta (CAMK2D); or Phosphodiesterase 1 (PDE1).
[00404] In one embodiment, the target gene is selected from the group consisting of myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Collagen Type XIII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYSI), survival of motor neuron 1 (SMN1), and alpha-glucosidase (GAA).
[00405] In one embodiment, the target gene is selected from the group consisting of adrenoceptor beta 1 (ADRBl); calcium voltage-gated channel subunit alphal C (CACNA1C); calcium voltage-gated channel subunit alphal G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type l(AGTRl); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium/cahnodulin dependent protein kinase II delta (CAMK2D); or Phosphodiesterase 1 (PDE1).
[00406] In one embodiment, the target gene is selected from the group consisting of MUC5B, TSLP, IL33, ALOX15, AGER(RAGE), MUC5AC, and STAT6.
[00407] In one embodiment, the target gene is selected from the group consisting of Delta 4- Desaturase, Sphingolipid 1 (DEGS1); leptin; folliculin (FLCN); Zinc Finger Protein 423 (ZFP423); Cyclin Dependent Kinase 6 (CDK6); Regulatory Associated Protein Of MTOR Complex 1 (RPTOR); Mechanistic Target Of Rapamycin Kinase, (mTOR); Forkhead Box Pl (FOXP1); Phosphodiesterase 3B (PDE3B); and Activin A Receptor Type 1C (ACVR1C).
[0400] In one embodiment, the target gene is selected from the group consisting of TTR for hATTR (CNS, ocular and systemic), myocilin (MYOC), Ras homolog family member A (RhoA), SSB (small RNA binding exonuclease protection factor La), optineurin, Carbonic Anhydrase 2 (CA2), Rho associated coiled-coil containing protein kinase 1 (ROCK1), Rho associated coiled- coil containing protein kinase 2 (ROCK2), Angiopoietin-Like 7 (ANGPTL7), and cytochrome P450 1B1 (CYP1B1).
[0401] In another embodiment, the target gene is selected from the group consisting of PPARG, ADIPOQ, CD36, LPL, ADAMTS9, RASD1, GYS2, CAT, DPYS, MLXIPL, VEGFA, HLA- DQA1, LIPA, CTSC, FCGR2A, GBE1, SH2B3, CTSK, CDKN2B, ELN, ARG1, HHEX, TCF7L2, CYP2A6, ALDH2, ACADS, GLYCTK, LDLR, HAL, ACER3, SLC7A7. PTPN22, CDKN1C, LEPR, SNAI2, PGM1, IGF2BP2, TTPA, ATP7B, ASPA, ADRB3, MAN2B1, RCAN1, PIGL, TBX1, LMNB1, FBP1, ETFA, LMNA, LAT2, PRKAG2, SELENBP1, TKT, PCSK1, PSAP, NDN, ACY1, SATB2, CYP21A2, POMC, CDC73, CTSH, CFTR, CTSA, G6PD, EXT1, EXT2, CPT1A, SEMA5A, WFS1, KIT, ACAT1, GGCX, FKBP6, PPARGC1B, DGCR6, HMGCS2, PEPD, WRN, LCAT, KLF13, SLC16A2, DHCR7, ITPR3, CLDN4, FZD9, SLC30A2, APOA5, HADHA, CDKAL1, PTPN2, LIPC, CD226, PON1, MCCC1, EIF2AK3, GYG1, BCL7B, AGL, VKORC1, BAZ1B, NAGS, ASL, STAR, ACP2, POLG, GAA, ALDH3A2, MANBA, ARSA, AGA, CYP27B1, CPS1, DLAT, DCXR, EIF4H, DYRK1 A, GTF2I, LAMP2, CTH, EPO, FLAD1, AKT2, WAC, GLB1, RFC2, BACH2, D2HGDH, GHRL, TBL2, RRM2B, PRKACA, DLD, NEU1, ADSL, SLC22A5, ADCY10, INSR, HSD17B10, DGCR8, NPAP1, OXCT1, SDC3, HMGCL, PGAP1, MCCC2, LMF1, PIGM, UCP3, PAH, VPS33A, BCS1L, PDP1, AHCY, ALDH18A1, ENO3, MTTP, MAT1A, GNPTAB, PHGDH, BCAT2, CBS, HDAC4, LIG3, PSAT1, HGD, CTNND2, PDHB, PDHA1, NADK2, UPB1, PKLR, BCKDK, MEN1, GALT, LIMK1, SLC39A4, KCNJ11, PDHX, ACAD8, GSS, CHRNA7, SLC6A9, ERBB3, GLUD1, GSR, OAT, SLC6A8, CLIP2, STX1A, CARTPT, SLC25A15, DGCR2, LIPT2, NR5A1, DNM1L, PHEX, SLC30A9, B3GAT3, SLC34A3, SLC12A3, EPX, SARS2, CAPN10, ASNS, ALDOB, AGRP, MFF, GK, APOC2, CLDN3, HPRT1, PFKM, AMACR, SNRPN, HNF1B, L2HGDH, SORD, IDH2, TPMT, CYP2C19, TERT, MC4R, TMPRSS15, SLCO1B3, FGF23, PAX4, SLC30A8, MTNRIB, SI, SLCO1B1, andNR0B2.
Reactive phosphorous group
[00408] Embodiments of the various aspects described herein include a reactive phosphorus group. Without wishing to be bound by a theory, reactive phosphorus groups are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and contain phosphorus atoms in Pin or Pv valence state including, but not limited to, phosphoramidite, H- phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. Reactive phosphorous group in the form of phosphoramidites (PIII chemistry) as reactive phosphites are a preferred reactive phosphorous group for solid phase oligonucleotide synthesis. The intermediate phosphite compounds are subsequently oxidized to the Pv state using known methods to yield phosphodiester or phosphorothioate internucleoside linkages.
[00409] In some embodiments, the reactive phosphorous group is -P(ORP1)N(RP2)2, - P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, - P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3. [00410] In some embodiments, RP1 is an optionally substituted C1-6alkyl. For example, RP1 is a C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1- C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)- alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m— (CH2)p— OH, CH2— [CH(OH)]m— (CH2)p— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. In some embodiments, RP1 is a C1-6alkyl, optionally substituted with a CN or -SC(O)Ph. For example, RP1 is cyanoethyl (-CH2CH2CN).
[00411] In some embodiments, each RP2 is independently optionally substituted C1-6alkyl. For example, each RP2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl. It is noted that when two or more RP2 groups are present in the reactive phosphorous group, they can be same or different. Thus, in some none-limiting examples, when two or more RP2 groups are present, the RP2 groups are different. In some other non-limiting examples, when two or more RP2 groups are present, the RP2 groups are same. In some embodiments, each RP2 is isopropyl.
[00412] In some embodiments, both RP2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4- diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like, each of which can be optionally substituted with 1, 2 or 3 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1- C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)- C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m — (CH2)p — OH, CH2 — [CH(OH)]m — (CH2)p — NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00413] In some embodiments, RP1 and one of RP2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4- diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like, each of which can be optionally substituted with 1, 2 or 3 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1- C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)- C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m — (CH2)p — OH, CH2 — [CH(OH)]m — (CH2)p — NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00414] In the reactive phosphorous groups, each RP3 is independently optionally substituted C1-3oalkyl substituted C1-C30alkyl, optionally substituted C2-C30alkenyl, or optionally substituted C2-C30alkynyl (e.g., optionally substituted C1-C10alkyl, optionally substituted C2-C10alkenyl, or optionally substitutedC2-C10alkynyl). For example, RP3 can be a C1-6alkyl, optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1- C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O( C1-C8)alkyl (i.e., C1-C8alkoxy), O( C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, atyl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)- C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m — (CH2)p — OH, CH2 — [CH(OH)]m — (CH2)p — NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, RP3 is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl or hexyl, each of which can be optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-Cealkoxy.
[00415] In some embodiments, the reactive phosphorous group is -P(ORP1)(N(RP2)2). For example, the reactive phosphorous group is -P(ORP1)(N(RP2)2), where RP1 is 2-cyanoethyl (- CH2CH2CN) and each RP2 is isopropyl.
Hydroxyl protecting groups
[00416] Some embodiments of the various aspects described herein include a hydroxyl protecting group. Hydroxyl protecting groups include, but are not limited to, -ROP1, -N(ROP2)2, -C(=O)SROP1, -C(=O)ROP1, -CO2ROP1, -C(=O)N(ROP2)2, -C(=NROP2)ROP1, -C(=NROP2)OROP1, -C(=NROP2)N(ROP2)2, -CH2OC(=O)ROP1, -S(=O)ROP1, -SO2RO1*1, -Si(ROP1)3, -P(ROP3)2, -P(ROP3)+ 3 X-, -P(OROP3)2, -P(OROP3)3 X-, -P(=O)(ROP1)2, -P(=O)(OROP3)2, and -P(=O)(N(ROP2)2)2; wherein each X- is a counterion; each ROP1 is independently C1-10 alkyl, C1- io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10alkenyl, heteroC2. loalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two ROP1 groups are joined to form a 3-14 membered heteroreacticvvecyclyl or 5-14 membered heteroaryl ring; each ROP2 is hydrogen, -OH, -OROP1, -N(ROP3)2, -CN, -C(=O)ROP1, -C(=O)N(ROP3)2, -CO2ROP1, -SO2ROP1, -C(=NROP3)OROP1, -C(=NROP3)N(ROP3)2, -SO2N(ROP3)2, -SO2ROP3, -SO2OROP3, -SOROP1, -C(=S)N(ROP3)2, -C(=O)SROP3, -C(=S)SROP3, -P(=O)(ROP1)2, -P(=O)(OROP3)2, -P(=O)(N(ROP3)2)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two ROP2 groups are joined to form a 3- 14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each ROP3 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2. io alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two ROP3 groups are joined to form a 3-14 membered heterocyclyl or 5- 14 membered heteroaryl ring; and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of ROP1, ROP2 and ROP3 can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1- C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1- C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m — (CH2)p — OH, CH2 — [CH(OH)]m — (CH2)p — NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4,
5 or 6.
[00417] Hydroxyl protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5th Edition, John Wiley
6 Sons, 2014, incorporated herein by reference.
[00418] Exemplary hydroxyl protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boe), methoxymethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacohnethyl (GUM), t-butoxymethyl, pivaloyloxymethyl (POM), acetyloxymethyl, (AM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3 -bromotetrahydropyranyl, tetrahydrothiopyranyl, 1 -methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxyethyl, l-(2-chloroethoxy)ethyl, 1 -methyl- 1-methoxyethyl, 1 -methyl- 1-benzyloxyethyl, 1- methyl-l-benzyloxy-2-fhioroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6- dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenyhnethyl, α- naphthyldiphenyhnethyl, p-methoxyphenyldiphenyhnethyl, di(p- methoxyphenyl)phenyhnethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenyhnethyl, 4,4',4"-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4, 4', 4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4- methoxyphenyl)-r-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, l,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formyle, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, methoxyacetyl, triphenylmethoxyacetyl, phenoxyacetyl, p-chlorophenoxyacetyl, 3-phenylpropionyl, 4- oxopentanoyl (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenyhnethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2- trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4- bis(1,1- dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N',N'- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
[00419] In some embodiments, hydroxyl protecting group is benzyl, benzoyl, 2,6- dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4 '-dimethoxytrityl. In some embodiments, hydroxyl protecting group is pivaloyloxymethyl.
[00420] The terms “protected hydroxyl” and “protected hydroxyl” as used herein mean a group of the formula -ORPro, wherein RPro is an oxygen protecting group as defined herein.
Amine protecting groups
[00421] Some embodiments of the various aspects described herein include an amine protecting group (also referred to as an amino protecting group herein). Amine protecting groups include, but are not limited to, -OH, -ORNP1, -N(RNP2)2, -C(=O)RNP1, -C(=O)N(RNP2)2, -CO2RNP1, -SO2RNP1, - C(=NRNP2)RNP1, -C(=NRNP2)ORNP1, -C(=NRNP2)N(RNP2)2, -SO2N(RNP2)2, -SO2RNP2, -SO2ORNP2, - SORNP1, -C(=S)N(RNP2)2, -C(=O)SRNP2, -C(=S)SRNP2, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2- io alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, where each RNP1 is independently C1-10 alkyl, C1-10 perhaloalkyl, C2- 10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3- 10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two RNPI groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RNP2 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2- 10 alkynyl, heteroC1-10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP3 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of RNP1 and RNP2 can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(0H)]m— (CH2)p— OH, CH2— [CH(0H)]m— (CH2)p— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00422] Amine protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
[00423] Exemplary amide (e.g., -C(=O)RNP1) based amine protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N- benzoylphenylalanyl derivative, benzamide, p- phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'- dithiobenzyloxy acylaminojacetamide, 3-(p- hydroxylphenyljpropanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxyjpropanamide, 2-methyl-2-(o- phenylazophenoxyjpropanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o- nitrocinnamide, N-acetyhnethionine derivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.
[00424] Exemplary carbamate (e.g., -C(=O)ORNP1) based amine protecting groups include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfojfluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (l-adamantyl)-l -methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t- BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-t- butylphenyl)- 1 -methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'- pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boe), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1- isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coe), 4-nitrocinnamyl carbamate (Noe), 8-quinolyl carbamate, N-hydroxylpiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p- bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2- methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p- toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), 1,1- dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxylboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)- 6-chromonylmethyl carbamate (Tcroc), m- nitrophenyl carbamate, 3, 5 -dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy- 6-nitrobenzyl carbamate, phenyl(o- nitrophenyl)methyl carbamate, t-arnyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2- dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3- (N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2- pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1 -methylcyclohexyl carbamate, 1 -methyl- 1- cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1- methyl- l-(p- phenylazophenyl)ethyl carbamate, 1 -methyl- 1 -phenylethyl carbamate, 1- methyl- 1 -(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6- trimethylbenzyl carbamate.
[00425] Exemplary sulfonamide (e.g., -S(=O)2RNP1) based amine protecting groups include, but are not limited to, such as p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, - trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4- methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4- methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4- methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
[00426] Additional exemplary amine protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N - phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N- 2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-l,3,5-triazacyclohexan-2-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N- allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(l- isopropyl-4- nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N- di(4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N-2- picolylamino N'- oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl] methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N - isopropylidenediamine, N-p- nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2- hydroxylphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l- cyclohexenyl)amine, N-borane and N-diphenylborinic acid derivative, N- [phenyl(pentNPlcylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N- nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Opp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3- nitropyridinesulfenamide (Npys).
Thiol protecting groups
[00427] Some embodiments of the various aspects described herein include a thiol protecting group. Thiol protecting groups include, but are not limited to, -RSP1, -N(RSP2)2, -C(=O)SRSP1, - C(=O)RSP1, -CO2RSP1, -C(=O)N(RSP2)2, -C(=NRSP2)RSP1, -C(=NRSP2)ORSP1, -C(=NRSP2)N(RSP2)2, -S(=O)RSP1, -SO2RSP1, -Si(RSP1)3, -P(RSP3)2, -P(RSP3)+ 3 X-, -P(ORSP3)2, -P(ORSP3)+ 3 X-, - P(=O)(RSP1)2, -P(=O)(ORSP3)2, and-P(=O)(N(RSP2) 2)2, wherein: X" is a counterion; each RSP1 is independently C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two RSP1 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; each RSP2 is hydrogen, -OH, -ORSP1, -N(RSP3)2, -CN, -C(=O)RSP1, -C(=O)N(RSP3)2, -CO2RSP1, -SO2RSP1, -C(=NRSP3)ORSP1, -C(=NRSP3)N(RSP3)2, -SO2N(RSP3)2, -SO2RSP3, -SO2ORSP3, -SORSP1, -C(=S)N(RSP3)2, -C(=O)SRSP3, -C(=S)SRSP3, -P(=O)(RSP1)2, -P(=O)(ORSP3)2, -P(=O)(N(RSP3)2)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP2 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RSP3 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi. 10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP3 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of RSP1, RSP2 and RSP3 can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1- C8jalkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(0H)]m— (CH2)p— OH, CH2— [CH(0H)]m— (CH2)p— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00428] Sulfur protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
Definitions
[00429] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[00430] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
[00431] Further, the practice of the present invention can employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ecL, 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). [00432] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[00433] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. In some embodiments of the various aspects described herein, the term “about” when used in connection with percentages can mean ±5%. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
[00434] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective components) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
[00435] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00436] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00437] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [00438] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
[00439] As used herein, the terms “siRNA”, and “iRNA agent” are used interchangeably to refer to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.
[00440] As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target gene, e.g., mRNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
[00441] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
[00442] As used herein, the term “substantially complementary”, with respect to a nucleotide sequence in relation to a reference nucleotide sequence means a nucleotide sequence having a percentage of identity between the substantially complementary nucleotide sequence and the exact complementary sequence of said reference of at least at least 80%. e.g., at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (i.e., exactly complementary). Preferably identity is assessed over a length of at least 15, e.g., at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides.
[00443] The term “off-target” and the phrase “off-target effects” refer to any instance in which an effector molecule against a given target causes an unintended affect by interacting either directly or indirectly with another target sequence, a DNA sequence or a cellular protein or other moiety. For example, an “off-target effect” may occur when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of an siRNA.
[00444] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,
“reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00445] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
[00446] As used herein, a “terminal” of a strand refers to position 1, counting from the nearest end of the strand. For example, a 5’-terminal refers to position 1, counting from the 5’-end of the strand. Similarly, a 3 ’-terminal refers to position 1, counting from the 3 ’-end of the strand.
[00447] As used herein, a “terminal region” of a strand refers to positions 1-4, e.g., positions 1, 2, 3, and 4, counting from the nearest end of the strand. For example, a 5 ’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 5 ’-end of the strand. Similarly, a 3’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 3’-end of the strand.
[00448] For example, a 5 ’-terminal region for the antisense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the antisense strand. A preferred 5 ’-terminal region for the antisense strand is positions 1 , 2 and 3 counting from the 5 ’ -end of the antisense strand. A 3 ’ -terminal region for the antisense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand. A preferred 3 ’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 3’- end of the antisense strand.
[00449] Similarly, a 5 ’-terminal region for the sense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the sense strand. A preferred 5 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 5 ’-end of the sense strand. A 3 ’-terminal region for the sense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand. A preferred 3 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 3 ’-end of the sense strand.
[00450] As used herein, a “central region” of a strand refers to positions 5-17, e.g., positions 6- 16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5 ’-end of the strand. For example, the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand. A preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5’-end of the sense strand. A more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5 ’-end of the sense strand. A preferred central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 15 16 and 17, counting from 5’-end of the antisense strand. A more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5’- end of the antisense strand.
[00451] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g. animal or a plant). As used herein, the term “ex vivo” refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube). As used herein, the term "in vivo" refers to events that occur within an organism (e.g. animal, plant, and/or microbe).
[00452] As used herein, the term "subject" or "patient" refers to any organism to which a composition disclosed herein can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female.
[00453] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders. In addition, compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.
[00454] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment. Alternatively, a subject can also be one who has not been previously diagnosed. A “subject in need” of testing for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
[00455] In some embodiments, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species. In some embodiments, the subject can be of European ancestry. In some embodiments, the subject can be of African American ancestry. In some embodiments, the subject can be of Asian ancestry.
[00456] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.
[00457] As used herein, the term “parenteral administration,” refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
[00458] As used herein, the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein. [00459] As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.
[00460] As used herein, the term “dosage unit” refers to a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial comprising lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
[00461] By the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) it is meant that the severity of the subject’s condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
[00462] The terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present invention.
[00463] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.
[00464] A glycolic nucleic acid (GNA) is wherein B is a modified or unmodified nucleobase, and * is R, S, or racemic.
[00465] The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1 ’, C2’, C3’, C4’ or 04’) are independently or in combination absent from the nucleotide. In some embodiments,
wherein B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.
[00466] Unlocked nucleic acid (UNA) modification encompasses monomers with bonds between C1’-C4’ being removed (i.e. the covalent carbon-oxygen-carbon bond between the Cl’ and C4’ carbons). In another example, the C2’-C3’ bond (i.e. the covalent carbon-carbon bond between the C2’ and C3’ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage. In some embodiments, UNA has the structure: where B is a modified or unmodified nucleobase; R, R’, R”, R’” and R”” are independently H, OH, CH3, CH2CH3, O- alkyl, NH2, NHMe or NMei; and each * is independently R, S, or racemic. In some embodiments, the UNA modification is where B is a modified or unmodified nucleobase and R is H, OH or O-alkyl.
[00467] Modified unlocked nucleic acid (mUNA) modification include, but are not limited to the following: wherein Base is a modified or unmodified nucleobase, and * is R, S, or racemic.
[00468] In some embodiments, the UNA modification is selected from the group consisting of:
[00469] The term “abasic modification” refers to a nucleotide or analog thereof that does not have a nucleobase. Some exemplary abasic modifications include, but are not limited to, the following:
wherein R is H, Me, Et or OMe; R’ is H, Me, Et or OMe; R” is H, Me, Et or OMe; and * represents either R, S or racemic.
[00470] In some embodiments the thermally destabilizing modification is selected from the group consisting of: wherein B is a modified or unmodified nucleobase, and * is R, S, or racemic. [00471] As used herein, the 2 ’-5 ’ RNA is where Base is a modified or unmodifed nucleobase.
[00472] As used herein TNA is a nucleotide comprising a threose sugar instead of a ribose sugar, where its 3 ’-position is linked to the 3’-positoon of the nucleotide upstream of it, and its 2’-psotion is linked to the 5’-positoon of the nucleotide downstream of it. TNA has the structure
Some exemplary TNA modifications include, but are not limited to, the following: where B is a modified or unmodified nucleobase.
[00473] As used herein, a Hyp-spacer modification comprises a hydroxyprolinol monomer, e.g., where R is H or a modified or unmodified nucleobase.
[00474] Additional exemplary sugar modifications include, but are not limited to the following: where B is a modified or unmodified nucleobase, and R is H or C1- Cealkyl (e.g., methyl or ethyl),
[00475] Exemplary, nucleotides with impaired W-C H-bonding to complementary base on opposing strand include, but are not limited to, nucleotides comprising a nucleobase independently selected from the following:
[00476] Exemplary non-canonical bases with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand, include, but are not limited to, inosine, nebularine, 2-aminopurine, 2,4-difhiorotoluene, 5 -nitroindole, 3-nitropyrrole, 4-fluoro-6- methylbenimidazole and 4-methylbenzimidazole.
[00477] Exemplary α-nucleotides include, but are not limited to, B is a modified or unmodified nucleobase, and R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.
[00478] Exemplary phosphate modifications known to decrease the thermal stability of dsNA duplexes compared to natural phosphodiester linkages include, but are not limited to, the following: where the alkyl for the R group can be a C1-C6alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.
[00479] As used herein, the term “bridged nucleic acid” includes, but is not limited to, nucleotides that comprise a five-membered or six-membered bridged structure with a fixed 3'-endo confirmation, also known as the north confirmation. The bridged structure connects the 2'-arbon (e.g., 2’-oxygen) of the ribose sugar to the 4' carbon of the ribose sugar. Various different bridge structures are possible containing carbon, oxygen, nitrogen, and hydrogen atoms. [00480] In some embodiments, the BNA is locked nucleic acid (LNA). As used herein, the term "locked nucleic acid" (LNA) generally refers to a class of BNAs, where the ribose ring is "locked" with a methylene bridge connecting the 2' oxygen of the ribose sugar to the 4' carbon of the ribose sugar.
[00481] As used herein, the term “aliphatic” means a saturated or unsaturated and straight, branched, and/or cyclic hydrocarbon having the defined number of carbon atom. Examples include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, and cycloalkylalkynyl, having the defined number of carbon atoms.
[00482] As used herein, the term “alkyl” refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms. The alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl. Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.
[00483] A “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH2 group to an NH group or an O group). The term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof. In certain embodiments, the heteroatom(s) is placed at any interior position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, - CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2- S-CH2-CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, - CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In some embodiments, up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3
[00484] As used herein, the term “alkenyl” refers to an alkyl group containing at least one carbon-carbon double bond. The alkenyl group can be optionally substituted with one or more “alkyl group substituents.” Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-l-yl andheptadec-8,ll-dien-l-yl.
[00485] As used herein, the term “alkynyl” refers to an alkyl group containing a carbon-carbon triple bond. The alkynyl group can be optionally substituted with one or more “alkyl group substituents.” Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl. Useful alkynyl groups include the lower alkynyl groups.
[00486] As used herein, the term “cycloalkyl” refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and/or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
[00487] “Heterocyclyl” refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent. Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4- morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3- dioxanyl, 1,4-dioxanyland the like.
[00488] “Aryl” refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms. The aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and — NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl.
[00489] “Heteroaryl” refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively. [00490] Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4- oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H- pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1, 3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent. [00491] As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term “halogen radioisotope” or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.
[00492] A “halogen-substituted moiety” or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.
[00493] The term “haloalkyl” as used herein refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different. The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (C1-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like). [00494] As used herein, the term “amino” means -NH2. The term “alkylamino” means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl). The term “dialkylamino” means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -N(alkyl)(alkyl). The term “alkylamino” includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.” The term “arylamino” means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and — N(aryl)2. The term “heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example — NHheteroaryl, and — N(heteroaryl)2. Optionally, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like. Exemplary alkylamino includes, but is not limited to, NH(Cl-ClOalkyl), such as — NHCH3, — NHCH2CH3, — NHCH2CH2CH3, and — NHCH(CH3)2. Exemplary dialkylamino includes, but is not limited to, — N(Cl-C10alkyl)2, such as N(CH3)2, — N(CH2CH3)2, — N(CH2CH2CH3)2, and — N(CH(CH3)2)2.
[00495] The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms ( — N — ) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl. For example, an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.
[00496] The terms “hydroxyl” and “hydroxyl” mean the radical — OH.
[00497] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O- alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described above for alkyl. Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n- propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O- hexyl, O- cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.
[00498] As used herein, the term “carbonyl” means the radical — C(O) — . It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.
[00499] As used herein, the term “oxo” means double bonded oxygen, i.e., =0.
[00500] The term “carboxy” means the radical — C(O)O — . It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., carboxyl group.
[00501] The term “ester” refers to a chemical moiety with formula -C(=O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl.
[00502] The term “cyano” means the radical — CN.
[00503] The term “nitro” means the radical — NO2.
[00504] The term, “heteroatom” refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include — N=, — NRN — , — N+(O-)=, — O — , — S — or — S(O)2 — , — OS(O)2 — , and — SS — , wherein RN is H or a further substituent.
[00505] The terms “alkylthio” and “thioalkoxy” refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur. In preferred embodiments, the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups.
[00506] The term “sulfinyl” means the radical — SO — . It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.
[00507] The term “sulfonyl” means the radical — SO2 — . It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.
[00508] The term “thiocarbonyl” means the radical — C(S) — . It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.
[00509] “Acyl” refers to an alkyl-CO — group, wherein alkyl is as previously described. Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
[00510] “Aroyl” means an aryl-CO — group, wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.
[00511] “Arylthio” refers to an aryl-S — group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio. [00512] “Aralkyl” refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
[00513] “Aralkyloxy” refers to an aralkyl-0 — group, wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.
[00514] “Aralkylthio” refers to an aralkyl-S — group, wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.
[00515] “Alkoxycarbonyl” refers to an alkyl-0 — CO — group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
[00516] “Aryloxycarbonyl” refers to an aryl-0 — CO — group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
[00517] “Aralkoxycarbonyl” refers to an aralkyl-0 — CO — group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
[00518] “Carbamoyl” refers to an H2N — CO — group.
[00519] “Alkylcarbamoyl” refers to a R'RN — CO — group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.
[00520] “Dialkylcarbamoyl” refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.
[00521] “Acyloxy” refers to an acyl-0 — group, wherein acyl is as previously described. “Acylamino” refers to an acyl-NH — group, wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH — group, wherein aroyl is as previously described.
[00522] The term “optionally substituted” means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified. The term “substituents” refers to a group “substituted” on a substituted group at any atom of the substituted group. Suitable substituents include, without limitation, halogen, hydroxyl, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxylalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In some cases, two substituents, together with the carbons to which they are attached to can form a ring.
[00523] For example, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from OH, CN, - SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(Cl-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(Cl-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1- C8)alkyl, 0(Cl-C8)alkyl, O(Cl-C8)haloalkyl, (C2 C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m— (CH2)p— OH, CH2— [CH(OH)]m— (CH2)p— NH2 or CH2-aryl-alkoxy; “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00524] In some embodiments, an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.
[00525] In some embodiments, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl (e.g., alkyl) is optionally substituted with 1, 2 or 3 substituents selected independently from the group consisting of halogen, -OR22’ -N(R22)2, -SR22, -C(O)OR22, -C(O)N(R22)2, wherein R22 is hydrogen or C1-3alkyl (e.g., 2,2,2-trifhioroethyl, l,3-dimethoxyprop-2-yl). For example, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl (e.g., alkyl) is optionally substituted with one or two substituents selected independently from the group consisting of halogen, -OR22, -N(R22)2, -SR22, -C(O)OR22, -C(O)N(R22)2, wherein R22 is hydrogen or C1-3alkyl (e.g., 2,2,2-trifhioroethyl, 1 ,3-dimethoxyprop-2-yl).
[00526] An “isocyanato” group refers to a NCO group.
[00527] A “thiocyanato” group refers to a CNS group.
[00528] An “isothiocyanate” group refers to a NCS group.
[00529] “Alkoyloxy” refers to a RC(=O)O- group.
[00530] “Alkoyl” refers to a RC(=O)- group.
[00531] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., provided herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. The invention is further illustrated by the following example, which should not be construed as further limiting.
[00532] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00533] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00534] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
EXAMPLES
[00535] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
Example 1: Synthesis of monomers
Synthesis of compound 9
[00536] Compound 9 was synthesized according to Scheme 1.
[00537] Compound 2: To a solution of compound 1 (60.0 g, 534 mmol, 1.00 eq.) in ACN (420 mL) and Py (210 mL) was added BzCI (165 g, 1176 mmol, 136 mL, 2.20 eq.) at 20 °C. The mixture was stirred at 20 °C for 16 hrs. The crude product was triturated with DCM (1900 mL) and H2O (1900 ml) at 20 °C for 30 min. The precipitate was filtered, and compound 2 (78.0 g, 332 mmol, 62.1% yield) was obtained as a white solid. 1H NMR DMSO-d6400 MHz δ 11.60 (s, 1H), 7.96- 7.94 (m, 2H), 7.78-7.75 (m, 1H), 7.66-7.57 (m, 3H), 5.74 (d, J= 8.0 Hz, 1H).
[00538] Compound 3: To a mixture of compound 2 (54.0 g, 249 mmol, 1.00 eq.), PPh3 (78.5 g, 298 mmol, 1.20 eq.) and compound 2A (6.30 g, 324 mmol, 46.0 mL, 1.30 eq.) in THF (545 mL) was added DEAD (52.1 g, 298 mmol, 54.4 mL, 1.20 eq.) at 0 °C. The resulting mixture was degassed and purged with N2 for 3 times. And then the reaction was stirred at 20 °C for 2 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 3 (crude) was obtained as a yellow solid, which was used to the next step without further purification.
[00539] Compound 4: To a solution of compound 3 (60.0 g, 174 mmol, 1.00 eq.) andNHsH2O (273 g, 1.95 mol, 300 mL, 25% purity, 11.1 eq.) in MeOH (300 mL). The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 100: 1 to 0: 1). Compound 4 (62.6 g, 82.4%) was obtained as a white solid. 1H NMR DMSO- d6400 MHz δ 11.20 (s, 1H), 7.60 (d, J= 8.0 Hz, 1H), 5.54-5.52 (m, 1H), 4.05-3.96 (m, 3H), 3.82-3.61 (m, 2H), 3.50-3.46 (m, 1H), 1.83-1.74 (m, 2H), 1.30-1.24 (m, 6H).
[00540] Compound 5: To a solution of compound 5 (62.0 g, 258 mmol, 1.00 eq.) in MeOH (2331 mL) was added I2 (23.3 g, 91.8 mmol, 1%, w/v). The mixture was stirred at 20 °C for 16 h. The reaction mixture was quenched by addition NaHSO3 (100 mL) at 20°C, and then filtered and concentrated under reduced pressure to give a residue. Compound 5 (92.6 g, crude) was obtained as a dark red oil, which was used to the next step without further purification. 1H NMR DMSO- d6 400 MHz δ 11.17 (s, 1H), 7.58 (d, .7= 8.0 Hz, 1H), 5.5 l(d, J= 8.0 Hz, 1H), 3.83-3.59 (m, 2H), 3.38 (s, 1H), 3.31-3.18 (m, 2H), 1.80-1.72 (m, 1H), 1.51-1.42 (m, 1H).
[00541] Compound 6: To a solution of 5 (51.6 g, 257 mmol, 1.00 eq.) in pyridine (1842 mL) was added TrtCl (114 g, 412 mmol, 1.60 eq.) and DMAP (3.15 g, 25.7 mmol, 0.10 eq.) at 20 °C. The mixture was stirred at 80 °C for 16 h. The reaction mixture was diluted with DCM (1000 mL) and extracted with H2O (1500 X 3 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 100: 1 to 0: 1). Compound 6 (45.2 g, 93.0 mmol, 36.1% yield, 91.5% purity) was obtained as a yellow solid. 1H NMR: DMSO- d6 400 MHz δ 11.19 (s, 1H), 7.54 (d, J= 8.0 Hz, 1H), 7.40-7.22 (m, 16H), 5.52-5.49 (m, 1H), 4.92 (d, J= 8.0 Hz, 1H), 3.83-3.76 (m, 1H), 3.68-3.56 (m, 2H), 3.00-2.97 (m, 1H), 2.81-2.77 (m,lH), 1.87- 1.79 (m, 1H), 1.57-1.54 (m, 1H).
[00542] Compound 7: Added NaH (14.1 g, 352 mmol, 60.0% purity, 3.00 eq.) portion wise to THF (129 mL). Added a solution of compound 6 (52.0 g, 117 mmol, 1.00 eq.) and compound 6A (56.7 g, 176 mmol, 45.4 mL, 1.50 eq.) in THF (998 mL) to the above suspension at -20 °C. The mixture was stirred at 20 °C for 17 h. The reaction mixture was quenched by addition of EtOH (40.0 mL) at 0 °C, and stirred for 30 min. Then the mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Ethyl acetate: Methanol = 100: 1 to 0: 1). Compound 7 (42.0 g), was obtained as a white solid. 1H NMR DMSO- d6400 MHz δ 11.21 (s, 1H), 7.57 (d, J= 8.0 Hz, 1H), 7.40-7.24 (m, 15H), 5.51- 5.49 (m, 1H), 4.09-4.00 (m, 4H), 3.90-3.59 (m, 4H), 3.56-3.44 (m, 1H), 3.13-3.02 (m, 2H), 1.81- 1.76 (m,2H), 1.26-1.19 (m, 6H).
[00543] Compound 8: To a solution of compound 7 (29.0 g, 48.9 mmol, 1.00 eq.) in DCM (2320 mL) was bubbled HCl (g) at 0 °C for 30 mins. The crude product was triturated with Petroleum ether at 20 °C for 20 mins. The residue was purified by prep-HPLC (neutral condition: column: Welch Xtimate C18 250 * 100mm # lOum; mobile phase: [Water-ACN]; B%: 1% - 25%, 20 min). Compound 8 (7.20 g, 19.4 mmol, 39.7% yield) was obtained as a colorless oil. 1H NMR DMSO- d6400 MHz δ 11.22 (s, 1H), 7.64 (d, J= 8.0 Hz, 1H), 5.52 (d, J= 4.0 Hz, 1H), 4.09-3.94 (m, 5H), 3.85-3.64 (m, 3H), 3.48-3.32 (m, 4H), 1.84-1.64 (m,2H), 1.26-1.22 (m, 6H).
[00544] Compound 9: To a solution of compound 8 (8.00 g, 22.8 mmol, 1.00 eq.) in DCM (80.0 mL) was added compound 8A (10.3 g, 34.2 mmol, 10.8 mL, 1.50 eq.) and DCI (2.97 g, 25.1 mmol, 1.10 eq.). The mixture was stirred at 20 °C for 1 h. The reaction mixture was diluted with DCM (10.0 mL) and washed with NaHCO3 (600 ml, 100 mL x 6). The organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was diluted with MTBE (150 mL) and washed with H2O and DMF (H2O: DMF = 1: 1, 900 mL, 90 mL x 10). And then washed with brine (360 mL, 90mL x 4), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 100: 1 to 0: 1). (1.50 g, 2.48 mmol, 10.8% yield) was obtained as a colorless oil. 1H NMR DMSO- d.6400 MHz δ 11.20 (s, 1H), 7.65-7.62 (m, 1H), 5.53 (d, J= 8.0 Hz, 1H), 4.09-3.94 (m, 7H), 3.88-3.52 (m, 11H), 2.77- 2.74 (m, 2H), 1.87-1.73 (m,2H), 1.26-1.11 (m, 18H). MS (ESI+APCI) calculated for C22H39N4O8P2 [M-H]- m/z = 549.2249, found 549.3.
Synthesis of compound 17
[00545] Compound 17 was ynthesized according to Scheme 2. Scheme 2
[00546] Compound 11: To a solution of methyl cis- l-Boc-4-hydroxy-D-prolinate (1 g, 4.08 mmol) (10) in EtOH (30 mL) was added NaBH4 (926 mg, 24.5 mmol) at 0 °C and the mixture was stirred at room temperature overnight. The reaction mixture was cooled to 0 °C and carefully quenched by adding dropwise AcOH. The mixture was then concentrated under vacuum. The residue was partitioned with CH2Cl2 and saturated NaHCO3 (aq.) and the product was extracted with CH2CI2 and EtOAc. The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50-90% ethyl acetate in hexane) to obtain compound 11 as a white solid (728 mg, 82%). 1H NMR (600 MHz, DMSO-d6) δ 5.12-5.10 (m, 1H), 4.89 (t, J= 5.3 Hz, 1H), 4.15 (brs, 1H), 3.73-3.68 (m, 1H), 3.57-3.53 (m, 2H), 3.44-3.40 (m, 1H), 3.06 (dd, J= 21.7, 11.2 Hz, 1H), 2.07-2.04 (m, 1H), 1.82 (dd, J= 36.9, 13.3 Hz 1H), 1.39 (s, 9H). MS (ESI+APCI) calculated for C10H18NO4 [M-H]- m/z = 216.12, found 216.0.
[00547] Compound 12: To a solution of 11 (700 mg, 3.22 mmol) in 1,4-dioxane (20 mL) was added dropwise HCl (4 M in 1,4-dioxane: 10 mL) at room temperature and the mixture was stirred overnight. The reaction mixture was filtrated to obtain the precipitate. The white solid was washed with 1,4-dioxane and CH2Cl2to obtain 12 as a white solid (348 mg, 70%). 1H NMR (600 MHz, DMSO-d6) δ 9.71 (brs, 1H), 8.85 (brs, 1H), 5.33 (brs, 2H), 4.36-4.33 (m, 1H), 3.63-3.60 (m, 2H), 3.59-3.55 (m, 1H), 3.17-3.12 (m, 1H), 3.01-2.97 (m, 1H), 2.18-2.13 (m, 1H), 1.60-1.56 (m, 1H). MS (ESI+APCI) calculated for C5H12NO2 [M+H]+ m/z = 118.09, found 118.2.
[00548] Compound 13: To a suspension of 12 (345 mg, 2.25 mmol) in diethyl phosphite (405 μL, 3.14 mmol) was added 37 wt% aqueous formaldehyde (0.310 mL, 3.82 mmol) and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum and the crude residue was purified by column chromatography on silica gel (0-20% MeOH in CH2CI2) to obtain 13, which contained some impurities, as a colorless oil. The obtained compound was used for the next reaction without any further purification. 1H NMR (600 MHz, DMSO-d6) δ 5.42 (brs, 2H), 4.40-4.34 (m, 1H), 4.16-4.09 (m, 4H), 3.95-3.90 (m, 1H), 3.80-3.79 (m, 1H), 3.71-3.69 (m, 2H), 3.61-3.48 (m, 2H), 3.39-3.33 (m, 1H), 2.37-2.32 (m, 1H), 1.59-1.56 (m, 1H), 1.29-1.27 (m, 6H). 31P NMR (243 MHz, DMSO-d6) δ 17.35. MS (ESI+APCI) calculated for C10H23NO5P [M+H]+ m/z = 268.13, found 268.2.
[00549] Compound 14: To a solution of 13 (500 mg, 1.87 mmol) in pyridine (15 mL) was added DMTrCl (697 mg, 2.06 mmol) and the mixture was stirred at room temperature for 1 h. The reaction was quenched with MeOH and the reaction mixture was concentrated under vacuum. The residue was dissolved in ethyl acetate and washed with saturated NaHCO3 (aq.), water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-5% MeOH in ethyl acetate) to obtain 14 as a brown form (63% yield over 2 steps). 1H NMR (600 MHz, DMSO-d6) δ 7.40-7.38 (m, 2H), 7.32-7.29 (m, 2H), 7.26- 7.21 (m, 5H), 6.90-6.88 (m, 4H), 4.58-4.57 (m, 1H), 4.14-4.12 (m, 1H), 3.97-3.92 (m, 4H), 3.74 (s, 6H), 3.41 (dd, J= 17.5, 15.2 Hz, 1H), 3.12-3.06 (m, 2H), 3.00 (dd, J= 9.5, 4.9 Hz, 1H), 2.75- 2.66 (m, 2H), 2.47 (dd, J= 10.3, 5.8 Hz, 1H), 2.13 (dt, J= 13.2, 7.5 Hz, 1H), 1.26-1.22 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H). 31PNMR (243 MHz, DMSO-d6) δ 24.75, 24.72, 24.68, 24.65, 24.62, 24.58. MS (ESI+APCI) calculated for C31H40NNaO7P [M+Na]+ m/z = 592.24, found 592.2.
[00550] Compound 15: To a suspension of 14 (550 mg, 0.966 mmol), N3 -benzoyluracil (313 mg, 1.45 mmol) and Ph3P (633 mg, 2.41 mmol) in THF (9 mL) was added dropwise a solution of di- tert-butyl azodicarboxylate (DBAD: 556 mg, 2.41 mmol) in THF (3 mL) at 0°C and the mixture was stirred at room temperature for 8 h. The reaction mixture was concentrated under vacuum and the residue was partitioned in ethyl acetate and saturated NaHCO3 (aq.). The organic layer was washed with water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-5% MeOH in ethyl acetate) to obtain 15 as an orange form (243 mg, 33%). 1H NMR (600 MHz, DMSO-d6) δ 8.01-7.99 (m, 2H), 7.96 (d, J= 8.1 Hz, 1H), 7.80-7.77 (m, 1H), 7.62-7.59 (m, 2H), 7.40-7.38 (m, 2H), 7.33-7.30 (m, 2H), 7.27-7.21 (m, 5H), 6.90-6.89 (m, 4H), 5.89 (d, J= 8.1 Hz, 1H), 4.90-4.85 (m, 1H), 3.98- 3.92 (m, 4H), 3.73 (s, 6H), 3.49 (dd, J= 9.6, 7.0 Hz, 1H), 3.27-3.19 (m, 2H), 3.06-3.02 (m, 2H), 2.86 (dd, J= 15.1, 6.8 Hz, 1H), 2.76 (dd, J= 9.6, 7.5 Hz, 1H), 2.17-2.12 (m, 1H), 2.00-1.95 (m, 1H), 1.19 (t, J = 7.0 Hz, 3H), 1.19 (t, J = 7.0 Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ 24.41, 24.38, 24.35, 24.31, 24.28, 24.25. MS (ESI+APCI) calculated for C42H46N3NaO9PNa [M+Na]+m/z = 790.29, found 790.2.
[00551] Compound 16: Compound 15 (600 mg, 0.781 mmol) was dissolved in 80% aqueous AcOH and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-10% MeOH in ethyl acetate) to obtain compound 16 as a white form (332 mg, 91%). 1H NMR (600 MHz, DMSO-d6) δ 8.00-7.96 (m, 3H), 7.80-7.78 (m, 1H), 7.62-7.59 (m, 2H), 5.88 (d, J= 8.2 Hz, 1H), 4.88-4.83 (m, 1H), 4.64 (t, J= 52 Hz, 1H), 4.05-4.00 (m, 4H), 3.46-3.42 (m, 3H), 3.35-3.30 (m, 1H), 3.04 (dt, J= 13.9, 5.2 Hz, 1H), 2.97 (dd, J= 15.3, 7.9 Hz, 1H), 2.79 (dd, J= 9.6, 7.2 Hz, 1H), 2.10-2.00 (m, 2H), 1.24 (t, J= 7.0 Hz, 6H).31P NMR (243 MHz, DMSO-d6) δ 25.01, 24.98, 24.95, 24.92, 24.88, 24.85. MS (ESI+APCI) calculated for C21H29N3O7P [M+H]+ m/z = 466.17, found 466.2. [00552] Compound 17: A solution of 16 (350 mg, 0.752 mmol) and DIPEA (0.393 mL, 2.26 mL) in CH2CI2 was added dropwise 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.185 mL, 0.827 mmol) at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction was quenched with saturated NaHCO3 (aq.) and the reaction mixture was stirred for 10 min. The organic layer was washed with saturated NaHCO3 (aq.), water and brine and dried with Na2SO4 and concentrated under vacuum. The crude residue was dissolved in small amount of 5% Et3N in CH2CI2 and loaded on a pre-equilibrated (10% Et3N in hexane) ISCO silica gel cartridge and then purified by silica gel column chromatography (0-100% ethyl acetate (contained 10% Et3N) in hexane (contained 10% Et3N)) to obtain 17 as a sticky yellow oil (359 mg, 72%). 1H NMR (600 MHz, CD3CN) δ 7.98- 7.80 (m, 2H), 7.78-7.73 (m, 2H), 7.58-7.55 (m, 2H), 5.78 (d, J= 8.2 Hz, 1H), 4.99-4.94 (m, 1H), 4.10-4.05 (m, 4H), 3.85-3.80 (m, 1H), 3.78-3.71 (m, 1.5H), 3.67-3.65 (m, 1H), 3.63-3.58 (m, 2H), 3.48 (dd, J= 9.9, 6.9 Hz, 1H), 3.31 (sept, J= 4.6 Hz, 1H), 3.27-3.22 (m, 1H), 3.14-3.05 (m, 1.5H), 2.93 (dd, J= 9.9, 5.8 Hz, 1H), 2.66-2.62 (m, 2H), 2.22-2.16 (m, 1H), 2.14-2.09 (m, 1H), 1.30-1.27 (m, 6H), 1.18-1.17 (m, 12H). 31P NMR (243 MHz, CD3CN) δ 147.98, 147.94, 147.91, 147.88, 147.84, 147.54, 147.50, 147.47, 147.44, 147.40, 24.28, 24.24, 24.20, 24.15, 24.11, 24.07. MS (APCI) calculated for C30H46N5O8P2 [M+H]+ m/z = 666.28, found 666.6.S
Synthesis of compound 21
[00553] Compound 21 is synthesized according to Scheme 3
[00554] Compound 19: A solution of compound 18 (15 g, 29.85 mmol) and diethoxyphosphorylmethyl-4-methylbenzenesulfonate (11.54 g, 35.82 mmol, 9.24 mL) in anhydrous THF (300 mL) was cooled to -20°C, then mixed with NaH (60% in oil dispersion) (3.43 g, 89.54 mmol). The resulting mixture was allowed to reach room temperature and stirred for 20h. The reaction mixture was quenched with sat. aqueous NH4Cl and diluted with EtOAc (300 mL). The organic layer was washed with water and brine. The residue was purified by ISCO automated column (220g) using 0-100% EtOAc in hexanes as eluant to give compound 19 (13 g, 66%). 1H NMR (600 MHz, DMSO) δ 11.22 (s, 1H), 7.41 - 7.21 (m, 11H), 6.94 - 6.87 (m, 4H), 4.02 - 3.94 (m, 4H), 3.90 - 3.69 (m, 12H), 3.18 (dd, J = 10.7, 3.2 Hz, 1H), 2.93 (dd, J = 10.6, 4.4 Hz, 1H), 1.69 (d, J = 1.2 Hz, 3H), 1.19 (td, J = 7.1, 1.8 Hz, 6H). 31P NMR (243 MHz, DMSO) δ 20.98. MS (ESI+APCI) calculated for C34H41N2O9P [M+Na]+ m/z = 675.245, found 675.2.
[00555] Compound 20: A solution of p-toluenesulfonic acid monohydrate (2.99 g, 15.74 mmol) in MeOH/DCM (500 mL) was added to a solution of compound 19 (7.9 g, 12.10 mmol) in a 1:1 mixture of MeOH/DCM (500 mL) at 0°C. . After 30 min at 0°C, solid NaHCO3 was added until the orange color of the solution disappeared. The resulting mixture was filtered, the filtrate was combined with silica gel, and the volatiles were evaporated to dryness. The residue was purified by ISCO automated column (120g) using 0-10% MeOH in DCM as eluant to give compound 20 (2.4 g, 56%). 1H NMR (600 MHz, DMSO) δ 11.24 (s, 1H), 7.40 - 7.36 (m, 1H), 4.84 (t, J = 5.5 Hz, 1H), 4.01 - 3.93 (m, 5H), 3.89 (dd, J = 14.1, 3.4 Hz, 1H), 3.83 - 3.76 (m, 1H), 3.68 - 3.62 (m, 1H), 3.59 - 3.54 (m, 1H), 3.50 (dt, J = 11.6, 5.0 Hz, 1H), 3.44 (ddd, J = 11.7, 5.8, 4.6 Hz, 1H), 2.29 (s, 1H), 1.74 (d, J = 1.2 Hz, 3H), 1.24 - 1.16 (m, 6H). 31P NMR (243 MHz, DMSO) 21.40. MS (ESI+APCI) calculated for C13H24N2O7P [M+H]+ m/z = 351.1316, found 351.2. [00556] Compound 21: Compound 20 (2 g, 5.71 mmol) was suspended in acetonitrile (3 mL) and stirred for 5 min. DIPEA (3.98 mL, 22.84 mmol), 3-[chloro- (diisopropylamino)phosphanyl]oxypropanenitrile (1.91 mL, 8.56 mmol) and 1 -Methylimidazole (0.45 mL, 5.71 mmol) were added sequentially to the previous suspension and stirred for 20 min. The mixture was evaporated to dryness and the residue was diluted with EtOAc (100 mL) and washed with a saturated solution of NaHCO3 (x2) and brine. The residue was purified by ISCO automated column using 0-100% EtOAc in hexanes eluant to give compound 21 (2.46 g, 78%). 31P NMR (243 MHz, DMSO) 8 147.67, 147.53, 20.93. MS (ESI+APCI) calculated for C13H24N2O7P [M-H]- m/z = 549.2249, found 549.2.
[00557] Compound 22: The synthesis of compound 22 has been reported: J. Org. Chem. 2016, 81, 6, 2261.
[00558] Compound 23: The synthesis of compound 23 has been reported: J. Org. Chem. 2016, 81, 6, 2261.
[00559] Compound 24: The synthesis of compound 24 has been reported: Nucleosides &
Nucleotides (1997), 16(10 & 11), 1933-1950.
[00560] Compound 25: The synthesis of compound 24 has been reported: Rosier, A.;
Panayotou, G.; Hornby, D. P.; Barlow, T.; Brown, T.; Pearl, L. H.; Sawa, R.; Blackbum, G. M.
Nucleosides, Nucleotides & Nucleic Acids 2000, 19, 1505.
Synthesis of compound 33
[00561] Compound 33 was synthesized according to Scheme 4
[00562] Compound 27: A solution of compound 261 (5 g, 12.4 mmol) in pyridine (100 mL) was added 4,4 '-dimethoxytrityl chloride (4.21 g, 12.4 mmol) and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate and saturated aqueous NaHCO3. The organic layer was washed with water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (45-100% ethyl acetate in hexane) to obtain compound 27 (5.8 g, 66%) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.43 (d, J= 1.8 Hz, 1H), 7.98 (d, J= 8.1 Hz, 1H), 7.40-7.39 (m, 2H), 7.30-7.19 (m, 7H), 6.88-6.85 (m, 4H), 5.90 (d, J= 5.5 Hz, 1H), 5.73 (dd, J= 8.1, 2.3 Hz, 1H), 5.25 (t, J= 4.9 Hz, 1H), 4.37 (d, J= 5.5 Hz, 1H), 3.96 (dd, J= 11.6, 4.6 Hz, 1H), 3.90 (t, J= 5.5 Hz, 1H), 3.72 (s, 6H), 3.48 (d, J= 10.7 Hz, 1H), 3.41 (dd, J = 11.6, 5.0 Hz, 1H), 3.23 (s, 3H), 2.80 (d, J= 10.7 Hz, 1H), 0.65 (s, 9H), -0.05 (s, 3H), -0.17 (s, 3H). MS (ESI+APCI) calculated for C38H48N2NaO9Si [M+Na]+ m/z = 727.30, found 727.1.
[00563] Compound 28: To a solution of compound 27 (1 g, 1.42 mmol) and imidazole (483 mg, 7.09 mmol) in DMF (10 mL) was added tert-butyldiphenylsilyl chloride (0.728 mL, 2.84 mmol) and the mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with diethyl ether and washed with saturated aqueous NaHCO3. The organic layer was further washed with water and brine and dried over Na2SO4 and then concentrated under vacuum. The residue was dissolved in 80% aqueous acetic acid (10 mL) and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-40% ethyl acetate in hexane) to obtain compound 28 (533 mg, 59% yield). 1H NMR (600 MHz, DMSO-d6) δ 11.42 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.66-7.65 (m, 2H), 7.63-7.61 (m, 2H), 7.50-7.47 (m, 2H), 7.46-7.42 (m, 4H), 5.92 (d, J= 5.0 Hz, 1H), 5.21 (d, J= 8.0 Hz, 1H), 4.52 (dd, J= 6.8, 4.8 Hz, 1H), 4.49 (d, J= 5.6 Hz, 1H), 3.96 (t, J= 5.3 Hz, 1H), 3.93 (d, J= 11.0 Hz, 1H), 3.76 (d, J= 11.0 Hz, 1H), 3.68 (dd, J = 12.2, 4.8 Hz, 1H), 3.43 (dd, J= 12.2, 6.8 Hz, 1H), 3.30 (s, 3H), 1.05 (s, 9H), 0.87 (s, 9H), 0.07 (s, 6H). MS (ESI+APCI) calculated for C33H49N2O7S12 [M+H]+ m/z = 641.31, found 641.0.
[00564] Compound 29: Compound 28 (1.5 g, 2.34 mmol), imidazole (191 mg, 2.81 mmol), triphenylphosphine (1.84 g, 7.02 mmol) and I2 (653 mg, 2.57 mmol) was dissolved in THF (15 mL). The mixture was stirred and heated in a microwave reactor at 150 °C for 6 h. The reaction was cooled to 0 °C and then quenched by addition of saturated NaHCO3 (aq.) and saturated Na2SiO3 (aq.). The reaction mixture was diluted with ethyl acetate. The organic layer was washed with saturated NaHCO3 (aq.), water and brine and dried with Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-40% ethyl acetate in hexane) to obtain compound 29 (1.34 g, 76%) as a brown form. 1H NMR (600 MHz, DMSO-d6) 8 11.43 (s, 1H), 7.69-7.62 (m, 5H), 7.51-7.48 (m, 2H), 7.46-7.43 (m, 4H), 5.84 (d, J = 4.8 Hz, 1H), 5.26 (d, J = 8.0 Hz, 1H), 4.55 (d, J = 5.5 Hz, 1H), 4.04 (dd, J = 5.5, 4.8 Hz, 1H), 3.98 (d, J = 11.0 Hz, 1H), 3.69 (d, J = 11.0 Hz, 1H), 3.62 (d, J = 11.0 Hz, 1H), 3.35-3.33 (m, 1H), 3.30 (s, 3H), 1.04 (s, 9H), 0.87 (s, 9H), 0.08 (s, 3H), 0.08 (s, 3H). MS (ESI+APCI) calculated for C33H48IN2O6Si2 [M+H]+ m/z = 751.21, found 751.2.
[00565] Compound 30: To a suspension of 10% palladium on carbon (50 mg) and compound 29 (1.6 g, 2.13 mmol) in EtOH (20 mL) was added ammonium formate (672 mg, 10.7 mmol) and the mixture was refluxed overnight. The reaction mixture was filtered through a celite pad rinsing with EtOH and the filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-40% ethyl acetate in hexane) to obtain compound 30 (1.28 g, 96%) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.64-7.59 (m, 4H), 7.48-7.41 (m, 6H), 5.85 (d, J = 4.1 Hz, 1H), 5.22 (d, J = 8.0 Hz, 1H), 4.34 (d, J = 5.8 Hz, 1H), 3.91 (dd, J = 5.8, 4.1 Hz, 1H), 3.72 (d, J = 11.1 Hz, 1H), 3.56 (d, J = 11.1 Hz, 1H), 3.31 (s, 3H), 1.12 (s, 3H), 1.03 (s, 9H), 0.86 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H). MS (ESI+APCI) calculated for C33H49N2O6Si2 [M+H]+ m/z = 625.31, found 625.4.
[00566] Compound 31: To a solution of compound 30 (1.3 g, 2.08 mmol) in THF (20 mL) was added triethylamine trihydrofluoride (3.39 mL, 20.8 mmol) and trimethylamine (2.90 mL, 20.8 mmol) at room temperature and the mixture was refluxed for 48 h. The reaction was cooled to room temperature and then concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-10% MeOH in CH2CI2) to obtain compound 31 (550 mg, 97%) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.97 (d, J= 8.1 Hz, 1H), 5.91 (d, J = 6.5 Hz, 1H), 5.67 (dd, J= 8.1, 2.0 Hz, 1H), 5.26 (t, J= 52 Hz, 1H), 5.07 (d, J= 6.5 Hz, 1H), 4.11 (t, J = 5.2 Hz, 1H), 3.94 (dd, J= 6.5, 5.2 Hz, 1H), 3.42-3.36 (m, 2H), 3.31 (s, 3H), 1.08 (s, 3H). MS (ESI+APCI) calculated for C11H17N2O6 [M+H]+ m/z = 273.11, found 273.2.
[00567] Compound 32: To a solution of compound 31 (400 mg, 1.47 mmol) in pyridine (18 mL) was added 4,4 '-dimethoxytrityl chloride (498 mg, 1.47 mmol) and the mixture was stirred at room temperature for 3 h. The reaction was quenched by adding MeOH and concentrated under vacuum. The residue was dissolved in ethyl acetate and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50-80% ethyl acetate in hexane) to obtain compound 32 (763 mg, 90%) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.38 (s, 1H), 7.62 (d, J= 8.1 Hz, 1H), 7.39-7.37 (m, 2H), 7.35-7.32 (m, 2H), 7.26-7.24 (m, 5H), 6.92-6.91 (m, 4H) 5.84 (d, J = 4.7 Hz, 1H), 5.31 (d, J= 8.1 Hz, 1H), 5.20 (d, J= 6.7 Hz, 1H), 4.22 (t, J= 6.2 Hz, 1H), 3.85 (dd, J= 5.6, 4.7 Hz, 1H), 3.75 (s, 6H), 3.36 (s, 3H), 3.13 (d, J= 10.0 Hz, 1H), 3.02 (d, J= 10.0 Hz, 1H), 1.14 (s, 3H). MS (ESI+APCI) calculated for C32H34N2NaO8 [M+Na]+ m/z = 597.22, found 597.2.
[00568] Compound 33: To a solution of 32 (750 mg, 1.31 mmol), 1 -methylimidazole (10.4 μL, 0.131 mmol) and diisopropylethylamine (0.682 mL, 3.92 mmol) in CH2CI2 (13 mL) was added dropwise 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.349 mL, 1.57 mmol) atO °C and the mixture was stirred at room temperature for 1 h. The reaction was quenched by adding saturated NaHCO3 (aq.) and the organic layer washed with saturated NaHCO3 (aq.) and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-50% ethyl acetate in hexane) to obtain compound 33 (828 mg, 82%) as a white form. 1H NMR (600 MHz, CD3CN) δ 9.07 (brs, 1H), 7.83 (d, J= 8.2 Hz, 0.5H), 7.76 (d, J= 8.2 Hz, 0.5H), 7.50-7.45 (m, 2H), 7.38-7.27 (m, 7H), 6.92-6.89 (m, 4H), 5.91-5.90 (m, 1H), 5.18-5.15 (m, 1H), 4.64 (dd, J= 10.4, 5.8 Hz, 0.5H), 4.57 (dd, J= 10.4, 5.8 Hz, 0.5H), 3.97 (dd, J= 5.8, 3.0 Hz, 0.5H), 3.92-3.88 (m, 1H), 3.80-3.79 (m, 7H), 3.71-3.63 (m, 2.5H), 3.51 (s, 1.5H), 3.49 (s, 1.5H), 3.29-3.20 (m, 2H), 2.72-2.69 (m, 1H), 2.56-2.54 (m, 1H), 1.24-1.18 (m, 12H), 1.10 (s, 1.5H), 1.09 (s, 1.5H). 31P NMR (243 MHz, CD3CN) δ 149.80, 149.60. MS (ESI+APCI) calculated for C41H52N4O9P [M+H]+ m/z = 775.35, found 775.4.
Synthesis of compound 37
[00569] Compound 37 was synthesized according to Scheme 5
[00570] Compound 34: Compound 27 (500 mg, 0.709 mmol), imidazole (145 mg, 2.13 mmol), triphenylphosphine (223 mg, 0.851 mmol) and h (198 mg, 0.780 mmol) was dissolved in THF (5 mL). The mixture was stirred and heated in a microwave reactor at 100 °C for 2 h. The reaction was cooled to room temperature and quenched by addition of saturated NaHCO3 (aq.) and saturated Na2SiO3 (aq.) and diluted with ethyl acetate. The organic layer was washed with saturated NaHCO3 (aq.), water and brine and dried with Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-40% ethyl acetate in hexane) to obtain compound 34 (313 mg, 54%) as a yellow form. 1H NMR (600 MHz, DMSO-d6) δ 11.54 (s, 1H), 7.88 (d, J= 8.1 Hz, 1H), 7.43-7.41 (m, 2H), 7.31-7.26 (m, 6H), 7.22-7.19 (m, 1H), 6.87-6.85 (m, 4H), 5.94 (d, J= 7.1 Hz, 1H), 5.79 (d, J= 8.1 Hz, 1H), 4.36 (d, J= 5.4 Hz, 1H), 4.23 (dd, J= 7.1, 5.4 Hz, 1H), 3.72-3.70 (m, 7H), 3.62 (d, J= 10.1 Hz, 1H), 3.51 (d, J= 10.9 Hz, 1H), 3.23 (s, 3H), 3.08 (d, J= 10.9 Hz, 1H), 0.65 (s, 9H), -0.03 (s, 3H), -0.14 (s, 3H). MS (ESI+APCI) calculated for C38H47lN2NaO8Si [M+Na]+ m/z = 837.20, found 837.2.
[00571] Compound 35: Under hydrogen atmosphere, a suspension of 10% palladium on carbon (50 mg), compound 34 (900 mg, 1.10 mmol) and triethylamine (30.8 μL, 0.221 mmol) in EtOH (10 mL) was stirred at room temperature for 3 h. The reaction mixture was filtered through a celite pad rinsing with EtOH and the filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (30-80% ethyl acetate in hexane) to obtain compound 35 (721 mg, 95%). 1H NMR (600 MHz, DMSO-d6) δ 11.43 (d, J= 2.2 Hz, 1H), 7.71 (d, J= 8.1 Hz, 1H), 7.42-7.40 (m, 2H), 7.31-7.24 (m, 6H), 7.21-7.19 (m, 1H), 6.89-6.86 (m, 4H), 5.87 (d, J= 4.9 Hz, 1H), 5.73 (dd, J= 8.1, 2.2 Hz, 1H), 4.14 (d, J= 5.7 Hz, 1H), 3.92 (t, J= 5.7 Hz, 1H), 3.73-3.72 (m, 6H), 3.44 (d, J= 10.5 Hz, 1H), 3.21 (s, 3H), 2.70 (d, J= 10.5 Hz, 1H), 1.39 (s, 3H), 0.65 (s, 9H), -0.04 (s, 3H), -0.12 (s, 3H). MS (ESI+APCI) calculated for CssILsNiNaOsSi [M+Na]+ m/z = 711.31 , found 711.4.
[00572] Compound 36: To a solution of compound 35 (800 mg, 1.16 mmol) in THF (10 mL) was added triethylamine trihydrofluoride (1.89 mL, 11.6 mmol) and trimethylamine ( 1.62 mL, 11.6 mmol) at room temperature and the mixture was refluxed for 48 h. The reaction was cooled to room temperature and then concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50-100% ethyl acetate in hexane) to obtain compound 36 (608 mg, 91%) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.40 (d, J= 1.9 Hz, 1H), 7.67 (d, J= 8.1 Hz, 1H), 7.43-7.42 (m, 2H), 7.31-7.26 (m, 6H), 7.22-7.20 (m, 1H), 6.89-6.88 (m, 4H), 5.85 (d, J = 6.6 Hz, 1H), 5.71 (dd, J= 8.1, 1.9 Hz, 1H), 5.16 (d, J= 5.0 Hz, 1H), 4.07-4.03 (m, 2H), 3.73 (s, 6H), 3.25 (s, 3H), 3.20 (d, J= 9.8 Hz, 1H), 3.00 (d, J= 9.8 Hz, 1H), 1.38 (s, 3H). MS (ESI+APCI) calculated for C32H34N2NaO8 [M+Na]+ m/z = 597.22, found 597.2.
[00573] Compound 37: To a solution of compound 36 (1 g, 1.74 mmol), 1 -methylimidazole (13.8 μL, 0.174 mmol) and diisopropylethylamine (0.909 mL, 5.22 mmol) in CH2CI2 (15 mL) was added dropwise 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.427 mL, 1.91 mmol) at 0 °C and the mixture was stirred at room temperature for 1 h. The reaction was quenched by adding saturated NaHCO3 (aq.) and the organic layer washed with saturated NaHCO3 (aq.) and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50% ethyl acetate in hexane) to obtain compound 37 (952 mg, 71%) as a white form. 1H NMR (600 MHz, CD3CN) δ 9.07 (brs, 1H), 7.48-7.41 (m, 3H), 7.35-7.29 (m, 6H), 7.23-7.20 (m, 1H), 6.88-6.85 (m, 4H), 5.82 (d, J= 6.0 Hz, 0.4H), 5.78 (d, J= 3.2 Hz, 0.6H), 5.70 (d, J= 8.1 Hz, 0.4H), 5.67 (d, J= 8.1 Hz, 0.6H), 4.16 (dd, J= 11.6, 5.2 Hz, 0.4H), 4.08 (dd, J = 10.9, 6.1 Hz, 0.6H), 3.97 (dd, J= 6.1, 5.2 Hz, 0.4H), 3.97 (dd, J= 6.1, 3.2 Hz, 0.6H), 3.77-3.76 (m, 6H), 3.74-3.61 (m, 2H), 3.56-3.51 (m, 1H), 3.49-3.45 (m, 1.2H), 3.42-3.38 (m, 0.8H), 3.31 (s, 1.8H), 3.30 (s, 1.2H), 3.02 (d, J= 10.6 Hz, 0.4H), 2.87 (d, J= 10.6 Hz, 0.6H), 2.57-2.52 (m, 1.2H), 2.45-2.41 (m, 0.4H), 2.34-2.29 (m, 0.4H), 1.55 (s, 1.2H), 1.52 (s, 1.8H), 1.10-1.09 (m, 6H), 0.98 (d, J= 6.8 Hz, 2H), 0.95 (d, J= 6.8 Hz, 4H). 31PNMR (243 MHz, CD3CN) δ 150.46, 149.11. MS (ESI+APCI) calculated for C41H51N4NaO9P [M+Na]+ m/z = 797.33, found 797.4.
Synthesis of compound 51
[00574] Compound 51 was synthesized according to Scheme 6.
[00575] Compound 39: Add MeOH (2500 mL) to the reaction flask under nitrogen. Add Compound 38 (340 g, 2.26 mol, 1.00 eq) under stirring, (the system is in a suspended state) at 15 °C. Dropwise add H2SO4 (56.6 g, 566 mmol, 30.8 mL, 98% purity, 0.25 eq), stirring at 30 °C and reacting for 20 hours after the dropwise addition (the system gradually dissolves). TLC (DCM/MeOH = 10/1, (reactant) Rf= 0.1, (product) Rf= 0.7) shows the reaction complete. Add 100 g of sodium carbonate to the reaction system, stir to pH=7-8, filter (diatomaceous earth filtration), and concentrate the filtrate under reduced pressure at 35 °C to 40 °C to dryness. Without purification. Obtain Compound 39 (410 g, crude) as a yellow oil. About 80.0%. 1H NMR: MeOD 400 MHz 4.78 (m, 2H), 3.96-3.65 (m, 13H), 3.50-3.38 (m, 3H).
[00576] Compound 40: Note: Four batches in parallel. (125 g x 4). Add Compound 39 (125 g, 761 mmol, 1.00 eq) dissolve in Py (1250 mL) and cooled to 0 °C. Add BzCl (857 g, 6.10 mol, 708 mL, 8.01 eq) slowly. Warm the reaction to 20 °C and stir 12 hrs. TLC (Petroleum ether/Ethyl acetate = 5/1, (product) Rf = 0.5) shows the reaction complete. Poured into water (1000 mL), extracted DCM (2000 mL x 3). Wash the combined organic extracts with 10% HCl (aq) (5 x 1000 mL) and evaporated under reduced pressure. Combine with four batches. Purify the crude by silica gel chromatography (SiO2, Petroleum ether/Ethyl acetate = 30/1 to 0/1). Obtain Compound 40 (1.40 kg, 2.06 mol, 67.5% yield) as yellow oil. About 70% TLC purity.
[00577] Compound 41: Dissolve Compound 40 (130 g, 272 mmol, 1.00 eq) in EtOAc (2000 mL). Add then AC2O (91.9 g, 900 mmol, 84.5 mL, 3.30 eq) and H2SO4 (5.35 g, 54.5 mmol, 2.91 mL, 0.20 eq) at 25 °C. Stir at 25 °C for 12 hrs. TLC (Petroleum ether/Ethyl acetate = 5/1, (product) Rf = 0.45) showed the reaction complete. The residue was diluted with EtOAc (200 mL) and washed withSat.NaHCO3 solution (500 mL x 2), water (300 mL) and brine (300 mL x 2). Organic layer was separated, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness. Obtain Compound 41 (170 g, crude) as yellow oil.
[00578] Compound 42: Note: Two batches in parallel (10.0 g + 80.0 g). Add compound 41 (80.0 g, 158 mmol, 1.00 eq) and uracil (23.1 g, 206 mmol, 1.30 eq) into ACN (800 mL). Add BSA (129 g, 634 mmol, 156 mL, 4.00 eq) into the mixture at 20 °C. The solution in to an ice bath, slowly add TMSOTf (56.3 g, 253 mmol, 45.8 mL, 1.60 eq) at 0 °C -5 °C. Stir at 85 °C for 3 hrs. TLC (Petroleum ether/Ethyl acetate = 1/2, Rf (compound 5) = 0.5) showed the reaction complete. Combine work up with the other batch. Quench residue with H2O (500 mL). Extract with EtOAc (500 mL) and washed with Sat.NaHCO3 solution (500 mL x 2) and brine (500 mL x 2). Separated organic layer, dried over with anhydrous Na2SO4, filtered and evaporated to dryness. (200 g crude) Purified the residue by column chromatography (SiO2, Petroleum ether/Ethyl acetate= 30/1 to 0/1). Obtain compound 42 (150 g, 83.0 %. as white solid).
[00579] Compound 43: Note: Four batches in parallel. (5.00 g +45.0 g x 3). Add compound 42 (45.0 g, 80.8 mmol, 1.00 eq) in MeOH (450 mL) and CHCI3 (90 mL). Add MeONa (4.37 g, 80.8 mmol, 1.00 eq) into the mixture. Stir at 30 °C for 12 hrs. Concentrate under reduced pressure. Triturate it with EtOAc (300 mL) at 25 °C for 30 min and collect filter cake. Repeat the operation above again. Triturate it with ACN (300 mL) at 25 °C for 30 min and collect filter cake. Filter the reaction solution and collect the cake. Concentrate under reduced pressure. Obtain the compound 43 (76.0 g, 99.6%) as white solid. 1H NMR: D2O 400 MHz 5 11.29 (s, 1H), 7.79 (d, J= 8.0 Hz, 1H), 5.62-5.61 (m, 1H), 4.95-4.93 (m, 1H), 4.01-4.05 (m, 1H), 3.53-3.64 (m, 3H), 1.53-1.57 (m, 3H), 1.34-1.25 (m, 4H), 1.14-1.11 (m, 3H), 0.86 (t, J= 7.6 Hz, 6H).
[00580] Compound 44: Note: Three batches in parallel. (5.00 g + 35.0 g x 2). Add compound 43 (35.0 g, 143 mmol, 1.00 eq) into pyridine (350 mL) and DMF (175 mL). Add TIPDSCh (49.7 g, 157 mmol, 50.4 mL, 1.10 eq) into the mixture at 15-20 °C. Stir at 15-20°C (external temperature) for 12 hrs. Quench the reaction with water (400 mL). Extracted the mixture with EtOAc (500 mL x 2). Wash with salt water (50 mL) and derived with Na2SO4, filtered and the filtrate was evaporated to dryness. Purified the residue by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 30/1 to 0/1). Obtain the compound 44 (65.9 g, 95.0%) as yellow oil foam. 1H NMR: CDCI3 400 MHz 5 0.84 - 1.12 (m, 31 H), 2.81 (s, 2 H), 2.89 (s, 2 H),3.82 - 4.02 (m, 3 H),4.29 - 4.35 (m, 3 H), 5.48 - 5.51 (d, 1 H),5.68-5.73(d, 1 H),7.21-7.44(d, 1 H),7.94 (s, 1 H),10.07(s, 1 H).
[00581] Compound 45: Note: Four batches in parallel. (5.00 g + 25.0 g + 20.0 g x 2). Add compound 44 (20.0 g, 41.0 mmol, 1.00 eq) andDMAP (9.99 g, 81.7 mmol, 1.99 eq) into ACN (400 mL) at 15-20 °C. Add PhOCSCl (7.80 g, 45.2 mmol, 6.25 mL, 1.10 eq) into the mixture at 15-20 °C. Stir at 15-20 °C (external temperature) for 12 hrs. Extracted the mixture with EtOAc (200 mL x 2).Wash with salt water (400 mL) and dried with Na2SO4, filtered and the filtrate was evaporated to dryness. Purified the residue by column chromatography (SiO2, Petroleum ether/Ethyl acetate= 30/1 to 0/1). Obtain the compound 45 (48.0 g, 93%) as yellow solid.
[00582] Compound 46: Note: Four batches in parallel. (5.00 g + 30.0 g + 20.0 g x 2). Add compound 45 (20.0 g, 32.1 mmol, 1.00 eq) into toluene (300 mL) at 20 °C (reactor 1). Add AIBN (1.05 g, 6.42 mmol, 0.2 eq) into toluene (300 mL) at 20 °C (reactor 2). Add Bu3SnH (74.7 g, 256 mmol, 68.0 mL, 8.00 eq) into the reaction mixture of AIBN (1.05 g, 6.42 mmol, 0.20 eq) and Tol. (300 mL) at 15-20 °C. Add the mixture of the reactor 2 into the mixture of reactor 1 by drops 70- 75 °C (internal temperature). Stir at 80 °C (external temperature) for 1 hr. Quench the reaction with NH4F.aq (400mL). Extracted the mixture with EtOAc (500 mL x 2). Wash with salt water (50 mL) and dried with Na2SO4, filtered and the filtrate was evaporated to dryness. Purified the residue by column chromatography (SiO2, Petroleum ether/Ethyl acetate= 30/1 to 0/1). Obtain the compound 46 (53.0 g, 90% TLC purity) as white foam. 1H NMR: CDCI3 400 MHz 5 0.90 - 1.11 (m, 23 H), 2.19 (dt, .7=13.91, 6.07 Hz, 1 H), 2.87 (dt, .7=14.03, 7.23 Hz, 1 H), 3.83 (dd, J=12.78, 8.01 Hz, 1 H) ,4.02 - 4.12 (m, 2 H), 4.60 (q, .7=6.60 Hz, 1 H), 5.79 (dd, .7=8.07, 1.96 Hz, 1 H), 6.20 (t, .7=6.05 Hz, 1 H), 7.74 (d, .7=8.19 Hz, 1 H) ,9.22 (br s, 1 H).
[00583] Compound 47: Note: Three batches in parallel. (16 g x 3). Add Compound 46 (16.0 g, 33.9 mmol, 1.00 eq) in MeOH (1200 mL) at 15 °C. Add NH4F (12.5 g, 339 mmol, 10.0 eq) to the mixture at 20 °C. Stir at 80 °C for 4 hrs. Concentrate the combined organic phase in vacuum at 40 °C to give a residue. Filter and collect filter liquor. Purify the crude by silica gel chromatography (Dichloromethane/Methanol = 100/1 to 20/1). Obtain Compound 47 (22.0 g, 91.5 mmol, 89.8% yield, 95.0% purity) as white solid. About 95.0% TLC purity.
[00584] Compound 48: Note: Three batches in parallel. (6.00 g x 3). Add compound 47 (6.00 g, 26.2 mmol, 1.00 eq) into pyridine (60 mL) at 20 °C. Add DMTrCl (10.6 g, 31.5 mmol, 1.20 eq) into the reaction mixture (dissolve DMTrCl in Py (60 mL)) at 0-10 °C. Stir at 15-20 °C for 3 hrs. Quench the reaction with NaHCO3 (aq, 500 mL). Extracted the mixture with EtOAc (500 mL x 2). Wash with salt water (50.0 mL) and dried with Na2SO4, filtered and the filtrate was evaporated to dryness. Purified the residue by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 30/1 to 0/1). Obtain the compound 48 (39.7 g, 97.5%) as white foam. 1H NMR: CDCl3400 MHz δ ppm 1.18 (t, .7=7.13 Hz, 1 H), 1.97 (s, 1 H), 2.25 (br d, J=14.88 Hz, 1 H), 2.63 (dt, .7=14.66, 6.49 Hz, 1 H), 3.03 - 3.16 (m, 2 H), 3.65 (br s, 1 H), 3.70 (s, 6 H), 4.33 - 4.44 (m, 2 H), 5.54 (d, J=8.13 Hz, 1 H), 6.11 (d, .7=6.50 Hz, 1 H), 6.76 (d, .7=8.88 Hz, 4 H), 7.10 - 7.17 (m, 1 H), 7.18 - 7.25 (m, 8 H), 7.33 (d, .7=7.50 Hz, 2 H), 7.56 - 7.68 (m, 2 H), 8.53 (dd, .7=5.63, 1.50 Hz, 1 H), 9.61 (br s, 1 H).
[00585] Compound 49: Note: Three batches in parallel. (5.00 g x 3). A mixture of compound 48 (5.00 g, 9.42 mmol, 1.00 eq) in dry ACN (20 mL) was cooled to 0-5 °C and degassed and purged with Ar for 3 times, and NaH (1.70 g, 42.4 mmol, 60.0% purity, 4.50 eq) was added at 0 °C, the mixture was stirred at 18 °C for 4 hrs under Ar atmosphere. A DMF (10.0 mL) solution of (Diethoxyphosphoryl)methyl 4-methylbenzenesulfonate (12.1 g, 37.7 mmol, 9.68 mL, 4.00 eq) (pre-dried by co-evaporation with dry ACN (20.0 mL x 3)) was slowly added in the mixture, the mixture was stirred at this temperature for another 4 hrs at 18 °C. Quench the reaction with H2O (50 mL). Extracted the mixture with EtOAc (50.0 mL x 2). Wash with salt water (50.0 mL) and dried with Na2SO4, filtered and the filtrate was evaporated to dryness. Purified the residue by column chromatography (SiOi, Ethyl acetate/Acetone = 30/1 to 0/1). Obtain the compound 49 (28.0 g, 92.8%) as yellow foam. 1H NMR: DMSO-d6 400 MHz 8 1.20 - 1.35 (m, 11 H), 1.45 (s, 0 H), 2.04 (s, 3 H), 2.09 - 2.28 (m, 1 H), 2.66 - 2.85 (m, 2 H), 2.94 (s, 1 H), 3.04 - 3.22 (m, 2 H) ,3.76 - 3.94 (m, 8 H), 3.97 - 4.21 (m, 9 H), 4.59 (br t, .7=4.06 Hz, 1 H), 5.61 (d, .7=8.13 Hz, 1 H), 6.28 (dd, J=7.57, 1.94 Hz, 1 H), 6.96 (d, .7=8.63 Hz, 4 H), 7.26 - 7.34 (m, 5 H), 7.36 - 7.45 (m, 4 H), 7.73 (d, .7=8.13 Hz, 1 H), 11.4 (br s, 1 H).
[00586] Compound 50: Add compound 49 (12.0 g, 17.6 mmol, 1.0 eq) into DCM (84.0 mL) at 20 °C. Add dodecane- 1 -thiol (5.35 g, 26.4 mmol, 6.33 mL, 1.50 eq) at 20 °C. Add TFA (3.02 g, 26.4 mmol, 1.96 mL, 1.50 eq) at 20 °C. Stir at 20 °C for 2 hrs. Add H2O (50 mL) to the mixture. Extract the solution with DCM (50.0 mL x 2).Wash the organic layer with NaHCCL (50 mL).Purified the residue by column chromatography (SiO2, Petroleum ether /Ethyl acetate = 30/1 to 0/1). Obtain the compound 50 (4.00 g, 96.8%) as yellow oil. 1H NMR: CDCI3400 MHz 8 1.27 (td, .7=7.03, 1.91 Hz, 6 H), 2.16 (br d, J=15.02 Hz, 1 H), 2.56 - 2.72 (m, 1 H), 3.54 - 3.64 (m, 3 H),
3.67 - 3.78 (m, 2 H), 4.01 - 4.16 (m, 4 H), 4.20 (br d, .7=6.20 Hz, 1 H), 4.44 (t, J=4.11 Hz, 1 H), 5.65 (d, .7=8.23 Hz, 1 H), 6.25 (dd, J=7.51, 1.91 Hz, 1 H), 7.62 (d, .7=8.11 Hz, 1 H), 9.27 (br s, 1 H).
[00587] Compound 51: 2-Cyanoethyl tetraisopropylphosphorodiamidite (1.58 g, 5.25 mmol,
1.67 mL) and IH-Tetrazole (0.45 M, 2.33 mL) were added to a solution of compound 50 (1.32 g, 3.50 mmol) in MeCN (18 mL) . After stirring for 3h, the volatiles evaporated to dryness, and the residue re-disolved in EtOAc, washed with sat. bicarb, water and brine. The organic layer was dried over Na2SO4, filtered and the filtrate was evaporated to dryness. Purified the residue by column chromatography using 0-60% (3 : 1 EtO Ac/EtOH) in hexanes as eluant to give compound 51 ( 1.45g, 71%). 31PNMR (243 MHz, DMSO) δ 147.67, 147.59, 20.91.
Synthesis of compound 64
[00588] Compound 64 was synthesized according to Scheme 7. Scheme 7
[00589] Compound 53: Note: Three batches in parallel. (150 g x 3). Add Compound 52 (150 g, 438 mmol, 1.00 eq) in Ca(OH)2 (0.37 M, 1500 mL, 1.26 eq) at 15°C. The suspension was degassed under vacuum and purged with N2 (15Psi) several times. Stir at 90°C for 12 hrs. Combine with three batches. Filter and collect filter liquor. (Remove excess insoluble impurities). Used to the next. Obtain Compound 53 (900 g, crude) as brown solid.
[00590] Compound 54: Note: Three batches in parallel. (300 g x 3). Only Compound 53 (300 g, 1.36 mol, 1.00 eq, Ca) for purification material. Purification by prep-HPLC (cation exchange resin, H2O). Concentrated in vacuum at 60 °C to give a yellow oil. Combine with three batches. Purify the crude by silica gel chromatography (Dichloromethane /Methanol = 20/1 to 3/1). Concentrated in vacuum at 40 °C to give a yellow oil. Obtain Compound 54 (180 g, 943 mmol, 23.0% yield, 85.0% purity) as yellow oil. 85.0% 1H NMR (ET73917-92-P1A) purity. 1H NMR: DMSO-d6400 MHz 5 5.88 (s, 1H), 5.72 (s, 1H), 5.21 (s, 1H), 5.08-5.00 (m, 3H), 4.54-4.52 (m, 1H), 4.39-4.38 (m, 1H), 3.60-3.56 (m, 4H), 3.45-3.31 (m, 5H), 2.43-2.41 (m, 1H), 2.26-2.22 (m, 1H), 1.97-1.92 (m, 2H).
[00591] Compound 55: Note: Three batches in parallel. (172 g x3). Add Compound 54 (172 g, 1.06 mol, 1.00 eq) in DMP (438 g, 4.21 mol, 516 mL, 3.97 eq) in acetone (2550 mL) at 25°C. Add TSOH.H2O (8.17 g, 42.9 mmol, 4.05e-2 eq) into the mixture at 25°C. Stir at 25°C for Ihr. TLC (Dichloromethane /Methanol = 15/1, (reactant) Rf = 0.20, (product) Rf = 0.60) shows Compound 55 formed. Add 17 mL TEA to the mixture, pH=8. Combine with three batches. Concentrated in vacuum. Purify the crude by silica gel chromatography (SiO2, Dichloromethane /Methanol =30/1 to 10/1). Obtain Compound 55 (310 g, 1.23 mol, 38.5% yield, 80.0 % purity) as yellow oil. About 80.0% 1H NMR purity. 1H NMR: CDCl3400 MHz 5 1.31 - 1.47 (m, 10 H), 2.29 - 2.45 (m, 4 H), 3.29 (br s, 1 H), 3.57 (dd, .7=12.6, 3.36 Hz, 1 H), 3.62 - 3.69 (m, 1 H), 3.82 - 3.95 (m, 2 H), 4.04 - 4.11 (m, 2 H), 4.13 - 4.19 (m, 1 H), 4.30 (d, .7=9.29 Hz, 1 H), 4.41 - 4.50 (m, 1 H), 4.64 - 4.70 (m, 1 H).
[00592] Compound 56: Note: Three batches in parallel. (30.0 g x 3). Add Compound 55 (30.0 g, 148 mmol, 1.00 eq) in THF (300 mL) at 20°C. Dropwise add LAH (2.5 M, 59.3 mL, 1.00 eq) to the mixture at 0-5°C. Stir at 20°C for 2 hrs. TLC (Dichloromethane/Methanol = 15/1, (reactant) Rf = 0.60, (product) Rf = 0.30) shows the reaction complete. Combine with three batches. Add water (36 mL), and 10% NaOH (55.0 mL) and water (110 mL) to the solution at 0-10°C. Filter and collect filter liquor. Concentrated in vacuum. Obtain Compound 56 (105 g, crude) as yellow oil. [00593] Compound 57: Note: Two batches in parallel. (55.0 g x 2). Add slowly a solution of NaIO4 (68.5 g, 320 mmol, 17.7 mL, 1.20 eq) in H2O (385 mL) to a solution of Compound 56 (55.0 g, 266 mmol, 1 eq) in MeOH (660 mL) at 20°C. Stir at 20°C for Ihr. TLC (Dichloromethane/Methanol = 15/1, (reactant) Rf = 0.30, (product) Rf = 0.60) shows the reaction complete. Combine with two batches. Add Sat.NaHCO3 (250 mL) to the mixture. Filter and collect filter liquor. Concentrated in vacuum. Obtain Compound 57 (100 g, crude) as yellow oil.
[00594] Compound 58: Note: Three batches in parallel. (30.0 g x 3). Add Compound 57 (30.0 g, 172 mmol, 1 eq) and DMAP (600 mg, 4.91 mmol, 2.85e-2 eq) in Pyr (300 mL) at 20°C.Dropwise add Ac2O (26.3 g, 258 mmol, 24.2 mL, 1.50 eq) to the mixture at 0°C. Stir at 20°C for 4 hrs. TLC (Petroleum ether/ Ethyl acetate = 5/1, (product) Rf = 0.20) shows the reaction complete. Poured into water (1000 mL), extracted EtOAc (500 mL x 3). Organic layer was separated, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness. The residue was to purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=30/l to 3/1). Obtain Compound 58 (32g) as yellow oil, about 95.0% 1H NMR purity. 1H NMR: CDCI3400 MHz 6.35 (m, 1H), 4.07 (m, 4H), 2.51 (dd, .7=14.07, 5.57 Hz, 1 H), 2.31 (d, .7=3.88 Hz, 1 H), 1.99 - 2.16 (m, 3 H), 1.38 (d, .7=8.00 Hz, 6 H).
[00595] Compound 58B: The suspension of uracil (8.73 g, 77.8 mmol, 1.20 eq), HMDS (560 mL), (NH4)2SO4 (750 mg, 5.68 mmol, 424 μL, 8.75e-2 eq) (catalytic amount) at 20°C. Under a nitrogen atmosphere for 4 h at 135°C. Used to the next. Remove the excess HMDS under high vacuum. Obtain Compound 58B (16.6 g, crude) as white solid.
[00596] Compound 59: Note: Three batches in parallel. (10.0 g x2 +14.0 g). The suspension of Compound 58B (16.6 g, 64.7 mmol, 1.00 eq) in ACN (200 mL) at 20°C. Add a solution of the Compound 58 (14.0 g, 64.7 mmol, 1.00 eq) in dry ACN (200 mL), and add TMSOTf (15.8 g, 71.2 mmol, 12.8 mL, 1.10 eq) at 20°C. Stir resulting reaction mixture for 1 h at 20°C. TLC (Petroleum ether: Ethyl acetate = 1/3, (product) Rf = 0.35, (product) Rf = 0.30) shows the reaction complete.. Residue was diluted with DCM (500 mL) and washed withSat.NaHCO3 solution (300 mLx 2). Organic layer was separated, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness. Purify the crude by silica gel chromatography (Petroleum ether: Ethyl acetate =20/lto 0/1). Concentrated in vacuum at 40 °C to give a yellow oil. Obtain Compound 59 13.0 g as yellow oil, 95. % HNMR purity. Obtain Compound 59 (59.0 g) as yellow oil, about 50.0% TLC purity. 1H NMR: DMSO-d6400 MHz 5 1.74 - 1.98 (m, 1 H), 2.01 - 2.20 (m, 2 H), 2.46 - 2.50 (m, 1 H), 3.15 - 3.27 (m, 3 H), 3.34 - 3.48 (m, 4 H), 3.61 - 3.71 (m, 1 H), 3.82 (d, J=9.05 Hz, 1 H), 3.91 (d, J=9.17 Hz, 1 H), 4.07 (d, .7=8.92 Hz, 1 H), 5.65 (t, .7=7.58 Hz, 2 H), 6.02 (dd, .7=7.82, 2.45 Hz, 1 H), 6.17 (t, .7=7.15 Hz, 1 H), 7.65 (d, .7=8.07 Hz, 1 H), 7.89 (d, .7=8.07 Hz, 1 H). [00597] Compound 60: Note: Three batches in parallel. (40.0 g +10.0 g +13.0 g). Add Compound 59 (40.0 g, 149 mmol, 1.00 eq) in AcOH (960 mL) and H2O (240 mL) at 20°C. Stir at 80°C for 6 hrs. The solvent was concentrated and evaporated with toluene (100 mL) twice. The solvent was concentrated and evaporated with Py. (200 mL) eight times. Obtain Compound 60(crude) (92.0 g) as yellow oil.
[00598] Compound 61: Note: Two batches in parallel. (41.0 g x 2). Add Compound 60 (41.0 g, 179 mmol, 1.0 eq) into Py (200 mL) at 20°C. Add DMTC1 (42.6 g, 125 mmol, 0.70 eq) into the reaction mixture (dissolve DMTrCl in Py (200 mL)) at 0-10°C. Stir at 20°C for 1 hr. Quench the reaction with NaHCO3 (aq, 500mL). Extracted the mixture with EtOAc (1000 mLx 2). Wash with salt water (lOOOmL) and dried with Na2SO4, filtered and the filtrate was evaporated to dryness. Purification by Prep-HPLC (column: Welch Xtimate C18250*100mm#10um; mobile phase: [H2O (10mM NH4HCO3)-ACN]; gradient: 35%-70% B over 20.0 min). Concentrate to dryness in vacuum. Compound 61 (20.0 g, 35.8 mmol, 19.9% yield, 95.0% purity) as yellow oil. About 95.0% HNMR purity. 1H NMR: ET73917-168-P1ADMSO-d6,400 MHz δ 1.96 (d, .7=14.3 Hz, 1 H), 2.06 (s, 2 H), 2.43 - 2.48 (m, 1 H), 2.96 - 3.08 (m, 2 H), 3.73 (s, 6 H), 3.78 (d, 7=9.13 Hz, 1 H), 4.00 (d, 7=9.01 Hz, 1 H), 5.37 (br s, 1 H), 5.63 (d, 7=8.13 Hz, 1 H), 6.05 (dd, 7=7.82, 2.19 Hz, 1 H), 6.90 (d, 7=8.88 Hz, 4 H), 7.20 - 7.34 (m, 7 H), 7.41 (d, 7=7.50 Hz, 2 H), 7.88 (d, 7=8.13 Hz, 1 H), 11.24 (br s, 1 H).
[00599] Compound 62: Note: Five batches in parallel. (5.00 g x 5). A mixture of Compound 61 (5.00 g, 9.42 mmol, 1.00 eq) in dry ACN (25 mL) was cooled to 0-5 °C and degassed and purged with Ar for 3 times, and NaH (1.70 g, 42.4 mmol, 60.0% purity, 4.50 eq) was added at 0 °C, the mixture was stirred at 20 °C for 2 h under Ar atmosphere. A DMF (50.0 mL) solution of (Diethoxyphosphoryl)methyl 4-methylbenzenesulfonate (12.1 g, 37.7 mmol, 9.68 mL, 4.00 eq) (pre-dried by co-evaporation with dry ACN (20.0 mL x 3)) was slowly added in the mixture stirred at 20 °C and the mixture was stirred at this temperature for another 0.5 h. Poured into water (50 mL), extracted with ethyl acetate (50.0 mLx 3). Collect organic layer. Dry the organic layer with Na2SO4 and filter. Purify the crude by silica gel chromatography (SiO2, Petroleum ether/Ethyl acetate=30/l to 0/1). Obtain Compound 62, 27.0 g as yellow oil. About 95.0% TLC purity.
[00600] Compound 63: Note: Two batches in parallel. (10.0 g x 2). Add Compound 62 (10.0 g, 14.6 mmol, 1.00 eq) to DCM (100 mL). Add dodecane- 1 -thiol (4.46 g, 22.0 mmol, 5.28 mL, 1.50 eq) to the mixture. Add TFA (2.51 g, 22.0 mmol, 1.64 mL, 1.50 eq) to the mixture. Stir at 25 °C for 2 hrs. Purify the crude by silica gel chromatography (SiO2, Dichloromethane/ Methanol=30/lto 3/1). Purify by SFC (column: DAICEL CHIRALPAK AD (250mm*50mm, lOum); mobile phase: [CO2-EtOH]; B%:28%, isocratic elution mode). Concentrate in vacuum. Combine with other batch. Compound 63 obtained (5.66 g) as yellow oil. 1H NMR: DMSO-76, 400 MHz 5 1.22 (t, .7=7.07 Hz, 6 H), 1.99 - 2.10 (m, 1 H), 2.37 - 2.46 (m, 1 H), 3.54 - 3.64 (m, 2 H), 3.68 - 3.82 (m, 2 H), 3.84 - 3.94 (m, 1 H), 4.04 (quin, .7=7.32 Hz, 4 H), 4.15 (d, .7=10.01 Hz, 1 H), 5.04 (br s, 1 H), 5.53 (dd, .7=8.07, 2.06 Hz, 1 H), 6.05 (dd, .7=8.00, 2.88 Hz, 1 H), 7.79 (d, .7=8.13 Hz, 1 H), 11.26 (s, 1 H)
[00601] Compound 64: 2-Cyanoethyl tetraisopropylphosphorodiamidite (262.92 mg, 872.29 μmol, 277.05 μL) and IH-Tetrazole (0.45 M, 387.69 μL) were added to a solution of compound 63 (220 mg, 581.53 μmol) in MeCN (2.72 mL). After stirring for 3h, the volatiles were evaporated to dryness, and the residue redissolved in EtOAc and washed with saturated bicarbonate solution, water, and brine. Dry the organic layer with Na2SO4 and filtered. The residue was purified by ISCO automated column using 0-60% (3:1 EtOAc/EtOH) in hexanes as eluant to give compound 64 (294 mg, 87%).
Synthesis of compound 77
[00602] Compound 77 was synthesized according to Scheme 8.
[00603] Compound 66: Note: Three batches in parallel. (250 g x 3). To a mixture of Compound 65 (250 g, 918.27 mmol) and methanol (58.85 g, 1.84 mol) in dichloroethane (1250 mL) was added BF3 Et2O (84.71 g, 596.88 mmol) at 0°C. The mixture was stirred at 20°C for 1 hr. The TLC showed the starting material was consumed and a main point was detected. The crude batches were combined and work-up, purification. The mixture was cooled to 0°C and NaHCO3 solution (1000 mL) was added dropwised. The mixture was separated. The organic phase was washed with water (2 x 750 mL) and brine (1000 mL), dried and concentrated to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=100/l to 4/1) to give Compound 66 (650 g, 81.51% yield, 90% purity) as a colorless oil. 1H NMR: (CHLOROFORM- d, 400 MHz) δ 5.8-5.9 (m, 2H), 5.32 (d, 1H, .7=9.6 Hz), 4.93 (s, 1H), 4.2-4.3 (m, 1H), 4.2-4.2 (m, 1H), 4.08 (ddd, 1H, J=2.3, 5.4, 9.5 Hz), 3.46 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H).
[00604] Compound 67: To a solution of Compound 66 ( 125 g, 511.79 mmol) in methanol (1500 mL) was added Pd/C (25 g, 23.49 mmol, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20°C for 12 hrs. HNMR showed the starting material was consumed. Another three scales were combined. The suspension was filtered through celite and the filter cake was washed with methanol (2 x 500 mL). The combined filtrates were concentrated to dryness to give Compound 67 (410 g, 81.33% yield) as a yellow oil. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 4.6-4.8 (m, 2H), 4.23 (dd, 1H, .7=5.3, 12.0 Hz), 4.08 (dd, 1H, .7=2.0, 12.0 Hz), 3.87 (ddd, 1H, .7=2.0, 5.2, 9.9 Hz), 3.34 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.9-2.0 (m, 1H), 1.7-1.9 (m, 3H).
[00605] Compound 68: Note: Two batches in parallel. (175 g x 2)To a solution of Compound 67 (175 g, 710.64 mmol) and lH-pyrimidine-2, 4-dione (88.42 g, 788.81 mmol) in acetonitrile (2000 mL) was added HMDS (143.37 g, 888.30 mmol) and TMSC1 (101.91 g, 938.05 mmol) in sequence at 20°C under Ar. After the reaction mixture was stirred for 30 min, SnCl4 (259.19 g, 994.90 mmol) was added. The mixture was heated to 45°C and stirred for 16 hr. The LCMS showed the starting material was consumed and a new peak with desired MS was detected. The batches were combined for work-up and purification. The reaction mixture was diluted with ethyl acetate (2500 mL) and washed with water (2 x 2500 mL), NaHCCL (2 x 1000 mL). The aqueous phase was extracted by ethyl acetate (2 x 2500 mL). The combined organic phase was washed with brine (2 x 1000 mL), dried over Na2SO4 and concentrated in vacuum to give the crude. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=100/l to 0/1) to give Compound 67 (151 g, 30.93% yield, 95% purity) as a white solid. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 10.13 (br s, 1H), 7.3-7.4 (m, 1H), 5.6-5.8 (m, 2H), 4.66 (dt, 1H, .7=5.0, 9.8 Hz), 4.1-4.2 (m, 1H), 4.0-4.1 (m, 1H), 3.7-3.9 (m, 1H), 2.28 (br d, 1H, .7=2.4 Hz), 2.0- 2.1 (m, 7H), 1.6-1.7 (m, 2H).
[00606] Compound 69: A mixture of Compound 68 (175 g, 536.31 mmol) in NH3/MeOH (7 M, 3500 mL) was stirred at 20°C for 16 hrs. The LCMS showed the starting material was consumed and a main peak with desired MS was detected. The reaction mixture was concentrated in vacuum to give Compound 69 (130 g, 95.07% yield, 95% purity) as a yellow solid. 1H NMR: (CHLOROFORM-d, 400 MHz) 5 9.67 (br s, 1H), 7.4-7.5 (m, 1H), 5.70 (br s, 2H), 3.86 (br t, 2H, J=4.0 Hz), 3.6-3.8 (m, 2H), 3.5-3.6 (m, 2H), 1.6-1.8 (m, 4H).
[00607] Compound 70: To a solution of Compound 69 (130 g, 536.68 mmol) in dimethylformamide (1250 mL) was added imidazole (91.34 g, 1.34 mol) and TBSC1 (202.23 g, 1.34 mol). The mixture was stirred at 20°C for 20 hrs. The LCMS showed the starting material was consumed and a main peak with desired MS was detected. The reaction mixture was poured into ice water (1500 mL) and stirred at 20°C for 2 hrs. The mixture was filtered and the filter cake was concentrated in vacuum. The crude product was triturated with H2O (2000 mL) at 20°C for 120 min. The mixture was filtered and the filter cake was concentrated in vacuum to give Compound 70 (230 g, 91.04% yield) as a white solid. 1H NMR: (CHLOROFORM-d, 400 MHz) 5 8.76 (br s, 1H), 7.42 (d, 1H, .7=8.1 Hz), 5.6-5.8 (m, 2H), 3.7-3.9 (m, 3H), 3.4-3.4 (m, 1H), 2.1-2.2 (m, 1H), 1.9-2.0 (m, 1H), 1.7-1.8 (m, 1H), 1.5-1.6 (m, 1H), 0.89 (d, 18H, J=5.5 Hz), 0.0-0.1 (m, 12H).
[00608] Compound 71: To a solution of Compound 70 (230 g, 488.58 mmol) in THF (11500 mL) and H2O (11500 mL) was added TFA (278.55 g, 2.44 mol). The mixture was stirred at 20°C for 20 hrs. The LCMS showed the starting material was consumed. The reaction mixture was quenched by saturated NaHCCL solution (1500 mL) at 0°C, and then diluted with ethyl acetate (2000 mL). The combined organic layers were washed with H2O (2 x 1000 mL), brine (2 x 1000 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with petroleum ether (1000 mL) at 20 C for 30 min. The mixture was filtered and the filter cake was concentrated in vacuum to give the Compound 71 (111 g, 63.73% yield) as a white solid. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 9.21 (br s, 1H), 7.41 (d, 1H, .7=8.1 Hz), 5.74 (br d, 2H, J=9.9 Hz), 3.88 (dd, 1H, J=2.3, 11.8 Hz), 3.70 (dd, 1H, .7=5.6, 11.8 Hz), 3.5-3.6 (m, 2H), 2.1-2.2 (m, 2H), 1.99 (br dd, 1H, J=2.3, 12.2 Hz), 1.6-1.8 (m, 2H), 0.89 (s, 9H), 0.09 (s, 6H).
[00609] Compound 72: To a solution of Compound 7 (50 g, 140.26 mmol) in acetonitrile (1250 mL) was added IBX (98.19 g, 350.64 mmol). The mixture was stirred at 85°C for 3 hrs. LCMS showed the starting material was consumed. The reaction mixture was cooled to 20°C and filtered. The filtrate was concentrated in vacuum to give Compound 72 (53 g, 95.94% yield, 90% purity) as a white solid. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 9.74 (s, 1H), 9.15 (br s, 1H), 7.45 (d, 1H, .7=8.1 Hz), 5.7-5.9 (m, 2H), 4.04 (d, 1H, J=9.5 Hz), 3.78 (dt, 1H, J=4.7, 10.0 Hz), 2.2-2.3 (m, 1H), 2.0-2.1 (m, 1H), 1.8-1.9 (m, 1H), 1.6-1.7 (m, 1H), 0.89 (s, 9H), 0.09 (d, 6H, .7=12.3 Hz). [00610] Compound 73 : To a solution of Compound 72 (45 g, 126.95 mmol) in dichloromethane (450 mL) was added 0,0-Diethyl P-[(triphenylphosphoranylidene)methyl]phosphonothioate (65.27 g, 152.34 mmol). The mixture was stirred at 25°C for 12 hrs. The LCMS showed the starting material was consumed. The mixture was concentrated in vacuum to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=100/1 to 2/1) to give Compound 73 (10 g, 14.83% yield, 95% purity) as a yellow solid. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 8.25 (br s, 1H), 7.45 (d, 1H, .7=8.1 Hz), 6.0-6.2 (m, 1H), 5.7-5.8 (m, 2H), 4.1-4.2 (m, 4H), 4.0-4.1 (m, 1H), 3.4-3.5 (m, 1H), 2.1-2.2 (m, 1H), 2.0-2.0 (m, 1H), 1.7- 1.8 (m, 2H), 1.32 (dt, 6H, .7=2.6, 7.1 Hz), 0.9-0.9 (m, 9H), 0.10 (d, 6H, .7=12.1 Hz).
[00611] Compound 74:To a solution of Me3SOI (13.08 g, 59.45 mmol) in dimethyl sulfoxide (200 mL) was added NaH (2.38 g, 59.45 mmol) at 20°C under N2. The mixture was stirred for 30 min until a uniform solution was formed. The solution was then added to another solution of Compound 73 (10 g, 19.82 mmol) in dimethyl sulfoxide (200 mL). Then the reaction was stirred at 25°C. The reaction was held at 25°C for 15.5 hrs until complete conversion of starting material to a more polar intermediate. The LCMS showed the starting material was consumed. The reaction was cooled to room temperature and poured into ice water (500 mL), extracted with ethyl acetate (3 x 250 mL). The combined organic layers were washed with water (3 x 100 mL) and brine (2 x 100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to dryness. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate=100/l to 4/1) to give Compound 74 (6.8 g, 66.16% yield) as a white solid. 1H NMR: (CHLOROFORM- d, 400 MHz) δ 8.80 (br s, 1H), 7.26 (d, 1H, .7=8.1 Hz), 5.60 (dd, 1H, .7=1.9, 8.1 Hz), 5.4-5.5 (m, 1H), 3.9-4.0 (m, 4H), 3.4-3.5 (m, 1H), 2.85 (dd, 1H, J=7.3, 8.6 Hz), 1.8-2.0 (m, 2H), 1.5-1.6 (m, 1H), 1.4-1.5 (m, 2H), 1.17 (t, 3H, .7=7.1 Hz), 1.10 (t, 3H, .7=7.1 Hz), 1.0-1.1 (m, 2H), 0.8-0.9 (m, 1H), 0.76 (s, 9H), -0.01 (d, 6H, .7=10.3 Hz).
[00612] Compound 75: To a solution of Compound 74 (6.8 g, 13.11 mmol) in tetrahydrofuran (70 mL) was added HCl (11.5 mL) at 0°C. The solution was allowed to warm to 20°C and stirred for 1 hr. The LCMS showed the starting material was consumed. The solution was concentrated to dryness under vacuum at 20°C and then subjected to co-evaporation with acetonitrile (2 x 100 mL) to give Compound 75 (5.2 g, 93.17% yield, 95% purity) as a white solid. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 8.9-9.1 (m, 1H), 7.3-7.5 (m, 1H), 5.5-5.9 (m, 2H), 4.10 (br d, 4H, .7=1.8 Hz), 3.58 (br s, 1H), 3.24 (br s, 1H), 2.0-2.3 (m, 3H), 2.02 (br s, 1H), 1.67 (br d, 3H, J=l.l Hz), 1.30 (br s, 6H), 1.19 (br s, 2H).
[00613] Compound 76: To a solution of Compound 75 (5 g, 12.36 mmol) in tetrahydrofuran (50 mL) and H2O (50 mL) was added Oxone (6.24 g, 37.09 mmol) at 20°C. The mixture was stirred at 20°C for 12 hrs. LCMS showed the reaction was completed. The mixture was extracted with ethyl acetate (7 x 100 mL). The combined organic phase was dried over Na2SO4, filtered and filtrate was concentrated to give crude product. The residue was purified by column chromatography (SiO2, ethyl acetate/MeOH= 100/1 to 10/1) to give Compound 76 (3.5 g, 72.90% yield) as white solid. 1H NMR: (CHLOROFORM-d, 400 MHz) δ 9.09 (br s, 1H), 7.33 (d, 1H, .7=8.1 Hz), 5.74 (d, 1H, .7=8.1 Hz), 5.6-5.7 (m, 1H), 4.0-4.2 (m, 4H), 3.5-3.6 (m, 1H), 3.15 (dd, 1H, J=5.9, 9.0 Hz), 2.21 (td, 1H, .7=4.0, 8.4 Hz), 1.9-2.1 (m, 1H), 1.5-1.8 (m, 3H), 1.3-1.4 (m, 6H), 1.1- 1.2 (m, 1H), 1.0-1.1 (m, 1H), 0.8-0.9 (m, 1H).
[00614] Compound 77: To a solution of Compound 76 (3.5 g, 9.01 mmol) and 3- bis(diisopropylamino) phosphanyloxypropanenitrile (5.43 g, 18.02 mmol) in dichloromethane (50 mL) was added lH-imidazole-4,5-dicarbonitrile (851.47 mg, 7.21 mmol) in portions at 25°C. The mixture was stirred at 25 °C for 16 hr. LCMS showed the starting material was consumed and a main peak with desired mass was detected. The reaction mixture was purified directly by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 50-100% Ethyl acetate/Petroleum ethergradient @ 50 mL/min) to give a crude product. The crude product was triturated with MTBE/Hexane (50 mL= 1 : 1) to give compound 77 (3.1 g, 56.45% yield, 96.6% purity) as white solid. 1H NMR: (DMSO-d6, 400 MHz) δ 11.36 (s, 1H), 7.7-7.8 (m, 1H), 5.62 (d, 1H, .7=8.1 Hz), 5.56 (br d, 1H, J=10.4 Hz), 3.9-4.0 (m, 4H), 3.5-3.8 (m, 5H), 3.0-3.1 (m, 1H), 2.7 - 2.9 (m, 2H), 2.1-2.3 (m, 1H), 1.5-2.0 (m, 3H), 1.3-1.5 (m, 1H), 1.1-1.2 (m, 18H), 0.8-1.0 (m, 3H). LCMS: m/z 649.2 (M+H) +, RT: 2.606 min. LC/MS (The gradient was 5%B in 0.40min and 5-95% B in 2.60 min, hold on 95% B in l.OOmin, and then 95-5%B in O.Olmin, the flow rate was 1.0 mL/min. Mobile phase A was 0.04% Trifluoroacetic Acid in water, mobile phase B was 0.02% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Kinetex Cl 8 2.1*50mm,5um. Detection methods are diode array (DAD), and evaporative light scattering detection (ELSD). MS mode was positive electrospray ionization.MS range was 100-1000.
Synthesis of compound 83
[00615] Compound 83 was synthesized according to Scheme 9.
[00616] Compound 79: A mixture of compound 78 ,1H-pyrimidine-2, 4-dione (11.5 g, 102 mmol, 1.10 eq) (co-evaporated with ACN (20.0 mL x 3) was added BSA (41.7 g, 205 mmol, 50.6 mL, 2.20 eq) in ACN (250 mL) degassed and purged with N2 for 3 times, the mixtrue was stirred at 60 °C for 0.5 h. The mixture cooled to 0 °C was added TMSOTf (33.1 g, 149 mmol, 26.9 mL, 1.60 eq) degassed and purged with N2 for 3 times, and then the mixture was stirred at 60 °C for 2 h under N2 atmosphere. The mixture was poured into aq.sat.NaHCO3 (500.0 mL), extracted with ethyl acetate (500.0 mL, 300.0 mL, 200.0 mL). The combined organic layers were washed with brine 400.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Compound 79 (54.0 g, crude) obtained as a yellow foam was used to next step without purification.
[00617] Compound 80: To a solution of compound 79 (54.0 g, 97.0 mmol, 1.00 eq) in THF (540 mL) was added TBAF (1.00 M, 97.0 mL, 1.00 eq) at 0 °C. The mixture was stirred at 0 -5 °C for 2 h. The reaction mixture was quenched by addition H2O 500.0 mL at 15 °C, extracted with ethyl acetate (500.0 mL x 3). The combined organic layers were washed with brine (500.0 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue dissolved in solvent (ethyl acetate/petroleum ether = 1/2) was stirred for 0.5 h, filtered, and collected the filter-cake. After dried under vacuum, compound 80 (16.5 g, 50.6 mmol, 52.2% yield, 97.7% purity) was obtained as a light yellow solid. 1H NMR: (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.02-8.00 (m, 2H), 7.79-7.77 (d, J = 8.00 Hz, 1H), 7.73-7.68 (m, 1H), 7.57-7.53 (m, 2H), 5.79- 5.93 (m, 2H), 5.70-5.68 (d, J = 8.00 Hz, 1H), 5.28 (s, 1H), 4.37 (s, 1H), 4.19-4.11 (m, 2H). [00618] Compound 81: A mixture of compound 80 (10.0 g, 31.4 mmol, 1.00 eq) in dry ACN (40.0 mL) was cooled to 0-5 °C and degassed and purged with Ar for 3 times, and NaH (2.51 g, 62.8 mmol, 60.0% purity, 2.00 eq) was added at 0 °C, the mixture was stirred at 18 °C for 4 h under Ar atmosphere. A DMF (20.0 mL) solution of diethoxyphosphorylmethyl 4- methylbenzenesulfonate (20.2 g, 62.8 mmol, 16.1 mL, 2.00 eq) (pre-dried by co-evaporation with dry ACN (20.0 mL x 3)) was slowly added in the mixture stirred at 18 °C and the mixture was stirred at this temperature for another 4 h. The reaction mixture was quenched by addition aq. saturated NH4Cl (300 mL) at 0 °C and extracted with ethyl acetate (200.0 mL x 3). The combined organic layers were washed with brine (300.0 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Compound 4 (24.0 g, crude) was obtained as a yellow oil which was used in next step without purification.
[00619] Compound 82:To a solution of compound 81 (9.60 g, 20.5 mmol, 1.00 eq) in ACN (21.0 mL) was added LiOH.H2O (774 mg, 18.4 mmol, 0.900 eq) in MeOH (21.0 mL) and H2O (8.00 mL), the mixture was stirred at 18 °C for 24 h. The reaction mixture was quenched by addition formic acid ~0.4 mL (pH 7-8) at 0 °C, filtered and concentrated under reduced pressure to give a residue, The residue was purified by prep-HPLC ( neutral condition, column: Welch Xtimate C18 250*100mm#10um; mobile phase: [H2O(10 mM NH4HCO3)-ACN]; gradient:5%-30% B over 20.0 min). Compound 82 (6.00 g, 15.5 mmol, 75.5% yield, 94.0% purity) was obtained as a white solid. 1H NMR: (400 MHz, DMSO-d6) δ 7.63-7.61 (d, J = 8.40 Hz, 1H), 5.77 (s, 1H), 5.61-5.59 (d, J = 8.00 Hz, 1H), 4.20-3.93 (m, 10H), 1.24-1.21 (t, J = 14.0 Hz, 6H). 31PNMR: ET73037-40- P1 Al (400 MHz, DMSO-d6) δ 20.4
[00620] Compound 83: To a solution of compound 82(4.50 g, 12.3 mmol, 1.00 eq) (pre-dried by co-evaporation with dry ACN (20.0 mL x 3)) in dichloromethane (45.0 mL) was added 3- bis(diisopropylamino)phosphanyloxypropanenitrile (7.45 g, 24.7 mmol, 7.85 mL, 2.00 eq), then lH-imidazole-4,5-dicarbonitrile (2.63 g, 22.2 mmol, 1.80 eq) at 0 °C. The mixture was degassed and purged with N2 for 3 times. The mixture was stirred at 18 °C for 2 h. The reaction mixture was diluted with dichloromethane (20.0 mL). The organic layers were washed with aq.sat.NaHCO3 (30.0 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 5/1 to 0/1, contain 0.5% TEA). Compound 83 (4.00 g, 7.09 mmol, 57.4% yield, 99.3% purity) was obtained as a colorless oil. 1H NMR: (400 MHz, CDCl3) δ 9.82 (s, 1H), 7.58-7.53 (dd, J = 8.00 Hz, 12.4 Hz, 1H), 5.77-5.86 (d, J = 4.80 Hz, 1H), 5.68-5.64 (t, J = 8.00 Hz, 1H), 4.47-4.42 (m, 1H), 4.35-4.32 (m, 1H), 4.22-4.10 (m, 7H), 4.05-3.97 (m, 1H) 3.86-3.60 (m, 2H), 3.55-3.45 (m, 2H), 2.64-2.58 (m, 2H), 1.33-1.29 (m, 6H), 1.14-1.11 (m, 9H), 1.08-1.06 (d, J = 6.80 Hz, 1H). 31PNMR: (400 MHz, CDCl3) δ 150.08,149.89; 20.00,19.92. Synthesis of compound 88
[00621] Compound 88 was synthesized according to Scheme 10.
[00622] Compound 85: To a solution of compound 84 (7 g, 18.8 mmol, 1 eq) in ACN (140 mL) was added IBX (6.31 g, 22.6 mmol, 1.2 eq). The mixture was stirred at 80 °C for Ihr. The reaction mixture was filtered and concentrated under reduced pressure to give compound 85 (7 g, crude) was obtained as white solid.
[00623] Compound 86: To a solution of compound 85a (10.2 g, 26.5 mmol, 1.4 eq) in THF (70 mL) was added NaH (907 mg, 22.7 mmol, 60% purity, 1.2 eq) at -10~0°C. The mixture was stirred at -10~0 °C for 0.5 h. A solution of compound 85 (7 g, 18.9 mmol, 1 eq) in THF (70 mL) was added dropwise to above mixture at -10~0 °C and stirred for 2 h. The reaction mixture was quenched by sat NH4Cl aq. (500 mL), and extracted with EA (150 mL * 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10/l to 1/1). Compound 86 (4.9 g, 9.71 mmol, 51% yield) was obtained as a white foam. 1H NMR (400 MHz, CDCl3) 9.55 (s, 1H), 7.27-7.23 (m, 1H), 6.98-6.93 (m, 1H), 6.57-6.53 (d, J=16 Hz, 1H), 5.82-5.78(m, 2H), 4.58 (s, 1H), 4.10-3.86 (m, 4H), 3.53 (s, 3H), 2.04-2.00(m, 1H), 0.98-0.92 (m,15H), 0.12 (s, 6H).
[00624] Compound 87: To a solution of compound 86 (4.9 g, 9.71 mmol, 1 eq) in HCOOH (50 mL) was added H2O (50 mL). The mixture was stirred at 20 °C for 24hr. The reaction mixture was quenched sat Na2CO3 aq. (500 mL) and extracted with solvent EA (100 mL * 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10/l to 1/1). Compound 6(3.2 g, 8.20 mmol, 84% yield) was obtained as a white foam. 1H NMR: (400 MHz, CDCl3) 9.80 (s, 1H), 7.29- 7.27 (d, J=8 Hz, 1H), 7.11-7.06 (dd, J=4 Hz, 16Hz, 1H), 6.61-6.57 (dd, J=4 Hz, 16Hz, 1H), 5.90(s, 1H), 5.85-5.83 (d, J=8 Hz, 1H), 4.55-4.54 (m, 1H), 4.14-3.97 (m, 1H), 3.96-3.93(m, 3H), 3.64 (s,3H), 2.08-2.01 (m, 1H), 1.00-0.98 (d, J=8 Hz, 6H).
[00625] Compound 88: To a solution of compound 87 (1.5 g, 3.84 mmol, 1 eq) in DCM (20 mL) was added 3-bis(diisopropylamino)phosphanyloxypropanenitrile (1.62 g, 5.38 mmol, 1.71 mL, 1.4 eq) and DCI (545 mg, 4.61 mmol, 1.2 eq). The mixture was stirred at 15 °C for 2h. The reaction mixture was diluted with DCM (30 mL), was washed with sat NaHCO3 aq(30 mL*3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Kromasil Eternity XT 250*80mm*10um;mobile phase: [water( NH4HCO3)-ACN];gradient:40%-70% B over 30 min ). Target 1 (1.4 g, 2.37 mmol, 62% yield) was obtained as a light yellow foam. 1H NMR (400 MHz, DMSO-d6) 11.44 (s, 1H), 7.77-7.73 (m, 1H), 7.06-6.88 (m, 2H), 5.80-5.79 (m, 1H), 5.67-5.65(d, J=8 Hz, lH),4.66-4.58(m, 2H), 4.22-4.12 (m, 1H), 3.92-3.89 (m, 2H),3.80- 3.60(m, 4H), 3.42-3.38 (m,3H), 2.82-2.79 (m, 2H), 1.95-1.94 (m,lH), 1.73-1.42(m, 12H), 0.91- 0.89(m, 6H). 3 IP NMR (162 MHz, DMSO-d6) 149.51, 149.20. MS (ESI+APCI) calculated for C24H40N4O9PS [M+H]+ m/z = 591.22, found 591.1.
Synthesis of compound 91
[00626] Compound 91 was synthesized according to Scheme 11. [00627] Compound 89: Sodium hydride (oil dispersion) 60% dispersion in mineral oil (1.59 g, 41.48 mmol) was added to a cooled (-20 C) solution of compound 84 (5.15 g, 13.83 mmol) and di(ethoxy)phosphorylmethyl 4-methylbenzenesulfonate (5.35 g, 16.59 mmol, 4.28 mL) in THF (125 mL). The resulting mixture was allowed to reach r.t. and stirred for 20h. The reaction mixture was quenched with saturated aqueous NH4Cl and diluted with EtOAc (200 mL). The organic layer was washed with water and brine. Silica-gel was added to the organic layer and the solvent removed under reduced pressure. The crude residue was purified by column chromatography on silica gel (50-100% EtOAc in hexanes) to obtain compound 89 (3.5 g, 48%). 1H NMR (600 MHz, DMSO) δ 11.37 (s, 1H), 7.86 (d, J = 8.1 Hz, 1H), 5.82 (d, J = 4.4 Hz, 1H), 5.62 (d, J = 8.1 Hz, 1H), 4.26 (t, J = 4.9 Hz, 1H), 4.09 - 4.03 (m, 4H), 3.95 (dq, J = 6.0, 2.8 Hz, 1H), 3.93 - 3.88 (m, 2H), 3.82 (t, J = 4.6 Hz, 1H), 3.79 (dd, J = 10.9, 2.9 Hz, 1H), 3.66 (dd, J = 10.9, 3.0 Hz, 1H), 3.35 (s, 3H), 1.24 (td, J = 7.1, 1.3 Hz, 6H), 0.88 (s, 9H), 0.09 (d, J = 1.4 Hz, 6H). MS (ESI+APCI) calculated for C10H18NO4 [M-H]- m/z = 216.12, found 216.0.
[00628] Compound 90: Compound 89 (3.5 g, 6.70 mmol) was dissolved in a 1:1 mixture of formic acid/water (50 mL) and heated to 60 °C for 3 h. The volatiles were evaporated to dryness and the residue purified by column chromatography on silica gel (0-8% MeOH in DCM) to obtain compound 3 (1.98 g, 72%). 1H NMR (600 MHz, DMSO) δ 11.38 - 11.35 (m, 1H), 7.85 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 4.9 Hz, 1H), 5.62 (dd, J = 8.1, 2.2 Hz, 1H), 5.30 (d, J = 5.9 Hz, 1H), 4.13 - 4.03 (m, 6H), 3.97 (q, J = 3.3 Hz, 1H), 3.91 (d, J = 8.5 Hz, 2H), 3.80 - 3.76 (m, 2H), 3.68 (dd, J = 10.9, 3.3 Hz, 1H), 3.36 (s, 3H), 1.27 - 1.23 (m, 6H). 3 IP NMR (243 MHz, DMSO) δ 20.80.
[00629] Compound 91: Compound 90 (1.1 g, 2.69 mmol) was dissolved in EtOAc (14 mL) and cooled in an ice bath. To the previous solution, diisopropylethylamine (1.04 g, 8.08 mmol, 1.41 mL), 1 -Methylimidazole (221.16 mg, 2.69 mmol, 214.51 μL) , and 2-Cyanoethyl N,N- diisopropylchlorophosphoramidite (956.37 mg, 4.04 mmol, 902.23 μL) were added sequentially. After stirring for 30 min, the mixture was diluted with more EtOAc and washed with sat bicarb (x2), and brine. The combined organic phase was dried over Na2SO4, filtered and filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (50-100% EtOAc in hexanes) to obtain compound 91 (1.63 g, 98%). 31P NMR (243 MHz, DMSO) 8 149.60, 148.74, 20.74, 20.61. MS (ESI+APCI) calculated for C25H45N4NaO11P2 [M+Na]+ m/z = 631.227, found 631.2.
Synthesis of compound 96
[00630] Compound 96 was synthesized according to Scheme 12.
[00631] Compound 93: 4,4-dimethoxytrityl chloride (1.04 g, 3.07 mmol) and triethylamine (620.35 mg, 6.13 mmol, 854.48 μL) were added to a stirred solution of alpha-deoxy thymidine (495 mg, 2.04 mmol) in pyridine (10 mL). After stirring for 5h, the volatiles were evaporated to dryness, the residue dissolved in EtOAc, washed with aqueous saturated sodium bicarbonate (x2), and brine. The organic phase was dried over Na2SO4, filtered and filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using 0-80% EtOAc in hexanes as eluent to give compound 93 (540 mg, 48%). 1H NMR (600 MHz, DMSO) δ 11.28 (s, 1H), 7.76 (d, J = 1.4 Hz, 1H), 7.38 (dd, J = 8.4, 1.4 Hz, 2H), 7.32 (t, J = 7.8 Hz, 2H), 7.28 - 7.21 (m, 5H), 6.92 - 6.88 (m, 4H), 6.21 (dd, J = 7.6, 3.6 Hz, 1H), 5.39 (d, J = 3.4 Hz, 1H), 4.29 (td, J = 4.5, 2.3 Hz, 1H), 4.19 (dq, J = 6.0, 2.9 Hz, 1H), 3.74 (s, 6H), 3.32 (s, 2H), 3.06 (dd, J = 10.2, 4.0 Hz, 1H), 2.96 (dd, J = 10.2, 4.9 Hz, 1H), 2.62 - 2.56 (m, 1H), 1.94 (dt, J = 14.1, 3.2 Hz, 1H), 1.79 (d, J = 1.2 Hz, 3H).
[00632] Compound 94: Sodium hydride (in oil dispersion) 60% dispersion in mineral oil (113.98 mg, 2.97 mmol, 60% purity) was added to a -20 °C solution of nucleoside (540 mg, 991.56 μmol) and di(ethoxy)phosphorylmethyl 4-methylbenzenesulfonate (383.51 mg, 1.19 mmol, 306.81 μL) in THF (10 mL). The resulting mixture was allowed to reach r.t. and stirred for 20h. The rxn mixture was quenched with saturated aqueous NH4Cl and diluted with EtOAc (100 mL). The organic layer was washed with water and brine. The organic phase was dried over Na2SO4, filtered and filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using 20-100%EtOAc in hexanes as eluent to give compound 94 (379 mg, 55%). 1H NMR (600 MHz, DMSO) δ 11.28 (s, 1H), 7.57 (d, J = 1.3 Hz, 1H), 7.40 - 7.36 (m, 2H), 7.33 (dd, J = 8.4, 6.8 Hz, 2H), 7.27 - 7.21 (m, 5H), 6.93 - 6.89 (m, 4H), 6.28 (dd, J = 8.1, 2.7 Hz, 1H), 4.50 (t, J = 4.6 Hz, 1H), 4.12 (d, J = 6.1 Hz, 1H), 4.05 - 3.97 (m, 4H), 3.85 (qd, J = 13.8, 9.5 Hz, 2H), 3.74 (s, 6H), 3.32 (s, 27H), 3.11 - 3.00 (m, 2H), 2.67 (dt, J = 14.7, 7.2 Hz, 1H), 2.11 (d, J = 15.0 Hz, 1H), 1.80 (d, J = 1.2 Hz, 3H), 1.19 (td, J = 7.1, 2.4 Hz, 6H). 31P NMR (243 MHz, DMSO) δ 20.98.
[00633] Compound 95: p-Toluenesulfonic acid (110.78 mg, 709.22 μmol) was added to a solution of nucleoside (379 mg, 545.55 μmol) in MeOH/DCM (25 mL). After 10 min, solid NaHCO3 was added until the orange color of the solution disappeared. The resulting mixture was filtered, and the filtrate was combined with silica gel, and the volatiles were evaporated. The residue was purified by ISCO automated column using 0-6% MeOH in DCM as eluant to give compound 95 (144 mg 67%). 1H NMR (600 MHz, DMSO) δ 11.24 (s, 1H), 7.57 (q, J= 1.2 Hz, 1H), 6.19 (dd, J= 8.0, 2.3 Hz, 1H), 4.95 (t, J= 5.5 Hz, 1H), 4.40 - 4.37 (m, 1H), 4.13 (dt, J= 6.4, 1.1 Hz, 1H), 4.03 (dqd, J= 8.3, 7.1, 2.6 Hz, 4H), 3.92 - 3.82 (m, 2H), 3.45 - 3.36 (m, 2H), 3.32 (s, 7H), 2.62 (ddd, J= 14.6, 8.1, 6.3 Hz, 1H), 2.10 - 2.02 (m, 1H), 1.80 (d, J= 1.2 Hz, 3H), 1.22 (td, J= 7.1, 1.7 Hz, 6H). 31PNMR (243 MHz, DMSO) δ 20.51.
[00634] Compound 96: Diisopropylethylamine (142.30 mg, 1.10 mmol, 191.78 μL) , 1- Methylimidazole (30.13 mg, 367.03 μmol, 29.23 μL) , and 2-Cyanoethyl N,N- diisopropylchlorophosphoramidite (130.30 mg, 550.54 μmol, 122.93 μL) were added to a solution of compound 95 (144 mg, 367.03 μmol) in EtOAc (3mL). After 1 h, the mixture was diluted with EtOAC, and washed with sat bicarb and brine. The organic phase was dried over Na2SO4, filtered and filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using 20-100%EtOAc in hexanes as eluent to give compound 96 (163 mg, 74%). 1H NMR (600 MHz, DMSO) δ 11.29 - 11.21 (m, 1H), 7.61 - 7.50 (m, 1H), 6.25 - 6.18 (m, 1H), 4.60 - 4.51 (m, 1H), 4.17 (dd, J = 12.9, 6.2 Hz, 1H), 4.07 - 3.99 (m, 4H), 3.94 - 3.84 (m, 2H), 3.81 - 3.70 (m, 2H), 3.69 - 3.54 (m, 4H), 2.82 - 2.77 (m, 2H), 2.69 - 2.60 (m, 2H), 2.11 - 2.06 (m, 1H), 1.79 (s, 3H), 1.28 - 1.08 (m, 20H). 31PNMR (243 MHz, DMSO) δ 147.19, 147.11, 20.37. MS (ESI+APCI) calculated for C24H42N4NaO9P2 [M+Na]+ m/z = 615.23, found 615.2.
Synthesis of compound 100
[00635] Compound 100 was synthesiszed according to Scheme 13.
[00636] Compound 97: Compound 84 was dissolved in MeCN (150 mL) followed by addition of water (150 mL). To the previous solution, (Diacetoxyiodo)benzene (8.65 g, 26.85 mmol) and TEMPO (419.48 mg, 2.68 mmol) were added sequentially, and the resulting mixture was stirred at r.t overnight (open flask)). The solvent was removed under reduced pressure and co-evaporated with toluene (x2). The residue was partially purified by trituration with 150 ml EtiO and filtered through a fritted glass funnel to give compound 97 (3.49 g, 67%) as a white solid. The product was used in the next step without further purification.
[00637] Compound 98: Compound 97 (1.26 g, 3.26 mmol) in DCM (20ml) was added HBTU (1.85 g, 4.89 mmol) and Diisopropylethylamine (1.26 g, 9.78 mmol, 1.70 mL), after stirring for 5 min to activate the acid, Diethyl P-(aminomethyl)phosphonate (708.40 mg, 4.24 mmol, 644.00 μL) was added. After stirring overnight, the mixture was diluted with DCM and washed with saturated aqueous sodium bicarbonate and brine. The organic phase was dried over Na2SO4, filtered and filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using 0-8% MeOH in DCM as eluent to give compound 98 (1.73 g, 97%). 1H NMR (600 MHz, DMSO) δ 11.42 (d, J = 2.2 Hz, 1H), 8.91 (t, J = 6.1 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 5.95 (d, J = 6.0 Hz, 1H), 5.72 (dd, J = 8.1, 2.2 Hz, 1H), 4.40 (d, J = 3.0 Hz, 1H), 4.34 (dd, J = 4.4, 3.0 Hz, 1H), 4.06 - 3.99 (m, 5H), 3.63 (dd, J = 11.8, 6.1 Hz, 2H), 1.29 - 1.20 (m, 9H), 0.89 (s, 9H), 0.10 (d, J = 6.1 Hz, 6H). 31P NMR (243 MHz, DMSO) δ 22.26.
[00638] Compound 99: Compound 98 (1.73 g, 3.23 mmol) was dissolved in a 1:1 mixture of formic acid/Water (40 mL) and heated to 60°C for 2h. The volatiles were evaporated to dryness and the residue was purified using 0-10% MeOH in DCM to give compound 99 (1.07 g, 78%). 1H NMR (600 MHz, DMSO) δ 11.40 (d, J = 2.2 Hz, 1H), 8.83 (t, J = 6.1 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H), 6.02 (d, J = 6.5 Hz, 1H), 5.75 (s, 1H), 5.72 (dd, J = 8.1, 2.2 Hz, 1H), 5.68 (d, J = 5.6 Hz, 1H), 4.43 (d, J = 2.5 Hz, 1H), 4.19 (ddd, J = 5.5, 4.4, 2.6 Hz, 1H), 4.06 - 3.99 (m, 4H), 3.93 (dd, J = 6.5, 4.4 Hz, 1H), 3.70 - 3.57 (m, 2H), 3.32 (s, 4H), 1.23 (td, J = 7.0, 1.2 Hz, 6H). 31P NMR (243 MHz, DMSO) δ 22.36.
[00639] Compound 100: Diisopropylethylamine (816.23 mg, 6.32 mmol, 1.10 mL), 1- Methylimidazole (172.84 mg, 2.11 mmol, 167.64 μL), and 2-Cyanoethyl N,N- diisopropylchlorophosphoramidite (747.39 mg, 3.16 mmol, 705.08 μL) were added to a solution of compound 99 (887 mg, 2.11 mmol) in EtOAc (36mL) and MeCN (3 mL). The initial cloudy mixture turned into a clear solution after 30 min. The solution was then diluted with EtOAc, and washed with aqueous sodium bicarbonate (x2) and brine. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using 20-100% EtOAc in hexanes as eluent to give compound 100 (1.13g, 85%). 1H NMR (600 MHz, DMSO) δ 11.48 - 11.40 (m, 1H), 8.93 - 8.86 (m, 1H), 8.17 - 8.08 (m, 1H), 6.02 - 5.94 (m, 1H), 5.75 - 5.68 (m, 1H), 4.58 - 4.50 (m, 1H), 4.47 - 4.40 (m, 1H), 4.12 (ddd, J= 17.9, 6.0, 4.4 Hz, 1H), 4.06 - 3.98 (m, 3H), 3.85 - 3.55 (m, 5H), 3.38 (s, 1H), 3.33 (s, 1H), 2.83 - 2.79 (m, 1H), 1.26 - 1.10 (m, 12H). 31PNMR (243 MHz, DMSO) δ 150.21, 148.91, 21.69, 21.65. MS (ESI+APCI) calculated for C24H41N5NaO10P2 [M+Na]+ m/z = 644.22, found 644.2.
Synthesis of compound 103
[00640] Compound 103 was synthesized according to Scheme 14.
[00641] Compound 102: To a clear solution of compound 101 (1.1 g, 1.09 mmol) in THF (20 mL) was added TBAF (371.20 mg, 1.42 mmol) and the reaction mixture was kept at stirring at 22 °C for 4 hr. All the volatile matters were removed under vacuum pump and the residue thus obtained was purified by flash column chromatography (gradient: 0-3% MeOH in DCM) to afford compound 102 (0.79 g, 81% yield) as white foam. 1H NMR (600 MHz, DMSO-d6) δ 11.43 (d, J = 2.1 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.34 - 7.14 (m, 18H), 6.87 - 6.80 (m, 7H), 5.80 (d, J = 7.0 Hz, 1H), 5.44 (dd, J = 8.1, 2.0 Hz, 1H), 5.27 (d, J = 5.6 Hz, 1H), 4.21 (t, J = 5.3 Hz, 1H), 3.76 - 3.70 (m, 12H), 3.65 - 3.57 (m, 1H), 3.53 (d, J = 9.5 Hz, 1H), 3.40 (d, J = 10.2 Hz, 1H), 3.18 (s, 3H), 3.17 -3.11 (m, 2H)ppm. 13CNMR(151 MHz, DMSO-d6) δ 163.28, 158.59, 158.54, 158.40, 151.03, 145.50, 145.09, 140.69, 136.16, 135.81, 135.72, 135.35, 130.36, 130.30, 130.23, 128.35,
128.19. 128.14. 127.26. 126.95. 113.67. 113.65. 113.54. 113.49. 102.76. 86.75. 86.56, 85.56, 85.17, 81.76, 69.43, 67.49, 63.78, 63.12, 57.85, 55.51, 55.48, 55.45, 55.41 ppm.
[00642] Compound 103: To a clear solution of compound 102 (0.78 g, 873.48 μmol) in DCM (15.03 mL) at 22 °C was added N-Methyl imidazole (107.57 mg, 1.31 mmol, 104.33 μL) and Diisopropylethylamine (564.44 mg, 4.37 mmol, 760.70 μL). The reaction mixture was stirred for 5 minutes at 22 °C and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (413.47 mg, 1.75 mmol, 390.06 μL) was added slowly into it. Reaction was kept for stirring at 22 °C and TLC was checked after 0.5 hr. Reaction mixture was diluted with dichloromethane (20 mL) and washed with 10% NaHCO3 solution (2x20 mL). Organic layer separated, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness. The crude mass obtained was purified by flash column chromatography (gradient: 40-60% EtOAc in hexane) to afford compound 103 (0.74 g, 78% yield) as white foam. 1H NMR (600 MHz, CD3CN) δ 9.04 (s, 1H), 7.76 - 7.55 (m, 1H), 7.47 - 7.40 (m, 2H), 7.32 - 7.08 (m, 11H), 6.91 - 6.75 (m, 8H), 5.83 (dd, J = 6.0, 4.5 Hz, 1H), 5.31 - 5.08 (m, 1H), 4.67 - 4.47 (m, 1H), 3.80 - 3.74 (m, 10H), 3.73 - 3.37 (m, 6H), 3.36 - 3.26 (m, 3H), 3.14 - 2.95 (m, 1H), 2.61 - 2.55 (m, 1H), 2.36 - 2.27 (m, 1H), 1.10 (dd, J = 15.9, 6.8 Hz, 6H), 0.99 - 0.77 (m, 6H) ppm. 13C NMR (151 MHz, CD3CN) δ 171.65, 163.82, 163.71, 159.74, 159.68, 159.51, 159.45, 151.34, 151.24, 146.09, 146.00, 145.84, 145.80, 141.35, 141.06, 137.23, 137.17, 136.58,
136.56, 136.48, 136.40, 136.17, 136.11, 131.37, 131.28, 131.26, 131.17, 131.03, 130.92, 130.80, 130.70, 129.11, 129.05, 128.98, 128.95, 128.93, 128.90, 128.83, 128.78, 128.76, 128.73, 127.97, 127.96, 127.64, 119.59, 119.17, 114.14, 114.10, 114.00, 113.94, 113.90, 102.91, 102.59, 88.50,
88.13, 88.08, 88.06, 87.81, 86.99, 86.46, 86.39, 84.46, 82.87, 82.85, 72.20, 72.13, 71.93, 71.86,
65.13, 64.43, 63.88, 63.49, 60.94, 59.55, 59.54, 59.31, 59.18, 59.06, 59.02, 58.92, 55.91, 55.89,
55.85, 55.81, 55.32, 43.91, 43.83, 43.76, 25.17, 25.12, 24.76, 24.71, 24.65, 21.13, 21.03, 20.99,
20.89, 20.84 ppm. 3 IP NMR (243 MHz, CD3CN) δ 150.08, 149.40 ppm.
Synthesis of compound 105
[00643] Compound 105 was synthesized according to Scheme 15.
[00644] Compound 105: To a clear solution of compound 104 (10 g) in MeCN (20V) was added DBU (2 eq.) and the resulting solution was stirred at 20 °C for 4h. The reaction was diluted with DCM (10V) and 5% aqueous sodium bicarbonate (7V). The organic layer was separated, washed with brine (x2) and evaporated to dryness. The residue was purified by Prep-HPLC to give compound 105 (3g). 1H NMR (300 MHz, DMSO) δ 7.62 (dd, J = 8.1, 2.4 Hz, 1H), 6.13 (dd, J = 5.8, 3.4 Hz, 1H), 5.71 (d, J = 8.0 Hz, 1H), 5.66 - 5.51 (m, 5H), 4.87 (dd, J = 9.0, 5.3 Hz, 1H), 4.61 (dq, J = 27.7, 7.6 Hz, 1H), 4.29 - 4.17 (m, 1H), 3.86 - 3.51 (m, 5H), 3.36 (d, J = 12.7 Hz, 5H), 2.87 - 2.57 (m, 5H), 2.50 (p, J = 1.9 Hz, 2H), 1.26 - 1.04 (m, 36H). 31P NMR (121 MHz, DMSO) δ 149.83, 149.70, 27.42.
Synthesis of compound 109
[00645] Compound 109 was synthesized according to Scheme 16. [00646] Compound 106: To a suspension of methyltriphenylphosphonium bromide (1.08 g, 2.97 mmol) in THF (1.6 mL) was added potassium tert-butoxide (340mg, 2.97 mmol). The bright yellow suspension was stirred at 0 °C for 1 hr. Compound 85 was dissolved in THF (2 mL), transferred into a dropping funnel and slowly added to the solution of ylide at 0 °C. The mixture was then vigorously stirred at 0 °C for 10 minutes and then at 22 °C for 3 hr. The mixture was diluted with DCM (30 mL) and organic layer was washed with saturated NH4Cl solution (30 mL). The organic layer was then dried using Na2SO4, filtered, and concentrated in vacuo. The crude compound was purified by flash column chromatography using 0-50% EtOAc/Hex. LRMS (ESI) calculated for C17H28N2O5Si [M+H]+ m/z = 368.51, found 369.2. 1H NMR (600 MHz, CDCl3) δ 9.42 (s, 1H), 7.29 (d, J=8.1 Hz, 1H), 5.81 (ddd, J=6.2 Hz, 1H), 5.73 (d, J =1.8 , 1H), 5.68 (d, J=8.5 Hz, 2H), 5.36 (dt, J=17.2 Hz, 1H), 5.26 (d, J = 10.6 Hz, 1H), 4.33 (t, J = 7.2 Hz, 1H), 3.81 (dd, J = 4.9 Hz, 1H), 3.62 (dd, J=3.1 Hz, 1H), 3.46 (s, 3H), 0.81 (s, 9H), 0.01 (d, J = 10.8 Hz, 6H). [00647] Compound 107: Compound 106 (150 mg, 0.4 mmol) was dissolved in dichloromethane (DCM) (6.67 mL) and stirred at room temperature. Diethyl allylphosphonate was added portion wise, followed by the Grubbs (2nd gen) catalyst. The solution was then stirred at reflux overnight. Afterwards, the solution was dry loaded using silica gel and purified by flash column chromatography using 0-15% MeOH/DCM. LRMS (ESI) calculated for C22H39N2O8PSi [M+H]+ m/z = 518.62, found 519.2. 1H NMR (600 MHz, DMSO) δ 11.40 (s, 1H), 7.62 (d, J=8.0 Hz, 1H), 5.82-5.76 (m, 2H), 5.7-5.63 (m, 2H), 4.19 (t, J=6.3 Hz, 1H), 4.12 (t, J=5.6 Hz, 1H), 3.99 (m, 4H), 3.89 (t, J = 3.9 Hz, 1H), 3.35 (s, 3H), 3.32 (s, 1H), 2.67 (m, 2H), 1.22 (dt, J = 7.1 Hz, 6H), 0.87 (s, 9H), 0.07 (d, J = 5.5 Hz, 6H). 3 IP NMR (600 MHz, DMSO) δ 25.9.
[00648] Compound 108: Compound 107 (100 mg, 0.19 mmol) was dissolved in tetrahydrofuran (THF) (0.6 mL) and stirred at room temperature. Concentrated aqueous HCl (1 mL) was then added portion wise. The solution was then stirred at room temperature for 1 hour. Afterwards, the solution was concentrated and purified by flash column chromatography using 0- 10% MeOH/DCM. LRMS (ESI) calculated for C16H25N2O8P [M+H]+ m/z = 404.36, found 405.4. 1H NMR (600 MHz, DMSO) δ 11.39 (s, 1H), 7.59 (d, J=8.0 Hz, 1H), 5.86-5.78 (m, 2H), 5.7-5.6 (m, 2H), 4.21 (t, J=6.3 Hz, 1H), 3.99 (m, 5H), 3.83 (t, J = 3.9 Hz, 1H), 3.37 (s, 3H), 2.68 (dd, J = 21.0 Hz, 2H), 1.22 (t, J = 7.1 Hz, 6H). 3 IP NMR (600 MHz, MeOH) δ 27.89.
[00649] Compound 109: Compound 108 (1.14 g, 2.82 mmol) was dissolved in dry Ethyl Acetate (EtOAc) (28.2 mL) and stirred at room temperature under an inert atmosphere. Diethyllsopropylamine (DIPEA) (1.96 mL, 11.28 mmol) was added portion wise, followed by N- methyllmidazole (0.224 mL, 2.82 mmol). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (1.26 mL, 5.64 mmol) was then added dropwise. The reaction was then stirred at room temperature for 1 hour. Afterwards, the solution was diluted using Ethyl Acetate. The organic layer was then washed with saturated sodium bicarbonate and brine, dried using magnesium sulfate (MgSO4), and concentrated in vacuo. The compound was then purified via flash column chromatography using triethylamine-neutralized silica with 0-10% MeOH/DCM to give compound 109. LRMS (ESI) calculated for C25H42N4O9P2 [M+H]+ m/z = 604.58, found 404.4. 1H NMR (600 MHz, DMSO) 8 11.39 (s, 1H), 7.64 (dd, J = 8.0 Hz, 1H), 5.88-5.8 (m, 2H), 5.75 - 5.63 (m, 2H), 4.44 - 4.33 (m, 1H), 4.29-4.16 (m, 1H), 4.14-4.06 (m, 1H), 4.05-3.95 (m, 4H), 3.83-3.66 (m, 2H), 3.65-3.55 (m, 2H), 3.39 (s, 1H), 3.35 (s, 2H), 3.33 (s, 1H), 2.81-2.76 (m, 2H), 2.73-2.59 (m, 2H), 1.25-1.12 (m, 18H). 31P NMR (600 MHz, DMSO) δ 149.29, 149.19, 26.44, 26.29
Synthesis of compound 115
[00650] Compound 115 was synthesized according to Scheme 17.
[00651] Compound 111: Compound 110 (100 mg, 0.203 mmol) was dissolved in 80% aqueous acetic acid (3 mL) and heated at 90 °C for 18 h. Solvent was removed under reduced pressure. The residue was co-evaporated with toluene and the obtained residue was dissolved in pyridine (3 mL). The solution was cooled in an ice-water bath and acetic anhydride (0.211 mL, 2.23 mmol) was added. The reaction mixture was removed from the ice bath and stirred at room temperature for 12 h. The reaction mixture was concentrated under vacuum and the residue was diluted with EtOAc and washed with water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-5% MeOH in ethyl acetate) to obtain compound 111 (100.6 mg, 92% yield) as a colorless oil. 1H NMR (600 MHz, DMSO-d6) δ 7.93-7.91 (m, 3 H), 7.85-7.85 (m, 1 H), 7.54-7.50 (m, 2 H), 7.46-7.45 (m, 1 H), 6.26 (d, J = 4.6 Hz, 0.1 H), 6.02 (s, 0.9 H), 5.44 (dd, J = 4.8, 5.0 Hz, 0.1 H), 5.33 (d, J = 4.3 Hz, 0.9 H), 4.80-4.75 (m, 2 H), 4.52 (d, J = 4.3 Hz, 0.9 H), 4.40 (d, J = 5.0 Hz, 0.1 H), 4.20-4.17 (m, 1 H), 4.08-4.02 (m, 5 H), 3.85 (d, J = 10.0 Hz, 0.1 H), 3.75 (d, J = 10.0 Hz, 0.9 H), 2.36-2.29 (m, 1 H), 2.22-2.19 (m, 1 H), 2.09-2.04 (m, 6 H), 1.21-1.18 (m, 6 H). 31P NMR (243 MHz, DMSO-d6) δ 22.33, 22.33. MS (ESI+APCI) calculated for C26H34O10P [M+H]+ m/z = 537.19, found 537.2.
[00652] Compound 112: To a suspension of uracil (32.8 mg, 0.293 mmol) in CH3CN (1 mL) was added dropwise N,O-bis(trimethylsilyl)acetamide (0.144 mL, 0.586 mmol) and the mixture was stirred at room temperature for 5 h. To the reaction mixture was added a solution of compound 111 (110 mg, 0.205 mmol) in CH3CN (1 mL) and the mixture was cooled in an ice-water bath. To the mixture was added dropwise tin tetrachloride (18.9 μL, 0.161 mmol). The reaction mixture was refluxed for 1 h and cooled to room temperature and quenched by adding a saturated NaHCO3 (aq.). The product was extracted with ethyl acetate and the combined organic layer was washed with water, brine, dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50-100% ethyl acetate in hexane) to obtain compound 112 (85.1 mg, 49% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.43 (d, J = 2.2 Hz, 1 H), 7.93-7.91 (m, 3 H), 7.86-7.86 (m, 1 H), 7.70 (d, J = 8.1 Hz, 1 H), 7.54-7.47 (m, 3 H), 5.81 (d, J = 3.2 Hz, 1 H), 5.64 (dd, J = 8.1, 2.2 Hz, 1 H), 5.58 (dd, J = 5.3, 3.2 Hz, 1 H), 4.79 (d, J = 11.9 Hz, 1 H), 4.73 (d, J = 11.9 Hz, 1 H), 4.61 (d, J = 5.3 Hz, 1 H), 4.26-4.23 (m, 1 H), 4.04—4.01 (m, 5 H), 3.85 (d, J = 10.0 Hz, 1 H), 2.44-2.39 (m, 1 H), 2.35-2.28 (m, 1 H), 2.08 (s, 3 H), 1.20-1.16 (m, 6 H). 3 IP NMR (243 MHz, DMSO-d6) δ 22.49. MS (ESI+APCI) calculated for C28H34N2O10P [M+H]+ m/z = 589.20, found 589.2.
[00653] Compound 113: To a solution of compound 112 (1.8 g, 3.06 mmol) and 1,8- diazabicyclo[5.4.0]undec-7-ene (1.37 mL, 9.18 mmol) in DMF (30 mL) was added dropwise benzyl chloromethyl ether (60% NMR purity: 1.06 mL, 4.59 mmol) at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction was diluted with ethyl acetate and washed with saturated aqueous NaHCO3 (aq.), water and brine and then dried over Na2SO4 and concentrated under vacuum. The residue was dissolved in MeOH (30 mL) and K2CO3 (1.27 g, 9.18 mmol) was added to the solution. The mixture was stirred at room temperature for 1 h and then diluted with ethyl acetate and water. The organic layer was washed with water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-5% MeOH in ethyl acetate) to obtain compound 113 (1.88 g, 92% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 7.92-7.89 (m, 4 H), 7.79 (d, J = 8.1 Hz, 1 H), 7.54-7.51 (m, 3 H), 7.34-7.28 (m, 5 H), 5.86-5.79 (m, 3 H), 5.33 (dd, J = 14.2, 9.8 Hz, 1 H), 5.01 (d, J = 11.9 Hz, 1 H), 4.74 (d, J = 11.9 Hz, 1 H), 4.60-4.56 (m, 3 H), 4.29-4.22 (m, 2 H), 4.05-4.01 (m, 5 H), 3.84 (d, J = 10.0 Hz, 1 H), 2.51-2.48 (m, 1 H), 2.32-2.25 (m, 1 H), 1.20-1.16 (m, 6 H). 31P NMR (243 MHz, DMSO-d6) δ 22.70. MS (ESI+APCI) calculated for C34H40N2010P [M+H]+ m/z = 667.24, found 667.4. [00654] Compound 114: To a solution of compound 113 (700 mg, 1.05 mmol) in methyl iodide (6.54 mL, 105 mmol) was added silver oxide (1.22 g, 5.25 mmol) and the suspension was stirred at 45 °C for 48 h. The reaction mixture was filtrated through a pad of celite and washed the celite pad thoroughly with CH2Cl2. The filtrate was concentrated under vacuum. The residue was dissolved in CH2Cl2 (10 mL) and 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ: 625.29 mg, 2.75 mmol) and water (49.6 μL, 2.75 mmol) and the mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with ethyl acetate and saturated NaHCO3 (aq.). The organic layer was washed with saturated NaHCO3 (aq.), water, saturated NH4Cl (aq.), and water and dried over Na2SO4 and concentrated under vacuum. The residue was dissolved in EtOH (10 mL) and the solution was added to a suspension of 10% palladium on carbon (68.9 mg) in EtOH (5 mL). Under hydrogen atmosphere, the suspension was stirred at room temperature for 3 h. The reaction mixture was filtered through a celite pad rinsing with EtOH and the filtrate was concentrated under vacuum under. The residue was dissolved in MeOH (10 mL) and K2CO3 (358 mg, 2.59 mmol) was added. The mixture was stirred at room temperature for 30 min and then concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-20% MeOH in CH2Cl2) to obtain compound 114 (292 mg, 66% yield) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 11.40 (d, J = 2.2 Hz, 1 H), 7.72 (d, J = 8.1 Hz, 1 H), 5.84 (d, J = 5.6 Hz, 1 H), 5.65 (dd, J = 8.1, 2.2 Hz, 1 H), 5.59 (d, J = 5.6 Hz, 1 H), 4.33 (t, J = 5.1 Hz, 1 H), 4.23-4.20 (m, 1 H), 4.09-4.00 (m, 6 H), 3.70 (d, J = 9.7 Hz, 1 H), 3.34 (s, 3 H), 2.43-2.38 (m, 1 H), 2.28-2.20 (m, 1 H), 1.25-1.22 (m, 6 H). 3 IP NMR (243 MHz, DMSO-d6) δ 22.78. MS (ESI+APCI) calculated for C16H26N2O9P [M+H]+ m/z = 421.14, found 421.1.
[00655] Compound 115: To a solution of compound 114 (290 mg, 0.690 mmol) and 1- methylimidazole (5.49 μL, 68.99 μmol) in ethyl acetate (7 mL) was added diisopropylethylamine (DIPEA: 0.361 mL, 2.07 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.462 mL, 2.07 mmol) at 0 °C and the mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of saturated NaHCO3 (aq.) and diluted with ethyl acetate. The organic layer was washed with saturated NaHCO3(aq.), water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel. The column was eluted with isocratic 60% ethyl acetate in hexane (1% Et3N) followed by a gradient of 60 to 100% ethyl acetate in hexane (1% Et3N) to obtain compound 115 (300 mg, 70% yield) as a white form. 1H NMR (600 MHz, CD3CN) δ 9.03 (brs, 1H), 7.42-7.40 (m, 1H), 5.85- 5.80 (m, 1H), 5.64-5.62 (m, 1H), 4.51 (dd, J = 11.0, 4.7 Hz, 0.65H), 4.40 (dd, J = 9.5, 4.7 Hz, 0.35H), 4.26-4.23 (m, 1H), 4.14-4. 08 (m, 5H), 4.00-3.99 (m, 0.65H), 3.97-3.95 (m, 0.35H), 3.91- 3.82 (m, 2H), 3.78-3.74 (m, 1H), 3.67-3.60 (m, 2H), 3.48 (s, 1.05H), 3.43 (s, 1.95H), 2.71-2.65 (m, 2H), 2.60-2.34 (m, 2H), 1.31-1.26 (m, 6H), 1.21-1.18 (m, 12H). 31P NMR (243 MHz, CD3CN) δ 150.58, 149.73, 22.17, 22.03. MS(ESI+APCI) calculated for C25H41N4O10 [M-H]- m/z = 619.23, found 619.3.
Synthesis of compound 121
[00656] Compound 121 was synthesized according to Scheme 18.
[00657] Compound 116: Compound 116 (1 g, 2.03 mmol) was dissolved in 80% aqueous acetic acid (20 mL) and heated at 90 °C for 18 h. Solvent was removed under reduced pressure. The residue was co-evaporated with toluene and the obtained residue was dissolved in pyridine (20 mL). The solution was cooled in an ice-water bath and acetic anhydride (2.11 mL, 22.3 mmol) was added. The reaction mixture was removed from the ice bath and stirred at room temperature for 12 h. The reaction mixture was concentrated under vacuum and the residue was diluted with EtOAc and washed with water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-5% MeOH in ethyl acetate) to obtain compound 117 (1.05 g, 96% yield) as a colorless oil. 1H NMR (600 MHz, DMSO-d6) δ 7.93-7.85 (m, 4H), 7.53-7.50 (m, 3H), 6.24 (d, J = 4.7 Hz, 0.1H), 6.00 (d, J = 1.3 Hz, 0.9H), 5.38 (dd, J = 5.4, 4.7 Hz, 0.1H), 5.31 (dd, J = 4.4, 1.3 Hz, 0.9H), 4.77-4.77 (m, 2 H), 4.50 (d, J = 4.4 Hz, 0.9H), 4.33 (d, J = 5.4 Hz, 0.1H), 4.13-3.99 (m, 6H), 3.79 (d, J = 9.7 Hz, 1H), 2.47-2.42 (m, 1H), 2.26-2.19 (m, 1H), 2.07-2.04 (m, 6H), 1.22-1.20 (m, 6H). 31P NMR (243 MHz, DMSO-d6) δ 21.57, 21.57. MS (ESI+APCI) calculated for C26H34O10P [M+H]+ m/z = 537.19, found 537.2. [00658] Compound 117: To a suspension of uracil (298 mg, 2.66 mmol) in CH3CN (18 mL) was added dropwise N,O-bis(trimethylsilyl)acetamide (1.31 mL, 5.33 mmol) and the mixture was stirred at room temperature for 5 h. To the reaction mixture was added a solution of compound 116 (1 g, 1.86 mmol) in CH3CN (10 mL) and the mixture was cooled in an ice-water bath. To the mixture was added dropwise tin tetrachloride (171 μL, 1.46 mmol). The reaction mixture was refluxed for 1 h and cooled to room temperature and quenched by adding a saturated NaHCO3 (aq.). The product was extracted with ethyl acetate and the combined organic layer was washed with water, brine, dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50-100% ethyl acetate in hexane) to obtain compound 117 (620 mg, 40% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.42 (d, J = 2.2 Hz, 1H), 7.93-7.91 (m, 3H), 7.87-7.87 (m, 1 H), 7.68 (d, J = 8.1 Hz, 1H), 7.53-7.50 (m, 3H), 5.81 (d, J = 4.4 Hz, 1H), 5.67 (dd, J = 8.1, 2.2 Hz, 1H), 5.62 (t, J = 4.7 Hz, 1H), 4.80 (d, J = 11.8 Hz, 1H), 4.77 (d, J = 11.8 Hz, 1H), 4.55 (d, J = 5.1 Hz, 1H), 4.13 (t, J = 8.6 Hz, 1H), 4.05- 3.99 (m, 4H), 3.97 (d, J = 9.8 Hz, 1H), 3.85 (d, J = 9.8 Hz, 1H), 2.51-2.47 (m, 1H), 2.32-2.25 (m, 1H), 2.06 (s, 3 H), 1.21-1.18 (m, 6H). 31PNMR (243 MHz, DMSO-d6) δ 21.50. MS (ESI+APCI) calculated for C28H34N2O10P [M+H]+ m/z = 589.20, found 589.2.
[00659] Compound 118: To a solution of compound 117 (560 mg, 0.952 mmol) and 1,8- diazabicyclo[5.4.0]undec-7-ene (0.426 mL, 2.85 mmol) in DMF (9 mL) was added dropwise benzyl chloromethyl ether (60% NMR purity: 0.199 mL, 1.43 mmol) at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction was diluted with ethyl acetate and washed with saturated aqueous NaHCO3 (aq.), water and brine and then dried over Na2SO4 and concentrated under vacuum. The residue was dissolved in MeOH (9 mL) and K2CO3 (395 mg, 2.85 mmol) was added to the solution. The mixture was stirred at room temperature for 1 h and then diluted with ethyl acetate and water. The organic layer was washed with water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-5% MeOH in ethyl acetate) to obtain compound 118 (602 mg, 95% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 7.93-7.90 (m, 4H), 7.74 (d, J = 8.2 Hz, 1H), 7.57-7.49 (m, 3H), 7.34-7.26 (m, 5H), 5.86-5.84 (m, 3H), 5.33 (dd, J = 14.8, 9.8 Hz, 1H), 5.05 (d, J = 12.0 Hz, 1H), 4.78 (d, J = 12.0 Hz, 1H), 4.66-4.63 (m, 2H), 4.59 (s, 2H), 4.20 (d, J = 4.3 Hz, 1H), 4.12 (dd, J = 9.7, 8.2 Hz, 1H), 4.07-4.00 (m, 5H), 3.78 (d, J = 9.7 Hz, 1H), 2.55-2.50 (m, 1H), 2.31-2.24 (m, 1H), 1.22-1.18 (m, 6 H). 31P NMR (243 MHz, DMSO-d6) δ 21.69. MS (ESI+APCI) calculated for C34H40N2010P [M+H]+ m/z = 667.24, found 667.4.
[00660] Compound 119: To a solution of compound 118 (600 mg, 0.900 mmol) in methyl iodide (5.60 mL, 90.0 mmol) was added silver oxide (1.04 g, 4.50 mmol) and the suspension was stirred at 45 °C for 48 h. The reaction mixture was filtrated through a pad of celite and washed the celite pad thoroughly with CH2Cl2. The filtrate was concentrated under vacuum. The residue was dissolved in CH2Cl2 (10 mL) and 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ: 500 mg, 2.20 mmol) and water (39.7 μL, 2.20 mmol) and the mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with ethyl acetate and saturated NaHCO3 (aq.). The organic layer was washed with saturated NaHCO3 (aq.), water, saturated NH4Cl (aq.), and water and dried over Na2SO4 and concentrated under vacuum. The residue was dissolved in EtOH (10 mL) and the solution was added to a suspension of 10% palladium on carbon (39.4 mg) in EtOH (5 mL). Under hydrogen atmosphere, the suspension was stirred at room temperature for 3 h. The reaction mixture was filtered through a celite pad rinsing with EtOH and the filtrate was concentrated under vacuum under. The residue was dissolved in MeOH (10 mL) and K2CO3 (307 mg, 2.22 mmol) was added. The mixture was stirred at room temperature for 30 min and then concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0-20% MeOH in CH2Cl2) to obtain compound 119 (225 mg, 60% yield) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 5.84 (d, J = 6.6 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 5.60 (d, J = 5.1 Hz, 1H), 4.24 (dd, J = 5.1, 2.2 Hz, 1H), 4.20 (dd, J = 6.6, 2.2 Hz, 1H), 4.12 (dd, J = 9.6, 8.0 Hz, 1H), 4.06-4.02 (m, 4H), 3.98 (d, J = 9.6 Hz, 1H), 3.66 (d, J = 9.6 Hz, 1H), 3.22 (s, 3H), 2.51-2.46 (m, 1H), 2.22-2.15 (m, 1H), 1.23-1.19 (m, 6H). 31P NMR (243 MHz, DMSO-d6) δ 21.79. MS (ESI+APCI) calculated for C16H26N2O9P [M+H]+ m/z = 421.14, found 421.2.
[00661] Compound 120: To a solution of compound 119 (275 mg, 0.654 mmol) and 1- methylimidazole (5.21 μL, 65.4 μmol) in CH2Cl2 (10 mL) was added diisopropylethylamine (DIPEA: 0.342 mL, 1.96 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.437 mL, 1.96 mmol) at 0 °C and the mixture was stirred at room temperature for 3 h. The reaction was quenched by addition of saturated NaHCO3 (aq.) and diluted with CH2Cl2. The organic layer was washed with saturated NaHCO3(aq.), water and brine and dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel. The column was eluted with isocratic 60% ethyl acetate in hexane (5% Et3N) followed by a gradient of 60 to 100% ethyl acetate in hexane (5% Et3N) to obtain compound 120 (118 mg, 29% yield) as a white form. 1H NMR (600 MHz, CD3CN) δ 9.15 (brs, 1H), 7.39-7.38 (m, 1H), 5.80-5.78 (m, 1H), 5.66-5.63 (m, 1H), 4.51-4.38 (m, 1H), 4.12-4.01 (m, 6H), 3.99-3.97 (m, 1H), 3.90-3.85 (m, 2H), 3.81-3.61 (m, 3H), 3.48-3.47 (m, 2H), 3.44-3.42 (m, 1H), 2.84-2.66 (m, 2H), 2.63-2.35 (m, 2H), 1.28-1.26 (m, 6H), 1.23-1.18 (m, 12H). 31P NMR (243 MHz, CD3CN) δ 150.65, 149.95, 21.12, 21.07. MS (ESI+APCI) calculated for C25H41N4O10 [M-H]- m/z = 619.23, found 619.2.using Ethyl Acetate. The organic layer was then washed with saturated sodium bicarbonate and brine, dried using magnesium sulfate (MgSO4), and concentrated in vacuo. The compound was then purified via flash column chromatography using triethylamine-neutralized silica with 0-10% MeOH/DCM. LRMS (ESI) calculated for C25H42N4O9P2 [M+H]+ m/z = 604.58, found 404.4. 1H NMR (600 MHz, DMSO) δ 11.39 (s, 1H), 7.64 (dd, J = 8.0 Hz, 1H), 5.88-5.8 (m, 2H), 5.75 - 5.63 (m, 2H), 4.44 -4.33 (m, 1H), 4.29-4.16 (m, 1H), 4.14-4.06 (m, 1H), 4.05-3.95 (m, 4H), 3.83- 3.66 (m, 2H), 3.65-3.55 (m, 2H), 3.39 (s, 1H), 3.35 (s, 2H), 3.33 (s, 1H), 2.81-2.76 (m, 2H), 2.73-
2.59 (m, 2H), 1.25-1.12 (m, 18H).
Synthesis of compound 126
[00662] Compound 116 was synthesized according to Scheme 19.
[00663] Compound 122: Palladium, 10% on carbon is added to a solution of compound 121 in MeOH. Under hydrogen atmosphere, the suspension is stirred at room temperature for 1 h. The reaction mixture is filtered through a celite pad rinsing with EtOH and the filtrate is concentrated under vacuum to give compound 122. The product is used without further purification.
[00664] Compound 123: Compound 122 is dissolved in piperidine and the resulting solution is stirred at r.t. for 48h and 24 h at 50 °C. The volatiles are evaporated to dryness and the residue is purified by flash chromatography using 0-10% MeOH in DCM as eluent to give compound 123. The product is dissolved in DCM and washed with IM HCl.
[00665] Compound 124: Compound 123 is dissolved in DCM and dimethylformamide. The solution is cooled in an ice bath, followed by the dropwise addition of a solution of oxalyl chloride in DCM. After 4 h, the volatiles are evaporated to dryness, and co-evaporated with MeCN. The residue is dissolved in MeCN, cooled to 0 °C, and combined with methanesulfonamide and 1,8- Diazabicyclo[5.4.0]undec-7-ene and stirred overnight. The solution is evaporated to dryness, then the residue is dissolved in DCM and washed with IM HCl and brine. The solvent is removed under reduced pressure and the residue is purified using flash chromatography using 0-10% DCM in MeOH as eluent to give compound 124.
[00666] Compound 125: Compound 124 is dissolved in a 1:1 solution of formic acid in water and the resulting mixture is warmed to 50 °C for 3h. The volatiles are removed under reduced pressure and the residue co-evaporated with toluene. The crude residue is purified by flash chromatography using 2-20% MeOH in DCM as eluent to give compound 125.
[00667] Compound 126: Diphenyl phosphite is added to a stirred solution of compound 125 in pyridine. After stirring for 30 min, a 1:1 mixture of NEt3/H2O (5 mL) is added and stirred for an additional 30 min. The solvent is removed under reduced pressure and the residue is co-evaporated with MeCN. The crude residue is purified by flash chromatography using 5% MeOH in DCM to 40% (DCM +1% formic acid) as eluent to give compound 126.
Synthesis of compound 132
[00668] Compound 131 was synthesized according to Scheme 20.
[00669] Compound 128: Imidazole and tert-Butyldimethylsilyl chloride are added to a solution of compound 127 in DMF and the resulting mixture is stirred for 24h. The volatiles are evaporated to dryness, and the residue dissolved partitioned in EtOAc and sat bicarb. The layers are separated and the aq layer is extracted back with EtOAc, dried over Na2SO4, evaporated to dryness. The residue is purified by ISCO automated column using 20-100% EtOAc in hexanes as eluant to give compound 128.
[00670] Compound 129:Compound 128 and Lithium iodide are dissolved in pyridine and heated to reflux overnight. The mixture is then evaporated to dryness and the residue dissolved in a small amount of DCM and pre-adsorbed in silica. The residue is purified by flash chromatography using 20-100% (3:1) EtOAc/EtOH mixture in hexanes as eluent to give compound 129.
[00671] Compound 130: Compound 129 is dissolved in DCM and dimethylformamide. The solution is cooled in an ice bath, followed by the dropwise addition of a solution of oxalyl chloride in DCM. After 4 h, the volatiles are evaporated to dryness, and co-evaporated with MeCN. The residue is dissolved in MeCN, cooled to °C, and combined with methanesulfonamide and 1,8- Diazabicyclo[5.4.0]undec-7-ene and stirred overnight. The solution is evaporated to dryness, and the residue is dissolved in DCM and washed with IM HCl (x2) and brine. The solvent is evaporated to dryness and the residue was purified by flash chromatography using 0-10% MeOH in DCM as eluent to give compound 130.
[00672] Compound 131: Compound 130 is dissolved in THF and combined with Triethylamine trihydrofluoride and triethylamine. The resulting mixture is stirred overnight, then the volatiles are removed under reduced pressure and the residue is purified by flash chromatography using 5-20% MeOH in DCM as eluent to give compound 131.
[00673] Compound 132: Diphenyl phosphite is added to a solution of compound 131 in pyridine. After 30 min, a 1 : 1 mixture of NEt3/water is added and stirred for an additional 30 min. The solvent is then removed under reduced pressure. The residue is purified by flash chromatography using 5-40% MeOH in DCM (+1% formic acid) to give compound 132.
Synthesis of compound 134
[00674] Compound 134 was synthesized according to Scheme 21.
[00675] Compound 134: Sodium azide (1.14 g, 17.57 mmol) was added to a solution of compound 133 (2 g, 13.51 mmol, 1.45 mL) in MeCN (45 mL). After stirring for 12 h at r.t., the mixture was diluted with EtOAc (200 mL) and mixed with water (50 mL). The organic phase was washed with brine and dried over Na2SO4 and evaporated to dryness to give compound 134 (1.95 g, 98%). The crude product was used without further purification. 1H NMR (600 MHz, CDCl3) δ 2.78 (tt, J = 8.0, 4.7 Hz, 1H), 1.43 - 1.37 (m, 2H), 1.24 - 1.19 (m, 2H).
Synthesis of compound 136
[00676] Compound 136 was synthesized according to Scheme 22.
[00677] Compound 136: Sodium azide (493.31 mg, 7.59 mmol) was added to a solution of compound 135 (0.95 g, 5.84 mmol) in MeCN (24 mL). After stirring for 12 h at r.t., the mixture was diluted with EtOAc (200 mL) and mixed with water (50 mL). The organic phase was washed with brine and dried over Na2SO4 and evaporated to dryness to give compound 136 (626 mg, 66%). The crude product was used without further purification.
Synthesis of compound 138
[00678] Compound 138 was synthesized according to Scheme 23.
[00679] Compound 138: Sodium azide (1.48 g, 0.798 mmol) was added to a solution of compound 137 (2.74 g, 17.47 mmol) in MeCN (70 mL). After stirring for 12 h at r.t., the mixture was diluted with EtOAc (50 mL) and mixed with water (10 mL). The organic phase was washed with brine and dried over Na2SO4 and evaporated to dryness to give compound 138 (2.58 g, 94%). The crude product was used without further purification.
Synthesis of compound 142
[00680] Compound 142 was synthesized according to Scheme 24.
[00681] Compound 139: To a solution of compound 850 (52.5 g, 108.83 mmol, 1 eq) in DMF (260 mL) was added NaN3 (7.22 g, 111.01 mmol, 1.02 eq). The mixture was stirred at 60 °C for 12 hrs. LCMS showed starting material was consumed completely and one main peak with desired m/z was detected. The reaction mixture was poured into cold water (600 ml) and extracted with EtOAc (3 x 500 mL).The combined organic layers were washed with brine (6 x 100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude. The crude product was triturated with petroleum ether: ethyl acetate = 10:1 to give compound 139 (37.5 g, 86.68% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.37 - 11.46 (m, 1 H) 7.72 (d, J=8.00 Hz, 1 H) 5.80 (d, J=4.50 Hz, 1 H) 5.69 (dd, J=8.00, 2.13 Hz, 1 H) 4.29 (t, J=5.25 Hz, 1 H) 3.97 (t, J=4.88 Hz, 1 H) 3.88 - 3.94 (m, 1 H) 3.53 - 3.67 (m, 2 H) 3.34 (s, 3 H) 0.88 (s, 9 H) 0.10 (s, 6 H).
[00682] Compound 140: To a solution of compound 139 (20 g, 50.31 mmol, 1 eq) in toluene (80 mL) was added diethyl ethynylphosphonate (8.16 g, 50.31 mmol, 1 eq) and [RuCl(Cp)(PPh3)2] (1.10 g, 1.51 mmol, 0.03 eq) .The mixture was stirred at 80 °C for 48 hrs. LC-MS showed Reactant 1 was consumed completely and desired mass was detected. Additional one reaction in 20 g scale was set up as described above. The reaction mixture was concentrated under reduced pressure to remove toluene and give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=3/l to 1/3) to give compound 140 (15 g, 54.8% yield) as a white solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.38 (br s, 1 H) 7.99 (s, 1 H) 7.20 (d, J=8.13 Hz, 1 H) 5.79 (d, J=3.50 Hz, 1 H) 5.72 (dd, J=8.13, 2.13 Hz, 1 H) 4.98 - 5.07 (m, 1 H) 4.87 - 4.97 (m, 1 H) 4.49 (q, J=5.88 Hz, 1 H) 4.28 (t, J=5.57 Hz, 1 H) 4.09 - 4.22 (m, 4 H) 3.80 - 3.88 (m, 1 H) 3.49 (s, 3 H) 1.34 (d, J=8.38 Hz, 6 H) 0.92 (s, 9 H) 0.09 (d, J=11.51 Hz, 6 H).
[00683] Compound 141: To a solution of compound 140 (15 g, 26.80 mmol, 1 eq) in THF (168 mL) was added HCl/dioxane (4 M, 56 mL, 8.36 eq). The mixture was stirred at 25 °C for 2hr. LCMS showed starting material was consumed completely and one main peak with desired m/z was detected. Additional one reaction in 10 g scale was set up as described above. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by flash silica gel chromatography (Biotage®; 80 g Agela® Silica Flash Column, Eluent of 0-100% Ethyl acetate/ Petroleum ether gradient @ 100 mL/min) to give compound 141 (10 g, 94.24% yield) as a colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.47 (br s, 1 H) 7.98 (s, 1 H) 7.38 (d, J=8.13 Hz, 1 H) 5.80 (d, J=1.63 Hz, 1 H) 5.73 (d, J=8.13 Hz, 1 H) 5.03 - 5.10 (m, 2 H) 4.44 (dt, J=7.91, 5.30 Hz, 1 H) 4.10 - 4.23 (m, 5 H) 3.82 (dd, J=5.63, 1.63 Hz, 1 H) 3.59 (s, 3 H) 1.34 (q, J=6.80 Hz, 6 H).
[00684] Compound 142: To a solution of compound 141 (16 g, 35.93 mmol, 1 eq) in DCM (160 mL) was added 3-bis(diisopropylamino)phosphanyloxypropanenitrile (21.66 g, 71.85 mmol, 2 eq) and lH-imidazole-4,5-dicarbonitrile (3.39 g, 28.74 mmol, 0.8 eq) at 0°C. The mixture was stirred at 25 °C for 2 hrs. LCMS showed starting material was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The reaction solution was purified by flash silica gel chromatography (Biotage®; 40 g Agela® Silica Flash Column, Eluent of 0-100% Ethyl acetate/ Petroleum ether gradient @ 75 mL/min) to give Compound 142 (2.62 g, 11.30% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.42 (br s, 1 H) 8.20 (d, J=2.25 Hz, 1 H) 7.74 (dd, J=13.51, 8.13 Hz, 1 H) 5.80 (dd, J=11.26, 5.63 Hz, 1 H) 5.68 (dd, J=8.07, 2.06 Hz, 1 H) 4.84 - 5.01 (m, 2 H) 4.53 - 4.61 (m, 1 H) 4.37 - 4.51 (m, 1 H) 4.18 - 4.35 (m, 1 H) 4.03 - 4.16 (m, 4 H) 3.52 - 3.84 (m, 4 H) 3.38 (d, J=13.38 Hz, 3 H) 2.74 - 2.85 (m, 2 H) 2.07 (s, 1 H) 1.21 - 1.29 (m, 6 H) 1.10 - 1.20 (m, 9 H) 1.06 (d, J=6.75 Hz, 3 H).
Synthesis of compounds 146, 147, 148, and 149.
[00685] Compounds 146, 147, 148 and 149 were synthesized according to Scheme 25.
[00686] Compound 143: To a solution of compound 106 (2.0 g, 5.43 mmol) in THF (25 mL) was added 9-borabicyclo[3.3.1]nonane (3.97 g, 32.56 mmol, 4.44 mL) at 0 °C. The mixture was allowed to warm and stirred at 22 °C for 20 hrs. Then the reaction mixture was cooled, and methanol (MeOH) (20 mL) was added dropwise. When the gas evolution ceased, water (30 mL) was added followed by sodium perborate tetrahydrate (20.88 g, 130.26 mmol). The resulting mixture was stirred for 30 hr vigorously at 0 °C and then filtered. Filtrate was washed with EtO Ac (50 mL). The organic layer was further washed with brine (40 mL), dried over anhydrous Na2SO4, filtered and filtrate was evaporated to dryness. Crude residue thus obtained was purified by column chromatography (gradient: 20-75% EtOAc in hexane) to afford compound 143 (1.57 g, 75% yield) as white solid. 1H NMR (400 MHz, CDCl3) δ 9.23 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 5.85 - 5.52 (m, 2H), 4.11 (ddd, J = 9.1, 7.4, 3.8 Hz, 1H), 3.94 - 3.75 (m, 4H), 3.73 (dd, J = 5.2, 2.3 Hz, 1H), 2.15 - 1.95 (m, 2H), 1.85 (dddd, J = 14.4, 9.1, 6.9, 5.4 Hz, 1H), 1.73 (q, J = 6.4, 6.0 Hz, 2H), 0.91 (s, 9H), 0.10 (d, J = 3.3 Hz, 6H) ppm. 13C NMR (126 MHz, CDCl3) δ 163.26, 150.04, 140.10, 102.76, 90.26, 83.16, 82.14, 74.68, 60.44, 58.53, 35.63, 25.93, 25.84, 18.29, -4.38, -4.69 ppm. HRMS calc, for C17H30N2O6SiNa [M + Na]+ 409.1771, found 409.1767.
[00687] Compound 144: To a clear solution of compound 143 (1.0 g, 2.59 mmol) in DCM (30 mL) was added Dess-Martin Periodinane (1.37 g, 3.23 mmol) at 0 °C. The resulting mixture was stirred for 2 hr at 22 °C and then cooled again to 0 °C. The reaction mixture was diluted with NaHCO3 solution (25 mL). The organic layer was separated and washed with 5% sodium thiosulfate solution (25 mL). The resulting organic layer was separated, dried over anhydrous Na2SO4, filtered and filtrate was evaporated to dryness to afford white foam of aldehyde 144 (0.93 g, 99%) which was used for next step without further purification. 1H NMR (600 MHz, DMSO- d6) δ 11.40 (d, J = 2.1 Hz, 1H), 9.66 (t, J = 1.8 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 5.78 (d, J = 4.5 Hz, 1H), 5.66 (dd, J = 8.0, 2.1 Hz, 1H), 4.26 (dt, J = 7.4, 5.5 Hz, 1H), 4.18 (t, J = 5.2 Hz, 1H), 3.94 (t, J = 4.8 Hz, 1H), 3.33 (s, 3H), 2.87 - 2.79 (m, 2H), 0.88 (s, 9H), 0.10 (d, J = 5.7 Hz, 6H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 201.08, 163.02, 150.38, 141.22, 102.24, 87.70, 80.93, 78.21, 72.97, 57.53, 46.20, 25.65, 17.79, -4.83, -4.93 ppm.
[00688] Compound 145: To a solution of compound 144 in THF is added a solution of 0,0- Diethyl P-[(triphenylphosphoranylidene)methyl]phosphonothioate in THF drop-wise at 5 °C. Then, the reaction is allowed to warm to 25 °C and stirred for 1 h. The reaction mixture is concentrated under reduced pressure. The residue is purified by column chromatography to get compound 145 as a mixture of isomers.
[00689] Compounds 146, 147, 148 and 149: To a solution of MesSOI in DMSO is added sodium hydride as a THF slurry. The resulting mixture is stirred at 25 °C for 15 minutes and the resulting white solution is charged to a flask containing compound 145. The yellow solution is stirred for 2 hours and then heated to 50 °C for 2 hours. The reaction mixture is cooled to 5 °C and quenched with ice. The product is extracted with ethyl acetate and the organic layer is washed with water. The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue is dissolved in a 1:1 mixture of formic acid and water and allowed to stir overnight, then the volatiles are removed under reduced pressure. The crude residue is dissolved in a 1:1 mixture of THF/water and cooled to 0°C. Oxone is added to the solution and the resulting solution stirred for 2h. The reaction mixture is diluted with DCM and washed with water and brine. The organic layer is dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue is purified by reverse phase chromatography to give four compounds 146, 147, 148, and 149.
Synthesis of compound 150
[00690] Compound 142 is synthesized according to Scheme 26. [00691] Compound 150: To a solution of compound 146 in DCM was added 3- bis(diisopropylamino)phosphanyloxypropanenitrile and DCI. The mixture was stirred at 15 °C for 2h. The reaction mixture was diluted with DCM, was washed with sat NaHCO3 aq. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography to give compound 150.
Synthesis of compound 151
[00692] Compound 151 is synthesized according to Scheme 27.
[00693] Compound 151: To a solution of compound 147 in DCM was added 3- bis(diisopropylamino)phosphanyloxypropanenitrile and DCI. The mixture was stirred at 15 °C for 2h. The reaction mixture was diluted with DCM, was washed with sat NaHCO3 aq. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography to give compound 151.
Synthesis of compound 152
[00694] Compound 152 is synthesized according to Scheme 28. [00695] Compound 152: To a solution of compound 148 in DCM was added 3- bis(diisopropylamino)phosphanyloxypropanenitrile and DCI. The mixture was stirred at 15 °C for 2h. The reaction mixture was diluted with DCM, was washed with sat NaHCO3 aq. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography to give compound 152.
Synthesis of compound 153
[00696] Compound 153 is synthesized according to Scheme 29.
[00697] Compound 153: To a solution of compound 149 in DCM was added 3- bis(diisopropylamino)phosphanyloxypropanenitrile and DCI. The mixture was stirred at 15 °C for 2h. The reaction mixture was diluted with DCM, was washed with sat NaHCO3 aq. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography to give compound 153.
Synthesis of compound 165:
[00698] Compound 165 is synthesized according to Scheme 30. [00699] Compound 163: To a clear solution of compound 85 (0.7 g, 1.89 mmol) in DCM (15 mL) and acetic acid (5 mL) was added O-[di(ethoxy)phosphorylmethyl]hydroxylamine (692.09 mg, 3.78 mmol) in single portion. To this reaction mixture was stirred at 22 °C for 10 hr. The reaction mixture was diluted with DCM (15 mL) and then washed with water (20 mL) and brine (2x20 mL). The organic layer was deparated, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness under high vacuum pump. The crude compound was purified by flash column chromatography (gardient: 30-80% EtOAc in hexane followed by 5% MeOH in DCM) to afford compound 163(0.65 g, 64% yield) as a transparent sticky gum as mixture of cis and trans isomers. 1H NMR (600 MHz, DMSO-d6) δ 11.40 - 11.28 (m, 1H), 7.77 (d, J= 8.1 Hz, OH), 7.69 (dd, J= 7.7, 4.3 Hz, 1H), 7.13 (d, J= 5.9 Hz, OH), 5.77 (dd, J= 11.0, 6.0 Hz, 1H), 5.58 (ddd, J= 7.9, 5.3, 2.1 Hz, 1H), 4.74 (dd, J= 5.9, 2.3 Hz, OH), 4.40 - 4.30 (m, 3H), 4.18 (dd, J= 7.3, 4.8 Hz, 1H), 4.01 - 3.91 (m, 7H), 3.22 (d, J= 17.5 Hz, 4H), 1.14 (dd, J= 7.8, 6.4 Hz, 6H), 0.79 (d, J= 9.6 Hz, 9H), 0.06 - -0.05 (m, 6H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 163.01, 162.89, 150.70, 150.63, 150.46, 149.60, 141.65, 141.09, 102.48, 102.25, 87.67, 87.10, 81.08, 80.65, 80.47, 77.76, 72.32, 72.28, 69.66, 68.18, 67.73, 67.13, 66.67, 61.89, 61.85, 61.80, 61.76, 61.73, 61.56, 61.52, 61.40, 61.36, 60.78, 60.74, 57.88, 57.62, 54.92, 25.59, 25.55, 17.78, 17.77, 16.34, 16.30, 16.26, - 4.86, -4.94, -5.19 ppm. 31P NMR (243 MHz, DMSO-d6) δ 19.87, 19.55 ppm.
[00700] Compound 164: To a clear solution of compound 163 (1.0 g, 1.87 mmol) in THF (20 mL) was added hydrochloric acid, ACS grade 36-38% (1.36 g, 37.34 mmol, 1.70 mL) in single portion and stirred at 22°C for 0.5 hr. TLC was checked and all the volatile matters were removed under high vacuum pump. Crude compound thus obtained was purified by flash column chromatography (gradient: 0-10% MeOH in DCM) to afford compound 164 (0.53 g, 67% yield) as white foam. 1H NMR (600 MHz, DMSO-d6) δ 11.42 (dd, J= 17.8, 2.2 Hz, 1H), 7.77 (dd, J = 11.6, 7.6 Hz, 2H), 7.25 - 7.10 (m, 1H), 5.89 (d, J= 5.3 Hz, 1H), 5.67 (dd, J= 8.0, 2.3 Hz, 1H), 4.53 - 4.41 (m, 2H), 4.31 (dd, J= 7.2, 4.5 Hz, 1H), 4.26 (t, J= 4.7 Hz, 1H), 4.14 - 4.01 (m, 5H), 3.98 (t, J= 5.1 Hz, 1H), 3.36 (s, 3H), 1.24 (td, J= 7.1, 1.0 Hz, 6H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 163.00, 150.50, 150.01, 140.97, 128.91, 128.22, 125.33, 102.26, 87.15, 81.23, 80.72, 70.76, 67.68, 66.63, 61.98, 61.94, 61.91, 61.87, 57.66, 57.40, 16.31, 16.28 ppm. 31P NMR (243 MHz, DMSO-d6) δ 19.72, 19.64 ppm.
[00701] Compound 165: To a clear solution of compound 164 (0.5 g, 1.19 mmol) and 5- (ethylthio)-lH-tetrazole (154.47 mg, 1.19 mmol) in anhydrous DCM (10 mL) was added 2- cyanoethyl-N,A,A',N'-tetraisopropylphosphorodiamidite (715.36 mg, 2.37 mmol, 753.81 μL). The reaction mixture was stirred at 22 °C for 1 hr. TLC analysis confirmed formation of the product. The reaction mixture was filtered, concentrated, and the residue thus obtained was purified by flash column chromatography (gradient: 0-5% MeOH in DCM) to afford compound 165 (0.51 g, 69% yield) as yellowish white hygroscopic foam. 1H NMR (600 MHz, CD3CN) δ 9.04 (s, 1H), 7.66 - 7.63 (m, 1H), 7.62 - 7.57 (m, 1H), 5.91 (dd, J= 16.3, 5.2 Hz, 1H), 5.75 - 5.58 (m, 1H), 4.69 -4.46 (m, 2H), 4.44 - 4.39 (m, 2H), 4.16 - 4.02 (m, 6H), 3.97 - 3.80 (m, 1H), 3.71 - 3.61 (m, 2H), 3.54 - 3.37 (m, 5H), 2.80 - 2.65 (m, 3H), 1.29 (tdd, J= 6.7, 3.6, 1.6 Hz, 6H), 1.24 - 1.16 (m, 22H) ppm. 13C NMR (151 MHz, CD3CN) δ 163.73, 163.71, 151.45, 151.39, 150.54, 150.10, 149.83, 142.01, 141.67, 141.64, 119.65, 119.58, 103.64, 103.40, 103.29, 103.24, 89.76, 89.49, 88.88, 82.39, 82.37, 82.05, 81.93, 81.89, 81.14, 81.12, 80.89, 80.86, 73.93, 73.82, 73.43, 73.31, 69.59, 69.32, 68.25,
63.33, 63.31, 63.29, 63.27, 63.23, 59.97, 59.85, 59.69, 59.35, 59.22, 59.15, 59.11, 59.01, 58.99,
58.71, 58.69, 46.26, 46.22, 45.99, 45.95, 44.25, 44.20, 44.17, 44.14, 44.09, 44.06, 27.23, 24.95,
24.94, 24.90, 24.85, 24.81, 23.34, 23.24, 23.17, 23.16, 23.10, 23.08, 23.02, 22.55, 22.01, 21.02,
20.98, 20.93, 20.62, 20.57, 20.36, 16.84, 16.81 ppm. 31P NMR (243 MHz, CD3CN) δ 151.01, 150.84, 150.12, 149.79, 19.38, 19.28, 19.07, 18.92 ppm.
Synthesis of compound 168
[00702] Compound 168 is synthesized according to Scheme 31.
[00703] Compound 166: To a clear solution of compound 163 (1.59 g, 2.97 mmol) in AcOH (5.0 mL) and DCM (20.0 mL) at 15 °C was added sodium cyanoborohydride (485.03 mg, 7.72 mmol) in single portion and stirred for 1 hr. To this resulting reaction mixuture, formaldehyde (356.59 mg, 11.87 mmol, 329.26 μL) was added slowly and stirred for 1 hr. To this mixuture was added sodium cyanoborohydride (485.03 mg, 7.72 mmol) and stirred for 2 hr at 15 °C. Reaction mixture was diluted with DCM (20 mL) and washed with brine (30 mL). The organic layer was separated, dried over anhydrous Na2SO4 , filtered and filtrate was evaporated to dryness. The crude residue thus obtained, was purified by combiflash chromatorgraphy (gradient: 40-95% EtOAc in hexane followed by 5% MeOH in DCM) to afford compound 166 (1.39 g, 85% yield) as hygroscopic transparent gum. 1H NMR (600 MHz, CDCl3) δ 9.49 (s, 1H), 7.41 (d, J= 8.1 Hz, 1H), 5.76 (d, J= 8.1 Hz, 1H), 5.74 (d, J= 2.2 Hz, 1H), 4.23 - 4.09 (m, 7H), 3.97 - 3.90 (m, 1H), 3.76 (dd, J= 5.3, 2.3 Hz, 1H), 3.51 (d, J= 0.7 Hz, 3H), 3.07 - 3.02 (m, 1H), 2.96 (s, 1H), 2.73 (s, 3H), 1.34 (t, J= 7 A Hz, 6H), 0.91 (d, J= 0.8 Hz, 9H), 0.10 (d, J= 52 Hz, 6H) ppm. 31P NMR (243 MHz, CDCl3) δ 20.25 ppm.
[00704] Compound 167: To a clear solution of compound 166 (1.0 g, 1.81 mmol) in THF (20.50 mL) was added hydrochloric acid, ACS grade 36-38% (1.32 g, 36.26 mmol, 1.65 mL) in single portion and stirred at 22°C for 0.5 hr. TLC was checked and all the volatile matters were removed under high vacuum pump. Crude compound thus obtained was purified by flash column chromatography (gradient: 0-10% MeOH in DCM) to afford compound 167 (0.4 g, 50% yield) as white hygroscopic foam. 1H NMR (600 MHz, DMSO-d6) δ 11.39 (dd, J= 7.8, 2.2 Hz, 1H), 7.64 (d, J= 8.1 Hz, 1H), 5.87 - 5.80 (m, 1H), 5.68 - 5.62 (m, 1H), 4.17 - 3.95 (m, 9H), 3.90 (dt, J = 15.0, 5.3 Hz, 1H), 3.34 (s, 4H), 2.95 (dd, J= 13.5, 5.4 Hz, 1H), 2.86 (dd, J= 13.5, 7.1 Hz, 1H), 2.61 (s, 3H), 1.29 - 1.19 (m, 6H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 162.99, 150.52, 140.94, 102.19, 86.59, 81.26, 81.07, 70.12, 66.21, 65.18, 62.28, 61.72, 61.67, 57.56, 57.50, 45.73, 16.28, 16.24 ppm. 31P NMR (243 MHz, DMSO-d6) δ 20.31 ppm.
[00705] Compound 168: To a clear solution of compound 167 (0.33 g, 754.49 μmol) and 5- (ethylthio)-lH-tetrazole (98.21 mg, 754.49 μmol) in anhydrous DCM (10 mL) was added 2- cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (454.82 mg, 1.51 mmol, 479.26 μL). The reaction mixture was stirred at 22 °C for 1 hr. TLC analysis confirmed formation of the product. The reaction mixture was filtered, concentrated, and the residue thus obtained was purified by flash column chromatography (gradient: 5% MeOH in DCM) to afford compound 167 (0.33 g, 69% yield) as white hygroscopic foam. 1H NMR (600 MHz, CD3CN) δ 8.96 (s, 1H), 7.43 (dd, J= 8.1, 3.7 Hz, 1H), 5.84 (td, J= 8.1, 4.9 Hz, 1H), 5.64 (dd, J= 8.1, 4.3 Hz, 1H), 4.31 -4.19 (m, 2H), 4.17 - 3.99 (m, 7H), 3.96 - 3.61 (m, 5H), 3.49 - 3.38 (m, 3H), 3.08 - 2.88 (m, 2H), 2.75 - 2.63 (m, 5H), 1.28 (dtd, J= 5.3, 3.4, 1.6 Hz, 5H), 1.20 (ddt, J= 6.8, 4.6, 2.6 Hz, 13H) ppm. 13C NMR (151 MHz, CD3CN) δ 163.75, 151.33, 151.26, 141.31, 103.12, 103.01, 89.52, 88.81, 82.94, 82.41, 73.83, 73.73, 73.62, 67.75, 66.71, 63.39, 63.24, 63.07, 63.05, 63.03, 63.01, 62.99, 59.74, 59.62, 59.28, 59.15, 58.88, 58.79, 58.77, 46.40, 44.14, 44.12, 44.06, 44.03, 25.01, 24.96, 24.94, 24.90, 24.89, 24.84, 21.00, 20.95, 16.83, 16.80, 16.79 ppm. 31P NMR (243 MHz, CD3CN) δ 149.63, 149.32, 19.82, 19.74 ppm.
Synthesis of compound 171:
[00706] Compound 171 is synthesized according to Scheme 32.
[00707] Compound 850: To a solution of compound 84 (10 g, 26.9 mmol, 1 eq) in dry THF (150 mL) was added imidazole (4.57 g, 67.1 mmol, 2.5 eq), PPh3 (10.6 g, 40.3 mmol, 1.5 eq) and 12 (10.2 g, 40.3 mmol, 8.11 mL, 1.5 eq) at 0°C. The mixture was stirred at 20°C for 18hr. The reaction mixture was quenched by 10% NaS2SO3 aq. (300 mL), and extracted with EA (150 mL*3), the combined organic layer was washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10/l to 1/1). Compound 850 (10 g, 20.7 mmol, 77% yield) was obtained as a white solid. 1H NMR: (400 MHz, CDCl3) 9.63 (s, 1H), 7.70- 7.68 (d, J=8 Hz, 1H), 5.84-5.83 (d, J=4 Hz, 1H), 5.80-5.78 (m, 1H), 4.00-3.99 (m, 1H), 3.81-3.77 (m, 2H), 3.59-3.58 (m, 1H), 3.57 (s, 3H), 3.51-3.41 (m, 1H), 0.90 (s, 9H), 0.15-0.12 (d, J=12 Hz, 6H).
[00708] Compound 169: To a solution of isobutyl methanesulfonate (11.0 g, 72.6 mmol, 3.5 eq) and DMPU (12.0 g, 93.3 mmol, 11.2mL, 4.5 eq) in THF (50 mL) at -70 °C was added n-BuLi (2.5 M, 29.9 mL, 3.6 eq) under N2 atmosphere. The mixture was stirred at -70 °C for Ihr. A solution of compound 850 (10 g, 20.7 mmol, 1 eq) in THF (100 mL) was added dropwise to above mixture at -70 °C. The reaction mixture was stirred at -50 °C for 2 h. The reaction mixture was quenched by sat NH4Cl aq (500 mL), and extracted with EtOAc (150 mL*3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=l/O to 1/1). Compound 169 (5.3 g, 10.5 mmol, 50% yield) was obtained as a light yellow foam. 1H NMR: (400 MHz, CDCl3) 9.98 (s, 1H), 7.29-7.27 (d, J=8 Hz, 1H), 5.81-5.79 (d, J=8 Hz, 1H), 5.69-5.68 (d, J=4 Hz, 1H), 4.02-3.96 (m, 4H), 3.94-3.84 (m, 1H), 3.51 (s, 3H), 3.35-3.22 (m, 2H), 2.30-2.02 (m, 3H), 0.98-0.95 (d, J=12 Hz, 6H), 0.94-0.92 (d, J=12 Hz, 9H), 0.12-0.08 (d, J=16 Hz, 6H).
[00709] Compound 170: To a solution of compound 169 (5.3 g, 10.5 mmol, 1 eq) in HCOOH (50 mL) was added H2O (50 mL). The mixture was stirred at 20 °C for 24hr. The reaction mixture was quenched sat NaHCO3 aq (500 mL) and extracted with solvent EA (100 mL * 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=O/l to 1/1). Compound 170 (3 g, 73.08% yield) was obtained as a white foam. 1H NMR (400 MHz, CDCl3) 9.98 (s, 1H), 7.24-7.22 (d, J=8 Hz, 1H), 5.75-5.73 (d, J=8 Hz, 1H), 5.71-5.70 (d, J=4 Hz, 1H), 3.94-3.92 (d, J=8 Hz, 2H), 3.89- 3.83 (m, 3H), 3.52 (s, 3H), 3.25-3.24 (m, 2H), 3.02-3.00(d, J=8 Hz, 1H), 2.31-1.95 (m,3H), 0.95- 0.92 (d, J=12 Hz, 6H).
[00710] Compound 171: To a solution of compound 170 (2 g, 5.10 mmol, 1 eq) in DCM (20 mL) was added 3-bis(diisopropylamino)phosphanyloxypropanenitrile (2.15 g, 7.14 mmol, 2.27 mL, 1.4 eq) and DCI (722 mg, 6.12 mmol, 1.2 eq). The mixture was stirred at 15 °C for 2hr. The reaction mixture was diluted with DCM (30 mL), then washed with sat NaHCO3 aq. (50 mL*3), the organic layers was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Kromasil Eternity XT250*80mm*10um; mobile phase: [H2O-1M Ammonia Bicarbonate]; gradient: 35%-65% B over 30 min) to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=3/1 to 2/1). Compound 171 (1.1 g, 1.86 mmol, 36% yield) was obtained as off-white foam. 1H NMR: (400 MHz, DMSO-d6) 11.40 (s, 1H), 7.70-7.67 (m, 1H), 5.76-5.75 (d, J=4 Hz, 1H), 5.68-5.64 (m, 1H), 4.32-4.10 (m, 1H), 4.00-3.97 (m, 4H), 3.81-3.61 (m, 4H), 3.41-3.35 (m, 5H), 2.81-2.78 (m,2H), 2.09-1.93 (m,3H), 1.17-1.15 (d, J=8 Hz, 12H), 0.92-0.91(d, J=4 Hz, 6H). 31P NMR (162 MHz, DMSO-d6) 149.20. MS (ESI+APCI) calculated for C24H42N4O9PS [M+H]+ m/z = 593.24, found 593.2.
Synthesis of compound 175
[00711] Compound 175 is synthesized according to Scheme 33.
[00712] Compound 172: To a clear solution of compound 144 (1.0 g, 2.59 mmol) in DCM (30 mL) was added carbon tetrabromide (1.72 g, 5.17 mmol) and triphenylphosphine (2.71 g, 10.35 mmol) in single portion and reaction mixture was stirred for 16 hr at 22°C. The reaction mixture was diluted with DCM (20 mL) and washed by water (2x30 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered and filtrate was evaporated to dryness. Crude compound thus obtained was purified by flash colum chromatorgraphy to afford 172 (0.83 g, 59% yield) as white foam. 1H NMR (600 MHz, DMSO-d6) δ 11.41 (d, J = 2.2 Hz, 1H), 7.68 (dd, J = 8.1, 1.3 Hz, 1H), 6.67 (td, J = 7.1, 1.3 Hz, 1H), 5.75 (dd, J = 4.5, 1.3 Hz, 1H), 5.67 (ddd, J = 8.1, 2.3, 1.2 Hz, 1H), 4.18 (td, J = 5.4, 1.3 Hz, 1H), 3.95 - 3.90 (m, 1H), 3.85 (dtd, J = 7.1, 5.6, 1.3 Hz, 1H), 3.33 (d, J = 1.3 Hz, 3H), 2.48 - 2.43 (m, 1H), 2.39 (dtd, J = 14.8, 7.3, 1.3 Hz, 1H), 0.88 (d, J = 1.4 Hz, 9H), 0.10 (dd, J = 6.4, 1.3 Hz, 6H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 163.03, 150.37, 141.36, 134.87, 102.20, 90.39, 87.83, 81.18, 81.14, 72.71, 57.53, 36.06, 25.64, 17.78, -4.73, -5.01 ppm.
[00713] Compound 173: Compound 172 (0.65 g, 1.20 mmol), diethylphosphite (332.27 mg, 2.41 mmol, 309.95 μL) and propylene oxide (209.61 mg, 3.61 mmol, 252.54 μL) in DMF (15 mL) was added dropwise at 22°C. The solution was stirred for 16 hr at 90°C. The solution was evaporated under high vacuum and coevaporated with EtOH. Crude compound was purified by flash column chromatography (gradient: 40-90% EtOAc in hexane) to afford compound 173 (0.31 g, 49% yield) as yellow foam. 1H NMR (600 MHz, DMSO-d6) δ 11.43 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 5.82 (d, J = 4.8 Hz, 1H), 5.67 (dd, J = 8.1, 2.3 Hz, 1H), 4.19 (t, J = 4.9 Hz, 1H), 4.09 - 3.98 (m, 5H), 3.93 (q, J = 5.5 Hz, 1H), 3.33 (s, 3H), 2.98 (ddd, J = 17.7, 5.7, 4.2 Hz, 1H), 2.84 (ddd, J = 17.7, 6.0, 4.2 Hz, 1H), 1.25 (t, J = 7.1 Hz, 6H), 0.88 (s, 9H), 0.11 (d, J = 5.9 Hz, 6H) ppm. 13C NMR (151 MHz, DMS0-d6) δ 162.83, 150.26, 140.46, 102.16, 98.86, 98.52, 87.09, 80.65, 80.23, 73.48, 71.95, 71.52, 62.58, 62.56, 62.54, 62.52, 57.52, 25.52, 25.48, 22.24, 22.22, 17.62, 15.76, 15.72, -5.00, -5.10 ppm. 31PNMR (243 MHz, DMSO-d6) δ -7.83 ppm.
[00714] Compound 174: To a clear solution of compound 173 (0.85 g, 1.65 mmol) in THF (6 mL) was added hydrochloric acid, 36% w/w aq. soln. (1.5 mL) and reaction mixture was stirred at 22°C for 1 hr. TLC was checked and all the volatile matters were removed under high vacuum pump. The dark red residue thus obtained was purified by flash column chromatography (gradient: 0-10% MeOH in DCM) to afford compound 174 (0.57 g, 86% yield) as white foam. 1H NMR (600 MHz, DMSO-d6) δ 11.44 - 11.41 (m, 1H), 7.67 (d, J = 8.1 Hz, 1H), 5.84 (d, J = 4.9 Hz, 1H), 5.66 (dd, J = 8.1, 1.7 Hz, 1H), 5.43 (d, J = 6.1 Hz, 1H), 4.07 - 4.02 (m, 4H), 4.02 - 3.99 (m, 1H), 3.93 (ddd, J = 7.1, 5.8, 3.5 Hz, 2H), 3.36 (s, 3H), 2.96 (dt, J = 17.7, 4.6 Hz, 1H), 2.86 (ddd, J = 17.7, 6.5, 4.2 Hz, 1H), 1.25 (td, J = 7.0, 1.0 Hz, 6H) ppm. 13C NMR (151 MHz, DMSO-d6) δ 162.95, 150.41, 140.56, 102.28, 99.67, 99.33, 86.93, 81.29, 80.49, 80.48, 73.18, 71.23, 70.92, 62.75, 62.72,
57.73, 22.70, 22.67, 15.91, 15.86 ppm. 31PNMR (243 MHz, DMSO-d6) δ -7.69 ppm.
[00715] Compound 175: To a clear solution of compound 174 (0.40 g, 994.20 μmol) and 5- (ethylthio)-lH-tetrazole (129.41 mg, 994.20 μmol) in anhydrous DCM (20 mL) was added 2- cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (599.32 mg, 1.99 mmol, 631.53 μL). The reaction mixture was stirred at 22 °C for 1 hr. TLC analysis confirmed formation of the product. The reaction mixture was filtered, concentrated, and the residue thus obtained was purified by flash column chromatography (gradient: 0-5% MeOH in DCM) to afford compound 175 (0.32 g, 53% yield) as white foam. 1H NMR (600 MHz, CD3CN) δ 9.01 (s, 1H), 7.57 (t, J = 8.2 Hz, 1H), 5.85 (dd, J = 4.0, 2.3 Hz, 1H), 5.68 (dd, J = 8.2, 3.1 Hz, 1H), 4.34 - 4.06 (m, 6H), 4.00 - 3.93 (m, 1H), 3.89 - 3.62 (m, 4H), 3.50 - 3.37 (m, 3H), 2.97 (ddt, J = 18.0, 7.4, 4.4 Hz, 1H), 2.83 - 2.74 (m, 1H), 2.70 - 2.62 (m, 2H), 1.30 (dddd, J = 8.5, 7.1, 1.4, 0.7 Hz, 6H), 1.24 - 1.18 (m, 13H) ppm. 13C NMR (151 MHz, CD3CN) δ 163.32, 163.27, 150.87, 150.80, 140.65, 140.59, 119.23, 119.20, 102.93, 102.85, 99.41, 99.07, 98.90, 98.55, 89.05, 88.49, 82.51, 82.49, 82.05, 82.02, 80.24, 80.22, 80.21, 79.72, 79.70, 79.68, 79.66, 74.87, 74.55, 73.52, 73.41, 73.09, 72.98, 72.92, 63.67, 63.64,
59.44, 59.32, 58.83, 58.73, 58.70, 58.63, 58.62, 58.52, 58.50, 54.91, 45.57, 45.53, 43.77, 43.74,
43.69, 43.66, 24.60, 24.57, 24.55, 24.52, 24.50, 24.45, 24.40, 23.14, 23.11, 22.91, 22.89, 22.76,
22.74, 22.68, 22.67, 20.57, 20.52, 16.05, 16.03, 16.01, 15.99 ppm. 31PNMR (243 MHz, CD3CN) δ 150.17, 149.94, -7.72, -7.87 ppm.
Synthesis of compound 178
[00716] Compound 178 is synthesized according to Scheme 34.
[00717] Compound 176: To a solution of compound 143 (7.8 g, 20 mmol, 1.0 eq) in THF (60 mL) at room temperature, pyridine (20 ml), DMTrCl(13.4 g, 40 mmol, 2.0 eq) and AgNO3 (6.8 g,40 mmol, 2.0 eq) were added. After stirring for 16h at 50 °C in N2, diluted with EtOAc, washed with NaHCO3(aq) and brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by column chromatography on silica (hexane/EtOAc =1/1) to obtain Compound 176 (6.0 g, 44% yield) as a white solid. MS (M-H)': 687.1
[00718] Compound 177: To a clear solution of compound 176 (6.0 g, 9.0 mmol, 1.0 eq) in THF (50 mL) at 22 °C, tetrabutylammonium fluoride, 1 M in THF (13.5 mmol, 13.5 mL,1.5eq), was added slowly in a single portion and then stirred for 3 h. All the volatile matters were removed under high vacuum, and the residue thus obtained was purified by column chromatography (gradient: 0-10% MeOH in DCM) to afford Compound 177 (2.8 g, % 54%yield) as a white solid. MS (M-Na+): 597.2
[00719] Compound 178: To a solution of Compound 177 ( 4.0 g, 6.97 mmol, 1.0 eq) in DCM (60 mL) at room temperature, DIEA (9.0 g, 69.7 mmol, 10.0 eq ), 2-cyanoethyl-N,N,N',N'- tetraisopropylphosphorodiamidite (21.0 g, 69.7 mmol, 10.0 eq ) and CH2N4 (2.44 g, 34.9 mmol, 5.0 eq ) were added. After stirring for 0.5h at in N2, diluted with EtOAc, washed with NaHCO3(aq) and brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by PRE-HPLC to obtain compound 178 (2.5 g, 46% yield) as a white solid. 1H NMR (600 MHz, CD3CN) δ 9.05 (s, 1H), 7.43 (tt, J = 8.1, 1.3 Hz, 2H), 7.34 - 7.26 (m, 7H), 7.23 (ddd, J = 10.5, 7.7, 5.0 Hz, 2H), 6.86 (ddt, J = 6.8, 5.4, 1.5 Hz, 5H), 5.80 (dd, J = 4.7, 3.6 Hz, 1H), 5.57 (dd, J = 8.1, 1.3 Hz, 1H), 4.23 - 4.10 (m, 2H), 3.87 - 3.74 (m, 8H), 3.72 - 3.61 (m, 2H), 3.46 - 3.35 (m, 3H), 3.17 (dt, J = 8.1, 6.1 Hz, 2H), 2.69 - 2.59 (m, 2H), 2.12 - 2.01 (m, 1H), 1.88 (qd, J = 9.2, 4.2 Hz, 1H), 1.25 - 1.14 (m, 17H) ppm. 13C NMR (151 MHz, CD3CN) δ 163.76, 163.74, 159.59, 151.33, 151.31, 146.39, 146.36, 141.00, 140.95, 137.29, 137.24, 137.20, 130.93, 130.91, 130.88, 128.95, 128.91, 128.80, 128.79, 127.76, 119.61, 119.56, 114.00, 113.99, 103.09, 103.05, 88.77, 88.41, 87.00, 86.99, 82.93, 82.90, 82.51, 82.48, 81.68, 81.66, 81.26, 81.23, 75.16, 75.06, 74.98, 74.86, 60.96, 60.84, 60.74, 59.81, 59.69, 59.23, 59.10, 58.89, 58.87, 58.68, 58.66, 55.87, 44.16, 44.10, 44.08, 44.02, 34.40, 34.29, 25.02, 24.97, 24.93, 24.91, 24.88, 24.86, 21.15, 21.04, 20.99, 20.94 ppm. 31PNMR (243 MHz, CD3CN) δ 149.70, 149.53 ppm.
Synthesis of compound 184
[00720] Compound 184 is synthesized according to Scheme 35
[00721] Compound 180: A mixture of compound 179 (45 g, 65.5 mmol), TBSC1 (29.6 g, 196.3 mmol), imidazole (20 g, 294.1 mmol), KI (16.3 g, 98.1 mmol) in DMF (500ml) was stirred at room temperature for 16 hours. Upon completion, Pour the reaction mixture into ice water, filtered. The cake was dried over to afford the product (64.6 g, crude.) as a white solid. MS(M+H+): 802.5.
[00722] Compound 181: To a solution of compound 180 (64.6 g, 80.6 mmol) in DCM (500 ml) was added Et3SiH (23.3 g, 200.8 mmol) and TFA (9.2 g, 80.7 mmol) at OoC. The mixture was stirred at room temperature for 2 hours. Upon completion. The reaction mixture was washed with NaHCO3 (3 x 300 mL) and brine (3 X 300 mL). The organic layer with dried over Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (silica gel, 0-50% EA / PE) to give compound 181 (27.2 g, 54.5 mmol, 70.0% yield) as a white solid. MS(M-H+): 500.
[00723] Compound 182: To a solution of NaH (2.4 g, 60 mmol, 60% purity) in THF (30 ml) was added a solution of compound 181 and (diethoxyphosphoryl)methyl 4- methylbenzenesulfonate in THF (10 mL) dropwise. The mixture was stirred at room temperature for 16 hours. Upon completion, the reaction mixture was quenched by addition of EtOH (30ml) at 0 oC Then the mixture was concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (silica gel, 0-4% MeOH / DCM) to give compound 182 (5 g, 7.6 mmol, 38.0% yield) as a yellow solid. MS(M+H+): 650.5
[00724] Compound 183: To a solution of compound 182 (5 g, 7.7 mmol) in THF (50 ml) was added IM TBAF (11.6 ml, 11.6 mmol). The mixture was stirred at room temperature for 2 hours. Upon completion. The reaction mixture was diluted with EA (100 ml), washed with NH4Cl. The organic layer with dried over Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by silica gel column chromatography (silica gel, 0-3% MeOH/DCM) to give compound 183 (3.3 g, 6.1 mmol, 79.2%yield) as a yellow solid. MS(M+H+): 536.3
[00725] Compound 184: The solution of compound 183 (3.3 g, 6.1 mmol), 2,3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (2.8 g, 9.3 mmol) in DCM (30 ml) was added DIPEA (1.6 g, 12.4 mmol) and IH-Tetrazole (0.86 g, 12.2mmol) was stirred at room temperature for 3 hours. Upon completion. The mixture was washed with NH4Cl(aq) (60 mL X2), brine (60 mL X 3), the organic layer was dried over Na2SO4, filtered and concentrated, the crude was purified by chromatography (30% to 70%, ACN in 0.1%NH4HCO3/water) to give compound 184 (1.307 g, 1.7mmol, 27.8%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.72 (d, J = 3.5 Hz, 1H), 8.08 - 8.01 (m, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.6 Hz, 2H), 6.18 (dd, J = 10.0, 6.0 Hz, 1H), 4.66 (d, J = 7.6 Hz, 2H), 4.29 (d, J = 29.4 Hz, 1H), 3.94 (ddddd, J = 64.4, 53.8, 49.4, 8.4, 5.9 Hz, 12H), 3.41 - 3.33 (m, 4H), 2.83 (dt, J = 12.5, 6.2 Hz, 2H), 1.24 - 1.13 (m, 18H). MS(M+H+): 736.2
Synthesis of compound 1007
[00726] Compound 1007 is synthesized according to Scheme 36.
[00727] Compound 186: Diethyl P-(2-hydroxyethyl)phosphonate (14.84 g, 77.42 mmol, 13.62 mL, 95% purity) and Boron trifluoride etherate (28.91 g, 203.73 mmol, 25.14 mL) were added sequentially to a solution of compound 185 (20.8 g, 40.75 mmol) in DCM (180 mL). After stirring for 20h, the mixture was diluted with DCM and stirred with aqueous saturated bicarbonate (100 mL) and 50 ml of brine. The layers were separated, and the organic layer was washed with water, and brine Dried over sodium sulphate. Purification 0-100% EtOAc (330 g silica cartridge) to give compound 186 (19.06 g, 73%). 1H NMR (600 MHz, DMSO) δ 8.04 - 8.00 (m, 2H), 7.75 - 7.70 (m, 2H), 7.62 - 7.57 (m, 2H), 7.36 - 7.25 (m, 5H), 6.28 (d, J = 6.5 Hz, 1H), 5.95 (d, J = 8.2 Hz, 1H), 5.49 - 5.46 (m, 1H), 5.42 (d, J = 0.9 Hz, 1H), 5.37 - 5.30 (m, 2H), 4.60 (s, 2H), 4.41 (dd, J = 6.6, 4.4 Hz, 1H), 4.02 (dddd, J = 14.1, 11.9, 7.1, 3.6 Hz, 5H), 3.89 (ddt, J = 12.9, 10.1, 7.3 Hz, 1H), 3.76 (ddt, J = 16.9, 10.1, 6.8 Hz, 1H), 2.22 (dt, J = 17.5, 7.1 Hz, 2H), 1.23 (td, J = 7.1, 4.9 Hz, 7H). [00728] Compound 187: Palladium, 10% on carbon, (641.29 mg, 602.60 umol,) was added to a solution of compound 186 (19.06 g, 30.13 mmol) in MeOH (300 mL) and stirred overnight. The Pd was filtered off and rinsed with more MeOH (100 mL). The resulting solution was combined with potassium carbonate, anhydrous, 99% (12.49 g, 90.39 mmol) and stirred overnight. The next day, silica gel was added to the mixture and evaporated to dryness. The solid residue was loaded into an empty cartridge and purified using 0-8% MeOH in DCM to give compound 187 (10.57 g, 85%). 1H NMR (600 MHz, DMSO) δ 11.41 (d, J = 2.3 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 6.14 (d, J = 6.8 Hz, 1H), 5.59 (d, J = 4.9 Hz, 1H), 4.95 (s, 1H), 4.08 (t, J = 4.6 Hz, 1H), 4.04 - 3.96 (m, 4H), 3.94 (dd, J = 6.8, 4.1 Hz, 1H), 3.80 (ddt, J = 13.1, 10.0, 7.3 Hz, 1H), 3.63 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 3.29 (s, 3H), 2.19 - 2.11 (m, 2H), 1.23 (td, J = 7.0, 1.7 Hz, 6H).
[00729] Compound 1007: 2-Cyanoethyl tetraisopropylphosphorodiamidite (10.43 g, 34.60 mmol, 10.99 mL) and IH-Tetrazole (0.45 M, 15.38 mL) were added to a solution of compound 187 (9.42 g, 23.07 mmol) in MeCN (100 mL). After stirring for 5h, the volatiles evaporated to dryness, and the residue was re-disolved in EtOAc, washed with sat. bicarb, water and brine. The resulting residue was purified by silica gel column chromatography using 20-100% EtOAc as eluent to give compound 188 (8.1g, 57%). 1H NMR (600 MHz, DMSO) δ 11.44 (dd, J = 6.8, 2.2 Hz, 1H), 7.62 (dd, J = 8.2, 1.6 Hz, 1H), 6.10 (dd, J = 6.9, 4.8 Hz, 1H), 5.76 (dt, J = 8.1, 1.9 Hz, 1H), 5.07 (d, J = 38.1 Hz, 1H), 4.32 (ddd, J = 26.9, 10.3, 4.1 Hz, 1H), 4.14 -4.05 (m, 1H), 4.05 - 3.94 (m, 4H), 3.86 - 3.77 (m, 3H), 3.74 - 3.55 (m, 4H), 3.45 (dp, J = 18.2, 6.8 Hz, 1H), 3.34 (s, 1H), 3.32 (s, 3H), 3.28 (s, 1H), 2.85 - 2.76 (m, 2H), 2.15 (ddt, J= 14.3, 10.2, 7.4 Hz, 2H), 1.27 - 1.10 (m, 20H). 3 IP NMR (243 MHz, DMSO) δ 150.72, 149.77, 28.09, 28.02.
Synthesis of compound 1008:
[00730] Compound 1008 is synthesized according to Scheme 37.
[00731] Compound 188: To a solution of compound 84 (15.0 g, 40.2 mmol, 1.0 eq) in THF (150 mL) was added NaH (4.83 g, 120.8 mmol, 3.0 eq) portionwise at OoC. After stirred at OoC for 30 mins, diethyl vinylphosphonate (33.0 g, 201.3 mol, 5.0 eq) was slowly added. The mixture was stirred at OoC for 30 mins, warmed to rt for 16 hours under N2. The mixture was quenched with NH4Cl aq, extracted with EA (100 mL) twice, washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (DCM : MeOH = 20 : 1) to give 3 (16.2 g, crude) as a colorless oil. LC/MS (ESI, m/z): [(M+H] + = 537.3. [00732] Compound 189: To a solution of compound 188 (16.2 g, 22.3 mol, 1.0 eq) in MeOH (160 mL) was added KF (6.5 g, 111.8 mmol, 5.0 eq) at rt. The mixture was stirred at 70oC for 16 hours under N2. The mixture was concentrated under vacuum and purified by prep-HPLC (0.1%NH3.H2O/MeCN/H2O) to give compound 189 (2.5 g, 19.6% yield) as a white solid. LC/MS (ESI, m/z): [(M+H] + = 423.2.
[00733] Compound 1008: To a solution of compound 189 (2.0 g, 4.73 mmol, 1.0 eq) in DCM (20 mL) was added 2,3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (1.85 g, 6.15 mmol, 0.3 eq) andDIEA(916 mg, 7.10 mmol, 1.5 eq), followed by IH-tetrazole (497g, 7.10 mmol, 1.5 eq) portion wise. The mixture was stirred at rt for 2 hours under N2. The reaction mixture was added water, extracted with DCM (20 mL) twice, washed with brine (500 mL), dried over anhydrous Na2SO4, concentrated and purified by prep-HPLC (0.1%NH3.H2O/MeCN/H2O) to give compound 1008 (400 mg, 13.6% yield) as a colorless oil. 1H NMR (400 MHz, MeOD-d4) 5 7.98 (dd, J = 8.1, 2.4 Hz, 1H), 6.00 - 5.95 (m, 1H), 5.76 - 5.72 (m, 1H), 4.54 - 4.37 (m, 1H), 4.32 -4.18 (m, 1H), 4.18 -4.05 (m, 4H), 4.03 -4.00 (m, 1H), 3.95 - 3.59 (m, 8H), 3.54 - 3.45 (m, 3H), 2.82 - 2.68 (m, 2H), 2.30 - 2.11 (m, 2H), 1.36 - 1.18 (m, 18H).
Synthesis of compound 196
[00734] Compound 196 is synthesized according to Scheme 38. Scheme 38
[00735] Compound 190: To a solution of compound 1 (1 g, 2.68 mmol) in CH2Cl2 (25 mL) was added Dess-Martin periodinane (1.59 g, 3.76 mmol) at 0 °C and the mixture was stirred at room temperature for 3 h. The reaction was quenched by adding 1 : 1 mixture of saturated Na2S2O3 (aq.) and saturated NaHCO3 (aq.) and the organic layer was washed with saturated NaHCO3 (aq.) and brine. The organic layer was dried over Na2SO4 and concentrated under vacuum. The residue was dissolved in CH2Cl2 (30 mL) and ethyl triphenylphosphoranylideneacetate (1.87 g, 5.36 mmol) was added at room temperature. The mixture was stirred at this temperature overnight. The reaction mixture was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (40-100% ethyl acetate in hexane) to obtain compound 190 (1.09 g, 92% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.42 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 6.94 (dd, J = 15.6, 6.7 Hz, 1H), 6.08 (dd, J = 15.6, 1.3 Hz, 1H), 5.80 (d, J = 3.6 Hz, 1H), 5.67 (dd, J = 8.1, 2.3 Hz, 1H), 4.39 (td, J = 6.7, 1.3 Hz, 1H), 4.29 (dd, J = 6.4, 5.3 Hz, 1H), 4.18-4.13 (m, 2H), 3.97 (dd, J = 5.3, 3.6 Hz, 1H), 3.37 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 0.87 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H). MS (ESI+APCI) calculated for C20H33N2O7Si [M+H]+ m/z = 441.2057, found 441.2.
[00736] Compound 191: To a solution of compound 190 (1.5 g, 3.40 mmol) in CH3CN (30 mL) was added NaBH4 (644 mg, 17.0 mmol) and the mixture was stirred at room temperature overnight. To the reaction mixture was added NaBH4 (644 mg, 17.0 mmol) and the mixture was heated up to 90 °C and refluxed for 10 min. To the refluxing mixture was added slowly MeOH (5 mL) over 30 min and the mixture was refluxed for 2 h. The reaction was cooled in an ice-water bath and then quenched by adding saturated NH4Cl (aq.) and diluted with ethyl acetate. The organic layer was washed with water and brine and dried over Na2SO4 and then concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (60-100% ethyl acetate in hexane) to obtain compound 191 (836 mg, 61% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.38 (d, J = 1.9 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 5.76 (d, J = 4.4 Hz, 1H), 5.67 (dd, J = 8.1, 1.9 Hz, 1H), 4.42 (t, J = 5.1 Hz, 1H), 4.06 (t, J = 5.3 Hz, 1H), 3.86 (t, J = 4.4 Hz, 1H), 3.74 (dt, J = 8.2, 5.3 Hz, 1H), 3.42-3.39 (m, 2H), 3.32 (s, 3H), 1.72-1.66 (m, 1H), 1.60-1.43 (m, 3H), 0.88 (s, 9H), 0.09-0.09 (m, 6H). MS (ESI+APCI) calculated for C18H33N2O6Si [M+H]+ m/z = 401.2108, found 401.2.
[00737] Compound 192: To a solution of triphenylphosphine (917 mg, 3.50 mmol) in THF (20 mL) was added dropwise diisopropyl azodicarboxylate (688 μL, 3.50 mmol) at 0°C and the mixture was stirred at 0 °C for 30 min. To the mixture was a solution of compound 191 (700 mg, 1.75 mmol) and thioacetic acid (498 μL, 6.99 mmol) in THF (10 mL) was added dropwise at 0 °C and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (30-70% ethyl acetate in hexane) to obtain compound 192 (699 mg, 87% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.38 (d, J = 2.3 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 5.74 (d, J = 4.0 Hz, 1H), 5.66 (dd, J = 8.1, 2.3 Hz, 1H), 4.06 (t, J = 5.6 Hz, 1H), 3.85 (dd, J = 5.4, 4.0 Hz, 1H), 3.74-3.70 (m, 1H), 3.32 (s, 3H), 2.90-2.83 (m, 2H), 2.31 (s, 3H), 1.71-1.56 (m, 4H), 0.87 (s, 9H), 0.08-0.08 (m, 6H). MS (ESI+APCI) calculated for C20H35N2O6SSi [M+H]+ m/z = 459.1985, found 459.0.
[00738] Compound 193: To a mixture of CH3CN (15 mL) and 2.0 M aqueous HCl solution (3 mL) was added chlorosuccinimide (873 mg, 6.54 mmol) at -10 °C. To the mixture was added dropwise a solution of compound 192 (750 mg, 1.64 mmol) in CH2Cl2 (5 mL) at -10 °C and the resulting mixture was stirred at -10 °C for 6 h. After addition of EtOAc and ice, the organic layer was washed with water and brine and dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (30-70% ethyl acetate in hexane) to obtain compound 193 (551 mg, 70%) as a white form. 1H NMR (600 MHz, CDCl3) δ 9.31 (brs, 1H), 7.25 (s, 1H), 5.77 (d, J = 8.1 Hz, 1H), 5.66 (d, J = 2.1 Hz, 1H), 3.93 (ddd, J = 9.5, 7.5, 3.1 Hz, 1H), 3.85 (dd, J = 7.5, 5.2 Hz, 1H), 3.79-3.76 (m, 3H), 3.50 (s, 3H), 2.27- 2.19 (m, 2H), 2.00-1.94 (m, 1H), 1.81-1.75 (m, 1H), 0.89 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H). MS (ESI+APCI) calculated for C18H32ClN2O7SSi [M+H]+ m/z = 483.1388, found 483.0.
[00739] Compound 194: To a solution of isobutanol (344 μL, 3.73 mmol) and triethylamine (519 μL, 3.73 mmol) in CH2Cl2 (10 mL) was added dropwise a solution of compound 193 (600 mg, 1.24 mmol) in CH2Cl2 (3 mL) at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (30-70% ethyl acetate in hexane) to obtain compound 194 (614 mg, 1.18 mmol, 95% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.31 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 5.66 (d, J = 3.9 Hz, 1H), 5.56 (dd, J = 8.1, 2.2 Hz, 1H), 4.00 (t, J = 5.7 Hz, 1H), 3.87 (d, J = 6.4 Hz, 2H), 3.77 (dd, J = 5.7, 3.9 Hz, 1H), 3.69-3.66 (m, 1H), 3.41-3.31 (m, 5H), 1.96-1.89 (m, 1H), 1.84-1.75 (m, 3H), 1.72-1.65 (m, 1H), 0.91-0.88 (m, 15H), 0.09 (s, 3H), 0.09 (s, 3H). MS (ESI+APCI) calculated for C22H41N2O8SSi [M+H]+ m/z = 521.2353, found 521.2.
[00740] Compound 195:To a solution of compound 194 (600 mg, 1.15 mmol) in THF (5 mL) was added dropwise triethylamine trihydrofluoride (1.88 mL, 11.5 mmol) and the mixture was stirred at room temperature for 24 h. The reaction mixture was concentrated under vacuum and the crude residue was purified by column chromatography on silica gel (30-80% ethyl acetate, contained 5% MeOH, in hexane) to obtain compound 195 (423 mg, 90% yield) as a white form. 1H NMR (600 MHz, DMSO-d6) δ 11.39 (d, J = 2.3 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 5.76 (d, J = 4.3 Hz, 1H), 5.64 (dd, J = 8.1, 2.3 Hz, 1H), 5.19 (d, J = 6.5 Hz, 1H), 3.96 (d, J = 6.4 Hz, 2H), 3.90 (q, J = 5.9 Hz, 1H), 3.82 (t, J = 4.3 Hz, 1H), 3.76-3.73 (m, 1H), 3.39-3.34 (m, 5H), 1.93 (hept, J = 6.5 Hz, 1H), 1.83- 1.67 (m, 4H), 0.90 (d, J = 6.5 Hz, 6H). MS (ESI+APCI) calculated for C16H27N2O8S [M+H]+ m/z = 407.1488, found 407.0.
[00741] Compound 196: To a solution of compound 195 (450 mg, 1.11 mmol), 1- methylimidazole (8.82 μL, 111 μmol) and diisopropylethylamine (482 μL, 2.77 mmol) in CH2Cl2 (10 mL) was added dropwise 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (272 μL, 1.22 mmol) at 0 °C and the mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of saturated NaHCO3 (aq.) and diluted with ethyl acetate. The organic layer was washed with saturated NaHCO3 (aq.), water and brine and dried over Na2SO4 and then concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50% ethyl acetate in hexane) to obtain compound 196 (570 mg, 85% yield) as a white form. 1H NMR (600 MHz, CD3CN) δ 9.04 (brs, 1H), 7.38 (d, J = 8.1 Hz, 1H), 5.82-5.81 (m, 1H), 5.65- 5.63 (m, 1H), 4.13 (dt, J = 10.4, 5.3 Hz, 0.5H), 4.07-4.03 (m, 1H), 4.00-3.95 (m, 2.5H), 3.90-3.77 (m, 2.5H), 3.74- 3.61 (m, 2.5H), 3.45 (s, 1.5H), 3.41 (s, 1.5H), 3.24-3.19 (m, 2H), 2.69-2.66 (m, 2H), 2.02-1.86 (m, 4H), 1.79-1.72 (m, 1H), 1.20-1.17 (m, 12H), 0.95 (d, J = 2.5 Hz, 3H), 0.94 (d, J = 2.4 Hz, 3H). 3 IP NMR (243 MHz, CD3CN) δ 149.71, 149.46. MS (ESI+APCI) calculated for C25H44N4O89PS [M+H]+ m/z = 607.2567, found 607.2.
Synthesis of compound 210
[00742] Compound 210 is synthesized according to Scheme 39.
[00743] Compound 201: Aluminium foil (343.0 g, 4.2 mol) and dry 2-methoxyethanol (9000 mL) were heated, under reflux until all of the aluminium had been consumed. Compound 200 (958 g, 12.71 mol) was added and the reactants were heated, under reflux, for 16 hours. Absolute ethanol (9000 mL), followed by water (762.8 mL, 42.3 mol) and Celite were added to the cooled products. The resulting mixture was heated, under reflux, for 60 minutes and was then filtered. The residue was washed with ethanol (2 X 1000mL). The combined filtrate and washings were evaporated under reduced pressure. The material was purified by column chromatography on silica gel (10-0% PE in EA) to give 201 (488 g, 38.0% yield, 4.84 mmol) as a white solid. LC/MS (ESI, m/z): [(M+H] + = 303.2.
[00744] Compound 202: To the solution of compound 201 (488 g, 1.61 mol), imidazole (578.0 g, 8.05 mol) and Nal (241.3 g, 1.61 mol) in DMF (2500 mL) was added TBSC1 (970.6 g, 6.44 mmol) at ice-bath, stirred at room temperature for 16 hours. Diluted with water (4 L), washed with water (2 L X 3), brine (2L X 3), dried over Na2SO4, concentrated under vacuum to give 202 (800.0 g, 1.49 mol, 93% yield) as a yellow oil. LC/MS (ESI, m/z): [(M+H] + = 531.1.
[00745] Compound 203: A solution of compound 202 (800.0 g, 1.5 mol) in a mixture solvent of TFA/H2O = 1/1 (4.0 L) and THF (4 L) was stirred at OoC for 60 min. The resulting mixture was added con.NH3.H2O to pH = 7, and then extracted with EA (1.0 L X 2). The organic layer was washed with brine, dried over sodium sulfate and removed to give the residue was purified by column chromatography on silica gel (10-0% PE in EA) to give 203 (412 g, 65.6% yield, 0.98 mol) as a white solid. LC/MS (ESI, m/z): [(M+H] + = 417.3
[00746] Compound 204: The solution of compound 203 (412.0 g, 0.98 mol) , DBU (298.4 g, 196 mol) in DMF (4000 mL) ,BOM-C1 was added at 0 °C, was stirred at room temperature for 2 hours. After the reaction mixture was completed, diluted with EA (200 mL X 2). The filtrate was concentrated under vacuum, the residue was diluted with EA (12 L), washed with Water (2000 mL X 3), brine (2000 mL X 2), dried over Na2SO4, concentrated under vacuum to give 204 (300 g, crude) as a yellow oil. LC/MS (ESI, m/z): [(M+H] + = 537.4
[00747] Compound 205: To the solution of NaH (67.2 g, 1.68 mol, 60%) in THF (1500 mL) , added compound 204 (300 g, 0.56 mol) and DESMP(270.7 g, 0.84 mol) in THF (1500 mL) at - 20 °C under N2, was stirred 16 hour at room temperature. After the reaction mixture was completed, the reaction mixture was diluted with EtOH (1.5 L), concentrated, the crude was purified by column chromatography on silica gel (50-100% EA in PE, then 0-5% MeOH in DCM) to give compound 205 (164.0 g, 42.7% yield, 0.23 mol) as a white oil. LC/MS (ESI, m/z): [(M+H] + = 687
[00748] Compound 206: To the solution of compound 205 (60 g, 0.09 mol) and pyridine (56.0 g, 0.71 mol) in DCM (1.0 L) was added TMSBr (107.0 g, 0.70 mol) at OoC, and stirred at room temperature for 8 hours. The reaction mixture was quenched with water (300 mL), brine (300 mL), extracted with DCM (1 L X 3), EA (IL X 2). The organic layers were dried over Na2SO4, filtered and concentrated under vacuum to give compound 206 (48.4 g, crude) as a brown solid. LC/MS (ESI, m/z): [(M+H]+ = 631.43.
[00749] Compound 207: To the solution of compound 206 (48.4 g, 0.08 mol) in DMF (500 mL) was added DIPEA (50.0 g, 0.39 mol) and chloromethyl pivalate (46.0 g, 0.31 mol), stirred at 70 oC for 8 hours. After the reaction mixture was completed, the mixture was diluted with EA (2L), washed with water (500 mL X 3), brine (500 mL X 3), dried over Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography on silica gel (0-3% MeOH in DCM) to give compound 207 (26.0 g, 39.4% yield, 30.26 mmol) as a yellow oil. LC/MS (ESI, m/z): [(M+H] + = 859.2.
[00750] Compound 208: To the solution of compound 207 (25.0 g, 29.10 mmol) and FA (3 mL) in MeOH (300 mL) was added Pd/C (10%, 2.5 g), and stirred at room temperature for 16 hours under H2(1 atm). After the reaction mixture was completed, the reaction mixture was filtered, the filtrate was concentrated to give compound 208 (22.0 g, 100% yield) as a colorless oil. LC/MS (ESI, m/z): [(M+H] + = 739.3.
[00751] Compound 209: The solution of compound 208 (21.0 g, 28.42 mol) in water (200 mL) and FA (200 mL) was stirred at 40 oC for 2 hours. After the reaction mixture was completed, the mixture was adjust pH= 8 with NH3/H2O at OoC, then stirred for 30 min. Extracted with EA (2 L), washed with brine (1.0 L X2), the organic layer was dried over Na2SO4, filtered, concentrated under vacuum. The resulting residue was purified by column chromatography on silica gel (0~7% MeOH in DCM) to give 209 (15.0 g, 84.6% yield, 24.04 mmol) as a colorless oil. LC/MS (ESI, m/z): [(M+H]+ = 625.3
[00752] Compound 210: To a solution of 209 (15.0 g, 24.04 mmol) and IH-tetrazole (3.37 g, 48.14 mmol) in DCM (200 mL) was added DIPEA (6.2 g, 48.06 mmol) and 3- ((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (10.8 g, 35.88 mmol), the mixture was stirred at room temperature for 2 hours. The mixture was washed with NH4Cl(aq) (200 mL X 3), brine (200 mL X 3), the organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by reversed-phase chromatography (30% to 80%, ACN in 0.1%NH4HCO3/water) to give Compound 210 (10 g, 50.5% yield, 12.14 mmol) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 7.77 (dd, J = 8.1, 2.7 Hz, 1H), 5.86 (t, J = 5.2 Hz, 1H), 5.65 (ddd, J = 10.7, 6.3, 3.1 Hz, 5H), 4.40 - 4.24 (m, 1H), 4.19 - 4.04 (m, 2H), 4.03 - 3.94 (m, 2H), 3.85 - 3.55 (m, 8H), 3.47 - 3.40 (m, 2H), 3.26 - 3.17 (m, 3H), 2.79 (dt, J = 5.6, 3.9 Hz, 2H), 1.19 - 1.12 (m, 30H).LC/MS (ESI, m/z): [M+H]+= 825.3
Synthesis of compound 214v
[00753] Compound 214 is synthesized according to Scheme 40 Scheme 40
[00754] Compound 212: To a solution of compound 211 in THF is added NaH followed by addition of DESMP in THF at -20 °C under N2. After stirring overnight, the reaction mixture is completed, the reaction mixture is diluted with EtOH, concentrated, the crude is purified by column chromatography on silica to give compound 212.
[00755] Compound 213: A solution of compound 212 in water and formic acid is stirred at 40 oC for 2 hours, then the mixture is adjusted to pH= 8 with NH3/H2O at OoC, then stirred for 30 min. Extracted with ethyl acetate, washed with brine, the organic layer is dried over Na2SO4, filtered, concentrated under vacuum. The resulting residue is purified by column chromatography on silica gel to give compound 213.
[00756] Compound 214: To a solution of 213 and IH-tetrazole in DCM is added DIPEA and 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile, the mixture is stirred at room temperature for 2 hours. The mixture is washed with NH4Cl(aq), brine, the organic layer is dried over Na2SO4, filtered and concentrated under vacuum. The residue is purified flash chromatography to give Compound 21
Synthesis of compound 218
[00757] Compound 218 is synthesized according to Scheme 41.
[00758] Compound 216: To a solution of compound 215 in THF is added NaH followed by addition of DESMP in THF at -20 °C under N2. After stirring overnight, the reaction mixture is completed, the reaction mixture is diluted with EtOH, concentrated, the crude is purified by column chromatography on silica to give compound 216.
[00759] Compound 217: A solution of compound 216 in water and formic acid is stirred at 40 oC for 2 hours, then the mixture is adjusted to pH= 8 with NH3/H2O at OoC and stirred for 30 min. Extracted with ethyl acetate, washed with brine, the organic layer is dried over Na2SO4, filtered, concentrated under vacuum. The resulting residue is purified by column chromatography on silica gel to give compound 217.
[00760] Compound 218: To a solution of 217 and IH-tetrazole in DCM is added DIPEA and 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile, the mixture is stirred at room temperature for 2 hours. The mixture is washed with NH4Cl(aq), brine, the organic layer is dried over Na2SO4, filtered and concentrated under vacuum. The residue is purified flash chromatography to give Compound 218.
Synthesis of compound 225
[00761] Compound 225 is synthesized according to Scheme 42
[00762] Compound 220: To a mixture of compound 2 (73.0 g, 0.33 mol, 1.00 eq.) in THF (800 mL) was added PPh3 (106.4 g, 0.40 mol, 1.20 eq.) and (R)-2-(2,2-dimethyl-l,3-dioxolan-4- yl)ethan-l-ol (49.4 g, 0.33 mol, 1.00 eq.), followed by DEAD (76.5 g, 0.43mol, 1.30 eq) dropwise at OoC. The reaction was stirred at rt for 2 h under N2 atmosphere. The mixture was concentrated to give the crude. The crude product was triturated with MeOH (300 ml) for 30 min. The precipitate was filtered to give compound 220 (70.8 g, 60.9 % yield, 0.21 mol) as a white solid. LC/MS (ESI, m/z): [(M +H)] + = 345.3.
[00763] Compound 221: To a solution of compound 220 (70.8 g, 0.21 mol, 1.00 eq.) in MeOH (300 mL) was added NH3.H2O (300 mL). The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated to give a residue. The residue was purified by column chromatography (DCM: MeOH = 20:1) to give compound 221 (62.3 g, crude) as a white solid. LC/MS (ESI, m/z): [(M +H)] + = 241.2.
[00764] Compound 222: To a solution of compound 221 (62.3 g, 260 mmol, 1.00 eq) in MeOH (600 mL) was added TosOH (4.5 g, 26.0 mmol, 0.1 eq). The mixture was stirred at 20 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure to give Compound 222 (50.2 g, crude) as a white solid, which was used to the next step without further purification. LC/MS (ESI, m/z): [(M +H)] + = 201.0. [00765] Compound 223: To a solution of compound 222 (50.2 g, 0.25 mol, 1.00 eq) in Pyridine (600 mL) was added TrtCl (111.6 g, 0.40 mol, 1.60 eq) and DMAP (3.06 g, 25.1 mmol, 0.10 eq). The mixture was stirred at 80 °C for 16 h under N2 atmosphere. The reaction mixture was concentrated to give a residue. The residue was purified by column chromatography (DCM: MeOH = 10:1) to give compound 223 (53 g, 47.8% yield, 0.118 mol) as a colorless oil. LC/MS (ESI, m/z): [(M +Na)] + = 465.2.
[00766] Compound 224: To a solution of NaH (14.2 g, 0.35 mol, 60.0% purity, 3.00 eq) in THF (400 mL) was added a solution of compound 223 (52.3 g, 118.3 mmol, 1.00 eq) and diethyl (p- Toluenesulfonyloxymethyl)phosphonate (57.15 g, 0.177 mol, 1.50 eq) in THF (400 mL) dropwise at -20 °C. The mixture was stirred at rt for 16 h under N2 atmosphere. The reaction mixture was quenched by addition of EtOH (40.0 mL) at 0 °C and stirred for 30 min. Then the mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (DCM: MeOH = 20:1) to give compound 224 (56 g, 80.0% yield, 0.094 mol) as a colorless oil. LC/MS (ESI, m/z): [(M +Na)] + = 615.3.
[00767] Compound 225: To a solution of compound 224 (56.0 g, 60.8 mmol, 1.00 eq) in 1,4- dioxane (150 mL) was HCl/dioxane (50 mL, 4 M). The reaction mixture was stirred at rt for Ih. The mixture was concentrated and purified by prep-HPLC (H2O) to give Compound 8 (15.0 g, 45.3% yield) as a colorless oil. LC/MS (ESI, m/z): [(M +H)] + = 351.3.
Compound 501
[00768] Compound 501 was synthesized according to Scheme 43A
[00769] Compound 500: Compound 90 (200 mg, 0.49 mmol) was dissolved in 4 mL of anhydrous acetonitrile and pyridine (658 mg, 670μL, 8.33 mmol) under argon. The mixture was cooled to 0-5 °C under ice bath and then added with iodotrimethylsilane (1.37 g, 954μL, 6.86mmol). The reaction mixture warmed to room temperature and stirred for 3 hours. The volatiles in reaction mixture were evaporated under vacuum and the residue was purified by flash chromatography using 10-30% MeOH/DCM to give compound 500 (89 mg, 51%). 1H NMR (600 MHz, MeOD) δ 8.64 - 8.58 (m, IH), 8.16 - 8.04 (m, IH), 7.66 - 7.59 (m, IH), 6.00 - 5.88 (m, 1H), 5.73 (d, J = 8.1 Hz, 1H), 4.33 (t, J= 5.1 Hz, 1H), 4.08 - 3.98 (m, 1H), 3.90 - 3.80 (m, 2H), 3.78 - 3.67 (m, 2H), 3.48 (s, 3H). 31P NMR (243 MHz, MeOD) δ 17.53.
[00770] Compound 501: Compound 500 (20 mg) was dissolved in 4 mL of anhydrous DMF under argon. The solution was added tributylamine (42 mg, 0.228 mmol) and stirred for 5 min. And then was added with carbonyl-diimidazole (CDI) (102 mg, 0.628 mmol) and the reaction mixture was stirred at room temperature for 18 hours. To the reaction mixture was added Tris(tetrabutylammonium) hydrogen pyrophosphate (1.03 g, 1.14 mmol) and the reaction mixture was stirred for another 3.5 hours. The solvent was evaporated to dryness and the residue was purified by RP-CombFlash with C18 column to remove excess reagents. The result crude product (triethylamine salt) was further purified by Ion Exchange Chromatography (IEX) to obtain desired compound as TEA salt form. The pure fractions were pooled together as TEA salt and lyophilized three times before passing through Na-exchange resin to obtain as Na salt of the compound 501 (2.32mg, 8%). 1H NMR (600 MHz, D2O) δ 7.97 (d, J = 8.1 Hz, 1H), 5.93 (d, J = 4.4 Hz, 1H), 5.87 (d, J = 8.1 Hz, 1H), 4.43 (t, J = 5.2 Hz, 1H), 4.11 (dt, J = 5.5, 2.9 Hz, 1H), 4.00 (t, J = 4.8 Hz, 1H), 3.85 (dd, J = 11.2, 2.6 Hz, 1H), 3.79 (d, J = 8.7 Hz, 2H), 3.73 (dd, J = 11.3, 3.2 Hz, 1H), 3.40 (d, J = 0.6 Hz, 3H). 3 IP NMR (243 MHz, D2O) δ 8.26, -9.55, -22.92.)
Compound 503
[00771] Compound 503 was synthesized according to Scheme 43B
[00772] Compound 502: Compound 174 (330 mg) was dissolved in 4 mL of anhydrous acetonitrile and pyridine (658 mg, 670μL, 8.33 mmol) under argon. The mixture was cooled to 0- 5 °C under ice bath and then added with iodotrimethylsilane (1.37 g, 954μL, 6.86mmol). The reaction mixture warmed to room temperature and stirred for 3 hours. The volatiles in reaction mixture were evaporated under vacuum and the residue was purified by flash chromatography using 10-30% MeOH/DCM to give compound 502 (145mg, 51%).1H NMR (600 MHz, MeOD) δ 8.77 - 8.69 (m, 1H), 7.92 - 7.82 (m, 2H), 5.91 (d, J= 3.7 Hz, 1H), 5.86 - 5.76 (m, 1H), 4.18 (t, J = 5.9 Hz, 1H), 3.99 (dt, J= 6.2, 4.3 Hz, 1H), 3.86 (dd, J= 5.6, 3.7 Hz, 1H), 3.49 (d, J= 0.8 Hz, 3H), 2.87 (dt, J= 17.8, 4.3 Hz, 1H), 2.78 - 2.67 (m, 1H). 31PNMR (243 MHz, MeOD) δ -10.45. [00773] Compound 503: Compound 502 (30 mg) was dissolved in 4 mL of anhydrous DMF under argon. The solution was added tributylamine (42 mg, 0.228 mmol) and stirred for 5 min and then was added with carbonyl-diimidazole (CDI) (102 mg, 0.628 mmol) and the reaction mixture was stirred at room temperature for 18 hours. To the reaction mixture was added Tris(tetrabutylammonium) hydrogen pyrophosphate (1.03 g, 1.14 mmol) and the reaction mixture was stirred for another 3.5 hours. The solvent was evaporated to dryness and the residue was purified by RP-CombFlash with C18 column to remove excess reagents. The result crude product (triethylamine salt) was further purified by Ion Exchange Chromatography (IEX) to obtain desired compound as TEA salt form. The pure fractions were pooled together as TEA salt and lyophilized three times before passing through Na-exchange resin to obtain as Na salt of the compound 503 (6.46mg, 15%). 1H NMR (600 MHz, D2O) δ 7.78 (d, J = 8.0 Hz, 1H), 6.01 - 5.82 (m, 2H), 4.37 (t, J = 5.8 Hz, 1H), 4.07 (q, J = 5.3 Hz, 1H), 4.00 (dd, J = 5.7, 4.0 Hz, 1H), 3.40 (s, 3H), 2.79 (qt, J = 17.9, 4.5 Hz, 2H). 31PNMR (243 MHz, D2O) δ -7.45, -21.18, -22.91.
Example 2. Synthesis of siRNA with 5’ modifications
General Conditions for Solid-Phase Oligonucleotide Synthesis:
[00774] All oligonucleotides were synthesized on a MerMade-12 DNA/RNA synthesizer. Solvents/reagents purchased commercially were used as received. 500-A controlled pore glass (CPG) solid supports were obtained from Prime Synthesis 2’-OMe, 2’-F nucleoside and 2-O-C16 cytidine 3’-phosphoramidites were purchased from Hogene. The 2’-OMe-uridine-5’-bis-POM-(E)- vinyl phosphate (VP) 3’-phosphoramidite was purchased from Wuxi AppTec. 2’-O-C22 cytidine phosphoramidite was purchased.
[00775] After the synthesis, the oligonucleotides were cleaved from the support and deprotected using 28-30% ammonium hydroxide solution containing 5% (v/v) diethylamine (DEA) at 60 °C for 5h (O’Shea et al, Tetrahedron, 2018). For amino containing oligonucleotides, the CPG solid support was incubated with a mixture of ammonia and methylamine (50%-50%, v/v, AMA) 35 °C for 3 h. Purification was performed by ion-exchange chromatography with TSKGel SuperQ- 5PW(20) (Sigma) using an appropriate gradient of mobile phase (buffer A: 20 mM sodium phosphate, 15% ACN, pH 8.5; buffer B: 1 M NaBr, 20 mM sodium phosphate, 15% ACN, pH 8.5). The purified oligonucleotides were then desalted by size exclusion chromatography (SEC) using a custom packed with Sephadex G25 (GE Healthcare) and water as an eluent. The purity and molecular weight were determined using reverse phase HPLC and mass spectrometry, respectively. The lyophilized sense and antisense strands were first resuspended in distilled and deionized water. The concentrations of each strand were determined by determining the absorbance at 260 nm. Equimolar amounts of both strands were mixed and heated at 90oC, followed by slow cooling to form the duplex. The duplexes were then analyzed by mass spectrometry and tested for endotoxin as well as osmolality.Oligonucleotides containing a GalNAc ligand were synthesized using a solid supported trivalent GalNAc ligand (L96). The synthesis of this solid support has been described previously.
[00776] Ethyl protecting groups on phosphonates were removed with 0.18M trimethylsilyl iodide solution (2% pyridine in acetonitrile) for 1 hour.
Procedure for reverse H-phosphonate coupling of nucleoside 225
[00777] 5’DMTr group removed from precursor strand on automated synthesizer with 10% dichloroacetic acid in toluene, support washed with acetonitrile, dried with argon, and solid support transferred to a peptide synthesis vessel. Added IM diphenyl phosphite in pyridine and reacted for 30 minutes. Removed diphenyl phosphite solution, washed with acetonitrile, purged with argon and added 0.1M triethylammonium bicarbonate in water, reacted for 1 hour. Removed TEAB solution, washed with acetonitrile and purged vessel with argon.
[00778] Prepared solution of alcohol 225 (6eq), 120mM in 1:1 Acetonitrile:Pyridine as well as 6eq, 120mM solution of HATU in 1:1 Acetonitrile :Pyridine. Added nucleoside and activator solutions to solid support simultaneously and mixed for 1 hour. Removed coupling solution, rinsed with acetonitrile and purged with argon. Thiolation was performed with 0.5 M elemental sulfur in pyridine. Thiolation solution was removed after 10 minutes and CPG was washed with acetonitrile and dried under argon.
General procedure of the introduction of a 5’-alkylsulfonyl phosphoroamidate group
[00779] Synthesis of 5 ’-methylsulfonyl phosphoramidate containing oligonucleotides was carried out according to Scheme 44.
[00780] The antisense oligonucleotide was synthesized following standard protocols using phosphoramidite chemistry. After the last monomer was coupled and the oligo was still attached to the solid support, the corresponding 5’-ODMT protecting group was cleavage using a 3% dichloroacetic acid in DCM solution, the resin was washed with MeCN followed by coupling with Bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite (0.15M in CAN) using ETT as activator, 1:1 by volume with the amidite monomer. The resin was washed with MeCN followed by incubation with a 0.5M solution of the corresponding alkyl sulfonyl azide in acetonitrile at 30 °C for Ih. The resin was washed with MeCN and the oligo was cleaved from support and deprotected with 5% DEA in aqueous ammonia solution at 35 °C for 24h. The oligonucleotide was purified using a column packed with TSK-gel SuperQ-5PW (20) resin with Buffer A comp: 20mM sodium phosphate, 10% ACN in Water, Buffer B comp: 20mM sodium phosphate, IM sodium bromide, 10% ACN in water, Buffer pH 11, gradient 23-48% in 120 minutes. Desalted by size exclusion, Cytiva HiPrep 26/10 desalting column.
[00781] This strategy was used to incorporate modifications (u5ms), (u5mRs), (Pn2)Y392, (Pn2)Y469, (Pn3)us, (Pn4)us, (Pn5)us, Y476, (Pn2)Y338, and Y378 at the 5 ’-position of the antisense strand using the corresponding sulfonyl azide as oxidizing reagent.
[00782] Exemplary synthesis of 5 ’-modified oligonucleotides via H-phosphonate coupling is shown in Scheme 45.
[00783] The antisense oligonucleotide was synthesized following standard protocols using phosphoramidite chemistry. After the last monomer was coupled and the oligo was still attached to the solid support, the corresponding 5’-0DMT protecting group was cleavage using a 3% dichloroacetic acid in DCM solution, the resin was washed with MeCN. A solution of the nucleoside H-phosphonates (triethylammonium salts) in MeCN was added together with a solution of PivCl. After 5 min of coupling, the column was flushed with MeCN. A solution of iodine in pyridine was added to oxidize the H-phosphonate. The oligo deprotected under standard conditions.
On-support synthesis of oligo modified with Y303 modification
[00784] On-support synthesis of 5 ’-sulfonamide containing oligonucleotide was according to
Scheme 46.
[00785] An oligo containing a 5’-NH-monometoxytrityl-2’OMe-uridine nucleotide was synthesized on K&A automated synthesizer using PAC protected amidites. The oligo still bound to the solid support was treated with 10% v/v Piperidine/ Acetonitrile for 20 minutes with subsequent removal of MMTr group with 3% v/v DCA/DCM, support washed with MeCN. The solid support was transferred to a 25mL peptide synthesis vessel, added 5-10mL of 0.5M SO3-Pyin 1:1 Py:DMF, put in incubator and shaken at 40 for 24h. The supernatant was removed, and the solid support was washed with DMF. The oligo was cleaved from support and deprotected with 5% DEA in aqueous ammonia solution at r.t for Ih. The oligo was purified by anion exchange prep HPLC (Column packed with TSK-gel SuperQ-5PW (20) resin using Buffer A comp: 20mM sodium phosphate, 10% ACN in Water, Buffer B comp: 20mM sodium phosphate, IM sodium bromide, 10% ACN in water. Buffer pH 11 , gradient 25-50% in 150 minutes. The oligo was desalted by size exclusion chromatography (Cytiva HiPrep 26/10 desalting column).
Table 1. Exemplary Antisense Strands
Table 2. Exemplary Sense strands
Abbreviations used in the sequences are described in Table 14
Table 3. siRNA used for in vivo and in vitro studies.
Example 3: In vitro evaluation of siRNA conjugates
[00786] Cell Culture and Transfection: For transfection assay, 4.9 μL of Opti-MEM, 0.1 μL of Lipofectamine RNAiMax and 5 μL of 10x stock siRNA duplex in DPBS were added, following with incubation at room temperature for 15 minutes. When incubation time is up, 40 μL of BioIVT INVITROGRO CP Rodent Medium containing approximately 7.5x103 primary mouse hepatocyte cells were then added. The following reagents were added for free uptake assay: 5 μL of Opti- MEM, 5 μL of 10x stock siRNA duplex in DPBS and 40 μL of BioIVT INVITROGRO CP Rodent Medium containing approximately 7.5x103 primary mouse hepatocyte. Cells were incubated for 24 hours for transfection assay or 48 hours for free uptake assay at 37°C and 5% carbon dioxide prior to RNA purification. Each duplex was tested in four independent transfections.
[00787] Total RNA Isolation: RNA was isolated using an automated protocol on the HighRes Integration system using Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen™, Catalog No. 61012). Briefly, 70 μL of Lysis/Binding Buffer was added and shaken for 20 minutes. Ten μL of lysis buffer containing 3 μL of magnetic beads were then added to the plates. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were cap: ured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 90 μL Wash Buffer A and once with 90 μL Wash Buffer B. Beads were then washed with 90 μL Elution Buffer, re-captured, and the supernatant was removed.
[00788] Complementary DNA Synthesis: Complementary DNA (cDNA) was synthesized using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems™, Catalog No. 4374967) according to the manufacturer’s recommendations. A master mix containing 1 μL 1 OX Buffer, 0.4 μL 25X deoxyribonucleotide triphosphate, 1 μL 1 OX Random primers, 0.5 μL Reverse Transcriptase, 0.5 μL RNase inhibitor, and 6.6 μL of water per reaction was added to RNA isolated above. The plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours incubation at 37°C.
[00789] Quantitation of Messenger RNA by RT-qPCR: APP, SOD1, or TTR mRNA levels were quantified by performing RT-qPCR analysis. cDNA (1.25 μL) were added to a RT-qPCR master mix containing 0.5 μL of 20x human glycerol-3-phosphate dehydrogenase (GAPDH) TaqMan probe (Thermofisher, Cat# 4326317E) and 0.5 μL 20* specific Taqman (ThermoFisher Mm01344172_ml for APP or Mm01344233_g1 for SOD1 or Mm00443267_ml for TTR), 3 μLof Nuclease-free water, and 5 μL TaqMan™ Fast Advanced Master Mix for qPCR (Applied Biosystems™ Catalog No 4444558) per well in 384 well plates. The RT-qPCR assay was carried out in a QuantStudio 7 Pro Real-Time PCR Systems (ThermoFisher Scientific (Catalog No. A43055) using the TaqMan gene expression assay.
[00790] Data Analysis: To calculate relative fold change, real-time data were analyzed using the Delta-Delta Threshold Cycle (Relative Quantification) (AACt[RQ]) method [Schmittgen 2008] and normalized to control assays performed using cells transfected with 10 nmol/L AD 1955 (nontarget control), or DPBS for the free uptake assay. For all samples, APP, SOD1 or TTR mRNA levels were first normalized to GAPDH as a reference gene. Data are expressed as the percentage of APP, SOD 1 or TTR mRNA remaining and error is expressed as standard deviation (SD), derived from the 4 transfection or free uptake replicates. Means and SDs were calculated on CODEX LIMS. [00791] GraphPad Prism, Version 10.4.1 (GraphPad Software [San Diego, CA]), was used for graphical presentation of the concentration-response data. The IC50 values were obtained from curves fitted using a 4-parameter model. No statistical analysis was conducted.
Results
[00792] Results are summarized in Tables 4-9
Table 4. Dose-dependent reduction of TTR mRNAin primary mouse hepatocytes by free uptake
Table 5. Dose-dependent reduction of TTR mRNAin primary mouse hepatocytes by transfection
Table 6. Dose-dependent reduction of SOD1 mRNA in primary mouse hepatocytes by transfection
Table 7. Dose-dependent reduction of SOD1 mRNA in primary mouse hepatocytes by free uptake
Table 8. IC50 of selected siRNA.
Table 9. IC50 of selected siRNA
Example 4. In vivo evaluation of siRNA conjugates
[00793] In vivo mouse Study: All studies were conducted using protocols consistent with local, state, and federal regulations, as applicable, and were approved by the Institutional Animal Care and Use Committee (IACUC) at Alnylam Pharmaceuticals. Only female C57BL/6 mice (Charles River Laboratories) of 6 -8 weeks old mice used. Mice received subcutaneous administration of test article solutions at a dose volume of 10 μL/g. There are 3 mice for each group and mice were given a single subcutaneous (s.c.) administration of siRNA at 0.3mg/kg at day 0. Plasma samples were collected by using EDTA collection tube at days 0 (pre-dose); 7; 14; 21; 28; etc. Mouse TTR protein levels were determined by using Mouse Prealbumin (TTR) ELISA kit (Catalog #: 41- PALMS-E01, from Alpco) in accordance with the manufacturer’s protocol, and data were normalized to pre-bleed target protein levels.
[00794] In vivo study NHP (CNS): All studies were conducted using protocols consistent with local, state, and federal regulations, as applicable, and were approved by the Institutional Animal Care and Use Committee (IACUC) at Alnylam Pharmaceuticals. Female cynomolgus monkey (Charles River laboratories) were used. Monkeys received intrathecal administration of test article solutions at a fixed dose volume of 2 mL. There were 3 monkeys for each group and monkeys were given a single intrathecal (i.t.) administration of siRNA at a fixed dose of 30 mg at day 0 (APP siRNA), and 20 mg at day 29 (MAP2). Monkeys were euthanized on day 91 and tissue samples were harvested and flash frozen.
[00795] Soluble APP-α and β (sAPPα/β) Protein from CSF : sAPPα/β were measured from NHP CSF Samples using a commercially available ELISA from Meso Scale Diagnostics (Catalog # K15120E) in accordance with the manufacturer’s protocol. Samples post-dose were normalized to each individual animal pre-dose value to obtain the fraction sAPPα/β remaining.
[00796] Tissue APP mRNA: Tissue samples were harvested and flash frozen. Frozen tissue samples were disrupted by cryogenic grinding using the SPEX SamplePrep Geno/Grinder Automated Tissue Homogenizer and Cell Lyser with 3/8 inch (9.5 mm) diameter grinding balls per general recommendations from the manufacturer. RNA was extracted from approximately 20mg of tissue using MACHEREY-NAGEL NucleoMag® RNA kit according to manufacturer instructions. Taqman probes were used to measure target mRNA, APP (Assay ID: Mf01552291_ml) and MAP2 (Assay ID: Mf01103242_gl), and normalized to housekeeping genes GAPDH (Mf04392546_gl) and EIF3F (Mf02846493_gl). Values were normalized relative to the average of control animals.
[00797] Results of in vivo studies are shown in FIGS. 1-12.
Example 5. DNA polymerase inhibition assay for Y510s and Y486s phosphate analogs
[00798] The materials for the assay were purchased from commercial sources: Human DNA Polymerase Alpha Assay Kit Plus (ProFoldin Catalog # HDPA100KE1, Human DNA Polymerase Beta Assay Kit Plus (Profildin Catalog # DPB100KE), Human DNA Polymerase Gamma Assay Kit Plus (Profoldin Catalog # DPG100KE), dNTP Set (Thermo Scientific, Catalog # RO 182), 3H dTTP (VWR Catalog Number 101973-576), Aphidicolin (catalog# 5047440001, Sigma), ddNTP (catalog# 373278001, Millipore) DNA template and Primer (IDT). The assay was performed in a MicroBeta2 (with 96-well plate reader, Rewity) instrument.
[00799] The assay was performed as follows: Prepared a premix composed of 34 pl of H2O, 5 pl of 10 x assay buffer, 0.5 pl of 100 x DNA and 0.5 pl of 100 x human DNA polymerase. Add 40 pl of the premix into the wells with 5 pl of the compound monomer (or control ddNTP) in 96-well plastic plate. Incubated the mixture for 10 min in 37 C incubator. Add 2.5 pl of 20 x dNTP (dATP, dCTP, dGTP ImM. dTTP luM) and 2.5 pl of 3H-dTTP (luCi ) into each well Incubate the plate at 37C for 60 min. Take out the plate and add 50pl of 20% TCA to final concentration of 10% TCA. Put the plate in 4C fridge (or on the ice) for 40 minutes. The synthesized DNA is precipitated at 4C. Transfer all (lOOul) reaction solution to a new 96-well plate with fiber glass filter. Spin down at 3000rpm for 5 min. The plate will be extensively washed by 10% TCA twice, then 70% isopropanol for one time. Put the plate in the incubator. Dry plate at 600C for 60 min. Add 20ul of MicroScint in each dry well. Read plate in MicroBeta2 Microplate counter (30 second per well).
[00800] The results of DNA polymerase inhibition for compound 501 (Y487 triphosphate) are shown in Table 10.
Table 10. IC50 values for compound 501, 503, and selected positive and negative controls. Example 6 - siRNAs with antisense strands with 5’-(E)-vinyl phosphonate-α-L-LNA elicit stronger RNAi-mediated gene silencing than those with 5’-(E)-vinyl phosphonate-LNA
[00801] Conformationally constrained nucleotides, LNA or α-L-LNA, at the 5' terminus of the antisense strand impeded gene silencing of small interfering RNA (siRNA) by hindering phosphorylation, thereby deterring loading into the RNA-induced silencing complex. Installation of a phosphate mimic, (E)- vinyl phosphonate (VP), improved activity considerably. Gene silencing was more efficient when the antisense strand of the siRNA was modified with 5'-VP-α-L-LNA, which adopts a C3’-exo (South) conformation, than when the antisense strand was modified with 5 -VP-LNA, which adopts a C3'-endo (North) pucker. These data underscore the critical role of conformation of nucleotides in RNA interference.
[00802] Therapeutics that act through the RNA interference (RNAi) pathway are a promising class of medicines, as demonstrated by the FDA approvals of several small interfering RNA (siRNA) drugs. Chemistry has played a crucial role in advancing these therapeutics as modified nucleotides are necessary to enhance metabolic stability, potency, and delivery to target tissues.1, 2 [00803] The potency of an siRNA is influenced by the efficiency of loading of the antisense strand of the siRNA into the RNA-induced silencing complex (RISC); a 5’ phosphate is necessary for interaction with the MID domain of Ago2, the endonuclease component of RISC1, 2,3. Oligonucleotides with 2 ’-OH or 2’-O-methyl (2’-OMe) sugars are rapidly phosphorylated, but a synthetic phosphate mimic such as 5-(E)-vinyl phosphonate (5'- VP) must be incorporated in order for siRNAs with antisense strands ending with modifications such as 2’-O-methoxyethyl RNA, 2’- 5’ DNA, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA) RNA, or 2'-deoxy-2'-α-F-2'-β-C-methyl (gem-2'-F/Me) RNA to be loaded into RISC (Fig. 13, 1-V).4-9 Recently, we evaluated siRNAs with antisense strands modified at the 5' termini with the rigid bicyclic 2' -fluorinated Northem- methanocarbacyclic (FNMC) sugar (Fig. 13, VI). siRNAs with the FNMC modification were not active even with the 5 ’-VP.10 A key conclusion of our analysis of siRNAs modified with various sugar analogues was that a South-type (e.g., C2’-endo) conformation is critical for recognition by Ago2 and stable binding. 3’4, 6-10
[00804] The locked nucleic acid (LNA) modification has a methylene bridge that links the 2' oxygen to the 4' carbon of the RNA pentose ring, which fixes the pentose ring in the C3'-endo conformation (Fig. 14).11’ 12 A diastereomer of LNA, α-L-LNA, locks the pentose ring in the C3'- exo conformation (Fig. 14).13-18 The sugar conformations of LNA and α-L-LNA have been unequivocally established to be conformationally restricted in the C3'-endo and C3'-exo conformations, respectively, by NMR spectroscopy.19, 20 When incorporated into oligonucleotides, both LNA and α-L-LNA modifications improve affinity for RNA and nuclease stability, and both modifications have been well-studied in the context of RNase H, splice modulation, miRNA antisense oligonucleotides, and aptamers.11'18 In this example, the inventors investigated for the first time the impact of C3'-endo (North) LNA and C3'-exo (South) α-L-LNA incorporated at the 5' terminus of the antisense strand, with and without 5'-VP, on the RNAi-based gene silencing activity of siRNA.
Scheme 47. Synthesis of 5'-VP-LNA-U phosphoramidite (5).
[00805] To synthesize the 5'-VP-LNA phosphoramidite, the 5'-OH group of commercially available nucleoside I21 was oxidized by Dess-Martin periodinane to obtain 2, which was then reacted with bis-pivaloyloxymethyl (POM) reagent to afford the POM-protected 3 through a Wittig reaction (Scheme 46). After deprotection of the tert-butyldimethylsilyl (TBS) group from 3 followed by the phosphitylation reaction afforded the 5'-VP-LNA-U phosphoramidite building block 5. Similarly, 5'-VP-α-L-LNA-U phosphoramidite (12) was prepared from the corresponding α-L-LNA-U nucleoside (6) (Scheme 47). Reaction of 6, prepared as described,22 with TBS-chloride and imidazole in DMF, followed by treatment with aqueous trifluoroacetic acid in THF, afforded alcohol 8. Oxidation of 8 using Dess-Martin periodinane provided aldehyde 9, which was then converted to the POM-protected 10. Removal of the 3'-O-TBDMS group from 10 followed by phosphitylation afforded phosphoramidite 12. For both 5 and 12, the Z-isomers were obtained as minor side products. As Z-isomers usually exhibit poor RNAi activity,4, 6-8 they were not included in this study.
[00806] POM-protected monomers 5 and 12, as well as standard LNA VII23, α-L-LNA VIII (Scheme 47, ESI), 2’-F-RNA, and 2’-OMe-RNA phosphoramidites, were used to synthesize oligonucleotides via automated solid-phase oligonucleotide synthesis. When monomers 5 and 12 were incorporated, the final detritylation step was omitted. A one -step cleavage and deprotection was performed using aqueous ammonia containing 3% diethylamine (DEA). The crude oligonucleotides were purified by anion exchange HPLC and characterized by mass spectroscopy. The monomers were incorporated at the 5' termini of antisense strands of siRNAs targeting mouse transthyretin (Ttr) (Table 11, Table 12). The sense strand was conjugated at the 3' terminus to N- acetylgalactosamine (GalNAc). Strands were chemically modified as previously described.1, 2
Scheme 48. Synthesis of 5'-VP-α-L-LNA-U phosphoramidite (12).
[00807] si-1 and si-2 cany LNA (VII) and α-L-LNA U (VIII) monomers, respectively, at the 5' end of the antisense strand; the 5'-most linkage is a natural phosphodiester. Controls included the siRNAs with antisense strands with a 5'-terminal 2’-OMe with phosphodiester or phosphorothioate linkages between the two 5' terminal residues (si-3 and si-4, respectively). In mouse primary hepatocytes under free uptake conditions, si-1 and si-2 were considerably less potent than the control si-4 (Fig. 15A). The abilities of these siRNAs to reduce levels of serum Thin mice after a single subcutaneous dose of 1 mg/kg were also evaluated. Only si-4 reduced levels of Ttr significantly (Fig. 15B). The phosphodiester linkage between N1 and N2 in si-3 likely led to degradation and inactivity. We reason that kinase-catalyzed installation of phosphate groups on the bulky and conformationally restricted LNA and α-L-LNA 5'-terminal residues of si-1 and si-2 was impeded, resulting in diminished activity.
[00808] To test this hypothesis, the inventors incorporated LNA and α-L-LNA monomers functionalized with the phosphate-mimicking 5'-VP at the 5' termini of antisense stands of siRNAs. Under free-uptake conditions in primary mouse hepatocytes, the activities of siRNAs with antisense strands modified with 5’-VP-LNA and 5’-VP-α-L-LNA (si-5 and si-6, respectively) were comparable to the controls in which the 5' termini are 2'-OMe with a phosphodiester or phosphorothioate linkage (si-7 and si-8, respectively) (Fig. 16A). In mice at a dose of 1.0 mg/kg, potencies of the siRNAs were similar, although si-5 appeared to be slightly less potent (Fig. 18). An additional experiment at a dose of 0.4 mg/kg confirmed that si-6, with the antisense strand modified with 5’-VP-α-L-LNA was more potent than si-5, modified with 5 -VP-LNA (Fig. 13B). Potencies of si-6, si-7, and si-8 were comparable. That si-7, with a 5'-VP-2'-OMe with a phosphodiester linkage, was active is possibly due to the metabolic stability provided by the 5'-VP. [00809] Next, the inventors used computational modelling to evaluate how antisense strands modified with 5'-VP-LNA or 5'-VP-α-L-LNA interact with the Ago2 MID domain. A strand modified with 5'-VP-2’-OMe served as the reference structure (Fig. 17A)3,24 (see ESI for details). The 5'-VP-2’-OMe nucleotide adopts the C2’-endo sugar pucker as does the uridine in the parent structure.3 The model of a strand with a 5 ’-phosphate α-L-LNA computed as a prelude to the 5'-VP model had a torsion angle P of -179.5°, ideal for replacing the phosphate with the 5'-VP, and the 5'-VP-α-L-LNA residue maintained all hydrogen-bonding interactions seen in the model with 5'- VP-2’-OMe (Fig. 17B). The α-L-LNA pucker is C3’-exo (South), a neighbour range of C2'-endo in the pseudorotation phase cycle and therefore consistent with the preferred conformation for binding to the Ago2 MID domain.1 This analysis is in complete agreement with the solution NMR analysis of α-L-LNA.25
[00810] The precursor model with 5 ’-phosphate LNA has a torsion angle P of +88° (sc+), not ideal for incorporation of 5'-VP (Fig. 20A), and a North C3’-endo sugar pucker that required conformational adjustments within the nucleotide to accommodate the tight turn between the first two residues of the antisense strand necessary to establish optimal interactions with basic side chains of the MID and PIWI domains. Installing an 5'-VP moiety on the LNA residue and energy minimizing the model showed that it is possible to insert the phosphonate close to the locations of the corresponding moieties in the 5'-VP-2’-OMe model (Fig. 17C). However, the location of the phosphate in the 5'-LNA model is not as close to that in the crystal structure of Ago2 in complex with miR-20a as are the phosphonates in the 5'-VP-2’-OMe and 5'-VP-α-L-LNA models (Fig. 20A- 20B). Crucially, the locked sugar of LNA has non-optimal contacts as short as 2.9 A between its O2’-C4” bridge and Ago2 main chain atoms of amino acids C546 and V547, which are located in a β-strand. As the overlay between the 5'-VP-α-L-LNA and 5'-VP-LNA models illustrates, a slight movement of the amide moiety away from the locked sugar bridge in the latter does not fully alleviate the short contacts that are below the sum of van der Waals radii of atoms involved (Fig.
17D).
[00811] In summary, the inventors report syntheses of two new phosphoramidites, 5 and 12, and their incorporation into oligonucleotides to introduce 5’-VP-LNA and 5’-VP-α-L-LNA modifications. When conformationally restricted LNA and α-L-LNA nucleotides were positioned at the 5’ end of the antisense strand, gene silencing activity was poor as was the case for the conformationally constrained FNMC modification.10 The introduction of the 5'- VP modification in combination with α-L-LNA resulted in activity comparable to the siRNAs modified with a 5'-VP- 2'-0Me. The siRNA modified with 5’-VP-LNA was less potent akin to VP-FNMC. Molecular modelling studies showed that the 5'-VP-α-L-LNA fits well within the Ago2 MID domain, maintaining all hydrogen-bonding interactions observed in the crystal structure with an unmodified RNA. Thus, the presence of conformationally constrained nucleotides at the 5' terminus of the antisense strand impedes RNAi activity but installation of 5'-VP may result in RNAi activity if the 5’ nucleotide preorganizes the 5'-VP for favourable interaction with the MID domain. The α-L- LNA has a South pucker, the preferred conformation for Ago2 binding, whereas LNA, which has a rigid C3'-endo North pucker like FNMC, does not. Modification of the antisense strand with 5'- VP-α-L-LNA has the potential to expand the therapeutic utility of siRNAs. Furthermore, the 5'-VP modification was shown to provide sufficient metabolic stability to enable an antisense strand modified with 5'-VP-2'-OMe with a terminal phosphodiester linkage to attain comparable RNAi activity to an siRNA with a phosphorothioate linkage at that position.
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Supporting Information for Example 6
Experimental
[00812] General conditions: TLC was performed on Merck silica gel 60 plates coated with F254. Compounds were visualized under UV light (254 nm) or after spraying with the p- anisaldehyde staining solution followed by heating. Flash column chromatography was performed using a Teledyne ISCO Combi Flash system with pre-packed RediSep Flash-Prep-HPLC (IntelFlash-1) C18 Column packed with silica gel. All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercially available reagents and solvents were purchased from Sigma-Aldrich unless otherwise stated and were used as received. ESI-MS spectra were recorded on a Waters QTof Premier instrument using the direct flow injection mode. 1H NMR spectra were recorded at 300, 400, 500 and 600 MHz. 13C NMR spectra were recorded at 101 and 151 MHz. 31P NMR spectra were recorded at 161 , 202 and 243 MHz. Chemical shifts are given in ppm referenced to the solvent residual peak (DMSO-d6 - 1H: δ at 2.50 ppm and 13C δ at 39.5 ppm; CDCl3 - 1H: δ at 7.26 ppm and 13C δ at 77.16 ppm; CD3CN - 1H: δ at 1.94 ppm and 13C δ at 1.32 ppm respectively). Coupling constants are given in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), septet (sept), broad signal (brs), or multiplet (m). 31P NMR spectra were recorded under proton- decoupled mode.
[00813] Synthesis and characterization of building blocks
Scheme 19: Synthesis of phosphoramidite S2 for building block VIII
[00814] l-((lS,3R,4S,7R)-l-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-7-hydroxy- 2,5-dioxabicyclo[2.2.1]heptan-3-yl)pyrimidine-2,4(lH,3H)-dione SI: To a clear solution of 61 (0.4 g, 1.56 mmol) in pyridine (10 mL) was added 4,4'-dimethoxytrityl chloride (634.78 mg, 1.87 mmol) in two portions. Reaction mixture was stirred at for 16 hr, diluted with DCM (20 mL) and then quenched with 10% NaHCO3 (20 mL). Organic layer was washed with brine (2 x 20 mL), separated, dried over anhydrous Na2SO4 and filtered. Filtrate was evaporated under high vacuum pump and crude mass obtained, was purified by flash column chromatography (gradient: 0-5% MeOH in DCM) to afford SI (0.7 g, 80% yield). 1H NMR (600 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.42 (dd, J= 8.2, 1.4 Hz, 2H), 7.36 - 7.18 (m, 7H), 6.95 - 6.88 (m, 4H), 5.99 (s, 1H), 5.95 (d, J= 4.4 Hz, 1H), 5.69 (dd, J= 8.1, 1.4 Hz, 1H), 4.41 (d, J= 4.4 Hz, 1H), 4.23 (s, 1H), 4.12 (d, J= 8.4 Hz, 1H), 3.94 (d, J= 8.4 Hz, 1H), 3.74 (s, 6H), 3.35 - 3.27 (m, 2H) ppm. 13C NMR (151 MHZ, DMSO-d6) δ 163.3, 158.1, 150.4, 144.7, 140.5, 135.3, 135.2, 129.8, 129.8, 127.9, 127.7, 126.7, 113.3, 100.5, 89.2, 86.6, 85.4, 78.7, 72.9, 72.2, 60.1, 59.8, 55.0 ppm. HRMS (ESI+) m/z calcd for C31H31N2O8 [M + H]+ 559.2080, found 559.2089. [00815] 3-[[(4S,6R)-4-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4- dioxopyrimidin-l-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy- (diisopropylamino)phosphanyI] oxy propanenitrile.
[00816] Compound S1 (0.7 g, 1.12 mmol) was dissolved in dichloromethane (DCM) (10 mL) and the resultant clear solution was added N-methyl imidazole (NMI) (256.35 mg, 3.12 mmol, 248.88 μL) and diisopropylethylamine (DIPEA) (1.01 g, 7.81 mmol, 1.36 mL) in single portions. After stirring the reaction mixture for 5 minutes at 22 °C, 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite (739.01 mg, 3.12 mmol, 697.18 μL) was added and continued stirring for 1 hr and TLC was checked. Starting material was consumed and reaction mixture was diluted with DCM (15 mL). DCM layer was washed with 10% NaHCO3 (2 x 25 mL) solution, and brine (30 mL). Organic layer was separated, dried over anhydrous Na2SO4, filtered and filtrate was evaporated at 36°C to afford crude compound which was purified by flash chromatography (gradient: 35-80% EtOAc in hexane) to obtain S2 (0.8 g, 84%) as white foam. 1H NMR (500 MHz, CD3CN) δ 9.16 (s, 1H), 7.80 (dd, J= 8.2, 3.4 Hz, 1H), 7.50 - 7.43 (m, 2H), 7.38 - 7.27 (m, 7H), 7.23 (ddt, J= 9.3, 7.1, 2.9 Hz, 1H), 6.91 - 6.82 (m, 4H), 6.03 (t, J= 1.1 Hz, 1H), 5.68 (dd, J= 8.2, 2.7 Hz, 1H), 4.70 - 4.49 (m, 1H), 4.48 (t, J= 1.3 Hz, 1H), 4.22 - 4.11 (m, 1H), 4.01 - 3.87 (m, 1H), 3.82 - 3.68 (m, 8H), 3.65 - 3.46 (m, 3H), 3.44 - 3.35 (m, 2H), 2.62 (t, J= 6.0 Hz, 1H), 2.47 (t, J = 6.0 Hz, 1H), 1.15 - 1.06 (m, 11H), 0.98 (d, J = 6.8 Hz, 2H) ppm. 13C NMR (101 MHz, CD3CN) 8 164.1, 159.7, 159.7, 151.4, 145.9, 145.9, 141.6, 141.5, 136.6, 136.6, 136.5, 136.4, 131.1, 131.1, 131.0, 129.0, 128.9, 128.9, 127.9, 127.9, 119.5, 119.3, 114.1, 114.1, 90.6, 90.6, 90.4, 90.38, 88.2, 88.2, 87.0, 86.9, 79.4, 79.4, 79.0, 78.9, 75.8, 75.7, 75.3, 75.1, 74.1, 74.0, 61.2, 61.0, 61.0, 59.68, 59.5, 59.5, 59.3, 55.9, 55.9, 55.3, 44.2, 44.2, 44.1, 44.1, 25.0, 24.9, 24.9, 24.8, 24.7, 21.1, 21.0, 20.9, 20.9, 20.9 ppm. 31P NMR (202 MHz, CD3CN) δ 151.02, 150.40 ppm. HRMS (ESC) m/z calcd for C40H48N4O9P [M + H]+ 759.3159, found 759.3164.
[00817] (lS,3R)-7-[(tert-butyldimethylsilyl)oxy]-3-(2,4-dioxo-3H-pyrimidin-l-yl)-2,5- dioxabicyclo [2.2.1]heptane-l-carbaldehyde 9: To a clear solution of commercially available 12 (20 g, 53.984 mmol) in EtOAc (600.0 mL) at 0 °C was added a solution of Dess-Martin periodinane (114.49 g, 269.922 mmol) in DMSO (300 mL) slowly. The resulting solution was stirred for 8 hr at 22 °C. The reaction mixture was then added to 10% sodium hyposulfite solution (1500 mL). The resulting mixture was extracted with ethyl acetate (3 x 1000 mL). The combined organic layers were washed with water (2 x 1000 mL) and brine (2 x1000 mL). The organic layer was separated, dried over anhydrous Na2SO4 and concentrated to afford 2 (20 g, crude, quantitative) of as a white solid which was used for the next step without further purification.
[00818] [[(E)-2-[(lS,3R)-7-[(tert-butyldimethylsilyl)oxy]-3-(2,4-dioxo-3H-pyrimidin-l-yl)- 2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl([(2,2-dimethylpropanoyl)oxy]methoxy)phosphoryl] oxy] methyl 2,2-dimethylpropanoate 3: To a mixture of 2 (20.0 g, 54.28 mmol) and potassium carbonate (22.51 g, 162.839 mmol) in DMF (200 mL) was added bis-POM reagent (24.01 g, 37.991 mmol) at 0°C. The resulting solution was stirred overnight at 22 °C. LCMS analysis confirmed the formation of the product (E/Z=4:l). The reaction was then quenched by the addition of saturated NH4Cl (500 mL) at 0°C and then extracted with EtOAc (3 x 200 mL). Combined organic layer was washed with water (2 x 200 mL) and brine (2 x 200 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The residue was purified by flash-prep-HPLC [(CombiFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=50/50 increasing to ACN/H2O=95/5 within 30 min] to afford 3 (8.5 g, 23% yield) of as a oil and corresponding Z-isomer 3Z (1.5g, 4% yield).
[00819] Data for 3: 1H NMR (300 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 6.87 (dd, J= 24.6, 17.5 Hz, 1H), 6.27 (dd, J= 20.9, 17.5 Hz, 1H), 5.69 - 5.40 (m, 6H), 4.40 (s, 1H), 4.16 - 3.98 (m, 2H), 3.73 (d, J= 8.1 Hz, 1H), 1.16 (d, J= 1.8 Hz, 18H), 0.85 (s, 9H), 0.07 (d, J = 3.5 Hz, 6H) ppm. HRMS (ESI+) m/z calcd for C29H48N2O12PSi [M + H]+ 675.2714, found 675.2709.
[00820] HRMS (ESI+) m/z calcd for C24H40N7O4Si [M + H]+ 518.2911, found 518.2923.
[00821] Data for 3Z: 1H NMR (400 MHz, DMSO-d6) δ 11.38 (d, J= 2.2 Hz, 1H), 7.86 (d, J= 8.1 Hz, 1H), 6.61 (dd, J= 52.7, 14.5 Hz, 1H), 6.18 (dd, J= 18.3, 14.4 Hz, 1H), 5.70 - 5.50 (m, 6H), 4.39 (s, 1H), 4.12 (s, 1H), 4.02 (dd, J= 14.8, 7.6 Hz, 1H), 3.89 (d, J= 8.0 Hz, 1H), 1.17 (d, J = 1.4 Hz, 18H), 0.87 (s, 9H), 0.10 (d, J = 2.4 Hz, 6H) ppm. HRMS (ESI+) m/z calcd for C29H48N2O12PSi [M + H]+ 675.2714, found 675.2711.
[00822] ([[(2,2-dimethylpropanoyl)oxy]methoxy((E)-2-[(lS,3R)-3-(2,4-dioxo-3H-pyrimidin- l-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl)phosphoryl]oxy)methyl-2,2- dimethylpropanoate 4: Compound 3 (10.30 g)was suspended in 50% aqueous formic acid (500.00 mL) at 0°C. The resulting solution was stirred for 36 h at 35°C in an oil bath. The pH of the solution was adjusted to 7 with saturated aqueous sodium bicarbonate at 0°C. The resulting solution was extracted with ethyl acetate (3 x 800 mL) and the combined organic layer washed with water (2 x 200 mL) and brine (2 x 200 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated. The residue was purified by flash-prep-HPLC [(CombiFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=15/85 increasing to ACN/H2O=95/5 within 30 min] to afford 4 (4.7 g, 55% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.62 (d, J= 8.1 Hz, 1H), 6.92 (dd, J= 24.6, 17.5 Hz, 1H), 6.41 - 6.09 (m, 2H), 5.77 - 5.47 (m, 6H), 4.37 (s, 1H), 4.20 - 4.04 (m,lH), 3.92 (d, J= 4.6 Hz, 1H), 3.72 (d, J = 8.0 Hz, 1H), 1.18 (s, 18H) ppm. HRMS (ESI+) m/z calcd for C23H34N2O12P [M + H]+ 561.1849, found 561.1851.
[00823] ([[(2,2-dimethylpropanoyl)oxy]methoxy((Z)-2-[(lS,3R)-3-(2,4-dioxo-3H-pyrimidin- l-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl)phosphoryl]oxy)methyl-2,2- dimethylpropanoate 4Z: Into a 250-mL round-bottom flask purged and maintained with an inert atmosphere of argon, was placed 3Z (1.70 g).This was followed by the addition of 50% formic acid(80.00 mL) at 22 °C. The resulting solution was stirred for 40 h at 35 °C in an oil bath. The pH was adjusted to 7 with saturated aqueous sodium bicarbonate at 0°C. The resulting solution was extracted with 3x150 mL of ethylacetate and organic layers combined. The organic solution was washed with 2x100 ml of water and 2x100 mL of brine. The solution was dried over anhydrous sodium sulfate and concentrated. The residue was purified by Flash-Prep-HPLC [(CombiFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=5/95 increasing to ACN/H2O=95/5 within 30 min] to afford 4Z (0.52 g, % yield) of as a solid. 1H NMR (300 MHz, CD3CN) δ 9.11 (s, 1H), 8.13 - 7.84 (m, 1H), 6.79 - 6.46 (m, 1H), 6.09 (m, 1H), 5.63 (m, 6H), 4.39 (s, 1H), 4.24 - 4.08 (m, 1H), 4.03 (s, 1H), 3.95 (d, J= 8.5 Hz, 1H), 1.23 (p, J= 1.9 Hz, 18H) ppm. HRMS (ESI+) m/z calcd for C23H34N2O12P [M + H]+ 561.1849, found 561.1844.
[00824] [[(E)-2-[(lS,3R)-7-[[(2-cyanoethoxy)(diisopropylamino)phosphanyl]oxy]-3-(2,4- dioxo-3H-pyrimidin-l-yl)-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl([(2,2- dimethylpropanoyl)oxy] methoxy) phosphoryl] oxy Jmethy 1-2, 2-dimethylpr op ano ate 5: To a solution of 3-[[bis(diisopropylamino)phosphanyl]oxy]propanenitrile (4.62 g, 15.344 mmol) in DCM (43.00 mL) was added a solution of 1H-imidazole-4,5-dicarbonitrile (DCI) (1.36 g, 11.508 mmol) at 22 °C. After stirring for 10 min, a solution of 4 (4.30 g, 7.672 mmol) was added at 22 °C. The resulting solution was stirred for 1 hr at 22 °C. The reacting solution was diluted with DCM (300 mL) and washed with saturated aqueous NaHCO3 (100 mL) and brine (200 mL). The organic layer was dried over anhydrous Na2SO4 filtered and the filtrate was concentrated. The residue was purified by flash-prep-HPLC [(CombiFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=20/80 increasing to ACN/H2O=95/5 within 40 min] to afford 5 (5.4 g, 93% yield) as a solid. 1H NMR (600 MHz, CD3CN) δ 9.04 (s, 1H), 7.51 (dd, J= 8.2, 4.3 Hz, 1H), 7.04 - 6.85 (m, 1H), 6.34 - 6.23 (m, 1H), 5.68 - 5.56 (m, 6H), 4.52 (d, J= 11.7 Hz, 1H), 4.18 -4.04 (m, 2H), 3.87 - 3.77 (m, 2H), 3.74 (dtd, J= 10.7, 5.4, 2.4 Hz, 1H), 3.62 (tdd, J= 11.3, 8.9, 5.7 Hz, 2H), 2.77 - 2.61 (m, 2H), 1.26 - 1.12 (m, 31H) ppm. 13C NMR (151 MHz, CD3CN) δ 177.6, 177.6, 177.5,
163.9, 150.9, 143.7, 143.7, 143.3, 143.2, 139.9, 139.8, 122.0, 121.6, 120.7, 120.4, 119.6, 119.5,
102.4, 102.3, 88.7, 88.6, 87.6, 87.6, 87.5, 87.4, 87.4, 87.4, 87.2, 87.2, 82.8, 82.7, 82.7, 82.7, 80.9,
80.9, 80.6, 80.6, 76.4, 76.4, 76.2, 76.1, 74.5, 74.3, 59.6, 59.5, 59.4, 59.4, 44.2, 44.2, 44.2, 44.1,
39.4, 39.4, 27.1, 27.1, 25.0, 24.9, 24.8, 24.8, 24.8, 24.7, 21.0, 20.9 ppm. 31P NMR (243 MHz, CD3CN) δ 149.45, 149.31, 15.80, 15.49 ppm. HRMS (ESI+) m/z calcd for C32H51N4O13P2 [M + H]+ 761.2928, found 761.2916.
[00825] [[(Z)-2-[(lS,3R)-7-[[(2-cyanoethoxy)(diisopropylamino)phosphanyl]oxy]-3-(2,4- dioxo-3H-pyrimidin-l-yl)-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl([(2,2- dimethylpropanoyl)oxy] methoxy)phosphoryl]oxy]methyl 2,2-dimethylpropanoate 5Z; To roundbottom under an inert atmosphere of argon, was placed 3- [[bis(diisopropylamino)phosphanyl]oxy]propanenitrile (537.03 mg, 1.784 mmol) and DCI (126.32 mg, 1.070 mmol) in DCM (5.00 mL) at 22 °C. After 10 min to this reaction mixture was added 4Z (500.00 mg, 0.892 mmol, 1.00 equiv) at 22 °C. The resulting solution was stirred for 1 hr at 22 °C. The reacting solution was diluted with 300 mL of DCM and washed with 2x150 ml of water and 2x150 mL of brine. The solution was dried over anhydrous sodium sulfate and concentrated. The residue was purified by Flash-Prep-HPLC [(CombiFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=2O/8O increasing to ACN/H2O=95/5 within 48 min] to afford 5Z (0.230 g, 34.0% yield) as a white solid. 1H NMR (600 MHz, CD3CN) δ 9.07 - 8.89 (m, 1H), 7.97 (dd, J= 9.3, 8.2 Hz, 1H), 6.82 - 6.49 (m, 1H), 6.09 (ddt, J= 17.4, 14.5, 1.4 Hz, 1H), 5.66 - 5.53 (m, 6H), 4.54 (d, J= 17.5 Hz, 1H), 4.13 - 4.07 (m, 1H), 4.05 (dd, J= 8.2, 2.6 Hz, 1H), 3.99 (dd, J= 8.2, 4.4 Hz, 1H), 3.62 (dh, J= 10.4, 6.7 Hz, 2H), 2.64 (dt, J= 9.7, 5.9 Hz, 2H), 1.22 - 1.18 (m, 19H), 1.18 - 1.13 (m, 14H) ppm. 13C NMR (151 MHz, CD3CN) δ 177.7, 177.7, 177.7, 163.9, 151.0, 141.7, 141.35, 141.0, 140.9, 119.5, 119.5, 102.0, 102.0, 89.4, 89.3, 87.7, 87.6, 87.6, 87.4, 87.4, 87.4, 83.2, 83.2, 82.7, 82.7, 82.7, 80.0, 80.0, 79.9, 79.8, 76.2, 76.1, 76.0, 75.9, 74.1, 74.0, 59.7, 59.6, 59.5, 59.5, 44.2, 44.2, 44.2, 44.1, 39.4, 27.10, 24.85, 24.83, 24.80, 24.78, 24.75, 24.74, 24.68, 21.03, 20.98, 20.93 ppm. 31PNMR (243 MHz, CD3CN) δ 149.60, 149.16, 13.96, 13.76 ppm. HRMS (ESI+) m/z calcd for C32H51N4O13P2 [M + H]+ 761.2928, found 761.2922.
[00826] l-[(lS,3R)-7-[(tert-butyldimethylsilyl)oxy]-l-[[(tert-butyldimethylsilyl)oxy]methyl]- 2, 5-dioxabicyclo[2.2.1]heptan-3-yl]-3H-pyrimidine-2, 4-dione 7: To a clear solution of 61 (1.90 g, 7.416 mmol) in DMF (4.0 mL) was added imidazole (1.77 g, 25.955 mmol) at 22 °C. After stirring for 5 min, tert-butyldimethylsilyl chloride (TBSC1) (2.79 g, 18.51 mmol) was added at 22 °C. The resulting solution was stirred overnight at 22 °C. The reaction was then quenched by the addition of saturated NaHCO3 solution (100 mL). After stirring for 10 min, the resulting solution was extracted with EtOAc (2 x 100 mL). The organic layer was washed with water (100 mL) and brine (100 mL). The organic solution was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under high vacuum pump. The residue thus obtained was purified by flash column chromatography (gradient: 30-75% EtOAc in hexane) to afford 7 (3.11 g, 87% yield) of as a white foam. 1H NMR (300 MHz, DMSO-d6) δ 11.36 (d, J= 2.3 Hz, 1H), 7.73 (d, J= 8.1 Hz, 1H), 5.93 (s, 1H), 5.62 (dd, J= 8.1, 2.1 Hz, 1H), 4.49 (s, 1H), 4.21 (s, 1H), 3.87 (d, J= 9.3 Hz, 4H), 0.87 (d, J= 1.5 Hz, 18H), 0.13 (d, J= 3.7 Hz, 6H), 0.05 (s, 6H) ppm. HRMS (ESI+) m/z calcd for C22H41N2O6Si2 [M + H]+ 485.2503, found 485.2500.
[00827] l-[(lR,3R)-7-[(tert-butyldimethylsilyl)oxy]-l-(hydroxymethyl)-2,5- dioxabicyclo[2.2.1]heptan-3-yl]-3H-pyrimidine-2, 4-dione 8: To a clear solution of 7 (2.80 g, 5.776 mmol) in THF (30 mL) under inert atmosphere was added 50% aqueous TFA (15 mL) dropwise with stirring at 0°C. The resulting solution was stirred for 2 hr at 0 °C. The reaction was then quenched by the addition of saturated NaHCO3 solution (200 mL) at 0 °C. The resulting mixture was extracted with EtOAc (3 x 200 mL). The organic layer was washed with water (100 mL) and brine (100 mL). The organic solution was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under high vacuum pump. The residue thus obtained was purified by flash column chromatography (gradient: 0-5% MeOH in DCM) to afford 8 (1.68 g, 79% yield) of as a white foam. 1H NMR: (300 MHz, DMSO-d6) δ 11.40 (d, J= 2.3 Hz, 1H), 7.81 (d, J= 8.1 Hz, 1H), 5.95 (s, 1H), 5.65 (dd, J= 8.1, 2.1 Hz, 1H), 5.02 (s, 1H), 4.50 (s, 1H), 4.22 (s, 1H), 4.02 - 3.86 (m, 2H), 3.70 (s, 2H), 0.90 (s, 9H), 0.15 (d, J= 2.3 Hz, 6H) ppm. HRMS (ESI+) m/z calcd for Ci6H27N2O6Si [M + H]+ 371.1638, found 371.1641.
[00828] (lR,3R)-7-[(tert-butyldimethylsilyl)oxy]-3-(2,4-dioxo-3H-pyrimidin-l-yl)-
2,5dioxabicyclo [2.2.1]heptane-l-carbaldehyde 9: To a solution of 8 (1.70 g, 4.589 mmol) in anhydrous EtOAc (75 mL) under inert atmosphere was added Dess-Martin periodinane (5.84 g, 13.769 mmol) in DMSO (15 mL) dropwise at 0 °C .The resulting solution was stirred for 3 hr at 0 °C. To the reaction mixture was then added 10% aqueous sodium hyposulfite (200 mL) and was extracted with EtOAc (3 x 200 mL). The organic solution was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under high vacuum pump to afford 9 (1.7 g, crude, quantitative) as white solid which was used for the next step without further purification.
[00829] [[(E)-2-[(lR,3R)-7-[(tert-butyldimethylsilyl)oxy]-3-(2,4-dioxo-3H-pyrimidin-l-yl)- 2,5dioxabicyclo[2.2.1]heptan-l- yl]ethenyl([(2,2dimethylpropanoyl)oxy]methoxy)phosphoryl]oxy]methyl2,2- dimethylpropanoate 10: To a suspension of 9 (1.80 g, 4.885 mmol) in DMF (30 mL) at 0 °C was added potassium carbonate (2.03 g, 14.656 mmol). To this reaction mixture was added bis-POM reagent (3.71 g, 5.862 mmol) at 0°C. The resulting solution was stirred overnight at 22 °C. LCMS analysis confirmed the formation of the product (E/Z=4: 1). The reaction was then quenched by the addition of saturated ammonium chloride (200 mL) at 0°C. The resulting solution was extracted with of ethyl acetate (200 mL). The organic layer was washed with water (100 mL) and brine (100 mL). The organic solution was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under high vacuum pump. The residue was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=10:90 increasing to ACN/H2O=95:5 within 60 min. This resulted in major E-isomer 10 (1.5 g, 45% yield) as a white solid along with the Z-isomer 10Z (0.3 g, 9% yield) as white solid. 1H NMR(300 MHZ, DMSO-d6) δ 11.44 (s, 1H), 7.81 (d, J= 8.2 Hz, 1H), 6.74 (dd, J= 24.4, 17.4 Hz, 1H), 6.23 (dd, J = 21.4, 17.4 Hz, 1H), 6.10 (s, 1H), 5.70 - 5.57 (m, 5H), 4.62 (s, 1H), 4.37 (s, 1H), 4.06 (dd, J = 42.2, 8.6 Hz, 2H), 1.19 (d, J= 5.6 Hz, 18H), 0.89 (s, 9H), 0.14 (d, J= 3.1 Hz, 6H) ppm. HRMS (ESI+) m/z calcd for C29H48N2O12PSi [M + H]+ 675.2714, found 675.2724.
[00830] Data for 10Z: 1H NMR (400 MHz, DMSO-d6) δ 11.41 (d, J= 2.2 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 6.68 (dd, J= 52.8, 14.1 Hz, 1H), 6.20 (dd, J= 17.9, 14.1 Hz, 1H), 6.00 (s, 1H), 5.66 - 5.51 (m, 5H), 4.74 (s, 1H), 4.33 (s, 1H), 4.16 (d, J= 8.4 Hz, 1H), 3.98 (d, J= 8.4 Hz, 1H), 1.15 (d, J = 1.1 Hz, 18H), 0.88 (s, 9H), 0.15 (d, J = 3.1 Hz, 6H) ppm. HRMS (ESI+) m/z calcd for C29H48N2O12PSi [M + H]+ 675.2714, found 675.2734. [00831] (2,2-dimethylpropanoyl)oxy]methoxy((E)-2-[(lR,3R)-3-(2,4-dioxo-3H-pyrimidin-l- yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl)phosphoryl]oxy)methyl-2,2- dimethylpro panoate 11: A solution containing mixture of 10 (1.60 g, 2.371 mmol) in 120 mL of 50% aqueous formic acid was stirred for 2 days at 35 °C. The reaction was then quenched by the addition of 200 mL of saturated sodium bicarbonate at 0 °C. The resulting solution was extracted with EtO Ac (2 x 300 mL). The organic layer was washed with water (100 mL) and brine (100 mL). The organic solution was separated, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under high vacuum pump. The residue was purified by Flash-Prep-HPLC to afford 11 (0.72 g, 54% yield) as white solid. Purification conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, ACN/H20=10:90 increasing to ACN/H2O=95:5 within 60 min. 1H NMR (300 MHz, DMSO-d6) δ 11.44 - 11.38 (m, 1H), 7.81 (d, J= 8.1 Hz, 1H), 6.81 (dd, J= 24.4, 17.4 Hz, 1H), 6.41 - 6.11 (m, 2H), 6.02 (s, 1H), 5.70 - 5.57 (m, 5H), 4.38 (d, J= 9.0 Hz, 2H), 4.12 (d, J= 8.5 Hz, 1H), 3.94 (d, J = 8.5 Hz, 1H), 1.19 (s, 18H) ppm. HRMS (ESI+) m/z calcd for C23H33N2O12PNa [M + Na]+ 583.1669, found 583.1688.
[00832] ([[(2,2-dimethylpropanoyl)oxy]methoxy((Z)-2-[(lR,3R)-3-(2,4-dioxo-3H-pyrim,idin- l-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl)phosphoryl]oxy)methyl-2,2- dimethylpropanoate 11Z: A solution containing mixture of 10Z (300.00 mg, 0.445 mmol, 1.00 equiv), in 120 mL of 50% formic acid. The resulting solution was stirred for 2 days at 35 °C. The reaction was then quenched by the addition of 200 mL of ice water. The resulting solution was extracted with 2 x 300 mL of ethyl acetate. The organic solution was washed with 2 x 200 mL of water and 2 x 200 mL of brine. The solution was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by Flash-Prep-HPLC [(IntelFlash-1): Column, C18 silica gel; mobile phase, ACN/H20=10:90 increasing to ACN/H2O=95:5 within 60 min] to afford 11Z (0.14 g, 56% yield) as a white solid. 1H NMR (300 MHz, CD3CN) δ 8.34 (d, J = 8.2 Hz, 1H), 6.72 (dd, J= 52.7, 14.2 Hz, 1H), 6.10 (dd, J = 17.2, 14.2 Hz, 1H), 5.96 (s, 1H), 5.65 - 5.50 (m, 5H), 4.53 (s, 1H), 4.43 (s, 1H), 4.19 (d, J = 8.7 Hz, 1H), 4.07 (d, J = 8.7 Hz, 1H), 1.21 (d, J = 2.7 Hz, 18H) ppm. HRMS (ESI+) m/z calcd for C23H34N2O12P [M + H]+ 561.1849, found 561.1855. [00833] [[(E)-2-[(lR,3R)-7-[[(2-cyanoethoxy)(diisopropylamino)phosphanyl]oxy]-3-(2,4- dioxo-3H-pyrimidin-l-yl)-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl([(2,2- dimethylpropanoyl) oxy /meth oxy)phosphoryl] oxy /methyl 2,2-dimethylpropanoate 12: To a well stirred reaction mixture of DCI (227.56 mg, 1.927 mmol) and 3- [[bis(diisopropylamino)phosphanyl]oxy]propanenitrile (774.39 mg, 2.569 mmol) in DCM (5 mL) under an inert atmosphere was added 11 (720.00 mg, 1.285 mmol) dissolved in DCM (2 mL). The resulting solution was stirred for 1 hr at 22 °C. The reaction was then quenched by the addition of ice water (50 mL). The resulting solution was extracted with ethyl acetate (20 mL). The organic layer was washed with water (50 mL), separated, dried over anhydrous Na2SO4, then concentrated. The residue was purified by Flash-Prep-HPLC to afford 12 (0.7 g, 72% yield) as a white solid. Purification conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=10:90 increasing to ACN/H2O=95:5 within 50 min. 1H NMR (600 MHz, CD3CN) δ 8.98 (s, 1H), 7.69 (dd, J= 8.2, 5.7 Hz, 1H), 6.95 - 6.77 (m, 1H), 6.26 - 6.16 (m, 1H), 6.01 (d, J= 8.8 Hz, 1H), 5.67 - 5.57 (m, 5H), 4.59 (d, J= 10.8 Hz, 1H), 4.55 - 4.47 (m, 1H), 4.18 (dd, J= 8.8, 5.4 Hz, 1H), 3.93 (dd, J= 8.8, 6.6 Hz, 1H), 3.91 - 3.75 (m, 2H), 3.65 (dqt, J= 13.7, 6.8, 3.8 Hz, 2H), 2.75 - 2.64 (m, 2H), 1.25 - 1.15 (m, 33H) ppm. 13C NMR (151 MHz, CD3CN) δ 177.6, 177.5, 163.8, 151.2, 143.7, 143.6, 143.3, 143.2, 141.2, 141.2, 122.0, 121.7, 120.7, 120.4, 119.5, 101.5, 101.5, 89.2, 89.2, 89.0, 89.0, 88.9, 88.9, 88.8, 88.8, 88.2, 88.1, 82.8, 82.7, 82.7, 82.7, 82.7, 82.7, 82.7, 80.5, 80.5, 80.2, 80.2, 79.1, 78.9, 78.9, 78.8, 75.2, 75.0, 59.9, 59.7, 59.6, 44.3, 44.3, 44.3, 44.2, 39.4, 27.1, 27.1, 25.0, 25.0, 24.9, 24.8, 24.8, 24.8, 24.7, 21.0, 20.9, 20.9 ppm. 31P NMR (243 MHz, CD3CN) δ 149.66, 149.51, 15.99, 15.75 ppm. HRMS (ESI+) m/z calcd for C32H5IN4O13P2 [M + H]+ 761.2928, found 761.2932.
[00834] [[(Z)-2-[(lR,3R)-7-[[(2-cyanoethoxy)(diisopropylamino)phosphanyl]oxy]-3-(2,4- dioxo-3H-pyrimidin-l-yl)-2,5-dioxabicyclo[2.2.1]heptan-l-yl]ethenyl([(2,2- dimethylpropanoyl)oxy/ m ethoxy) phosphoryl] oxy /methy 1-2, 2-dimethylpr op ano ate 12Z: Under inert atmosphere of argon, to a solution of DCI (35.40 mg, 0.300 mmol) in DCM (0.7 mL) was added 3-[[bis(diisopropylamino)phosphanyl]oxy]propanenitrile (150.57 mg, 0.500 mmol) and stirred for 10 min. To this reaction mixture was added HZ (140.00 mg, 0.250 mmol) in DCM (2 mL). The resulting solution was stirred for 2 hr at 22 °C. The reaction was then quenched by the addition of 20 mL of ice water. The resulting solution was extracted with 3 x 20 mL of ethyl acetate. The organic solution was washed with 2 x 50mL of water. Then the solution was dried over anhydrous sodium sulfate and concentrated. The residue was purified by Flash-Prep-HPLC [(IntelFlash-1): Column, C18 silica gel; mobile phase, ACN/H2O=10:90 increasing to ACN/H2O=95:5 within 60 min] to afford 12Z (86 mg, 45% yield) as a white solid. 1H NMR (600 MHz, CD3CN) δ 9.12 - 8.81 (m, 1H), 8.30 (dd, J= 15.3, 8.2 Hz, 1H), 6.90 - 6.60 (m, 1H), 6.10 (ddd, J= 17.5, 14.1, 3.6 Hz, 1H), 6.02 - 5.97 (m, 1H), 5.64 - 5.52 (m, 6H), 4.70 (t, J= 9.1 Hz, 1H), 4.62 - 4.50 (m, 1H), 4.24 (d, J= 8.7 Hz, 1H), 4.05 (dd, J= 8.7, 2.8 Hz, 1H), 3.92 - 3.75 (m, 2H), 3.65 (dh, J= 10.4, 6.7 Hz, 2H), 2.68 (q, J= 6.0 Hz, 2H), 1.26 - 1.17 (m, 35H) ppm. 13C NMR (151 MHz, CD3CN) δ 177.6, 177.6, 164.0, 151.2, 142.4, 142.2, 142.2, 142.1, 124.7, 124.3, 123.5, 123.1, 119.5, 119.5, 101.2, 101.2, 89.2, 89.2, 88.4, 88.3, 88.3, 88.3, 88.1, 88.1, 88.1, 88.0, 82.9,
82.9, 82.8, 82.8, 82.8, 82.7, 82.7, 79.7, 79.7, 79.4, 79.4, 78.4, 78.3, 78.1, 78.0, 74.4, 74.2, 59.8,
59.7, 59.7, 59.6, 44.3, 44.3, 44.2, 44.2, 39.4, 39.4, 27.1, 24.9, 24.9, 24.9, 24.8, 24.8, 24.7, 21.0,
21.0, 20.9 ppm. 31PNMR (243 MHz, CD3CN) δ 149.68, 149.24, 13.76, 13.55 ppm. HRMS (ESI+) m/z calcd for C32H51N4O13P2 [M + H]+ 761.2928, found 761.2940.
[00835] Oligonucleotide synthesis and purification: Sense strands were synthesized on L96- tri-N-acetylgalactosamine (GalNAc)-cluster3 immobilized on controlled pore glass (CPG) solid support. On the other hand, antisense strands were assembled on 2'-O-methyl-uridine loaded CPG- solid support, available from LGC Biosearch Technologies (Petaluma, CA, USA) (porosity 616 A, loading 87 pmol/g). The single strands were synthesized on K&A H-8-SE synthesizer. A solution of 0.5 M 5-(5'-ethylthio)-lH-tetrazole in acetonitrile (CH3CN) was used as the activator. The solutions of commercially available phosphoramidites and synthesized phosphoramidities were used at 0.1 M in anhydrous CH3CN or CH2Cl2.The oxidizing reagent was 0.05 M I2 in THF/pyridine/H2O. 100 mM solution of 3-Amino-l,2,4-dithiazole-5-thione (or Xanthane hydride obtained from TCI Chemicals, Germany) dissolved in acetonitrile-pyridine (2:3 v/v) was employed as sulfurizing agent . The detritylation reagent was 3% trichloroacetic acid in CH2CI2. Waiting times for coupling, capping, oxidation, and sulfurization step were 450 s, 25 s, 80 s, and 300 s, respectively. After completion of the automated synthesis, the oligonucleotide was manually released from the solid support and deprotected using AMA (1:1 (v/v) mixture of concentrated aqueous ammonia and 40% aqueous methylamine, both available from Sigma Aldrich). For 5’-VP- modified antisense strands, cleavage from solid support and quantitative deprotection was achieved using 3% DEA in concentrated aqueous NH3, following published protocol (Tetrahedron 2018, 74, 6182)
[00836] After filtration through a 0.45-pm nylon filter, oligonucleotides were purified by ion exchange HPLC using a Dionex DNA Pae 100 (9 x 250 mm) (ThermoFisher, Dreieich, Germany). Appropriate gradients of mobile phase (eluent A: 20 mM TRIS buffer, 20% CH3CN, pH 7.4; eluent B: 500 mM NaCIO4 in eluent A) were employed. Oligonucleotides were desalted using sizeexclusion chromatography using a column packed with Sephadex G25 (GE Healthcare) and water as an eluent. Oligonucleotides were then quantified by measuring the absorbance at 260 nm. Extinction coefficients were calculated using the following extinction coefficients for each residue: A, 13.86; T/U, 7.92; C, 6.57; and G, 10.53 M-1cm-1. The identities of modified oligonucleotides were verified by mass spectrometry. Sequences and mass spectroscopy data are shown in Table 9. Purities were evaluated by analytical reverse-phase HPLC. For reverse-phase HPLC, a C-18 column was used with a gradient of 2-29% buffer B (eluent A: 95 mM hexafluoroisopropanol, 16.3 mM TEA, 0.05 mM EDTA; eluent B: MeOH) over 39 min.
Table 12: Table characterization
[00837] In vitro siRNA activity: The in vitro activity of modified and control duplexes was evaluated for gene silencing in cell culture by targeting the target TTR. Primary mouse hepatocytes (PMH) were plated in 96 well format for free uptake with hepatocyte plating medium (Primacyt, Germany). lOx stocks were prepared to create free uptake final concentrations of 100, 10, InM. Each duplex was tested in quadruplicate. Cells were incubated 48 hours for free uptake at 37°C and subsequently lysed for mRNA quantification using the Quantigene Singleplex assay system (Thermo), according to the manufacurers protocol. Probesets for murine TTR and GAPDH were custom designed by Thermo. Luciferase signal was read on a Victor light plate luminometer (Perkin Elmer) For each well, TTR mRNA level was normalized to the respective GAPDH mRNA level. The activity of a given siRNA was expressed as percent of TTR mRNA concentration (normalized to GAPDH mRNA) in treated cells, relative to the TTR mRNA concentration averaged across control wells.
[00838] Evaluation of silencing in mice. All studies were conducted following the animal welfare regulations of the state of Bavaria (Germany) and the European Union. Protocols were approved by the government of lower Franconia. Mice received a single subcutaneous injection of 1 mg/kg siRNA, prepared in an injection volume of 5 μL/g body weight in PBS. At the indicated time pre- or post-dosing, blood was obtained by puncturing the facial vein. TTR protein was quantified by ELISA from serum isolated from whole blood. The ELISA was performed according to the manufacturer’s protocol (ALPCO, 41-PALMS-E01) after a 4000-fold dilution of the serum samples. TTR protein concentration was calculated from a standard curve, and each data point is the average of all the mice within each cohort (n = 3).
[00839] Modeling studies: In Figs. 17A-17D, models were built based on the crystal structure of the complex between miR-20a and human Ago2, PDB ID 4f3t.4 The build/modify options in UCSF Chimera5 were used to install the modified residues. All models were energy-minimized with the AMBER ffl4 force field6 as implemented in UCSF Chimera until conversion. The 5 -VP- 2'-OMe nucleotide adopts the C2'-endo sugar pucker as does the uridine in the parent structure.4 This computational model was overlaid on the crystal structure of an RNA carrying the 5’-VP bound to Ago2, PDB ID 5t7b.7
References
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6. D. A. Case, T. E. Cheatham lii, T. Darden, H. Gohlke, R. Luo, K. M. Merz Jr, A. Onufriev, C. Simmerling, B. Wang and R. J. Woods, J. Comut. Chem., 2005, 26, 1668-1688.
7. E. Elkayam, C. R. Faehnle, M. Morales, J. Sun, H. Li and L. Joshua-Tor, Mol Cell, 2017, 67, 646-658. Example 7 - Evaluation of siRNA comprising exemplary compounds
[00840] Activity of double-stranded RNAs comprising some exemplary compounds described herein was evaluated in vivo in CNS via mouse ICV.
[00841] The siRNA used in this study are shown in Table 13.
[00842] The results are shown in FIGS. 29 and 30.
Table 14: Abbreviations used in sequences
[00843] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

CLAIMS What is claimed is: 1. A compound of Formula (I), (I), or (II): or a salt thereof, wherein: n is an integer selected from 1 - 3; A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2, wherein * is the bond to E; E is a bond or -CH2-; B is an optionaly modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionaly replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; one of R2’and R3’is hydrogen, halogen, or -OR20, alkyl, branched alkyl, aminoC1- 6alkyl(e.g., branched aminoC1-6alkyl), C2-6alkenyl, C2-6alkynyl, C1-6alkyl ester, C1-6alkylthio (e.g., branched C1-6alkylthio), C1-6alkylamino (e.g., branched N-C1- 6alkylamino), C2-6alkenylthio (e.g., branched C2-6alkenylthio), N- C2- 6alkenylamino (e.g., branched N- C2-6alkenylamino), C2-6alkylthioester, N-C1- 6alkylcarbamyl, wherein: R20 is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); the other of R2’and R3’is -OR30, wherein R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; provided that: (a) Q is not , wherein * is the bond to the phosphorous atom; (b) when X is O or S, and each Rp is ORO, wherein each RO is hydrogen or an oxygen protecting group, then Q is not , wherein * is the bond to the phosphorous atom; and (c) the compound is not of the formula, . 2. The compound of claim 1, wherein Q is , , , where * is the bond to the phosphorous atom. 3. The compound of claim 1 or 2, wherein Q is , , , , , , , , , , , , , . 4. The compound of claim 1, wherein the compound is of Formula (Ia): . 5. The compound of claim 4, wherein the compound is of formula, , , r Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. 6. The compound of claim 5, wherein the compound is of formula, . 7. The compound of claim 5, wherein the compound is of formula, . 8. The compound of claim 7, wherein the compound is of formula, , . 9. The compound of claim 5, wherein the compound is of formula, .
10. The compound of claim 5, wherein the compound is of formula, . 11. The compound of claim 5, wherein the compound is of formula, 12. The compound of claim 1, wherein the compound is of Formula (Ib), . 13. The compound of claim 1, wherein the compound is of Formula (Ia), (Ib), (Ic) or (Id): a 14. The compound of claim 1, wherein the compound is of the formula, r .
15. The compound of claim 1, wherein the compound is of the formula, 16. The compound of claim 1, wherein the compound is of Formula (IIa), , wherein n is 1, 2 or 3. 17. The compound of any one of claims 1-16, wherein X is O. 18. The compound of any one of claims 1-16, wherein X is S. 19. The compound of any one of claims 1-18, wherein at least one RP is -ORO, optionaly, each RP is independently -ORO. 20. The compound of claim 19, wherein each RO is independently H, methyl, ethyl, propyl, 1- methylethyl, butyl, or tert-butyl, optionaly each RO is independently H, methyl, or ethyl. 21. The compound of claim 20, wherein each RO is independently a hydroxyl protecting group. 22. The compound of claim 21, wherein each RO is independently pivaloyloxymethyl (POM), ethyl, methyl, isopropyl, tert-butyl, trihaloalkyl, benzyl, nitrobenzyl, chlorobenzyl, fluorenyl-9-methyl, 2-cyanoethyl, 2-chlorophenyl, 2,2,2-25-trihalogen-1,1-dimethylethyl, 5-chloroquin-8-yl, 2-methylthioethyl, or 2-methylthioethyl, optionaly each RO is independently POM or ethyl. 23. The compound of any one of claims 1-22, wherein at least one RP is -SRS. 24. The compound of claim 23, wherein each RS is independently H, methyl, ethyl, propyl, or 1-methylethyl, optionaly each RS is independently H, methyl or ethyl.
25. The compound of claim 24, wherein each RS is independently a thiol protecting group. 26. The compound of any one of claims 1-25, wherein at least one RP is -N(RN)2 or - N(RN)S(O)2R2S (e.g, -NHSO2CH3. 27. The compound of any one of claims 1-18, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound. 28. The compound of any one of claims 1-27, wherein B is a modified or protected nucleobase. 29. The compound of claim 28, wherein B is a protected nucleobase comprising at least one amine or hydroxyl protecting group. 30. The compound of any one of claims 1-29, wherein R2’ is hydrogen or halogen (e.g., fluoro). 31. The compound of any one of claims 1-29, wherein R2’ is -OR20. 32. The compound of claim 31, wherein R20 is optionaly substituted C1-6alkyl. 33. The compound of claim 32, wherein R20 is methyl, ethyl, propyl, 2-methoxyethyl, 1,3- dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1- yl, optionaly, R20 is methyl or 2-(N-methylamino)-2-oxoethyl. 34. The compound of claim 31, wherein R20 is optionaly substituted C2-6alkenyl. 35. The compound of claim 31, wherein R20 is optionaly substituted C2-6alkynyl (e.g., propargyl). 36. The compound of any one of claims 30-35, wherein R3’ is -OR30, wherein R30 is hydrogen, a hydroxyl protecting group, or a reactive phosphorous group. 37. The compound of claim 36 wherein R30 is hydrogen or a hydroxyl protecting group. 38. The compound of claim 37, wherein R30 is hydrogen.
39. The compound of claim 37, R30 is a hydroxyl protecting group. 40. The compound of claim 39, wherein the hydroxyl protecting group is t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS. 41. The compound of claim 36, wherein R30 is a reactive phosphorous group. 42. The compound of claim 41, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 43. The compound of claim 42, wherein the reactive phosphorous group is -P(ORP)N(RP2)2, -P(SRP)N(RP2)2, -P(O)(ORP)N(RP2)2, -P(S)(ORP)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP)N(RP2)2, -P(O)(ORP)H, -P(S)(ORP)H, -P(O)(SRP)H, -P(O)(ORP)RP3, -P(S)(ORP)RP3, or -P(O)(SRP)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl(e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 44. The compound of claim 43, wherein the reactive phosphorous group is -P(ORP)N(RP2)2. 45. The compound of claim 43 or 44, wherein RP is C1-6alkyl substituted with cyano or - SC(O)Ph. 46. The compound of any one of claims 43-45, wherein RP is –CH2CH2CN.
47. The compound of any one of claims 43-46, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 48. The compound of any one of claims 43-47, wherein each RP2 is isopropyl. 49. The compound of any one of claims 43-48, wherein RP3 is an optionaly substituted C1- C6alkyl, (e.g., methyl). 50. The compound of any one of claims 30-35, wherein R3’ is -OR30, wherein R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 51. The compound of claim 50, wherein the R30’ is a bond to an oligonucleotide. 52. The compound of claim 51, wherein R3’ is connected to the oligonucleotide via a phosphodiester or modified internucleotide linkage (e.g., phosphorothioate). 53. The compound of claim 51 or 52, wherein R3’ is connected to the 5’-terminal of the oligonucleotide (e.g., 5’-hydroxyl at the 5’-terminal of the oligonucleotide). 54. The compound of any one of claims 1-29, wherein R3’ is hydrogen or halogen (e.g., F). 55. The compound of any one of claims 1-29, wherein R3’ is -OR20. 56. The compound of claim 55, wherein R20 is optionaly substituted C1-6alkyl. 57. The compound of claim 56, wherein R20 is methyl, ethyl, propyl, 2-methoxyethyl, 1,3- dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1- yl, optionaly, R20 is methyl or 2-(N-methylamino)-2-oxoethyl. 58. The compound of claim 55, wherein R20 is optionaly substituted C2-6alkenyl. 59. The compound of claim 55, wherein R20 is optionaly substituted C2-6alkenyl (e.g., propargyl). 60. The compound of any one of claims 54-59, wherein R2’ is -OR30, wherein R30 is hydrogen, a hydroxyl protecting group, or a reactive phosphorous group. 61. The compound of claim 60, wherein R30 is hydrogen or a hydroxyl protecting group.
62. The compound of claim 61, wherein R30 is hydrogen. 63. The compound of claim 62, wherein R30 is a hydroxyl protecting group. 64. The compound of claim 63, wherein the hydroxyl protecting group is t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS. 65. The compound of claim 60, wherein R30 is a reactive phosphorous group. 66. The compound of claim 65, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 67. The compound of claim 66, wherein the reactive phosphorous group is -P(ORP)N(RP2)2, -P(SRP)N(RP2)2, -P(O)(ORP)N(RP2)2, -P(S)(ORP)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP)N(RP2)2, -P(O)(ORP)H, -P(S)(ORP)H, -P(O)(SRP)H, -P(O)(ORP)RP3, -P(S)(ORP)RP3, or -P(O)(SRP)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 68. The compound of claim 67, wherein the reactive phosphorous group is -P(ORP)N(RP2)2. 69. The compound of claim 66 or 68, wherein RP is C1-6alkyl substituted with cyano or - SC(O)Ph.
70. The compound of any one of claims 67-69, wherein RP is –CH2CH2CN. 71. The compound of any one of claims 67-70, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 72. The compound of any one of claims 67-71, wherein each RP2 is isopropyl. 73. The compound of any one of claims 67-72, wherein RP3 is an optionaly substituted C1- C6alkyl, (e.g., methyl). 74. The compound of any one of claims 54-59, wherein R2’ is -OR30, wherein R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 75. The compound of claim 74, wherein R30 is a bond to an oligonucleotide. 76. The compound of claim 75, wherein R2’ is connected to the oligonucleotide via a phosphodiester or modified internucleotide linkage (e.g., phosphorothioate). 77. The compound of claim 75 or 76, wherein R2’ is connected to the 5’-terminal of the oligonucleotide (e.g., 5’-hydroxyl at the 5’-terminal of the oligonucleotide). 78. The compound of any one of claims 1-16, wherein: X is O; each RP is independently -ORO or is -N(RN)2; R3’ is -OR30, wherein R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. 79. The compound of claim 78, wherein R30 is hydrogen or hydroxyl protecting group. 80. The compound of claim 78, wherein R30 is a reactive phosphorous group. 81. The compound of claim 80, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 82. The compound of claim 81, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 83. The compound of claim 82, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2. 84. The compound of claim 82 or 83, wherein RP1 is C1-6alkyl substituted with cyano or - SC(O)Ph. 85. The compound of any one of claims 82-84, wherein RP1 is –CH2CH2CN. 86. The compound of any one of claims 82-85, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 87. The compound of any one of claims 82-86, wherein each RP2 is isopropyl. 88. The compound of any one of claims 82-87, wherein the reactive phosphorous group is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. 89. The compound of any one of claims 1-16, wherein: X is O; each RP is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, POM, or NHSO2CH3; and R3’ is R30, where R30 is -P(ORP)N(RP2)2, where RP is –CH2CH2CN, and each RP2 is isopropyl. 90. The compound of any one of claims 75-86, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound. 91. The compound of any one of claims 1-16, wherein: X is O; each RP is independently -ORO or is -N(RN)2; R2’ is hydrogen, F, or -OR20, wherein R20 is hydrogen, optionaly substituted C1-6alkyl, (e.g., methyl, 2- methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl, or 3-oxo- 3-(N-methylamino)prop-1-yl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); and R3’ is -OR30, wherein R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. 92. The compound of claim 91, wherein R30 is hydrogen or hydroxyl protecting group. 93. The compound of claim 91, wherein R30 is a reactive phosphorous group. 94. The compound of claim 93, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 95. The compound of claim 94, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.
96. The compound of claim 95, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2. 97. The compound of claim 95 or 96, wherein RP1 is C1-6alkyl substituted with cyano or - SC(O)Ph. 98. The compound of any one of claims 95-97, wherein RP1 is –CH2CH2CN. 99. The compound of any one of claims 95-98, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 100. The compound of any one of claims 95-99, wherein each RP2 is isopropyl. 101. The compound of any one of claims 95-100, wherein the reactive phosphorous group is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. 102. The compound of any one of claims 95-101, wherein R2’ is hydrogen, F, or -OR20, wherein R20 is methyl or 2-methoxyethyl.9 103. The compound of any one of claims 95-102, wherein each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl. 104. The compound of any one of claims 1-16, wherein: X is O; each RP is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, POM or NHSO2CH3; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, methyl, 2-methoxyethyl, 1,3- dmethoxyprop-2-yl, or 2-(N-methylamino); and R3’ is R30, where R30 is -P(ORP)N(RP2)2, where RP is –CH2CH2CN, and each RP2 is isopropyl. 105. The compound of any one of claims 91-104, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound.
106. The compound of any one of claims 1-16, wherein: Q is , , , , where * is the bond to the phosphorous atom; X is O; each RP is -ORO; R2’ is hydrogen, F, or -OR20, wherein R20 is hydrogen, optionaly substituted C1-6alkyl, (e.g., methyl, 2- methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl, or 3-oxo- 3-(N-methylamino)prop-1-yl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); and R3’ is -OR30, wherein R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. 107. The compound of claim 106, wherein R30 is hydrogen or hydroxyl protecting group. 108. The compound of claim 106, wherein R30 is a reactive phosphorous group. 109. The compound of claim 108, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 110. The compound of claim 109, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 111. The compound of claim 110, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2. 112. The compound of claim 110 or 111, wherein RP1 is C1-6alkyl substituted with cyano or - SC(O)Ph. 113. The compound of any one of claims 110-112, wherein RP1 is –CH2CH2CN. 114. The compound of any one of claims 110-113, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 115. The compound of any one of claims 110-114, wherein each RP2 is isopropyl. 116. The compound of any one of claims 110-115, wherein the reactive phosphorous group is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. 117. The compound of any one of claims 110-116, wherein R2’ is hydrogen, F, or -OR20, wherein R20 is methyl or 2-methoxyethyl.9 118. The compound of any one of claims 110-117, wherein each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl. 119. The compound of any one of claims 110-118, wherein: Q is , , , , , , , , , , , , , , , , ,; X is O; each RP is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, POM or NHSO2CH3; R2’ is hydrogen, F, or -OR20, where R20 is hydrogen, methyl, 2-methoxyethyl, 1,3- dmethoxyprop-2-yl, or 2-(N-methylamino); and R3’ is R30, where R30 is -P(ORP)N(RP2)2, where RP is –CH2CH2CN, and each RP2 is isopropyl. 120. The compound of any one of claims 106-119, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound. 121. The compound of claim 1, wherein the compound is selected from the group consisting of:
,
, ,
, ,
, 122. An oligonucleotide, having a 5’-terminal phosphate mimic comprising the structure: , wherein: * is a carbon atom in a sugar moiety of the 5’-terminal nucleotide (e.g., C4’ of a ribose); A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2, wherein * is the bond to E; E is a bond or -CH2-; X is O or S; Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: eachRO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl.
123. The oligonucleotide of claim 122, wherein the 5’-terminal modification comprises the structure: , . 124. The oligonucleotide of claim 123, wherein the 5’-terminal modification comprises the structure: , , , 125. An oligonucleotide, having a 5’-terminal phosphate mimic comprising the structure: or , wherein: X is O or S; Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, or 2-(2-methyl)cyclopropyl)ethyl; each RP is independently -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, or 2-(2- methyl)cyclopropyl)ethyl, wherein: one or two methylene groups in Q5 are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionaly replaced with -N=; and RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC), wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl. 126. The oligonucleotide of any one of claims 122-125, wherein the 5’-terminal modification is comprised in a nucleotide comprising a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, optionaly ribofuranose) or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, alopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose) sugar, and alpha and beta, D and L, deoxy, and modified derivates thereof. 127. An oligonucleotide, wherein the 5’-terminal nucleotide has the structure: , wherein: n is an integer selected from 1 - 3; A is -C(*)(H)-, -CH2C(*)(H)-, or -C(*)(H)CH2, wherein * is the bond to E; E is a bond or -CH2-; B is an optionaly modified nucleobase (e.g., uracil); Q is propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2- ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2- methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3-cyclopropyl)propyl, (2- ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2- methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RN)-, wherein RN is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q are both replaced with O or S; one methine in Q is optionaly replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORP1, -SRP1, -N(RP1)2, or -N(RP1)S(O)2RS, wherein: each RP1 is independently hydrogen, C1-3alkyl, or a hydroxy protecting group; and RS is C1-3alkyl; one of R2’and R3’is hydrogen, halogen, or -OR20, wherein: R20 is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); the other of R2’and R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide); provided that: (a) Q is not , wherein * is the bond to the phosphorous atom; (b) when X is O or S, and each Rp is ORO, wherein each RO is hydrogen or an oxygen protecting group, then Q is not , wherein * is the bond to the phosphorous atom; and (c) the 5’-terminal nucleotide is not of the formula, . 128. The oligonucleotide of claim 127, wherein the 5’-terminal nucleotide has the structure: . 129. The oligonucleotide of claim 128, wherein the 5’-terminal nucleotide has the structure: , , r , wherein: Q1 is -O-, -S-, or -N(RN)-, wherein: RN is hydrogen, methyl, C1-3alkoxy, or C1-3acyl. 130. The oligonucleotide of claim 128, wherein the 5’-terminal nucleotide has the structure: . 131. The oligonucleotide of claim 128, wherein the 5’-terminal nucleotide has the structure: . 132. The oligonucleotide of claim 130, wherein the 5’-terminal nucleotide has the structure: r . 133. The oligonucleotide of claim 128, wherein the 5’-terminal nucleotide has the structure: .
134. The oligonucleotide of claim 128, wherein the 5’-terminal nucleotide has the structure: . 135. The compound of claim 128, wherein the compound is of formula, 136. The oligonucleotide of claim 127, wherein the 5’-terminal nucleotide has the structure: . 137. The oligonucleotide of claim 127, wherein the 5’-terminal nucleotide has the structure: . 138. The compound oligonucleotide of claim 127, wherein the 5’-nucleotide has the structure: . 139. The compound oligonucleotide of claim 127, wherein the 5’-nucleotide has the structure: r . 140. The oligonucleotide of claim 127, wherein the 5’-terminal nucleotide has the structure: , wherein n is 1, 2 or 3. 141. The oligonucleotide of any one of claims 127-140, wherein R3’ is -OR30. 142. An oligonucleotide, wherein the oligonucleotide is a compound of any one of claims 50-53 or 74-77. 143. The oligonucleotide of any one of claims 122-142, wherein the oligonucleotide is from 10 to 50 nucleotides (e.g., from 15 to 40 nucleotides) in length, wherein the compound of Formula (I) is one nucleotide. 144. The oligonucleotide of claim 143, wherein the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, optionaly, the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. 145. The oligonucleotide of any one of claims 122-144, wherein the oligonucleotide comprises at least one) nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications). 146. The oligonucleotide of claim 145, wherein the oligonucleotide comprises at least one nucleic acid modification selected from the group consisting of nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and any combinations thereof.
147. The oligonucleotide of any one of claims 122-146, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-OMe nucleotides. 148. The oligonucleotide of any one of claims 122-147, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermaly destabilizing modification of the duplex. 149. The oligonucleotide of claim 148, wherein said thermaly destabilizing modification of the duplex is located at position 4, 5, 6, 7, or 8, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the thermaly destabilizing modification of the duplex is located at position 6, 7, or 8, counting from the 5’-end of the oligonucleotide, preferably the thermaly destabilizing modification of the duplex is located at position 7, counting from the 5’-end of the oligonucleotide. 150. The oligonucleotide of any one of claims 122-149, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-F nucleotides. 151. The oligonucleotide of claim 150, wherein the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 14 and 16, counting from the 5’-end of the oligonucleotide, preferably the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 9, 14 and 16, or at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the oligonucleotide. 152. The compound of any one of claims 122-151, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-deoxy (2’-H) nucleotides. 153. The oligonucleotide of claim 152, wherein the oligonucleotide comprises a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of oligonucleotide, preferably, the oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, or at least at positions 2, 5, 7, and 12, or at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’-end of the oligonucleotide.
154. The oligonucleotide of any one of claims 122-153, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-natural or modified nucleobases. 155. The oligonucleotide of any one of claims 122-154, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleoside linkages. 156. The oligonucleotide of claim 122-155, wherein the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at nucleotide position 1 from the 5’-end of the oligonucleotide; and the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide. 157. The oligonucleotide of any one of claims 122-156, wherein the oligonucleotide is covalently linked to a support, e.g., a solid support. 158. A double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand is substantialy complementary to the antisense strand, and wherein one of the sense or the antisense strand is an oligonucleotide of any one of claims 122-157. 159. The dsRNA of claim 158, wherein the antisense strand is the oligonucleotide of any one of claims 119-146. 160. The dsRNA of any one of claims 158-159, wherein the dsRNA is capable of inducing RNA interference. 161. A method of reducing the expression of a target gene in a subject, comprising administering to the subject either: (i) a double-stranded RNA according to any one of claims 158-160, wherein the antisense strand is substantialy complementary to a target gene; or (i) an oligonucleotide according to any one of claims 122-157, wherein the oligonucleotide is substantialy complementary to a target gene. 162. A composition comprising a compound of any one of claims 1-121, an oligonucleotide of any one of claims 122-157, or a dsRNA of any one of claims 158-160.
163. A kit comprising a compound of any one of claims 1-121, an oligonucleotide of any one of claims 122-157, or a dsRNA of any one of claims 158-160. 164. A cel comprising a compound of any one of claims 1-121, an oligonucleotide of any one of claims 122-157, or a dsRNA of any one of claims 158-160. 165. The cel of claim 164, wherein the cel is in in vivo. 166. A compound of the Formula (IV): or a salt thereof, wherein: M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6- membered sugar; B is an optionaly modified nucleobase (e.g., uracil); Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2- methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2-propylcyclopropyl, (3- cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2- methyl)cyclopropylethyl, 2-methylcyclobutyl, 3-methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q4 are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionaly replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; one of R2’ and R3’is hydrogen, halogen, -OR20, alkyl, branched alkyl, alkyl amine, branched alkyl amine, alkenyl, alkynyl, alkyl ester, S-alkyl, N-alkyl, branched N- alkyl, branched S-alkyl, S-alkenyl, N-alkenyl, branched N-alkenyl, branched S- alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , or , wherein: R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amin, and R20 is hydrogen, hydroxyl protecting group, optionaly substituted alkyl (e.g., optionaly substituted C1-6alkyl, (e.g., methyl, 2- methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2- oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3-(N- methylamino)prop-1-yl), optionaly substituted branched alkyl, optionaly substituted alkenyl (e.g., optionaly substituted C2- 6alkenyl), optionaly substituted branched alkenyl, or optionaly substituted alkynyl (e.g., optionaly substituted C2-6alkynyl (e.g., propargyl)); the other of R2’and R3’is -OR30, wherein R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 167. The compound of claim 166, wherein the compound is of Formula (V): or a salt thereof. 168. The compound of claim 167, wherein: R2’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and R3’is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 169. The compound of claim 167, wherein: R3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., - OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and R2’is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 170. A compound of claim 166, wherein the compound is of Formula (VI): or a salt thereof, wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; YA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 171. The compound of claim 170, wherein YA is O, and the compound is of Formula (VI): or a salt thereof. 172. The compound of claim 169, wherein XA is O, and the compound is of Formula (VII) or a salt thereof. 173. The compound of any one of claims 170-172, wherein the compound is of compound of Formula (IX): or a salt thereof, wherein: B is an optionaly modified nucleobase (e.g., uracil); Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, or cyclopropylmethyl, wherein: one or two methylene groups in Q4 are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionaly replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. . The compound of claim 166, wherein the compound is of Formula (X): or a salt thereof, wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R2’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amine, alkenyl, alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2- (methylamino)-2-oxoethyl], -O-(2-[N,N-dimethyl)aminooxy]ethyl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, or N-alkyl ester; and R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide 175. The compound of claim 174, wherein XA is O. 176. The compound of claim 174 or 175, wherein R2’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, or N-alkyl ester. 177. The compound of claim 166, wherein the compound is of Formula (XI): wherein: XA is O, S SO2CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R2’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl),S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 178. The compound of claim 177, wherein XA is O. 179. The compound of claim 177 or 178, wherein: R2’ alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. 180. The compound of claim 166, wherein the compound is of formulae (XI)-(XIV): , or a salet thereof, wherein: XA is O, S SO2CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R2’ is H, halogen, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and RA is methyl, vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 181. The compound of claim 180, wherein XA is O. 182. The compound of claim 180 or 181, wherein: R2’ is F, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., - OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. 183. The compound of any one of claims 166-182, wherein Q4 is , , , , , w 1 here * is the bond to the phosphorous atom, and Q is - O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, optionaly, Q4 is , where * is the bond to the phosphorous atom. 184. The compound of any one of claims 166-183, wherein Q4 is , , , , , , , , , , , , , , , optionaly, optionaly. Q4 is , where * is the bond to the phosphorous atom. 185. The compound of any one of claims 166-184, wherein X is O. 186. The compound of any one of claims 166-184, wherein X is S. 187. The compound of any one of claims 166-186, wherein at least one RP is -ORO, optionaly, each RP is independently -ORO.
188. The compound of claim 187, wherein each RO is independently H, methyl, ethyl, propyl, 1-methylethyl, butyl, or tert-butyl, optionaly each RO is independently H, methyl, or ethyl. 189. The compound of claim 188, wherein each RO is independently a hydroxyl protecting group. 190. The compound of claim 189, wherein each RO is independently pivaloyloxymethyl (POM), ethyl, methyl, isopropyl, tert-butyl, trihaloalkyl, benzyl, nitrobenzyl, chlorobenzyl, fluorenyl-9-methyl, 2-cyanoethyl, 2-chlorophenyl, 2,2,2-25-trihalogen-1,1-dimethylethyl, 5-chloroquin-8-yl, 2-methylthioethyl, or 2-methylthioethyl, optionaly each RO is independently POM or ethyl. 191. The compound of any one of claims 166-190, wherein at least one RP is -SRS. 192. The compound of claim 191, wherein each RS is independently H, methyl, ethyl, propyl, or 1-methylethyl, optionaly each RS is independently H, methyl or ethyl. 193. The compound of claim 192, wherein each RS is independently a thiol protecting group. 194. The compound of any one of claims 166-193, wherein at least one RP is -N(RN)2 or - N(RN)S(O)2R2S (e.g, -NHSO2CH3. 195. The compound of any one of claims 166-194, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound, optionaly, is *-P(O)(OPOM)2, wherein * represents the bond to the remainder of the compound. 196. The compound of any one of claims 166-195, wherein B is a modified or protected nucleobase. 197. The compound of claim 196, wherein B is a protected nucleobase comprising at least one amine or hydroxyl protecting group.
198. The compound of any one of claims 166-197, wherein R3’ is -OR30, wherein R30 is hydrogen, a hydroxyl protecting group, or a reactive phosphorous group. 199. The compound of claim 198, wherein R30 is hydrogen or a hydroxyl protecting group. 200. The compound of claim 199, wherein R30 is hydrogen. 201. The compound of claim 200, R30 is a hydroxyl protecting group. 202. The compound of claim 201, wherein the hydroxyl protecting group is t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS. 203. The compound of claim 198, wherein R30 is a reactive phosphorous group. 204. The compound of claim 203, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 205. The compound of claim 204, wherein the reactive phosphorous group is -P(ORP)N(RP2)2, -P(SRP)N(RP2)2, -P(O)(ORP)N(RP2)2, -P(S)(ORP)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP)N(RP2)2, -P(O)(ORP)H, -P(S)(ORP)H, -P(O)(SRP)H, -P(O)(ORP)RP3, -P(S)(ORP)RP3, or -P(O)(SRP)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2-C30alkenyl, or optionaly substituted C2-C30alkynyl(e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2-C10alkynyl); each RP is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 206. The compound of claim 205, wherein the reactive phosphorous group is -P(ORP)N(RP2)2.
207. The compound of claim 205 or 206, wherein RP is C1-6alkyl substituted with cyano or - SC(O)Ph. 208. The compound of any one of claims 205-207, wherein RP is –CH2CH2CN. 209. The compound of any one of claims 205-208, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 210. The compound of any one of claims 205-209, wherein each RP2 is isopropyl. 211. The compound of any one of claims 205-210, wherein RP3 is an optionaly substituted C1- C6alkyl, (e.g., methyl). 212. The compound of any one of claims 166-197, wherein R3’ is -OR30, wherein R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 213. The compound of claim 212, wherein the R30’ is a bond to an oligonucleotide. 214. The compound of claim 213, wherein R3’ is connected to the oligonucleotide via a phosphodiester or modified internucleotide linkage (e.g., phosphorothioate). 215. The compound of claim 213 or 214, wherein R3’ is connected to the 5’-terminal of the oligonucleotide (e.g., 5’-hydroxyl at the 5’-terminal of the oligonucleotide). 216. The compound of claim any one of claims 172 or 182-184, wherein the compound is of Formula (IX): , or a salt thereof, where: X is O; each RP is independently -ORO or is -N(RN)2; and R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.
217. The compound of claim 216, wherein R30 is hydrogen or hydroxyl protecting group. 218. The compound of claim 217, wherein R30 is a reactive phosphorous group. 219. The compound of claim 218, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 220. The compound of claim 219, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 221. The compound of claim 220, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2. 222. The compound of claim 220 or 221, wherein RP1 is C1-6alkyl substituted with cyano or - SC(O)Ph. 223. The compound of any one of claims 221-222, wherein RP1 is –CH2CH2CN. 224. The compound of any one of claims 222-223, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 225. The compound of any one of claims 220-224, wherein each RP2 is isopropyl.
226. The compound of any one of claims 220-225, wherein the reactive phosphorous group is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. 227. The compound of any one of claims 216-226, wherein each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl 228. The compound of any one of claims 172 or 182-184, wherein the compound is of Formula (IX): or a salt thereof, where: X is O; each RP is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, POM, or NHSO2CH3; and R3’ is -OR30, wherein: where R30 is -P(ORP)N(RP2)2, where RP is –CH2CH2CN, and each RP2 is isopropyl. 229. The compound of any one of claims 216-228, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound, optionaly, is *-P(O)(OPOM)2, wherein * represents the bond to the remainder of the compound. 230. The compound of any one of claims 172 or 182-184, wherein the compound is of Formula (IX): ), or a salt thereof, where: Q is ethenylene; X is O; each RP is independently -ORO or is -N(RN)2; and R3’ is -OR30, wherein: R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group. 231. The compound of claim 230, wherein R30 is hydrogen or hydroxyl protecting group. 232. The compound of claim 231, wherein R30 is a reactive phosphorous group. 233. The compound of claim 232, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 234. The compound of claim 233, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.
235. The compound of claim 234, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2. 236. The compound of claim 234 or 235, wherein RP1 is C1-6alkyl substituted with cyano or - SC(O)Ph. 237. The compound of any one of claims 234-236, wherein RP1 is –CH2CH2CN. 238. The compound of any one of claims 234-237, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl. 239. The compound of any one of claims 234-238, wherein each RP2 is isopropyl. 240. The compound of any one of claims 234-239, wherein the reactive phosphorous group is - P(ORP1)N(RP2)2, where RP1 is –CH2CH2CN, and each RP2 is isopropyl. 241. The compound of any one of claims 234-240, wherein each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl. 242. The compound of any one of claims 172 or 182-184, wherein: or a salt thereof, where: Q is ethenylene; X is O; each RP is -ORO, where each RO is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, POM or NHSO2CH3; R3’ is R30, where R30 is -P(ORP)N(RP2)2, where RP is –CH2CH2CN, and each RP2 is isopropyl. 243. The compound of any one of claims 230-242, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound. 244. The compound of claim 166, wherein the compound is selected from the group consisting of: 245. An oligonucleotide, wherein the 5’-terminal nucleotide has the structure: or a salt thereof, wherein: M is a monocyclic or bicyclic ring (such as C3-8cycloalkyl (e.g., cyclohexyl, cyclopentyl or cyclobutyl) or heterocyclyl (e.g., piperidinyl, piperazinyl, morpholinyl, pyrolidinyl, or tetrahydrothienyl), a 5-membered sugar, or a 6-membered sugar; B is an optionaly modified nucleobase (e.g., uracil); Q4 is ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, or cyclopropylmethyl, wherein: one or two methylene groups in Q4 are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q4 are both replaced with O or S; one methine in Q4 is optionaly replaced with -N=; X is O or S; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN) N 2, or -N(R)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; one of R2’ and R3’is hydrogen, halogen, -OR20, absent, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, S-alkyl, N-alkyl, branched N-alkyl, branched S-alkyl, S-alkenyl, N-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein: R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amin, and R20 is hydrogen, hydroxyl protecting group, optionaly substituted alkyl (e.g., optionaly substituted C1-6alkyl, (e.g., methyl, 2- methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2- oxoethyl, 2-[N,N-dimethyl)aminooxy]ethyl, or 3-oxo-3-(N- methylamino)prop-1-yl), optionaly substituted branched alkyl, optionaly substituted alkenyl (e.g., optionaly substituted C2- 6alkenyl), optionaly substituted branched alkenyl, or optionaly substituted alkynyl (e.g., optionaly substituted C2-6alkynyl (e.g., propargyl)); the other of R2’and R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 246. The oligonucleotide of claim 245, wherein the 5’-terminal nucleotide has the structure: (Formula V). 247. The oligonucleotide of claim 246, wherein: R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., - OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 248. The compound of claim 246, wherein: R3’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., - OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 249. The oligonucleotide of claim 245, wherein the 5’-terminal nucleotide has the structure: wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; YA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 250. The oligonucleotide of claim 249, wherein the 5’-terminal nucleotide has the structure: wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 251. The oligonucleotide of claim 249, wherein the 5’-terminal nucleotide has the structure: wherein: YA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; and R3’is hydrogen, halogen, or -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 252. The oligonucleotide of any one of claims 249-251, wherein the 5’-terminal nucleotide has the structure: wherein: R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 253. The oligonucleotide of claim 245, wherein the 5’-terminal nucleotide has the structure: or a salt thereof, wherein: XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R2’ is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, R3’is hydrogen, halogen, or -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 254. The oligonucleotide of claim 252, wherein XA is O. 255. The oligonucleotide of claim 252 or 253, wherein: R2’ is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -0-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-l-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, or N-alkyl ester.
256. The oligonucleotide of claim 245, wherein the 5 ’-terminal nucleotide has the structure: wherein:
XA is O, S SO2CH2, NHRS or N(CO)RS, wherein Rs is vinyl, ethynyl, allyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl arnin, alkenyl, or alkyl ester;
Y is H, OH, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -0-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-l-yl)), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and
R3’is -OR30, wherein:
R30 is a bond to an oligonucleotide (e.g., to an intemucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).
257. The oligonucleotide of claim 255, wherein XA is O.
258. The oligonucleotide of claim 244 or 256, wherein:
R2’is alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or allyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, - O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. 259. The oligonucleotide of claim 245, wherein the 5’-terminal nucleotide is of formulae (XI)- (XIV): r wherein: XA is O, S SO2CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; YA is H, halogen, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., -OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine; and RA is methyl, vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; R3’is hydrogen, halogen, or -OR30, wherein R3’is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 260. The compound of claim 259, wherein XA is O. 261. The compound of claim 259 or 260, wherein: R2’ is F, alkyl (e.g., methyl), branched alkyl, alkyl amine, branched alkyl amine, alkenyl (e.g., vinyl or alyl), alkynyl (e.g., ethynyl or propargyl), alkyl ester, O-alkyl (e.g., - OMe, -O-2-methoxyethyl, -O-[2-(methylamino)-2-oxoethyl], -O-(2-[N,N- dimethyl)aminooxy]ethyl) or 3-oxo-3-(N-methylamino)prop-1-yl), S-alkyl, N-alkyl, branched O-alkyl, branched N-alkyl, branched S-alkyl, O-alkenyl, S-alkenyl, N- alkenyl, branched O-alkenyl, branched N-alkenyl, branched S-alkenyl, O-alkyl ester, S-alkyl ester, N-alkyl ester, , wherein R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amine. 262. The oligonucleotide of any one of claims 245-261, wherein Q4 is , , , , , where * is the bond to the phosphorous atom, and Q1 is -O-, -S-, or -N(RNQ)-, wherein: RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, optionaly, Q4 is , where * is the bond to the phosphorous atom. 263. The oligonucleotide of any one of claims 245-261, wherein Q4 is , , , , , , , , , , , , , , , optionaly, optionaly. Q4 is , where * is the bond to the phosphorous atom. 264. The oligonucleotide of any one of claims 245-263, wherein X is O. 265. The oligonucleotide of any one of claims 245-263, wherein X is S. 266. The oligonucleotide of any one of claims 245-265, wherein at least one RP is -ORO, optionaly, each RP is independently -ORO. 267. The oligonucleotide of claim 266, wherein each RO is independently H, methyl, ethyl, propyl, 1-methylethyl, butyl, or tert-butyl, optionaly each RO is independently H, methyl, or ethyl. 268. The oligonucleotide of claim 267, wherein each RO is independently a hydroxyl protecting group. 269. The oligonucleotide of claim 268, wherein each RO is independently pivaloyloxymethyl (POM), ethyl, methyl, isopropyl, tert-butyl, trihaloalkyl, benzyl, nitrobenzyl, chlorobenzyl, fluorenyl-9-methyl, 2-cyanoethyl, 2-chlorophenyl, 2,2,2-25-trihalogen-1,1-dimethylethyl, 5-chloroquin-8-yl, 2-methylthioethyl, or 2-methylthioethyl, optionaly each RO is independently POM or ethyl.
270. The oligonucleotide of any one of claims 245-269, wherein at least one RP is -SRS. 271. The oligonucleotide of claim 270, wherein each RS is independently H, methyl, ethyl, propyl, or 1-methylethyl, optionaly each RS is independently H, methyl or ethyl. 272. The oligonucleotide of claim 270, wherein each RS is independently a thiol protecting group. 273. The oligonucleotide of any one of claims 245-272, wherein at least one RP is -N(RN)2 or - N(RN)S(O)2R2S (e.g, -NHSO2CH3). 274. The oligonucleotide of any one of claims 245-273, wherein is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OMe)2, *-P(O)(OEt)2, *-P(O)(OPOM)2, *- P(O)(OH)OMe, *-P(O)(CH3)OH, or *-P(O)(OH)N(H)SO2CH3, wherein * represents the bond to the remainder of the compound, optionaly, is *-P(O)(OPOM)2, wherein * represents the bond to the remainder of the compound. 275. The oligonucleotide of any one of claims 245-274, wherein B is a modified or protected nucleobase. 276. The oligonucleotide of claim 275, wherein B is a protected nucleobase comprising at least one amine or hydroxyl protecting group. 277. An oligonucleotide, wherein the oligonucleotide is a compound of any one of claims 212- 215 or 245-278. 278. The oligonucleotide of any one of claims 245-277, wherein the oligonucleotide is from 10 to 50 nucleotides (e.g., from 15 to 40 nucleotides) in length, wherein the nucleotide of Formula (IV) (e.g., one of formulae (V)-(XIV) is one nucleotide. 279. The oligonucleotide of claim 278, wherein the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, optionaly, the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length.
280. The oligonucleotide of any one of claims 245-279, wherein the oligonucleotide comprises at least one) nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications). 281. The oligonucleotide of claim 280, wherein the oligonucleotide comprises at least one nucleic acid modification selected from the group consisting of nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and any combinations thereof. 282. The oligonucleotide of any one of claims 245-281, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-OMe nucleotides. 283. The oligonucleotide of any one of claims 245-282, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermaly destabilizing modification of the duplex. 284. The oligonucleotide of claim 283, wherein said thermaly destabilizing modification of the duplex is located at position 4, 5, 6, 7, or 8, counting from the 5’-end of the oligonucleotide, where the nucleotide of Formula (IV) (e.g., one of formulae (V)-(XIV) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the thermaly destabilizing modification of the duplex is located at position 6, 7, or 8, counting from the 5’-end of the oligonucleotide, preferably the thermaly destabilizing modification of the duplex is located at position 7, counting from the 5’-end of the oligonucleotide. 285. The oligonucleotide of any one of claims 245-284, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-F nucleotides. 286. The oligonucleotide of claim 284, wherein the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 14 and 16, counting from the 5’-end of the oligonucleotide, the nucleotide of Formula (IV) (e.g., one of formulae (V)-(XIV) is at position 1 from the 5’- end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 14 and 16, counting from the 5’-end of the oligonucleotide, preferably the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 9, 14 and 16, or at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the oligonucleotide. 287. The compound of any one of claims 245-286, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-deoxy (2’-H) nucleotides.
288. The oligonucleotide of claim 287, wherein the oligonucleotide comprises a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the oligonucleotide, where the nucleotide of Formula (IV) (e.g., one of formulae (V)-(XIV) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of oligonucleotide, preferably, the oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, or at least at positions 2, 5, 7, and 12, or at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’-end of the oligonucleotide. 289. The oligonucleotide of any one of claims 245-288, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-natural or modified nucleobases. 290. The oligonucleotide of any one of claims 245-289, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleoside linkages. 291. The oligonucleotide of claim 245-289, wherein the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 5’-end of the oligonucleotide, where the nucleotide of Formula (IV) (e.g., one of formulae (V)-(XIV) is at nucleotide position 1 from the 5’-end of the oligonucleotide; and the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide. 292. The oligonucleotide of any one of claims 245-291, wherein the oligonucleotide is covalently linked to a support, e.g., a solid support. 293. A double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand is substantialy complementary to the antisense strand, and wherein one of the sense or the antisense strand is an oligonucleotide of any one of claims 245-292. 294. The dsRNA of claim 293, wherein the antisense strand is the oligonucleotide of any one of claims 245-292. 295. The dsRNA of claim 293 or 294, wherein the dsRNA is capable of inducing RNA interference.
296. A method of reducing the expression of a target gene in a subject, comprising administering to the subject either: (i) a double-stranded RNA according to any one of claims 293-295, wherein the antisense strand is substantialy complementary to a target gene; or (i) an oligonucleotide according to any one of claims 245-292, wherein the oligonucleotide is substantialy complementary to a target gene. 297. A composition comprising a compound of any one of claims 166-244, an oligonucleotide of any one of claims 245-292, or a dsRNA of any one of claims 293-295. 298. A kit comprising a compound of any one of claims 166-244, an oligonucleotide of any one of claims 245-292, or a dsRNA of any one of claims 293-295. 299. A cel comprising a compound of any one of claims 166-244, an oligonucleotide of any one of claims 245-292, or a dsRNA of any one of claims 293-295. 300. The cel of claim 299, wherein the cel is in in vivo. 301. A compound of the Formula (XVII) or Formula (XX): ), or a salt thereof, wherein: n8 is an integer selected from 1 - 3 (e.g., 1 or 2); B is an optionaly modified nucleobase (e.g., uracil); Q5 is methylene, ethylene, ethenylene, propylene, propenylene, propynylene, methylcyclopropyl, cyclopropylmethyl, 2-ethylcyclopropyl, (2-cyclopropyl)ethyl, methyl-(2-methyl)cyclopropyl, (2-methyl)cyclopropylmethyl, 2- propylcyclopropyl, (3-cyclopropyl)propyl, (2-ethyl)cyclopropylmethyl, cyclopropyl, 2-(2-methyl)cyclopropylethyl, 2-methylcyclobutyl, 3- methylcyclobutyl or cyclobutylmethyl, wherein: one or two methylene groups in Q5 are optionaly and independently replaced with -C(O)-, -S(O)2-, -O-, -S-, or -N(RNQ)-, wherein RNQ is hydrogen, methyl, C1-3alkoxy, or C1-3acyl, provided that: no two consecutive methylene groups in Q5 are both replaced with O or S; one methine in Q5 is optionaly replaced with -N=; X is O or S; XA is O, S, SO2, CH2, NHRS’ or N(CO)RS’, wherein RS’ is vinyl, ethynyl, alyl, propargyl, alkyl, branched alkyl, alkyl amine, branched alkyl amin, alkenyl, or alkyl ester; each RP is independently C1-3alkyl, -ORO, -SRS, -N(RN)2, or -N(RN)S(O)2R2S, wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RS is independently hydrogen, C1-3alkyl, or a thiol protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and R2S is C1-3alkyl; RPS is C1-3alkyl, -ORO, -N(RN)2, -N(RN)S(O)2R2S, -N(RN)P(O)(ORO)(RPC), or - N=P(ORO)2(RPC), wherein: each RO is independently hydrogen, C1-6alkyl, or a hydroxyl protecting group; each RN is independently hydrogen, C1-3alkyl, or an amine protecting group; and RPC is C1-6alkyl (e.g., C1-3alkyl or methyl); and R2S is C1-3alkyl; R4’ is C1-6alkyl (e.g., methyl),r C1-6alkoxy (e.g., methoxy), hydrogen; one of R2’and R3’is hydrogen, halogen, -OR20, alkyl, branched alkyl, aminoC1- 6alkyl(e.g., branched aminoC1-6alkyl), C2-6alkenyl, C2-6alkynyl, C1-6alkyl ester, C1-6alkylthio (e.g., branched C1-6alkylthio), C1-6alkylamino (e.g., branched N- C1- 6alkylamino), C2-6alkenylthio (e.g., branched C2-6alkenylthio), N- C2- 6alkenylamino (e.g., branched N- C2-6alkenylamino), C2-6alkylthioester, N-C1- 6alkylcarbamyl, ,wherein: R20 is hydrogen, hydroxyl protecting group, optionaly substituted alkyl, (e.g., optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl, 2-[N,N- dimethyl)aminooxy]ethyl, or 3-oxo-3-(N-methylamino)prop-1-yl), optionaly substituted branched alkyl, optionaly substituted alkenyl (e.g., optionaly substituted C2-6alkenyl,) or optionaly substituted alkynyl, (e.g., optionaly substituted C2-6alkynyl (e.g., propargyl); and R1 and R2 independently are alkyl, branched alkyl, alkyl ester or alkyl amin, and the other of R2’and R3’is -OR30, wherein R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. 302. The compound of claim 301, where the compound is selected from the group consisting of:
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