WO2022103842A1 - Oligonucleotides, reagents and preparation thereof - Google Patents

Oligonucleotides, reagents and preparation thereof Download PDF

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Publication number
WO2022103842A1
WO2022103842A1 PCT/US2021/058786 US2021058786W WO2022103842A1 WO 2022103842 A1 WO2022103842 A1 WO 2022103842A1 US 2021058786 W US2021058786 W US 2021058786W WO 2022103842 A1 WO2022103842 A1 WO 2022103842A1
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Prior art keywords
formula
group
salt
independently
compound
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English (en)
French (fr)
Inventor
Wuming YAN
Xuan Zhou
Xianglin Shi
Firoz Antia
William F. Kiesman
Yannick FILLON
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Biogen MA Inc
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Biogen MA Inc
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Priority to EP21841044.7A priority Critical patent/EP4244232A1/en
Priority to KR1020237019471A priority patent/KR20230106665A/ko
Priority to JP2023528215A priority patent/JP2023551647A/ja
Priority to CN202180089208.3A priority patent/CN116940582A/zh
Priority to MX2023005467A priority patent/MX2023005467A/es
Priority to US18/036,459 priority patent/US20230406878A1/en
Priority to AU2021377229A priority patent/AU2021377229A1/en
Priority to IL302825A priority patent/IL302825A/en
Priority to CA3198433A priority patent/CA3198433A1/en
Publication of WO2022103842A1 publication Critical patent/WO2022103842A1/en
Anticipated expiration legal-status Critical
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/192—Radicals derived from carboxylic acids from aromatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/82—Benzo [b] furans; Hydrogenated benzo [b] furans with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/83—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • C07H21/04—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 deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • OLIGONUCLEOTIDES REAGENTS, AND PREPARATION THEREOF RELATED APPLICATION
  • This application claims the benefit of the filing date, under 35 U.S.C. ⁇ 119(e), of U.S. Provisional Application No.63/112,281, filed on November 11, 2020, the entire contents of which is incorporated herein by reference.
  • FIELD OF THE INVENTION [02] The present invention relates to oligonucleotides, reagents, and methods for preparing oligonucleotides.
  • BACKGROUND Oligonucleotides are short DNA or RNA oligomers that can be chemically synthesized for a wide range of applications.
  • oligonucleotides are synthesized by a solid phase automated synthesizer utilizing phosphoramidite chemistry, limited to a scale of less than 2 moles.
  • the solid phase synthesis is insufficient for the production of materials needed for clinical development and commercialization of oligonucleotide drugs in large indications.
  • the solid phase synthesis often requires the use of excess reagents and consequently increases the cost associated with the production of the target oligonucleotides.
  • One aspect of the present disclosure is directed to a compound of Formula I’ or B: I’ B or a salt thereof; wherein ring A, A 1 , A 2 , A 3 , R1, R2, P1, Y, e, and f are defined below.
  • One aspect of the present disclosure is directed to a nucleotide or an oligonucleotide represented by Formula III or IIIP, or a salt thereof, wherein R 31 , R 32 , R 34 , R 35 , R 36 , q, X, and Z are defined below.
  • One aspect of the present disclosure is directed to a nucleotide or an oligonucleotide represented by Formula III' or IIIP',
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula (V), or a salt thereof, comprising the steps of: 1) deprotecting a compound of formula (VA): or a salt thereof, to form a compound of formula (VB): salt thereof; 2) reacting the compound of formula (VB), or a salt thereof, with a compound of formula (VC): or a salt thereof, to form a compound of formula (VD), salt thereof; 3) sulfurizing or oxidizing the compound of formula (VD), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (VE): salt thereof; 4) deprotecting the compound of formula (VE), or a salt thereof to form a compound of formula (VF):
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula(V'), , or a salt thereof, comprising the steps of: 1) deprotecting a compound of formula (VA): or a salt thereof, to form a compound of formula (VB): salt thereof; 2) reacting the compound of formula (VB), or a salt thereof, with a compound of formula (VC'): or a salt thereof, to form a compound of formula (VD'), (VD'), or a salt thereof; 3) sulfurizing or oxidizing the compound of formula (VD'), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (VE'): (VE'), or a salt thereof; 4) deprotecting the compound of formula (VE'), or a salt thereof to form a compound of formula (VF'):
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula (V-C1) or (V-C2),
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula (V-C1) or (V-C2), or a salt thereof, comprising the steps of: 1) reacting the compound of formula (VB), , or a salt thereof, with a reagent of formula (VR1) or (VR2),
  • V-CR3 a compound of formula (V-CR3) or (V-CR4), or a salt thereof; 2) reacting the compound of formula (V-CR3) or (V-CR4), or a salt thereof, with a compound of formula (VG): , or a salt thereof, and a base, to form the compound of formula (V-C1) or (V-C2), wherein R 31 , R 32 , R 34 , R 35 , R 36 , q, X, and Z are defined below.
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula (VBZ), or a salt thereof, comprising the steps of: 1) reacting the compound of formula (VBZ-1), or a salt thereof, with a compound of formula (VBZ-2): or a salt thereof, to form a compound of formula (VBZ-3),
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula (V), , or a salt thereof, comprising the steps of: a) coupling a nucleotide of formula (V-1): salt thereof, with an oligonucleotide fragment of formula (V-2): salt thereof, in a solution to form an oligonucleotide fragment of formula (V-3), salt thereof; and b) sulfurizing or oxidizing the oligonucleotide of formula (V-3), or a salt thereof, to form an oligonucleotide of formula (V):
  • One aspect of the present disclosure is directed to a process for preparing an oligonucleotide fragment of formula (V*), , or a salt thereof, comprising the steps of: a) coupling a nucleotide of formula (V-1): salt thereof, with an oligonucleotide fragment of formula (V-2'): salt thereof, in a solution to form an oligonucleotide fragment of formula (V-3'), salt thereof; and b) sulfurizing or oxidizing the oligonucleotide of formula (V-3'), or a salt thereof, to form the oligonucleotide of formula (V*) or a salt thereof; wherein R 31 , R 32 , R 34 , R 35 , R 36 , q, X, and Z are defined below.
  • One aspect of the present disclosure is directed to a process for preparing a target oligonucleotide of formula (VI) or (VI-1), or a salt thereof, comprising a) coupling an oligonucleotide fragment of formula (F1) or (F1-1):
  • One aspect of the present disclosure is directed to a process for preparing a target oligonucleotide of formula (VI') or (VI'-1),
  • FIG.1 shows a retro-synthesis scheme for preparing oligonucleotide I.
  • FIG.2 shows a synthetic scheme for preparing oligonucleotide fragment A.
  • FIG.3 shows a synthetic scheme for preparing oligonucleotide fragment B from reagent M19.
  • FIG.4 shows a synthetic scheme for preparing oligonucleotide fragment C.
  • FIG.5 shows a synthetic scheme for preparing oligonucleotide fragment D.
  • FIG.6 shows a synthetic scheme for preparing oligonucleotide fragment E.
  • FIG.7 shows a synthetic scheme for preparing oligonucleotide fragment F.
  • FIG.8 shows a synthetic scheme for preparing oligonucleotide fragment J.
  • FIG.9 shows a synthetic scheme for preparing oligonucleotide fragment K.
  • FIG.10 shows a synthetic scheme for preparing oligonucleotide fragment O.
  • FIG.11 shows synthetic scheme for preparing oligonucleotide fragment B from reagent M40.
  • FIG.13 shows a synthetic scheme for preparing oligonucleotide I on a large scale. DETAILED DESCRIPTION [031] Reagents for facilitating the preparation of oligonucleotides, especially in large scale are described.
  • the synthetic processes based on reagents of the present disclosure produce protected target oligonucleotides on a large-scale with high purity without the need for chromatographic purification from the assembly of oligonucleotide fragments. Further, the protected target oligonucleotides can be easily deprotected selectively based on the conditions of the present disclosure. After deprotection and standard downstream purification, high purity ASO oligonucleotides suitable for therapeutic uses are obtained. Accordingly, the novel reagents and synthetic processes of the present disclosure provide great advantages over traditional preparation of oligonucleotides. Definitions [032]
  • the term “nucleobase” means the heterocyclic base portion of a nucleoside.
  • Nucleobases may be naturally occurring or may be modified.
  • a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a nucleobase of another nucleic acid.
  • the nucleobase is a heterocyclic base, typically purines and pyrimidines.
  • a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp.
  • nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
  • nucleoside means a compound comprising a heterocyclic base moiety and a sugar moiety, which can be modified at the 2’-end.
  • nucleotide means a nucleoside comprising a phosphate or thiophosphate or dithiophosphate linking group.
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
  • RNA ribonucleosides
  • DNA deoxyribonucleosides
  • target oligonucleotide refers to the oligonucleotide product that can be prepared based on the reagents and the processes of the present disclosure. In certain embodiments, the target oligonucleotide comprises at least 10 or at least 15 nucleotides.
  • the target oligonucleotide has 10 to 500, 15 to 500, 15 to 200, 15 to 100, 15 to 50, 15 to 40, 15 to 30 or 16 to 30 nucleotides.
  • oligonucleotide fragments refers to short oligonucleotides that are assembled to make the target oligonucleotide. In certain embodiments, the oligonucleotide fragment has 3 to 10, 3 to 8, 3 to 6 or 4 to 6 nucleotides. In certain embodiments, the oligonucleotide fragment has 4 or 5 nucleotides.
  • alkyl refers to a fully saturated branched or unbranched hydrocarbon moiety. In some embodiments, the alkyl comprises 1 to 30 carbon atoms, 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some embodiments, an alkyl comprises from 6 to 20 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso- propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3- methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.
  • Carbocyclyl refers to a saturated or unsaturated monocyclic, bicyclic or tricyclic (e.g., fused, bridged or spiro ring systems) ring system which has from 4- to 12- ring members, all of which are carbon.
  • the term “carbocyclyl” encompasses cycloalkyl groups, cycloalkenyl group and aromatic groups (i.e., aryl).
  • Cycloalkyl refers to completely saturated monocyclic hydrocarbon groups of 3-7 carbon atoms, including cyclopropyl, cyclobutyl, cyclpentyl, cyclohexyl and cyclopentyl; and “cycloalkyenyl” refers to unsaturated non-aromatic monocyclic hydrocarbon groups of 3-7 carbon atoms, including cyclpenteneyl, cyclohexenyl and cyclopentenyl. [040]
  • aryl refers to monocyclic, bicyclic or tricyclic aromatic hydrocarbon groups having from 6 to 14 carbon atoms in the ring portion.
  • aryl refers to monocyclic and bicyclic aromatic hydrocarbon groups having from 6 to 10 carbon atoms.
  • Representative examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracenyl.
  • aryl also refers to a bicyclic or tricyclic group in which at least one ring is aromatic and is fused to one or two non-aromatic hydrocarbon ring(s). Nonlimiting examples include tetrahydronaphthalene, dihydronaphthalenyl and indanyl.
  • bridged ring system is a ring system that has a carbocyclyl or heterocyclyl ring wherein two non-adjacent atoms of the ring are connected (bridged) by one or more (preferably from one to three) atoms selected from C, N, O, or S.
  • a bridged ring system may have from 6-7 ring members.
  • spiro ring system is a ring system that has two rings each of which are independently selected from a carbocyclyl or a heterocyclyl, wherein the two ring structures having one ring atom in common. Spiro ring systems have from 5 to 7 ring members.
  • heterocyclyl refers to a saturated or unsaturated, monocyclic or bicyclic (e.g., bridged or spiro ring systems) ring system which has from 3- to 7-ring members, or 3- to 6- ring members or 5- to 7- ring members, at least one of which is a heteroatom, and up to 4 (e.g., 1, 2, 3, or 4) of which may be heteroatoms, wherein the heteroatoms are independently selected from O, S and N, and wherein C can be oxidized (e.g., C(O)), N can be oxidized (e.g., N(O)) or quaternized, and S can be optionally oxidized to sulfoxide and sulfone.
  • C can be oxidized
  • N can be oxidized
  • S can be optionally oxidized to sulfoxide and sulfone.
  • heteroaryl refers to an aromatic 5 or 6 membered monocyclic ring system, having 1 to 4 heteroatoms independently selected from O, S and N, and wherein N can be oxidized (e.g., N(O)) or quaternized, and S can be optionally oxidized to sulfoxide and sulfone.
  • a heterocyclyl is a 3-to 7-membered saturated monocyclic or a 3-to 6-membered saturated monocyclic or a 5-to 7-membered saturated monocyclic ring.
  • a heterocyclyl is a 3-to 7-membered monocyclic or a 3-to 6-membered monocyclic or a 5-to 7-membered monocyclic ring.
  • a heterocyclyl is a 6 or-7-membered bicyclic ring.
  • the heterocyclyl group can be attached at a heteroatom or a carbon atom.
  • heterocyclyls include aziridinyl, oxiranyl, thiiranyl, oxaziridinyl, dioxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuranyl, thiolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, oxathiolanyl, piperidinyl, tetrahydropyranyl, thianyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl, dithianyl, trioxanyl, trithianyl, azepanyl, oxepanyl, thiepanyl, dihydro
  • bicyclic heterocyclic ring systems include 3-azabicyclo[3.1.0]hexanyl, 3- azabicyclo[3.1.1]heptanyl, 2-azaspiro[3.3]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, and 5- azaspiro[2.3]hexanyl.
  • "Halogen” or “halo” may be fluoro, chloro, bromo or iodo.
  • a “hydroxyl protecting group” refers to a group that is suitable for protecting a hydroxyl group, -OH, from reacting with other reagents.
  • the hydroxyl protecting groups can be selected from, for example, acetyl (Ac); benzoyl (Bz); benzyl (Bn); ⁇ -methoxyethoxymethyl ether (MEM); methoxymethyl ether (MOM); methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT); 4,4′-dimethoxytrityl (DMT); methoxyethyl (MOE); p-methoxybenzyl ether (PMB); methylthiomethyl ether; pivaloyl (Piv); tetrahydropyranyl (THP); tetrahydrofuran (THF); silyl ether (including, but not limited to, trimethylsilyl (TMS), ter
  • the hydroxyl protecting group protects the 3’ –hydroxyl of a nucleoside (referred to as 3’-hydroxyl protecting group).
  • the 3’- hydroxyl protecting groups include a silyl hydroxyl protecting group, such as trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyl tri(trimethylsilyl)silyl, t- butylmethoxyphenylsilyl, and t-butoxydiphenylsilyl.
  • the 3’- hydroxyl protecting group is TBDPS. In certain embodiments, the 3’-hydroxyl protecting group is a large hydrophobic protecting group (LHPG), such as those described herein.
  • LHPG large hydrophobic protecting group
  • the suffic “yl” added to the end of a chemical name indicates that the named moiety is bonded to the molecule at point.
  • the suffix “ene” added to the end of a chemical name indicates that the named moiety is bonded to the molecule at two points.
  • the hydroxyl protecting group protects the 5’ –hydroxyl of a nucleoside (referred to as 5’-hydroxyl protecting group).
  • Exemplary 5’-hydoxyl groups include, but are not limited to those as described herein (e.g., R 35 in any of the aspects or embodiments).
  • 5’-hydoxyl protecting group is an acid-labile 4,4'- dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT or DMTr) protecting group.
  • the 5’-hydroxyl protecting group is a large hydrophobic protecting group (LHPG), such as those described herein.
  • selective precipitation refers to a purification method that separates the desired product from one or more impurities in a solution by adding the solution to a solvent that precipitates out the product; while leaving the one or more impurities in the solution.
  • the solvent can be added to the solution comprising the crude product and the one or more impurities to precipitate out the product.
  • the desired compound or oligonucleotide of the present disclosure comprises a hydrophobic group (e.g., hydrophobic 3’-hydroxyl protecting group or hydrophobic 5’-hydroxyl protecting group (e.g., LHPG group described herein)) and the addition of a polar solvent (e.g.
  • the desired compound or oligonucleotide of the present disclosure can be purified by adding a co-solvent or solvent mixture (e.g., heptane, tert-butylmethylether (TBME or MBTE), heptane/MBTE mixture (e.g.
  • an organic solvent e.g., dichloromethane (DCM) or ethylacetate (EtOAc)
  • the solution comprising the crude product and the one or more impurities can be added to the non-polar or less polar solvent or solvent mixture to precipitate out the product.
  • Suitable co-solvent can be determined based on the hydrophobicity of the product. In certain embodiments, the co-solvent is less polar than the organic solvent the product is dissolved in.
  • extraction refers to a purification method that separates the desired product from one or more impurities in a solution by contacting the solution with a solvent that the product is soluble in; while the one or more impurities are insoluble.
  • the solution containing the product and one or more impurities can be contacted with a solvent that the one or more impurities are soluble in; while the product is insoluble.
  • the solution e.g., a reaction mixture or a solution of crude product
  • an organic solvent e.g., DCM, EtOAc or THF
  • an organic solvent mixture e.g., water or an aqueous solution (e.g., NaHCO3/H2O solution or NaCl/H2O solution) to remove hydrophilic impurities.
  • base refers to a substance that can produce hydroxide ion (OH-) in water solutions or a substance that can donate a pair of nonbonding electrons.
  • exemplary bases include, but are not limited to, alkaline hydroxide, alkaline earth hydroxide, alkylamines (e.g., tert-butylamine, sec-butylamine, trimethylamine, triethylamine, diisopropylethylamine, 2-methylpropan-2-amine), 8-diazabicyclo[5.4.0]undec-7-ene (DBU), imidazole, N-methylimidazole, pyridine and 3-picoline.
  • salt refers to an organic or inorganic salt of a compound, nucleotide or oligonucleotide described herein.
  • the salt is a pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • the salt of the compound, nucleotide or oligonucleotide described herein is a sodium salt, a potassium salt or an ammonium salt.
  • the salt is a sodium salt or ammonium salt.
  • the Reagents of the disclosure serve as a protecting group protecting a 3’-hydroxyl group of a nucleotide/oligonucleotide fragment.
  • the nucleotide, oligonucleotide fragment, or target oligonucleotide which is protected by the Reagents of the disclosure can be selectively precipitated from the reaction mixture. As such, the nucleotide, oligonucleotide fragment, or target oligonucleotide is easily collected by filtration without chromatograph.
  • the present disclosure provides a compound of Formula I’ or B: or a salt thereof, wherein: one of A 1 , A 2 and A 3 is Y A and the others are H; is a single bond or a double bond; Y A is Y-(CH2)a1CH2O(CH2)a2-, wherein a1 and a2 are each independently 0 or an integer from 1 to 10; ring A is phenyl, 8- to 10-membered bicyclic aryl, 5- to 6-membered heteroaryl having 1 to 3 heteroatoms independently selected from oxygen, nitrogen, and sulfur, or 7- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from oxygen, nitrogen, and sulfur; Y is H, halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more
  • the present disclosure provides a compound of formula I’ I’ or a salt thereof, wherein: ring A is phenyl, 8- to 10-membered bicyclic aryl, 5- to 6-membered heteroaryl having 1 to 3 heteroatoms independently selected from oxygen, nitrogen, and sulfur, or 7- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms selected from oxygen, nitrogen, and sulfur; Y is H, halogen, OR 1A , NR 2A R 3A , SR 4A , CR 5A R 6A R 7A , or a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms; wherein R 1A , R 2A , R 3A , R 4A , R 5A , R 6A , and R 7A is independently C1-6alkyl, C1-6alkenyl, C1- 6 alkynyl, phenyl, OR 8A ,
  • the present disclosure provides a compound of formula B: B or a salt thereof. The remainder of the variables in formula B are described in the first embodiment.
  • the present disclosure provides a compound of formula B-1 or B-2: , B-1 B-2 or a salt thereof. The remainder of the variables in formula B are described in the third embodiment.
  • the present disclosure provides a compound of formula I’ or B or a salt thereof, Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms. The remainder of the variables in formula I’ or B are described in the first, second, third or fourth embodiment.
  • the present disclosure provides a compound of formula I’ or a salt thereof, wherein ring A is phenyl or naphthalenyl. The remainder of the variables in Formula I’ are described in the second and/or fifth embodiments.
  • the present disclosure provides a compound of formula I’ or B or a salt thereof, wherein P1 is a silyl hydroxyl protecting group selected from the following:
  • the present disclosure provides a compound of formula I’ or B or a salt thereof, wherein P 1 is selected from the group consisting of -O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and –O-TBDAS: remainder of the variables in Formula I’ are described in any one of the first through seventh embodiments.
  • the present disclosure provides a compound of formula I or Ia: or a salt thereof; wherein P1 is selected from the group consisting of -O-TBDPS, -O-TBoDPS, and –O- TBDAS: , , .
  • P1 is selected from the group consisting of -O-TBDPS, -O-TBoDPS, and –O- TBDAS: , , .
  • the remainder of the variables in Formula I or Ia are described in the first, second, and/or fifth through eighth embodiments.
  • the present disclosure provides a compound of Formulae I’, B, or Formula I or a salt thereof, wherein Y is represented by Formula A: wherein: represents the point of attachment for Y; W is represented by Formula A1, A2, A2-1, A2-2, A3, A3-1, or A3-2:
  • the present disclosure provides a compound of Formula I’, B, or Formula I or a salt thereof, wherein the TBDAS group is: , wherein s is an integer from 1 to 30.
  • the remainder of the variables in Formula I, Ia, B, or Formula I’ are described in any one of the seventh through tenth embodiments.
  • the present disclosure provides a compound of Formula I’, B or Formula I or Ia or a salt thereof, wherein P1 is –O-TBDPS.
  • the present disclosure provides a compound of Formula I, Ia, B, or I’ or a salt thereof, wherein W is represented by Formula A1: , wherein R w is C n H 2n+1 ; and n is an integer from 1 to 30.
  • W is represented by Formula A1: , wherein R w is C n H 2n+1 ; and n is an integer from 1 to 30.
  • R w is C n H 2n+1
  • n is an integer from 1 to 30.
  • the remainder of the variables in Formula I, B, or I’ are described in the tenth embodiment.
  • the present disclosure provides a compound of Formula I, Ia, B, or I’ or a salt thereof, wherein R w is selected from a group consisting of C 12 H 25 , C 18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C 28 H 57 .
  • R w is selected from a group consisting of C 12 H 25 , C 18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C 28 H 57 .
  • the remainder of the variables in Formula I, Ia B, or I’ are described in any one of the tenth through thirteenth embodiments.
  • the remainder of the variables in Formula I, Ia, B, or I’ are described in any one of the tenth through fourteenth embodiments.
  • the present disclosure provides a compound of Formula I, Ia, B, or I’ or a salt thereof, wherein U is a bond, CH 2 , CH 2 CH 2 , carbonyl, triazolylene, piperazinylene, .
  • the present disclosure provides a compound of Formula I, Ia, B, or I’ or a salt thereof, wherein U-V is selected from the group consisting of: wherein R8 is H or C1-6alkyl.
  • the remainder of the variables in Formula I, B, or I’ are described in any one of the tenth through sixteenth embodiments.
  • the present disclosure provides a compound of Formula I or Ia or Formula I’ or B or a salt thereof, wherein Y is selected from the groups consisting of
  • R 8 is H or C 1-6 alkyl; and m is an integer from 1 to 5.
  • R 8 is H or C 1-6 alkyl; and m is an integer from 1 to 5.
  • the remainder of the variables in Formula I’, B, or Formula I or Ia are described in any one of the first through twelfth embodiments.
  • the present disclosure provides a compound of Formula I or Ia or Formula I’ or a salt thereof, wherein R 1 and R 2 are independently H or CH 3 .
  • the remainder of the variables in Formula I or Ia or Formula I’ are described in the first, second, and/or fifth through eighteenth embodiments.
  • R1 and R2 are both H.
  • R 1 and R 2 are both CH 3 .
  • the present disclosure provides a compound of Formula I’ or Formula B or a salt thereof, wherein e is 0, 1, or 2; and f is 0, 1, or 2.
  • the remainder of the variables in Formula I’ are described in the first, second, third, and/or fifth through nineteenth embodiments.
  • the present disclosure provides a compound of Formula I, Ia, I’, or B, or a salt thereof, wherein R8 is H or C1-4alkyl.
  • the remainder of the variables in Formula I, Ia, I’, or B are described in any one of the tenth through twentieth embodiments.
  • the present disclosure provides a compound of Formula II or IIa: or a salt thereof, wherein: t is an integer from 10 to 30; is selected from the group consisting of wherein R8 is H or C1-6alkyl.
  • t is an integer from 10 to 30; is selected from the group consisting of wherein R8 is H or C1-6alkyl.
  • the present disclosure provides a compound of Formula II or a salt thereof that is selected from the group consisting of or a salt thereof.
  • the present disclosure provides the following compound: ,or a salt thereof.
  • the present disclosure provides a compound selected from one of the following formulae:
  • a1 and a2 are each an integer from 1 to 6, 1 to 5, or 1 to 4.
  • the present disclosure provides the compounds depicted in Table 1 and prepared in the Exemplification, both the neutral form and salts thereof. Table 1
  • the present disclosure describes a nucleotide or an oligonucleotide protected by a 3’-hydroxyl protecting group described herein.
  • the 3’- hydroxyl protecting group is derived from the Regent described above.
  • the protected nucleotide or oligonucleotide is separated by selective precipitation.
  • the protected nucleotide or oligonucleotide is soluble in a non-polar organic solvent such as dichloromethane but precipitate in a polar organic solvent such as acetonitrile.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III or IIIP,
  • R 31 for each occurrence, is independently a nucleobase, wherein the NH 2 of the nucleobase, if present, is optionally protected by an amine protecting group;
  • R 32 for each occurrence, is independently selected from the group consisting of H, halo, OH, and C 1-6 alkoxy optionally substituted with C 1-6 alkoxy; wherein the OH group is optionally protected by a hydroxyl protecting group;
  • R 34 for each occurrence, is independently H or forms a ring with the alkoxy group of R 32 ;
  • R 35 is a hydroxyl protecting group;
  • R 36 for each occurrence, is independently H, C1-6alkyl group, C2-6alkenyl group, phenyl or benzyl group, each of which is optionally substituted with –CN, -NO 2 or halogen; or q is an integer from 1 to 20;
  • X for each occurrence, is independently O or S;
  • Z is a group represented by Formula I
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III' or IIIP', or a salt thereof, wherein: Q is a hydroxyl protecting group; is a nucleobase comprising a NH2 group which is modified by Z; R 31 , for each occurrence, is independently a nucleobase, wherein the NH 2 of the nucleobase, if present, is optionally protected by an amine protecting group; R 32 , for each occurrence, is independently selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy; wherein the OH group is optionally protected by a hydroxyl protecting group; R 34 , for each occurrence, is independently H or forms a ring with the alkoxy group of R 32 ; R 35 is a hydroxyl protecting group; R 36 , for each occurrence, is independently H, C 1-6 alkyl group, C
  • the hydroxyl protecting group of R 32 is a silyl protecting group.
  • the silyl protecting group is selected from the group consisting of trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyl tri(trimethylsilyl)silyl, t-butylmethoxyphenylsilyl, and t-butoxydiphenylsilyl.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP', or a salt thereof, wherein Z is a group represented by Formula I * ,
  • Z is a group represented by Formula I * .
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is a group represented by Formula B * ,
  • Z is a group represented by Formula B * .
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is a group represented by Formula B-1 * or B-2 * : B-1 * B-2 *
  • Z is a group represented by Formula B-1 * or B-2 * : B-1 * B-2 *
  • the remainder of the variables in Formula III, III', IIIP, or IIIP', are described in the twenty- seventh and/or twenty-eighth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the twenty-seventh and/or twenty-eighth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein ring A is phenyl or naphthalenyl.
  • ring A is phenyl or naphthalenyl.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the twenty-seventh, twenty-eighth, and/or thirty-second embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein P1 is a silyl hydroxyl protecting group selected from the following:
  • R 5 , R 6 and R 7 are each independently H, C 1-30 alkyl, or C 1-30 alkoxy.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through thirty-third embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein P 1 is selected from the group consisting of –O-TBDMS, -O-TIPS, -O-TBDPS, -O-TBoDPS, and – O-TBDAS: , -O-TIPS , , , and .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through thirty-fourth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is a group represented by by Formula I** or Ia ** : or salt thereof, wherein P1 is selected from the group consisting of -O-TBDPS, -O-TBoDPS, and –O-TBDAS: , , and .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through thirty-fifth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein the TBDAS group is: , wherein s is an integer from 1 to 30.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the thirty-fourth through thirty-seventh embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein P 1 is TBDPS.
  • P 1 is TBDPS.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through thirty-seventh embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein W is represented by Formula A1: , wherein Rw is CnH2n+1; and n is an integer from 1 to 30.
  • W is represented by Formula A1: , wherein Rw is CnH2n+1; and n is an integer from 1 to 30.
  • Rw is CnH2n+1
  • n is an integer from 1 to 30.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the thirty-seventh embodiment.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Rw is selected from a group consisting of C 12 H 25 , C 18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C28H57.
  • Rw is selected from a group consisting of C 12 H 25 , C 18 H 37 , C 20 H 41 , C 22 H 45 , C 24 H 49 , C 26 H 53 , and C28H57.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the thirty-seventh and/or fortieth embodiments.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the thirty-seventh through forty-first embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein U is a bond, CH2, CH2CH2, carbonyl, triazolylene, piperazinylene, , or .
  • U is a bond, CH2, CH2CH2, carbonyl, triazolylene, piperazinylene, , or .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the thirty-seventh through forty-second embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein U-V is selected from the group consisting of wherein R8 is H or C1-6alkyl.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the thirty-seventh through the forty-first embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Y is selected from the groups consisting of: wherein R 8 is H or C 1-6 alkyl; and m is an integer from 1 to 5.
  • R 8 is H or C 1-6 alkyl
  • m is an integer from 1 to 5.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R1 and R 2 are independently H or CH 3 .
  • R1 and R 2 are independently H or CH 3 .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through forty-fifth embodiments.
  • R1 and R2 are both H.
  • R1 and R2 are both CH 3 .
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein e is 0, 1, or 2; and f is 0, 1, or 2.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through the forty-sixth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof. wherein R8 is H or C 1-4 alkyl.
  • R8 is H or methyl.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is represented by Formula II* or IIa * , IIa * Wherein: t is an integer from 10 to 30; is selected from the group consisting of
  • R8 is H or C1-6alkyl.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the twenty-seventh and/or twenty-eighth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the forty- ninth embodiment.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the twenty- seventh and/or twenty-eighth embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein Z is .
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in the twenty- seventh and/or twenty-eighth embodiments.
  • a1 and a2 are each an integer from 1 to 6, 1 to 5, or 1 to 4.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein when X is S, the phosphorothiolate group has S-configuration as shown below: R-configuration as shown below: wherein indicates the connection point to 3’-OH group and indicates the connection point to 5’-OH group.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through the fifty-second embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R 31 , for each occurrence, is adenine (A), guanine (G), thymine (T), cytosine (C), or uracil (U).
  • R 31 for each occurrence, is adenine (A), guanine (G), thymine (T), cytosine (C), or uracil (U).
  • R 31 for each occurrence, is adenine (A), guanine (G), thymine (T), cytosine (C), or uracil (U).
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through the fifty-third embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R 32 , for each occurrence, is independently H, F, Cl, Br, I, or –OCH2CH2OMe.
  • R 32 for each occurrence, is independently H or –OCH2CH2OMe.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R 34 , is H.
  • R 34 is H.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through the fifty-third embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R 35 is 4,4’-dimethoxytrityl.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R 36 is –CH2CH2CN.
  • R 36 is –CH2CH2CN.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through the fifty-third embodiments.
  • the present disclosure provides a nucleotide or oligonucleotide represented by Formula III, III', IIIP, or IIIP' or a salt thereof, wherein R 32 is –OCH2CH2OMe.
  • R 32 is –OCH2CH2OMe.
  • the remainder of the variables in Formula III, III', IIIP, or IIIP' are described in any one of the twenty-seventh through the fifty-third embodiments. 3.
  • the present disclosure describes a process of preparing an oligonucleotide fragment bearing a hydroxyl protecting group (e.g., a hydrophobic hydroxyl protecting group) at the 3’-end (when the fragment bears a hydrophobic hydroxyl protecting group, it can be referenced herein as the “3’-fragment”) or an amino protecting group at a nucleobase (when the nucleobase comprises a NH 2 group. It can be referenced herein as the "nucleobase SiLHPG fragment").
  • a hydroxyl protecting group e.g., a hydrophobic hydroxyl protecting group
  • an amino protecting group at a nucleobase when the nucleobase comprises a NH 2 group. It can be referenced herein as the "nucleobase SiLHPG fragment”).
  • the methods of the present disclosure for synthesizing the 3’-fragment or the nucleobase SiLHPG fragment can be used to prepare an oligonucleotide fragment having 3 to 20 (e.g., 3 to 10, 3 to 8, 3 to 5 or 4 to 5) nucleotides with high purity without chromatographic purification.
  • a hydrophobic 3’-hydroxyl protecting group is used, which facilitates the separation of the oligonucleotide fragment product by selective precipitation.
  • a hydrophobic amino protecting group is used, which facilitates the separation of the oligonucleotide fragment product by selective precipitation.
  • the liquid phase process comprises (1) 5’-OH deprotection step, (2) coupling step, and (3) oxidation or sulfurization step, wherein the steps (1), (2) and (3) are repeated until the desired number of nucleotides are linked together to form the 3’-oligonucleotide fragment.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V),
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V'), , or a salt thereof, comprising the steps of: 1) deprotecting a compound of formula (VA): or a salt thereof, to form a compound of formula (VB): salt thereof; 2) reacting the compound of formula (VB), or a salt thereof, with a compound of formula (VC'): , or a salt thereof, to form a compound of formula (VD'), (VD'), or a salt thereof; 3) sulfurizing or oxidizing the compound of formula (VD'), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (VE'): (VE'), or a salt thereof; 4) deprotecting the compound of formula (VE'), or a salt thereof to form a compound of formula (VF'):
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V-C1) or (V-C2),
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V-C1) or (V-C2), or a salt thereof, comprising the steps of: 1) reacting the compound of formula (VB), or a salt thereof, with a reagent of formula (VR1) or (VR2), to form a compound of formula (V-CR3) or (V-CR4), or a salt thereof; 2) reacting the compound of formula (V-CR3) or (V-CR4), or a salt thereof, with a compound of formula (VG):
  • reaction of reagent (VR1) with the compound of formula (VB) forms the compound of formula (V-CR3), which reacts with the compound of formula (VG) to form the compound of formula (V-C1).
  • reaction of reagent (VR2) with the compound of formula (VB) forms the compound of formula (V-CR4), which reacts with the compound of formula (VG) to form the compound of formula (V-C2).
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (VBZ), or a salt thereof, comprising the steps of: 1) reacting the compound of formula (VBZ-1), or a salt thereof, with a compound of formula (VBZ-2): or a salt thereof, to form a compound of formula (VBZ-3), salt thereof; 2) sulfurizing or oxidizing the compound of formula (VBZ-3), or a salt thereof, with a sulfurization or oxidation agent to form a compound of formula (VBZ), or a salt thereof; wherein: Q is a hydroxyl protecting group; is a nucleobase comprising a NH2 group which is modified by Z; and R 31 , R 32 , , R 36 , R 37a , R 37b , q, X and Z are as described above for formula (V) in the fifty-fourth embodiment.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (VBZ) or a salt thereof described in the fifty-eighth embodiment, wherein the compound of formula VBZ-1 is prepared by 1) reacting the compound of formula (VBZ-4), or a salt thereof, with Z-OH to form a compound of formula VBZ-5, or a salt thereof; 2) deprotecting the compound of formula (VBZ-5) to form the compound of formula (VBZ-1).
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V), (V'), (V-C1), (V-C2), or (VBZ) or a salt thereof described in the fifty-fourth through fifty-ninth embodiment, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • the remainder of the variables in Formula (V), (V'), (V-C1), (V-C2), or (VBZ) are described in any one of the fifty-fourth through the fifty-ninth embodiments.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V), (V'), (V-C1), (V-C2), or (VBZ) or a salt thereof described in the fifty-fourth through fifty-ninth embodiment, wherein no chromatography is used for purifying the reaction product of any one of steps 1), 2), 3) and 4).
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V), (V'), (V-C1), (V-C2), or (VBZ) or a salt thereof described in the fifty-fourth through fifty-ninth embodiment, wherein the reaction product of any one of steps 1), 2), 3) and 4) is purified by selective precipitation.
  • the selective precipitation of the reaction product of any one of steps 1), 2), 3) and 4) or a salt thereof can be achieved by adding acetonitrile to a solution of the crude product in DCM.
  • the solution of the crude product can be added to acetonitrile to precipitate out the desired product.
  • the reaction product of any one of steps 1), 2), 3) and 4) or a salt thereof is purified by extracting a solution comprising the reaction product of any one of steps 1), 2), 3) and 4) or a salt thereof in an organic solvent (MBTE, EtOAc, heptane/MBTE mixture, DCM, etc.) with an aqueous solution (e.g., NaHCO 3 /H 2 O or NaCl/H 2 O) in addition to selective precipitation.
  • an organic solvent e.g., EtOAc, heptane/MBTE mixture, DCM, etc.
  • an aqueous solution e.g., NaHCO 3 /H 2 O or NaCl/H 2 O
  • the extraction is carried out before selective precipitation.
  • the extraction is carried out after selective precipitation.
  • the selective precipitation of the reaction product of any one of steps 1), 2), 3) and 4) or a salt thereof can be achieved by adding heptane or a heptane/MBTE mixture to a solution of the crude product in DCM or EtOAc.
  • the solution of the crude product can be added to heptane or a heptane/MBTE mixture to precipitate out the desired product.
  • a heptane/MBTE mixture with a suitale volume ratio (e.g., a volume ratio described herein) can be used.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V) , or a salt thereof, comprising the steps of: a) coupling a nucleotide of formula (V-1): salt thereof, with an oligonucleotide fragment of formula (V-2):
  • R 31 for each occurrence, is independently a nucleobase, wherein the NH2 of the nucleobase, if present, is protected by an amine protecting group;
  • R 32 for each occurrence, is independently selected from the group consisting of H, halo, OH, and C1-6alkoxy optionally substituted with C1-6alkoxy; wherein the OH group is optionally protected by a hydroxyl protecting group;
  • R 34 for each occurrence, is independently H or forms a ring with the alkoxy group of R 32 ;
  • R 35 is a hydroxyl protecting group;
  • R 36 for each occurrence, is independently C 1-6 alkyl group, C 2-6 alkenyl group, phenyl or benzyl group, each of which is optionally substituted with –CN, -NO2 or halogen; or
  • R 37a and R 37b are independently C1-6alkyl;
  • q is an integer from 1 to 20;
  • X for each occurrence, is independently O or S;
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V) or (V*) or a salt thereof described in the sixty-third or sixty-fourth embodiment, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V) or a salt thereof described in the fifty-fourth or the sixty-third embodiments, further comprising deprotecting the fragment of formula (V) to form deprotected fragment of formula (VH):
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V') or a salt thereof described in the fifty- fifth embodiment, further comprising deprotecting the fragment of formula (V') to form deprotected fragment of formula (VH'): salt thereof.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V-C1) or (V-C2), or a salt thereof described in the fifty-sixth or the fifty-seventh embodiment, further comprising deprotecting the fragment of formula (V-C1) or (V-C2) to form a deprotected fragment of formula (V-C3) or (V-C4):
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (VBZ), or a salt thereof described in the fifty-eighth embodiment, further comprising deprotecting the fragment of formula (VBZ) to form deprotected fragment of formula (VBZ-6):
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V*), or a salt thereof described in the sixty-fourth embodiment, further comprising deprotecting the fragment of formula (V*) to form a salt thereof.
  • the present disclosure provides a process for preparing an oligonucleotide fragment of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*), or a salt thereof described in the fifty-fourth through sixty-fourth embodiment, further comprising desilylation of the fragment of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) to form the fragment of formula (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), or (V*-2), respectively:
  • the desilylation reaction forms a compound of formula (VBZ-7’): preparing the fragment of formula (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), or (V*-2) described in any one of the seventy-first embodiment, wherein the desilylation reaction is carried out by reacting the compound of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) with HF in the presence of a base.
  • the present disclosure provides a process described in the seventy-second embodiment, wherein the base is imidazole or pyridine, wherein the imidazole or pyridine are optionally substituted.
  • the pyridine and/or imidazole is each independently substituted with one to three substituents selected from halogen, C1-6alkyl, C1-6alkoxy, -OH, and C1-6haloalkyl.
  • the present disclosure provides a process described in the seventy-third embodiment, wherein the desilylation reaction is carried out by reacting the compound of formula (V), (V'), (V-C1), (V-C2), (VBZ), or (V*) with HF in the presence of pyridine and imidazole.
  • the present disclosure provides a process described in the seventy-fourth embodiment, wherein the molar ratio of imidazole to HF is in the range of 0.5:1 to 10:1.
  • the present disclosure provides a process described in the seventy-fifth embodiment, wherein the molar ratio of imidazole to HF is in the range of 1.1:1 to 5:1.
  • the present disclosure provides a process described in the seventy-sixth embodiment, wherein the molar ratio of imidazole to HF is in the range of 2:1.
  • the present disclosure provides a process described in the seventy-fourth through seventy-seventh embodiments, wherein the molar ratio of pyridine to HF is in the range of 100:1 to 1:1.
  • the present disclosure provides a process described in the seventy-fourth through seventy-seventh embodiments, wherein the molar ratio of pyridine to HF is in the range of 1:1.
  • the present disclosure provides a process described in any one of the fifty-fourth through seventy-first embodiments, wherein the fragment for formula (V), (V'), (V-C1), (V-C2), (VBZ), (V*), (VH), (VH'), (V-C3), (V-C4), (VBZ-6), (V*-1), (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), (VBZ-7’) or (V*-2) is not purified by chromatography.
  • the present disclosure provides a process described in the eightieth embodiments, wherein the fragment for formula (V), (V'), (V-C1), (V-C2), (VBZ), (V*), (VH), (VH'), (V-C3), (V-C4), (VBZ-6), (V*-1), (VJ), (VJ'), (V-C5), (V-C6), (VBZ-7), (VBZ-7’) or (V*-2) is purified by selective precipitation and/or extraction.
  • the present disclosure provides a process described in any one of the fifty-fourth through eighty-first embodiments, wherein q is 2 to 5.
  • the present disclosure provides a process described in any one the eighty-second embodiment, wherein q is 4.
  • the variables R 31 , R 32 , R 34 , R 35 , R 36 , q, and/or Z are described in the second aspect or any one of embodiments described therein (e.g., the twenty-seventh to thirty-third embodiments).
  • the 5’-OH deprotection step is a detritylation method for removing a 5’-trityl group. It is discovered that when the detritylation reaction is carried out under anhydrous or substantially anhydrous conditions, significant reduction of side reactions (e.g., deamination of nucleobase cytosine or 5-methylcytosine or their derivatives commonly used for oligonucleotide synthesis) can be achieved.
  • the present detritylation method also involves the addition of a cation scavenger to facilitate the completion of the reaction.
  • Water level of the detritylation reaction can be controlled by the use of drying agent (e.g., molecular sieves), azeoptropic distillation or other suitable methods known in the art.
  • drying agent e.g., molecular sieves
  • azeoptropic distillation e.g., azeoptropic distillation
  • solvents, acids, and other reagents used in the detritylation reaction, substrates to be subjected to detritylation reaction, and the reaction vessels can be dried to meet the residual water levels prior to use for the detritylation reaction.
  • R 36 is one of the following: See Nat Biotechnol.2017 Sep;35(9):845-851; J. Org. Chem.1999, 64, 7515-7522; Biopolymers (Peptide Science), 2001, 60, 3, each of which is incorporated herein by reference.
  • the 5’-OH deprotection (or detritylation) reaction is carried out in the presence of a drying agent.
  • drying agent is selected from calcium chloride, potassium chloride, sodium sulfate, calcium sulfate, magnesium sulfate and molecular sieves.
  • the drying agent is molecular sieves.
  • the size of molecular sieves is 3 ⁇ or 4 ⁇ .
  • the size of molecular sieves is 3 ⁇ .
  • the anhydrous or substantially anhydrous solution for the deprotection reaction is obtained by removing water using azeotropic distillation prior to the deprotection reaction.
  • solvents, acids or acid solutions, and other reagents or solutions comprising the reagents to be used in the detritylation reaction, substrates or substrate solutions to be subjected to detritylation reaction, and the reaction vessels can be dried individually or combined prior to the detritylation reaction.
  • the deprotection reaction is carried out in the presence of a scavenger selected from a cation scavenger comprising a –SH group, a silane scanveger (such as HSiPh3, HSiBu3, triisopropylsilane etc.), siloxane, polystyrene, furan, pyrrole and indole.
  • a scavenger selected from a cation scavenger comprising a –SH group, a silane scanveger (such as HSiPh3, HSiBu3, triisopropylsilane etc.), siloxane, polystyrene, furan, pyrrole and indole.
  • the deprotection reaction is carried out in the presence of a scavenger selected from 1-dodecanethiol, cyclohexanethiol, 1-octanethiol, triisopropylsilane, indole, 2,3-dimethylfuran, diphenylsilane, 2-mercaptoimidazole, diphenylmethylsilane, phenylsilane, 5-methoxyindole, methylphenylsilane, chlorodimethylsilane, 1,1,3,3- tetramethyldisiloxane, 1-thioglycerol, triphenylsilane, tert-butyldimethylsilane, butylsilane, methyldiethoxysilane, 1,1,3,3,5,5-hexamethyltrisiloxane, hexylsilane, (mercaptomethyl)polystyrene, or dimethylphenylscavenger selected from
  • the cation scavenger is a compound of formula RSH, wherein R is an alkyl, a cycloalkyl, a heterocycloalkyl, an aryl or a heteroaryl group, each of which is optionally substituted.
  • R 35 is a 4,4’-dimethoxytrityl (DMT) group.
  • the deprotection reaction is carried out by reacting the compound of formula (VA) with a detritylation reagent. Any suitable detritylation reagent can be used.
  • the detritylation reagent is a strong organic acid.
  • the detritylation reagent is selected from CF3COOH, CCl 3 COOH, CHCl 2 COOH, CH 2 ClCOOH, H 3 PO 4 , methanesulfonic acid (MSA), benzenesulfonic acid (BSA), CClF2COOH, CHF2COOH, PhSO2H (phenylsulfinic acid) etc.
  • the detritylation reagent is CH2ClCOOH.
  • the detritylation reagent is CF 3 COOH.
  • the detritylation reagent is CHCl2COOH.
  • the detritylation reagent is citric acid. In certain embodiments, the detritylation reagent is saturated citric acid solution.
  • the coupling reaction of step 2) can be carried out in the presence of an activator described herein (e.g. activators described in the thirty-ninth embodiment). In certain embodiments, the activator is 4,5-dicyanoimidazole (DCI) or 5-ethylthio-1H-tetrazole (ETT).
  • DCI 4,5-dicyanoimidazole
  • ETT 5-ethylthio-1H-tetrazole
  • the sulfurization reaction of step 3 is carried out using a sulfurizing agent, such as 3-amino- 1,2,4-dithiazole-5-thione (xanthane hydride or ADTT), 3-(N,N-dimethylamino- methylidene)amino)-3H-1,2,4-dithiazole (DDTT), phenylacetyl disulfide (PADS), 3H-1,2- benzodithiol-3-one 1,1-dioxide (Beaucage Reagent), or phenyl-3H-1,2,4-dithiazol-3-one (POS).
  • a sulfurizing agent such as 3-amino- 1,2,4-dithiazole-5-thione (xanthane hydride or ADTT), 3-(N,N-dimethylamino- methylidene)amino)-3H-1,2,4-dithiazole (DDTT), phenylacetyl disulfide (PADS), 3H-1,
  • the sulfurizing agent is DDTT.
  • the sulfurizing agent is xanthane hydride.
  • the sulfurization reaction is carried out in the presence of a base as described herein.
  • the base is pyridine or imidazole.
  • the sulfurization reaction of step 3) is carried out in the presence of DDTT and 4,5-dicyanoimidazole (DCI).
  • the oxidation reaction of step 3) is carried out by using standard oxidizing agents known in the literature.
  • Exemplary oxidizing agent include, but are not limited to, tert-butylhydroperoxide ( t-BuOOH), (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO), (1R)-( ⁇ )-(10- camphorsulfonyl)oxaziridine (enantiomer of CSO), I2, and iodine-pyridine-water oxidizer solution.
  • the oxidizing agent is t-BuOOH.
  • the coupling/oxidation/detritylation steps are carried out in a one pot reaction.
  • the oxidation reagents in the one pot reaction is BPO or tBuOOH: . 4. Process to Prepare Target Oligonucleotides [0169]
  • the present disclosure describes a process for preparing target oligonucleotides, wherein the target oligonucleotide is assembled in the direction of the 3’- terminal to the 5’-terminal (3’-5’ direction).
  • the process of the present disclosure is successfully used to synthesize target oligonucleotides in large quantities.
  • high purity protected target oligonucleotide can be obtained by the methods of the present disclosure without chromatographic purification.
  • the process described herein involves step by step addition of oligonucleotide fragments in liquid (solution) phase to synthesize the target oligonucleotide. For example, 5-mer and 4-mer fragments are coupled first to synthesize a 9-mer fragment which is further reacted with another 5-mer fragment to synthesize 14-mer oligonucleotide.
  • the 14-mer oligonucleotide can be further coupled with another fragment until the desired length of the target oligonucleotide is obtained.
  • nucleobase LHPG fragment is first coupled with 5-mer fragment to form a 10-mer fragment having 3’-LHPG group or nucleobase LHPG group, which is then further reacted with a 4-mer fragment to form a 14-mer fragment, which is in turn coupled with another 4-mer fragment to form the target 18-mer oligonucleotide.
  • the 3’-end fragment having n nucleotides e.g.5-mer fragment
  • the nucleobase LHPG fragment having n nucleotides is synthesized by coupling a single nucleotide having the LHPG group at the nucleobase with a fragment having n-1 nucleotides (e.g., 4-mer fragment).
  • a fragment having n-1 nucleotides e.g., 4-mer fragment.
  • the present disclosure provides a process for preparing an oligonucleotide of formula (VI) or (VI-1), or a salt thereof, comprising a) coupling an oligonucleotide fragment of formula (F1) or (F1-1):
  • Q is a hydroxyl protecting group
  • R 31 for each occurrence, is independently a nucleobase, wherein the NH2 of the nucleobase, if present, is protected by an amine protecting group
  • R 32 for each occurrence, is independently selected from the group consisting of H, halo, OH, and C 1-6 alkoxy optionally substituted with C 1-6 alkoxy; wherein the OH group is optionally protected by a hydroxyl protecting group
  • R 34 for each occurrence, is independently H or forms a ring with the alkoxy group of R 32
  • R 35 is a hydroxyl protecting group
  • the present disclosure provides a process for preparing an oligonucleotide of formula (VI), (VI'), (VI-1), or (VI'-1) described in the eighty-fourth or eighty-fifth embodiment, wherein Y is a hydrophobic group comprising one or more aliphatic hydrocarbon group having 10 or more carbon atoms.
  • the present disclosure provides a process for preparing an oligonucleotide of formula (VI), (VI'), (VI-1), or (VI'-1) described in the eighty-fourth or eighty-fifth embodiment, further comprising step c) deprotecting the oligonucleotide of formula (VI), (VI'), (VI-1), or (VI'-1) to form an oligonucleotide of formula (VII), (VII-1), (VII'), or (VII'-1) : or a salt thereof.
  • the present disclosure provides a process for preparing an oligonucleotide of formula (VII), (VII-1), (VII'), or (VII'-1) described in the eighty-seventh embodiment, wherein starting from oligonucleotide of formula (VII), (VII-1), (VII'), or (VII'-1), the process further comprises repeating steps a), b) and c) for 1 to 10 times, followed by steps a) and b) to form the target oligonucleotide with desired length.
  • the present disclosure provides a process described in the eighty-eighth embodiment, wherein the process further comprises repeating steps a), b) and c) for 1 to 3 times followed by steps a) and b) to form the target oligonucleotide with desired length.
  • the present disclosure provides a process described in the eighty-fourth through eighty-ninth embodiments, wherein o is an integer from 2 to 20.
  • the present disclosure provides a process described in the ninetieth embodiment, wherein o is an integer from 2 to 5.
  • the present disclosure provides a process described in the ninety-first embodiment, wherein o is 4.
  • the present disclosure provides a process described in the third or fourth aspect (e.g., any one of the fifty-fourth through ninety-second embodiments), wherein Z is a group represented by Formula I * , [0181]
  • the present disclosure provides a process described in the third or fourth aspect (e.g., any one of the fifty-fourth through ninety-second embodiments), wherein Z is a group represented by Formula B * , [0182]
  • the present disclosure provides a process described in the third or fourth aspect (e.g., any one of the fifty-fourth through ninety-second embodiments), wherein Z is a group represented by Formula B-1 * or B-2 * : B-1 * B-2 * [0183]
  • Formula B-1 * or B-2 * B-1 * B-2 *
  • the present disclosure provides a process described in the third or fourth aspect (e.g., any one of the fifty-fourth through ninety-six embodiments), wherein P 1 is a silyl hydroxyl protecting group selected from the following: ,
  • the present disclosure provides a process described in the third or fourth aspect (e.g., any one of the fifty-fourth through ninety-seventh embodiments), wherein P 1 is selected from the group consisting of –O-TBDMS, -O-TIPS, - O-TBDPS, -O-TBoDPS, and –O-TBDAS: , -O-TIPS , , , and .
  • the present disclosure provides a process described in the third or fourth aspect (e.g., any one of the fifty-fourth through ninety-third embodiments), wherein Z is a group represented by Formula I ** or Ia ** : or a salt thereof; wherein P1 is selected from the group consisting of -O-TBDPS, -O-TBoDPS, and –O- TBDAS: independently H, C 1-30 alkyl, or C 1-30 alkoxy.
  • the present disclosure provides a process described in any one of the ninety-seventh through hundredth embodiments, wherein the TBDAS group is: , wherein s is an integer from 1 to 30.
  • the present disclosure provides a process described in the fifty-fourth through hundredth embodiments, wherein P 1 is TBDPS.
  • W is represented by Formula A1: , wherein Rw is CnH2n+1; n is an integer from 1 to 30.
  • the present disclosure provides a process described in the one hundredth through one hundred-third embodiments, wherein Rw is selected from a group consisting of C12H25, C18H37, C20H41, C22H45, C24H49, C26H53, and C 28 H 57 .
  • the present disclosure provides a process described in the hundredth through one hundred-fourth embodiments, wherein V is a bond
  • a one hundred-sixth embodiment the present disclosure provides a process described in the fifty-fourth through hundredth embodiments, wherein Y is selected from the groups consisting of wherein R8 is H or C1-6alkyl; and m is an integer from 1 to 5.
  • the present disclosure provides a process described in the third or fourth aspect (e.g., the fifty-fourth through one hundred-sixth embodiments), wherein R 1 and R 2 are independently H or CH 3 .
  • R 1 and R 2 are both H. In another specific embodiment, R 1 and R 2 are both CH 3 .
  • the present disclosure provides a process described in the third or fourth aspect (e.g., the fifty-fourth through one hundred-seventh embodiments), wherein e is 0, 1, or 2; and f is 0, 1, or 2.
  • the present disclosure provides a process described in the third or fourth aspect (e.g., the fifty-fourth through one hundred-eighth embodiments), wherein e is 1; and f is 1.
  • the present disclosure provides a process described in the third or fourth aspect (e.g., the fifty-fourth through one hundred-eighth embodiments), wherein e is 0; and f is 1 or e is 1; and f is 0. [0198] In a one hundred-eleventh embodiment, the present disclosure provides a process described in the third or fourth aspect (e.g., the fifty-fourth through one hundred-tenth embodiments), wherein R8 is H or C1-4alkyl.
  • the present disclosure provides a process described in the third or fourth aspect (e.g., the fifty-fourth through one hundred-eleventh embodiments), wherein Z is represented by Formula II * or IIa * , IIa * wherein t is an integer from 10 to 30; is selected from the group consisting of wherein R 8 is H or C 1-6 alkyl.
  • Z is:
  • the present disclosure a process described in the third or fourth aspect (e.g., the fifty-fourth through ninety-third embodiments), wherein Z is .
  • the present disclosure a process described in the third or fourth aspect (e.g., the fifty-fourth through ninety-third embodiments), wherein Z is
  • the present disclosure provides a nucleotide or oligonucleotide described in the twenty-seventh through fifty-third embodiments or a process described in the fifty-fourth through one hundred-twenty first embodiments, wherein each R 32 is independently selected from the group consisting of H, F, -OCH 3 , –OCH2CH2OCH3,and -OTBDMS; and each R 34 is independently H or forms a ring with the alkoxy group of R 32 , wherein the ring is a 5-membered ring.
  • the present disclosure provides a nucleotide or oligonucleotide described in the twenty-seventh through fifty-third embodiments or a process described in the fifty-fourth through one hundred-twenty first embodiments, wherein each R 34 is independently H or together with the alkoxy group of R 32 form –CH2-O-.
  • the present disclosure provides a nucleotide or oligonucleotide described in the twenty-seventh through fifty-third embodiments or a process described in the fifty-fourth through one hundred-twenty first embodiments, wherein each R 32 is independently selected from H or –OCH 2 CH 2 OMe; each R 34 is H; each R 35 is a 4,4’-dimethoxytirtyl group; R 36 is –CH 2 CH 2 CN; and R 37a and R 37b are both -CH(CH3)2.
  • the present disclosure provides a process described in the fifty-fifth, sixty-fourth, or eighty-fifth embodiment, wherein the salt of the compound of formula (VD'), (V-2'), or (F2') is selected from trimethyl amine salt, triethyl amine salt, and triisopropyl amine salt.
  • the present disclosure provides a process described in one hundred-twenty sixth the embodiment, wherein the salt of the compound of formula (VD'), (V-2'), or (F2') is triethyl amine salt.
  • the present disclosure provides a nucleotide or oligonucleotide described in the second aspect (e.g., the twenty-eighth embodiment) or a process described in the third or fourth aspect (e.g., any one of the fifty- eighth, fifty-ninth, sixty-ninth, and seventy-first through ninety-second embodiments), wherein the adenine, cytosine, or guanine.
  • the present disclosure provides a nucleotide or oligonucleotide described in the second aspect (e.g., the twenty-eighth embodiment) or a process described in the third or fourth aspect (e.g., any one of the fifty- eighth, fifty-ninth, sixty-ninth, and seventy-first through ninety-second embodiments), wherein the Q is a silyl protecting group.
  • the present disclosure provides a nucleotide or oligonucleotide described in the second aspect (e.g., the twenty-eighth embodiment) or a process described in the third or fourth aspect (e.g., any one of the fifty-eighth, fifty-ninth, sixty-ninth, and seventy-first through ninety-second embodiments), wherein the Q is selected from the group consisting of trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t- butylmethylsilyl tri
  • the present disclosure provides a nucleotide or oligonucleotide described in the second aspect (e.g. twenty-eighth embodiment) or a process described in the third or fourth aspect (e.g., any one of the fifty-eighth, fifty-ninth, sixty-ninth, and seventy-first through ninety-second embodiments), wherein the Q is t- butyldiphenylsilyl.
  • the variables R 31 , R 32 , R 34 , R 35 , R 36 , q, and/or Z are described in the second aspect or any one of embodiments described therein (e.g., the twenty-seventh to fifty-third embodiments).
  • the 5’-OH deprotection (or detritylation) step, the coupling step, and the oxidation or sulfurization step are carried out under conditions described in the third aspect or any one of embodiments described therein (e.g., the fifty-fourth to eighty-third embodiments).
  • the phosphorothiolate group can have S-configuration, R-configuration or a mixture thereof (e.g., a racemic mixture).
  • the reaction mixture was quenched by addition brine (200 mL), and adjusted to pH ⁇ 2 with KHSO4 aqueous solution (200 mL, 1.00 M). Then it was extracted with DCM (300 mL) and washed with brine (100 mL x 2 ), dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • the crude product was re-dissolve in DCM (50.0 mL) and dropped into MeOH (300 mL) with vigorous stirring. Desired product was precipitated out and filtered. Compound SiLHPG M22 (4.5 g, 88.2% yield) was obtained as a white solid.
  • reaction was clean according to TLC.
  • the reaction mixture was diluted with NMI (9.00 g, 109.72 mmol, 8.76 mL, 8.00 eq).
  • the crude was dropped into ACN (900 ml, 30 V) with vigorous stirring. Desired product was precipitated out.
  • Compound M19-Fragment III-C (23.00 g, 13.22 mmol, 96.42% yield) was obtained as a white solid.
  • M22-Fragment I-U-DMTr (5.20 g, crude) was obtained as a white solid.
  • General procedure for preparation of compound M22-Fragment I-U [0341] To a solution of M22-Fragment I-U-DMTr (5.10 g, 2.61 mmol) in DCM (30.0 mL) was added dodecane-1-thiol (1.58 g, 7.83 mmol, 1.88 mL) and TFA (2.38 g, 20.9 mmol, 1.55 mL). The mixture was stirred at 0°C for 1 h.
  • Oligonucleotide fragment C (3.90 g, 90.3% yield and 84.2% purity) was obtained as a white solid.
  • Example 10 Synthesis of Oligonucleotide Fragment D [0350] a. Scheme for Synthesis of Oligonucleotide Fragment D [0351] Fragment D was synthesized according to synthetic scheme depicted in FIG.5. [0352] b. Procedures for Synthesis of Oligonucleotide Fragment D [0353] Procedures for synthesizing oligonucleotide fragment D were similar to those described for the synthesis of oligonucleotide fragment A.
  • Example 11 Synthesis of Oligonucleotide Fragment E [0354] a.
  • Fragment G was synthesized according to synthetic scheme depicted below. [0378] b. Procedures for Synthesis of Oligonucleotide Fragment G [0379] Compound E-1 (500 mg, 292 umol, 1.00 eq) with dry DCM (4.0ml) and ACN (2.00 mL) were concentrated under reduced pressure to remove water three times. To a solution of compound E-1 (500 mg, 292 umol, 1.00 eq) in DCM (4.00 mL) was added 3A MS (500 mg) in one portion at 25°C under Ar and stir for 0.5 hr.
  • Fragment G (700 mg, 266 umol, 91.1% yield) was obtained as a white solid. Mass calcd for C150H216N10O22PSSi2+ [M+H+]: 2628.5043, found: 2628.5959.
  • Example 14 Synthesis of Oligonucleotide Fragment H [0380] a. Scheme for Synthesis of Oligonucleotide Fragment H [0381] Fragment H was synthesized according to the synthetic scheme depicted below:
  • Fragment L [0411] b. Procedures for Synthesis of Oligonucleotide Fragment L [0412] General procedure for preparation of Fragment L [0413] A mixture of compound L-1 (0.5 g, 0.70 mmol, 3.00 eq), pivaloyl chloride (85 mg, 0.70 mmol, 3.0 eq) in pyridine (5 mL) was stirred at 0 °C for 1 h, compound E-1 (402 mg, 235.1 mmol, 1.0 eq) was added, and the reaction mixture was stirred 0 °C for 2.0 h.
  • Chiral oligonucleotide Fragment N was synthesized according to the synthetic scheme depicted below: [0423] General procedure for preparation of Compound N-2 [0424] Compound M19-Fragment I-U (200 mg, 117 umol, 1.00 eq) with dry DCM (1.0 ml) and DCM (3.0 mL) were concentrated under reduced pressure to remove water two times.
  • the scheme which shows the reaction product 3 and by-products 1 and 2, is depicted in FIG.12.
  • the performance of each oxidation reagent is summarized in Table 2 below.
  • DCM/ACN 2:1 (6V) was added to the RBF, followed by 3A MS (5%) at 25-30 °C for 1h.
  • Compound I-1 (1.5 eq) was then added to a second RBF under Ar and was dried three times by co-evaporating with (ACN 4 v) at 25-30°C.
  • DCM (2V) was added to the second RBF and the resulting solution in the second RBF was added to the first RBF dropwise at 20 ⁇ 25 °C, followed by the addition DCI (2 eq). The resulting mixture was stirred at 25-30°C for 1h. A sample was taken for analysis. Then to the reaction mixture was added DDTT (2eq). The mixture was stirred at 25-30°C for 0.5h.
  • Step 2 detritylation of compound I-3
  • Compound I-3 (1 eq) was added into a RBF under Ar, followed by DCM (7 V) at 0- 5°C under Ar and 3A MS (5%) at 20-25°C for 1h.
  • Step compound I-6 I-6 Compound I-4 (1 eq) was added to a first RBF under Ar. It was dried three time by co-evaporating with DCM/ACN(3:1,4 v) at 25-30°C. Then DCM/ACN (2:1, 6V) was added to the RBF, followed by 3A MS (5%) at 25-30 for 1h. Compound I-5 was added (1.5 eq) to a second RBF under Ar. The mixture was dried three times by co-evaporating with ACN (4 v) at 25-30°C and then DCM (2V) was added.
  • the resulting solution in the second RBF was transferred to the first RBF dropwise at 20 ⁇ 25 °C, followed by the addition of DCI (2 eq).
  • the resulting mixture was stirred at 25-30°C for 1h.
  • a sample taken out for analysis.
  • DDTT (2eq) was added to the RBF .
  • the resulting mixture was stirred at 25-30°C for 0.5h.
  • a sample was taken out for analysis.
  • the reaction mixture was filtered to remove 3 ⁇ MS, washed by DCM (2 V x 2).
  • the resulting solution was slowly added into ACN (50 V) at 20 °C for 0.5 h. Solid was collected by filtration and washed by ACN (5 V x 2) to give compound I-6 as a white solid.
  • Step 4 detritylation of compound I-6
  • Compound I-6 (1 eq) was added to a round bottom flask (RBF) under Ar, followed by DCM (7 V) at 0-5°C under Ar and 3A MS (5%) at 20-25°C for 1h. Then to the mixture was added C 12 H 25 SH (3 eq), followed by TCA (12 eq) dropwise at 0-5°C for 2h. A sample was taken out for analysis. Then Py (15 eq) was added to the RBF at 0-5°C. The mixture was filtered to remove 3 ⁇ MS and washed with DCM (2 V x 2). The pH value was adjusted to 7 ⁇ 8 by adding NaHCO 3 (4% wt , 10 V).
  • oligonucleotide I Characterization of oligonucleotide I: The mixture of oligonucleotide I (100.0 mg) and 30% NH4OH (2 mL) in a 4 mL pressure flask was stirred at 65 o C for 4 h. The resulting compound was checked in LCMS. The structure of oligonucleotide I was confirmed by LCMS. HRMS calcd for C 231 H 318 N 53 O 118 P 17 S 15 /4- [M]/4: 1682.0, found: 1682.1.

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US20250136632A1 (en) * 2022-10-27 2025-05-01 Sumitomo Chemical Company, Limited Method for producing oligonucleotide
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WO2025235761A1 (en) 2024-05-09 2025-11-13 Biogen Ma Inc. Reagents and processes for preparing oligonucleotides

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