Classifications

• • 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
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
  • C07F9/6581—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and nitrogen atoms with or without oxygen or sulfur atoms, as ring hetero atoms
  • C07F9/6584—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and nitrogen atoms with or without oxygen or sulfur atoms, as ring hetero atoms having one phosphorus atom as ring hetero atom
  • C07F9/65842—Cyclic amide derivatives of acids of phosphorus, in which one nitrogen atom belongs to the ring
  • C07F9/65844—Cyclic amide derivatives of acids of phosphorus, in which one nitrogen atom belongs to the ring the phosphorus atom being part of a five-membered ring which may be condensed with another ring system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
  • C07H1/06—Separation; Purification
• • 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
  • 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
  • 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
  • C07H19/16—Purine 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
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
  • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• • 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

Definitions

• Oligonucleotides are useful for many purposes, including treating various diseases. There is a need for efficient synthetic methods for oligonucleotides.
• Phosphoramidites are important reagents for oligonucleotide synthesis. Many technologies for preparing phosphoramidites suffer from low yields and/or purities, which may significantly increase the cost of oligonucleotide preparation. In some embodiments, the present disclosure encompasses the recognition of a source of a problem with many technologies for phosphoramidite preparation.
• a material amount e.g., containing water in an amount of equal to or more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold in mole (equivalents) of desired phosphoramidite products; molar ratios between water and desired phosphoramidite products (equivalents) are generally more than 2, 5, 10, 50, or 100) of an aqueous solvent (optionally comprising one or more solutes) to wash crude preparations of phosphoramidite products in order, e.g., to quench certain unreacted reagents (e.g., a phosphorochloridite (Cl-P(-O-)-N-)), to remove one or more unreacted reagents (e.g., a base), to remove one or more reaction byproducts formed during synthesis of a material amount (e.g., containing water in an
• the present disclosure recognizes that exposure to a material amount of aqueous solvents and/or extraction processes can decrease yields and/or purity of desired phosphoramidite products.
• the present disclosure encompasses certain surprising findings, including that replacing exposure to aqueous washes and/or extractions with filtration to remove solid reaction products can unexpectedly increase yields and/or purity of phosphoramidite products.
• filtration can increase yields by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to aqueous washes/extractions.
• filtration can be performed under vacuum and/or under inert gas.
• phosphoramidite decomposition is greatly decreased using filtration when compared to contact with aqueous washes and extractions.
• removal of extraction processes particularly those employing halogenated solvents such as chloroform, additionally avoid generation of organic wastes, providing substantial environmental and economic benefits.
• the present disclosure provides a method for preparing a phosphoramidite, comprising steps of:
• the present disclosure provides improvement comprising:
• the present disclosure provides improvement comprising:
• a preparation comprising the phosphoramidite product and the one or more reaction byproducts to filtration without adding a material amount of any aqueous or halogenated solvent to the preparation after the phosphoramidite product has been formed.
• the present disclosure provides improvement comprising:
• technologies provided in the present disclosure greatly decreased decomposition of desired phosphoramidite products during their isolation after formation.
• the present disclosure provides G phosphoramidite monomers without O 6 protection for oligonucleotide synthesis.
• oligonucleotide preparation including stereoselective oligonucleotide preparation which can provide powerful therapeutics but can be expensive.
• the present disclosure provides a compound of formula I, described infra.
• provided compounds are phosphoramidites useful for oligonucleotide preparation, including stereoselective preparation of chirally controlled oligonucleotide compositions.
• the present disclosure provides methods for oligonucleotide synthesis using provided phosphoramidites, e.g., those of formula I. In some embodiments, provided methods can be used for preparing chirally controlled oligonucleotide compositions.
• the present disclosure provides the following example embodiments:
• a method for preparing a phosphoramidite comprising steps of:
• each R is independently hydrogen, or an optionally substituted group selected from Ci -30 aliphatic, Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6-3 o aryl, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• silylating reagent is selected from chlorotnmethylsilane, chlorotriethylsilane, tert-tutyldimethylsilyl chloride, chloro- decyl-dimethylsilane, and chlorodimethylphenethylsilane.
• nucleoside has the structure of formula I-a:
• BA is an optionally substituted group selected from Ci-3o cycloaliphatic, C 6- 3o aryl, C3-30
• heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety;
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0- -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R')
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SEC, -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6- 3o aryl C 6- 3o aryl
• a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• each of R 1 , R2 , and R 3 is independently R', or two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci -30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')- -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')- -N(R')
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci -3 o
• Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• BA is an optionally substituted group selected from Ci-3o cycloaliphatic, C 6- 3o aryl, C3-30
• heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 5 - 3 0 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety and a modified nucleobase moiety;
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• -0-L-N(R') 2 , or R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R2 , and R 3 is independently R', or two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci -30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• BA is an optionally substituted group selected from Ci -3 o cycloaliphatic, C 6-3 o aryl, C 3-30 heterocyclyl, C 5-3 o heteroaryl, and a natural nucleobase moiety.
• BA is an optionally substituted group selected from Ci -3 o cycloaliphatic, C 6-3 o aryl, C 3-30 heterocyclyl, C 5-3 o heteroaryl, and a natural nucleobase moiety.
• nucleobase selected from
• Ring A comprises a ring
• Ring A comprises a ring
• the phosphoramidite can deliver diastereoselectivity greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% at the newly formed P-chiral center, optionally with greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% yield.
• oligonucleotide synthesis method is one described in WO/2011/005761, WO/2013/012758, WO/2014/012081, WO/2015/107425, WO/2010/064146, WO/2014/010250, WO/2011/108682, WO/2012/039448, or WO/2012/073857.
• reaction forming the preparation prior to filtration comprises the use of a reduced temperature.
• reaction forming the preparation prior to filtration comprises reacting residual phosphorochloridite with water.
• the purified phosphoramidite product has a purity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
• the purified phosphoramidite product has a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
• the purified phosphoramidite product has a purity of 95%, 96%, 97%, 98%, 99% or more.
• a method of preparing a phosphoramidite that includes a step of separating a phosphoramidite product from one or more reaction byproducts of the product's synthesis, the improvement comprising:
• a method of preparing a phosphoramidite that includes a step of separating a phosphoramidite product from one or more reaction byproducts of the product's synthesis, the improvement comprising:
• a method of preparing a phosphoramidite that includes a step of separating a phosphoramidite product from one or more reaction byproducts of the product's synthesis, the improvement comprising:
• phosphoramidite has a purity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
• phosphoramidite has a purity of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
• phosphoramidite has a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
• phosphoramidite has a purity of 95%, 96%, 97%, 98%, 99% or more.
• any one of embodiments 116-136 wherein a material amount is about or more than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more (v/v) of the preparation comprising the phosphoramidite product and the one or more reaction byproducts.
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R'
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R2 , and R 3 is independently R', wherein two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci -30
• Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• BA is optionally substituted , wherein O 6 is not substituted;
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0- -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R')
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SEC, -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R , R , and R is independently R', wherein two or three of R , R , and R are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6 -3o aryl C 6 -3o aryl
• a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0- -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R')
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SEC, -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R , R , and R is independently ', wherein two or three of R , R , and R are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6 -3o aryl C 6 -3o aryl
• a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• a compound or a salt thereof, wherein the compound is an oligonucleotide comprising
• BA one or more base moieties BA, wherein BA is optionally protected , wherein O 6 is not protected.
• BA is optionally substituted , wherein O is not substituted.
• oligonucleotide comprises one or more moieties having the structure of BA-SU-, wherein:
• SU is -L-O- or , wherein SU is connected to a phosphorus atom through the 3'- oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R'
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6 -3o aryl C 6 -3o aryl
• a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• oligonucleotide comprises one or more moieties having the structure of i-L-@-(R s )t , wherein:
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci -30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• oligonucleotide comprises about or more than 5, 6, 7, 8, 9, 10, 11, 12, 14, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 bases.
• oligonucleotide comprises about or more than 5, 6, 7, 8, 9, 10, 11, 12, 14, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 chiral linkage phosphorus.
• oligonucleotide comprises about or more than 5, 6, 7, 8, 9, 10, 11, 12, 14, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50 chirally controlled internucleotidic linkages.
• the phosphoramidite can deliver diastereoselectivity greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% at the newly formed P-chiral center, optionally with greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% yield. 229.
• oligonucleotide synthesis method is one described in WO/2011/005761, WO/2013/012758, WO/2014/012081, WO/2015/107425, WO/2010/064146, WO/2014/010250, WO/2011/108682, WO/2012/039448, or WO/2012/073857.
• a method for preparing an oligonucleotide comprising reacting a compound of any one of embodiments 1-253 to incorporate a nucleoside into an oligonucleotide.
• a method for preparing an oligonucleotide comprising providing a compound of any one of embodiments 1-253.
• oligonucleotide is WV- 1510, WV-2378, WV-2380, WV-2417, WV-2418, WV-2601, WV-2602, WV-1092, WV-2528, WV-2531, WV-3047, or WV-3153.
• oligonucleotide is capable of hybridizing with a transcript of dystrophin, Malatl, myostatin, Huntingtin, a myostatin receptor, c-Myc, ActRIIB, ActRIIA, SMN2, K-Ras, beta-catenin, dystrophia myotonica protein kinase (DMPK), C9orf72, alpha-ENaC, beta-ENaC, ApoE4, ApoC3, FGF23, an epidermal growth factor receptor, Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2), Proprotein convertase subtilisin/kexin type 9 (PCSK9), SMAD7 or KRT14 (Keratin 14), estrogen receptor (ER) and ERBB2, calcium signal transducer 2 (TACSTD2) or a mutant thereof.
• DMPK dystrophia myotonica protein kinase
• PCSK9 Proprotein convertase subtilisin/kexin type 9
• oligonucleotide is capable of hybridizing with a transcript of dystrophin or a mutant thereof.
• the oligonucleotide is capable of hybridizing with a transcript of mutant dystrophin, and causes skip of one or more exons to produce a protein with higher activity than the protein produced absent skip of the one or more exons.
• Aliphatic means a straight-chain ⁇ i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation, or combinations thereof.
• aliphatic groups contain 1- 100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms.
• aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof.
• Alkyl As used herein, the term "alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone ⁇ e.g., Ci-C 2 o for straight chain, C 2 -C 20 for branched chain), and alternatively, about 1- 10.
• cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure.
• an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms ⁇ e.g., C 1 -C 4 for straight chain lower alkyls).
• alkenyl As used herein, the term “alkenyl” refers to an alkyl group, as defined herein, having one or more double bonds.
• Alkynyl As used herein, the term “alkynyl” refers to an alkyl group, as defined herein, having one or more triple bonds.
• Aryl The term “aryl” used alone or as part of a larger moiety as in “aralkyl,”
• aralkoxy refers to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic.
• an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members.
• an aryl group is a biaryl group.
• aryl may be used interchangeably with the term “aryl ring.”
• aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents.
• aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
• an aryl group has a radical or point of attachment on an aromatic ring.
• Chiral control refers to an ability to control the stereochemical designation of a chiral linkage phosphorus in a chiral intemucleotidic linkage within an oligonucleotide.
• a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in some embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation as exemplified in the present disclosure.
• oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry at a chiral intemucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral intemucleotidic linkage.
• the stereochemical designation of each chiral linkage phosphorus in a chiral intemucleotidic linkage within an oligonucleotide is controlled.
• Chirally controlled oligonucleotide composition refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids) which share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides share the same stereochemistry at one or more chiral intemucleotidic linkages (chirally controlled intemucleotidic linkages), and the level of the plurality of oligonucleotides in the composition is pre-determined.
• each chiral intemucleotidic linkage is a chiral controlled intemucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition.
• not all chiral intemucleotidic linkages are chiral controlled intemucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition.
• a chirally controlled oligonucleotide composition comprises predetermined levels of individual oligonucleotide or nucleic acids types. For instance, in some embodiments a chirally controlled oligonucleotide composition comprises one oligonucleotide type.
• a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types.
• Cycloaliphatic refers to saturated or partially unsaturated aliphatic monocyclic, bicyclic, or poly cyclic ring systems having, e.g., from 3 to 30, members, wherein the aliphatic ring system is optionally substituted.
• Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl.
• the cycloalkyl has 3-6 carbons.
• cycloaliphatic may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring.
• a carbocyclic group is bicyclic.
• a carbocyclic group is tricyclic.
• a carbocyclic group is polycyclic.
• cycloaliphatic refers to a monocyclic C3-C6 hydrocarbon, or a C 8 -Cio bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule, or a C 9 -Ci 6 tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
• Halogen means F, CI, Br, or I.
• Heteroaliphatic is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms is replaced with a heteroatom ⁇ e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).
• Heteroalkyl The term "heteroalkyl” is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom ⁇ e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).
• heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl- substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.
• Heteroaryl The terms “heteroaryl” and “heteroar-,” used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom.
• a heteroaryl group is a group having 5 to 10 ring atoms ⁇ i.e., monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms.
• a heteroaryl group has 6, 10, or 14 ⁇ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
• Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyndazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
• a heteroaryl is a heterobiaryl group, such as bipyridyl and the like.
• heteroaryl and hetero- as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
• Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one.
• heteroaryl group may be monocyclic, bicyclic or polycyclic.
• heteroaryl may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted.
• heteroarylkyl refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.
• Heteroatom means an atom that is not carbon or hydrogen.
• a heteroatom is oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl); etc.).
• Heterocyclyl As used herein, the terms “heterocycle,” “heterocyclyl,”
• heterocyclic radical and “heterocyclic ring” are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms.
• a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
• nitrogen includes substituted nitrogen.
• the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), H (as in pyrrolidinyl), or + R (as in N-substituted pyrrolidinyl).
• a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
• saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
• heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
• Oligonucleotide type is used to define an oligonucleotide that has a particular base sequence, pattern of backbone linkages ⁇ i.e., pattern of internucleotidic linkage types, for example, phosphate, phosphorothioate, etc.), pattern of backbone chiral centers ⁇ i.e. pattern of linkage phosphorus stereochemistry (RpASp)), and pattern of backbone phosphorus modifications ⁇ e.g., pattern of "-XLR 1 " groups in formula I).
• oligonucleotides of a common designated "type" are structurally identical to one another.
• Partially unsaturated refers to a moiety that includes at least one double or triple bond.
• the term “partially unsaturated” is intended to encompass groups having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties.
• composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
• active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
• compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: 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; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
• oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
• 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.
• composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
• compositions that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
• Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
• pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
• nontoxic acid addition salts which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
• pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate
• pharmaceutically acceptable salts include, but are not limited to, nontoxic base addition salts, such as those formed by acidic groups of provided compounds ⁇ e.g., phosphate linkage groups of oligonucleotides, phosphorothioate linkage groups of oligonucleotides, etc.) with bases.
• Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like.
• pharmaceutically acceptable salts are ammonium salts.
• pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
• Predetermined By predetermined (or pre-determined) is meant deliberately selected, for example as opposed to randomly occurring or achieved without control.
• compositions that permit selection of particular chemistry and/or stereochemistry features to be incorporated into oligonucleotide compositions, and further permits controlled preparation of oligonucleotide compositions having such chemistry and/or stereochemistry features.
• Such provided compositions are "predetermined” as described herein. Compositions that may contain certain oligonucleotides because they happen to have been generated through a process that cannot be controlled to intentionally generate the particular chemistry and/or stereochemistry features is not a "predetermined" composition. In some embodiments, a predetermined composition is one that can be intentionally reproduced ⁇ e.g., through repetition of a controlled process).
• a predetermined level of a plurality of oligonucleotides in a composition means that the absolute amount, and/or the relative amount (ratio, percentage, etc.) of the plurality of oligonucleotides in the composition is controlled.
• protecting group refers to temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
• protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
• Si protecting group is a protecting group comprising a Si atom, such as Si-trialkyl ⁇ e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl ⁇ e.g., t- butyldiphenylsilyl), or Si-aryl-dialkyl ⁇ e.g., Si-phenyldialkyl).
• a Si protecting group is attached to an oxygen atom.
• the field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). Such protecting groups (and associated protected moieties) are described in detail below.
• Protected hydroxyl groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.
• Examples of suitably protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
• suitable esters include formates, acetates, proprionates, pentanoates, crotonates, and benzoates.
• esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benznylbenzoate, 2,4,6-trimethylbenzoate.
• Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p- nitrobenzyl carbonate.
• Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilyl ethers.
• alkyl ethers examples include methyl, benzyl, p-methoxybenzyl, 3,4- dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof.
• Alkoxyalkyl ethers include acetals such as methoxy methyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.
• Suitable arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O- nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.
• Suitable mono-protected amines further include, but are not limited to, aralkylamines, carbamates, allyl amines, amides, and the like.
• suitable mono- protected amino moieties include t-butyloxycarbonylamino (- HBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxocarbonylamino (- HCBZ), allylamino, benzylamino (- HBn), fluorenylmethylcarbonyl (- HFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, tri chloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, t- butyldipheny
• Suitable di-protected amines include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides, such as phthalimide, maleimide, succinimide, and the like. Suitable di-protected amines also include pyrroles and the like, 2,2,5,5-tetramethyl- [l,2,5]azadisilolidine and the like, and azide.
• Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Suitable protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes, semicarbazones, and derivatives thereof.
• Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Suitable protected carboxylic acids further include, but are not limited to, optionally substituted Ci_ 6 aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, and the like. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, wherein each group is optionally substituted. Additional suitable protected carboxylic acids include oxazolines and ortho esters.
• Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates, and the like.
• examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioester, to name but a few.
• compounds of the disclosure may contain optionally substituted and/or substituted moieties.
• substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
• an "optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
• Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
• stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
• Suitable monovalent substituents include halogen; -(CH 2 )o-4R°; -(CH 2 ) 0 ⁇ OR°;
• Suitable monovalent substituents on R° are independently halogen, -(CH 2 ) 0 _ 2 R*, -(haloR*), -(CH 2 ) 0 _ 2 OH, -(CH 2 ) 0 _ 2 OR*, -(CH 2 ) 0 _ 2 CH(OR*) 2 ; - 0(haloR*), -CN, -N 3 , -(CH 2 ) 0 _ 2 C(O)R*, -(CH 2 ) 0 _ 2 C(O)OH, -(CH 2 ) 0 _ 2 C(O)OR*, -(CH 2 ) 0 _ 2 SR*, -(CH 2 )o_ 2 SH, -(CH 2 )o_ 2 NH 2 , -(CH 2 ) 0 _ 2 NHR*, -
• Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: -0(CR * 2 ) 2 _ 3 0-, wherein each independent occurrence of R * is selected from hydrogen, Ci_ 6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• Suitable substituents on the aliphatic group of R * include halogen, -R*, -(haloR*),
• each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_ 4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0 _iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• suitable substituents on a substitutable nitrogen include -
• each R ⁇ is independently hydrogen, Ci-e aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5- 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R ⁇ , taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono
• Suitable substituents on the aliphatic group of R ⁇ are independently halogen, -
• Unsaturated means that a moiety has one or more units of unsaturation.
• Phosphoramidites have a variety of applications; for example, nucleoside phosphoramidites are widely used as monomers for oligonucleotide synthesis. Many technologies for preparing phosphoramidites, however, suffer from low yields and/or purity.
• the present disclosure encompasses the recognition that in some instances, a low yield and/or purity can be due to the purification process when a phosphoramidite product is separated from other substances, e.g., reaction byproducts formed during formation of the phosphoramidite product, e.g., work-ups after formation of the phosphoramidite product.
• work-ups in typical conventional preparations comprise washing a preparation comprising a phosphoramidite product and one or more reaction byproducts of the product's synthesis using a material amount of an aqueous solvent (optionally having one or more solute, e.g., NaHC0 3 , NaCl, etc), followed by extraction(s) of the resulting aqueous phase using an organic extraction solvent in order to recover phosphoramidite lost during the washes.
• extractions are performed with a halogenated solvent.
• a halogenated solvent is chloroform.
• the present disclosure recognizes that exposure to material amounts of water and/or extractions with organic solvents increase degradation of phosphoramidite products, lower yields and/or purity, and/or produce excessive waste, all of which increase production cost of phosphoramidites and oligonucleotides synthesized therefrom.
• the present disclosure proposes that, in some instances, phosphoramidites may be lost due to exposure to air and/or water during aqueous work-ups and/or extractions.
• the present disclosure provides technologies that enable the removal of exposure to material amounts of water and/or extractions, thereby significantly improving yields and/or purity of phosphoramidite products.
• provided technologies use filtration to remove (a fraction of, most of, or all of) one or more reaction byproducts.
• reaction byproducts removed by filtration in provided methods were removed (partially, mostly, or completely) by the aqueous work-ups and/or extractions.
• provided technologies significantly improve yields and/or purity of phosphoramidites, thereby lowering cost and improving efficiency. Such lowered cost and improved efficiency may be particularly important for preparation of chirally controlled oligonucleotide compositions, the preparation of which requires multiple-step preparation of chiral phosphoramidites, for example, those illustrated in the present disclosure.
• Preparation of a phosphoramidite starts with formation of the phosphoramidite through reactions that provide a preparation comprising a phosphoramidite product and one or more reaction byproducts of the product's synthesis.
• the preparation comprises a solution and one or more solids.
• the solution comprises the desired phosphoramidite products, while the one or more solids are mostly reaction products to be separated from the desired phosphoramidite products.
• the solution also comprises one or more reaction byproducts, including one or more that exist in the solids, as the solution is a saturated solution for one or more compounds in the solids.
• provided technologies comprise providing a silylating reagent.
• a silylating reagent has the structure of (R) 3 SiX, wherein each R is independently as described in the present disclosure, and X is halogen.
• a silylating reagent has the structure of (R) 3 SiCl, wherein each R is independently as described in the present disclosure.
• a silylating reagent has the structure of (R) 3 SiCl, wherein each R is independently as described in the present disclosure and is not -H.
• each R is independently optionally substituted aliphatic or aryl.
• each R is independently optionally substituted alkyl or aryl.
• various silylating reagents can be used in accordance with the present disclosure.
• a silylating reagent is chlorotrimethylsilane.
• a silylating reagent is chlorotriethylsilane.
• a silylating reagent is tert-tutyldimethylsilyl chlorid.
• a silylating reagent is chloro- decyl-dimethylsilane.
• a silylating reagent is chlorodimethylphenethylsilane.
• a silylating reagent is chloro triisopropylsilane.
• a silylating reagent protects a functional group of a compound, e.g., a starting material.
• a silylating reagent protects a nucleobase.
• a silylating reagent protects G.
• a silylating reagent protects O 6 of G.
• the present disclosure provides methods, comprising using a silylating reagent to protect, in some embodiments, transiently protect, a compound, e.g., protection of a nucleobase, such as O 6 of G.
• provided technologies comprises protecting a nucleobase using a silylating reagent.
• such a step is performed before reacting a nucleobase-containing compound with an amino alcohol- or chiral auxiliary-containing compound.
• a protection by a silylating reagent is transient in that, as shown in the Examples, a final phosphoramidite product comprises no silyl protection group.
• no specific de-protection step is performed for removing a silyl protection group.
• protection, de- protection, and phosphoramidite formation are all performed in one pot.
• the present disclosure provides filtration to effectively remove byproducts in solids.
• a provided filtrate contains a reduced level of one or more reaction byproducts relative to a phosphoramidite product as compared to that level present in the preparation prior to the filtration step.
• a reduced level is measured by molar ratio.
• a reduced level is due to removal of the solids, which comprise mostly byproducts, and retention of the solution as a filtrate, which contains most of the desired phosphoramidite product.
• a phosphoramidite product forms in the presence of a base.
• a base is used to neutralize an acid generated during formation of a phosphoramidite.
• a base is converted into a salt thereof.
• a salt precipitates out as a solid.
• one or more solids in a provided method comprise a salt of a base.
• a base has the formula of N(R) 3 , wherein each R is independently hydrogen, or an optionally substituted group selected from Ci -3 o aliphatic, Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6-3 o aryl, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• R is hydrogen, or optionally substituted Ci -6 aliphatic.
• a base is Et 3 N.
• a base is DIEA.
• a base is DIPEA.
• a salt is formed between a base and HC1.
• a salt is N(R) 3 HC1.
• other salts may form depending on the reagents and/or methods used for forming a phosphoramidite.
• phosphoramidites are formed by reactions between nucleosides and phosphorochloridites.
• phosphoramidites are formed by reactions between nucleosides and amino alcohols, such as chiral reagents described in US 201 1/0294124, US 2015/021 1006, US 2015/0197540, and WO 2015/107425, each of which is incorporated herein by reference.
• nucleosides and/or amino alcohols are optionally derivatized, protected, and/or activated for formation of phosphoramidites.
• phosphoramidites are formed by reactions between compounds comprising nucleoside moieties and compounds comprising amino alcohol moieties.
• chiral auxiliaries are amino alcohols.
• phosphoramidites may be protected, for example, as protected for oligonucleotide synthesis.
• a provided nucleoside has the structure of formula I-a:
• BA is an optionally substituted group selected from Ci -3 o cycloaliphatic, C 6-3 o aryl, C 3-30
• heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety;
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— , -C(R') 2 - -Cy- -0-, S ⁇ SS- -N(R')-, -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R')
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci -30
• Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• a provided phosphorochloridite has the structure of formula I-b:
• each of R 1 , R2 , and R 3 is independently R', or two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci -3 o aliphatic and Ci -3 o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— , -C(R') 2 - -Cy- -0-, S ⁇ SS- -N(R')-, -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')- -N(R')C(0)- -N(R')C(0)0- -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')- -N(
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6-
• heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci -30
• Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• a provided phosphoramidite has the structure of formula I:
• BA is an optionally substituted group selected from Ci -3 o cycloaliphatic, C 6-3 o aryl, C 3-30
• heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 5-3 o heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety;
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci.6 alkylene, Ci -6 alkenylene,— C ⁇ C— , -C(R') 2 - "Cy- "0-, S ⁇ -S-S- -N(R')-, -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R , R , and R is independently R', or two or three of R , R , and R are taken together with their intervening atoms to fo
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6 -3o aryl C 6 -3o aryl
• a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• BA is optionally substituted Ci-3o cycloaliphatic. In some embodiments, BA is optionally substituted C 6- 3o aryl. In some embodiments, BA is optionally substituted C3-30 heterocyclyl. In some embodiments, BA is optionally substituted C5-30 heteroaryl. In some embodiments, BA is an optionally substituted natural base moiety. In some embodiments, BA is an optionally substituted modified base moiety. BA is an optionally substituted group selected from Ci -3 o cycloaliphatic, C 6- 3o aryl, C 3- 3o heterocyclyl, and C 5- 3 0 heteroaryl. In some embodiments, BA is an optionally substituted group selected from Ci -3 o cycloaliphatic, C 6- 3o aryl, C3-30 heterocyclyl, C5-30 heteroaryl, and a natural nucleobase moiety.
• BA is connected to SU through an aromatic ring. In some embodiments, BA is connected to SU through a heteroatom. In some embodiments, BA is connected to SU through a ring heteroatom of an aromatic ring. In some embodiments, BA is connected to SU through a ring nitrogen atom of an aromatic ring.
• BA is a natural nucleobase moiety. In some embodiments,
• BA is an optionally substituted natural nucleobase moiety. In some embodiments, BA is a substituted natural nucleobase moiety.
• BA is an optionally substituted group, which group is
• BA is an optionally substituted group which group is selected
• BA is an optionally substituted group, which group is formed by removing a -H
• BA is an
• BA is or
• BA is an optionally substituted purine base residue. In some embodiments, BA is a protected purine base residue. In some embodiments, BA is an optionally substituted adenine residue. In some embodiments, BA is a protected adenine residue. In some embodiments, BA is an optionally substituted guanine residue. In some embodiments, BA is a protected guanine residue. In some embodiments, provided technologies provide surprisingly improved yields and/or purity for preparation of purine phosphoramidites, which can be particularly challenging to prepare and often suffer from low yields and/or purity.
• BA is a protected base residue as used in oligonucleotide preparation.
• BA is a base residue illustrated in US 2011/0294124, US 2015/0211006, US 2015/0197540, and WO 2015/107425, each of which is incorporated herein by reference.
• modified nucleobases are suitable for use in accordance with the present disclosure in formula I.
• Example modified bases include but are not limited to those limited in WO/2011/005761, WO/2013/012758, WO/2014/012081, WO/2015/107425, WO/2010/064146, WO/2014/010250, WO/2011/108682, WO/2012/039448, and WO/2012/073857, the modified nucleobases of each of which are hereby incorporated by reference.
• BA is a substituted nucleobase so that the phosphoramidite is properly protected with one or more protecting groups and can be used for oligonucleotide synthesis.
• Suitable protecting groups for nucleobases are widely known in the art, including those useful for oligonucleotide synthesis, and can be used in accordance with the present disclosure.
• a protecting group is acetyl (Ac), phenylacetyl, benzoyl (Bz), isobutyryl (iBu), phenoxyacetyl (Pac), isopropyl-Pac, tertbutyl-Pac, alkyl-Pac, dimethylformamidine (DMF), or dialkylformamidine.
• a protecting group is phthalimido, 9-fludrenylmethoxycarbonyl (FMOC), triphenylmethylsulfenyl, t-BOC, 4,4'- dimethoxytrityl (DMTr), 4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or 9- (p-methoxyphenyl)xanthin-9-yl (MOX).
• the present disclosure encompasses the recognition that, surprisingly, guanine-containing phosphoramidites can be prepared with satisfactory yields and
• BA is
• the present disclosure provides a compound of formula I or a salt thereof, wherein:
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C 1 .6 alkylene, Ci -6 alkenylene,— C ⁇ C— , -C(R') 2 - "Cy- "0-, S ⁇ -S-S- -N(R')-, -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered
• heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R2 , and R 3 is independently R', wherein two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci-3o
• Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6 -3o aryl C 6 -3o aryl
• a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• BA is protected , wherein O 6 is not protected.
• the present disclosure provides a compound of formula I or a salt thereof wherein:
• BA is optionally substituted , wherein O is not substituted;
• SU is -L-O- or , wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci -6 alkenylene,— C ⁇ C— , -C(R') 2 - -Cy- -0-, S ⁇ SS- -N(R')-, -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- " S(0) 2 -, -S(0) 2 N(R')-, -N(R')
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R2 , and R 3 is independently R', wherein two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or: two or more R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci -30
• Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• C 6-3 o aryl a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon
• a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• BA is substituted , wherein O is not substituted.
• the present disclosure provides a compound of formula I or a salt thereof, wherein:
• L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci -3 o aliphatic and Ci -3 o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci.
• Ci -6 alkylene Ci -6 alkenylene,— C ⁇ C— , -C(R') 2 - "Cy- "0-, S ⁇ -S-S- -N(R')-, -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')- -N(R')C(0)- -N(R')C(0)0- -OC(0)N(R')-, -S(O)- "S(0) 2 -, -S(0) 2 N(R')-, -N(R')S(0) 2 - -SC(O)- -C(0)S- -OC(O)-, or -C(0)0-;
• R 5s is R' or -OR'
• R 2s is -F, -CN, -N 3 , -NO, -N0 2 , -R' -OR', -SR', -N(R') 2 , -O-L-OR', -O-L-SR', or
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5;
• -Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R2 , and R 3 is independently R', wherein two or three of R 1 , R2 , and R 3 are taken together with their intervening atoms to form:
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently R' or -L-R';
• t 0-5;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R, or:
• R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
• each R is independently hydrogen, or an optionally substituted group selected from Ci -30
• BA is . In some embodiments, BA is
• a provided compound is a phosphoramidite.
• a provided phosphoramidite is selected from:
• a provided phosphoramidite is selected from compounds
• a provided nucleoside is selected from compounds 9-16, 49-52, 49a- 52a, 69-72, 89-92, and 109-112.
• a provided phosphorochloridite is selected from compound 5-8.
• a chiral auxiliary useful for preparing provided phosphorochloridite and/or phosphoramidite is selected from compounds 1-4.
• a provided phosphoramidite is selected from compounds
• a provided phosphoramidite is selected from compounds 21-24. In some embodiments, a provided phosphoramidite is selected from compounds 25-28. In some embodiments, a provided phosphoramidite is selected from compounds 29-32. In some embodiments, a provided phosphoramidite is selected from compounds 33-36. In some embodiments, a provided phosphoramidite is selected from compounds 37-40. In some embodiments, a provided phosphoramidite is selected from compounds 41-44. In some embodiments, a provided phosphoramidite is selected from compounds 45-48. In some embodiments, a provided phosphoramidite is selected from compounds 53-56. In some embodiments, a provided phosphoramidite is selected from compounds 53a-56a.
• a provided phosphoramidite is selected from compounds 57-60. In some embodiments, a provided phosphoramidite is selected from compounds 57a-60a. In some embodiments, a provided phosphoramidite is selected from compounds 61-64. In some embodiments, a provided phosphoramidite is selected from compounds 61a-64a. In some embodiments, a provided phosphoramidite is selected from compounds 65-68. In some embodiments, a provided phosphoramidite is selected from compounds 65a-68a. In some embodiments, a provided phosphoramidite is selected from compounds 73-76. In some embodiments, a provided phosphoramidite is selected from compounds 77-80.
• a provided phosphoramidite is selected from compounds 81-84. In some embodiments, a provided phosphoramidite is selected from compounds 85-88. In some embodiments, a provided phosphoramidite is selected from compounds 93-96. In some embodiments, a provided phosphoramidite is selected from compounds 97-100. In some embodiments, a provided phosphoramidite is selected from compounds 101-104. In some embodiments, a provided phosphoramidite is selected from compounds 105-108. In some embodiments, a provided phosphoramidite is selected from compounds 113-116. In some embodiments, a provided phosphoramidite is selected from compounds 117-120.
• a provided phosphoramidite is selected from compounds 121-124. In some embodiments, a provided phosphoramidite is selected from compounds 125-128. In some embodiments, a provided phosphoramidite is selected from compounds 113-128. In some embodiments, a provided phosphoramidite is compound 113. In some embodiments, a provided phosphoramidite is compound 114. In some embodiments, a provided phosphoramidite is compound 115. In some embodiments, a provided phosphoramidite is compound 116. In some embodiments, a provided phosphoramidite is compound 117. In some embodiments, a provided phosphoramidite is compound 118. In some embodiments, a provided phosphoramidite is compound 119.
• a provided phosphoramidite is compound 120. In some embodiments, a provided phosphoramidite is compound 121. In some embodiments, a provided phosphoramidite is compound 122. In some embodiments, a provided phosphoramidite is compound 123. In some embodiments, a provided phosphoramidite is compound 124. In some embodiments, a provided phosphoramidite is compound 125. In some embodiments, a provided phosphoramidite is compound 126. In some embodiments, a provided phosphoramidite is compound 127. In some embodiments, a provided phosphoramidite is compound 128.
• SU is a sugar moiety as used in oligonucleotides.
• SU is a modified sugar moiety as used in oligonucleotides.
• a modified sugar moiety is a 2'-OMe, 2'-F, or 2'-MOE sugar moiety.
• a modified sugar moiety is a sugar moiety a LNA sugar moiety or a cET sugar moiety.
• SU is a sugar moiety or modified sugar moiety in natural or unnatural nucleosides, nucleotides, and/or oligonucleotides.
• nucleotides are comprised of ribose sugars linked to the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U).
• modified nucleotides wherein a phosphate group or linkage phosphorus in the nucleotides can be linked to various positions of a sugar or modified sugar.
• the phosphate group or linkage phosphorus can be linked to the 2', 3', 4' or 5' hydroxyl moiety of a sugar or modified sugar.
• Nucleotides that incorporate modified nucleobases as described herein are also contemplated in this context. In some embodiments, nucleotides or modified nucleotides comprising an unprotected -OH moiety are used in accordance with methods of the present disclosure.
• SU is -L-O- .
• L is -Cy-
• L is optionally substituted 3-30 membered carbocyclylene.
• L is optionally substituted 6-30 membered arylene.
• L is optionally substituted 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur.
• L is optionally substituted 5- 30 membered heteroarylene having 1-5 heteroatoms independently selected from oxygen, nitrogen and sulfur.
• L is optionally substituted 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• L is optionally substituted 3-30 membered heterocyclylene having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is optionally substituted 5-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is optionally substituted 5-30 membered heterocyclylene having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, L is optionally substituted 5-10 membered heterocyclylene having one oxygen atom. In some embodiments, L is optionally substituted 5- membered heterocyclylene having one oxygen atom.
• L is optionally substituted 6-membered heterocyclylene having one oxygen atom. In some embodiments, L is optionally substituted 5-10 membered bicyclic heterocyclylene having one or two oxygen atoms. In some embodiments, L is optionally substituted 7-10 membered bicyclic heterocyclylene having one or two oxygen atoms. In some embodiments, L is optionally substituted 7-10 membered bicyclic heterocyclylene having two oxygen atoms. In some embodiments, L is optionally substituted 7-membered bicyclic heterocyclylene having two oxygen atoms.
• SU is a sugar moiety used in oligonucleotide synthesis.
• a person of ordinary skill in the art understands that phosphoramidites with a variety of sugar moieties can benefit from improved yields and/or purity when provided technologies are utilized for their preparation.
• SU is an optionally substituted saturated monocyclic, bicyclic or polycyclic saturated aliphatic ring wherein one or more methylene units are replaced with -0-.
• SU is a ribose or deoxyribose moiety found in natural DNA or RNA molecules.
• SU is a modified sugar having the structure of:
• R 5s is -OR'; and R 2s is -F, -CN, -N 3 , -NO, -N0 2 -R ⁇ -OR', -SR', -N(R') 2 ,
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5.
• R 2s is -H.
• R 2s is -F.
• R 2s is -OMe.
• R 2s is -OCH 2 CH 2 OMe.
• R 2s is L connecting C2 with CI, C2, C3, C4 or C5.
• R 2s is L connecting C2 with and C4.
• L is optionally substituted (C2)-0-CH 2 - In some
• L is (C2)-0— CH -.
• a sugar moiety e.g., of an
• oligonucleotide has the structure , wherein R 2s is as described in the present disclosure.
• CI is bonded to a nucleobase, and each of C3 and C5 is independently bonded to an intemucleotidic linkage, a 5'-end group, or a 3'-end group.
• an oligonucleotide comprises one or erein R 2s is -H, and one or more R zs is L connecting , C3, C4 or C5. In some embodiments, an oligonucleotide comprises
• R 2s is -H, and one or wherein R s is L conn H 2 - In some embodiments,
• an ol 2 R , z 2 s s is -H, and one or more
• a modified sugar contains one or more substituents at the
• R 2s including one of the following: -F, -CN, -N 3 , -NO, -N0 2 , -OR', -SR', or -N(R') 2 , wherein each R' is independently as defined above and described herein; -0-(Ci-io alkyl), -S-(Ci.io alkyl), -NH-(Ci.i 0 alkyl), or -N(Ci-i 0 alkyl) 2 ; -O-(C 2- i 0 alkenyl), -S-(C 2- i 0 alkenyl), -NH-(C 2- i 0 alkenyl), or -N(C 2 -io alkenyl) 2 ; -O-(C 2- i 0 alkynyl), -S-(C 2- i 0 alkynyl), - NH-(C 2- io alky
• alkyl 10 alkyl), -O-(Ci.i 0 alkylene)-NH-(Ci.io alkyl) or -0-(Ci-io alkylene)-NH(Ci-i 0 alkyl) 3 ⁇ 4 -NH-(Ci-i 0 alkylene)-0- (Ci-io alkyl), or -N(Ci-io alkyl)-(Ci-io alkylene)-0-(Ci-io alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or un substituted.
• substituents include, and are not limited to, -0(CH 2 ) n OCH 3 , and -0(CH 2 ) n NH 2 , wherein n is from 1 to about 10, MOE, DMAOE, and DMAEOE.
• a modified sugar comprises one or more groups selected from a substituted silyl group, an RNA cleaving group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, a group for improving the pharmacodynamic properties of a nucleic acid, or other substituents having similar properties.
• modifications are made at one or more of the the 2', 3', 4', 5', or 6' positions of the sugar or modified sugar, including the 3' position of the sugar on the 3 '-terminal nucleotide or in the 5' position of the 5'- terminal nucleotide.
• the 2' -OH of a ribose is replaced with a group including one of the following: -H, -F; -CF 3 , -CN, -N 3 , -NO, -N0 2 , -OR', -SR', or -N(R') 2 , wherein each R' is independently as defined above and described herein; -O-(Ci-Ci 0 alkyl), -S-(Ci-Ci 0 alkyl), -NH-(Ci-Ci 0 alkyl), or -N(Ci-Ci 0 alkyl) 2 ; -O-(C 2 -Ci 0 alkenyl), -S-(C 2 -Ci 0 alkenyl), - NH-(C 2 -Cio alkenyl), or -N(C 2 -Ci 0 alkenyl) 2 ; -O-(C 2
• the 2' -OH is replaced with -H (deoxyribose). In some embodiments, the 2' -OH is replaced with -F. In some embodiments, the 2' -OH is replaced with -OR' . In some embodiments, the 2' -OH is replaced with -OMe. In some embodiments, the 2' -OH is replaced with -OCH 2 CH 2 OMe.
• Modified sugars also include sugar moieties of locked nucleic acids (LNAs).
• LNAs locked nucleic acids
• two substituents on sugar carbon atoms are taken together to form a bivalent moiety.
• two substituents are on two different sugar carbon atoms.
• a formed bivalent moiety has the structure of -L- as defined herein.
• R 2s is -L-.
• -L- is -0 ⁇ CH 2 -, wherein -CH 2 - is optionally substituted.
• -L- is -0 ⁇ CH 2 -.
• -L- is -O-CH(Et)-.
• -L- is between C2 and C4 of a sugar moiety.
• a locked nucleic acid has the structure indicated below. A locked nucleic acid of the structure below is indicated, wherein Ba represents a nucleobase or modified nucleobase as described herein, and wherein R 2s is -OCH 2 C4'-.
• a modified sugar is an ENA.
• a modified sugar is any of those found in an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol, threose, 2'fluoroarabinose, or cyclohexene.
• a modified sugar is a sugar in tricyclo-DNA (tcDNA).
• tcDNA tricyclo-DNA
• an oligonucleotide comprises a LNA sugar or a tcDNA sugar.
• Modified sugars include sugar mimetics such as cyclobutyl or cyclopentyl moieties in place of the pentofuranosyl sugar.
• Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, US Patent Nos. : 4,981,957; 5, 1 18,800; 5,319,080; and 5,359,044.
• Some modified sugars that are contemplated include sugars in which the oxygen atom within the ribose ring is replaced by nitrogen, sulfur, selenium, or carbon.
• a modified sugar is a modified ribose wherein the oxygen atom within the ribose ring is replaced with nitrogen, and wherein the nitrogen is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc.).
• an alkyl group e.g., methyl, ethyl, isopropyl, etc.
• Non-limiting examples of modified sugars include glycerol, which form glycerol nucleic acid (GNA) analogues.
• GNA glycerol nucleic acid
• FNA GNA derived analogue, flexible nucleic acid
• modified sugars include hexopyranosyl to 4'), pentopyranosyl (4' to 2'), pentopyranosyl (4' to 3 '), or tetrofuranosyl (3 ' to 2') sugars, some embodiments, a hexopyranosyl (6' to 4') sugar is of any one in the following formulae:
• X s corresponds to a P-modification group and Ba is defined as BA.
• a pentopyranosyl (4' to 2') sugar is of any one in the following formulae:
• X s corresponds to a P-modification group and Ba is defined as BA.
• a pentopyranosyl (4' to 3') sugar is of any one in the following formulae:
• X s corresponds to a P-modification group and Ba is defined as BA.
• a tetrofuranosyl (3' to 2') sugar is of either in the following formulae:
• a modified sugar is of any one in the following formulae:
• X s corresponds to a P-modification group and Ba is defined as BA.
• a sugar mimetic is as illustrated below, X s corresponds to a P-modification group and Ba is defined as BA, and X 1 is selected from -S-, -Se- -CH 2 _, -
• only purine residues are modified (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more] of the purine residues are modified).
• only pyrimidine residues are modified (e.g., about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more] of the pyridimine residues are modified). In some embodiments, both purine and pyrimidine residues are modified.
• Modified sugars and sugar mimetics can be prepared by methods known in the art, including, but not limited to: A. Eschenmoser, Science (1999), 284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75: 1416-1477; M. Egli et al, I. Am. Chem. Soc. (2006), 128(33): 10847-56; A. Eschenmoser in Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V. Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p.293; K.-U.
• a modified sugar is any of those described in PCT Publication No. WO2012/030683, incorporated herein by reference, and depicted in the Figures 26-30 of the present application.
• a modified sugar is any modified sugar described in any of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997 Chem. Eur. J. 3 : 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo.
• Example sugars are also described in US 20110294124, US 20120316224, US
• a modified sugar moiety is an optionally substituted pentose or hexose moiety. In some embodiments, a modified sugar moiety is an optionally substituted pentose moiety. In some embodiments, a modified sugar moiety is an optionally substituted hexose moiety. In some embodiments, a modified sugar moiety is an optionally substituted ribose or hexitol moiety. In some embodiments, a modified sugar moiety is an optionally substituted ribose moiety. In some embodiments, a modified sugar moiety is an optionally substituted hexitol moiety.
• an example modified internucleotidic linkage and/or sugar is selected from:
• R 1 is R as defined and described.
• R 2 is R.
• R e is R.
• R e is H, CH 3 , Bn, COCF 3 , benzoyl, benzyl, pyren-l-ylcarbonyl, pyren-l-ylmethyl, 2-aminoethyl.
• an example modified internucleotidic linkage and/or sugar is selected from those described in Ts'o et al. Ann. N. Y. Acad. Sci. 1988. 507, 220; Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994.
• one or more hydroxyl group in a sugar moiety is optionally and independently replaced with halogen, -R' -N(R') 2 , -OR', or -SR', wherein each R' is independently as defined above and described herein.
• SU is In some embodiments, SU is
• SU is -HTM . In some embodiments, SU is . In some embodiments, SU is .
• L is a covalent bond. In some embodiments, L is not a covalent bond. In some embodiments, L is a bivalent, optionally substituted, linear or branched group selected from Ci-3o aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci -6 alkylene, Ci -6 alkenylene, — C ⁇ C— , -C(R') 2 - -Cy- -0-, S ⁇ SS- -N(R')- -C(0)- -C(S)-, -C(NR')-, -C(0)N(R')-, -N(R')C(0)N(R')-, -N(R')C(0)-, -N(R')C(0)0- -OC(0)N(R')-, -S(
• L is a bivalent, optionally substituted, linear or branched Ci -30 heteroaliphatic group. In some embodiments, L comprises one or more Si.
• R 5s is R' . In some embodiments, R 5s is -OR' . In some embodiments, R 5s is a protected hydroxyl group suitable for oligonucleotide synthesis. In some embodiments, R 5s is -OR', wherein R' is optionally substituted Ci -6 aliphatic. In some embodiments, R 5s is DMTrO- Example protecting groups are widely known in the art for use in accordance with the present disclosure. For additional examples, see Greene, T. W.; Wuts, P. G. M.
• R 2s is -H. In some embodiments, R 2s is -F. In some embodiments, R 2s is -CN. In some embodiments, R 2s is -N 3 . In some embodiments, R 2s is -NO. In some embodiments, R 2s is -N0 2 . In some embodiments, R 2s is -R'. In some embodiments, R 2s is -OR' . In some embodiments, R 2s is -OR', wherein R' is optionally substituted Ci -6 aliphatic. In some embodiments, R 2s is -OMe. In some embodiments, R 2s is - SR' .
• R 2s is -N(R') 2 . In some embodiments, R 2s is -O-L-OR'. In some embodiments, R 2s is -O-L-OR', wherein L is optionally substituted Ci -6 alkylene, and R' is optionally substituted Ci -6 aliphatic. In some embodiments, R 2s is -0-(optionally substituted Ci. 6 alkylene)-OR' . In some embodiments, R 2s is -0-(optionally substituted Ci -6 alkylene)-OR', wherein R' is optionally substituted Ci -6 alkyl. In some embodiments, R 2s is -OCH 2 CH 2 OMe.
• R 2s is -O-L-SR' . In some embodiments, R 2s is -0-L-N(R') 2 . In some embodiments, R 2s is L connecting C2 with CI, C2, C3, C4 or C5. In some embodiments, R 2s is L connecting C2 with CI . In some embodiments, R 2s is L connecting C2 with C2. In some embodiments, R 2s is L connecting C2 with C3. In some embodiments, R 2s is L connecting C2 with C4. In some embodiments, R 2s is L connecting C2 with C5. In some embodiments, R 2s (C2)-0-(optionally substituted methylene)-(C4).
• R 2s (C2)-0-(ethylmethylene)-(C4). In some embodiments, R 2s (C2)-0-((R)- ethylmethylene)-(C4). In some embodiments, R 2s (C2)-0-((S)-ethylmethylene)-(C4). In some embodiments, R 2s comprises a chiral carbon in R configuration. In some embodiments, R 2s comprises a chiral carbon in S configuration.
• CI is connected to BA.
• -Cy- is an optionally substituted bivalent 3-30 membered carbocyclylene. In some embodiments, -Cy- is an optionally substituted bivalent 6-30 membered arylene. In some embodiments, -Cy- is an optionally substituted bivalent 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur. In some embodiments, -Cy- is an optionally substituted bivalent 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, -Cy- is an optionally substituted bivalent 5-30 membered heteroarylene having 1-5 heteroatoms independently selected from oxygen, nitrogen and sulfur. In some embodiments, -Cy- is an optionally substituted bivalent 3-30 membered heterocyclylene having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• R 1 is R' .
• R 2 is R'.
• R 3 is R' .
• each of R 1 , R 2 , and R 3 is independently R', or two or three l 2 3
• R , and R are taken together with their intervening atoms to form— (3 ⁇ 4— (R s ' of R ,
• valence of Ring A is t+1. In some embodiments, Ring A is bivalent. In some embodiments, Ring A is trivalent. In some embodiments, Ring A is polyvalent having a valence greater than 3.
• R 1 is R. In some embodiments, R 1 is R, wherein R is not hydrogen. In some embodiments, R 1 is optionally substituted Ci-io aliphatic. In some embodiments, R 1 is optionally substituted Ci-io alkyl. In some embodiments, R 1 is optionally substituted methyl. In some embodiments, R 1 is optionally substituted ethyl. In some embodiments, R 1 is -0(CH 2 ) 2 CN.
• R 2 is R. In some embodiments, R 2 is R, wherein R is not hydrogen. In some embodiments, R 2 is optionally substituted Ci-io aliphatic. In some embodiments, R 2 is optionally substituted Ci-io alkyl. In some embodiments, R 2 is optionally substituted methyl. In some embodiments, R 2 is optionally substituted ethyl. In some embodiments, R 2 is isopropyl.
• R 3 is R. In some embodiments, R 3 is R, wherein R is not hydrogen. In some embodiments, R 3 is optionally substituted Ci-io aliphatic. In some embodiments, R 3 is optionally substituted Ci-io alkyl. In some embodiments, R 3 is optionally substituted methyl. In some embodiments, R 3 is optionally substituted ethyl. In some embodiments, R 3 is isopropyl.
• R 2 and R 3 are the same. In some embodiments, R 2 and R 3 are different.
• R 1 and R 2 are taken together with their intervening atoms f (R s )t
• R 1 and R 3 are taken together with their intervening atoms to form ⁇ - ⁇ . In some embodiments, R 2 and R 3 are taken together
• R 1 , R 2 and R 3 are
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or poly cyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or poly cyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
• Ring A is an optionally substituted multivalent, monocyclic, bicyclic or poly cyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0 heteroatom.
• Ring A is an optionally substituted multivalent monocyclic saturated 5-7 membered ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms. In some embodiments, Ring A is an optionally substituted multivalent monocyclic saturated 5-membered ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms. In some embodiments, Ring A is an optionally substituted multivalent monocyclic saturated 6-membered ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms. In some embodiments, Ring A is an optionally substituted multivalent monocyclic saturated 7-membered ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms.
• Ring A is an optionally substituted multivalent, bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• Ring A is an optionally substituted multivalent, bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
• Ring A is a multivalent, bicyclic and saturated 8-10 membered bicyclic ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms. In some embodiments, Ring A is a multivalent, bicyclic and saturated 8-membered bicyclic ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms. In some embodiments, Ring A is a multivalent, bicyclic and saturated 9-membered bicyclic ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms.
• Ring A is a multivalent, bicyclic and saturated 10-membered bicyclic ring having the intervening nitrogen, phosphorus and oxygen atoms and no additional heteroatoms.
• Ring A is bicyclic and comprises a 5-membered ring fused to a 5-membered ring.
• Ring A is bicyclic and comprises a 5-membered ring fused to a 6-membered ring.
• the 5-membered ring comprises the intervening nitrogen, phosphorus and oxygen atoms as ring atoms.
• Ring A comprises a ring system having the
• Ring A is an optionally substituted multivalent, polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• Ring A is an optionally substituted multivalent, polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
• Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-10 membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-9 membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-8 membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms.
• Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-7 membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-6 membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms.
• Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 6-membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 7-membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms.
• Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 8-membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 9- membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 10-membered monocyclic ring whose ring atoms comprise the intervening nitrogen, phosphorus and oxygen atoms.
• Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 5-membered ring whose ring atoms consist of the intervening nitrogen, phosphorus and oxygen atoms and carbon atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 6-membered ring whose ring atoms consist of the intervening nitrogen, phosphorus and oxygen atoms and carbon atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 7-membered ring whose ring atoms consist of the intervening nitrogen, phosphorus and oxygen atoms and carbon atoms.
• Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 8-membered ring whose ring atoms consist of the intervening nitrogen, phosphorus and oxygen atoms and carbon atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 9-membered ring whose ring atoms consist of the intervening nitrogen, phosphorus and oxygen atoms and carbon atoms. In some embodiments, Ring A is monocyclic, bicyclic or polycyclic and comprises an optionally substituted 10-membered ring whose ring atoms consist of the intervening nitrogen, phosphorus and oxygen atoms and carbon atoms.
• Ring A comprises a ring system having the backbone
• R s is R' . In some embodiments, R s is R. In some embodiments, R s is optionally substituted Ci -30 heteroaliphatic. In some embodiments, R s comprises one or more silicon atoms. In some embodiments, R s is -CH 2 Si(Ph) 2 CH 3 . [00132] In some embodiments, R s is -L-R' . In some embodiments, R s is -L-R' wherein
• -L- is a bivalent, optionally substituted Ci -30 heteroaliphatic group.
• R s is -CH 2 Si(Ph) 2 CH 3 .
• t is 0. In some embodiments, t is 1-5. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5.
• R' is -R. In some embodiments, R' is -C(0)R. In some embodiments, R' is -C0 2 R. In some embodiments, R' is -S0 2 R. In some embodiments, two or more R' are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or poly cyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R' is hydrogen. In some embodiments, R' is not hydrogen.
• R is hydrogen. In some embodiments, R is not hydrogen.
• R is an optionally substituted group selected from Ci -3 o aliphatic, Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6-3 o aryl, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• R is hydrogen or an optionally substituted group selected from Ci -2 o aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1- 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted Ci -3 o aliphatic. In some embodiments, R is optionally substituted Ci -20 aliphatic. In some embodiments, R is optionally substituted CMS aliphatic. In some embodiments, R is optionally substituted Ci-io aliphatic. In some embodiments, R is optionally substituted Ci -6 aliphatic. In some embodiments, R is optionally substituted Ci -6 alkyl. In some embodiments, R is optionally substituted hexyl, pentyl, butyl, propyl, ethyl or methyl. In some embodiments, R is optionally substituted hexyl. In some embodiments, R is optionally substituted pentyl.
• R is optionally substituted butyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is hexyl. In some embodiments, R is pentyl. In some embodiments, R is butyl. In some embodiments, R is propyl. In some embodiments, R is ethyl. In some embodiments, R is methyl. In some embodiments, R is isopropyl. In some embodiments, R is ⁇ -propyl. In some embodiments, R is tert-butyl. In some embodiments, R is sec-butyl. In some embodiments, R is «-butyl.
• R is optionally substituted C3-30 cycloaliphatic. In some embodiments, R is optionally substituted C 3-20 cycloaliphatic. In some embodiments, R is optionally substituted C3-10 cycloaliphatic. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl.
• R is an optionally substituted 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 6- membered saturated or partially unsaturated carbocyclic ring.
• R is an optionally substituted 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is optionally substituted cycloheptyl. In some embodiments, R is cycloheptyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.
• R is Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is Ci-2o heteroaliphatic having 1-10 heteroatoms. In some embodiments, R is Ci-20 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus or silicon, optionally including one or more oxidized forms of nitrogen, sulfur, phosphorus or selenium. In some embodiments, R is Ci-3o heteroaliphatic comprising 1-
• R is optionally substituted C 6- 3o aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is substituted phenyl.
• R is an optionally substituted 8-10 membered bicyclic saturated, partially unsaturated or aryl ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated ring. In some embodiments, R is an optionally substituted 8- 10 membered bicyclic partially unsaturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic aryl ring. In some embodiments, R is optionally substituted naphthyl.
• R is optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
• R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is selected from optionally substituted pyrrolyl, furanyl, or thienyl.
• R is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom, and an additional heteroatom selected from sulfur or oxygen.
• Example R groups include but are not limited to optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.
• R is an optionally substituted 5-membered heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• Example R groups include but are not limited to optionally substituted triazolyl, oxadiazolyl or thiadiazolyl.
• R is an optionally substituted 5-membered heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• Example R groups include but are not limited to optionally substituted tetrazolyl, oxatriazolyl and thiatriazolyl.
• R is an optionally substituted 6-membered heteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having four nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having three nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having two nitrogen atoms.
• R is an optionally substituted 6-membered heteroaryl ring having one nitrogen atom.
• Example R groups include but are not limited to optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.
• R is 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
• R is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen or sulfur.
• R is optionally substituted 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted indolinyl.
• R is optionally substituted isoindolinyl.
• R is optionally substituted 1, 2, 3, 4-tetrahydroquinolinyl.
• R is optionally substituted 1, 2, 3, 4-tetrahydroisoquinolinyl.
• R is an optionally substituted azabicyclo[3.2.1]octanyl.
• R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6- fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted l,4-dihydropyrrolo[3,2-£]pyrrolyl, 4H- furo[3,2-£]pyrrolyl, 4H-thieno[3,2-£]pyrrolyl, furo[3,2-£]furanyl, thieno[3,2-£]furanyl, thieno[3,2-£]thienyl, lH-pyrrolo[l,2-a]imidazolyl, pyrrolo[2, l-b]oxazolyl or pyrrolo[2,l- b]thiazolyl.
• R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted dihydropyrroloimidazolyl, lH-furoimidazolyl, ⁇ H- thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, IH- imidazoimidazolyl, imidazooxazolyl or imidazo[5,l-b]thiazolyl.
• R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6- fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo[£]thienyl.
• R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted azaindolyl.
• R is optionally substituted benzimidazolyl.
• R is optionally substituted benzothiazolyl.
• R is optionally substituted benzoxazolyl.
• R is an optionally substituted indazolyl.
• R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted oxazolopyridiyl, thiazolopyridinyl or imidazopyridinyl.
• R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl or imidazopyridazinyl.
• R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is an optionally substituted 6,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinolinyl. In some embodiments, R is optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted quinazolinyl, phthalazinyl, quinoxalinyl or naphthyridinyl.
• R is an optionally substituted 6,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted pyridopyrimidinyl, pyridopyridazinyl, pyridopyrazinyl, or benzotriazinyl.
• R is an optionally substituted 6,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R is optionally substituted pyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl, pyridazinopyridazinyl, pyrimidopyridazinyl or pyrimidopyrimidinyl.
• R is an optionally substituted 6,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• R 1 , R 2 , R 3 elements for example, chiral centers.
• Phosphoramidites comprising such asymmetric moieties can, among other things, be used for preparing chirally controlled oligonucleotide compositions, such as those described in WO/2014/012081, WO/2015/107425, etc.
• such phosphoramidites when used in oligonucleotide synthesis, can deliver diastereoselectivity greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% at the newly formed P-chiral center, optionally with greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% yield.
• diastereoselectivity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g.
• the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage
• the 5'-nucleoside of the dimer is the same as the nucleoside to the 5'-end of the chiral internucleotidic linkage
• the 3 '-nucleoside of the dimer is the same as the nucleoside to the 3 '-end of the chiral internucleotidic linkage.
• diastereoselectivity of the underlined coupling or linkage in NNN sfNNG* SGNNNNNNN can be assessed from coupling two G moieties under the same or comparable conditions, e.g., monomers, chiral auxiliaries, solvents, activators, temperatures, etc.
• At least one R s is not hydrogen. In some embodiments, each R s is not hydrog ;en. In some embodiments, R s is optionally substituted phenyl. In some embodiments, R s i s
• R S optionally substituted Ci -6 aliphatic.
• R or ⁇ - ⁇ , is
• phosphoramidites can be prepared from nucleoside and chiral auxiliaries, including those comprising an amino group and a hydroxyl group as illustrated in the examples.
• Suitable chiral auxiliaries include but are not limited to those described in WO/2011/005761, WO/2013/012758, WO/2014/012081, WO/2015/107425, WO/2010/064146, WO/2014/010250, WO/2011/108682, WO/2012/039448, or WO/2012/073857, the chiral auxiliaries of each of which are hereby incorporated by reference.
• provided technologies have been applied to prepare phosphoramidites using chiral auxiliaries of various structures, for example, those demonstrated in the Examples.
• a phosphoramidite is one described in WO/2011/005761,
• the present disclosure provides a method comprising an oligonucleotide preparation method described in one of these applications, wherein the phosphoramidites are purified using a method described herein.
• a phosphoramidite is:
• a reaction forming a preparation prior to filtration is a multi-step reaction.
• Provided methods may comprise use of a temperature higher and/or lower than room temperature.
• provided methods e.g., in a reaction forming a phosphoramidite, comprise use of a lowered temperature, such as a temperature equal to or lower than about -78, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, 0, 5, 10, 15, or 20 °C.
• provided methods e.g., in a reaction forming a phosphoramidite, comprise use of an elevated temperature, such as a temperature equal to or more than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140 or 150 °C.
• provided methods comprise a temperature increase from a lowered temperature to another lowered temperature.
• provided methods comprise a temperature increase from a lowered temperature to room temperature.
• provided methods comprise a temperature increase from room temperature to an elevated temperature.
• provided methods comprise a temperature increase from a lowered temperature to an elevated temperature.
• provided methods comprise a temperature decrease from an elevated temperature to another elevated temperature. In some embodiments, provided methods comprise a temperature decrease from an elevated temperature to room temperature. In some embodiments, provided methods comprise a temperature decrease from room temperature to a lowered temperature. In some embodiments, provided methods comprise a temperature decrease from an elevated temperature to a lowered temperature.
• reactions for forming phosphoramidites are performed in a solvent comprising ether.
• reactions for forming phosphoramidites are performed in a solvent comprising THF.
• reactions for forming phosphoramidites are performed in THF.
• salts formed when forming phosphoramidites have a lower solubility than phosphoramidites; they precipitate from the preparation and can be removed by filtration as described herein.
• provided methods use water to quench unreacted reagents, for example, phosphorochloridite.
• a desiccant is Na 2 S0 4 .
• a desiccant is MgS0 4 .
• a limited amount is about or less than 1 mole equivalent of phosphoramidites (i.e., molar ratio of water : phosphoramidite is about or less than 1 : 1).
• a limited amount is about or less than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mole equivalents of phosphoramidites.
• a material amount is about or more than 1, 2, 3, 4, 5, 6, 7,
• phosphoramidites i.e., molar ratio of water : phosphoramidite is about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mole equivalents of phosphoramidites (i.e., molar ratio of water : phosphoramidite is about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 : 1).
• a material amount is about or more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 2 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 3 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 4 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 5 mole equivalents of phosphoramidites.
• a material amount is about or more than 6 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 7 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 8 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 9 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 10 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 20 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 50 mole equivalents of phosphoramidites. In some embodiments, a material amount is about or more than 100 mole equivalents of phosphoramidites.
• a material amount is about or more than 0.5%, 1%, 2%,
• a material amount is about or more than 0.5% (v/v). In some embodiments, a material amount is about or more than 1% (v/v). In some embodiments, a material amount is about or more than 2% (v/v). In some embodiments, a material amount is about or more than 3% (v/v).
• a material amount is about or more than 4% (v/v). In some embodiments, a material amount is about or more than 5% (v/v). In some embodiments, a material amount is about or more than 6% (v/v). In some embodiments, a material amount is about or more than 7% (v/v). In some embodiments, a material amount is about or more than 8% (v/v). In some embodiments, a material amount is about or more than 9% (v/v). In some embodiments, a material amount is about or more than 10% (v/v). In some embodiments, a material amount is about or more than 15%> (v/v). In some embodiments, a material amount is about or more than 20% (v/v).
• a material amount is about or more than 30%> (v/v). In some embodiments, a material amount is about or more than 40%) (v/v). In some embodiments, a material amount is about or more than 50%> (v/v). In some embodiments, a material amount is about or more than 60%> (v/v). In some embodiments, a material amount is about or more than 70% (v/v). In some embodiments, a material amount is about or more than 80%> (v/v). In some embodiments, a material amount is about or more than 90%) (v/v). In some embodiments, a material amount is about or more than 100% (v/v). In some embodiments, a material amount is about or more than 200%> (v/v). In some embodiments, a material amount is about or more than 250%) (v/v). In some embodiments, a material amount is about or more than 300%) (v/v).

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