Oligonucleotide synthesis using cyclic-phosphorous nucleosides

Abstract

The present disclosure provides methods for preparing oligonucleotides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using cyclic-phosphate or cyclic-thiophosphate nucleosides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using nucleophiles selected from substituted thiol nucleophiles, substituted nitrogen-based nucleophiles, substituted phosphine nucleophiles, halides, pseudohalides, azides, and substituted lithium-based nucleophiles.

Description

OLIGONUCLEOTIDE SYNTHESIS USING CYCLIC-PHOSPHOROUS NUCLEOSIDES TECHNICAL FIELD

[0001] The present disclosure provides methods for preparing oligonucleotides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using cyclic-phosphate or cyclic-thiophosphate nucleosides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using nucleophiles selected from substituted thiol nucleophiles, substituted nitrogen-based nucleophiles, substituted phosphine nucleophiles, halides, pseudohalides, azides, and substituted lithium-based nucleophiles. DISCLOSURE

[0002] The demand for synthetic oligonucleotides is steadily increasing as advances in DNA and RNA technology continue to expand through the scientific and medical communities. Innovations in molecular biology, RNA-based therapeutics, DNA-based diagnostics, genetic engineering and treatments, and other similar methods continue to present a high demand for synthesized oligonucleotides that can be used to amplify, detect, analyze, quantify, modify, or target natural nucleic acids. However, current methods for synthesizing oligonucleotides (such as automated solid phase synthesis using phosphoramidite chemistry) can be expensive, limited in scalability, limited in portability (e.g., due to reliance on specialized packed bed reactors), and often require complex workflow systems.

[0003] A need therefore exists for oligonucleotide synthesis methods which are efficient, scalable, easily performed in batch or flow reactors, and reliably provide short, medium, or long- chain oligonucleotides of interest, including oligonucleotides for direct use or for use as synthetic building blocks. SUMMARY

[0004] In certain embodiments, the present disclosure provides a method for preparing a dinucleotide of Formula (vi.b) or a salt or solvate thereof,.

[0005] e present disclosure provides a method for preparing adinucleotide of Formula (vi.b) or a salt or solvate thereof, wherein:R1and R2are independently chosen from hydrogen, hydroxy, halogen, N3, (C1-C20) alkyl, and (C1-C20) alkoxy; optionally wherein R1and R2are independently chosen from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B1and B2are nucleobases, which may optionally be protected; and R4is hydrogen or a protecting group; the method comprising: (i) providing the cyclic phosphorus dinucleotide of Formula (iv) of claim 1, wherein R4is a protecting group: dus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) to form a dinucleotide of Formula (vi.b):;protecting groups.

[0006] In certain embodiments, the present disclosure provides a method for preparing a dinucleotide of Formula (vi) or a salt or solvate thereof, .ents, the present disclosure provides a method for preparing a dinucleotide of Formula (vi) or a salt or solvate thereof, ; wherein:R1and R2are independently chosen from hydrogen, hydroxy, halogen, N3, (C1-C20) alkyl, and (C1-C20) alkoxy; optionally wherein R1and R2are independently chosen from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B1and B2are nucleobases, which may optionally be protected; and R4is hydrogen or a protecting group; the method comprising: (a) reacting a nucleoside of Formula (i) with P(Y)R3, wherein Y is O or S, and wherein each R is independently selected from Cl, Br, (C1-C6) alkoxy, (C6-C12) aryloxy, S-(C1-C6) alkyl, O-aryl, and S-aryl; R is Cl, Br, or (C1-C6) alkoxy, thereby forming a cyclic phosphorous nucleoside of Formula (ii): ; e of Formula (ii) with a nucleoside ofFormula (iii) to form a cyclic phosphorus dinucleotide of Formula (iv), wherein R4is a protecting group: ;e (N) of Formula (v) to form a dinucleotide of Formula (vi.b): ;protecting groups.

[0008] In certain embodiments, the cyclic phosphorus dinucleotide of Formula (iv) is obtained as a mixture of stereoisomers, and wherein the stereoisomers are separated prior to the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v).

[0009] In certain embodiments, the nucleophile (N) of Formula (v) is a thiolate. In certain embodiments, the thiolate is selected from:, [00 nembodiments, the halide is selected from lithium iodide (LiI), lithium bromide (LiBr), and lithium chloride (LiCl).

[0011] In certain embodiments, the nucleophile (N) of Formula (v) is an azide. In certain embodiments, the azide is sodium azide (NaN3).

[0012] In certain embodiments, the nucleophile (N) of Formula (v) is an amine. In certain embodiments, the amine is selected from: , [00phosphine. In certain embodiments, the tri-substituted phosphine is selected from: . [00de of Formula (iv) with a nucleophile (N) of Formula (v) is carried out in a solvent system comprising: tetrahydrofuran (THF); 2-methyltetrahydrofuran (2Me-THF); diethyl ether; 1,4-dioxane; dimethylsulfoxide (DMSO); dimethylformamide (DMF), N-methylpyrrolidone (NMP); dimethylacetamide (DMA); 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 7-Methyl-1,5,7- triazabicyclo[4.4.0]dec-5-ene (mTBD), dichloromethane (DCM); acetonitrile (MeCN or ACN); methyl tert-butyl ether (MTBE); tertAmyl alcohol; or toluene.

[0015] In certain embodiments, the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) is carried out in the presence of a base comprising: n-butyllithium (n-BuLi); lithium tertbutoxide (LiOtBu); lithium bis(trimethylsilyl)amide (LiHMDS); potassium bis(trimethylsilyl)amide (KHMDS); lithium tetramethylpiperidide (LiTMP); or Li-RZ, where RZ is alkyl or aryl; sodium t-butoxide (NaOtBu) + lithium chloride (LiCl); potassium t-butoxide (KOtBu) + lithium chloride (LiCl); triethylamine (Et3N); Diazabicycloundecene (DBU); 4-Dimethylaminopyridine (DMAP); S-collidine; triazabicyclodecene (TBD); N-methyl-triazabicyclodecene (mTBD); or a combination thereof. In certain embodiments, the base is lithium tertbutoxide.

[0016] In certain embodiments, the base is pre-mixed with the nucleophile (N) of Formula (v) prior to the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v).

[0017] In certain embodiments, the dinucleotide of Formula (vi.b) is isolated by reacting with a silyl chloride.

[0018] In certain embodiments, R2is an alkoxy group linked to the adjacent O to form a ketal protecting group with R4.

[0019] In certain embodiments, R4is chosen from H, a silyl group, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl (TBS, TBDMS), t-butyldiphenylsilyl, triisopropylsilyl, or a ketal protecting group formed with R2.

[0020] In certain embodiments, each nucleobase is chosen from pyrimidines and purines.

[0021] In certain embodiments, each nucleobase is protected with one or more protecting groups independently chosen from dimethyl formamide (DMF), formamidine, DMF- formamidine, N-methylpyrrolidin-2-ylidenyl (PyA), pivaloyloxymethyl (POM), phthalimido, chloro, bromo, acyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM).

[0022] In certain embodiments, the method is a liquid phase process. In certain embodiments, the method is conducted using continuous flow. In certain embodiments, the method is carried out in a flow reactor. In certain embodiments, the method is performed at ambient temperature.

[0023] In certain embodiments, the present disclosure provides a dinucleotide produced by the methods of present disclosure.

[0024] In certain embodiments, the present disclosure provides a dinucleotide of the following formula:erein: Y is O or S; R1and R2are independently chosenfrom hydrogen, hydroxy, halogen, N3, (C1-C20) alkyl, and (C1-C20) alkoxy (optionally wherein R1and R2are independently chosen from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE)); R4is H or a protecting group; and B1and B2are nucleobases, which may optionally be protected.

[0025] In certain embodiments, the present disclosure provides a solution comprising a dinucleotide of the present disclosure.

[0026] In certain embodiments, the present disclosure provides for the use of a dinucleotide of the present disclosure in the synthesis of an oligonucleotide or polynucleotide. In certain embodiments, the present disclosure provides for the use of a dinucleotide of the present disclosure as a blockmer amidite in solid phase ligation, chemical ligation, or other synthesis method of an oligonucleotide or polynucleotide. In certain embodiments, the present disclosure provides for the use of a dinucleotide of the present disclosure in an enzymatic ligation reaction for the synthesis of an oligonucleotide or polynucleotide. DETAILED DESCRIPTION OLIGONUCLEOTIDE SYNTHESIS Oligonucleotides

[0027] The present disclosure provides methods for preparing oligonucleotides. The present disclosure also provides oligonucleotides prepared by the methods of the present disclosure. Oligonucleotides are polymers of linked nucleotides, typically less than about 100 nucleotides in length. In certain embodiments, oligonucleotides of the present disclosure include at least 2 nucleotides. Thus, in certain embodiments, the oligonucleotides of the present invention include 2 to 100 nucleotides and all whole-number integer ranges from (e.g., 2 to 100, 2 to 50, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 3 nucleotides, 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 6 nucleotides, etc.). In certain embodiments, the oligonucleotide includes 2 nucleotides (i.e., a dinucleotide). In certain embodiments, the oligonucleotide includes3 nucleotides (i.e., a trinucleotide). In certain embodiments the oligonucleotide includes 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0028] In certain embodiments, oligonucleotides of the present disclosure include from 3 to 100 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 50 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 30 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 20 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 15 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 10 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 8 nucleotides. In certain embodiments, the oligonucleotide includes from 3 to 6 nucleotides. In certain embodiments, the oligonucleotide includes 3 nucleotides (i.e., a trinucleotide).

[0029] In certain embodiments, oligonucleotides of the present disclosure include from 5 to 20 nucleotides. In certain embodiments, oligonucleotides of the present disclosure include from 10 to 20 nucleotides. In certain embodiments, oligonucleotides of the present disclosure include from 15 to 20 nucleotides. In certain embodiments, the oligonucleotide includes 3 nucleotides. In certain embodiments, the oligonucleotide includes 5 nucleotides. In certain embodiments, the oligonucleotide includes 6 nucleotides. In certain embodiments, the oligonucleotide includes 7 nucleotides. In certain embodiments, the oligonucleotide includes 8 nucleotides. In certain embodiments, the oligonucleotide includes 9 nucleotides. In certain embodiments, the oligonucleotide includes 10 nucleotides. In certain embodiments, the oligonucleotide includes 11 nucleotides. In certain embodiments, the oligonucleotide includes 12 nucleotides. In certain embodiments, the oligonucleotide includes 13 nucleotides. In certain embodiments, the oligonucleotide includes 14 nucleotides. In certain embodiments, the oligonucleotide includes 15 nucleotides. In certain embodiments, the oligonucleotide includes 16 nucleotides. In certain embodiments, the oligonucleotide includes 17 nucleotides. In certain embodiments, the oligonucleotide includes 18 nucleotides. In certain embodiments, the oligonucleotide includes 19 nucleotides. In certain embodiments, the oligonucleotide includes 20 nucleotides.

[0030] The term "nucleotide" is defined herein (see below) as an organic compound having a nucleoside and a phosphate group. The term "nucleoside" is defined herein (see below) as an organic compound having a nucleobase (e.g., adenine, cytosine, guanine, thymine, or uracil) covalently attached to a pentose sugar (e.g., ribose or 2'-deoxyribose). The term "nucleobase" is defined herein (see below) as a heterocyclic moiety of a nucleoside. In certain embodiments, anucleobase of the present disclosure is a canonical nucleobase. In certain embodiments, the nucleobase is a purine base -- adenine (A) or guanine (G). In certain embodiments, the nucleobase is a pyrimidine base -- thymine (T), cytosine (C), or uracil (U).

[0031] In certain embodiments, a nucleobase of the present disclosure is a noncanonical nucleobase (i.e., not a canonical nucleobase). Examples of noncanonical nucleobases include, but are not limited to: 3-methylcytosine; 5-methylcytosine (5-me-C); 5-ethylcytosine; 5-ethyluracil; 5-hydroxymethyl cytosine; 7-methylguanine; 1-methylguanine; 2-methylguanine; 8- methylguanine; 2,2-dimethylguanine; 1-methyladenine; 2-methyladenine; N6-methyladenine; 7- methyladenine; N,N-dimethyladenine; alkyl derivatives (e.g., 6-methyl, 2-propyl) of adenine and guanine; 5-propyluracil; xanthine; hypoxanthine; 2-aminoadenine; 2-aminopyridine; 2- aminopurine; 2,6-diaminopurine; 8-aminoguanine; 2-thiouracil; 5-methyl-2-thiouracil; 4- thiouracil; 2-thiothymine; 2-thiocytosine; 6-thiopurine; 8-thioguanine; 5-halouracil; cytosine; alkynyl derivatives (e.g., 5-propynyl) of uracil and cytosine; 6-azo uracil; 5-uracil (pseudouracil); 8-substituted (e.g., 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-halo) adenines and guanines; 5-substituted (e.g., 5-chloro, 5-bromo, 5-trifluoromethyl) uracils and cytosines; 2- F-adenine; 8-azaguanine; 8-azaadenine; 7-deazaguanine; 7-deazaadenine; 3-deazaguanine; 3- deazaadenine; tricyclic pyrimidines; phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin- 2(3H)-one); phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one); substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin- 2(3H)-one); carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one); pyridoindole cytidine (H- pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one); 2-pyridone; N6-isopentyladenine; 2-methylthio- N6-isopentyladenine; 8-bromoadenine; 8-bromoguanine; 8-chloroguanine; 5-bromouracil; 5- fluorouracil; 5-chlorouracil; 5-iodouracil; 4-acetylcytosine; 5-methoxyuracil; 5- hydroxymethyluracil; 5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5- (carboxymethylaminomethyl)-uracil; 5-(2-bromovinyl)uracil; uracil-5-oxyacetic acid; uracil-5- oxyacetic acid methyl ester; pseudouracil, l-methylpseudouracil; queosine; inosine; 1- methylinosine; and 6-hydroxyaminopurine. Examples of noncanonical nucleobases also include, but are not limited to: methylated purines or pyrimidines, alkylated purines or pyrimidines, acylated purines or pyrimidines, halogenated purines or pyrimidines, deazapurines, alkylated riboses, diaminopurine, inosine, and thiolated purines or pyrimidines.

[0032] In certain embodiments, the nucleobase is unprotected. In certain embodiments, the nucleobase is protected by a protecting group. The term “protecting group” is defined herein (see below) as a chemical moiety that protects reactive or labile groups on a molecule from undergoing undesired chemical reactions during synthetic procedures or other chemical reactions. A nucleobase protecting group refers to a protecting group applied to one or more functional groups of a nucleobase to prevent side reactions at the nucleobase during the synthesis of oligonucleotides. In certain embodiments, nucleobase protecting groups are removed at the end of the oligonucleotide synthesis. Nucleobase protecting groups can include exocyclic amino groups or lactam groups. In certain embodiments, a nucleobase protecting group is applied to the N6-amino group of an adenine moiety. In certain embodiments, a nucleobase protecting group is applied to N4-amino group of a cytosine moiety. In certain embodiments, a nucleobase protecting group is applied to the N2-amino group, the N1-lactam (i.e., cyclic amide) group, and / or the O6-lactam groups of a guanine moiety. In certain embodiments, a nucleobase protecting group is applied to the N3-lactam group and / or the O4-lactam group of a uracil or thymine moiety. In certain embodiments, the nucleobase protecting group is base-labile. Examples of base-labile protecting group include FMOC (fluorenylmethyloxycarbonyl). In certain embodiments, the nucleobase protecting group is acid labile. Examples of acid-labile protecting group include triarylmethyl protecting groups, 4, 4'-dimethoxytriphenyl methyl (DMT), and BOC carbamate (tert-butoxycarbonyl), In certain embodiments, the nucleobase protecting group is labile with a reagent other than bases or acids.

[0033] In certain embodiments, the nucleobase protecting group is an amino protecting group. As used herein, an amino protecting group refers to a protecting group which can be used to protect an amino group on a molecule. Examples of amino protecting groups include, but are not limited to, N-methylpyrrolidin-2-ylidenyl (PyA), pivaloyloxymethyl (POM), 2- trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t- butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrophenylacetyl, 2- nitrobenzenesulfonyl, phthalimido, dithiasuccinoyl, methyl carbamate, ethyl carbamate, 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-phenylethylcarbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2- trichloroethyl carbamate (TCBOC), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t- Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N- hydroxypiperidinyl carbamate, alkyldithio carbamate, p-methoxybenzyl carbamate (Moz), p- nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p- toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1- dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6- chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1- methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p- phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, dimethyl formamide (DMF), formamidine, DMF-formamidine,acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3- phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p- hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5- triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5- dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4- methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′- oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p- nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2- hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1- cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N- [phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamid.

[0034] In certain embodiments, the nucleobase protecting group is a keto / carboxylic acid protecting group. As used herein, a keto protecting group, a carboxylic acid protecting group, or a keto / carboxylic acid protecting group refers to a protecting group which can be used to protect a keto or carboxylic acid group on a molecule. Examples of keto / carboxylic acid protecting groups include, but are not limited to: silyl protecting groups such as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl (TBS, TBDMS), t-butyldiphenylsilyl, or triisopropylsilyl; alkyl protecting groups such as methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, or tetrahydropyran-2-yl; alkenyl protecting groups such as allyl; aryl protecting groups such as optionally substituted phenyl, biphenyl, or naphthyl; and arylalkyl protecting groups such as substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), or 2- and 4-picolyl.

[0035] In certain embodiments, the nucleobase protecting group is a hydroxyl protecting group. As used herein, a hydroxyl protecting group refers to a protecting group which can be used to protect a hydroxyl group on a molecule. Examples of protecting groups that can be used as a hydroxyl protecting group include, but are not limited to, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, t-butyl, t-butoxymethyl, methoxymethyl, bis(2- acetoxyethoxy)methyl (ACE), [(triisopropylsilyl)oxy]methyl (TOM), pivaloyl, benzoyl, p- phenylbenzoyl, monomethoxytrityl (MMTr), dimethoxytrityl (DMT), 4,4′,4″-trimethoxytrityl (TMTr), 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), substituted pixyl, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2- trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin- 4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o- nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2- picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′- bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S- dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl(DEIPS), dimethylthexylsilyl, t- butyldimethylsilyl (TBS, TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl)ethyl (TSE), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1- dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4- nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6- trimethylphenyl, 2-(2-nitrophenyl)ethyl, sulfate, methanesulfonate (mesylate), benzylsulfonate, tosylate (Ts), 2-cyanoethyl (CE or Cne), 3,5-dichlorophenyl, 2,4-dimethylphenyl, butylthiocarbonyl, 4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2- (dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9- phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0036] In certain embodiments, the nucleobase protecting group is a thiol protecting group. As used herein, a thiol protecting group refers to a protecting group which can be used to protect a thiol group on a molecule. Examples of protecting groups that can be used as a thiol protecting group include, but are not limited to, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, t-butyl, t-butoxymethyl, methoxymethyl, bis(2-acetoxyethoxy)methyl (ACE), [(triisopropylsilyl)oxy]methyl (TOM), pivaloyl, benzoyl, p-phenylbenzoyl, monomethoxytrityl (MMTr), dimethoxytrityl (DMT), 4,4′,4″-trimethoxytrityl (TMTr), 1(2-fluorophenyl)-4- methoxypiperidin-4-yl (FPMP), substituted pixyl, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p- AOM), guaiacolmethyl (GUM), 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p- methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6- dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N- oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a- naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″- tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4- methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10- oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBS, TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4- methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2- trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl (TSE), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4- azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate,chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o- (methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4- nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6- trimethylphenyl, 2-(2-nitrophenyl)ethyl, sulfate, methanesulfonate (mesylate), benzylsulfonate, tosylate (Ts), 2-cyanoethyl (CE or Cne), 3,5-dichlorophenyl, 2,4-dimethylphenyl, butylthiocarbonyl, 4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2- (dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9- phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0037] In certain embodiments, the nucleobase protecting group includes a protecting group for protecting 1,2- or 1,3-diols. Examples of protecting groups for protecting 1,2- or 1,3-diols include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p- methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene ortho ester, 1-(N,N- dimethylamino)ethylidene derivative, α-(N,N′-dimethylamino)benzylidene derivative, 2- oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3- tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

[0038] In certain embodiments, each nucleobase is protected with one or more protecting groups independently chosen from dimethyl formamide (DMF), N-methylpyrrolidin-2-ylidenyl (PyA), pivaloyloxymethyl (POM), formamidine, DMF-formamidine, phthalimido, chloro, bromo, acyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM). Cyclic-phosphorous nucleosides

[0039] In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using cyclic-phosphorous nucleosides, or a salt or solvate thereof. As usedherein, a cyclic-phosphorous nucleoside refers to a nucleoside which includes a P(Y)R3 group bound to the 3' and 5' oxygens of a nucleoside sugar to form a compound of Formula (ii): or a ereof, wherein: Y is O or S; each R is independently selected from Cl, Br,(C1-C6) alkoxy, (C6-C12) aryloxy, S-(C1-C6) alkyl, O-aryl, and S-aryl; R1is chosen from hydrogen, hydroxy (protected), halogen (including fluorine), N3, (C1-C20) alkyl, and (C1-C20) alkoxy (including methoxy and -O-methoxyethyl); and B1is a nucleobase, which may be optionally protected. In certain embodiments, R1is (C1-C16) alkyl or (C1-C16) alkoxy. In certain embodiments, R1is (C1-C12) alkyl or (C1-C12) alkoxy. In certain embodiments, R1is (C1-C8) alkyl or (C1-C8) alkoxy. In certain embodiments, R1is (C1-C4) alkyl or (C1-C4) alkoxy. In certain embodiments, R1is fluorine. In certain embodiments, R1is methoxy. In certain embodiments, R1is O-methoxyethyl (MOE).

[0040] As used herein, a cyclic-phosphate nucleoside refers to a cyclic-phosphorous nucleoside in which Y is O. As used herein, a cyclic-thiophosphate nucleoside refers to a cyclic- phosphorous nucleoside in which Y is S.

[0041] In certain embodiments, the present disclosure provides methods for preparing a cyclic-phosphorous nucleoside of Formula (ii), or a salt or solvate thereof. In certain embodiments, the method for preparing a cyclic-phosphorous nucleoside of Formula (ii) comprises: (a) reacting a nucleoside of Formula (i) with P(Y)R3, wherein Y is O or S, and wherein each R is independently selected from Cl, Br, (C1-C6) alkoxy, (C6-C12) aryloxy, S-(C1- C6) alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphorous nucleoside of Formula (ii) .

[004] n certa n em o ments, t e present sclosure provides a cyclic-phosphorous nucleoside of Formula (ii), or a salt or solvate thereof.

[0043] In certain embodiments, the nucleobase of the cyclic phosphorous nucleoside is a canonical nucleobase. In certain embodiments, the nucleobase is a purine base -- adenine (A) or guanine (G). In certain embodiments, the nucleobase is a pyrimidine base -- thymine (T), cytosine (C), or uracil (U). In certain embodiments, the nucleobase of the cyclic phosphorous nucleoside is a noncanonical nucleobase.

[0044] In certain embodiments, the nucleobase of the cyclic-phosphorous nucleoside is unprotected. In certain embodiments, the nucleobase of the cyclic-phosphorous nucleoside is protected with a nucleobase protecting group. In certain embodiments, the nucleobase of the cyclic-phosphorous nucleoside is protected with an amino nucleobase protecting group. In certain embodiments, the nucleobase of the cyclic-phosphorous nucleoside is protected with a keto / carboxylic acid nucleobase protecting group. In certain embodiments, the nucleobase of the cyclic-phosphorous nucleoside is protected with a hydroxyl nucleobase protecting group. In certain embodiments, the nucleobase of the cyclic-phosphorous nucleoside is protected with a thiol nucleobase protecting group. In certain embodiments, the nucleobase of the cyclic- phosphorous nucleoside is protected with one or more protecting groups independently chosen from dimethyl formamide (DMF), formamidine, DMF-formamidine, phthalimido, chloro, bromo, acyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM).

[0045] In certain embodiments, the cyclic phosphorus nucleoside of Formula (ii) is obtained as a mixture of stereoisomers. In certain embodiments, the stereoisomers are separated prior to subsequent reaction steps. Dinucleotides

[0046] In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using a dinucleotide which comprises a cyclic-phosphorous nucleoside of the present disclosure, or a salt or solvate thereof. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using a dinucleotide of Formula (iv) or a salt or solvate thereof:wherein: Y is O or S; R1and R2are independently chosen from hydrogen, hydroxy (protected), halogen (including fluorine), N3, (C1-C20) alkyl, and (C1-C20) alkoxy group (including methoxy and -O-methoxyethyl); B1and B2are nucleobases, which may optionally be protected with one or more protecting groups; and R4is hydrogen or a protecting group. In certain embodiments, R1and R2are independently chosen from (C1-C16) alkyl and (C1-C16) alkoxy. In certain embodiments, R1and R2are independently chosen from (C1-C12) alkyl and (C1-C12) alkoxy. In certain embodiments, R1and R2are independently chosen from (C1-C8) alkyl and (C1-C8) alkoxy. In certain embodiments, R1and R2are independently chosen from (C1-C4) alkyl and (C1- C4) alkoxy. In certain embodiments, R1and R2are fluorine. In certain embodiments, R1and R2are methoxy. In certain embodiments, R1and R2are O-methoxyethyl (MOE).

[0047] In certain embodiments, the present disclosure provides a method for preparing a dinucleotide of Formula (iv) or a salt or solvate thereof, wherein the method comprises: (a) reacting a nucleoside of Formula (i) with P(Y)R3, wherein Y is O or S, and wherein each R is independently selected from Cl, Br, (C1-C6) alkoxy, (C6-C12) aryloxy, S-(C1-C6) alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphorous nucleoside of Formula (ii): nd (b)ula (ii) with a nucleoside of Formula (iii) to form a cyclic phosphorus dinucleotide of Formula (iv), wherein R4is a protecting group: . In cera n em o men s, e me o ur er compr ses removng one or more o e protecting groups.

[0048] In certain embodiments, the cyclic phosphorus chloride nucleoside of Formula (ii) is obtained as a mixture of stereoisomers. In certain embodiments, the stereoisomers are separatedprior to the step of reacting the cyclic phosphorous nucleoside of Formula (ii) with a nucleoside of Formula (iii).

[0049] In certain embodiments, the present disclosure provides a dinucleotide of Formula (iv) or a salt or solvate thereof.

[0050] In certain embodiments, R2is an alkoxy group linked to the adjacent 3' oxygen to form a ketal protecting group with R4.

[0051] In certain embodiments, R4is hydrogen. In certain embodiments, R4is a protecting group. In certain embodiments, R4is a hydroxyl protecting group. In certain embodiments, R4is chosen from a silyl group, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl (TBS, TBDMS), t- butyldiphenylsilyl, or triisopropylsilyl. In certain embodiments, R4forms a ketal protecting group with R2.

[0052] In certain embodiments, the cyclic phosphorus dinucleotide of Formula (iv) is obtained in an (R) stereoisomer configuration. In certain embodiments, the cyclic phosphorus dinucleotide of Formula (iv) is obtained in an (S) stereoisomer configuration. In certain embodiments, the cyclic phosphorus dinucleotide of Formula (iv) is obtained as a mixture of stereoisomers. In certain embodiments, the cyclic phosphorus dinucleotide of Formula (iv) is obtained as a mixture of (R) and (S) stereoisomers. In certain embodiments, the stereoisomers are separated prior to subsequent synthesis steps. In certain embodiments, the stereoisomers are separated prior to the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile, such as a substituted benzyl alcohol nucleophile of Formula (v).

[0053] In certain embodiments, the mixture of stereoisomers comprises at least about 10% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 20% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 30% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 40% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 50% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 60% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 70% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 75% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 80% of(R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 85% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 90% of (R) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 95% of (R) configuration product.

[0054] In certain embodiments, the mixture of stereoisomers comprises at least about 10% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 20% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 30% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 40% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 50% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 60% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 70% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 75% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 80% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 85% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 90% of (S) configuration product. In certain embodiments, the mixture of stereoisomers comprises at least about 95% of (S) configuration product.

[0055] In certain embodiments, the mixture of stereoisomers comprises a ratio of (R) configuration product to (S) configuration product. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 1.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 2.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 3.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 4.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 5.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 6.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 7.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 8.0. In certain embodiments, the mixture ofstereoisomers has an (R) / (S) ratio of at least about 9.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 10.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 11.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 12.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 13.0. In certain embodiments, the mixture of stereoisomers has an (R) / (S) ratio of at least about 14.0.

[0056] In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 1.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 2.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 3.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 4.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 5.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 6.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 7.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 8.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 9.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 10.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 11.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 12.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 13.0. In certain embodiments, the mixture of stereoisomers has an (S) / (R) ratio of at least about 14.0. Nucleophiles

[0057] In certain embodiments, the present disclosure provides methods for preparing dinucleotides using a nucleophile (N). Nucleophiles for use in the disclosure can include functional groups with high polarizability and low electronegativity, including functional groups associated with a lone pair of electrons that is either delocalized or in a large orbital. Examples of nucleophiles include thiolates, halides, pseudohalides, azides, enolates, tri-substituted phosphines (such as triphenylphosphine), and amines.

[0058] In certain embodiments, the nucleophile is a thiolate. In certain embodiments, the nucleophile is a thiolate selected from:, , thenucleophile is an iodide, such as lithium iodide (LiI). In certain embodiments, the nucleophile is bromide, such as lithium bromide (LiBr). In certain embodiments, the nucleophile is a chloride, such as lithium chloride (LiCl).

[0060] In certain embodiments, the nucleophile is an azide. In certain embodiments, the nucleophile is sodium azide (NaN3).

[0061] In certain embodiments, the nucleophile is an amine. In certain embodiments, the nucleophile is an amine selected from: andembodiments, the nucleophile is a tri-substituted phosphine selected from: nd

[0063] In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using cyclic-phosphorous nucleosides and nucleophiles. In certain embodiments, the present disclosure provides methods for preparing a dinucleotide of Formula (vi.b) or a salt or solvate thereof: Nucleophile R4Y O O B1whe R2are independently chosen from hydrogen, hydroxy (protected),halogen (including fluorine), N3, (C1-C20) alkyl, and (C1-C20) alkoxy (including methoxy and -O- methoxyethyl); B1and B2are nucleobases, which may optionally be protected; and R4is hydrogen or a protecting group; n is chosen from 1, 2, 3, 4, and 5. In certain embodiments, R1and R2are independently chosen from (C1-C16) alkyl and (C1-C16) alkoxy. In certain embodiments, R1and R2are independently chosen from (C1-C12) alkyl and (C1-C12) alkoxy. In certain embodiments, R1and R2are independently chosen from (C1-C8) alkyl and (C1-C8) alkoxy. In certain embodiments, R1and R2are independently chosen from (C1-C4) alkyl and (C1- C4) alkoxy. In certain embodiments, R1and R2are fluorine. In certain embodiments, R1and R2are methoxy. In certain embodiments, R1and R2are -O-methoxyethyl (MOE).

[0064] In certain embodiments, the present disclosure provides a method for preparing a dinucleotide of Formula (vi.b) or a salt or solvate thereof, wherein the method comprises: providing the cyclic phosphorus dinucleotide of Formula (iv), wherein R4is a protecting group; and reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) to form a dinucleotide of Formula (vi.b): . Incerta n embod ments, t e met od urt er compr ses remov ng one or more o t e protecting groups from the dinucleotide of Formula (vi.b).

[0065] In certain embodiments, the present disclosure provides a method for preparing a dinucleotide of Formula (vi.b) or a salt or solvate thereof, wherein the method comprises: (a) reacting a nucleoside of Formula (i) with P(Y)R3, wherein Y is O or S, and wherein each R is independently selected from Cl, Br, (C1-C6) alkoxy, (C6-C12) aryloxy, S-(C1-C6) alkyl, O-aryl, and S-aryl , thereby forming a cyclic phosphorous nucleoside of Formula (ii): ; (b)Formula (ii) with a nucleoside of Formula (iii) to form a cyclic phosphorus dinucleotide of Formula (iv), wherein R4is a protecting group: nd (c)Formula (v) to form a dinucleotide of Formula (vi.b): . In c, g cting groups from the dinucleotide of Formula (vi.b).

[0066] In certain embodiments, the present disclosure provides a dinucleotide of Formula (vi.b) or a salt or solvate thereof.

[0067] In certain embodiments, the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) is carried out in a solvent system. In certainembodiments, the solvent comprises: tetrahydrofuran (THF); 2-methyltetrahydrofuran (2Me- THF); diethyl ether; 1,4-dioxane; dimethylsulfoxide (DMSO), dimethylformamide (DMF), N- methylpyrrolidone (NMP), dimethylacetamide (DMA), 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (mTBD), acetonitrile (MeCN or ACN), methyl tert-butyl ether (MTBE), tertAmyl alcohol, toluene, or a mixture thereof.

[0068] In certain embodiments, the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) is carried out in the presence of a base. In certain embodiments, the base comprises: n-butyllithium (n-BuLi); lithium tertbutoxide (LiOtBu); lithium bis(trimethylsilyl)amide (LiHMDS); lithium tetramethylpiperidide (LiTMP); or Li-RZ, where RZis alkyl or aryl; sodium t-butoxide (NaOtBu) + lithium chloride (LiCl); potassium t-butoxide (KOtBu) + lithium chloride (LiCl); triethylamine, Diazabicycloundecene (DBU); 4-Dimethylaminopyridine (DMAP); triazabicyclodecene (TBD); N-methyl- triazabicyclodecene (mTBD); or a combination thereof. In certain embodiments, the base is pre- mixed with the substituted benzyl alcohol nucleophile of Formula (v) prior to the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a substituted benzyl alcohol nucleophile of Formula (v). In certain embodiments, the base is lithium tertbutoxide (LiOtBu).

[0069] In certain embodiments, the dinucleotide of Formula (vi.b) is isolated by reacting with a silyl chloride.

[0070] In certain embodiments, dinucleotides of the present disclosure can be used in subsequent oligonucleotide or polynucleotide synthesis.

[0071] In certain embodiments of the present disclosure, the oligonucleotide synthesis method is a liquid phase process (i.e., conducted in liquid phase). As used herein, a liquid phase process refers to a process in which all reaction components (e.g., starting materials, reagents, catalysts, bases) are dissolved fully in a solvent mixture, such that there are no solid particles present.

[0072] In certain embodiments, the method is conducted using continuous flow. As used herein, continuous flow refers to a process in which starting materials, reagents, catalysts, bases, and all other reaction components are continuously added to a reactor while the product, side- products, unreacted starting materials, and other reaction components are continuously removed from the reactor.

[0073] In certain embodiments, the method is carried out in a flow reactor. As used herein, flow reactor refers to a pipe, a tube, a continuously stirred tank reactor (CSTR), a cascade ofmultiple CSTRs, an intermittently filled and emptied batch reactor, or other reactors which can be operated continuously. In certain embodiments, the method is carried out in a flow reactor at ambient temperature (e.g., 20-25 ºC). In certain embodiments, the method is carried out in a flow reactor with a reaction time between 45-120 minutes. In certain embodiments, the method is carried out in a flow reactor with a reaction time between 45-60 minutes. DEFINITIONS

[0074] Unless stated otherwise, the following terms and phrases have the meanings described below. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present disclosure.

[0075] Approximately / About: As used herein, the terms "approximately" and "about" are used interchangeably herein and refer to a value that is within + / - 10% of the recited value as applied to one or more values of interest. In certain embodiments, the term refers to a range of values that fall within + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, or less of the stated reference value, unless otherwise expressly stated or otherwise clearly evident from the context.

[0076] Complementary: As used herein, the terms "complementary" or "complementarity" refer a structural relationship between two nucleotides, nucleosides, or nucleobases (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand such as a hairpin), wherein the relationship permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the canonical Watson-Crick manner (i.e., adenine pairing with thymine or uracil, and guanine pairing with cytosine), or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity.

[0077] Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers a nucleotide having a hydrogen in place of a hydroxyl at the 2' position of its pentose sugar when compared with a ribonucleotide. A modified deoxyribonucleotide has one or more modifications or substitutions of atoms other than hydroxyl at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0078] Double-stranded oligonucleotide: As used herein, the terms "double-stranded oligonucleotide" or "ds oligonucleotide" refer to an oligonucleotide that is in a duplex form. The complementary base-pairing of duplex region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. Likewise, complementary base-pairing of duplex region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. Moreover, complementary base-pairing of duplex region(s) of a ds oligonucleotide can be formed from single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. A ds oligonucleotide can include two covalently separate nucleic acid strands that are fully duplexed with one another. A ds oligonucleotide can include two covalently separate nucleic acid strands that are partially duplexed (e.g., having overhangs at one or both ends). A ds oligonucleotide can include an antiparallel sequence of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

[0079] Duplex: As used herein, the terms "duplex" and "duplex region" in reference to nucleic acids (e.g., oligonucleotides), refer to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides, whether formed by two covalently separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin). A duplex may form despite not having full complementarity between the two strands, or when an abasic moiety is present.

[0080] Modified internucleotide linkage: As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage is a non-naturally occurring linkage.

[0081] Modified nucleotide: As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide is a non-naturally occurring nucleotide. A modified nucleotide can have, for example, one or more chemical modifications in its sugar, nucleobase, and / or phosphate group.Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[0082] Nucleobase: As used herein, the term "nucleobase" refers to a heterocyclic moiety of a nucleoside (e.g., adenine in adenosine, cytosine in cytidine, guanine in guanosine, thymine in thymidine, and uracil in uridine). A nucleobase can be a "canonical nucleobase" or "primary nucleobase," which are used interchangeably herein and include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). A nucleobase can be a "noncanonical nucleobase," which include synthetic or natural nucleobases which are not a canonical nucleobase.

[0083] Nucleoside: As used herein, the term "nucleoside" refers to an organic compound having a nucleobase -- for example, adenine, cytosine, guanine, thymine, or uracil -- covalently attached to a pentose sugar -- for example, ribose or 2'-deoxyribose.

[0084] Nucleotide: As used herein, the term "nucleotide" refers to a nucleoside in a phosphorylated form (e.g., a phosphate ester or thiophosphate ester of a nucleoside). A nucleotide can serve as a monomeric unit of nucleic acid polymers (e.g., oligonucleotides) such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0085] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a polymer of linked nucleotides, each of which can include a canonical or noncanonical nucleobase. An oligonucleotide is typically less than about 100 nucleotides in length. An oligonucleotide may be single-stranded (ss) or double stranded (ds). An oligonucleotide may or may not have duplex regions. Oligonucleotides can include dinucleotide or trinucleotide polymers. As used herein, the term "polynucleotide" refers to a polymer of linked nucleotides which can be more than about 100 nucleotides in length. As used herein, the term dinucleotide includes two linked nucleosides with a one or more phosphate-based linkages (e.g., a dinucleoside with two nucleosides and a cyclic phosphorus, phosphate ester, or thiophosphate ester linkage). As used herein, the term trinucleotide includes three linked nucleosides with two or more phosphate-based linkages (e.g., a trinucleoside with three nucleosides and cyclic phosphorus, phosphate ester, or thiophosphate ester linkages).

[0086] Phosphate analog: As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). An oligonucleotide can have a phosphate analog at a 4'-carbon position of the sugar (referred to as a "4'-phosphate analog") or at a 5'-terminal nucleotide in place of a 5'-phosphate. An example of a 4'-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'-carbon) or analog thereof. See, e g., Intl. Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., Intl. Patent Application No. WO 2011 / 133871; US Patent No.8,927,513; and Prakash et al. (2015) Nuc. Acids Res.43:2993-3011).

[0087] Protecting group: As used herein, the terms "protecting group" or "protective group" refer to a chemical moiety which is known in the art to protect reactive or labile groups on a molecule (e.g., hydroxyl, amino, and thiol groups) against undesired reactions during synthetic procedures and other chemical reactions. Protecting groups are typically used selectively and / or orthogonally to protect labile sites in a molecule from undergoing undesired modifications during chemical reactions at other reactive sites within the molecule, and can then be removed to leave the labile group unprotected and available for further reactions.

[0088] Ribonucleotide: As used herein, the term "ribonucleotide" refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions in or of the nucleobase, sugar, or phosphate group.

[0089] Substituted benzyl alcohol nucleophile: As used herein, the terms "substituted benzyl alcohol nucleophile" or "BnOH" refer to a benzyl alcohol which includes at least one substitution on the benzene ring. A substituted benzyl alcohol nucleophile can include a primary benzyl alcohol (Bn-CH2-OH), a secondary benzyl alcohol (Bn-CH(R7)-OH), or a tertiary benzyl alcohol (Bn-C(R7)(R8)-OH).

[0090] Strand: As used herein, the term "strand" refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5' end and a 3' end).

[0091] Synthetic: As used in the context of a product herein (e.g., synthetic oligonucleotide, synthetic nucleobase), the term "synthetic" refers to a nucleic acid or other compound that is artificially synthesized (e.g., using a machine such as, for example, a solid phase nucleic acidsynthesizer) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the nucleic acid or other compound. General Considerations

[0092] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, one or more equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description or the following Examples.

[0093] At various places in the present disclosure, substituents, or properties of compounds of the present disclosure are disclosed in groups or in ranges. It is intended that the present disclosure comprise each and every individual or sub-combination of the members of such groups and ranges, and that such groups or ranges include the endpoints. By way of nonlimiting example, if a group or range is from about 1 to about 10, then the group or range includes both the value of about 1 and the value of about 10.

[0094] Articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that comprise "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure can include embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure can include embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0095] The term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" and "consisting essentially of" is also encompassed and disclosed.

[0096] The abbreviation, "e.g.," is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g.," is synonymous with the term "for example." The abbreviation, "i.e.," is derived from the Latin id est, and is used herein to indicate a non-limiting rewording or clarification. Thus, the abbreviation "i.e.," is synonymous with the term "that is."

[0097] Any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Any particular embodiment of the agents, methods, and / or compositions of the present disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0098] The present specification will control in instances where publications, patent applications, patents, and other references mentioned herein are incorporated by reference and are in conflict with the present specification.

[0099] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES

[0100] Unless noted otherwise, all reactions in the following Examples were set up inside of a chemical fume hood and run under a nitrogen or argon atmosphere. Anhydrous acetonitrile (MeCN) was purchased from Sigma-Aldrich. HPLC-grade toluene was purchased from Sigma- Aldrich and was further purified by successive filtration through neutral alumina and CuO columns under argon pressure. Reactions were run in oven-dried reaction tubes (Fisherbrand, 16 x 125 mm, product no.1495935A) sealed with a screw cap (Thermo Fisher Scientific, catalog no. B7995-18), and fitted with a Teflon-lined septum (Thermo Fisher Scientific, catalog no. C47995-15). Solvents used for extractions, crystallizations, and column chromatography were purchased from Sigma-Aldrich as ACS grade, expect for hexanes, which were HPLC grade. Lithium tert-butoxide (LiOt-Bu) was purchased from Alfa Aesar and stored in a nitrogen-filled glovebox. All preparative reactions were set up in the nitrogen filled glovebox and closed reaction vials were taken outside and stirred for noted time. Screening of reaction conditions and mechanistic experiments were carried out with the aid of a nitrogen-filled glovebox. Organic compounds were purified by flash chromatography using Silicycle SiliaFlash P60 (230–400 mesh) silica gel either manually or using a CombiFlash NextGen 300 automated chromatography system.

[0101] For general analytics, CDCl3 and d6-DMSO were purchased from Cambridge Isotope Labs. NMR spectra were collected on Bruker Avance III HD 400 or 500 MHz spectrometer.1H (CDCl3: δ 7.26; d6-DMSO: δ 2.50) and13C NMR shifts (CDCl3: δ 77.16; d6-DMSO: δ 39.52) were referenced to residual solvent peaks. The following abbreviations were used to characterize multiplicities: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, p = pentet, sept= septet, m = multiplet.13C and19F spectra were obtained with1H decoupling. Gas chromatography (GC) analyses were performed on an Agilent 7890A gas chromatograph with an FID detector using a J&W DB-1 column (10 m, 0.1 mm I.D.). LC / MS was recorded on an Agilent 6120 Quadrupole LC / MS. Elemental analyses were performed by Atlantic Microlabs Inc., Norcross, GA, USA. High-resolution mass spectra were recorded on a JEOL AccuTOF LC- Plus 46 DART system and on an Agilent Technologies 6545 Q-TOF LC / MS system. IR spectra were recorded on a Nicolet iS5 spectrometer equipped with an iD5 diamond laminate ATR accessory from Thermo Fisher Scientific. IR spectra were acquired from neat samples. Melting points were obtained using a Stanford Research Systems EZ-Melt melting point apparatus. Example 1. Preparation of substrates -- C, A, G, U a. Synthesis of 3’,5’-Bis-TBS-protected nucleosides (GP1)nucleosides 'B' (U, C, A or G, 1 equiv.) and imidazole (4 equiv.) and dried in vacuo by using standard Schlenk line technique. This mixture was dissolved in anhydrous DMF (0.5-1 M, depending on solubility) and t-butyldimethylsilyl chloride (3 equiv.) was added. The reaction mixture was stirred for 18 hours at room temperature under an inert atmosphere. Upon completion of reaction (monitored by TLC or LC-MS analysis), reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layer was washed with brine (100 mL) and dried over anhydrous Na2SO4 and evaporated. The residue was purified by silica gel column chromatography and eluted with ethyl acetate / hexane, 6:4. Fractions containing the product were pooled and evaporated. b. Synthesis of Benzyl-protected nucleosides (GP2)

[0103] A 100-mL round bottom flask equipped with a magnetic stir bar was charged with Bis-TBS-protected cytidine (1 equiv.) and dry DMF (0.3 M). The resulting solution was cooled down to 0 °C and NaH was added. The resulting solution was stirred for 5 minutes and benzyl bromide was added dropwise at 0 °C. The progress of reaction was monitored by LC-MS analysis. After 45 minutes, second portion of NaH was added and stirred for additional 1 hour at 0 °C. The reaction mixture was warmed to RT and stirred for 30 minutes before quenched with NH4Cl (50 mL). The resulting mixture was extracted with EtOAc (100 mL×3). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography with hexanes / ethyl acetate as eluants (0-100% gradient) by using a Combi-Flash NextGen 300 automated chromatography system. c. Synthesis of 3’,5’-dihydroxy nucleoside via tbs-deprotection (GP3)is- TBS-multibenzylprotected nucleoside (4 mmol) and dry THF (20 ml). The reaction mixture was cooled to 0oC and tetrabutylammonium fluoride (TBAF, 1M solution in THF, 8.2 mmol, 8.2 mL) was added dropwise. The reaction was stirred for 1 hour at 0oC. The reaction was monitored via LC-MS. Upon completion of the reaction, it was quenched with NH4Cl (10 mL) and diluted with water (10 mL). The resulting mixture was extracted with ethyl acetate (100 mL x3). The mixture was then concentrated under reduced pressure. The crude product was purified by flash chromatography on a silica gel column using methylene chloride-methanol (0 to 5% gradient) (for C) or hexane-ethyl acetate (0 to 100% gradient, for A, G) to give the final product compound (87-97% yield) as a white solid.d. Synthesis of 3-OTBS-5-OH- nucleosides via selective deprotection of 5’-Siloxy of bis- TBS-nucleoside (GP4) G;1 equiv.) were dissolved in THF (0.2 M) and treated with TFA (5 equiv.) in H2O (TFA:H2O, 1:1 mixture) at 0oC. The reaction was monitored via LC-MS. Upon completion after 4-6 hours, the reaction was diluted with equal volume water and pH adjusted to 7 to 8 with solid sodium bicarbonate. The aqueous layer was extracted with ethyl acetate (3x150 mL) and washed with brine (100 mL). The combined organic layer was dried over sodium sulfate and concentrated with the aid of rotavapor. The residue purified by silica gel column chromatography and eluted with hexane-ethyl acetate [0 to 100% gradient] to give 5’-OH, 3-TBS-nucleoside (70-80% yields). e. One pot-procedure to synthesize 3-OTBS-5-OH-nucleosides from nucleoside (GP5) 1-((2R,3R,4R,5R)-4-((tert-lyl)oxy)-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, S1

[0106] To a solution of 2’-O-methyl uridine (10 g, 39 mmol) in DMF (30 mL) was added imidazole (5.80 g, 85.0 mmol) and the solution was cooled to 5 °C. To the solution was added TBSCl (12.8 g, 85.0 mmol) slowly over 15 minutes. The reaction was then warmed to 23 °C andfound to reach completion within 20 hours. The reaction was added to a separatory funnel containing 100 mL ethyl acetate and washed 5 x 250 mL with water to remove the DMF. The organic layer was dried over sodium sulfate and concentrated to dryness. The white solids were redissolved in dichloromethane (100 mL) and treated with TFA (18.2 g, 160 mmol). After 20 hours, the reaction was poured into a flask containing 300 mL water and pH adjusted to pH = 8 with solid sodium bicarbonate. The aqueous layer was extracted with dichloromethane (3x150 mL). The dichloromethane layer was dried over sodium sulfate and concentrated to 30 mL or until solids begin to precipitate. The concentrated solution was then transferred to a flask equipped with overhead stirring and treated with 70 mL hexanes. The solution was aged for 3 hours and the solids were collected via filtration funnel and the cake was rinsed with 100 mL hexanes. The solids were dried in a vacuum oven resulting white solids (9.9 g, 68%). The crude solids were found to be 95% pure by HPLC and used without further purification. The identity was confirmed with proton NMR and MS analysis and found to be consistent with data in literature (Chem. Commun. 2021, 57 (55), 6808-6811).

[0107] 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.68 (d, J = 8.1 Hz, 1H), 5.75 (dd, J = 8.1, 2.3 Hz, 1H), 5.68 (d, J = 4.1 Hz, 1H), 4.38 (t, J = 5.1 Hz, 1H), 4.09 (dt, J = 5.0, 2.2 Hz, 1H), 4.06 – 3.95 (m, 2H), 3.85 – 3.72 (m, 1H), 3.51 (s, 3H), 2.61 – 2.52 (m, 1H), 0.94 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H).

[0108] 13C NMR (101 MHz, CDCl3) δ 163.44, 150.24, 142.10, 102.31, 91.11, 85.33, 82.55, 69.57, 61.06, 58.47, 25.71, 25.65, 18.15, -4.72, -4.82. f. Benzyloxymethyl acetal (BOM)-protection of nucleoside (GP6) [01q pp g g .0 mL of DMF, was added 1-(4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione (7.11 g, 25.0 mmol). The resulting solution was treated with DBU (4.85 mL, 32.5 mmol, 1.3 equiv.) and BOMCl (4.50 mL, 32.5 mmol, 1.3 equiv.) at 0 °C for 40 minutes. The progress of reaction was monitored by TLC analysis. Upon completion, thereaction was quenched with H2O (50 mL), and the resulting mixture was extracted with EtOAc (150 mL×4). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography with DCM / MeOH as eluants (0-5% MeOH-DCM gradient) by using a Combi- Flash NextGen 300 automated chromatography system. g. DMF-formamidine protection of NH2of nucleoside (GP7) hadenosine-ketal (1 equiv) or 2'-OMe-cytidine (1 equiv), and then l, l-dimethoxy-N,N- dimethylmethanamine (5 equiv, 32.5 mmol) in DMF (10 mL, 0.65 M). After stirring for 18 hours at room temperature, the reaction mixture was concentrated to dryness (20 x 2 mL toluene was added to aid the evaporation of DMF under reduced pressure) and the crude white solid triturated with ether (3 x 100 mL). The solid was collected by filtration and dried under high vacuum for 12 hours to yield >90% pure product (90-95%) as off-white solid. h. N-Alkylations of uridine1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-3-(3-of DMF was added 2’-O-methyl uridine (5.96 g, 23.069 mmol, 1.0 equiv.), 1-bromo-3- methylbut-2-ene (4.77 mL, 6.15 mmol, 1.79 equiv.) and potassium carbonate (6.35 g, 45.96 mmol, 2.0 equiv.). The reaction mixture was allowed to stir at 60 ºC for 4h before it was diluted with 30 mL of water and extracted with dichloromethane (3×20 mL). The organic layer was collected, dried over magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography with acetone / hexane = 1:1 as eluent followed by drying under vacuum overnight to give titled compound as a white solid with a yield of 54%. The identity was confirmed with proton NMR and MS analysis and found to be consistent with data in literature (J. Am. Chem. Soc. 2017, 139, 5467−5473).

[0112] 1H NMR (500 MHz, CDCl3) δ 7.73 (d, J = 8.1 Hz, 1H), 5.81 (d, J = 3.1 Hz, 1H), 5.78 (d, J = 8.1 Hz, 1H), 5.24 (td, J = 7.7, 7.0, 1.6 Hz, 1H), 4.61 – 4.48 (m, 2H), 4.35 (dt, J = 7.2, 5.6 Hz, 1H), 4.08 – 3.98 (m, 3H), 3.88 (ddd, J = 12.7, 6.1, 2.8 Hz, 1H), 3.61 (s, 3H), 2.74 (d, J = 6.2 Hz, 1H), 2.71 (d, J = 7.3 Hz, 1H), 1.82 (d, J = 1.4 Hz, 3H), 1.72 (d, J = 1.6 Hz, 3H).

[0113] 13C NMR (126 MHz, CDCl3) δ 162.65, 150.90, 139.01, 137.41, 118.28, 102.23, 90.19, 84.85, 82.98, 68.61, 61.32, 58.87, 39.41, 25.85, 18.25. i. Bis-Boc protection of Cytidinek. Phthalimido protection of Adenosinel. Guanosine protectionExample 2. Spectra of specific monomers a. DMF-A-diol

[0114] 1H NMR (500 MHz, DMSO) δ 8.92 (t, J = 0.6 Hz, 1H), 8.51 (s, 1H), 8.42 (s, 1H), 6.06 (d, J = 5.9 Hz, 1H), 5.33 – 5.26 (m, 2H), 4.41 – 4.31 (m, 2H), 3.98 (q, J = 3.6 Hz, 1H), 3.68 (ddd, J = 12.0, 4.9, 3.8 Hz, 1H), 3.57 (ddd, J = 12.0, 6.5, 3.8 Hz, 1H), 3.31 (s, 3H), 3.20 (d, J = 0.5 Hz, 3H), 3.13 (d, J = 0.6 Hz, 3H).

[0115] 13C NMR (126 MHz, DMSO) δ 159.37, 158.09, 151.90, 151.11, 141.33, 125.77, 86.28, 85.63, 82.47, 68.74, 61.36, 57.48, 54.92, 40.69, 34.58. b. 3-TBS-G-DMF-AMIDE

[0116] 1H NMR (500 MHz, CDCl3) δ 8.46 (s, 1H), 7.71 (s, 1H), 7.16 – 7.13 (m, 1H), 5.80 (d, J = 7.4 Hz, 1H), 4.50 (dd, J = 4.9, 1.4 Hz, 1H), 4.42 (dd, J = 7.5, 4.9 Hz, 1H), 4.15 (p, J = 1.4 Hz, 1H), 3.90 (dd, J = 12.6, 2.1 Hz, 1H), 3.68 (dd, J = 12.6, 1.5 Hz, 1H), 3.27 (s, 3H), 3.18 (t, J = 2.2 Hz, 3H), 3.12 – 3.05 (m, 3H), 0.93 (t, J = 1.1 Hz, 9H), 0.12 (dt, J = 8.5, 1.0 Hz, 6H). c. 3-TBS-ABn2

[0117] 1H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 7.79 (s, 1H), 7.36 – 7.24 (m, 10H), 5.86 (d, J = 8.0 Hz, 1H), 5.80 – 4.75 (m, 4H), 4.72 (dd, J = 8.0, 4.6 Hz, 1H), 4.61 (d, J = 4.6 Hz, 1H),4.22 (d, J = 1.6 Hz, 1H), 3.97 (dd, J = 13.1, 1.6 Hz, 1H), 3.74 (d, J = 13.0 Hz, 1H), 3.29 (s, 3H), 0.96 (s, 9H), 0.16 (d, J = 8.6 Hz, 6H). d. 3-TBS-T-BOM

[0118] 1H NMR (400 MHz, CDCl3) δ 7.38 – 7.16 (m, 7H), 6.10 (t, J = 6.7 Hz, 1H), 5.44 (s, 2H), 4.65 (s, 2H), 4.43 (dt, J = 6.8, 3.7 Hz, 1H), 3.87 (dd, J = 9.8, 3.4 Hz, 2H), 3.71 (ddd, J = 12.1, 6.4, 3.5 Hz, 1H), 2.36 – 2.10 (m, 3H), 0.85 (s, 9H).

[0119] 13C NMR (101 MHz, CDCl3) δ 163.54, 151.13, 138.11, 135.70, 128.41, 127.80, 127.75, 110.46, 87.68, 87.61, 72.37, 71.71, 70.66, 62.24, 40.69, 25.84, 18.08, 13.40, -4.55, -4.72. e. 3-TBS-N-Allyl-U-diol

[0120] 1H NMR (400 MHz, CDCl3) δ 7.24 (s, 1H), 5.38 – 5.30 (m, 2H), 4.80 (tp, J = 6.9, 1.4 Hz, 1H), 4.16 – 4.02 (m, 2H), 3.90 (dt, J = 7.2, 5.6 Hz, 1H), 3.66 – 3.55 (m, 3H), 3.43 (ddd, J = 12.6, 6.2, 2.8 Hz, 1H), 3.17 (s, 3H), 2.23 (d, J = 7.2 Hz, 1H), 2.16 (s, 1H), 1.37 (s, 3H), 1.28 (q, J = 1.1 Hz, 3H).

[0121] 13C NMR (101 MHz, CDCl3) δ 162.73, 162.62, 150.89, 139.25, 137.06, 118.38, 101.85, 90.10, 85.06, 83.28, 69.56, 60.66, 58.46, 39.30, 36.56, 31.49, 25.76, 18.19, 18.15, -4.68, -4.79. f. Adenosine-N-Formamidine

[0122] 1H NMR (500 MHz, CDCl3) δ 8.93 (s, 1H), 8.47 (s, 1H), 7.90 (s, 1H), 6.57 – 6.52 (m, 1H), 5.86 (d, J = 4.9 Hz, 1H), 5.20 (t, J = 5.4 Hz, 1H), 5.10 (dd, J = 5.9, 1.3 Hz, 1H), 4.51 (q, J = 1.6 Hz, 1H), 3.96 (dd, J = 12.7, 1.5 Hz, 1H), 3.80 – 3.73 (m, 1H), 3.19 (s, 3H), 1.62 (s, 3H), 1.35 (s, 3H).

[0123] 13C NMR (126 MHz, CDCl3) δ 160.43, 158.43, 152.16, 150.14, 141.38, 127.69, 114.04, 94.41, 86.12, 83.05, 81.81, 63.53, 41.48, 35.36, 27.75, 25.34. Example 3. Synthesis of cyclic-chlorophosphate nucleosides (GP8) a. Study of conditions for synthesis of cyclic chlorophosphite

[0124] An oven-dried 100 mL Schlenk flask equipped with a magnetic stir bar, was charged with protected diol-nucleosides (1 equiv., 2 mmol), DMAP (0.1-0.2 equiv., 60-120 mg, 0.25-0.5 mmol) in DCM (18 mL, 0.1 M). To the resulting suspension triethylamine or S-Collidine (2.2 equiv, 4.4 mmol in 1 mL DCM) was added via syringe. The resulting solution was cooled to -20 °C, and freshly distilled POCl3 (1.05 equiv., 2.1 mmol in 1 mL DCM) was added in one portion. After letting the solution stir for one hour, it was warmed to 0 °C and stirred overnight. The diastereoselectivity of the crude product was monitored by taking aliquots of reaction mixture and by analyzing with31P NMR. The reaction mixture was further stirred at room temperature for 2-4 hours until the diastereomeric ratio (dr) of crude product reached to >20:1. Once the crude dr achieved >20:1, reaction mixture was diluted with 10 mL of freshly distilled diethyl ether to precipitate the amine salt. The resulting suspension was filtered via frit filter funnel and the residue was rinsed with diethyl ether (10x2 mL). The filtrate was concentrated with the aid of rotary evaporator. The crude product was purified via silica gel (crude product was loaded on column by dissolving in minimum amount of DCM, height, ~2-inch, diameter, ~0.9-inch ) column chromatography with hexanes: ethyl acetate (0 to 80% gradient) by using a Combi-Flash NextGen 300 automated chromatography system.

[0125] Variations in synthesis conditions are summarized in Table 1. Resulting cyclic- chlorophosphate nucleoside yields and diastereomeric ratios (dr) are also summarized in Table 1.Table 1 - Cyclic-chlorophosphate nucleoside synthesis conditionsprocedures in Examples 3a, but using various nucleosides. Resulting cyclic-chlorophosphate nucleosides with their yield and diastereomeric ratio (dr) were as follows:Example 4. Synthesis of specific cyclic-chlorophosphate nucleosides a. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxidotetrahydro- 4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione sized by following general procedure GP8 using U-BOM(1.85 g, 4.9 mmol, 1 equiv.), triethylamine (1.09 g, 10.76 mmol, 2.2 equiv.), DMAP (120 mg, 0.098 mmol, 0.2 equiv.) and POCl3 (824 mg, 5.37 mmol, 1.1 equiv.), in DCM (35 mL, 0.14M). Column conditions. SiO2-25g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 80%), 1380 mg of desired product isolated as white solid. Yield = 62%, dr = 38:1.

[0128] 31P NMR (162 MHz, CDCl3) δ -1.12, -1.72.

[0129] 1H NMR (400 MHz, CDCl3) δ 7.41 – 7.27 (m, 5H), 7.13 (d, J = 8.1 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 5.50 – 5.41 (m, 2H), 5.30 (q, J = 0.9 Hz, 1H), 4.92 (ddd, J = 9.9, 5.1, 3.5 Hz, 1H), 4.78 – 4.64 (m, 3H), 4.46 (ddd, J = 11.2, 9.7, 1.7 Hz, 1H), 4.32 (d, J = 5.1 Hz, 1H), 4.25 (td, J = 10.3, 4.7 Hz, 1H), 3.57 (s, 3H).

[0130] 13C NMR (101 MHz, CDCl3) δ 162.22, 150.38, 141.12, 137.83, 128.52, 128.01, 127.82, 103.03, 97.69, 80.02, 79.95, 79.55, 79.46, 72.67, 70.93, 70.83, 70.51, 70.30, 70.24, 59.87.

[0131] HR-MS is not determined as the compound hydrolyzed in the process of analysis. b. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-fluoro-2-oxidotetrahydro-4H- furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione

[0132] The compound was synthesized by following general procedure GP8 using U-BOM (900 mg, 2.46 mmol, 1 equiv.), triethylamine (522 mg, 5.17 mmol, 2.1 equiv.), DMAP (60 mg, 0.049 mmol, 0.2 equiv.) and POCl3 (414 mg, 2.70 mmol, 1.1 equiv.), in DCM (25 mL, 0.1M). Column conditions. SiO2-10g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 80%), 530 mg of desired product isolated as white solid. Yield = 48%, dr = 18:1.

[0133] 31P NMR (162 MHz, CDCl3) δ -1.62, -2.04.

[0134] 1H NMR (400 MHz, CDCl3) δ 7.38 – 7.27 (m, 5H), 7.12 (d, J = 8.0 Hz, 1H), 5.82 (d, J = 8.0 Hz, 1H), 5.65 – 5.36 (m, 3H), 5.31 (s, 1H), 5.30 (s, 2H), 5.15 (ddt, J = 22.9, 8.6, 4.0 Hz, 1H), 4.76 (dd, J = 9.7, 4.6 Hz, 1H), 4.69 (s, 2H), 4.51 (t, J = 10.0 Hz, 1H), 4.24 (td, J = 10.3, 4.6 Hz, 1H), 4.12 (q, J = 7.1 Hz, 1H).

[0135] HR-MS is not determined as the compound hydrolyzed in the process of analysis. c. 1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)-4-(dibenzylamino)pyrimidin-2(1H)-oned by following general procedure GP8 using C-Bn2 (870 mg, 2.46 mmol, 1 equiv.), triethylamine (443 mg, 4.37 mmol, 2.2 equiv.), DMAP (24 mg, 0.02 mmol, 0.1 equiv.) and POCl3 (335 mg, 2.19 mmol, 1.1 equiv.), in DCM (21 mL, 0.1M). Column conditions. SiO2-10g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 100%), 304 mg of desired product isolated as white solid. Yield = 30%, dr = 22:1.

[0137] 31P NMR (162 MHz, CDCl3) δ -0.96, -1.11.

[0138] 1H NMR (400 MHz, CDCl3) δ 7.34 – 7.17 (m, 12H), 7.07 (d, J = 7.3 Hz, 2H), 5.78 (d, J = 7.7 Hz, 1H), 5.26 (d, J = 14.4 Hz, 1H), 5.18 (ddd, J = 9.6, 5.0, 3.6 Hz, 1H), 5.02 – 4.90 (m, 2H), 4.63 (ddd, J = 27.1, 9.6, 4.7 Hz, 1H), 4.51 – 4.39 (m, 5H), 4.21 (td, J = 10.3, 4.7 Hz, 1H), 3.53 (s, 3H).

[0139] 13C NMR (101 MHz, CDCl3) δ 164.53, 154.59, 144.22, 136.64, 135.55, 129.25, 128.82, 128.65, 128.03, 127.91, 126.26, 98.34, 92.95, 80.30, 80.23, 79.84, 79.75, 77.36, 71.24, 71.14, 70.40, 70.35, 59.70, 50.85, 50.57.

[0140] HR-MS is not determined as the compound hydrolyzed in the process of analysis. d. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7aS)-2-chloro-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)-5-methylpyrimidine-2,4(1H,3H)-dionenthesized by following general procedure GP8 using T-BOM (2.5 g, 7.18 mmol, 1 equiv.), triethylamine (1.598 g, 15.79 mmol, 2.2 equiv.), DMAP (175 mg, 1.43 mmol, 0.1 equiv.) and POCl3 (1.21 g, 7.89 mmol, 1.1 equiv.), in DCM (60 mL, 0.12M). Column conditions. SiO2-25g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 85%), 1700 mg of desired product isolated as white solid. Yield = 55%, dr = 16:1.

[0142] 31P NMR (162 MHz, CDCl3) δ -1.48, -1.98.

[0143] 1H NMR (400 MHz, CDCl3) δ 7.40 – 7.28 (m, 5H), 7.28 – 7.22 (m, 1H), 6.92 (q, J = 1.2 Hz, 1H), 5.97 (dd, J = 8.8, 3.0 Hz, 1H), 5.48 (d, J = 1.7 Hz, 2H), 5.04 – 4.91 (m, 1H), 4.79 – 4.61 (m, 3H), 4.53 (ddd, J = 10.6, 9.7, 1.7 Hz, 1H), 3.95 (dddd, J = 10.7, 9.1, 4.6, 0.7 Hz, 1H), 2.74 – 2.53 (m, 2H), 1.95 (d, J = 1.2 Hz, 3H).

[0144] 13C NMR (126 MHz, CDCl3) δ 163.13, 150.39, 137.95, 135.67, 128.47, 127.92, 127.82, 111.49, 88.37, 79.12, 79.07, 77.36, 73.71, 73.66, 72.57, 71.07, 70.99, 70.75, 34.70, 34.62.

[0145] HR-MS is not determined as the compound hydrolyzed in the process of analysis.2 4 R R R R 2 hl hl H i l h h d 4H Me-A(6Cl)-diol (1050 mg, 3.5 mmol, 1 equiv.), triethylamine (777 mg, 7.68 mmol, 2.2 equiv.), DMAP (43 mg, 1.43 mmol, 0.1 equiv.) and POCl3(589 mg, 3.84 mmol, 1.1 equiv.), in DCM (30 mL, 0.12M). Column conditions. SiO2-10 g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 85%), 620 mg of desired product isolated as white solid. Yield = 47%, dr = 10:1.

[0147] 31P NMR (162 MHz, CDCl3) δ -1.16, -1.76.

[0148] 1H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 8.18 (s, 1H), 6.01 (s, 1H), 5.60 (dt, J = 8.9, 4.2 Hz, 1H), 4.90 – 4.37 (m, 4H), 3.63 (s, 3H).

[0149] 13C NMR (101 MHz, CDCl3) δ 152.55, 144.61, 132.93, 91.84, 80.24, 80.15, 80.03, 79.97, 71.01, 70.91, 70.64, 70.58, 60.20.

[0150] HR-MS is not determined as the compound hydrolyzed in the process of analysis. f. (2S,4aR,6R,7R,7aR)-2-chloro-6-(6-(dibenzylamino)-9H-purin-9-yl)-7-methoxytetrahydro-(2.2 g, 4.77 mmol, 1 equiv.), triethylamine (1.061 g, 10.49 mmol, 2.2 equiv.), DMAP (58 mg, 0.47 mmol, 0.1 equiv.) and POCl3(804 mg, 5.24 mmol, 1.1 equiv.), in DCM (45 mL, 0.11M). Column conditions. SiO2-25g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 100%), 1220 mg of desired product isolated as white solid. Yield = 47%, dr = 18:1.

[0152] 31P NMR (203 MHz, CDCl3) δ -1.01, -1.26.

[0153] 1H NMR (500 MHz, CDCl3) δ 8.37 (s, 1H), 7.73 (s, 1H), 7.33 – 7.24 (m, 11H), 5.90 (s, 1H), 5.81 (ddd, J = 9.7, 4.9, 3.6 Hz, 1H), 5.00 (s, 2H), 4.70 (ddd, J = 27.2, 9.6, 4.7 Hz, 1H), 4.63 (d, J = 4.9 Hz, 1H), 4.51 (ddd, J = 11.0, 9.6, 1.6 Hz, 1H), 4.38 (td, J = 10.3, 4.6 Hz, 1H), 3.60 (s, 3H).

[0154] 13C NMR (101 MHz, CDCl3) δ 152.41, 150.19, 138.39, 128.84, 127.95, 127.69, 120.56, 91.79, 80.31, 80.22, 80.12, 80.05, 71.22, 71.12, 70.53, 70.47, 60.00.

[0155] HR-MS is not determined as the compound hydrolyzed in the process of analysis. g. 1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- g U-Allyl-diol (650 mg, 2 mmol, 1 equiv.), S-collidine (507 mg, 4.18 mmol, 2.1 equiv.), DMAP (49 mg, 0.4 mmol, 0.2 equiv.) and POCl3 (804 mg, 5.24 mmol, 1.1 equiv.), in DCM (20 mL, 0.1M). Column conditions. SiO2-10 g column, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 100%), 400 mg of desired product isolated as white solid. Yield = 49%, dr = 14:1.

[0157] 31P NMR (162 MHz, CDCl3) δ -1.08, -1.71.

[0158] 1H NMR (400 MHz, CDCl3) δ 7.12 (d, J = 8.1 Hz, 1H), 5.81 (d, J = 8.0 Hz, 1H), 5.35 (d, J = 1.1 Hz, 1H), 5.20 (tdd, J = 6.8, 2.8, 1.4 Hz, 1H), 4.93 (ddd, J = 9.8, 5.1, 3.5 Hz, 1H), 4.72 (ddd, J = 27.1, 9.7, 4.7 Hz, 1H), 4.57 – 4.42 (m, 3H), 4.36 (d, J = 5.0 Hz, 1H), 4.26 (td, J = 10.3, 4.7 Hz, 1H), 3.58 (s, 3H), 1.81 (s, 3H), 1.72 (d, J = 1.2 Hz, 3H).

[0159] HR-MS is not determined as the titled compound hydrolyzed in the process of analysis.Example 5. Synthesis of cyclic dinucleotides a. Multi-pot synthesis – Method A (GP9)

[0160] Inside the N2-filled glovebox, a 20 mL reaction tube A equipped with a magnetic stir- bar, was charged with LiOtBu (1 equiv., 1 mmol), and the reaction tube was then taken out of the glovebox, put under nitrogen atmosphere via the Schlenk-line technique and kept at -20 °C with aid of a cryocooler. A stock of solution with 5’-OH-nucleoside (1 equiv., 1 mmol) in Toluene (5 mL, 0.2 M) was added to the reaction tube A. The resulting reaction was stirred at -20 °C for 30 min.

[0161] Another 20 mL reaction tube B equipped with magnetic stir bar was charged with cyclic-chlorophosphate nucleosides (1.2 equiv., 1.2 mmol) and 5 mL of MeCN. The reaction mixture was cooled to -20 °C with aid of a cryocooler. The pre-stirred solution of alkoxide from reaction tube A was transferred to a round bottom flask via syringe dropwise over the period of 5 minutes while stirring under nitrogen atmosphere. The resulting reaction mixture was stirred for 18 hours at -20 °C. In cases where incomplete conversion was observed, the reaction was warmed to room temperature for 30 minutes to ensure 100% conversion. The reaction was tracked by31P NMR. Upon completion of the reaction, the crude reaction mixture was concentrated under reduced pressure and the crude product was purified via Combi-Flash NextGen 300 automated chromatography system with hexane: ethyl acetate (0% to 100% gradient). Alternatively, DCM: Acetonitrile (2% isopropanol) or DCM: Ethyl acetate (2% MeOH) was used for more polar compounds. For adenosine and cytidine containing cyclic dinucleotide, DCM: MeOH was used as an eluant (0% to 10%). b. Multi-pot synthesis – Method B (GP10)

[0162] A solution of 5’-OH-nucleoside (1 equiv., 1 mmol) and triethylamine (1.1 equiv., 1.1 mmol) in dichloromethane (2.5 mL) was added to reaction tube A containing a solution of POCl3 (1 equiv., 1 mmol) in DCM (2.5 mL), dropwise over a period of 25 minutes while stirring at -20oC. The resulting solution was stirred for 30 minutes.

[0163] Another reaction tube B equipped with magnetic stir bar was charged with protected diol-nucleosides (1 equiv., 1 mmol) and DMAP (2.2 equiv., 2.2 mmol) in DCM (5 mL, 0.2M). The resulting suspension was cooled to -78oC. The pre-stirred solution from reaction tube A was cannula transferred to reaction tube B. The resulting mixture was stirred for an additional 1 hour at -78oC and slowly warmed to room temperature, while stirring for another 1 hour. The reactionwas monitored by31P NMR and upon completion, it was diluted with diethyl ether to precipitate the salts and filtered with frit filter funnel. The resulting filtrate was concentrated under reduced pressure. The crude product was purified via Combi-Flash NextGen 300 automated chromatography system with silica gel column with hexanes:acetone (0% to 100% gradient, Crude product was loaded on the column by dissolving in minimum amount of DCM (height, ~3-inch, diameter, ~0.9-inch). Alternatively, DCM:Acetone was used for more polar compounds. For adenosine containing cyclic dinucleotide, DCM:MeOH was used as an eluant (0% to 5%) c. Study of reaction conditions for synthesizing cyclic dinucleotidesmple 5b, but using various synthesis conditions as summarized in Table 2. Resulting cyclic dinucleotides yields (based on31P NMR) are also summarized in Table 2. Table 2 – Cyclic dinucleotide synthesis conditions[0Examples 5b, but using various nucleosides. Resulting cyclic dinucleotides with their yield and / or diastereomeric ratio (dr) were as follows [yields and dr are reported for isolated materials; structure of major diastereomer shown][ ] souton o - -nuceos e ( equv., mmo) an tret yamne ( . equv., .1 mmol) in dichloromethane (2.5 mL) was added to reaction tube A containing a solution of POCl3(1 equiv., 1 mmol) in DCM (2.5 mL), dropwise over a period of 25 minutes while stirring at -20oC. The resulting solution was stirred for 30 minutes.

[0167] Another reaction tube B equipped with magnetic stir bar was charged with diol (1 equiv., 1 mmol) and DMAP (2.2 equiv., 2.2 mmol) in DCM (5 mL, 0.2M). The resulting suspension was cooled to -78oC. The pre-stirred solution from reaction tube A was cannula transferred to reaction tube B. The resulting mixture was stirred for additional 1 hour at -78oC and slowly warmed to room temperature while stirring for another 1 hour. The reaction was monitored by31P NMR and upon completion, it was diluted with diethyl ether to precipitate the salts and filtered with frit filter funnel. The resulting filtrate was concentrated under reduced pressure. The crude product was purified via Combi-Flash NextGen 300 automated chromatography system with silica gel column with hexanes:acetone (0% to 100% gradient). Crude product was loaded on column by dissolving in minimum amount of DCM, height, ~3- inch, diameter, ~0.9-inch. Alternatively, DCM:Acetone can be used for more polar compounds. For adenosine containing cyclic dinucleotide, DCM:MeOH was used as an eluant (0% to 5%). f. Guanosine dimer synthesisExample 6. Synthesis of specific cyclic dinucleotides a. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2- oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione ing general procedure GP10 using 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3-OTBS-U-BOM, 400 mg, 0.81 mmol, 1 equiv.), LiOtBu (72 mg, 0.89 mmol, 1.1 equiv.), 3-((benzyloxy)methyl)-1- ((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-Cl-CP, 410 mg, 0.89 mmol, 1.1 equiv.), and in Toluene:ACN (1:1). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 100%), desired product was isolated as white solid in two fractions. Yield = F2, 66%, dr = 20:1, F1, 17%, 15:1. Overall yield = 83%.

[0169] 31P NMR (162 MHz, CDCl3) δ -3.76.

[0170] 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 8.2 Hz, 1H), 7.44 – 7.26 (m, 10H), 7.14 (d, J = 8.2 Hz, 1H), 5.91 (d, J = 2.0 Hz, 1H), 5.81 (t, J = 8.5 Hz, 2H), 5.60 – 5.43 (m, 5H), 4.80 (ddd, J = 10.0, 5.1, 1.3 Hz, 1H), 4.73 (s, 4H), 4.70 – 4.59 (m, 1H), 4.59 – 4.48 (m, 2H), 4.39 – 4.23 (m, 2H), 4.22 – 4.11 (m, 3H), 3.69 (dd, J = 4.2, 2.0 Hz, 1H), 3.59 (s, 3H), 3.59 (s, 3H), 0.94 (s, 9H), 0.14 (d, J = 2.3 Hz, 6H).

[0171] 13C NMR (101 MHz, CDCl3) δ 162.66, 162.15, 150.94, 150.38, 139.34, 138.23, 137.96, 137.93, 128.50, 128.47, 127.94, 127.88, 127.84, 127.73, 102.93, 102.10, 95.36, 89.13, 83.36, 81.44, 81.36, 80.31, 80.23, 78.07, 78.03, 77.36, 72.67, 72.44, 70.99, 70.93, 70.61, 70.41, 69.39, 69.31, 69.15, 66.51, 66.45, 59.45, 58.58, 25.77, 18.20, -4.53, -4.83.

[0172] HR-MS (Q-TOF, ESI) calculated for [C42H55N4O15PSi [M+H+]: 937.3063, found 937.3009. b. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-fluoro-2- oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dioneng general procedure GP10 using 3-OTBS- U-BOM (200 mg, 0.41 mmol, 1 equiv.), LiOtBu (34 mg, 0.43 mmol, 1.05 equiv.), F-U-BOM-Cl- CP (200 mg, 0.45 mmol, 1.1 equiv.), and in Toluene: ACN (1:1). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 100%), desired product was isolated as white solid. Yield = 77%, dr = 5:1.

[0174] 31P NMR (162 MHz, CDCl3) δ -4.29, -6.86.

[0175] 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.2 Hz, 1H), 7.40 – 7.27 (m, 12H), 7.08 (d, J = 8.2 Hz, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.82 – 5.72 (m, 3H), 5.53 – 5.33 (m, 8H), 4.70 (d, J = 4.8 Hz, 4H), 4.58 – 4.45 (m, 3H), 4.36 (ddd, J = 11.6, 6.2, 2.3 Hz, 2H), 4.28 – 4.10 (m, 4H), 3.70 (dd, J = 4.5, 2.2 Hz, 1H), 3.56 (s, 4H), 0.92 (d, J = 1.7 Hz, 10H), 0.12 (d, J = 1.2 Hz, 6H).

[0176] HR-MS (Q-TOF, ESI) calculated for C41H52FN4O14PSi [M+H+]: 903.3044, found 903.3038.c. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((2R,4aR,6R,7R,7aR)-6-(4-(dibenzylamino)-2-oxopyrimidin-1(2H)-yl)-7-methoxy-2- oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione neral procedure GP10 using 3-OTBS-U-BOM (200 mg, 0.41 mmol, 1 equiv.), LiOtBu (36 mg, 0.44 mmol, 1.1 equiv.), C-Bn2-Cl-CP (235 mg, 0.45 mmol, 1.12 equiv.), and in Toluene:ACN (1:1). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM-EA (1% MeOH) gradient (0% to 100%), desired product was isolated as white solid. Yield = 250 mg, 63%, dr = 10:1.

[0178] 31P NMR (162 MHz, CDCl3) δ -3.55, -6.36.

[0179] 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.2 Hz, 1H), 7.40 – 7.26 (m, 15H), 7.12 (d, J = 7.3 Hz, 2H), 5.90 (d, J = 2.1 Hz, 1H), 5.82 (dd, J = 9.0, 8.0 Hz, 2H), 5.75 (s, 1H), 5.54 – 5.40 (m, 2H), 5.03 (q, J = 14.7 Hz, 2H), 4.75 – 4.58 (m, 4H), 4.53 (s, 1H), 4.53 – 4.43 (m, 4H), 4.34 (ddd, J = 11.3, 9.6, 5.5 Hz, 2H), 4.25 – 4.11 (m, 3H), 3.69 – 3.61 (m, 4H), 3.61 – 3.51 (m, 4H), 0.91 (s, 10H), 0.11 (d, J = 2.4 Hz, 6H).

[0180] 13C NMR (101 MHz, CDCl3) δ 164.50, 164.36, 162.75, 162.67, 154.70, 150.98, 141.29, 138.20, 137.98, 136.76, 135.65, 129.25, 128.80, 128.61, 128.45, 128.00, 127.87, 127.84, 126.29, 102.14, 94.62, 92.69, 88.94, 83.37, 81.53, 81.45, 80.63, 80.56, 78.09, 78.05, 72.42, 70.95, 70.89, 70.40, 69.70, 69.62, 69.13, 66.43, 66.38, 59.29, 58.55, 58.53, 50.79, 50.49, 25.81, 25.77, 18.19, -4.45, -4.54, -4.73, -4.84.

[0181] HR-MS (Q-TOF, ESI) calculated for C48H60N5O13PSi, [M+H+]: 974.3768, found 974.3761.d. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- BS-U-BOM (400 mg, 0.81 mmol, 1 equiv.), LiOtBu (72 mg, 0.89 mmol, 1.1 equiv.), A(6Cl)-Cl-CP (371 mg, 0.97 mmol, 1.2 equiv.), and in Toluene:ACN (1:1). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA gradient (0% to 100%), desired product was isolated as white solid in two fractions. Yield = 500 mg, 74%, dr = 15:1.

[0183] 31P NMR (162 MHz, CDCl3) δ -4.02, -6.82.

[0184] 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 0.6 Hz, 1H), 8.17 (d, J = 1.4 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.39 – 7.27 (m, 5H), 6.02 (d, J = 0.9 Hz, 1H), 5.89 (d, J = 1.9 Hz, 1H), 5.82 (d, J = 8.2 Hz, 1H), 5.57 – 5.41 (m, 3H), 4.71 (s, 2H), 4.69 – 4.49 (m, 5H), 4.37 (ddd, J = 10.9, 9.5, 5.5 Hz, 2H), 4.21 – 4.13 (m, 2H), 3.72 – 3.66 (m, 1H), 3.59 (s, 4H), 3.57 (s, 3H), 0.92 (s, 9H), 0.13 (d, J = 1.3 Hz, 6H).

[0185] 13C NMR (101 MHz, CDCl3) δ 162.65, 152.70, 152.22, 150.93, 150.79, 144.26, 138.22, 137.94, 132.72, 128.47, 127.88, 127.78, 102.11, 91.65, 89.25, 83.38, 81.41, 81.33, 80.57, 80.49, 78.39, 72.45, 71.37, 71.31, 70.41, 69.54, 69.46, 69.18, 66.54, 66.48, 59.87, 58.62, 25.78, 18.22, -4.51, -4.82.

[0186] HR-MS (Q-TOF, ESI) calculated for C35H46ClN6O12PSi, [M+Na+]: 859.2261, found 859.2256.e. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert- butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purin-9-yl)-4-methoxytetrahydrofuran-2- yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6- yl)pyrimidine-2,4(1H,3H)-dione general procedure GP10 using 3-OTBS-A-Bn (350 mg, 0.6 mmol, 1 equiv.), LiOtBu (59 mg, 0.73 mmol, 1.2 equiv.), U-BOM-Cl-CP (335 mg, 0.73 mmol, 1.2 equiv.), and Toluene:ACN (1:1, 7 mL). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM:MeOH gradient (0% to 5%), desired product was isolated as white solid. Yield = 530 mg, 87%, dr = 22:1.

[0188] 31P NMR (162 MHz, CDCl3) δ -3.99, -6.90.

[0189] 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.95 (s, 1H), 7.36 – 7.21 (m, 17H), 6.92 (d, J = 8.1 Hz, 1H), 6.11 (d, J = 3.8 Hz, 1H), 5.64 (d, J = 8.1 Hz, 1H), 5.42 (d, J = 1.9 Hz, 3H), 4.96 (s, 2H), 4.74 – 4.63 (m, 4H), 4.55 – 4.37 (m, 3H), 4.36 – 4.16 (m, 4H), 4.04 (d, J = 5.0 Hz, 1H), 3.52 (s, 3H), 3.50 (s, 3H), 0.94 (s, 9H), 0.17 (d, J = 2.9 Hz, 6H).

[0190] 13C NMR (101 MHz, CDCl3) δ 162.17, 155.13, 152.92, 150.85, 150.29, 139.56, 137.95, 137.64, 128.73, 128.50, 128.03, 127.93, 127.72, 127.49, 120.36, 102.68, 95.58, 87.27, 82.53, 82.23, 82.15, 80.19, 80.12, 78.22, 78.18, 72.63, 70.62, 70.56, 70.39, 69.16, 69.08, 66.89, 66.84, 59.41, 58.77, 25.89, 18.28, -4.55, -4.72.

[0191] HR-MS (Q-TOF, ESI) calculated for C49H60N7O12PSi, [M+H+]: 998.388, found 998.3893.f. 3-((benzyloxy)methyl)-1-((2R,4S,5R)-5-((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)- 2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran- 2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione wing general procedure GP10 using 3-OTBS-T-BOM (250 mg, 0.52 mmol, 1 equiv.), LiOtBu (51 mg, 0.63 mmol, 1.2 equiv.), U-BOM-Cl-CP (289 mg, 0.63 mmol, 1.2 equiv.), and Toluene:ACN (1:1, 6 mL). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex:EA gradient (0% to 100%), desired product was isolated as white solid. Yield = 420 mg, 89%, dr = 22:1.

[0193] 31P NMR (162 MHz, CDCl3) δ -3.60, -6.41.

[0194] 1H NMR (400 MHz, CDCl3) δ 7.41 – 7.31 (m, 10H), 7.29 (d, J = 7.1 Hz, 3H), 7.13 (d, J = 8.2 Hz, 1H), 6.34 (t, J = 6.7 Hz, 1H), 5.80 (dd, J = 8.1, 0.7 Hz, 1H), 5.57 – 5.42 (m, 6H), 4.82 – 4.70 (m, 6H), 4.65 (ddd, J = 17.7, 9.7, 5.3 Hz, 2H), 4.58 – 4.38 (m, 4H), 4.38 – 4.23 (m, 3H), 4.16 – 4.03 (m, 2H), 3.58 (d, J = 0.7 Hz, 3H), 2.34 (ddd, J = 13.6, 6.1, 3.4 Hz, 1H), 2.16 – 2.04 (m, 2H), 2.01 – 1.96 (m, 3H), 0.92 (s, 9H), 0.12 (d, J = 1.7 Hz, 6H).

[0195] 13C NMR (126 MHz, CDCl3) δ 163.50, 163.43, 162.23, 151.17, 151.15, 151.09, 151.07, 150.44, 150.39, 139.53, 139.40, 138.04, 137.99, 137.89, 137.86, 135.54, 135.25, 133.72, 128.50, 128.44, 128.42, 127.95, 127.84, 127.81, 127.72, 110.88, 110.70, 110.46, 102.85, 95.31, 95.23, 87.18, 87.15, 86.99, 85.74, 85.69, 85.66, 85.64, 85.59, 85.54, 85.37, 80.64, 80.60, 80.22, 80.21, 80.16, 80.15, 79.75, 79.71, 78.10, 78.07, 78.04, 72.65, 72.41, 72.40, 72.38, 71.48, 70.71, 70.69, 70.66, 70.59, 70.53, 69.57, 69.51, 69.44, 69.38, 63.65, 63.38, 62.47, 62.17, 62.09, 59.67, 59.40, 59.38, 26.04, 26.03, 25.89, 25.82, 25.77, 13.40, 13.39, 13.34, -2.83, -3.46, -5.28, -5.37.

[0196] HR-MS (Q-TOF, ESI) calculated for C42H55N4O14PSi, [M+H+]: 899.3295, found 899.3288.g. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((2R,4aR,6R,7R,7aR)-6-(6-(dibenzylamino)-9H-purin-9-yl)-7-methoxy-2-oxidotetrahydro- 4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-3-methoxytetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione ng general procedure GP10 using 3-OTBS-U-BOM (400 mg, 0.81 mmol, 1 equiv.), LiOtBu (78 mg, 0.97 mmol, 1.2 equiv.), A-Bn2-Cl-CP (528 mg, 0.97 mmol, 1.2 equiv.), and Toluene:ACN (1:1, 8 mL). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex:EA gradient (0% to 100%), desired product was isolated as white solid. Yield = 680 mg, 87%, dr = 30:1.

[0198] 1H NMR (500 MHz, CDCl3) δ 8.38 (s, 1H), 7.73 (s, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 7.1 Hz, 2H), 7.34 – 7.20 (m, 16H), 5.94 – 5.88 (m, 2H), 5.84 (d, J = 8.2 Hz, 1H), 5.67 (ddd, J = 10.0, 5.2, 1.1 Hz, 1H), 5.48 (q, J = 9.7 Hz, 3H), 4.97 (s, 2H), 4.70 (s, 2H), 4.67 – 4.57 (m, 2H), 4.53 (dtt, J = 9.9, 6.3, 3.5 Hz, 2H), 4.38 – 4.28 (m, 2H), 4.18 (qd, J = 7.5, 3.8 Hz, 2H), 3.66 (dd, J = 4.7, 2.1 Hz, 1H), 3.57 (s, 3H), 3.56 (s, 3H), 0.91 (s, 10H), 0.12 (s, 6H).

[0199] 31P NMR (203 MHz, CDCl3) δ -4.06, -6.54.

[0200] 13C NMR (126 MHz, CDCl3) δ 162.57, 155.06, 153.17, 150.87, 150.28, 138.06, 137.86, 137.56, 128.65, 128.35, 127.75, 127.73, 127.38, 120.32, 102.05, 91.52, 88.88, 83.32, 81.41, 81.34, 80.45, 80.39, 78.48, 78.45, 77.24, 72.32, 71.06, 71.01, 70.29, 69.73, 69.66, 69.05, 66.21, 66.16, 59.57, 58.49, 25.72, 25.67, 18.10, -4.65, -4.91.

[0201] HR-MS (Q-TOF, ESI) calculated for C49H60N7O12PSi, [M+Na+]: 998.388, found 998.3887.h. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7aS)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-OTBS-U-BOM (270 mg, 0.61 mmol, 1 equiv.), LiOtBu (54 mg, 0.67 mmol, 1.1 equiv.), T- BOM-Cl-CP (330 mg, 0.67 mmol, 1.1 equiv.), and Toluene:ACN (1:1, 6 mL). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, MeOH:DCM gradient (0% to 5%), desired product was isolated as white solid. Yield = 500 mg, 91%, dr = 5:1.

[0203] 1H NMR (500 MHz, CDCl3) δ 7.61 – 7.55 (m, 1H), 7.43 – 7.29 (m, 10H), 6.92 (d, J = 1.4 Hz, 1H), 6.28 (dd, J = 8.7, 2.7 Hz, 1H), 4.99 – 4.82 (m, 1H), 4.73 (d, J = 7.2 Hz, 4H), 4.67 – 4.47 (m, 4H), 4.40 – 4.29 (m, 1H), 4.22 – 4.11 (m, 2H), 4.00 – 3.84 (m, 1H), 3.73 (q, J = 1.9 Hz, 1H), 3.59 (s, 3H), 2.68 – 2.40 (m, 2H), 2.04 – 1.88 (m, 4H), 0.94 (s, 12H), 0.14 (d, J = 5.4 Hz, 8H).

[0204] 31P NMR (162 MHz, CDCl3) δ -3.64, -6.68.

[0205] 13C NMR (126 MHz, CDCl3) δ 163.02, 162.61, 150.89, 150.67, 138.42, 138.01, 137.95, 133.83, 128.48, 128.45, 127.90, 127.86, 127.81, 127.77, 111.85, 102.01, 89.68, 86.08, 83.23, 81.41, 81.34, 77.36, 77.33, 77.30, 74.20, 74.15, 72.53, 72.46, 70.85, 70.40, 69.58, 69.52, 69.28, 66.84, 66.79, 58.61, 35.57, 35.50, 25.84, 25.78, 18.22, 13.42, -4.46, -4.80.

[0206] HR-MS (Q-TOF, ESI) calculated for Chemical Formula: C42H55N4O14PSi, [M+Na+]:921.3114, found 921.3110.-- n ( mg, . mmo , equ v.), t u ( mg, . mmo , . equ v.), n - - (452 mg, 0.83 mmol, 1.2 equiv.), and Toluene:ACN (1:1, 7 mL). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, MeOH:DCM gradient (0% to 5%), desired product was isolated as white solid. Yield = 650 mg, 86%, dr = 14:1.

[0208] 31P NMR (162 MHz, CDCl3) δ -4.02, -6.62.

[0209] 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.41 (s, 1H), 8.00 (s, 1H), 7.60 (s, 1H), 7.38 – 7.20 (m, 25H), 6.15 (d, J = 3.9 Hz, 1H), 5.87 (s, 1H), 5.61 (dd, J = 9.5, 5.1 Hz, 2H), 5.50 (s, 4H), 5.01 (s, 5H), 4.78 (t, J = 5.3 Hz, 1H), 4.62 – 4.24 (m, 10H), 3.56 (s, 3H), 3.55 (s, 3H), 1.00 (s, 9H), 0.22 (d, J = 1.3 Hz, 6H).

[0210] 13C NMR (101 MHz, CDCl3) δ 155.17, 155.14, 153.19, 152.92, 150.84, 150.37, 137.69, 137.66, 128.74, 128.69, 128.65, 127.96, 127.47, 127.42, 120.42, 120.39, 91.55, 87.33, 82.51, 82.31, 82.23, 80.56, 80.48, 78.65, 78.61, 77.36, 70.85, 70.77, 70.42, 69.61, 69.53, 66.77, 66.72, 59.57, 58.78, 51.04, 49.43, 25.90, 18.29, -4.55, -4.70.

[0211] HR-MS (Q-TOF, ESI) calculated for C56H65N10O9PSi, [M+Na+]:1103.4335, found 1103.433.j. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,3aS,9aR)-3-((tert- H-y g, . , q . , g, . , . q . , Cl-CP (296 mg, 0.65 mmol, 1.1 equiv.), and Toluene:ACN (1:1, 9 mL). Column conditions: Reverse phase C-18 column, Next-Gen automated system, wet loading by dissolving in minimum amount of DMSO, Water:ACN gradient (10% to 100%), desired product was isolated as white solid. Yield = 230 mg, 51%, dr >99:1.

[0213] 31P NMR (203 MHz, CD3CN) δ -5.19.

[0214] 31P NMR (203 MHz, CDCl3) δ -4.64.

[0215] 1H NMR (500 MHz, CD3CN) δ 7.54 (d, J = 7.4 Hz, 1H), 7.41 – 7.25 (m, 6H), 6.23 (d, J = 5.8 Hz, 1H), 5.90 (d, J = 7.5 Hz, 1H), 5.71 (d, J = 8.1 Hz, 2H), 5.37 (s, 2H), 5.20 (dd, J = 5.8, 1.6 Hz, 1H), 4.64 (s, 2H), 4.63 – 4.58 (m, 1H), 4.57 (ddd, J = 3.6, 1.6, 0.6 Hz, 1H), 4.39 (dt, J = 10.3, 9.1 Hz, 1H), 4.29 (dtd, J = 5.4, 3.7, 1.7 Hz, 1H), 4.23 (td, J = 10.3, 5.7 Hz, 1H), 4.15 – 4.02 (m, 2H), 3.98 (ddd, J = 11.8, 6.8, 5.3 Hz, 1H), 3.52 (s, 3H), 2.13 (s, 4H), 0.93 (s, 8H), 0.20 (s, 3H), 0.17 (s, 3H).

[0216] 13C NMR (126 MHz, CD3CN) δ 172.57, 163.53, 161.05, 151.93, 141.24, 139.63, 136.85, 129.46, 128.74, 128.70, 110.64, 102.86, 94.76, 90.99, 90.10, 86.94, 86.87, 81.21, 81.15, 79.10, 79.06, 77.56, 72.77, 71.47, 71.26, 71.19, 70.67, 70.61, 67.64, 67.59, 59.60, 26.14, 18.74, - 4.56, -4.65.

[0217] HR-MS (Q-TOF, ESI) calculated for C33H43N4O13PSi, [M+Na+]: 785.2226, found 785.2221.k. 1-((2R,4S,5S)-4-azido-5-((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-3-((benzyloxy)methyl)-5- methylpyrimidine-2,4(1H,3H)-dione owing general procedure GP10 using 2’-N3(300 mg, 0.77 mmol, 1 equiv.), LiOtBu (74 mg, 0.92 mmol, 1.2 equiv.), U-BOM-Cl-CP (426 mg, 0.93 mmol, 1.2 equiv.), and Toluene:ACN (1:1, 8 mL). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, hexanes: ethyl acetate gradient (0% to 100%), desired product eluted at 100% ethyl acetate and was isolated as white solid. Yield = 520 mg, 83%, dr = 30:1.

[0219] 31P NMR (203 MHz, CDCl3) δ -3.76, -6.49.

[0220] 1H NMR (500 MHz, CDCl3) δ 7.40 – 7.23 (m, 11H), 7.11 (d, J = 8.2 Hz, 1H), 6.18 (t, J = 6.4 Hz, 1H), 5.78 (d, J = 8.1 Hz, 1H), 5.50 (d, J = 13.7 Hz, 3H), 5.46 (d, J = 1.8 Hz, 2H), 4.78 (ddd, J = 10.1, 5.1, 1.3 Hz, 1H), 4.70 (d, J = 2.3 Hz, 4H), 4.67 – 4.58 (m, 1H), 4.54 – 4.43 (m, 2H), 4.43 – 4.31 (m, 2H), 4.27 (td, J = 10.3, 5.4 Hz, 1H), 4.11 (t, J = 6.2 Hz, 1H), 4.05 (dq, J = 5.5, 2.8 Hz, 1H), 3.56 (s, 3H), 2.49 (ddd, J = 14.0, 6.5, 4.8 Hz, 1H), 2.33 (ddd, J = 13.9, 7.5, 6.3 Hz, 1H), 1.96 (s, 3H).

[0221] 13C NMR (126 MHz, CDCl3) δ 163.41, 162.13, 150.91, 150.37, 139.35, 138.04, 137.90, 134.06, 128.51, 128.45, 128.03, 127.95, 127.87, 127.83, 127.79, 127.73, 127.71, 127.70, 127.68, 110.84, 102.95, 95.33, 85.98, 82.28, 82.23, 80.26, 80.20, 78.13, 78.09, 77.41, 77.16, 76.91, 72.66, 72.64, 72.43, 70.84, 70.79, 70.69, 70.60, 69.50, 69.44, 67.86, 67.81, 60.09, 59.43, 37.73, 13.32.

[0222] HR-MS (Q-TOF, ESI) calculated for C36H40N7O13P, [M+H+]: 810.2494, found 810.2495.l. (E)-N'-(9-((2R,3R,4R,5R)-5-((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)-2,4-dioxo- 3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3- methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N,N-dimethylformimidamide y following general procedure GP10 using 3-OTBS-DMF-A-Bn2 (275 mg, 0.61 mmol, 1 equiv.), LiOtBu (54 mg, 0.67 mmol, 1.1 equiv.), U-BOM- Cl-CP (308 mg, 0.67 mmol, 1.1 equiv.), and in Toluene:ACN (1:1). Column conditions: SiO2- 10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM-MeOH gradient (0% to 5%), desired product was isolated as white solid. Yield = 460 mg, 86%, dr = 15:1. Mass was confirmed via LC-MS.

[0224] 31P NMR (162 MHz, CDCl3) δ -3.87, -6.91.

[0225] 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 8.55 (d, J = 1.1 Hz, 1H), 8.05 (s, 1H), 7.38 – 7.22 (m, 7H), 6.05 (d, J = 3.4 Hz, 1H), 5.73 (dd, J = 8.2, 1.1 Hz, 1H), 5.48 – 5.39 (m, 2H), 5.37 (s, 1H), 4.79 (t, J = 5.6 Hz, 1H), 4.74 – 4.63 (m, 3H), 4.55 – 4.44 (m, 2H), 4.41 – 4.18 (m, 4H), 4.06 (d, J = 5.1 Hz, 1H), 3.97 (td, J = 10.2, 5.8 Hz, 1H), 3.49 (dd, J = 5.5, 1.1 Hz, 6H), 3.21 (dd, J = 17.4, 1.2 Hz, 6H), 0.95 (d, J = 1.1 Hz, 9H), 0.18 (d, J = 5.6 Hz, 6H).

[0226] 13C NMR (101 MHz, CDCl3) δ 162.32, 160.03, 158.62, 153.08, 151.27, 150.33, 141.00, 140.56, 137.90, 128.49, 128.44, 127.93, 127.74, 126.62, 102.54, 95.87, 87.59, 82.39, 81.83, 81.75, 79.90, 79.83, 78.29, 78.25, 77.36, 72.61, 70.52, 70.35, 70.32, 70.27, 69.06, 68.98, 66.41, 66.35, 59.39, 58.87, 41.46, 35.23, 25.88, 18.25, -4.57, -4.81.m. (E)-N'-(9-((3aR,4R,6R,6aR)-6-((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4- yl)-9H-purin-6-yl)-N,N-dimethylformimidamide ollowing general procedure GP10 using KETAL-DMF-A-5'OH (215 mg, 0.59 mmol, 1 equiv.), LiOtBu (52 mg, 0.65 mmol, 1.1 equiv.), U-BOM- Cl-CP (299 mg, 0.65 mmol, 1.1 equiv.), and in Toluene:ACN (1:1). Column conditions: SiO2- 10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM:MeOH gradient (0% to 10%), desired product was isolated as white solid. Yield = 82%, dr = 23:1. Mass was confirmed via LC-MS.

[0228] 31P NMR (162 MHz, CDCl3) δ -4.24, -6.93.

[0229] 1H NMR (400 MHz, CDCl3) δ 9.02 – 8.99 (m, 1H), 8.60 (s, 1H), 8.04 (s, 1H), 7.39 – 7.29 (m, 5H), 7.19 (d, J = 8.2 Hz, 1H), 6.18 (d, J = 2.3 Hz, 1H), 5.77 (d, J = 8.1 Hz, 1H), 5.55 – 5.48 (m, 1H), 5.46 (d, J = 1.3 Hz, 2H), 5.38 (d, J = 1.0 Hz, 1H), 5.14 (dd, J = 6.2, 3.0 Hz, 1H), 4.71 (s, 2H), 4.59 – 4.27 (m, 5H), 4.04 (d, J = 5.1 Hz, 1H), 3.94 (td, J = 10.3, 5.6 Hz, 1H), 3.51 (s, 3H), 3.27 (d, J = 0.6 Hz, 3H), 3.22 (s, 3H), 1.65 (s, 3H), 1.43 (s, 3H).n. (E)-N'-(1-((3aR,4R,6R,6aR)-6-((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxidotetrahydro-4H- furo[3,2d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N,N-dimethylformimidamide eneral procedure GP10 withmodification using dmf-C-5'-OH (250 mg, 0.74 mmol, 1 equiv.), LiOtBu (65 mg, 0.81 mmol, 1.1 equiv.), 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxidotetrahydro- 4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-Cl-CP, 373 mg, 0.81 mmol, 1.1 equiv.),, and in Toluene:ACN (1:5). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM:MeOH gradient (0% to 10%), desired product was isolated as pale-white solid. Yield =280 mg, 50%, dr = 14:1. Mass was confirmed via LC-MS.

[0231] 31P NMR (162 MHz, CDCl3) δ -3.94, -6.65.

[0232] 1H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 7.75 (s, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.39 – 7.21 (m, 8H), 6.08 (d, J = 7.2 Hz, 1H), 5.80 (d, J = 8.2 Hz, 1H), 5.69 (s, 1H), 5.58 (d, J = 1.6 Hz, 1H), 5.53 – 5.41 (m, 2H), 5.17 (dd, J = 6.3, 1.6 Hz, 1H), 4.95 (dd, J = 6.5, 3.1 Hz, 1H), 4.69 (s, 3H), 4.64 – 4.39 (m, 5H), 4.26 (td, J = 10.2, 5.6 Hz, 1H), 4.00 (d, J = 5.1 Hz, 1H), 3.55 (s, 3H), 3.33 (d, J = 13.6 Hz, 7H), 3.18 (s, 3H), 3.15 – 3.08 (m, 3H), 2.84 (s, 1H), 1.56 (s, 3H), 1.35 (s, 3H).

[0233] 13C NMR (101 MHz, CDCl3) δ 172.40, 162.37, 159.02, 157.08, 156.12, 150.55, 144.57, 139.32, 137.96, 128.49, 127.91, 127.82, 127.77, 114.15, 103.19, 102.88, 98.00, 94.07, 87.01, 86.95, 85.05, 81.72, 80.48, 80.40, 77.98, 77.94, 77.36, 72.56, 70.73, 70.67, 70.59, 69.48, 69.40, 69.31, 69.25, 59.35, 46.60, 41.84, 39.27, 35.70, 35.56, 27.16, 25.33.o. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((3aR,4R,6R,6aR)-6-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H- furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione wing general procedure GP10 using Ketal-U-BOM (300 mg, 0.74 mmol, 1 equiv.), LiOtBu (65 mg, 0.82 mmol, 1.1 equiv.), U-BOM-Cl-CP (374 mg, 0.82 mmol, 1.1 equiv.), and in Toluene:ACN (1:1, 8 mL). Column conditions: SiO2- 10g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM:MeOH gradient (0% to 5%), desired product was isolated as white solid. Yield = 550 mg, 90%, dr = 15:1.

[0235] 31P NMR (203 MHz, CDCl3) δ -3.92, -6.77.

[0236] 1H NMR (500 MHz, CDCl3) δ 7.43 – 7.29 (m, 10H), 7.10 (d, J = 8.1 Hz, 1H), 5.83 – 5.75 (m, 2H), 5.72 (d, J = 2.2 Hz, 1H), 5.59 – 5.43 (m, 5H), 4.92 (ddd, J = 23.6, 6.5, 2.5 Hz, 2H), 4.78 – 4.67 (m, 5H), 4.50 – 4.36 (m, 4H), 4.35 – 4.21 (m, 1H), 4.10 (d, J = 5.1 Hz, 1H), 3.58 (s, 3H), 1.61 (s, 3H), 1.39 (s, 3H).

[0237] 13C NMR (126 MHz, CDCl3) δ 162.48, 162.05, 150.82, 150.25, 140.40, 139.25, 137.84, 137.81, 128.38, 128.36, 127.82, 127.77, 127.67, 127.66, 127.61, 114.63, 102.76, 102.26, 102.20, 95.24, 95.20, 85.49, 85.43, 84.60, 80.61, 80.14, 80.08, 78.00, 77.96, 77.23, 72.53, 72.43, 70.64, 70.59, 70.47, 70.45, 69.21, 69.15, 68.44, 68.40, 59.39, 27.13, 25.29.

[0238] HR-MS (Q-TOF, ESI) calculated for C38H43N4O15P, [M+Na+]:849.2355, found 849.236. Example 7. Ring-opening of cyclic dinucleotide with nucleophiles a. General study considerations

[0239] Unless otherwise stated, all reactions in Example 7 were set up inside of a chemical fume hood and run under a nitrogen or argon atmosphere. Anhydrous acetonitrile (MeCN) waspurchased from Sigma-Aldrich. Anhydrous toluene was purchased from Sigma-Aldrich. Reactions were run in oven-dried reaction tubes (Fisherbrand, 16 x 125 mm, product no. 1495935A) sealed with a screw cap (Thermo Fisher Scientific, catalog no. B7995-18) fitted with a Teflon-lined septum (Thermo Fisher Scientific, catalog no. C47995-15). Solvents used for extractions, and column chromatography were purchased from Sigma-Aldrich as ACS grade, except for hexanes, which was HPLC grade. Organic compounds were purified by flash chromatography using Silicycle, SiliaFlash P60 (230–400 mesh) silica gel either manually or using a Combi-Flash Biotage, automated chromatography system.

[0240] For compound analysis, CDCl3and CD3OD were purchased from Cambridge Isotope Labs. NMR spectra were collected on Bruker Avance III HD 400 or 500 MHz spectrometer.1H (CDCl3: δ 7.26; d4-MeOD: δ 3.35) and13C NMR shifts (CDCl3: δ 77.16; d4-MeOD: δ 49.3) were referenced to residual solvent peaks. The following abbreviations were used to characterize multiplicities: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, p = pentet, sept = septet, m = multiplet.13C and19F spectra were obtained with1H decoupling. LC / MS was recorded on an Agilent LC / MSD XT (single quad). b. Ring Opening Reaction at 5' C of cyclic phosphate

[0241] An oven-dried reaction tube containing a Teflon-coated magnetic stir bar was charged with base (0.05 mmol, 1.5-2 equiv.), 4-bromobenzylthiol (0.025 mmol, 1.0-2.0 equiv.) and 0.25 mL of anhydrous acetonitrile. The mixture was left stirring for 20 minutes at RT and cooled to -20 ºC or 0 ºC over the period of 10 min. Then, a solution of cyclic dinucleotide (0.025 mmol, 1 equiv.) in solvent (0.25 mL) was added in one portion and left stirring at that temperature for 7 to 18 hrs. The reaction was quenched with a solution of acetic acid (0.05 mmol, 2.0 equiv.) in MeCN (0.5 mL) at -20 ºC. Upon stirring for 5 minutes, a solution of internal standard, PO(OPh)3(0.025 mmol, 1.0 equiv.) in MeCN (0.5 mL) was added and NMR yields and regioselectivities were recorded. Experiments and results are tabulated as follows:Br SH UBOMEntry Cyclic diNu Base (equiv., pKa) Temp,Time Conversion Yield1. UUBOM LiOtBu (1.5, 17) -20 to 0, 16h 100% 112%2. UTBOM LiOtBu (1.5, 17) -20 to 0, 16h 100% 109%3. UUBOM TBD (0.3, 15.2) -20 to 0, 16h 30% 30%4. UUBOM DBU (1.5, 13.5) -20 to 0, 16h 80% 80%5. UUBOM DBU (1.5, 13.5) 0 to RT, 7h 98% 116%6. UUBOM NEt3 (1.5, 10) 0 to RT, 18h <5% <5%Yields and selectivities are determined via 31P-NMR by using (PhO)3PO as an internal standardc. General procedure (GP12)

[0242] Inside an N2-filled glovebox, a reaction tube, equipped with a magnetic stir-bar, was charged with thiol, base, and anhydrous solvent (0.1 M). The resulting reaction mixture was stirred at ambient temperature for 3 mins (unless otherwise stated) to preactivate or deprotect thiol, then cyclic dinucleotide was added to the reaction tube in one portion. The resulting reaction mixture was stirred at ambient temperature for around 18 hrs. The reaction was tracked by31P NMR and TLC. Upon completion of the reaction, the reaction was quenched with AcOH (1 equiv more than base used) and concentrated under reduced pressure. The crude product was purified via Combi-Flash NextGen 300 automated chromatography system with conditions indicated. d. General procedure (GP13)

[0243] Under an osphere, a reaction tube, equipped with a magnetic stir-bar, wascharged with thiol, base, and anhydrous solvent (0.1 M). The resulting reaction mixture wasdegassed by vacuum / N2 cycle three times using standard Schlenk technique and stirred at ambient temperature for 3 mins (unless otherwise stated) to preactivate or deprotect thiol, then cyclic dinucleotide was added to the reaction tube in one portion. The resulting reaction mixture was degassed again by vacuum / N2 cycle three times using standard Schlenk technique and stirred at ambient temperature for around 18 hrs. The reaction was tracked by31P NMR and TLC. Upon completion of the reaction, the reaction was quenched with AcOH (1 equiv more than base used) and concentrated under reduced pressure. The crude product was purified via Combi-Flash NextGen 300 automated chromatography system with condition indicated. e. General procedure (GP14)

[0244] An oven-dried 20 ml Scintillation vial containing a Teflon-coated magnetic stir bar was charged with nucleophiles (1.5-2 equiv.), cyclic dinucleotides (1 equiv.) and solvent (0.2 M). The mixture was left stirring at RT or 60oC until the reaction went to completion. Upon completion as per LC-MS analysis, the reaction was quenched with acetic acid (3.0 equiv.) and concentrated under reduced pressure. The crude product was purified via Biotage automated chromatography system with condition indicated. f. Tested substrates Thiolates, halides, and azides Nucleophile TBSOOOO UBOMRSH / Base (1.5 equiv. each)TBSOOMOBOM), .)Example 8. Synthesis of specific 5'C modified dinucleotides a. (2S,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (((mercapto(methoxy)phosphorothioyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- ((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) hydrogen phosphatefollowing general procedure 14 (GP14) of Example 7. To 20 mL Scintillation vial equipped with magnetic stir was added 3- ((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4- methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione, UU-BOM (500 mg, 1 equiv., 0.55 mmol), and potassium O-methyl phosphorotrithioate (MeOPS3K2) (194 mg, 1.5 equiv, 0.82 mmol) in 5 mL of anhydrous MeOH was added and vial was closed by screw cap. The reaction was stirred at 40oC for 20 hours. Upon completion of reaction, the product was concentrated to white yellow solid. The crude product was washed with diethyl ether / hexane to get rid of residual reactants. The product was isolated as a white solid with yield of 520 mg, 88%.

[0246] 31P NMR (162 MHz, MeOD) δ 118.31, -1.08.

[0247] 1H NMR (400 MHz, MeOD) δ 8.03 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.36 – 7.20 (m, 10H), 6.03 (d, J = 4.9 Hz, 1H), 5.94 (dd, J = 13.2, 6.3 Hz, 2H), 5.81 (d, J = 8.1 Hz, 1H), 5.49 – 5.37 (m, 4H), 4.70 – 4.55 (m, 7H), 4.44 (dt, J = 9.2, 4.6 Hz, 2H), 4.28 – 4.19 (m, 1H), 4.11 (dt, J = 9.6, 4.4 Hz, 3H), 3.90 (t, J = 4.9 Hz, 1H), 3.63 – 3.50 (m, 6H), 3.49 – 3.33 (m, 6H), 3.22 (td, J = 13.3, 7.5 Hz, 1H), 0.93 (s, 9H), 0.16 (d, J = 3.3 Hz, 6H).

[0248] 13C NMR (101 MHz, MeOD) δ 164.76 (d, J = 2.2 Hz), 152.77, 152.53, 141.36, 139.31 (d, J = 5.7 Hz), 129.34, 129.15, 128.89 – 128.60 (m), 103.08, 102.53, 89.43, 89.04, 84.25, 83.25 (d, J = 3.5 Hz), 75.97 (d, J = 5.4 Hz), 73.17, 71.87 – 71.39 (m), 65.69 (d, J = 5.6 Hz), 58.86 (d, J = 11.4 Hz), 53.27 (d, J = 9.1 Hz), 39.01 (d, J = 3.5 Hz), 26.30, 18.96, -4.45.

[0249] HR-MS (Q-TOF, ESI) calculated for C43H60N4O16P2S3Si, [M+Na+]: 1097.2303, found 1097.2279. b. (2S,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (iodomethyl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)- 2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4- methoxytetrahydrofuran-2-yl)methyl) hydrogen phosphatewing general procedure 14 (GP14) in Example 7. To 20 mL Scintillation vial equipped with magnetic stir was added 3- ((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4- methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione, UU-BOM (500 mg, 1 equiv., 0.55 mmol), and lithium iodide (91 mg, 1.25 equiv., 0.68 mmol) in 5 mL of anhydrous ACN was added and vial was closed by screw cap. The reaction was stirred at 50 ◦C for 20 hours. Upon completion of reaction, the reaction was quenched with AcOH (197 mg, 3 equiv.) and concentrated to thick oil. The crude thick oil was then purified on pre-packed 10 g silica gel biotage-column system by using DCM:MeOH gradient (0 to 20%). The desired product was eluted with ~10% MeOH. The product was isolated as a white solid with yield of 550 mg, 96%.

[0251] 31P NMR (162 MHz, cd3od) δ -1.03.

[0252] 1H NMR (400 MHz, cd3od) δ 8.01 (dd, J = 8.2, 0.9 Hz, 1H), 7.71 (dd, J = 8.2, 0.9 Hz, 1H), 7.34 – 7.21 (m, 9H), 6.01 (d, J = 4.5 Hz, 1H), 5.94 – 5.85 (m, 2H), 5.76 (dd, J = 8.2, 0.9 Hz, 1H), 5.46 – 5.39 (m, 4H), 4.65 (d, J = 5.3 Hz, 4H), 4.47 (dt, J = 8.4, 5.8 Hz, 1H), 4.41 (t, J = 4.8 Hz, 1H), 4.27 – 4.19 (m, 1H), 4.16 (dt, J = 6.3, 3.1 Hz, 1H), 4.14 – 4.03 (m, 3H), 3.86 (t, J = 4.7 Hz, 1H), 3.70 (dd, J = 11.2, 3.3 Hz, 1H), 3.52 (d, J = 0.9 Hz, 4H), 3.47 (d, J = 0.9 Hz, 3H), 0.94 (s, 9H), 0.15 (s, 6H).

[0253] 13C NMR (101 MHz, MeOD) δ 164.93, 164.75, 152.80, 152.44, 141.49, 141.37, 139.60, 139.57, 129.53, 128.92, 128.83, 103.12, 102.80, 90.25, 89.44, 85.51, 85.42, 84.59, 83.55, 83.50, 83.22, 83.19, 76.34, 76.29, 73.39, 73.36, 71.88, 71.69, 65.64, 65.58, 59.24, 59.00, 26.48, 19.17, 5.97, 5.94, -4.25, -4.32.

[0254] HR-MS (Q-TOF, ESI) calculated for C42H56IN4O15PSi, [M+Na+]: 1065.2186, found 1065.2182. c. ((2S,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- (((((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- ((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl diethylcarbamodithioate [] e compoun was syn es ze y o ow ng general procedure 14 (GP14) in Example 7. To 20 mL Scintillation vial equipped with magnetic stir was added 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4- methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione, UU-BOM (1000 mg, 1 equiv., 1.09 mmol), Diethylammonium N,N-diethyldithiocarbamate (304 mg, 1.5 equiv.1.37 mmol) in 5 mL of DMF was added and vial was closed by screw cap. The reaction was stirred at 50oC for 18 hours. Upon completion of reaction, the reaction was quenched with AcOH (197 mg, 3 equiv.) and concentrated to thick oil. The crude thick oil was then purified on pre-packed 10 g silica gel biotage-column system by using DCM:MeOH gradient (0 to 10%). The unreacted starting material was eluted at around 5% of MeOH followed by purified product (10% MeOH). The product was isolated as a white solid with yield of 1120 mg, 96%.

[0256] 31P NMR (162 MHz, cd3od) δ -1.21.

[0257] 1H NMR (400 MHz, cd3od) δ 8.03 (dd, J = 8.2, 1.1 Hz, 1H), 7.67 (dd, J = 8.2, 1.0 Hz, 1H), 7.36 – 7.19 (m, 9H), 6.05 – 5.96 (m, 1H), 5.91 – 5.82 (m, 2H), 5.73 (d, J = 8.1 Hz, 1H), 5.51 – 5.36 (m, 4H), 4.65 (d, J = 7.3 Hz, 4H), 4.61 – 4.54 (m, 1H), 4.45 – 4.35 (m, 2H), 4.29 – 4.20 (m, 1H), 4.11 (dt, J = 8.3, 3.3 Hz, 3H), 4.03 (dt, J = 13.6, 4.9 Hz, 3H), 3.88 (t, J = 4.7 Hz, 1H), 3.81 (d, J = 7.4 Hz, 2H), 3.75 – 3.64 (m, 1H), 3.54 (d, J = 1.0 Hz, 3H), 3.46 (d, J = 0.9 Hz, 3H), 3.03 (qd, J = 7.4, 1.2 Hz, 4H), 1.30 (dd, J = 7.3, 1.0 Hz, 5H), 1.28 – 1.18 (m, 6H), 0.93 (d, J = 1.0 Hz, 9H), 0.18 – 0.11 (m, 6H).

[0258] 13C NMR (101 MHz, MeOD) δ 195.47, 164.74, 152.68, 152.25, 141.24, 139.43, 139.40, 129.34, 128.73, 128.68, 102.93, 102.36, 90.68, 89.16, 85.51, 84.43, 83.04, 82.40, 82.34, 76.35, 76.29, 73.19, 73.15, 71.69, 71.62, 71.58, 65.46, 59.03, 58.79, 50.93, 47.96, 43.51, 40.33, 26.30, 18.98, 12.90, 11.80, 11.60, -4.43, -4.48.

[0259] HR-MS (Q-TOF, ESI) calculated for C47H66N5O15PS2Si, [M+Na+]: 1086.3396 found 1086.3386.d. (2S,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-Example 7. To 20 mL Scintillation vial equipped with magnetic stir was added 3- ((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4- methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione, UU-BOM (600 mg, 1 equiv., 0.65 mmol) and ammonium O,O-diethyl S-hydrogen phosphorodithioate (200 mg, 1.5 equiv.0.98 mmol) in 5 mL of DMF was added and vial was closed by screw cap. The reaction was stirred at 50oC for 18h. Upon completion of reaction, the reaction was quenched with AcOH and concentrated to thick oil. The crude thick oil was then purified on pre-packed 10 g silica gel biotage-column system by using DCM:MeOH gradient (0 to 20%). The unreacted starting material was eluted at around 5% of MeOH followed by purified product (10% MeOH). The product was isolated as white solid in total yield 580 mg, 80% yield.

[0261] 31P NMR (162 MHz, cd3od) δ 93.81, -1.18.

[0262] 1H NMR (400 MHz, cd3od) δ 8.00 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.38 – 7.19 (m, 9H), 6.00 (d, J = 4.3 Hz, 1H), 5.91 – 5.82 (m, 2H), 5.75 (s, 1H), 5.49 – 5.37 (m, 4H),4.65 (d, J = 8.2 Hz, 4H), 4.52 – 4.44 (m, 1H), 4.39 (t, J = 4.9 Hz, 1H), 4.31 (ddd, J = 9.6, 6.5, 3.4 Hz, 1H), 4.26 – 4.04 (m, 8H), 4.04 – 3.95 (m, 2H), 3.85 (t, J = 4.6 Hz, 1H), 3.54 (d, J = 0.8 Hz, 3H), 3.47 (d, J = 0.8 Hz, 3H), 3.22 (ddd, J = 15.6, 13.9, 8.7 Hz, 1H), 1.31 (ddd, J = 7.5, 6.7, 3.0 Hz, 6H), 1.26 (t, J = 7.1 Hz, 4H), 0.93 (d, J = 0.8 Hz, 9H), 0.16 (d, J = 2.5 Hz, 6H).

[0263] 13C NMR (101 MHz, MeOD) δ 164.66, 164.54, 152.59, 152.16, 141.29, 141.24, 141.10, 139.38, 139.33, 129.31, 128.71, 128.64, 128.62, 128.53, 102.90, 102.42, 90.85, 89.57, 89.22, 85.25, 85.01, 84.40, 82.96, 82.66, 75.96, 75.91, 73.23, 73.18, 73.15, 71.67, 71.59, 71.40, 69.42, 65.39, 65.33, 65.28, 65.22, 65.18, 62.60, 62.54, 58.99, 58.79, 36.40, 26.27, 18.95, 16.49, 16.40, 16.26, 16.17, -4.47, -4.52.

[0264] HR-MS (Q-TOF, ESI) calculated for C46H66N4O15P2S2Si, [M+Na+]: 1123.3001, found 1123.2995. e. (2S,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-Example 7. To 20 mL reaction vial equipped with magnetic stir was added LiOtBu (1.25 equiv.) and diphenylphosphinodithioic acid (178 mg, 0.71 mmol, 1.3 equiv.) and 3 mL of ACN and reaction vial was closed. After stirring for 15 minutes, a solution of 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2- yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6- yl)pyrimidine-2,4(1H,3H)-dione, UU-BOM (500 mg, 0.546 mmol, 1 equiv.) in 2 mL of ACN was added and vial was closed by screw cap. The reaction was stirred at RT for 18 hours. Upon completion of reaction, the reaction mixture was concentrated and the crude thick oil was then purified on pre-packed 10 g silica gel biotage-column system by using DCM:MeOH gradient (0 to 20%). The unreacted starting material was eluted at around 5% of MeOH followed by purified product (10% MeOH). The product was isolated in two fractions (F1100 mg) and F2 (500 mg) and NMR analysis was done. Both fractions contain the desired product with >95% purity. Total yield = 600 mg.

[0266] 31P NMR (162 MHz, cd3od) δ 5.57, -1.20.

[0267] 1H NMR (400 MHz, cd3od) δ 7.99 (dd, J = 8.2, 0.9 Hz, 1H), 7.90 (dddt, J = 29.3, 14.4, 8.2, 1.2 Hz, 4H), 7.63 – 7.52 (m, 1H), 7.52 – 7.38 (m, 6H), 7.35 – 7.19 (m, 9H), 5.99 (dd, J = 4.5, 0.9 Hz, 1H), 5.85 (dd, J = 8.1, 0.9 Hz, 1H), 5.69 (dd, J = 3.6, 0.9 Hz, 1H), 5.66 (dd, J = 8.2, 0.9 Hz, 1H), 5.44 – 5.34 (m, 4H), 4.64 (d, J = 5.5 Hz, 4H), 4.47 (dt, J = 8.4, 5.8 Hz, 1H), 4.38 (t, J = 4.8 Hz, 1H), 4.31 – 4.15 (m, 2H), 4.06 (tt, J = 7.3, 2.9 Hz, 2H), 4.00 (t, J = 4.4 Hz, 1H), 3.88 – 3.81 (m, 1H), 3.49 (d, J = 1.0 Hz, 3H), 3.46 (d, J = 0.9 Hz, 3H), 3.37 – 3.2 (m, 1H), 0.93 (d, J = 1.0 Hz, 9H), 0.14 (s, 6H).

[0268] 13C NMR (101 MHz, MeOD) δ 164.72, 164.64, 152.67, 152.11, 141.35, 141.20, 139.42, 139.39, 133.20, 133.12, 132.87, 132.76, 132.37, 132.26, 129.83, 129.70, 129.61, 129.48, 129.35, 129.32, 128.75, 128.71, 128.69, 128.65, 102.97, 102.34, 90.44, 89.17, 85.44, 85.35, 84.42, 83.04, 83.01, 82.73, 76.16, 76.11, 73.18, 73.16, 71.68, 71.60, 71.50, 65.48, 65.43, 58.98, 58.81, 34.96, 26.31, 18.98, -4.45.

[0269] HR-MS (Q-TOF, ESI) calculated for C54H66N4O15P2S2Si, [M+Na+]: 1187.3103, found 1187.3096.f. ((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- ((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl ((2S,3S,4R,5R)-2- (bromomethyl)-5-(4-(dibenzylamino)-2-oxopyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran- 3-yl) hydrogen phosphateprocedure 14 (GP14) in Example 7. To 20 mL Scintillation vial equipped with magnetic stir was added 3 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((4aR,6R,7R,7aR)-6-(4-(dibenzylamino)-2-oxopyrimidin-1(2H)-yl)-7-methoxy-2- oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, UC-Bn2 (500 mg, 1 equiv., 0.513 mmol) and lithium bromide (67 mg, 1.5 equiv.0.77 mmol) in 5 mL of anhydrous acetonitrile was added and vial was closed by screw cap. The reaction was stirred at 50oC for 18 hours. Upon completion of reaction, the reaction was quenched with AcOH (92 mg, 1.54 mmol, 3 equiv.) and concentrated to thick oil. The crude thick oil was then purified on pre-packed 10 g silica gel biotage-column system by using DCM:MeOH gradient (0 to 20%). The unreacted starting material was eluted at around 5% of MeOH followed by purified product (9-10% MeOH). The product was isolated in two fractions and NMR analysis was done. Both fractions are desired product with purity >95%.510 mg, 94% yield.

[0271] 31P NMR (162 MHz, cd3od) δ -1.67.

[0272] 13C NMR (100 MHz, cd3od) δ 164.86, 163.77, 156.82, 151.73, 141.88, 140.33, 138.55, 129.08, 128.81, 128.45, 127.84, 127.77, 126.84, 101.97, 93.69, 89.36, 88.38, 84.49,84.41, 83.49, 82.81, 82.78, 81.65, 81.59, 74.13, 74.08, 72.29, 70.77, 70.70, 64.63, 58.26, 57.91, 32.83, 25.44, 18.12, -5.28, -5.36. g. ((2S,3S,3aS,9aR)-3-(((((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)oxy)-6-oxo-2,3,3a,9a-tetrahydro-6H- furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-2-yl)methyl diethylcarbamodithioateng general procedure 14 (GP14) in Example 7.: To 20 mL Scintillation vial equipped with magnetic stir was added 3- ((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-methoxy-5- ((((4aR,5aR,11aS,11bR)-2-oxido-9-oxo-4a,5a,11a,11b-tetrahydro-4H,9H- [1,3,2]dioxaphosphinino[4'',5'':4',5']furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-2- yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione, cyclo-UU (400 mg, 1 equiv., 0.52 mmol) and Diethylammonium N,N-diethyldithiocarbamate (146 mg, 1.25 equiv., 0.66 mmol) in 5 mL of anhydrous acetonitrile was added and vial was closed by screw cap. The reaction was stirred at 50oC for 18 hours. Upon completion of reaction, the reaction was quenched with AcOH (92 mg, 1.54 mmol, 3 equiv.) and concentrated to thick oil. The crude thick oil was then purified on pre-packed 10 g silica gel biotage-column system by using DCM:MeOH gradient (0 to 20%). The unreacted starting material was eluted at around 5% of MeOH followed by purified product (9-10% MeOH). The product was isolated in twofractions and NMR analysis was done. Both fractions are desired product with purity >95%.500 mg, 96% yield.

[0274] 31P NMR (162 MHz, cd3od) δ -1.91.

[0275] 13C NMR (100 MHz, cd3od) δ 219.59, 194.07, 174.11, 163.25, 160.34, 151.34, 150.93, 139.94, 138.01, 137.38, 127.95, 127.34, 127.29, 108.71, 101.08, 90.00, 89.26, 86.72, 86.63, 81.52, 81.20, 81.14, 76.73, 74.88, 71.81, 70.26, 64.44, 57.69, 41.98, 38.92, 24.79, 17.39, 11.52, 10.43, 10.20, -6.02, -6.09. h. (2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (((4-bromophenyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) phosphate BOM Br O N S O Oesized by following general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), DBU (5.7 mg, 0.038 mmol, 1.5 equiv.), 4-bromobenzenethiol (7.1 mg, 0.038 mmol, 1.5 equiv.), and in MeCN (0.25 mL). Column conditions: SiO2-25g column, Next- Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as a white foamy solid (27.7 mg, 99%).

[0277] 31P NMR (202 MHz, DMSO) δ -1.31.

[0278] 1H NMR (500 MHz, DMSO) δ 8.12 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.36 – 7.22 (m, 12H), 5.89 (d, J = 5.2 Hz, 1H), 5.82 (d, J = 5.1 Hz, 1H), 5.76 – 5.72 (m, 2H), 5.34 – 5.25 (m, 4H), 4.58 (s, 2H), 4.57 (s, 2H), 4.47 (br s, 1H), 4.33 (t, J = 4.4 Hz, 1H), 4.20 – 4.16 (m, 1H), 4.01 (t, J = 5.2 Hz, 1H), 3.96 (d, J = 3.9 Hz, 1H), 3.92 (br s, 1H), 3.89 (t, J = 5.0 Hz, 1H), 3.84 (br d, J = 11.9 Hz, 1H), 3.54 (dd, J = 14.1, 3.4 Hz, 1H), 3.36 (s, 3H, assumed; partially obscured by solvent peak), 3.31 (s, 3H), 3.31 – 3.29 (m, 1H), 0.87 (s, 9H), 0.08 (s, 6H).

[0279] 13C NMR (126 MHz, CDCl3) δ 151.51, 151.11, 139.81, 138.34, 137.74, 137.56, 135.01, 132.31, 130.23, 128.53, 120.34, 102.83, 102.04, 89.55, 87.53, 83.18, 83.07, 83.03, 82.55, 82.05, 73.78, 72.46, 72.40, 70.45, 70.39, 69.89, 64.22, 58.90, 58.47, 35.83, 29.43, 25.78, 18.18.

[0280] HR-MS (Q-TOF, ESI) calculated for C48H6079BrN4O15PSSi [M+Na+]: 1125.2358, found 1125.2359. i. (2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (((4-bromobenzyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) phosphatesynthesized by following general procedure 1 (GP1) in Example 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), DBU (5.7 mg, 0.038 mmol, 1.5 equiv.), (4-bromophenyl)methanethiol(7.7 mg, 0.038 mmol, 1.5 equiv.), and in MeCN (0.25 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as a white foamy solid (27.8 mg, 99%).

[0282] 31P NMR (202 MHz, DMSO) δ -1.47.

[0283] 1H NMR (500 MHz, DMSO) δ 8.12 (d, J = 7.7 Hz, 1H), 7.72 (dd, J = 8.1, 1.7 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.36 – 7.21 (m, 12H), 5.90 (d, J = 5.2 Hz, 1H), 5.88 – 5.84 (m, 1H), 5.79 (d, J = 8.1 Hz, 1H), 5.74 – 5.69 (m, 1H), 5.35 – 5.26 (m, 4H), 4.58 (s, 4H), 4.48 – 4.42 (m, 1H), 4.33 (s, 1H), 4.25 – 4.20 (m, 1H), 4.03 – 3.92 (m, 3H), 3.90 (t, J = 5.0 Hz, 1H), 3.87 – 3.74 (m, 3H), 3.37 (s, 3H, assumed; partially obscured by solvent peak), 3.31 (s, 3H, assumed; partially obscured by solvent peak), 2.86 (dd, J = 14.3, 4.2 Hz, 1H), 2.73 (dd, J = 14.4, 7.4 Hz, 1H), 0.87 (s, 9H), 0.08 (s, 6H).

[0284] 13C NMR (126 MHz, DMSO) δ 161.89, 161.78, 150.92, 150.76, 140.09, 139.84, 138.05, 138.03, 137.98, 131.22, 128.20, 128.02, 127.49, 127.37, 127.35, 119.89, 101.49, 101.40, 87.73, 86.90, 84.46, 82.86, 82.06, 80.33, 72.94, 71.09, 71.07, 70.32, 70.08, 70.06, 63.73, 57.57, 57.54, 34.88, 32.86, 25.64, 17.78, -4.82, -4.85.

[0285] HR-MS (Q-TOF, ESI) calculated for C49H6279BrN4O15PSSi [M+Na+]: 1139.2515, found 1139.2516. j. (2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-

[0286] The compound was synthesized by following general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), DBU (5.7 mg, 0.038 mmol, 1.5 equiv.), methyl (tert-butoxycarbonyl)-L- cysteinate (Boc-Cys-OMe, 8.9 mg, 0.038 mmol, 1.5 equiv.), and in MeCN (0.25 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as a white foamy solid (26.5 mg, 92%).

[0287] 31P NMR (202 MHz, DMSO) δ -1.63.

[0288] 1H NMR (500 MHz, DMSO) δ 8.14 (t, J = 8.2 Hz, 1H), 7.75 (dd, J = 11.8, 8.1 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.36 – 7.23 (m, 10H), 5.90 (dd, J = 5.3, 1.4 Hz, 1H), 5.85 (t, J = 5.2 Hz, 1H), 5.78 (dd, J = 8.2, 2.8 Hz, 1H), 5.74 (dd, J = 8.8, 2.0 Hz, 1H), 5.34 – 5.27 (m, 4H), 4.58 (s, 2H), 4.58 (s, 2H), 4.43 (br s, 1H), 4.35 (t, J = 3.9 Hz, 1H), 4.19 – 4.10 (m, 2H), 4.02 – 3.98 (m, 1H), 3.97 (br s, 1H), 3.93 (br s, 1H), 3.90 (t, J = 5.2 Hz, 1H), 3.84 (d, J = 11.8 Hz, 1H), 3.59 (s, 3H), 3.37 (s, 3H, assumed; partially obscured by solvent peak), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 2.99 (dd, J = 13.3, 4.6 Hz, 1H), 2.94 – 2.87 (m, 2H), 2.86 – 2.77 (m, 1H), 1.39 – 1.27 (m, 9H, Boc rotamers), 0.88 (s, 9H), 0.09 (s, 6H).

[0289] 13C NMR (126 MHz, DMSO) δ 171.66, 161.92, 161.79, 155.44, 155.31, 150.96, 150.79, 140.11, 139.86, 139.83, 138.07, 138.04, 128.22, 127.51, 127.38, 101.49, 101.44, 87.67, 86.85, 84.49, 83.49, 83.29, 82.09, 80.30, 78.35, 72.94, 71.09, 71.07, 70.33, 70.07, 63.70, 57.60, 57.56, 55.85, 53.64, 53.53, 51.96, 51.90, 33.93, 33.50, 33.08, 29.62, 28.14, 25.66, 17.80, -4.80, - 4.84.

[0290] HR-MS (Q-TOF, ESI) calculated for C51H72N5O19PSSi [M+Na+]: 1172.3941, found 1172.3956.k. (2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-Example 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), LiOtBu (3.2 mg, 0.040 mmol, 1.6 equiv.), O,O-diethyl S-hydrogen phosphorodithioate (7.5 mg, 0.040 mmol, 1.6 equiv.), and in PhMe (0.25 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as a white foamy solid (26.9 mg, 97%).

[0292] 31P NMR (202 MHz, DMSO) δ 92.47, -1.48.

[0293] 1H NMR (500 MHz, DMSO) δ 8.12 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.36 – 7.22 (m, 10H), 5.90 (d, J = 5.2 Hz, 1H), 5.83 (d, J = 5.1 Hz, 1H), 5.80 (d, J = 8.2 Hz, 1H), 5.74 (d, J = 8.2 Hz, 1H), 5.37 – 5.26 (m, 4H), 4.58 (s, 2H), 4.58 (s, 2H), 4.44 – 4.39 (m, 1H), 4.35 (t, J = 4.3 Hz, 1H), 4.19 – 4.03 (m, 6H), 3.99 – 3.93 (m, 2H), 3.91 (t, J = 5.0 Hz, 1H), 3.88 – 3.83 (m, 1H), 3.38 (s, 3H, assumed; partially obscured by solvent peak), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 3.32 – 3.28 (m, 1H, assumed; partially obscured by solvent peak), 3.17 (td, J = 13.6, 9.1 Hz, 1H), 1.26 (t, J = 7.0 Hz, 6H), 0.88 (s, 9H), 0.10 (s, 6H).

[0294] 13C NMR (126 MHz, DMSO) δ 161.89, 161.77, 150.92, 150.73, 140.12, 140.10, 138.06, 138.02, 128.21, 127.78, 127.49, 127.37, 127.36, 101.49, 101.40, 88.11, 86.92, 84.42,82.04, 81.90, 80.26, 73.28, 73.24, 71.09, 71.07, 70.29, 70.07, 63.82, 63.78, 63.74, 57.63, 57.60, 34.94, 34.91, 25.65, 17.78, 16.30, 15.61, 15.55, -4.79, -4.83.

[0295] HR-MS (Q-TOF, ESI) calculated for C46H66N4O17P2S2Si [M+Na+]: 1123.3001, found 1123.3005. l. (2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (5,8,11,14-tetraoxa-2-thiaheptadec-16-yn-1-yl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- ((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) phosphatewing general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), DBU (11.5 mg, 0.075 mmol, 3 equiv.), 3,6,9,12-tetraoxapentadec-14- yne-1-thiol (18.7 mg, 0.075 mmol, 3 equiv.), and in MeCN (0.25 mL). Column conditions: SiO2- 25 g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as a colorless clear gum (26.9 mg, 92%).

[0297] 31P NMR (202 MHz, DMSO) δ -1.42.

[0298] 1H NMR (500 MHz, DMSO) δ 8.14 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.36 – 7.24 (m, 10H), 5.90 (d, J = 5.3 Hz, 1H), 5.86 (d, J = 5.1 Hz, 1H), 5.81 (d, J = 8.2 Hz, 1H), 5.76 – 5.72 (m, 1H), 5.34 – 5.27 (m, 4H), 4.59 (s, 2H), 4.58 (s, 2H), 4.43 – 4.37 (m, 1H), 4.35 (t, J = 4.3 Hz, 1H), 4.17 – 4.12 (m, 3H), 3.99 – 3.92 (m, 3H), 3.91 (t, J = 5.1 Hz, 1H), 3.86 – 3.81 (m, 1H), 3.56 – 3.49 (m, 20H), 3.43 – 3.41 (m, 1H), 3.38 (s, 3H, assumed; partially obscured by solvent peak), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 2.99 – 2.88 (m, 2H), 2.79 – 2.65 (m, 3H), 0.88 (s, 9H), 0.10 (s, 6H).

[0299] 13C NMR (126 MHz, DMSO) δ 161.92, 161.79, 150.95, 150.77, 140.14, 139.75, 138.06, 138.04, 128.21, 127.50, 127.38, 127.37, 101.50, 101.41, 87.58, 86.88, 84.50, 83.51, 82.08, 80.50, 80.47, 80.34, 77.13, 77.11, 72.99, 72.11, 71.08, 70.64, 70.35, 70.29, 70.17, 70.07, 70.00, 69.79, 69.76, 69.74, 69.57, 69.52, 69.49, 68.53, 68.50, 63.70, 57.60, 57.56, 57.51, 57.49, 33.85, 31.68, 31.62, 30.25, 25.66, 17.79, -4.80, -4.84.

[0300] HR-MS (Q-TOF, ESI) calculated for [C53H75N4O19PSSi [M+Na+]: 1185.4145, found 1185.4157. m. (2S,3S,4R,5R)-2-(13-Azido-5,8,11-trioxa-2-thiatridecyl)-5-(3-((benzyloxy)methyl)-2,4-

[0301] The compound was synthesized by following general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (TBD, 14.0 mg, 0.10 mmol, 4 equiv.), S-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl) ethanethioate (22.3 mg, 0.084 mmol, 3.2 equiv.), and in MeCN (0.25 mL). Acetyl-protected thiol and TBD were stirred for 15 mins before addition of U-BOM-PO-U-BOM. Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA- MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as an amber clear gum (26.6 mg, 92%).

[0302] 31P NMR (202 MHz, DMSO) δ -1.49.

[0303] 1H NMR (500 MHz, DMSO) δ 8.14 (d, J = 8.2 Hz, 1H), 7.85 – 7.78 (m, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.36 – 7.23 (m, 10H), 6.14 (br s, 1H), 5.90 (d, J = 5.4 Hz, 1H), 5.85 (d, J = 5.0 Hz, 1H), 5.80 (d, J = 8.1 Hz, 1H), 5.74 (d, J = 8.2 Hz, 1H), 5.35 – 5.26 (m, 4H), 4.58 (s, 2H), 4.58 (s, 2H), 4.39 (dt, J = 8.9, 5.0 Hz, 1H), 4.35 (t, J = 4.2 Hz, 1H), 4.15 (dt, J = 8.5, 4.4 Hz, 1H), 4.00 – 3.92 (m, 3H), 3.91 (t, J = 5.1 Hz, 1H), 3.86 – 3.80 (m, 1H), 3.59 – 3.48 (m, 12H), 3.37 (s, 3H, assumed; partially obscured by solvent peak), 3.33 (s, 3H), 3.19 – 3.15 (m, 2H), 3.08 (td, J = 5.9, 2.6 Hz, 1H), 3.01 – 2.88 (m, 3H), 2.79 – 2.63 (m, 2H), 1.80 – 1.74 (m, 3H), 1.53 (m, 1H), 0.88 (s, 9H), 0.10 (s, 6H).

[0304] 13C NMR (126 MHz, DMSO) δ 169.26, 168.91, 161.92, 161.80, 155.44, 150.95, 150.78, 140.13, 139.76, 138.07, 138.05, 128.22, 127.51, 127.38, 127.37, 101.50, 101.42, 87.59, 86.90, 84.50, 84.45, 83.53, 83.50, 82.09, 80.50, 80.47, 73.04, 73.00, 71.09, 70.34, 70.07, 70.02, 69.82, 69.79, 69.76, 69.68, 69.60, 69.58, 69.53, 69.27, 69.17, 68.64, 63.71, 60.26, 57.61, 57.57, 55.85, 50.00, 44.94, 44.29, 38.57, 37.87, 36.18, 33.85, 31.64, 29.61, 27.44, 25.66, 22.71, 22.57, 22.04, 17.79, -4.81, -4.84.

[0305] HR-MS (Q-TOF, ESI) calculated for [C50H72N7O18PSSi [M+Na+]: 1172.4054, found 1172.4058.n. (2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (((3-carboxypropyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) phosphate hesized by following general procedure 12 (GP12) inExample 7, using 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-U-BOM, 23.0 mg, 0.025 mmol, 1 equiv.), DBU (23.0 mg, 0.15 mmol, 6 equiv.), 4-mercaptobutanoic acid (9.1 mg, 0.075 mmol, 3 equiv.), and in MeCN (0.25 mL). Column conditions: SiO2-25g column, Next- Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH- H2O-AcOH gradient (100:0:0:0 to 84:10:5:1), desired product was isolated as a colorless clear gum (25.6 mg, 98%).

[0307] 31P NMR (202 MHz, DMSO) δ -1.89.

[0308] 1H NMR (500 MHz, DMSO) δ 8.10 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.35 – 7.23 (m, 10H), 5.91 (d, J = 5.4 Hz, 1H), 5.88 (d, J = 5.4 Hz, 1H), 5.84 – 5.76 (m, 2H), 5.34 – 5.26 (m, 4H), 4.58 (s, 2H), 4.57 (s, 2H), 4.48 (br s, 1H), 4.34 (t, J = 4.2 Hz, 1H), 4.18 (br s, 1H), 4.04 – 4.00 (m, 1H), 3.99 – 3.94 (m, 2H), 3.90 (t, J = 5.1 Hz, 1H), 3.86 (br d, J = 11.4 Hz, 1H), 3.36 (s, 3H, assumed; partially obscured by solvent peak), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 2.96 (d, J = 13.9 Hz, 1H), 2.81 (dd, J = 14.2, 7.4 Hz, 1H), 2.56 (t, J = 7.2 Hz, 2H), 2.24 – 2.12 (m, 2H), 1.79 – 1.68 (m, J = 6.9 Hz, 2H), 0.87 (s, 9H), 0.09 (s, 6H).

[0309] 13C NMR (126 MHz, DMSO) δ 161.96, 161.80, 150.98, 150.85, 140.04, 139.82, 138.07, 138.04, 128.22, 127.51, 127.39, 101.55, 87.34, 86.82, 84.40, 82.97, 82.03, 80.32, 73.03, 71.09, 71.08, 70.33, 70.08, 63.75, 57.60, 57.52, 55.85, 34.45, 33.43, 29.62, 25.65, 22.46, 17.79, - 4.82, -4.86.

[0310] HR-MS (Q-TOF, ESI) calculated for [C46H63N4O17PSSi [M+H+]: 1135.3489, found 1135.3482. o. (2S,3S,4R,5R)-2-(((4-Bromophenyl)thio)methyl)-5-(4-(dibenzylamino)-2-oxopyrimidin-Example 7, using 1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5- (6-(dibenzylamino)-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2- oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)-4-(dibenzylamino)pyrimidin- 2(1H)-one (C-Bn2-PO-A-Bn2, 100.0 mg, 0.095 mmol, 1 equiv.), DBU (21.6 mg, 0.14 mmol, 1.5 equiv.), 4-bromobenzenethiol (26.8 mg, 0.14 mmol, 1.5 equiv.), and in MeCN (0.95 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM-MeOH gradient (100:0 to 90:10), desired product was isolated as a light-yellow foamy solid (114.0 mg, 97%).

[0312] 31P NMR (202 MHz, DMSO) δ -1.08.

[0313] 1H NMR (500 MHz, DMSO) δ 8.39 (s, 1H), 8.28 (s, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.36 – 7.20 (m, 22H), 7.10 (d, J = 7.4 Hz, 2H), 6.03 (d, J = 6.4 Hz, 1H), 6.01 (d, J = 7.7 Hz, 1H), 5.93 (d, J = 4.5 Hz, 1H), 5.49 (br s, 2H), 4.89 (br s, 2H), 4.84 (s, 2H), 4.77 (br s, 1H), 4.67 (t, J = 5.8 Hz, 1H), 4.60 – 4.51 (m, 2H), 4.35 (br s, 1H), 4.29 (t, J = 4.8 Hz, 1H), 4.06 – 3.98 (m, 1H), 3.98 – 3.92 (m, 1H), 3.91 – 3.85 (m, 1H), 3.83 (t, J = 4.8 Hz, 1H), 3.47 (dd, J = 13.9, 4.1 Hz,1H), 3.38 (m, 1H, assumed; partially obscured by solvent peak), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 3.30 (s, 3H, assumed; partially obscured by solvent peak), 0.85 (s, 9H), 0.06 (s, 6H).

[0314] 13C NMR (126 MHz, DMSO) δ 163.64, 154.39, 154.19, 152.25, 150.68, 142.70, 138.87, 137.37, 136.71, 135.87, 131.57, 129.83, 128.68, 128.54, 128.45, 127.54, 127.37, 127.24, 127.11, 126.35, 119.19, 118.42, 91.94, 85.26, 82.76, 82.52, 80.28, 80.24, 73.49, 70.48, 68.51, 64.21, 57.49, 55.85, 50.51, 49.60, 45.43, 35.28, 32.12, 30.70, 29.61, 25.65, 21.15, 17.79, -4.81, - 4.85.

[0315] HR-MS (Q-TOF, ESI) calculated for C61H7079BrN8O10PSSi [M+H+]: 1245.3699, found 1245.3701. p. (2S,3S,4R,5R)-5-(6-(Benzyloxy)-2-(dibenzylamino)-9H-purin-9-yl)-2-(((4- bromobenzyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl (((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) phosphatesynthesized by following general procedure 12 (GP12) in Example 7, using 1-((2R,3R,4R,5R)-5-((((2R,4aR,6R,7R,7aR)-6-(6-(benzyloxy)-2- (dibenzylamino)-9H-purin-9-yl)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3- methoxytetrahydrofuran-2-yl)-3-((benzyloxy)methyl)pyrimidine-2,4(1H,3H)-dione (G-Bn3-PO- U-BOM, 100.0 mg, 0.091 mmol, 1 equiv.), DBU (20.7 mg, 0.14 mmol, 1.5 equiv.), (4- bromophenyl)methanethiol (27.6 mg, 0.14 mmol, 1.5 equiv.), and in MeCN (0.91 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving inminimum amount of DCM, DCM-MeOH gradient (100:0 to 90:10), desired product was isolated as a light-yellow foamy solid (96.5 mg, 82%).

[0317] 31P NMR (202 MHz, DMSO) δ -1.93.

[0318] 1H NMR (500 MHz, DMSO) δ 8.40 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.34 – 7.15 (m, 20H), 6.94 – 6.88 (m, 2H), 5.95 (d, J = 6.2 Hz, 1H), 5.89 (d, J = 5.3 Hz, 1H), 5.81 (d, J = 8.1 Hz, 1H), 5.43 (d, J = 16.0 Hz, 1H), 5.39 (d, J = 15.8 Hz, 1H), 5.31 (d, J = 9.9 Hz, 1H), 5.29 (d, J = 9.8 Hz, 1H), 4.57 (s, 2H), 4.53 – 4.46 (m, 2H), 4.25 (app s, 5H), 4.10 – 4.07 (m, 1H), 4.04 – 3.97 (m, 2H), 3.94 – 3.88 (m, 1H), 3.79 (d, J = 13.2 Hz, 1H), 3.74 (d, J = 13.2 Hz, 1H), 3.36 (s, 3H, assumed; partially obscured by solvent peak), 3.10 (s, 3H), 2.87 (dd, J = 14.3, 4.2 Hz, 1H), 2.74 (dd, J = 14.2, 7.4 Hz, 1H), 0.90 (s, 9H), 0.11 (s, 6H).

[0319] 13C NMR (126 MHz, DMSO) δ 161.77, 157.43, 156.54, 150.79, 146.55, 139.82, 138.44, 138.02, 137.97, 137.02, 136.73, 131.22, 131.17, 128.30, 128.26, 128.20, 127.49, 127.35, 127.31, 126.82, 126.05, 120.22, 119.88, 101.60, 87.56, 85.11, 84.81, 82.96, 82.52, 80.28, 73.06, 71.10, 71.06, 70.11, 68.51, 64.39, 57.58, 57.40, 55.85, 55.32, 47.13, 35.01, 33.02, 32.12, 29.61, 25.65, 17.83, -4.71, -4.83.

[0320] HR-MS (Q-TOF, ESI) calculated for C63H7379BrN7O13PSSi [M+H+]: 1306.3750, found 1306.3753. q. (2S,3S,4R,5R)-5-(6-(Benzyloxy)-2-(dibenzylamino)-9H-purin-9-yl)-2-((((R)-2-((tert- butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)thio)methyl)-4-methoxytetrahydrofuran-3- yl (((2R,3R,4R,5R)-5-(6-(benzyloxy)-2-(dibenzylamino)-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) phosphate

[0321] The compound was synthesized by following general procedure 12 (GP12) in Example 7, using (2R,4aR,6R,7R,7aR)-6-(6-(benzyloxy)-2-(dibenzylamino)-9H-purin-9-yl)-2- (((2R,3R,4R,5R)-5-(6-(benzyloxy)-2-(dibenzylamino)-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxytetrahydro-4H- furo[3,2-d][1,3,2]dioxaphosphinine 2-oxide (G-Bn3-PO-G-Bn3, 100.0 mg, 0.077 mmol, 1 equiv.), DBU (17.7 mg, 0.12 mmol, 1.5 equiv.), methyl (tert-butoxycarbonyl)-L-cysteinate (Boc- Cys-OMe, 27.3 mg, 0.12 mmol, 1.5 equiv.), and in MeCN (0.77 mL). Column conditions: SiO2- 25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, DCM-MeOH gradient (100:0 to 90:10), desired product was isolated as a light yellow foamy solid (102.0 mg, 86%).

[0322] 31P NMR (202 MHz, DMSO) δ -1.89.

[0323] 1H NMR (500 MHz, DMSO) δ 8.41 (s, 1H), 8.18 (s, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.27 – 7.14 (m, 22H), 7.14 – 7.09 (m, 4H), 6.95 (d, J = 6.1 Hz, 2H), 6.91 – 6.87 (m, 2H), 5.97 – 5.93 (m, 1H), 5.91 – 5.86 (m, 1H), 5.50 – 5.34 (m, 4H), 4.69 (br s, 1H), 4.64 – 4.47 (m, 2H), 4.43 – 4.37 (m, 1H), 4.34 (br s, 1H), 4.31 – 4.18 (m, 9H), 4.18 – 4.12 (m, 1H), 4.10 (br s, 1H), 4.05 – 3.99 (m, 1H), 3.95 (br s, 1H), 3.51 (s, 1H), 3.49 (s, 1H), 3.37 (s, 3H, assumed; partially obscured by solvent peak), 3.36 (s, 3H, assumed; partially obscured by solvent peak), 3.23 – 3.17 (m, 3H), 3.12 (s, 3H), 3.02 – 2.72 (m, 4H), 1.37 – 1.25 (m, 9H, Boc rotamers), 0.91 (s, 9H), 0.13 (s, 6H).

[0324] 13C NMR (126 MHz, DMSO) δ 171.62, 171.56, 157.41, 156.73, 156.69, 156.52, 155.44, 155.33, 146.57, 146.40, 146.36, 139.02, 138.95, 138.46, 137.01, 136.75, 136.63, 128.35, 128.28, 128.27, 128.13, 128.08, 127.30, 126.83, 126.81, 126.02, 125.96, 120.89, 120.86, 120.22, 85.78, 85.16, 84.74, 84.67, 84.41, 82.50, 80.36, 78.31, 73.45, 71.10, 68.52, 64.37, 57.41, 55.85, 55.36, 55.14, 53.72, 53.44, 51.88, 51.80, 47.23, 47.08, 34.45, 33.78, 33.48, 32.89, 32.13, 29.61, 28.12, 27.85, 25.66, 17.84, -4.69, -4.83.

[0325] HR-MS (Q-TOF, ESI) calculated for C79H94N11O15PSSi [M+H+]: 1528.6231, found 1528.6230.r ((2R3R4R5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purin-9-yl)-4-Example 7, using (2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6- (dibenzylamino)-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)-6-(6- (dibenzylamino)-9H-purin-9-yl)-7-methoxytetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinine 2- oxide (A-Bn2-PO-A-Bn2, 100.0 mg, 0.092 mmol, 1 equiv.), DBU (84.5 mg, 0.55 mmol, 6 equiv.), 4-mercaptobutanoic acid (33.3 mg, 0.28 mmol, 3 equiv.), and in MeCN (0.93 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O-AcOH gradient (100:0:0:0 to 84:10:5:1), desired product was isolated as a white foamy solid (96.9 mg, 87%).

[0327] 31P NMR (202 MHz, DMSO) δ -2.23.

[0328] 1H NMR (500 MHz, DMSO) δ 12.35 (br s, 1H), 8.65 (s, 1H), 8.40 (s, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 7.32 – 7.19 (m, 20H), 6.11 (d, J = 6.6 Hz, 1H), 6.05 (d, J = 6.5 Hz, 1H), 5.48 (br s, 4H), 4.88 (br s, 4H), 4.77 (br s, 1H), 4.68 (t, J = 5.7 Hz, 1H), 4.61 (s, 1H), 4.54 (t, J = 5.6 Hz, 1H), 4.32 (br s, 1H), 4.14 – 4.10 (m, 1H), 4.00 (br s, 1H), 3.95 (br s, 1H), 3.32 (s, 3H, assumed; partially obscured by solvent peak), 3.28 (s, 3H, assumed; partially obscured by solvent peak), 2.93 (dd, J = 13.8, 5.4 Hz, 1H), 2.85 (dd, J = 14.2, 7.4 Hz, 1H), 2.50 – 2.43 (m, 2H, assumed; partially obscured by solvent peak), 2.33 – 2.23 (m, 1H), 2.23 – 2.15 (m, 1H), 1.73 – 1.63 (m, 2H), 0.90 (s, 9H), 0.86 – 0.79 (m, 1H), 0.12 (s, 6H).

[0329] 13C NMR (126 MHz, DMSO) δ 172.55, 154.66, 154.62, 152.70, 151.33, 151.20, 139.37, 139.30, 138.38, 138.04, 129.00, 128.97, 127.84, 127.53, 119.66, 119.41, 85.81, 85.71,85.30, 84.41, 82.81, 80.62, 73.82, 71.57, 68.98, 65.07, 58.02, 57.89, 56.30, 51.22, 49.07, 34.13, 33.33, 32.58, 31.70, 30.07, 26.14, 25.11, 21.60, 18.29, -4.28, -4.41.

[0330] HR-MS (Q-TOF, ESI) calculated for C60H73N10O11PSSi [M+H+]: 1201.4761, found 1201.4766. s. ((2R,3R,4R,5R)-5-(6-(Benzyloxy)-2-(dibenzylamino)-9H-purin-9-yl)-3-((tert-Example 7, using 1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(6-(benzyloxy)-2- (dibenzylamino)-9H-purin-9-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2- yl)methoxy)-7-methoxy-2-oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)-3- ((benzyloxy)methyl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PO-G-Bn3, 100.0 mg, 0.091 mmol, 1 equiv.), LiOtBu (11.6 mg, 0.14 mmol, 1.6 equiv.), O,O-diethyl S-hydrogen phosphorodithioate (27.0 mg, 0.14 mmol, 1.6 equiv.), and in PhMe (0.91 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, EA-MeOH-H2O gradient (100:0:0 to 85:10:5), desired product was isolated as an off-white foamy solid (99.2 mg, 85%).

[0332] 31P NMR (202 MHz, DMSO) δ 92.62, -1.53.

[0333] 1H NMR (500 MHz, DMSO) δ 8.20 (s, 1H), 8.14 (d, J = 8.2 Hz, 1H), 7.36 – 7.17 (m, 14H), 7.17 – 7.13 (m, 4H), 6.97 – 6.92 (m, 2H), 5.91 (d, J = 5.2 Hz, 1H), 5.89 (d, J = 5.9 Hz, 1H), 5.73 (d, J = 7.9 Hz, 1H), 5.46 (d, J = 15.9 Hz, 1H), 5.41 (d, J = 15.9 Hz, 1H), 5.32 (d, J = 9.8 Hz, 1H), 5.29 (d, J = 9.8 Hz, 1H), 4.65 (br s, 1H), 4.57 (s, 2H), 4.49 – 4.43 (m, 1H), 4.36 (t, J= 4.2 Hz, 1H), 4.32 – 4.27 (m, 1H), 4.26 (s, 3H), 4.10 – 3.97 (m, 6H), 3.95 – 3.88 (m, 2H), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 3.32 – 3.29 (m, 1H), 3.27 (s, 3H), 3.23 – 3.16 (m, 1H), 1.18 (m, 6H), 0.87 (s, 9H), 0.09 (s, 6H).

[0334] 13C NMR (126 MHz, DMSO) δ 161.87, 157.33, 156.87, 150.92, 146.42, 140.15, 139.02, 138.03, 137.01, 136.63, 128.36, 128.27, 128.23, 128.19, 127.48, 127.36, 127.33, 126.85, 125.99, 120.87, 101.40, 86.95, 85.79, 84.45, 82.61, 82.01, 80.39, 80.35, 73.40, 71.06, 70.36, 70.06, 63.86, 63.73, 63.71, 63.69, 63.66, 57.59, 55.15, 47.19, 35.27, 35.24, 25.65, 17.78, 15.54, 15.51, 15.48, 15.45, -4.78, -4.82.

[0335] HR-MS (Q-TOF, ESI) calculated for C60H77N7O15P2S2Si [M+H+]: 1290.4236, found 1290.4234. Example 9. Synthesis of cyclic-phosphorothioate nucleosides a. Synthesis of Cl-cyclic phosphorothioate with PSCl3nucleoside diol BOM-U (1 mmol, 1.0 eq) in DCM (5 mL) was prepared. The solution was charged with a 2 mL solution of lithium tert-butoxide in 1 M THF (2.05 mmol, 2.05 eq) over a period of 5 minutes under Schlenk condition at 0 °C. After the addition, the mixture was warmed to ambient temperature overnight. An aliquot of crude product mixture was concentrated to monitor by LCMS and NMR for conversion, which observed two diastereomers P1 and P2 by P NMR with dr = 1.7:1 to 1.3:1.

[0337] NMR data of the two isomers:31P NMR (162 MHz, CDCl3) δ 61.23 (P1), 57.83 (P2). 1H NMR (400 MHz, CDCl3) P1, δ 7.40 – 7.27 (m, 5H), 7.11 (dd, J = 8.1, 0.8 Hz, 1H), 5.78 (dd, J = 8.2, 0.8 Hz, 1H), 5.46 (d, J = 1.0 Hz, 2H), 5.42 (s, 1H), 4.97 – 4.88 (m, 1H), 4.86 – 4.66 (m, 2H), 4.71 (s, 2H), 4.64 – 4.46 (m, 1H), 4.22 – 4.13 (m, 1H), 3.61 (d, J = 0.8 Hz, 3H).1H NMR (400 MHz, CDCl3) P2, δ 7.42 – 7.30 (m, 5H), 7.18 – 7.09 (m, 1H), 5.82 (d, J = 8.1 Hz, 1H), 5.49(d, J = 3.5 Hz, 2H), 5.40 – 5.35 (m, 1H), 4.86 (dt, J = 9.9, 5.0 Hz, 1H), 4.74 (s, 2H), 4.71 – 4.62 (m, 1H), 4.53 (ddd, J = 10.8, 9.6, 3.0 Hz, 1H), 4.27 (dd, J = 8.8, 4.9 Hz, 2H), 3.62 (s, 3H).

[0338] Purification method (a): Crude product was precipitated with 0.1 mL MTBE at 0 °C while stirring. The filtrate was concentrated and vacuumed for NMR and analysis.

[0339] Purification method (b): Crude product was concentrated by rotavap and then purified by wet loading (dissolved in DCM) to silica gel column EA / Hex 0-100% to give two diastereomers P1 and P2 with 40% combined isolation yield.

[0340] Reaction conditions and Cr d.r. results are shown in Table 8 [Cr dr is P(R):P(S) by P NMR of crude reaction. 4a and 4b are two time points of same batch. Reaction 1 used purification method (a). Reaction 2-4 used purification method (b)]. Table 8 - Synthesis of Cl-cyclic phosphorothioateProcedure 2b. Synthesis of Cl-cyclic-2'-methoxy-G-phosphorothioate (1-benzyl-9-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)-2-(dibenzylamino)-1,9-dihydro-6H-purin-6-one)

[0343] Under schlenk line N2, 1-benzyl-2-(dibenzylamino)-9-((2R,3R,4R,5R)-4-hydroxy-5- (hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (5.677 g, 1 Eq, 10.00 mmol) dissolved in DCM (50 mL) at RT then slowly cooled to -20 °C over 90 minutes, then freshly distilled phosphorothioyl trichloride (1.778 g, 1.06 mL, 1.05 Eq, 10.50 mmol) was added by syringe needle dropwise, at 1:50pm followed by addition of THF (9.3 mL)2- methylpropan-2-ol, lithium salt (2.2 M in THF) (1.662 g, 2.05 Eq, 20.50 mmol) by syringe needle over 10min. after 30 min, ~50 uL crude sample was collected by syringe for analysis, crude PNMR show two main product isomer peaks dr ~ 2.4:1. Starting material was mostly consumed after 30 minutes (with 1% remained detected on UV, integration determined by uncorrected LCMS) and other small peaks of impurities earlier eluted than product. Reaction was stopped after 1 h. Crude reaction solution was concentrated to remove most of solvent before redissolved in DCM then loaded on silica gel column (40g), which eluted with EA / Hex (0-80%) by combi-flash system, affording 3.56g product as light beige to white solid (isolated yield 54%, dr 2.2:1).

[0344] 13C NMR (101 MHz, CDCl3) δ 157.50, 145.65, 138.96, 136.14, 135.61, 135.50, 128.78, 128.76, 128.73, 128.68, 128.21, 128.02, 127.91, 127.73, 127.46, 126.45, 126.39, 91.10, 90.81, 79.86, 78.80, 78.73, 77.36, 77.25, 77.04, 76.73, 71.73, 71.61, 69.27, 69.13, 59.64, 59.62, 55.59, 55.50, 48.29c. Synthesis of alkoxyl-cyclic phosphorothioate via PSORCl2 75.7 mg, 1 eq, 0.2 mmol) and DMAP (63.5 mg,2.6 eq, 520 μmol) in 1.8 mL DCM (2 mL) were added O-ethyl phosphorodichloridothioate (37.6 mg, 1.05 eq, 210 μmol) in 0.2 mL DCM slowly under schlenk line at -20 °C stirring for 20 hour with the aid of a cryocooler. The reaction was raised to room temperature and stirred for 3 hours. Crude product was concentrated and redissolved in 0.5 mL DCM for loading onto a 5g biotage silica gel column, then purified with elute EA / Hex 0-100%, and eluted at ~30% EA / Hex to give two diastereomers P1 and P2 (dr of P1:P2 = 2:1) with isolate yield 40%.

[0346] NMR data of the two isomers:31P NMR (162 MHz, CDCl3) δ 66.52 (P1), 61.80 (P2).1H NMR (400 MHz, CDCl3) P1, δ 7.42 – 7.27 (m, 6H), 7.18 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 8.2 Hz, 1H), 5.69 (t, J = 0.9 Hz, 1H), 5.51 (s, 2H), 4.74 (s, 2H), 4.69 – 4.47 (m, 3H), 4.44 – 4.24 (m, 3H), 4.01 (d, J = 4.9 Hz, 1H), 3.62 (s, 3H).Example 10. Synthesis of cyclic thioate dinucleotides via cyclic chlorophosphorothioate a. Cyc U*U (Procedure 1)que, a 20 mL oven dried reaction tube A equipped with a magnetic stir bar, was charged with LiOtBu (48.7 mg, 1.5 eq, 609 μmol). The stock of solution of 3'-TBS-5'-OH nucleoside (200 mg, 1 eq, 406 μmol) in toluene (2 mL) was added to the reaction tube A, put and kept it at -20oC with aid of a cryocooler, stirred for 30 minutes.

[0348] Solution B: Cyclic chlorophosphorothioate monomer PS-Cl UBOM (dr Rp:Sp=1.2:1, 231 mg, 1.0 eq, 487 μmol) was dissolved in acetonitrile (2 mL) in an oven dried flask at -20 °C with cyrocooler under Schlenk-line condition. solution A was added into B over 5 minutes by syringe needle dropwise, stirred for 20 hour. Aliquot of 50 uL was collected by syringe and concentrated for31P NMR and LCMS analysis to confirm reaction completion and dr (Rp:Sp = 13:1) of crude reaction. Crude reaction solution was concentrated to remove most of solvent before loading on silica gel column (5 g), which eluted with EA / Hex (0-60%) by combi-flash system, affording 150 mg major isomer "Rp" as white solid (isolated yield 40%, dr 16:1).

[0349] 31P NMR (162 MHz, CDCl3) δ 66.20 (Rp, less polar), 62.00 (Sp, more polar).

[0350] 1H NMR (400 MHz, CDCl3) δ 7.73 (dd, J = 8.2, 5.5 Hz, 1H), 7.44 – 7.25 (m, 10H), 7.13 (s, 1H), 5.93 (d, J = 1.8 Hz, 1H), 5.90 – 5.78 (m, 2H), 5.64 – 5.44 (m, 5H), 4.74 (d, J = 1.5 Hz, 4H), 4.71 – 4.50 (m, 4H), 4.47 – 4.38 (m, 1H), 4.30 – 4.06 (m, 5H), 3.68 (q, J = 2.0 Hz, 1H), 3.64 – 3.51 (m, 6H), 0.93 (d, J = 2.7 Hz, 9H), 0.17 – 0.09 (m, 6H).

[0351] 13C NMR (101 MHz, CDCl3) δ 171.16, 162.62, 162.08, 150.86, 150.28, 138.70, 138.21, 137.86, 128.38, 128.35, 127.85, 127.75, 127.73, 127.62, 102.80, 101.88, 94.29, 88.80, 83.39, 81.34, 81.24, 80.43, 80.34, 77.35, 77.03, 76.71, 72.54, 72.31, 71.21, 71.15, 70.47, 70.26, 69.08, 69.00, 66.40, 66.35, 60.41, 59.24, 58.43, 25.68, 21.07, 18.08, 14.22, -4.61, -4.86.

[0352] HRMS (ESI+): m / z calcd. for C42H65N4O14PSSi[M+H]+: 930.29; found 930.31 (Rp), 930.33 (Sp). b. Cyc U*U (Procedure 2)L reaction flask A equipped with a magnetic stir bar, was charged with LiOtBu (96.1 mg, 1.5 Eq, 1.20 mmol) cooled to -20oC with aid of a cryocooler. The stock of solution of 3'-TBS-5'-OH nucleoside (394 mg, 1 Eq, 800 μmol) in toluene (4 mL) was added to the reaction tube A via syringe needle slowly, stirred for 30 minutes at -20oC with aid of a cryocooler, stirred for 30 minutes.

[0354] Solution B: Cyclic chlorophosphorothioate monomer PS-Cl UBOM (dr Rp:Sp=1:1, 500 mg, 70% Wt, 0.921 Eq, 737 μmol) was dissolved in acetonitrile (4 mL) at -20 °C with cyrocooler under Schlenk-line condition. solution A was added into B over 5 minutes by syringe needle dropwise, stirred for 20 hour, 50 uL was concentrated to test P NMR and LCMS to confirm reaction completion and product dr (Rp:Sp = 27:1) of crude reaction.

[0355] Reactant cyclic chloro-phosphorothioate monomer PS-Cl UBOM used in procedure 2 was not purified by silica gel column chromatography. The synthesis and quenching method is as follows.

[0356] Under schlenk line N2, diol-U-BOM (3.784 g, 1 Eq, 10.00 mmol) and phosphorothioyl trichloride (1.778 g, 1.06 mL, 1.05 Eq, 10.50 mmol) dissolved in DCM (48 mL) at 0 °C stirred for 20 minutes, followed by addition of THF (20 mL)lithium 2- methylpropan-2-olate (1 M THF solution) (1.601 g, 2.0 Eq, 20.00 mmol) by syringe and needle, warm from 0 °C to 23 °C over 4 hours. Reaction completed and was concentrated by rotovap. The condensed crude product solution was slowly added mixture of Heptane / MTBE / EA = 50 mL / 50 mL / 30 mL to partially precipitate salt impurity and other insoluble byproduct. The filtrate solution was concentrated and vacuumed to give light brown solid 5.6 g. Based on theoretical yield 4.75 g and actual mass 5.6 g after precipitation, there could be ~0.80 g mixture of LiOH, LiOtBu and LiCl = 10-15 mmol ~ 1-1.5 eq. 10 mmol calculated based on MW 80 of LiOtBu. c. Two-step addition (General)reaction tube A equipped with a magnetic stir bar, was charged with LiOtBu (1.5 eq). The stock of solution of 3'-TBS-5'-OH nucleoside (1 eq) in toluene (0.2 M) was added to the reaction tube A, put and kept it at -20oC with aid of a cryocooler, stirred for 30 minutes.

[0358] Solution B: Cyclic chlorophosphorothioate monomer (dr Rp:Sp~1:1, 1.0 eq) was dissolved in acetonitrile (0.2 M) in an oven dried flask at -20 °C with cyrocooler under Schlenk- line condition. Solution A was added into B over 5 minutes by syringe needle dropwise, stirred for 20 hours. Aliquot of 50 uL was collected by syringe and concentrated for31P NMR and LCMS analysis to confirm reaction completion and dr of crude reaction. Crude reaction solution was concentrated to remove most of solvent and dissolved in minimum DCM before loading on silica gel column, which eluted with EA / Hex (0-60%) by combi-flash system, affording major isomer "Rp" as white solid, dr ~ 4:1 to 20:1.ol) were mixed in a heat gun vacuum dried glass vessel at room temp followed by fully dissolving in anhydrous DCM (0.1 M) then cooled to -20C under N2 with aid of cryocooler. Lithium 2- methylpropan-2-olate (2.2 M in THF) (1.5 Eq) was added into the reaction vessel by syringe needle dropwise over 5min. Then kept the temperature and stirred for 1-20 hours under N2 until reaction completed. LCMS was used to monitor conversion. After 1 h, Aliquot of 50 uL was collected by syringe and concentrated for31P NMR and LCMS analysis to confirm reaction completion as 82% crude yield and cr dr (Rp:Sp) of crude reaction. Crude reaction solution was concentrated to remove most of solvent and dissolved in minimum DCM before loading on silica gel column, which eluted with EA / Hex (0-60%) by combi-flash system, affording 90 mg major isomer "Rp" as white solid (isolated yield 82%, dr 10:1).

[0360] The one-pot two-step addition is scalable from milligram scale to gram scale, with good conversion and moderate-to-high diastereoselectivity.Table 10 – One-pot addition

[0361] B15'-OH-3'TBS mA ((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6- (dibenzylamino)-9H-purin-9-yl)-4-methoxytetrahydrofuran-2-yl)methanol (57.6 mg, 1 Eq, 100 μmol) and B2 Cl-PS-cyclic mU monomer 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2- chloro-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine- 2,4(1H,3H)-dione (57.0 mg, 1.2 Eq, 120 μmol) were mixed in a heat gun vacuum dried glass vessel at room temp followed by fully dissolving in anhydrous DCM (2 mL) then cooled to -20 oC under N2 with aid of cryocooler. Lithium 2-methylpropan-2-olate (2.2 M in THF) (12.0 mg, 68 μL, 1.5 Eq, 150 μmol) was added into the reaction vessel by syringe needle dropwise over 5min. Then kept the temperature and stirred for 2 hrs under N2until reaction completed. LCMS was used to monitor conversion. After 1 h, Aliquot of 50 uL was collected by syringe and concentrated for 31P NMR and LCMS analysis to confirm reaction completion as 82% crude yield and cr dr (Rp:Sp = 10:1) of crude reaction. Crude reaction solution was concentrated to remove most of solvent and dissolved in minimum DCM before loading on silica gel column (5g), which eluted with EA / Hex (0-60%) by combi-flash system, affording 90 mg major isomer "Rp" as white solid (isolated yield 82%, dr 10:1).31P NMR (162 MHz, CDCl3) Rp 66.40, Sp isomer not confirmed due to trace amount detected may get confirmation after scale-up.Example 11. Spectra of dinucleotides a. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert- butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purin-9-yl)-4-methoxytetrahydrofuran-2- yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6- yl)pyrimidine-2,4(1H,3H)-dione :1

[0363] 31P NMR (162 MHz, CDCl3) δ 66.27 (Rp)

[0364] 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.04 (s, 1H), 7.37 (t, J = 5.6 Hz, 4H), 7.35 – 7.27 (m, 32H), 6.16 (d, J = 4.2 Hz, 2H), 5.75 (d, J = 8.1 Hz, 2H), 5.57 (s, 2H), 5.47 (s, 3H), 4.76 – 4.64 (m, 6H), 4.49 – 4.38 (m, 5H), 4.32 – 4.24 (m, 3H), 4.06 (d, J = 4.9 Hz, 2H), 3.64 – 3.50 (m, 8H), 2.07 (s, 2H), 0.98 (s, 9H), 0.19 (t, J = 1.2 Hz, 6H). b. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((2S,4aR,6R,7R,7aR)-6-(6-(dibenzylamino)-9H-purin-9-yl)-7-methoxy-2- sulfidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-3- methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione,

[0366] 31P NMR (162 MHz, CDCl3) δ 65.97 (Rp),e. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-fluorotetrahydrofuran-2-yl)methoxy)-7-methoxy-2- sulfidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (6-MHz, CDCl3) δ 8.43 (s, 1H), 8.01 (s, 1H), 7.58 (s, 1H), 7.36 – 7.19 (m, 21H), 7.13 (dt, J = 7.3, 2.9 Hz, 6H), 6.18 (d, J = 4.0 Hz, 1H), 5.67 (d, J = 15.5 Hz, 1H), 5.61 (s, 2H), 5.32 (s, 1H), 5.23 (ddd, J = 9.5, 5.1, 2.8 Hz, 1H), 4.72 – 4.60 (m, 2H), 4.50 – 4.42 (m, 2H), 4.42 – 4.37 (m, 3H), 4.34 – 4.10 (m, 6H), 3.94 (d, J = 5.1 Hz, 1H), 3.54 (s, 3H), 3.35 (s, 3H), 0.98 (s, 9H), 0.20 (s, 6H).

[0371] 13C NMR (100 MHz, CDCl3) δ 158.20, 157.51, 154.96, 152.72, 150.71, 145.82, 138.93, 137.71, 137.39, 136.23, 136.14, 136.05, 128.66, 128.60, 128.01, 127.87, 127.82, 127.55, 127.35, 127.33, 126.42, 126.36, 122.31, 120.20, 91.02, 87.12, 82.65, 82.41, 82.32, 80.17, 80.08, 77.97, 77.92, 77.37, 77.25, 77.05, 76.73, 70.88, 70.82, 70.43, 69.27, 69.19, 67.42, 59.30, 58.62, 56.06, 55.77, 53.45, 48.11, 25.78, 25.78, 25.78, 18.17, -4.64, -4.76.. , , . , , . . , , . , . , . , , . . , 5H), 7.05 (dd, J = 6.6, 3.0 Hz, 1H), 5.93 (d, J = 1.6 Hz, 1H), 5.82 (d, J = 8.2 Hz, 1H), 5.68 (d, J = 14.2 Hz, 2H), 5.62 (s, 1H), 5.60 – 5.45 (m, 2H), 5.32 (s, 2H), 5.31 – 5.26 (m, 1H), 4.73 (s, 2H), 4.64 (dd, J = 11.4, 6.6 Hz, 1H), 4.52 (ddd, J = 22.9, 9.7, 4.9 Hz, 1H), 4.44 (s, 1H), 4.40 (s, 2H), 4.38 – 4.31 (m, 1H), 4.27 (d, J = 14.9 Hz, 3H), 4.24 – 4.10 (m, 5H), 4.01 (d, J = 5.2 Hz, 1H), 3.68 (q, J = 1.7 Hz, 1H), 3.61 (s, 3H), 3.46 (s, 1H), 3.40 (s, 2H), 0.94 (s, 9H), 0.15 (d, J = 2.1 Hz, 6H). h.1-benzyl-9-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(1-benzyl-2-(dibenzylamino)-6-Example 12. Ring-opening of cyclic thioate dinucleotide with nucleophiles OBOMbromophenyl)methanethiol (60.9 mg, 1.5 eq, 300 μmol) in 1 ml ACN and added mTBD (1- methyl-2,3,4,6,7,8-hexahydro-1H-pyrimido[1,2-a]pyrimidine) (45.9 mg, 43.0 μL, 1.5 eq, 300 μmol) in 1 mL ACN, stirred for 20min under N2 at 23 °C.

[0375] Solution B: 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (186 mg, 1 Eq, 200 μmol).

[0376] Solution A was slowly added to B by syringe needle over 5 minutes at RT. After 1 hour, approximately 71% conversion to desired product was achieved, which was monitored by LCMS. Reaction completed overnight. Crude yield 86% by uncorrected LCMS. Ring open product diastereomer ratio was determined by crude 31P NMR and crude LCMS (crude Rp / Sp = 5:1).31P NMR (162 MHz, CDCl3) δ 56.28 (Rp), 56.21(Sp). Procedure 2added mTBD (5.9 mg, 5.5 μL, 1.7 eq, 39 μmol) in DMF (0.3 mL, ACN, 0.3 mL), stirred for 5 min under N2 at 50oC. Solution B: 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2- (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2- sulfidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (21 mg, 1 eq, 23 μmol) in DMF (0.3 mL, ACN, 0.3 mL) heat to 50 °C, reaction finished in 3 h, crude yield 60% by uncorrected LCMS. Ring open product diastereomer ratio (Rp:Sp = 11:1) was determined by crude LCMS and 31P NMR (162 MHz, CDCl3) δ 53.92, 92.02 (Rp); 54.13, 92.11 (Sp).Procedure 3μmol) and added mTBD (5.9 mg, 5.5 μL, 1.7 eq, 38 μmol) in DMF (0.3 mL), stirred for 5 min under N2. Solution B: 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (21 mg, 1 eq, 23 μmol) in DMF (0.3 mL) heat to 50 °C, solution A was dropwise added to solution B under N2, 90% conversion of starting material after 4 h. crude yield 53% determined by uncorrected LCMS. Ring open product diastereomer ratio (Rp:Sp = 5.5:1) was determined by crude 31P NMR (162 MHz, CDCl3) δ 56.72, 26.97 (Rp); 56.49, 26.64 (Sp). Procedure 4d added DBU (26 mg, 26 μL, 1.7 eq, 0.17 mmol)in ACN (1 mL), stirred for 10 min under N2 at rt. Solution B: 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (93 mg, 1 eq, 0.10 mmol) in ACN (1 mL), solution A was dropwise added to solution B under N2, after overnight reaction, crudeyield 60% by uncorrected LCMS. Ring open product diastereomer ratio (Rp:Sp = 7:1) was determined by crude 31P NMR.31P NMR (162 MHz, CDCl3) δ 57.37 (Rp), 57.08 (Sp). Procedure 5bromophenyl)methanethiol (14 mg, 1.4 eq, 69 μmol)in ACN (0.5 mL) and added mTBD (11 mg, 10 μL, 1.5 eq, 72 μmol), stirred for 20min under N2 at 23 °C. Solution B: 1-((2R,3R,4R,5R)-5- ((((2S,4aR,6R,7R,7aR)-6-(1-benzyl-2-(dibenzylamino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-7- methoxy-2-sulfidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-yl)oxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)-3-((benzyloxy)methyl)pyrimidine- 2,4(1H,3H)-dione (54 mg, 1 eq, 48 μmol) was dissolved in ACN (0.5 mL).0.4ml of Solution A was slowly added to solution B by syringe needle over 5 min at rt under N2. After reaction stirred overnight, 4-BrBnSH (8mg 0.8eq) and mTBD (8 uL 1.2 eq) was dissolved in 0.2 mL ACN under N2 stirred for 20min before adding into reaction mixture by syringe needle. After 3.5 hours, remaining starting material was mostly consumed, crude yield was 40% by uncorrected LCMS. Ring open product diastereomer ratio was determined by crude 31P NMR and LCMS (Rp / Sp = 27:2).31P NMR (162 MHz, CDCl3) δ 56.29 (Rp), 53.93 (Sp).Example 13. Synthesis of specific 5'C modified dinucleotides a. O-((2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (((4-bromobenzyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl) O-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) S-hydrogen phosphorothioatesynthesized by following general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PS-U-BOM (d / r = 4:1), 24.0 mg, 0.026 mmol, 1 equiv.), DBU (5.9 mg, 0.039 mmol, 1.5 equiv.), (4- bromophenyl)methanethiol (7.9 mg, 0.039 mmol, 1.5 equiv.), and in MeCN (0.26 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA-MeOH gradient (100:0:0 to 0:90:10), desired product was isolated as an off-white solid (major diastereomer, 21.5 mg, 74%).

[0382] 31P NMR (202 MHz, DMSO) δ 53.78.

[0383] 1H NMR (500 MHz, DMSO) δ 8.17 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.36 – 7.22 (m, 12H), 5.90 (d, J = 5.2 Hz, 1H), 5.88 (d, J = 5.2 Hz, 1H), 5.80 (d, J = 8.1 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.34 – 5.26 (m, 4H), 4.74 – 4.66 (m, 1H), 4.58 (d, J = 4.9 Hz, 4H), 4.38 (t, J = 4.2 Hz, 1H), 4.33 – 4.28 (m, 1H), 4.07 – 4.00 (m, 3H), 3.99 – 3.93 (m, 2H), 3.82 (d, J = 13.2 Hz, 1H), 3.77 (d, J = 13.1 Hz, 1H), 3.37 (s, 3H, assumed;partially obscured by solvent peak), 3.33 (s, 3H, assumed; partially obscured by solvent peak), 2.92 (dd, J = 14.3, 3.8 Hz, 1H), 2.78 (dd, J = 14.3, 7.6 Hz, 1H), 0.87 (s, 9H), 0.10 (s, 3H), 0.10 (s, 3H).

[0384] 13C NMR (126 MHz, DMSO) δ 161.89, 161.75, 150.94, 150.78, 140.21, 139.77, 138.07, 138.03, 131.24, 128.21, 127.49, 127.39, 127.36, 119.89, 101.58, 101.41, 87.70, 86.97, 84.19, 84.14, 82.78, 82.76, 82.07, 80.02, 79.98, 73.11, 71.10, 71.08, 70.42, 70.08, 63.57, 57.65, 57.57, 55.84, 34.99, 32.91, 29.61, 25.67, 17.79, -4.78, -4.84.

[0385] HR-MS (Q-TOF, ESI) calculated for C49H6279BrN4O14PS2Si [M+Na+]: 1155.2286, found 1155.2274. b. O-((2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 2-(((4-bromophenyl)thio)methyl)-4-methoxytetrahydrofuran-3-yl) O-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) S-hydrogen phosphorothioateesized by following general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PS-U-BOM (d / r = 4:1), 24.0 mg, 0.026 mmol, 1 equiv.), DBU (5.9 mg, 0.039 mmol, 1.5 equiv.), 4-bromobenzenethiol (7.3 mg, 0.039 mmol, 1.5 equiv.), and in MeCN (0.26 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM,Hex-EA-MeOH gradient (100:0:0 to 0:90:10), desired product was isolated as an off-white solid (major diastereomer, 22.5 mg, 78%).

[0387] 31P NMR (202 MHz, DMSO) δ 54.05.

[0388] 1H NMR (500 MHz, DMSO) δ 8.17 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.36 – 7.24 (m, 12H), 5.90 (d, J = 5.0 Hz, 1H), 5.83 (d, J = 5.2 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.35 – 5.24 (m, 4H), 4.72 (dt, J = 10.8, 5.2 Hz, 1H), 4.58 (s, 2H), 4.57 (s, 2H), 4.37 (t, J = 4.3 Hz, 1H), 4.28 – 4.23 (m, 1H), 4.06 (t, J = 5.6 Hz, 1H), 4.02 (d, J = 4.5 Hz, 2H), 3.97 – 3.91 (m, 2H), 3.61 (dd, J = 14.1, 2.2 Hz, 1H), 3.37 (s, 3H, assumed; partially obscured by solvent peak), 3.34 – 3.32 (m, 4H, assumed; partially obscured by solvent peak), 0.87 (s, 9H), 0.10 (s, 6H).

[0389] 13C NMR (126 MHz, DMSO) δ 161.89, 161.72, 150.91, 150.72, 140.17, 139.82, 138.07, 138.02, 135.83, 131.69, 129.85, 128.21, 127.49, 127.38, 127.36, 118.45, 101.44, 101.38, 87.81, 87.01, 84.08, 84.02, 82.10, 81.82, 80.07, 80.04, 73.14, 72.28, 71.09, 71.07, 70.33, 70.06, 68.21, 63.51, 62.97, 57.68, 57.65, 34.50, 29.61, 25.66, 17.78, -4.79, -4.85.

[0390] HR-MS (Q-TOF, ESI) calculated for C48H6079BrN4O14PS2Si [M+Na+]: 1141.2120, found 1141.2112. c. Methyl S-(((2S,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)-3-(((((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2- yl)methoxy)(mercapto)phosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl)-N-(tert- butoxycarbonyl)-L-cysteinatep y thesized by following general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PS-U-BOM (d / r = 4:1), 24.0 mg, 0.026 mmol, 1 equiv.), DBU (5.9 mg, 0.039 mmol, 1.5 equiv.), methyl (tert- butoxycarbonyl)-L-cysteinate (Boc-Cys-OMe, 9.1 mg, 0.039 mmol, 1.5 equiv.), and in MeCN (0.26 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA-MeOH gradient (100:0:0 to 0:90:10), desired product was isolated as a white foamy solid (major diastereomer, 21.3 mg, 71%).

[0392] 31P NMR (202 MHz, DMSO) δ 53.92.

[0393] 1H NMR (500 MHz, DMSO) δ 8.16 (d, J = 8.2 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.37 – 7.19 (m, 11H), 5.90 (d, J = 5.1 Hz, 1H), 5.87 (d, J = 5.2 Hz, 1H), 5.79 (dd, J = 8.3, 2.6 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.37 – 5.25 (m, 4H), 4.69 – 4.63 (m, 1H), 4.59 (s, 2H), 4.58 (s, 2H), 4.40 – 4.35 (m, 1H), 4.28 – 4.22 (m, 1H), 4.17 (q, J = 8.2 Hz, 1H), 4.08 – 3.99 (m, 3H), 3.94 (t, J = 5.1 Hz, 2H), 3.64 – 3.57 (m, 3H), 3.37 (s, 3H), 3.34 (s, 3H, assumed; partially obscured by solvent peak), 3.03 – 2.73 (m, 4H), 1.39 – 1.31 (m, 9H, Boc rotamers), 0.88 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H).

[0394] 13C NMR (126 MHz, DMSO) δ 171.63, 171.61, 161.88, 161.75, 155.36, 155.32, 150.93, 150.77, 140.17, 139.78, 138.07, 138.03, 128.20, 127.49, 127.38, 127.36, 101.55, 101.41, 87.71, 87.65, 86.96, 84.15, 84.08, 83.55, 83.23, 82.07, 80.03, 78.40, 73.23, 71.10, 71.07, 70.38, 70.07, 63.53, 57.66, 57.59, 53.69, 53.45, 51.96, 51.92, 33.96, 33.61, 33.56, 33.42, 29.61, 28.15, 27.90, 25.66, 17.79, -4.78, -4.80, -4.85.

[0395] HR-MS (Q-TOF, ESI) calculated for C51H72N5O18PS2Si [M+Na+]: 1188.3713, found 1188.3695.d.4-((((2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 3-(((((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- ((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2- yl)methoxy)(mercapto)phosphoryl)oxy)-4-methoxytetrahydrofuran-2- yl)methyl)thio)butanoic acidhesized by following general procedure 13 (GP13) in Example 7, using 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PS-U-BOM (d / r = 4:1), 24.0 mg, 0.026 mmol, 1 equiv.), DBU (23.6 mg, 0.15 mmol, 6 equiv.), 4-mercaptobutanoic acid (9.3 mg, 0.077 mmol, 3 equiv.), and in MeCN (0.26 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA- MeOH-AcOH gradient (100:0:0:0 to 0:89:10:1 to 0:0:100:0), desired products were isolated as colorless clear gum (major diastereomer, 17.0 mg, 63%, mixture of major and minor diastereomers, 6.1 mg (after removing silica by filtration), combined 23.1 mg, 85%).

[0397] Reporting the major diastereomer

[0398] 31P NMR (202 MHz, CD3CN) δ 57.08.

[0399] 1H NMR (500 MHz, CD3CN) δ 8.03 (br s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.35 – 7.23 (m, 10H), 5.93 (t, J = 4.8 Hz, 2H), 5.82 (br d, J = 7.6 Hz, 1H), 5.72 (app d, J = 8.2 Hz, 1H), 5.39 – 5.30 (m, 4H), 4.94 (br s, 1H), 4.62 (s, 2H), 4.61 (s, 2H), 4.39 – 4.25 (m, 3H), 4.14 – 4.04 (m, 2H), 3.96 (s, 1H), 3.82 (t, J = 4.7 Hz, 1H), 3.45 (s, 3H), 3.42 (s, 3H), 3.14 – 3.05 (m, 1H), 2.87 –2.79 (m, 1H), 2.72 (d, J = 6.2 Hz, 1H), 2.63 – 2.55 (m, 2H), 2.30 – 2.23 (m, 1H), 1.85 (br s, 2H), 1.65 – 1.53 (m, 1H), 0.90 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H).

[0400] 13C NMR (126 MHz, CD3CN) δ 163.74, 163.30, 152.32, 152.21, 140.67, 140.05, 139.34, 139.30, 129.21, 128.55, 128.52, 128.48, 118.26, 102.81, 102.64, 89.09, 88.70, 84.58, 84.00, 83.21, 82.04, 75.01, 72.48, 72.44, 71.25, 71.19, 58.76, 34.38, 30.29, 26.12, 18.66, -4.45.

[0401] HR-MS (Q-TOF, ESI) calculated for C46H63N4O16PS2Si [M+H+]: 1151.3260, found 1151.3302. e. O-((2S,3S,4R,5R)-5-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- (5,8,11,14-tetraoxa-2-thiaheptadec-16-yn-1-yl)-4-methoxytetrahydrofuran-3-yl) O- (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3- ((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) S-hydrogen phosphorothioatewing general procedure 12 (GP12) in Example 7, using 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PS-U-BOM (d / r = 4:1), 24.0 mg, 0.026 mmol, 1 equiv.), DBU (11.8 mg, 0.077 mmol, 3 equiv.), 3,6,9,12-tetraoxapentadec-14-yne-1-thiol (19.2 mg, 0.077 mmol, 3 equiv.), and in MeCN (0.26 mL). Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA-MeOH gradient (100:0:0 to 0:90:10 to 0:0:100), desired products were isolated as colorless clear solid (major diastereomer, 13.6 mg, 45%, mixture of major and minor diastereomers, 4.5 mg (after removing silica by filtration), combined 18.1 mg, 60%).

[0403] Reporting the major diastereomer

[0404] 31P NMR (202 MHz, DMSO) δ 53.76.

[0405] 1H NMR (500 MHz, DMSO) δ 8.19 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.37 – 7.22 (m, 10H), 5.90 (d, J = 5.2 Hz, 1H), 5.87 (d, J = 5.2 Hz, 1H), 5.81 (d, J = 8.1 Hz, 1H), 5.71 (d, J = 8.2 Hz, 1H), 5.34 – 5.26 (m, 4H), 4.64 (dt, J = 10.6, 5.0 Hz, 1H), 4.58 (s, 2H), 4.57 (s, 2H), 4.39 (t, J = 4.2 Hz, 1H), 4.24 (dt, J = 8.1, 4.1 Hz, 1H), 4.14 – 4.12 (m, 2H), 4.03 – 3.91 (m, 5H), 3.58 – 3.49 (m, 20H), 3.41 (t, J = 2.5 Hz, 1H), 3.37 (s, 3H), 3.34 (s, 3H, assumed; partially obscured by solvent peak), 3.02 (dd, J = 14.2, 3.5 Hz, 1H), 2.94 (dd, J = 14.3, 7.8 Hz, 1H), 2.80 – 2.67 (m, 3H), 0.88 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H).

[0406] 13C NMR (126 MHz, DMSO) δ 161.87, 161.74, 150.93, 150.76, 140.24, 139.68, 138.07, 138.03, 128.20, 127.48, 127.38, 127.35, 101.55, 101.38, 87.54, 86.90, 84.25, 84.19, 83.42, 82.08, 80.33, 80.16, 80.12, 77.10, 73.06, 72.28, 72.10, 71.08, 71.06, 70.64, 70.40, 70.28, 70.06, 69.99, 69.78, 69.76, 69.73, 69.56, 69.51, 69.48, 68.52, 68.49, 63.39, 57.64, 57.56, 57.50, 57.48, 34.51, 33.76, 31.67, 31.58, 30.24, 25.66, 17.78, -4.79, -4.86.

[0407] HR-MS (Q-TOF, ESI) calculated for C53H75N4O18PS2Si [M+Na+]: 1201.3917, found 1201.3910.f. O-((2S,3S,4R,5R)-2-(13-Azido-5,8,11-trioxa-2-thiatridecyl)-5-(3-((benzyloxy)methyl)-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl) O-(((2R,3R,4R,5R)- 5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert- butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) S-hydrogen phosphorothioatewing general procedure 13 (GP13) in Example 7, using 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)- 4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-sulfidotetrahydro-4H-furo[3,2- d][1,3,2]dioxaphosphinin-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-PS-U-BOM (d / r = 4:1), 24.0 mg, 0.026 mmol, 1 equiv.), 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (TBD, 14.4 mg, 0.10 mmol, 4 equiv.), S-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl) ethanethioate (22.9mg, 0.082 mmol, 3.2 equiv.), and in MeCN (0.26 mL). Acetyl-protected thiol and TBD were stirred for 15 mins before addition of U-BOM-PS-U-BOM. Column conditions: SiO2-25g column, Next-Gen automated system, wet loading by dissolving in minimum amount of DCM, Hex-EA-MeOH gradient (100:0:0 to 0:90:10 to 0:0:100), desired products were isolated as colorless clear solid (major diastereomer, 19.8 mg, 66%, mixture of major and minor diastereomers, 4.6 mg (after removing silica by filtration), combined 24.4 mg, 81%).

[0409] Reporting the major diastereomer

[0410] 31P NMR (202 MHz, DMSO) δ 53.80.

[0411] 1H NMR (500 MHz, DMSO) δ 8.18 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.37 – 7.21 (m, 10H), 5.90 (d, J = 5.2 Hz, 1H), 5.88 (d, J = 5.2 Hz, 1H), 5.81 (d, J = 8.1 Hz, 1H), 5.72 (d, J = 7.8 Hz, 1H), 5.34 – 5.26 (m, 4H), 4.68 – 4.61 (m, 1H), 4.58 (s, 2H), 4.57 (s, 2H), 4.38 (t, J = 4.3 Hz, 1H), 4.28 – 4.22 (m, 1H), 4.05 – 3.98 (m, 3H), 3.97 – 3.90 (m, 2H), 3.60 – 3.50 (m, 12H), 3.37 (s, 3H, assumed; partially obscured by solvent peak), 3.34 (s, 3H, assumed; partially obscured by solvent peak), 3.02 (dd, J = 14.3, 3.5 Hz, 1H), 2.94 (dd, J = 14.3, 7.8 Hz, 1H), 2.79 – 2.65 (m, 2H), 0.88 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H).

[0412] 13C NMR (126 MHz, DMSO) δ 161.89, 161.75, 150.94, 150.78, 140.23, 139.69, 138.08, 138.04, 128.21, 127.49, 127.38, 127.36, 101.57, 101.40, 87.52, 86.94, 84.17, 83.44, 83.41, 82.09, 80.15, 80.11, 73.13, 71.09, 71.07, 70.40, 70.07, 70.02, 69.82, 69.79, 69.68, 69.53, 69.26, 63.46, 57.65, 57.57, 50.00, 33.78, 31.61, 29.61, 25.66, 17.79, -4.78, -4.85.

[0413] HR-MS (Q-TOF, ESI) calculated for C50H72N7O17PS2Si [M+Na+]: 1188.3825, found 1188.3844.

Claims

CLAIMS We claim:

1. A method for preparing a dinucleotide of Formula (vi.b) or a salt or solvate thereof, wherein:Y is O or S; R1and R2are independently chosen from hydrogen, hydroxy, halogen, N3, (C1-C20) alkyl, and (C1-C20) alkoxy; optionally wherein R1and R2are independently chosen from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B1and B2are nucleobases, which may optionally be protected; and R4is hydrogen or a protecting group; the method comprising: (i) providing the cyclic phosphorus dinucleotide of Formula (iv) of claim 1, wherein R4is a protecting group: nd (ii) reacting the cyclic phospv) with a nucleophile (N) of Formula (v) to form a dinucleotide of Formula (vi.b):; wh inggroups.

2. A method for preparing a dinucleotide of Formula (vi) or a salt or solvate thereof, ; wherein:Y is O or S; R1and R2are independently chosen from hydrogen, hydroxy, halogen, N3, (C1-C20) alkyl, and (C1-C20) alkoxy; optionally wherein R1and R2are independently chosen from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B1and B2are nucleobases, which may optionally be protected; and R4is hydrogen or a protecting group; the method comprising: (a) reacting a nucleoside of Formula (i) with P(Y)R3, wherein Y is O or S, and wherein each R is independently selected from Cl, Br, (C1-C6) alkoxy, (C6-C12) aryloxy, S-(C1-C6) alkyl, O-aryl, and S-aryl; R is Cl, Br, or (C1-C6) alkoxy, thereby forming a cyclic phosphorous nucleoside of Formula (ii): ;(b) reacting the cyclic phosphorous nucleoside of Formula (ii) with a nucleoside of Formula (iii) to form a cyclic phosphorus dinucleotide of Formula (iv), wherein R4is a protecting group: and ( ofFormula (v) to form a dinucleotide of Formula (vi.b): ;ecting groups.

3. The method according to claim 2, wherein the cyclic phosphorus dinucleotide of Formula (iv) is obtained as a mixture of stereoisomers, and wherein the stereoisomers are separated prior to the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v).

4. The method according to any one of claims 1 to 3, wherein the nucleophile (N) of Formula (v) is a thiolate; optionally a thiolate selected from:,5. The method according to any one of claims 1 to 3, wherein the nucleophile (N) of Formula (v) is a halide; optionally a halide selected from lithium iodide (LiI), lithium bromide (LiBr), and lithium chloride (LiCl).

6. The method according to any one of claims 1 to 3, wherein the nucleophile (N) of Formula (v) is an azide; optionally sodium azide (NaN3).

7. The method according to any one of claims 1 to 3, wherein the nucleophile (N) of Formula (v) is an amine; optionally an amine selected from: nd8. The method according to any one of claims 1 to 3, wherein the nucleophile (N) of Formula (v) is a tri-substituted phosphine; optionally a tri-substituted phosphine selected from: .

9. The method according to any one of claims 1 to 8, wherein the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) is carried outin a solvent system comprising: tetrahydrofuran (THF); 2-methyltetrahydrofuran (2Me- THF); diethyl ether; 1,4-dioxane; dimethylsulfoxide (DMSO); dimethylformamide (DMF), N-methylpyrrolidone (NMP); dimethylacetamide (DMA); 1,8-Diazabicyclo[5.4.0]undec-7- ene (DBU), 7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (mTBD), dichloromethane (DCM); acetonitrile (MeCN or ACN); methyl tert-butyl ether (MTBE); tertAmyl alcohol; or toluene.

10. The method according to any one of claims 1 to 9, wherein the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v) is carried out in the presence of a base comprising: n-butyllithium (n-BuLi); lithium tertbutoxide (LiOtBu); lithium bis(trimethylsilyl)amide (LiHMDS); potassium bis(trimethylsilyl)amide (KHMDS); lithium tetramethylpiperidide (LiTMP); or Li-RZ, where RZ is alkyl or aryl; sodium t- butoxide (NaOtBu) + lithium chloride (LiCl); potassium t-butoxide (KOtBu) + lithium chloride (LiCl); triethylamine (Et3N); Diazabicycloundecene (DBU); 4- Dimethylaminopyridine (DMAP); S-collidine; triazabicyclodecene (TBD); N-methyl- triazabicyclodecene (mTBD); or a combination thereof.

11. The method according to claim 10, wherein the base is pre-mixed with the nucleophile (N) of Formula (v) prior to the step of reacting the cyclic phosphorus dinucleotide of Formula (iv) with a nucleophile (N) of Formula (v).

12. The method according to claim 10 or claim 11, wherein the base is lithium tertbutoxide.

13. The method according to any one of claims 1 to 12, wherein the dinucleotide of Formula (vi.b) is isolated by reacting with a silyl chloride.

14. The method of any one of claims 1 to 13, wherein R2is an alkoxy group linked to the adjacent O to form a ketal protecting group with R4.

15. The method of any one of claims 1 to 14, wherein R4is chosen from H, a silyl group, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl (TBS, TBDMS), t-butyldiphenylsilyl, triisopropylsilyl, or a ketal protecting group formed with R2.

16. The method of any one of claims 1 to 15, wherein each nucleobase is chosen from pyrimidines and purines.

17. The method of any one of claims 1 to 16, wherein each nucleobase is protected with one or more protecting groups independently chosen from dimethyl formamide (DMF), formamidine, DMF-formamidine, N-methylpyrrolidin-2-ylidenyl (PyA), pivaloyloxymethyl (POM), phthalimido, chloro, bromo, acyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM).

18. The method of any one of claims 1 to 17, wherein the method is a liquid phase process.

19. The method of any one of claims 1 to 18, wherein the method is conducted using continuous flow.

20. The method of any one of claims 1 to 19, wherein the method is carried out in a flow reactor.

21. The method of any one of claims 1 to 20, wherein the method is performed at ambient temperature.

22. A dinucleotide produced by the method of any one of claims 1 to 21.

23. A dinucleotide of the following formula: or a salt or solvate thereof, wherendependently chosen from hydrogen, hydroxy, halogen, N3, (C1-C20) alkyl, and (C1-C20) alkoxy (optionally wherein R1and R2are independently chosen from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE)); R4is H or a protecting group; and B1and B2are nucleobases, which may optionallybe protected.

24. A solution comprising a dinucleotide of claim 23.

25. Use of a dinucleotide of claim 23 , in the synthesis of an oligonucleotide or polynucleotide.

26. Use of a dinucleotide of claim 23, as a blockmer amidite in solid phase, chemical ligation, or other synthesis method of an oligonucleotide or polynucleotide.

27. Use of a dinucleotide of claim 23, in an enzymatic ligation reaction for the synthesis of an oligonucleotide or polynucleotide.