Oligonucleotide synthesis using nucleoside cyclophosphate

Abstract

The present disclosure provides methods of making oligonucleotides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using nucleoside cyclophosphate. In certain embodiments, the present disclosure provides methods of making oligonucleotides using substituted benzyl alcohol nucleophiles. In certain embodiments, the present disclosure provides methods of making oligonucleotides using nucleoside cyclophosphate and a substituted benzyl alcohol nucleophile.

Description

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 500,726, filed May 8, 2023, and European Patent Application No. 24382484.4, filed May 3, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure provides methods for preparing oligonucleotides. In some embodiments, this disclosure provides methods for preparing oligonucleotides using cyclic-phosphorous nucleosides. In some embodiments, this disclosure provides methods for preparing oligonucleotides using substituted benzyl alcohol nucleophiles. In some embodiments, this disclosure provides methods for preparing oligonucleotides using cyclic-phosphorous nucleosides and substituted benzyl alcohol nucleophiles. Background Technology

[0003] With the continued advancements in DNA and RNA technologies in science and medicine, the demand for synthetic oligonucleotides is steadily increasing. Innovations in molecular biology, RNA-based therapies, DNA-based diagnostics, gene editing and therapy, and other similar approaches continue to drive high demand for synthetic oligonucleotides that can be used to amplify, detect, analyze, quantify, modify, or target native nucleic acids. However, current methods for synthesizing oligonucleotides (e.g., automated solid-phase synthesis using phosphoramide chemistry) can be costly, have limited scalability and portability (e.g., due to reliance on specific packed-bed reactors), and typically require complex workflow systems.

[0004] Therefore, there is a need for an efficient, scalable, and easily implemented oligonucleotide synthesis method in batch or flow reactors, which should reliably provide short, medium, or long oligonucleotides of interest containing oligonucleotides that can be used directly or as synthetic structural units. Summary of the Invention

[0005] Details of various embodiments of this disclosure are set forth in the description below. In some embodiments, this disclosure provides methods for preparing oligonucleotides. In some embodiments, this disclosure provides methods for preparing dinucleotides. In some embodiments, this disclosure provides methods for preparing trinucleotides. In some embodiments, this disclosure provides methods for preparing oligonucleotides with a length of 2-20 nucleotides.

[0006] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (iv) or a salt or solvate thereof. Where: Y is O or S, R 1 and R 2Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and methoxyethyl groups); B 1 and B 2 It is a nucleobase, which may optionally be protected by one or more protecting groups; and R 4 It is hydrogen or a protecting group.

[0007] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (iv) or a salt or solvate thereof, wherein the method comprises: (a) React the nucleoside of formula (i) with P(Y)R3, where Y is O or S, and each R is independently selected from Cl, Br, (C1-C6)alkoxy, (C6-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): ;and (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: In some embodiments, the method further includes removing one or more protecting groups.

[0008] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof: Where: Y is O or S; R 1 and R 2 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); B 1 and B 2 It is a nucleobase, which can be optionally protected; R 4 It is hydrogen or a protecting group; n is selected from 1, 2, 3, 4, and 5; each R 5 Independently, it is a substituent on the benzyl protecting group of the benzyl ring; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 Alkyl and aryl groups.

[0009] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof, wherein the method comprises: providing a cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group; and it reacts the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 Alkyl and aryl groups. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

[0010] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof, wherein the method comprises: (a) React the nucleoside of formula (i) with P(Y)R3, where Y is O or S, and each R is independently selected from Cl, Br, (C1-C6)alkoxy, (C6-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: ; and (c) React the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 Alkyl and aryl groups. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

[0011] In some embodiments, this disclosure provides a method for preparing oligonucleotides of formula (vii): Where: each Y is independently O or S; R 1 R 2 and R 3 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); R 4 It is an H or protecting group; n is selected from 1, 2, 3, 4 and 5; each R 5 Independently, each R is a substituent on the benzyl protecting group of the benzyl ring; 7 and R 8 Independently selected from hydrogen, (C1-C 20 )alkyl and aryl; B 1 B 2 and B 3 It is a nucleobase, which may optionally be protected; m is selected from integers in the range of 1 to 18; and X is hydrogen, a protecting group, or .

[0012] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (vii), wherein the method comprises: providing a dinucleotide of formula (vi); and repeating the reactions of steps (a), (b), and (c) of the method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof once or more (as disclosed herein) to obtain an oligonucleotide of formula (vii): In a repeated step (b), the cyclic phosphorus nucleoside of formula (ii) is reacted with the 5'-terminal nucleoside of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

[0013] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (vii), 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-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: (c) React the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 (a) Alkyl and aryl; and (d) Repeat the reactions of steps (a), (b), and (c) one or more times to obtain the oligonucleotide of formula (vii): In a repeated step (b), the cyclic phosphorus nucleoside of formula (ii) is reacted with the 5'-terminal nucleoside of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

[0014] In some embodiments, this disclosure provides a method for preparing oligonucleotides of formula (1) or their salts or solvates. Each Y is independently O or S; R 1 R 2 and R 3 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); B 1 B 2 and B 3 It is a nucleobase, which can be optionally protected; R 4 It is hydrogen or a protecting group; m is an integer selected from the range of 1 to 18; and X is hydrogen, a protecting group, or .

[0015] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (1) or a salt or solvate thereof, wherein the method comprises: providing a dinucleotide of formula (vi) or an oligonucleotide of formula (vii); and (ii) hydrogenating a dinucleotide of formula (vi) or an oligonucleotide of formula (vii); wherein the method produces an oligonucleotide of formula (1) or a salt or solvate thereof. In some embodiments, the method further comprises removing one or more protecting groups.

[0016] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (1) 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-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: (c) React the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 (d) Alkyl and aryl groups; optionally, the reactions in steps (a), (b) and (c) are repeated once or more to obtain the oligonucleotide of formula (vii): In step (b), the cyclic phosphorus nucleotide of formula (ii) is reacted with the 5'-terminal nucleotide of the oligonucleotide; and (e) the product of step (c) or step (d) is hydrogenated; wherein the method produces the oligonucleotide of formula (1) or a salt or solvate thereof. In some embodiments, the method further includes the removal of one or more protecting groups.

[0017] In some embodiments of this disclosure, the cyclic phosphorus dinucleotide of formula (iv) is obtained as a mixture of stereoisomers. In some embodiments, the stereoisomers are isolated prior to the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v).

[0018] In some embodiments of this disclosure, R 5 Each instance is an ortho- or para-substituent on the benzyl group. In some embodiments, R 5 Each instance is an ortho-substituent on a benzyl group. In some embodiments, R 5Each instance is a para-substituent on a benzyl group. In some embodiments, R 5 Each instance is independently selected from halogens, (C1-C2) 20 )alkyl, (C1-C 20 )alkoxy, trifluoromethyl, and phenyl. In some embodiments, R 5 Each instance is independently selected from phenyl, methyl, methoxy, fluorine, chlorine, and CF3. In some embodiments, R 5 Each instance is selected from chlorine and CF3. In some embodiments, R 5 It is a fused benzene ring that forms the naphthalene ring system. In some embodiments, the substituted benzyl alcohol nucleophile of formula (v) is: .

[0019] In certain embodiments of this disclosure, the reaction of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in a solvent system. In some embodiments, the solvent includes: tetrahydrofuran (THF); 2-methyltetrahydrofuran (2Me-THF); diethyl ether; 1,4-dioxane; dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylacetamide (DMA), dichloromethane (DCM), acetonitrile (MeCN), methyl tert-butyl ether (MTBE), tert-amyl alcohol, toluene, or mixtures thereof.

[0020] In certain embodiments of this disclosure, the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in the presence of a base. In some embodiments, the base comprises: n-butyllithium (n-BuLi); lithium tert-butoxide (LiOtBu); lithium bis(trimethylsilyl)amide (LiHMDS); lithium tetramethylpiperidine (LiTMP); or Li-R Z , where R Z It is alkyl or aryl; sodium tert-butoxide (NaOtBu) + lithium chloride (LiCl); potassium tert-butoxide (KOtBu) + lithium chloride (LiCl); triethylamine, diazabicycloundecene (DBU), triazabicyclodecene (TBD), or combinations thereof. In some embodiments, the base is premixed with the substituted benzyl alcohol nucleophile of formula (v) prior to the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v). In some embodiments, the base is lithium tert-butoxide (LiOtBu).

[0021] In some embodiments of this disclosure, the dinucleotide of formula (vi) is isolated by reacting with silyl chloride.

[0022] In some embodiments of this disclosure, the cyclophosphochloronucleotide of formula (ii) is obtained as a mixture of stereoisomers. In some embodiments, the stereoisomers are separated prior to the step of reacting the cyclophosphonucleotide of formula (ii) with the nucleoside of formula (iii).

[0023] In some embodiments of this disclosure, R 2 It is connected to the adjacent O to connect with R. 4 Alkoxy groups that form ketal protecting groups.

[0024] In some embodiments of this disclosure, R 4 Selected from H, silyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl, triisopropylsilyl, or with R 2 The ketal protecting group formed.

[0025] In some embodiments of this disclosure, each nucleobase is selected from pyrimidine and purine. In some embodiments, each nucleobase is protected by one or more protecting groups. In some embodiments, each nucleobase is protected by one or more protecting groups independently selected from dimethylformamide (DMF), formamidinium, DMF-formamidinium, phthalimide, chlorine, bromine, acyl, allyl, benzyl, tert-butoxycarbonyl (Boc), and benzyloxymethyl (BOM).

[0026] In some embodiments of this disclosure, the oligonucleotide synthesis method is a liquid-phase process (i.e., carried out in the liquid phase). In some embodiments, the method is carried out using a continuous flow. In some embodiments, the method is carried out in a flow reactor.

[0027] In some embodiments, this disclosure provides oligonucleotides (e.g., dinucleotides, trinucleotides) produced by the methods of this disclosure.

[0028] In some embodiments, this disclosure provides cyclic phosphate nucleosides of formula (ii): Or its salts or solvates, wherein: Y is O or S; each R is independently selected from Cl, Br, (C1-C6)alkoxy, (C6-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl; R 1 Selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); and B 1 These are nucleobases, which may optionally be protected. In some embodiments, this disclosure provides cyclic phosphononucleotides of formula (ii) produced by the methods of this disclosure.

[0029] In some embodiments, this disclosure provides a cyclic phosphorus dinucleotide of formula (iv): Or its salt or solvate, wherein: Y is O or S; R 1 and R 2 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); R 4 It is H or a protecting group; and B 1 and B 2 It is a nucleobase, which may optionally be protected. In some embodiments, this disclosure provides a cyclic phosphorus dinucleotide of formula (iv) produced by the methods of this disclosure.

[0030] In some embodiments, this disclosure provides a dinucleotide of formula (vi): Or its salt or solvate, wherein: Y is O or S; R 1 and R 2 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); R 4 It is an H or protecting group; each R 5 Independently, it is a substituent on the benzyl protecting group of the benzyl ring; R 7 and R 8 Independently selected from hydrogen, (C1-C 20 )alkyl and aryl; B 1 and B 2 n is a nucleobase, which may optionally be protected; and n is selected from 1, 2, 3, 4, and 5. In some embodiments, this disclosure provides a dinucleotide of formula (vi) produced by the methods of this disclosure.

[0031] In some embodiments, this disclosure provides oligonucleotides of formula (vii): Or its salt or solvate, wherein: each Y is independently O or S; R 1 R 2 and R 3 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20)alkoxy groups (including methoxy and -O-methoxyethyl); R 4 It is an H or protecting group; n is selected from 1, 2, 3, 4 and 5; each R 5 Independently, each R is a substituent on the benzyl protecting group of the benzyl ring; 7 and R 8 Independently selected from hydrogen, (C1-C 20 )alkyl and aryl; B 1 B 2 and B 3 It is a nucleobase, which may optionally be protected; m is selected from integers in the range of 1 to 18; and X is hydrogen, a protecting group, or In some embodiments, this disclosure provides oligonucleotides of formula (vii) produced by the methods of this disclosure.

[0032] In some embodiments, this disclosure provides oligonucleotides of formula (1) or salts or solvates thereof. Where: each Y is independently O or S; R 1 R 2 and R 3 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); B 1 B 2 and B 3 It is a nucleobase, which can be optionally protected; R 4 It is hydrogen or a protecting group; m is an integer selected from the range of 1 to 18; and X is hydrogen, a protecting group, or In some embodiments, this disclosure provides oligonucleotides of formula (1) produced by the methods of this disclosure.

[0033] In some embodiments, this disclosure provides a solution containing the nucleosides of this disclosure (e.g., cyclic phosphate nucleosides). In some embodiments, this disclosure provides a solution containing the dinucleotides of this disclosure. In some embodiments, this disclosure provides a solution containing the trinucleotides of this disclosure. In some embodiments, this disclosure provides a solution containing the oligonucleotides of this disclosure.

[0034] In some embodiments, the oligonucleotides of this disclosure can be used for subsequent synthetic applications of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of this disclosure can be used as block polymers in the solid-phase synthesis of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of this disclosure can be used as phosphorous acid block polymers in the solid-phase synthesis of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of this disclosure can be used in enzymatic ligation reactions in the synthesis of oligonucleotides or polynucleotides. Attached Figure Description

[0035] Figure 1A and Figure 1B An example of an enzymatic ligation reaction for generating nonamers from short polymers is demonstrated. With advancements in DNA and RNA technologies, the demand for synthetic oligonucleotides is steadily increasing.

[0036] Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E and Figure 2F The results show the dinucleotide screening-plate Spotfire output overlap 2HR; PhMe (toluene) results.

[0037] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E and Figure 3F The results of dinucleotide screening-plate Spotfire output overlap 2HR; ACN are shown.

[0038] Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E and Figure 4F The results show the dinucleotide screening-plate Spotfire output overlap of 20HR; PhMe (toluene).

[0039] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E and Figure 5F The results of dinucleotide screening-plate Spotfire output overlap 20HR; ACN are shown.

[0040] Figure 6 A general overview of one embodiment of the solution-phase method for oligonucleotide synthesis disclosed herein is presented.

[0041] Figure 7A general overview of one embodiment of the continuous flow oligonucleotide synthesis system disclosed herein is presented. Detailed Implementation

[0042] This disclosure provides a method for preparing oligonucleotides. This disclosure also provides oligonucleotides prepared by the method of this disclosure. Oligonucleotides are typically linked nucleotide polymers with a length of less than about 100 nucleotides. In some embodiments, the oligonucleotides of this disclosure contain at least two nucleotides. Therefore, in some embodiments, the oligonucleotides of this disclosure contain 2 to 100 nucleotides and all integers within that range (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 some embodiments, the oligonucleotide contains two nucleotides (i.e., a dinucleotide). In some embodiments, the oligonucleotide contains three nucleotides (i.e., a trinucleotide). In some implementations, the oligonucleotide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0043] In some embodiments, the oligonucleotides of this disclosure comprise 3 to 100 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 50 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 30 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 20 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 15 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 10 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 8 nucleotides. In some embodiments, the oligonucleotides comprise 3 to 6 nucleotides. In some embodiments, the oligonucleotides comprise 3 nucleotides (i.e., trinucleotides).

[0044] In some embodiments, the oligonucleotides of this disclosure comprise 5 to 20 nucleotides. In some embodiments, the oligonucleotides of this disclosure comprise 10 to 20 nucleotides. In some embodiments, the oligonucleotides of this disclosure comprise 15 to 20 nucleotides. In some embodiments, the oligonucleotide comprises 3 nucleotides. In some embodiments, the oligonucleotide comprises 5 nucleotides. In some embodiments, the oligonucleotide comprises 6 nucleotides. In some embodiments, the oligonucleotide comprises 7 nucleotides. In some embodiments, the oligonucleotide comprises 8 nucleotides. In some embodiments, the oligonucleotide comprises 9 nucleotides. In some embodiments, the oligonucleotide comprises 10 nucleotides. In some embodiments, the oligonucleotide comprises 11 nucleotides. In some embodiments, the oligonucleotide comprises 12 nucleotides. In some embodiments, the oligonucleotide comprises 13 nucleotides. In some embodiments, the oligonucleotide comprises 14 nucleotides. In some embodiments, the oligonucleotide comprises 15 nucleotides. In some embodiments, the oligonucleotide comprises 16 nucleotides. In some embodiments, the oligonucleotide comprises 17 nucleotides. In some embodiments, the oligonucleotide comprises 18 nucleotides. In some embodiments, the oligonucleotide comprises 19 nucleotides. In some implementations, the oligonucleotide contains 20 nucleotides.

[0045] The term "nucleotide" is defined herein (see below) as an organic compound containing a nucleoside and a phosphate group. The term "nucleoside" is defined herein (see below) as an organic compound containing a nucleobase (e.g., adenine, cytosine, guanine, thymine, or uracil) covalently linked to a pentose sugar (e.g., ribose or 2'-deoxyribose). The term "nucleobase" is defined herein (see below) as the heterocyclic portion of a nucleoside. In some embodiments, the nucleobases of this disclosure are canonical nucleobases. In some embodiments, the nucleobases are purine bases—adenine (A) or guanine (G). In some embodiments, the nucleobases are pyrimidine bases—thymine (T), cytosine (C), or uracil (U).

[0046] In some embodiments, the nucleosides of this disclosure are atypical nucleosides (i.e., not typical nucleosides). Examples of atypical nucleosides include, but are not limited to: 3-methylcytosine; 5-methylcytosine (5-me-C); 5-ethylcytosine; 5-ethyluracil; 5-hydroxymethylcytosine; 7-methylguanine; 1-methylguanine; 2-methylguanine; 8-methylguanine; 2,2-dimethylguanine; 1-methyladenine; 2-methyladenine; N6-methyladenine; 7-methyladenine; N,N-dimethyladenine; alkyl (e.g., 6-methyl, 2-propyl) derivatives 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-Thiothymidine; 2-Thiocytosine; 6-Thiopurine; 8-Thioguanine; 5-Halogenated uracil; cytosine; alkynyl (e.g., 5-propynyl) derivatives of uracil and cytosine; 6-Azouracil; 5-uracil (pseudouracil); 8-substituted (e.g., 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy, 8-halogenated) adenine and guanine; 5-substituted (e.g., 5-chloro, 5-bromo, 5-trifluoromethyl) uracil and cytosine; 2-F-adenine; 8-azaguanine; 8-azaadenine; 7-deadenine; 7-deadenine Purines; 3-Denitroguanine; 3-Denitroadenine; Tricyclic pyrimidines; Phenomazincytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one); Phenomhiazincytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one); Substituted phenomazincytidines (e.g., 9-(2-aminoethoxy)-1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one); Carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one); Pyridoindolcytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidindol-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; 1-Methylpseudouracil; Queosine; Inosine; 1-Methylinosine and 6-Hydroxyaminopurine.Examples of atypical nucleobases include, but are not limited to: methylated purines or pyrimidines, alkylated purines or pyrimidines, acylated purines or pyrimidines, halogenated purines or pyrimidines, denitrified purines, alkylated ribose, diaminopurines, inosine, and thiolated purines or pyrimidines.

[0047] In some embodiments, the nucleobase is not protected. In some embodiments, the nucleobase is protected by a protecting group. The term "protecting group" is defined herein (see below) as a chemical part that protects a reactive or unstable group on a molecule from unwanted chemical reactions during the synthetic procedure or other chemical reactions. A nucleobase protecting group is a protecting group applied to one or more functional groups of a nucleobase to prevent side reactions at the nucleobase during oligonucleotide synthesis. In some embodiments, the nucleobase protecting group is removed at the end of oligonucleotide synthesis. The nucleobase protecting group may contain an exocyclic amino or lactam group. In some embodiments, the nucleobase protecting group is applied to the N6-amino group of the adenine moiety. In some embodiments, the nucleobase protecting group is applied to the N4-amino group of the cytosine moiety. In some embodiments, the nucleobase protecting group is applied to the N2-amino, N1-lactam (i.e., cyclic amide) group, and / or O6-lactam group of the guanine moiety. In some embodiments, the nucleoprotecting group is applied to the N3-lactam and / or O4-lactam groups of the uracil or thymine moiety. In some embodiments, the nucleoprotecting group is base-labile. Examples of base-labile protecting groups include FMOC (fluorenylmethoxycarbonyl). In some embodiments, the nucleoprotecting group is acid-labile. Examples of acid-labile protecting groups include triarylmethyl protecting groups, 4,4'-dimethoxytriphenylmethyl (DMT), and BOC carbamate (tert-butyloxycarbonyl). In some embodiments, the nucleoprotecting group is unstable to reagents other than bases or acids.

[0048] In some embodiments, the nucleobase protecting group is an amino protecting group. As used herein, an amino protecting group refers to a protecting group that can be used to protect an amino group on a molecule. Examples of amino protecting groups include, but are not limited to, N-methylpyrrolidone-2-ylene (PyA), neopentyloxymethyl (POM), 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), formyl, acetyl, trihaloacetyl, benzoyl, nitrobenzyl, 2-nitrobenzenesulfonyl, phthalimide, dithiasuccinyl, methyl carbamate, ethyl carbamate, 9-(2-sulfonyl)fluorenyl methyl carbamate, and amino groups. 9-(2,7-dibromo)fluorenyl methyl formate, 2,7-di-tert-butyl-[9-(10,10-di-side-oxy-10,10,10,10-tetrahydrothiopheneyl)]methyl formate (DBD-Tmoc), 4-methoxybenzoyl methyl formate (Phenoc), 2,2,2-trichloroethyl formate (Troc), 2-phenylethyl formate (hZ), 1-(1-adamantyl)-1-methylethyl formate (Adpoc), 1,1-dimethyl-2-haloethyl formate, 1,1-dimethyl-2,2-dibromoethyl formate (DB-t-BOC), 1,1-dimethyl-2,2-dibromoethyl formate 2-Trichloroethyl ester (TCBOC), 1-(3,5-di-tert-butylphenyl)-1-methylethyl ester of carbamate (t-Bumeoc), 2-(2′ and 4′ pyridyl)ethyl ester of carbamate (Pyoc), 2-(N,N-dicyclohexylformamido)ethyl ester of carbamate, 1-adamantane ester of carbamate (Adoc), vinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinoline carbamate, N-hydroxyhexahydropyridine carbamate, alkyl dithiocarbamate, p-methoxybenzyl carbamate (Moz), ammonia p-Nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinyl benzyl carbamate (Msz), 9-anthrayl methyl carbamate, diphenyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonyl ethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithiaalkyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphoethyl carbamate (Peoc), 2-triphenylphosphoisopropyl carbamate (Ppoc), 1,1-Dimethyl-2-cyanoethyl ester, m-chloro-p-acylbenzyl ester of carbamate, p-(dihydroxyboryl)benzyl ester of carbamate, 5-benzisoxazolyl methyl ester of carbamate, 2-(trifluoromethyl)-6-chromone methyl ester of carbamate (Tcroc), m-nitrophenyl ester of carbamate, 3,5-dimethoxybenzyl ester of carbamate, o-nitrobenzyl ester of carbamate, 3,4-dimethoxy-6-nitrobenzyl ester of carbamate, phenyl(o-nitrophenyl)methyl ester of carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, tert-amyl ester of carbamate, S-benzyl thiocarbamate, p-cyanobenzyl ester of carbamate, cyclobutyl ester of carbamate, cyclohexyl ester of carbamate Cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decoxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylformamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylformamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinic acid carbamate, p-(p'methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1- (3,5-Dimethoxyphenyl)ethyl ester, 1-methyl-1-(p-phenylazophenyl)ethyl ester, 1-methyl-1-phenylethyl ester, 1-methyl-1-(4-pyridyl)ethyl ester, phenyl ester, p-(phenylazo)benzyl ester, 2,4,6-tri-tert-butylphenyl ester, 4-(trimethylammonium)benzyl ester, 2,4,6-trimethylbenzyl ester, formamide, dimethylformamide (DMF), formamide, DMF-formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridineamide, 3-pyridylformamide, N-benzoylphenylpropionamide derivative, benzamide, p-phenylbenzamide o-Nitrophenylacetamide, o-Nitrophenoxyacetamide, acetylacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazoline-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-Tetramethyldisiloxane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridinone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-sideoxy-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptanylamine, N-triphenylmethylamine (Tr), N- [(4-Methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluoreneamine (PhF), N-2,7-dichloro-9-fluorenemethyleneamine, N-ferroceneylmethylamino (Fcm), N-2-methylpyridinylamino N′ oxide, N-1,1-dimethylthiomethyleneamine, N-benzylamine, N-p-methoxybenzylamine, N-diphenylmethyleneamine, N-[(2-pyridyl)trimethylmethyl]methyleneamine, N-(N′,N′dimethylaminomethylene)amine, N,N′isopropylidene diamine, N-p-nitrobenzylamine, N-salicylamine, N-5-chlorosalicylamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N- (5,5-Dimethyl-3-sideoxy-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitramine, amine N oxide, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkyl aminophosphate, dibenzyl aminophosphate, diphenyl aminophosphate, benzyl aminophosphate, phenylenesulfinamide, o-nitrobenzenesulfinamide (Nps), 2,4-dinitrobenzenesulfinamide, pentachlorobenzenesulfinamide, 2-nitro-4-methoxybenzenesulfinamide, triphenylmethylsulfinamide, 3-nitropyridinesulfinamide (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-pentamethylsomn-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracitesulfonamide, 4-(4′,8′-Dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylmethylsulfonamide.

[0049] In some embodiments, the nucleobase protecting group is a ketone / carboxylic acid protecting group. As used herein, a ketone protecting group, a carboxylic acid protecting group, or a ketone / carboxylic acid protecting group refers to a protecting group that can be used to protect a ketone or carboxylic acid group on a molecule. Examples of ketone / carboxylic acid protecting groups include, but are not limited to: silyl protecting groups (e.g., trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl or triisopropylsilyl); alkyl protecting groups (e.g., methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, triphenylmethyl, tert-butyl or tetrahydropyran-2-yl); alkenyl protecting groups (e.g., allyl); aryl protecting groups (e.g., optionally substituted phenyl, biphenyl or naphthyl); and arylalkyl protecting groups (e.g., 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-methylpyridinyl).

[0050] In some embodiments, the nucleobase protecting group is a hydroxyl protecting group. As used herein, a hydroxyl protecting group refers to a protecting group that can be used to protect a hydroxyl group on a molecule. Examples of protecting groups that can be used as hydroxyl protecting groups include, but are not limited to, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, bis(2-acetoxyethoxy)methyl (ACE), [(triisopropylsilyl)oxy]methyl (TOM), neopentanoyl, benzoyl, p-phenylbenzoyl, monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMT), 4,4′,4″-trimethoxytriphenylmethyl (TMTr), 1-(2-fluorophenyl)-4-methoxyhexahydropyridin-4-yl (FPMP), substituted pixyl, methyl, methoxymethyl (MOM), and methylthiomethyl (MTM). ), tert-butylthiomethyl, (phenyl dimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), 4-pentenoxymethyl (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-methoxytetrahydro Thiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxyhexahydropyridin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanolbenzofuran-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-(phenylhydroselenoyl)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-methylpyridinyl, 4-methylpyridinyl, 3-methyl-2-methylpyridinyl N-oxide, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzocycloheptanyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromobenzoylmethyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-Dichlorophthaliminophenyl)methyl, 4,4′,4″-tris(acetylpropionyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xantholyl, 9-(9-phenyl-10-oxa)anthrayl, 1,3-benzodithiocyclopentan-2-yl, benzisothiazolyl S,S-dioxy, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEI) PS), dimethyl tert-hexylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylenedithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantate, crotonate, 4 -Methoxycrotonate, Benzoate, p-Phenylbenzoate, 2,4,6-Trimethylbenzoate (trimethylbenzoate), Alkyl Carbonate Methyl Ester, 9-Fluorenylmethyl Carbonate (Fmoc), Alkyl Carbonate Ethyl Ester, Alkyl Carbonate 2,2,2-Trichloroethyl Ester (Troc), 2-(Trimethylsilyl)ethyl (TSE), 2-(Trimethylsilyl)ethyl Carbonate (TMSEC), 2-(Phenylsulfonyl)ethyl Carbonate (Psec), 2-(Triphenylphosphono)ethyl Carbonate (Peoc), Alkyl Carbonate Isobutyl Ester, Alkyl Carbonate Vinyl Ester, Alkyl Carbonate Allyl Ester, Alkyl Carbonate p-Nitrophenyl Ester, Alkyl Carbonate Benzyl Ester, Alkyl Carbonate p- Methoxybenzyl ester, alkyl carbonate ester 3,4-dimethoxybenzyl ester, alkyl carbonate ester o-nitrobenzyl ester, alkyl carbonate ester p-nitrobenzyl ester, alkyl thiocarbonate ester S-benzyl ester, 4-ethoxy-1-naphthyl carbonate, dithiocarbonate methyl ester, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-Dimethylpropyl)phenoxyacetic acid ester, dichlorophenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthaleneate, nitrate ester, N,N,N′,N′-tetramethylphosphonamide alkyl ester, N-phenylcarbamate alkyl ester, borate ester, dimethylthiophosphoryl, 2,4-dinitrophenylsulfinic acid alkyl ester, 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrobenzenesulfonyl)ethyl, 2-nitrophenyl, 4-nitrophenyl, 2,4, 6-Trimethylphenyl, 2-(2-nitrophenyl)ethyl, sulfate, methanesulfonate, benzylsulfonate, toluenesulfonate (Ts), 2-cyanoethyl (CE or Cne), 3,5-dichlorophenyl, 2,4-dimethylphenyl, butyl thiocarbonyl, 4,4′,4″-tris(benzoyloxy)triphenylmethyl, diphenylaminoformyl, acetylacetyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptm), 9-phenylxanthine-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0051] In some embodiments, the nucleobase protecting group is a thiol protecting group. As used herein, a thiol protecting group refers to a protecting group that can be used to protect thiols on a molecule. Examples of protecting groups that can be used as thiol protecting groups include, but are not limited to, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, bis(2-acetoxyethoxy)methyl (ACE), [(triisopropylsilyl)oxy]methyl (TOM), neopentanoyl, benzoyl, p-phenylbenzoyl, monomethoxytriphenylmethyl (MMTr), dimethoxytriphenylmethyl (DMT), 4,4′,4″-trimethoxytriphenylmethyl (TMTr), 1(2-fluorophenyl)-4-methoxyhexahydropyridin-4-yl (FPMP), substituted pixyl, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl , (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), 4-pentenoxymethyl (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-methoxyhexahydropyridin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanolbenzofuran-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-(phenylhydroselenoyl)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-methylpyridinyl, 4-methylpyridinyl, 3-methyl-2-methylpyridinyl N-oxide, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzocycloheptanyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromobenzoylmethoxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidephenyl)methyl, 4,4′,4″-tris(acetylpropionyloxyphenyl)methyl, 4,4′,4″-Tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xantholyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiocyclopentan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltert-hexylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl (TBDPS) Tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (trimethylbenzyl ester), alkyl methyl carbonate, carbonate 9 -Fluorenylmethyl ester (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-(triphenylphosphono)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, thioalkyl S-benzyl carbonate, 4-ethoxy-1- Naphthyl ester, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, dichlorophenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthyl ester, nitrate, N,N,N′,N′-Tetramethylphosphinodiazide alkyl ester, N-phenylcarbamate alkyl ester, borate ester, dimethylphosphinothioate, 2,4-dinitrophenylsulfinic acid alkyl ester, 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrobenzenesulfonyl)ethyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, sulfate ester, methanesulfonate ester (methanesulfonate), benzyl Sulfonates, toluenesulfonates (Ts), 2-cyanoethyl (CE or Cne), 3,5-dichlorophenyl, 2,4-dimethylphenyl, butylthiocarbonyl, 4,4′,4″-tris(benzoyloxy)triphenylmethyl, diphenylaminoformyl, acetylacetyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthine-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0052] In some embodiments, the nucleobase protecting group comprises a protecting group for protecting 1,2- or 1,3-diol. Examples of protecting groups for protecting 1,2- or 1,3-diol include methylene acetal, ethylene acetal, 1-tert-butyl ethylene ketal, 1-phenyl ethylene ketal, (4-methoxyphenyl)ethylene acetal, 2,2,2-trichloroethylene acetal, acetal ketal, cyclopentylene ketal, cyclohexylene ketal, cycloheptylene ketal, benzylene acetal, p-methoxybenzylene acetal, 2,4-dimethoxybenzylene ketal, 3,4-dimethoxybenzylene acetal, 2-nitrobenzylene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1 -Methoxyethylidene, 1-ethoxyethylidene, 1,2-dimethoxyethylidene, α-methoxybenzylidene, 1-(N,N-dimethylamino)ethylene derivative, α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylene, di-tert-butylsilyl (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxane) derivative (TIPDS), tetra-tert-butoxydisiloxane-1,3-diethylene derivative (TBDS), cyclic carbonates, cyclic borates, ethyl borate, and phenyl borate.

[0053] In some embodiments, each nucleobase is independently protected by one or more protecting groups selected from dimethylformamide (DMF), N-methylpyrrolidone-2-ylene (PyA), neopentyloxymethyl (POM), formamidinium, DMF-formamidinium, phthalimide, chlorine, bromine, acyl, allyl, benzyl, tert-butoxycarbonyl (Boc), and benzyloxymethyl (BOM).

[0054] Cyclochloronucleotide In some embodiments, this disclosure provides a method for preparing oligonucleotides using cyclic phosphate nucleosides or their salts or solvates. As used herein, a cyclic phosphate nucleoside refers to a nucleoside containing a P(Y)R3 group that binds to the 3' and 5' oxygen atoms of a nucleoside sugar to form a compound of formula (ii): Or its salts or solvates, wherein: Y is O or S; each R is independently selected from Cl, Br, (C1-C6)alkoxy, (C6-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl; R 1 Selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); and B 1 It is a nucleobase, which may optionally be protected. In some embodiments, R 1 It is (C1-C) 16 )alkyl and (C1-C 16 )alkoxy. In some embodiments, R 1 It is (C1-C) 12 )alkyl and (C1-C 12 )alkoxy. In some embodiments, R 1 It is (C1-C8)alkyl and (C1-C8)alkoxy. In some embodiments, R 1 It is (C1-C4)alkyl and (C1-C4)alkoxy. In some embodiments, R 1 It is fluorine. In some implementations, R 1 It is a methoxy group. In some embodiments, R 1 It is O-methoxyethyl (MOE).

[0055] In some embodiments, this disclosure provides a method for preparing a cyclic phosphononucleotide of formula (ii) or a salt or solvate thereof. In some embodiments, the method for preparing a cyclic phosphononucleotide 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-C6)alkoxy, (C1 ... 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii). .

[0056] In some embodiments, this disclosure provides cyclic phosphononucleotides of formula (ii) or their salts or solvates.

[0057] In some embodiments, the cyclic phosphorus nucleoside has a typical nucleobase. In some embodiments, the nucleobase is a purine base—adenine (A) or guanine (G). In some embodiments, the nucleobase is a pyrimidine base—thymine (T), cytosine (C), or uracil (U). In some embodiments, the cyclic phosphorus nucleoside has an atypical nucleobase.

[0058] In some embodiments, the nucleobases of the cyclic phosphate nucleoside are not protected. In some embodiments, the nucleobases of the cyclic phosphate nucleoside are protected by nucleobase protecting groups. In some embodiments, the nucleobases of the cyclic phosphate nucleoside are protected by amino nucleobase protecting groups. In some embodiments, the nucleobases of the cyclic phosphate nucleoside are protected by ketone / carboxylic acid nucleobase protecting groups. In some embodiments, the nucleobases of the cyclic phosphate nucleoside are protected by hydroxy nucleobase protecting groups. In some embodiments, the nucleobases of the cyclic phosphate nucleoside are protected by one or more protecting groups independently selected from dimethylformamide (DMF), formamidinium, DMF-formamidinium, phthalimide, chlorine, bromine, acyl, allyl, benzyl, tert-butoxycarbonyl (Boc), and benzyloxymethyl (BOM).

[0059] In some embodiments, the cyclophosphochloronucleotide of formula (ii) is obtained as a mixture of stereoisomers. In some embodiments, the stereoisomers are separated prior to subsequent reaction steps.

[0060] Dinucleotide In some embodiments, this disclosure provides methods for preparing oligonucleotides using dinucleotides comprising the cyclic phosphate nucleoside of this disclosure or its salts or solvates. In some embodiments, this disclosure provides methods for preparing oligonucleotides using dinucleotides of formula (iv) or their salts or solvates: Where: Y is O or S; R 1 and R 2 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); B 1 and B 2 It is a nucleobase, which may optionally be protected by one or more protecting groups; and R 4 It is hydrogen or a protecting group. In some embodiments, R 1 and R 2 Independently selected from (C1-C 16 )alkyl and (C1-C 16 )alkoxy. In some embodiments, R1 and R 2 Independently selected from (C1-C 12 )alkyl and (C1-C 12 )alkoxy. In some embodiments, R 1 and R 2 Independently selected from (C1-C8) alkyl and (C1-C8) alkoxy groups. In some embodiments, R 1 and R 2 Independently selected from (C1-C4)alkyl and (C1-C4)alkoxy. In some embodiments, R 1 and R 2 It is fluorine. In some implementations, R 1 and R 2 It is a methoxy group. In some embodiments, R 1 and R 2 It is O-methoxyethyl (MOE).

[0061] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (iv) or a salt or solvate thereof, wherein the method comprises: (a) React the nucleoside of formula (i) with P(Y)R3, where Y is O or S, and each R is independently selected from Cl, Br, (C1-C6)alkoxy, (C6-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): ;and (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: .

[0062] In some embodiments, the method further includes removing one or more protecting groups.

[0063] In some embodiments, the cyclophosphochloronucleotide of formula (ii) is obtained as a mixture of stereoisomers. In some embodiments, the stereoisomers are separated prior to the step of reacting the cyclophosphonucleotide of formula (ii) with the nucleoside of formula (iii).

[0064] In some embodiments, this disclosure provides a dinucleotide of formula (iv) or a salt or solvate thereof.

[0065] In some implementations, R 2 It is connected to the adjacent 3' oxygen to connect with R 4 Alkoxy groups that form ketal protecting groups.

[0066] In some implementations, R 4 It is hydrogen. In some implementations, R 4 It is a protecting group. In some embodiments, R 4 It is a hydroxyl protecting group. In some embodiments, R 4 Selected from silicon-based, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl, or triisopropylsilyl. In some embodiments, R 4 With R 2 It forms a ketal protecting group.

[0067] In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) is obtained in the (R) stereoisomer configuration. In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) is obtained in the (S) stereoisomer configuration. In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) is obtained as a mixture of stereoisomers. In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) is obtained as a mixture of (R) and (S) stereoisomers. In some embodiments, the stereoisomers are separated prior to subsequent synthetic steps. In some embodiments, the stereoisomers are separated prior to the reaction step of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v).

[0068] In some embodiments, the mixture of stereoisomers comprises at least about 10% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 20% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 30% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 40% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 50% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 60% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 70% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 75% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 80% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 85% (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 90% of the (R) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 95% of the (R) configuration product.

[0069] In some embodiments, the mixture of stereoisomers comprises at least about 10% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 20% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 30% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 40% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 50% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 60% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 70% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 75% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 80% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 85% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 90% (S) configuration product. In some embodiments, the mixture of stereoisomers comprises at least about 95% (S) configuration product.

[0070] In some embodiments, the mixture of stereoisomers comprises a ratio of (R) configuration product to (S) configuration product. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 1.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 2.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 3.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 4.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 5.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 6.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 7.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 8.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 9.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 10.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 11.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 12.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 13.0. In some embodiments, the (R) / (S) ratio of the mixture of stereoisomers is at least about 14.0.

[0071] In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 1.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 2.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 3.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 4.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 5.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 6.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 7.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 8.0. In some embodiments, the (S) / (R) ratio of the stereoisomer mixture is at least about 9.0. In some embodiments, the (S) / (R) ratio of the mixture of stereoisomers is at least about 10.0. In some embodiments, the (S) / (R) ratio of the mixture of stereoisomers is at least about 11.0. In some embodiments, the (S) / (R) ratio of the mixture of stereoisomers is at least about 12.0. In some embodiments, the (S) / (R) ratio of the mixture of stereoisomers is at least about 13.0. In some embodiments, the (S) / (R) ratio of the mixture of stereoisomers is at least about 14.0.

[0072] Substituted benzyl alcohol nucleophiles In some embodiments, this disclosure provides a method for preparing oligonucleotides using a substituted benzyl alcohol nucleophile. As used herein, a substituted benzyl alcohol nucleophile or “BnOH” refers to a benzyl alcohol containing at least one substituent on a benzene ring. Substituted benzyl alcohol nucleophiles may include primary benzyl alcohol (Bn-CH2-OH), secondary benzyl alcohol (Bn-CH(R)), etc. 7 )-OH) or tertiary benzyl alcohol (Bn-C(R) 7 (R) 8 (-OH). In some embodiments, this disclosure provides a method for preparing oligonucleotides using a substituted benzyl alcohol nucleophile of formula (v): Where: n is selected from 1, 2, 3, 4, and 5; each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 Alkyl and aryl groups.

[0073] In some implementations, R5 Each instance is an ortho- or para-substituent on the benzyl group. In some embodiments, R 5 Each instance is an ortho-substituent on a benzyl group. In some embodiments, R 5 Each instance is a para-substituent on a benzyl group. In some embodiments, R 5 Each instance is independently selected from halogens, (C1-C2) 20 )alkyl, (C1-C 20 Alkyl, trifluoromethyl, and phenyl (substituted or unsubstituted). In some embodiments, R 5 It is a fused benzene ring that forms a naphthalene ring system. In some embodiments, R 5 It is (C1-C) 16 )alkyl and (C1-C 16 )alkoxy. In some embodiments, R 5 It is (C1-C) 12 )alkyl and (C1-C 12 )alkoxy. In some embodiments, R 5 It is (C1-C8)alkyl and (C1-C8)alkoxy. In some embodiments, R 5 It consists of (C1-C4)alkyl and (C1-C4)alkoxy groups.

[0074] In some embodiments, the substituted benzyl alcohol nucleophile of formula (v) is: .

[0075] In some embodiments, this disclosure provides methods for preparing oligonucleotides using cyclic phosphate nucleosides and substituted benzyl alcohol nucleophiles. In some embodiments, this disclosure provides methods for preparing dinucleotides of formula (vi) or their salts or solvates: Where: Y is O or S; R 1 and R 2 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); B 1 and B 2 It is a nucleobase, which can be optionally protected; R 4 It is hydrogen or a protecting group; n is selected from 1, 2, 3, 4, and 5; each R 5 Independently, it is a substituent on the benzyl protecting group of the benzyl ring; R 6 It is hydrogen or a protecting group; and R 7 and R8 Independently selected from hydrogen, (C1-C 20 )alkyl and aryl. In some embodiments, R 1 and R 2 Independently selected from (C1-C 16 )alkyl and (C1-C 16 )alkoxy. In some embodiments, R 1 and R 2 Independently selected from (C1-C 12 )alkyl and (C1-C 12 )alkoxy. In some embodiments, R 1 and R 2 Independently selected from (C1-C8) alkyl and (C1-C8) alkoxy groups. In some embodiments, R 1 and R 2 Independently selected from (C1-C4)alkyl and (C1-C4)alkoxy. In some embodiments, R 1 and R 2 It is fluorine. In some implementations, R 1 and R 2 It is a methoxy group. In some embodiments, R 1 and R 2 It is O-methoxyethyl (MOE).

[0076] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof, wherein the method comprises: providing a cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group; and it reacts the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 Alkyl and aryl groups. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

[0077] In some embodiments, this disclosure provides a method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof, wherein the method comprises: (a) React the nucleoside of formula (i) with P(Y)R3, where Y is O or S, and each R is independently selected from Cl, Br, (C1-C6)alkoxy, (C6-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: ; and (c) React the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 Alkyl and aryl groups. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

[0078] In some embodiments, this disclosure provides a dinucleotide of formula (vi) or a salt or solvate thereof.

[0079] In some implementations, R 6 It is hydrogen. In some implementations, R 6 It is a protecting group. In some embodiments, R 6 It is a hydroxyl protecting group. In some embodiments, R 6 Selected from silicon-based, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl or triisopropylsilyl.

[0080] In some embodiments, the reaction of the cyclic phosphorus dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) forms a dinucleotide having [3'-5']-linked bonds (e.g., the dinucleotide of formula (vi)). In some embodiments, the reaction of the cyclic phosphorus dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) forms a dinucleotide having [5'-5']-linked bonds. In some embodiments, the reaction of the cyclic phosphorus dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) forms a mixture of isomers (e.g., constitutional isomers). In some embodiments, the isomer mixture comprises a mixture of [3'-5']-linked and [5'-5']-linked stereoisomers. In some embodiments, the isomers are isolated prior to subsequent synthetic steps.

[0081] In some embodiments, the isomer mixture comprises at least about 10% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 20% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 30% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 40% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 50% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 60% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 70% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 75% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 80% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 85% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 90% of the [3'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 95% of the [3'-5']-linked product.

[0082] In some embodiments, the isomer mixture comprises at least about 10% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 20% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 30% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 40% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 50% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 60% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 70% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 75% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 80% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 85% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 90% of the [5'-5']-linked product. In some embodiments, the isomer mixture comprises at least about 95% of the [5'-5']-linked product.

[0083] In some embodiments, the isomer mixture comprises [3'-5']-bondings and [5'-5']-bondings in an [S] ratio. In some embodiments, the [S] ratio of the isomer mixture is at least about 1.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 2.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 3.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 4.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 5.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 6.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 7.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 8.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 9.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 10.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 11.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 12.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 13.0. In some embodiments, the [S] ratio of the isomer mixture is at least about 14.0.

[0084] In some embodiments, the reaction of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in a solvent system. In some embodiments, the solvent includes: tetrahydrofuran (THF); 2-methyltetrahydrofuran (2Me-THF); diethyl ether; 1,4-dioxane; dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylacetamide (DMA), dichloromethane (DCM), acetonitrile (MeCN), methyl tert-butyl ether (MTBE), tert-amyl alcohol, toluene, or mixtures thereof.

[0085] In some embodiments, the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in the presence of a base. In some embodiments, the base comprises: n-butyllithium (n-BuLi); lithium tert-butoxide (LiOtBu); lithium bis(trimethylsilyl)amide (LiHMDS); lithium tetramethylpiperidine (LiTMP); or Li-R Z , where R Z It is alkyl or aryl; sodium tert-butoxide (NaOtBu) + lithium chloride (LiCl); potassium tert-butoxide (KOtBu) + lithium chloride (LiCl); triethylamine, diazabicycloundecene (DBU), triazabicyclodecene (TBD), or combinations thereof. In some embodiments, the base is premixed with the substituted benzyl alcohol nucleophile of formula (v) prior to the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v). In some embodiments, the base is lithium tert-butoxide (LiOtBu).

[0086] In some implementations, the dinucleotide of formula (vi) is isolated by reacting with silyl chloride.

[0087] Protected oligonucleotides In some embodiments, this disclosure provides a method for preparing phosphate-protected oligonucleotides. In some embodiments, this disclosure provides a method for preparing the oligonucleotide of formula (vii): Where: each Y is independently O or S; R 1 R 2 and R 3 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); R 4 It is an H or protecting group; n is selected from 1, 2, 3, 4 and 5; each R 5 Independently, each R is a substituent on the benzyl protecting group of the benzyl ring; 7 and R8 Independently selected from hydrogen, (C1-C 20 )alkyl and aryl; B 1 B 2 and B 3 It is a nucleobase, which may optionally be protected; m is selected from integers in the range of 1 to 18; and X is hydrogen, a protecting group, or In some implementations, R 1 R 2 and R 3 Independently selected from (C1-C 16 )alkyl and (C1-C 16 )alkoxy. In some embodiments, R 1 R 2 and R 3 Independently selected from (C1-C 12 )alkyl and (C1-C 12 )alkoxy. In some embodiments, R 1 R 2 and R 3 Independently selected from (C1-C8) alkyl and (C1-C8) alkoxy groups. In some embodiments, R 1 R 2 and R 3 Independently selected from (C1-C4)alkyl and (C1-C4)alkoxy. In some embodiments, R 1 R 2 and R 3 It is fluorine. In some implementations, R 1 R 2 and R 3 It is a methoxy group. In some embodiments, R 1 R 2 and R 3 It is O-methoxyethyl (MOE).

[0088] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (vii), wherein the method comprises: providing a dinucleotide of formula (vi); and repeating the reactions of steps (a), (b), and (c) of the method for preparing a dinucleotide of formula (vi) or a salt or solvate thereof once or more (as disclosed herein) to obtain an oligonucleotide of formula (vii): In a repeated step (b), the cyclic phosphorus nucleoside of formula (ii) is reacted with the 5'-terminal nucleoside of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

[0089] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (vii), 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-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: (c) React the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 (a) Alkyl and aryl; and (d) Repeat the reactions of steps (a), (b), and (c) one or more times to obtain the oligonucleotide of formula (vii): In a repeated step (b), the cyclic phosphorus nucleoside of formula (ii) is reacted with the 5'-terminal nucleoside of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

[0090] In some embodiments, this disclosure provides oligonucleotides of formula (vii) or their salts or solvates.

[0091] In some embodiments, X is hydrogen. In some embodiments, the R6 of the terminal nucleoside is deprotected and X is hydrogen. In some embodiments, X is a protecting group. In some embodiments, X is a hydroxyl protecting group. In some embodiments, X is selected from silyl, acyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl, or triisopropylsilyl.

[0092] In some embodiments, X is a cyclic phosphate nucleoside. , where Y is O or S. In some embodiments, X is a cyclic phosphate nucleoside, which can be used to continue the elongation of oligonucleotides by repeating the reactions in steps (a), (b) and (c) of the method for preparing dinucleotides of formula (vi) as disclosed herein.

[0093] Synthetic oligonucleotides In some embodiments, this disclosure provides methods for preparing oligonucleotides. In some embodiments, this disclosure provides methods for preparing oligonucleotides of formula (1) or their salts or solvates. Each Y is independently O or S; R 1 R 2 and R 3 Independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), N3, (C1-C) 20 )alkyl and (C1-C 20 )alkoxy groups (including methoxy and -O-methoxyethyl); B 1 B 2 and B 3 It is a nucleobase, which can be optionally protected; R 4 It is hydrogen or a protecting group; m is an integer selected from the range of 1 to 18; and X is hydrogen, a protecting group, or In some implementations, R 1 R 2 and R 3 Independently selected from (C1-C 16 )alkyl and (C1-C 16 )alkoxy. In some embodiments, R 1 R 2 and R 3 Independently selected from (C1-C 12 )alkyl and (C1-C 12 )alkoxy. In some embodiments, R 1 R 2 and R 3 Independently selected from (C1-C8) alkyl and (C1-C8) alkoxy groups. In some embodiments, R 1 R 2 and R 3 Independently selected from (C1-C4)alkyl and (C1-C4)alkoxy. In some embodiments, R 1 R 2 and R 3 It is fluorine. In some implementations, R 1 R 2 and R 3 It is a methoxy group. In some embodiments, R 1 R2 and R 3 It is O-methoxyethyl (MOE).

[0094] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (1) or a salt or solvate thereof, wherein the method comprises: providing a dinucleotide of formula (vi) or an oligonucleotide of formula (vii); and (ii) hydrogenating a dinucleotide of formula (vi) or an oligonucleotide of formula (vii); wherein the method produces an oligonucleotide of formula (1) or a salt or solvate thereof. In some embodiments, the method further comprises removing one or more protecting groups.

[0095] In some embodiments, this disclosure provides a method for preparing an oligonucleotide of formula (1) 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-C 12 ) aryloxy, S-(C1-C6)alkyl, O-aryl and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reacting the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein R 4 It is a protecting group: (c) React the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Among them: each R 5 Independently, it is a substituent on the benzyl alcohol nucleophile ring; n is selected from 1, 2, 3, 4, and 5; R 6 It is hydrogen or a protecting group; and R 7 and R 8 Independently selected from hydrogen, (C1-C 20 (d) Alkyl and aryl groups; optionally, the reactions in steps (a), (b) and (c) are repeated once or more to obtain the oligonucleotide of formula (vii): In step (b), the cyclic phosphorus nucleotide of formula (ii) is reacted with the 5'-terminal nucleotide of the oligonucleotide; and (e) the product of step (c) or step (d) is hydrogenated; wherein the method produces the oligonucleotide of formula (1) or a salt or solvate thereof. In some embodiments, the method further includes the removal of one or more protecting groups.

[0096] In some implementations, the oligonucleotide is a block polymer. As used herein, the term "block polymer" refers to an oligonucleotide comprising at least two consecutive nucleotide units that share a common structural feature (i.e., chemical or stereochemical feature) at the junction of nucleobases, sugars, and / or nucleotides. At least two consecutive nucleotide units sharing a common structural feature may be referred to as a "block".

[0097] In some embodiments, the oligonucleotides of this disclosure can be used for subsequent oligonucleotide or polynucleotide synthesis applications. In some embodiments, the oligonucleotides of this disclosure can be used as block polymers of phosphorous amide in the solid-phase synthesis of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of this disclosure can be used in enzymatic ligation reactions in the synthesis of oligonucleotides or polynucleotides.

[0098] In some embodiments, this disclosure provides a polynucleotide of formula (1) or a salt or solvate thereof, wherein m is selected from an integer in the range of 0 to 100. In some embodiments, m is selected from an integer in the range of 1 to 18. In some embodiments, m is selected from an integer in the range of 1 to 3.

[0099] In some embodiments of this disclosure, the oligonucleotide synthesis method is a liquid-phase process (i.e., carried out in a 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 completely dissolved in a solvent mixture such that no solid particles are present.

[0100] In some implementations, the method is carried out using a continuous flow. As used herein, continuous flow refers to a process in which starting materials, reagents, catalysts, bases, and all other reactants are continuously added to the reactor while products, byproducts, unreacted starting materials, and other reactants are continuously removed from the reactor.

[0101] In some embodiments, the method is carried out in a flow reactor. As used herein, a flow reactor refers to a pipe, tube, continuous stirred tank reactor (CSTR), multiple CSTRs in series, batch reactor with intermittent filling and emptying, glass microreactor, or other continuously operable reactor. In some embodiments, the method is carried out in a flow reactor at ambient temperature (e.g., 20-25°C). In some embodiments, the method is carried out in a flow reactor for a reaction time between 45 and 120 minutes. In some embodiments, the method is carried out in a flow reactor for a reaction time between 45 and 60 minutes.

[0102] limited Unless otherwise stated, the following terms and phrases have the meanings set forth below. These limitations are not intended to be restrictive in nature, but rather to provide clarity in understanding certain aspects of this disclosure.

[0103] Approximately / About: As used herein, the terms “approximately” and “about” are used interchangeably and refer to a value applied to one or more values ​​of interest that is within + / -10%. In some embodiments, unless explicitly stated otherwise or clearly apparent from the context, the term refers to a range of values ​​within + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, or less of the reference value.

[0104] Complementarity: As used herein, the term “complementarity” or “complementarity” refers to 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 structure), where this relationship allows the two nucleotides to form base pairs with each other. For example, a purine nucleotide on a nucleic acid complementary to a pyrimidine nucleotide on an opposing nucleic acid can pair together by forming hydrogen bonds. Complementary nucleotides can pair in the typical Watson-Crick manner (i.e., adenine pairs with thymine or uracil, and guanine pairs with cytosine) or in any other manner that allows for the formation of a stable duplex. Similarly, two nucleic acids can have multiple nucleotide regions that are complementary to each other to form complementary regions.

[0105] Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers to a nucleotide whose pentose sugar has a hydrogen atom at the 2' position instead of a hydroxyl group, compared to a ribonucleotide. Modified deoxyribonucleotides have one or more atomic modifications or substitutions at the 2' position other than a hydroxyl group, including modifications or substitutions in the nucleotide base, sugar, or phosphate group.

[0106] Double-stranded oligonucleotides: As used herein, the term "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide in double-stranded form. A ds oligonucleotide double-stranded region can be formed between antiparallel nucleotide sequences of covalently separated nucleic acid strands by complementary base pairing. Similarly, a ds oligonucleotide double-stranded region can also be formed between antiparallel nucleotide sequences of covalently linked nucleic acid strands by complementary base pairing. Furthermore, complementary base pairings of a ds oligonucleotide double-stranded region can also be formed from a single nucleic acid strand capable of self-folding (e.g., through a hairpin structure) to provide complementary antiparallel nucleotide sequences for base pairing. A ds oligonucleotide may comprise two fully double-stranded, covalently separated nucleic acid strands. A ds oligonucleotide may also comprise two partially double-stranded, covalently separated nucleic acid strands (e.g., with overhangs at one or both ends). A ds oligonucleotide may contain partially complementary antiparallel nucleotide sequences and therefore may contain one or more mismatches, including internal or terminal mismatches.

[0107] Double strands: As used herein, the terms “double strand” and “double strand region” for nucleic acids (e.g., oligonucleotides) refer to a structure formed by complementary base pairing of two antiparallel nucleotide sequences, whether it is formed by two covalently separated nucleic acid chains or by a single folded chain (e.g., by a hairpin structure). Double strands can form even if the two chains are not perfectly complementary or in the presence of a baseless nucleotide.

[0108] Modified internucleotide linkages: As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage with one or more chemical modifications compared to a reference internucleotide linkage having a phosphodiester bond. Modified internucleotide linkages are non-natural linkages.

[0109] Modified nucleotides: As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide, and these nucleotides are selected from: adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides, and thymidine deoxyribonucleotides. Modified nucleotides are non-natural nucleotides. Modified nucleotides may have one or more chemical modifications, for example, in their sugar, nucleotide, and / or phosphate groups. Additionally or alternatively, modified nucleotides may have one or more chemical groups conjugated to a corresponding reference nucleotide.

[0110] Nucleobase: As used herein, the term "nucleobase" refers to the heterocyclic moiety of a nucleoside (e.g., adenine in adenosine, cytosine in cytidine, guanine in guanosine, thymine in thymidine, and uracil in uridine). Nucleobases can be "typical nucleobases" or "primary nucleobases," and these terms are used interchangeably herein, including purine bases adenine (A) and guanine (G) and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Nucleobases can also be "atypical nucleobases," which are synthetic or native nucleobases that contain atypical nucleobases.

[0111] Nucleoside: As used herein, the term “nucleoside” refers to an organic compound having a nucleobase (such as adenine, cytosine, guanine, thymine, or uracil) covalently linked to a pentose sugar (such as ribose or 2'-deoxyribose).

[0112] Nucleotides: As used herein, the term "nucleotide" refers to a phosphorylated form of a nucleoside (e.g., a phosphate ester or thiophosphate ester of a nucleoside). Nucleotides can serve as monomeric units of nucleic acid polymers (e.g., oligonucleotides) (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).

[0113] Oligonucleotides: As used herein, the term "oligonucleotide" refers to a polymer of linked nucleotides, each polymer containing typical or atypical nucleobases. Oligonucleotides are typically less than about 100 nucleotides in length. Oligonucleotides can be single-stranded (ss) or double-stranded (ds). Oligonucleotides may or may not have double-stranded regions. Oligonucleotides may comprise dinucleotide or trinucleotide polymers. As used herein, the term "polynucleotide" refers to a polymer of linked nucleotides that may be longer than about 100 nucleotides in length. As used herein, the term "dinucleotide" comprises two linked nucleosides connected by one or more phosphate-based bonds (e.g., a dinucleotide with two nucleosides and a cyclic phosphorus, phosphate, or thiophosphate bond). As used herein, the term "trinucleotide" comprises three linked nucleosides connected by two or more phosphate-based bonds (e.g., a trinucleotide with three nucleosides and a cyclic phosphorus, phosphate, or thiophosphate bond).

[0114] Phosphate ester analogs: As used herein, a “phosphate ester analog” refers to a chemical group that mimics the electrostatic and / or steric properties of a phosphate group. Examples of phosphate ester analogs include, but are not limited to, 5' phosphate esters, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). Oligonucleotides may have a phosphate ester analog (referred to as a “4'-phosphate ester analog”) at the 4'-carbon position of the sugar, or may substitute the 5'-phosphate ester at the 5'-terminal nucleotide. An example of a 4'-phosphate ester analog is oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to a sugar group (e.g., at its 4'-carbon) or an analog thereof. See, for example, International Patent Application No. WO 2018 / 045317. Other modifications to the 5' end of oligonucleotides have been developed (see, for example, International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nuc. AcidsRes. 43:2993-3011).

[0115] Protecting group: As used herein, the term "protecting group" (or "protective group") refers to a group known in the art that protects a reactive or unstable group (e.g., hydroxyl, amino, and thiol groups) on a molecule from undesirable chemical modifications during synthetic procedures and other chemical reactions. Protecting groups are typically used selectively and / or orthogonally to protect unstable sites in a molecule from undesirable modifications during chemical reactions at other reactive sites within the molecule. The protecting group can then be removed, leaving the unstable group unprotected and usable for other reactions.

[0116] Ribonucleotide: As used herein, the term "ribonucleotide" refers to a nucleotide whose pentose sugar is ribose and which contains a hydroxyl group at the 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions at an atom other than hydrogen at the 2' position, including modifications or substitutions to or on a nucleotide base, sugar, or phosphate group.

[0117] Substituted benzyl alcohol nucleophiles: As used herein, the term “substituted benzyl alcohol nucleophile” or “BnOH” refers to a benzyl alcohol containing at least one substituent on a benzene ring. Substituted benzyl alcohol nucleophiles may include primary benzyl alcohol (Bn-CH2-OH), secondary benzyl alcohol (Bn-CH(R7)-OH) or tertiary benzyl alcohol (Bn-C(R7)(R8)-OH).

[0118] Chain: As used herein, the term "chain" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds or thiophosphate bonds). A chain may have two free ends (e.g., a 5' end and a 3' end).

[0119] Synthesis: As used in the context of the products of this document (e.g., synthetic oligonucleotides, synthetic nucleobases), the term “synthesis” means artificially synthesized (e.g., using a machine, such as a solid-phase nucleic acid synthesizer) or nucleic acids or other compounds that are not derived from natural sources (e.g., cells or organisms) that typically produce nucleic acids or other compounds.

[0120] General considerations Those skilled in the art will be able to recognize or determine one or more equivalent forms of the specific embodiments of this disclosure described herein through routine experimentation. The scope of this disclosure is not intended to be limited to the foregoing description or the following examples.

[0121] At various locations within this disclosure, substituents or properties of the compounds of this disclosure are disclosed within groups or ranges. This disclosure is intended to cover every individual or subcombination of members in these groups and ranges, and these groups or ranges include endpoint values. By way of a non-limiting example, if a group or range is from about 1 to about 10, then the group or range includes values ​​of about 1 and about 10.

[0122] In this document, for example, “a,” “an,” or “the” may refer to one or more, unless the context otherwise indicates or specifies. A claim or statement containing “or” between one or more members of a group is deemed to satisfy a requirement if one, more, or all of the group members are present in, used in, or otherwise associated with a given product or process, unless the context otherwise indicates or specifies. This disclosure may include embodiments in which exactly one group member is present in, used in, or otherwise associated with a given product or process. This disclosure may include embodiments in which one or more or all of the group members are present in, used in, or otherwise associated with a given product or process.

[0123] The term “comprising” is intended to be an open-ended expression, allowing, but not requiring, the inclusion of additional elements or steps. When the term “comprising” is used herein, the terms “consisting of” and “substantially consisting of” are also covered and disclosed.

[0124] The abbreviation “eg” comes from the Latin “exempli gratia” and is used in this text to indicate a non-restrictive instance. Therefore, the abbreviation “eg” is a synonym of the term “for example”. The abbreviation “ie” comes from the Latin “id est” and is used in this text to indicate a non-restrictive paraphrase or explanation. Therefore, the abbreviation “ie” is a synonym of the term “that is”.

[0125] Any particular embodiment of the present disclosure that falls into the prior art is expressly excluded from any one or more claims. Regardless of the existence of prior art, any particular embodiment of the agents, methods, and / or compositions of the present disclosure is excluded from any one or more claims for any reason.

[0126] This specification will be limited to incorporating references to publications, patent applications, patents and other references mentioned herein by way of citation, and to handling any conflicting information herein.

[0127] The chapter titles, materials, methods, and examples are for illustrative purposes only and are not intended to be restrictive. Example

[0128] Unless otherwise stated, all reactions in the following examples were performed in a chemical fume hood under a nitrogen or argon atmosphere. Anhydrous acetonitrile (MeCN) was purchased from Sigma-Aldrich. HPLC-grade toluene was purchased from Sigma-Aldrich and further purified by continuous filtration through a neutral alumina and CuO column under argon pressure. Reactions were carried out in oven-dried reaction tubes (Fisherbrand, 16 × 125 mm, product number 1495935A) sealed with a screw cap (Thermo Fisher Scientific, catalog number B7995-18) and fitted with a Teflon-lined diaphragm (Thermo Fisher Scientific, catalog number C47995-15). Solvents used for extraction, crystallization, and column chromatography were all ACS-grade from Sigma-Aldrich, except for hexane, which was HPLC-grade. Lithium tert-butoxide (LiOt-Bu) was purchased from Alfa Aesar and stored in a nitrogen-filled glove box. All preparative reactions were conducted in a nitrogen-filled glove box, with the sealed reaction vials removed and stirred for the specified duration. Reaction conditions and mechanistic experiments were screened using the nitrogen-filled glove box. Organic compounds were purified by rapid column chromatography using Silicycle SiliaFlash P60 (230-400 mesh) silica gel, either manually or using a CombiFlash NextGen 300 automated chromatography system.

[0129] For general analysis, CDCl3 and d6-DMSO were purchased from Cambridge Isotope Labs. NMR spectra were acquired on a Bruker Avance III HD 400 or 500 MHz spectrometer. 1 H (CDCl3: δ 7.26; d6-DMSO: δ2.50) and 13 10⁻⁶ C NMR shifts (CDCl₃: δ 77.16; d⁶-DMSO: δ 39.52) are referenced to the residual solvent peak. The following abbreviations are used to indicate multiplicity: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, p = quintet, sept = septet, m = multiplet. 1 H decoupling obtains 13 C and 19F-spectroscopy. Gas chromatography (GC) analysis was performed using a J&W DB-1 column (10 m, 0.1 mm ID) on an Agilent 7890A gas chromatograph equipped with an FID detector. LC / MS was recorded on an Agilent 6120 quadrupole LC / MS. Elemental analysis was performed by Atlantic Microlabs Inc., Norcross, GA, USA. High-resolution mass spectra were recorded on a JEOL AccuTOF LC-Plus 46 DART system and an Agilent Technologies 6545 Q-TOF LC / MS system. IR spectra were recorded on a Nicolet iS5 spectrometer from Thermo Fisher Scientific equipped with an iD5 diamond indentation ATR accessory. IR spectra were obtained from purified samples. Melting points were determined using a Stanford Research Systems EZ-Melt melting point apparatus.

[0130] Example 1. Preparation of the matrix – C, A, G, U a. Synthesis of 3',5'-bis-TBS protected nucleosides (GP1) Nucleoside 'B' (U, C, A, or G, 1 equivalent) and imidazole (4 equivalents) were charged into a 50 mL round-bottom flask equipped with a magnetic stir bar and dried under vacuum using a standard Schlenk line technique. The mixture was dissolved in anhydrous DMF (0.5–1 M, depending on solubility) and tert-butyldimethylchlorosilane (3 equivalents) was added. The reaction mixture was stirred at room temperature under an inert atmosphere for 18 hours. After the reaction was complete (monitored by TLC or LC-MS analysis), the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography, eluting with ethyl acetate / hexane (6:4). The fraction containing the product was collected and evaporated.

[0131] b. Synthesis of benzyl-protected nucleosides (GP2) To a 100 mL round-bottom flask equipped with a magnetic stir bar, add bis-TBS-protected cytidine (1 equivalent) and anhydrous DMF (0.3 M). Cool the resulting solution to 0 °C and add NaH. Stir the resulting solution for 5 min, then add benzyl bromide dropwise at 0 °C. Monitor the reaction progress by LC-MS. After 45 min, add the second portion of NaH and stir for another 1 h at 0 °C. Heat the reaction mixture to room temperature and stir for 30 min, then quench with NH4Cl (50 mL). Extract the resulting mixture with EtOAc (100 mL × 3). Wash the combined organic layers with brine (100 mL), dry to Na2SO4, filter, and concentrate under vacuum. Purify the crude product using silica gel column chromatography with hexane / ethyl acetate as eluent (0-100% gradient) on a Combi-Flash NextGen 300 automated chromatography system.

[0132] c. Synthesis of 3',5'-dihydroxynucleosides protected by TBS (GP3) To a 100 mL round-bottom flask equipped with a magnetic stir bar, add 4 mmol of bis-TBS-polybenzyl-protected nucleoside and 20 mL of anhydrous THF. Cool the reaction mixture to 0 °C and add 8.2 mmol of tetrabutylammonium fluoride (TBAF, 1 M solution in THF, 8.2 mL) dropwise. Stir the reaction mixture at 0 °C for 1 hour. Monitor the reaction by LC-MS. After the reaction is complete, quench with 10 mL of NH4Cl and dilute with 10 mL of water. Extract the mixture with ethyl acetate (100 mL × 3). Then concentrate the mixture under reduced pressure. Purify the crude product by rapid silica gel column chromatography eluting with dichloromethane-methanol (0 to 5% gradient) (for C) or hexane-ethyl acetate (0 to 100% gradient, for A, G) to give the final product compound as a white solid (yield 87-97%).

[0133] d. Synthesis of 3-OTBS-5-OH-nucleoside via selective deprotection of the 5'-siloxy group of bis-TBS-nucleoside (GP4) The isolated or crude bis-TBS protected nucleosides (U, dibenzyl-C, -A, and tribenzyl-G; 1 equivalent) were dissolved in THF (0.2 M) and treated with TFA (5 equivalents) in H2O (TFA:H2O, 1:1 mixture) at 0 °C. The reaction was monitored by LC-MS. After the reaction was completed in 4–6 h, the reaction solution was diluted with an equal volume of water and the pH was adjusted to 7–8 with solid sodium bicarbonate. The aqueous layer was extracted with ethyl acetate (3 × 150 mL) and washed with brine (100 mL). The combined organic layers were dried over sodium sulfate and concentrated with the aid of a rotary evaporator. The residue was purified by silica gel column chromatography eluting with hexane-ethyl acetate [0–100% gradient] to give 5'-OH, 3-TBS-nucleosides (70–80% yield).

[0134] e. One-pot procedure for the synthesis of 3-OTBS-5-OH-nucleosides from nucleosides (GP5) 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione, S1 Imidazole (5.80 g, 85.0 mmol) was added to a DMF (30 mL) solution of 2'-O-methyluridine (10 g, 39 mmol), and the solution was cooled to 5°C. TBSCl (12.8 g, 85.0 mmol) was slowly added to the solution over 15 minutes. The reaction mixture was then heated to 23°C, and the reaction was observed to be complete within 20 hours. The reaction mixture was transferred to a separatory funnel containing 100 mL of ethyl acetate and washed with water (5 × 250 mL) to remove DMF. The organic layer was dried over sodium sulfate and concentrated to dryness. The white solid was redissolved in dichloromethane (100 mL) and treated with TFA (18.2 g, 160 mmol). After 20 hours, the reaction mixture was poured into a flask containing 300 mL of water, and the pH was adjusted to pH 8 with solid sodium bicarbonate. The aqueous layer was extracted with dichloromethane (3 × 150 mL). The dichloromethane layer was dried with sodium sulfate and concentrated to 30 mL or until a solid began to precipitate. The concentrated solution was then transferred to a flask equipped with a top stirrer and treated with 70 mL of hexane. The solution was allowed to stand for 3 hours, and the solid was collected through a filter funnel and the filter cake was washed with 100 mL of hexane. The solid was dried in a vacuum oven to give a white solid (9.9 g, 68%). The purity of the crude solid was found to be 95% by HPLC and it was used directly without further purification. Its structure was confirmed by proton NMR and MS analysis and found to be consistent with the data in the literature (Chem. Commun. 2021, 57(55), 6808-6811).

[0135] 1 H 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).

[0136] 13 C 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.

[0137] f. Benzyloxymethyl acetal (BOM) protection of nucleosides (GP6) 1-(4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (7.11 g, 25.0 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar and containing 50.0 mL of DMF. The resulting solution was treated with DBU (4.85 mL, 32.5 mmol, 1.3 equivalents) and BOMCl (4.50 mL, 32.5 mmol, 1.3 equivalents) at 0 °C for 40 min. The reaction progress was monitored by TLC. After the reaction was complete, the reaction was terminated with H2O (50 mL), and the resulting mixture was extracted with EtOAc (150 mL × 4). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography using a Combi-Flash NextGen 300 automated chromatography system with DCM / MeOH as the eluent (0-5% MeOH-DCM gradient).

[0138] g. DMF-formamidin protection of nucleoside NH2 (GP7) Adenosine-ketal (1 equivalent) or 2'-OMe-cytidine (1 equivalent) was added to a 100 mL RB flask equipped with a magnetic stir bar and dried in an oven. Then, 1,1-dimethoxy-N,N-dimethylmethylamine (5 equivalents, 32.5 mmol) was added in N,N-dimethylformamide (DMF, 10 mL, 0.65 mol). After stirring at room temperature for 18 hours, the reaction mixture was concentrated to dryness (20 × 2 mL toluene was added to aid in the evaporation of DMF under reduced pressure), and a coarse white solid was ground with diethyl ether (3 × 100 mL). The solid was collected by filtration and dried under high vacuum for 12 hours to give a grayish-white solid product with a purity >90% (yield 90-95%).

[0139] h. N-alkylation of uridine 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-3-(3-methylbut-2-en-1-yl)pyrimidin-2,4(1H,3H)-dione 2'-O-methyluridine (5.96 g, 23.069 mmol, 1.0 equivalent), 1-bromo-3-methylbut-2-ene (4.77 mL, 6.15 mmol, 1.79 equivalent), and potassium carbonate (6.35 g, 45.96 mmol, 2.0 equivalent) were added to a 50 mL round-bottom flask equipped with a magnetic stir bar and containing 30.0 mL of DMF. The reaction mixture was stirred at 60 °C for 4 hours, then 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 using acetone / hexane = 1:1 as eluent. The purified solution was then dried under vacuum overnight to give the title compound as a white solid in 54% yield. Its identity was confirmed by proton NMR and MS analysis, and it was found to be consistent with the data in the literature (J. Am. Chem. Soc. 2017, 139, 5467-5473).

[0140] 1 H 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 = 7.3Hz, 1H), 1.82 (d, J = 1.4 Hz, 3H), 1.72 (d, J = 1.6 Hz, 3H)。

[0141] 13 C 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。

[0142] i. Bi-Boc protection of cytidine j. Bi-Boc protection of adenosine k. Phthalimide protection of adenosine l. Guanosine protection Example 2. Spectra of a specific monomer a. DMF-A-diol 1 H 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 = 0.6Hz, 3H)。

[0143] 13 C 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。

[0144] b. 3-TBS-G-DMF-amide 1 H 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)。

[0145] c. 3-TBS-ABn2 1 H 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, 1.6Hz, 1H), 3.74 (d, J = 13.0 Hz, 1H), 3.29 (s, 3H), 0.96 (s, 9H), 0.16 (d, J = 8.6Hz, 6H)。

[0146] d. 3-TBS-T-BOM 1 H 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)。

[0147] 13 C 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。

[0148] e. 3-TBS-N-Allyl-U-diol 1 H 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)。

[0149] 13 C 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。

[0150] f. Adenosine-N-methylammonium 1 H 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).

[0151] 13 C 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.

[0152] Example 3. Synthesis of cyclochlorophosphate nucleoside (GP8) a. Study on the synthesis conditions of cyclochlorophosphite In a 100 mL Schlenk flask, dried in an oven and equipped with a magnetic stir bar, add a protected solution of diol-nucleoside (1 equivalent, 2 mmol), DMAP (0.1–0.2 equivalent, 60–120 mg, 0.25–0.5 mmol), and 18 mL of DCM (0.1 M, 0.1 M). Add triethylamine or S-Collidine (2.2 equivalent, 4.4 mmol, dissolved in 1 mL of DCM) to the resulting suspension using a syringe. Cool the solution to -20 °C and add freshly distilled POCl3 (1.05 equivalent, 2.1 mmol, dissolved in 1 mL of DCM) in a single batch. Stir the solution for 1 hour, then heat to 0 °C and stir overnight. Aliquots of the reaction mixture are collected and analyzed using… 31 P NMR analysis was performed to monitor the diastereoselectivity of the crude product. The reaction mixture was stirred further at room temperature for 2–4 hours until the diastereomeric ratio (dr) of the crude product reached >20:1. Once the dr of the crude product reached >20:1, the reaction mixture was diluted with 10 mL of freshly distilled diethyl ether to precipitate the amine salt. The resulting suspension was filtered through a glass frit funnel and the residue was washed with diethyl ether (10 × 2 mL). The filtrate was concentrated with the aid of a rotary evaporator. The crude product was purified by silica gel column chromatography (the crude product was dissolved in a minimal amount of DCM before loading onto the column, approximately 2 inches high and 0.9 inches in diameter) using a Combi-Flash NextGen 300 automated chromatography system, with a hexane:ethyl acetate gradient (0 to 80%).

[0153] The variations in synthetic conditions are summarized in Table 1. The yields and diastereomeric ratios (dr) of the obtained cyclochlorophosphate nucleosides are also summarized in Table 1.

[0154] Table 1 - Synthesis conditions of cyclochlorophosphate nucleosides b. Research on nucleosides used in the synthesis of cyclochlorophosphite Cyclochlorophosphate nucleosides were synthesized according to the general procedure in Example 3a, but using different nucleosides. The obtained cyclochlorophosphate nucleosides and their yields and diastereomeric ratios (dr) are as follows: c. Synthesis of guanosine cyclic phosphate Example 4. Synthesis of specific cyclochlorophosphate nucleosides a. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro- 4H-furano[3,2-d][1,3,2]dioxopyrazine-6-yl)pyrimidin-2,4(1H,3H)-dione The compound was synthesized in DCM (35 mL, 0.14 M) using U-BOM (1.85 g, 4.9 mmol, 1 equivalent), triethylamine (1.09 g, 10.76 mmol, 2.2 equivalent), DMAP (120 mg, 0.098 mmol, 0.2 equivalent), and POCl3 (824 mg, 5.37 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-25 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 80%), yielding 1380 mg of the desired product as a white solid. Yield = 62%, dr = 38:1.

[0155] 31 P NMR (162 MHz, CDCl3) δ -1.12, -1.72.

[0156] 1 H 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).

[0157] 13 C 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.

[0158] HR-MS was not performed because the compound was hydrolyzed during the analysis.

[0159] b. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-fluoro-2-oxotetrahydro-4H-furyl [3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione The compound was synthesized in DCM (25 mL, 0.1 M) using U-BOM (900 mg, 2.46 mmol, 1 equivalent), triethylamine (522 mg, 5.17 mmol, 2.1 equivalent), DMAP (60 mg, 0.049 mmol, 0.2 equivalent), and POCl3 (414 mg, 2.70 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 80%), yielding 530 mg of the desired product as a white solid. Yield = 48%, dr = 18:1.

[0160] 31 P NMR (162 MHz, CDCl3) δ -1.62, -2.04.

[0161] 1 H 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).

[0162] HR-MS was not performed because the compound was hydrolyzed during the analysis.

[0163] c. 1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1, [3,2]dioxophosphazenecyclohexane-6-yl)-4-(dibenzylamino)pyrimidine-2(1H)-one The compound was synthesized in DCM (21 mL, 0.1 M) using C-Bn2 (870 mg, 2.46 mmol, 1 equivalent), triethylamine (443 mg, 4.37 mmol, 2.2 equivalent), DMAP (24 mg, 0.02 mmol, 0.1 equivalent), and POCl3 (335 mg, 2.19 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), yielding 304 mg of the desired product as a white solid. Yield = 30%, dr = 22:1.

[0164] 31 P NMR (162 MHz, CDCl3) δ -0.96, -1.11.

[0165] 1 H 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).

[0166] 13 C 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.

[0167] HR-MS was not performed because the compound was hydrolyzed during the analysis.

[0168] d. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7aS)-2-chloro-2-oxotetrahydro-4H-furano[3,2- [d][1,3,2]dioxophosphazenecyclohexane-6-yl)-5-methylpyrimidine-2,4(1H,3H)-dione The compound was synthesized in DCM (60 mL, 0.12 M) using T-BOM (2.5 g, 7.18 mmol, 1 equivalent), triethylamine (1.598 g, 15.79 mmol, 2.2 equivalent), DMAP (175 mg, 1.43 mmol, 0.1 equivalent), and POCl3 (1.21 g, 7.89 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-25 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 85%), yielding 1700 mg of the desired product as a white solid. Yield = 55%, dr = 16:1.

[0169] 31 P NMR (162 MHz, CDCl3) δ -1.48, -1.98.

[0170] 1 H 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).

[0171] 13 C 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.

[0172] HR-MS was not performed because the compound was hydrolyzed during the analysis.

[0173] e. (2S,4aR,6R,7R,7aR)-2-chloro-6-(6-chloro-9H-purin-9-yl)-7-methoxytetrahydro-4H-furan [3,2-d][1,3,2]dioxophosphazenecyclohexane 2-oxide The compound was synthesized in DCM (30 mL, 0.12 M) using 2-OMe-A(6Cl)-diol (1050 mg, 3.5 mmol, 1 equivalent), triethylamine (777 mg, 7.68 mmol, 2.2 equivalent), DMAP (43 mg, 1.43 mmol, 0.1 equivalent), and POCl3 (589 mg, 3.84 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 85%), yielding 620 mg of the desired product as a white solid. Yield = 47%, dr = 10:1.

[0174] 31 P NMR (162 MHz, CDCl3) δ -1.16, -1.76.

[0175] 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).

[0176] 13 C 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.

[0177] HR-MS was not performed because the compound was hydrolyzed during the analysis.

[0178] f. (2S,4aR,6R,7R,7aR)-2-chloro-6-(6-(dibenzylamino)-9H-purin-9-yl)-7-methoxytetracycline Hydrogen-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane 2-oxide The compound was synthesized in DCM (45 mL, 0.11 M) using A-Bn2 (2.2 g, 4.77 mmol, 1 equivalent), triethylamine (1.061 g, 10.49 mmol, 2.2 equivalent), DMAP (58 mg, 0.47 mmol, 0.1 equivalent), and POCl3 (804 mg, 5.24 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-25 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), yielding 1220 mg of the desired product as a white solid. Yield = 47%, dr = 18:1.

[0179] 31 P NMR (203 MHz, CDCl3) δ -1.01, -1.26.

[0180] 1 H 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).

[0181] 13 C 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.

[0182] HR-MS was not performed because the compound was hydrolyzed during the analysis.

[0183] g. 1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1, [3,2]dioxaphosphazenecyclohexane-6-yl)-3-(3-methylbut-2-en-1-yl)pyrimidin-2,4(1H,3H)-dione The title compound was synthesized in DCM (20 mL, 0.1 M) using U-allyl-diol (650 mg, 2 mmol, 1 equivalent), S-coradine (507 mg, 4.18 mmol, 2.1 equivalent), DMAP (49 mg, 0.4 mmol, 0.2 equivalent), and POCl3 (804 mg, 5.24 mmol, 1.1 equivalent) according to the general procedure GP8. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), yielding 400 mg of the desired product as a white solid. Yield = 49%, dr = 14:1.

[0184] 31 P NMR (162 MHz, CDCl3) δ -1.08, -1.71.

[0185] 1 H 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).

[0186] HR-MS was not performed because the title compound was hydrolyzed during the analysis.

[0187] Example 5. Synthesis of Cyclic Dinucleotides a. Multi-pot synthesis - Method A (GP9) In a glove box filled with N2, LiOtBu (1 equivalent, 1 mmol) was added to a 20 mL reaction tube A equipped with a magnetic stir bar. The reaction tube was then removed from the glove box and placed under a nitrogen atmosphere using the Schlenk line technique, maintained at -20°C with the aid of a refrigerator. A stock solution of 5'-OH-nucleoside (1 equivalent, 1 mmol) in toluene (5 mL, 0.2 M) was added to reaction tube A. The resulting reaction solution was stirred at -20°C for 30 minutes.

[0188] Add 1.2 equivalents (1.2 mmol) of cyclochloronucleotide phosphate and 5 mL of MeCN to another 20 mL reaction tube B equipped with a magnetic stir bar. Cool the reaction mixture to -20°C with the aid of a refrigerator. While stirring under a nitrogen atmosphere, transfer the pre-stirred solution of the alkoxide in reaction tube A dropwise over 5 minutes using a syringe. Stir the resulting reaction mixture at -20°C for 18 hours. If incomplete conversion is observed, warm the reaction mixture to room temperature over 30 minutes to ensure 100% conversion. 31 The reaction was tracked by P NMR. After the reaction was complete, the crude reaction mixture was concentrated under reduced pressure and purified by a Combi-Flash NextGen 300 automated chromatography system using hexane:ethyl acetate (0% to 100% gradient). Alternatively, for more polar compounds, DCM:acetonitrile (containing 2% isopropanol) or DCM:ethyl acetate (containing 2% MeOH) could be used. For adenosine and cytidine containing cyclic dinucleotides, DCM:methanol (0% to 10%) was used as the eluent.

[0189] b. Multi-pot synthesis - Method B (GP10) Under stirring conditions at -20°C, a solution of 5'-OH-nucleoside (1 equivalent, 1 mmol) and triethylamine (1.1 equivalent, 1.1 mmol) in dichloromethane (2.5 mL) was added dropwise over a 25-minute period to reaction tube A containing a solution of DCM (2.5 mL) containing POCl3 (1 equivalent, 1 mmol). The resulting solution was stirred for 30 minutes.

[0190] Add the protected diol-nucleoside (1 equivalent, 1 mmol) and DMAP (2.2 equivalents, 2.2 mmol) in DCM (5 mL, 0.2 M) to another reaction tube B equipped with a magnetic stir bar. Cool the resulting suspension to -78°C. Transfer the pre-stirred solution from reaction tube A to reaction tube B through a sleeve. Stir the resulting mixture at -78°C for 1 hour, then slowly heat to room temperature while stirring for another hour. Monitor the reaction by 31P NMR. After the reaction is complete, dilute with diethyl ether to precipitate the salt and filter through a glass frit funnel. Concentrate the filtrate under reduced pressure. Purify the crude product using a Combi-Flash NextGen 300 automated chromatography system with a silica gel column and a hexane:acetone gradient (0% to 100%, with the crude product dissolved in a minimum amount of DCM and loaded onto the column, approximately 3 inches high and 0.9 inches in diameter). Alternatively, for more polar compounds, use a DCM:acetone system. For adenosine containing cyclic dinucleotides, use DCM:MeOH as the eluent (0% to 5%).

[0191] c. Study on reaction conditions for the synthesis of cyclic dinucleotides Cyclic dinucleotides were synthesized according to the general procedure of Example 5b, but under various synthetic conditions as outlined in Table 2. The yields of the obtained cyclic dinucleotides (based on...) 31 P NMR is also summarized in Table 2.

[0192] Table 2 - Conditions for Cyclic Dinucleotide Synthesis d. Research on nucleosides used for the synthesis of cyclic dinucleotides Cyclic dinucleotides were synthesized according to the general procedure of Example 5b, but using different nucleosides. The resulting cyclic dinucleotides and their yields and / or diastereomeric ratios (dr) are as follows [report the yields and dr of the isolated materials; show the structures of the major diastereomers]. e. One-pot synthesis - Method C (GP11) Under stirring conditions at -20°C, a solution of 5'-OH-nucleoside (1 equivalent, 1 mmol) and triethylamine (1.1 equivalent, 1.1 mmol) in dichloromethane (2.5 mL) was added dropwise to reaction tube A containing a solution of POCl3 (1 equivalent, 1 mmol) in DCM (2.5 mL) over a 25-minute period. The resulting solution was stirred for 30 minutes.

[0193] Add diol (1 equivalent, 1 mmol) and DMAP (2.2 equivalents, 2.2 mmol) in DCM (5 mL, 0.2 M) to another reaction tube B equipped with a magnetic stir bar. Cool the resulting suspension to -78°C. Transfer the pre-stirred solution from reaction tube A to reaction tube B through a sleeve. Stir the resulting mixture at -78°C for another hour, then slowly raise the temperature to room temperature while continuing to stir for another hour. 31 The reaction was monitored by P NMR. After the reaction was complete, the solution was diluted with diethyl ether to precipitate the salt and filtered through a glass frit funnel. The resulting filtrate was concentrated under reduced pressure. The crude product was purified using a silica gel column on a Combi-Flash NextGen 300 automated chromatography system with a hexane:acetone gradient (0% to 100%). The crude product was dissolved in a minimal amount of DCM and then loaded onto a column approximately 3 inches high and 0.9 inches in diameter. Alternatively, for more polar compounds, DCM:acetone can be used. For adenosine containing cyclic dinucleotides, DCM:MeOH was used as the eluent (0% to 5%).

[0194] f. Synthesis of guanosine dimer Example 6. Synthesis of a specific cyclic dinucleotide a. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ... (Oxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methyl (Oxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazene Cyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione By 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 equivalent), LiOtBu (72 mg, 0.89 mmol, 1.1 equivalent), 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-Cl-CP, 410 mg, 0.89 mmol, 1.1 equivalent), LiOtBu (72 ... The compound was synthesized in toluene:ACN (1:1) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), yielding the desired product as a white solid in two fractions. Yields = F2: 66%, dr = 20:1; F1: 17%, 15:1. Overall yield = 83%.

[0195] 31 P NMR (162 MHz, CDCl3) δ -3.76.

[0196] 1 H 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).

[0197] 13 C 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.

[0198] HR-MS (Q-TOF, ESI) calculated values ​​[C 42 H 55 N4O 15 PSi [M+H + ]: 937.3063, measured value 937.3009.

[0199] b. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ... (Oxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methyl (Oxytetrahydrofuran-2-yl)methoxy)-7-fluoro-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane Alkyl-6-pyrimidin-2,4(1H,3H)-dione The compound was synthesized using 3-OTBS-U-BOM (200 mg, 0.41 mmol, 1 equivalent), LiOtBu (34 mg, 0.43 mmol, 1.05 equivalent), and FU-BOM-Cl-CP (200 mg, 0.45 mmol, 1.1 equivalent) in toluene:ACN (1:1) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), yielding the desired product as a white solid. Yield = 77%, dr = 5:1.

[0200] 31P NMR (162 MHz, CDCl3) δ -4.29, -6.86.

[0201] 1 H 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).

[0202] HR-MS (Q-TOF, ESI) calculated value C 41 H 52 FN4O 14 PSi [M+H + ]: 903.3044, measured value 903.3038.

[0203] 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-oxotetracycline) Hydrogen-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-2-yl)oxy)methyl)-3-methoxytetrahydrofuran-2- 2,4(1H,3H)-pyrimidine-2,4(1H,3H)-dione The compound was synthesized using 3-OTBS-U-BOM (200 mg, 0.41 mmol, 1 equivalent), LiOtBu (36 mg, 0.44 mmol, 1.1 equivalent), and C-Bn2-Cl-CP (235 mg, 0.45 mmol, 1.12 equivalent) in toluene:ACN (1:1) according to a general procedure. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, DCM-EA (containing 1% MeOH) gradient (0% to 100%), yielding the desired product as a white solid. Yield = 250 mg, 63%, dr = 10:1.

[0204] 31 P NMR (162 MHz, CDCl3) δ -3.55, -6.36.

[0205] 1 H 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, 8.0Hz, 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, 5.5Hz, 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).

[0206] 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.

[0207] HR-MS (Q-TOF, ESI) calculated value C 48 H 60 N5O 13 PSi, [M+H + ]: 974.3768, measured value 974.3761.

[0208] d. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((2R,4aR,6R,7R,7aR)-6-(6-chloro-9H-purin-9-yl)-7-methoxy-2-oxotetrahydro-4H-furano[3, 2-d][1,3,2]dioxophosphazenecyclohexane-2-yl)oxy)methyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-Diketone The compound was synthesized using 3-OTBS-U-BOM (400 mg, 0.81 mmol, 1 equivalent), LiOtBu (72 mg, 0.89 mmol, 1.1 equivalent), and A(6Cl)-Cl-CP (371 mg, 0.97 mmol, 1.2 equivalent) in toluene:ACN (1:1) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), yielding the desired product as a white solid in two fractions. Yields: 500 mg, 74%, dr = 15:1.

[0209] 31 P NMR (162 MHz, CDCl3) δ -4.02, -6.82.

[0210] 1 H 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).

[0211] 13 C 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.

[0212] HR-MS (Q-TOF, ESI) calculated value C 35 H 46 ClN6O 12 PSi, [M+Na + ]: 859.2261, measured value 859.2256.

[0213] e. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert-butyl) (dimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy (-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4 (1H,3H)-dione The compound was synthesized using 3-OTBS-A-Bn (350 mg, 0.6 mmol, 1 equivalent), LiOtBu (59 mg, 0.73 mmol, 1.2 equivalent), U-BOM-Cl-CP (335 mg, 0.73 mmol, 1.2 equivalent), and toluene:ACN (1:1, 7 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, DCM:MeOH gradient (0% to 5%), yielding the desired product as a white solid. Yield = 530 mg, 87%, dr = 22:1.

[0214] 31 P NMR (162 MHz, CDCl3) δ -3.99, -6.90.

[0215] 1 H 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).

[0216] 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.

[0217] HR-MS (Q-TOF, ESI) calculated value C 49 H 60 N7O 12 PSi, [M+H + ]: 998.388, measured value 998.3893.

[0218] f. 3-((benzyloxy)methyl)-1-((2R,4S,5R)-5-(((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)-1-((2R,4S,5R)-5-((((2R,4aR,6R,7R,7aR)-6-(3-(benzyloxy)methyl)-1-((2R (Oxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxotetrahydro-4H-furano[3, 2-d][1,3,2]dioxophosphanecyclohexane-2-yl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran- 2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione The compound was synthesized using 3-OTBS-T-BOM (250 mg, 0.52 mmol, 1 equivalent), LiOtBu (51 mg, 0.63 mmol, 1.2 equivalent), U-BOM-Cl-CP (289 mg, 0.63 mmol, 1.2 equivalent), and toluene:ACN (1:1, 6 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient elution (0% to 100%), yielding the desired product as a white solid. Yield = 420 mg, 89%, dr = 22:1.

[0219] 31 P NMR (162 MHz, CDCl3) δ -3.60, -6.41.

[0220] 1 H 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, 5.3Hz, 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)。

[0221] 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.

[0222] HR-MS (Q-TOF, ESI) calculated value C 42 H 55 N4O 14 PSi, [M+H + ]: 899.3295, measured value 899.3288.

[0223] g. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((2R,4aR,6R,7R,7aR)-6-(6-(dibenzylamino)-9H-purine-9-yl)-7-methoxy-2-oxotetrahydro-4H-furyl) [3,2-d][1,3,2]dioxophosphazenecyclohexane-2-yl)oxy)methyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione The compound was synthesized using 3-OTBS-U-BOM (400 mg, 0.81 mmol, 1 equivalent), LiOtBu (78 mg, 0.97 mmol, 1.2 equivalent), A-Bn2-Cl-CP (528 mg, 0.97 mmol, 1.2 equivalent), and toluene:ACN (1:1, 8 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient elution (0% to 100%), yielding the desired product as a white solid. Yield = 680 mg, 87%, dr = 30:1.

[0224] 31 P NMR (203 MHz, CDCl3) δ -4.06, -6.54.

[0225] 13 C 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.

[0226] HR-MS (Q-TOF, ESI) calculated value C 49 H 60 N7O 12 PSi, [M+Na + ]: 998.388, measured value 998.3887.

[0227] h. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7aS)-2-(((2R,3R,4R,5R)-5-(3- ... (2H)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxy Tetrahydrofuran-2-yl)methoxy)-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6- 5-methylpyrimidine-2,4(1H,3H)-dione The compound was synthesized using 3-OTBS-U-BOM (270 mg, 0.61 mmol, 1 equivalent), LiOtBu (54 mg, 0.67 mmol, 1.1 equivalent), T-BOM-Cl-CP (330 mg, 0.67 mmol, 1.1 equivalent), and toluene:ACN (1:1, 6 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading via a minimum amount of DCM dissolved in the desired product, using a methanol:dichloromethane gradient (0% to 5%), yielding a white solid. Yield = 500 mg, 91%, dr = 5:1.

[0228] 1 H 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). 31 P NMR (162 MHz, CDCl3) δ -3.64, -6.68.

[0229] 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.

[0230] HR-MS (Q-TOF, ESI) calculated chemical formula: C 42 H 55 N4O 14 PSi, [M+Na + ]:921.3114, measured value 921.3110.

[0231] i. AA-Bn2-dimer--(2R,4aR,6R,7R,7aR)-2-[[((2R,3R,4R,5R)-3-[(tert-butyldimer ...2R,3R,4R,5R)-3-[(tert-butyldimer)-2-[ [Methylsilyl)oxy]-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy]-6- (6-(dibenzylamino)-9H-purine-9-yl)-7-methoxytetrahydro-4H-furano[3,2-d][1,3,2]dioxopyrazine Alkane 2-oxides The compound was synthesized using 3-OTBS-A-Bn2 (400 mg, 0.69 mmol, 1 equivalent), LiOtBu (67 mg, 0.83 mmol, 1.2 equivalent), ABn2-Cl-CP (452 ​​mg, 0.83 mmol, 1.2 equivalent), and toluene:ACN (1:1, 7 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, gradient elution of MeOH:DCM (0% to 5%), yielding the desired product as a white solid. Yield = 650 mg, 86%, dr = 14:1.

[0232] 31 P NMR (162 MHz, CDCl3) δ -4.02, -6.62.

[0233] 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).

[0234] 13 C 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. HR-MS (Q-TOF, ESI) calculated value C 56 H 65 N 10 O9PSi, [M+Na + ]:1103.4335, Measured value 1103.433.

[0235] j. 3-((benzyloxy)methyl)-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,3aS,9aR)-3-((tert-) Butyldimethylsilyl)oxy)-6-oxo-2,3,3a,9a-tetrahydro-6H-furano[2',3':4,5]oxazolo[3,2-a] Pyrimidin-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxopyrazine-cyclohexane- 6-yl)pyrimidine-2,4(1H,3H)-dione The compound was synthesized using 5'-OH-cyclo-U (200 mg, 0.59 mmol, 1 equivalent), LiOtBu (52 mg, 0.65 mmol, 1.1 equivalent), U-BOM-Cl-CP (296 mg, 0.65 mmol, 1.1 equivalent), and toluene:ACN (1:1, 9 mL) according to the general procedure GP10. Column conditions: reversed-phase C-18 column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DMSO, water:ACN gradient (10% to 100%), to obtain the desired product as a white solid. Yield = 230 mg, 51%, dr > 99:1.

[0236] 31 P NMR (203 MHz, CD3CN) δ -5.19.

[0237] 31 P NMR (203 MHz, CDCl3) δ -4.64.

[0238] 1 H 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).

[0239] 13 C 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.

[0240] HR-MS (Q-TOF, ESI) calculated value C 33 H 43 N4O 13 PSi, [M+Na + ]: 785.2226, measured value 785.2221.

[0241] k. 1-((2R,4S,5S)-4-azido-5-((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl) 1,2-di(2H)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-1-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1, 3,2]dioxophosphazenecyclohexane-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-3-((benzyloxy)methyl)-5-methylpyrimidine- 2,4(1H,3H)-dione The compound was synthesized using 2'-N3 (300 mg, 0.77 mmol, 1 equivalent), LiOtBu (74 mg, 0.92 mmol, 1.2 equivalent), U-BOM-Cl-CP (426 mg, 0.93 mmol, 1.2 equivalent), and toluene:ACN (1:1, 8 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, hexane:ethyl acetate gradient (0% to 100%), elution with 100% ethyl acetate, and separation of the desired product as a white solid. Yield = 520 mg, 83%, dr = 30:1.

[0242] 31 P NMR (203 MHz, CDCl3) δ -3.76, -6.49.

[0243] 1 H 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 = 2.3Hz, 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)。

[0244] 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.

[0245] HR-MS (Q-TOF, ESI) calculated value C 36 H 40 N7O 13 P, [M+H + ]: 810.2494, measured value 810.2495.

[0246] l. (E)-N'-(9-((2R,3R,4R,5R)-5-(((((2R,4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl 1,2-di(2H)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-1-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1, 3,2]dioxophosphazenecyclohexane-2-yl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran (2-yl)-9H-purin-6-yl)-N,N-dimethylformamidin The compound was synthesized in toluene:ACN (1:1) using 3-OTBS-DMF-A-Bn2 (275 mg, 0.61 mmol, 1 equivalent), LiOtBu (54 mg, 0.67 mmol, 1.1 equivalent), and U-BOM-Cl-CP (308 mg, 0.67 mmol, 1.1 equivalent) according to the general procedure GP10. Column conditions: A SiO2-10 g column was used in the Next-Gen automated system. The product was wet-loaded by dissolution in a minimal amount of DCM, followed by gradient elution (0% to 5%) of DCM-MeOH to obtain a white solid. Yield = 460 mg, 86%, dr = 15:1. Quality was confirmed by LC-MS.

[0247] 31 P NMR (162 MHz, CDCl3) δ -3.87, -6.91.

[0248] 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, 1.1Hz, 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)。

[0249] 13 C 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。

[0250] 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-oxotetrahydro-4H-furano[3,2-d] [1,3,2]dioxacyclohexane-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3 ... Cyclopenten-4-yl)-9H-purin-6-yl)-N,N-dimethylformamidin The compound was synthesized using ketal-DMF-A-5'OH (215 mg, 0.59 mmol, 1 equivalent), LiOtBu (52 mg, 0.65 mmol, 1.1 equivalent), and U-BOM-Cl-CP (299 mg, 0.65 mmol, 1.1 equivalent) in toluene:ACN (1:1) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, DCM:MeOH gradient (0% to 10%), yielding the desired product as a white solid. Yield = 82%, dr = 23:1. Quality was confirmed by LC-MS.

[0251] 31 P NMR (162 MHz, CDCl3) δ -4.24, -6.93.

[0252] 1 H 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).

[0253] 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-oxotetrahydro-4H-furano[3,2-d] [1,3,2]dioxacyclohexane-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3 ... Cyclopenten-4-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N,N-dimethylformamidin The compound was synthesized using dmf-C-5'-OH (250 mg, 0.74 mmol, 1 equivalent), LiOtBu (65 mg, 0.81 mmol, 1.1 equivalent), 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-Cl-CP, 373 mg, 0.81 mmol, 1.1 equivalent) in toluene:ACN (1:5) according to the modified general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, DCM:MeOH gradient (0% to 10%), yielding the desired product as a white solid. Yield = 280 mg, 50%, dr = 14:1. Quality was confirmed by LC-MS.

[0254] 31 P NMR (162 MHz, CDCl3) δ -3.94, -6.65.

[0255] 1 H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 7.75 (s, 1H), 7.44 (d, J = 7.2Hz, 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).

[0256] 13 C 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.

[0257] 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-dimethyltetrahydrofurano[3,4-d] [1,3]dioxacyclopenten-4-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]di Oxyphosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione The compound was synthesized using ketal-U-BOM (300 mg, 0.74 mmol, 1 equivalent), LiOtBu (65 mg, 0.82 mmol, 1.1 equivalent), and U-BOM-Cl-CP (374 mg, 0.82 mmol, 1.1 equivalent) in toluene:ACN (1:1, 8 mL) according to the general procedure GP10. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, DCM:MeOH gradient (0% to 5%), yielding the desired product as a white solid. Yield = 550 mg, 90%, dr = 15:1.

[0258] 31 P NMR (203 MHz, CDCl3) δ -3.92, -6.77.

[0259] 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).

[0260] 13 C 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.

[0261] HR-MS (Q-TOF, ESI) calculated value C 38 H 43 N4O 15 P, [M+Na + ]:849.2355, Measured value 849.236.

[0262] Example 7. Regioselective ring-opening of dinucleotide cyclic phosphates a. General procedure for regioselective ring-opening reactions of cyclic dinucleotides (GP12) In a nitrogen-filled glove box, lithium tert-butoxide (LiOtBu) (0.05 mmol, 1.5–2 equivalents), substituted benzyl alcohol nucleophile (BnOH) (0.025 mmol, 1.0 equivalent), and 0.25 mL of solvent were placed into an oven-dried reaction tube with a Teflon-coated magnetic stir bar and then removed from the glove box. The mixture was stirred at RT for 20 min and cooled to -20 °C over 10 min. Subsequently, a solution of cyclic dinucleotide (1) (0.025 mmol, 1 equivalent) in 0.25 mL of solvent was added in one go at 20 °C, and the mixture was stirred at this temperature for 3–4 h. The reaction was terminated at -20 °C with a solution of acetic acid (0.05 mmol, 2.0 equivalent) in 0.5 mL of MeCN. After stirring for 5 minutes, a solution of internal standard PO(OPh)3 (0.025 mmol, 1.0 equivalent) in MeCN (0.5 mL) was added, and the NMR yield and regioselectivity were recorded. Results for various BnOH and solvents are listed in Table 3. Table 3 - Ring-opening of dinucleotide cyclic phosphates (*-BOM is the protecting group on N-uridine; S represents the ratio of [3'-5']-bonds to [5'-5']-bonds; yield and selectivity are determined via...) 31 (P-NMR measurement) b. General procedure for regioselective ring-opening reactions of cyclic dinucleotides (GP13) Unless otherwise specified, the reaction conditions are: 1.5 eq LiOtBu, 1 eq CF3BnOH, 1 eq (0.025 mmol) 1-Cy-Nu at 20ºC for 3 h. S represents the ratio of [3-5]-bonding (2) to [5-5]-bonding (3). via 31 P NMR was used with (PhO)₃PO as an internal standard to determine conversion, yield, and selectivity. In a nitrogen-filled glove box, LiOtBu (0.037 mmol, 1.5 equivalents) and 2-CF3BnOH (as a substituted benzyl alcohol nucleophile BnOH) (0.05 mmol, 2.0 equivalents, stock solution in 0.25 mL anhydrous toluene (0.2 M)) were loaded into an oven-dried reaction tube with a Teflon-coated force stir bar and removed from the glove box. The mixture was stirred at room temperature for 20 minutes and cooled to -20 to -25°C over 10 minutes. Then, a solution of the protected cyclic dinucleotide (0.025 mmol, 1 equivalent) in toluene (0.35 mL) was added in a single batch at -20 to -25°C, and the mixture was stirred at this temperature for 3–4 hours. The reaction was terminated at -20°C with a solution of acetic acid (0.05 mmol, 2.0 equivalents) in MeCN (0.5 mL). After stirring for 5 minutes, a solution of internal standard PO(OPh)3 (0.025 mmol, 1.0 equivalent) in MeCN (0.5 mL) was added, and the NMR yield was recorded. The obtained dinucleotides and their yields and diastereomeric ratios (dr) are as follows (S represents the ratio of [3'-5'] linkage to [5'-5'] linkage; yield and selectivity are determined by...). 31 P-NMR, using PO(OPh)3 as an internal standard for determination.

[0263] c. A general procedure for selective ring-opening reaction of cyclic dinucleotide regions followed by capture with TBSCl (GP14) Step I: In a nitrogen-filled glove box, add LiOtBu (0.18 mmol, 1.5 equivalents) and 2-CF3BnOH (as a substituted benzyl alcohol nucleophile BnOH) (0.24 mmol, 2.0 equivalents, 1 mL in anhydrous toluene (0.24 M) stock solution) to an oven-dried reaction tube with a Teflon-coated magnetic stir bar, and then remove it from the glove box. Stir the mixture at room temperature for 20 minutes, then cool it to -20 to -25 °C over 10 minutes. Subsequently, add a solution of protected cyclic dinucleotide (0.12 mmol, 1 equivalent) in toluene (2 mL) at -20 °C in one step, and stir at this temperature for 4 hours.

[0264] Step II: In a 4 mL vial, prepare a solution of TBSCl (0.36 mmol, 3 equivalents) and imidazole (0.72 mmol, 6 equivalents) in 0.5 mL MeCN, and add this solution to the reaction tube from Step I using a syringe. Stir the resulting mixture at -20°C for 1 hour, then slowly raise the temperature to 0°C and stir overnight (16–18 hours). Further warm the reaction mixture to room temperature and monitor by LC-MS to ensure a conversion >90%. After completion, add a few drops of methanol to quench excess TBS-imidazolium salt. Dilute the reaction mixture with dichloromethane and transfer to a round-bottom flask. Evaporate the crude mixture to dryness using a rotary evaporator and purify it using a Combi-Flash NextGen 300 automated chromatography system with a silica gel column and a DCM-EtOAc (1% MeOH) gradient. The obtained cyclic dinucleotides and their yields and diastereomeric ratios (dr) are as follows: d. General procedure for regioselective ring-opening reactions of cyclic dinucleotides using TBD catalysts Inside a nitrogen-filled glove box, LiOtBu (0.18 mmol, 1.5 equivalents) and 2-CF3BnOH (as a substituted benzyl alcohol nucleophile BnOH) (0.24 mmol, 2.0 equivalents, stock solution in 1 mL anhydrous toluene (0.24 M)) were placed into an oven-dried reaction tube with a Teflon-coated magnetic stir bar and then removed from the glove box. The mixture was stirred at room temperature for 20 minutes and then cooled to -20 to -25 °C over 10 minutes. Next, a 2 mL solution of the protected cyclic dinucleotide (0.12 mmol, 1 equivalent) in toluene was added in a single batch at -20 °C, and the mixture was stirred at this temperature for 4 hours.

[0265] e. General steps for regioselective ring-opening reactions of cyclic dinucleotides Reaction conditions: At 20ºC, for 4 hours.

[0266] S represents the ratio of [3-5]-bonding (2) to [5-5]-bonding (2').

[0267] via 31 P NMR uses (PhO)3PO as an internal standard to determine conversion, yield, and selectivity.

[0268] f. General steps for regioselective ring-opening reactions of cyclic dinucleotides There was no reaction without imidazole. After adding 3 eq. TBSCL + 6 eq. imidazole, the reaction was observed by HPLC with 90% conversion and 75% NMR yield. The [5-5-] bonds remained unreacted.

[0269] g. General steps for regioselective ring-opening reactions of cyclic dinucleotides h. Ring-opening of guanosine dimer Example 8. Synthesis of Cyclic Trimers a. General steps for one-pot synthesis of cyclic trimers (GP15) Step I: In a nitrogen-filled glove box, add 0.18 mmol (1.5 equivalents) of 2-CF3BnOH (as the substituted benzyl alcohol nucleophile BnOH) and 0.18 mmol (1.5 equivalents) of LiOtBu dissolved in anhydrous toluene (1 mL) to reaction tube A, which has been dried in an oven and contains a Teflon-coated magnetic stir bar. Remove the tube from the glove box. Stir the mixture at room temperature for 30 minutes, then cool it to -25°C. Next, add a solution of protected cyclic dinucleotide (0.12 mmol (1 equivalent) in toluene (2 mL) at -25°C in one step, and stir at this temperature for 4 hours.

[0270] Step II: Add Cl-CP (0.18 mmol, 1.5 equivalents) dissolved in a solvent (e.g., MeCN (0.2 mL)) to another reaction tube B, and remove the reaction tube from the glove box. Incubate the reaction tube at -25°C for 10 minutes, then, with the aid of a syringe, transfer the pre-stirred alkoxide solution from reaction tube A dropwise to reaction tube B. Alternatively, with the aid of a syringe, transfer the pre-stirred Cl-CP solution from reaction tube B dropwise to reaction tube A. Stir the reaction mixture at -25°C for 18 hours. Dilute the reaction mixture with dichloromethane and transfer it to a round-bottom flask. Evaporate the crude mixture to dryness with the aid of a rotary evaporator and purify it by column chromatography using a DCM-EtOAc (1% MeOH) gradient.

[0271] Several different reaction conditions and substrate concentrations were investigated for the synthesis of cyclic trimers. The results are shown in Table 4: Table 4 - Reaction conditions and substrate concentrations for the synthesis of cyclic trimers b. A general procedure for one-pot synthesis of trinucleotide block copolymers (GP16) Step I: In a nitrogen-filled glove box, add 0.18 mmol (1.5 equivalents) of 2-CF3BnOH (as the substituted benzyl alcohol nucleophile BnOH) and 0.18 mmol (1.5 equivalents) dissolved in anhydrous toluene (0.18 M, 1 mL) to reaction tube A, which has been dried in an oven. Then remove the tube from the glove box. Stir the mixture at room temperature for 30 minutes, then cool it to -25°C. Next, add a solution of the protected cyclic dinucleotide (0.12 mmol, 1 equivalent) in toluene (2 mL) at -25°C in one step, and stir at this temperature for 4 hours.

[0272] Step II: Add 0.6 mL of MeCN containing Cl-CP (0.18 mmol, 1.5 equivalents) to another reaction tube B, and remove the reaction tube from the glove box. Incubate the reaction tube at -25°C for 10 minutes, then, with the aid of a syringe, transfer the pre-stirred alkoxide solution from reaction tube A dropwise into reaction tube B. Stir the reaction solution at -25°C for 18 hours.

[0273] Step III: Add 0.36 mmol (3 equivalents) of 2-CF3BnOH (as the substituted benzyl alcohol nucleophile BnOH) dissolved in 1 mL of toluene and LiOtBu (0.30 mmol, 2.5 equivalents) to reaction tube C, stir for 30 minutes, and then add dropwise to reaction tube B at -30°C. Stir the reaction solution at -30°C for 5 hours.

[0274] Step IV: Add a solution of TBSCl (0.72 mmol, 6 equivalents) and imidazole (1.44 mmol, 12 equivalents) in 1 mL MeCN to reaction tube C. Stir the resulting mixture at -30°C for 1 hour, then slowly raise the temperature to 0°C and stir overnight (16–18 hours). Further raise the reaction mixture to room temperature and monitor by LC-MS to ensure a conversion >90%. After completion, add a few drops of methanol to quench excess TBS-imidazolium salt. Dilute the reaction mixture with dichloromethane and transfer to a round-bottom flask. Evaporate the crude mixture to dryness using a rotary evaporator and purify by column chromatography using a DCM-EtOAc (1% MeOH) gradient. Dissolve the crude product in a minimal amount of DCM and load it onto a column packed with 10 g of silica gel.

[0275] c. A general procedure for the four-step one-pot synthesis of trinucleotide block copolymers This report describes the separation yield of a one-pot, four-step sequence. (PhO)₃PO was used as an internal standard, based on... 31 P NMR report NMR yield. Method A: The compounds were separated by silica gel column chromatography using a DCM:EtOAc (2% MeOH) gradient and by a C-18 reversed-phase column.

[0276] d. Separation of trimer block copolymers The crude trimeric block copolymer product was purified by reversed-column chromatography on an automated system. The obtained trinucleotide block copolymers and their yields are as follows: For reversed column chromatography, the compounds do not decompose, but the separation is not baseline to baseline. Preparing a 0.1% buffer solution in water with NH4OC(O)H or NH4OAc can aid in separation.

[0277] Example 9. Synthesis of ring-opening TBS-protected dimers a. Phosphate (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester(((2R,3R,4R, 5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl) 2-(trifluoromethyl)benzyl) ester (4-methoxytetrahydrofuran-2-yl)methyl) ester The compound was synthesized according to GP14 (two-step, one-pot sequence), using UU-Cy-Nu (117 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-trifluoromethylbenzyl alcohol [2-CF3BnOH, 43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] dissolved in 2 mL toluene:ACN (5:1) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (49 mg, 0.72 mmol, 6 equivalents) dissolved in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate (0% to 100%), yielding the desired product as a white solid. Yield = 84 mg, 61%, dr = 29:1.

[0278] 31 P NMR (162 MHz, CDCl3) δ -1.91 (major), -2.49 (minor).

[0279] 1 H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.2 Hz, 1H), 7.56 (dd, J = 7.8, 4.7Hz, 2H), 7.45 (dd, J = 25.5, 8.0 Hz, 2H), 7.35 (t, J = 7.7 Hz, 1H), 7.30 – 7.11(m, 10H), 5.93 (d, J = 3.7 Hz, 1H), 5.75 (d, J = 2.2 Hz, 1H), 5.61 (d, J = 8.2 Hz,1H), 5.54 (d, J = 8.2 Hz, 1H), 5.36 (qd, J = 9.7, 7.4 Hz, 4H), 5.27 – 5.14 (m,3H), 4.77 (q, J = 5.5 Hz, 1H), 4.59 (d, J = 3.2 Hz, 4H), 4.28 (ddd, J = 11.7, 6.1,2.2 Hz, 1H), 4.20 – 4.10 (m, 2H), 4.02 (qd, J = 7.5, 3.6 Hz, 2H), 3.90 (dd, J =11.9, 1.9 Hz, 1H), 3.80 (t, J = 4.3 Hz, 1H), 3.70 (dd, J = 11.9, 1.7 Hz, 1H),3.53 (dd, J = 4.7, 2.2 Hz, 1H), 3.43 (s, 3H), 3.36 (s, 3H), 0.83 (s, 9H), 0.78(s, 9H), 0.03 (s, 6H), -0.03 (d, J = 10.0 Hz, 6H)。

[0280] 13C NMR (101 MHz, CDCl3) δ 162.62, 162.49, 151.06, 150.91, 138.23,138.06, 137.98, 137.96, 133.61, 132.52, 129.65, 129.04, 128.45, 128.44,128.08, 127.86, 127.82, 126.40, 126.34, 125.53, 102.20, 102.13, 89.27, 87.24,83.30, 82.92, 82.83, 82.62, 81.59, 81.51, 73.79, 73.74, 72.39, 70.40, 70.38, 69.46, 66.17, 65.97, 65.92, 61.54, 58.58, 58.57, 25.99, 25.74, 18.47, 18.17, -4.55, -4.88, -5.45.

[0281] HR-MS (Q-TOF, ESI) calculated value C 56 H 76 F3N4O 16 Psi2, [M+Na + ]:1227.4377, measured value 1227.4368.

[0282] b. Phosphate ((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl ester ((2R,3R,4R, 5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(4-(dibenzylamino)-2-oxopyrimidin-1(2H)-yl)-4- 2-(trifluoromethyl)benzyl methoxytetrahydrofuran-3-yl) ester The compound was synthesized using a stock solution of UC-Cy-Nu (117 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, in 1 mL of anhydrous toluene (0.24 M)] dissolved in 2 mL of toluene:ACN (5:1) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (49 mg, 0.72 mmol, 6 equivalents) dissolved in 0.5 mL of MeCN (for step 2), according to GP14 (two-step, one-pot reaction sequence). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM:ethyl acetate (containing 2% MeOH) gradient (0% to 100%), yielding the desired product as a white solid. Yield = 84 mg, 61%, dr = 20:1.

[0283] 31 P NMR (203 MHz, CDCl3) δ -1.91.

[0284] 19 F NMR (471 MHz, CDCl3) δ -59.76.

[0285] 1 H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 8.1 Hz, 2H), 7.50 (dd, J = 19.6, 7.9 Hz, 2H), 7.39 (t, J = 7.7 Hz, 1H), 7.34 – 7.16 (m,16H), 7.09 (s, 1H), 6.03 (d, J = 2.0 Hz, 1H), 5.80 (d, J = 2.3 Hz, 1H), 5.67 (d, J = 7.8 Hz, 1H), 5.57 (d, J = 8.2 Hz, 1H), 5.45 – 5.35 (m, 2H), 5.28 (d, J = 6.4Hz, 2H), 4.98 (q, J = 14.2 Hz, 2H), 4.80 (ddd,J = 7.4, 6.1, 4.9 Hz, 1H), 4.64(s, 2H), 4.43 (s, 2H), 4.31 (ddd, J = 11.6, 5.9, 2.0 Hz, 1H), 4.22 – 4.14 (m,2H), 4.10 – 4.01 (m, 2H), 3.99 (dd, J = 12.0, 2.1 Hz, 1H), 3.94 (dd, J = 4.9, 2.1Hz, 1H), 3.76 – 3.69 (m, 1H), 3.61 – 3.51 (m, 2H), 3.50 (s, 3H), 3.46 (s,3H), 0.82 (s, 9H), 0.78 (s, 9H).

[0286] 13 C NMR (126 MHz, CDCl3) δ 164.28, 162.53, 155.41, 150.97, 141.16,138.08, 137.99, 137.21, 136.23, 133.66, 132.56, 129.46, 129.18, 128.96,128.71, 128.46, 127.84, 127.67, 126.50, 126.35, 126.31, 125.28, 123.10,102.13, 91.85, 89.04, 88.14, 83.35, 82.53, 81.91, 81.83, 81.68, 81.62, 77.36, 72.92, 72.88, 72.40, 70.37, 69.40, 66.06, 65.80, 65.75, 60.80, 58.56, 58.50, 50.81, 50.31, 25.95, 25.88, 25.75, 18.41, 18.18, -4.56, -4.88, -5.43, -5.46.

[0287] HR-MS (Q-TOF, ESI) calculated value C 62 H 81 F3N5O 14 PSi2, [M+Na + ]:1286.4900, measured value 1286.4894.

[0288] c. Phosphate ((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine) (2R,3R,4R,5R)-2-((tert-butyldimethylsilyl)oxy (dibenzylamino)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-3-yl)2-(trifluoromethyl)benzyl ester The compound was synthesized according to GP14 (two-step, one-pot reaction sequence) using a stock solution (0.24 M) of UU-Cy-Nu (130 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents) dissolved in 2 mL toluene and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, in 1 mL anhydrous toluene (0.24 M)] (for step 1) and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (49 mg, 0.72 mmol, 6 equivalents) dissolved in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM:ethyl acetate (1% MeOH) gradient (0% to 100%), yielding the desired product as a white solid. Yield = 120 mg, 72%, dr = 50:1.

[0289] 19 F NMR (471 MHz, CDCl3) δ -59.86.

[0290] 31 P NMR (203 MHz, CDCl3) δ -1.77.

[0291] 1 H NMR (500 MHz, CDCl3) δ 8.38 (s, 1H), 8.36 (s, 1H), 7.96 (s, 1H),7.93 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 7.8Hz, 1H), 7.37 – 7.21 (m, 25H), 6.17 (d, J = 6.6 Hz, 1H), 6.10 (d, J = 4.1 Hz,1H), 5.49 (s, 3H), 5.36 (d, J = 6.3 Hz, 2H), 5.16 (ddd, J = 7.2, 4.7, 2.5 Hz,1H), 4.96 (s, 3H), 4.59 (qd,J = 4.9, 2.6 Hz, 2H), 4.46 (ddd, J = 10.9, 5.8, 3.8Hz, 1H), 4.38 (ddd, J = 13.4, 5.4, 3.5 Hz, 2H), 4.34 – 4.23 (m, 2H), 3.88 –3.75 (m, 2H), 3.49 (s, 3H), 3.40 (s, 3H), 0.93 (s, 9H), 0.89 (s, 9H), 0.14(s, 3H), 0.12(s, 3H).

[0292] 13 C NMR (126 MHz, CDCl3) δ 155.08, 155.05, 152.93, 152.81, 151.30,150.76, 137.90, 137.48, 136.94, 134.08, 132.39, 129.41, 128.71, 128.53,128.07, 128.02, 127.39, 126.08, 126.04, 125.29, 123.11, 120.46, 120.17,87.48, 85.43, 84.14, 84.09, 82.75, 82.68, 82.58, 82.08, 82.05, 77.36, 75.70, 75.66, 70.53, 66.68, 65.92, 62.66, 58.86, 58.71, 26.10, 25.86, 25.84, 18.46, 18.26, -4.55, -4.74, -5.25, -5.42.

[0293] HR-MS (Q-TOF, ESI) calculated value C 70 H 86 F3N 10 O 10 PSi2[M+Na + ]: 1393.5649, measured value 1393.5642.

[0294] d. Phosphoric acid ((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl ester ((2R,3R,4R, 5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(6-chloro-9H-purin-9-yl)-4-methoxytetrahydrofuran- 3-yl)2-(trifluoromethyl)benzyl ester The compound was synthesized according to GP14 (two-step, one-pot sequence) using UA(6Cl)-Cy-Nu (101 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, stock solution (0.24 M) in 1 mL anhydrous toluene] (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, DCM: ethyl acetate (1% MeOH) gradient (0% to 100%), yielding the desired product as a white solid. Yield = 80 mg, 59%, dr = 17:1.

[0295] 31 P NMR (203 MHz, CDCl3) δ -1.71, -2.12.

[0296] 19 F NMR (471 MHz, CDCl3) δ -59.70.

[0297] 1 H NMR (500 MHz, CDCl3) δ 8.75 (s, 1H), 8.47 (s, 1H), 7.66 (dd, J = 8.1,2.2 Hz, 2H), 7.61 – 7.53 (m, 2H), 7.45 (t, J = 7.7 Hz, 1H), 7.38 – 7.29 (m,4H), 7.29 – 7.22 (m, 2H), 6.24 (d, J = 6.2 Hz, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.69 (d, J = 8.2 Hz, 1H), 5.50 – 5.30 (m, 4H), 5.12 (ddd, J = 7.2, 4.7, 2.7 Hz,1H), 4.69 (s, 2H), 4.51 – 4.39 (m, 3H), 4.27 (ddd, J= 11.6, 5.2, 2.9 Hz, 1H), 4.19 – 4.08 (m, 3H), 3.97 (dd, J = 11.6, 2.8 Hz, 1H), 3.86 (dd, J = 11.6, 2.4 Hz, 1H), 3.65 (dd, J = 4.4, 2.1 Hz, 1H), 3.54 (s, 3H), 3.38 (s, 3H), 0.95 (s, 9H), 0.93 – 0.85 (m, 10H), 0.18 – 0.05 (m, 12H).

[0298] 13 C NMR (126 MHz, CDCl3) δ 162.51, 152.43, 151.70, 151.40, 150.92,143.39, 138.06, 137.94, 133.62, 133.55, 132.49, 132.21, 129.68, 129.06,128.46, 128.12, 127.86, 127.82, 126.45, 126.41, 126.37, 126.32, 123.08,102.19, 89.37, 85.94, 84.67, 84.63, 83.35, 83.15, 83.12, 81.54, 81.47, 77.36, 75.60, 75.55, 72.42, 70.38, 69.45, 66.26, 65.91, 65.87, 62.66, 60.53, 58.90, 58.60, 26.12, 26.08, 25.75, 25.74, 18.53, 18.20, -4.52, -4.84, -5.23, -5.36.

[0299] HR-MS (Q-TOF, ESI) calculated value C 49 H 67 ClF3N6O 13 PSi2, [M+Na + ]: 1149.3575, measured value 1149.3568.

[0300] e. Phosphate ((3aR,4R,6R,6aR)-6-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-yl)methyl ester, ((2R,3R,4R, 5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(((tert-butyldimethylsilyl) 4-(trifluoromethyl)benzyl)-4-methoxytetrahydrofuran-3-yl) ester (2-(trifluoromethyl)benzyl) ester The compound was synthesized according to GP14 (two-step, one-pot sequence), using katal-UU-Cy-Nu (100 mg, 0.12 mmol, dr 10:1), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] in toluene (1.6 mL) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, hexane:ethyl acetate gradient (0% to 100%), yielding the desired product as a white solid. Yield = 107 mg, 79%, dr => 99:1.

[0301] 31 P NMR (203 MHz, CDCl3) δ -2.26, -2.31.

[0302] 19 F NMR (471 MHz, CDCl3) δ -59.73, -59.85.

[0303] 1 H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 8.0 Hz,2H), 7.59 – 7.53 (m, 1H), 7.43 (q, J = 7.2 Hz, 1H), 7.39 – 7.23 (m, 12H), 7.16(d, J = 8.1 Hz, 1H), 6.05 (d, J = 4.0 Hz, 1H), 5.73 – 5.64 (m, 2H), 5.61 (d, J =2.0 Hz, 1H), 5.49 (d, J = 9.8 Hz, 1H), 5.48 – 5.42 (m, 2H), 5.37 (d, J = 9.8 Hz, 1H), 5.32 (d, J= 6.5 Hz, 2H), 4.93 – 4.78 (m, 4H), 4.70 (s, 2H), 4.67 (s, 2H), 4.38 – 4.26 (m, 4H), 3.97 (dd, J = 11.8, 1.9 Hz, 1H), 3.92 (t, J = 4.4 Hz, 1H), 3.80 (dd, J = 11.9, 1.7 Hz, 1H), 3.46 (s, 3H), 1.55 (s, 3H), 1.34 (s, 3H), 0.92 (s, 9H), 0.12 (s, 6H).

[0304] 13 C NMR (126 MHz, CDCl3) δ 162.65, 162.47, 151.11, 150.83, 140.82,138.30, 137.99, 137.96, 133.86, 133.80, 132.47, 129.43, 128.78, 128.44,127.86, 127.85, 127.83, 127.75, 126.24, 126.19, 114.79, 102.45, 102.20,95.72, 87.20, 85.96, 85.91, 84.56, 84.53, 83.04, 82.98, 82.67, 82.65, 80.91, 77.36, 73.79, 73.75, 72.47, 72.37, 70.44, 70.41, 67.70, 67.66, 66.05, 66.01, 65.98, 61.71, 58.60, 27.21, 26.00, 25.37, 18.46, -5.47.

[0305] HR-MS (Q-TOF, ESI) calculated value C 52 H 64 F3N4O 16 PSi, [M+Na + ]: 1139.3668, measured value 1139.3661.

[0306] f. Phosphate (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester(((2R,3S, 5R)-5-(3-((benzyloxy)methyl)-5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldi) methylsilyl)oxy)tetrahydrofuran-2-yl)methyl) ester (2-(trifluoromethyl)benzyl) ester The compound was synthesized according to GP14 (two-step, one-pot sequence), using UT-Cy-Nu (110 mg, 0.12 mmol, dr 5:1), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] in toluene (1.6 mL) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, hexane:ethyl acetate gradient (0% to 100%), yielding the desired product as a white solid. Yield = 112 mg, 85%, dr = > 99:1.

[0307] 31 P NMR (203 MHz, CDCl3) δ -1.97, -2.42.

[0308] 19 F NMR (471 MHz, CDCl3) δ -59.77.

[0309] 1 H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 8.2 Hz, 1H), 7.65 (dd, J = 7.8, 3.7Hz, 2H), 7.55 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.40 – 7.35 (m, 4H), 7.35 – 7.27 (m, 6H), 7.25 (s, 1H), 6.31 (dd, J = 7.2, 6.2 Hz, 1H), 6.05 (d, J =3.9 Hz, 1H), 5.71 (d, J = 8.2 Hz, 1H), 5.52 – 5.42 (m, 4H), 5.34 (d, J = 6.5 Hz, 2H), 4.87 (q, J = 5.3 Hz, 1H), 4.70 (d, J= 7.3 Hz, 4H), 4.35 (dt, J = 7.0, 3.6 Hz, 1H), 4.28 (dt, J = 5.6, 1.8 Hz, 1H), 4.24 (dd, J = 6.2, 3.5 Hz, 2H), 4.00 (dt, J =8.8, 2.1 Hz, 2H), 3.94 (t, J = 4.3 Hz, 1H), 3.81 (dd, J = 11.9, 1.7 Hz, 1H), 3.47(s, 3H), 2.25 (ddd, J = 13.5, 6.2, 3.6 Hz, 1H), 2.01 (dt, J = 13.6, 6.9 Hz, 1H), 1.86 (d, J = 1.2 Hz, 3H), 0.93 (s, 9H), 0.88 (s, 10H), 0.13 (s, 6H), 0.07 (d, J =4.7 Hz, 6H).

[0310] HR-MS (Q-TOF, ESI) calculated value C 56 H 76 F3N4O 15 PSi2, [M+Na + ]: 1211.4427, measured value 1211.4438.

[0311] g. Phosphate (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl ester(((2R,3R,4R,5R)- 5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy 2-(trifluoromethyl)benzyl) ester (4-methoxytetrahydrofuran-2-yl)methyl) ester The compound was synthesized using a GP14 (two-step, one-pot reaction sequence) specifically using F-UU-Cy-Nu (108 mg, 0.12 mmol, dr 5:1), LiOtBu (11.5 mg, 0.14 mmol, 1.2 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] dissolved in toluene (2 mL) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) dissolved in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, hexane:ethyl acetate gradient (0% to 100%), yielding the desired product as a white solid. Yield = 97mg, 75%, dr > 99:1.

[0312] 31 P NMR (203 MHz, CDCl3) δ -2.03, -2.07.

[0313] 19 F NMR (471 MHz, CDCl3) δ -59.64, -202.82 (dt, J = 51.4, 15.3 Hz).

[0314] 1 H NMR (500 MHz, CDCl3) δ 7.74 (d, J = 8.2 Hz, 1H), 7.69 – 7.54 (m, 3H), 7.50 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.39 – 7.22 (m, 11H), 6.09(dd, J = 15.2, 2.6 Hz, 1H), 5.84 (d, J = 2.1 Hz, 1H), 5.71 (d, J = 8.2 Hz, 1H), 5.65 (d, J = 8.2 Hz, 1H), 5.51 – 5.41 (m, 4H), 5.34 – 5.27 (m, 3H), 5.00 – 4.86(m, 2H), 4.70 (d, J= 3.6 Hz, 4H), 4.40 (ddd, J = 11.6, 6.2, 2.3 Hz, 1H), 4.31 –4.21 (m, 2H), 4.17 – 4.00 (m, 3H), 3.83 (dd, J = 12.0, 1.9 Hz, 1H), 3.64 (dd, J =4.9, 2.2 Hz, 1H), 3.53 (s, 3H), 0.91 (s, 9H), 0.89 (s, 9H), 0.12 (d, J = 1.3Hz, 6H), 0.09 (s, 3H), 0.06 (s, 3H).

[0315] 13 C NMR (126 MHz, CDCl3) δ 162.49, 162.39, 150.88, 150.84, 138.05,137.98, 137.95, 133.23, 133.16, 132.58, 129.98, 129.22, 128.44, 127.85,127.82, 127.78, 126.51, 126.47, 126.42, 102.48, 102.13, 92.37, 92.35, 90.82,90.80, 89.38, 88.08, 87.82, 83.28, 82.31, 82.24, 81.45, 81.39, 72.80, 72.76, 72.68, 72.64, 72.51, 72.39, 70.47, 70.37, 69.43, 66.50, 66.22, 66.17, 60.95, 58.58, 25.97, 25.72, 18.48, 18.17, -4.55, -4.92, -5.44, -5.46.

[0316] HR-MS (Q-TOF, ESI) calculated value C 55 H 73 F4N4O 15 PSi2, [M+Na + ]: 1215.4177, measured value 1215.4171.

[0317] h. Phosphate ((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl ester ((2R,3S,5R)- 5-(3-((benzyloxy)methyl)-5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(((tert-butyldimethyl) (3-yl)-(trifluoromethyl)benzyl) ester The compound was synthesized according to GP-14 (two-step, one-pot sequence) using UT-Cy-Nu (110 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] dissolved in toluene (2 mL) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) dissolved in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, hexane:ethyl acetate gradient (0% to 100%), yielding the desired product as a white solid. Yield = 115 mg, 79%, dr = >99:1.

[0318] 31 P NMR (203 MHz, CDCl3) δ -1.88.

[0319] 19 F NMR (471 MHz, CDCl3) δ -59.55.

[0320] 1 H NMR (500 MHz, CDCl3) δ 7.74 – 7.69 (m, 1H), 7.69 – 7.58 (m, 2H),7.55 (d, J = 8.2 Hz, 1H), 7.52 – 7.46 (m, 1H), 7.43 (q, J = 1.2 Hz, 1H), 7.41 –7.37 (m, 4H), 7.37 – 7.28 (m, 5H), 7.28 – 7.23 (m, 2H), 6.37 (dd, J = 9.1, 5.2Hz, 1H), 5.89 (d, J = 2.1 Hz, 1H), 5.72 (d, J = 8.2 Hz, 1H), 5.53 – 5.44 (m, 4H), 5.32 (qd, J = 12.7, 7.0 Hz, 2H), 5.01 (t, J = 5.8 Hz, 1H), 4.71 (s, 4H), 4.42(ddd,J = 11.6, 6.0, 2.2 Hz, 1H), 4.29 (q, J = 1.9 Hz, 1H), 4.25 (ddd, J = 11.6,5.3, 3.1 Hz, 1H), 4.20 – 4.11 (m, 2H), 3.90 (dd, J = 11.5, 2.1 Hz, 1H), 3.85(dd, J = 11.5, 2.2 Hz, 1H), 3.68 (dd, J = 4.6, 2.1 Hz, 1H), 3.56 (s, 3H), 2.53 –2.46 (m, 1H), 2.06 (dddd, J = 15.3, 9.1, 5.6, 2.1 Hz, 1H), 1.93 (d, J = 1.2 Hz,3H), 0.92 (d, J = 0.7 Hz, 19H), 0.13 (d, J = 1.1 Hz, 6H), 0.12 (d, J = 4.3 Hz, 6H).

[0321] 13 C NMR (126 MHz, CDCl3) δ 163.46, 162.48, 150.97, 150.88, 138.10, 137.94, 133.65, 133.40, 133.33, 132.57, 130.03, 129.19, 128.4, 128.37, 128.26, 127.80, 127.75, 127.72, 126.48, 126.44, 126.39, 110.57, 102.12, 89.40, 85.96, 85.92, 85.40, 83.27, 81.45, 81.38, 79.70, 79.66, 72.36, 72.32, 70.67, 70.36, 69.45, 66.21, 65.97, 65.93, 63.38, 58.55, 39.39, 39.35, 25.97, 25.72, 18.35, 18.16, 13.31, -4.54, -4.87, -5.33, -5.46.

[0322] HR-MS (Q-TOF, ESI) Calcd for C56 H 76 F3N4O 15 PSi2, [M+Na + ]: 1211.4427, measured value 1211.4420.

[0323] i. Phosphate (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester(((2R,3R,4R, 5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(((E)-(dimethylamino)methylene)amino)-9H-purine-9- 2-(trifluoromethyl)benzyl) ester (4-methoxytetrahydrofuran-2-yl)methyl) ester The compound was synthesized according to GP-14 (two-step, one-pot reaction sequence) using N-dmf-3-TBS-AU-BOM-Cy-Nu (105 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] dissolved in toluene (2 mL) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) dissolved in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by a minimum amount of DCM, DCM:MeOH gradient (0% to 5%), yielding the desired product as a white solid. Yield = 90 mg, 64%, dr = 10:1.

[0324] 31 P NMR (162 MHz, CDCl3) δ -2.10, -2.33.

[0325] 19 F NMR (471 MHz, CDCl3) δ -59.82.

[0326] 1 H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.48 (s, 1H), 8.01 (s, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 7.9 Hz, 2H), 7.56 – 7.46 (m, 1H), 7.45 –7.20 (m, 8H), 6.05 – 5.99 (m, 2H), 5.69 (d, J = 8.2 Hz, 1H), 5.47 (q,J = 9.8 Hz,2H), 5.36 – 5.27 (m, 2H), 4.85 (q, J = 5.3 Hz, 1H), 4.71 (s, 2H), 4.57 (q, J =5.0 Hz, 1H), 4.45 – 4.36 (m, 2H), 4.33 – 4.20 (m, 3H), 3.94 – 3.85 (m, 2H), 3.74 (dd, J = 12.0, 1.7 Hz, 1H), 3.45 (s, 3H), 3.40 (s, 3H), 3.24 (s, 3H), 3.18(s, 3H), 0.91 (s, 10H), 0.90 (s, 8H), 0.10 (d, J = 6.8 Hz, 7H), 0.08 (d, J = 1.0Hz, 6H).

[0327] 13 C NMR (101 MHz, CDCl3) δ 162.70, 159.93, 158.36, 158.31, 152.84,151.30, 151.09, 140.79, 138.44, 138.02, 133.89, 133.80, 132.42, 129.51,128.65, 128.42, 127.87, 127.80, 127.56, 126.89, 126.18, 126.13, 126.07,102.10, 87.63, 87.26, 82.99, 82.92, 82.80, 82.74, 82.72, 82.20, 73.73, 73.68, 72.36, 70.67, 70.40, 67.03, 66.97, 65.93, 61.61, 58.65, 58.58, 41.39, 35.26, 25.98, 25.82, 18.43, 18.23, -4.58, -4.82, -5.48, -5.50.

[0328] HR-MS (Q-TOF, ESI) calculated value C 63 H 81 F3N7O 13 PSi2, [M+Na + ]: 1310.5013, measured value 1310.5008.

[0329] j. Cyclo-UU-BOM-TBS-TRAP The compound was synthesized according to GP-14 (two-step, one-pot sequence) using cyclo-UU-Cy-Nu (90 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents), and 2-CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL anhydrous toluene stock solution (0.24 M)] in toluene:ACN (5:1, 2 mL) (for step 1), and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) in 0.5 mL MeCN (for step 2). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate gradient (0% to 100%), yielding the desired product as a white solid. Yield = 70 mg, 56%, dr => 99:1.

[0330] 31 P NMR (162 MHz, CDCl3) δ -2.50.

[0331] 19 F NMR (376 MHz, CDCl3) δ -59.62.

[0332] 1 H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.2 Hz, 1H), 7.66 (dq, J = 15.0, 7.7 Hz, 3H), 7.46 (t, J = 7.6 Hz, 1H), 7.42 – 7.26 (m, 7H), 6.12 (dd, J = 19.4, 5.3 Hz, 2H), 6.04 (d, J = 7.5 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.48 (q, J= 9.7Hz, 2H), 5.36 – 5.27 (m, 2H), 5.12 – 5.06 (m, 1H), 4.88 – 4.80 (m, 1H), 4.69(s, 2H), 4.56 – 4.50 (m, 1H), 4.36 – 4.26 (m, 2H), 4.07 – 3.82 (m, 5H), 3.40(d, J = 2.1 Hz, 3H), 0.92 (s, 10H), 0.89 (s, 9H), 0.13 (dd, J = 10.4, 8.5 Hz, 12H).

[0333] 13 C NMR (101 MHz, CDCl3) δ 171.16, 162.54, 159.36, 151.11, 138.38,137.85, 134.33, 133.52, 133.43, 132.59, 129.85, 128.78, 128.33, 127.76,127.72, 126.07, 126.02, 110.85, 102.22, 89.82, 88.77, 86.73, 86.52, 86.45,83.33, 83.26, 82.51, 82.48, 76.42, 74.70, 74.64, 72.23, 70.31, 66.14, 65.90, 65.85, 62.10, 58.50, 25.90, 25.54, 18.32, 17.87, -4.93, -5.08, -5.55, -5.58.

[0334] HR-MS (Q-TOF, ESI) calculated value C 47 H 64 F3N4O 14 PSi2, [M+Na + ]: 1075.3539, measured value 1075.3505.

[0335] Example 10. Removal of protecting groups by hydrogenation a. General procedure for removing protecting groups via hydrogenation (GP17) To an oven-dried reaction tube fitted with a Teflon-coated magnetic stir bar, add BOM- and benzyl-protected [3'-5'] and [5'-5'] dinucleotide conjugates (1 equivalent), methanol (10:1, 0.05 M), and HCO2H (0.5%). Add 10% Pd-C (10%) under N2 conditions. Introduce hydrogen (H2) into the reaction mixture using a double-folded balloon. Monitor the reaction by LC-MS. After the reaction is complete, filter the reaction mixture through a diatomaceous earth mat and wash with MeOH. Evaporate all volatiles under vacuum. Add a solution of internal standard PO(OPh)3 (0.025 mmol, 1.0 equivalent) in acetonitrile (MeCN, 0.5 mL) and record the NMR yield.

[0336] The results of the hydrogenation rate study of the protecting group (PG) are listed in Table 5 (BOM as the nucleoside protecting group): Table 5 - Hydrogenation rate of protecting group (PG) b. Deprotection of dinucleotide block copolymer AA Table 6 - Deprotection of dinucleotide block copolymers c. Deprotection of the trinucleotide block copolymer UUU d. Deprotection of the trinucleotide block copolymer AAA e. Deprotection screening of novel nucleosides and dimers Filter settings: In a glove box filled with N2, solid nucleosides are added to a dispenser head for dispensing. Nucleotides with a syrupy viscosity are dispensed as a stock solution into acetonitrile, which is then evaporated under an N2 stream.

[0337] A solid matrix was added to the dispensing head and dispensed using a Quantos (based on 5 mg, adjusting the 3'-5' and 5'-5' dinucleotide mixture to dispense 5 mg of the desired isomer). Pd / C (Johnson Matthey JM 10R39) was dispensed using a Quantos at a loading of 10 mol%. Subsequently, the solvent was added cleanly to the reaction wells, or, in the case of a mixed solvent, pre-mixed the solvent and then dispensed from the Falcon tube into appropriate vials using a single-channel pipette. A stitched pad with cross-slits added using an Exacto-type flat-blade tool was fitted into the tray, and the tray was removed from the glove box, placed in the CAT96 device, and then sealed. The CAT96 was programmed to run for 3 hours at RT and a 30 psi hydrogen (H2) atmosphere, followed by running the program at a constant stirring rate of 300 rpm.

[0338] Preparation of analytical samples: Unseal the tray and remove 20 µL aliquots from each vial, then use a multichannel pipette to add them to appropriately labeled 0.45 µm filter vials. Filter the sample into a 96-well collection tray and use a multichannel pipette to add 10 µL aliquots from each well to the corresponding well of the appropriately labeled 96-well UPLC sample tray.

[0339] Prepare a 20 mL 2.5 mM stock solution of internal standard IS-01 (13.6 mg) using MeOH. Add 400 µL of the stock solution to each filter vial using a multichannel pipette, inserting the top of the vial to filter the sample. Analyze the sample using LCMS (using the OA high-pH rapid method with a 2.0 µL injection volume). Each sample is assigned a batch number reflecting its position on the reaction plate and directly correlated with its position on the analysis plate, e.g., CAX-D01021-012-XXX, where XXX contains A01-H12.

[0340] The screening results are listed in Tables 7A and 7B (X = mixture of partially deprotected products; 0 = mixture of completely and partially deprotected products; + = more than 95% converted to completely deprotected products).

[0341] Table 7A - Screening Results for Additive-Free Screening Table 7B - Screening results using 0.5 mol% formic acid additive Example 11. Synthesis of Cyclic Trimer: UUU a. Phosphate (2R,3R,4R,5R)-5-[3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl]-2-[[[(4aR,6R,7R,7aR)-6-[3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl]-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-2-yl]oxy] [Methyl]-4-methoxytetrahydrofuran-3-yl ester [[((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] Ester (2-(trifluoromethyl)benzyl) ester The compound was synthesized according to GP-15 (two-step, one-pot sequence), using a stock solution of 2-CF3BnOH [58 mg, 0.33 mmol, 1.5 equivalents, in 2 mL of anhydrous toluene (0.33 M)] in toluene (3 mL) for step 1, and U-BOM-Cl-CP [160 mg, 0.35 mmol, 1.6 equivalents] in 1.2 mL MeCN for step 2. Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolving in a minimal amount of DCM, DCM:MeCN (2% iPrOH) gradient (0% to 80%). Fractions were analyzed by LC-MS, and the fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Total yield (two steps) = 180 mg, 57%.

[0342] 31 P NMR (203 MHz, CDCl3) δ -1.87, -4.01.

[0343] 19 F NMR (471 MHz, CDCl3) δ -59.70.

[0344] 1 H NMR (500 MHz, CDCl3) δ 7.67 (dd, J = 11.6, 7.8 Hz, 2H), 7.60 (q, J =7.5 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.52 – 7.41 (m, 3H), 7.40 – 7.27 (m,15H), 7.11 (d, J = 8.2 Hz, 1H), 5.93 (d, J = 3.5 Hz, 1H), 5.91 – 5.86 (m, 1H), 5.86 – 5.74 (m, 3H), 5.66 (d, J = 8.2 Hz, 1H), 5.55 – 5.40 (m, 8H), 5.35 (d,J = 6.5 Hz, 2H), 4.86 (q, J = 6.1 Hz, 1H), 4.78 (dd, J = 10.0, 5.1 Hz, 1H), 4.74 – 4.59 (m, 8H), 4.52 – 4.43 (m, 3H), 4.42 (dd, J = 9.4, 2.9 Hz, 1H), 4.38 (dt, J = 6.1, 2.8 Hz, 1H), 4.27 (ddd, J = 10.5, 9.0, 5.4 Hz, 2H), 4.18 – 4.06 (m, 4H), 4.00 (dd, J = 5.2, 3.5 Hz, 1H), 3.67 (dd, J = 4.4, 2.3 Hz, 1H), 3.56 (s, 3H), 3.52 (s, 3H), 3.48 (s, 3H), 0.89 (s, 9H), 0.08 (d, J = 11.2 Hz, 6H).

[0345] 13 C NMR (126 MHz, CDCl3) δ 162.58, 162.45, 162.17, 151.00, 150.96, 150.39, 139.62, 138.38, 138.02, 137.96, 137.92, 132.60, 129.62, 129.11, 128.51, 128.48, 127.95, 127.90, 127.89, 127.87, 127.84, 127.75, 126.43, 102.97, 102.68, 102.09, 95.60, 89.41, 88.52, 83.24, 81.64, 81.47, 80.39, 80.22, 80.16, 78.09, 73.18, 72.66, 72.47, 72.44, 70.84, 70.78, 70.61, 70.47, 70.41, 69.55, 66.34, 59.46, 58.72, 58.58, 53.57, 31.07, 18.20, -4.54, -4.85.

[0346] HR-MS (Q-TOF, ESI) Calcd for C 68H 81 F3N6O 24 P2Si, [M+H + ]: 1513.4572, measured value 1513.4565.

[0347] BS-trap-UUU-BOM-[3'-5']-trimer-blockpolymer b. Phosphate (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-((((((2R,3R,4R,5R)-5-(3-(((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl)oxy)((2-(tri) fluoromethyl)benzyl)oxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester (((2R,3R,4R,5R)-5- (3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy 2-(trifluoromethyl)benzyl) ester (4-methoxytetrahydrofuran-2-yl)methyl) ester The compound was synthesized using the following items according to GP-16 (4-step, one-pot sequence): UU-Cy-Nu (110 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalent), and 2-CF3BnOH [32 mg, 0.18 mmol, 1.5 equivalent, stock solution in 1 mL anhydrous toluene (0.18 M)] in toluene (2 mL) (for step 1), and U-BOM-Cl-CP (88 mg, 0.19 mmol, 1.6 equivalent) in MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equivalent) and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equivalent, stock solution in 1 mL anhydrous toluene (0.36 M)] in MeCN (for step 3), and TBSCl (109 mg, 0.72 mmol, 3 equivalent) in 1 mL MeCN (109 mg, 0.72 mmol, 1.5 equivalent). 98 mg (1.44 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:ethyl acetate (2% MeOH) gradient (0% to 80%). Fractions were analyzed by LC-MS, and fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Overall yield (4-step sequence) = 106 mg, 49%.

[0348] 31 P NMR (203 MHz, CDCl3) δ -1.66, -2.20.

[0349] 1 H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 8.2 Hz, 1H), 7.65 (dd, J= 11.7,7.7 Hz, 5H), 7.58 (td, J = 7.8, 4.5 Hz, 3H), 7.51 (d, J = 8.2 Hz, 1H), 7.47 –7.40 (m, 4H), 7.38 – 7.28 (m, 14H), 7.28 – 7.23 (m, 6H), 6.06 (d, J = 4.3 Hz,1H), 5.92 (d, J = 4.2 Hz, 1H), 5.83 (d, J = 2.2 Hz, 1H), 5.74 – 5.61 (m, 4H),5.52 – 5.29 (m, 12H), 4.90 – 4.81 (m, 2H), 4.69 (d, J = 4.9 Hz, 7H), 4.47 –4.18 (m, 7H), 4.17 – 4.05 (m, 3H), 3.98 (dd, J = 11.9, 2.0 Hz, 1H), 3.91 – 3.87(m, 2H), 3.82 (dd, J = 11.9, 1.7 Hz, 1H), 3.66 – 3.61 (m, 1H), 3.52 (s, 3H),3.43 (s, 3H), 3.42 (s, 3H), 0.93 (s, 9H), 0.88 (s, 9H), 0.13 (s, 6H), 0.09(s, 3H), 0.06 (s, 3H)。

[0350] 13C NMR (126 MHz, CDCl3) δ 162.61, 162.50, 162.30, 151.12, 151.02,150.89, 138.26, 138.22, 137.97, 137.96, 137.90, 133.42, 132.64, 132.59,130.12, 129.64, 129.24, 129.14, 128.46, 128.45, 127.87, 127.85, 127.83,126.48, 126.44, 126.40, 102.72, 102.32, 102.11, 89.45, 88.31, 87.02, 83.23, 83.11, 83.05, 82.61, 81.54, 81.48, 81.33, 81.31, 80.60, 74.35, 74.31, 73.46, 73.42, 72.43, 72.39, 70.46, 70.43, 70.38, 69.46, 66.52, 66.33, 66.16, 66.12, 65.97, 61.85, 58.72, 58.62, 58.56, 25.99, 25.73, 18.46, 18.17, -4.56, -4.88, -5.46.

[0351] HR-MS (Q-TOF, ESI) calculated value C 82 H 102 F6N6O 25 P2Si2, [M+Na + ]: 1825.5705, measured value 1825.5695.

[0352] c. Phosphate ((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine) (2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-) (((tert-butyldimethylsilyl)oxy)methyl)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran (2-(trifluoromethyl)benzyl)oxy)phosphoyl)oxy)methyl)-5-(6-(dibenzylamino)-9H-purine) (2-(trifluoromethyl)benzyl) ester (-9-yl)-4-methoxytetrahydrofuran-3-yl) ester The compound was synthesized using the following items according to GP-16 (4-step, one-pot sequence): UU-Cy-Nu (130 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalent), and 2-CF3BnOH [32 mg, 0.18 mmol, 1.5 equivalent, 1 mL anhydrous toluene stock solution (0.18 M)] in toluene (2 mL) (for step 1), and A-Bn 2-Cl-CP (104 mg, 0.19 mmol, 1.6 equivalent) in MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equivalent) and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equivalent, 1 mL anhydrous toluene stock solution (0.36 M)] in 1 mL MeCN (for step 3), and TBSCl (109 mg, 0.72 mmol, 1.5 equivalent) in MeCN (for step 2). 98 mg (1.44 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:ethyl acetate (2% MeOH) gradient (0% to 80%). Fractions were analyzed by LC-MS, and fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Overall yield (4-step sequence) = 100 mg, 40%. Quality was confirmed by LC-MS.

[0353] 31 P NMR (203 MHz, CD3CN) δ -1.90, -2.28.

[0354] 19 F NMR (471 MHz, CD3CN) δ -59.96, -60.01.

[0355] 1 H NMR (500 MHz, CD3CN) δ 8.26 (d, J = 2.9 Hz, 1H), 8.24 (s, 2H), 8.07(s, 1H), 8.02 (d, J = 4.8 Hz, 1H), 8.00 (s, 1H), 7.66 (ddd, J = 10.3, 5.9, 2.3Hz, 4H), 7.53 (q, J = 8.1 Hz, 2H), 7.42 (dt, J= 15.1, 7.6 Hz, 2H), 7.31 – 7.12(m, 35H), 6.08 (d, J = 4.4 Hz, 1H), 6.05 (d, J = 6.5 Hz, 1H), 6.01 (d, J = 5.8 Hz,1H), 5.45 (s, 4H), 5.29 (t, J = 7.8 Hz, 6H), 5.13 (ddd, J = 7.5, 4.8, 2.8 Hz,2H), 4.91 (s, 5H), 4.69 (dt, J = 16.8, 5.0 Hz, 3H), 4.43 – 4.30 (m, 7H), 4.19(dd, J = 7.1, 3.8 Hz, 2H), 3.76 – 3.67 (m, 2H), 3.37 (s, 3H), 3.32 (s, 3H), 3.31 (s, 3H), 0.91 (s, 9H), 0.83 (s, 9H), 0.11 (d, J = 3.7 Hz, 6H), -0.00 (d, J =1.3 Hz, 6H)。

[0356] 13C NMR (126 MHz, CD3CN) δ 169.09, 155.37, 155.34, 155.31, 152.94,152.89, 152.80, 151.58, 151.32, 151.26, 138.77, 138.68, 138.28, 134.48,133.24, 130.58, 130.53, 129.46, 129.41, 129.06, 129.02, 128.99, 128.09,128.03, 127.64, 127.61, 126.64, 126.59, 126.55, 125.96, 123.78, 120.59,120.52, 120.32, 117.86, 87.36, 86.87, 85.87, 84.38, 83.37, 82.96, 81.98,81.07, 75.72, 74.97, 70.92, 67.71, 67.09, 66.33, 62.93, 58.67, 58.53, 58.37,31.77, 25.85, 25.81, 25.64, 18.50, 18.24, -4.93, -5.12, -5.67, -5.76。

[0357] d. Phosphate (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-((((((2R,3R,4R,5R)-5-(3-(((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1 (2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl)oxy)((2-(tri) fluoromethyl)benzyl)oxy)phosphoryl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester (((2R,3R,4R,5R)-3- ((tert-butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl) 2-(trifluoromethyl)benzyl ester (methyl) ester The compound was synthesized using the following items according to GP-16 (4-step, one-pot sequence): Bn2-AU-Cy-Nu (120 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalent), and 2-CF3BnOH [32 mg, 0.18 mmol, 1.5 equivalent, 1 mL anhydrous toluene stock solution (0.18 M)] in toluene (2 mL) (for step 1) and U-BOM-Cl-CP (88 mg, 0.19 mmol, 1.6 equivalent) in 0.6 mL MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equivalent) and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equivalent, 1 mL anhydrous toluene stock solution (0.36 M)] in 1 mL MeCN (for step 3) and TBSCl (109 mg, 0.72 mmol, 3 equivalent, 1 mL anhydrous toluene stock solution (0.36 M)] in 1 mL MeCN (for step 3). 1.44 mmol (6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:ethyl acetate (2% MeOH) gradient (0% to 80%). Fractions were analyzed by LC-MS, and fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Overall yield (4-step sequence) = 60 mg, 26%.

[0358] Alternatively, the crude product was purified using reversed-phase C-18 column chromatography with the aid of a next-generation automated system. The crude product was dissolved in a minimal amount of DMSO and then loaded onto a pre-packed 25g C-18 column, eluted with a water:ACN gradient (0% to 100%). Fractions were analyzed by LC-MS, and the fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Overall yield (4-step reaction sequence) = 105 mg, 46%. Quality was confirmed by LC-MS.

[0359] 31 P NMR (203 MHz, CDCl3) δ -1.85, -2.30.

[0360] 1 H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 4.2 Hz, 1H), 7.90 – 7.84 (m, 1H), 7.62 (ddd, J = 17.2, 7.7, 1.3 Hz, 4H), 7.54 (dd, J= 8.3, 6.7 Hz, 1H), 7.50 –7.24 (m, 26H), 6.07 (dd, J = 11.4, 4.0 Hz, 2H), 5.75 – 5.61 (m, 2H), 5.52 – 5.36 (m, 6H), 5.36 – 5.22 (m, 5H), 5.02 – 4.79 (m, 4H), 4.85 (ddt, J = 17.5, 7.1, 5.0 Hz, 2H), 4.74 – 4.63 (m, 5H), 4.37 – 4.19 (m, 7H), 4.00 – 3.92 (m, 1H), 3.92 – 3.78 (m, 3H), 3.47 (s, 3H), 3.43 (s, 3H), 3.39 (s, 3H), 0.91 (d, J = 2.7 Hz, 18H), 0.13 – 0.09 (m, 12H)。

[0361] 13C NMR (126 MHz, CDCl3) δ 162.62, 162.34, 155.06, 152.78, 151.10,151.05, 151.03, 150.65, 138.31, 138.23, 137.98, 137.96, 137.92, 137.58,133.64, 133.48, 133.42, 132.60, 132.46, 130.03, 129.51, 129.11, 128.75,128.71, 128.69, 128.43, 127.98, 127.84, 127.81, 127.41, 126.39, 126.35,126.22, 126.18, 126.13, 126.09, 125.27, 125.24, 123.09, 123.07, 120.41,102.67, 102.27, 88.06, 87.58, 87.01, 83.12, 83.06, 82.65, 82.63, 82.59,82.40, 82.34, 81.37, 81.34, 80.69, 80.64, 74.32, 74.28, 73.39, 73.36, 72.37,70.50, 70.42, 67.03, 66.99, 66.42, 66.12, 66.08, 61.85, 58.74, 58.71, 58.68,58.66, 58.61, 25.98, 25.81, 18.44, 18.39, 18.22, -4.58, -4.82, -5.46, -5.49。

[0362] e. Phosphoric acid (2R,3R,4R,5R)-2-[[[[(2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo 3,4-Dihydropyrimidin-1(2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran- 3-yl)oxy]((2-(trifluoromethyl)benzyl)oxy)phosphoyl]oxy]methyl]-5-(6-chloro-9H-purine-9-yl)-4- Methoxytetrahydrofuran-3-yl ester, (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydro Pyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) ester, (2- (trifluoromethyl)benzyl) ester The compound was synthesized according to GP-16 (4-step, one-pot sequence) using the following: UA(6Cl)-Cy-Nu (101 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalence) and 2-CF3BnOH [32 mg, 0.18 mmol, 1.5 equivalence, 1 mL anhydrous toluene stock solution (0.18 M)] in toluene (2 mL) (for step 1), and U-BOM-Cl-CP (89 mg, 0.19 mmol, 1.6 equivalence) in 0.6 mL MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equivalence) and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equivalence, 1 mL anhydrous toluene stock solution (0.36 M)] in 1 mL MeCN (for step 3), and TBSCl (109 mg, 0.36 mmol, 3 equivalence, 1 mL anhydrous toluene stock solution (0.36 M)] in 1 mL MeCN (for step 3). (mg, 0.72 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) (for step 4). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimal amount of DCM, DCM:ethyl acetate (2% MeOH) gradient (0% to 80%). Fractions were analyzed by LC-MS, and fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Overall yield (4-step sequence) = 100 mg, 40%, dr 4:1. Quality was confirmed by LC-MS.

[0363] 31 P NMR (203 MHz, CDCl3) δ -1.16 (P1, minor diastereomer), -1.51 (P1, major diastereomer), -2.36 (P2, major diastereomer), -2.67 (P2, minor diastereomer).

[0364] diastereomer mixtures 1 H NMR (500 MHz, CDCl3): δ 8.69 (s, 1H), 8.32 (s, 1H), 7.88 – 7.78 (m, 2H), 7.65 (q, J= 7.4 Hz, 4H), 7.62 – 7.48 (m, 8H), 7.48 – 7.39 (m, 4H), 7.39 – 7.26 (m, 16H), 7.26 – 7.18 (m, 3H), 6.16 – 6.02(m, 3H), 5.87 – 5.81 (m, 2H), 5.76 – 5.68 (m, 2H), 5.66 (dd, J = 8.2, 1.6 Hz,1H), 5.57 – 5.25 (m, 15H), 5.18 (tdd, J = 7.8, 4.8, 2.7 Hz, 2H), 4.94 – 4.83(m, 2H), 4.73 – 4.62 (m, 8H), 4.56 – 4.48 (m, 2H), 4.48 – 4.32 (m, 5H), 4.32– 4.23 (m, 3H), 4.18 – 4.03 (m, 6H), 4.02 – 3.89 (m, 3H), 3.81 (dd, J = 11.9, 1.7 Hz, 1H), 3.66 (p, J = 2.5 Hz, 2H), 3.52 (s, 6H), 3.44 (s, 4H), 3.34 (s,4H), 0.89 (d, J = 3.6 Hz, 27H), 0.16 – 0.04 (m, 18H).

[0365] diastereomer mixtures 13C NMR (126 MHz, CDCl3) δ 162.61, 162.46, 152.33,152.31, 151.69, 151.65, 151.42, 151.17, 151.11, 150.88, 150.86, 144.05,144.01, 138.35, 138.26, 138.22, 138.20, 137.96, 137.89, 137.76, 133.41,132.51, 132.44, 132.41, 129.80, 129.78, 129.57, 129.10, 129.04, 128.97,128.94, 128.42, 128.41, 127.83, 127.78, 126.34, 125.23, 123.05, 120.87,102.46, 102.28, 102.09, 102.04, 89.59, 89.47, 88.28, 87.02, 86.75, 86.32,83.26, 83.22, 83.13, 83.07, 82.88, 82.59, 82.57, 82.13, 82.08, 82.03, 81.49,81.43, 81.00, 80.93, 80.90, 75.08, 74.67, 74.63, 74.49, 74.45, 72.41, 72.38,72.33, 70.40, 70.36, 69.46, 69.44, 68.25, 66.37, 66.32, 66.09, 66.05, 62.07,61.89, 60.49, 59.02, 58.95, 58.91, 58.86, 58.61, 58.55, 25.96, 25.93, 25.71,25.70, 21.16, 18.41, 18.16, 14.31, -4.56, -4.90, -5.50, -5.54, -5.57。

[0366] f. Phosphoric acid (2R,3R,4R,5R)-2-[[[[(2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo 3,4-Dihydropyrimidin-1(2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran- 3-yl)oxy]((2-(trifluoromethyl)benzyl)oxy)phosphoyl]oxy]methyl]-5-(6-(dibenzylamino)-9H-purine- 9-yl)-4-methoxytetrahydrofuran-3-yl ester [[((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] Ester (2-(trifluoromethyl)benzyl) ester The compound was synthesized using the following ingredients according to GP-16 (4-step, one-pot sequence): UA-Bn2-Cy-Nu (120 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalent), and 2-CF3BnOH [32 mg, 0.18 mmol, 1.5 equivalent, 1 mL anhydrous toluene stock solution (0.18 M)] in toluene (2 mL) (for step 1), and U-BOM-Cl-CP (88 mg, 0.19 mmol, 1.6 equivalent) in MeCN (0.6 mL) (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equivalent) and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equivalent, 1 mL anhydrous toluene stock solution (0.36 M)] in MeCN (1 mL) (for step 3), and TBSCl (109 mg, 0.72 mmol, 1.5 equivalent, 1 mL anhydrous toluene stock solution (0.36 M)] in MeCN (1 mL). 98 mg (1.44 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) (for step 4). The crude product was purified by reversed-phase C-18 column chromatography with the aid of the Next-Gen automated system. The crude product was dissolved in a minimal amount of DMSO and loaded onto a pre-packed 25 g C-18 column using a water:ACN gradient (0% to 100%). The fractions were analyzed by LC-MS, and the fractions containing the desired product were combined and concentrated to give the title compound as a white solid with a purity of 90%. Overall yield (4-step sequence) = 100 mg, 44%. Quality was confirmed by LC-MS.

[0367] 31 P NMR (203 MHz, CD3CN) δ -1.95, -2.21.

[0368] 19 F NMR (471 MHz, CD3CN) δ -59.86, -59.99.

[0369] 1 H NMR (500 MHz, CD3CN) δ 8.26 (s, 1H), 8.06 (s, 1H), 7.78 (dd, J = 8.3, 2.8 Hz, 1H), 7.66 (ddd, J = 30.5, 14.5, 7.6 Hz, 5H), 7.57 – 7.41 (m, 4H), 7.35– 7.16 (m, 21H), 6.08 (d, J = 4.4 Hz, 1H), 6.00 (d, J= 5.2 Hz, 1H), 5.85 (d, J =4.9 Hz, 1H), 5.72 – 5.65 (m, 1H), 5.61 (dd, J = 8.1, 5.0 Hz, 1H), 5.45 (s, 1H), 5.39 – 5.31 (m, 6H), 5.28 (dd, J = 17.2, 8.8 Hz, 3H), 4.95 – 4.87 (m, 2H), 4.67– 4.53 (m, 5H), 4.40 – 4.22 (m, 7H), 4.18 (q, J = 4.8 Hz, 1H), 4.00 – 3.85 (m,3H), 3.81 (dd, J = 11.9, 2.2 Hz, 1H), 3.41 – 3.31 (m, 9H), 0.93 – 0.86 (m, 18H), 0.13 – 0.08 (m, 12H).

[0370] Example 12. Synthesis of Cyclic Nucleotide Imide Compounds Program A In a glove box filled with N2, 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-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy 7-Methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (0.200 g, 250 μmol, 1.0 equivalent), 3-((bis(diisopropylamino)phosphoryl)oxy)propionitrile (103 μL, 325 μmol, 1.3 equivalent), and a stir bar. The reaction mixture in container A was sealed, then removed from the glove box, and DCM (4 mL) was added to the container. The mixture was then cooled to 0 °C over 10 minutes.

[0371] In a separate vial (container B) outside the glove box, 29.3 mg (225 μmol, 0.9 equivalents) of solid 5-(ethylthio)-1H-tetrazole was added, followed by purging of container B with nitrogen five times. Then, 1.2 mL (47 mmol) of DCM was added to container B, and the solution in container B was transferred dropwise to container A at 0°C. The reaction mixture in container A was heated to room temperature and stirred for 3 hours, or until the starting material was exhausted. Container A was then left open to air to evaporate the solvent. The compound was purified by column chromatography using 100% ethyl acetate. 0.138 g of the product mixture was obtained as a pale yellow oily N,N-diisopropylphosphonamide 2-cyanoethyl ester. The actual amount of product in the mixture was 60 mg, 60 μmol, with a yield of 24%. It was observed that at 14 ppm, N,N-diisopropylphosphonamide 2-cyanoethyl ester (hydrolyzed N,N,N′,N′-tetraisopropylphosphonamide 2-cyanoethyl ester) was difficult to separate from the product.

[0372] 31 P NMR (203 MHz, CD3CN) δ -1.11.

[0373] Program B Add 0.200 g, 250 μmol, 1.0 equivalent 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-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidin-2,4(1H,3H)-dione and 103 μL, 325 μL of 3-((bis(diisopropylamino)phosphono)oxy)propionitrile to a heat-dried round-bottom flask (container A). μmol (1.3 equivalents) of desiccant was added to the reaction vessel along with a stir bar. The vessel A was then sealed and purged with N2 five times. Anhydrous DCM (4 mL) was then added to vessel A, and the mixture was cooled to 0°C over a 10-minute period.

[0374] In a separate vial (container b), 29.3 mg (225 μmol, 0.9 equivalents) of solid 5-(ethylthio)-1H-tetrazole was added, followed by purging with N2 and spraying five times. Then, 1.2 mL (47 mM) of DCM was added to container b, and the solution in container b was transferred dropwise to container a at 0 °C. Container a was heated to room temperature and stirred for 3 hours or until the starting material was exhausted. The reaction progress was monitored by LCMS. Container a was then left open to air to evaporate the solvent. The compound was purified by column chromatography using 100% ethyl acetate.

[0375] Example 13. Synthesis of Cyclotetramer a. Linear synthesis of fragments in the solution phase b. Synthesis via solution-phase imide chemistry using aggregated dimer coupling. Add 22 mg, 20 ml of (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl ester (i.e., (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) ester (2-(trifluoromethyl)benzyl) ester (20 mg) to vial (container A). μmol, 1.0 equivalent), stir bar, sieve bag, and (2R,3R,4R,5R)-5-(3-(((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((((4aR,6R,7R,7aR)-6-(3-(((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-2-yl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl) (20 mg, 20 μmol, 1.0 equivalent). The reaction vessel A was then sealed, purged with nitrogen, and sprayed five times. Add anhydrous DCM (4 mL) to container A and cool the reaction solution to 0°C within 10 minutes.

[0376] Add 2.3 mg, 18 μmol, 0.9 equivalents of 5-(ethylthio)-1H-tetrazole to a separate vial (container B) and seal the container. Then purge container B with N2 and spray five times. Next, add DCM (total 1 mL, 4 mmol) to container B. Then, transfer the contents of container B dropwise to container A at 0 °C. After the addition is complete, heat container A to room temperature and stir for 3 hours or until the starting material is exhausted. Monitor the reaction progress using LCMS.

[0377] c. Synthesis using imide derivatives of cyclic dinucleotides d. Polymerization reactions with longer segments in the solution phase The process provides a conversion rate of over 95%, and LC integration confirms that the crude yield of the tetramer product is approximately 22%.

[0378] e. Synthesis of longmers 150 mg, 1 equivalent, 187 μmol 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-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidin-2,4(1H,3H)-dione (187 μmol) was added to an 8 mL vial and stirred with a stir bar. The vial was then purged with nitrogen and sprayed three times. Anhydrous pyridine (1.48 g, 1.52 mL, 100 equivalent, 18.7 mmol) was then added to the container. Diphenylphosphonate (65.8 mg, 53.8 µL, 1.5 equivalents, 281 μmol) was added, and the reaction mixture was stirred at room temperature and monitored by LCMS. After the reaction was complete, the crude yield was calculated to be 52% by LCMS. The reaction contents were then used in subsequent reaction steps.

[0379] A 0.25 mL aliquot of the reaction product containing phosphonic acid (2R,3R,4R,5R)-5-(3-(((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((((4aR,6R,7R,7aR)-6-(3-(((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxopyrazine-2-yl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester phenyl ester was taken and concentrated by rotary evaporator to remove most of the remaining pyridine. Then, 0.5 mL of anhydrous DCM was added to dissolve the crude residue, and excess benzyl alcohol (15 µL) was added to the mixture. The reaction mixture was stirred overnight at RT. After 18 hours, the starting material was completely consumed, and the product was generated. The crude yield was observed to be 43% by LCMS.

[0380] Example 14. Synthesis of cyclopentamers using a one-pot solution-phase chemical method Step I: Dry the 50 mL flask (container A) equipped with a stir bar twice under vacuum using a hot air gun. Then purge and spray the container three times with N2. After the flask has cooled, add (22 µL, 0.16 mmol, 1.5 equivalents) of (2-(trifluoromethyl)phenyl)methanol under N2. Next, weigh lithium tert-butoxide (LitBuO, 13 mg, 0.16 mmol, 1.5 equivalents) into a separate vial, purge and spray three times with N2, and then add toluene (0.9 mL) to the vial. Add the solution from the vial to container A, stir the reaction mixture at room temperature for 30 minutes, and then cool to -25°C.

[0381] Add 99 mg, 0.11 mmol, 1.0 equivalent 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-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidin-2,4(1H,3H)-dione to a separate vial, then purge with nitrogen and spray three times. Add toluene (1.8 mL) to the vial, then add the resulting solution in one go to container A at -25°C. The reaction vessel A was stirred at this temperature for 4 hours.

[0382] Step II: 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (74 mg, 0.16 mmol, 1.5 equivalents) was added to a separate vial and then sealed. The vial was purged with nitrogen and sprayed three times, followed by the addition of anhydrous acetonitrile (0.53 mL). The resulting solution was then added dropwise to container A at -25°C. The reaction vessel was stirred at -25°C for 18 hours, after which an aliquot of the reaction mixture was analyzed to observe the completion of the reaction.

[0383] Step III: Lower the cooler temperature to -25°C. Dry the 50 mL round-bottom flask (container B) with a stir bar twice under vacuum using a hot air gun. Then purge and spray the container three times with N2. After the flask has cooled, add (22 µL, 0.16 mmol, 1.5 equivalents) of (2-(trifluoromethyl)phenyl)methanol under N2. Next, weigh potassium tert-butoxide (LitBuO, 13 mg, 0.16 mmol, 1.5 equivalents) into a vial, purge and spray the vial three times with N2, and then add toluene (0.9 mL) to the vial. After 30 minutes, add the contents of container B to container A at -30°C. Then stir the reaction mixture at -25°C for 5 hours. Afterward, take an aliquot of the reaction mixture for analysis to observe the completion of the reaction.

[0384] Step IV: Dry the 100 mL flask (container C) equipped with a stir bar twice under vacuum using a hot air gun. After container C has cooled, add 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (89 mg, 0.19 mmol, 1.8 equivalents) to the flask and seal the container. Purge and spray reaction container C three times with nitrogen, then add anhydrous acetonitrile (0.53 mL). Subsequently, cool container C to -25°C over 10 minutes. Next, transfer the contents of reaction container A dropwise to reaction container C, and then stir at -25°C for 18 hours. Eighteen hours later, equal portions of the reaction sample were taken to observe the reaction.

[0385] Step V: The vial equipped with a stir bar was dried twice under vacuum using a hot air gun. After the vial cooled, LitBuO (22 mg, 0.27 mmol, 2.5 equivalents) was weighed into the vial, and then purged with N2 and sprayed three times. Toluene (0.9 mL) was added to the vial, followed by (2-(trifluoromethyl)phenyl)methanol (43 µL, 0.32 mmol, 3.0 equivalents), and the solution was stirred for 30 minutes. Then, the contents of the vial were transferred to container C at -25°C. The reaction mixture was stirred at -25°C for 4 hours. Aliquots were taken from container C to monitor the reaction progress.

[0386] Step VI: Dry the 100 mL flask (container C) equipped with a stir bar twice under vacuum using a hot air gun. After container D has cooled, add 89 mg (0.19 mmol, 1.8 equivalents) of 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione to the flask and seal the container. Purge and spray reaction vessel D three times with N2, and add anhydrous acetonitrile (0.53 mL). Then, cool container D to -25°C over 10 minutes. Transfer the contents of reaction vessel C dropwise to reaction vessel D and stir at -25°C for 18 hours. Eighteen hours later, equal portions of the reaction sample were taken to observe the reaction.

[0387] Example 15. Templated enzymatic ligation of nonamers using trimers Figure 1A and Figure 1B Examples of enzymatic ligation reactions from short polymers to nonamellars are shown. Each reaction was controlled to ensure enzyme activity.

[0388] a. Reaction setup procedure: Two LCMS vials were used as reaction vessels. Reaction vessel A was the enzyme-free control, and reaction vessel B was the enzymatic reaction. Each vessel was filled with HO-UUU-OH (32.51 mmol / L), Phos-CCC-OH (25.97 mmol / L), nonamer directing agent (25.02 mmol / L), Tris-HCl (pH 7.5, 1000 mmol / L), magnesium chloride solution (1000 mmol / L), potassium chloride solution (3000 mmol / L), ATP (100 mmol / L), dithiothreitol (DTT, 100 mmol / L), and an enzyme-free aqueous solution. Finally, the enzyme was added to vessel B.

[0389] Add the following to reaction vessel A: HO-UUU-OH (6.2 µL, 1.0 equivalent 32.51 mmol solution), Phos-CCC-OH (15.4 µL, 2.0 equivalent 25.97 mmol solution), nonamer directing agent (8.0 µL, 1.0 equivalent 25.02 mmol solution), Tris-HCl (pH 7.5) (25.0 µL, 125 equivalent 1000 mmol solution), magnesium chloride (MgCl2) solution (5.0 µL, 25 equivalent 1000 mmol solution), potassium chloride (KCl) solution (16.7 µL, 250 equivalent 3000 mmol solution), adenosine triphosphate (ATP) (10.0 µL, 5.0 equivalent 100 mmol solution), and dithiothreitol (DTT) (5.0 µL, 2.4 equivalent 100 mmol solution). mmol solution) and nuclease-free water (408.7 µL).

[0390] The following substances were added to reaction vessel B: HO-UUU-OH (6.2 µL, 1.0 equivalent of 32.51 mmol solution), Phos-CCC-OH (15.4 µL, 2.0 equivalent of 25.97 mmol solution), 9-mer directing agent (8.0 µL, 1.0 equivalent of 25.02 mmol solution), Tris-HCl (pH 7.5) (25.0 µL, 125 equivalent of 1000 mmol solution), magnesium chloride (MgCl2) solution (5.0 µL, 25 equivalent of 1000 mmol solution), potassium chloride (KCl) solution (16.7 µL, 250 equivalent of 3000 mmol solution), adenosine triphosphate (ATP) (10.0 µL, 5.0 equivalent of 100 mmol solution), dithiothreitol (DTT) (5.0 µL, 2.4 equivalent of 100 mmol solution), and nuclease-free water (408.7 µL). Finally, add Codexis 3.007 enzyme (2.1 µL, 0.0625 equivalent of 5.91 mmol solution).

[0391] The two containers were then added to a hot mixer, heated at 37°C, and stirred at 350 rpm for 18 hours.

[0392] b. Reaction termination procedure: Eighteen hours later, 25 µL aliquots from both reaction vessel A and reaction vessel B were added to separate LCMS vials. Subsequently, EDTA (125 µL of 26.7 mmol solution) was added to each aliquot to terminate the reaction (aimed at a 5:1 aliquot ratio). The solutions were then analyzed by LCMS, revealing evidence of both enzyme ligation and control reactions.

[0393] Crude yield was determined by integrating the nonamer directing agent as an internal standard using a denaturing UPLC method and comparing the ratio of product to nonamer directing agent.

[0394] Example 16. Synthesis of Cyclic Phosphophosphate Nucleosides a. Synthesis of cyclic thiophosphates using Cl⁻ from PSCl₃ Program 1 A stirred solution of thiophosphoryl chloride (1.05 mmol, 1.05 equivalent) and N-protected nucleoside diol BOM-U (1 mmol, 1.0 equivalent) in DCM (5 mL) was prepared. Under Schlenk conditions, 2 mL of lithium tert-butoxide solution (2.05 mmol, 2.05 equivalent) in 1 M THF was added to the solution over a 5-minute period at 0 °C. After the addition was complete, the mixture was heated to ambient temperature and left overnight. Aliquots of the crude product mixture were concentrated, and the conversion was monitored by LCMS and NMR. PNMR revealed the presence of two diastereomers, P1 and P2, with dr ratios ranging from 1.7:1 to 1.3:1.

[0395] NMR data for the two isomers: 31 P NMR (162 MHz, CDCl3) δ 61.23 (P1), 57.83 (P2). 1 H 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, δ7.42 – 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).

[0396] Purification method (a): The crude product was precipitated at 0°C using 0.1 mL of MTBE with stirring. The filtrate was concentrated and subjected to vacuum before NMR analysis.

[0397] Purification method (b): The crude product was concentrated by rotary evaporator and then purified by wet loading (dissolved in DCM) on a silica gel column EA / Hex 0-100% to obtain two diastereomers, P1 and P2, with a combined separation yield of 40%.

[0398] The reaction conditions and Cr dr results are listed in Table 8 [Cr dr is the crude reaction P(R):P(S) determined by P NMR. 4a and 4b are two time points of the same batch. Reaction 1 was purified using method (a). Reaction 2-4 were purified using method (b)].

[0399] Table 8 - Synthesis of Cl-cyclic thiophosphates Program 2 Under Schlenk conditions, in an ice bath or refrigerator at 0 to -20°C, a feed solution of lithium tert-butoxide in 1 M THF or MeTHF solvent (2.05 equivalents) was added to a stirred solution of thiophosphoryl chloride (1.05 equivalents) and N-protected nucleoside diol N-Ar protected nucleoside base (1.0 equivalents) in DCM (x mL) over a 5-minute period. After 1–20 hours, the conversion and dr of the crude reaction mixture were monitored by LCMS and NMR.

[0400] 31P NMR (162 MHz, CDCl3), mA: δ 60.82 (Rp), 58.44 (Sp). mC: δ 61.50(Rp), 58.90 (Sp). fU: δ 60.35 (Rp), 57.42 (Sp). mG: 61.86 (Rp), 57.32 (Sp). fALCMS.

[0401] Range of nucleobases b. Synthesis of chlorocyclic 2'-methoxy-G-thiophosphate 1-Benzyl-9-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-thiooxytetrahydro-4H-furano[3,2-d][1,3,2]dioxopyrazine-6-yl)-2-(dibenzylamino)-1,9-dihydro-6H-purine-6-one) Under N2 conditions via the Schlenk line, 1-benzyl-2-(dibenzylamino)-9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-1,9-dihydro-6H-purine-6-one (5.677 g, 1 equivalent, 10.00 mmol) was dissolved in DCM (50 mL) at RT and then slowly cooled to -20 °C for 90 minutes. Freshly distilled thiophosphoryl chloride (1.778 g, 1.06 mL, 1.05 equivalent, 10.50 mmol) was then added dropwise using a syringe needle. Subsequently, at 1:50 p.m., 2-methylprop-2-ol lithium salt (2.2 M in THF) (1.662 g, 2.05 equivalent, 20.50 mmol) was added over 10 minutes using a syringe needle to THF (9.3 mL). Approximately 50 µL of crude sample was collected using a syringe for analysis. The crude PNMR showed two major product isomer peaks with a ratio of approximately 2.4:1. After 30 minutes, most of the starting material was consumed (1% was still detectable under UV detection, and the integral was determined by uncorrected LCMS), and smaller peaks of other impurities eluted earlier than the products. The reaction was stopped after 1 hour. The crude reaction solution was concentrated to remove most of the solvent, then redissolved in DCM, and loaded onto a 40 g silica gel column eluted with EA / Hex (0-80%) (using a combi-flash system) to give 3.56 g of a pale yellow to white solid product (54% separation yield, isomer ratio 2.2:1).

[0402] 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, 48.29.

[0403] c. Synthesis of alkoxy-cyclic thiophosphates via PSORCl2 Under a Schlenk line, N-protected nucleoside diol BOM-U (75.7 mg, 1 equivalent, 0.2 mmol) and 4-dimethylaminopyridine (DMAP, 63.5 mg, 2.6 equivalent, 520 μmol) dissolved in 1.8 mL DCM (total 2 mL) were slowly added to O-ethylthiophosphoric dichloride (37.6 mg, 1.05 equivalent, 210 μmol) dissolved in 0.2 mL dichloromethane. The mixture was stirred at -20 °C for 20 hours with the aid of a refrigerator. The reaction solution was then heated to room temperature and stirred for 3 hours. The crude product was concentrated and redissolved in 0.5 mL of dichloromethane, loaded onto a 5 g Biotage silica column, and then purified with EA / Hex 0-100% eluent. The product was eluted with approximately 30% EA / Hex to give two diastereomers, P1 and P2, with a separation yield of 40% (dr = 2:1 for P1:P2).

[0404] NMR data for the two isomers: 31 P 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).

[0405] Example 17. Synthesis of cyclic thioester dinucleotides via cyclic chlorothiophosphates a. Cyc U*U (Program 1) Solution A: Under a nitrogen atmosphere, LiOtBu (48.7 mg, 1.5 equivalents, 609 μmol) was added to a 20 mL reaction tube A equipped with a magnetic stir bar and dried in an oven using the Schlenk line technique. A stock solution of 3'-TBS-5'-OH nucleoside (200 mg, 1 equivalent, 406 μmol) in toluene (2 mL) was added to reaction tube A, and the mixture was allowed to stand and maintained at -20°C with the aid of a refrigerator, and stirred for 30 minutes.

[0406] Solution B: Under Schlenk line conditions, the cyclochlorothiophosphate monomer PS-Cl UBOM (dr Rp:Sp = 1.2:1, 231 mg, 1.0 equivalent, 487 μmol) was dissolved in acetonitrile (2 mL) in an oven-dried flask at -20°C using a refrigeration unit. Solution A was added dropwise to B over 5 minutes using a syringe needle, and the mixture was stirred for 20 hours. 50 µL aliquots were collected using a syringe, concentrated, and then... 31 P NMR and LCMS analyses were performed to confirm the completion of the reaction and the crude reaction yield (Rp:Sp = 13:1). The crude reaction solution was concentrated to remove most of the solvent and then loaded onto a silica gel column (5 g) eluted with EA / Hex (0-60%) via a combi-flash system to give 150 mg of the major isomer “Rp” as a white solid (40% separation yield, dr 16:1).

[0407] 31 P NMR (162 MHz, CDCl3) δ 66.20 (Rp, low polarity), 62.00 (Sp, high polarity).

[0408] 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).

[0409] 13 C 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.

[0410] HRMS (ESI+): m / z Calculated value C42H65N4O14PSSi[M+H]+: 930.29; Measured values ​​930.31 (Rp), 930.33 (Sp).

[0411] b. Cyc U * U (Program 2) Solution A: Under a nitrogen atmosphere, LiOtBu (96.1 mg, 1.5 equivalents, 1.20 mmol) was added to a 20 mL reaction flask A equipped with a magnetic stir bar using the Schlenk line technique, and the mixture was cooled to -20 °C with the aid of a refrigerator. A stock solution of 3'-TBS-5'-OH nucleoside (394 mg, 1 equivalent, 800 μmol) in toluene (4 mL) was slowly added to reaction tube A through a syringe needle, and the mixture was stirred at -20 °C for 30 minutes with the aid of a refrigerator.

[0412] Solution B: Under Schlenk line conditions, the cyclochlorothiophosphate monomer PS-Cl UBOM (dr Rp:Sp = 1:1, 500 mg, 70 wt%, 0.921 equivalents, 737 μmol) was dissolved in acetonitrile (4 mL) at -20 °C using a refrigerator. Solution A was added dropwise to B over 5 minutes using a syringe, and the mixture was stirred for 20 hours. The solution was then concentrated to 50 µL and analyzed by P NMR and LCMS to confirm the completion of the reaction and the crude product dr (Rp:Sp = 27:1).

[0413] The reactant used in Procedure 2, the cyclochloro-thiophosphate monomer PS-Cl UBOM, was not purified by silica gel column chromatography. Its synthesis and termination methods are as follows.

[0414] Under Schulenk line N2, diol-U-BOM (3.784 g, 1 equivalent, 10.00 mmol) and thiophosphoryl chloride (1.778 g, 1.06 mL, 1.05 equivalent, 10.50 mmol) were dissolved in DCM (48 mL) at 0 °C and stirred for 20 min. Then, 20 mL of lithium 2-methylpropane-2-ol (1 M THF solution) (1.601 g, 2.0 equivalent, 20.00 mmol) was added via syringe and needle, and the temperature was increased from 0 °C to 23 °C over 4 hours. After the reaction was complete, the solution was concentrated using a rotary evaporator. A mixture of heptane / MTBE / EA = 50 mL / 50 mL / 30 mL was slowly added to the concentrated crude product solution to precipitate salt impurities and other insoluble byproducts. The filtrate was concentrated and vacuum-sealed to give 5.6 g of a light brown solid. Based on the theoretical yield of 4.75 g and the actual mass of 5.6 g, the precipitate may contain a mixture of ~0.80 g of LiOH, LiOtBu, and LiCl, totaling approximately 10⁻¹⁵ mmol to 1-1.5 eq. Calculate 10 mmol based on the MW 80 of LiOtBu.

[0415] c. Two-step addition (standard) Solution A: Under a nitrogen atmosphere, LiOtBu (1.5 equivalents) was added to a 20 mL reaction tube A equipped with a magnetic stir bar using the Schlenk line technique. A stock solution of 3'-TBS-5'-OH nucleoside (1 equivalent) in toluene (0.2 M) was added to reaction tube A, and the mixture was allowed to stand at -20°C with the assistance of a refrigerator, and stirred for 30 minutes.

[0416] Solution B: Under Schlenk line conditions, the cyclochlorothiophosphate monomer (dr Rp:Sp ~ 1:1, 1.0 eq) was dissolved in acetonitrile (0.2 M) at -20 °C in an oven-dried flask using a refrigeration system. Solution A was added dropwise to solution B over 5 minutes using a syringe needle, and the mixture was stirred for 20 hours. 50 µL aliquots were collected using a syringe and concentrated for further processing. 31 PNMR and LCMS analyses were performed to confirm the completion of the reaction and the dr value of the crude reaction. The crude reaction solution was concentrated to remove most of the solvent, dissolved in a minimal amount of DCM, and then loaded onto a silica gel column eluted with EA / Hex (0-60%) via a combi-flash system to obtain the major isomer "Rp" as a white solid (dr ~ 4:1 to 20:1).

[0417] Table 9 - Two-Step Addition d. One-pot addition In a vacuum-dried glass container using a hot air gun, B1 5'-OH-3'TBS-monomer (1 equivalent) and B2 Cl-PS-cyclic monomer (1.2 equivalent, 120 μmol) were mixed at room temperature and then completely dissolved in anhydrous DCM (0.1 M). The mixture was then cooled to -20°C under N2 with the aid of a refrigerator. Lithium 2-methylpropane-2-ol (2.2 M, dissolved in THF) (1.5 equivalent) was added dropwise to the reaction vessel over 5 minutes using a syringe needle. The temperature was then maintained and the mixture was stirred under N2 for 1 to 20 hours until the reaction was complete. The conversion was monitored using LCMS. After 1 hour, 50 µL aliquots were collected using a syringe, concentrated, and then subjected to further processing. 31 P NMR and LCMS analysis confirmed the completion of the reaction, with a crude yield of 82% and an enantiomeric excess ratio of cr dr, Rp:Sp. The crude reaction solution was concentrated to remove most of the solvent and dissolved in a minimal amount of DCM, then loaded onto a silica gel column eluted with EA / Hex (0-60%) via a combi-flash system to give 90 mg of the major isomer "Rp" as a white solid (82% yield, enantiomeric excess ratio 16:1).

[0418] Table 10 – One-Pot Addition Method e. Ring PS mAmU In a vacuum-dried glass container using a hot air gun, B1 5'-OH-3'TBS mA ((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methanol (57.6 mg, 1 equivalent, 100 μmol) and B2 Cl-PS-cyclomU monomer 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-thioanion tetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (57.0 mg, 1.2 equivalent, 120 μmol) were mixed. The mixture was stirred at room temperature and then completely dissolved in anhydrous dichloromethane (DCM) (2 mL). The solution was then cooled to -20 °C under nitrogen (N2) protection with the aid of a refrigeration unit. Lithium 2-methylprop-2-ol (2.2 M dissolved in tetrahydrofuran (THF)) (12.0 mg, 68 μL, 1.5 equivalents, 150 μmol) was added dropwise to the reaction vessel over 5 minutes using a syringe needle. The temperature was then maintained and the mixture was stirred under nitrogen protection for 2 hours until the reaction was complete. The conversion was monitored using liquid chromatography-mass spectrometry (LCMS). After 1 hour, 50 μL aliquots were collected and concentrated using a syringe. Analysis by 31P nuclear magnetic resonance (31P NMR) and LCMS confirmed the completion of the reaction, with a crude yield of 82% and a crude reaction Cr dr (Rp:Sp = 10:1). The crude reaction solution was concentrated to remove most of the solvent and dissolved in a minimal amount of DCM. It was then loaded onto a 5g silica column eluted with EA / Hex (0-60%) via a combi-flash system, yielding 90 mg of the major isomer "Rp" as a white solid (82% separation yield, diastereoselectivity ratio dr 10:1). The chemical shift of Rp was 66.40 in 31p NMR spectroscopy (162 MHz, deuterated chloroform CDCl3). Since trace detection was not confirmed, the Sp isomer was confirmed upon amplification.

[0419] Example 18. Dinucleotide Spectroscopy a. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert-butyl) (dimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy (-7-methoxy-2-thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4 (1H,3H)-dione CR dr=17:1, after purification dr>30:1.

[0420] 31 P NMR (162 MHz, CDCl3) δ 66.27 (Rp).

[0421] 1 H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.04 (s, 1H), 7.37 (t, J = 5.6Hz, 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).

[0422] b. 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- ((((2S,4aR,6R,7R,7aR)-6-(6-(dibenzylamino)-9H-purine-9-yl)-7-methoxy-2-thiotetrahydro-4H-furyl) [3,2-d][1,3,2]dioxophosphazenecyclohexane-2-yl)oxy)methyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione CR dr=29:1, after purification dr = 20:1.

[0423] 31 P NMR (162 MHz, CDCl3) δ 65.97 (Rp).

[0424] c. (2S,4aR,6R,7R,7aR)-2-[[((2R,3R,4R,5R)-3-[(tert-butyldimethylsilyl)oxy]- 5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy]-6-(6-(dibenzylamino)- 9H-purin-9-yl)-7-methoxytetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane 2-sulfide 31 P NMR (162 MHz, CDCl3) δ 66.18 (Rp) cr dr = 14:1, 25:1.

[0425] d. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert-butyl)) (dimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy (-7-fluoro-2-thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine 31 P NMR (162 MHz, CDCl3) δ 66.03 (Rp), 61.49(Sp)。

[0426] e. 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3- ... (Oxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-fluoro Tetrahydrofuran-2-yl)methoxy)-7-methoxy-2-thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxopyroxicyclohexane Alkyl-6-pyrimidin-2,4(1H,3H)-dione 31 P NMR (162 MHz, CDCl3) δ 66.36 (Rp)。

[0427] f.1-Benzyl-9-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl) (dibenzylamino)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy 2-Thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)-2-(dibenzylamino)-1,9-di Hydrogen-6H-purine-6-one 31 P NMR (162 MHz, CDCl3) δ 66.21(Rp), 62.05(Sp). Yield 62%. 1 H NMR (400MHz, 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)。

[0428] 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。

[0429] g. 1-((2R,3R,4R,5R)-5-((((2S,4aR,6R,7R,7aR)-6-(1-benzyl-2-(dibenzylamino)- 6-oxo-1,6-dihydro-9H-purine-9-yl)-7-methoxy-2-thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxo Phosphacyclohexane-2-yl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)- 3-((benzyloxy)methyl)pyrimidine-2,4(1H,3H)-dione 31 P NMR (162 MHz, CDCl3) δ 65.72, 61.88. 1 H NMR (400 MHz, CDCl3) δ 7.80– 7.71 (m, 1H), 7.70 (s, 1H), 7.43 – 7.30 (m, 5H), 7.28 (dd, J = 6.4, 3.7 Hz,11H), 7.18 – 7.10 (m, 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-(Dibenzylamine) 9-((tert-butyldimethylsilyl)oxy)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran (2-yl)methoxy)-7-methoxy-2-thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxopyrazine-6- 2-(dibenzylamino)-1,9-dihydro-6H-purine-6-one 31 P NMR (162 MHz, CDCl3) δ 66.73 (Rp).

[0430] Example 19. Nucleophilic ring-opening reaction of a cyclic dinucleotide thioester at position P with a benzyl alkoxide. a. Schematic 1. General catalytic conditions flow chart for U*U Solution A: Under N2, cuprous chloride (CuCl) (0.25-1 equivalents) (green powder) was added to a vial that had been dried with a hot air gun and then dissolved in Solvent-1 (0.05 M) at room temperature. Then, alkali (1.5-3 equivalents) was added, followed by benzyl alcohol (2 equivalents) or other benzyl alcohol (e.g., CF3BnOH) after 10 minutes. The mixture was stirred at room temperature under N2 for 20 minutes.

[0431] Solution B: Under Schlenk line, 13:45 of 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-thiotetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidin-2,4(1H,3H)-dione (93 mg, 1 equivalent, 0.10 mmol) was dissolved in toluene-2 ​​(1.3 mmol) at 23 °C. Solution A was added dropwise to solution B over 5 minutes via a syringe needle (mL). The reaction was completed within 20 hours as monitored by LCMS.

[0432] Ring-opening reactions were screened among 34 reactions using cyc U*U with different dr ratios (8:1 or 13:1) to optimize conditions. In one screening run of these 34 reactions, the modified conditions were: MTBD (1.5 equivalents), BnOH or 2-CF3-BnOH (2 equivalents), CuCl (0.25 equivalents), using toluene, MTBE, and acetonitrile as solvents, and reaction time t = 20-40 hours (starting material conversion 95%, [S] < 1:1 to [S] ~ 5:1).

[0433] A second round of screening was conducted using cyc U*U, dr=4:1, 2-CF3-BnOH, and various Lewis acids, bases, and solvent combinations to further optimize the process.

[0434] b. Schematic 2. Detailed procedure for U*U Solution A: Cuprous chloride (CuCl) (2.5 mg, 0.64 μL, 0.25 equivalents, 25 μmol) (green powder) was added to a vial dried with a hot air gun and dissolved in toluene-1 (0.7 mL) at room temperature. Then, mTBD (1-methyl-2,3,4,6,7,8-hexahydro-1H-pyrimidino[1,2-a]pyrimidin) (23 mg, 22 μL, 1.5 equivalents, 0.15 mmol) was added. After 10 minutes, benzyl alcohol (22 mg, 21 μL, 2 equivalents, 0.20 mmol) or other benzyl alcohol (e.g., CF3BnOH) was added and stirred at room temperature under N2 for 20 minutes.

[0435] Solution B: Under Schlenk line, at 23°C, 13:45 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-thioanionic tetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphacyclohexane-6-yl)pyrimidin-2,4(1H,3H)-dione (93 mg, 1 equivalent, 0.10 mmol) was dissolved in toluene-2 ​​(1.3 mmol) at 1.3 mmol / L. Solution A was added dropwise to solution B over 5 minutes via a syringe needle (mL). The reaction was completed within 20 hours as monitored by LCMS.

[0436] c. General library scope programs d. First screening Alkali selected R1 and R2 are selected from Naphthyl Disc Design The first screening procedure Inside the glove box container, according to the tray design, use a manual pipette to dispense each cyclic dinucleotide matrix as a stock solution into the corresponding reaction vials in columns A and C (using toluene) and columns E and G (using CAN). Purge the toluene stock solutions with a nitrogen stream. Using Quantos, dispense the solid base and additives into the corresponding alkoxide vials in columns B, D, and F of the 96-vial tray.

[0437] Prepare the alkoxide stock solution by following these steps: (i) adding an appropriate solvent to a suitable vial containing a solid base / additive using a manual pipette; (ii) adding a solid alcohol (weighed in a glove box) to a suitable vial; and (iii) adding a liquid MTBD base to a suitable vial using a manual pipette. Then, add the appropriate solvent to the matrix vials in columns A, C, and E. Seal the vials using an appropriate spiral pattern and stir at 800 rpm in a glove box at RT, allowing at least 30 minutes from the time of final addition to the alkoxide vials for benzyl alkoxide formation and redissolving of the matrix mixture.

[0438] Unseal the pan, and then use a manual pipette to transfer the alkoxide solution (each type / reaction vial) dropwise (approximately 10 seconds / drop) into the corresponding reaction vial. Reseal the pan using the appropriate spiral pattern and stir at 800 rpm in a glove box at room temperature.

[0439] The culture dish was unsealed at 2, 4, 20, 27, and 66 hours, and samples were taken as follows: (1) 20–60 mL of 2.5 mM stock solution containing internal standard TPP (triphenyl phosphate) was collected using a CAN; (2) 20 µL of sample from each well was aliquoted into 0.45 µm filter vials using a single-channel pipette; (3) 400 µL of internal standard stock solution was added to each well using a multi-channel pipette; (4) the sample was analyzed using UPLC high pH_NoMeOH method-Dreadnought (0.5 µL injection volume); and (5) each sample was assigned a batch number reflecting its position in the reaction and analysis dishes (CAX-D00157-067-XXX, where XXX is from A01 to E10). The solvent in each well dried out several times. In these cases, 100 µL of appropriate solvent was added to reconstitute the reaction. Before final sample preparation, add 100 µL of appropriate solvent to all reaction wells again.

[0440] The screening conditions and results are listed in Table 11 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 equivalents), ArOh (2 equivalents)). [s] shows the proportion of [3-5]:[5-5] regioisomeric products.

[0441] Table 11 - Screening criteria and results ii. Second screening The second screening procedure Using a manual pipette, dispense the cyclic dinucleotide substrate as a stock solution into the corresponding reaction vials in columns A and C (using toluene) and columns E and G (using ACN). Purge the stock solution under a nitrogen stream. Then, using Quantos, dispense the solid base and additive into the corresponding alkoxide forming vials in columns B, D, F, and H of the 1 mL × 96 vial pan.

[0442] Prepare an alkoxide stock solution and stir for 20 minutes. Dissolve the matrix in a suitable solvent and stir for 20 minutes. Then, add the alkoxide solution of B dropwise to A, and follow the same procedure for D to C, F to E, and H to G. Use a suitable spiral-patterned sealed disc and stir at 800 rpm in a glove box at RT, allowing at least 30 minutes from the final addition of the alkoxide to the vial to form the benzyl alkoxide and redissolve the matrix mixture.

[0443] Unseal the dish and then use a manual pipette to transfer the alkoxide solution (one / reaction vial) dropwise into the corresponding reaction vial (approximately 10 seconds per addition). Reseal the dish with the appropriate spiral pattern and stir at 800 rpm in a glove box at RT. Unseal the dish at 2 hours and 20 hours for sampling and analysis.

[0444] Market observation During the reaction, substances were observed and identified by UPLC: (i) retention time was determined using the XBRIDGE POS SCAN LONG method (260 nm); and (ii) Waters XBridge C18, 2.5 µm, 3.0 × 75 mm, at 40 °C; A: 5 mM Am formate in water; B: ACN (acetonitrile); gradient: B increased from 0% to 95% in 13.50 min, and held at 95% B for 2.25 min. Total run time was 15.75 min; flow rate was 0.8 mL / min.

[0445] Screening resulted in different conversions to DP 3'-5', with 5'-5' DP being the predominant isomer in most cases. Two (2) time points were selected: 2 hours and 20 hours (the latter being the endpoint). The first time point, at 2 hours, allowed for possible interconversion between 3'-5' (kinetic product) and 5'-5' (thermodynamic product). The second time point, at 20 hours, observed more frequent interconversions between 3'-5' (kinetic product) and 5'-5' (thermodynamic product) in some cases. No internal standard was used in the reaction.

[0446] Figures 2A-2F The results shown are from dinucleotide screening-plate Spotfire output overlap 2HR; PhMe (toluene) conditions. Figures 3A-3F The results shown are from dinucleotide screening-plate Spotfire output overlap 2HR; under ACN conditions. Figures 4A-4F The results shown are from dinucleotide screening - plate-site Spotfire output overlap 20HR; PhMe (toluene) conditions. Figures 5A-5FThe results shown are from dinucleotide screening - plate-site Spotfire output overlap 20HR; under ACN conditions.

[0447] The screening conditions and results using toluene are listed in Table 12 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 equivalents), Lewis acid (0.25 equivalents)). [s] indicates the ratio of [3-5]:[5-5] regioisomers. This screening was performed using starting material with a dr ratio of 4:1.

[0448] Table 12 - Screening criteria and results (toluene) The screening conditions and results using ACN are listed in Table 13 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 equivalents), Lewis acid (0.25 equivalents)). [s] represents the ratio of [3-5]:[5-5] regioisomers. The screening was performed using starting material with a dr ratio of 4:1.

[0449] Table 13 - Screening Criteria and Results (ACN) iii. Nucleobase combination screening - The screening conditions and results are listed in Table 14 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 equivalents), Lewis acid (0.25 equivalents)). [s] indicates the ratio of [3-5]:[5-5] regioisomer products.

[0450] Table 14 - Screening Criteria and Results Example 20. Spectroscopy of open-ring dinucleotides a. Thiophosphate O-((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine Pyridine-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) ester O-(((2R,3R,4R,5R)-5-(3-((benzyloxy) (2H)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxy O-(2-(trifluoromethyl)benzyl) ester (2-yl)tetrahydrofuran-2-yl)methyl) ester [3'-3'] 31 P NMR (162 MHz, CDCl3) δ 68.11 (Rp), 67.23 (Sp) [5'-5'] 31 P NMR (162 MHz, CDCl3) δ 68.57 (Rp), 68.28 (Sp) DP accuracy: 1106.34 (1107.35 MH+), measurement quality: 1107.46 MH+.

[0451] b. Thiophosphate O-((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) ester O-(((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(6-(dibenzylamino)-9H-purine-9-yl)-4-methoxytetrahydrofuran-2-yl)methyl) ester O-(2-(trifluoromethyl)benzyl) ester 31 P NMR (162 MHz, CDCl3): [3'-5'], δ 67.92 (Rp), 67.61 (Sp); [5'-5'], 68.95.

[0452] c. Thiophosphate O-((((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydro Pyrimidine-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) ester O- ((2R,3R,4R,5R)-5-(6-(dibenzylamino)-9H-purine-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3- O-(2-(trifluoromethyl)benzyl) ester 31 P NMR (162 MHz, CDCl3): [3'-5'], δ67.66 (Rp), 67.52 (Sp). [5'-5'],68.75.

[0453] d. O-benzyl thiophosphate O-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3, 4-Dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) ester 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) ester 31 P NMR (162 MHz, CDCl3): [3'-5'], δ 68.38 (Rp), 67.73 (Sp). [5'-5'], 68.94.

[0454] Example 21. Synthesis of trimer thiophosphates from open-ring thiodinucleotides Under Schlenk line conditions and in a N2 atmosphere, lithium 2-methyl-2-propoxide (6.0 mg, 7.0 μL, 1.5 equivalent, 75 °C) was added to reaction tube A, which had been dried with a hot air gun. A stirring rod was added to a stock solution of O-benzyl thiophosphate O-((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) ester 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) ester (0.10 g, 50 wt%, 1 equivalent, 50 μmol) in toluene (1 mL) at -20 °C for 10 minutes.

[0455] To another reaction tube B, Cl-CP (0.18 mmol, 1.5 equivalents) and 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-thioanion tetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (36 mg, 1.5 equivalents, 75 μmol) in MeCN (0.6 mL) were added. The reaction tubes were kept at -25 °C for 10 minutes, and the pre-stirred alkoxide solution from reaction tube A was transferred dropwise to reaction tube B with syringe assistance. The reaction mixture was stirred at -20 °C for 18 hours, then heated to room temperature and stirred for 4 hours. LC-MS showed the major product and the expected product peak of the isomer at MH+ 1477.3. Based on uncorrected UPLC, the crude yield of the reaction was approximately (below the LCMS screenshot, assuming the newly formed dr is ~ 56:25:12:3:2:1.6, limited by the retention time and characterization features of the cyclic dinucleotide), with a large amount of PS-Cl remaining unreacted. LCMS showed separation of the starting material and six product isomers, and MS-ID was confirmed by EIC (non-HRMS).

[0456] Example 22. Liquid-phase synthesis of oligonucleotides using flow technology A general overview of liquid-phase methods for oligonucleotide synthesis, as follows: Figure 6 As shown.

[0457] An overview of the continuous flow oligonucleotide synthesis system is as follows: Figure 7 As shown. Figure 7 The device shown is a glass microreactor (“200 µL flow reactor”), but any type of reactor known in the art and suggested herein may also be used.

[0458] The study was conducted to compare the performance and efficiency of oligonucleotide synthesis using batch or flow systems as disclosed in this disclosure. Using a flow system, 12 test reactions were performed at different temperatures (-20°C, 0°C, ambient temperature (approximately 20-25°C), and 45°C) and different residence times (30, 40, or 50 minutes). The solvent (toluene) and concentration were kept constant in the batch program. Analysis by 31PNMR and LCMS revealed that a 50-minute residence time at ambient temperature resulted in complete conversion with the same selectivity as the batch reaction under low-temperature conditions.

[0459] Prepare a solution of LiOtBu (0.148 M, 1 equivalent) and 2-CF3BnOH (0.2 M) in toluene [Solution A], bubble with nitrogen, and store as a stock solution in a 100 mL glass bottle. Prepare a solution of the cyclic dinucleotide in toluene (0.07 M) [Solution B], bubble with nitrogen, and store as a stock solution in a 100 mL glass bottle. Use methanol as a quenching agent, bubble with nitrogen, and store in a third 100 mL glass bottle. Connect all solutions to nitrogen lines saturated with toluene (Solution A and Solution B) or methanol, respectively.

[0460] Each bottle was connected to a VICI M6HP pump via 1 / 16-inch outside diameter (OD) tubing. Solutions A and B were fed into a 200 µL MR-LAB-MS flow reactor (from Little Things Factory). The temperature was controlled and maintained at 22°C using a PolarBear device (from Uniqsis Ltd.).

[0461] At the reactor outlet, the reaction mixture is mixed using a PEEK tee and rapidly cooled with methanol. This line is then connected to a fraction collector to automatically discard or collect the fraction from which the reaction has been terminated.

[0462] The first step was to flush the reactor with toluene to ensure complete cleanliness of the reactor and piping. Then, after priming the pumps and piping with reagents and quenching solution, the flow rates of the three pumps were set to 1.7 µL / min for solution A, 2.3 µL / min for solution B (producing a 50-minute residence time), and 6.6 µL / min for methanol, respectively. The reaction outlet mixture was discarded three hours before the start of the experiment (unsteady operation phase). Upon reaching steady state, two fractions were collected: a first fraction corresponding to a collection time of 2.5 hours and a theoretical product mass of 26.7 mg, and a second fraction corresponding to a theoretical product mass of 21.4 mg and a collection time of 2 hours.

[0463] The two fractions were combined, and the distillate was evaporated under vacuum to obtain a mass of 48 mg. The crude product was analyzed by LCMS to determine the selectivity ratio between the isomers and their purity.

[0464] Table 15 provides a summary of some details for comparison.

[0465] Table 15 .

Claims

1. A method of preparing a dinucleotide of formula (iv) or a salt or solvate thereof: wherein: Y is O or S; R 1 and R 2 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 and R 2 are independently selected from hydrogen, fluorine, methoxy, and -0-methoxyethyl (MOE); B 1 and B 2 is a nucleobase, which can optionally be protected by one or more protecting groups; and R 4 is 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 the group consisting of Cl, Br, (C1-C6)alkoxy, (C6-C 12 )aryloxy, S-(C1-C6)alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphonucleoside of formula (ii) (b) reacting a cyclic phosphorinucleoside of formula (ii) with a nucleoside of formula (iii) to form a cyclic phosphodi-nucleotide of formula (iv), wherein R 4 is a protecting group: ； wherein the method further comprises optionally removing one or more protecting groups.

2. A method of preparing a dinucleotide of formula (vi) or a salt or solvate thereof: wherein: Y is O or S; R 1 and R 2 are independently selected from the group consisting of hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B 1 and B 2 is a nucleobase, which can optionally be protected; R 4 is hydrogen or a protecting group; n is selected from 1, 2, 3, 4, and 5; Each R 5 Substituents on the benzene ring that are independently protected by the benzyl group; R 6 is hydrogen or a protecting group; and R 7 and R 8 are independently selected from hydrogen, (Ci-C 20 )alkyl and aryl; the method comprising: (i) providing a cyclic phosphodiester nucleotide of formula (iv) as defined in claim 1, wherein R 4 is a protecting group: (ii) reacting a cyclic phosphodiester dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) to form a dinucleotide of formula (vi): wherein: each R 5 independently is a substituent on the phenyl ring of the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4, and 5; R 6 is hydrogen or a protecting group; and R 7 and R 8 are independently selected from hydrogen, (Ci-C 20 )alkyl, and aryl; wherein the method further comprises optionally removing one or more protecting groups.

3. A method of preparing a dinucleotide of formula (vi) or a salt or solvate thereof: wherein: Y is O or S; R 1 and R 2 are independently selected from the group consisting of hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B 1 and B 2 is a nucleobase, which can optionally be protected; R 4 is hydrogen or a protecting group; n is selected from 1, 2, 3, 4, and 5; Each R 5 Substituents on the benzene ring that are independently protected by the benzyl group; R 6 is hydrogen or a protecting group; and R 7 and R 8 are independently selected from the group consisting of hydrogen, (Ci-C 20 )alkyl and aryl; 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 the group consisting of Cl, Br, (C1-C6)alkoxy, (C6-C 12 )aryloxy, S-(C1-C6)alkyl, O-aryl, and S-aryl; R is Cl, Br, or (C1-C6)alkoxy, to form a cyclic phosphonucleoside of formula (ii): (b) reacting a cyclic phosphorinucleoside of formula (ii) with a nucleoside of formula (iii) to form a cyclic phosphodi-nucleotide of formula (iv), wherein R 4 is a protecting group: ; and (c) reacting a cyclic phosphodiester dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) to form a dinucleotide of formula (vi): wherein: each R 5 independently is a substituent on the phenyl ring of the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4, and 5; R 6 is hydrogen or a protecting group; and R 7 and R 8 are independently selected from hydrogen, (Ci-C 20 )alkyl, and aryl; wherein the method further comprises optionally removing one or more protecting groups.

4. A method of preparing an oligonucleotide of formula (vii), wherein: X is hydrogen, a protecting group or ; each Y is independently O or S; R 1 , R 2 , and R 3 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 , R 2 , and R 3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); R 4 is H or a protecting group; n is selected from 1, 2, 3, 4, and 5; Each R 5 Substituents on the benzene ring that are independently protected by the benzyl group; each R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 7 and R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 8 each R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 20 )alkyl, and aryl; B 1 , B 2 and B 3 is a nucleobase, which can optionally be protected; and m is selected from an integer in the range of 1 to 18; the method comprising: (i) providing a dinucleotide of formula (vi) as claimed in claim 2 or claim 3; and (ii) repeating the reactions of steps (a), (b), and (c) in claim 3 one or more times to obtain an oligonucleotide of formula (vii); wherein, in repeating step (b), a cyclic phosphonucleoside of formula (ii) is reacted with the 5’ terminal nucleoside of the dinucleotide or oligonucleotide; wherein the method further comprises optionally removing one or more protecting groups.

5. A method of preparing an oligonucleotide of formula (vii), wherein: X is hydrogen, a protecting group or each Y is independently O or S; R 1 , R 2 , and R 3 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 , R 2 , and R 3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); R 4 is H or a protecting group; n is selected from 1, 2, 3, 4, and 5; Each R 5 Substituents on the benzene ring that are independently protected by the benzyl group; each R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 7 and R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 8 each R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 20 each R is independently selected from hydrogen, ( B 1 , B 2 and B 3 is a nucleobase, which can optionally be protected; and m is selected from an integer in the range of 1 to 18; 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 the group consisting of Cl, Br, (C1-C6)alkoxy, (C6-C 12 )aryloxy, S-(C1-C6)alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphonucleoside of formula (ii): (b) reacting a cyclic phosphorinucleoside of formula (ii) with a nucleoside of formula (iii) to form a cyclic phosphodi-nucleotide of formula (iv), wherein R 4 is a protecting group: ； (c) reacting a cyclic phosphodiester dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) to form a dinucleotide of formula (vi): ； wherein: each R 5 independently is a substituent on the phenyl ring of the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4, and 5; R 6 is hydrogen or a protecting group; and R 7 and R 8 are independently selected from hydrogen, (Ci-C 20 )alkyl, and aryl; and (d) repeating the reactions of steps (a), (b), and (c) one or more times to obtain an oligonucleotide of formula (vii); wherein, in repeating step (b), a cyclic phosphonucleoside of formula (ii) is reacted with the 5’ terminal nucleoside of the dinucleotide or oligonucleotide; wherein the method further comprises optionally removing one or more protecting groups.

6. A method of preparing an oligonucleotide of formula (1) or a salt or solvate thereof, wherein: X is hydrogen, a protecting group or each Y is independently O or S; R 1 , R 2 , and R 3 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 , R 2 , and R 3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B 1 , B 2 and B 3 is a nucleobase, which can optionally be protected; R 4 is hydrogen or a protecting group; and m is selected from an integer in the range of 1 to 18; the method comprising: (i) providing a dinucleotide of formula (vi) as claimed in claim 2 or claim 3, or an oligonucleotide of formula (vii) as claimed in claim 4 or claim 5; (ii) hydrogenating the dinucleotide of formula (vi) or the oligonucleotide of formula (vii); and (iii) optionally removing one or more protecting groups; wherein the method produces an oligonucleotide of formula (1) or a salt or solvate thereof.

7. A method of preparing an oligonucleotide of formula (1), or a salt or solvate thereof, wherein: X is hydrogen, a protecting group or each Y is independently O or S; R 1 , R 2 , and R 3 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 , R 2 , and R 3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B 1 , B 2 and B 3 is a nucleobase, which can optionally be protected; R 4 is hydrogen or a protecting group; and m is selected from an integer in the range of 1 to 18; 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 the group consisting of Cl, Br, (C1-C6)alkoxy, (C6-C 12 )aryloxy, S-(C1-C6)alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphonucleoside of formula (ii): (b) reacting a cyclic phosphorinucleoside of formula (ii) with a nucleoside of formula (iii) to form a cyclic phosphodi-nucleotide of formula (iv), wherein R 4 is a protecting group: ； (c) reacting a cyclic phosphodi nucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) to form a di nucleotide of formula (vi): wherein: each R 5 independently is a substituent on the phenyl ring of the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4, and 5; R 6 is hydrogen or a protecting group; and R 7 and R 8 are independently selected from hydrogen, (Ci-C 20 )alkyl, and aryl; and (d) optionally, repeating the reactions of steps (a), (b) and (c) one or more times to obtain an oligonucleotide of formula (vii); wherein, in repeating step (b), a cyclic phosphonucleoside of formula (ii) is reacted with a 5' terminal nucleoside of the oligonucleotide; (e) hydrogenating the product of step (c) or step (d); and (f) optionally, removing one or more protecting groups; wherein the method produces an oligonucleotide of formula (1), or a salt or solvate thereof.

8. The method of any one of claims 2 to 7, wherein the cyclic phosphodi nucleotide of formula (iv) is obtained as a mixture of stereoisomers, and wherein the stereoisomers are separated prior to the step of reacting the cyclic phosphodi nucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v).

9. The method of any one of claims 2 to 8, wherein each instance of R 5 is an ortho or para substituent on the benzyl group; optionally wherein each instance of R 5 is an ortho substituent on the benzyl group.

10. The method of any one of claims 2 to 9, wherein each instance of R 5 is independently selected from halogen, (Ci-C 20 )alkyl, (Ci-C 20 )alkoxy, trifluoromethyl, and phenyl; optionally wherein each R 5 is independently selected from phenyl, methyl, methoxy, fluoro, chloro, and CF3; optionally wherein R 5 is selected from chloro and CF3.

11. The method of any one of claims 2 to 8, wherein R 5 is a fused benzene ring forming a naphthalene ring system.

12. The method of any one of claims 2 to 8, wherein the substituted benzyl alcohol nucleophile of formula (v) is: 。 13. The method of any one of claims 2 to 12, wherein the step of reacting the cyclic phosphodi nucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is performed in a solvent system comprising tetrahydrofuran (THF), 2-methyltetrahydrofuran (2Me-THF), diethyl ether, 1,4-dioxane, dimethylsulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylacetamide (DMA), dichloromethane (DCM), acetonitrile (MeCN), methyl tert-butyl ether (MTBE), tert-amyl alcohol, or toluene.

14. The method of any one of claims 2 to 13, wherein the step of reacting the cyclic phosphodi nucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in the presence of a base selected from: n-butyllithium (n-BuLi); lithium tert-butoxide (LiOtBu); lithium bis(trimethylsilyl)amide (LiHMDS); potassium bis(trimethylsilyl)amide (KHMDS); lithium tetramethylpiperidide (LiTMP); or Li-R Z wherein R Z is an alkyl or aryl group; sodium tert-butoxide (NaOtBu) + lithium chloride (LiCl); potassium tert-butoxide (KOtBu) + lithium chloride (LiCl); triethylamine (Et3N); diazabicycloundecene (DBU); 4-dimethylaminopyridine (DMAP); S-collidine; and triazabicyclodecene (TBD).

15. The method of claim 14, wherein the base is pre-mixed with the substituted benzyl alcohol nucleophile of formula (v) prior to the step of reacting the cyclic phosphodi nucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v).

16. The method of claim 14 or claim 15, wherein the base is lithium tert-butoxide.

17. The method of any one of claims 2 to 16, wherein the di nucleotide of formula (vi) is isolated by reaction with a silicon-based chloride.

18. The method of any one of claims 1 to 17, wherein the cyclic phosphonochloridate of formula (ii) is obtained as a mixture of stereoisomers, and wherein the stereoisomers are separated prior to the step of reacting the cyclic phosphonucleoside of formula (ii) with the nucleoside of formula (iii).

19. The method of any one of claims 1 to 18, wherein R 2 is an alkoxy group attached to an adjacent O to form a ketal protecting group with R4.

20. The method of any one of claims 1 to 19, wherein R 4 is selected from H, silyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl, triisopropylsilyl, or 2 ketals.

21. The method of any one of claims 1 to 20, wherein each nucleobase is selected from a pyrimidine and a purine.

22. The method of any one of claims 1 to 21, wherein each nucleobase is protected by one or more protecting groups independently selected from dimethylformamide (DMF), formamidine, DMF-formamidine, N-methylpyrrolidin-2-ylidene (PyA), pivaloyloxymethyl (POM), phthalimido, chloro, bromo, acyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM).

23. The method of any one of claims 1 to 22, wherein the method is a liquid phase process.

24. The method of any one of claims 1 to 23, wherein the method is carried out using continuous flow.

25. The method of any one of claims 1 to 24, wherein the method is carried out in a flow reactor.

26. The method of claim 24 or claim 25, wherein the method is carried out at ambient temperature.

27. The method of any one of claims 24 to 26, wherein the reaction time is reduced compared to the same reaction in a batch reactor; optionally wherein the reaction time in a flow reactor is 45-120 minutes, optionally 45-60 minutes.

28. A dinucleotide or oligonucleotide produced by the method of any one of claims 1 to 25.

29. A cyclic phosphonucleoside of the formula: or a salt or solvate thereof, wherein: Y is O or S; each R is independently selected from Cl, Br, (Ci-C6)alkoxy, (C6-C 12 )aryloxy, S-(Ci-C6)alkyl, O-aryl, and S-aryl; R 1 is selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy (optionally wherein R 1 is selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE)); and B 1 is a nucleobase, which can optionally be protected.

30. A cyclic phosphorodinucleotide of the formula: or a salt or solvate thereof, wherein: Y is O or S; R 1 and R 2 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy (optionally wherein R 1 and R 2 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE)); R 4 is H or a protecting group; and B 1 and B 2 are nucleobases, which can optionally be protected.

31. A dinucleotide of the formula: or a salt or solvate thereof, wherein: Y is O or S; R 1 and R 2 are independently selected from the group consisting of hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy (optionally wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE)); R 4 is H or a protecting group; each R 5 is independently a substituent on the phenyl ring of a benzyl protecting group; each R 7 and R 8 are independently selected from the group consisting of hydrogen, (Ci-C 20 )alkyl, and aryl; B 1 and B 2 are nucleobases, which can optionally be protected; and n is selected from 1, 2, 3, 4, and 5.

32. An oligonucleotide of the formula or a salt or solvate thereof, wherein: X is hydrogen, a protecting group or each Y is independently O or S; R 1 , R 2 , and R 3 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 , R 2 , and R 3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); R 4 is H or a protecting group; n is selected from 1, 2, 3, 4 and 5; Each R 5 Substituents on the benzene ring that are independently protected by the benzyl group; each R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 7 and R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 8 each R is independently selected from hydrogen, (Ci-C6)alkyl, and aryl; 20 each R is independently selected from hydrogen, ( B 1 , B 2 and B 3 is a nucleobase, which can optionally be protected; and m is an integer in the range of 1 to 18.

33. An oligonucleotide of the formula or a salt or solvate thereof, wherein: X is hydrogen, a protecting group or each Y is independently O or S; R 1 , R 2 , and R 3 are independently selected from hydrogen, hydroxyl, halogen, N3, (Ci-C 20 )alkyl, and (Ci-C 20 )alkoxy; optionally wherein R 1 , R 2 , and R 3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); B 1 , B 2 and B 3 is a nucleobase, which can optionally be protected; R 4 is hydrogen or a protecting group; and m is an integer in the range of 1 to 18.

34. A solution comprising: (i) a nucleoside of claim 29; (ii) a dinucleotide of claim 28, claim 30 or claim 31; and / or (iii) an oligonucleotide of claim 28, claim 32 or claim 33.

35. Use of a dinucleotide or oligonucleotide of any one of claims 28 and 30 to 34 in the synthesis of an oligonucleotide or polynucleotide.

36. Use of a dinucleotide or oligonucleotide of any one of claims 28 and 30 to 34 as a blockmer blockmer phosphoramidite in solid phase, chemical ligation or other synthesis methods of oligonucleotides or polynucleotides.

37. Use of a dinucleotide or oligonucleotide of any one of claims 26 and 28 to 32 in an enzymatic ligation reaction for the synthesis of an oligonucleotide or polynucleotide.

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