TWI904659B - Oligonucleotide synthesis using cyclic-phosphorous nucleosides - Google Patents

Abstract

The present disclosure provides methods for preparing oligonucleotides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using cyclic-phosphorous nucleosides. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using substituted benzyl alcohol nucleophiles. In certain embodiments, the present disclosure provides methods for preparing oligonucleotides using cyclic-phosphorous nucleosides and substituted benzyl alcohol nucleophiles.

Description

Oligonucleotide synthesis using cyclic phosphate nucleosides

This invention provides methods for preparing oligonucleotides. In some embodiments, this invention provides methods for preparing oligonucleotides using cyclic phosphate nucleosides. In some embodiments, this invention provides methods for preparing oligonucleotides using substituted benzyl alcohol nucleophiles. In some embodiments, this invention provides methods for preparing oligonucleotides using both cyclic phosphate nucleosides and substituted benzyl alcohol nucleophiles.

As advancements in DNA and RNA technologies continue to expand in the scientific and medical fields, 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 phosphorylamine chemistry) are expensive, have limited scalability and portability (e.g., due to reliance on specific packed-bed reactors), and typically require complex workflow systems.

Therefore, there is a need for efficient, scalable, and easily implemented oligonucleotide synthesis methods in batch or flow reactors, and such methods should reliably provide short-chain, medium-chain, or long-chain oligonucleotides of interest containing oligonucleotides used directly or as synthetic structural units.

Details of various embodiments of the present invention are described below. In some embodiments, the present invention provides a method for preparing oligonucleotides. In some embodiments, the present invention provides a method for preparing dinucleotides. In some embodiments, the present invention provides a method for preparing trinucleotides. In some embodiments, the present invention provides a method for preparing oligonucleotides with a length of 2-20 nucleotides.

In some embodiments, the present invention provides a method for preparing the dinucleotide of formula (iv) or its salt or solvent. Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and methoxyethyl); ^B1 and ^B2 are nucleobases that may be protected by one or more protecting groups as appropriate; and ^R4 is hydrogen or a protecting group.

In some embodiments, the present invention provides a method for preparing a dinucleotide of formula (iv) or a salt or solvent thereof, wherein the method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming a cyclic 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 ^R4 is a protecting group: In some embodiments, the method further includes removing one or more protective groups.

In some embodiments, the present invention provides a method for preparing the dinucleotide of formula (vi) or its salt or solvent: Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^B1 and ^B2 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl.

In some embodiments, the present invention provides a method for preparing a dinucleotide of formula (vi) or a salt or solvent thereof, wherein the method comprises: providing a cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group; and reacting the cyclic phosphorus dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4, and 5; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl, and aryl. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

In some embodiments, the present invention provides a method for preparing a dinucleotide of formula (vi) or a salt or solvent thereof, wherein the method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: ; and (c) reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4, and 5; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl, and aryl. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

In some embodiments, the present invention provides a method for preparing oligonucleotides of formula (vii): Wherein: each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^R4 is H or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group's benzyl ring; each ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; ^B1 , ^B2 and ^B3 are nucleobases that may be protected as appropriate; m is an integer between 1 and 18; and X is hydrogen, a protecting group or .

In some embodiments, the present invention provides a method for preparing an oligonucleotide of formula (vii), wherein the method comprises: providing a dinucleotide of formula (vi); and repeating (as disclosed herein) one or more reactions of steps (a), (b), and (c) of the method for preparing a dinucleotide of formula (vi) or a salt or solvent thereof to obtain the oligonucleotide of formula (vii): In step (b), the cyclic phosphorus nucleotide of formula (ii) reacts with the 5'-terminal nucleotide of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

In some embodiments, the present invention provides a method for preparing the oligonucleotide of formula (vii), wherein the method comprises: (a) reacting the nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming the cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: (c) Reaction of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; and (d) repeating steps (a), (b) and (c) one or more times to obtain the oligonucleotide of formula (vii): In step (b), the cyclic phosphorus nucleotide of formula (ii) reacts with the 5'-terminal nucleotide of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

In some embodiments, the present invention provides a method for preparing oligonucleotides of formula (1) or their salts or solvents. Wherein: each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^B1 , ^B2 and ^B3 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; m is an integer from 0 to 18; and X is hydrogen, a protecting group or .

In some embodiments, the present invention provides a method for preparing an oligonucleotide of formula (1) or a salt or solvent thereof, wherein the method comprises: providing a dinucleotide of formula (vi) or an oligonucleotide of formula (vii); and hydrogenating the dinucleotide of formula (vi) or the oligonucleotide of formula (vii); wherein the method produces an oligonucleotide of formula (1) or a salt or solvent thereof. In some embodiments, the method further comprises removing one or more protecting groups.

In some embodiments, the present invention provides a method for preparing an oligonucleotide of formula (1) or a salt or solvent thereof, wherein the method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: (c) Reaction of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; (d) repeat steps (a), (b) and (c) one or more times as appropriate 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 its salt or solvent. In some embodiments, the method further includes the removal of one or more protecting groups.

In some embodiments of the present invention, the cyclic phosphorus dinucleotide of formula (iv) is obtained as a mixture of stereoisomers. 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).

In some embodiments of the present invention, ^R5 in each case is an ortho- or para-substituent on the benzyl group. In some embodiments, ^R5 in each case is an ortho-substituent on the benzyl group. In some embodiments, ^R5 in each case is a para-substituent on the benzyl group. In some embodiments, ^R5 in each case is independently selected from halogen, ( _C1 - _C20 )alkyl, ( _C1 - _C20 )alkoxy, trifluoromethyl, and phenyl. In some embodiments, ^R5 in each case is independently selected from phenyl, methyl, methoxy, fluorine, chlorine, and _CF3 . In some embodiments, ^R5 in each case is independently selected from chlorine and _CF3 . In some embodiments, ^R5 is a fused benzyl ring forming a naphthalene ring system. In some embodiments, the substituted benzyl alcohol nucleophile of formula (v) is: or .

In certain embodiments of the present invention, the reaction step 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 certain embodiments, the solvent includes: tetrahydrofuran (THF), 2-methyltetrahydrofuran (2Me-THF), diethyl ether, 1,4-dioxane, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), dimethylacetamide (DMA), acetonitrile (MeCN), methyl tributyl ether (MTBE), tripentyl alcohol, toluene, or mixtures thereof.

In certain embodiments of the present invention, the reaction step of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in the presence of an alkali. In certain embodiments, the alkali includes: n-butyllithium (n-BuLi); lithium tributoxide (LiOtBu); bis(trimethylsilyl)acetamide lithium (LiHMDS); lithium tetramethylhexahydropyridine (LiTMP); or Li- _RZ (where _RZ is alkyl or aryl); sodium tributoxide (NaOtBu) + lithium chloride (LiCl); potassium tributoxide (KOtBu) + lithium chloride (LiCl); triethylamine; diazabiscycloundecene (DBU); triazabiscyclodecene (TBD); or combinations thereof. In some embodiments, the alkali is premixed with the substituted benzyl alcohol nucleophile of formula (v) prior to the reaction step of the cyclic phosphorus dinucleotide of formula (iv) and the substituted benzyl alcohol nucleophile of formula (v). In some embodiments, the alkali is lithium tributoxide (LiOtBu).

In some embodiments of the present invention, the dinucleotide of formula (vi) is separated by reaction with silicon chloride.

In some embodiments of the present invention, the cyclophosphochloronucleotide of formula (ii) is obtained as a mixture of stereoisomers. In some embodiments, the stereoisomers are separated prior to the reaction step of the cyclophosphonucleotide of formula (ii) and the nucleoside of formula (iii).

In some embodiments of the present invention, ^R2 is an alkoxy group attached to an adjacent O to form a ketal protective group with ^R4 .

In certain embodiments of the present invention, ^R4 is selected from H, silica, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl, triisopropylsilyl, or a ketal protecting group formed with ^R2 .

In some embodiments of the present invention, 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 independently by one or more protecting groups selected from dimethylformamide (DMF), formamidinium, DMF-formamidinium, phthalimide, chlorine, bromine, acetyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM).

In some embodiments of the present invention, 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 process. In some embodiments, the method is carried out in a flow reactor.

In some embodiments, the present invention provides oligonucleotides (e.g., dinucleotides, trinucleotides) produced by the method of the present invention.

In some embodiments, the present invention provides a cyclic phosphate nucleoside of formula (ii): B1 is a nucleobase of formula (ii) produced by the method of the invention, wherein: Y is S or O; each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl and S-aryl; ^R1 is selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 )alkyl and ( _C1 - _C20 )alkoxy (including methoxy and -O-methoxyethyl); and ^B1 is a nucleobase that may be protected. In some embodiments, the invention provides a cyclic phosphorus nucleoside of formula (ii) produced by the method of the invention.

In some embodiments, the present invention provides a cyclic phosphorus dinucleotide of formula (iv): ; or its salts or solvents, wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^R4 is H or a protecting group; and ^B1 and ^B2 are nucleobases that may be protected. In some embodiments, the present invention provides a cyclic phosphorus dinucleotide of formula (iv) produced by the method of the present invention.

In some embodiments, the present invention provides the dinucleotide of formula (vi): Or its salts or solvents, wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^R4 is H or a protecting group; each ^R5 is independently a substituent on the benzyl protecting group; ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; ^B1 and ^B2 are nucleobases which may be protected; and n is selected from 1, 2, 3, 4 and 5. In some embodiments, the present invention provides a cyclic phosphorus dinucleotide of formula (vi) produced by the method of the present invention.

In some embodiments, the present invention provides oligonucleotides of formula (vii): Or its salts or solvents, wherein: each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^R4 is H or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group of the benzyl ring; each ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; ^B1 , ^B2 and ^B3 are nucleobases which may be protected as appropriate; m is an integer from 1 to 18; and X is hydrogen, a protecting group or In some embodiments, the present invention provides oligonucleotides of formula (vii) produced by the method of the present invention.

In some embodiments, the present invention provides an oligonucleotide of formula (1) or a salt or solvent thereof. Wherein: each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^B1 , ^B2 and ^B3 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; m is an integer from 0 to 18; and X is hydrogen, a protecting group or In some embodiments, the present invention provides oligonucleotides of formula (1) produced by the method of the present invention.

In some embodiments, the present invention provides a solution comprising the present invention nucleosides (e.g., cyclic phosphate nucleosides). In some embodiments, the present invention provides a solution comprising the present invention dinucleotides. In some embodiments, the present invention provides a solution comprising the present invention trinucleotides. In some embodiments, the present invention provides a solution comprising the present invention oligonucleotides.

In some embodiments, the oligonucleotides of the present invention can be used in subsequent oligonucleotide or polynucleotide synthesis applications. In some embodiments, the oligonucleotides of the present invention can be used as block polymers in solid-phase synthesis of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of the present invention can be used as acetylated block polymers in solid-phase synthesis of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of the present invention can be used in enzymatic ligation reactions for the synthesis of oligonucleotides or polynucleotides.

Related applications

This application claims priority over U.S. Provisional Application No. 63/500,726, filed May 8, 2023, and European Application No. 24,382,484.4, filed May 3, 2024, which are incorporated herein by reference. Oligonucleotide Synthesis

This invention provides a method for preparing oligonucleotides. This invention also provides oligonucleotides prepared by the method of this invention. Oligonucleotides are polymers of linked nucleotides with a length generally less than about 100 nucleotides. In some embodiments, the oligonucleotides of this invention contain at least two nucleotides. Therefore, in some embodiments, the oligonucleotides of this invention contain all integers in the range of 2 to 100 nucleotides (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 embodiments, the oligonucleotide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

In some embodiments, the oligonucleotide of the present invention contains 3 to 100 nucleotides. In some embodiments, the oligonucleotide contains 3 to 50 nucleotides. In some embodiments, the oligonucleotide contains 3 to 30 nucleotides. In some embodiments, the oligonucleotide contains 3 to 20 nucleotides. In some embodiments, the oligonucleotide contains 3 to 15 nucleotides. In some embodiments, the oligonucleotide contains 3 to 10 nucleotides. In some embodiments, the oligonucleotide contains 3 to 8 nucleotides. In some embodiments, the oligonucleotide contains 3 to 6 nucleotides. In some embodiments, the oligonucleotide contains 3 nucleotides (i.e., trinucleotides).

In some embodiments, the oligonucleotide of the present invention contains 5 to 20 nucleotides. In some embodiments, the oligonucleotide of the present invention contains 10 to 20 nucleotides. In some embodiments, the oligonucleotide of the present invention contains 15 to 20 nucleotides. In some embodiments, the oligonucleotide contains 3 nucleotides. In some embodiments, the oligonucleotide contains 5 nucleotides. In some embodiments, the oligonucleotide contains 6 nucleotides. In some embodiments, the oligonucleotide contains 7 nucleotides. In some embodiments, the oligonucleotide contains 8 nucleotides. In some embodiments, the oligonucleotide contains 9 nucleotides. In some embodiments, the oligonucleotide contains 10 nucleotides. In some embodiments, the oligonucleotide contains 11 nucleotides. In some embodiments, the oligonucleotide contains 12 nucleotides. In some embodiments, the oligonucleotide contains 13 nucleotides. In some embodiments, the oligonucleotide contains 14 nucleotides. In some embodiments, the oligonucleotide contains 15 nucleotides. In some embodiments, the oligonucleotide contains 16 nucleotides. In some embodiments, the oligonucleotide contains 17 nucleotides. In some embodiments, the oligonucleotide contains 18 nucleotides. In some embodiments, the oligonucleotide contains 19 nucleotides. In some embodiments, the oligonucleotide contains 20 nucleotides.

The term "nucleotide" is defined herein (see below) as an organic compound having a nucleoside and a phosphate group. The term "nucleoside" is defined herein (see below) as an organic compound having a nucleobase (e.g., adenine, cytosine, guanine, thymine, or uracil) covalently 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 nucleobase of the present invention is 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 nucleobase of the present invention is an atypical nucleobase (i.e., not a typical nucleobase). Examples of atypical nucleobases 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 groups of adenine and guanine (e.g., 6-methyl, 2-methyladenine ... 5-propyl uracil; xanthine; hypoxanthine; 2-aminoadenine; 2-aminopyridine; 2-aminopurine; 2,6-diaminopurine; 8-aminoguanine; 2-thiouracil; 5-methyl-2-thiouracil; 4-thiouracil; 2-thiothymine; 2-thiocytosine; 6-thiopurine; 8-thioguanine; 5-halogenated uracil; cytosine; uracil and alkynyl (e.g., 5-propynyl) derivatives of cytosine; 6-azouracil; 5-uracil (pseudouracil) ); 8-substituted (e.g., 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-halogenated) adenine and guanine; 5-substituted (e.g., 5-chloro, 5-bromine, 5-trifluoromethyl) uracil and cytosine; 2-F-adenine; 8-azaguanine; 8-azaadenine; 7-deazaguanine; 7-deazaadenine; 3-deazaguanine; 3-deazaadenine; tricyclic pyrimidine; phenoxazine-cytidine (1H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)) - ketone); phenothiazincytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one); substituted phenothiazincytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one); carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one); pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one); 2-pyridinone; N ₆ -Isopentyladenine; 2-Methylthio-N- ₆ -Isopentyladenine; 8-Bromoadenine; 8-Bromoguanine; 8-Chloroglucinine; 5-Bromouracil; 5-Fluorouracil; 5-Chlorouracil; 5-Iodouracil; 4-Ethylcytosine; 5-Methoxyuracil; 5-Hydromethyluracil; 5-(Carboxyhydroxymethyl)uracil; 5-(Methylaminomethyl)uracil; 5-(Carboxymethylaminomethyl)uracil; 5-(2-Bromovinyl)uracil; Uracil-5-oxyacetic acid; Uracil-5-oxyacetic acid methyl ester; Pseudorazine; 1-Methylpseudorazine; Glucoside; Inosine; 1-Methylinosine and 6-Hydroaminopurine. Examples of atypical nucleobases also include, but are not limited to: methylated purines or pyrimidines, alkylated purines or pyrimidines, acetylated purines or pyrimidines, halogenated purines or pyrimidines, denitrified purines, alkylated ribose, diaminopurines, inosine, and thiolated purines or pyrimidines.

In some embodiments, the nucleobase is unprotected. 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 reactive or unstable groups on a molecule from unwanted chemical reactions during the synthetic process or other chemical reactions. A nucleobase protecting group is a protecting group applied to one or more functional groups of the 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 group or an endoamino 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 group, N1-lactam (i.e., cyclolactam) group, and/or O6-lactam group of the guanine moiety. In some embodiments, the nucleobase protecting group is applied to the N3-lactam and/or O4-lactam groups of the uracil or thymine moiety. In some embodiments, the nucleobase protecting group is base-instable. Examples of base-instable protecting groups include FMOC (fumonisylmethyloxycarbonyl). In some embodiments, the nucleobase protecting group is acid-instable. Examples of acid-instability protecting groups include triarylmethyl protecting groups, 4,4'-dimethoxytriphenylmethyl (DMT), and BOC carbamate (terbutoxycarbonyl). In some embodiments, nucleobase protecting groups are unstable with reagents other than bases or acids.

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-ene (PyA), neopentyloxymethyl (POM), 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tributoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-furoylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cb). z), methyl, acetylated, trihalomethanel, benzoyl, nitrophenylacetylated, 2-nitrobenzenesulfonyl, phenyldimethylimino, dithiosuccinyl, methyl carbamate, ethyl carbamate, 9-(2-sulfonyl)enylmethyl carbamate, 9-(2,7-dibromo)enylmethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxy-10,10, 10,10-Tetrahydrothiotonyl)methyl ester (DBD-Tmoc), 4-methoxybenzomethyl ester of carbamate (Phenoc), 2,2,2-trichloroethyl ester of carbamate (Troc), 2-phenylethyl ester of carbamate (hZ), 1-(1-adamantyl)-1-methylethyl ester of carbamate (Adpoc), 1,1-dimethyl-2-halogenated ethyl ester of carbamate, 1,1 1,1-Dimethyl-2,2-dibromoethyl ester (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylmethoxyamino)ethyl carbamate, 1-Dalmatian carbamate (Adoc), Vinyl carbamate (Voc), 1-Isopropyl allyl carbamate (Ipaoc), Cinnamyl carbamate (Coc), 4-Nitrocinnamyl carbamate (Noc), 8-Quinolinyl carbamate, N-Hydroxyhexahydropyridyl carbamate, Alkyl dithiocarbamate, p-Methoxybenzyl carbamate (Moz), p-Nitrocarbamate Benzyl ester, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinyl benzyl carbamate (Msz), 9-anthraylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithiaalkyl)]carbamate Methyl ester (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphoethyl carbamate (Peoc), 2-triphenylphosphoisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acetylated benzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, carbamate 5-Benzoxazolyl methyl ester, 2-(trifluoromethyl)-6-chromone methyl ester (Tcroc), m-nitrophenyl ester of carbamate, 3,5-dimethoxybenzyl ester of carbamate, ortho-nitrobenzyl ester of carbamate, 3,4-dimethoxy-6-nitrobenzyl ester of carbamate, phenyl (ortho-nitrophenyl) methyl ester of carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative Biological, N′-phenylaminothiocarbonyl derivatives, tripentyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decoxybenzyl carbamate, 2,2-dimethoxycarbonyl vinyl carbamate, ortho(N,N-dimethylformamide)benzyl carbamate, carbamic acid 1,1-Dimethyl-3-(N,N-dimethylformamide)propyl ester, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isoniazid carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, amine 1-Methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, aminomethyl 2,4,6-tri-tert-butylphenyl ester, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, methylamine, dimethylformamide (DMF), formamidinium, DMF-formamidinium, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridinamide, 3-pyridylmethylamine, N-benzoylphenylpropylaminoacetyl derivative, benzoylamine, p- Phenylacetamide, anthraquinone acetamide, anthraquinone phenoxyacetamide, acetylacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(anthraquinone acetamide), 2-methyl-2-(anthraquinone phenoxy)propionamide, 2-methyl-2-(anthraquinone azophenoxy)propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, anthraquinone cinnamamide, N-acetylated methionine derivatives, anisonitrobenzoylamine, anisonitrobenzoylamine, 4,5-diphenyl-3-oxazoline-2-one, N-anisophenyldimethylimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisiloxazonicyclopentane adduct (STABASE), 5-substituted derivatives 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-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-ethoxypropylamine, N-(1-isopropyl-4-nitro-2-sideoxy-3-pyridone) (3-yl)amine, tetramethylammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptaylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylenylammonylamine (PhF), N-2,7-dichloro-9-enylammonylamine, 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′-isopropylamine, N-p-nitrobenzylamine, N-salicylateamine, N-5-chlorosalicylateamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-dimethyl-3-phenyl)-phenylmethyleneamine -Side-oxy-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, benzosulfinamide, nitrobenzylaminosulfinamide (Nps), 2, 4-Dinitrobenzenesulfinamide, pentachlorobenzenesulfinamide, 2-nitro-4-methoxybenzenesulfinamide, triphenylmethylbenzenesulfinamide, 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-pentamethyl-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracitesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzylmethylsulfonamide.

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, tributyldimethylsilyl (TBS, TBDMS), tributyldiphenylsilyl, or triisopropylsilyl); alkyl protecting groups (e.g., methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, triphenylmethyl, tributyl, or tetrahydropyran-2-yl). ; alkenyl protecting groups (e.g., allyl); aryl protecting groups (e.g., substituted phenyl, biphenyl, or naphthyl, depending on the case); and arylalkyl protecting groups (e.g., substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, orthonitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl) or 2- and 4-methylpyridinyl).

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 hydroxyl groups 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), neopentyl, benzoyl, p-phenylbenzoyl, monomethoxytriphenylmethyl (MMTr), and dimethoxytriphenylmethyl (D). MT), 4,4′,4″-trimethoxytriphenylmethyl (TMTr), 1(2-fluorophenyl)-4-methoxyhexahydropyridin-4-yl (FPMP), substituted pixyl, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tributylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-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-di Nitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, ortho-nitrobenzyl, p-nitrobenzyl, p-halogenated benzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-methylpyridinyl, 4-methylpyridinyl, 3-methyl-2-methylpyridinyl N-oxy, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxy Phenylacetyl diphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromobenzoxylmethyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorobenzyliminophenyl)methyl, 4,4′,4″-tris(acetylacetyloxyphenyl)methyl, 4,4′,4″-tris(benzoxyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′ ,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xanthyl, 9-(9-phenyl-10-ephthyl)anthrayl, 1,3-benzodithiocyclopentane-2-yl, benzoisothiazolyl S,S-dioxy, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl ( IPDMS), diethyl isopropyl silica (DEIPS), dimethyl trihexyl silica, tributyldimethyl silica (TBS, TBDMS), tributyldiphenyl silica (TBDPS), tribenzyl silica, tri-p-xylyl silica, triphenyl silica, diphenylmethyl silica (DPMS), tributylmethoxyphenyl silica (TBMPS), formate, benzoylformate, acetate, chloroacetate Dichloroacetic acid ester, trichloroacetic acid ester, trifluoroacetic acid ester, methoxyacetic acid ester, triphenylmethoxyacetic acid ester, phenoxyacetic acid ester, p-chlorophenoxyacetic acid ester, 3-phenylpropionate, 4-epoxyvalerate (acetylpropionate), 4,4-(epoxyethyldithio)valerate (acetylpropionyldithioacetal), neovalerate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4- 6-Trimethylbenzoate (carrageenan), alkyl carbonate methyl ester, 9-furomethyl carbonate (Fmoc), alkyl carbonate ethyl ester, alkyl carbonate 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl (TSE), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphino)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 3,4-dimethoxybenzyl ester, alkyl carbonate orthonitrobenzyl ester, alkyl carbonate p-nitrobenzyl ester, thioalkyl carbonate S-benzyl ester, 4-ethoxy-1-naphthyl carbonate, dithiocarbonate Methyl benzoate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, dibromomethyl benzoate, 2-methylbenzenesulfonate, 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, omega-(methoxycarbonyl)benzoate, α-naphthaleneate, nitrate ester, N,N,N′,N′-tetramethylphosphodiamide alkyl ester, N-phenylaminocarboxylic acid alkyl ester, borate ester, dimethylthiophosphoryl, 2,4-dinitrophenylsulfinic acid alkyl Esters, 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, sulfates, methanesulfonates (methanesulfonates), benzylsulfonates, toluenesulfonates (Ts), 2-cyanoethyl (CE or Cne) 3,5-Dichlorophenyl, 2,4-Dimethylphenyl, Butylthiocarbonyl, 4,4′,4″-Triphenyl(benzoyloxy)triphenylmethyl, Diphenylaminomethyl, Acetylpropyl, 2-(Dibromomethyl)benzoyl (Dbmb), 2-(Isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-Phenylon-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

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), neopentyl, benzoyl, p-phenylbenzoyl, monomethoxytriphenylmethyl (MMTr), and dimethoxytriphenylmethyl (D MT), 4,4′,4″-trimethoxytriphenylmethyl (TMTr), 1(2-fluorophenyl)-4-methoxyhexahydropyridin-4-yl (FPMP), substituted pixyl, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tributylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-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-di Nitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, ortho-nitrobenzyl, p-nitrobenzyl, p-halogenated benzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-methylpyridinyl, 4-methylpyridinyl, 3-methyl-2-methylpyridinyl N-oxy, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxy Phenylacetyl diphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromobenzoxylmethyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorobenzyliminophenyl)methyl, 4,4′,4″-tris(acetylacetyloxyphenyl)methyl, 4,4′,4″-tris(benzoxyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′ ,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xanthyl, 9-(9-phenyl-10-ephthyl)anthrayl, 1,3-benzodithiocyclopentane-2-yl, benzoisothiazolyl S,S-dioxy, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl ( IPDMS), diethyl isopropyl silica (DEIPS), dimethyl trihexyl silica, tributyldimethyl silica (TBS, TBDMS), tributyldiphenyl silica (TBDPS), tribenzyl silica, tri-p-xylyl silica, triphenyl silica, diphenylmethyl silica (DPMS), tributylmethoxyphenyl silica (TBMPS), formate, benzoylformate, acetate, chloroacetate Dichloroacetic acid ester, trichloroacetic acid ester, trifluoroacetic acid ester, methoxyacetic acid ester, triphenylmethoxyacetic acid ester, phenoxyacetic acid ester, p-chlorophenoxyacetic acid ester, 3-phenylpropionate, 4-epoxyvalerate (acetylpropionate), 4,4-(epoxyethyldithio)valerate (acetylpropionyldithioacetal), neovalerate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4- 6-Trimethylbenzoate (carrageenan), alkyl carbonate methyl ester, 9-furomethyl carbonate (Fmoc), alkyl carbonate ethyl ester, alkyl carbonate 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl (TSE), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphino)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 3,4-dimethoxybenzyl ester, alkyl carbonate orthonitrobenzyl ester, alkyl carbonate p-nitrobenzyl ester, thioalkyl carbonate S-benzyl ester, 4-ethoxy-1-naphthyl carbonate, dithiocarbonate Methyl benzoate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, dibromomethyl benzoate, 2-methylbenzenesulfonate, 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, omega-(methoxycarbonyl)benzoate, α-naphthaleneate, nitrate ester, N,N,N′,N′-tetramethylphosphodiamide alkyl ester, N-phenylaminocarboxylic acid alkyl ester, borate ester, dimethylthiophosphoryl, 2,4-dinitrophenylsulfinic acid alkyl Esters, 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, sulfates, methanesulfonates (methanesulfonates), benzylsulfonates, toluenesulfonates (Ts), 2-cyanoethyl (CE or Cne) 3,5-Dichlorophenyl, 2,4-Dimethylphenyl, Butylthiocarbonyl, 4,4′,4″-Triphenyl(benzoyloxy)triphenylmethyl, Diphenylaminomethyl, Acetylpropyl, 2-(Dibromomethyl)benzoyl (Dbmb), 2-(Isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-Phenylon-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

In some embodiments, the nucleobase protecting group includes a protecting group for protecting 1,2- or 1,3-diol. Examples of protecting groups used to protect 1,2- or 1,3-diols include methylene acetal, ethyl acetal, 1-tert-butyl ethyl acetal, 1-phenyl ethyl acetal, (4-methoxyphenyl)ethyl acetal, 2,2,2-trichloroethyl acetal, acetone, cyclopentyl acetal, cyclohexyl acetal, cycloheptyl acetal, benzyl acetal, p-methoxybenzyl acetal, 2,4-dimethoxybenzyl acetal, 3,4-dimethoxybenzyl acetal, 2-nitrobenzyl acetal, methoxymethylene acetal, ethoxymethylene acetal, and dimethoxybenzyl acetal. Methyl ester, 1-methoxyethylidene, 1-ethoxyethylidene, 1,2-dimethoxyethylidene, α-methoxybenzyl ester, 1-(N,N-dimethylamino)ethylidene derivative, α-(N,N′-dimethylamino)benzyl ester, 2-oxocyclopentanediyl ester, di-tert-butylsilyl (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxane) derivative (TIPDS), tetra-tert-butoxydisiloxane-1,3-diethylene derivative (TBDS), cyclic carbonate, cyclic Ester, Ethyl ester and Phenyl ester.

In some embodiments, each nucleobase is independently protected by one or more protecting groups selected from dimethylformamide (DMF), N-methylpyrrolidin-2-ylene (PyA), neopentyloxymethyl (POM), formamidinium, DMF-formamidinium, phthalimide, chlorine, bromine, acetyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM). Cyclochlorophosphate nucleoside

In some embodiments, the present invention provides a method for preparing oligonucleotides using cyclic phosphate nucleosides or their salts or solvents. As used herein, a cyclic phosphate nucleoside refers to a nucleoside comprising 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 solvents, wherein: Y is O or S; each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl; ^R1 is selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 )alkyl, and ( _C1 - _C20 )alkoxy (including methoxy and -O-methoxyethyl); and ^B1 is a nucleobase that may be protected. In some embodiments, ^R1 is ( _C1 - _C16 )alkyl or ( _C1 - _C16 )alkoxy. In some embodiments, ^R1 is a ( _C1 - _C12 )alkyl or ( _C1 - _C12 )alkoxy. In some embodiments, ^R1 is a ( _C1 - _C8 )alkyl or ( _C1 - _C8 )alkoxy. In some embodiments, ^R1 is a ( _C1 - _C4 )alkyl or ( _C1 - _C4 )alkoxy. In some embodiments, ^R1 is fluorine. In some embodiments, ^R1 is methoxy. In some embodiments, ^R1 is O-methoxyethyl (MOE).

In some embodiments, the present invention provides a method for preparing a cyclic phosphorus nucleoside of formula (ii) or its salt or solvent. In some embodiments, the method for preparing a cyclic phosphorus nucleoside of formula (ii) comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, orthoaryl, and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii). .

In some embodiments, the present invention provides a cyclic phosphate nucleoside of formula (ii) or a salt or solvent thereof.

In some embodiments, the nucleobase of the cyclic phosphate nucleoside is 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 nucleobase of the cyclic phosphate nucleoside is an atypical nucleobase.

In some embodiments, the nucleobase of the cyclic phosphorus nucleoside is unprotected. In some embodiments, the nucleobase of the cyclic phosphorus nucleoside is protected by a nucleobase protecting group. In some embodiments, the nucleobase of the cyclic phosphorus nucleoside is protected by an amino nucleobase protecting group. In some embodiments, the nucleobase of the cyclic phosphorus nucleoside is protected by a ketone/carboxylic acid nucleobase protecting group. In some embodiments, the nucleobase of the cyclic phosphorus nucleoside is protected by a hydroxyl nucleobase protecting group. In some embodiments, the nucleobase of the cyclic phosphorus nucleoside is protected by a thiol nucleobase protecting group. In some embodiments, the nucleobase of the cyclic phosphate nucleoside is protected independently by one or more of the following protecting groups: dimethylformamide (DMF), formamidinium, DMF-formamidinium, phthalimide, chlorine, bromine, acetyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM).

In some embodiments, the cyclophosphochloronucleotide of formula (ii) is obtained as a mixture of stereoisomers. In some embodiments, the stereoisomers are separated before subsequent reaction steps. Dinucleotide

In some embodiments, the present invention provides a method for preparing oligonucleotides using a dinucleotide comprising the cyclic phosphate nucleoside of the present invention or its salt or solvent. In some embodiments, the present invention provides a method for preparing oligonucleotides using a dinucleotide of formula (iv) or its salt or solvent: Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^B1 and ^B2 are nucleobases that may be protected by one or more protecting groups; and ^R4 is hydrogen or a protecting group. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C16 ) alkyl and ( _C1 - _C16 ) alkoxy. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C12 ) alkyl and ( _C1 - _C12 ) alkoxy. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C8 )alkyl and ( _C1 - _C8 )alkoxy. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C4 )alkyl and ( _C1 - _C4 )alkoxy. In some embodiments, ^R1 and ^R2 are fluorine. In some embodiments, ^R1 and ^R2 are methoxy. In some embodiments, ^R1 and ^R2 are O-methoxyethyl (MOE).

In some embodiments, the present invention provides a method for preparing a dinucleotide of formula (iv) or a salt or solvent thereof, wherein the method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming a cyclic 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 ^R4 is a protecting group: In some embodiments, the method further includes removing one or more protective groups.

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 reaction step of the cyclophosphonucleotide of formula (ii) with the nucleoside of formula (iii).

In some embodiments, the present invention provides a dinucleotide of formula (iv) or a salt or solvent thereof.

In some embodiments, ^R2 is an alkoxy group attached to an adjacent 3' oxygen to form a ketal protective group with ^R4 .

In some embodiments, ^R4 is hydrogen. In some embodiments, ^R4 is a protecting group. In some embodiments, ^R4 is a hydroxyl protecting group. In some embodiments, ^R4 is selected from silyl, trimethylsilyl, triethylsilyl, tributyldimethylsilyl (TBS, TBDMS), tributyldiphenylsilyl, or triisopropylsilyl. In some embodiments, ^R4 and ^R2 form a ketal protecting group.

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

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

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

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

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 stereoisomer mixture is at least about 10.0. In some embodiments, the (S)/(R) ratio of the stereoisomer mixture is at least about 11.0. In some embodiments, the (S)/(R) ratio of the stereoisomer mixture is at least about 12.0. In some embodiments, the (S)/(R) ratio of the stereoisomer mixture is at least about 13.0. In some embodiments, the (S)/(R) ratio of the stereoisomer mixture is at least about 14.0. Substituted benzyl alcohol nucleophile.

In some embodiments, the present invention provides a method for preparing oligonucleotides using a substituted benzyl alcohol nucleophile. As used herein, a substituted benzyl alcohol nucleophile or "BnOH" refers to benzyl alcohol containing at least one substituent on a benzene ring. The substituted benzyl alcohol nucleophile may comprise primary benzyl alcohol (Bn-CH₂- _OH ), secondary benzyl alcohol (Bn-CH( ^R₇ )-OH), or tertiary benzyl alcohol (Bn-C( ^R₇ )( ^R₈ )-OH). In some embodiments, the present invention provides a method for preparing oligonucleotides using the substituted benzyl alcohol nucleophile of formula (v): Wherein: n is selected from 1, 2, 3, 4 and 5; each R ⁵ is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; and R ⁷ and R ⁸ are independently selected from hydrogen, (C ₁ -C ₂₀ ) alkyl and aryl.

In some embodiments, ^R5 in each case is an ortho- or para-substituent on the benzyl group. In some embodiments, ^R5 in each case is an ortho-substituent on the benzyl group. In some embodiments, ^R5 in each case is a para-substituent on the benzyl group. In some embodiments, ^R5 in each case is independently selected from halogens, ( _C1 - _C20 )alkyl, ( _C1 - _C20 )alkoxy, trifluoromethyl, and phenyl (substituted or unsubstituted). In some embodiments, ^R5 is a fused phenyl ring forming a naphthalene ring system. In some embodiments, ^R5 is a ( _C1 - _C16 )alkyl or ( _C1 - _C16 )alkoxy. In some embodiments, ^R5 is a ( _C1 - _C12 )alkyl or ( _C1 - _C12 )alkoxy. In some embodiments, ^R5 is a ( _C1 - _C8 )alkyl or ( _C1 - _C8 )alkoxy. In some embodiments, ^R5 is a ( _C1 - _C4 )alkyl or ( _C1 - _C4 )alkoxy.

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

In some embodiments, the present invention provides a method for preparing oligonucleotides using cyclic phosphate nucleosides and substituted benzyl alcohol nucleophiles. In some embodiments, the present invention provides a method for preparing dinucleotides of formula (vi) or their salts or solvents: Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^B1 and ^B2 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C16 )alkyl and ( _C1 - _C16 )alkoxy. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C12 )alkyl and ( _C1 - _C12 )alkoxy. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C8 )alkyl and ( _C1 - _C8 )alkoxy. In some embodiments, ^R1 and ^R2 are independently selected from ( _C1 - _C4 )alkyl and ( _C1 - _C4 )alkoxy. In some embodiments, ^R1 and ^R2 are fluorine. In some embodiments, ^R1 and ^R2 are methoxy. In some embodiments, ^R1 and ^R2 are -O-methoxyethyl (MOE).

In some embodiments, the present invention provides a method for preparing a dinucleotide of formula (vi) or a salt or solvent thereof, wherein the method comprises: providing a cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group; and reacting the cyclic phosphorus dinucleotide of formula (iv) with a substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4, and 5; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl, and aryl. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

In some embodiments, the present invention provides a method for preparing a dinucleotide of formula (vi) or a salt or solvent thereof, wherein the method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: ; and (c) reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4, and 5; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl, and aryl. In some embodiments, the method further includes removing one or more protecting groups from the dinucleotide of formula (vi).

In some embodiments, the present invention provides a dinucleotide of formula (vi) or a salt or solvent thereof.

In some embodiments, ^R6 is hydrogen. In some embodiments, ^R6 is a protecting group. In some embodiments, ^R6 is a hydroxyl protecting group. In some embodiments, ^R6 is selected from silyl, trimethylsilyl, triethylsilyl, tributyldimethylsilyl (TBS, TBDMS), tributyldiphenylsilyl, or triisopropylsilyl.

In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) reacts with the substituted benzyl alcohol nucleophile of formula (v) to form a dinucleotide having [3'-5']-linked bonds (e.g., the dinucleotide of formula (vi)). In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) reacts with the substituted benzyl alcohol nucleophile of formula (v) to form a dinucleotide having [5'-5']-linked bonds. In some embodiments, the cyclic phosphorus dinucleotide of formula (iv) reacts with the substituted benzyl alcohol nucleophile of formula (v) to form a mixture of isomers (e.g., forming isomers). In some embodiments, the mixture of isomers comprises a mixture of [3'-5']-linked and [5'-5']-linked stereoisomers. In some embodiments, isomers are isolated prior to the subsequent synthesis step.

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

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

In some embodiments, the isomer mixture includes [3'-5']-linked and [5'-5']-linked [S] ratios. 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.

In some embodiments, the reaction step of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is performed 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), acetonitrile (MeCN), methyl tributyl ether (MTBE), tripentyl alcohol, toluene, or mixtures thereof.

In some embodiments, the reaction step of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) is carried out in the presence of an alkali. In some embodiments, the alkali includes: n-butyllithium (n-BuLi); lithium tributoxide (LiOtBu); bis(trimethylsilyl)acetamide lithium (LiHMDS); lithium tetramethylhexahydropyridine (LiTMP); or Li- _RZ (where _RZ is alkyl or aryl); sodium tributoxide (NaOtBu) + lithium chloride (LiCl); potassium tributoxide (KOtBu) + lithium chloride (LiCl); triethylamine; diazabiscycloundecene (DBU); triazabiscyclodecene (TBD); or combinations thereof. In some embodiments, the alkali is premixed with the substituted benzyl alcohol nucleophile of formula (v) prior to the reaction step of the cyclic phosphorus dinucleotide of formula (iv) and the substituted benzyl alcohol nucleophile of formula (v). In some embodiments, the alkali is lithium tributoxide (LiOtBu).

In some embodiments, the dinucleotide of formula (vi) is isolated by reaction with silicon chloride. Protected oligonucleotides

In some embodiments, the present invention provides a method for preparing phosphate-protected oligonucleotides. In some embodiments, the present invention provides a method for preparing the oligonucleotide of formula (vii): Wherein: each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^R4 is H or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group's benzyl ring; each ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; ^B1 , ^B2 and ^B3 are nucleobases that may be protected as appropriate; m is an integer between 1 and 18; and X is hydrogen, a protecting group or In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C16 )alkyl and ( _C1 - _C16 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C12 )alkyl and ( _C1 - _C12 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C8 )alkyl and ( _C1 - _C8 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C4 )alkyl and ( _C1 - _C4 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are fluorine. In some embodiments, ^R1 , ^R2 , and ^R3 are methoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are -O-methoxyethyl (MOE).

In some embodiments, the present invention provides a method for preparing an oligonucleotide of formula (vii), wherein the method comprises: providing a dinucleotide of formula (vi); and repeating (as disclosed herein) one or more reactions of steps (a), (b), and (c) of the method for preparing a dinucleotide of formula (vi) or a salt or solvent thereof to obtain the oligonucleotide of formula (vii): In step (b), the cyclic phosphorus nucleotide of formula (ii) reacts with the 5'-terminal nucleotide of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

In some embodiments, the present invention provides a method for preparing the oligonucleotide of formula (vii), wherein the method comprises: (a) reacting the nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming the cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: (c) Reaction of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; and (d) repeating steps (a), (b) and (c) one or more times to obtain the oligonucleotide of formula (vii): In step (b), the cyclic phosphorus nucleotide of formula (ii) reacts with the 5'-terminal nucleotide of a dinucleotide or oligonucleotide. In some embodiments, the method further includes the removal of one or more protecting groups.

In some embodiments, the present invention provides oligonucleotides of formula (vii) or their salts or solvents.

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, acetyl, trimethylsilyl, triethylsilyl, tributyldimethylsilyl (TBS, TBDMS), tributyldiphenylsilyl, or triisopropylsilyl.

In some embodiments, X-series cyclic phosphate nucleosides In this context, Y is either O or S. In some embodiments, X is a cyclic phosphate nucleoside, which can be used to further elongate the oligonucleotide by repeating (as disclosed herein) the reactions of steps (a), (b), and (c) in the method for preparing the dinucleotide of formula (vi). The resulting oligonucleotide...

In some embodiments, the present invention provides a method for preparing oligonucleotides. In some embodiments, the present invention provides a method for preparing oligonucleotides of formula (1) or their salts or solvents. Wherein: each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl (protected), halogen (including fluorine), _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy (including methoxy and -O-methoxyethyl); ^B1 , ^B2 and ^B3 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; m is an integer from 0 to 18; and X is hydrogen, a protecting group or In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C16 )alkyl and ( _C1 - _C16 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C12 )alkyl and ( _C1 - _C12 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C8 )alkyl and ( _C1 - _C8 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are independently selected from ( _C1 - _C4 )alkyl and ( _C1 - _C4 )alkoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are fluorine. In some embodiments, ^R1 , ^R2 , and ^R3 are methoxy groups. In some embodiments, ^R1 , ^R2 , and ^R3 are -O-methoxyethyl (MOE).

In some embodiments, the present invention provides a method for preparing an oligonucleotide of formula (1) or a salt or solvent thereof, wherein the method comprises: providing a dinucleotide of formula (vi) or an oligonucleotide of formula (vii); and hydrogenating the dinucleotide of formula (vi) or the oligonucleotide of formula (vii); wherein the method produces an oligonucleotide of formula (1) or a salt or solvent thereof. In some embodiments, the method further comprises removing one or more protecting groups.

In some embodiments, the present invention provides a method for preparing an oligonucleotide of formula (1) or a salt or solvent thereof, wherein the method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: (c) Reaction of the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile's benzyl ring; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; (d) repeat steps (a), (b) and (c) one or more times as appropriate 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 its salt or solvent. In some embodiments, the method further includes the removal of one or more protecting groups.

In some embodiments, oligonucleotides are block copolymers. As used herein, the term block copolymer refers to an oligonucleotide comprising at least two consecutive nucleotide units that share a common structural feature (i.e., chemical or stereochemical) at the nucleotide, sugar, and/or internucleotide bond sites. At least two consecutive nucleotide units that share a common structural feature may be referred to as a "block".

In some embodiments, the oligonucleotides of the present invention can be used in subsequent oligonucleotide or polynucleotide synthesis applications. In some embodiments, the oligonucleotides of the present invention can be used as block polymeric amides in the solid-phase synthesis of oligonucleotides or polynucleotides. In some embodiments, the oligonucleotides of the present invention can be used in enzymatic ligation reactions for the synthesis of oligonucleotides or polynucleotides.

In some embodiments, the invention provides a polynucleotide of formula (1) or a salt or solvent thereof, wherein m is selected as an integer between 0 and 100. In some embodiments, m is selected as an integer between 1 and 18. In some embodiments, m is selected as an integer between 1 and 3.

In some embodiments of the present invention, 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, alkalis) are completely dissolved in a solvent mixture so that no solid particles are present.

In some embodiments, the method is implemented using a continuous flow. As used herein, continuous flow refers to a process in which starting materials, reagents, catalysts, alkalis and all other reactive components are continuously added to the reactor while products, byproducts, unreacted starting materials and other reactive components are continuously removed from the reactor.

In some embodiments, the method is carried out in a fluidized bed reactor. As used herein, a fluidized bed reactor refers to a pipe, tube, continuous stirred tank reactor (CSTR), multiple cascaded CSTRs, batch reactor with intermittent filling and emptying, glass microreactor, or other reactor capable of continuous operation. In some embodiments, the method is carried out in a fluidized bed reactor at an ambient temperature (e.g., 20-25°C). In some embodiments, the method is carried out in a fluidized bed reactor with a reaction time between 45 and 120 minutes. In some embodiments, the method is carried out in a fluidized bed reactor with a reaction time between 45 and 60 minutes. Definitions

Unless otherwise stated, the following terms and phrases have the following meanings. These definitions are not intended to be restrictive and are intended to provide a clearer understanding of certain aspects of the invention.

Approximately /About : As used herein, the terms “approximately” and “about” are used interchangeably and refer to values within +/- 10% of one or more values of interest. In some embodiments, unless explicitly stated otherwise or 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 values.

Complementarity : As used herein, the term "complementarity" or "complementarity" refers to a structural relationship between two nucleotides, nucleosides, or nucleosides ( e.g. , on two opposing nucleic acids or on opposing regions of a single nucleic acid strand (e.g., a hair clip)) that allows the two nucleotides to form base pairs with each other. For example, a purine nucleotide of a nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid can pair together by forming a hydrogen bond base. 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 double strand. Similarly, two nucleic acids can have multiple nucleotide regions that complement each other to form complementary regions.

Deoxyribonucleotide : As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen atom at the 2' position of its pentose sugar in place of a hydroxyl group, compared to a ribonucleotide. Modified deoxyribonucleotides have one or more atoms modified or substituted at the 2' position, other than a hydroxyl group, including modifications or substitutions of a nucleotide, sugar, or phosphate group or thereof.

Bis-stranded oligonucleotides : As used herein, the term "bis-stranded oligonucleotide" or "ds-oligonucleotide" refers to an oligonucleotide in bis-stranded form. Complementary base pairs of ds-oligonucleotide bis-stranded regions can be formed between antiparallel nucleotide sequences of covalently separated nucleic acid chains. Similarly, complementary base pairs of ds-oligonucleotide bis-stranded regions can be formed between antiparallel nucleotide sequences of covalently linked nucleic acid chains. Furthermore, complementary base pairs of ds-oligonucleotide bis-stranded regions can be formed from a single nucleic acid chain that can be folded ( e.g., via hairpins) to provide complementary antiparallel nucleotide sequences with base pairs together. A ds-oligonucleotide may comprise two covalently separated nucleic acid chains that are fully bistranded. ds oligonucleotides may comprise two covalently separated nucleic acid chains that are partially double-stranded ( e.g., with overhangs at one or both ends). ds oligonucleotides may comprise partially complementary antiparallel nucleotide sequences and therefore may have one or more mismatches, including internal mismatches or terminal mismatches.

Bistanza : As used herein, the terms "bistanza" and "bistanza region" for nucleic acids ( e.g. , oligonucleotides) refer to a structure formed by the complementary base pairing of two antiparallel nucleotide sequences, whether formed from two covalently separated nucleic acid chains or from a single folded chain ( e.g. , via a hairpin). Bistanzas can also be formed even if there is no perfect complementarity between the two chains or in the presence of abase-free nucleotides.

Modified nucleotide linkages : As used herein, the term "modified nucleotide linkage" refers to a nucleotide linkage that has one or more chemical modifications compared to a reference nucleotide linkage with a phosphodiester bond. Modified nucleotide linkages are non-natural linkages.

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, selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. 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. In addition, or alternatively, modified nucleotides may have one or more chemical moieties coupled to a corresponding reference nucleotide.

Nucleobase : As used herein, the term "nucleobase" refers to the heterocyclic portion 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," which are used interchangeably herein and include purine bases adenine (A) and guanine (G) and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Nucleobases can also be "atypical nucleobases," which include synthetic or natural nucleobases that are not typical nucleobases.

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

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 monomers of nucleic acid polymers ( e.g., oligonucleotides) (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).

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

Phosphate ester analogues : As used herein, "phosphate ester analogue" refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. Examples of phosphate ester analogues include, but are not limited to, 5' phosphonates, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). Oligonucleotides may have a phosphate ester analogue (referred to as "4'-phosphate ester analogue") at the 4'-carbon position of the sugar or replace the 5'-phosphate ester at the 5'-terminal nucleotide. An example of a 4'-phosphate ester analogue is oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon) or an analogue thereof. See, for example, International Patent Application Publication 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. Acids Res. 43:2993-3011).

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

Ribonucleotide : As used herein, the term "ribonucleotide" refers to a nucleotide whose pentose sugar is ribose and which contains a hydroxyl group at its 2' position. Modified ribonucleotides are ribonucleotides with one or more modified or substituted atoms other than hydrogen at the 2' position, which include modifications or substitutions of nucleotides, sugars, or phosphate groups.

Substituted benzyl alcohol nucleophiles : As used herein, the term "substituted benzyl alcohol nucleophile" or "BnOH" refers to benzyl alcohol containing at least one substituent on the 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).

Chain : As used herein, the term "chain" refers to a single, continuous nucleotide sequence 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).

Synthesis : As used in the context of the products described herein (e.g., synthetic oligonucleotides, synthetic nucleotides), the term "synthesis" means artificial synthesis (e.g., using machinery, such as a solid-phase nucleic acid synthesizer) or other 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. General considerations

Those skilled in the art can recognize or identify one or more equivalent forms of the specific embodiments of the invention described herein using only conventional experiments. The scope of the invention is not intended to limit the above description or the following examples.

At various locations within this invention, substituents or properties of the compounds of the invention are disclosed in groups or ranges. This invention is intended to include each individual or sub-combination of members of such groups and ranges, and such groups or ranges include endpoints. By way of non-limiting examples, 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.

In this document, for example, "a" ("a", "an") or "the" ("the") may mean one or more, unless the context indicates otherwise. A claim or statement including "or" among one or more members of a group is deemed satisfied if one, more, or all of them are present in, used in, or otherwise associated with the given product or process, unless the context indicates otherwise. This invention may include embodiments in which exactly one group member is present in, used in, or otherwise associated with the given product or process. This invention may include embodiments in which one or more or all of them are present in, used in, or otherwise associated with the given product or process.

The term "including" is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term "including" is used in this document, it also covers and discloses the terms "consisting of" and "substantially consisting of".

The abbreviation "eg" is derived from the Latin word "exempli gratia" and is used in this text to indicate a non-restrictive example. Therefore, the abbreviation "eg" is a synonym of the term "for example." The abbreviation " ie " is derived from the Latin word " 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."

Any particular embodiment of the present invention within the prior art may be expressly excluded from any one or more technical solutions. For any reason, whether or not with respect to the prior art, any particular embodiment of the present invention, method and/or composition may be excluded from any one or more technical solutions.

This specification is limited to the inclusion of any publications, patent applications, patents and other references mentioned herein by way of citation, and any conflict with this specification.

Chapter titles, materials, methods, and examples are for illustrative purposes only and are not intended to be restrictive. Examples

Unless otherwise stated, all reactions in the following examples were carried out 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 under argon pressure by continuous filtration through a neutral alumina and CuO column. Reactions were carried out in oven-dried reaction tubes (Fisherbrand, 16 × 125 mm, product number 1495935A) sealed with nuts (Thermo Fisher Scientific, catalog number B7995-18) and fitted with Teflon-lined diaphragms (Thermo Fisher Scientific, catalog number C47995-15). Solvents used for extraction, crystallization, and column chromatography were purchased from Sigma-Aldrich in ACS grade, except for hexane, which was HPLC grade. Lithium tributoxide (LiOt-Bu) was purchased from Alfa Aesar and stored in a nitrogen-filled glove box. All preparation reactions were set up in a nitrogen-filled glove box, and the sealed reaction vials were removed and stirred for the specified times. Screening of reaction conditions and mechanisms was conducted with the aid of a nitrogen-filled glove box. Organic compounds were purified by rapid chromatography using a Silicycle SiliaFlash P60 (230-400 sieve) silicone manual or CombiFlash NextGen 300 automated chromatography system.

For general analysis, _CDCl₃ and _d₆ -DMSO were purchased from Cambridge Isotope Labs. NMR spectra were collected on a Bruker Avance III HD 400 or 500 MHz spectrometer. ^¹H ( _CDCl₃ : δ 7.26; _d₆ -DMSO: δ 2.50) and ^¹³C NMR shifts ( _CDCl₃ : δ 77.16; _d₆- DMSO: δ 39.52) were referenced to residual solvent peaks. Multiplicity was characterized using the following abbreviations: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = triplet, p = quintet, sept = septet, m = multiplet. ^¹³C and ^¹⁹F spectra were obtained by ^¹H decoupling. Gas chromatography (GC) analysis was performed using a J&W DB-1 column (10 m, 0.1 mm ID) on an Agilent 7890A GC analyzer with an FID detector. LC/MS was recorded on an Agilent 6120 quadrupole LC/MS. Elemental analysis was performed using 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. Infrared spectroscopy (IR) spectra were recorded on a Nicolet iS5 spectrometer from Thermo Fisher Scientific equipped with an iD5 diamond lamination ATR accessory. IR spectra were obtained from clean samples. Melting point was obtained using the Stanford Research Systems EZ-Melt melting point apparatus. Example 1. Preparation of 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 added to a 50 mL round-bottom flask equipped with a magnetic stir bar and dried under vacuum using the standard Schlenk line technique. The mixture was dissolved in anhydrous DMF (0.5–1 M, depending on solubility) and tert-butyldimethylsilicon chloride (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 layer was washed with brine (100 mL) and dried over _{anhydrous Na₂SO₄} and evaporated. The residue was purified by silicone column chromatography and eluted with ethyl acetate/hexane (6:4). The fraction containing the product was collected and evaporated. b. Synthesis of benzyl-protected nucleosides (GP2)

Bis-TBS-protected cytidine (1 equivalent) and anhydrous DMF (0.3 M) were added to a 100-mL round-bottom flask equipped with a magnetic stir bar. The resulting solution was cooled to 0°C and NaH was added. The solution was stirred for 5 minutes, and benzyl bromide was added dropwise at 0°C. The reaction process was monitored by LC-MS. After 45 minutes, a second portion of NaH was added and the mixture was stirred for another hour at 0°C. The reaction mixture was heated to RT and stirred for 30 minutes, then suddenly cooled with _NH4Cl (50 mL). The mixture was extracted with EtOAc (100 mL × 3). The combined organic layer was washed with brine (100 mL), dried over _Na2SO4 , _filtered , and concentrated under vacuum. The crude product was purified using a Combi-Flash NextGen 300 automated chromatography system via silicone column chromatography with hexane/ethyl acetate as eluent (0-100% gradient). c. Synthesis of 3',5'- dihydroxynucleotides deprotected 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, cool 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. The crude product was purified by rapid chromatography on a silicone column eluted with dichloromethane-methanol (0 to 5% gradient) (for C) or hexane-ethyl acetate (0 to 100% gradient for A and G) to give a white solid final product compound (87-97% yield). d. Synthesis of 3-OTBS-5-OH- nucleoside of the 5'- siloxy group of the selectively deprotected bis -TBS- nucleoside (GP4)

Separated 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. When complete after 4–6 hours, 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 layer was dried over sodium sulfate and concentrated using a rotary evaporator. The residue was purified by silicone column chromatography eluted with hexane-ethyl acetate [0 to 100% gradient] to give 5'-OH, 3-TBS-nucleoside (70-80% yield). e. A pot-based procedure (GP5) for the synthesis of 3-OTBS-5-OH- nucleoside from nucleosides . 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 solution of 2'-O-methyluridine (10 g, 39 mmol) in DMF (30 mL), 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 added 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 solution 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 over sodium sulfate and concentrated to 30 mL or until the 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 aged for 3 hours, and the solid was collected through a filter funnel and the cake was washed with 100 mL of hexane. The solid was dried in a vacuum oven to obtain a white solid (9.9 g, 68%). HPLC analysis showed that the crude solid was 95% pure and was used without further purification. The identity was confirmed using proton NMR and MS analysis and found to be consistent with the data in the literature ( Chem. Commun. 2021, 57 (55), 6808-6811).

¹ H NMR (400 MHz, CDCl ₃ ) 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).

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. 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 equivalence) and BOMCl (4.50 mL, 32.5 mmol, 1.3 equivalence) at 0 °C for 40 min. The reaction was monitored by TLC. After completion, the reaction was terminated with _H₂O (50 mL) and the resulting mixture was extracted with EtOAc (150 mL × 4). The combined organic layer was washed with brine (100 mL), dried over _{Na₂SO₄ ,} filtered, and concentrated under vacuum. The crude product was purified using a Combi-Flash NextGen 300 automated chromatography system via silicone column chromatography with DCM/MeOH as the eluent (0-5% MeOH-DCM gradient). g. DMF- formamidinium protection of nucleoside _NH₂ (GP7)

Adenosine-ketal (1 equivalent) or 2'-OMe-cytidine (1 equivalent) and 1,1-dimethoxy-N,N-dimethylmethylamine (5 equivalents, 32.5 mmol) in DMF (10 mL, 0.65 M) were added to a 100-mL RB flask equipped with a magnetic stir bar and dried in an oven. After stirring at room temperature for 18 hours, the reaction mixture was concentrated to dryness (20 × 2 mL toluene was added to help the DMF evaporate under reduced pressure) and ground with diethyl ether (3 × 100 mL) to obtain a coarse white solid. The solid was collected by filtration and dried under high vacuum for 12 hours to obtain a grayish-white solid >90% pure product (90-95%). 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 h, then diluted with 30 mL of water and extracted with dichloromethane (3 × 20 mL). The organic layer was collected, dried with magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography using acetone/hexane (1:1) as the eluent. It was then dried overnight under vacuum to obtain a white solid, 54% yield of the title compound. Proton NMR and MS analysis confirmed its identity and showed consistency with data from the literature ( J. Am. Chem. Soc. 2017 , 139, 5467-5473 ).

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

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. i. Bi -Boc protection of cytidine j. Bi -Boc protection of adenosine k. Adenosine phenoxyimino group protection l. Guinea glycoside protection Example 2. Spectrum 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 = 0.6 Hz, 3H).

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. b. 3-TBS-G-DMF -amide

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.46 (s, 1H), 7.71 (s, 1H), 7.16 - 7.13 (m, 1H), 5.80 (d, J = 7.4 Hz, 1H), 4.50 (dd, J = 4.9, 1.4 Hz, 1H), 4.42 (dd, J = 7.5, 4.9 Hz, 1H), 4.15 (p, J = 1.4 Hz, 1H), 3.90 (dd, J = 12.6, 2.1 Hz, 1H), 3.68 (dd, J = 12.6, 1.5 Hz, 1H), 3.27 (s, 3H), 3.18 (t, J = 2.2 Hz, 3H), 3.12 - 3.05 (m, 3H), 0.93 (t, J = 1.1 Hz, 9H), 0.12 (dt, J = 8.5, 1.0 Hz, 6H). c. 3-TBS-ABn ₂

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

¹ H NMR (400 MHz, CDCl ₃ ) 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).

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. e. 3-TBS-N -allyl -U- diol

¹ H NMR (400 MHz, CDCl ₃ ) 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).

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. f. adenosine -N -methylamidine

¹ H NMR (500 MHz, CDCl ₃ ) 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).

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. Example 3. Synthesis of cyclochlorophosphate nucleosides (GP8) a. Study on the synthetic conditions of cyclochlorophosphites

Add protected diol-nucleoside (1 equivalent, 2 mmol) and DMAP (0.1–0.2 equivalent, 60–120 mg, 0.25–0.5 mmol) to DCM (18 mL, 0.1 M) in an oven-dried 100 mL Schulenck flask equipped with a magnetic stir bar. Add triethylamine or S-Collidine (2.2 equivalent, 4.4 mmol, in 1 mL DCM) to the resulting suspension via syringe. Cool the resulting solution to -20°C and add freshly distilled _POCl₃ (1.05 equivalent, 2.1 mmol, in 1 mL DCM) in a single batch. After stirring the solution for 1 hour, heat it to 0°C and stir overnight. The diastereoselectivity of the crude product was monitored by taking aliquots of the reaction mixture and analyzing them using ^31P NMR. The reaction mixture was further stirred 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 salts. The suspension obtained by filtering through a glass frit funnel was rinsed with diethyl ether (10 × 2 mL) to remove the residue. The filtrate was concentrated with the aid of a rotary evaporator. The crude material was purified by chromatography using a Combi-Flash NextGen 300 automated chromatography system with a silicone column (the crude material was loaded onto the column by dissolving it in a minimum amount of DCM, with a height of ~2 inches and a diameter of ~0.9 inches) and a hexane:ethyl acetate (0 to 80% gradient).

The variations in synthetic conditions are summarized in Table 1. The yields and diastereomeric ratios (dr) of the obtained cyclochloronucleotides are also summarized in Table 1. Table 1 - Synthetic Conditions of Cyclochloronucleotides b. Research on nucleosides used in the synthesis of cyclochlorophosphites

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] dioxaphosphane-cyclohexane -6 -yl ) pyrimidine -2,4(1H,3H) -dione

The compound was synthesized 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) in DCM (35 mL, 0.14 M) according to standard GP8 procedure. Column conditions: SiO2-25 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 80%), resulting in the separation of 1380 mg of the desired product as a white solid. Yield = 62%, dr = 38:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -1.12, -1.72.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS was not determined because the compound was hydrolyzed during the analysis. b. 3-(( benzyloxy ) methyl )-1-((2S,4aR,6R,7R,7aR)-2- chloro - 7- fluoro - 2-oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxaphosphanecyclohexane -6 -yl ) pyrimidine -2,4(1H,3H) -dione

The compound was synthesized 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) in DCM (25 mL, 0.1 M) according to standard GP8 procedure. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 80%), separating 530 mg of the desired product as a white solid. Yield = 48%, dr = 18:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -1.62, -2.04.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

HR-MS was not determined because the compound was hydrolyzed during the analysis. c. 1-((2S,4aR,6R,7R,7aR)-2- chloro - 7- methoxy - 2- oxotetrahydro - 4H -furano [ 3,2-d][1,3,2] dioxaphosphanecyclohexane -6 -yl )-4-( dibenzylamino ) pyrimidin -2(1H) -one

The compound was synthesized 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) in DCM (21 mL, 0.1 M) according to standard GP8 procedure. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 100%), 304 mg of the desired product as a white solid was isolated. Yield = 30%, dr = 22:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -0.96, -1.11.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS was not determined because the compound was hydrolyzed during the analysis. d. 3-(( benzyloxy ) methyl )-1-((2S,4aR,6R,7aS)-2- chloro -2 -oxotetrahydro - 4H - furano [ 3,2-d][1,3,2] dioxaphosphanecyclohexane- 6 -yl )-5 -methylpyrimidine -2,4(1H,3H) -dione

The compound was synthesized 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) in DCM (60 mL, 0.12 M) according to standard GP8 procedure. Column conditions: SiO2-25 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 85%), separating 1,700 mg of the desired product as a white solid. Yield = 55%, dr = 16:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -1.48, -1.98.

¹ H NMR (400 MHz, CDCl ₃ ) 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 (dddd, 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS was not determined because the compound was hydrolyzed during the analysis. e. (2S,4aR,6R,7R,7aR)-2- chloro -6-(6- chloro -9H- purin- 9- yl )-7- methoxytetrahydro - 4H -furano [ 3,2-d][1,3,2] dioxophosphoruscyclohexane 2- oxide

The compound was synthesized 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) in DCM (30 mL, 0.12 M) according to the standard GP8 procedure. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 85%), 620 mg of the desired product as a white solid was isolated. Yield = 47%, dr = 10:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -1.16, -1.76.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS was not determined because the compound was hydrolyzed during the analysis. f. (2S,4aR,6R,7R,7aR)-2- chloro -6-(6-( dibenzylamino )-9H- purin- 9 -yl )-7- methoxytetrahydro -4H -furano [ 3,2-d][1,3,2] dioxophosphoruscyclohexane 2- oxide

The compound was synthesized 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) in DCM (45 mL, 0.11 M) according to the standard GP8 procedure. Column conditions: SiO2-25 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 100%), separating 1220 mg of the desired product as a white solid. Yield = 47%, dr = 18:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.01, -1.26.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS was not determined because the compound was hydrolyzed during the analysis. g. 1-((2S,4aR,6R,7R,7aR)-2- chloro - 7-methoxy -2- oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxaphosphanecyclohexane -6 -yl )-3-(3 - methylbut -2- en -1- yl ) pyrimidin -2,4(1H,3H) -dione

The title compound was synthesized 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) in DCM (20 mL, 0.1 M) according to standard GP8 procedure. Column conditions: SiO2-10 g column, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 100%), separating 400 mg of the desired product as a white solid. Yield = 49%, dr = 14:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -1.08, -1.71.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

HR-MS was not performed because the title compound was hydrolyzed during the analysis. Example 5. Synthesis of cyclic dinucleotides a. Multipot synthesis - Method A (GP9)

In a glovebox 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 glovebox and placed under a nitrogen atmosphere using the Schulenk line technique, maintained at -20°C with the aid of a refrigeration unit. 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 min.

Cyclochloronucleotide phosphate (1.2 equivalents, 1.2 mmol) and 5 mL MeCN were added to another 20 mL reaction tube B equipped with a magnetic stir bar. The reaction mixture was cooled to -20°C with the aid of a refrigeration unit. While stirring under a nitrogen atmosphere, the pre-stirred alkoxide solution from reaction tube A was transferred dropwise over 5 minutes to a round-bottom flask using a syringe. The resulting reaction mixture was stirred at -20°C for 18 hours. If incomplete conversion was observed, the reaction mixture was heated to room temperature over 30 minutes to ensure 100% conversion. The reaction was monitored by ³¹ pNMR. After the reaction is complete, the crude reaction mixture is concentrated under reduced pressure and purified by a Combi-Flash NextGen 300 automated chromatography system using hexane:ethyl acetate (0% to 100% gradient). Alternatively, DCM:acetonitrile (2% isopropanol) or DCM:ethyl acetate (2% MeOH) is used for more polar compounds. For adenosine and cytidine containing cyclic dinucleotides, DCM:MeOH is used as the eluent (0% to 10%). b. Multipot Synthesis - Method B (GP10)

Under stirring 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.

Protected diol-nucleoside (1 equivalent, 1 mmol) and DMAP (2.2 equivalent, 2.2 mmol) in DCM (5 mL, 0.2 M) were added to another reaction tube B equipped with a magnetic stir bar. The resulting suspension was cooled to -78°C. The pre-stirred solution in reaction tube A was transferred to reaction tube B. The resulting mixture was stirred at -78°C for 1 hour and then slowly heated to room temperature while stirring for another hour. The reaction was monitored by ³¹ 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 filtrate was concentrated under reduced pressure. The crude compound was purified using a Combi-Flash NextGen 300 automated chromatography system with a silicone column and a hexane:acetone gradient (0% to 100%, loaded onto the column by dissolving in a minimum amount of DCM, height ~3 inches, diameter ~0.9 inches). Alternatively, DCM:acetone was used for more polar compounds. For adenosine containing cyclic dinucleotides, DCM:MeOH was used as the eluent (0% to 5%). c. Study of reaction conditions for the synthesis of cyclic dinucleotides.

Cyclic dinucleotides were synthesized according to the general procedure of Example 5b, but using the various synthetic conditions summarized in Table 2. The yields of the obtained cyclic dinucleotides (based on ³¹ p NMR) are also summarized in Table 2. Table 2 - Synthetic Conditions of Cyclic Dinucleotides d. Research on the synthesis of nucleosides of cyclic dinucleotides

Cyclic dinucleotides were synthesized according to the general procedure in Example 5b, but using different nucleosides. The resulting cyclic dinucleotides and their yields and/or diastereomeric ratios (dr) are shown below [Report the yields and dr of the separated materials; show the structures of the major diastereomers]. e. One-pot synthesis - Method C (GP11)

Under stirring 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.

Diol (1 equivalent, 1 mmol) and DMAP (2.2 equivalents, 2.2 mmol) in DCM (5 mL, 0.2 M) were added to another reaction tube B equipped with a magnetic stir bar. The resulting suspension was cooled to -78°C. The pre-stirred solution in reaction tube A was transferred to reaction tube B. The resulting mixture was stirred at -78°C for 1 hour and then slowly heated to room temperature while stirring for another hour. The reaction was monitored by ³¹ 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 filtrate was concentrated under reduced pressure. The crude compound was purified using a silicone column with a hexane:acetone gradient (0% to 100%) via a Combi-Flash NextGen 300 automated chromatography system. The crude compound was loaded onto the column by dissolving it in a minimal amount of DCM to a height of ~3 inches and a diameter of ~0.9 inches. Alternatively, DCM:acetone can be used for more polar compounds. For adenosine containing cyclic dinucleotides, DCM:MeOH (0% to 5%) was used as the eluent. f. Synthesis of guanosine dimers 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-(( benzyloxy ) methyl )-2,4 -dioxy- 3,4 -dihydropyrimidine -1(2H) -yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2 -yl ) methoxy )-7- methoxy -2- oxotetrahydro - 4H - furano [3,2-d][1,3,2] dioxaphosphacyclohexane -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) and LiOtBu (72 mg, 0.89 mmol, The compound was synthesized from 1.1 equivalents of 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphanecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (U-BOM-Cl-CP, 410 mg, 0.89 mmol, 1.1 equivalents) in toluene:ACN (1:1) according to the general procedure GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 100%), separating two fractions of the desired white solid product. Yield = F2:66%, dr = 20:1; F1:17%, 15:1. Total yield = 83%.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.76.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: [C ₄₂ H ₅₅ N ₄ O ₁₅ PSi [M+H ⁺ ]: 937.3063, experimental value: 937.3009. b. 3-(( benzyloxy ) methyl )-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 3,4 -dihydropyrimidine -1(2H) -yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2- yl ) methoxy )-7- fluoro -2- oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxophosphoruscyclohexane -6 -yl ) pyrimidine -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 F-U-BOM-Cl-CP (200 mg, 0.45 mmol, 1.1 equivalent) in toluene:ACN (1:1) according to the standard procedure GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 100%), separation of the desired product as a white solid. Yield = 77%, dr = 5:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -4.29, -6.86.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

HR-MS (Q-TOF, ESI) calculated value: C ₄₁ H ₅₂ FN ₄ O ₁₄ PSi [M+H ⁺ ]: 903.3044, experimental value: 903.3038. c. 3-(( benzyloxy ) methyl )-1-((2R,3R,4R,5R)-4-(( tert-butyldimethylsilyl ) oxy )-5-((((2R,4aR,6R,7R,7aR)-6-(4-( dibenzylamino )-2- sideoxypyrimidin -1(2H) -yl )-7- methoxy -2- oxotetrahydrofuran -4H -furano [3,2-d][1,3,2] dioxophosphorus-cyclohexane -2- yl ) oxy ) methyl )-3- methoxytetrahydrofuran -2 -yl ) pyrimidin -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 standard GP procedures. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM-EA (1% MeOH) gradient (0% to 100%), separation of the desired white solid product. Yield = 250 mg, 63%, dr = 10:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.55, -6.36.

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

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₄₈ H ₆₀ N ₅ O ₁₃ PSi, [M+H ⁺ ]: 974.3768, experimental value: 974.3761. 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- oxotetrahydrofuran -4H -furano [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 (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 standard procedure GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, Hex-EA gradient (0% to 100%), separating two fractions of the desired white solid product. Yields: 500 mg, 74%, dr = 15:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -4.02, -6.82.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₃₅ H ₄₆ ClN ₆ O ₁₂ PSi, [M+Na ⁺ ]: 859.2261, experimental value: 859.2256. e. 3-(( benzyloxy ) methyl )-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-3-(( tert-butyldimethylsilyl ) oxy )-5-(6-( dibenzylamino )-9H- purine -9- yl )-4- methoxytetrahydrofuran -2- yl ) methoxy )-7- methoxy -2- oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxophosphoric cyclohexane -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 standard GP10 procedures. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM:MeOH gradient (0% to 5%), separation of the desired white solid product. Yield = 530 mg, 87%, dr = 22:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.99, -6.90.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₄₉ H ₆₀ N ₇ O ₁₂ PSi, [M+H ⁺ ]: 998.388, experimental value: 998.3893. f. 3-(( benzyloxy ) methyl )-1-((2R,4S,5R)-5-((((2R,4aR,6R,7R,7aR)-6-(3-(( benzyloxy ) methyl )-2,4 -dioxy - 3,4 -dihydropyrimidine -1(2H) -yl )-7- methoxy -2- oxotetrahydro -4H- furano [3,2-d][1,3,2] dioxophosphorus-cyclohexane -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 standard GP10 procedures. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), separation of the desired white solid product. Yield = 420 mg, 89%, dr = 22:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.60, -6.41.

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

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₄₂ H ₅₅ N ₄ O ₁₄ PSi, [M+H ⁺ ]: 899.3295, experimental value: 899.3288. g. 3-(( benzyloxy ) methyl )-1-((2R,3R,4R,5R)-4-(( tert-butyldimethylsilyl ) oxy )-5-((((2R,4aR,6R,7R,7aR)-6-(6-( dibenzylamino )-9H- purine -9 -yl )-7- methoxy - 2 -oxotetrahydrofuran - 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 standard GP10 procedures. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, Hex-EA gradient (0% to 100%), separation of the desired white solid product. Yield = 680 mg, 87%, dr = 30:1.

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

³¹ P NMR (203 MHz, CDCl ₃ ) δ -4.06, -6.54.

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₄₉ H ₆₀ N ₇ O ₁₂ PSi, [M+Na ⁺ ]: 998.388, experimental value: 998.3887. h. 3-(( benzyloxy ) methyl )-1-((2R,4aR,6R,7aS)-2-(((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 3,4- dihydropyrimidin -1(2H) -yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2- yl ) methoxy )-2- oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxophosphoruscyclohexane- 6- yl )-5 -methylpyrimidin -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 standard GP10 procedures. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, MeOH:DCM gradient (0% to 5%), separation of the desired white solid product. Yield = 500 mg, 91%, dr = 5:1.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

^31P NMR (162 MHz, CDCl3) δ -3.64, -6.68.

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated values: Chemical formula: C ₄₂ H ₅₅ N ₄ O ₁₄ PSi, [M+Na ⁺ ]: 921.3114, Experimental value: 921.3110. i. AA-Bn2- dimer --(2R,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- purine -9- yl )-7- methoxytetrahydrofuran - 4H -furano [3,2-d][1,3,2] dioxophosphoruscyclohexane 2- oxide

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 standard GP10 procedure. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, MeOH:DCM gradient (0% to 5%), separation of the desired white solid product. Yield = 650 mg, 86%, dr = 14:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -4.02, -6.62.

¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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 O ₉ PSi, [M+Na ⁺ ]: 1103.4335, experimental value: 1103.433. j. 3-(( benzyloxy ) methyl )-1-((2R,4aR,6R,7R,7aR)-2-(((2R,3R,3aS,9aR)-3-(( tert-butyldimethylsilyl ) oxy )-6- sideoxy- 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] dioxophosphoric cyclohexane -6 -yl ) pyrimidin -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 standard GP10 procedure. 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%), separation of the desired white solid product. Yield = 230 mg, 51%, dr > 99:1.

³¹ P NMR (203 MHz, CD ₃ CN) δ -5.19.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -4.64.

¹ H NMR (500 MHz, CD ₃ CN) 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).

¹³ C NMR (126 MHz, CD ₃ CN) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₃₃ H ₄₃ N ₄ O ₁₃ PSi, [M+Na ⁺ ]: 785.2226, experimental value: 785.2221. k. 1-((2R,4S,5S)-4- azono- 5-((((2R,4aR,6R,7R,7aR)-6-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidine -1(2H) -yl )-7- methoxy -2- oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxophosphorus-cyclohexane -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 standard GP10 procedure. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimal amount of DCM, hexane:ethyl acetate gradient (0% to 100%), eluted with 100% ethyl acetate, and the desired white solid product was separated. Yield = 520 mg, 83%, dr = 30:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -3.76, -6.49.

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

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₃₆ H ₄₀ N ₇ O ₁₃ P, [M+H ⁺ ]: 810.2494, experimental value: 810.2495. l. (E)-N'-(9-((2R,3R,4R,5R)-5-((((2R,4aR,6R,7R,7aR)-6-(3-(( benzyloxy ) methyl )-2,4- dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-7- methoxy -2- oxotetrahydro -4H- furano [3,2-d][1,3,2] dioxophosphorus-cyclohexane -2- yl ) oxy ) methyl )-4-(( tert-butyldimethylsilyl ) oxy )-3- methoxytetrahydrofuran -2- yl )-9H- purine -6 -yl )-N,N- dimethylformamide

The compound was synthesized 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) in toluene:ACN (1:1) according to the standard procedure GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM-MeOH gradient (0% to 5%), separation of the desired white solid product. Yield = 460 mg, 86%, dr = 15:1. Quality was confirmed by LC-MS.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.87, -6.91.

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

¹³ C NMR (101 MHz, CDCl ₃ ) δ 162.32, 160.03, 158.62, 153.08, 151.27, 150.33, 141.00, 140.56, 137.90, 128.49, 128.44, 127.93, 127.74, 126.62, 102.54, 95.87, 87.59, 82.39, 81.83, 81.75, 79.90, 79.83, 78.29, 78.25, 77.36, 72.61, 70.52, 70.35, 70.32, 70.27, 69.06, 68.98, 66.41, 66.35, 59.39, 58.87, 41.46, 35.23, 25.88, 18.25, -4.57, -4.81. m. (E)-N'-(9-((3aR,4R,6R,6aR)-6-((((2R,4aR,6R,7R,7aR)-6-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-7- methoxy -2- oxotetrahydro -4H- furano [3,2-d][1,3,2] dioxophosphorus-cyclohexane -2- yl ) oxy ) methyl )-2,2 -dimethyltetrahydrofurano [3,4-d][1,3] dioxocyclopenten -4- yl )-9H- purine -6 -yl )-N,N- dimethylformamidinium

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 standard procedure GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM:MeOH gradient (0% to 10%), separating the desired product as a white solid. Yield = 82%, dr = 23:1. Quality was confirmed by LC-MS.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -4.24, -6.93.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.02 - 8.99 (m, 1H), 8.60 (s, 1H), 8.04 (s, 1H), 7.39 - 7.29 (m, 5H), 7.19 (d, J = 8.2 Hz, 1H), 6.18 (d, J = 2.3 Hz, 1H), 5.77 (d, J = 8.1 Hz, 1H), 5.55 - 5.48 (m, 1H), 5.46 (d, J = 1.3 Hz, 2H), 5.38 (d, J = 1.0 Hz, 1H), 5.14 (dd, J = 6.2, 3.0 Hz, 1H), 4.71 (s, 2H), 4.59 - 4.27 (m, 5H), 4.04 (d, J = 5.1 Hz, 1H), 3.94 (td, J = 10.3, 5.6 Hz, 1H), 3.51 (s, 3H), 3.27 (d, J = 0.6 Hz, 3H), 3.22 (s, 3H), 1.65 (s, 3H), 1.43 (s, 3H). n. (E)-N'-(1-((3aR,4R,6R,6aR)-6-((((2R,4aR,6R,7R,7aR)-6-(3-(( benzyloxy ) methyl )-2,4- di-side-oxy -3,4 -dihydropyrimidin -1(2H) -yl )-7- methoxy -2- oxotetrahydro -4H -furano [3,2d][1,3,2] dioxophosphorus-cyclohexane -2 -yl ) oxy ) methyl )-2,2 -dimethyltetrahydrofurano [3,4-d][1,3] dioxocyclopenten -4- yl )-2- side-oxy -1,2 -dihydropyrimidin -4- yl )-N,N- dimethylformamidinium

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]dioxaphosphanecyclohexane-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 a modified general procedure GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:MeOH gradient (0% to 10%), separating the desired white solid product. Yield = 280 mg, 50%, dr = 14:1. Quality confirmed by LC-MS.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.94, -6.65.

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

¹³ C NMR (101 MHz, CDCl ₃ ) δ 172.40, 162.37, 159.02, 157.08, 156.12, 150.55, 144.57, 139.32, 137.96, 128.49, 127.91, 127.82, 127.77, 114.15, 103.19, 102.88, 98.00, 94.07, 87.01, 86.95, 85.05, 81.72, 80.48, 80.40, 77.98, 77.94, 77.36, 72.56, 70.73, 70.67, 70.59, 69.48, 69.40, 69.31, 69.25, 59.35, 46.60, 41.84, 39.27, 35.70, 35.56, 27.16, 25.33. o. 3-(( benzyloxy ) methyl )-1-((2R,4aR,6R,7R,7aR)-2-(((3aR,4R,6R,6aR)-6-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidine -1(2H) -yl )-2,2 -dimethyltetrahydrofurano [3,4-d][1,3] dioxocyclopenten -4- yl ) methoxy )-7- methoxy -2- oxotetrahydro -4H -furano [3,2-d][1,3,2] dioxophosphacyclohexane -6 -yl ) pyrimidine -2,4(1H,3H) -dione

The compound was synthesized using ketone-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 standard procedure of GP10. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM:MeOH gradient (0% to 5%), separation of the desired white solid product. Yield = 550 mg, 90%, dr = 15:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -3.92, -6.77.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₃₈ H ₄₃ N ₄ O ₁₅ P, [M+Na ⁺ ]: 849.2355, experimental value: 849.236. 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, 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 added to an oven-dried reaction tube containing a Teflon-coated magnetic stir bar, and then removed from the glove box. The mixture was stirred at RT for 20 minutes and cooled to -20°C over a 10-minute interval. Then, cyclic dinucleotide (1) (0.025 mmol, 1 equivalent) was added in a single step to the solution in the solvent (0.25 mL) at 20°C, and the mixture was stirred at that temperature for 3–4 hours. The reaction was terminated at -20°C with a solution of acetic acid (0.05 mmol, 2.0 equivalent) in MeCN (0.5 mL). After stirring for 5 minutes, an internal standard PO(OPh) ₃ solution (0.025 mmol, 1.0 equivalent) in MeCN (0.5 mL) was added, and NMR yields and regioselectivity were recorded. Results for various BnOH and solvents are shown in Table 3: Table 3 - Ring-opening of cyclic phosphates of dinucleotides (* - BOM is the protecting group on N -uridine; S represents the ratio of [3'-5']-linkage to [5'-5']-linkage; yield and selectivity were determined by ³¹ p-NMR) b. General procedure for regioselective ring-opening reactions of cyclic dinucleotides (GP13)

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, 0.25 mL in anhydrous toluene (0.2 M) stock solution) were added to an oven-dried reaction tube containing a Teflon-coated magnetic stir bar. The mixture was then removed from the glove box. The mixture was stirred at room temperature for 20 minutes and cooled to -20 to -25°C over a 10-minute interval. Then, a solution of protected cyclic dinucleotide (0.025 mmol, 1 equivalent) in toluene (0.35 mL) was added in a single addition 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 acetic acid (0.05 mmol, 2.0 equivalence) in 0.5 mL of MeCN. After stirring for 5 minutes, internal standard PO(OPh) ₃ (0.025 mmol, 1.0 equivalence) was added to 0.5 mL of MeCN and the NMR yield was recorded. The resulting 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 were determined by ³¹ p-NMR using PO(OPh) ₃ as an internal standard). c. General procedure for regioselective ring-opening reaction of cyclic dinucleotides followed by capture with TBS-Cl (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 stock solution in anhydrous toluene (0.24 M)) to an oven-dried reaction tube containing a Teflon-coated magnetic stir bar, and then remove it from the glove box. Stir the mixture at room temperature for 20 minutes and then cool it to -20 to -25°C over a 10-minute interval. Then, add a single solution of protected cyclic dinucleotide (0.12 mmol, 1 equivalent) in toluene (2 mL) at -20°C and stir at that temperature for 4 hours.

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 inject this solution into 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 heat the reaction solution to room temperature and monitor by LC-MS to ensure a conversion rate >90%. After completion, add a few drops of methanol to quench any excess TBS-imidazolium salt. Dilute the reaction solution with dichloromethane and transfer to a round-bottom flask. The crude mixture was evaporated to dryness using a rotary evaporator and purified by a Combi-Flash NextGen 300 automated chromatography system using a silicone column with a DCM-EtOAc (1% MeOH) gradient. The resulting cyclic dinucleotides and their yields and diastereomeric ratios (dr) are as follows: d. General procedure for the regioselective ring-opening reaction of cyclic dinucleotides catalyzed by TBD

In 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, 1 mL stock solution in anhydrous toluene (0.24 M)) were added to an oven-dried reaction tube containing 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 a 10-minute interval. Then, a solution of protected cyclic dinucleotide (0.12 mmol, 1 equivalent) in toluene (2 mL) was added in a single batch at -20°C and stirred at that temperature for 4 hours. e. General procedure for the regioselective ring-opening reaction of cyclic dinucleotides f. General procedure for the regioselective ring-opening reaction of cyclic dinucleotides g. General procedure for the regioselective ring-opening reaction of cyclic dinucleotides h. Ring-opening of guanosine dimer Example 8. Synthesis of Cyclotrimers a. General procedure for one-pot synthesis of cyclotrimers (GP15)

Step I : In a nitrogen-filled glove box, add 0.18 mmol (1.5 equivalence) of 2- _CF3BnOH (as a substituted benzyl alcohol nucleophile BnOH) and 0.18 mmol (1.5 equivalence) of LiOtBu dissolved in anhydrous toluene (1 mL) to reaction tube A containing a Teflon-coated magnetic stir bar, and then remove it from the glove box. Stir the mixture at room temperature for 30 minutes and then cool to -25°C. Then, at -25°C, add a single solution of protected cyclic dinucleotide (0.12 mmol (1 equivalence) in toluene (2 mL) and stir at that temperature for 4 hours.

Step II : Add Cl-CP (0.18 mmol, 1.5 equivalence) in 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, and use a syringe to transfer the pre-stirred alkoxide solution from reaction tube A dropwise to reaction tube B. Alternatively, use a syringe to transfer the pre-stirred Cl-CP solution from reaction tube B dropwise to reaction tube A. Stir the reaction solution at -25°C for 18 hours. Dilute the reaction solution with dichloromethane and transfer it to a round-bottom flask. The crude mixture was evaporated to dryness with the aid of a rotary evaporator and purified by column chromatography using a DCM-EtOAc (1% MeOH) gradient.

This study investigated several different reaction conditions and matrix concentrations used to synthesize cyclic trimers. The results are shown in Table 4: Table 4 – Reaction conditions and matrix concentrations for the synthesis of cyclic trimers. b. General procedure for one-pot synthesis of trinucleotide block copolymers (GP16)

Step I : In a nitrogen-filled glove box, add 2- _CF3BnOH (as the substituted benzyl alcohol nucleophile BnOH) (0.18 mmol, 1.5 equivalent) and LiOtBu (0.18 mmol, 1.5 equivalent) dissolved in anhydrous toluene (0.18 M, 1 mL) to an oven-dried reaction tube A containing a Teflon-coated magnetic stir bar, and then remove it from the glove box. Stir the mixture at room temperature for 30 minutes and then cool to -25°C. Then, at -25°C, add a single solution of protected cyclic dinucleotide (0.12 mmol, 1 equivalent) in toluene (2 mL) and stir at that temperature for 4 hours.

Step II : Add 0.6 mL of MeCN (Cl-CP, 0.18 mmol, 1.5 equivalence) to another reaction tube B and remove the reaction tube from the glove box. Incubate the reaction tube at -25°C for 10 minutes, and 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.

Step III : Add 0.36 mmol (3 equivalents) of 2- _CF3 BnOH (as a substituted benzyl alcohol nucleophile BnOH) and 0.30 mmol (2.5 equivalents) of LiOtBu in 1 mL of toluene to reaction tube C and stir for 30 minutes. Then, add the mixture dropwise to reaction tube B at -30°C. Stir the reaction mixture at -30°C for 5 hours.

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 in one step. Stir the resulting mixture at -30°C for 1 hour and slowly raise it to 0°C, stirring overnight (16-18 hours). Further heat the reaction solution to room temperature and monitor by LC-MS to ensure a conversion rate >90%. After completion, add a few drops of methanol to quench excess TBS-imidazolium salt. Dilute the reaction solution with dichloromethane and transfer it 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. The crude compound was loaded onto a 10g silicone column by dissolving it in a minimum amount of DCM. c. General procedure for the four-step one-pot synthesis of trinucleotide block copolymers.

Reported separation yields of a one-pot, four-step sequence. NMR yields were reported based on ^31P NMR using (PhO) _₃PO as an internal standard. Method A: Compounds were separated by silicone column chromatography using a DCM:EtOAc (2% MeOH) gradient, followed by separation using a C-18 reversed-phase column. d. Separation of trimer block copolymers .

The crude trinucleotide block copolymer product was purified by reversed-phase column chromatography on an automated system. The obtained trinucleotide block copolymers and their yields are as follows:

For reversed column chromatography, the compound does not decompose, but the separation is not baseline to baseline. Adding a 0.1% buffer solution of _NH₄OC (O)H or _NH₄OAc in water can help with separation. Example 9. Synthesis of a ring-opening TBS -protected dimer a. Phosphate (2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 3,4 -dihydropyrimidin -1(2H) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl )-4- methoxytetrahydrofuran -3- yl ester (((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 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 GP14 (2-step, one-pot sequence) using UU-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, 1 mL stock solution (0.24 M) in anhydrous toluene] (for step 1) in 2 mL of toluene:ACN (5:1) and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (49 mg, 0.72 mmol, 6 equivalents) in 0.5 mL of MeCN. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate (0% to 100%), separation of the desired white solid product. Yield = 84 mg, 61%, dr = 29:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -1.91 (major), -2.49 (minor).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.77 (d, J = 8.2 Hz, 1H), 7.56 (dd, J = 7.8, 4.7 Hz, 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₅₆ H ₇₆ F ₃ N ₄ O ₁₆ Psi ₂ , [M+Na ⁺ ]: 1227.4377, experimental value: 1227.4368. b . Phosphate ((2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4 -di-oxy- 3,4 -dihydropyrimidin -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 -di-oxypyrimidin -1( 2H ) -yl )-4- methoxytetrahydrofuran -3- yl ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP14 (2-step, one-pot sequence) using 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, 1 mL stock solution (0.24 M) in anhydrous toluene] (for step 1) in 2 mL of toluene:ACN (5:1) and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (49 mg, 0.72 mmol, 6 equivalents) in 0.5 mL of MeCN. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:ethyl acetate (2% MeOH) gradient (0% to 100%), separating the desired white solid product. Yield = 84 mg, 61%, dr = 20:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.91.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.76.

¹ H NMR (500 MHz, CDCl ₃ ) 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 = 6.4 Hz, 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, 2.1 Hz, 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₆₂ H ₈₁ F ₃ N ₅ O ₁₄ PSi ₂ , [M+Na ⁺ ]: 1286.4900, experimental value: 1286.4894. c. Phosphate ((2R,3R,4R,5R)-3-(( tert-butyldimethylsilyl ) oxy )-5-(6-( dibenzylamino )-9H- purine -9- yl )-4- methoxytetrahydrofuran -2 -yl ) methyl ester ((2R,3R,4R,5R)-2-((( tert-butyldimethylsilyl ) oxy ) methyl )-5-(6-( dibenzylamino )-9H- purine -9- yl )-4- methoxytetrahydrofuran -3- yl ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP14 (2-step, one-pot sequence) using UU-Cy-Nu (130 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equivalents) in 2 mL toluene and 2- _CF3BnOH [43 mg, 0.24 mmol, 2.0 equivalents, 1 mL stock solution (0.24 M) in anhydrous toluene] (for step 1) and TBSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (49 mg, 0.72 mmol, 6 equivalents) in 0.5 mL MeCN. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:ethyl acetate (1% MeOH) gradient (0% to 100%), separating the desired white solid product. Yield = 120 mg, 72%, dr = 50:1.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.86.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.77.

¹ H NMR (500 MHz, CDCl ₃ ) 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 = 7.8 Hz, 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, 3.8 Hz, 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₇₀ H ₈₆ F ₃ N ₁₀ O ₁₀ PSi ₂ [M+Na ⁺ ]: 1393.5649, experimental value: 1393.5642. d. Phosphate ((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4- dioxy -3,4- dihydropyrimidin -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 ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP14 (2-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, 1 mL of a stock solution (0.24 M) in 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. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:ethyl acetate (1% MeOH) gradient (0% to 100%), separating the desired product as a white solid. Yield = 80 mg, 59%, dr = 17:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.71, -2.12.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.70.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₄₉ H ₆₇ ClF ₃ N ₆ O ₁₃ PSi ₂ , [M+Na ⁺ ]: 1149.3575, experimental value: 1149.3568. e. Phosphate ((3aR,4R,6R,6aR)-6-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-2,2 -dimethyltetrahydrofurano [3,4-d][1,3] dioxanecyclopenten- 4- yl ) methyl ester ((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl )-4- methoxytetrahydrofuran -3- yl ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP14 (2-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 of a stock solution (0.24 M) in 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: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate gradient (0% to 100%), separation of the desired white solid product. Yield = 107 mg, 79%, dr = >99:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -2.26, -2.31.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.73, -59.85.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₅₂ H ₆₄ F ₃ N ₄ O ₁₆ PSi, [M+Na ⁺ ]: 1139.3668, experimental value: 1139.3661. f. Phosphate (2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl ) -4- methoxytetrahydrofuran -3- yl ester (((2R,3S,5R)-5-(3-(( benzyloxy ) methyl )-5- methyl -2,4 -dioxy -3,4- dihydropyrimidin -1(2H) -yl )-3-(( tert-butyldimethylsilyl ) oxy ) tetrahydrofuran -2- yl ) methyl ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP14 (2-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 of a stock solution (0.24 M) in 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. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate gradient (0% to 100%), separation of the desired white solid product. Yield = 112 mg, 85%, dr = >99:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.97, -2.42.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.77.

¹ H NMR (500 MHz, CDCl ₃ ) δ 7.87 (d, J = 8.2 Hz, 1H), 7.65 (dd, J = 7.8, 3.7 Hz, 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).

HR-MS (Q-TOF, ESI) calculated value: C ₅₆ H ₇₆ F ₃ N ₄ O ₁₅ PSi ₂ , [M+Na ⁺ ]: 1211.4427, experimental value: 1211.4438. g. Phosphate (2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4- dihydropyrimidin -1(2H) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl )-4- fluorotetrahydrofuran -3- yl ester (((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -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 GP14 (2-step, _one -pot sequence) 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 stock solution (0.24 M) in 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: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate gradient (0% to 100%), separation of the desired white solid product. Yield = 97 mg, 75%, dr = >99:1.

^31P NMR (203 MHz, CDCl ₃ ) δ -2.03, -2.07.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.64, -202.82 (dt, J = 51.4, 15.3 Hz).

¹ H NMR (500 MHz, CDCl ₃ ) 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 = 1.3 Hz, 6H), 0.09 (s, 3H), 0.06 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₅₅ H ₇₃ F ₄ N ₄ O ₁₅ PSi ₂ , [M+Na ⁺ ]: 1215.4177, experimental value: 1215.4171. h. Phosphate ((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4- dioxy -3,4- dihydropyrimidin -1(2H) -yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2- yl ) methyl ester ((2R,3S,5R)-5-(3-(( benzyloxy ) methyl )-5- methyl -2,4 -dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl ) tetrahydrofuran -3- yl ) ester 2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP-14 (2-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 of a stock solution (0.24 M) in 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. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate gradient (0% to 100%), separation of the desired white solid product. Yield = 115 mg, 79%, dr = >99:1.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.88.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.55.

¹ H NMR (500 MHz, CDCl ₃ ) 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, 5.2 Hz, 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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, 128.41, 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.

HR-MS (Q-TOF, ESI) calculated value: C ₅₆ H ₇₆ F ₃ N ₄ O ₁₅ PSi ₂ , [M+Na ⁺ ]: 1211.4427, experimental value: 1211.4420. i. Phosphate (2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4- dihydropyrimidin -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- yl )-4- methoxytetrahydrofuran -2 -yl ) methyl ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized according to GP- ₁₄ (2-step, one-pot 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 stock solution (0.24 M) in 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. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, DCM:MeOH gradient (0% to 5%), separating the desired white solid product. Yield = 90 mg, 64%, dr = 10:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ -2.10, -2.33.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.82.

¹ H NMR (400 MHz, CDCl ₃ ) 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 = 1.0 Hz, 6H).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₆₃ H ₈₁ F ₃ N ₇ O ₁₃ PSi ₂ , [M+Na ⁺ ]: 1310.5013, experimental value: 1310.5008. j. Cyclo-UU-BOM-TBS-TRAP

The compound was synthesized according to GP-14 (2-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 of a stock solution (0.24 M) in anhydrous toluene] (for step 1) and BSCl (55 mg, 0.36 mmol, 3 equivalents) and imidazole (50 mg, 0.72 mmol, 6 equivalents) in 0.5 mL MeCN. Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolving in a minimum amount of DCM, hexane:ethyl acetate gradient (0% to 100%), separation of the desired white solid product. Yield = 70 mg, 56%, dr = >99:1.

^31P NMR (162 MHz, CDCl3) δ -2.50.

¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -59.62.

¹ H NMR (400 MHz, CDCl ₃ ) 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 = 9.7 Hz, 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).

¹³ C NMR (101 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₄₇ H ₆₄ F ₃ N ₄ O ₁₄ PSi ₂ , [M+Na ⁺ ]: 1075.3539, experimental value: 1075.3505. Example 10. Removal of protecting groups by hydrogenation a. General procedure for removing protecting groups by hydrogenation (GP17)

A reaction tube containing a Teflon-coated magnetic stir bar, after being dried in an oven, was loaded with BOM- and benzyl-protected [3'-5'] and [5'-5'] dinucleotide bonds (1 equivalent), methanol (10:1, 0.05 M), and _HCO₂H₂ (0.5%). 10% Pd-C (10%) was added under _N₂ . _H₂ gas was introduced into the reaction mixture using a double-folded balloon. The reaction was monitored by LC-MS. After completion, the reaction mixture was filtered through a diatomaceous earth mat and washed with MeOH. All volatiles were evaporated under vacuum. An internal standard PO₄(OPh) _₃ (0.025 mmol, 1.0 equivalent) was added to a solution in MeCN (0.5 mL), and the NMR yield was recorded.

The results of the study on the hydrogenation rate of the protecting group (PG) are shown in Table 5 (BOM as the nucleoside protecting group): Table 5 - Hydrogenation rate of the protecting group (PG) b. Deprotection of dinucleotide block copolymers AA Table 6 - Deprotection of Dinucleotide Block Molecules 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 :

Solid nucleosides are added to a dispenser head for dispensing in a glove box filled with _N2 . Nucleotides with syrup viscosity are dispensed as a reserve solution into acetonitrile, which is then evaporated under a stream _{of N2} .

Add the solid matrix to the dispensing head and dispense on a Quantos (5 mg base, adjusting the 3'-5' and 5'-5' dinucleotide mixture to dispense 5 mg of the desired isomer). Dispense Pd/C (Johnson Matthey JM 10R39) at a 10 mol% loading on the Quantos. Then, add the solvent cleanly to the reaction wells, or, in the case of mixtures, premix the solvent first and then dispense the solvent from the Falcon tube into the appropriate vials using a single-channel pipette. Assemble the tray with a cross-stitched gasket added using an Exacto flat-blade tool, remove the tray from the glove box, place it in the CAT96 equipment, and then seal it. The CAT96 program was programmed to run for 3 hours at RT and 30 psi _H2 atmosphere, followed by a constant stirring rate of 300 rpm. Sample preparation for analysis:

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

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

The screening results are shown in Tables 7A and 7B (X = mixture of partially deprotected species; 0 = mixture of fully and partially deprotected products; + = 95+% converted to fully deprotected products). Table 7A – Screening Results without Additives nucleosides EtOH IPA THF EtOAc 10:1 EtOH: _H₂O 10:1 IPA: _H2O 3' OTBS mU ^BOM + + + + + + 3' OTBS fG ^Bn3 X X X X X X 3' OTBS fU ^BOM + + 0 + + 0 3' OTBS mA ^Bn2 X X X X X X 3' OTBS mC ^Bn2 X X X X X X 3' OTBS mG ^Bn3 X X X X X X mU ^BOM mU ^BOM dimer-OH 0 0 0 0 0 0 mG ^Bn3 mG ^Bn3 dimer-OH 0 0 0 0 0 0 Table 7B - Screening Results with 0.5 mol% Formic Acid Additive nucleosides EtOH IPA THF EtOAc 10:1 EtOH: _H₂O 10:1 IPA: _H2O 3' OTBS mU ^BOM + + + + + + 3' OTBS fG ^Bn3 X X X X X X 3' OTBS fU ^BOM 0 0 0 + + + 3' OTBS mA ^Bn2 X X X X X X 3' OTBS mC ^Bn2 X X X X X X 3' OTBS mG ^Bn3 X X X X X X mU ^BOM mU ^BOM dimer-OH 0 0 0 0 0 0 mG ^Bn3 mG ^Bn3 dimer-OH 0 0 0 0 0 0 Example 11. Synthesis of Cyclic Trimers : Cyclic Trimers : UUU a. Phosphophosphate (2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4- di-epoxy -3,4 -dihydropyrimidin -1(2H) -yl )-2-((((4aR,6R,7R,7aR)-6-(3-(( benzyloxy ) methyl )-2,4- di-epoxy -3,4 -dihydropyrimidin -1(2H) -yl )-7- methoxy -2- oxotetrahydro -4H -furano [3,2-d [1,3,2] dioxophosphorus- 2- yl ) oxy ) methyl )-4- methoxytetrahydrofuran -3- yl ester (((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 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 (2-step, one-pot sequence) using UU-Cy-Nu (200 mg, 0.22 mmol), LiOtBu (26 mg, 0.33 mmol, 1.5 equivalent) in toluene (3 mL), and 2-CF3BnOH [58 mg, 0.33 mmol, 1.5 equivalent, 2 mL stock solution (0.33 M) in anhydrous toluene] (for step 1) and U-BOM-Cl-CP (160 mg, 0.35 mmol, 1.6 equivalent) in 1.2 mL MeCN (for step 2). Column conditions: SiO2-10g column, Next-Gen automated system, wet loading by dissolution in a minimum amount of DCM, DCM:MeCN (2% iPrOH) gradient (0% to 80%). Fractions were analyzed by LC-MS, and fractions containing the desired product were concentrated to provide a white solid title compound with 90% purity. Overall yield (2 steps) = 180 mg, 57%.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.87, -4.01.

¹⁹ F NMR (471 MHz, CDCl ₃ ) δ -59.70.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₆₈ H ₈₁ F ₃ N ₆ O ₂₄ P ₂ Si, [M+H ⁺ ]: 1513.4572, experimental value: 1513.4565. TBS - trap - UUU - BOM - [3'-5']- trimer - block polymer b . phosphate (2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4- dioxy -3,4 -dihydropyrimidine -1( 2H ) -yl )-2-(((((2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4 -dioxy - 3,4- dihydropyrimidine -1( 2H ) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl )-4- methoxytetrahydrofuran- 3- yl ) oxy )((2-( trifluoromethyl ) benzyl ) oxy ) phosphoyl ) oxy ) methyl )-4- methoxytetrahydrofuran -3- yl ester (((2R ) ,3 R ,4 R ,5 R )-5-(3-(( benzyloxy ) methyl )-2,4 - dioxy- 3,4- dihydropyrimidin -1(2 H ) -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 steps, one-pot sequence): UU-Cy-Nu (110 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 stock solution (0.18 M) in anhydrous toluene] in toluene (2 mL) (for step 1); and U-BOM-Cl-CP (88 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 stock solution (0.18 M) in anhydrous toluene] in 2 mL of toluene (for step 1); and U-BOM-Cl-CP (88 mg, 0.19 mmol, 1.6 equivalence) in _0.6 mL MeCN (for step 2); and LiOtBu (24 mg, 0.30 mmol, 2.5 equivalence) and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equivalence, 1 mL stock solution (0.18 M) in 2 mL of anhydrous toluene (2 mL) (for step 2). [0.36 M stock solution in anhydrous toluene] (for step 3) and TBSCl (109 mg, 0.72 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) in 1 mL MeCN (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution 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 concentrated to provide a white solid title compound with 90% purity. Overall yield (4-step sequence) = 106 mg, 49%.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.66, -2.20.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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.

HR-MS (Q-TOF, ESI) calculated value: C ₈₂ H ₁₀₂ F ₆ N ₆ O ₂₅ P ₂ Si ₂ , [M+Na ⁺ ]: 1825.5705, experimental value: 1825.5695. c . Phosphate ((2R , 3R , 4R , 5R ) -3-(( tert-butyldimethylsilyl ) oxy )-5-(6-( dibenzylamino )-9H - purine - 9- yl )-4- methoxytetrahydrofuran - 2-yl ) methyl ester (( 2R , 3R , 4R ,5R ) -2-(((((2R , 3R , 4R , 5R ) -2-((( tert-butyldimethylsilyl ) oxy ) methyl )-5-(6-( dibenzylamino )-9H - purine - 9 -yl )-4- methoxytetrahydrofuran- 3 -yl ) oxy )((2-( trifluoromethyl ) benzyl ) oxy ) phosphatyl ) oxy ) methyl )-5-(6-( dibenzylamino ) -9H -purine - 9- yl )-4- methoxytetrahydrofuran -3- yl ) ester (2-( trifluoromethyl ) benzyl ) ester

The compound was synthesized using the following ingredients 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 equiv.), and 2-CF3BnOH [32 mg, 0.18 mmol, 1.5 equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in toluene (2 mL) (for step 1); and A- _Bn2 -Cl-CP (104 mg, 0.19 mmol, 1.6 equiv.) in 0.6 mL MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equiv.), and ₂ -CF3BnOH [64 mg, 0.36 mmol, 3 equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in 2 mL of toluene (for step 1); and A-Bn2-Cl-CP (104 mg, 0.19 mmol, 1.6 equiv.) in 0.6 mL of MeCN (for step 2); and LiOtBu (24 mg, 0.30 mmol, 2.5 equiv.), and 2- _CF3BnOH [64 mg, 0.36 mmol, 3 equiv., 1 mL stock solution (0.18 M) in 2 mL of anhydrous toluene (2 mL) (for step 2). [0.36 M stock solution in anhydrous toluene] (for step 3) and TBSCl (109 mg, 0.72 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) in 1 mL MeCN (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution 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 concentrated to provide a white solid title compound with 90% purity. Overall yield (4-step sequence) = 100 mg, 40%. Quality confirmed by LC-MS.

³¹ P NMR (203 MHz, CD ₃ CN) δ -1.90, -2.28.

¹⁹ F NMR (471 MHz, CD ₃ CN) δ -59.96, -60.01.

¹ H NMR (500 MHz, CD ₃ CN) 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, 2.3 Hz, 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).

¹³ C NMR (126 MHz, CD ₃ CN) 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. d . Phosphophosphate (2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4 - dioxy -3,4 -dihydropyrimidine -1( 2H )-yl )-2-((((( 2R , 3R ,4R ,5R) -5-(3-(( benzyloxy ) methyl )-2,4-dioxy-3,4- dihydropyrimidine -1( 2H ) -yl)-2-((( tert-butyldimethylsilyl)oxy ) methyl )-4- methoxytetrahydrofuran- 3-yl)oxy ) ((2-( trifluoromethyl)benzyl)oxy)phosphatyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidine-1(2H ) -yl ) -2 - ( ( ( tert - butyldimethylsil ... hydropyrimidine - 1 ( 2H ) -yl )-2-(((tert-butyldimethylsilyl ) oxy ) methyl ) -4 - methoxytetrahydrofuran - 3 -yl ) -4- methoxytetrahydrofuran -3-yl) -5 - ((( 2R , 3R ,4R , 5R ) -5-(3-((ben 3-(( tert-butyldimethylsilyl ) oxy )-5-(6-( dibenzylamino )-9H - purine - 9 -yl )-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): Bn2-AU-Cy-Nu (120 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equiv.), and 2- _CF3BnOH [32 mg, 0.18 mmol, 1.5 equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in toluene (2 mL) (for step 1); and U-BOM-Cl-CP (88 mg, 0.19 mmol, 1.6 equiv.) in 0.6 mL MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equiv.), and 2-CF3BnOH [64 mg, 0.36 mmol, 3 equiv., _{1 mL} stock solution (0.18 M) in anhydrous toluene] in 2 mL of toluene (for step 1). [0.36 M stock solution in anhydrous toluene] (for step 3) and TBSCl (109 mg, 0.72 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) in 1 mL MeCN (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution 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 concentrated to provide a white solid title compound with 90% purity. Overall yield (4-step sequence) = 60 mg, 26%.

Alternatively, the crude compound was purified by reverse-phase C-18 column chromatography with the assistance of the Next-Gen automated system. The crude compound was loaded into a pre-packed 25 g C-18 column using a water:ACN gradient (0% to 100%) by dissolving it in a minimal amount of DMSO. Fractions were analyzed by LC-MS, and fractions containing the desired product were concentrated to provide a white solid title compound with 90% purity. Overall yield (4-step sequence) = 105 mg, 46%. Quality was confirmed by LC-MS.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.85, -2.30.

¹ H NMR (500 MHz, CDCl ₃ ) 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).

¹³ C NMR (126 MHz, CDCl ₃ ) 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. e . Phosphate (2R , 3R , 4R , 5R ) -2-(((((2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy )methyl) -2,4-dioxy-3,4-dihydropyrimidine-1(2H)-yl) -2-(((tert-butyldimethylsilyl )oxy) methyl )-4- methoxytetrahydrofuran - 3-yl)oxy)((2-(trifluoromethyl)benzyl)oxy)phosphatyl)oxy)methyl)-5-(6 - chloro - 9H -purine-9-yl)-4-methoxytetrahydrofuran-3-yl ester((( 2R,3R,4R,5R)-5-(3-((benzyloxy)methyl) -2,4 - dioxy - 3,4 - dihydropyrimidine - 1 ( 2H) -yl ) -yl ) -2 - ((( tert- butyldimethylsilyl ) oxy ) methyl ... ) - yl )-2-(((2R, 3R , 4R,5R )-5-(3- ( ( benzyloxy ) methyl )-2,4- dioxy -3,4-dihydropyrimidine -1(2H) -yl) -2 - ((( 2R , 3R , 4R ,5R ) -5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 - dihydropyrimidine -1( 2H )-yl)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy ) -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(6Cl)-Cy-Nu (101 mg, 0.12 mmol), LiOtBu (15 mg, 0.18 mmol, 1.5 equiv.), and 2- _CF3BnOH [32 mg, 0.18 mmol, 1.5 equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in toluene (2 mL) (for step 1); and U-BOM-Cl-CP (89 mg, 0.19 mmol, 1.6 equiv.) in 0.6 mL MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equiv.), and 2- _CF3BnOH [64 mg, 0.36 mmol, 3 equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in 0.6 mL MeCN (for step 2); [0.36 M stock solution in anhydrous toluene] (for step 3) and TBSCl (109 mg, 0.72 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) in 1 mL MeCN (for step 4). Column conditions: SiO2-10 g column, Next-Gen automated system, wet loading by dissolution 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 concentrated to provide a white solid title compound with 90% purity. Overall yield (4-step sequence) = 100 mg, 40%, dr 4:1. Quality was confirmed by LC-MS.

³¹ P NMR (203 MHz, CDCl ₃ ) δ -1.16 (P1, minor diastereomer), -1.51 (P1, major diastereomer), -2.36 (P2, major diastereomer), -2.67 (P2, minor diastereomer).

^¹H NMR (500 MHz, _CDCl₃ ) of diastereomer mixtures: 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).

^13C NMR (126 MHz, _CDCl₃ ) values of diastereomer mixtures: 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. f . Phosphate (2R , 3R , 4R , 5R ) -2-(((((2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4 - dioxy -3,4 - dihydropyrimidin -1( 2H ) -yl )-2-((( tert-butyldimethylsilyl ) oxy ) methyl ) -4- methoxytetrahydrofuran -3- yl ) oxy )((2-( trifluoromethyl ) benzyl ) oxy ) phosphatyl ) oxy ) methyl )-5-(6-( dibenzylamino )-9H - purine- 9 - yl )-4- methoxytetrahydrofuran -3- yl ester (((2R , 3R , 4R , 5R ) -2- ... 5-(3-(( benzyloxy ) methyl )-2,4 -dioxy - 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 equiv.), and 2- _CF3BnOH [32 mg, 0.18 mmol, 1.5 equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in toluene (2 mL) (for step 1); and U-BOM-Cl-CP (88 mg, 0.19 mmol, 1.6 equiv.) in 0.6 mL MeCN (for step 2); LiOtBu (24 mg, 0.30 mmol, 2.5 equiv.), and 2-CF3BnOH [64 mg, 0.36 mmol, ₃ equiv., 1 mL stock solution (0.18 M) in anhydrous toluene] in 0.6 mL MeCN (for step 2). [0.36 M stock solution in anhydrous toluene] (for step 3) and TBSCl (109 mg, 0.72 mmol, 6 equivalents) and imidazole (98 mg, 1.44 mmol, 12 equivalents) in 1 mL MeCN (for step 4). The crude extract was purified by reverse-phase C-18 column chromatography with the aid of the Next-Gen automated system. The crude extract was loaded into a pre-packed 25 g C-18 column using a water:ACN gradient (0% to 100%) by dissolving in a minimal amount of DMSO. Fractions were analyzed by LC-MS, and fractions containing the desired product were concentrated to provide a white solid title compound with 90% purity. Overall yield (4-step sequence) = 100 mg, 44%. Quality is verified by LC-MS.

³¹ P NMR (203 MHz, CD ₃ CN) δ -1.95, -2.21.

¹⁹ F NMR (471 MHz, CD ₃ CN) δ -59.86, -59.99.

¹ H NMR (500 MHz, CD ₃ CN) 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). Example 12. Synthesis of Cyclic Nucleotide Acidylamines Program A

In a glove box filled with _N2 , 0.200 g, 250 μmol, 1.0 μL of 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidine-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (0.00 g, 250 μmol, 1.0 μL) was added to a vial (container A). eq.), 3-((bis(diisopropylamino)phosphono)oxy)propionitrile (103 uL, 325 umol, 1.3 eq.) and a stirring rod. Seal the reaction solution in container A, then remove it from the glove box and add DCM (4 mL) to the container, then cool to 0°C over a 10-minute interval.

In a separate vial (container B) outside the glove box, 29.3 mg (225 μmol, 0.9 eq.) of solid 5-(ethylthio)-1H-tetrazole was added, and container B was then purged with _N2 and sprayed 5 times. DCM (total 1.2 mL, 47 mM) was then added to container B, and the solution in container B was transferred dropwise to container A at 0°C. The reaction solution 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 exposed 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 separated by a pale yellow oily N,N-diisopropylphosphonamide 2-cyanoethyl ester. The actual amount of the product in the mixture was 60 mg, 60 μmol, and 24% yield. It was observed that N,N-diisopropylphosphonamide 2-cyanoethyl ester (hydrolyzed N,N,N′,N′-tetraisopropylphosphonamide 2-cyanoethyl ester) was difficult to separate from the product at 14 ppm.

³¹ p NMR (203 MHz, CD ₃ CN) δ -1.11. Program B

Add 0.200 g, 250 μmol, 1.0 μL of 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-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]dioxaphosphacyclohexane-6-yl)pyrimidin-2,4(1H,3H)-dione (0.00 g, 250 μmol, 1.0 μL) to a heat-dried round-bottom flask (container A). Add 103 μL (325 μmol, 1.3 eq.) of 3-((bis(diisopropylamino)phosphono)oxy)propionitrile (1.3 eq.) to the reaction vessel along with a dry sieve and a stirring rod. Then, seal container A, purge with _N₂ and spray 5 times. Next, add anhydrous DCM (4 mL) to container A and cool to 0°C over a 10-minute interval.

In a separate vial (container b), solid 5-(ethylthio)-1H-tetrazole (29.3 mg, 225 μmol, 0.9 eq.) was added, followed by purging with _N2 and spraying five times. Then, DCM (total 1.2 mL, 47 mM) was added to container B, and the solution in container B was then 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 was monitored by LCMS. Container A was then exposed to air to evaporate the solvent. The compound was purified by column chromatography using 100% ethyl acetate. Example 13. Synthesis of Cyclotetramers a. Linear Synthesis of Fragments in Solution Phase b. Synthesis via solution-phase amide chemistry using polymeric dimer coupling

Add 22 mg, 20 μmol, 1.0 μL of (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl ester (((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) ester (2-(trifluoromethyl)benzyl) ester (2-(trifluoromethyl)benzyl) ester (2.0 μL) to vial (container A). eq.), stirring rod, sieve and (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-di-oxy-3,4-dihydropyrimidine-1(2H)-yl)-2-((((4aR,6R,7R,7aR)-6-(3-((benzyloxy)methyl)-2,4-di-oxy-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(2-cyanoethyl) (20 mg, 20 umol, 1.0 equivalent). Then seal reaction vessel A, and purge with _N2 and spray 5 times. Add anhydrous DCM (4 mL) to vessel A and cool the reaction solution to 0°C over a 10-minute period.

Add 5-(ethylthio)-1H-tetrazole (2.3 mg, 18 μmol, 0.9 equivalence) to a separate vial (container B) and seal the container. Then purge container B with _N2 and spray 5 times. Then 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 addition, heat container A to room temperature and stir for 3 hours or until the starting material is exhausted. Monitor the reaction progress by LCMS. c. Synthesis using cyclodinucleotide amides d. Longer segments in the solution phase will aggregate into

The process provides a tetramer product with a conversion rate of >95% and a crude yield of ~22% as confirmed by LC integration. e. Long-polymer synthesis

Add 150 mg, 1 Eq, 187 μmol of 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidine-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione to an 8 mL vial, stirring with a stirring rod, then purging with _N2 and spraying 3 times. Anhydrous pyridine (1.48 g, 1.52 mL, 100 Eq, 18.7 mmol) was then added to the container. Diphenyl phosphonate (65.8 mg, 53.8 μL, 1.5 Eq, 281 μmol) was added, and the reaction mixture was stirred at room temperature and monitored by LCMS. After the reaction was complete, a crude yield of 52% was calculated by LCMS. The reaction mixture was then used in subsequent reaction steps.

Earn 0.25 Aliquots of the reaction product of phosphonic acid (2R,3R,4R,5R)-5-(3-(((benzyloxy)methyl)-2,4-di-oxy-3,4-dihydropyrimidin-1(2H)-yl)-2-((((4aR,6R,7R,7aR)-6-(3-(((benzyloxy)methyl)-2,4-di-oxy-3,4-dihydropyrimidin-1(2H)-yl)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-2-yl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester phenyl ester were prepared and concentrated by rotary evaporation 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 to produce the product. A crude yield of 43% was observed by LCMS. Example 14. Synthesis of Cyclopentamers Using One-Pot Solution Phase Chemistry

Step I : Dry the 50 mL flask (container A) containing the stirring rod twice under vacuum using a hot air gun. Then purge the container with _Ne and spray it three times. After the flask has cooled, add (22 μL, 0.16 mmol, 1.5 equivalence) methanol under _N2 . Then, weigh LitBuO (13 mg, 0.16 mmol, 1.5 equivalence) into a separate vial, purge it with _N2 and spray it three times, and then add toluene (0.9 mL) to the vial. Add the solution from the vial to container A, and stir the reaction mixture at room temperature for 30 min, then cool to -25°C.

Add 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidine-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (99 mg, 0.11 mmol, 1.0 equivalent)) to a separate vial, then purge with _N2 and spray 3 times. Add 1.8 mL of toluene to the vial, and then add the resulting solution to container A in one go at -25°C. Stir container A at this temperature for 4 hours.

Step II : 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-oxotetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (74 mg, 0.16 mmol, 1.5 equivalents) was placed in a separate vial and sealed. The vial was purged with _N2 and sprayed three times, then anhydrous acetonitrile (0.53 mL) was added. 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 solution was analyzed to observe the completion of the reaction.

Step III : Lower the cooler temperature to -25°C. Dry the 50 mL RBF (container B) containing the stirring rod twice under vacuum using a hot air gun. Then purge the container with _N2 and spray three times. After cooling the flask, add (22 μL, 0.16 mmol, 1.5 equivalence) methanol under _N2 . Next, weigh LitBuO (13 mg, 0.16 mmol, 1.5 equivalence) into a vial, purge with _N2 and spray three times, and 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. Then, equal portions of the reaction sample were taken for analysis to observe the completion of the reaction.

Step IV : Dry the 100 mL flask (container C) containing the stirring rod twice under vacuum using a hot air gun. After container C 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]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione to the flask and seal the container. Purge and spray reaction vessel C three times with _N2 , and then add anhydrous acetonitrile (0.53 mL) to the container. Then, container C was cooled to -25°C for 10 minutes. The contents of reaction container A were then transferred dropwise to reaction container C, and stirred at -25°C for 18 hours. After 18 hours, equal portions of the reaction were taken to observe the reaction.

Step V : Dry the vial containing the stirring rod twice under vacuum using a hot air gun. After the vial has cooled, weigh LitBuO (22 mg, 0.27 mmol, 2.5 equivalences) into the vial, then purge with _N2 and spray three times. Add toluene (0.9 mL) to the vial, followed by (2-(trifluoromethyl)phenyl)methanol (43 μL, 0.32 mmol, 3.0 equivalences) and the solution, and stir for 30 minutes. Then, add the contents of the vial to container C at -25°C. Stir the reaction solution at -25°C for 4 hours. Take an aliquot from container C to monitor the reaction progress.

Step VI : Dry the 100 mL flask (container C) containing the stirring rod 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]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione to the flask and seal the container. Purge and spray reaction container D three times with _N2 , and add anhydrous acetonitrile (0.53 mL) to the container. Then cool container D to -25°C for 10 minutes. The contents of reaction vessel C were then transferred dropwise to reaction vessel D, and stirred at -25°C for 18 hours. After 18 hours, aliquots of the reaction mixture were taken to observe the reaction status. Example 15. Preparation of nonameliton templated enzyme ligation using trimer.

Figures 1A and 1B illustrate examples of enzymatic ligation reactions that generate nonamelisers from short polymers. Each reaction is controlled by reaction control to ensure enzyme activity. a. Reaction setup procedure :

Two LCMS vials were used as reaction vessels. Reaction vessel A was the enzyme-free control reaction, and reaction vessel B was the enzymatic reaction reaction. Each vessel was filled with HO-UUU-OH (32.51 mM), Phos-CCC-OH (25.97 mM), 9-mer inhibitor (25.02 mM), Tris-HCl pH 7.5 (1000 mM), MgCl2 solution (1000 mM), KCl solution (3000 mM), ATP (100 mM), DTT (100 mM), and a nuclease-free aqueous solution, respectively. Finally, the solution was added to vessel B.

The following substances were added to reaction vessel A: HO-UUU-OH (6.2 μL, 1.0 equivalent of 32.51 mM solution), Phos-CCC-OH (15.4 μL, 2.0 equivalent of 25.97 mM solution), 9-mercury conduction (8.0 μL, 1.0 equivalent of 25.02 mM solution), Tris-HCl pH 7.5 (25.0 μL, 125 equivalent of 1000 mM solution), _MgCl2 solution (5.0 μL, 25 equivalent of 1000 mM solution), KCl solution (16.7 μL, 250 equivalent of 3000 mM solution), ATP (10.0 μL, 5.0 equivalent of 100 mM solution), and DTT (5.0 μL, 2.4 equivalent of 100 mM solution). mM solution and nuclease-free water (408.7 uL).

The following substances were added to reaction vessel B: HO-UUU-OH (6.2 uL, 1.0 equivalent of 32.51 mM solution), Phos-CCC-OH (15.4 uL, 2.0 equivalent of 25.97 mM solution), 9-mercury conduction (8.0 uL, 1.0 equivalent of 25.02 mM solution), Tris-HCl pH 7.5 (25.0 uL, 125 equivalent of 1000 mM solution), _MgCl2 solution (5.0 uL, 25 equivalent of 1000 mM solution), KCl solution (16.7 uL, 250 equivalent of 3000 mM solution), ATP (10.0 uL, 5.0 equivalent of 100 mM solution), and DTT (5.0 uL, 2.4 equivalent of 100 mM solution). The solution contained mM solution and nuclease-free water (408.7 uL). Finally, Codexis 3.007 enzyme (2.1 uL, 0.0625 equivalent of 5.91 mM solution) was added.

Then add both containers to the hot mixer and heat at 37°C, stirring at 350 rpm for 18 hours. b. Reaction termination procedure :

After 18 hours, 25 μL aliquots from both reaction vessels A and B were added to separate LCMS vials. Then, 125 μL of 26.7 mM EDTA solution was added to each aliquot to terminate the reaction (aiming to use a 5:1 aliquot ratio). The solutions were then analyzed by LCMS, and the results demonstrated evidence of both the enzyme ligation reaction and the control reaction.

Crude yield was determined by integrating the 9-mer leader as an internal standard using a denaturing UPLC method and comparing the product-to-9-mer leader ratio.

[0399] Example 16. Synthesis of cyclic thiophosphate nucleosides a. Synthesis procedure of Cl- cyclic thiophosphate using _PSCl3 1

A stirred solution of thiophosphoric chloride (1.05 mmol, 1.05 eq) and N-protected nucleoside diol BOM-U (1 mmol, 1.0 eq) in DCM (5 mL) was prepared. After a 5-minute interval at 0°C under Schlenk conditions, 2 mL of lithium tributoxide in 1 M THF (2.05 mmol, 2.05 eq) was added to the solution. Following the addition, the mixture was heated to ambient temperature overnight. Aliquots of the crude mixture were concentrated for conversion monitoring by LCMS and NMR. Two diastereomers, P1 and P2, with dr ratios ranging from 1.7:1 to 1.3:1 were observed by ρ NMR.

NMR data for the two isomers: ³¹ P NMR (162 MHz, CDCl ₃ ) δ 61.23 (P1), 57.83 (P2). ¹ H NMR (400 MHz, CDCl ₃ ) 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). ¹ H NMR (400 MHz, CDCl ₃ ) P2, δ 7.42 - 7.30 (m, 5H), 7.18 - 7.09 (m, 1H), 5.82 (d, J = 8.1 Hz, 1H), 5.49 (d, J = 3.5 Hz, 2H), 5.40 - 5.35 (m, 1H), 4.86 (dt, J = 9.9, 5.0 Hz, 1H), 4.74 (s, 2H), 4.71 - 4.62 (m, 1H), 4.53 (ddd, J = 10.8, 9.6, 3.0 Hz, 1H), 4.27 (dd, J = 8.8, 4.9 Hz, 2H), 3.62 (s, 3H).

Purification method (a) : The crude product was precipitated with 0.1 mL MTBE at 0 °C while stirring. The filtrate was concentrated and subjected to NMR analysis under vacuum.

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

The reaction conditions and Cr dr results are shown in Table 8 [by P NMR, Cr dr is the crude reaction P(R):P(S). 4a and 4b are two time points from the same batch. Reaction 1 was purified using method (a). Reactions 2-4 were purified using method (b)]. Table 8 Synthesis of -Cl-cyclic thiophosphates reaction solvent Total concentration temperature scale Cr dr Conversion rate Separation yield 1 DCM 0.15 M 0℃ to RT 0.53 mmol 13:1 26% 20% 2 DCM/THF 1:1 0.25 M 0℃ to RT 0.5 mmol 1.2:1 NA NA 3 DCM/THF 2:1 0.17 M 0℃ to RT 0.5 mmol 0.9:1 NA NA 4 DCM/THF 5:2 0.15 M 0℃, 1.5 h 1.0 mmol 1.3:1 99% 41% 5 DCM/THF 5:2 0.15 M -20℃, 20 h 0.13 mmol NA twenty three% NA 6 DCM/THF 5:2 0.15 M 0℃, 20 h 0.5 mmol 1.8:1 97% 41% 7 DCM/THF 5:2 0.15 M 0℃, 1 h 2.64 mmol 1.7:1 97% 56% 8 DCM/THF 5:2 0.15 M 0℃ to RT 3.0 mmol 1:2:1 99% 52% Program 2

After a 5-minute interval under Schlenk conditions at 0 to -20°C in an ice bath or refrigerator, lithium tributoxide in 1 M THF or MeTHF solvent (2.05 eq) was added to a stirred solution of thiophosphoric chloride (1.05 eq) and N-protected nucleoside diol N-Ar-protected nucleobase (1.0 eq) in DCM (x mL). The conversion and dr of the crude reaction mixture were monitored by LCMS and NMR after 1–20 hours.

^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). fA LCMS nucleobase range b. Synthesis of Cl- cyclo -2'- methoxy -G- thiophosphate ( 1-benzyl-9-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-thioionic tetrahydro-4H-furano[3,2-d][1,3,2]dioxophosphazenecyclohexane-6-yl)-2-(dibenzylamino)-1,9-dihydro-6H-purine-6-one)

Under Schulenck line N2, 1-benzyl-2-(dibenzylamino)-9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (5.677 g, 1 Eq, 10.00 mmol) was dissolved in DCM (50 mL) at RT, and then slowly cooled to -20°C for 90 min. Freshly distilled thiophosphoric chloride (1.778 g, 1.06 mL, 1.05 Eq, 10.50 mmol) was then added dropwise using a syringe. After 30 min, it was added to THF (9.3 mg/mL) at a concentration of 1:50 pm over 10 min. 2-Methylprop-2-ol lithium salt (2.2 M in THF) (1.662 g, 2.05 Eq, 20.50 mmol) was collected in a syringe (~50 μL). The crude sample was analyzed, and the crude PNMR showed two peaks of the main product isomers with a dr ratio of ~2.4:1. The starting material was largely exhausted after 30 min (1% was still detectable on UV, and the integral was determined by uncorrected LCMS), and other smaller peaks of impurities eluted earlier than the products. The reaction was stopped after 1 h. The crude reaction solution was concentrated to remove most of the solvent, then dissolved in DCM, and then loaded onto a silicone column (40g) eluted with EA/Hex (0-80%) using a combi-flash system, thereby providing 3.56g of pale yellow to white solid product (separation yield 54%, dr 2.2:1).

¹³ C NMR (101 MHz, CDCl ₃ ) 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. c. Synthesis of alkoxy - cyclic thiophosphates from _PSORCl2

Under Schulenk line conditions, N-protected nucleoside diol BOM-U (75.7 mg, 1 eq, 0.2 mmol) and DMAP (63.5 mg, 2.6 eq, 520 μmol) in 1.8 mL DCM (2 mL) were slowly added to O-ethylthiophosphoric acid dichloro(37.6 mg, 1.05 eq, 210 μmol) in 0.2 mL DCM, and stirred for 20 hours at -20°C with the aid of a refrigeration unit. 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 DCM and loaded onto a 5 g Biotage silicone column. It was then purified with EA/Hex eluent from 0 to 100% and eluted with ~30% EA/Hex to obtain two diastereomers, P1 and P2, in 40% yield (dr = 2:1 for P1:P2).

NMR data for the two isomers: ³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.52 (P1), 61.80 (P2). ^¹H NMR (400 MHz, _CDCl₃ ) P₁, δ 7.42–7.27 (m, 6H), 7.18 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 8.2 Hz, 1H), 5.69 (t, J = 0.9 Hz, 1H), 5.51 (s, 2H), 4.74 (s, 2H), 4.69–4.47 (m, 3H), 4.44–4.24 (m, 3H), 4.01 (d, J = 4.9 Hz, 1H), 3.62 (s, 3H). Example 17. Synthesis of cyclothioester dinucleotide a from cyclochlorothiophosphate . Cyc U*U ( Program 1)

Solution A: Under a nitrogen atmosphere, LiOtBu (48.7 mg, 1.5 eq, 609 μmol) was added to a 20 mL oven-dried reaction tube A equipped with a magnetic stir bar using the Schulnk line technique. A stock solution of 3'-TBS-5'-OH nucleoside (200 mg, 1 eq, 406 μmol) in toluene (2 mL) was added to reaction tube A. The mixture was then placed in a cooler and maintained at -20°C for 30 minutes with stirring.

Solution B: Under Schulencke line conditions, the cyclochlorophosphate monomer PS-Cl UBOM (dr Rp:Sp = 1.2:1, 231 mg, 1.0 eq, 487 μmol) was dissolved in acetonitrile (2 mL) in an oven-dried flask at -20°C. 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 and concentrated for ³¹ p NMR and LCMS analysis to confirm the completion of the reaction and the crude reaction dr (Rp:Sp = 13:1). The crude reaction solution was concentrated to remove most of the solvent and then loaded onto a silicone column (5 g) eluted with EA/Hex (0-60%) using a combi-flash system to provide 150 mg of the white solid major isomer "Rp" (separation yield 40%, dr 16:1).

³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.20 (Rp, lower polarity), 62.00 (Sp, higher polarity).

¹ H 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).

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.

HRMS (ESI+): m / z calculated value: C42H65N4O14PSSi[M+H]+: 930.29; experimental values: 930.31 (Rp), 930.33 (Sp). b . Cyc U * U ( Program 2)

Solution A: Under a nitrogen atmosphere, LiOtBu (96.1 mg, 1.5 Eq, 1.20 mmol) was added to a 20 mL reaction flask A equipped with a magnetic stir bar using the Schulenk line technique, and cooled to -20°C with the aid of a refrigeration unit. A stock solution of 3'-TBS-5'-OH nucleoside (394 mg, 1 Eq, 800 μmol) in toluene (4 mL) was slowly added to reaction tube A using a syringe needle, and the mixture was stirred at -20°C for 30 minutes with the aid of a refrigeration unit.

Solution B: The cyclochlorophosphate monomer PS-Cl UBOM (dr Rp:Sp = 1:1, 500 mg, 70% Wt, 0.921 Eq, 737 μmol) was dissolved in acetonitrile (4 mL) at -20 °C using a chiller under Schulenck line conditions. Solution A was added dropwise to B over 5 minutes using a syringe needle, 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).

The cyclochloro-thiophosphate monomer PS-Cl UBOM used in Procedure 2 was not purified by silicone column chromatography. The synthesis and termination methods are as follows.

Under Schulencke _N2 conditions, diol-U-BOM (3.784 g, 1 Eq, 10.00 mmol) and thiophosphoric chloride (1.778 g, 1.06 mL, 1.05 Eq, 10.50 mmol) were dissolved in DCM (48 mL) at 0 °C and stirred for 20 minutes. Then, 20 mL of 2-methylprop-2-ol lithium (1 M THF solution) (1.601 g, 2.0 Eq, 20.00 mmol) was added using a syringe and needle. The mixture was heated from 0 °C to 23 °C over 4 hours. The reaction was completed and concentrated using a rotary evaporator. A mixture of heptane/MTBE/EA (50 mL/50 mL/30 mL) was slowly added to a concentrated crude product solution to partially 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 a theoretical yield of 4.75 g and an actual mass of 5.6 g, ~0.80 g of a mixture of LiOH, LiOtBu, and LiCl (10-15 mmol ~ 1-1.5 eq) may be present after precipitation. Based on a MW 80 of LiOtBu, 10 mmol is calculated. c. Two-step addition ( general )

Solution A: Under a nitrogen atmosphere, LiOtBu (1.5 eq) was added to a 20 mL reaction tube A equipped with a magnetic stir bar using the Schulenk line technique. A stock solution of 3'-TBS-5'-OH nucleoside (1 eq) in toluene (0.2 M) was added to reaction tube A, and the mixture was placed in a cooler and kept at -20°C for 30 minutes.

Solution B: Under Schulencke conditions, 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. 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 and concentrated for ³¹ p NMR and LCMS analysis to confirm the completion of the reaction and the preparation of the crude dr. The crude reaction solution is concentrated to remove most of the solvent and dissolved in a minimum amount of DCM. It is then loaded onto a silicone column eluted with EA/Hex (0-60%) using a combi-flash system to provide the white solid major isomer "Rp" (dr ~ 4:1 to 20:1). Table 9 - Two-step addition d. One-pot addition

In a vacuum-dried glass container, B1 5'-OH-3'TBS-monomer (1 eq) and B2 Cl-PS-cyclic monomer (1.2 Eq, 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 refrigeration unit. Lithium 2-methylprop-2-ol (2.2 M in THF) (1.5 Eq) 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 1–20 hours until the reaction was complete. The conversion rate was monitored using LCMS. One hour later, 50 μL aliquots were collected by syringe and concentrated for ³¹ p NMR and LCMS analysis to confirm the completion of the reaction with an 82% crude yield and the crude reaction Cr dr (Rp:Sp). The crude reaction solution was concentrated to remove most of the solvent and dissolved in a minimum amount of DCM, then loaded onto a silicone column eluted with EA/Hex (0-60%) using a combi-flash system, providing 90 mg of the white solid major isomer "Rp" (82% separation yield, dr 16:1). Table 10 - One-pot addition e. 簵 PS mAmU

In a glass container that has been vacuum-dried with 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 Eq, 100 μmol) and B2 Cl-PS-cyclomU monomer 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-thioionic tetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphanecyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (57.0 mg, 1.2 Eq, 120 μmol) was mixed at room temperature and then completely dissolved in anhydrous DCM (2 mL), and then cooled to -20 ^° C under N2 with the aid of a refrigeration unit. 2-Methylprop-2-lithium (2.2 M in THF) (12.0 mg, 68 μL, 1.5 Eq, 150 μmol) was added dropwise to the reaction vessel over 5 min using a syringe. The temperature was then maintained and the mixture was stirred under _N2 for 2 h until the reaction was complete. The conversion rate was monitored using LCMS. After 1 h, 50 μL aliquots were collected using a syringe and concentrated for ³¹ p NMR and LCMS analysis to confirm the completion of the reaction with an 82% crude yield 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 silicone column eluted with EA/Hex (0-60%) using a combi-flash system, yielding 90 mg of the white solid major isomer "Rp" (82% separation yield, dr 10:1). ^31P NMR (162 MHz, _CDCl₃ ) showed Rp at 66.40. The Sp isomer, previously unconfirmed due to trace detection, was confirmed upon scale-up. Example 18. Spectrum of a dinucleotide a. 3-(( benzyloxy ) methyl )-1-((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 )-7- methoxy -2- thiotetrahydro -4H - furano [3,2-d][1,3,2] dioxaphosphacyclohexane- 6 -yl ) pyrimidine -2,4(1H,3H) -dione

cr dr=17:1, purified dr > 30:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.27 (Rp).

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

cr dr=29:1, purified dr = 20:1.

³¹ P NMR (162 MHz, CDCl ₃ ) δ 65.97 (Rp), c . (2 S ,4 aR , 6 R ,7 R ,7 aR )-2-(((2 R ,3 R ,4 R ,5 R )-3-(( tert-butyldimethylsilyl ) oxy )-5-(6-( dibenzylamino )-9H - purine - 9 -yl )-4- methoxytetrahydrofuran -2 -yl ) methoxy )-6-(6-( dibenzylamino ) -9H - purine -9 - yl )-7- methoxytetrahydrofuran - 4H - furano [ 3,2- d ][1,3,2] dioxophosphoruscyclohexane 2- sulfide

³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.18 (Rp) cr dr = 14:1, 25:1. d. 3-(( benzyloxy ) methyl )-1-((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 )-7- fluoro -2- thioiono -tetrahydro -4H -furano [3,2-d][1,3,2] dioxophosphoric cyclohexane -6- yl ) pyrimidine -2,4(1H,3H) -dione

³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.03 (Rp), 61.49 (Sp). e . 3-(( benzyloxy ) methyl )-1-((2S , 4aR , 6R , 7R , 7aR ) -2-(((2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4- dioxy- 3,4 - dihydropyrimidine -1( 2H ) -yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- fluorotetrahydrofuran -2- yl ) methoxy )-7- methoxy -2 - thioionyltetrahydro - 4H - furano [ 3,2 -d ][1,3,2] dioxophosphoric cyclohexane -6- yl ) pyrimidine -2,4( 1H , 3H ) -dione

³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.36 (Rp). f . 1- benzyl -9-(( 2S , 4aR , 6R , 7R , 7aR )-2-(((2R , 3R , 4R , 5R ) -3-(( tert-butyldimethylsilyl ) oxy )-5-(6-( dibenzylamino )-9H - purin - 9- yl )-4- methoxytetrahydrofuran -2- yl ) methoxy )-7- methoxy- 2 -thioionyltetrahydro - 4H - furano [3,2 - d ][1,3,2] dioxophosphoric cyclohexane -6- yl )-2-( dibenzylamino )-1,9- dihydro - 6H - purin -6 -one

³¹ P NMR (162 MHz, CDCl ₃ ) δ 66.21 (Rp), 62.05 (Sp). Yield 62%. ¹ H NMR (400 MHz, CDCl ₃ ) 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).

¹³ C NMR (100 MHz, CDCl ₃ ) 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. g. 1-((2R,3R,4R,5R)-5-((((2S,4aR,6R,7R,7aR)-6-(1- benzyl -2-( dibenzylamino )-6- sideoxy -1,6 -dihydro -9H - purin- 9- yl )-7- methoxy -2- thioionyltetrahydro - 4H -furano [3,2-d][1,3,2] dioxophosphorus-cyclohexane -2- yl ) oxy ) methyl )-4-(( tert-butyldimethylsilyl ) oxy )-3- methoxytetrahydrofuran -2- yl )-3-(( benzyloxy ) methyl ) pyrimidin -2,4(1H,3H) -dione

³¹ P NMR (162 MHz, CDCl ₃ ) δ 65.72, 61.88. ¹ H NMR (400 MHz, CDCl ₃ ) 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-( dibenzylamino )-6- sideoxy -1,6 -dihydro - 9H - purin -9- yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2- yl ) methoxy )-7- methoxy - 2- thioionyltetrahydro - 4H - furano [ 3,2 -d ][1,3,2] dioxophosphorylcyclohexane -6 -yl )-2-( dibenzylamino )-1,9- dihydro - 6H - purin -6 -one

³¹ p NMR (162 MHz, _CDCl₃ ) δ 66.73 (Rp). Example 19. Nucleophilic ring-opening of a p - terminated cyclic dinucleotide thioester with a benzyl alcohol . α. Reaction Graph 1. General catalytic program for U*U.

Solution A: Add copper chloride (CuCl) (0.25-1 Eq) (raw powder) to a vial that has been dried with a hot air gun under _N2 , and then dissolve it with solvent-1 (0.05 M) at room temperature. Then add alkali (1.5-3 Eq), and after 10 minutes add phenylmethanol (2 Eq) or other benzyl alcohol (e.g., _CF3BnOH ). Stir at RT under _N2 for 20 minutes.

Solution B: 13:45 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidine-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-thioionyltetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (93 mg, 1 Eq, 0.10) was added under Schulnk line. Solution A (mmol) was dissolved in toluene-2 (1.3 mL) at 23 °C, and then added dropwise to solution B over 5 minutes using a syringe needle. The reaction was completed within 20 hours after LCMS monitoring.

Ring-opening reactions were screened out from 34 reactions using cyc U*U with different dr (8:1 or 13:1) to optimize conditions. In the 34 reactions in one screening run, the improved conditions were: MTBD (1.5 eq), BnOH or 2-CF3-BnOH (2 eq), CuCl (0.25 eq), with toluene, MTBE and acetonitrile as solvents and t = 20-40 h (SM conversion 95%, [S] < 1:1 to [S] ~ 5:1).

A second round of screening was performed using cyc U*U, dr=4:1, 2- _CF3- BnOH, various Lewis acids, bases, and solvent combinations to further optimize the process. b. Reaction diagram 2. Detailed procedure for U*U

Solution A: Add copper chloride (CuCl) (2.5 mg, 0.64 μL, 0.25 Eq, 25 μmol) (raw powder) to a vial that has been dried with a hot air gun, and then dissolve it in toluene-1 (0.7 mL) at room temperature. Then add mTBD (1-methyl-2,3,4,6,7,8-hexahydro-1H-pyrimidino[1,2-a]pyrimidin) (23 mg, 22 μL, 1.5 Eq, 0.15 mmol), and after 10 minutes add phenylmethanol (22 mg, 21 μL, 2 Eq, 0.20 mmol) or other benzyl alcohol (e.g., _CF3BnOH ), and stir at RT for 20 min under _N2 .

Solution B: 13:45 3-((benzyloxy)methyl)-1-((2S,4aR,6R,7R,7aR)-2-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidine-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methoxy)-7-methoxy-2-thioionyltetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (93 mg, 1 Eq, 0.10) was added under Schulnk line. Solution A (mmol) was dissolved in toluene-2 (1.3 mL) at 23°C, and then added dropwise to solution B over 5 minutes using a syringe needle. The reaction was completed within 20 hours after LCMS monitoring. c. General library procedure d. First selection - Disk Design: The first screening process

In the glove box container, according to the tray design, manually pipette each cyclic dinucleotide matrix as a reserve solution into the appropriate reaction vials in columns A and C (using toluene) and columns E and G (using CAN). Purge the toluene reserve solution under nitrogen. Using Quantos, dispense the solid base and additives into the appropriate alkoxide-forming vials in columns B, D, and F of the 96-vial tray.

Prepare the alkoxide stock solution by the following steps: (i) adding an appropriate amount of solvent to the appropriate vial of solid alkali/additive using a manual pipette; (ii) adding solid alcohol (weighed in a glove box) to the appropriate vial; and (iii) adding liquid MTBD alkali to the appropriate vial using a manual pipette. Then add an appropriate amount of solvent to the matrix vials in columns A, C, and E. Seal the disc 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 to form the benzyl alkoxide and redissolve the matrix mixture.

Unseal the pan and then use a manual pipette to transfer the alkoxide solution (one type/reaction vial) to the corresponding reaction vial (drop by drop, ~10 seconds/add). Reseal the pan using the appropriate spiral pattern and stir at 800 rpm in the glove box at RT.

Unsealed the pan at 2, 4, 20, 27, and 66 hours and sampled as follows: (1) sampled 20–60 mL of 2.5 mM internal standard TPP (triphenyl phosphate) stock solution using CAN; (2) aliquoted 20 µL of sample from each well into a 0.45 µm filter vial using a single-channel pipette; (3) aliquoted 400 µL of internal standard stock solution into each well using a multi-channel pipette; (4) analyzed the sample using UPLC high pH NoMeOH method - Dreadnought (0.5 µL injection volume); and (5) assigned a batch number (CAX-D00157-067-XXX, where XXX is from A01 to E10) to each sample reflecting its position in the reaction pan and analysis pan. The solvent in each well dried out several times. In such cases, add 100 μL of a suitable solvent to the recombination reaction. Before final sample preparation, add another 100 μL of a suitable solvent to all reaction wells.

The screening conditions and results are shown in Table 11 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 eq), ArOh (2 eq)). [s] Plotting the ratio of isomer products in [3-5]: [5-5] regions. Table 11 - Screening Conditions and Results ii. Second Screening - The second screening process

Using a manual pipette, dispense the cyclic dinucleotide matrix as a stock solution into the appropriate reaction vials in columns A and C (using toluene) and columns E and G (using ACN). Purge the stock solution under a stream of _N2 . Then, using Quantos, dispense the solid base and additives into the appropriate alkoxide-forming vials in columns B, D, F, and H of a 1 mL × 96 vial tray.

Prepare a stock solution of the alkoxide 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 process for D to C, F to E, and H to G. Seal the disc using a suitable spiral pattern and stir at 800 rpm at RT in a glove box, allowing at least 30 minutes from the time of final addition to the alkoxide vial to form the benzyl alkoxide and redissolve the matrix mixture.

Unseal the pan and then use a manual pipette to transfer the alkoxide solution (one type/reaction vial) to the corresponding reaction vial (dropwise, ~10 seconds/addition). Reseal the pan using the appropriate spiral pattern and stir at 800 rpm at RT in a glove box. Unseal the pan at 2 and 20 hours for sampling and analysis. Pan observation .

Species were observed and identified in UPLC during the reaction: (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; gradient: 0–95% B for 13.50 min, followed by a 2.25 min hold at 95% B. Total run time: 15.75 min; flow rate: 0.8 mL/min.

The screening resulted in different transformations to DP 3'-5', and in most cases, 5'-5' DP was the dominant isomer. Two (2) time points were considered: 2 and 20 HR (the latter being the endpoint). The first time point was at 2 h, which may have allowed for the interconversion between 3'-5' (kinetic products) and 5'-5' (thermal products). The second time point was at 20 h, where in some cases, more interconversions between 3'-5' (kinetic products) and 5'-5' (thermal products) were observed. No internal standard was used in the reaction.

Figures 2A-2F show the results of the dinucleotide Screen-Plate Key Spotfire output overlap of 2HR; PhMe (toluene). Figures 3A-3F show the results of the dinucleotide Screen-Plate Key Spotfire output overlap of 2HR; ACN. Figures 4A-4F show the results of the dinucleotide Screen-Plate Key Spotfire output overlap of 20HR; PhMe (toluene). Figures 5A-5F show the results of the dinucleotide Screen-Plate Key Spotfire output overlap of 20HR; ACN.

The screening conditions and results using toluene are shown in Table 12 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 eq), Lewis acid (0.25 eq)). [s] plots the ratios of isomer products in [3-5]:[5-5]. This screening was performed using starting material with a dr ratio of 4:1. Table 12 - Screening Conditions and Results ( Toluene )

The screening conditions and results using ACN are shown in Table 13 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 eq), Lewis acid (0.25 eq)). [s] plots the ratios of isomer products in [3-5]:[5-5]. This screening was performed using starting material with a dr ratio of 4:1. Table 13 - Screening Conditions and Results (ACN) iii. Nucleobase combination screening -

The screening conditions and results are shown in Table 14 (baseline reaction conditions: room temperature, toluene (0.05 M), reaction base (1.5 eq), Lewis acid (0.25 eq)). [s] plots the ratios of isomer products in [3-5]: [5-5]. Table 14 - Screening Conditions and Results Example 20. Spectral analysis of open-ring dinucleotides a. Phosphothiophosphate O-((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 3,4 -dihydropyrimidin -1(2H) -yl )-2-( hydroxymethyl )-4- methoxytetrahydrofuran -3 -yl ) ester O-(((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidin -1(2H) -yl )-3-(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2 -yl ) methyl ) ester O-(2-( trifluoromethyl ) benzyl ) 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 Accurate Mass: 1106.34 (1107.35 MH+), Measured Mass: 1107.46 MH+ b . Phosphophosphate O -((2R , 3R , 4R , 5R ) -5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -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, CDCl ₃ ): [3'-5'], δ 67.92 (Rp), 67.61 (Sp); [5'-5'], 68.95 c. Thiophosphate O-(((2R,3R,4R,5R)-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 -dihydropyrimidin -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- yl ) ester O-(2-( trifluoromethyl ) benzyl ) ester

³¹ P NMR (162 MHz, CDCl ₃ ): [3'-5'], δ 67.66 (Rp), 67.52 (Sp). [5'-5'], 68.75 d . O - benzyl thiophosphate O - ((2 R ,3 R ,4 R ,5 R )-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy- 3,4-dihydropyrimidine - 1 (2 H ) -yl )-2-( hydroxymethyl )-4- methoxytetrahydrofuran -3- yl ) ester O -(((2 R ,3 R ,4 R ,5 R )-5-(3-(( benzyloxy ) methyl )-2,4 -dioxy -3,4 - dihydropyrimidine -1(2 H) -yl) ) -3 -(( tert-butyldimethylsilyl ) oxy )-4- methoxytetrahydrofuran -2 -yl ) methyl ) ester

³¹ P NMR (162 MHz, CDCl ₃ ): [3'-5'], δ 68.38 (Rp), 67.73 (Sp). [5'-5'], 68.94 Example 21. Synthesis of trimer thiophosphates from self-opening ring-opening thiodinucleotides

Under Schulenk line conditions, lithium 2-methyl-2-propoxide (6.0 mg, 7.0 μL, 1.5 Eq, 75 °C) was charged into reaction tube A (dried by a hot air gun) under _N2 conditions. μmol) and a stirring rod were added, followed by the addition of O-benzyl thiophosphate O-((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) ester O-(((2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl) ester (0.10 g, 50% Wt, 1 Eq, 50) at -20°C for 10 minutes. A reserve solution of μmol in toluene (1 mL).

Add Cl-CP (0.18 mmol, 1.5 Eq) and 3-((benzyloxy)methyl)-1-((4aR,6R,7R,7aR)-2-chloro-7-methoxy-2-thioionic tetrahydro-4H-furano[3,2-d][1,3,2]dioxaphosphacyclohexane-6-yl)pyrimidine-2,4(1H,3H)-dione (36 mg, 1.5 Eq, 75 μmol) to another reaction tube B. Incubate the reaction tubes at -25°C for 10 minutes, and with the aid of a syringe, transfer the pre-stirred alkoxide solution from reaction tube A dropwise into reaction tube B. The reaction solution was stirred at -20°C for 18 hours, then heated to room temperature and stirred for 4 hours. LCMS showed the expected product peaks of the major product and isomers at MH+ 1477.3. Based on the uncorrected UPLC crude yield of approximately (LCMS screenshot below, assuming the newly formed dr ~ 56:25:12:3:2:1.6, defined by the retention time and characterization features of the cyclic dinucleotide), a large amount of PS-Cl remained unreacted. LCMS showed the separation of the starting material and 6 product isomers, and MS-ID was confirmed by EIC (not HRMS). Example 22. Liquid-phase synthesis of oligonucleotides using flow technology.

A general overview of liquid-phase methods used for oligonucleotide synthesis is presented in Figure 6.

An overall overview of the continuous flow oligonucleotide synthesis system is presented in Figure 7. Figure 7 shows a glass microreactor (“200 μl flow reactor”), but any type of reactor known in the industry and recommended herein can be used.

The study aimed to compare the performance and efficiency of oligonucleotide synthesis using a batch system or a flow system. Twelve test reactions were performed using a flow system at different temperatures (-20°C, 0°C, ambient temperature (~20-25°C), and 45°C) and different residence times (30, 40, or 50 minutes). The solvent (toluene) and concentration were constant in the batch process. Analysis by ^31P NMR and LCMS revealed that performing the reaction at ambient temperature with a residence time of 50 minutes allowed for complete conversion with the same selectivity as in the batch under low-temperature conditions.

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 reserve solution in a 100 ml glass bottle. Prepare a solution of cyclic dinucleotide in toluene (0.07 M) [Solution B], bubble with nitrogen, and store as a reserve solution in a 100 ml glass bottle. Use methanol as a coolant, bubble with nitrogen, and store in a third 100 ml glass bottle. Connect all solutions accordingly to nitrogen lines saturated with toluene (Solutions A and B) or methanol.

Each bottle was connected to the VICI M6HP pump via a 1/16’’ O.D. pipe. 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 by a Polar Bear device (from Uniqsis Ltd.).

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

The first step was to flush the reactor with toluene to ensure complete cleaning of the reactor and piping. Then, after priming the pumps and lines with reagents and a cooling 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-min residence time), and 6.6 µL/min for methanol, respectively. During the first three hours of the experiment, the reaction outlet mixture was discarded (unstable operation). Upon reaching steady-state conditions, two fractions were collected: a first fraction corresponding to a theoretical mass of 26.7 mg of product at a collection time of 2.5 hours, and a second fraction corresponding to a theoretical mass of 21.4 mg of product at a collection time of 2 hours.

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

Table 15 summarizes some details for comparison. batch Flow Isomer control 85:15 85:15 Low temperature conditions -20℃ (Required) Ambient temperature Response time 4-6 hours (27 mg dosage) 45-60 minutes Reproducibility medium high Safety Hazards medium Low

Figures 1A and 1B illustrate examples of enzymatic ligation reactions that generate nonamers from short polymers. With advancements in DNA and RNA technology, the demand for synthetic oligonucleotides is gradually increasing.

Figures 2A, 2B, 2C, 2D, 2E, and 2F show the results of the dinucleotide Screen-Plate Key Spotfire output overlay 2HR; PhMe (toluene).

Figures 3A, 3B, 3C, 3D, 3E and 3F show the results of the dinucleotide Screen-Plate Key Spotfire output overlapping 2HR;ACN.

Figures 4A, 4B, 4C, 4D, 4E and 4F show the results of dinucleotide Screen-Plate Key Spotfire output overlap 20HR; PhMe (toluene).

Figures 5A, 5B, 5C, 5D, 5E, and 5F show the results of the dinucleotide Screen-Plate Key Spotfire output overlap of 20 HR; ACN.

Figure 6 presents an overview of one embodiment of the solution-phase method for synthesizing oligonucleotides according to the present invention.

Figure 7 presents an overview of one embodiment of the continuous flow oligonucleotide synthesis system of the present invention.

Claims (27)

A method for preparing a dinucleotide of formula (iv) or a salt thereof, Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 )alkyl and ( _C1 - _C20 )alkoxy; where applicable, ^R1 and ^R2 are independently selected from hydrogen, fluorine, methoxy and -O-methoxyethyl (MOE); ^B1 and ^B2 are nucleobases that may be protected by one or more protecting groups; and ^R4 is hydrogen or a protecting group; the method comprises: (a) reacting the nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6) )alkyl, O-aryl and S-aryl, thereby forming a cyclic 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 ^R4 is a protecting group: The method further includes removing one or more of the protective groups as appropriate. A method for preparing a dinucleotide of formula (vi) or a salt thereof. Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 )alkyl and ( _C1 - _C20 )alkoxy; where applicable, ^R1 and ^R2 are independently selected from hydrogen, fluorine, methoxy and -O-methoxyethyl (MOE); ^B1 and ^B2 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _{C20)alkyl ...} )alkyl and aryl; the method includes: (i) providing a cyclic phosphorus dinucleotide of formula (iv) as claimed in claim 1, wherein ^R4 is a protecting group: ; and (ii) reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; wherein the method further includes, as appropriate, removing one or more of the protecting groups. A method for preparing a dinucleotide of formula (vi) or a salt thereof. Wherein: Y is O or S; ^R1 and ^R2 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 )alkyl and ( _C1 - _C20 )alkoxy; where ^R1 and ^R2 are independently selected from hydrogen, fluorine, methoxy and -O-methoxyethyl (MOE); ^B1 and ^B2 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; n is selected from 1, 2, 3, 4 and ⁵ ; each R5 is independently a substituent on the benzyl protecting group; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20) alkyl ... The method comprises: (a) reacting a nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, wherein R is Cl, Br, or ( _C1 - _C6 )alkoxy, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 is a protecting group: ; and (c) reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) to form the dinucleotide of formula (vi): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; wherein the method further includes, as appropriate, removing one or more of the protecting groups. A method for preparing oligonucleotides (vii), Among them: X-series hydrogen, protective groups or Each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy; where applicable, ^R1 , ^R2 and ^R3 are independently selected from hydrogen, fluorine, methoxy and -O-methoxyethyl (MOE); ^R4 is H or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group; each ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; ^B1 , ^B2 and B ³ is a nucleobase that may be protected as appropriate; and m is an integer between 1 and 18; the method includes: (i) providing a dinucleotide of formula (vi) as in claim 2 or 3; and (ii) repeating the reactions of steps (a), (b) and (c) of claim 3 one or more times to obtain an oligonucleotide of formula (vii); wherein, in the repeated step (b), the cyclic phosphorus nucleoside of formula (ii) reacts with the 5' terminal nucleoside of the dinucleotide or oligonucleotide; wherein the method further includes removing one or more of the protecting groups as appropriate. A method for preparing oligonucleotides (vii), Among them: X-series hydrogen, protective groups or Each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy; where applicable, ^R1 , ^R2 and ^R3 are independently selected from hydrogen, fluorine, methoxy and -O-methoxyethyl (MOE); ^R4 is H or a protecting group; n is selected from 1, 2, 3, 4 and 5; each ^R5 is independently a substituent on the benzyl protecting group; each ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; ^B1 , ^B2 and B ³ is a nucleobase that may be protected as appropriate; and m is an integer between 1 and 18; the method comprises: (a) reacting the nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 )alkoxy, ( _C6 - _C12 )aryloxy, S-( _C1 - _C6 )alkyl, O-aryl, and S-aryl, thereby forming the cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 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): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4 and 5; ^R6 is hydrogen or a protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; and (d) repeating steps (a), (b) and (c) one or more times to obtain the oligonucleotide of formula (vii); wherein, in the repeated step (b), the cyclic phosphorus nucleoside of formula (ii) reacts with the 5'-terminal nucleoside of the dinucleotide or oligonucleotide; wherein the method further includes, as appropriate, removing one or more of the protecting groups. A method for preparing an oligonucleotide of formula (1) or a salt thereof, Among them: X-type hydrogen, protective groups or Each Y is independently O or S; ^R1 , ^R2 , and ^R3 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 ) alkyl, and ( _C1 - _C20 ) alkoxy; where applicable, ^R1 , ^R2 , and ^R3 are independently selected from hydrogen, fluorine, methoxy, and -O-methoxyethyl (MOE); ^B1 , ^B2 , and ^B3 are nucleobases that may be protected, where applicable; R ⁴ is a hydrogen or a protecting group; and m is an integer between 1 and 18; the method includes: (i) providing a dinucleotide of formula (vi) as in claim 2 or 3 or an oligonucleotide of formula (vii) as in claim 4 or 5; (ii) hydrogenating the dinucleotide of formula (vi) or the oligonucleotide of formula (vii); and (iii) removing one or more of the protecting groups as appropriate; wherein the method produces the oligonucleotide of formula (1) or a salt thereof. A method for preparing an oligonucleotide of formula (1) or a salt thereof, Among them: X-type hydrogen, protective groups or Each Y is independently O or S; ^R1 , ^R2 and ^R3 are independently selected from hydrogen, hydroxyl, halogen, _N3 , ( _C1 - _C20 ) alkyl and ( _C1 - _C20 ) alkoxy; where applicable, ^R1 , ^R2 and ^R3 are independently selected from hydrogen, fluorine, methoxy and -O-methoxyethyl (MOE); ^B1 , ^B2 and ^B3 are nucleobases that may be protected; ^R4 is hydrogen or a protecting group; and m is an integer from 1 to 18; the method comprises: (a) reacting the nucleoside of formula (i) with P(Y) _R3 , wherein Y is O or S, and wherein each R is independently selected from Cl, Br, ( _C1 - _C6 ) alkoxy, (C6-C12) alkyl, ( _C1 - _{C20 ...} ) aryloxy group, S-( _C1 - _C6 )alkyl group, O-aryl group and S-aryl group, thereby forming a cyclic phosphorus nucleoside of formula (ii): (b) Reaction of the cyclic phosphorus nucleotide of formula (ii) with the nucleotide of formula (iii) to form the cyclic phosphorus dinucleotide of formula (iv), wherein ^R4 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): Wherein: each ^R5 is independently a substituent on the benzyl alcohol nucleophile; n is selected from 1, 2, 3, 4 and 5; ^R6 is a hydrogen or protecting group; and ^R7 and ^R8 are independently selected from hydrogen, ( _C1 - _C20 ) alkyl and aryl; (d) repeat steps (a), (b) and (c) one or more times as appropriate to obtain the oligonucleotide of formula (vii): ; wherein, in repeated step (b), the cyclic phosphorus nucleotide of formula (ii) reacts with the 5'-terminal nucleotide of the oligonucleotide; (e) hydrogenates the product of step (c) or step (d); and (f) removes one or more of the protecting groups as appropriate; wherein the method produces the oligonucleotide of formula (1) or a salt thereof. The method of any of claims 2 to 7, wherein the cyclic phosphorus dinucleotide of formula (iv) is obtained as a mixture of stereoisomers, and wherein the stereoisomers are separated prior to the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v). The method of any of claims 2 to 7, wherein R ⁵ in each case is an ortho or para substituent on the benzyl group; or, as appropriate, wherein R ⁵ in each case is an ortho substituent on the benzyl group. The method of any of claims 2 to 7, wherein ^R5 in each case is independently selected from halogen, ( _C1 - _C20 )alkyl, ( _C1 - _C20 )alkoxy, trifluoromethyl and phenyl; wherein, as appropriate, each ^R5 is independently selected from phenyl, methyl, methoxy, fluorine, chlorine and _CF3 ; wherein, as appropriate, ^R5 is selected from chlorine and _CF3 . The method of any of claims 2 to 7, wherein R ⁵ is a fused benzene ring forming a naphthalene ring system. As in any of the methods in claims 2 to 7, the substituted benzyl alcohol nucleophile of formula (v) is: or . The method of any of claims 2 to 7, wherein the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) in a solvent system is performed, the solvent system comprising: tetrahydrofuran (THF); 2-methyltetrahydrofuran (2Me-THF); diethyl ether; 1,4-dioxane; dimethyl sulfoxide (DMSO); N-methylpyrrolidone (NMP); dimethylacetamide (DMA); dichloromethane (DCM); acetonitrile (MeCN); methyl tributyl ether (MTBE); tripentyl alcohol or toluene. The method of any of claims 2 to 7, wherein the step of reacting the cyclic phosphorus dinucleotide of formula (iv) with the substituted benzyl alcohol nucleophile of formula (v) in the presence of an alkali selected from: n-butyllithium (n-BuLi); lithium tributoxide (LiOtBu); bis(trimethylsilyl)acetamide lithium (LiHMDS); bis(trimethylsilyl)acetamide potassium (KHMDS); tetramethylhexahydropyridine lithium (LiTMP) or Li- _RZ , wherein _RZ is alkyl or aryl; sodium tributoxide (NaOtBu) + lithium chloride (LiCl); potassium tributoxide (KOtBu) + lithium chloride (LiCl); triethylamine (Et3 ₎ N); diazabicycloundecene (DBU); 4-dimethylaminopyridine (DMAP); S-collidine and triazabicyclodecene (TBD). The method of claim 14, wherein 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). The method of claim 14, wherein the alkali is lithium tributanol. The method of any of claims 2 to 7, wherein the dinucleotide of formula (vi) is separated by reaction with silicon chloride. The method of any one of claims 1 to 7, wherein the cyclophosphochloronucleotide of formula (ii) is obtained as a mixture of stereoisomers, and wherein the stereoisomers are separated prior to the step of reacting the cyclophosphonucleotide of formula (ii) with the nucleotide of formula (iii). The method of any of claims 1 to 7, wherein ^R2 is an alkoxy group connected to an adjacent O to form a ketal protective group with R4. The method of any one of claims 1 to 7, wherein R ⁴ is selected from H, silica, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBS, TBDMS), tert-butyldiphenylsilyl, triisopropylsilyl or a ketal protecting group formed with R ² . The method of any of the claims 1 to 7, wherein each nucleobase is selected from pyrimidine and purine. The method of any of claims 1 to 7, wherein each nucleobase is protected independently by one or more of the following protecting groups: dimethylformamide (DMF), formamidinium, DMF-formamidinium, N-methylpyrrolidin-2-ylene (PyA), neopentyloxymethyl (POM), phthalimide, chlorine, bromine, acetyl, allyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxymethyl (BOM). The method described in any of the requests 1 to 7, wherein the method is a liquid phase method. The method of any of the requests 1 to 7, wherein the method is implemented using a continuous flow. The method of any of claims 1 to 7, wherein the method is implemented in a flow reactor. The method described in claim 24, wherein the method is performed at ambient temperature. The method of claim 24, wherein the reaction time is reduced compared to the same reaction in a batch reactor; wherein the reaction time in the flow reactor is 45-120 minutes, or 45-60 minutes.