Novel phosphorus(V)-based reagents, methods for their preparation, and their use in the preparation of stereodefined organophosphorus(V) compounds

Abstract

The present invention relates to novel phosphorus(V) (P(V)) reagents and methods for their preparation, as well as methods for preparing nucleoside phosphorothioate compounds by using the novel reagents. TIFF2025533488000214.tif40133

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international patent application claiming priority to U.S. Provisional Patent Application No. 63 / 376,249, filed September 19, 2022, the disclosure of which is incorporated by reference in its entirety.

[0002] The present invention provides novel phosphorus(V) (P(V)) reagents [ka] and methods for preparing enantiomerically enriched (e.g., homochiral, optically pure, or single isomer) p-chiral nucleoside phosphorothioate compounds by using novel (P(V)) reagents. [Background technology]

[0003] Organophosphorus compounds have a wide range of uses, including as therapeutic and diagnostic agents, pest and insect control agents, among many other applications. Organophosphorus compounds are generally classified based on the oxidation state of the phosphorus atom: +5 (phosphorus(V)) or +3 (phosphorus(III)). Organothiophosphates (phosphorus(V) compounds containing sulfur attached to phosphorus) are a subclass of organophosphate compounds in which at least one oxygen atom in the phosphate is replaced with sulfur. In some circumstances, the asymmetry induced at phosphorus results in chiral organothiophosphate compounds, making this class of compounds particularly suitable for therapeutic, diagnostic, research, and other applications.

[0004] Well-known and widely used examples of organic thiophosphates are nucleic acids containing thiophosphates, such as phosphorothioate backbones.The use of poly(nucleic acids), such as dinucleotides, containing the natural phosphodiester backbones of DNA or RNA is limited by their instability to nucleases.Nucleoside phosphorothioates have one of the non-bridging oxygen atoms in phosphodiester bond replaced with a sulfur atom.Therefore, the nucleoside phosphorothioates containing phosphorothioate backbones have higher nuclease resistance and cell membrane permeability than dinucleotides with phosphodiester backbones.

[0005] Due to the chiral nature of the phosphorus atom in some organothiophosphates, two stereoisomers (R P and S P Diastereoisomers (isomeric forms) may exist. Therefore, diastereoisomers exist in P-chiral nucleoside phosphorothioates, posing a serious problem in the development of such drugs. It is known that the properties of oligonucleotides, including binding affinity, sequence-specific binding to complementary RNA, and stability against nucleases, are affected by the configuration at the phosphorus atom. Furthermore, it has been suggested that homochiral isomers may have different properties (solubility, stability, activity, pharmacokinetics, etc.). Therefore, it is highly desirable to prepare nucleoside phosphorothioates with specific stereochemical configurations. Summary of the Invention [Means for solving the problem]

[0006] The compounds of the present disclosure are represented by the following structure: [ka]

[0007] One embodiment of the present disclosure is Compound 1: [ka] Compound 2 or Compound 3: [ka] and in the presence of an acid.

[0008] In another embodiment, the present disclosure provides a method of making compound 1, comprising the step of: [ka] with P2S5 in the presence of a base.

[0009] In another embodiment, the present disclosure provides a method of making Compound 2 or Compound 3, comprising producing Compound 5 or Compound 6: [ka] each with hydrogen in the presence of a catalyst.

[0010] In some embodiments, the present disclosure provides a method of making a nucleoside diphosphorothioate or a salt thereof, comprising: (a) Compound 7, 8, or 9: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , and R 5 each independently represents hydrogen, CD3, CF3, straight or branched C1-C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 , R 7 , and R 8 each independently represents a C, ...20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiodiphosphate transfer reagent; (b) reacting a chiral thiodiphosphate transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside diphosphorothioate; and (c) deprotecting the protected nucleoside diphosphorothioate to form a nucleoside diphosphorothioate.

[0011] In some embodiments, the nucleoside is a protected nucleoside.

[0012] In some embodiments, the nucleoside is an unprotected nucleoside.

[0013] In some embodiments, the present disclosure provides a method of making a nucleoside triphosphorothioate or a salt thereof, comprising: (a) Compound 20 or Compound 21: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , and R 5 each independently represents hydrogen, CD3, CF3, straight or branched C1-C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 CD3, CF3, straight or branched chain C1 to C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiotriphosphate transfer reagent; (b) reacting a chiral thiotriphosphate transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside triphosphorothioate; and (c) deprotecting the protected nucleoside triphosphorothioate to form the nucleoside triphosphorothioate. The present invention provides a method comprising:

[0014] In some embodiments, the nucleoside is a protected nucleoside.

[0015] In some embodiments, the nucleoside is an unprotected nucleoside.

[0016] In some embodiments, the present disclosure provides Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: [ka] (In the formula, R 9 each independently represents hydrogen, acetyl, branched or linear C2-C 20 alkanoyl, benzoyl, aryloyl, acryloyl, or heteroaryloyl).

[0017] In some embodiments, the present disclosure provides a nucleoside diphosphorothioate selected from the group consisting of: [ka] [ka] [ka] wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0018] In some embodiments, the present disclosure provides a nucleoside triphosphorothioate selected from the group consisting of: [ka] [ka] [ka] wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0019] Additional embodiments and advantages of the disclosure will be set forth in part in the description that follows, and in part will arise from the description, or may be learned by practice of the disclosure. The embodiments and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0020] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0021] The accompanying drawings incorporated herein, form a part of this specification, and illustrate embodiments of the present disclosure. Together with the description, the drawings further serve to explain the principles of the disclosed embodiments and to enable one skilled in the relevant art to make and use the disclosed embodiments. The drawings are intended to be illustrative, not limiting. [Brief explanation of the drawings]

[0022] [Figure 1] As an illustration of LC traces showing diastereomeric purity for diphosphorothioates of the present disclosure, a representative LC trace for compound 50 is shown. [Figure 2] As an illustration of LC traces showing diastereomeric purity for triphosphorothioates of the present disclosure, a representative LC trace for compound 55 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions set forth below. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed unless otherwise indicated. In this application, the use of "or" or "and" means "and / or" unless specifically stated otherwise. Furthermore, the use of the terms "including" and other forms such as "include," "includes," and "included" is not limiting.

[0024] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0025] Whenever an embodiment is described herein with the term "comprising," otherwise similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0026] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is described, each intervening integer and fraction between the upper and lower limits of that stated range is also to be understood as specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may independently be included in or excluded from the range, and each range in which either one, neither, or both limits are included is also encompassed within the scope of the invention. When a value is explicitly recited, it is to be understood that values ​​that are approximately the same quantity or amount as the recited value are also within the scope of the invention. When a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, when different elements or groups of elements are individually disclosed, combinations of those elements are also disclosed. Where multiple options are disclosed for any element of the invention, examples of the invention with each option individually or in any combination with other options are also hereby disclosed; more than one element of the invention may have such an exclusion, and all combinations of elements with such an exclusion are hereby disclosed.

[0027] The present disclosure is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of hydrogen include 1 H (hydrogen), 2 H (deuterium) and 3 These are generally represented as D for deuterium and T for tritium. In this application, CD3 represents a methyl group in which all hydrogen atoms are deuterium. Carbon isotopes include 13 C and 14C. Isotopically labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described herein, using an appropriate isotopically labeled reagent in place of the originally used non-labeled reagent.

[0028] In this disclosure, the term "compound" is meant to include all stereoisomers and isotopes of the depicted structure. As used herein, the term "stereoisomer" refers to any geometric isomer (e.g., cis and trans isomers), enantiomer, or diastereomer of a compound. The present disclosure encompasses all stereoisomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds and means for resolving them into their component enantiomers or stereoisomers are well known. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, compounds, salts, or complexes of the present disclosure can be prepared in combination with solvents or water molecules by conventional methods to form solvates and hydrates.

[0029] In this disclosure, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers and / or double bonds and therefore exist as stereoisomers, e.g., double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present invention, the chemical structures depicted herein, and thus the compounds of the invention, encompass all of the corresponding stereoisomers, i.e., both stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), and mixtures of enantiomers and stereoisomers, e.g., racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallizing the compounds as chiral salt complexes, or crystallizing the compounds in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0030] In this disclosure, the term "stereoisomer" refers to all possible different isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may possess, in particular all possible stereochemical and conformational isomeric forms, all diastereomers, enantiomers, and / or conformational isomers of the basic molecular structure. Some compounds of the present disclosure may exist in different tautomeric forms, all of which are included within the scope of the present disclosure.

[0031] In this disclosure, the term "enantiomer" refers to each individual optically active form of a compound of the present invention having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and no more than 10% of the other enantiomer), at least 90%, or at least 98%.

[0032] In this disclosure, the term "diastereomers" means stereoisomers that are not mirror images of each other and are not superimposable with respect to one another.

[0033] In this disclosure, the term "nucleic acid" encompasses poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N- or C-glycosides of nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges. The term encompasses nucleic acids containing any combination of nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. The prefix "poly-" refers to a nucleic acid containing from about 1 to about 10,000 nucleotide monomer units, and the prefix "oligo-" refers to a nucleic acid containing from about 1 to about 200 nucleotide monomer units. The term "nucleic acid" can also encompass cyclic dinucleotides (CDNs).

[0034] In this disclosure, the terms "nucleobase" and "nucleoside base moiety," used interchangeably, refer to the portion of a nucleic acid that participates in hydrogen bonding to link one nucleic acid strand to a complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T).

[0035] In the present disclosure, the term "nucleobase" includes modified nucleobases. Examples of nucleobases include, but are not limited to, adenine, guanine, uracil, cytosine, and thymine. Examples of nucleobases also include modified nucleobases, such as heterocyclic compounds that can function as nucleobases, including certain "universal bases" that are not nucleobases in the most classical sense, but function as nucleobases.

[0036] In this disclosure, the term "nucleoside" refers to the compound glycosylamine, in which a nucleobase (a nitrogenous base such as adenine, guanine, thymine, uracil, 5-methyluracil, etc.) is covalently linked to a five-carbon sugar (ribose or deoxyribose) or modified sugar.

[0037] In the present disclosure, the term "sugar" refers to a monosaccharide in closed and / or open ring form. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, morpholino, carbocyclic analogs, hexopyranose moieties, and bicyclic sugars, such as those found in locked nucleic acids. Examples of locked nucleic acids include, but are not limited to, those disclosed in WO 2016 / 079181.

[0038] In this disclosure, the term "modified sugar" refers to a moiety that can replace a sugar and mimic the spatial arrangement, electronic properties, or some other physicochemical property of the sugar.

[0039] In this disclosure, the term "nucleotide" refers to a moiety in which a nucleobase is covalently linked to a sugar or modified sugar, which is covalently linked to a modified phosphorus atom moiety, such as a phosphate or thiophosphate group.

[0040] In the present disclosure, the term "purified," when used in reference to nucleic acids, refers to something that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that makes another substance unsuitable, impure, or inferior. Thus, a purified oligonucleotide exists in a form or configuration that is different from that which existed before it was subjected to the purification method.

[0041] In this disclosure, the term "about" encompasses the range of experimental error that occurs in any measurement.

[0042] In this disclosure, the term "hydrocarbon," as used herein, refers to any chemical structure containing hydrogen and carbon atoms.

[0043] In this disclosure, the term "alkyl," used by itself or as part of another group, refers to an unsubstituted straight or branched chain aliphatic hydrocarbon. In one embodiment, an alkyl group is C 1~20 In one embodiment, the alkyl group is C 1~10 In another embodiment, the alkyl group is C 1~6 In another embodiment, the alkyl group is C 1~4 Non-limiting exemplary C 1~20 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl. 1~10 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. 1~6 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, pentyl, and hexyl.1~4 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and iso-butyl.

[0044] In this disclosure, the term "alkanoyl," as used by itself or as part of another group, refers to an optionally substituted alkyl attached to a terminal ketone group. Non-limiting exemplary alkanoyl groups include: [ka] is.

[0045] In this disclosure, the term "cycloalkyl," used by itself or as part of another group, refers to unsubstituted saturated and partially unsaturated, e.g., cycloalkyl groups containing one or two double bonds, of 3 to 12 carbon atoms (i.e., C 3~12 cycloalkyl) or a cyclic aliphatic hydrocarbon containing 1 to 3 rings having the specified number of carbon atoms. In one embodiment, a cycloalkyl group has two rings. In one embodiment, a cycloalkyl group has one ring. In another embodiment, a cycloalkyl group is saturated. In another embodiment, a cycloalkyl group is unsaturated. In another embodiment, a cycloalkyl group is C 3~8 In another embodiment, the cycloalkyl group is C 3~7 In another embodiment, the cycloalkyl group is C 5~7 In another embodiment, the cycloalkyl group is C 3~6 A cycloalkyl group. The term "cycloalkyl" includes groups in which the ring -CH- is replaced with -C(=O)-. Non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and cyclopentanone.

[0046] In this disclosure, the term "alkenyl," used by itself or as part of another group, refers to an alkyl containing one, two, or three carbon-carbon double bonds. In one embodiment, an alkenyl group is 2~12 In one embodiment, the alkenyl group is C 2~6 In another embodiment, the alkenyl group is C 2~4 Alkenyl groups. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

[0047] In this disclosure, the term "alkynyl," used by itself or as part of another group, refers to an alkyl containing one to three carbon-carbon triple bonds. In one embodiment, an alkynyl has one carbon-carbon triple bond. In one embodiment, an alkynyl group is C 2~12 In one embodiment, the alkynyl group is C 2~6 In another embodiment, the alkynyl group is C 2~4 Alkynyl groups. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.

[0048] In this disclosure, the term "aryl," as used by itself or as part of another group, refers to an unsubstituted monocyclic or bicyclic aromatic ring system. In one embodiment, an aryl group is C 6~14 In one embodiment, the aryl group is C 6~20 An aryl group. Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups. In one embodiment, the aryl group is phenyl or naphthyl.

[0049] In this disclosure, the term "aryloxy," used by itself or as part of another group, refers to an optionally substituted aryl attached to a terminal oxygen atom. Non-limiting exemplary aryloxy groups include PhO - is.

[0050] In this disclosure, the term "aryloyl," as used by itself or as part of another group, refers to an optionally substituted aryl attached to a terminal ketone group. Non-limiting exemplary aryloyl groups include: [ka] is.

[0051] In this disclosure, the term "heterocycle," "heterocyclyl," or "heterocyclic group" is intended to mean a stable 3-, 4-, 5-, 6-, or 7-membered mono- or bicyclic or 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered polycyclic heterocycle that is saturated, partially unsaturated, or fully unsaturated and contains carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, and S; and includes any polycyclic group in which any of the heterocycles defined above is fused to a benzene ring. The nitrogen and sulfur heteroatoms can optionally be oxidized (i.e., N→O and S(O)). p where p is 0, 1, or 2). The nitrogen atom can be substituted or unsubstituted (i.e., N or NR, where R is H or another substituent, as defined). A heterocycle can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. The heterocycles described herein can be substituted on a carbon or nitrogen atom if the resulting compound is stable. A nitrogen in a heterocycle can optionally be quaternized. When the total number of S and O atoms in a heterocycle exceeds 1, it is preferred that these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in a heterocycle is 1 or less. When the term "heterocycle" is used, it is intended to include heteroaryl.

[0052] Examples of heterocycles include acridinyl, azetidinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, and dihydrofuro[2,3-b]tetrahydrofuran. , furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, imidazolopyridinyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isothiazolopyridinyl, isoxazolyl, isoxazolopyridinyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2 ,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolopyridinyl, oxazolidinylperimidinyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolyl Nyl, pyrazolopyridinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2-pyrrolidonyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrazolyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,Examples of heterocyclic rings include, but are not limited to, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thiazolopyridinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl. Also included are fused ring and spiro compounds containing the above heterocycles.

[0053] As used herein, the term "aromatic heterocyclic group" or "heteroaryl" is intended to mean a stable monocyclic or polycyclic aromatic hydrocarbon containing at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen. Heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuryl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzodioxane. Heteroaryl groups are substituted or unsubstituted. The nitrogen atoms are substituted or unsubstituted (i.e., N or NR, where R is H or another substituent, as defined). The nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., N→O and S(O)). p where p is 0, 1 or 2).

[0054] Bridged rings are also included in the definition of heterocycle. A bridged ring occurs when one or more, preferably 1 to 3 atoms (i.e., C, O, N, or S) link two non-adjacent carbon or nitrogen atoms. Examples of bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. It should be noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge.

[0055] As used herein, the term "heteroaryl" or "heteroaromatic" refers to unsubstituted monocyclic and bicyclic aromatic ring systems in which at least one carbon atom of one of the rings is replaced with a heteroatom independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one embodiment, a heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one embodiment, a heteroaryl has 3 heteroatoms. In another embodiment, a heteroaryl has 2 heteroatoms. In another embodiment, a heteroaryl has 1 heteroatom. In another embodiment, a heteroaryl is a 5- to 14-membered heteroaryl. In another embodiment, a heteroaryl is a 5- to 10-membered heteroaryl. In another embodiment, a heteroaryl is a 5- or 6-membered heteroaryl. In another embodiment, a heteroaryl has 5 ring atoms, such as thienyl, a 5-membered heteroaryl having 4 carbon atoms and 1 sulfur atom. In another embodiment, a heteroaryl has 6 ring atoms, such as pyridyl, a 6-membered heteroaryl having 5 carbon atoms and 1 nitrogen atom. Non-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzoxazonyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 3H-indolyl, and indolyl. , indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, furazanyl, and phenoxazinyl.In one embodiment, heteroaryl is thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., 1H-pyrrol-2-yl and 1H-pyrrol-3-yl), imidazolyl (e.g., 2H-imidazol-2-yl and 2H-imidazol-4-yl), pyrazolyl (e.g., 1H-pyrazol-3-yl, 1H-pyrazol-4-yl, and 1H-pyrazol-5-yl), pyridyl (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl (e.g., pyrimidine- Examples of heteroaryl include 2-yl, pyrimidin-4-yl, and pyrimidin-5-yl), thiazolyl (e.g., thiazol-2-yl, thiazol-4-yl, and thiazol-5-yl), isothiazolyl (e.g., isothiazol-3-yl, isothiazol-4-yl, and isothiazol-5-yl), oxazolyl (e.g., oxazol-2-yl, oxazol-4-yl, and oxazol-5-yl), isoxazolyl (e.g., oxazol-3-yl, oxazol-4-yl, oxazol-5-yl), or indazolyl (e.g., 1H-indazol-3-yl). The term "heteroaryl" also includes possible N-oxides. A non-limiting exemplary N-oxide is pyridyl N-oxide.

[0056] In one embodiment, the heteroaryl is a 5- or 6-membered heteroaryl. In one embodiment, the heteroaryl is a 5-membered heteroaryl, i.e., the heteroaryl is a monocyclic aromatic ring system having 5 ring atoms, wherein at least one carbon atom of the ring is replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur. Non-limiting exemplary 5-membered heteroaryl groups include thienyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl.

[0057] In another embodiment, the heteroaryl is a 6-membered heteroaryl, e.g., the heteroaryl is a monocyclic aromatic ring system having 6 ring atoms, wherein at least one carbon atom of the ring is replaced with a nitrogen atom. Non-limiting exemplary 6-membered heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl.

[0058] In this disclosure, the term "heteroaryloyl" as used by itself or as part of another group refers to an optionally substituted heteroaryl attached to a terminal ketone group. Non-limiting exemplary heteroaryloyl groups include: [ka] is.

[0059] In the present disclosure, the term "halogen" is intended to include fluorine, chlorine, bromine and iodine.

[0060] In the present disclosure, the term "internucleoside linkage" refers to a naturally occurring or modified linkage between two adjacent nucleosides in an oligonucleotide or CDN. Naturally occurring RNA and DNA contain phosphorodiester internucleoside linkages. An example of a modified internucleoside linkage is a phosphorothioate linkage.

[0061] In this disclosure, the term "protecting group" refers to a group that protects a functional group from undesired chemical reactions, such as alcohol, amine, carbonyl, carboxylic acid, phosphate, or terminal alkyne. In some embodiments, the functional group is a nucleophile. Examples of alcohol protecting groups include, but are not limited to, acetyl (Ac), acryloyl, benzoyl (Bz), benzyl (Bn), 9-fluorenylmethyl (Fm), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl (MMT), p-methoxybenzyl ether (PMB), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl ether (TBDPS), tri-isopropylsilyloxymethyl (TOM), trityl (triphenylmethyl, Tr), pivaloyl (Piv), and the like. In one embodiment, the protecting group is 4,4'-dimethoxytrityl. Examples of amine protecting groups include, but are not limited to, carbobenzyloxy (Cbz), isobutyryl (iBu), p-methoxybenzylcarbonyl (MOZ), tert-butylcarbonyl (Boc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), tosyl (Ts), and the like. Examples of carbonyl protecting groups include, but are not limited to, acetals and ketals, acylals, dithianes, and the like. Examples of carboxylic acid protecting groups include, but are not limited to, methyl esters, benzyl esters, tert-butyl esters, silyl esters, orthoesters, oxazolines, and the like. Examples of phosphate protecting groups include, but are not limited to, 2-cyanoethyl, methyl, and the like. Examples of terminal alkyne protecting groups include, but are not limited to, propargyl and silyl groups. In one embodiment, a protecting group is used to protect the 5'-hydroxy group of a nucleoside used in the methods of the present disclosure. In one embodiment, the protecting group is DMT. In another embodiment, the protecting group is used to protect the nucleobase of the nucleoside used in the method of the present disclosure. In some embodiments, the protecting group is an amine protecting group.In one embodiment, the protecting group is Ac. In another embodiment, the protecting group is Bz. In yet another embodiment, the protecting group is iBu.

[0062] I. Compounds of the Present Disclosure The compounds of the present disclosure are represented by the following structure: [ka]

[0063] In one embodiment, the method of making a compound of the present disclosure comprises reacting Compound 1: [ka] A, Compound 2: [ka] in the presence of an acid.

[0064] In one embodiment, the method of making a compound of the present disclosure comprises reacting Compound 1: [ka] A, Compound 3: [ka] in the presence of an acid.

[0065] In some embodiments, the acid is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, acetic acid, and formic acid.

[0066] In some embodiments, the acid is trifluoroacetic acid.

[0067] In some embodiments, Compound 1 is selected from the group consisting of Compound 4: [ka] is formed by reacting with P2S5 in the presence of a base.

[0068] In some embodiments, the base is selected from the group consisting of tert-butylamine, triethylamine, pyridine, tri-n-propylamine, trimethylamine, 1,2-bicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0069] In some embodiments, the base is tert-butylamine.

[0070] In some embodiments, compound 2 is compound 5: [ka] is formed by reacting with hydrogen in the presence of a catalyst.

[0071] In some embodiments, compound 3 is compound 6: [ka] is formed by reacting with hydrogen in the presence of a catalyst.

[0072] In some embodiments, the catalyst is selected from the group consisting of platinum dioxide, palladium on carbon, platinum on carbon, Lindlar's catalyst, Raney nickel, nickel, rhodium on aluminum oxide, palladium, and platinum.

[0073] In some embodiments, the catalyst is platinum dioxide.

[0074] II. Methods for Making Nucleoside Diphosphorothioates or Nucleobase Diphosphorothioates In one embodiment, the present disclosure provides a method of making a nucleoside diphosphorothioate or a salt thereof, comprising: (a) Compound 7, 8, or 9: [ka] (In the formula, R 1 , R 2, R 3 , R 4 , and R 5 each independently represents hydrogen, CD3, CF3, straight or branched C1-C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 , R 7 , and R 8 each independently represents a C, ... 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiodiphosphate transfer reagent; (b) reacting a chiral thiodiphosphate transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside diphosphorothioate; and (c) deprotecting the protected nucleoside diphosphorothioate to form the nucleoside diphosphorothioate. The present invention provides a method comprising:

[0075] In some embodiments, the chiral thiodiphosphate transfer reagent in step (b) reacts with the nucleobase in the presence of a second base to form a protected nucleobase diphosphorothioate, and the protected nucleobase diphosphorothioate is then deprotected to form the nucleobase diphosphorothioate.

[0076] In some embodiments, the protected nucleoside diphosphorothioates or protected nucleobase diphosphorothioates are deprotected in an ammonia solution.

[0077] In some embodiments, the protected nucleoside diphosphorothioates or protected nucleobase diphosphorothioates are deprotected in acetic acid buffered tetra-n-butylammonium fluoride solution.

[0078] In some embodiments, the protected nucleoside diphosphorothioates or protected nucleobase diphosphorothioates are deprotected in an acetic acid solution.

[0079] In some embodiments, the nucleoside diphosphorothioates or nucleobase diphosphorothioates are purified by ion exchange chromatography.

[0080] In some embodiments, the nucleoside diphosphorothioates or nucleobase diphosphorothioates are purified by precipitation from a solution containing one of the following cations: lithium, sodium, potassium, calcium, and magnesium.

[0081] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, triethylamine, pyridine, 2,6-lutidine, and imidazole.

[0082] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0083] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0084] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0085] In some embodiments, the term "nucleoside" refers to the compound glycosylamine, in which a nucleobase (a nitrogenous base such as adenine, guanine, thymine, uracil, 5-methyluracil, etc.) is covalently linked to a five-carbon sugar (ribose or deoxyribose) or modified sugar.

[0086] In some embodiments, the term "sugar" refers to monosaccharides in closed and / or open ring form, including, but not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, morpholino, carbocyclic analogs, hexopyranose moieties, and bicyclic sugars.

[0087] In some embodiments, the term "modified sugar" refers to a moiety that can replace a sugar and mimic the spatial arrangement, electronic properties, or some other physicochemical property of the sugar.

[0088] In the present disclosure, the term "nucleobase" includes modified nucleobases. Examples of nucleobases include, but are not limited to, adenine, guanine, uracil, cytosine, and thymine. Examples of nucleobases also include modified nucleobases, such as heterocyclic compounds that can function as nucleobases, including certain "universal bases" that are not nucleobases in the most classical sense, but function as nucleobases.

[0089] In some embodiments, the nucleoside in step (b) is a protected nucleoside.

[0090] In some embodiments, the nucleoside in step (b) is an unprotected nucleoside.

[0091] In some embodiments, the nucleoside is Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: [ka] (In the formula, R 9 each independently represents hydrogen, acetyl, branched or linear C2-C 20 alkanoyl, benzoyl, aryloyl, acryloyl, or heteroaryloyl).

[0092] In some embodiments, the nucleoside is Compound 76, Compound 77, Compound 78, Compound 79, Compound 80, Compound 81, Compound 82, Compound 83, Compound 84, and Compound 85: [ka] (In the formula, R 9 each independently represents hydrogen, acetyl, branched or linear C2-C 20 alkanoyl, benzoyl, aryloyl, acryloyl, or heteroaryloyl).

[0093] In some embodiments, the nucleoside is Compound 86, Compound 87, Compound 88, Compound 89, and Compound 90: [ka] (In the formula, R 9 each independently represents hydrogen, acetyl, branched or linear C2-C 20alkanoyl, benzoyl, aryloyl, acryloyl, or heteroaryloyl; R 10 is a straight or branched chain C1 to C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, or straight or branched C2-C 12 alkynyl).

[0094] In some embodiments, the nucleoside diphosphorothioate is [ka] [ka] [ka] [ka] wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0095] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0096] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0097] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0098] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0099] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0100] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0101] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0102] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0103] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0104] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0105] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0106] In some embodiments, the nucleoside diphosphorothioate is a nucleobase selected from the group consisting of acyclovir: [ka] is.

[0107] In some embodiments, the nucleobase diphosphorothioate is [ka] wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0108] In some embodiments, the nucleobase diphosphorothioate is [ka] is.

[0109] In some embodiments, the present disclosure provides a method of making a nucleoside diphosphorothioate or a salt thereof, comprising: (a) Compound 92: [ka] (In the formula, Nu 1 is a nucleoside; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiodiphosphate transfer reagent; and (b) A chiral thiodiphosphate transfer reagent is reacted with compound 97 in the presence of a second base; [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , and R 5 each independently represents hydrogen, CD3, CF3, straight or branched C1-C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl) to form a protected nucleoside diphosphorothioate; and (c) deprotecting the protected nucleoside diphosphorothioate to form the nucleoside diphosphorothioate. The present invention provides a method comprising:

[0110] In some embodiments, Nu 1 is the nucleobase, then a protected nucleobase diphosphorothioate is generated in step (b) above, and a nucleobase diphosphorothioate is generated in step (c) above.

[0111] In some embodiments, the protected nucleoside diphosphorothioates or protected nucleobase diphosphorothioates are deprotected in an ammonia solution.

[0112] In some embodiments, the protected nucleoside diphosphorothioates or protected nucleobase diphosphorothioates are deprotected in acetic acid buffered tetra-n-butylammonium fluoride solution.

[0113] In some embodiments, the protected nucleoside diphosphorothioates or protected nucleobase diphosphorothioates are deprotected in an acetic acid solution.

[0114] In some embodiments, the nucleoside diphosphorothioates or nucleobase diphosphorothioates are purified by ion exchange chromatography.

[0115] In some embodiments, the nucleoside diphosphorothioates or nucleobase diphosphorothioates are purified by precipitation from a solution containing one of the following cations: lithium, sodium, potassium, calcium, and magnesium.

[0116] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, triethylamine, pyridine, 2,6-lutidine, and imidazole.

[0117] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0118] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0119] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0120] In some embodiments, compound 92 is [ka] (In the formula, Nu 1 is a nucleoside or nucleic acid base).

[0121] In some embodiments, compound 92 is (a) Reacting a nucleoside or nucleobase with iPrNP(OBn) to give compound 91: [ka] (In the formula, Nu 1 is a nucleoside or nucleobase); and (b) Prepared by reacting compound 91 with hydrogen in the presence of a catalyst.

[0122] In some embodiments, step (a) above is performed in the presence of 1-H-tetrazole. In some embodiments, step (a) above is performed in the presence of imidazole. In some embodiments, step (a) above is performed in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, step (a) above is performed in the presence of hydrogen peroxide. In some embodiments, step (a) above is performed in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, step (a) above is performed in the presence of a compound selected from the group consisting of 1-H-tetrazole, imidazole, and 5-phenyl-1-H-tetrazole, and a compound selected from the group consisting of hydrogen peroxide and tert-butyl hydroperoxide (TBHP). In some embodiments, step (a) above is performed in the presence of 1-H-tetrazole and hydrogen peroxide.

[0123] In some embodiments, the catalyst is selected from the group consisting of platinum dioxide, palladium on carbon, platinum on carbon, Lindlar's catalyst, Raney nickel, nickel, rhodium on aluminum oxide, palladium, and platinum.

[0124] In some embodiments, the catalyst is palladium on carbon.

[0125] In some embodiments, the nucleoside diphosphorothioate is [ka] (In the formula, Nu 1 is a nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium).

[0126] In some embodiments, Nu 1 is a protected nucleoside.

[0127] In some embodiments, Nu 1 is an unprotected nucleoside.

[0128] In some embodiments, the nucleoside is described above.

[0129] In some embodiments, the nucleoside diphosphorothioate is [ka] is.

[0130] III. Methods for Making Nucleoside Triphosphorothioates or Nucleobase Triphosphorothioates In one embodiment, the present disclosure provides a method of making a nucleoside triphosphorothioate or a salt thereof, comprising: (a) Compound 20 or Compound 21: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , and R 5 each independently represents hydrogen, CD3, CF3, straight or branched C1-C20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 CD3, CF3, straight or branched chain C1 to C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiotriphosphate transfer reagent; (b) reacting a chiral thiotriphosphate transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside triphosphorothioate; and (c) deprotecting the protected nucleoside triphosphorothioate to form the nucleoside triphosphorothioate. The present invention provides a method comprising:

[0131] In some embodiments, the chiral thiotriphosphate transfer reagent in step (b) reacts with the nucleobase in the presence of a second base to form a protected nucleobase triphosphorothioate, and the protected nucleobase triphosphorothioate is then deprotected to form the nucleobase triphosphorothioate.

[0132] In some embodiments, the protected nucleoside triphosphorothioate or protected nucleobase triphosphorothioate is deprotected in an ammonia solution.

[0133] In some embodiments, the protected nucleoside triphosphorothioates or protected nucleobase triphosphorothioates are deprotected in acetic acid buffered tetra-n-butylammonium fluoride solution.

[0134] In some embodiments, the protected nucleoside triphosphorothioates or protected nucleobase triphosphorothioates are deprotected in an acetic acid solution.

[0135] In some embodiments, the nucleoside triphosphorothioates or nucleobase triphosphorothioates are purified by ion exchange chromatography.

[0136] In some embodiments, the nucleoside triphosphorothioate or nucleobase triphosphorothioate is purified by precipitation from a solution containing one of the following cations: lithium, sodium, potassium, calcium, and magnesium.

[0137] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, and triethylamine.

[0138] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0139] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0140] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0141] In some embodiments, compound 20 is prepared by reacting compound 7 with iPr2NP(OFm)2.

[0142] In some embodiments, the reaction is carried out in the presence of 1-H-tetrazole. In some embodiments, the reaction is carried out in the presence of imidazole. In some embodiments, the reaction is carried out in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, the reaction is carried out in the presence of hydrogen peroxide. In some embodiments, the reaction is carried out in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, the reaction is carried out in the presence of a compound selected from the group consisting of 1-H-tetrazole, imidazole, and 5-phenyl-1-H-tetrazole, and a compound selected from the group consisting of hydrogen peroxide and tert-butyl hydroperoxide (TBHP). In some embodiments, the reaction is carried out in the presence of 1-H-tetrazole and hydrogen peroxide. In some embodiments, the reaction is carried out in the presence of 5-phenyl-1-H-tetrazole and tert-butyl hydroperoxide.

[0143] In some embodiments, compound 7 is [ka] is.

[0144] In some embodiments, compound 20 is [ka] is.

[0145] In some embodiments, the nucleoside in step (b) is a protected nucleoside.

[0146] In some embodiments, the nucleoside in step (b) is an unprotected nucleoside.

[0147] In some embodiments, the nucleoside is described above.

[0148] In some embodiments, the nucleoside triphosphorothioate is [ka] [ka] [ka] wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium. is selected from the group consisting of:

[0149] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0150] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0151] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0152] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0153] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0154] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0155] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0156] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0157] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0158] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0159] In some embodiments, the nucleoside triphosphorothioate is [ka] is.

[0160] In some embodiments, the present disclosure provides a method of making a nucleoside triphosphorothioate or a salt thereof, comprising: (a) Compound 93: [ka] (In the formula, Nu 1 is a nucleoside; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiotriphosphate transfer reagent; (b) A chiral thiotriphosphate transfer reagent is reacted with compound 97: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , and R 5 each independently represents hydrogen, CD3, CF3, straight or branched C1-C 20 Alkyl, straight or branched chain C2-C 12 Alkenyl, straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl) to form a protected nucleoside triphosphorothioate; (c) deprotecting the protected nucleoside triphosphorothioate to form the nucleoside triphosphorothioate. The present invention provides a method comprising:

[0161] In some embodiments, Nu 1 is the nucleobase, then a protected nucleobase triphosphorothioate is generated in step (b) above, and a nucleobase triphosphorothioate is generated in step (c) above.

[0162] In some embodiments, the protected nucleoside triphosphorothioate or protected nucleobase triphosphorothioate reagent is deprotected in an ammonia solution.

[0163] In some embodiments, the protected nucleoside triphosphorothioates or protected nucleobase triphosphorothioates are deprotected in acetic acid buffered tetra-n-butylammonium fluoride solution.

[0164] In some embodiments, the protected nucleoside triphosphorothioates or protected nucleobase triphosphorothioates are deprotected in an acetic acid solution.

[0165] In some embodiments, the nucleoside triphosphorothioates or nucleobase triphosphorothioates are purified by ion exchange chromatography.

[0166] In some embodiments, the nucleoside triphosphorothioate or nucleobase triphosphorothioate is purified by precipitation from a solution containing one of the following cations: lithium, sodium, potassium, calcium, and magnesium.

[0167] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, and triethylamine.

[0168] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0169] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0170] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0171] In some embodiments, compound 93 is selected from the group consisting of compound 92: [ka] with iPr2NP(OFm)2, where Nu 1 is a nucleoside or nucleobase; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium.

[0172] In some embodiments, the reaction is carried out in the presence of 1-H-tetrazole. In some embodiments, the reaction is carried out in the presence of imidazole. In some embodiments, the reaction is carried out in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, the reaction is carried out in the presence of hydrogen peroxide. In some embodiments, the reaction is carried out in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, the reaction is carried out in the presence of a compound selected from the group consisting of 1-H-tetrazole, imidazole, and 5-phenyl-1-H-tetrazole, and a compound selected from the group consisting of hydrogen peroxide and tert-butyl hydroperoxide (TBHP). In some embodiments, the reaction is carried out in the presence of 1-H-tetrazole and hydrogen peroxide. In some embodiments, the reaction is carried out in the presence of 5-phenyl-1-H-tetrazole and tert-butyl hydroperoxide.

[0173] In some embodiments, the nucleoside triphosphorothioate is [ka] (In the formula, Nu 1 is a nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium).

[0174] In some embodiments, Nu 1 is a protected nucleoside.

[0175] In some embodiments, Nu 1 is an unprotected nucleoside.

[0176] In some embodiments, the nucleoside is described above.

[0177] IV. METHODS OF MAKING DINUCLEOSIDES, DINUCLEOBASES, OR NUCLEOSIDE-NUCLEOBASE DIPHOSPHOROTHIOATES In one embodiment, the present disclosure provides a method of making a dinucleoside diphosphorothioate or a salt thereof, comprising: (a) Compound 92: [ka] (In the formula, Nu 1 is a nucleoside; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiodiphosphate transfer reagent; (b) reacting a chiral thiodiphosphate transfer reagent with a nucleoside in the presence of a second base to form a protected dinucleoside diphosphorothioate; and (c) deprotecting the protected dinucleoside diphosphorothioate to form a dinucleoside diphosphorothioate.

[0178] In some embodiments, the dinucleobase diphosphorothioate or salt thereof is Nu 1 is a nucleobase and a chiral thiodiphosphate transfer reagent is reacted with the nucleobase in step (b), it is prepared from the reaction described above.

[0179] In some embodiments, the nucleoside nucleobase diphosphorothioate or salt thereof is Nu 1 is a nucleoside and a chiral thiodiphosphate transfer reagent is reacted with the nucleobase in step (b), prepared from the reaction described above.

[0180] In some embodiments, the nucleoside nucleobase diphosphorothioate or salt thereof is Nu 1 is a nucleobase and a chiral thiodiphosphate transfer reagent is reacted with a nucleoside in step (b), it is prepared from the reaction described above.

[0181] In some embodiments, the protected dinucleoside diphosphorothioates, protected dinucleobase diphosphorothioates, or protected nucleoside nucleobase diphosphorothioates are deprotected in an ammonia solution.

[0182] In some embodiments, the protected dinucleoside diphosphorothioates, protected dinucleobase diphosphorothioates, or protected nucleoside nucleobase diphosphorothioates are deprotected in tetra-n-butylammonium fluoride solution buffered with acetic acid.

[0183] In some embodiments, the protected dinucleoside diphosphorothioates, protected dinucleobase diphosphorothioates, or protected nucleoside nucleobase diphosphorothioates are deprotected in an acetic acid solution.

[0184] In some embodiments, the dinucleoside diphosphorothioates, dinucleobase diphosphorothioates, or nucleoside nucleobase diphosphorothioates are purified by ion exchange chromatography.

[0185] In some embodiments, the dinucleoside diphosphorothioates, dinucleobase diphosphorothioates, or nucleoside nucleobase diphosphorothioates are purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0186] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, triethylamine, pyridine, 2,6-lutidine, and imidazole.

[0187] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0188] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0189] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0190] In some embodiments, Nu 1 is a protected nucleoside. In some embodiments, Nu 1 is an unprotected nucleoside.

[0191] In some embodiments, Nu 1 is a protected nucleobase. In some embodiments, Nu 1 is an unprotected nucleobase.

[0192] In some embodiments, the nucleoside in step (b) is a protected nucleoside. In some embodiments, the nucleoside in step (b) is an unprotected nucleoside.

[0193] In some embodiments, the nucleobase in step (b) is a protected nucleoside. In some embodiments, the nucleobase in step (b) is an unprotected nucleoside.

[0194] In some embodiments, the nucleoside in step (a) is the same as the nucleoside in step (b). In some embodiments, the nucleoside in step (a) is different from the nucleoside in step (b).

[0195] In some embodiments, the nucleobase in step (a) is the same as the nucleobase in step (b). In some embodiments, the nucleobase in step (a) is different from the nucleobase in step (b).

[0196] In some embodiments, the dinucleoside diphosphorothioate is [ka] (In the formula, Nu 1 and Nu 2 is a nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0197] In some embodiments, Nu 2 is a protected nucleoside.

[0198] In some embodiments, Nu 2 is an unprotected nucleoside.

[0199] In some embodiments, Nu 1 and Nu 2 are the same nucleoside.

[0200] In some embodiments, Nu 1 and Nu 2 are different nucleosides.

[0201] In some embodiments, the nucleoside is described above.

[0202] In some embodiments, the dinucleoside diphosphorothioate is [ka] is.

[0203] In some embodiments, the dinucleoside diphosphorothioate is [ka] is.

[0204] In some embodiments, the dinucleoside diphosphorothioate is [ka] is.

[0205] V. Methods for Making Dinucleoside Triphosphorothioates, Dinucleobase Triphosphorothioates, and Nucleoside Nucleobase Triphosphorothioates In one embodiment, the present disclosure provides a method of making a dinucleoside triphosphorothioate or a salt thereof, comprising: (a) Compound 93: [ka] (In the formula, Nu 1 is a nucleoside; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with a compound of the present disclosure in the presence of a first base to form a chiral thiotriphosphate transfer reagent; (b) reacting a chiral thiotriphosphate transfer reagent with a nucleoside in the presence of a second base to form a protected dinucleoside triphosphorothioate; and (c) deprotecting the protected dinucleoside triphosphorothioate to form the dinucleoside triphosphorothioate. The present invention provides a method comprising:

[0206] In some embodiments, the dinucleobase triphosphorothioate or salt thereof is Nu 1 is a nucleobase and a chiral thiotriphosphate transfer reagent is reacted with the nucleobase in step (b), it is prepared from the reaction described above.

[0207] In some embodiments, the nucleoside nucleobase triphosphorothioate or salt thereof is Nu 1 is a nucleoside and a chiral thiotriphosphate transfer reagent is reacted with the nucleobase in step (b), prepared from the reaction described above.

[0208] In some embodiments, the nucleoside nucleobase triphosphorothioate or salt thereof is Nu 1 is a nucleobase and a chiral thiotriphosphate transfer reagent is reacted with a nucleoside in step (b), prepared from the reaction described above.

[0209] In some embodiments, the protected dinucleoside triphosphorothioate, protected nucleobase triphosphorothioate, or protected nucleoside nucleobase triphosphorothioate is deprotected in an ammonia solution.

[0210] In some embodiments, the protected dinucleoside triphosphorothioate, protected dinucleobase triphosphorothioate, or protected nucleoside nucleobase triphosphorothioate is deprotected in tetra-n-butylammonium fluoride solution buffered with acetic acid.

[0211] In some embodiments, the protected dinucleoside triphosphorothioate, protected dinucleobase triphosphorothioate, or protected nucleoside nucleobase triphosphorothioate is deprotected in an acetic acid solution.

[0212] In some embodiments, the dinucleoside triphosphorothioates, dinucleobase triphosphorothioates, or nucleoside nucleobase triphosphorothioates are purified by ion exchange chromatography.

[0213] In some embodiments, the dinucleoside triphosphorothioate, dinucleobase triphosphorothioate, or nucleoside nucleobase triphosphorothioate is purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0214] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, and triethylamine.

[0215] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0216] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0217] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0218] In some embodiments, Nu 1 is a protected nucleoside. In some embodiments, Nu 1 is an unprotected nucleoside.

[0219] In some embodiments, Nu 1 is a protected nucleobase. In some embodiments, Nu 1 is an unprotected nucleobase.

[0220] In some embodiments, the nucleoside in step (b) is a protected nucleoside. In some embodiments, the nucleoside in step (b) is an unprotected nucleoside.

[0221] In some embodiments, the nucleobase in step (b) is a protected nucleobase. In some embodiments, the nucleobase in step (b) is an unprotected nucleobase.

[0222] In some embodiments, the nucleoside in step (a) is the same as the nucleoside in step (b). In some embodiments, the nucleoside in step (a) is different from the nucleoside in step (b).

[0223] In some embodiments, the nucleobase in step (a) is the same as the nucleobase in step (b). In some embodiments, the nucleobase in step (a) is different from the nucleobase in step (b).

[0224] In some embodiments, the dinucleoside triphosphorothioate is [ka] (In the formula, Nu 1 and Nu 2 is a nucleoside; Nu 3 is a cationic nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium. is selected from the group consisting of:

[0225] In some embodiments, Nu 2 is a protected nucleoside.

[0226] In some embodiments, Nu 2 is an unprotected nucleoside.

[0227] In some embodiments, Nu 3 is a protected nucleoside.

[0228] In some embodiments, Nu 3 is an unprotected nucleoside.

[0229] In some embodiments, Nu 1 and Nu 2 are the same nucleoside.

[0230] In some embodiments, Nu 1 and Nu 2 are different nucleosides.

[0231] In some embodiments, Nu 1 and Nu 3 are the same nucleoside.

[0232] In some embodiments, Nu 1 and Nu3 are different nucleosides.

[0233] In some embodiments, the nucleoside is described above.

[0234] In some embodiments, the dinucleoside triphosphorothioate is [ka] is.

[0235] VI. Methods for Making Capped Dinucleoside Triphosphorothioates In one embodiment, the present disclosure provides a method of making a capped dinucleoside triphosphorothioate or a salt thereof, comprising: (a) reacting a first nucleoside with Ψ in the presence of a first base; O reacting with a reagent to form a loaded nucleoside; [ka] (b) reacting the charged nucleoside with a second nucleoside in the presence of a second base to form a dinucleoside phosphate compound 94; [ka] (c) Compound 94 is reacted with Ψ in the presence of a third base. O Reacting with a reagent to form a loaded dinucleoside: [ka] (d) reacting the charged dinucleoside with water in the presence of a fourth base to form compound 95: [ka] (e) reacting compound 95 with iPrNP(OFm) to form compound 96: [ka] (f) reacting compound 96 with a compound of the present disclosure in the presence of a fifth base to form a chiral triphosphorothioate transfer reagent; (g) reacting a chiral triphosphorothioate transfer reagent with a third nucleoside in the presence of a sixth base to form a protected capped dinucleoside triphosphorothioate; and (h) deprotecting the protected capped dinucleoside triphosphorothioate to form a capped dinucleoside triphosphorothioate. a method comprising: In the formula, Nu 1 and Nu 2 is a nucleoside and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium.

[0236] In some embodiments, Ψ O The reagents are [ka] In some embodiments, Ψ O The reagents are [ka] is.

[0237] In some embodiments, the loaded nucleoside in step (a) is isolated before the next step.

[0238] In some embodiments, compound 94 in step (b) is isolated before the next step.

[0239] In some embodiments, the loaded dinucleoside in step (c) is not isolated prior to the next step.

[0240] In some embodiments, compound 95 in step (d) is isolated before the next step.

[0241] In some embodiments, compound 96 in step (e) is isolated before the next step.

[0242] In some embodiments, the chiral triphosphorothioate transfer reagent in step (f) is not isolated prior to the next step.

[0243] In some embodiments, the protected capped dinucleoside triphosphorothioate in step (g) is isolated by ion exchange chromatography prior to the next step.

[0244] In some embodiments, the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0245] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0246] In some embodiments, the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0247] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0248] In some embodiments, the third base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0249] In some embodiments, the third base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0250] In some embodiments, the fourth base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0251] In some embodiments, the fourth base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0252] In some embodiments, the fifth base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0253] In some embodiments, the fifth base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0254] In some embodiments, the sixth base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

[0255] In some embodiments, the sixth base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0256] In some embodiments, compound 95 reacts with iPr2NP(OFm)2 in the presence of 1-H-tetrazole. In some embodiments, compound 95 reacts with iPr2NP(OFm)2 in the presence of imidazole. In some embodiments, compound 95 reacts with iPr2NP(OFm)2 in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, compound 95 reacts with iPr2NP(OFm)2 in the presence of hydrogen peroxide. In some embodiments, compound 95 reacts with iPr2NP(OFm)2 in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, compound 95 reacts with iPr2NP(OFm)2 in the presence of a compound selected from the group consisting of 1-H-tetrazole, imidazole, and 5-phenyl-1-H-tetrazole, and a compound selected from the group consisting of hydrogen peroxide and tert-butyl hydroperoxide (TBHP). In some embodiments, compound 95 is reacted with iPrNP(OFm) in the presence of 1-H-tetrazole and hydrogen peroxide. In some embodiments, compound 95 is reacted with iPrNP(OFm) in the presence of 5-phenyl-1H-tetrazole and tert-butyl hydroperoxide.

[0257] In some embodiments, the protected capped dinucleoside triphosphorothioates are deprotected in an ammonia solution.

[0258] In some embodiments, the protected capped dinucleoside triphosphorothioates are deprotected in acetic acid buffered tetra-n-butylammonium fluoride solution.

[0259] In some embodiments, the protected capped dinucleoside triphosphorothioates are deprotected in an acetic acid solution.

[0260] In some embodiments, the capped dinucleoside triphosphorothioates are purified by reverse phase chromatography.

[0261] In some embodiments, the capped dinucleoside triphosphorothioates are purified by precipitation from a solution containing one of the following cations: lithium, sodium, potassium, calcium, and magnesium.

[0262] In some embodiments, Nu 1 is a protected nucleoside.

[0263] In some embodiments, Nu 1 is an unprotected nucleoside.

[0264] In some embodiments, Nu 2 is a protected nucleoside.

[0265] In some embodiments, Nu 2 is an unprotected nucleoside.

[0266] In some embodiments, the third nucleoside in step (g) is a protected nucleoside.

[0267] In some embodiments, the third nucleoside in step (g) is an unprotected nucleoside.

[0268] In some embodiments, the capped dinucleoside triphosphorothioate is [ka] (In the formula, Nu 1 , Nu 2 and Nu 3 is a nucleoside; Nu 4 is a cationic nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0269] In some embodiments, Nu 3is a protected nucleoside.

[0270] In some embodiments, Nu 3 is an unprotected nucleoside.

[0271] In some embodiments, Nu 4 is a protected nucleoside.

[0272] In some embodiments, Nu 4 is an unprotected nucleoside.

[0273] In some embodiments, Nu 1 , Nu 2 and Nu 3 are the same nucleoside.

[0274] In some embodiments, Nu 1 , Nu 2 and Nu 3 are different nucleosides.

[0275] In some embodiments, Nu 1 and Nu 2 is the same nucleoside, Nu 3 Nu 1 and Nu 2 It is a different nucleoside.

[0276] In some embodiments, Nu 1 and Nu 3 is the same nucleoside, Nu 2 Nu 1 and Nu 3 It is a different nucleoside.

[0277] In some embodiments, Nu 2 and Nu 3 is the same nucleoside, Nu 1 Nu 2 and Nu 3 It is a different nucleoside.

[0278] In some embodiments, Nu 1 , Nu 2 and Nu 4 are different nucleosides.

[0279] In some embodiments, Nu 1 and Nu 2 is the same nucleoside, Nu 4 Nu 1 and Nu 2 It is a different nucleoside.

[0280] In some embodiments, the nucleoside is described above.

[0281] In some embodiments, the capped dinucleoside triphosphorothioate is [ka] is. [Example]

[0282] The present invention is further defined in the following examples. It should be understood that the examples are given by way of illustration only. From the above description and examples, those skilled in the art can ascertain the essential features of the present invention and can make various changes and modifications to adapt the present invention to various uses and conditions without departing from the spirit and scope thereof. As a result, the present invention is not limited by the illustrative examples described below, but rather is defined by the claims appended hereto.

[0283] Example 1 Preparation of tetrafluoropyridine-4-thiol (compound 4) [ka] Tetrafluoropyridine-4-thiol was prepared according to the procedure disclosed by Dilman et al., Angew. Chem. Int. Ed. 2021, 60, 2849-2854. All steps should be carried out under a well-ventilated fume hood due to the large amount of H2S liberated during the reaction.

[0284] A 250 mL round-bottom flask equipped with a stir bar was charged with sodium hydrogen sulfide hydrate (48.2 g, 660 mmol, 2.2 equiv.) followed by the addition of MeOH (100 mL). The resulting suspension was stirred at room temperature until most of the solid dissolved. The flask was immersed in an ice / water bath, and pentafluoropyridine (33.0 mL, 300 mmol, 1.0 equiv.) was added slowly, maintaining the internal reaction temperature below 30 °C. The resulting viscous solution was stirred for 5 min, after which the volatile components were evaporated under reduced pressure. The residue was carefully treated with 4 M HCl solution (180 mL), and the product was extracted with hexane (100 mL, then 2 × 50 mL). The combined organic phases are dried using MgSO4, filtered, and the solvent is evaporated under reduced pressure (above 100 mbar, temperature 30°C) to give tetrafluoropyridine-4-thiol (compound 4; 52.2 g) as a colorless liquid that solidifies when stored at 0°C (yield = 95%). 19 Characterized by F NMR (376 MHz, CDCl3): δ -93.4--93.7 (m, 2F), -142.4--142.6 (m, 2F).

[0285] Example 2 Preparation of Compound 1 [ka] A flame-dried 1 L round-bottom flask equipped with a stir bar was charged with phosphorus pentasulfide (17.0 g, 75 mmol, 1.5 equiv.) followed by the addition of anhydrous DCM (135 mL). The batch was inert by flushing with argon for 2 minutes. Tetrafluoropyridine-4-thiol (compound 4; 21.0 g, 115 mmol, 2.0 equiv.) was then added, and the reaction flask was immersed in an ice / water bath. tert-Butylamine (18.4 mL, 150 mmol, 3.0 equiv.) was added to the reaction mixture (Caution: Highly exothermic reaction). The resulting suspension was allowed to warm to room temperature and stirred under an argon atmosphere for 16 hours. The reaction was carefully quenched with water (135 mL) (Caution: H2S is generated during this process), followed by the addition of hexane (135 mL). The resulting slurry was stirred for 30 minutes, after which the precipitate was filtered off and washed successively with water (60 mL), DCM / hexane (1:1; 3 x 60 mL), and hexane (60 mL). The filter cake was dried under reduced pressure for 16 hours to give 18.4 g of compound 1 as a white crystalline solid (yield = 60%). 1 H NMR(600MHz,(CD3)2CO):δ 7.93-7.65(m,3H),1.55(s,9H); 13 C NMR(150MHz,(CD3)2CO):δ 145.2-144.9(m),144.7-144.4(m),143.6-143.3(m),143.0-142.6(m),131.4-131.0(m),54.7,27.7; 19 F NMR(376MHz,CD3CN):δ -96.5--96.7(m,4F),-135.9--136.0(m,4F); 31 P NMR(162MHz,CD3CN):δ 92.5;HRMS(ESI-TOF)m / z:C 10 Calculated value for F8N2PS4 [MH] - : 458.8559, Found: 458.8562; Characterized by melting point 152-153°C.

[0286] Example 3 Preparation of Compound 3 [ka] A 250 mL round-bottom flask equipped with a stir bar was charged with (-)-cis-limonene oxide (compound 6; 12.2 mL, 75 mmol, 1.0 equiv.), and the atmosphere was replaced with argon. MeOH (50 mL) was added, followed by addition of PtO (surface area ≥ 60 m 2 84 mg, 0.37 mmol, 0.5 mol%) of HCl was added to the flask. The atmosphere in the flask was exchanged with H2, and the reaction vessel was equipped with a H2 balloon. The reaction mixture was stirred at room temperature for 3 hours, after which TLC showed complete conversion of the starting material. The crude reaction mixture was filtered through a pad of CELITE (diatomaceous earth), followed by several volumes of DCM. The resulting solution was concentrated under reduced pressure to approximately 12 mL (>100 mbar, temperature 35 °C) and used in the next step without further purification. Compound 3 was produced.

[0287] The second enantiomer of compound 2 was obtained via a similar procedure starting from (+)-cis-limonene oxide (compound 5). The synthesis of compounds 5 and 6 is disclosed in Steiner et al., Tetrahedron Asymmetry 2002, 13, 2359-2363.

[0288] Example 4 (+)-Ψ * Preparation of reagents [ka] A round-bottom flask equipped with a stir bar was charged with compound 1 (20.0 g, 37.5 mmol, 1.0 equiv.) followed by anhydrous DCM (75 mL). The reaction batch was inert by flushing with argon for 2 min, and the resulting suspension was cooled to -78 °C. MeOH (7.5 mL), crude epoxide compound 3 (12 mL; 2.0 equiv.), and TFA (12 mL; 3.0 equiv.) were then added successively, and the resulting clear solution was added over 10 min. The cooling bath was removed, and the reaction mixture was stirred for 1 h. 31P NMR showed complete conversion of starting material Compound 1 (see below). The reaction mixture was diluted with hexane (150 mL) and washed successively with water (75 mL), 10% aqueous KHPO (75 mL), and 10% aqueous KHPO (75 mL). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The resulting crude solid was redissolved in a minimum amount of DCM, and the resulting solution was diluted with MeOH (100 mL). Crystals appeared after the addition of MeOH, and the solution was left at room temperature for 1 h to complete the crystallization. The resulting slurry was filtered, and the filter cake was washed with cold MeOH. After drying under reduced pressure, Compound (+)-Ψ * The reagent was obtained as a white crystalline solid (8.9 g, dr>99:1, ee>99:1, yield=55%). * The reagents are 1 H NMR(600MHz,CDCl3):δ 4.51(ddd,J=12.8,6.1,3.7Hz,1H),2.28-2.23(m,1H),2.02(td,J=13.0,4.2Hz,1H),1.97-1.93(m,1 H),1.84-1.76(m,3H),1.67(s,3H),1.67-1.58(m,2H),1.03(d,J=6.6Hz,3H),0.97(d,J=6.6Hz,3H); 13 C NMR(150MHz,CDCl3):δ 144.7-144.5(m),143.9-143.5(m),143.1-142.8(m),142.1-141.8(m),86.6(d,J=3.3 Hz),66.2,40.9,33.2(d,J=8.8Hz),27.9(d,J=14.9Hz),27.0,23.4,22.4,22.0,21.1; 19 F NMR(376MHz,CDCl3):δ -91.1--91.3(m,2F),-135.1--135.3(m,2F); 31 P NMR(162MHz,CDCl3):δ 96.4;HRMS(ESI-TOF)m / z:C 15 H 19 F4NOPS3[M+H] + Calculated value for [α]: 432.0303, Measured value: 432.0290 D 20= +315.3 (c 1.01, CHCl3); characterized by melting point 131°C.

[0289] [ka] Compound (-)-Ψ * The reagent was obtained via a similar procedure starting from (+)-cis-limonene oxide (compound 2). All characterization data were identical except for the optical rotation. Compound (-)-Ψ * The reagent is [α] D 20 =-314.7 (c 1.01, CHCl3).

[0290] Example 5 Preparation of Compound 7-1 [ka] Step 1. Benzoylation A flame-dried round-bottom flask equipped with a stir bar was charged with 4-hydroxybenzaldehyde (24.4 g, 200 mmol, 1.0 equiv.). The solid substrate was dissolved in anhydrous THF (200 mL), followed by the addition of NEt (33.5 mL, 240 mmol, 1.2 equiv.). The reaction flask was immersed in an ice / water bath, and benzoyl chloride (23.2 mL, 200 mmol, 1.0 equiv.) was added over 3 min. The reaction mixture was allowed to warm to room temperature overnight. The mixture was then diluted with EtOAc and filtered through a pad of Celite. The filtrate was washed with saturated aqueous NH Cl, the organic layer was dried over Na SO , filtered, and the volatiles were removed under reduced pressure to give crude 4-formylphenyl benzoate (46 g), which was used in the next step without further purification.

[0291] Step 2: Reduction Crude 4-formylphenylbenzoate (46 g) was dissolved in THF (200 mL) and the reaction mixture was cooled to 0 °C. NaBH (11.3 g, 300 mmol, 3.0 equiv) was added in three portions and the mixture was allowed to warm to room temperature over 2 h. The reaction was then carefully quenched with saturated aqueous NH Cl and diluted with EtOAc. The organic phase was separated and the aqueous fraction was extracted with EtOAc. The combined organic layers were dried over Na SO , filtered, and concentrated under reduced pressure to give crude 4-(hydroxymethyl)phenylbenzoate (48 g), which was used in the next step without further purification.

[0292] Step 3. Phosphorylation Crude 4-(hydroxymethyl)phenylbenzoate (24 g) was dissolved in anhydrous THF (200 mL) and cooled to −78° C. Pyrophosphoryl chloride (13.8 mL, 300 mmol, 3.0 equiv.) was added dropwise over 10 min. The reaction mixture was stirred at −78° C. for 4 h, after which the reaction was quenched with water. The pH of the resulting solution was carefully adjusted to 8 with saturated aqueous NaHCO3. Concentrated aqueous HCl was added dropwise to the resulting suspension until the solution became clear. The reaction mixture was extracted with EtOAc, and the combined organic fractions were washed with water. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude solid compound 7-1. The solid was suspended in DCM, filtered, and the filter cake was washed with DCM. After drying under reduced pressure, phosphate compound 7-1 was obtained as a white crystalline solid (17.0 g, 55 mmol, yield = 55% over three steps). Compound 7-1 is 1 H NMR(600MHz,CD3OD):δ 8.20-8.16(m,2H),7.72-7.67(m,1H),7.59-7.54(m,2H),7.50(d,J=8.5Hz,2H),7.25(d,J=8.5Hz,2H),5.05(d,J=7.4Hz,2H); 13 C NMR(150MHz,CD3OD):δ 166.6,155.2,136.2(d,J=7.8Hz),135.0,131.04,131.02,130.7,129.9(d,J=3.1Hz),122.9,68.6(d,J=5.0Hz); 31P NMR(162MHz,CD3OD):δ -0.1;HRMS(ESI-TOF)m / z:C 14 H 12 O6P[MH] - Calculated for: 307.0371, Found: 307.0361; characterized by a melting point of 140-142°C.

[0293] Example 6 Preparation of Compound 22 [ka] iPr2NP(OFm)2 (compound 22) was prepared according to the reported procedure disclosed by Lambrecht et al., J. Am. Chem. Soc. 2015, 137, 3558-3564. A 500 mL flame-dried round-bottom flask equipped with a stir bar was evacuated and backfilled with argon (three times) and capped with a septum. Anhydrous THF (160 mL) was then added, followed by PCl3 (4.0 mL, 46 mmol, 1.0 equiv.). The resulting solution was cooled to 0 °C, and DIPEA (16.0 mL, 92 mmol, 2.0 equiv.) was added. Anhydrous diisopropylamine (12.0 mL, 87 mmol, 1.9 equiv.) was then added dropwise over 10 min, and the resulting suspension was stirred at 0 °C for 1 h. Another portion of DIPEA (16.0 mL, 92 mmol, 2.0 equiv.) was then added, followed by 9-fluorenylmethanol (17.9 g, 92 mmol, 2.0 equiv.). The reaction mixture was warmed to room temperature and stirred overnight under an argon atmosphere. The resulting suspension was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was diluted with DCM, loaded onto a pad of silica gel (18 × 4 cm), and flushed with hexane / EtOAc / NEt3 (100:5:1). The pure product ( 31 The fractions containing 22 (as determined by P NMR) were combined and concentrated under reduced pressure. After drying under reduced pressure, compound 22 was obtained as a yellow semi-solid (12.4 g; yield=52%), which was used in the next step without further purification. Compound 22 should be stored under an argon atmosphere at −20° C. Compound 22 can be prepared by 1H NMR(600MHz,CDCl3):δ 7.76-7.73(m,4H),7.67-7.64(m,4H),7.40-7.35(m,4H),7.31-7.26(m,4H),4.18(t,J=6.9Hz,2H),4.0 1(dt,J=9.9,6.8Hz,2H),3.81(dt,J=9.9,7.3Hz,2H),3.66(hept,J=6.8Hz,2H),1.16(d,J=6.8Hz,12H); 13 C NMR(150MHz,CDCl3):δ 145.1,144.8,141.5,141.4,127.54,127.50,127.0,126.9,125.6,125.3,120.0,11 9.9,66.1(d,J=17.1Hz),49.3(d,J=7.7Hz),43.2(d,J=12.1Hz),24.8(d,J=7.2Hz); 31 Characterized by P NMR (162 MHz, CDCl3): δ 146.0.

[0294] The NMR data were consistent with those previously reported in Lambrecht et al., J. Am. Chem. Soc. 2015, 137, 3558-3564.

[0295] Example 7 Preparation of Compound 20-1 [ka] A flame-dried round-bottom flask equipped with a stir bar was charged with freshly prepared iPrNP(OFm) (compound 22; 4.0 g, 7.7 mmol, 1.2 equiv.), followed by the addition of anhydrous MeCN (20 mL). To the resulting suspension, monophosphate compound 7-1 (2.0 g, 6.4 mmol, 1.0 equiv.) and triethylamine (0.89 mL, 6.4 mmol, 1.0 equiv.) were added sequentially. After stirring for 2 min, 5-phenyl-1H-tetrazole (1.4 g, 9.6 mmol, 1.5 equiv.) was added. The resulting mixture was stirred for 1 h under an argon atmosphere. tert-Butyl hydroperoxide (5.5 M in nonane; 2.3 mL, 12.8 mmol, 2.0 equiv.) was then added, and the reaction was stirred for an additional 1 h. The suspension was filtered through a pad of Celite and washed with EtOAc. The filtrate was loaded directly onto a chromatography column packed with silica gel (18 x 4 cm), and the column was flushed with 400 mL of EtOAc. The eluent was changed to MeOH / DCM (1:15), and chromatography was continued until all of the product had eluted from the silica (TLC: MeOH / DCM, 1:4, R f =0.8). The product-containing fractions were combined and concentrated under reduced pressure (temperature <35°C). The residue was treated with DCM, and the resulting suspension was filtered. The filter cake was washed with DCM, and the filtrate was concentrated under reduced pressure (temperature <35°C), and residual MeOH was removed by coevaporation with DCM. After drying under high vacuum, compound 20-1 was obtained as a white foam (3.5 g, yield = 65%). The pyrophosphate compound 20-1 can be stored at -20°C for several months without significant loss of purity.

[0296] Compound 20-1 is 1H NMR(600MHz,CDCl3):δ 8.86-8.78(m,2H),8.18-8.16(m,2H),7.68(ddt,J=8.7,7.6,1.0Hz,4H),7 .65-7.62(m,1H),7.52-7.49(m,6H),7.37-7.29(m,6H),7.20(tdd,J=7.4,4 .7,1.2Hz,4H),7.09-7.06(m,2H),4.97(d,J=8.0Hz,2H),4.32-4.23(m,4H ),4.14(t,J=7.1Hz,2H),3.12(hept,J=6.5Hz,2H),1.21(d,J=6.5Hz,12H); 13 C NMR(150MHz,CDCl3):δ 165.3,150.6,143.5,143.4,141.5,135.8(d,J=7.5Hz),133.8,130.4,129.7.129.0,128.8,127.96,127.95,127. 2,125.51,125.45,121.65,120.08,120.06,69.6(d,J=5.8Hz),67.9(d,J=5.8Hz),48.0(d,J=8.1Hz),46.8,19.1; 31 P NMR(162MHz,CD3OD):δ -11.6(d,J=20.0Hz),-13.4(d,J=20.0Hz);, HRMS(ESI-TOF)m / z:C 44 H 33 O9P2[M-iPr2NH2] - It was characterized by a calculated value of 743.1600 and an observed value of 743.1628.

[0297] Example 8 Stereocontrolled synthesis of α-thiodiphosphates General Procedure A: Stereocontrolled synthesis of α-thiodiphosphates [ka] α-Thiodiphosphate isomer R P is (+)-Ψ * It can be obtained from the reagent α-thiodiphosphate isomer S P is (-)-Ψ * It can be obtained from reagents.

[0298] A flame-dried 1-dram vial equipped with a stir bar was charged with monophosphate compound 7-1 (61.6 mg, 0.2 mmol, 1.0 equiv.). The vial was closed with a Teflon septum screw cap, evacuated, and backfilled with argon. Anhydrous MeCN (2.0 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 60 μL, 0.4 mmol, 2.0 equiv.) were added, and the mixture was stirred until the starting material was completely dissolved (approximately 5 min). Subsequently, 3 Å molecular sieves (60 mg) and Ψ * The reagent (128 mg, 0.3 mmol, 1.5 equiv.) was added, and the reaction was stirred at room temperature for 30 min. The protected nucleoside (0.5 mmol, 2.5 equiv.) was then added, followed by another portion of DBU (120 μL, 0.8 mmol, 4.0 equiv.), and the mixture was stirred for an additional 90 min. Upon completion of the reaction, the resulting mixture was filtered and concentrated to approximately 0.5 mL under reduced pressure. Concentrated aqueous NH3 (5.0 mL) was added to the residue, and the resulting mixture was stirred at room temperature (or 40 °C) for 16 h. The reaction mixture was then diluted with water and washed with EtOAc. The aqueous phase was concentrated under reduced pressure (temperature ≤ 40 °C). The remaining oily residue was dissolved in 2 mL of water and added dropwise to a solution of 0.2 M NaClO4 in acetone (40 mL). The resulting suspension was centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the pellet was washed twice with acetone. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, 0:100 to 30:70). The product-containing fractions were combined and the solvent was evaporated under reduced pressure (temperature ≤ 40 °C). The solid residue was dissolved in a minimum amount of water and lyophilized to give the pure nucleoside α-thiodiphosphate.

[0299] Example 9 Stereocontrolled synthesis of α-thiotriphosphates General Procedure B: Stereocontrolled synthesis of α-thiotriphosphates [ka] α-Thiotriphosphate isomer R Pis (+)-Ψ * It can be obtained from the reagent α-thiotriphosphate isomer S P is (-)-Ψ * It can be obtained from reagents.

[0300] A flame-dried 1-dram vial equipped with a stir bar was charged with pyrophosphate compound 20-1 (169 mg, 0.20 mmol, 1.0 equiv.). The vial was closed with a Teflon septum screw cap, evacuated, and backfilled with argon. Anhydrous MeCN (2.0 mL) and DBU (120 μL, 0.80 mmol, 4.0 equiv.) were added, and the mixture was stirred for 10 min. Subsequently, 3 Å molecular sieves (200 mg) and Ψ * The reagent (112 mg, 0.26 mmol, 1.3 equiv.) was added, and the reaction was stirred at room temperature for 15 min. The protected nucleoside (0.40 mmol, 2.0 equiv.) was then added, followed by another portion of DBU (180 μL, 1.2 mmol, 6.0 equiv.), and the mixture was stirred for 3–6 h. Upon completion of the reaction, the resulting mixture was filtered and concentrated to approximately 0.5 mL under reduced pressure. Concentrated aqueous NH3 (5.0 mL) was added to the residue, and the resulting mixture was stirred at room temperature (or 40 °C) for 16 h. The reaction mixture was then diluted with water and washed with EtOAc. The aqueous phase was concentrated under reduced pressure (40 °C). The remaining oily residue was dissolved in 2 mL of water and added dropwise to a solution of 0.2 M NaClO4 in acetone (40 mL). The resulting suspension was centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the pellet was washed twice with acetone. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, 0:100 to 50:50). The product-containing fractions were combined and the solvent was evaporated under reduced pressure (temperature 40 °C). The solid residue was dissolved in a minimum amount of water and lyophilized to give the pure nucleoside α-thiotriphosphate.

[0301] Example 10 Stereocontrolled synthesis of dinucleoside thiodiphosphates General Procedure C: Stereocontrolled Synthesis of Dinucleoside Thiodiphosphates [ka] Dinucleoside thiodiphosphate isomer R P is (+)-Ψ * It can be obtained from the reagents. Dinucleoside thiodiphosphate isomer S P is (-)-Ψ * It can be obtained from reagents.

[0302] A flame-dried 1-dram vial equipped with a stir bar was charged with the protected nucleoside monophosphate (0.2 mmol, 1.0 equiv.). The vial was closed with a Teflon septum screw cap, evacuated, and backfilled with argon. Anhydrous MeCN (2.0 mL) and DBU (60 μL, 0.4 mmol, 2.0 equiv.) were added, and the mixture was stirred until the starting material was completely dissolved (approximately 5 min). Subsequently, 3 Å molecular sieves (60 mg) and Ψ * The reagent (128 mg, 0.3 mmol, 1.5 equiv.) was added, and the reaction was stirred at room temperature for 30 min. The protected nucleoside (0.5 mmol, 2.5 equiv.) was then added, followed by another portion of DBU (120 μL, 0.8 mmol, 4.0 equiv.), and the mixture was stirred for an additional 2 h. Upon completion of the reaction, the resulting mixture was filtered and concentrated to approximately 0.5 mL under reduced pressure. Concentrated aqueous NH3 (5.0 mL) was added to the residue, and the resulting mixture was stirred at 40 °C for 16 h. The reaction mixture was then diluted with water and washed with EtOAc. The aqueous phase was concentrated under reduced pressure (temperature ≤ 40 °C). The remaining oily residue was dissolved in 2 mL of water and added dropwise to a solution of 0.2 M NaClO4 in acetone (40 mL). The resulting suspension was centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the pellet was washed twice with acetone. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, 0:100 to 30:70). The product-containing fractions were combined and the solvent was evaporated under reduced pressure (temperature ≤ 40 °C). The solid residue was dissolved in a minimum amount of water and lyophilized to give the pure dinucleoside thiodiphosphate.

[0303] Example 11 Preparation of commercially unavailable substrate compounds 10-1, 11-1, 12-1, 13-1, 14-1, 15-1, and 23 [ka] Compound 10-1 was prepared by the following procedure disclosed in Debarge et al., J. Org. Chem. 2011, 76, 105-126.

[0304] Compound 11-1 was prepared by the following procedure disclosed in Zhu et al., Synth. Commun. 2008, 38, 1346-1354.

[0305] Compound 12-1 was prepared by the following procedure disclosed in Debarge et al., J. Org. Chem. 2011, 76, 105-126.

[0306] Compound 13-1 was prepared by the following procedure disclosed in Zhu et al., Synth. Commun. 2008, 38, 1346-1354.

[0307] Compound 14-1 was prepared by the following procedure disclosed in Zhu et al., Synth. Commun. 2008, 38, 1346-1354.

[0308] Compound 15-1 was prepared by the following procedure disclosed in Zhu et al., Synth. Commun. 2008, 38, 1346-1354.

[0309] Compound 23 was prepared by the following procedure as disclosed in Saudi et al., Eur. J. Med. Chem. 2014, 76, 98-109.

[0310] Example 12 Preparation of commercially unavailable substrate compound 18-1 [ka] A round-bottom flask equipped with a stir bar was charged with deoxyguanosine (2.50 g, 9.4 mmol, 1.0 equiv.) followed by the addition of anhydrous DMF (25 mL). Imidazole (1.40 g, 20.6 mmol, 2.2 equiv.) and tert-butyldimethylsilyl chloride (1.55 g, 10.3 mmol, 1.1 equiv.) were then added, and the resulting suspension was stirred at room temperature for 15 min. The reaction mixture was then cooled to -10 °C. tert-Butyldimethylsilyl chloride (1.55 g, 10.3 mmol, 1.1 equiv.) was added, and the reaction mixture was stirred at 0 °C for 3 h. The reaction was quenched by the addition of water (400 mL), and the resulting suspension was filtered. The solid residue was washed successively with water and EtO and dried under reduced pressure to give 2.48 g of compound 24, which was used directly in the next step.

[0311] Compound 24 (2.48, 6.5 mmol, 1.0 equiv.) was suspended in anhydrous MeCN (50 mL), followed by the addition of anhydrous benzoyl peroxide (2.95 g, 13.0 mmol, 2.0 equiv.) and DMAP (0.16 g, 0.13 mmol, 0.2 equiv.). The reaction vessel was equipped with an air condenser, and the heterogeneous mixture was refluxed for 3 h. The reaction was then cooled to room temperature and quenched by the addition of saturated aqueous NaHCO3 solution. The solid residue was filtered off, washed successively with water and MeCN, and dried under reduced pressure to give 2.80 g of crude compound 25, which was used directly in the next step.

[0312] Crude compound 25 (2.80 g, 5.8 mmol, 1.0 equiv.) was suspended in THF (20 mL), followed by the addition of HO (5 mL) and TFA (5 mL). The reaction was stirred at room temperature for 90 min. The solution was then neutralized by careful addition of saturated aqueous NaHCO solution. The crude mixture was concentrated under reduced pressure to approximately 5 mL and filtered off. The solid residue was washed successively with water and EtO and dried under reduced pressure to give 1.92 g of pure compound 18-1 (yield = 55% for three steps). Physical state: white amorphous solid. Compound 18-1 was obtained as follows: 1H NMR(600MHz,DMSO-d6):δ 10.68(br s,1H),8.03(d,J=7.7Hz,2H),8.00(s,1H),7.72-7.68(m,1H),7.60-7.54(m,2H),6.48(br s,2H),6.23(dd,J=9.1,5.7Hz,1H),5.57(d,J=5.7Hz,1H),5.20(t,J=5.7Hz,1H),4.23-4.19 (m,1H),3.70-3.62(m,2H),2.91(ddd,J=14.6,9.1,5.9Hz,1H),2.58(dd,J=14.6,5.7Hz,1H); 13 C NMR(150MHz,DMSO-d6):δ 165.2,156.8,153.8,151.0,135.3,133.7,129.4,129.3,128.8,116.8,84.9,82.8,76.0,61.6,36.7;HRMS(ESI-TOF)m / z:C 17 H 18 N5O5[M+H] + It was characterized by a calculated value of 372.1303 and an observed value of 372.1285.

[0313] Example 13 Preparation of commercially unavailable substrate compound 17-1 [ka] A round-bottom flask equipped with a stir bar was charged with guanosine (14.2 g, 50 mmol, 1.0 equiv.) followed by the addition of anhydrous DMF (200 mL). The heterogeneous mixture was cooled to 0 °C, after which imidazole (10.2 g, 150 mmol, 3.0 equiv.) and tert-butyldimethylsilyl chloride (15.1 g, 100 mmol, 2.0 equiv.) were added. The reaction mixture was allowed to warm to room temperature overnight. The reaction was then quenched by the addition of water (800 mL), and the resulting suspension was filtered. The solid residue was washed successively with water and acetone and dried under reduced pressure to give 12.1 g of crude compound 26, which was used directly in the next step.

[0314] Crude compound 26 (12.1 g, 30.4 mmol, 1.0 equiv.) was suspended in anhydrous MeCN (150 mL), followed by the addition of anhydrous benzoyl peroxide (27.5 g, 121.6 mmol, 4.0 equiv.) and DMAP (0.74 g, 6.2 mmol, 0.2 equiv.). The reaction vessel was equipped with an air condenser, and the heterogeneous mixture was refluxed for 3 h. The reaction was then cooled to room temperature and quenched by the addition of saturated aqueous NaHCO3 solution. The solid residue was filtered off, washed successively with water and MeCN, and dried under reduced pressure to give 14.9 g of crude compound 27, which was used directly in the next step.

[0315] Crude compound 27 (14.9 g, 24.6 mmol, 1.0 equiv.) was suspended in THF (90 mL), followed by the addition of HO (22.5 mL) and TFA (22.5 mL). The reaction was stirred at room temperature for 90 minutes. The solution was then neutralized by careful addition of saturated aqueous NaHCO solution. The crude mixture was concentrated under reduced pressure to approximately 25 mL and filtered off. The solid residue was washed successively with water and EtO and dried under reduced pressure to give 11.5 g of pure compound 17-1 (yield = 47% for three steps). Physical state: white amorphous solid. Compound 17-1 was obtained by 1 H NMR(600MHz,DMSO-d6):δ 10.77(br s,1H),8.07(s,1H),7.94(dd,J=8.3,1.4Hz,2H),7.79(dd,J=8.4,1.4Hz,1H),7.68(tt,J=7 .4,1.4Hz,1H),7.61(tt,J=7.4,1.4Hz,1H),7.54-7.50(m,2H),7.43-7.39(m,2H),6.54(br s,2H),6.26(d,J=6.7Hz,1H),6.12(dd,J=6.7,5.5Hz,1H),5.84(dd,J=5.5, 2.8Hz,1H),5.51(t,J=5.5Hz,1H),4.49(q,J=3.4Hz,1H),3.84-3.75(m,2H); 13C NMR(150MHz,DMSO-d6):δ 164.9,164.4,157.0,154.2,151.2,135.6,134.0,133.9,129.32,129.30,128.91, 128.85,128.81,128.2,116.9,84.6,83.6,73.7,72.4,61.2;HRMS(ESI-TOF)m / z:C 24 H 22 N5O7[M+H] + It was characterized by a calculated value of 492.1514 and an observed value of 492.1493.

[0316] Example 14 Preparation of commercially unavailable substrate compounds 16-1 and 30 [ka] A round-bottom flask equipped with a stir bar was charged with 2-thiouridine (1.87 g, 7.2 mmol, 1.0 equiv.) followed by the addition of anhydrous pyridine (40 mL). The solution was cooled to 0 °C and then tert-butyldimethylsilyl chloride (1.42 g, 9.4 mmol, 1.3 equiv.) was added. The reaction mixture was allowed to warm to room temperature overnight. Benzoyl chloride (3.0 mL, 25.9 mmol, 3.6 equiv.) was then added, and the reaction was stirred for an additional 8 h. The reaction mixture was then diluted with DCM and washed with 1 M HCl and water. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (EtOAc / hexane / DCM; 0.5:50:50 to 2:50:50) to afford 3.21 g of a mixture of compounds 28 and 29 (a mixture of N- and S-benzoylated regioisomers), which was used directly in the next step.

[0317] A mixture of regioisomeric compounds 28 and 29 (3.21 g, 4.7 mmol, 1.0 equiv.) was dissolved in THF (84 mL). After cooling the solution to 0 °C, HO (21 mL) and TFA (21 mL) were added. The reaction was allowed to warm to room temperature over 3 h. The solution was then neutralized by careful addition of saturated aqueous NaHCO solution. The crude product was extracted with DCM, and the organic phase was dried over MgSO, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (EtOAc / hexane / DCM; 10:40:50 to 40:10:50) to give 1.77 g of a regioisomeric mixture of compounds 30 and 16-1 (2:1 ratio; the structures of the minor and major regioisomers have not been assigned) (yield = 43% over three steps). Physical state: white amorphous solid.

[0318] The regioisomeric mixture of compounds 30 and 16-1 is 1 H NMR(600MHz,DMSO-d6):δ 8.57(d,J=8.2Hz,1H;main),8.55(d,J=8.2Hz,1H;sub),8.00-7.79(m,6H;main+sub),7.75-7.36(m,9H;main+sub),7.21-7.17(m,1H;main+sub),6.5 2(d,J=8.2Hz,1H;main),6.46(d,J=8.2Hz,1H;sub),5.89-5.75(m,3H;main+sub),4.63(s,1H;main),4.62(s,1H;sub),3.94-3.83(m,2H;main+sub); 13C NMR(150MHz,DMSO-d6):δ 174.4 (secondary), 174.2 (main), 168.1 (secondary), 167.9 (main), 164.9 (main), 164.7 (main), 164.6 (secondary), 164.4 (secondary), 159.0 (secondary), 158.9 (main), 141.5 (main + sub), 1 35.0 (main + secondary), 133.91 (main), 133.90 (main + secondary), 138.85 (secondary), 130.8 (secondary), 130.6 (main), 130.20 (secondary), 130.15 (main), 129.5 (secondary), 129.4 (main), 129. 34 (main + sub), 129.28 (main), 129.20 (sub), 128.9 (main + sub), 128.82 (sub), 128.79 (main), 128.71 (main + sub), 128.4 (sub), 129.3 (main), 106.9 (main), 106. 8 (secondary), 89.5 (secondary), 89.4 (main), 84.0 (main), 83.9 (secondary), 74.9 (main), 74.7 (secondary), 71.6 (main), 71.1 (secondary), 60.2 (main), 60.1 (secondary); HRMS (ESI-TOF) m / z:C 30 H 24 N2O8SNa[M+Na] + It was characterized by a calculated value of 595.1151 and an observed value of 595.1138.

[0319] Example 15 Preparation of commercially unavailable substrate compound 31 [ka] A flame-dried round-bottom flask was charged with protected adenosine (compound 14-1; 2.05 g, 3.0 mmol, 1.0 equiv.) and 5-H-tetrazole (0.38 g, 5.4 mmol, 1.8 equiv.). The substrate was dissolved in a mixture of anhydrous MeCN (25 mL) and DCM (25 mL). iPrNP(OBn) (1.25 mL, 3.9 mmol, 1.3 equiv.) was added dropwise to the resulting mixture, and the reaction was stirred under an argon atmosphere for 1 h. The reaction mixture was then cooled to −40 °C, followed by the addition of 30% aqueous HO (10 mL), and the reaction was allowed to warm to room temperature over 1 h. The resulting solution was diluted with DCM, and the organic phase was washed successively with saturated aqueous NaHCO and brine. The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (EtOAc / Hexane / DCM; 10:40:50 to 20:30:50) to give 2.17 g of compound 31 (77% yield). Physical state: white amorphous solid. Compound 31 was obtained from 1 H NMR(600MHz,CDCl3):δ 8.62(s,1H),8.40(s,1H),7.97(dd,J=8.4,1.4Hz,2H),7.90(dd,J=8.4,1.4Hz,2H),7.88-7.84 (m,4H),7.58(tt,J=7.4,1.4Hz,1H),7.55(tt,J=7.4,1.4Hz,1H),7.49-7.46(m,2H),7.42-7.3 9(m,2H),7.38-7.32(m,10H),7.31-7.24(m,6H),6.50(d,J=5.7Hz,1H),6.09(t,J=5.7Hz,1H), 5.98(dd,J=5.7,4.0Hz,2H),5.11-5.02(m,4H),4.62-4.59(m,1H),4.37(dd,J=6.2,3.6Hz,2H); 13C NMR(150MHz,CDCl3):δ 172.4,165.4,165.0,153.1,152.6,152.3,143.4,135.60(d,J=6.5Hz),135.58 (d,J=6.5Hz),134.2,133.95,133.93,133.2,130.01,129.96,129.6,128.9,12 8.81,128.79,128.73,128.66,128.5,128.28,128.27,127.9,86.6,81.9(d,J= 8.0Hz),74.3,71.6,69.96(d,J=5.5Hz),69.94(d,J=5.5Hz),66.5(d,J=5.2Hz); 31 P NMR(162MHz,CDCl3):δ -1.0HRMS(ESI-TOF)m / z:C 52 H 43 N5O 11 P[M+H] + It was characterized by a calculated value of 944.2697 and an observed value of 944.2669.

[0320] Example 16 Preparation of commercially unavailable substrate compound 32 [ka] A round-bottom flask equipped with a stir bar was charged with protected adenosine phosphate (compound 31; 2.17 g, 2.3 mmol, 1.0 equiv.) and the atmosphere was exchanged for argon. MeOH (140 mL) was added, followed by Pd / C (10 wt. %; 325 mg). The atmosphere in the flask was exchanged for H2, and the reaction vessel was equipped with a H2 balloon. The reaction mixture was stirred at room temperature for 1 h, after which TLC showed complete conversion of the starting material. The crude reaction mixture was filtered through a pad of Celite, followed by several volumes of MeOH / DCM (1:1). The volatiles were removed under reduced pressure, and the residue was coevaporated twice with DCM to remove residual MeOH. After drying under reduced pressure, compound 32 was obtained as a white amorphous solid (1.55 g; yield = 88%). Physical state: white amorphous solid. Compound 32 was obtained as follows: 1H NMR (600 MHz, CD3OD): δ 8.81(s,1H),8.58(s,1H),8.00(d,J=6.8Hz,2H),7.81(d,J=7.0Hz,4H),7.76(d ,J=7.4Hz,2H),7.58(t,J=7.4Hz,1H),7.51-7.45(m,3H),7.41(t,J=7.8Hz,2H) ,7.34(t,J=7.7Hz,4H),7.27(t,J=7.8Hz,2H),6.67(d,J=5.8Hz,1H),6.21(t,J =5.8Hz,1H),6.08(dd,J=5.8,3.6Hz,1H),4.77-4.74(m,1H),4.47-4.38(m,2H); 13 C NMR(150MHz,CD3OD):δ 173.7,166.7,166.2,154.4,153.39,153.37,152.9,146.0,145.9,135.3,134.9,134.8,134.3,130.8,130.7, 130.5,130.2,129.8,129.71,129.69,129.6,128.9,87.9,83.5(d,J=8.0Hz),75.8,73.2,66.67(d,J=3.8Hz); 31 P NMR(162MHz,CD3OD):δ 0.4HRMS(ESI-TOF)m / z:C 38 H 29 N5O 11 P[MH] - It was characterized by a calculated value of 762.1601 and an observed value of 762.1625.

[0321] Example 17 Preparation of commercially unavailable substrate compound 33 [ka] A flame-dried round-bottom flask was charged with protected uridine (compound 11-1; 3.10 g, 5.6 mmol, 1.0 equiv.) and 5-H-tetrazole (0.71 g, 10.1 mmol, 1.8 equiv.). The substrate was dissolved in a mixture of anhydrous MeCN (45 mL) and DCM (45 mL). iPrNP(OBn) (2.30 mL, 7.3 mmol, 1.3 equiv.) was added dropwise to the resulting mixture, and the reaction was stirred under an argon atmosphere for 1 h. The reaction mixture was then cooled to −40 °C, followed by the addition of 30% aqueous HO (18 mL), and the reaction was allowed to warm to room temperature over 1 h. The resulting solution was diluted with DCM, and the organic phase was washed successively with saturated aqueous NaHCO and brine. The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (EtOAc / Hexane / DCM; 10:40:50 to 30:20:50) to give 3.88 g of compound 33 (86% yield). Physical state: white amorphous solid. Compound 33 was obtained from 1 H NMR(600MHz,CDCl3):δ 7.98(d,J=7.2Hz,2H),7.91(dd,J=8.4,1.2Hz,2H),7.87(d,J=7.3Hz,2H),7.68(d,J=8.3Hz,1H),7.61-7.56(m,2H),7.52(tt,J=7.3,1.2Hz,2H) ,7.43-7.30(m,16H),6.41(d,J=7.0Hz,1H),5.67-5.64(m,2H),5.46(t, J=6.5Hz,1H),5.19-5.08(m,4H),4.47-4.44(m,1H),4.37-4.30(m,2H); 13C NMR(150MHz,CDCl3):δ 168.4,165.42,165.40,161.7,149.7,139.2,130.49(d,J=5.7Hz),135.46(d,J= 6.2Hz),135.1,134.0,133.9,131.5,130.6,130.0,129.9,129.2,129.14,129.0 9,129.0,128.9,128.76,128.75,128.7,128.39,128.36,103.7,86.5,81.7(d,J =8.1Hz),73.7,71.6,70.17(d,J=5.4Hz),70.15(d,J=5.4Hz),66.6(d,J=5.2Hz); 31 P NMR(162MHz,CDCl3):δ -0.6;HRMS(ESI-TOF)m / z:C 44 H 38 N2O 12 P[M+H] + It was characterized by a calculated value of 817.2157 and an observed value of 817.2153.

[0322] Example 18 Preparation of commercially unavailable substrate compound 34 [ka] A round-bottom flask equipped with a stir bar was charged with protected uridine phosphate (compound 33; 3.85 g, 4.8 mmol, 1.0 equiv.) and the atmosphere was exchanged for argon. MeOH (100 mL) and EtOAc (100 mL) were added, followed by Pd / C (10 wt %; 675 mg). The atmosphere in the flask was exchanged with H2, and the reaction vessel was equipped with a H2 balloon. The reaction mixture was stirred at room temperature for 1.5 h, after which TLC showed complete conversion of the starting material. The crude reaction mixture was filtered through a pad of Celite, followed by several volumes of MeOH / DCM (1:1). The volatiles were removed under reduced pressure, and the residue was coevaporated twice with DCM to remove residual MeOH. After drying under reduced pressure, compound 34 was obtained as a white amorphous solid (2.82 g; yield = 93%). Physical state: white amorphous solid. Compound 34 was obtained as follows: 1H NMR(600MHz,(CD3)2CO):δ 9.38(br s,2H),8.18(d,J=7.5Hz,1H),8.03-7.98(m,4H),7.86(d,J=7.5Hz,2H),7. 66(t,J=7.4Hz,1H),7.61(t,J=7.4Hz,1H),7.54(t,J=7.4Hz,1H),7.49(t,J =7.7Hz,2H),7.43(t,J=7.7Hz,2H),7.32(t,J=7.7Hz,2H),6.48(d,J=6.6Hz ,1H),6.08(d,J=6.9Hz,1H),5.98-5.94(m,1H),5.84-5.79(m,1H),4.82(br s,1H),4.64-4.55(m,2H); 13 C NMR(150MHz,(CD3)2CO):δ 169.8,165.94,165.88,162.6,150.5,141.2,135.8,134.50,134.49,132.6,131.2,130.49,130.4 8,130.1,130.0,129.6,129.49,129.46,129.3,103.7,87.6,82.2(d,J=7.4Hz),75.0,72.7,66.9; 31 P NMR(162MHz,(CD3)2CO):δ 0.3HRMS(ESI-TOF)m / z:C 30 H 24 N2O 12 P[MH] - It was characterized by a calculated value of 635.1072 and an observed value of 635.1071.

[0323] Example 19 Preparation of commercially unavailable substrate compound 36 [ka] Following the procedure disclosed in J. Davisson et al., J. Org. Chem. 1987, 52, 1794-1801, guanosine (2.83 g, 10 mmol, 1.0 equiv.) was suspended in trimethyl orthoformate anhydride (25 mL), followed by the addition of pyridine hydrochloride (1.75 g, 15 mmol, 1.5 equiv.). The stirred suspension was treated with DMSO (3.5 mL), and the mixture was stirred at room temperature for 48 h. To the resulting cloudy suspension, MeOH (25 mL) and solid NaOMe (0.90 g, 16.5 mmol, 1.7 equiv.) were added sequentially, and the reaction was stirred for an additional 3 h. The mixture was then concentrated under reduced pressure, and the resulting thick suspension was treated with MeOH / Et2O (1:1). The resulting pale yellow solid was filtered off and dried under reduced pressure. The crude compound 35 was used directly in the next step.

[0324] Crude compound 35 was redissolved in anhydrous DMF (12 mL) and then NaHPO (2.84 g, 20 mmol, 2.0 equiv.) was added. Dimethyl sulfate (1.40 mL, 15 mmol, 1.5 equiv.) was added dropwise, and the reaction was stirred at room temperature for 1 h. The heterogeneous mixture was filtered through a pad of Celite, and the solid residue was washed with MeOH. The filtrate was concentrated under reduced pressure to approximately 10 mL and loaded directly onto silica gel. The crude material was purified by silica gel chromatography (DCM / MeOH; 100:0 to 80:20) to give 2.20 g of compound 36 (a 2:1 mixture of diastereoisomers). (Yield = 49% for two steps). Physical state: white amorphous solid. Compound 36 was obtained as follows: 11H NMR (600 MHz, D2O): δ 6.31 (d, J = 2.3 Hz, 1H; main), 6.23 (s, 1H; minor), 6.21 (d, J = 2.4 Hz, 1H; minor), 6.13 (s, 1H; main), 5.50 (dd, J = 6.2, 2.4 Hz, 1H; minor), 4.48 (dd, J = 6.8, 2.3 Hz, 1H; main), 5.15 (dd, J = 6.1, 2.4 Hz, 1H; minor), 5.09 (dd, J = 6.8, 2.6 Hz, 1H; main), 4.72 - 4.69 (m, 1H; main), 4.64 - 4.61 (m, 1H; minor), 4.11 (s, 3H; main + minor), 3.90 - 3.86 (m, 1H; main + minor), 3.86 - 3.81 (m, 1H; main + minor), 3.75 (s, 3H; main + minor), 3.49 (s, 3H; main), 3.40 (s, 3H; minor); 13 13C NMR (150 MHz, D2O): δ 150.5 (minor), 157.42 (minor), 157.37 (main), 157.29 (main), 149.4 (main + minor), 136.1 (t, 1:1:1, J 1 C-D = 35.1 Hz; main + minor), 118.4 (main), 117.3 (minor), 108.7 (main + minor), 92.6 (main), 91.9 (minor), 89.0 (main), 87.6 (minor), 84.3 (main), 83.6 (minor), 81.5 (main), 80.7 (minor), 61.4 (main), 61.2 (minor), 55.4 (main + minor), 52.6 (main), 51.6 (minor), 35.71 (minor), 35.70 (main); HRMS (ESI - TOF) m / z: C 13 H 18 N5O6[M] + characterized by a calculated value of 340.1252 and an observed value of 340.1264 for

[0325] [[ID=**15**]]Example 20 Preparation of Substrate Compound 39, Which Is Not Commercially Available

Chemical Structure

[0326] Guanosine dihydrogen monophosphate (compound 38) obtained as described above was suspended in a mixture of anhydrous trimethyl orthoformate (50 mL) and anhydrous DMF (50 mL). The resulting mixture was stirred overnight at room temperature. The reaction mixture was then concentrated under reduced pressure, followed by the addition of EtO. The resulting suspension was filtered to give an off-white solid, which was mixed with MeOH (100 mL) and triethylamine (20 mL). The solution was stirred overnight at 55 °C. The reaction was concentrated under reduced pressure, and the crude residue was purified by reverse-phase C18 silica gel chromatography (1 M TEAA / MeCN in water; 100:0 to 70:30) and lyophilized to give 7.13 g of compound 39 (a 9:1 mixture of diastereoisomers). (Yield = 47% for three steps). Physical state: white amorphous solid. Compound 39 was obtained as follows: 1 H NMR(600MHz,D2O):δ 7.97(s,1H; sub),7.95(s,1H;main),6.18(s,1H;sub),6.14(d,J=2.7Hz,1H;main),6.07(s,1H;main),6.02(d,J =2.9Hz,1H; sub), 5.40(dd,J=6.2,2.9Hz,1H; sub),5.35(dd,J=7.0,2.7Hz,1H;main),5.21(dd,J=6.2,2.8 Hz, 1H; sub), 5.15 (dd, J=7.0, 3.1Hz, main), 4.60 (q, J=4.3Hz, 1H; main), 4.51 (q, J=4.1Hz, 1H; sub), 4.10-4.0 5(m,1H;main+sub),4.05-3.99(m,1H;main+sub),3.45(s,3H;main),3.35(s,3H;sub),3.16(q,J=7.3Hz,12H;Et3NH + ), 1.24(t, J=7.3Hz, 18H; Et3NH + ); 13C NMR(150MHz,D2O):δ 158.6 (main + sub), 153.72 (sub), 153.66 (main), 151.1 (sub), 151.0 (main), 137.8 (main), 13 7.7 (sub), 118.6 (main), 117.3 (sub), 116.1 (main + sub), 90.1 (main), 89.1 (sub), 86.0 (d, J= 8.7Hz; main), 84.5 (d, J=8.8Hz; sub), 84.1 (main), 83.2 (sub), 81.4 (main), 80.7 (sub), 6 4.7 (d, J=4.8Hz; main), 64.5 (d, J=4.6 Hz; sub), 52.5 (main), 51.4 (sub), 46.6 (Et3NH + ), 8.2(Et3NH + ); 31 P NMR(162MHz,D2O):δ 0.6;HRMS(ESI-TOF)m / z:C 12 H 15 N5O9P[MH] - It was characterized by a calculated value of 404.0613 and an observed value of 404.0626.

[0327] Example 21 Preparation of commercially unavailable substrate compound 40 [ka] Compound 39 (2.61 g; 4.3 mmol; 1.0 equiv.) was dried by coevaporation with anhydrous DMF and dissolved in anhydrous DMF (12 mL). iPrNP(OFm) (3.35 g, 6.5 mmol, 1.5 equiv.) was added to the resulting solution, followed by 5-phenyl-1-H-tetrazole (0.94 g, 6.5 mmol, 1.5 equiv.), and the reaction was stirred at room temperature for 1 h. tert-Butyl hydroperoxide (5.5 M in nonane; 2.3 mL, 12.9 mmol, 3.0 equiv.) was then added, and the mixture was stirred for an additional 1 h. The reaction was quenched by the addition of EtO (100 mL). The solvent was decanted, and the remaining oily residue was washed twice with EtO. The residue was redissolved in DCM (10 mL), followed by the addition of EtO (100 mL). The resulting cloudy mixture was sonicated for 10 min to initiate precipitation. The precipitate was filtered off to give 4.05 g of crude compound 40 (quantitative). 3175% purity by weight as determined by P NMR (9:1 mixture of diastereoisomers) (NMR yield = 75%). Compound 40 can be used in the next step without further purification. Physical state: White amorphous solid. Compound 40 can be obtained by 1 H NMR (600MHz, DMSO-d6): δ 10.7 (br s, 1H; main + sub), 8.40 (br s, 2H; main + sub), 7.94 (s, 1H; main), 7.92 (s, 1H; sub), 7.86-7.81 (m, 4H; main + sub), 7.56- 7.48 (m, 4H; main + sub), 7.39-7.32 (m, 4H; main + sub), 7.26-7.18 (m, 4H; main + sub), 6.81 (br s,2H;iPr2NH2 + ),6.12(d,J=1.9Hz,1H;main),6.10(s,1H;secondary),6.02(d,J=2.4Hz,1H;secondary),6.01(s,1H;main),5.33(dd,J=7.2,3.5Hz,1H;secondary),5.26-5.19(m;2H main+1H sub),4.40-4.31(m,2H;main+sub),4.28-4.14(m,6H;main+sub),3.97-3.94(m,1H;sub),3.83-3.75(m,1H;main),3.30(s,3H;main),3.27(hept,J=6.5Hz,1H;iPr2NH2 + ),3.17(s,3H;sub),1.17(d,J=6.5Hz,12H;iPr2NH2 + ); 13C NMR(150MHz,DMSO-d6):δ 157.5 (sub), 156.8 (main), 153.9 (main + sub), 150.5 (sub), 150.4 (main), 143.4 (main + sub), 143.2 (main + sub), 140.79 (main + sub), 140.77 (main + sub), 140.76 (main + sub), 136.3 (sub), 136.2 (main), 129.4 (main + sub), 129.0 (main + sub), 127.6 (main + sub), 127.0 (main + sub), 126.8 (sub), 126.5 (main), 125.14 (main), 125.10 (sub) ,120.0(main+sub),118.2(main+sub),116.91(main),116.85(sub),89.6(main),88.6(sub),85.9(d,J=7.5Hz;main),84.7(d,J=8.1Hz;sub),84.2(main) ,83.0(sub),81.4(main),81.2(sub),68.3(main+sub),64.9(main+sub),54.9(main+sub),51.9(main),50.4(sub),47.3(d,J=7.1Hz;main+sub),46.1(iPr2NH2 + ), 18.7(iPr2NH2 + ); 31 P NMR(162MHz,DMSO-d6):δ -12.1(d,J=19.8Hz),-12.7(d,J=19.8Hz);HRMS(ESI-TOF)m / z:C 40 H 36 N5O 12 P2[MH] - It was characterized by a calculated value of 840.1841 and an observed value of 840.1861.

[0328] For the following Examples 22 to 79, the diastereomeric purity of the products was determined by NMR and LC-MS.

[0329] LC traces of the pure diastereoisomers were recorded from samples obtained after purification and lyophilization using one of methods 1, 2 or 3 described below.

[0330] The LC trace for the mixture of diastereoisomers is the same as the previously obtained R P and S PIt was obtained from a solution prepared by mixing isomers. In some cases, the samples of the compounds used to prepare such mixtures were partially hydrolyzed due to long-term storage, so the chromatogram of the isomer mixture does not reflect the purity of the compound obtained.

[0331] HPLC analysis was performed on a Waters Autopurification LC equipped with a Waters XBridge C18 column (4.6 x 150 mm, 3.5 μm). Solvent A: 0.1 M triethylammonium acetate (TEAA) in HO; Solvent B: MeCN; Flow rate: 1.5 mL / min; and Temperature: 25°C. Table 1 lists the HPLC gradient for Method 1. Table 2 lists the HPLC gradient for Method 2. Table 3 lists the HPLC gradient for Method 3.

[0332] [Table 1]

[0333] [Table 2]

[0334] [Table 3]

[0335] Example 22 5'-O-Azidothymidine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-41) [ka] Compound (R P )-41 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ *The compound was obtained from the reagent (128 mg, 0.3 mmol) and azidothymidine (133 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 52 mg of compound (R) after lyophilization. P )-41 was obtained (yield=53%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-41 is, 1 H NMR(600MHz,D2O):δ 7.79(s,1H),6.28(t,J=6.9Hz,1H),4.60(dt,J=6.7,3.5Hz,1H),4.29-4.20(m,3H),2.54-2.45(m,2H),1.95(s,3H); 13 C NMR(150MHz,D2O):δ 166.5,151.7,137.3,111.8,84.9,83.0(d,J=9.8Hz),65.7(d,J=5.8Hz),61.0,36.2,11.7; 31 P NMR(162MHz,D2O):δ 41.1(d,J=29.9Hz),-6.7(d,J=29.9Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O9P2S[MH] - Calculated for: 441.9987, Found: 441.9979; Retention time: 11.57 min (Method 1).

[0336] Example 23 5'-O-Azidothymidine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-41) [ka] Compound (S) was prepared according to General Procedure A. P )-41 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ *The product was obtained from the reagent (128 mg, 0.3 mmol) and azidothymidine (133 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 49 mg of compound (S) after lyophilization. P )-41 was obtained (yield=50%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-41 is, 1 H NMR(600MHz,D2O):δ 7.78(s,1H),6.28(t,J=6.8Hz,1H),4.60(dt,J=6.9,3.7Hz,1H),4.28-4.21(m,3H),2.56-2.47(m,2H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.5,151.7,137.3,111.8,84.9,82.9(d,J=9.7Hz),65.4(d,J=6.1Hz),60.7,36.1,11.7; 31 P NMR(162MHz,D2O):δ 42.1(d,J=28.3Hz),-9.4(d,J=28.4Hz)HRMS(ESI-TOF)m / z:C 10 H 14 N5O9P2S[MH] - Calculated for: 441.9987, Found: 441.9979; Retention time: 10.59 min (Method 1).

[0337] Example 24 5'-O-Thymidine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-42) [ka] Compound (R P )-42 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ *The compound 10-1 was obtained from the reagent (128 mg, 0.3 mmol) and the protected thymidine compound 10-1 (225 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 44 mg of compound (R) after lyophilization. P )-42 was obtained (yield=47%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-42 is 1 H NMR(600MHz,D2O):δ 7.79(s,1H),6.35(t,J=6.9Hz,1H),4.67(dt,J=6.5,3.7Hz,1H),4.25-4.18(m,3H ),2.41(dt,J=13.8,6.9Hz,1H),2.35(ddd,J=13.8,6.9,3.0Hz,1H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.6,151.8,137.4,111.8,85.3(d,J=9.6Hz),84.9,70.9,65.4(d,J=5.9Hz),38.5,11.7; 31 P NMR(162MHz,D2O):δ 41.3(d,J=27.3Hz),-7.1(d,J=27.3Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N2O 10 P2S[MH] - Calculated for: 416.9923, Found: 416.9934; Retention time: 4.99 min (Method 1).

[0338] Example 25 5'-O-Thymidine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-42) [ka] Compound (S) was prepared according to General Procedure A. P )-42 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ *The compound 10-1 was obtained from the reagent (128 mg, 0.3 mmol) and the protected thymidine compound 10-1 (225 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 49 mg of compound (S) after lyophilization. P )-42 was obtained (yield=52%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-42 is 1 H NMR(600MHz,D2O)δ 7.77(s,1H),6.35(t,J=6.9Hz,1H),4.66(dt,J=6.2,3.2Hz,1H),4.26-4.17(m,3H ),2.41(dt,J=13.8,6.9Hz,1H),2.36(ddd,J=13.8,6.9,3.6Hz,1H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.6,151.8,137.4,111.8,85.2(d,J=9.5Hz),84.9,70.9,65.2(d,J=6.2Hz),38.3,11.7; 31 P NMR(162MHz,D2O):δ 42.1(d,J=29.4Hz),-9.3(d,J=29.4Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N2O 10 P2S[MH] - Calculated for: 416.9923, Found: 416.9934; Retention time: 4.21 min (Method 1).

[0339] Example 26 5'-O-uridine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-43) [ka] Compound (R P )-43 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ *The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 58 mg of compound (R) after lyophilization. P )-43 was obtained (yield 62%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-43 is 1 H NMR(600MHz,D2O):δ 8.05(d,J=8.1Hz,1H),5.96-5.94(m,2H),4.42(t,J=5.0Hz,1H),4.39(t,J=5.0Hz,1H),4.29-4.24(m,3H); 13 C NMR(150MHz,D2O)δ 166.3,151.8,141.9,102.6,88.4,83.2(d,J=9.7Hz),73.8,69.6,64.8(d,J=5.6Hz); 31 P NMR(162MHz,D2O):δ 41.1(d,J=30.9Hz),-6.8(d,J=30.9Hz)HRMS(ESI-TOF)m / z:C9H 13 N2O 11 P2S[MH] - Calculated for: 418.9715, Found: 418.9705; Retention time: 2.87 min (Method 1).

[0340] Example 27 5'-O-uridine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-43) [ka] Compound (S) was prepared according to General Procedure A. P )-43 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ *The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 60 mg of compound (S) after lyophilization. P )-43 was obtained (yield=64%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-43 is 1 H NMR(600MHz,D2O):δ 8.09(d,J=8.1Hz,1H),5.99-5.97(m,2H),4.42(t,J=4.8Hz,1H),4.40(t,J=4.8Hz,1H),4.31-4.29(m,1H),4.29-4.25(m,2H); 13 C NMR(150MHz,D2O):δ 166.3,151.8,142.0,102.6,88.5,83.1(d,J=9.5Hz),73.8,69.6,64.5(d,J=6.5Hz); 31 P NMR(162MHz,D2O)δ 41.7(d,J=28.5Hz),-9.7(d,J=28.5Hz);HRMS(ESI-TOF)m / z:C9H 13 N2O 11 P2S[MH] - Calculated for: 418.9715, Found: 418.9705; Retention time: 2.30 min (Method 1).

[0341] Example 28 5'-O-Deoxyadenosine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-44) [ka] Compound (R P )-44 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ *The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected deoxyadenosine compound 13-1 (281 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 60 mg of compound (R P )-44 was obtained (yield=63%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-44 is 1 H NMR(600MHz,D2O):δ 8.47(s,1H),8.11(s,1H),6.41(t,J=6.7Hz,1H),4.30-4.28(m,1H),4.22(ddd,J=11.2,7.4,3.7Hz,1H) ,4.16(ddd,J=11.2,6.6,3.8Hz,1H),2.79(dt,J=13.6,6.7Hz,1H),2.59(ddd,J=13.6,6.7,4.0Hz,1H); 13 C NMR(150MHz,D2O):δ 155.2,152.4,148.3,139.9,118.3,85.6(d,J=9.5Hz),83.5,71.0,65.1(d,J=6.0Hz),39.0; 31 P NMR(162MHz,D2O)δ 41.1(d,J=29.4Hz),-8.9(d,J=29.4Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O8P2S[MH] - Calculated for: 426.0038, Found: 426.0033; Retention time: 6.17 min (Method 1).

[0342] Example 29 5'-O-Deoxyadenosine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-44) [ka] Compound (S) was prepared according to General Procedure A. P)-44 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected deoxyadenosine compound 13-1 (281 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 55 mg of compound (S) after lyophilization. P )-44 was obtained (yield=57%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-44 is 1 H NMR(600MHz,D2O):δ 8.47(s,1H),8.08(s,1H),6.39(t,J=6.7Hz,1H),4.79-4.75(m,1H),4.31-4.29(m,1H),4.22(ddd,J=11.5,7.7,3 .9Hz,1H),4.16(ddd,J=11.5,6.6,3.7Hz,1H),2.77(dt,J=13.6,6.7Hz,1H),2.59(ddd,J=13.6,6.7,3.6Hz,1H); 13 C NMR(150MHz,D2O):δ 154.9,152.0,148.2,140.0,118.2,85.6(d,J=9.4Hz),83.7,71.2,65.4(d,J=6.3Hz),39.2; 31 P NMR(162MHz,D2O):δ 42.0(d,J=28.0Hz),-10.8(d,J=28.0Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O8P2S[MH] - Calculated for: 426.0038, Found: 426.0033; Retention time: 5.49 min (Method 1).

[0343] Example 30 5'-O-Adenosine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-45) [ka] Compound (R P )-45 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected adenosine compound 14-1 (341 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 55 mg of compound (R P )-45 was obtained (yield=56%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-45 is, 1 H NMR(600MHz,D2O):δ 8.54(s,1H),8.13(s,1H),6.08(d,J=5.4Hz,1H),4.79-4.74(m,1H),4.59(t,J=4.6Hz,1H),4.41-4.39(m,1H),4.31-4.25(m,2H); 13 C NMR(150MHz,D2O):δ 155.4,152.6,148.9,140.0,118.4,87.0,83.8(d,J=9.6Hz),74.4,70.4,65.1(d,J=5.6Hz); 31 P NMR(162MHz,D2O):δ 42.0(d,J=29.2Hz),-8.3(d,J=29.2Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O9P2S[MH] - Calculated for: 441.9987, Found: 441.9988; Retention time: 5.06 min (Method 1).

[0344] Example 31 5'-O-Adenosine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-45) [ka] Compound (S) was prepared according to General Procedure A. P)-45 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected adenosine compound 14-1 (342 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 52 mg of compound (S) after lyophilization. P )-45 was obtained (yield=53%, dr>20:1).

[0345] Preparative scale: Compound (S) was prepared according to general procedure A. P )-45 was reacted with the monophosphate precursor compound 7-1 (0.62 g, 2.0 mmol), (-)-Ψ * The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 620 mg of compound (S) after lyophilization. P )-45 was obtained (yield=63%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-45 is, 1 H NMR(600MHz,D2O)δ 8.55(s,1H),8.09(s,1H),6.06(d,J=5.3Hz,1H),4.74-4.72(m,1H),4.57(t,J=4.6 Hz,1H),4.40-4.37(m,1H),4.29(ddd,J=10.7,7.5,3.0Hz,1H),4.26-4.23(m,1H); 13 C NMR(150MHz,D2O):δ 155.1,152.4,148.6,140.0,118.2,87.0,83.6(d,J=9.4Hz),74.4,70.2,64.7(d,J=6.1Hz); 31 P NMR(162MHz,D2O):δ 42.0(d,J=29.1Hz),-8.1(d,J=29.1Hz);HRMS(ESI-TOF)m / z:C 10 H 14N5O9P2S[MH] - Calculated for: 441.9987, Found: 441.9988; Retention time: 3.64 min (Method 1).

[0346] Example 32 5'-O-Deoxycytidine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-46) [ka] Compound (R P )-46 was treated with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ * The compound 12-1 was obtained from the reagent (128 mg, 0.3 mmol) and the protected deoxycytidine compound 12-1 (217 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 38 mg of compound (R) after lyophilization. P )-46 was obtained (yield=42%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-46 is 1 H NMR(600MHz,D2O):δ 8.04(d,J=7.6Hz,1H),6.33(t,J=6.6Hz,1H),6.13(d,J=7.6Hz,1H),4.63(dt,J=6.6,3.5Hz ,1H),4.25-4.20(m,3H),2.42(ddd,J=13.9,6.6,4.1Hz,1H),2.33(dt,J=13.9,6.6Hz,1H); 13 C NMR(150MHz,D2O):δ 165.8,157.2,142.0,96.5,85.9,85.3(d,J=9.6Hz),70.6,65.1(d,J=5.8Hz),39.3; 31P NMR(162MHz,D2O):δ 42.2(d,J=28.5Hz),-10.0(d,J=28.5Hz);HRMS(ESI-TOF)m / z:C9H 14 N3O9P2S[MH] - Calculated for: 401.9926, Found: 401.9918; Retention time: 2.83 min (Method 1).

[0347] Example 33 5'-O-Deoxycytidine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-46) [ka] Compound (S) was prepared according to general procedure A with minor modifications. P )-46 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ * The compound 12-1 was obtained from the reagent (128 mg, 0.3 mmol) and the protected deoxycytidine compound 12-1 (217 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 41 mg of compound (S) after lyophilization. P )-46 was obtained (yield=45%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-46 is 1 H NMR(600MHz,D2O):δ 8.04(d,J=7.6Hz,1H),6.32(t,J=6.6Hz,1H),6.13(d,J=7.6Hz,1H),4.62(dt,J=6.7,3.5Hz ,1H),4.24-4.18(m,3H),2.42(ddd,J=13.9,6.6,4.1Hz,1H),2.32(dt,J=13.9,6.6Hz,1H); 13C NMR(150MHz,D2O):δ 165.8,157.1,142.0,96.5,85.9,85.3(d,J=9.7Hz),70.7,65.1(d,J=6.2Hz),39.3; 31 P NMR(162MHz,D2O):δ 42.2(d,J=29.1Hz),-9.7(d,J=29.1Hz);HRMS(ESI-TOF)m / z:C9H 14 N3O9P2S[MH] - Calculated for: 401.9926, Found: 401.9918; Retention time: 2.29 min (Method 1).

[0348] Example 34 5'-O-Cytidine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-47) [ka] Compound (R P )-47 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected cytidine compound 15-1 (277 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 35 mg of compound (R) after lyophilization. P )-47 was obtained (yield=37%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-47 is 1 H NMR(600MHz,D2O)δ 8.06(d,J=7.6Hz,1H),6.15(d,J=7.6Hz,1H),6.00(d,J=3.8Hz,1H),4.40(t,J=5.1Hz,1H),4.35-4.25(m,4H); 13C NMR(150MHz,D2O):δ 165.9,157.4,141.8,96.5,89.3,82.6(d,J=9.9Hz),74.2,69.2,64.6(d,J=5.9Hz); 31 P NMR(162MHz,D2O):δ 41.3(d,J=29.1Hz),-6.8(d,J=29.1Hz);HRMS(ESI-TOF)m / z:C9H 14 N3O 10 P2S[MH] - Calculated for: 417.9875, Found: 417.9983; Retention time: 5.75 min (Method 2).

[0349] Example 35 5'-O-Cytidine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-47) [ka] Compound (S) was prepared according to general procedure A with minor modifications. P )-47 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected cytidine compound 15-1 (277 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 32 mg of compound (S) after lyophilization. P )-47 was obtained (yield=34%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-47 is 1 H NMR(600MHz,D2O):δ 8.10(d,J=7.6Hz,1H),6.14(d,J=7.6Hz,1H),6.00(d,J=4.1Hz,1H),4.41(t,J=5.0Hz,1H),4.33(t,J=4.6Hz,1H),4.31-4.27(m,3H); 13C NMR(150MHz,D2O):δ 166.1,157.6,141.9,96.6,89.3,82.6(d,J=9.7Hz),74.3,69.2,64.2(d,J=6.6Hz); 31 P NMR(162MHz,D2O):δ 41.2(d,J=29.3Hz),-6.7(d,J=29.3Hz);HRMS(ESI-TOF)m / z:C9H 14 N3O 10 P2S[MH] - Calculated for: 417.9875, Found: 417.9983; Retention time: 4.97 min (Method 2).

[0350] Example 36 5'-O-Deoxyguanosine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-48) [ka] Compound (R P )-48 was reacted with the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (+)-Ψ * The compound 18-1 was obtained from the reagent (85 mg, 0.2 mmol) and the protected deoxyguanosine compound 18-1 (185 mg, 0.5 mmol). The coupling step was carried out with 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 32 mg of compound (R) after lyophilization. P )-48 was obtained (yield=33%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-48 is 1H NMR(600MHz,D2O):δ 8.14(s,1H),6.29(t,J=6.9Hz,1H),4.29-4.26(m,1H),4.21-4.18(m,2H),2.80(dt,J=13.8,6.9Hz,1H),2.51(ddd,J=13.8,6.9,3.5Hz,1H); 13 C NMR(150MHz,D2O):δ 158.8,153.8,151.3,137.7,116.1,85.5(d,J=9.5Hz),83.5,71.3,65.4(d,J=6.0Hz),38.6; 31 P NMR(162MHz,D2O):δ 42.3(d,J=28.5Hz),-10.2(d,J=28.5Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O9P2S[MH] - Calculated for: 441.9993, Found: 441.9999; Retention time: 9.03 min (Method 2).

[0351] Example 37 5'-O-Deoxyguanosine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-48) [ka] Compound (S) was prepared according to general procedure A with minor modifications. P )-48 was reacted with the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (-)-Ψ * The compound 18-1 was obtained from the reagent (85 mg, 0.2 mmol) and the protected deoxyguanosine compound 18-1 (185 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 31 mg of compound (S) after lyophilization. P)-48 was obtained (yield=31%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-48 is 1 H NMR(600MHz,D2O):δ 8.16(s,1H),6.29(t,J=6.9Hz,1H),4.29-4.26(m,1H),4.23-4.16(m,2H),2.79(dt,J=13.7,6.9Hz,1H),2.52(ddd,J=13.7,6.9,3.5Hz,1H); 13 C NMR(150MHz,D2O):δ 158.8,153.7,151.2,137.7,116.1,85.5(d,J=9.4Hz),83.6,71.4,65.4(d,J=6.2Hz),38.6; 31 P NMR(162MHz,D2O):δ 42.6(d,J=29.4Hz),-10.1(d,J=29.4Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O9P2S[MH] - Calculated for: 441.9993, Found: 441.9999; Retention time: 8.52 min (Method 2).

[0352] Example 38 5'-O-Guanosine Triammonium (R)-Diphosphoro-α-thioate (Compound (R P Preparation of )-49) [ka] Compound (R P )-49 was reacted with the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (+)-Ψ *The compound was obtained from the reagent (85 mg, 0.2 mmol) and the protected guanosine compound 17-1 (245 mg, 0.5 mmol). The coupling step was carried out with 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 34 mg of compound (R) after lyophilization. P )-49 was obtained (yield=33%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-49 is 1 H NMR(600MHz,D2O):δ 8.18(s,1H),5.92(d,J=5.6Hz,1H),4.58-4.54(m,1H),4.40-4.34(m,1H),4.29-4.24(m,2H); 13 C NMR(150MHz,D2O):δ 158.9,153.9,151.7,137.7,116.1,86.9,83.7(d,J=9.7Hz),73.9,70.4,65.2(d,J=5.8Hz); 31 P NMR(162MHz,D2O):δ 42.3(d,J=28.2Hz),-10.2(d,J=28.2Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O 10 P2S[MH] - Calculated for: 457.9942, Found: 457.9951; Retention time: 7.60 min (Method 2).

[0353] Example 39 5'-O-Guanosine Triammonium (S)-Diphosphoro-α-thioate (Compound (S P Preparation of )-49) [ka] Compound (S) was prepared according to general procedure A with minor modifications. P )-49 was reacted with the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (-)-Ψ *The product was obtained from the reagent (85 mg, 0.2 mmol) and the protected guanosine compound 17-1 (245 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 38 mg of the title compound after lyophilization (yield = 37%, dr > 20:1). Physical state: white amorphous solid. Compound (S P )-49 is 1 H NMR(600MHz,D2O):δ 8.21(s,1H),5.92(d,J=5.8Hz,1H),4.78-4.75(m,1H),4.57-4.55(m,1H),4.39-4.36(m,1H),4.30-4.23(m,2H); 13 C NMR(150MHz,D2O):δ 158.8,153.8,151.6,137.7,116.1,86.9,83.6(d,J=9.7Hz),73.9,70.4,65.1(d,J=6.1Hz); 31 P NMR(162MHz,D2O):δ 42.6(d,J=27.0Hz),-10.2(d,J=27.0Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O 10 P2S[MH] - Calculated for: 457.9942, Found: 457.9951; Retention time: 6.79 min (Method 2).

[0354] Example 40 5'-O-2-thiouridine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-50) [ka] Compound (R P )-50 was treated with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ *The product was obtained from the reagent (128 mg, 0.3 mmol) and a regioisomeric mixture of the protected 2-thiouridine derivatives Compounds 16-1 and 30 (286 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 35:65) to give 32 mg of the title compound after lyophilization (yield = 33%, dr > 20:1). Physical state: white amorphous solid. Compound (R P )-50 is 1 H NMR(600MHz,D2O):δ 8.26(d,J=8.2Hz,1H),6.63(d,J=2.8Hz,1H),6.27(d,J=8.2Hz,1H),4.46(dd,J=4.8,2.8Hz,1H),4.42-4.37(m,2H),4.35-4.28(m,2H); 13 C NMR(150MHz,D2O):δ 175.9,162.8,142.3,106.9,93.1,82.7(d,J=9.7Hz),74.6,68.3,63.9(d,J=5.7Hz); 31 P NMR(162MHz,D2O):δ 41.9(d,J=29.0Hz),-9.6(d,J=29.0Hz);HRMS(ESI-TOF)m / z:C9H 13 N2O 10 P2S2[MH] - Calculated for: 434.9487, Found: 434.9483; Retention time: 4.85 min (Method 1).

[0355] Example 41 5'-O-2-Thiouridine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-50) [ka] Compound (S) was prepared according to General Procedure A. P )-50 was treated with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ *The product was obtained from the reagent (128 mg, 0.3 mmol) and a regioisomeric mixture of the protected 2-thiouridine derivatives Compounds 16-1 and 30 (286 mg, 0.5 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 35:65) to give 33 mg of Compound (S) after lyophilization. P )-50 was obtained (yield=34%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-50 is 1 H NMR(600MHz,D2O):δ 8.30(d,J=8.1Hz,1H),6.64(d,J=2.3Hz,1H),6.28(d,J=8.1Hz,1H),4.46-4.43(m,1H),4.40-4.36(m,2H),4.35-4.28(m,2H); 13 C NMR(150MHz,D2O):δ 175.9,162.8,142.4,107.0,93.1,82.7(d,J=9.4Hz),74.6,68.4,63.7(d,J=6.1Hz); 31 P NMR(162MHz,D2O):δ 42.5(d,J=28.9Hz),-10.3(d,J=28.9Hz);HRMS(ESI-TOF)m / z:C9H 13 N2O 10 P2S2[MH] - Calculated for: 434.9487, Found: 434.9483; Retention time: 3.60 min (Method 1).

[0356] FIG. 1 illustrates the LC trace for compound 50.

[0357] Example 42 3'-O-Thymidine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-51) [ka] Compound (R P)-51 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected thymidine compound 23 (173 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 47 mg of compound (R P )-51 was obtained (yield=50%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-51 is, 1 H NMR(600MHz,D2O):δ 7.70(s,1H),6.34(t,J=6.8Hz,1H),5.05(ddt,J=10.7,7.0,3.8Hz,1H),4.27(q,J=3.8Hz,1H),3.88( d,J=3.8Hz,2H),2.63(ddd,J=14.3,6.8,3.8Hz,1H),2.49(ddd,J=14.3,7.0,6.8Hz,1H),1.90(s,3H); 13 C NMR(150MHz,D2O):δ 166.5,151.7,137.6,111.5,85.4(d,J=6.2Hz),84.8,74.6(d,J=5.7Hz),60.7,37.5(d,J=3.7Hz),11.5; 31 P NMR(162MHz,D2O):δ 41.0(d,J=28.5Hz),-8.3(d,J=28.5Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N2O 10 P2S[MH] - Calculated for: 416.9928, Found: 416.9926; Retention time: 7.96 min (Method 3).

[0358] Example 43 3'-O-Thymidine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-51) [ka] Compound (S) was prepared according to General Procedure A. P )-51 was reacted with the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ * Obtained from the reagent (128 mg, 0.3 mmol) and the protected thymidine compound 23 (173 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 50 mg of the title compound after lyophilization (yield = 53%, dr > 20:1). Physical state: white amorphous solid. Compound (S P )-51 is, 1 H NMR(600MHz,D2O):δ 7.69(d,J=1.2Hz,1H),6.35(t,J=7.0Hz,1H),5.05(ddt,J=10.4,7.0,3.6Hz,1H),4.26(dt,J=4.6,3.6Hz,1H),3.90(dd,J=12.7,3 .6Hz,1H),3.87(dd,J=12.7,4.6Hz,1H),2.65(ddd,J=14.3,7.0,3.6Hz,1H),2.46(dt,J=14.3,7.0Hz,1H),1.90(d,J=1.2Hz,3H); 13 C NMR(150MHz,D2O):δ 166.5,151.7,137.6,111.5,85.6(d,J=6.5Hz),85.0,75.1(d,J=6.1Hz),60.9,37.4(d,J=3.5Hz),11.5; 31 P NMR(162MHz,D2O):δ 41.7(d,J=27.8Hz),-10.2(d,J=27.8Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N2O 10 P2S[MH] - Calculated for: 416.9928, Found: 416.9926; Retention time: 8.22 min (Method 3).

[0359] Example 44 Preparation of 5'-O-adenosine triammonium diphosphoro-β-thioate (compound 52) [ka] Compound 52 was prepared according to general procedure A with minor modifications by reacting protected adenosine monophosphate compound 32 (152 mg, 0.2 mmol), (+)-Ψ * The compound 52 was obtained from the reagent (128 mg, 0.3 mmol) and 4-(hydroxymethyl)phenylbenzoate (114 mg, 0.5 mmol). The synthesis of 4-(hydroxymethyl)phenylbenzoate is described in Yang et al., Angew. Chem. Int. Ed. 2016, 55, 9080-9083. Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 42 mg of the title compound after lyophilization (yield = 43%). Physical state: white amorphous solid. Compound 52 was obtained from the following: 1 H NMR(600MHz,D2O):δ 8.55(s,1H),8.19(s,1H),6.11(d,J=5.5Hz,1H),4.68-4.66(m,1H),4.40-4. 38(m,1H),4.30(ddd,J=11.5,6.0,2.9Hz,1H),4.23(dt,J=11.5,3.4Hz,1H); 13 C NMR(150MHz,D2O):δ 155.3,152.5,148.9,140.0,118.4,86.9,83.9(d,J=8.8Hz),74.4,70.2,64.7(d,J=4.6Hz); 31 P NMR(162MHz,D2O):δ 33.5(d,J=30.6Hz),-11.5(d,J=30.6Hz);HRMS(ESI-TOF)m / z:C 10 H 14 N5O9P2S[MH] - Calculated for: 441.9987, Found: 441.9981; Retention time: 3.34 min (Method 1).

[0360] Example 45 5'-O-Azidothymidine triammonium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-53) [ka] Compound (R P )-53 was treated with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and azidothymidine (107 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 67 mg of compound (R) after lyophilization. P )-53 was obtained (yield=57%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-53 is 1 H NMR(600MHz,D2O):δ 7.80(s,1H),6.29(t,J=6.9Hz,1H),4.64(dt,J=6.7,3.4Hz,1H),4.34-4.25(m,3H),2.54-2.45(m,2H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.6,151.7,137.3,111.9,84.8,82.9(d,J=9.8Hz),66.1(d,J=6.1Hz),61.1,36.3,11.7; 31 P NMR(162MHz,D2O):δ 42.3(d,J=27.7Hz),-9.1(d,J=19.7Hz),-24.2(dd,J=27.7,19.7Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 12 P3S[MH] - Calculated for: 521.9651, Found: 521.9650; Retention time: 11.27 min (Method 1).

[0361] Example 46 5'-O-Azidothymidine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-53) [ka] Compound (S) was prepared according to general procedure B. P )-53 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The product was obtained from the reagent (112 mg, 0.26 mmol) and azidothymidine (107 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 70 mg of compound (S) after lyophilization. P )-53 was obtained (yield=59%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-53 is 1 H NMR(600MHz,D2O):δ 7.78(s,1H),6.28(t,J=6.9Hz,1H),4.61(dt,J=7.1,3.7Hz,1H),4.34-4.23(m,3H),2.55-2.45(m,2H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.6,151.7,137.3,111.8,84.9,82.8(d,J=9.8Hz),65.7(d,J=6.4Hz),60.8,36.2,11.7; 31 P NMR(162MHz,D2O):δ 43.3(d,J=26.8Hz),-9.1(d,J=20.0Hz),-23.6(dd,J=26.8,20.0Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 12 P3S[MH] - Calculated for: 521.9651, Found: 521.9650; Retention time: 10.43 min (Program: Method 1).

[0362] Example 47 5'-O-Thymidine triammonium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-54) [ka] Compound (R P )-54 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound 10-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected thymidine compound 10-1 (180 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 54 mg of compound (R) after lyophilization. P )-54 was obtained (yield=48%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-54 is 1 H NMR(600MHz,D2O):δ 7.80(s,1H),6.35(t,J=6.9Hz,1H),4.70(dt,J=6.5,3.4Hz,1H),4.30-4.26(m,2H),4.23-4.2 0(m,1H),2.41(ddd,J=13.9,7.5,6.9Hz,1H),2.35(ddd,J=13.9,6.9,3.6Hz,1H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.6,151.8,137.4,111.8,85.3(d,J=9.6Hz),84.9,70.8,65.7(d,J=6.2Hz),38.5,11.7; 31 P NMR(162MHz,D2O):δ 42.9(d,J=28.2Hz),-8.2(d,J=20.7Hz),-23.5(dd,J=28.2,20.7Hz);HRMS(ESI-TOF)m / z:C 10 H 16 N2O 13 P3S[MH] - Calculated for: 496.9586, Found: 496.9590; Retention time: 5.87 min (Method 1).

[0363] Example 48 5'-O-Thymidine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-54) [ka] Compound (S) was prepared according to general procedure B. P )-54 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected thymidine compound 10-1 (180 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 52 mg of compound (S) after lyophilization. P )-54 was obtained (yield=46%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-54 is 1 H NMR(600MHz,D2O):δ 7.77(s,1H),6.36(t,J=6.9Hz,1H),4.67(dt,J=6.2,3.2Hz,1H),4.29(ddd,J=11.9,7.6,4.0Hz,2H) ,4.25-4.19(m,2H),2.41(dt,J=13.9,6.9Hz,1H),2.35(ddd,J=13.9,6.9,3.2Hz,1H),1.96(s,3H); 13 C NMR(150MHz,D2O):δ 166.6,151.8,137.4,111.8,85.3(d,J=9.6Hz),85.0,70.9,65.6(d,J=6.7Hz),38.4,11.7; 31 P NMR(162MHz,D2O):δ 42.6(d,J=26.7Hz),-7.1(d,J=20.0Hz),-23.3(dd,J=26.7,20.0Hz);HRMS(ESI-TOF)m / z:C 10 H 16 N2O 13 P3S[MH] - Calculated for: 496.9586, Found: 496.9590; Retention time: 4.83 min (Method 1).

[0364] Example 49 5'-O-uridine triammonium (R)-triphosphoro-α-thioate (compound (R PPreparation of )-55) [ka] Compound (R P )-55 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * Obtained from the reagent (112 mg, 0.26 mmol) and the protected uridine compound 11-1 (222 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 53 mg of the title compound after lyophilization (yield = 47%, dr > 20:1). Physical state: white amorphous solid. Compound (R P )-55 is, 1 H NMR(600MHz,D2O):δ 8.04(d,J=8.1Hz,1H),5.99(d,J=5.1Hz,1H),5.97(d,J=8.1Hz,1H),4.46-4.43(m,1H),4.41(t,J=5.1Hz,1H),4.34-4.27(m,3H); 13 C NMR(150MHz,D2O):δ 166.2,151.8,141.9,102.6,88.2,83.2(d,J=9.7Hz),73.8,69.6,65.3(d,J=5.9Hz); 31 P NMR(162MHz,D2O):δ 43.1(d,J=27.7Hz),-8.9(d,J=20.0Hz),-23.6(dd,J=27.7,20.0Hz);HRMS(ESI-TOF)m / z:C9H 14 N2O 14 P3S[MH] - Calculated for: 498.9379, Found: 498.9383; Retention time: 3.67 min (Method 1).

[0365] Example 50 5'-O-uridine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-55) [ka] Compound (S) was prepared according to general procedure B. P )-55 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected uridine compound 11-1 (222 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 30:70) to give 52 mg of compound (S) after lyophilization. P )-55 was obtained (yield=46%, dr>20:1). Physical state: White amorphous solid. Compound S P )-55 is, 1 H NMR(600MHz,D2O):δ 8.10(d,J=8.1Hz,1H),6.00(d,J=5.1Hz,1H),5.98(d,J=8.1Hz,1H),4.45-4.43(m,1H),4.44(t,J=5.1Hz,1H),4.33-4.27(m,3H); 13 C NMR(150MHz,D2O):δ 166.3,151.8,142.1,102.6,88.3,83.2(d,J=9.5Hz),73.8,69.6,64.8(d,J=6.6Hz); 31 P NMR(162MHz,D2O):δ 43.4(d,J=27.1Hz),-8.7(d,J=19.9Hz),-23.5(dd,J=27.1,19.9Hz);HRMS(ESI-TOF)m / z:C9H 14 N2O 14 P3S[MH] - Calculated for: 498.9379, Found: 498.9383; Retention time: 2.91 min (Method 1).

[0366] FIG. 2 illustrates the LC trace for compound 55.

[0367] Example 51 5'-O-Deoxyadenosine triammonium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-56) [ka] Compound (R P )-56 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected deoxyadenosine compound 13-1 (225 mg, 0.4 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 54 mg of compound (R P )-56 was obtained (yield=47%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-56 is 1 H NMR(600MHz,D2O):δ 8.51(s,1H),8.17(s,1H),6.46(t,J=6.7Hz,1H),4.34-4.30(m,1H),4.27(ddd,J=10.9,7.2,3.5Hz,1H) ,4.22(ddd,J=10.9,6.5,3.6Hz,1H),2.82(dt,J=13.6,6.7Hz,1H),2.60(ddd,J=13.6,6.7,3.8Hz,1H); 13 C NMR(150MHz,D2O):δ 155.3,152.5,148.5,140.0,118.4,85.6(d,J=9.5Hz),83.6,71.0,65.6(d,J=6.2Hz),39.1; 31 P NMR(162MHz,D2O):δ 43.2(d,J=27.8Hz),-8.5(d,J=20.2Hz),-23.5(dd,J=27.8,20.2Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 11 P3S[MH] - Calculated for: 505.9702, Found: 505.9688; Retention time: 6.63 min (Method 1).

[0368] Example 52 5'-O-Deoxyadenosine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-56) [ka] Compound (S) was prepared according to general procedure B. P )-56 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The product was obtained from the reagent (112 mg, 0.26 mmol) and the protected deoxyadenosine compound 13-1 (225 mg, 0.4 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 59 mg of the title compound after lyophilization (yield = 51%, dr > 20:1). Physical state: white amorphous solid. Compound (S P )-56 is 1 H NMR(600MHz,D2O):δ 8.50(s,1H),8.11(s,1H),6.42(t,J=6.7Hz,1H),4.32-4.24(m,2H),4.21-4. 17(m,1H),2.80(dt,J=13.6,6.7Hz,1H),2.59(ddd,J=13.6,6.7,3.8Hz,1H); 13 C NMR(150MHz,D2O):δ 155.2,152.3,148.4,140.0,118.3,85.6(d,J=9.6Hz),83.6,71.0,65.5(d,J=6.4Hz),39.0; 31 P NMR(162MHz,D2O):δ 43.4(d,J=27.4Hz),-7.4(d,J=20.7Hz),-23.2(dd,J=27.4,20.7Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 11 P3S[MH] - Calculated for: 505.9702, Found: 505.9688; Retention time: 6.05 min (Method 1).

[0369] Example 53 5'-O-Adenosine triammonium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-57) [ka] Compound (R P )-57 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected adenosine compound 14-1 (273 mg, 0.4 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 59 mg of compound (R P )-57 was obtained (yield=50%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-57 is 1 H NMR(600MHz,D2O):δ 8.58(s,1H),8.21(s,1H),6.12(d,J=5.9Hz,1H),4.62(dd,J=5.1,3.6Hz,1H),4.43- 4.41(m,1H),4.34(ddd,J=10.4,7.5,2.7Hz,1H),4.28(ddd,J=11.9,6.1,2.7Hz,1H); 13 C NMR(150MHz,D2O):δ 155.5,152.7,149.0,140.0,118.5,86.7,83.9(d,J=9.7Hz),74.3,70.4,65.5(d,J=5.8Hz); 31 P NMR(162MHz,D2O):δ 43.4(d,J=27.6Hz),-10.9(d,J=19.6Hz),-24.1(dd,J=27.6,19.6Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 12 P3S[MH] - Calculated for: 521.9651, Found: 521.9662; Retention time: 5.85 min (Method 1).

[0370] Example 54 5'-O-Adenosine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-57) [ka] Compound (S) was prepared according to general procedure B. P )-57 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected adenosine compound 14-1 (273 mg, 0.4 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 61 mg of compound (S) after lyophilization. P )-57 was obtained (yield=52%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-57 is 1 H NMR(600MHz,D2O):δ 8.63(s,1H),8.18(s,1H),6.12(d,J=5.7Hz,1H),4.62-4.59(m,1H),4.43-4.40 (m,1H),4.35(ddd,J=11.0,7.7,3.0Hz,1H),4.28(ddd,J=11.7,5.5,3.0Hz,1H); 13 C NMR(150MHz,D2O):δ 155.4,152.6,148.9,140.1,118.4,86.8,83.8(d,J=9.5Hz),74.3,70.4,65.1(d,J=6.5Hz); 31 P NMR(162MHz,D2O):δ 43.4(d,J=26.9Hz),-8.5(d,J=20.1Hz),-23.5(dd,J=26.9,20.1Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 12 P3S[MH] - Calculated for: 521.9651, Found: 521.9662; Retention time: 4.52 min (Method 1).

[0371] Example 55 5'-O-Deoxycytidine triammonium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-58) [ka] Compound (R P )-58 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound 12-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected deoxycytidine compound 12-1 (174 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 40 mg of compound (R) after lyophilization. P )-58 was obtained (yield=36%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-58 is 1 H NMR(600MHz,D2O)δ 8.02(d,J=7.6Hz,1H),6.33(t,J=6.7Hz,1H),6.14(d,J=7.6Hz,1H),4.66-4.62(m,1H) ,4.30-4.24(m,2H),4.24-4.19(m,1H),2.44-2.38(m,1H),2.33(dt,J=13.9,6.7,1H); 13 C NMR(150MHz,D2O):δ 166.0,157.4,141.8,96.5,85.8,85.3(d,J=9.6Hz),70.4,65.4(d,J=6.0Hz),39.3; 31 P NMR(162MHz,D2O)δ 43.1(d,J=27.4Hz),-8.1(d,J=20.4Hz),-23.4(dd,J=27.4,20.4Hz);HRMS(ESI-TOF)m / z:C9H 15 N3O 12 P3S[MH] -Calculated for: 481.9589, Found: 481.9575; Retention time: 7.58 min (Method 2).

[0372] Example 56 5'-O-Deoxycytidine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-58) [ka] Compound (S) was prepared according to general procedure B with minor modifications. P )-58 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The compound 12-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected deoxycytidine compound 12-1 (174 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) to give 41 mg of compound (S) after lyophilization. P )-58 was obtained (yield=37%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-58 is 1 H NMR(600MHz,D2O)δ 8.07(d,J=7.6Hz,1H),6.34(t,J=6.6Hz,1H),6.16(d,J=7.6Hz,1H),4.64(dt,J=6.8,3.6H z,1H),4.31-4.21(m,3H),2.42(ddd,J=14.1,6.6,4.0Hz,1H),2.33(dt,J=14.1,6.6,1H); 13 C NMR(150MHz,D2O):δ 165.7,156.9,142.1,96.5,86.0,85.4(d,J=9.7Hz),70.7,65.4(d,J=6.3Hz),39.4; 31P NMR(162MHz,D2O):δ 43.4(d,J=27.1Hz),-9.9(d,J=19.9Hz),-23.9(dd,J=27.1,19.9Hz);HRMS(ESI-TOF)m / z:C9H 15 N3O 12 P3S[MH] - Calculated for: 481.9589, Found: 481.9575; Retention time: 6.66 min (Method 2).

[0373] Example 57 5'-O-Cytidine trisodium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-59) [ka] Compound (R P )-59 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound 15-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected cytidine compound 15-1 (222 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 45 mg of compound (R P )-59 was obtained. (Yield=38%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-59 is 1H NMR(600MHz,D2O):δ 8.06(d,J=7.6Hz,1H),6.15(d,J=7.6Hz,1H),6.01(d,J=4.3Hz,1H),4.47(t ,J=5.2Hz,1H),4.36(t,J=4.7Hz,1H),4.36-4.32(m,2H),4.31-4.28(m,1H); 13 C NMR(150MHz,D2O):δ 166.2,157.8,141.7,96.6,89.0,82.6(d,J=9.7Hz),74.2,69.1,64.9(d,J=5.6Hz); 31 P NMR(162MHz,D2O):δ 42.8(d,J=27.9Hz),-5.8(d,J=20.2Hz),-22.5(dd,J=27.9,20.2Hz);HRMS(ESI-TOF)m / z:C9H 15 N3O 13 P3S[MH] - Calculated for: 497.9538, Found: 497.9533; Retention time: 6.81 min (Method 2).

[0374] Example 58 5'-O-Cytidine trisodium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-59) [ka] Compound (S) was prepared according to general procedure B with minor modifications. P )-59 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ *The compound 15-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected cytidine compound 15-1 (222 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after workup was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 47 mg of compound (S P )-59 was obtained (yield=40%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-59 is 1 H NMR(600MHz,D2O):δ 8.13(d,J=7.6Hz,1H),6.17(d,J=7.6Hz,1H),6.02(d,J=4.3Hz,1H),4.45(t,J=5.2Hz,1H),4.37-4.32(m,3H),4.30(dt,J=5.2,2.5Hz,1H); 13 C NMR(150MHz,D2O):δ 166.2,157.8,141.9,96.7,89.1,82.7(d,J=9.7Hz),74.3,69.2,64.4(d,J=5.9Hz); 31 P NMR(162MHz,D2O):δ 43.2(d,J=27.1Hz),-8.7(d,J=19.3Hz),-23.5(dd,J=27.1,19.3Hz);HRMS(ESI-TOF)m / z:C9H 15 N3O 13 P3S[MH] - Calculated for: 497.9538, Found: 497.9533; Retention time: 5.79 min (Method 2).

[0375] Example 59 5'-O-Deoxyguanosine trisodium (R)-triphosphoro-α-thioate (compound (RP Preparation of )-60) [ka] Compound (R P )-60 was treated with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound 18-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected deoxyguanosine compound 18-1 (148 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 35 mg of compound (R P )-60 was obtained (yield=29%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-60 is, 1 H NMR(600MHz,D2O)δ 8.16(s,1H),6.32(t,J=6.8Hz,1H),4.31-4.28(m,1H),4.27-4.23(m,2H),2.83(dt,J=13.7,6.8Hz,1H),2.51(ddd,J=13.7,6.8,3.4Hz,1H); 13 C NMR(150MHz,D2O)δ 159.0,153.8,151.4,137.8,116.1,85.6(d,J=9.6Hz),83.6,71.2,65.7(d,J=6.1Hz),38.6; 31 P NMR(162MHz,D2O)δ 43.3(d,J=27.3Hz),-5.2(d,J=19.2Hz),-21.7(dd,J=27.3,19.2Hz)HRMS(ESI-TOF)m / z:C 10 H15 N5O 11 P3S[MH] - Calculated for: 505.9702, Found: 505.9688; Retention time: 9.78 min (Method 2).

[0376] Example 60 5'-O-Deoxyguanosine trisodium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-60) [ka] Compound (S) was prepared according to general procedure B with minor modifications. P )-60 was treated with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The compound 18-1 was obtained from the reagent (112 mg, 0.26 mmol) and the protected deoxyguanosine compound 18-1 (148 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 39 mg of compound (S P )-60 was obtained (yield=32%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-60 is, 1 H NMR(600MHz,D2O)δ 8.18(s,1H),6.30(t,J=6.8Hz,1H),4.83-4.79(m,1H),4.31-4.21(m,3H),2.81(dt,J=13.7,6.8Hz,1H),2.52(ddd,J=13.7,6.8,3.7Hz,1H); 13C NMR(150MHz,D2O)δ 159.1,153.9,151.3,137.9,116.2,85.6(d,J=9.4Hz),83.6,71.1,65.6(d,J=6.3Hz),38.6; 31 P NMR(162MHz,D2O)δ 43.4(d,J=27.5Hz),-5.8(d,J=20.1Hz),-22.4(dd,J=27.5,20.1Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 11 P3S[MH] - Calculated for: 505.9702, Found: 505.9688; Retention time: 9.17 min (Method 2).

[0377] Example 61 5'-O-guanosine trisodium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-61) [ka] Compound (R P )-61 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The product was obtained from the reagent (112 mg, 0.26 mmol) and the protected guanosine compound 17-1 (196 mg, 0.4 mmol). The coupling step was carried out with 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 39 mg of compound (R P)-61 was obtained (yield=31%, dr>20:1). Physical state: white amorphous solid. Compound (R P )-61 is, 1 H NMR(600MHz,D2O):δ 8.20(s,1H),5.93(d,J=5.9Hz,1H),4.64-4.62(m,1H),4.41-4.37(m,1H),4.33(ddd,J=10.8,7.9,2.7Hz,1H),4.27(ddd,J=10.8,5.9,3.0Hz,1H); 13 C NMR(150MHz,D2O)δ 159.1,154.0,151.7,137.7,116.1,86.6,83.7(d,J=9.3Hz),73.8,70.3,65.5(d,J=5.4Hz); 31 P NMR(162MHz,D2O)δ 43.0(d,J=28.6Hz),-5.8(d,J=19.9Hz),-22.4(dd,J=28.6,19.9Hz);HRMS(ESI-TOF)m / z:C 10 H 15 N5O 13 P3S[MH] - Calculated for: 537.9600, Found: 537.9592; Retention time: 8.92 min (Method 2).

[0378] Example 62 5'-O-Guanosine Trisodium (S)-triphosphoro-α-thioate (Compound (S P Preparation of )-61) [ka] Compound (S) was prepared according to general procedure B with minor modifications. P )-61 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ *The product was obtained from the reagent (112 mg, 0.26 mmol) and the protected guanosine compound 17-1 (196 mg, 0.4 mmol). The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 41 mg of compound (S P )-61 was obtained (yield=33%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-61 is, 1 H NMR(600MHz,D2O):δ 8.26(s,1H),5.93(d,J=6.0Hz,1H),4.82-4.79(m,1H),4.64-4.61(m,1H),4.41 -4.37(m,1H),4.34(ddd,J=10.8,7.6,3.0Hz,1H),4.27(dt,J=10.8,4.7Hz,1H); 13 C NMR(150MHz,D2O):δ 159.1,154.0,151.7,137.9,116.2,86.7,83.8(d,J=9.3Hz),73.7,70.4,65.1(d,J=6.7Hz); 31 P NMR(162MHz,D2O):δ 43.2(d,J=27.1Hz),-5.8(d,J=20.0Hz),-22.4(dd,J=27.1,20.0Hz);HRMS(ESI-TOF) m / z:C 10 H 15 N5O 13 P3S[MH] - Calculated for: 537.9600, Found: 537.9592; Retention time: 7.61 min (Method 2).

[0379] Example 63 5'-O-2-thiouridine trisodium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-62) [ka] Compound (R P )-62 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The product was obtained from the reagent (112 mg, 0.26 mmol) and a regioisomeric mixture of protected 2-thiouridine derivatives Compounds 16-1 and 30 (229 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 35 mg of Compound (R P )-62 was obtained (yield=29%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-62 is 1 H NMR(600MHz,D2O):δ 8.23(d,J=8.0Hz,1H),6.69(s,1H),6.25(d,J=8.0Hz,1H),4.48-4.40(m,3H),4.39-4.31(m,2H); 13 C NMR(150MHz,D2O):δ 176.5,164.5,142.2,106.8,93.1,82.7(d,J=9.7Hz),74.7,68.3,64.4(d,J=5.7Hz); 31 P NMR(162MHz,D2O):δ 42.6(d,J=28.3Hz),-6.0(d,J=20.3Hz),-22.7(dd,J=28.3,20.3Hz);HRMS(ESI-TOF) m / z:C9H 14 N2O 13 P3S2[MH] -Calculated for: 514.9150, Found: 514.9168; Retention time: 5.44 min (Method 1).

[0380] Example 64 5'-O-2-Thiouridine trisodium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-62) [ka] Compound (S) was prepared according to general procedure B. P )-62 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The product was obtained from the reagent (112 mg, 0.26 mmol) and a regioisomeric mixture of protected 2-thiouridine derivatives Compounds 16-1 and 30 (229 mg, 0.4 mmol). Deprotection was carried out at room temperature. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 40:60) followed by reverse-phase chromatography on C18 silica gel (1 M TEAA / MeCN in water, 100:0 to 95:5). Fractions containing the product were pooled and lyophilized. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone. After drying under high vacuum, 33 mg of Compound (S) was obtained. P )-62 was obtained (yield=27%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-62 is 1 H NMR(600MHz,D2O):δ 8.34(d,J=8.1Hz,1H),6.67(s,1H),6.28(d,J=8.1Hz,1H),4.48-4.43(m,2H),4.42-4.38(m,2H),4.35-4.31(m,1H); 13 C NMR(150MHz,D2O):δ 176.2,163.6,142.5,107.0,93.2,82.8(d,J=9.4Hz),74.7,68.2,63.7(d,J=6.8Hz); 31P NMR(162MHz,D2O):δ 42.9(d,J=27.1Hz),-6.2(d,J=20.0Hz),-22.6(dd,J=27.1,20.0Hz);HRMS(ESI-TOF)m / z:C9H 14 N2O 13 P3S2[MH] - Calculated for: 514.9150, Found: 514.9168; Retention time: 4.20 min (Method 1).

[0381] Example 65 3'-O-Thymidine triammonium (R)-triphosphoro-α-thioate (compound (R P Preparation of )-63) [ka] Compound (R P )-63 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected thymidine compound 23 (138 mg, 0.4 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 57 mg of compound (R P )-63 was obtained (yield=51%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-63 is 1 H NMR(600MHz,D2O)δ 7.69(s,1H),6.34(t,J=6.9Hz,1H),5.09(td,J=6.9,3.3Hz,1H),4.31(q,J=3.7Hz,1H),3.90 -3.84(m,2H),2.63(ddd,J=14.3,6.9,3.3Hz,1H),2.48(dt,J=14.3,6.9Hz,1H),1.90(s,3H); 13C NMR(150MHz,D2O)δ 166.5,151.7,137.6,111.5,85.5(d,J=5.8Hz),85.0,75.6(d,J=6.0Hz),61.0,37.5(d,J=3.9Hz),11.5; 31 P NMR(162MHz,D2O)δ 42.8(d,J=26.2Hz),-10.0(d,J=19.3Hz),-23.8(dd,J=26.2,19.3Hz);HRMS(ESI-TOF)m / z:C 10 H 16 N2O 13 P3S[MH] - Calculated for: 496.9551, Found: 496.9603; Retention time: 8.45 min (Method 3).

[0382] Example 66 3'-O-Thymidine triammonium (S)-triphosphoro-α-thioate (compound (S P Preparation of )-63) [ka] Compound (S) was prepared according to general procedure B. P )-63 was reacted with the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (−)-Ψ * The compound was obtained from the reagent (112 mg, 0.26 mmol) and the protected thymidine compound 23 (138 mg, 0.4 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 60 mg of compound (S) after lyophilization. P )-63 was obtained (yield 53%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-63 is 1H NMR(600MHz,D2O):δ 7.69(d,J=1.1Hz,1H),6.36(dd,J=7.1,6.4Hz,1H),5.10(ddt,J=10.0,6.4,3.1Hz,1H),4.29(q,J=3.6Hz,1H) ,3.91-3.85(m,2H),2.66(ddd,J=14.4,6.4,3.1Hz,1H),2.47(dt,J=14.4,7.1Hz,1H),1.90(d,J=1.1Hz,3H); 13 C NMR(150MHz,D2O)δ 166.5,151.7,137.7,111.5,85.7(d,J=6.3Hz),85.0,75.7(d,J=6.0Hz),61.0,37.4(d,J=3.7Hz),11.5; 31 P NMR(162MHz,D2O)δ 42.7(d,J=26.2Hz),-7.8(d,J=20.1Hz),-23.3(dd,J=26.2,20.1Hz);HRMS(ESI-TOF)m / z:C 10 H 16 N2O 13 P3S[MH] - Calculated for: 496.9551, Found: 496.9603; Retention time: 8.81 min (Method 3).

[0383] Example 67 P 1 ,P 2 -di-(5'-O-uridine)diammonium(R)-diphosphoro-1-thioate (compound (R P Preparation of )-64) [ka] Compound (R P )-64 was reacted with the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (+)-Ψ *The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 57 mg of compound (R P )-64 was obtained (yield=42%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-64 is 1 H NMR(600MHz,D2O)δ 8.03(d,J=8.0Hz,1H),7.96(d,J=8.2Hz,1H),6.00-5.94(m,4H),4.41-4.36(m,4H),4.36-4.27(m,4H),4.27-4.19(m,2H); 13 C NMR(150MHz,D2O)δ 165.65,165.64,151.27,151.26,141.4,141.3,102.24,102.16,88.0,87.9,82.8(d,J=9.4H z),82.6(d,J=9.7Hz),73.38,73.35,69.31,69.29,64.54(d,J=5.9Hz),64.53(d,J=6.5Hz); 31 P NMR(162MHz,D2O)δ 43.3(d,J=27.7Hz),-12.1(d,J=27.7Hz);HRMS(ESI-TOF)m / z:C 18 H 23 N4O 16 P2S[MH] - Calculated for: 645.0310, Found: 645.0323; Retention time: 4.85 min (Method 1).

[0384] Example 68 P 1 ,P 2 -Di-(5'-O-uridine)diammonium (S)-diphosphoro-1-thioate (compound (S P Preparation of )-64) [ka] Compound (S) was prepared according to general procedure C.P )-64 was reacted with the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (-)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 54 mg of compound (S) after lyophilization. P )-64 was obtained (yield=40%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-64 is 1 H NMR(600MHz,D2O):δ 8.01(dd,J=8.1,1.4Hz,1H),7.94(dd,J=8.1,1.4Hz,1H),5.98-5.94(m,4H), 4.42-4.39(m,1H),4.39-4.35(m,3H),4.32-4.24(m,6H),4.23-4.18(m,1H); 13 C NMR(150MHz,D2O):δ 165.63,165.62,151.2,141.4,141.3,102.3,102.2,87.83,87.77,82.7(d,J=9.5Hz) ,82.6(d,J=10.0Hz),73.4,73.3,69.29,69.28,64.7(d,J=6.0Hz),64.5(d,J=5.6Hz). 31 P NMR(162MHz,D2O)δ 43.1(d,J=26.3Hz),-12.1(d,J=26.3Hz);HRMS(ESI-TOF)m / z:C 18 H 23 N4O 16 P2S[MH] - Calculated for: 645.0310, Found: 645.0323; Retention time: 3.53 min (Method 1).

[0385] Example 69 P 1 -(5'-O-adenosine)-P 2 -(5'-O-uridine)diammonium (R)-diphosphoro-2-thioate (compound (R PPreparation of )-65) [ka] Compound (R P )-65 was reacted with the protected adenosine monophosphate compound 32 (152 mg, 0.2 mmol), (+)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 77 mg of compound (R P )-65 was obtained (yield=55%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-65 is, 1 H NMR(600MHz,D2O):δ 8.45(s,1H),8.19(s,1H),7.79(d,J=8.1Hz,1H),6.08(d,J=5.8Hz,1H),5.84(d,J=4.8Hz,1H),5.71(d,J=8.1Hz,1H),4. 79-4.76(m,1H),4.54(dd,J=5.0,3.7Hz,1H),4.40-4.37(m,1H),4.35(ddd,J=11.4,4.6,2.8Hz,1H),4.31-4.18(m,6H); 13 C NMR(150MHz,D2O):δ 165.7,155.1,152.4,151.4,148.9,141.3,139.9,118.4,102.2,88.3,86.9,83.8(d,J=9 .4Hz),83.0(d,J=9.7Hz),74.2,73.9,70.4,69.7,65.3(d,J=5.5Hz),64.8(d,J=6.5Hz); 31 P NMR(162MHz,D2O):δ 43.5(d,J=27.7Hz),-12.0(d,J=27.7Hz);HRMS(ESI-TOF)m / z:C 19 H 24 N7O 14 P2S[MH] -Calculated for: 668.0582, Found: 668.0587; Retention time: 7.82 min (Method 1).

[0386] Example 70 P 1 -(5'-O-adenosine)-P 2 -(5'-O-uridine)diammonium (S)-diphosphoro-2-thioate (compound (S P Preparation of )-65) [ka] Compound (S) was prepared according to general procedure C. P )-65 was reacted with the protected adenosine monophosphate compound 32 (152 mg, 0.2 mmol), (−)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 90 mg of compound (S) after lyophilization. P )-65 was obtained (yield=64%, dr>20:1).

[0387] Preparative scale: Compound (S) was prepared according to general procedure C. P )-65 was reacted with the protected adenosine monophosphate compound 32 (1.52 g, 2.0 mmol), (-)-Ψ * The compound was obtained from the reagent (1.28 g, 3.0 mmol) and the protected uridine compound 11-1 (2.78 g, 5.0 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 1.01 g of compound (S) after lyophilization. P )-65 was obtained (yield=72%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-65 is, 1H NMR(600MHz,D2O):δ 8.43(s,1H),8.14(s,1H),7.71(d,J=8.1Hz,1H),6.06(d,J=5.9Hz,1H),5.82(d,J=5.1Hz,1H),5.67(d ,J=8.1Hz,1H),4.56(dd,J=5.0,3.6Hz,1H),4.39-4.37(m,1H),4.31-4.28(m,1H),4.28-4.20(m,6H); 13 C NMR(150MHz,D2O):δ 165.6,155.0,152.3,151.4,148.9,141.1,139.8,118.3,102.2,88.1,86.7,83.8(d,J=9. 6Hz),83.0(d,J=10.1Hz),74.2,74.0,70.5,69.7,65.30(d,J=5.1Hz),64.26(d,J=5.4Hz); 31 P NMR(162MHz,D2O):δ 43.2(d,J=25.8Hz),-12.0(d,J=25.8Hz);HRMS(ESI-TOF)m / z:C 19 H 24 N7O 14 P2S[MH] - Calculated for: 668.0582, Found: 668.0587; Retention time: 5.20 min (Method 1).

[0388] Example 71 P 1 -(5'-O-adenosine)-P 2 -(5'-O-uridine)diammonium(R)-diphosphoro-1-thioate (compound (R P Preparation of )-66) [ka] Compound (R P )-66 was reacted with the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (+)-Ψ *The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected adenosine compound 14-1 (342 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 72 mg of compound (R P )-66 was obtained (yield=51%, dr>20:1). Physical state: White amorphous solid. Compound (R P )-66 is 1 H NMR(600MHz,D2O):δ 8.56(s,1H),8.16(s,1H),7.69(d,J=8.1Hz,1H),6.08(d,J=5.8Hz,1H),5.83(d,J=5.0Hz,1H),5.70(d,J=8.1Hz,1H),4.53(dd,J =5.0,3.6Hz,1H),4.42-4.39(m,1H),4.33(ddd,J=11.6,4.1,2.4Hz,1H),4.31-4.22(m,5H),4.17(ddd,J=11.6,5.3,2.6Hz,1H); 13 C NMR(150MHz,D2O)δ 165.6,155.1,152.4,151.4,148.9,141.0,140.0,118.3,102.3,88.2,87.0,83.8(d,J=9 .6Hz),83.1(d,J=9.4Hz),74.4,74.0,70.6,69.6,65.4(d,J=6.4Hz),65.0(d,J=5.2Hz); 31 P NMR(162MHz,D2O)δ 43.5(d,J=27.3Hz),-12.0(d,J=27.3Hz);HRMS(ESI-TOF)m / z:C 19 H 24 N7O 14 P2S[MH] - Calculated for: 668.0582, Found: 668.0557; Retention time: 7.11 min (Method 1).

[0389] Example 72 P 1 -(5'-O-adenosine)-P 2-(5'-O-uridine)diammonium (S)-diphosphoro-1-thioate (compound (S P Preparation of )-66) [ka] Compound (S) was prepared according to general procedure C. P )-66 was reacted with protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (-)-Ψ * The compound was obtained from the reagent (128 mg, 0.3 mmol) and the protected adenosine compound 14-1 (342 mg, 0.5 mmol). Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 20:80) to give 79 mg of compound (S) after lyophilization. P )-66 was obtained (yield=56%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-66 is 1 H NMR(600MHz,D2O)δ 8.51(s,1H),8.16(s,1H),7.66(d,J=8.1Hz,1H),6.07(d,J=5.9Hz,1H),5.82(d,J=5.1Hz,1H),5.69(d,J=8.1Hz,1H),4.7 9-4.76(m,1H),4.54(dd,J=4.9,3.3Hz,1H),4.42-4.39(m,1H),4.33-4.27(m,3H),4.26-4.20(m,3H),4.18-4.14(m,1H); 13 C NMR(150MHz,D2O)δ 165.6,155.1,152.4,151.4,149.0,141.0,139.9,118.3,102.3,88.1,86.7,83.8(d,J=1 0.0Hz),83.1(d,J=9.6Hz),74.3,73.9,70.6,69.6,65.7(d,J=5.9Hz),64.9(d,J=5.2Hz); 31 P NMR(162MHz,D2O)δ 43.1(d,J=26.1Hz),-12.1(d,J=26.1Hz);HRMS(ESI-TOF)m / z:C 19 H 24 N7O 14 P2S[MH]- Calculated for: 668.0582, Found: 668.0557; Retention time: 5.45 min (Method 1).

[0390] Example 73 P 1 -(5'-O-7-methylguanosine)-P 2 -(5'-O-guanosine) disodium (R)-triphosphoro-1-thioate (compound (R P Preparation of )-67) [ka] A flame-dried round-bottom flask equipped with a stir bar was charged with protected guanosine diphosphate compound 40 (1.26 g, 1.0 mmol, 1.0 equiv; 75 wt% purity), and the flask was capped with a septum. Anhydrous DMSO (10 mL) and DBU (0.90 mL, 6.0 mmol, 6.0 equiv) were added, and the mixture was stirred for 10 min. Subsequently, 3 Å molecular sieves (1.0 g) and (+)-Ψ *The reagent (1.08 g, 2.5 mmol, 2.5 equiv.) was added, and the reaction was stirred at room temperature for 1 h. Protected 7-methylguanosine compound 36 (0.90 g, 2.0 mmol, 2.0 equiv.) was then added, followed by another portion of DBU (1.05 mL, 7.0 mmol, 7.0 equiv.), and the mixture was stirred for an additional 5 h. Upon completion of the reaction, the resulting mixture was filtered, and the solid residue was washed with approximately 2 mL of DMSO. The filtrate was distributed among six 50 mL centrifuge tubes, each containing a solution of 0.2 M NaClO4 in acetone (40 mL). The resulting suspension was centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the pellet was washed twice with acetone. The solid residue from each centrifuge tube was redissolved in a minimal amount of water, combined, and purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, 0:100 to 40:60). The product-containing fractions were combined, and the solvent was evaporated under reduced pressure (temperature ≤ 40 °C). The residual solid was coevaporated three times with water to remove residual buffer. The residue was redissolved in 40 mL of 80% aqueous AcOH, and the resulting solution was stirred at 35 °C for 16 h. Subsequently, the volatiles were removed under reduced pressure, and the crude material was redissolved in 100 mL of a mixture of MeOH / HO / Et3N (20:5:1), and the solution was stirred at 30 °C for 16 h. The reaction mixture was concentrated under reduced pressure (temperature ≤ 40 °C), and the crude material was purified by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / MeCN in water, 100:0 to 95:5). The product-containing fractions were combined and lyophilized three times to remove residual buffer. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO in acetone and dried under high vacuum to give 370 mg of compound (R P )-67 was obtained (yield=43%, dr>20:1). Physical state: white amorphous solid. Compound (R P )-67 is 1H NMR(600MHz,D2O):δ 8.00(s,1H),5.86(d,J=2.2Hz,1H),5.78(d,J=6.1Hz,1H),4.60(t,J=5.6Hz,1H),4.49-4.46(m ,1H),4.46-4.42(m,2H),4.40-4.37(m,1H),4.36-4.29(m,4H),4.29-4.24(m,1H),4.05(s,3H); 13 C NMR(150MHz,D2O):δ 162.8,161.7,158.8,154.0,151.3,148.9.136.9,133.6(br),115.7,108.8,89.3,86.4,83.6(d, J=8.9Hz),82.9(d,J=9.7Hz),74.7,74.1,70.4,68.5,65.4(d,J=5.4Hz),63.9(d,J=6.5Hz),36.0; 31 P NMR(162MHz,D2O):δ 42.8(d,J=26.0Hz),-11.6(d,J=20.1Hz),-24.0(dd,J=26.0,20.1Hz);HRMS(ESI-TOF)m / z:C 21 H 28 N 10 O 17 P3S[M-2H] - Calculated for: 817.0573, Found: 817.0567; Retention time: 9.63 min (Method 2).

[0391] Example 74 P 1 -(5'-O-7-methylguanosine)-P 2 -(5'-O-guanosine) disodium (S)-triphosphoro-1-thioate (compound (S P Preparation of )-67) [ka] Compound (S P )-67 to (-)-Ψ * Using the reagent, (R P)-67 was obtained following a procedure similar to that for 67. After purification by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / MeCN in water, 100:0 to 95:5), followed by precipitation as the sodium salt and drying under high vacuum, 353 mg of compound (S P )-67 was obtained (yield=41%, dr>20:1). Physical state: White amorphous solid. Compound (S P )-67 is 1 H NMR(600MHz,D2O):δ 8.01(s,1H),5.88(d,J=3.0Hz,1H),5.79(d,J=5.9Hz,1H),4.66(t,J=5.5Hz,1H),4.5 2-4.48(m,2H),4.48-4.45(m,1H),4.41-4.32(m,4H),4.30-4.25(m,2H),4.06(s,3H); 13 C NMR(150MHz,D2O):δ 162.8,161.7,158.8,154.0,151.3,149.1.137.2,133.9(br),115.9,108.9,89.0,86.6,83.6(d, J=8.6Hz),83.3(d,J=9.7Hz),74.9,73.8,70.3,68.9,65.4(d,J=5.0Hz),64.4(d,J=5.7Hz),35.9; 31 P NMR(162MHz,D2O):δ 43.3(d,J=25.5Hz),-11.5(d,J=17.2Hz),-24.1(dd,J=25.5,27.2Hz);HRMS(ESI-TOF)m / z:C 21 H 28 N 10 O 17 P3S[M-2H] - Calculated for: 817.0573, Found: 817.0567; Retention time: 8.58 min (Method 2).

[0392] Example 75 Acycloguanosine triammonium (R)-diphosphoro-α-thioate (compound (R P Preparation of )-68) [ka] Compound (R P )-68 was synthesized by the reaction of the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (+)-Ψ using anhydrous DMF (2.0 mL) as the solvent. * The product was obtained from the reagent (85 mg, 0.2 mmol) and acyclovir (112 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 25:75) to give 42 mg of compound (R) after lyophilization. P )-68 was obtained (yield=47%, ee>20:1). Physical state: white amorphous solid. Compound (R P )-68 is 1 H NMR(600MHz,D2O):δ 7.91(s,1H),5.50(s,2H),4.11-4.04(m,2H),3.77(t,J=4.5Hz,2H); 13 C NMR(150MHz,D2O):δ 158.4,153.5,151.1,139.5,115.5,72.2,67.7(d,J=8.8Hz),64.4(d,J=5.9Hz); 31 P NMR(162MHz,D2O):δ 41.4(d,J=29.3Hz),-6.8(d,J=29.3Hz);HRMS(ESI-TOF)m / z:C8H 12 N5O8P2S[MH] - Calculated value for [α]: 399.9887, Measured value: 399.9879 D 25 Characterized by: = +10.6 (c 1.01, DMSO) (measured as the triethylammonium salt); retention time: 7.75 min (Method 2).

[0393] Example 76 Acycloguanosine triammonium (S)-diphosphoro-α-thioate (compound (S P Preparation of )-68) [ka] Compound (S) was prepared according to general procedure A with minor modifications. P )-68 was synthesized by the reaction of the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (−)-Ψ using anhydrous DMF (2.0 mL) as the solvent. * The product was obtained from the reagent (85 mg, 0.2 mmol) and acyclovir (112 mg, 0.5 mmol). The coupling step was carried out using 5.0 equivalents of DBU. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, 0:100 to 25:75) to give 46 mg of compound (S) after lyophilization. P )-68 was obtained (yield=51%, ee>20:1). All characterization data were consistent with that of compound (R P )-68. [α] D 25 =-11.0 (c 0.98, DMSO) (measured as the triethylammonium salt); retention time: 7.76 min (Method 2).

[0394] Example 77 Preparation of Compound 72 [ka] A round-bottom flask equipped with a stir bar was charged with 2'-O-methyladenosine (compound 69) (1.55 g, 5.5 mmol, 1.0 equiv.), followed by the addition of anhydrous DMF (55 mL). Imidazole (0.75 g, 11.0 mmol, 2.0 equiv.) and tert-butyldimethylsilyl chloride (1.0 g, 6.6 mmol, 1.2 equiv.) were added sequentially, and the reaction mixture was stirred at room temperature overnight. The reaction was then quenched by the addition of concentrated aqueous NaHCO3 and extracted with EtOAc. The organic phase was washed with water, brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude compound 70, which was used in the next step without further purification.

[0395] Crude compound 70 was redissolved in anhydrous DCM (30 mL) and then Ψ O The reagent (3.47 g, 8.3 mmol, 1.5 eq) was added. OThe synthesis of this reagent is described in Huang, et al., Science, 2021, 373, 1265-1270. Freshly dried 3 Å molecular sieves (3.0 g) were added, and the mixture was stirred for 5 minutes. The reaction mixture was then cooled to 0 °C, followed by the sequential addition of DIPEA (0.10 mL, 0.6 mmol, 0.1 equiv.) and DBU (1.30 mL, 8.8 mmol, 1.6 equiv.), and the reaction was stirred for 30 minutes under an argon atmosphere. The mixture was then diluted with EtOAc and filtered. The filtrate was washed successively with 10% aqueous KH2PO4 and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude compound 71, which was used in the next step without further purification.

[0396] Crude compound 71 was redissolved in anhydrous DMF (25 mL) and then protected guanosine compound 35 (3.58 g, 11.0 mmol, 2.0 equiv.) was added. The synthesis of protected guanosine compound 35 is described in J. Davisson, et al., J. Org. Chem. 1987, 52, 1794-1801. DBU (2.46 mL, 16.5 mmol, 3.0 equiv.) was added, and the reaction mixture was stirred for 30 min under an argon atmosphere. A 1.0 M solution of TBAF in THF (22.0 mL, 22.0 mmol, 4.0 equiv.) was then added, and the reaction mixture was stirred for an additional 4 h at room temperature. MeOH (25 mL) and EtN (5.0 mL) were then added sequentially, and the reaction mixture was stirred at 50 °C overnight. The resulting mixture was concentrated under reduced pressure, and the residue was suspended in water (50 mL). The resulting suspension was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was suspended in water, and the filtration process was repeated again. The filtrate was concentrated under reduced pressure, and the crude product was purified by reverse-phase C18 silica gel chromatography (1 M TEAA / MeCN in water; 100:0 to 80:20) and lyophilized to give 2.40 g of compound 72 (2:1 mixture of diastereoisomers). (Yield = 48% over 5 steps). Physical state: white amorphous solid.

[0397] Compound 72 is 1H NMR (600 MHz, D2O): δ 8.21 (s, 1H; main + secondary), 8.05 (s, 1H; secondary), 8.04 (s, 1H; main), 7.87 (s, 1H; secondary); 7.85 (s, 1H; main), 6.20 (s, 1H; secondary), 6.16 (s, 1H; main), 6.09 (s, 1H; main), 6.06 (s, 1H; secondary), 5.89 (d, J = 6.6 Hz, 1H; secondary), 5.86 (d, J = 6.3 Hz, 1H; main), 5.50 (d, J = 6.3 Hz, 1H; secondary), 5.46 (d, J = 6.9 Hz, 1H; main), 5.29 (dd, J = 6.1, 3.1 Hz, 1H; secondary), 5.19 ( dd, J = 6.9, 3.1 Hz, 1H; main), 4.66-4.62 (m, 1H; main), 4.54-4.50 (m, 1H; secondary), 4.36-4.30 (m, 1H; main + secondary), 4.20-4.11 (m, 2H; main + secondary), 4.06-4.03 (m, 1H; secondary), 4.00-3.96 (m, 1H; main), 3.66-3.59 (m, 1H; main + secondary), 3.57-3.48 (m, 1H; main + secondary), 3.46 (s, 3H; main), 3.38 (s, 3H; secondary), 3.29 (s, 3H; main + secondary), 3.16-3.10 (m, 8H; nBu4N + ),1.63-1.54(m,8H;nBu4N + ),1.36-1.27(m,8H;nBu4N + ),0.91(t,J=7.3Hz,12H;nBu4N + ); 13C NMR(150MHz,D2O)δ 159.2 (sub), 159.1 (main), 154.9 (main + sub), 154.1 (minor), 154.0 (main), 151.81 (sub), 151.79 (main), 150.4 (sub), 150.3 (main), 147.6 (main + sub), 1 40.0 (main + sub), 137.1 (main), 137.0 (sub), 118.5 (main + sub), 118.1 (main), 117.0 (sub), 116.0 (main), 115.9 (sub), 89.8 (main), 88.5 (sub), 86.19 (main), 8 6.15 (sub), 85.9 (d, J = 9.5 Hz; main), 84.7 (main + sub), 84.4 (d, J = 9.4 Hz; sub), 83.5 (main), 82.4 (sub), 81.24 (d, J = 5.5 Hz; main), 81.21 (d, J = 4. 7Hz; sub), 80.6(main), 80.1(sub), 72.7(d,J=5.5Hz;main+sub),65.4(d,J=5.1Hz;main),65.1(d,J=4.9Hz;sub),60.6(main+sub),57.6(br;nBu4N + ), 57.3 (main + sub), 52.0 (main), 51.0 (sub), 22.6 (nBu4N + ), 18.6(nBu4N + ), 12.3(nBu4N + ); 31 P NMR(162MHz,D2O)δ -1.1;HRMS(ESI-TOF) m / z:C 23 H 28 N 10 O 12 P[MH] - It was characterized by a calculated value of 667.1631 and an observed value of 667.1621.

[0398] Example 78 Preparation of Compound 73 [ka] Dinucleoside 72 (2.40 g, 2.6 mmol, 1.0 equiv.) was dried by coevaporation with anhydrous MeCN, and the substrate was dissolved in anhydrous MeCN (30 mL). O The reagent (2.22 g, 5.2 mmol, 2.0 equiv.) and 3 Å molecular sieves (3.0 g) were added and the resulting suspension was stirred for 5 min.O The synthesis of this reagent is described in Huang, et al., Science, 2021, 373, 1265-1270. DBU (0.77 mL, 5.2 mmol, 2.0 equiv.) was added dropwise, and the reaction was stirred at room temperature under an argon atmosphere for 45 minutes. Then, deionized water (3.0 mL) and DBU (2.32 mL, 15.6 mmol, 6.0 equiv.) were added sequentially, and the reaction was stirred for an additional 15 minutes. The mixture was diluted with water, filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was resuspended in water, and the filtration process was repeated again. The filtrate was concentrated under reduced pressure, and the crude product was purified by reverse-phase C18 silica gel chromatography (1 M TEAA / MeCN in water; 100:0 to 85:15) and lyophilized to give 1.56 g of compound 73 (a 2:1 mixture of diastereoisomers). (Yield = 63%). Physical state: White amorphous solid.

[0399] Compound 73 is 1 H NMR(600MHz,D2O):δ 8.39 (s, 1H; sub), 8.38 (s, 1H; main), 8.15 (s, 1H; main + sub), 7.90 (s, 1H; main + sub), 6.21 (s ,1H;sub),6.18(d,J=2.3Hz,1H;main),6.10(s,1H;main),6.08(d,J=2.3Hz,1H;sub), 5.98(d,J=7.1Hz,sub),5.97(d,J=6.8Hz;main),5.47(dd,J=6.2,2.3Hz,1H;sub), 5.43(dd,J=7.0,2.3Hz,1H;main),5.31(dd,J=6.2,3.6Hz,1H;sub),5.21(dd,J=7 .0,3.5Hz,1H;main),4.91-4.86(m,1H;main+sub),4.66(q,J=4.4Hz,1H;main),4.54(q ,J=4.2Hz,1H;sub),4.37-4.31(m,1H;main+sub),4.25-4.15(m,3H;main+sub),4.00-3. 92 (m, 1H; main + sub), 3.86-3.81 (m, 1H; sub), 3.80-3.75 (m, 1H; main), 3.47 (s, 3H; main), 3.37 (s, 3H; secondary), 3.35 (s, 3H; secondary), 3.34 (s, 3H; primary), 3.18 (q, J=7.3Hz, 12H; Et3NH +), 1.26 (t, J = 7.3 Hz, 18H; Et3NH + ); 13 13C NMR (150 MHz, D2O): δ 158.1 (main), 158.0 (sub), 154.5 (main + sub), 153.1 (sub), 153.0 (main), 151.8 (main + sub), 150.5 (sub), 150.3 (main), 148.3 (main + sub), 139.2 (main + sub), 137.6 (main), 137.5 (sub), 118.1 (main), 117.9 (main + sub), 116.9 (sub), 115.9 (main), 115.8 (sub), 89.9 (main), 88.6 (sub), 85.8 (d, J = 9.5 Hz; main), 84.6 (main + sub), 84.3 (d, J = 9.4 Hz; sub), 83.6 (main), 82.8 (d, J = 9.1 Hz; main + sub), 82.4 (sub), 82.1 (d, J = 5.5 Hz; main), 82.0 (d, J = 5.5 Hz; sub), 80.5 (main), 80.0 (sub), 72.71 (d, J = 5.7 Hz; main), 72.66 (d, J = 5.8 Hz; sub), 65.4 (d, J = 5.1 Hz; main), 65.1 (d, J = 5.2 Hz; sub), 63.7 (d, J = 4.7 Hz; main + sub), 57.2 (main + sub), 5 .0 (main), 50.9 (sub), 46.2 (Et3NH + ), 7.7 (Et3NH + ); 31 31P NMR (162 MHz, D2O) δ 0.1, -1.2; HRMS (ESI-TOF) m / z: C 23 H 29 N 10 O 15 P2 [M - H]<000101‌7>characterized by calculated value: 747.1294, measured value: 747.1287 for

[0400] Example 79<‌ Preparation of Compound 75

Chemical Structure

[0401] Compound 74 was used in the next step without further purification.

[0402] Compound 74 is 31 P NMR(162MHz,DMSO-d6):δ -1.9,-12.2(d,J=20.9Hz),-12.6(d,J=20.9Hz);HRMS(ESI-TOF)m / z:C 51 H 50 N 10 O 18 P3[MH] - It was characterized by a calculated value of 1183.2523 and an observed value of 1183.2497.

[0403] P 1 -{5'-O-[2'-O-methyladenylyl-(3',5')-guanosine]}-P 2 -(5'-O-7-methylguanosine) trisodium (R)-triphosphoro-3-thioate (compound (R P )-75) [ka] A flame-dried round-bottom flask equipped with a stir bar was charged with crude protected guanosine diphosphate compound 74 (1.82 g, 1.0 mmol, 1.0 equiv; 65 wt% purity) (obtained as described above), and the flask was capped with a septum. Anhydrous DMSO (10 mL) and DBU (1.05 mL, 7.0 mmol, 7.0 equiv) were added, and the mixture was stirred for 10 min. Subsequently, 3 Å molecular sieves (1.0 g) and (+)-Ψ *The reagent (1.08 g, 2.5 mmol, 2.5 equiv.) was added, and the reaction was stirred at room temperature for 1 h. Protected 7-methylguanosine compound 36 (0.90 g, 2.0 mmol, 2.0 equiv.) was then added, followed by another portion of DBU (1.05 mL, 7.0 mmol, 7.0 equiv.), and the mixture was stirred for an additional 5 h. Upon completion of the reaction, the resulting mixture was filtered, and the solid residue was washed with approximately 2 mL of DMSO. The filtrate was distributed among six 50 mL centrifuge tubes, each containing a solution of 0.2 M NaClO4 in acetone (40 mL). The resulting suspension was centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the pellet was washed twice with acetone. The solid residue from each centrifuge tube was redissolved in a minimal amount of water, combined, and purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, 0:100 to 40:60). The product-containing fractions were combined, and the solvent was evaporated under reduced pressure (temperature ≤ 40 °C). The residual solid was coevaporated three times with water to remove residual buffer. The residue was redissolved in 40 mL of 80% aqueous AcOH, and the resulting solution was stirred at 35 °C for 16 h. Subsequently, the volatiles were removed under reduced pressure, and the crude material was redissolved in 100 mL of a mixture of MeOH / HO / Et3N (20:5:1), and the solution was stirred at 30 °C for 16 h. The reaction mixture was concentrated under reduced pressure (temperature ≤ 40 °C), and the crude material was purified by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / MeCN in water, 100:0 to 95:5). The product-containing fractions were combined and lyophilized three times to remove residual buffer. The resulting solid was redissolved in a minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO in acetone. After drying under high vacuum, 522 mg of the title compound was obtained (yield from 72 = 38%, dr > 20:1). Physical state: white solid.

[0404] Compound (R P )-75 is 1H NMR(600MHz,D2O)δ 9.04(s,1H),8.36(s,1H),8.06(s,1H),7.95(s,1H),6.00(d,J=5.5Hz,1H),5.85-5.80(m,2H),4.97-4.92(m,1H),4.7 8-4.72(m,1H),4.54-4.48(m,3H),4.46-4.40(m,3H),4.37-4.31(m,3H),4.31-4.17(m,4H),4.02(s,3H),3.44(s,3H); 13 C NMR (150 MHz, DO) δ 158.0,156.3,155.9,154.7,153.1,152.3,150.9,148.4,148.0,138.9,137 .0,135.0(br),117.7,115.6,107.4,89.1,86.9,84.4,83.0(d,J=9.1Hz),82 .42(d,J=9.5Hz),82.40(d,J=9.6Hz),81.2(d,J=4.2Hz),74.3,72.9,72.1( d,J=4.9Hz),69.8,68.5,64.60(d,J=4.6Hz),63.5(d,J=6.2Hz),57.5,35.6; 31 P NMR(162MHz,D2O)δ 42.9(d,J=27.4Hz),-1.0,-11.9(d,J=18.7Hz),-24.2(dd,J=27.4,18.7Hz);HRMS(ESI-TOF)m / z:C 32 H 42 N 15 O 23 P4S[M-2H] - Calculated for: 1160.1254, Found: 1160.1228; Retention time: 8.43 min (Method 1).

[0405] P 1 -{5'-O-[2'-O-methyladenylyl-(3',5')-guanosine]}-P 2 -(5'-O-7-methylguanosine) trisodium (S)-triphosphoro-3-thioate (compound (S P )-75) [ka] Compound (SP )-75 on a 0.1 mmol scale and (-)-Ψ * Using the reagent, compound (R P )-75 was obtained following a procedure similar to that for 75. After purification by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / MeCN in water, 100:0 to 95:5), followed by precipitation as the sodium salt and drying under high vacuum, 43 mg of compound (S P )-75 was obtained (yield from 72=35%, dr>20:1). Physical state: white solid.

[0406] Compound (S P )-75 is 1 H NMR(600MHz,D2O):δ 9.16(s,1H),8.46(s,1H),8.18(s,1H),7.97(s,1H),6.04(d,J=4.5Hz,1H),5.88(s,1H),5.81(d,J=4.5Hz,1H),4.98- 4.91(m,1H),4.78-4.72(m,1H),4.61-4.56(m,1H),4.54-4.48(m,3H),4.45-4.17(m,10H),4.03(s,3H),3.47(s,3H); 13 C NMR(150MHz,D2O):δ 157.7,154.8,153.8,153.1,152.3,150.8,149.0,148.6,147.6,140. 0,137.0,136.1,117.7,115.3,107.2,89.1,87.0,85.0,83.4(d,J=9.3 Hz),83.0(d,J=8.8Hz),82.3(br),81.6(d,J=3.5Hz),74.4,73.0,71.9(d,J=4.8Hz),69.7,68.7,64.6(d,J=4.9Hz),64.4,63.8,57.5,35.6; 31 P NMR(162MHz,D2O):δ 43.2(d,J=26.6Hz),-1.0,-11.8(d,J=18.8Hz),-24.3(dd,J=26.6,18.8Hz);HRMS(ESI-TOF)m / z:C 32 H 42 N 15 O 23 P4S[M-2H] -Calculated for: 1160.1254, Found: 1160.1228; Retention time: 7.62 min (Method 1).

Claims

1. structure: 【Chemical Formula 1】 A compound having the formula:

2. 10. A method for making the compound of claim 1, comprising the step of: 【Chemistry 2】 Compound 2 or Compound 3: 【Chemistry 3】 in the presence of an acid.

3. 3. The method of claim 2, wherein the acid is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, acetic acid, and formic acid.

4. Compound 1 is converted to compound 4: 【Chemistry 4】 in the presence of a base ２ S ５ 3. The method of claim 2, wherein the compound is formed by reacting

5. 5. The method of claim 4, wherein the base is selected from the group consisting of tert-butylamine, triethylamine, pyridine, tri-n-propylamine, trimethylamine, 1,2-bicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene.

6. Compound 2 or Compound 3 is Compound 5 or Compound 6: 【Chemistry 5】 and each of the above with hydrogen in the presence of a catalyst.

7. 7. The method of claim 6, wherein the catalyst is selected from the group consisting of platinum dioxide, palladium on carbon, platinum on carbon, Lindlar's catalyst, Raney nickel, nickel, rhodium on aluminum oxide, palladium, and platinum.

8. 1. A method for making a nucleoside diphosphorothioate or a salt thereof, comprising: (a) Compound 7, 8, or 9: 【Chemistry 6】 (In the formula, R １ , R ２ , R ３ , R ４ , and R ５ each independently represents hydrogen, CD ３ , C.F. ３ , linear or branched C １ ~C ２０ Alkyl, straight or branched chain C ２ ~C １２ Alkenyl, straight or branched chain C ２ ~C １２ Alkynyl, aryl, heteroaryl, heterocycle, or C ３ ~C ８ is cycloalkyl; R ６ , R ７ , and R ８ Each of the ３ , C.F. ３ , linear or branched C １ ~C ２０ Alkyl, straight or branched chain C ２ ~C １２ Alkenyl, straight or branched chain C ２ ~C １２ Alkynyl, aryl, heteroaryl, heterocycle, or C ３ ~C ８ is cycloalkyl; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium. with the compound of claim 1 in the presence of a first base to form a chiral thiodiphosphate transfer reagent; (b) reacting the chiral thiodiphosphate transfer reagent with a protected or unprotected nucleoside in the presence of a second base to form a protected nucleoside diphosphorothioate; and (c) deprotecting the protected nucleoside diphosphorothioate to form a nucleoside diphosphorothioate. A method comprising:

9. 9. The method of claim 8, wherein the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, triethylamine, pyridine, 2,6-lutidine, and imidazole.

10. 9. The method of claim 8, wherein the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

11. 11. The method of claim 10, wherein the second base is 1,8-diazabicyclo(5.4.0)undec-7-ene.

12. The nucleoside is Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: 【Chemistry 7】 (In the formula, R ９ each independently represents hydrogen, acetyl, branched or linear C ２ ~C ２０ alkanoyl, benzoyl, aryloyl, acryloyl, or heteroaryloyl) 9. The method of claim 8, selected from the group consisting of:

13. The nucleoside diphosphorothioate is 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; Y is calcium or magnesium.

9. The method of claim 8, selected from the group consisting of:

14. The nucleoside diphosphorothioate is 【Chemistry 11】 The method of claim 13, wherein

15. The nucleoside diphosphorothioate is 【Chemistry 12】 The method of claim 13, wherein

16. The nucleoside diphosphorothioate is 【Chemistry 13】 The method of claim 13, wherein

17. The nucleoside diphosphorothioate is 【Chemistry 14】 The method of claim 13, wherein

18. The nucleoside diphosphorothioate is 【Chemistry 15】 The method of claim 13, wherein

19. The nucleoside diphosphorothioate is 【Chemistry 16】 The method of claim 13, wherein

20. The nucleoside diphosphorothioate is 【Chemistry 17】 The method of claim 13, wherein

21. The nucleoside diphosphorothioate is 【Chemistry 18】 The method of claim 13, wherein

22. The nucleoside diphosphorothioate is 【Chemistry 19】 The method of claim 13, wherein

23. The nucleoside diphosphorothioate is 【Chemistry 20】 The method of claim 13, wherein

24. 1. A method for making a nucleoside triphosphorothioate or a salt thereof, comprising: (a) Compound 20 or Compound 21: 【Chemical 21】 (In the formula, R １ , R ２ , R ３ , R ４ , and R ５ each independently represents hydrogen, CD ３ , C.F. ３ , linear or branched C １ ~C ２０ Alkyl, straight or branched chain C ２ ~C １２ Alkenyl, straight or branched chain C ２ ~C １２ Alkynyl, aryl, heteroaryl, heterocycle, or C ３ ~C ８ is cycloalkyl; R ６ is a CD ３ , C.F. ３ , linear or branched C １ ~C ２０ Alkyl, straight or branched chain C ２ ~C １２ Alkenyl, straight or branched chain C ２ ~C １２ Alkynyl, aryl, heteroaryl, heterocycle, or C ３ ~C ８ is cycloalkyl; Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with the compound of claim 1 in the presence of a first base to form a chiral thiotriphosphate transfer reagent; (b) reacting the chiral thiotriphosphate transfer reagent with a protected or unprotected nucleoside in the presence of a second base to form a protected nucleoside triphosphorothioate; and (c) deprotecting the protected nucleoside triphosphorothioate to form a nucleoside triphosphorothioate. A method comprising:

25. 25. The method of claim 24, wherein the first base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,1,3,3-tetramethylguanidine, lithium bis(trimethylsilyl)amide, lithium tert-butoxide, potassium bis(trimethylsilyl)amide, potassium tert-butoxide, sodium bis(trimethylsilyl)amide, sodium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, N-methylimidazole, N,N-diisopropylethylamine, and triethylamine.

26. 25. The method of claim 24, wherein the second base is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, and 1,1,3,3-tetramethylguanidine.

27. 27. The method of claim 26, wherein the second base is 1,8-diazabicyclo(5.4.0)undec-7-ene.

28. The nucleoside is Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: 【Chemical 22】 (In the formula, R ９ each independently represents hydrogen, acetyl, branched or linear C ２ ~C ２０ 25. The method of claim 24, wherein the aryl group is selected from the group consisting of alkanoyl, benzoyl, aryloyl, acryloyl, or heteroaryloyl.

29. The nucleoside triphosphorothioate is 【Chemical 23】 【Chemistry 24】 【Chemistry 25】 wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; Y is calcium or magnesium.

25. The method of claim 24, selected from the group consisting of:

30. The nucleoside triphosphorothioate is 【Chemical 26】 30. The method of claim 29, wherein:

31. The nucleoside triphosphorothioate is 【Chemical 27】 30. The method of claim 29, wherein:

32. The nucleoside triphosphorothioate is 【Chemical Formula 28】 30. The method of claim 29, wherein:

33. The nucleoside triphosphorothioate is 【Chemical 29】 30. The method of claim 29, wherein:

34. The nucleoside triphosphorothioate is 【Chemistry 30】 30. The method of claim 29, wherein:

35. The nucleoside triphosphorothioate is 【Chemical 31】 30. The method of claim 29, wherein:

36. The nucleoside triphosphorothioate is 【Chemical 32】 30. The method of claim 29, wherein:

37. The nucleoside triphosphorothioate is 【Chemical 33】 30. The method of claim 29, wherein:

38. The nucleoside triphosphorothioate is 【Chemical 34】 30. The method of claim 29, wherein:

39. The nucleoside triphosphorothioate is 【Chemistry 35】 30. The method of claim 29, wherein:

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