Novel phosphorus (V)-based reagents, method for preparation thereof and use thereof in preparation of stereoscopically defined organophosphorus (V) compounds

Abstract

The present invention relates to novel phosphorus (V) (P (V)) reagents and methods for preparing the same, as well as methods for preparing nucleoside phosphorothioate compounds by using the novel reagents. # imgabs0 #

Description

Cross - Reference to Related Applications

[0001] This application is an international patent application claiming priority to U.S. Provisional Application No. 63 / 376,249, filed on September 19, 2022, the disclosure of which is incorporated herein by reference in its entirety. Background Art Technical Field

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

[0003] Organophosphorus compounds have a wide range of applications as therapeutic and diagnostic agents, pest and insect control agents, along with many other applications. Organophosphorus compounds are generally classified based on the oxidation state of the phosphorus atom: +5 (phosphorus(V)) or +3 (phosphorus(III)). Organophosphorothioates (phosphorus(V) compounds containing sulfur attached to phosphorus) are a subclass of organophosphate compounds, where at least one oxygen atom in the phosphate ester is replaced by sulfur. In some cases, phosphorus - induced asymmetry gives rise to chiral organophosphorothioate compounds, making such compounds particularly suitable for therapeutic, diagnostic, research, and other applications.

[0004] Well - known and well - utilized examples of organophosphorothioates are nucleic acids containing a phosphorothioate (e.g., phosphorothioate) backbone. Due to the instability of poly(nucleic acids) (e.g., dinucleotides) containing the natural phosphodiester backbone of DNA or RNA to nucleases, their use is limited. In nucleoside phosphorothioates, one of the non - bridging oxygen atoms in the phosphodiester bond is replaced by a sulfur atom. Thus, compared to dinucleotides with a phosphodiester backbone, nucleoside phosphorothioates containing a phosphorothioate backbone have higher nuclease resistance and cell membrane permeability.

[0005] Due to the chiral nature of the phosphorus atom in some organophosphorothioates, two stereoisomers (R P - and S P - isomers) can exist. Thus, in p - chiral nucleoside phosphorothioates, there are diastereoisomers, leading to significant problems in the development of such reagents. It is known that the properties of oligonucleotides, including binding affinity, sequence - specific binding to complementary RNA, and stability to nucleases, are affected by the configuration of the phosphorus atom. In addition, it has been shown that homochiral isomers can have different properties (solubility, stability, activity, pharmacokinetics, etc.). Therefore, it is highly desirable to prepare nucleoside phosphorothioates with a specific stereochemical configuration. Summary of the Invention

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

[0007] One embodiment of the present disclosure relates to a method for preparing the compounds of the present disclosure, the method comprising reacting compound 1: with compound 2 or compound 3: in the presence of an acid.

[0008] In another embodiment, the present disclosure provides a method for preparing compound 1, the method comprising reacting compound 4: with P2S5 in the presence of a base.

[0009] In another embodiment, the present disclosure provides a method for preparing compound 2 or compound 3, the method comprising reacting compound 5 or compound 6: separately with hydrogen in the presence of a catalyst.

[0010] In some embodiments, the present disclosure provides a method for preparing a nucleoside dithiophosphate or a salt thereof, the method comprising: (a) reacting one of compounds 7, 8, or 9: wherein R 1 , R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, CD3, CF3, straight-chain or branched C1-C 20 alkyl, straight-chain or branched C2-C 12 alkenyl, straight-chain or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 , R 7 , and R 8 are each independently CD3, CF3, straight-chain or branched C1-C 20 alkyl, straight-chain or branched C2-C 12 alkenyl, straight-chain or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; React with the compounds of the present disclosure in the presence of a first base to form a chiral thiophosphoramidite transfer reagent; (b) React the chiral thiophosphoramidite transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside dithiophosphate; and (c) Deprotect the protected nucleoside dithiophosphate to form a nucleoside dithiophosphate.

[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 for preparing a nucleoside trithiophosphate or a salt thereof, the method comprising: (a) Reacting compound 20 or compound 21: wherein R 1 、R 2 、R 3 、R 4 、and R 5 are each independently hydrogen, CD3, CF3, linear or branched C1-C 20 alkyl, linear or branched C2-C 12 alkenyl, linear or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 is CD3, CF3, linear or branched C1-C 20 alkyl, linear or branched C2-C 12 alkenyl, linear or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; React with the compounds of the present disclosure in the presence of a first base to form a chiral thiophosphorotriester transfer reagent; (b) React the chiral thiophosphorotriester transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside trithiophosphate; and (c) Deprotect the protected nucleoside trithiophosphate to form a nucleoside trithiophosphate.

[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 nucleosides selected from the group consisting of: Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: wherein R 9 is independently hydrogen, acetyl, branched or straight-chain C2-C 20 alkanoyl, benzoyl, aroyl, acryloyl, or heteroaroyl.

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

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

[0019] Additional embodiments and advantages of the present disclosure will be set forth in part in the following description, and will be apparent from the description, or may be learned by practice of the present disclosure. The embodiments and advantages of the present 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 summary and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings incorporated herein form a part of the specification and illustrate embodiments of the present disclosure. The drawings, together with the description, further serve to explain the principles of the disclosed embodiments and to enable a person skilled in the relevant art to make and use the disclosed embodiments. These drawings are intended to be illustrative and not restrictive.

[0022] Figure 1 Depicted is a representative LC trace of Compound 50, as an illustration of an LC trace demonstrating the diastereomeric purity of the phosphorodithioates of the present disclosure.

[0023] Figure 2 Depicts a representative LC trace of Compound 55 as an illustration of an LC trace demonstrating the diastereomeric purity of the phosphorotrithioates of the present disclosure. Detailed Description Definitions

[0024] Unless otherwise indicated, the following terms used in this application (including the specification and claims) have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. Unless otherwise specified, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are employed. In this application, unless otherwise indicated, the use of "or" or "and" means "and / or". In addition, the use of the term "including" and other forms such as "include", "includes", and "included" is not restricted.

[0025] Unless otherwise defined, all technical and scientific 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, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in this disclosure for the skilled person.

[0026] When embodiments are described herein using the language "comprising" anywhere, embodiments that are otherwise similar and described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0027] Units, prefixes, and symbols are expressed in their accepted International System of Units (SI) form. Numerical ranges include the values defining the range. In the case of a series of values being recited, it is to be understood that each intervening integer value between the upper and lower limits of the recited range, each fraction thereof, and each subrange between such values are also specifically disclosed. The upper and lower limits of any range may independently be included in or excluded from the range, and each range including either limit, neither limit, or both limits is also covered within the invention. In the case where values are expressly recited, it is to be understood that values of approximately the same quantity or amount as the recited values are also within the scope of the invention. In the case of a disclosed combination, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the invention. Conversely, in the case of separately disclosed different elements or groups of elements, their combination is also disclosed. In the case where any element of the invention is disclosed as having a plurality of alternatives, instances where each such alternative of the invention, alone or in any combination with any other alternative, is excluded are hereby also disclosed; more than one element of the invention may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0028] This disclosure is intended to include all isotopes of atoms that are present in the compounds of the invention. Isotopes include those 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 may be represented as 1 H (hydrogen), 2 H (deuterium), and 3 H (tritium). They are also commonly represented as D for deuterium and T for tritium. In the present application, CD3 represents a methyl group in which all hydrogen atoms are deuterium. Isotopes of carbon include 13 C and 14 C. Isotopically labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described herein using appropriate isotopically labeled reagents in place of the unlabeled reagents otherwise employed.

[0029] In this disclosure, the term "compound" is intended to include all stereoisomers and isotopes of the depicted structures. As used herein, the term "stereoisomer" means any geometric isomer (e.g., cis and trans isomers), enantiomer, or diastereomer of a compound. This disclosure encompasses any and all stereoisomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and stereoisomers, such as racemates. Mixtures of enantiomers and stereoisomers of a compound and means for resolving them into their component enantiomers or stereoisomers are well known. "Isotope" refers 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. In addition, the compounds, salts, or complexes of this disclosure can be prepared by conventional methods in combination with solvent or water molecules to form solvates and hydrates.

[0030] In this disclosure, the term "isomer" means 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 thus exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the invention, the chemical structures depicted herein and thus the compounds of the invention encompass all corresponding stereoisomers, i.e., stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and stereoisomers, such as racemates. Mixtures of enantiomers and stereoisomers of the compounds of the 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, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthesis methods.

[0031] In this disclosure, the term "stereoisomer" refers to all possible different isomers and conformational forms that a compound can have (e.g., a compound having any of the formulas described herein), particularly all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers, and / or conformational isomers. Some compounds of this disclosure can exist in different tautomeric forms, which are all included within the scope of this disclosure.

[0032] In this disclosure, the term "enantiomer" means each individual optically active form of a compound of the 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 at most 10% of the other enantiomer), at least 90%, or at least 98%.

[0033] In this disclosure, the term "diastereomer" means stereoisomers that are not mirror images of each other and are non - superimposable on each other.

[0034] In this disclosure, the term "nucleic acid" encompasses poly - or oligoribonucleotides (RNA) and poly - or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N - glycosides 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 a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. The prefix "poly -" refers to nucleic acids containing from about 1 to about 10,000 nucleotide monomer units, and the prefix "oligo -" refers to nucleic acids containing from about 1 to about 200 nucleotide monomer units. The term "nucleic acid" may also encompass cyclic dinucleotides (CDN).

[0035] In this disclosure, the terms "nucleobase" and "nucleoside base moiety", which are used interchangeably, refer to the nucleic acid moiety that participates in hydrogen bonding to bind 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).

[0036] In this 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 serve as nucleobases, including certain "universal bases" that are not nucleobases in the most traditional sense but serve as nucleobases.

[0037] In this disclosure, the term "nucleoside" refers to the compound glucosylamine, in which a nucleobase (nitrogen - containing base, such as adenine, guanine, thymine, uracil, 5 - methyluracil, etc.) is covalently bound to a pentose sugar (ribose or deoxyribose) or a modified sugar.

[0038] In the present disclosure, the term "sugar" refers to monosaccharides in closed and / or open forms. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, morpholino sugars, 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.

[0039] In the present disclosure, the term "modified sugar" refers to a moiety that can replace a sugar. Modified sugars mimic the spatial arrangement, electronic properties, or some other physicochemical properties of sugars.

[0040] In the present disclosure, the term "nucleotide" refers to a moiety in which a nucleobase is covalently linked to a sugar or a modified sugar, and the sugar or modified sugar is covalently linked to a phosphate group or a modified phosphorus atom moiety, such as a phosphorothioate group.

[0041] In the present disclosure, when used in reference to nucleic acids, the term "purified" refers to a nucleic acid that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that renders another substance unsuitable, impure, or inferior. Thus, a purified oligonucleotide exists in a form or environment different from that in which it existed prior to being subjected to a purification method.

[0042] In the present disclosure, the term "about" encompasses the range of experimental error that occurs in any measurement.

[0043] In the present disclosure, the term "hydrocarbon" as used herein refers to any chemical structure containing hydrogen atoms and carbon atoms.

[0044] In the present disclosure, the term "alkyl", used by itself or as part of another group, refers to an unsubstituted straight-chain or branched-chain aliphatic hydrocarbon. In one embodiment, the alkyl is C 1-20 alkyl. In one embodiment, the alkyl is C 1-10 alkyl. In another embodiment, the alkyl is C 1-6 alkyl. In another embodiment, the alkyl is C 1-4 alkyl. Non-limiting exemplary C 1-20 alkyls include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl. Non-limiting exemplary C 1-10 alkyls include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Non-limiting exemplary C 1-6Alkyl includes methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, pentyl, and hexyl. Non-limiting exemplary C 1-4 Alkyl includes methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and isobutyl.

[0045] In the present disclosure, the term "alkanoyl", used by itself or as part of another group, refers to an optionally substituted alkyl attached to a terminal ketone group. Non-limiting exemplary alkanoyls are

[0046] In the present disclosure, the term "cycloalkyl", used by itself or as part of another group, refers to an unsubstituted saturated and partially unsaturated (e.g., containing one or two double bonds) cyclic aliphatic hydrocarbon containing one to three rings, the rings having three to twelve carbon atoms (i.e., C 3-12 cycloalkyl) or the specified number of carbon atoms. In one embodiment, the cycloalkyl has two rings. In one embodiment, the cycloalkyl has one ring. In another embodiment, the cycloalkyl is saturated. In another embodiment, the cycloalkyl is unsaturated. In another embodiment, the cycloalkyl is C 3-8 cycloalkyl. In another embodiment, the cycloalkyl is C 3-7 cycloalkyl. In another embodiment, the cycloalkyl is C 5-7 cycloalkyl. In another embodiment, the cycloalkyl is C 3-6 cycloalkyl. The term "cycloalkyl" includes groups in which the ring -CH2- is replaced by -C(=O)-. Non-limiting exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decahydronaphthalene, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and cyclopentanone.

[0047] In the present 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, the alkenyl is C 2-12 alkenyl. In one embodiment, the alkenyl is C 2-6 alkenyl. In another embodiment, the alkenyl is C 2-4 alkenyl. Non-limiting exemplary alkenyls include vinyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

[0048] In the present 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, the alkynyl has one carbon-carbon triple bond. In one embodiment, the alkynyl is C 2-12 alkynyl. In one embodiment, the alkynyl is C 2-6 alkynyl. In another embodiment, the alkynyl is C2-4 Alkynyl. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl.

[0049] In the present disclosure, the term "aryl", used by itself or as part of another group, refers to an unsubstituted monocyclic or bicyclic aromatic ring system. In one embodiment, aryl is C 6-14 Aryl. In one embodiment, aryl is C 6-20 Aryl. Non-limiting exemplary aryl groups include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylene, and fluorenyl. In one embodiment, aryl is phenyl or naphthyl.

[0050] In the present disclosure, the term "aryloxy", used by itself or as part of another group, refers to an optionally substituted aryl group attached to a terminal oxygen atom. Non-limiting exemplary aryloxy is PhO - .

[0051] In the present disclosure, the term "aroyl", used by itself or as part of another group, refers to an optionally substituted aryl group attached to a terminal ketone group. Non-limiting exemplary aroyl is

[0052] In the present disclosure, the terms "heterocycle", "heterocyclic group", or "heterocyclic moiety" are intended to mean a stable 3-, 4-, 5-, 6-, or 7-membered monocyclic 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 above-defined heterocycles is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O) p , where p is 0, 1, or 2). The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or another substituent, if defined). The heterocycle may be attached to its side group at any heteroatom or carbon atom to form a stable structure. If the resulting compound is stable, the heterocycles described herein may be substituted on a carbon or nitrogen atom. The nitrogen in the heterocycle may optionally be quaternized. Preferably, when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to each other. Preferably, the total number of S and O atoms in the heterocycle is not greater than 1. When the term "heterocycle" is used, it is intended to include heteroaryl.

[0053] Examples of heterocycles include, but are not limited to, acridinyl, azetidinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, imidazopyridinyl, 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, oxazolidinylpyrimidinyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidinone, 4-piperidinone, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolopyridinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridobenzimidazolyl, pyridothiazolyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2-pyrrolidinone, 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, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thiazolopyridinyl, thienothiazolyl, thienoxazolyl, thienobenzimidazolyl, 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-ring compounds containing, for example, the above heterocycles.

[0054] As used herein, the term "aromatic heterocyclic group" or "heteroaryl" is intended to mean a stable monocyclic and polycyclic aromatic hydrocarbon containing at least one heteroatom ring member such as sulfur, oxygen or nitrogen. Heteroaryl includes, but is not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolinyl, isoquinolinyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolyl and benzodioxanyl. Heteroaryl is substituted or unsubstituted. The nitrogen atom is substituted or unsubstituted (i.e., N or NR, where R is H or another substituent, if defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O) p , where p is 0, 1 or 2).

[0055] Bridged rings are also included in the definition of heterocycles. A bridged ring is formed when one or more, preferably one to three atoms (i.e., C, O, N, or S) connect 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 carbon-nitrogen groups. It should be noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents enumerated for that ring may also be present on the bridge.

[0056] In this disclosure, 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 by a heteroatom independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one embodiment, the heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur. In one embodiment, the heteroaryl has three heteroatoms. In another embodiment, the heteroaryl has two heteroatoms. In another embodiment, the heteroaryl has one heteroatom. In another embodiment, the heteroaryl is a 5- to 14-membered heteroaryl. In another embodiment, the heteroaryl is a 5- to 10-membered heteroaryl. In another embodiment, the heteroaryl is a 5- or 6-membered heteroaryl. In another embodiment, the heteroaryl has 5 ring atoms, such as thienyl, a 5-membered heteroaryl having four carbon atoms and one sulfur atom. In another embodiment, the heteroaryl has 6 ring atoms, such as pyridyl, a 6-membered heteroaryl having five carbon atoms and one nitrogen atom. Non-limiting exemplary heteroaryls include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuryl, benzoxazinonyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, and phenoxazinyl. In one embodiment, the 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., pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl), pyrimidinyl (e.g., pyrimidin-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., isoxazol-3-yl, isoxazol-4-yl, and isoxazol-5-yl), or indazolyl (e.g., 1H-indazol-3-yl). The term "heteroaryl" also includes possible N-oxides. Non-limiting exemplary N-oxides are pyridyl N-oxide.

[0057] 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 by a heteroatom independently selected from nitrogen, oxygen, and sulfur. Non-limiting exemplary 5-membered heteroaryls include thienyl, furyl, pyrrolyl, oxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl.

[0058] In another embodiment, the heteroaryl is a 6-membered heteroaryl. For example, the heteroaryl is a monocyclic aromatic ring system having 6 ring atoms, wherein at least one carbon atom of the ring is replaced by a nitrogen atom. Non-limiting exemplary 6-membered heteroaryls include pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl.

[0059] In the present disclosure, the term "heteroaroyl", used by itself or as part of another group, refers to an optionally substituted heteroaryl attached to a terminal ketone group. Non-limiting exemplary heteroaroyls are

[0060] In the present disclosure, the term "halogen" is intended to include fluorine, chlorine, bromine, and iodine.

[0061] 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 phosphodiester internucleoside linkages. Examples of modified internucleoside linkage groups are phosphorothioate linkages.

[0062] In the present disclosure, the term "protecting group" refers to a group that protects functional groups such as alcohols, amines, carbonyls, carboxylic acids, phosphate esters, terminal alkynes, etc. from unwanted chemical reactions. 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), triisopropylsilyloxymethyl (TOM), trityl (triphenylmethyl, Tr), pivaloyl (Piv), etc. In one embodiment, the protecting group is 4,4'-dimethoxytrityl. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl (Cbz), isobutyryl (iBu), p-methoxybenzylcarbonyl (MOZ), tert-butylcarbonyl (Boc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), tosyl (Ts), etc. Examples of carbonyl protecting groups include, but are not limited to, acetals and ketals, carbonates, dithianes, etc. Examples of carboxylic acid protecting groups include, but are not limited to, methyl esters, benzyl esters, tert-butyl esters, silyl esters, orthoesters, oxazolines, etc. Examples of phosphate ester protecting groups include, but are not limited to, 2-cyanoethyl, methyl, etc. Examples of terminal alkyne protecting groups include, but are not limited to, propargyl and silyl. In one embodiment, the protecting group is used to protect the 5'-hydroxy group of the 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 methods 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. I. Compounds of the Present Disclosure

[0063] The compounds of the present disclosure are represented by the following structure:

[0064] In one embodiment, a method for preparing the compounds of the present disclosure comprises reacting compound 1: with compound 2: in the presence of an acid.

[0065] In one embodiment, a method for preparing the compounds of the present disclosure comprises reacting Compound 1: with Compound 3: in the presence of an acid.

[0066] In some embodiments, the acid is selected from the group consisting of trifluoroacetic acid, dichloroacetic acid, acetic acid, and formic acid.

[0067] In some embodiments, the acid is trifluoroacetic acid.

[0068] In some embodiments, Compound 1 is formed by reacting Compound 4: with P2S5 in the presence of a base.

[0069] 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.

[0070] In some embodiments, the base is tert-butylamine.

[0071] In some embodiments, Compound 2 is formed by reacting Compound 5: with hydrogen in the presence of a catalyst.

[0072] In some embodiments, Compound 3 is formed by reacting Compound 6: with hydrogen in the presence of a catalyst.

[0073] In some embodiments, the catalyst is selected from the group consisting of platinum dioxide, palladium on carbon, platinum on carbon, Lindlar catalyst, Raney nickel, nickel, rhodium on alumina, palladium, and platinum.

[0074] In some embodiments, the catalyst is platinum dioxide. II. Method for Preparing Nucleoside Dithiophosphates or Nucleobase Dithiophosphates

[0075] In one embodiment, the present disclosure provides a method for preparing a nucleoside dithiophosphate or a salt thereof, the method comprising: (a) reacting one of Compounds 7, 8, or 9: wherein R 1 , R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, CD3, CF3, a straight or branched C1-C 20 alkyl, a straight or branched C2-C 12 alkenyl, a straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or a C3-C8 cycloalkyl; R 6 , R 7 , and R 8 are each independently CD3, CF3, a straight or branched C1-C 20 alkyl, a straight or branched C2-C 12 alkenyl, a straight or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or a C3-C8 cycloalkyl; and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; react with the compounds of the present disclosure in the presence of a first base to form a chiral thiophosphoramidite transfer reagent; (b) reacting the chiral thiophosphoramidite transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside phosphorodithioate; and (c) deprotecting the protected nucleoside phosphorodithioate to form a nucleoside phosphorodithioate.

[0076] In some embodiments, the chiral thiophosphoramidite transfer reagent in step (b) reacts with a nucleobase in the presence of a second base to form a protected nucleobase phosphorodithioate, and then the protected nucleobase phosphorodithioate is deprotected to form a nucleobase phosphorodithioate.

[0077] In some embodiments, the protected nucleoside phosphorodithioate or the protected nucleobase phosphorodithioate is deprotected in an ammonia solution.

[0078] In some embodiments, the protected nucleoside phosphorodithioate or the protected nucleobase phosphorodithioate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0079] In some embodiments, the protected nucleoside phosphorodithioate or the protected nucleobase phosphorodithioate is deprotected in an acetic acid solution.

[0080] In some embodiments, the nucleoside phosphorodithioate or the nucleobase phosphorodithioate is purified by ion exchange chromatography.

[0081] In some embodiments, the nucleoside dithiophosphates or nucleobase dithiophosphates are purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0082] 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-dimethylpyridine, and imidazole.

[0083] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0084] 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.

[0085] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0086] In some embodiments, the term "nucleoside" refers to the compound glucosylamine, in which a nucleobase (nitrogen-containing base such as adenine, guanine, thymine, uracil, 5-methyluracil, etc.) is covalently bonded to a pentose sugar (ribose or deoxyribose) or a modified sugar.

[0087] In some embodiments, the term "sugar" refers to monosaccharides in closed and / or open forms. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, morpholino sugar, carbocyclic analogs, hexopyranose moieties, and bicyclic sugars.

[0088] In some embodiments, the term "modified sugar" refers to a moiety that can replace a sugar. Modified sugars mimic the spatial arrangement, electronic properties, or some other physicochemical properties of sugars.

[0089] 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 serve as nucleobases, including certain "universal bases" that are not nucleobases in the most traditional sense but serve as nucleobases.

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

[0091] In some embodiments, the nucleoside in step (b) is an unprotected nucleoside.

[0092] In some embodiments, the nucleoside is selected from the group consisting of: Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: wherein R 9 is independently hydrogen, acetyl, a branched or straight-chain C2-C 20 alkanoyl, benzoyl, aroyl, acryloyl, or heteroaroyl.

[0093] In some embodiments, the nucleoside is selected from the group consisting of: Compound 76, Compound 77, Compound 78, Compound 79, Compound 80, Compound 81, Compound 82, Compound 83, Compound 84, and Compound 85: wherein R 9 is independently hydrogen, acetyl, a branched or straight-chain C2-C 20 alkanoyl, benzoyl, aroyl, acryloyl, or heteroaroyl.

[0094] In some embodiments, the nucleoside is selected from the group consisting of: Compound 86, Compound 87, Compound 88, Compound 89, and Compound 90: wherein R 9 is independently hydrogen, acetyl, a branched or straight-chain C2-C 20 alkanoyl, benzoyl, aroyl, acryloyl, or heteroaroyl; and R 10 is a straight-chain or branched C1-C 20 alkyl, a straight-chain or branched C2-C 12 alkenyl, or a straight-chain or branched C2-C 12 alkynyl.

[0095] In some embodiments, the nucleoside dithiophosphates are selected from the group consisting of: Wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0096] In some embodiments, the nucleoside dithiophosphate is

[0097] In some embodiments, the nucleoside dithiophosphate is

[0098] In some embodiments, the nucleoside dithiophosphate is

[0099] In some embodiments, the nucleoside dithiophosphate is

[0100] In some embodiments, the nucleoside dithiophosphate is

[0101] In some embodiments, the nucleoside dithiophosphate is

[0102] In some embodiments, the nucleoside dithiophosphate is

[0103] In some embodiments, the nucleoside dithiophosphate is

[0104] In some embodiments, the nucleoside dithiophosphate is

[0105] In some embodiments, the nucleoside dithiophosphate is

[0106] In some embodiments, the nucleoside dithiophosphate is

[0107] In some embodiments, the nucleobase is acyclovir:

[0108] In some embodiments, the nucleobase dithiophosphate is selected from the group consisting of: Wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0109] In some embodiments, the nucleobase dithiophosphate is

[0110] In some embodiments, the present disclosure provides a method for preparing a nucleoside dithiophosphate or a salt thereof, the method comprising: (a) reacting a compound 92: wherein Nu 1 is a nucleoside; and 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 dithiophosphate transfer reagent; and (b) reacting the chiral dithiophosphate transfer reagent with a compound 97 in the presence of a second base to form a protected nucleoside dithiophosphate; wherein R 1 、R 2 、R 3 、R 4 、and R 5 are each independently hydrogen, CD3, CF3, straight-chain or branched C1-C 20 alkyl, straight-chain or branched C2-C 12 alkenyl, straight-chain or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; and (c) deprotecting the protected nucleoside dithiophosphate to form a nucleoside dithiophosphate.

[0111] In some embodiments, Nu 1 is a nucleobase, then a protected nucleobase dithiophosphate is produced in step (b) above, and a nucleobase dithiophosphate is produced in step (c) above.

[0112] In some embodiments, the protected nucleoside dithiophosphate or the protected nucleobase dithiophosphate is deprotected in an ammonia solution.

[0113] In some embodiments, the protected nucleoside dithiophosphate or the protected nucleobase dithiophosphate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0114] In some embodiments, the protected nucleoside dithiophosphate or the protected nucleobase dithiophosphate is deprotected in an acetic acid solution.

[0115] In some embodiments, the nucleoside dithiophosphate or the nucleobase dithiophosphate is purified by ion exchange chromatography.

[0116] In some embodiments, the nucleoside dithiophosphates or nucleobase dithiophosphates are purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0117] 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-dimethylpyridine, and imidazole.

[0118] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0119] 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.

[0120] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0121] In some embodiments, compound 92 is: where Nu 1 is a nucleoside or a nucleobase.

[0122] In some embodiments, compound 92 is prepared by: (a) reacting a nucleoside or a nucleobase with iPr2NP(OBn)2 to form compound 91: where Nu 1 is a nucleoside or a nucleobase; and (b) reacting compound 91 with hydrogen in the presence of a catalyst.

[0123] In some embodiments, step (a) is carried out in the presence of 1-H-tetrazole. In some embodiments, step (a) is carried out in the presence of imidazole. In some embodiments, step (a) is carried out in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, step (a) is carried out in the presence of hydrogen peroxide. In some embodiments, step (a) is carried out in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, step (a) 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, step (a) is carried out in the presence of 1-H-tetrazole and hydrogen peroxide.

[0124] 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 alumina, palladium, and platinum.

[0125] In some embodiments, the catalyst is palladium on carbon.

[0126] In some embodiments, the nucleoside dithiophosphate is wherein Nu 1 is a nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0127] In some embodiments, Nu 1 is a protected nucleoside.

[0128] In some embodiments, Nu 1 is an unprotected nucleoside.

[0129] In some embodiments, the nucleoside is as described above.

[0130] In some embodiments, the nucleoside dithiophosphate is III. Method for Preparing Nucleoside Trithiophosphates or Nucleobase Trithiophosphates

[0131] In one embodiment, the present disclosure provides a method for preparing a nucleoside trithiophosphate or a salt thereof, the method comprising: (a) reacting compound 20 or compound 21: wherein R 1 、R 2 、R 3 、R 4 、and R5 Each independently is hydrogen, CD3, CF3, a linear or branched C1-C 20 alkyl group, a linear or branched C2-C 12 alkenyl group, a linear or branched C2-C 12 alkynyl group, aryl, heteroaryl, heterocycle, or a C3-C8 cycloalkyl group; R 6 is CD3, CF3, a linear or branched C1-C 20 alkyl group, a linear or branched C2-C 12 alkenyl group, a linear or branched C2-C 12 alkynyl group, aryl, heteroaryl, heterocycle, or a C3-C8 cycloalkyl group; and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; react with the compounds of the present disclosure in the presence of a first base to form a chiral thiophosphoramidite transfer reagent; (b) reacting the chiral thiophosphoramidite transfer reagent with a nucleoside in the presence of a second base to form a protected nucleoside trithiophosphate; and (c) deprotecting the protected nucleoside trithiophosphate to form a nucleoside trithiophosphate.

[0132] In some embodiments, the chiral thiophosphoramidite transfer reagent in step (b) is reacted with a nucleobase in the presence of a second base to form a protected nucleobase trithiophosphate, and then the protected nucleobase trithiophosphate is deprotected to form a nucleobase trithiophosphate.

[0133] In some embodiments, the protected nucleoside trithiophosphate or the protected nucleobase trithiophosphate is deprotected in an ammonia solution.

[0134] In some embodiments, the protected nucleoside trithiophosphate or the protected nucleobase trithiophosphate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0135] In some embodiments, the protected nucleoside trithiophosphate or the protected nucleobase trithiophosphate is deprotected in an acetic acid solution.

[0136] In some embodiments, the nucleoside trithiophosphate or the nucleobase trithiophosphate is purified by ion exchange chromatography.

[0137] In some embodiments, the nucleoside trithiophosphate or the nucleobase trithiophosphate is purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0138] 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.

[0139] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0140] 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.

[0141] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0142] In some embodiments, compound 20 is prepared by reacting compound 7 with iPr2NP(OFm)2.

[0143] In some embodiments, the above reaction is carried out in the presence of 1-H-tetrazole. In some embodiments, the above reaction is carried out in the presence of imidazole. In some embodiments, the above reaction is carried out in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, the above reaction is carried out in the presence of hydrogen peroxide. In some embodiments, the above reaction is carried out in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, the above 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 above reaction is carried out in the presence of 1-H-tetrazole and hydrogen peroxide. In some embodiments, the above reaction is carried out in the presence of 5-phenyl-1H-tetrazole and tert-butyl hydroperoxide.

[0144] In some embodiments, compound 7 is:

[0145] In some embodiments, compound 20 is:

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

[0147] In some embodiments, the nucleoside in step (b) is an unprotected nucleoside.

[0148] In some embodiments, the nucleoside is as described above.

[0149] In some embodiments, the nucleoside trithiophosphates are selected from the group consisting of: wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0150] In some embodiments, the nucleoside trithiophosphate is

[0151] In some embodiments, the nucleoside trithiophosphate is

[0152] In some embodiments, the nucleoside trithiophosphate is

[0153] In some embodiments, the nucleoside trithiophosphate is

[0154] In some embodiments, the nucleoside trithiophosphate is

[0155] In some embodiments, the nucleoside trithiophosphate is

[0156] In some embodiments, the nucleoside trithiophosphate is

[0157] In some embodiments, the nucleoside trithiophosphate is

[0158] In some embodiments, the nucleoside trithiophosphate is

[0159] In some embodiments, the nucleoside trithiophosphate is

[0160] In some embodiments, the nucleoside trithiophosphate is

[0161] In some embodiments, the present disclosure provides a method for preparing a nucleoside trithiophosphate or a salt thereof, the method comprising: (a) reacting a compound 93: wherein Nu 1 is a nucleoside; and 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 the chiral thiotriphosphate transfer reagent with a compound 97 in the presence of a second base to form a protected nucleoside trithiophosphate; wherein R 1 、R 2 、R 3 、R 4 、and R 5 are each independently hydrogen, CD3, CF3, straight-chain or branched C1-C 20 alkyl, straight-chain or branched C2-C 12 alkenyl, straight-chain or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; (c) deprotecting the protected nucleoside trithiophosphate to form a nucleoside trithiophosphate.

[0162] In some embodiments, Nu 1 is a nucleobase, then a protected nucleobase trithiophosphate is produced in step (b) above, and a nucleobase trithiophosphate is produced in step (c) above.

[0163] In some embodiments, the protected nucleoside trithiophosphate or the protected nucleobase trithiophosphate reagent is deprotected in an ammonia solution.

[0164] In some embodiments, the protected nucleoside trithiophosphate or the protected nucleobase trithiophosphate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0165] In some embodiments, the protected nucleoside trithiophosphate or the protected nucleobase trithiophosphate is deprotected in an acetic acid solution.

[0166] In some embodiments, the nucleoside trithiophosphate or the nucleobase trithiophosphate is purified by ion exchange chromatography.

[0167] In some embodiments, the nucleoside trithiophosphates or nucleobase trithiophosphates are purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0168] 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.

[0169] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0170] 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.

[0171] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0172] In some embodiments, compound 93 is prepared by reacting compound 92: with iPr2NP(OFm)2, where Nu 1 is a nucleoside or a nucleobase; and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium.

[0173] In some embodiments, the above reaction is carried out in the presence of 1-H-tetrazole. In some embodiments, the above reaction is carried out in the presence of imidazole. In some embodiments, the above reaction is carried out in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, the above reaction is carried out in the presence of hydrogen peroxide. In some embodiments, the above reaction is carried out in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, the above 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 above reaction is carried out in the presence of 1-H-tetrazole and hydrogen peroxide. In some embodiments, the above reaction is carried out in the presence of 5-phenyl-1H-tetrazole and tert-butyl hydroperoxide.

[0174] In some embodiments, the nucleoside trithiophosphate is wherein Nu 1 is a nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0175] In some embodiments, Nu 1 is a protected nucleoside.

[0176] In some embodiments, Nu 1 is an unprotected nucleoside.

[0177] In some embodiments, the nucleoside is as described above. IV. Method for Preparing Dinucleoside Dithiophosphate, Dinucleobase Dithiophosphate, or Nucleoside-Nucleobase Dithiophosphate

[0178] In one embodiment, the present disclosure provides a method for preparing a dinucleoside dithiophosphate or a salt thereof, the method comprising: (a) reacting a compound 92: wherein Nu 1 is a nucleoside; and 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 thiophosphonate transfer reagent; (b) reacting the chiral thiophosphonate transfer reagent with a nucleoside in the presence of a second base to form a protected dinucleoside dithiophosphate; and (c) deprotecting the protected dinucleoside dithiophosphate to form a dinucleoside dithiophosphate.

[0179] In some embodiments, when Nu 1 is a nucleobase and the chiral thiophosphonate transfer reagent reacts with the nucleobase in step (b), a dinucleobase dithiophosphate or a salt thereof is prepared from the above reaction.

[0180] In some embodiments, when Nu 1 is a nucleoside and the chiral thiophosphonate transfer reagent reacts with a nucleobase in step (b), a nucleoside-nucleobase dithiophosphate or a salt thereof is prepared from the above reaction.

[0181] In some embodiments, when Nu 1 is a nucleobase and the chiral thiophosphonate transfer reagent reacts with a nucleoside in step (b), a nucleoside-nucleobase dithiophosphate or a salt thereof is prepared from the above reaction.

[0182] In some embodiments, a protected dinucleoside dithiophosphate, a protected dinucleobase dithiophosphate, or a protected nucleoside nucleobase dithiophosphate is deprotected in an ammonia solution.

[0183] In some embodiments, a protected dinucleoside dithiophosphate, a protected dinucleobase dithiophosphate, or a protected nucleoside nucleobase dithiophosphate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0184] In some embodiments, a protected dinucleoside dithiophosphate, a protected dinucleobase dithiophosphate, or a protected nucleoside nucleobase dithiophosphate is deprotected in an acetic acid solution.

[0185] In some embodiments, a dinucleoside dithiophosphate, a dinucleobase dithiophosphate, or a nucleoside nucleobase dithiophosphate is purified by ion exchange chromatography.

[0186] In some embodiments, a dinucleoside dithiophosphate, a dinucleobase dithiophosphate, or a nucleoside nucleobase dithiophosphate is purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0187] 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-dimethylpyridine, and imidazole.

[0188] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0189] 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.

[0190] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

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

[0192] In some embodiments, Nu 1 is a protected nucleobase. In some embodiments, Nu 1 is an unprotected nucleobase.

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

[0194] In some embodiments, the nucleobase in step (b) is a protected nucleoside. In some embodiments, the nucleobase in step (b) is an unprotected nucleoside.

[0195] 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).

[0196] 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).

[0197] In some embodiments, the dinucleoside dithiophosphate is selected from the group consisting of: wherein Nu 1 and Nu 2 are nucleosides; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0198] In some embodiments, Nu 2 is a protected nucleoside.

[0199] In some embodiments, Nu 2 is an unprotected nucleoside.

[0200] In some embodiments, Nu 1 and Nu 2 are the same nucleoside.

[0201] In some embodiments, Nu 1 and Nu 2 are different nucleosides.

[0202] In some embodiments, the nucleoside is as described above.

[0203] In some embodiments, the dinucleoside dithiophosphate is

[0204] In some embodiments, the dinucleoside dithiophosphate is

[0205] In some embodiments, the dinucleoside dithiophosphate is V. Methods for Preparing Dinucleoside Trithiophosphates, Dinucleobase Trithiophosphates, and Nucleoside Nucleobase Trithiophosphates

[0206] In one embodiment, the present disclosure provides a method for preparing a dinucleoside trithiophosphate or a salt thereof, the method comprising: (a) reacting a compound 93: wherein Nu 1 is a nucleoside; and 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 thiophosphotriester transfer reagent; (b) reacting the chiral thiophosphotriester transfer reagent with a nucleoside in the presence of a second base to form a protected dinucleoside trithiophosphate; and (c) deprotecting the protected dinucleoside trithiophosphate to form a dinucleoside trithiophosphate.

[0207] In some embodiments, when Nu 1 is a nucleobase and the chiral thiophosphotriester transfer reagent reacts with the nucleobase in step (b), a dinucleobase trithiophosphate or a salt thereof is prepared from the above reaction.

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

[0209] In some embodiments, when Nu 1 is a nucleobase and the chiral thiophosphotriester transfer reagent reacts with a nucleoside in step (b), a nucleoside nucleobase trithiophosphate or a salt thereof is prepared from the above reaction.

[0210] In some embodiments, the protected dinucleoside trithiophosphate, the protected dinucleobase trithiophosphate, or the protected nucleoside nucleobase trithiophosphate is deprotected in an ammonia solution.

[0211] In some embodiments, the protected dinucleoside trithiophosphate, the protected dinucleobase trithiophosphate, or the protected nucleoside nucleobase trithiophosphate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0212] In some embodiments, a protected dinucleoside triphosphorothioate, a protected dinucleobase triphosphorothioate, or a protected nucleoside nucleobase triphosphorothioate is deprotected in an acetic acid solution.

[0213] In some embodiments, a dinucleoside triphosphorothioate, a dinucleobase triphosphorothioate, or a nucleoside nucleobase triphosphorothioate is purified by ion exchange chromatography.

[0214] In some embodiments, a dinucleoside triphosphorothioate, a dinucleobase triphosphorothioate, or a nucleoside nucleobase triphosphorothioate is purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0215] 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.

[0216] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0217] 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.

[0218] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

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

[0220] In some embodiments, Nu1 is a protected nucleobase. In some embodiments, Nu1 is an unprotected nucleobase.

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

[0222] In some embodiments, the nucleobase in step (b) is a protected nucleobase. In some embodiments, the nucleobase in step (b) is an unprotected nucleobase.

[0223] 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).

[0224] 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).

[0225] In some embodiments, the dinucleoside trithiophosphate is selected from the group consisting of: wherein Nu 1 and Nu 2 are nucleosides; Nu 3 is a cationic nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0226] In some embodiments, Nu 2 is a protected nucleoside.

[0227] In some embodiments, Nu 2 is an unprotected nucleoside.

[0228] In some embodiments, Nu 3 is a protected nucleoside.

[0229] In some embodiments, Nu 3 is an unprotected nucleoside.

[0230] In some embodiments, Nu 1 and Nu 2 are the same nucleoside.

[0231] In some embodiments, Nu 1 and Nu 2 are different nucleosides.

[0232] In some embodiments, Nu 1 and Nu 3 are the same nucleoside.

[0233] In some embodiments, Nu 1 and Nu 3 are different nucleosides.

[0234] In some embodiments, the nucleoside is as described above.

[0235] In some embodiments, the dinucleoside trithiophosphate is VI. Method for Preparing Capped Dinucleoside Trithiophosphate

[0236] In one embodiment, the present disclosure provides a method for preparing a capped dinucleoside trithiophosphate or a salt thereof, the method comprising: (a) reacting a first nucleoside with a Ψ O reagent in the presence of a first base to form a loaded nucleoside; (b) reacting the loaded nucleoside with a second nucleoside in the presence of a second base to form a dinucleoside phosphate compound 94: (c) reacting compound 94 with a Ψ O reagent in the presence of a third base to form a loaded dinucleoside: (d) reacting the loaded dinucleoside with water in the presence of a fourth base to form compound 95: (e) reacting compound 95 with iPr2NP(OFm)2 to form compound 96: (f) reacting compound 96 with a compound of the present disclosure in the presence of a fifth base to form a chiral trithiophosphate transfer reagent; (g) reacting the chiral trithiophosphate transfer reagent with a third nucleoside in the presence of a sixth base to form a protected capped dinucleoside trithiophosphate; and (h) deprotecting the protected capped dinucleoside trithiophosphate to form a capped dinucleoside trithiophosphate; wherein Nu 1 and Nu 2 are nucleosides; and Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium.

[0237] In some embodiments, the Ψ O reagent is In some embodiments, the Ψ O reagent is

[0238] In some embodiments, the loaded nucleoside in step (a) is separated before the next step.

[0239] In some embodiments, compound 94 in step (b) is separated before the next step.

[0240] In some embodiments, the loaded dinucleoside in step (c) is not separated before the next step.

[0241] In some embodiments, compound 95 in step (d) is separated before the next step.

[0242] In some embodiments, compound 96 in step (e) is separated before the next step.

[0243] In some embodiments, the chiral trithiophosphate transfer reagent in step (f) is not separated before the next step.

[0244] In some embodiments, the protected capped dinucleoside trithiophosphate in step (g) is separated by ion exchange chromatography before the next step.

[0245] 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.

[0246] In some embodiments, the first base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0247] 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.

[0248] In some embodiments, the second base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0249] 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.

[0250] In some embodiments, the third base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0251] 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.

[0252] In some embodiments, the fourth base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0253] 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.

[0254] In some embodiments, the fifth base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0255] 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.

[0256] In some embodiments, the sixth base is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0257] In some embodiments, compound 95 is reacted with iPr2NP(OFm)2 in the presence of 1-H-tetrazole. In some embodiments, compound 95 is reacted with iPr2NP(OFm)2 in the presence of imidazole. In some embodiments, compound 95 is reacted with iPr2NP(OFm)2 in the presence of 5-phenyl-1-H-tetrazole. In some embodiments, compound 95 is reacted with iPr2NP(OFm)2 in the presence of hydrogen peroxide. In some embodiments, compound 95 is reacted with iPr2NP(OFm)2 in the presence of tert-butyl hydroperoxide (TBHP). In some embodiments, compound 95 is reacted 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 iPr2NP(OFm)2 in the presence of 1-H-tetrazole and hydrogen peroxide. In some embodiments, compound 95 is reacted with iPr2NP(OFm)2 in the presence of 5-phenyl-1H-tetrazole and tert-butyl hydroperoxide.

[0258] In some embodiments, the protected capped dinucleoside trithiophosphate is deprotected in an ammonia solution.

[0259] In some embodiments, the protected capped dinucleoside trithiophosphate is deprotected in a tetrabutylammonium fluoride solution buffered with acetic acid.

[0260] In some embodiments, the protected capped dinucleoside trithiophosphate is deprotected in an acetic acid solution.

[0261] In some embodiments, the capped dinucleoside trithiophosphate is purified by reverse phase chromatography.

[0262] In some embodiments, the capped dinucleoside trithiophosphate is purified by precipitation from a solution containing one of the cations lithium, sodium, potassium, calcium, and magnesium.

[0263] In some embodiments, Nu 1 is a protected nucleoside.

[0264] In some embodiments, Nu 1 is an unprotected nucleoside.

[0265] In some embodiments, Nu 2 is a protected nucleoside.

[0266] In some embodiments, Nu 2 is an unprotected nucleoside.

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

[0268] In some embodiments, the third nucleoside in step (g) is an unprotected nucleoside.

[0269] In some embodiments, the capped dinucleoside trithiophosphate is selected from the group consisting of: wherein Nu 1 , Nu 2 and Nu 3 are nucleosides; Nu 4 is a cationic nucleoside; X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

[0270] In some embodiments, Nu 3 is a protected nucleoside.

[0271] In some embodiments, Nu 3 is an unprotected nucleoside.

[0272] In some embodiments, Nu 4 is a protected nucleoside.

[0273] In some embodiments, Nu 4 is an unprotected nucleoside.

[0274] In some embodiments, Nu 1 , Nu2 and Nu 3 are the same nucleoside.

[0275] In some embodiments, Nu 1 , Nu 2 and Nu 3 are different nucleosides.

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

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

[0278] In some embodiments, Nu 2 and Nu 3 are the same nucleoside, and Nu 1 is a nucleoside different from Nu 2 and Nu 3

[0279] In some embodiments, Nu 1 , Nu 2 and Nu 4 are different nucleosides.

[0280] In some embodiments, Nu 1 and Nu 2 are the same nucleoside, and Nu 4 is a nucleoside different from Nu 1 and Nu 2

[0281] In some embodiments, the nucleosides are as described above.

[0282] In some embodiments, the capped dinucleoside trithiophosphate is Example

[0283] ​​​​The present invention is further defined in the following examples. It should be understood that these examples are given by way of illustration only. From the above discussion and examples, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made without departing from its spirit and scope to adapt the present invention to various uses and conditions. Therefore, the present invention is not limited by the illustrative examples set forth hereinafter, but is defined by the appended claims. Example 1 Preparation of 4,4,5,5 - tetrafluoropyridine - 4 - thiol (Compound 4)

[0284] 4,4,5,5 - tetrafluoropyridine - 4 - thiol was prepared according to the procedure disclosed by Dilman et al., Angew. Chem. Int. Ed. [Angewandte Chemie International Edition] 2021, 60, 2849 - 2854. Since a large amount of H2S is released during the reaction, all steps should be carried out in a well - ventilated fume hood.

[0285] Sodium hydrosulfide hydrate (48.2 g, 660 mmol, 2.2 equiv) was charged into a 250 mL round - bottom flask equipped with a stir bar, followed by the addition of MeOH (100 mL), and 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 were dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure (> 100 mbar, temperature 30 °C) to give 4,4,5,5 - tetrafluoropyridine - 4 - thiol (Compound 4; 52.2 g) as a colorless liquid, which solidified upon storage at 0 °C (yield = 95%). The product was characterized by 19 19FNMR (376 MHz, CDCl3): δ - 93.4 - - 93.7 (m, 2F), - 142.4 - - 142.6 (m, 2F). Example 2 Preparation of Compound 1

[0286] Charge a flame-dried 1 L round-bottom flask equipped with a stir bar with phosphorus pentasulfide (17.0 g, 75 mmol, 1.5 equiv), and then add anhydrous DCM (135 mL). Inertize the batch by flushing with argon for 2 min. Subsequently, add tetrafluoropyridine-4-thiol (Compound 4; 21.0 g, 115 mmol, 2.0 equiv) and immerse the reaction flask in an ice / water bath. Add tert-butylamine (18.4 mL, 150 mmol, 3.0 equiv) to the reaction mixture (Note: The reaction is very exothermic). Warm the resulting suspension to room temperature and stir for 16 h under an argon atmosphere. Carefully quench the reaction with water (135 mL) (Note: H2S is evolved during this step), and then add hexane (135 mL). Stir the resulting slurry for 30 min, then filter off the precipitate and wash successively with water (60 mL), DCM / hexane (1:1; 3 × 60 mL), and hexane (60 mL). Dry the filter cake in vacuo for 16 h to afford 18.4 g of Compound 1 as a white crystalline solid (yield = 60%). The product was characterized by 1 1H NMR (600 MHz, (CD3)2CO): δ 7.93 - 7.65 (m, 3H), 1.55 (s, 9H); 13 13C NMR (150 MHz, (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 19F NMR (376 MHz, CD3CN): δ -96.5 - -96.7 (m, 4F), -135.9 - -136.0 (m, 4F); 31 31P NMR (162 MHz, CD3CN): δ 92.5; HRMS (ESI-TOF) m / z: C 10 8F8N2PS4 [M - H] - , calcd: 458.8559, found: 458.8562; m.p. 152 °C - 153 °C. Example 3 Preparation of Compound 3

[0287] Charge a 250 mL round-bottom flask equipped with a stir bar with (-)-cis-limonene oxide (Compound 6; 12.2 mL, 75 mmol, 1.0 equiv), and change the atmosphere to argon. Add MeOH (50 mL), then add PtO2 (surface area ≥ 60 m 2 / g; 84 mg, 0.37 mmol, 0.5 mol %). The atmosphere in the flask was changed to H2, and the reaction vessel was equipped with an H2 balloon. The reaction mixture was stirred at room temperature for 3 h, after which TLC indicated complete conversion of the starting material. The crude reaction mixture was passed through a CELITE pad and then filtered through several volumes of DCM. The resulting solution was concentrated in vacuo to approximately 12 mL (>100 mbar, temperature 35 °C) and used in the next step without any further purification. Compound 3 was produced.

[0288] The second enantiomer of compound 2 was obtained via a similar procedure starting from (+)-cis-limonene oxide (compound 5). The syntheses of compounds 5 and 6 are disclosed in Steiner et al., Tetrahedron Asymmetry 2002, 13, 2359 - 2363. Example 4 (+)-Ψ* Reagent Preparation

[0289] 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 rendered inert by flushing with argon for 2 min, and the resulting suspension was cooled to -78 °C. Subsequently, MeOH (7.5 mL), crude epoxide 3 (12 mL; 2.0 equiv), and TFA (12 mL; 3.0 equiv) were added successively, and the resulting clear solution was stirred for 10 min. The cooling bath was removed and the reaction mixture was stirred for 1 h. Thereafter, 31 P NMR indicated complete conversion of the starting material compound 1 (see below). The reaction mixture was diluted with hexanes (150 mL) and successively washed with water (75 mL), 10% aqueous K2HPO4 (75 mL), and 10% aqueous KH2PO4 (75 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The resulting crude solid was redissolved in the minimum amount of DCM, and the resulting solution was diluted with MeOH (100 mL). Crystals appeared upon addition of MeOH, and the solution was allowed to stand at room temperature for 1 h to complete crystallization. The resulting slurry was filtered and the cake was washed with cold MeOH. After drying in vacuo, the compound (+)-Ψ* reagent was obtained as a white crystalline solid (8.9 g, d.r. >99:1, ee >99:1, yield = 55%). The compound (+)-Ψ* reagent was characterized by 1HNMR(600MHz, CDCl3): δ 4.51 (ddd, J = 12.8, 6.1, 3.7 Hz, 1H), 2.28 - 2.23 (m, 1H), 2.02 (td, J = 13.0, 4.2 Hz, 1H), 1.97 - 1.93 (m, 1H), 1.84 - 1.76 (m, 3H), 1.67 (s, 3H), 1.67 - 1.58 (m, 2H), 1.03 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.6 Hz, 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 = 8.8 Hz), 27.9 (d, J = 14.9 Hz), 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: 432.0303, measured value: 432.0290; [α] D 20 = +315.3 (c 1.01, CHCl3); m.p. 131 °C.

[0290] Compound (-)-Ψ* reagent was obtained via a similar procedure starting from (+)-cis-limonene oxide (Compound 2). All the characterization data are the same except for the optical rotation. Compound (-)-Ψ* reagent is characterized by [α] D 20 = -314.7 (c 1.01, CHCl3). Example 5 Preparation of Compound 7-1 Step 1. Benzoylation

[0291] Charge a flame-dried round-bottom flask equipped with a stir bar with 4-hydroxybenzaldehyde (24.4 g, 200 mmol, 1.0 equiv). Dissolve the solid substrate in anhydrous THF (200 mL), followed by the addition of NEt3 (33.5 mL, 240 mmol, 1.2 equiv). Immerse the reaction flask in an ice / water bath and add benzoyl chloride (23.2 mL, 200 mmol, 1.0 equiv) over 3 min. Allow the reaction mixture to warm to room temperature overnight. Subsequently, dilute the mixture with EtOAc and filter through a pad of Celite. Wash the filtrate with saturated aqueous NH4Cl and dry the organic layer over Na2SO4, filter, and remove volatiles in vacuo to afford crude 4-formylphenyl benzoate (46 g), which is used in the next step without any further purification. Step 2. Reduction

[0292] Dissolve crude 4-formylphenyl benzoate (46 g) in THF (200 mL) and cool the reaction mixture to 0 °C. Add NaBH4 (11.3 g, 300 mmol, 3.0 equiv) in three portions and allow the mixture to warm to room temperature over 2 h. Subsequently, carefully quench the reaction with saturated aqueous NH4Cl and dilute with EtOAc. Separate the organic phase and extract the aqueous fraction with EtOAc. Combine the organic layers, dry over Na2SO4, filter, and concentrate in vacuo to afford crude 4-(hydroxymethyl)phenyl benzoate (48 g), which is used in the next step without any further purification. Step 3. Phosphorylation

[0293] Dissolve crude 4-(hydroxymethyl)phenyl benzoate (24 g) in anhydrous THF (200 mL) and cool to -78 °C. Add phosphorus oxychloride (13.8 mL, 300 mmol, 3.0 equiv) dropwise over 10 min. Stir the reaction mixture at -78 °C for 4 h, after which quench the reaction with water. Carefully adjust the pH of the resulting solution to 8 with saturated aqueous NaHCO3. Add concentrated aqueous HCl dropwise to the resulting suspension until the solution becomes clear. Extract the reaction mixture with EtOAc and wash the combined organic fractions with water. Combine the organic layers, dry over Na2SO4, filter, and concentrate in vacuo to afford a crude solid of compound 7-1. Suspend the solid in DCM, filter, and wash the filter cake with DCM. After drying under reduced pressure, obtain the phosphate ester compound 7-1 as a white crystalline solid (17.0 g, 55 mmol, yield over 3 steps = 55%). Compound 7-1 is characterized by 11H NMR (600 MHz, CD3OD): δ 8.20 - 8.16 (m, 2H), 7.72 - 7.67 (m, 1H), 7.59 - 7.54 (m, 2H), 7.50 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 5.05 (d, J = 7.4 Hz, 2H); 13 13C NMR (150 MHz, CD3OD): δ 166.6, 155.2, 136.2 (d, J = 7.8 Hz), 135.0, 131.04, 131.02, 130.7, 129.9 (d, J = 3.1 Hz), 122.9, 68.6 (d, J = 5.0 Hz); 31 31P NMR (162 MHz, CD3OD): δ -0.1; HRMS (ESI-TOF) m / z: C 14 H 12 O6P [M - H] - , calculated: 307.0371, found: 307.0361; m.p. 140 °C - 142 °C. Example 6 Preparation of Compound 22

[0294] 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, backfilled with argon (3 times) and capped with a septum. Subsequently, anhydrous THF (160 mL) was 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. Then, anhydrous diisopropylamine (12.0 mL, 87 mmol, 1.9 equiv) was added dropwise over 10 min and the resulting suspension was stirred at 0 °C for 1 h. Thereafter, another portion of DIPEA (16.0 mL, 92 mmol, 2.0 equiv) was 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 Celite pad and the filtrate was concentrated under reduced pressure. The residue was diluted with DCM, loaded onto a silica gel pad (18 × 4 cm) and eluted with hexane / EtOAc / NEt3 (100:5:1). The fractions containing the pure product (as determined by 31The (P NMR determination) was 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 any further purification. Compound 22 should be stored at -20 °C under an argon atmosphere. Compound 22 is characterized by 1 1H NMR (600 MHz, 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 = 6.9 Hz, 2H), 4.01 (dt, J = 9.9, 6.8 Hz, 2H), 3.81 (dt, J = 9.9, 7.3 Hz, 2H), 3.66 (hept, J = 6.8 Hz, 2H), 1.16 (d, J = 6.8 Hz, 12H); 13 13C NMR (150 MHz, CDCl3): δ 145.1, 144.8, 141.5, 141.4, 127.54, 127.50, 127.0, 126.9, 125.6, 125.3, 120.0, 119.9, 66.1 (d, J = 17.1 Hz), 49.3 (d, J = 7.7 Hz), 43.2 (d, J = 12.1 Hz), 24.8 (d, J = 7.2 Hz); 31 31P NMR (162 MHz, CDCl3): δ 146.0.

[0295] The NMR data is consistent with that previously reported in Lambrecht et al., J. Am. Chem. Soc. [Journal of the American Chemical Society] 2015, 137, 3558 - 3564. Example 7 Preparation of Compound 20-1

[0296] A flame-dried round-bottom flask equipped with a stir bar was charged with freshly prepared iPr2NP(OFm)2 (Compound 22; 4.0 g, 7.7 mmol, 1.2 equiv), followed by the addition of anhydrous MeCN (20 mL). To the resulting suspension was successively added the monophosphate compound 7-1 (2.0 g, 6.4 mmol, 1.0 equiv) and triethylamine (0.89 mL, 6.4 mmol, 1.0 equiv). After stirring for 2 min, 5-phenyl-1H-tetrazole (1.4 g, 9.6 mmol, 1.5 equiv) was added and the resulting mixture was stirred for 1 h under an argon atmosphere. Subsequently, tert-butyl hydroperoxide (5.5 M in decane; 2.3 mL, 12.8 mmol, 2.0 equiv) was 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 silica gel-packed chromatography column (18 × 4 cm), and the column was rinsed with 400 mL of EtOAc. The eluent was changed to MeOH / DCM (1:15), and chromatography was continued until all of the product was eluted from the silica (as indicated by TLC: MeOH / DCM, 1:4, R f = 0.8). The fractions containing the product were combined and concentrated in vacuo (temperature < 35 °C). The residue was treated with DCM and the resulting suspension was filtered. The filter cake was washed with DCM, the filtrate was concentrated in vacuo (temperature < 35 °C), and the residual MeOH was removed by co-evaporation 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 any significant loss of purity.

[0297] Compound 20-1 was characterized by 1 1H NMR (600 MHz, CDCl3): δ 8.86 - 8.78 (m, 2H), 8.18 - 8.16 (m, 2H), 7.68 (ddt, J = 8.7, 7.6, 1.0 Hz, 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, 1.2 Hz, 4H), 7.09 - 7.06 (m, 2H), 4.97 (d, J = 8.0 Hz, 2H), 4.32 - 4.23 (m, 4H), 4.14 (t, J = 7.1 Hz, 2H), 3.12 (hept, J = 6.5 Hz, 2H), 1.21 (d, J = 6.5 Hz, 12H); 13CNMR (150 MHz, CDCl3): δ 165.3, 150.6, 143.5, 143.4, 141.5, 135.8 (d, J = 7.5 Hz), 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 = 5.8 Hz), 67.9 (d, J = 5.8 Hz), 48.0 (d, J = 8.1 Hz), 46.8, 19.1; 31 PNMR (162 MHz, CD3OD): δ -11.6 (d, J = 20.0 Hz), -13.4 (d, J = 20.0 Hz); HRMS (ESI-TOF) m / z: C 44 H 33 O9P2[M-iPr2NH2] - , calculated: 743.1600, found: 743.1628. Example 8 Stereocontrolled synthesis of α-thiophosphonates General procedure A: Stereocontrolled synthesis of α-thiophosphonates

[0298] Isomer R of α-thiophosphonate P can be obtained from (+)-Ψ* reagent. Isomer S of α-thiophosphonate P can be obtained from (-)-Ψ* reagent.

[0299] Charge a flame-dried 1 dram vial equipped with a stir bar with monophosphate compound 7-1 (61.6 mg, 0.2 mmol, 1.0 equiv.). Seal the vial with a Teflon septum screw cap, evacuate and backfill with argon. Add anhydrous MeCN (2.0 mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 60 μL, 0.4 mmol, 2.0 equiv.), and stir the mixture until the starting material is completely dissolved (ca. 5 min). Subsequently, add A molecular sieve (60 mg) and Ψ* reagent (128 mg, 0.3 mmol, 1.5 equiv) were added and the reaction was stirred at room temperature for 30 min. Thereafter, the protected nucleoside (0.5 mmol, 2.5 equiv) was added, followed by a further portion of DBU (120 μL, 0.8 mmol, 4.0 equiv) and the mixture was stirred for an additional 90 min. After completion of the reaction, the resulting mixture was filtered and concentrated in vacuo to approximately 0.5 mL. Concentrated aqueous NH3 (5.0 mL) was added to the residue and the resulting mixture was stirred at room temperature (or at 40 °C) for 16 h. Subsequently, the reaction mixture was diluted with water and washed with EtOAc. The aqueous phase was concentrated in vacuo (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, from 0:100 to 30:70). The fractions containing the product were combined and the solvent was evaporated in vacuo (temperature ≤40 °C). The solid residue was dissolved in the minimum amount of water and lyophilized to afford the pure nucleoside α-thio diphosphate. Example 9 Stereocontrolled synthesis of α-thio triphosphates General procedure B: Stereocontrolled synthesis of α-thio triphosphates

[0300] Isomer R of α-thio triphosphate P can be obtained from the (+)-Ψ* reagent. Isomer S of α-thio triphosphate P can be obtained from the (-)-Ψ* reagent.

[0301] A flame-dried 1-dram vial equipped with a stir bar was charged with the pyrophosphate compound 20-1 (169 mg, 0.20 mmol, 1.0 equiv). The vial was sealed 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, Molecular sieve (200 mg) and Ψ* reagent (112 mg, 0.26 mmol, 1.3 equiv) and the reaction was stirred at room temperature for 15 min. Thereafter, the protected nucleoside (0.40 mmol, 2.0 equiv) was added, followed by another portion of DBU (180 μL, 1.2 mmol, 6.0 equiv) and the mixture was stirred for 3 to 6 h. After completion of the reaction, the resulting mixture was filtered and concentrated in vacuo to approximately 0.5 mL. Concentrated aqueous NH3 (5.0 mL) was added to the residue and the resulting mixture was stirred at room temperature (or at 40 °C) for 16 h. Subsequently, the reaction mixture was diluted with water and washed with EtOAc. The aqueous phase was concentrated in vacuo (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 pellets were washed twice with acetone. The crude product was purified by ion exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, from 0:100 to 50:50). The fractions containing the product were combined and the solvent was evaporated in vacuo (temperature 40 °C). The solid residue was dissolved in the minimum amount of water and lyophilized to afford the pure nucleoside α-thiotriphosphate. Example 10 Stereocontrolled synthesis of dinucleoside thiophosphates General procedure C: Stereocontrolled synthesis of dinucleoside thiophosphates

[0302] Isomer R of dinucleoside thiophosphate P can be obtained from the (+)-Ψ* reagent. Isomer S of dinucleoside thiophosphate P can be obtained from the (-)-Ψ* reagent.

[0303] Charge a flame-dried 1-dram vial equipped with a magnetic stir bar with the protected nucleoside monophosphate (0.2 mmol, 1.0 equiv). Seal the vial with a Teflon septum screw cap, evacuate and backfill with argon. Add anhydrous MeCN (2.0 mL) and DBU (60 μL, 0.4 mmol, 2.0 equiv) and stir the mixture until the starting material is completely dissolved (ca. 5 min). Subsequently, add Molecular sieve (60 mg) and Ψ* reagent (128 mg, 0.3 mmol, 1.5 eq) and the reaction was stirred at room temperature for 30 min. Thereafter, the protected nucleoside (0.5 mmol, 2.5 eq) was added, followed by another portion of DBU (120 μL, 0.8 mmol, 4.0 eq) and the mixture was stirred for an additional 2 h. After completion of the reaction, the resulting mixture was filtered and concentrated in vacuo to approximately 0.5 mL. Concentrated aqueous NH3 (5.0 mL) was added to the residue and the resulting mixture was stirred at 40 °C for 16 h. Subsequently, the reaction mixture was diluted with water and washed with EtOAc. The aqueous phase was concentrated in vacuo (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 pellets were washed twice with acetone. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, from 0:100 to 30:70). The fractions containing the product were combined and the solvent was evaporated in vacuo (temperature ≤40 °C). The solid residue was dissolved in the minimum amount of water and lyophilized to afford the pure dinucleoside thiophosphate. Example 11 Preparation of Non-Commercial Substrate Compounds 10-1, 11-1, 12-1, 13-1, 14-1, 15-1 and 23

[0304] Compound 10-1 was prepared by the following procedure disclosed by Debarge et al., J. Org. Chem. [Journal of Organic Chemistry] 2011, 76, 105-126.

[0305] Compound 11-1 was prepared by the following procedure disclosed by Zhu et al., Synth. Commun. [Synthetic Communications] 2008, 38, 1346-1354.

[0306] Compound 12-1 was prepared by the following procedure disclosed by Debarge et al., J. Org. Chem. [Journal of Organic Chemistry] 2011, 76, 105-126.

[0307] Compound 13-1 was prepared by the following procedure disclosed by Zhu et al., Synth. Commun. [Synthetic Communications] 2008, 38, 1346-1354.

[0308] Compound 14-1 was prepared by the following procedure disclosed by Zhu et al., Synth. Commun. 2008, 38, 1346-1354.

[0309] Compound 15-1 was prepared by the following procedure disclosed by Zhu et al., Synth. Commun. 2008, 38, 1346-1354.

[0310] Compound 23 was prepared by the following procedure disclosed by Saudi et al., Eur. J. Med. Chem. 2014, 76, 98-109. Example 12 Preparation of non-commercially available substrate compound 18-1

[0311] A round-bottomed 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). Subsequently, imidazole (1.40 g, 20.6 mmol, 2.2 equiv) and tert-butyldimethylchlorosilane (1.55 g, 10.3 mmol, 1.1 equiv) were added and the resulting suspension was stirred at room temperature for 15 min. Thereafter, the reaction mixture was cooled to -10 °C. A further portion of tert-butyldimethylchlorosilane (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 Et2O and dried under reduced pressure to afford 2.48 g of compound 24, which was used directly in the next step.

[0312] Compound 24 (2.48, 6.5 mmol, 1.0 equiv) was suspended in anhydrous MeCN (50 mL), followed by the addition of benzoic anhydride (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. Subsequently, the reaction was cooled to room temperature and quenched by the addition of saturated aqueous NaHCO3. The solid residue was filtered off, washed successively with water and MeCN, and dried under reduced pressure to afford 2.80 g of crude compound 25, which was used directly in the next step.

[0313] The crude compound 25 (2.80 g, 5.8 mmol, 1.0 equiv) was suspended in THF (20 mL), and then H2O (5 mL) and TFA (5 mL) were added. The reaction was stirred at room temperature for 90 min. Subsequently, the solution was neutralized by careful addition of saturated aqueous NaHCO3. The crude mixture was concentrated to approximately 5 mL under reduced pressure and filtered. The solid residue was washed successively with water and Et2O and dried under reduced pressure to afford 1.92 g of pure compound 18-1 (yield = 55% over 3 steps). Physical state: white amorphous solid. Compound 18-1 was characterized by 1 1H NMR (600 MHz, DMSO-d6): δ 10.68 (br s, 1H), 8.03 (d, J = 7.7 Hz, 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, 5.7 Hz, 1H), 5.57 (d, J = 5.7 Hz, 1H), 5.20 (t, J = 5.7 Hz, 1H), 4.23 - 4.19 (m, 1H), 3.70 - 3.62 (m, 2H), 2.91 (ddd, J = 14.6, 9.1, 5.9 Hz, 1H), 2.58 (dd, J = 14.6, 5.7 Hz, 1H); 13 13C NMR (150 MHz, 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] + , calculated: 372.1303, found: 372.1285. Example 13 Preparation of the non-commercially available substrate compound 17-1

[0314] Charge a round-bottomed flask equipped with a stir bar with guanosine (14.2 g, 50 mmol, 1.0 equiv), and then add anhydrous DMF (200 mL). Cool the heterogeneous mixture to 0 °C, and then add imidazole (10.2 g, 150 mmol, 3.0 equiv) and tert-butyldimethylchlorosilane (15.1 g, 100 mmol, 2.0 equiv). Allow the reaction mixture to warm to room temperature overnight. Subsequently, quench the reaction by adding water (800 mL), and filter the resulting suspension. Wash the solid residue successively with water and acetone, and dry under reduced pressure to afford 12.1 g of crude compound 26, which is used directly in the next step.

[0315] Suspend crude compound 26 (12.1 g, 30.4 mmol, 1.0 equiv) in anhydrous MeCN (150 mL), and then add benzoic anhydride (27.5 g, 121.6 mmol, 4.0 equiv) and DMAP (0.74 g, 6.2 mmol, 0.2 equiv). The reaction vessel is equipped with an air condenser, and reflux the heterogeneous mixture for 3 h. Then, cool the reaction to room temperature and quench by adding saturated aqueous NaHCO3. Filter off the solid residue, wash successively with water and MeCN, and dry under reduced pressure to afford 14.9 g of crude compound 27, which is used directly in the next step.

[0316] Suspend crude compound 27 (14.9 g, 24.6 mmol, 1.0 equiv) in THF (90 mL), and then add H2O (22.5 mL) and TFA (22.5 mL). Stir the reaction at room temperature for 90 min. Then, neutralize the solution by carefully adding saturated aqueous NaHCO3. Concentrate the crude mixture under reduced pressure to about 25 mL and filter. Wash the solid residue successively with water and Et2O, and dry under reduced pressure to afford 11.5 g of pure compound 17-1 (yield in 3 steps = 47%). Physical state: white amorphous solid. Compound 17-1 is characterized by 11H NMR (600 MHz, DMSO-d6): δ 10.77 (br s, 1H), 8.07 (s, 1H), 7.94 (dd, J = 8.3, 1.4 Hz, 2H), 7.79 (dd, J = 8.4, 1.4 Hz, 1H), 7.68 (tt, J = 7.4, 1.4 Hz, 1H), 7.61 (tt, J = 7.4, 1.4 Hz, 1H), 7.54 - 7.50 (m, 2H), 7.43 - 7.39 (m, 2H), 6.54 (br s, 2H), 6.26 (d, J = 6.7 Hz, 1H), 6.12 (dd, J = 6.7, 5.5 Hz, 1H), 5.84 (dd, J = 5.5, 2.8 Hz, 1H), 5.51 (t, J = 5.5 Hz, 1H), 4.49 (q, J = 3.4 Hz, 1H), 3.84 - 3.75 (m, 2H); 13 13C NMR (150 MHz, 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] + , calculated: 492.1514, found: 492.1493. Example 14 Preparation of Non-Commercial Substrate Compounds 16-1 and 30

[0317] Charge a round-bottom flask equipped with a stir bar with 2-thiouridine (1.87 g, 7.2 mmol, 1.0 equiv), and then add anhydrous pyridine (40 mL). Cool the solution to 0 °C, and then add tert-butyldimethylchlorosilane (1.42 g, 9.4 mmol, 1.3 equiv). Allow the reaction mixture to warm to room temperature overnight. Subsequently, add benzoyl chloride (3.0 mL, 25.9 mmol, 3.6 equiv) and stir the reaction for an additional 8 h. Thereafter, dilute the reaction mixture with DCM and wash with 1 M HCl and water. Dry the organic phase over MgSO4, filter, and concentrate under reduced pressure. Purify the crude product by silica gel chromatography (EtOAc / hexane / DCM; from 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 is used directly in the next step.

[0318] A mixture of regioisomer compounds 28 and 29 (3.21 g, 4.7 mmol, 1.0 equiv) was dissolved in THF (84 mL). The solution was cooled to 0 °C, and then H2O (21 mL) and TFA (21 mL) were added. The reaction was allowed to warm to room temperature over 3 h. Subsequently, the solution was neutralized by careful addition of saturated aqueous NaHCO3. The crude product was extracted with DCM, and 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; from 10:40:50 to 40:10:50) to afford 1.77 g of a regioisomer mixture of compound 30 and 16-1 (ratio 2:1; structures of the minor and major regioisomers not specified) (yield = 43% over 3 steps). Physical state: white amorphous solid.

[0319] The regioisomer mixture of compound 30 and 16-1 is characterized by 1 1H NMR (600 MHz, DMSO-d6): δ 8.57 (d, J = 8.2 Hz, 1H; major), 8.55 (d, J = 8.2 Hz, 1H; minor), 8.00 - 7.79 (m, 6H; major + minor), 7.75 - 7.36 (m, 9H; major + minor), 7.21 - 7.17 (m, 1H; major + minor), 6.52 (d, J = 8.2 Hz, 1H; major), 6.46 (d, J = 8.2 Hz, 1H; minor), 5.89 - 5.75 (m, 3H; major + minor), 4.63 (s, 1H; major), 4.62 (s, 1H; minor), 3.94 - 3.83 (m, 2H; major + minor); 1313C NMR(150MHz, DMSO-d6): δ 174.4 (minor), 174.2 (major), 168.1 (minor), 167.9 (major), 164.9 (major), 164.7 (major), 164.6 (minor), 164.4 (minor), 159.0 (minor), 158.9 (major), 141.5 (major + minor), 135.0 (major + minor), 133.91 (major), 133.90 (major + minor), 138.85 (minor), 130.8 (minor), 130.6 (major), 130.20 (minor), 130.15 (major), 129.5 (minor), 129.4 (major), 129.34 (major + minor), 129.28 (major), 129.20 (minor), 128.9 (major + minor), 128.82 (minor), 128.79 (major), 128.71 (major + minor), 128.4 (minor), 129.3 (major), 106.9 (major), 106.8 (minor), 89.5 (minor), 89.4 (major), 84.0 (major), 83.9 (minor), 74.9 (major), 74.7 (minor), 71.6 (major), 71.1 (minor), 60.2 (major), 60.1 (minor); HRMS(ESI-TOF) m / z: C 30 H 24 N2O8SNa [M+Na] + , calculated: 595.1151, found: 595.1138. Example 15 Preparation of Non-Commercial Substrate Compound 31

[0320] Charge a flame-dried round-bottom flask 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). Dissolve the substrate in a mixture of anhydrous MeCN (25 mL) and DCM (25 mL). Add iPr2NP(OBn)2 (1.25 mL, 3.9 mmol, 1.3 equiv) dropwise to the resulting mixture, and stir the reaction under an argon atmosphere for 1 h. Subsequently, cool the reaction mixture to -40 °C, then add 30% aqueous H2O2 (10 mL) and allow the reaction to warm to room temperature within 1 h. Dilute the resulting solution with DCM, and wash the organic phase successively with saturated aqueous NaHCO3 and brine. Dry the organic layer over MgSO4, filter and concentrate under reduced pressure. Purify the crude residue by silica gel chromatography (EtOAc / hexane / DCM; from 10:40:50 to 20:30:50) to afford 2.17 g of Compound 31 (yield = 77%). Physical state: white amorphous solid. Compound 31 is characterized by 1 1H NMR (600 MHz, CDCl3): δ 8.62 (s, 1H), 8.40 (s, 1H), 7.97 (dd, J = 8.4, 1.4 Hz, 2H), 7.90 (dd, J = 8.4, 1.4 Hz, 2H), 7.88 - 7.84 (m, 4H), 7.58 (tt, J = 7.4, 1.4 Hz, 1H), 7.55 (tt, J = 7.4, 1.4 Hz, 1H), 7.49 - 7.46 (m, 2H), 7.42 - 7.39 (m, 2H), 7.38 - 7.32 (m, 10H), 7.31 - 7.24 (m, 6H), 6.50 (d, J = 5.7 Hz, 1H), 6.09 (t, J = 5.7 Hz, 1H), 5.98 (dd, J = 5.7, 4.0 Hz, 2H), 5.11 - 5.02 (m, 4H), 4.62 - 4.59 (m, 1H), 4.37 (dd, J = 6.2, 3.6 Hz, 2H); 1313C NMR (150 MHz, CDCl3): δ 172.4, 165.4, 165.0, 153.1, 152.6, 152.3, 143.4, 135.60 (d, J = 6.5 Hz), 135.58 (d, J = 6.5 Hz), 134.2, 133.95, 133.93, 133.2, 130.01, 129.96, 129.6, 128.9, 128.81, 128.79, 128.73, 128.66, 128.5, 128.28, 128.27, 127.9, 86.6, 81.9 (d, J = 8.0 Hz), 74.3, 71.6, 69.96 (d, J = 5.5 Hz), 69.94 (d, J = 5.5 Hz), 66.5 (d, J = 5.2 Hz); 31 31P NMR (162 MHz, CDCl3): δ -1.0; HRMS (ESI-TOF) m / z: C 52 H 43 N5O 11 P [M + H] + , calculated: 944.2697, found: 944.2669. Example 16 Preparation of the non-commercially available substrate compound 32

[0321] A round-bottomed flask equipped with a stir bar was charged with the protected adenosine phosphate (Compound 31; 2.17 g, 2.3 mmol, 1.0 equiv), and the atmosphere was changed to argon. MeOH (140 mL) was added, followed by Pd / C (10% wt.; 325 mg). The atmosphere in the flask was changed to H2, and the reaction vessel was equipped with an H2 balloon. The reaction mixture was stirred at room temperature for 1 h, after which TLC indicated complete conversion of the starting material. The crude reaction mixture was filtered through a pad of Celite, followed by filtration with several volumes of MeOH / DCM (1:1). The volatiles were removed under reduced pressure, and the residue was co-evaporated with DCM twice 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 is characterized by 11H NMR (600 MHz, CD3OD): δ 8.81 (s, 1H), 8.58 (s, 1H), 8.00 (d, J = 6.8 Hz, 2H), 7.81 (d, J = 7.0 Hz, 4H), 7.76 (d, J = 7.4 Hz, 2H), 7.58 (t, J = 7.4 Hz, 1H), 7.51 - 7.45 (m, 3H), 7.41 (t, J = 7.8 Hz, 2H), 7.34 (t, J = 7.7 Hz, 4H), 7.27 (t, J = 7.8 Hz, 2H), 6.67 (d, J = 5.8 Hz, 1H), 6.21 (t, J = 5.8 Hz, 1H), 6.08 (dd, J = 5.8, 3.6 Hz, 1H), 4.77 - 4.74 (m, 1H), 4.47 - 4.38 (m, 2H); 13 13C NMR (150 MHz, 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 = 8.0 Hz), 75.8, 73.2, 66.67 (d, J = 3.8 Hz); 31 31P NMR (162 MHz, CD3OD): δ 0.4; HRMS (ESI - TOF) m / z: C 38 H 29 N5O 11 P [M - H] - , calculated: 762.1601, found: 762.1625. Example 17 Preparation of Non - Commercially Available Substrate Compound 33

[0322] 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). iPr2NP(OBn)2 (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. Subsequently, the reaction mixture was cooled to -40 °C, followed by the addition of 30% aqueous H2O2 (18 mL) and the reaction was allowed to warm to room temperature within 1 h. The resulting solution was diluted with DCM, and the organic phase was washed successively with saturated aqueous NaHCO3 and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (EtOAc / hexane / DCM; from 10:40:50 to 30:20:50) to afford 3.88 g of Compound 33 (yield = 86%). Physical state: white amorphous solid. Compound 33 was characterized by 1 1H NMR (600 MHz, CDCl3): δ 7.98 (d, J = 7.2 Hz, 2H), 7.91 (dd, J = 8.4, 1.2 Hz, 2H), 7.87 (d, J = 7.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 1H), 7.61 - 7.56 (m, 2H), 7.52 (tt, J = 7.3, 1.2 Hz, 2H), 7.43 - 7.30 (m, 16H), 6.41 (d, J = 7.0 Hz, 1H), 5.67 - 5.64 (m, 2H), 5.46 (t, J = 6.5 Hz, 1H), 5.19 - 5.08 (m, 4H), 4.47 - 4.44 (m, 1H), 4.37 - 4.30 (m, 2H); 13 13C NMR (150 MHz, CDCl3): δ 168.4, 165.42, 165.40, 161.7, 149.7, 139.2, 130.49 (d, J = 5.7 Hz), 135.46 (d, J = 6.2 Hz), 135.1, 134.0, 133.9, 131.5, 130.6, 130.0, 129.9, 129.2, 129.14, 129.09, 129.0, 128.9, 128.76, 128.75, 128.7, 128.39, 128.36, 103.7, 86.5, 81.7 (d, J = 8.1 Hz), 73.7, 71.6, 70.17 (d, J = 5.4 Hz), 70.15 (d, J = 5.4 Hz), 66.6 (d, J = 5.2 Hz); 31P NMR (162 MHz, CDCl3): δ -0.6; HRMS (ESI-TOF) m / z: C 44 H 38 N2O 12 P[M + H] + , calculated: 817.2157, found: 817.2153. Example 18 Preparation of Non-Commercial Substrate Compound 34

[0323] A round-bottomed 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 changed to argon. MeOH (100 mL) and EtOAc (100 mL) were added, followed by Pd / C (10% wt.; 675 mg). The atmosphere in the flask was changed to H2, and the reaction vessel was equipped with an H2 balloon. The reaction mixture was stirred at room temperature for 1.5 h, after which TLC indicated complete conversion of the starting material. The crude reaction mixture was filtered through a pad of Celite, followed by filtration through several volumes of MeOH / DCM (1:1). The volatiles were removed under reduced pressure, and the residue was co-evaporated with DCM twice 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 is characterized by 1 H NMR (600 MHz, (CD3)2CO): δ 9.38 (br s, 2H), 8.18 (d, J = 7.5 Hz, 1H), 8.03 - 7.98 (m, 4H), 7.86 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.61 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.49 (t, J = 7.7 Hz, 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.32 (t, J = 7.7 Hz, 2H), 6.48 (d, J = 6.6 Hz, 1H), 6.08 (d, J = 6.9 Hz, 1H), 5.98 - 5.94 (m, 1H), 5.84 - 5.79 (m, 1H), 4.82 (br s, 1H), 4.64 - 4.55 (m, 2H); 1313C NMR (150 MHz, (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, 130.48, 130.1, 130.0, 129.6, 129.49, 129.46, 129.3, 103.7, 87.6, 82.2 (d, J = 7.4 Hz), 75.0, 72.7, 66.9; 31 31P NMR (162 MHz, (CD3)2CO): δ 0.3; HRMS (ESI-TOF) m / z: C 30 H 24 N2O 12 P [M-H] - , calculated: 635.1072, found: 635.1071. Example 19 Preparation of Non-Commercial Substrate Compound 36

[0324] According to the procedure disclosed by Jo Davisson et al., J. Org. Chem. [Journal of Organic Chemistry] 1987, 52, 1794 - 1801, guanosine (2.83 g, 10 mmol, 1.0 equiv) was suspended in anhydrous trimethyl orthoformate (25 mL), and then pyridinium hydrochloride (1.75 g, 15 mmol, 1.5 equiv) was added. The stirred suspension was treated with DMSO (3.5 mL), and the mixture was stirred at room temperature for 48 h. MeOH (25 mL) and solid NaOMe (0.90 g, 16.5 mmol, 1.7 equiv) were sequentially added to the resulting turbid suspension, and the reaction was stirred for an additional 3 h. Subsequently, the mixture was concentrated under reduced pressure, and the resulting thick suspension was treated with MeOH / Et2O (1:1). The obtained pale yellow solid was filtered off and dried under reduced pressure. The crude compound 35 was used directly in the next step.

[0325] The crude compound 35 was redissolved in anhydrous DMF (12 mL), and then Na2HPO4 (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 Celite pad and the solid residue was washed with MeOH. The filtrate was concentrated to about 10 mL under reduced pressure and loaded directly onto silica gel. The crude product was purified by silica gel chromatography (DCM / MeOH; from 100:0 to 80:20) to afford 2.20 g of compound 36 (a 2:1 mixture of diastereomers). (Yield in 2 steps = 49%). Physical state: white amorphous solid. Compound 36 was characterized by 1 1H NMR (600 MHz, D2O): δ 6.31 (d, J = 2.3 Hz, 1H; major), 6.23 (s, 1H; minor), 6.21 (d, J = 2.4 Hz, 1H; minor), 6.13 (s, 1H; major), 5.50 (dd, J = 6.2, 2.4 Hz, 1H; minor), 4.48 (dd, J = 6.8, 2.3 Hz, 1H; major), 5.15 (dd, J = 6.1, 2.4 Hz, 1H; minor), 5.09 (dd, J = 6.8, 2.6 Hz, 1H; major), 4.72 - 4.69 (m, 1H; major), 4.64 - 4.61 (m, 1H; minor), 4.11 (s, 3H; major + minor), 3.90 - 3.86 (m, 1H; major + minor), 3.86 - 3.81 (m, 1H; major + minor), 3.75 (s, 3H; major + minor), 3.49 (s, 3H; major), 3.40 (s, 3H; minor); 13 13C NMR (150 MHz, D2O): δ 150.5 (minor), 157.42 (minor), 157.37 (major), 157.29 (major), 149.4 (major + minor), 136.1 (t, 1:1:1, J 1 C-D = 35.1 Hz; major + minor), 118.4 (major), 117.3 (minor), 108.7 (major + minor), 92.6 (major), 91.9 (minor), 89.0 (major), 87.6 (minor), 84.3 (major), 83.6 (minor), 81.5 (major), 80.7 (minor), 61.4 (major), 61.2 (minor), 55.4 (major + minor), 52.6 (major), 51.6 (minor), 35.71 (minor), 35.70 (major); HRMS (ESI-TOF) m / z: C13H18N5O6 [M] + , calcd: 340.1252, found: 340.1264. Example 20 Preparation of Non-Commercial Substrate Compound 39

[0326] Guanosine monophosphate disodium (Compound 37; 10 g, 25 mmol, 1.0 eq) was converted to dihydroguanosine monophosphate (Compound 38) according to the procedure disclosed in Lu et al., Org. Lett. [Organic Chemistry Letters] 2016, 18, 1724 - 1727. Compound 38 was used in the next step without any further purification.

[0327] The dihydroguanosine 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. Subsequently, the reaction mixture was concentrated under reduced pressure, and then Et2O was added. The resulting suspension was filtered to afford 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 chromatography (1 M TEAA / MeCN aqueous solution; from 100:0 to 70:30) and lyophilized to afford 7.13 g of Compound 39 (a 9:1 mixture of diastereomers). (Yield in 3 steps = 47%). Physical state: white amorphous solid. Compound 39 is characterized by 1 H NMR (600 MHz, D2O): δ 7.97 (s, 1H; minor), 7.95 (s, 1H; major), 6.18 (s, 1H; minor), 6.14 (d, J = 2.7 Hz, 1H; major), 6.07 (s, 1H; major), 6.02 (d, J = 2.9 Hz, 1H; minor), 5.40 (dd, J = 6.2, 2.9 Hz, 1H; minor), 5.35 (dd, J = 7.0, 2.7 Hz, 1H; major), 5.21 (dd, J = 6.2, 2.8 Hz, 1H; minor), 5.15 (dd, J = 7.0, 3.1 Hz, major), 4.60 (q, J = 4.3 Hz, 1H; major), 4.51 (q, J = 4.1 Hz, 1H; minor), 4.10 - 4.05 (m, 1H; major + minor), 4.05 - 3.99 (m, 1H; major + minor), 3.45 (s, 3H; major), 3.35 (s, 3H; minor), 3.16 (q, J = 7.3 Hz, 12H; Et3NH + ), 1.24 (t, J = 7.3 Hz, 18H; Et3NH + ); 1313C NMR (150 MHz, D2O): δ 158.6 (major + minor), 153.72 (minor), 153.66 (major), 151.1 (minor), 151.0 (major), 137.8 (major), 137.7 (minor), 118.6 (major), 117.3 (minor), 116.1 (major + minor), 90.1 (major), 89.1 (minor), 86.0 (d, J = 8.7 Hz; major), 84.5 (d, J = 8.8 Hz; minor), 84.1 (major), 83.2 (minor), 81.4 (major), 80.7 (minor), 64.7 (d, J = 4.8 Hz; major), 64.5 (d, J = 4.6 Hz; minor), 52.5 (major), 51.4 (minor), 46.6 (Et3NH + ), 8.2 (Et3NH + ); 31 31P NMR (162 MHz, D2O): δ 0.6; HRMS (ESI-TOF) m / z: C 12 H 15 N5O9P [M-H] - , calcd: 404.0613, found: 404.0626. Example 21 Preparation of Non-Commercial Substrate Compound 40

[0328] Compound 39 (2.61 g, 4.3 mmol; 1.0 equiv) was dried by co-evaporation with anhydrous DMF and dissolved in anhydrous DMF (12 mL). iPr2NP(OFm)2 (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. Subsequently, tert-butyl hydroperoxide (5.5 M in decane; 2.3 mL, 12.9 mmol, 3.0 equiv) was added and the mixture was stirred for an additional 1 h. The reaction was quenched by the addition of Et2O (100 mL). The solvent was decanted, and the remaining oily residue was washed twice with Et2O. The residue was redissolved in DCM (10 mL), followed by the addition of Et2O (100 mL). The resulting cloudy mixture was sonicated for 10 min to initiate precipitation. The precipitate was filtered off to afford 4.05 g of crude compound 40 (75% wt. purity as determined by 31P NMR) (9:1 mixture of diastereomers) (NMR yield = 75%). Compound 40 can be used in the subsequent step without any further purification. Physical state: white amorphous solid. Compound 40 is characterized by 31 31P NMR as described above.1 1H NMR (600 MHz, DMSO-d6): δ 10.7 (brs, 1H; major + minor), 8.40 (br s, 2H; major + minor), 7.94 (s, 1H; major), 7.92 (s, 1H; minor), 7.86 - 7.81 (m, 4H; major + minor), 7.56 - 7.48 (m, 4H; major + minor), 7.39 - 7.32 (m, 4H; major + minor), 7.26 - 7.18 (m, 4H; major + minor), 6.81 (br s, 2H; iPr2NH2 + ), 6.12 (d, J = 1.9 Hz, 1H; major), 6.10 (s, 1H; minor), 6.02 (d, J = 2.4 Hz, 1H; minor), 6.01 (s, 1H; major), 5.33 (dd, J = 7.2, 3.5 Hz, 1H; minor), 5.26 - 5.19 (m; 2H major + 1H minor), 4.40 - 4.31 (m, 2H; major + minor), 4.28 - 4.14 (m, 6H; major + minor), 3.97 - 3.94 (m, 1H; minor), 3.83 - 3.75 (m, 1H; major), 3.30 (s, 3H; major), 3.27 (hept, J = 6.5 Hz, 1H; iPr2NH2 + ), 3.17 (s, 3H; minor), 1.17 (d, J = 6.5 Hz, 12H; iPr2NH2 + ); 1313C NMR (150 MHz, DMSO-d6): δ 157.5 (minor), 156.8 (major), 153.9 (major + minor), 150.5 (minor), 150.4 (major), 143.4 (major + minor), 143.2 (major + minor), 140.79 (major + minor), 140.77 (major + minor), 140.76 (major + minor), 136.3 (minor), 136.2 (major), 129.4 (major + minor), 129.0 (major + minor), 127.6 (major + minor), 127.0 (major + minor), 126.8 (minor), 126.5 (major), 125.14 (major), 125.10 (minor), 120.0 (major + minor), 118.2 (major + minor), 116.91 (major), 116.85 (minor), 89.6 (major), 88.6 (minor), 85.9 (d, J = 7.5 Hz; major), 84.7 (d, J = 8.1 Hz; minor), 84.2 (major), 83.0 (minor), 81.4 (major), 81.2 (minor), 68.3 (major + minor), 64.9 (major + minor), 54.9 (major + minor), 51.9 (major), 50.4 (minor), 47.3 (d, J = 7.1 Hz; major + minor), 46.1 (iPr2NH2 + ), 18.7 (iPr2NH2 + ); 31 31P NMR (162 MHz, DMSO-d6): δ -12.1 (d, J = 19.8 Hz), -12.7 (d, J = 19.8 Hz); HRMS (ESI-TOF) m / z: C 40 H 36 N5O 12 P2 [M - H] - , calculated: 840.1841, found: 840.1861.

[0329] For the following Examples 22 - 79, the diastereomeric purity of the products was determined by NMR and LC-MS.

[0330] The LC traces of the pure diastereomers were recorded from samples obtained after purification and lyophilization using one of the methods 1, 2, or 3 described below.

[0331] The LC traces of the diastereomeric mixtures were obtained from solutions prepared by mixing the previously obtained R P and S P isomers. In several cases, the compound samples used to prepare such mixtures were partially hydrolyzed due to long-term storage, and thus the chromatograms of the isomeric mixtures do not reflect the purity of the compounds obtained.

[0332] HPLC analysis was performed on a Waters automated purification LC with a Waters XBridge C18 column (4.6 x 150 mm, 3.5 μm). Solvent A: 0.1 M triethylammonium acetate (TEAA) in H2O; 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. Table 1 Time (min) Solvent A (%) Solvent B (%) 0 98 2 15 92 8 17 5 90 Table 2 Time (min) Solvent A (%) Solvent B (%) 0 99 1 4 99 1 15 95 5 17 5 90 Table 3 Time (min) Solvent A (%) Solvent B (%) 0 99 1 4 99 1 15 92 5 17 5 90 Example 22 Preparation of 5'-O-azidothymidine triammonium (R)-diphosphate-α-thioate (Compound (R P )-41)

[0333] According to General Procedure A, Compound (R P )-41 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 20:80) to give 52 mg of Compound (R P )-41 after lyophilization (yield = 53%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-41 was characterized by 1 1H NMR (600 MHz, D2O): δ 7.79 (s, 1H), 6.28 (t, J = 6.9 Hz, 1H), 4.60 (dt, J = 6.7, 3.5 Hz, 1H), 4.29 - 4.20 (m, 3H), 2.54 - 2.45 (m, 2H), 1.95 (s, 3H); 13 13C NMR (150 MHz, D2O): δ 166.5, 151.7, 137.3, 111.8, 84.9, 83.0 (d, J = 9.8 Hz), 65.7 (d, J = 5.8 Hz), 61.0, 36.2, 11.7; 31PNMR (162 MHz, D2O): δ 41.1 (d, J = 29.9 Hz), -6.7 (d, J = 29.9 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O9P2S [M-H] - , calculated: 441.9987, found: 441.9979; retention time: 11.57 min (method 1). Example 23 Preparation of 5'-O-azidothymidine triammonium (S)-diphosphate-α-thioester (Compound (S P )-41)

[0334] According to General Procedure A, Compound (S P )-41 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 20:80) to give 49 mg of Compound (S P )-41 (yield = 50%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-41 is characterized by 1 1H NMR (600 MHz, D2O): δ 7.78 (s, 1H), 6.28 (t, J = 6.8 Hz, 1H), 4.60 (dt, J = 6.9, 3.7 Hz, 1H), 4.28 - 4.21 (m, 3H), 2.56 - 2.47 (m, 2H), 1.96 (s, 3H); 13 13C NMR (150 MHz, D2O): δ 166.5, 151.7, 137.3, 111.8, 84.9, 82.9 (d, J = 9.7 Hz), 65.4 (d, J = 6.1 Hz), 60.7, 36.1, 11.7; 31 PNMR (162 MHz, D2O): δ 42.1 (d, J = 28.3 Hz), -9.4 (d, J = 28.4 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O9P2S [M-H] - , calculated: 441.9987, found: 441.9979; retention time: 10.59 min (method 1). Example 24 Preparation of 5'-O-thymidine triammonium (R)-diphosphate-α-thioester (Compound (R P )-42)

[0335] According to General Procedure A, Compound (R P )-42 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* 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 (1M NH4HCO3 / water, from 0:100 to 20:80) to give 44 mg of Compound (R P )-42 (yield = 47%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-42 is characterized by 1 H NMR (600 MHz, D2O): δ 7.79 (s, 1H), 6.35 (t, J = 6.9 Hz, 1H), 4.67 (dt, J = 6.5, 3.7 Hz, 1H), 4.25 - 4.18 (m, 3H), 2.41 (dt, J = 13.8, 6.9 Hz, 1H), 2.35 (ddd, J = 13.8, 6.9, 3.0 Hz, 1H), 1.96 (s, 3H); 13 C NMR (150 MHz, D2O): δ 166.6, 151.8, 137.4, 111.8, 85.3 (d, J = 9.6 Hz), 84.9, 70.9, 65.4 (d, J = 5.9 Hz), 38.5, 11.7; 31 P NMR (162 MHz, D2O): δ 41.3 (d, J = 27.3 Hz), -7.1 (d, J = 27.3 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N2O 10 P2S [M-H] - , calculated: 416.9923, found: 416.9934; retention time: 4.99 min (Method 1). Example 25 Preparation of 5'-O-thymidine triammonium (S)-diphosphate-α-thioester (Compound (S P )-42)

[0336] According to General Procedure A, compound (S P )-42 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 20:80) to give 49 mg of compound (S P )-42 (yield = 52%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-42 was characterized by 1 1H NMR (600 MHz, D2O) δ 7.77 (s, 1H), 6.35 (t, J = 6.9 Hz, 1H), 4.66 (dt, J = 6.2, 3.2 Hz, 1H), 4.26 - 4.17 (m, 3H), 2.41 (dt, J = 13.8, 6.9 Hz, 1H), 2.36 (ddd, J = 13.8, 6.9, 3.6 Hz, 1H), 1.96 (s, 3H); 13 13C NMR (150 MHz, D2O): δ 166.6, 151.8, 137.4, 111.8, 85.2 (d, J = 9.5 Hz), 84.9, 70.9, 65.2 (d, J = 6.2 Hz), 38.3, 11.7; 31 31P NMR (162 MHz, D2O): δ 42.1 (d, J = 29.4 Hz), -9.3 (d, J = 29.4 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N2O 10 P2S [M - H] - , calculated: 416.9923, found: 416.9934; retention time: 4.21 min (Method 1). Example 26 Preparation of 5'-O-uridine triammonium (R)-diphosphate-α-thioate (compound (R P )-43)

[0337] According to General Procedure A, compound (R P)-43. Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1M NH4HCO3 / water, from 0:100 to 20:80) to give 58 mg of the compound (R P )-43 (yield = 62%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. The compound (R P )-43 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.05 (d, J = 8.1 Hz, 1H), 5.96 - 5.94 (m, 2H), 4.42 (t, J = 5.0 Hz, 1H), 4.39 (t, J = 5.0 Hz, 1H), 4.29 - 4.24 (m, 3H); 13 13C NMR (150 MHz, D2O) δ 166.3, 151.8, 141.9, 102.6, 88.4, 83.2 (d, J = 9.7 Hz), 73.8, 69.6, 64.8 (d, J = 5.6 Hz); 31 31P NMR (162 MHz, D2O): δ 41.1 (d, J = 30.9 Hz), -6.8 (d, J = 30.9 Hz); HRMS (ESI-TOF) m / z: C9H 13 N2O 11 P2S [M - H] - , calculated: 418.9715, found: 418.9705; retention time: 2.87 min (method 1). Example 27 Preparation of 5'-O-Uridine Triammonium (S)-Diphosphate-α-Thioester (Compound (S P )-43)

[0338] According to the general procedure A, the compound (S P )-43 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* 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 (1M NH4HCO3 / water, from 0:100 to 20:80) to give 60 mg of the compound (S P )-43 (yield = 64%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. The compound (S P )-43 is characterized by 11H NMR (600 MHz, D2O): δ 8.09 (d, J = 8.1 Hz, 1H), 5.99 - 5.97 (m, 2H), 4.42 (t, J = 4.8 Hz, 1H), 4.40 (t, J = 4.8 Hz, 1H), 4.31 - 4.29 (m, 1H), 4.29 - 4.25 (m, 2H); 13 13C NMR (150 MHz, D2O): δ 166.3, 151.8, 142.0, 102.6, 88.5, 83.1 (d, J = 9.5 Hz), 73.8, 69.6, 64.5 (d, J = 6.5 Hz); 31 31P NMR (162 MHz, D2O) δ 41.7 (d, J = 28.5 Hz), -9.7 (d, J = 28.5 Hz); HRMS (ESI-TOF) m / z: C9H 13 N2O 11 P2S [M-H] - , calculated value: 418.9715, found value: 418.9705; retention time: 2.30 min (method 1). Example 28 Preparation of 5'-O-deoxyadenosine triammonium (R)-diphosphate-α-thioate (Compound (R P )-44)

[0339] According to General Procedure A, Compound (R P )-44 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 30:70) to give 60 mg of Compound (R P )-44 after lyophilization (yield = 63%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-44 is characterized by 11H NMR (600 MHz, D2O): δ 8.47 (s, 1H), 8.11 (s, 1H), 6.41 (t, J = 6.7 Hz, 1H), 4.30 - 4.28 (m, 1H), 4.22 (ddd, J = 11.2, 7.4, 3.7 Hz, 1H), 4.16 (ddd, J = 11.2, 6.6, 3.8 Hz, 1H), 2.79 (dt, J = 13.6, 6.7 Hz, 1H), 2.59 (ddd, J = 13.6, 6.7, 4.0 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 155.2, 152.4, 148.3, 139.9, 118.3, 85.6 (d, J = 9.5 Hz), 83.5, 71.0, 65.1 (d, J = 6.0 Hz), 39.0; 31 31P NMR (162 MHz, D2O) δ 41.1 (d, J = 29.4 Hz), -8.9 (d, J = 29.4 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O8P2S [M-H] - , calculated value: 426.0038, measured value: 426.0033; retention time: 6.17 min (Method 1). Example 29 Preparation of 5'-O-deoxyadenosine triammonium (S)-diphosphate-α-thioate (Compound (S P )-44)

[0340] According to General Procedure A, Compound (S P )-44 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 30:70) to obtain 55 mg of Compound (S P )-44 (yield = 57%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-44 is characterized by 11H NMR (600 MHz, D2O): δ 8.47 (s, 1H), 8.08 (s, 1H), 6.39 (t, J = 6.7 Hz, 1H), 4.79 - 4.75 (m, 1H), 4.31 - 4.29 (m, 1H), 4.22 (ddd, J = 11.5, 7.7, 3.9 Hz, 1H), 4.16 (ddd, J = 11.5, 6.6, 3.7 Hz, 1H), 2.77 (dt, J = 13.6, 6.7 Hz, 1H), 2.59 (ddd, J = 13.6, 6.7, 3.6 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 154.9, 152.0, 148.2, 140.0, 118.2, 85.6 (d, J = 9.4 Hz), 83.7, 71.2, 65.4 (d, J = 6.3 Hz), 39.2; 31 31P NMR (162 MHz, D2O): δ 42.0 (d, J = 28.0 Hz), -10.8 (d, J = 28.0 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O8P2S [M-H] - , calculated: 426.0038, found: 426.0033; retention time: 5.49 min (method 1). Example 30 Preparation of 5'-O-adenosine triammonium (R)-diphosphate-α-thioate (Compound (R P )-45)

[0341] According to General Procedure A, Compound (R P )-45 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 30:70) to give 55 mg of Compound (R P )-45 after lyophilization (yield = 56%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-45 is characterized by 11H NMR (600 MHz, D2O): δ 8.54 (s, 1H), 8.13 (s, 1H), 6.08 (d, J = 5.4 Hz, 1H), 4.79 - 4.74 (m, 1H), 4.59 (t, J = 4.6 Hz, 1H), 4.41 - 4.39 (m, 1H), 4.31 - 4.25 (m, 2H); 13 13C NMR (150 MHz, D2O): δ 155.4, 152.6, 148.9, 140.0, 118.4, 87.0, 83.8 (d, J = 9.6 Hz), 74.4, 70.4, 65.1 (d, J = 5.6 Hz); 31 31P NMR (162 MHz, D2O): δ 42.0 (d, J = 29.2 Hz), -8.3 (d, J = 29.2 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O9P2S [M - H] - , calculated: 441.9987, found: 441.9988; retention time: 5.06 min (method 1). Example 31 Preparation of 5’-O-adenosine triammonium (S)-diphosphate-α-thioate (Compound (S P )-45)

[0342] According to General Procedure A, Compound (S P )-45 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 30:70) to give 52 mg of Compound (S P )-45 after lyophilization (yield = 53%, d.r. > 20:1).

[0343] Preparation scale: According to General Procedure A, Compound (S P)-45. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (1M NH4HCO3 / water, from 0:100 to 30:70) to give 620 mg of the compound (S P )-45 (yield = 63%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-45 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.55 (s, 1H), 8.09 (s, 1H), 6.06 (d, J = 5.3 Hz, 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, 3.0 Hz, 1H), 4.26 - 4.23 (m, 1H); 13 13C NMR (150 MHz, D2O): δ 155.1, 152.4, 148.6, 140.0, 118.2, 87.0, 83.6 (d, J = 9.4 Hz), 74.4, 70.2, 64.7 (d, J = 6.1 Hz); 31 31P NMR (162 MHz, D2O): δ 42.0 (d, J = 29.1 Hz), -8.1 (d, J = 29.1 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O9P2S [M - H] - , calculated: 441.9987, found: 441.9988; retention time: 3.64 min (Method 1). Example 32 Preparation of 5'-O-deoxycytidine triammonium (R)-diphosphate-α-thioate (Compound (R P )-46)

[0344] Compound (R P )-46 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* reagent (128 mg, 0.3 mmol) and the protected deoxycytidine compound 12-1 (217 mg, 0.5 mmol) according to the general procedure A with slight modification. 5.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. The crude product after work-up was neutralized with 10% aqueous AcOH and purified by ion-exchange chromatography on DEAE Sephadex (1M NH4HCO3 / water, from 0:100 to 30:70) to give 38 mg of the compound (RP )-46 (Yield = 42%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-46 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.04 (d, J = 7.6 Hz, 1H), 6.33 (t, J = 6.6 Hz, 1H), 6.13 (d, J = 7.6 Hz, 1H), 4.63 (dt, J = 6.6, 3.5 Hz, 1H), 4.25 - 4.20 (m, 3H), 2.42 (ddd, J = 13.9, 6.6, 4.1 Hz, 1H), 2.33 (dt, J = 13.9, 6.6 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 165.8, 157.2, 142.0, 96.5, 85.9, 85.3 (d, J = 9.6 Hz), 70.6, 65.1 (d, J = 5.8 Hz), 39.3; 31 31P NMR (162 MHz, D2O): δ 42.2 (d, J = 28.5 Hz), -10.0 (d, J = 28.5 Hz); HRMS (ESI-TOF) m / z: C9H 14 N3O9P2S [M - H] - , calculated: 401.9926, found: 401.9918; retention time: 2.83 min (Method 1). Example 33 5’-O-Deoxycytidine triammonium (S)-α-thiophosphorodiamidate (Compound (S P )-46) Preparation

[0345] Following a slightly modified General Procedure A, Compound (S P )-46 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* reagent (128 mg, 0.3 mmol), and the protected deoxycytidine compound 12-1 (217 mg, 0.5 mmol). 5.0 equivalents of DBU were used for the coupling step. 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, from 0:100 to 30:70) to give 41 mg of Compound (S P )-46 (Yield = 45%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-46 is characterized by 11H NMR (600 MHz, D2O): δ 8.04 (d, J = 7.6 Hz, 1H), 6.32 (t, J = 6.6 Hz, 1H), 6.13 (d, J = 7.6 Hz, 1H), 4.62 (dt, J = 6.7, 3.5 Hz, 1H), 4.24 - 4.18 (m, 3H), 2.42 (ddd, J = 13.9, 6.6, 4.1 Hz, 1H), 2.32 (dt, J = 13.9, 6.6 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 165.8, 157.1, 142.0, 96.5, 85.9, 85.3 (d, J = 9.7 Hz), 70.7, 65.1 (d, J = 6.2 Hz), 39.3; 31 31P NMR (162 MHz, D2O): δ 42.2 (d, J = 29.1 Hz), -9.7 (d, J = 29.1 Hz); HRMS (ESI-TOF) m / z: C9H 14 N3O9P2S [M - H] - , calculated: 401.9926, found: 401.9918; retention time: 2.29 min (method 1). Example 34 Preparation of 5'-O-Cytidine Triammonium (R)-Diphosphate-α-Thioate (Compound (R P )-47)

[0346] According to the general procedure A with slight modification, compound (R P )-47 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* reagent (128 mg, 0.3 mmol), and the protected cytidine compound 15-1 (277 mg, 0.5 mmol). 5.0 equivalents of DBU were used for the coupling step. 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, from 0:100 to 30:70) to give 35 mg of compound (R P )-47 after lyophilization (yield = 37%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-47 is characterized by 11H NMR (600 MHz, D2O) δ 8.06 (d, J = 7.6 Hz, 1H), 6.15 (d, J = 7.6 Hz, 1H), 6.00 (d, J = 3.8 Hz, 1H), 4.40 (t, J = 5.1 Hz, 1H), 4.35 - 4.25 (m, 4H); 13 13C NMR (150 MHz, D2O): δ 165.9, 157.4, 141.8, 96.5, 89.3, 82.6 (d, J = 9.9 Hz), 74.2, 69.2, 64.6 (d, J = 5.9 Hz); 31 31P NMR (162 MHz, D2O): δ 41.3 (d, J = 29.1 Hz), -6.8 (d, J = 29.1 Hz); HRMS (ESI-TOF) m / z: C9H 14 N3O 10 P2S [M - H] - , calculated: 417.9875, found: 417.9983; retention time: 5.75 min (method 2). Example 35 Preparation of 5'-O-Cytidine Triammonium (S)-Diphosphate-α-Thioate (Compound (S P )-47)

[0347] Following a slightly modified General Procedure A, Compound (S P )-47 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* reagent (128 mg, 0.3 mmol), and the protected cytidine compound 15-1 (277 mg, 0.5 mmol). 5.0 equivalents of DBU were used for the coupling step. 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, from 0:100 to 30:70) to give 32 mg of Compound (S P )-47 (yield = 34%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-47 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.10 (d, J = 7.6 Hz, 1H), 6.14 (d, J = 7.6 Hz, 1H), 6.00 (d, J = 4.1 Hz, 1H), 4.41 (t, J = 5.0 Hz, 1H), 4.33 (t, J = 4.6 Hz, 1H), 4.31 - 4.27 (m, 3H); 1313C NMR (150 MHz, D2O): δ 166.1, 157.6, 141.9, 96.6, 89.3, 82.6 (d, J = 9.7 Hz), 74.3, 69.2, 64.2 (d, J = 6.6 Hz); 31 31P NMR (162 MHz, D2O): δ 41.2 (d, J = 29.3 Hz), -6.7 (d, J = 29.3 Hz); HRMS (ESI-TOF) m / z: C9H 14 N3O 10 P2S [M-H] - , calculated value: 417.9875, measured value: 417.9983; retention time: 4.97 min (Method 2). Example 36 Preparation of 5'-O-deoxyguanosine triammonium (R)-diphosphate-α-thioate (Compound (R P )-48)

[0348] According to the slightly modified general procedure A, compound (R P )-48 was obtained from the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (+)-Ψ* reagent (85 mg, 0.2 mmol), and the protected deoxyguanosine compound 18-1 (185 mg, 0.5 mmol). 5.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to about 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 30:70) to obtain 32 mg of compound (R P )-48 (yield = 33%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-48 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.14 (s, 1H), 6.29 (t, J = 6.9 Hz, 1H), 4.29 - 4.26 (m, 1H), 4.21 - 4.18 (m, 2H), 2.80 (dt, J = 13.8, 6.9 Hz, 1H), 2.51 (ddd, J = 13.8, 6.9, 3.5 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 158.8, 153.8, 151.3, 137.7, 116.1, 85.5 (d, J = 9.5 Hz), 83.5, 71.3, 65.4 (d, J = 6.0 Hz), 38.6;31 PNMR (162 MHz, D2O): δ 42.3 (d, J = 28.5 Hz), -10.2 (d, J = 28.5 Hz); HRMS (ESI-TOF) m / z: C10H14N5O9P2S [M-H] - , calculated value: 441.9993, measured value: 441.9999; retention time: 9.03 min (Method 2). Example 37 Preparation of 5'-O-deoxyguanosine triammonium (S)-diphosphate-α-thioate (Compound (S P )-48)

[0349] According to the slightly modified general procedure A, Compound (S P )-48 was obtained from the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (-)-Ψ* reagent (85 mg, 0.2 mmol), and the protected deoxyguanosine compound 18-1 (185 mg, 0.5 mmol). A 5.0 equivalent of DBU was used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to about 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 30:70) to obtain 31 mg of Compound (S P )-48 (yield = 31%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-48 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.16 (s, 1H), 6.29 (t, J = 6.9 Hz, 1H), 4.29 - 4.26 (m, 1H), 4.23 - 4.16 (m, 2H), 2.79 (dt, J = 13.7, 6.9 Hz, 1H), 2.52 (ddd, J = 13.7, 6.9, 3.5 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 158.8, 153.7, 151.2, 137.7, 116.1, 85.5 (d, J = 9.4 Hz), 83.6, 71.4, 65.4 (d, J = 6.2 Hz), 38.6; 31 PNMR (162 MHz, D2O): δ 42.6 (d, J = 29.4 Hz), -10.1 (d, J = 29.4 Hz); HRMS (ESI-TOF) m / z: C10H14N5O9P2S [M-H] -, Calculated value: 441.9993, measured value: 441.9999; retention time: 8.52 min (Method 2). Example 38 Preparation of 5'-O-guanosine triammonium (R)-diphosphate-α-thioate (Compound (R P )-49)

[0350] According to a slightly modified general procedure A, Compound (R P )-49 was obtained from the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (+)-Ψ* reagent (85 mg, 0.2 mmol), and the protected guanosine compound 17-1 (245 mg, 0.5 mmol). 5.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to about 3 mL, neutralized with 10% aqueous AcOH, and purified directly by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 30:70) to give 34 mg of Compound (R P )-49 (yield = 33%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-49 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.18 (s, 1H), 5.92 (d, J = 5.6 Hz, 1H), 4.58 - 4.54 (m, 1H), 4.40 - 4.34 (m, 1H), 4.29 - 4.24 (m, 2H); 13 13C NMR (150 MHz, D2O): δ 158.9, 153.9, 151.7, 137.7, 116.1, 86.9, 83.7 (d, J = 9.7 Hz), 73.9, 70.4, 65.2 (d, J = 5.8 Hz); 31 31P NMR (162 MHz, D2O): δ 42.3 (d, J = 28.2 Hz), -10.2 (d, J = 28.2 Hz); HRMS (ESI-TOF) m / z: C10H14N5O10P2S [M-H] - , Calculated value: 457.9942, measured value: 457.9951; retention time: 7.60 min (Method 2). Example 39 Preparation of 5'-O-guanosine triammonium (S)-diphosphate-α-thioate (Compound (S P )-49)

[0351] Compound (S P )-49 was obtained from the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (-)-Ψ* reagent (85 mg, 0.2 mmol) and the protected guanosine compound 17-1 (245 mg, 0.5 mmol) according to the slightly modified general procedure A. 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 about 3 mL, neutralized with 10% aqueous AcOH and purified directly by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 30:70) to give 38 mg of the title compound after lyophilization (yield = 37%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-49 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.21 (s, 1H), 5.92 (d, J = 5.8 Hz, 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 13C NMR (150 MHz, D2O): δ 158.8, 153.8, 151.6, 137.7, 116.1, 86.9, 83.6 (d, J = 9.7 Hz), 73.9, 70.4, 65.1 (d, J = 6.1 Hz); 31 31P NMR (162 MHz, D2O): δ 42.6 (d, J = 27.0 Hz), -10.2 (d, J = 27.0 Hz); HRMS (ESI-TOF) m / z: C10H14N5O10P2S [M-H] - , calculated: 457.9942, found: 457.9951; retention time: 6.79 min (method 2). Example 40 Preparation of 5'-O-2-thiouridine triammonium (R)-diphosphate-α-thioester (Compound (R P )-50)

[0352] Compound (R P)-50. Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 35:65) to give 32 mg of the title compound after lyophilization (yield = 33%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-50 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.26 (d, J = 8.2 Hz, 1H), 6.63 (d, J = 2.8 Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 4.46 (dd, J = 4.8, 2.8 Hz, 1H), 4.42 - 4.37 (m, 2H), 4.35 - 4.28 (m, 2H); 13 13C NMR (150 MHz, D2O): δ 175.9, 162.8, 142.3, 106.9, 93.1, 82.7 (d, J = 9.7 Hz), 74.6, 68.3, 63.9 (d, J = 5.7 Hz); 31 31P NMR (162 MHz, D2O): δ 41.9 (d, J = 29.0 Hz), -9.6 (d, J = 29.0 Hz); HRMS (ESI-TOF) m / z: C9H 13 N2O 10 P2S2 [M - H] - , calculated: 434.9487, found: 434.9483; retention time: 4.85 min (Method 1). Example 41 5'-O-2-Thiouridine Triammonium (S)-Diphosphate-α-Thioester (Compound (S P )-50) Preparation

[0353] According to General Procedure A, compound (S P )-50 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 35:65) to give 33 mg of compound (S P )-50 (yield = 34%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P)-50 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.30 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 2.3 Hz, 1H), 6.28 (d, J = 8.1 Hz, 1H), 4.46 - 4.43 (m, 1H), 4.40 - 4.36 (m, 2H), 4.35 - 4.28 (m, 2H); 13 13C NMR (150 MHz, D2O): δ 175.9, 162.8, 142.4, 107.0, 93.1, 82.7 (d, J = 9.4 Hz), 74.6, 68.4, 63.7 (d, J = 6.1 Hz); 31 31P NMR (162 MHz, D2O): δ 42.5 (d, J = 28.9 Hz), -10.3 (d, J = 28.9 Hz); HRMS (ESI-TOF) m / z: C9H 13 N2O 10 P2S2 [M - H] - , calculated value: 434.9487, found value: 434.9483; retention time: 3.60 min (method 1).

[0354] Figure 1 Describes the LC trace of compound 50. Example 42 3’-O-Thymidine triammonium (R)-diphosphate-α-thioate (compound (R P )-51) preparation

[0355] According to the general procedure A, from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* reagent (128 mg, 0.3 mmol) and the protected thymidine compound 23 (173 mg, 0.5 mmol), compound (R P )-51 was obtained. Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 20:80) to give 47 mg of compound (R P )-51 after lyophilization (yield = 50%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-51 is characterized by 11H NMR (600 MHz, D2O): δ 7.70 (s, 1H), 6.34 (t, J = 6.8 Hz, 1H), 5.05 (ddt, J = 10.7, 7.0, 3.8 Hz, 1H), 4.27 (q, J = 3.8 Hz, 1H), 3.88 (d, J = 3.8 Hz, 2H), 2.63 (ddd, J = 14.3, 6.8, 3.8 Hz, 1H), 2.49 (ddd, J = 14.3, 7.0, 6.8 Hz, 1H), 1.90 (s, 3H); 13 13C NMR (150 MHz, D2O): δ 166.5, 151.7, 137.6, 111.5, 85.4 (d, J = 6.2 Hz), 84.8, 74.6 (d, J = 5.7 Hz), 60.7, 37.5 (d, J = 3.7 Hz), 11.5; 31 31P NMR (162 MHz, D2O): δ 41.0 (d, J = 28.5 Hz), -8.3 (d, J = 28.5 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N2O 10 P2S [M-H] - , calculated: 416.9928, found: 416.9926; retention time: 7.96 min (method 3). Example 43 Preparation of 3'-O-Thymidine Triammonium (S)-Diphosphate-α-Thioate (Compound (S P )-51)

[0356] According to General Procedure A, Compound (S P )-51 was obtained from the monophosphate precursor compound 7-1 (62 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 20:80) to give 50 mg of the title compound after lyophilization (yield = 53%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-51 is characterized by 11H NMR (600 MHz, D2O): δ 7.69 (d, J = 1.2 Hz, 1H), 6.35 (t, J = 7.0 Hz, 1H), 5.05 (ddt, J = 10.4, 7.0, 3.6 Hz, 1H), 4.26 (dt, J = 4.6, 3.6 Hz, 1H), 3.90 (dd, J = 12.7, 3.6 Hz, 1H), 3.87 (dd, J = 12.7, 4.6 Hz, 1H), 2.65 (ddd, J = 14.3, 7.0, 3.6 Hz, 1H), 2.46 (dt, J = 14.3, 7.0 Hz, 1H), 1.90 (d, J = 1.2 Hz, 3H); 13 13C NMR (150 MHz, D2O): δ 166.5, 151.7, 137.6, 111.5, 85.6 (d, J = 6.5 Hz), 85.0, 75.1 (d, J = 6.1 Hz), 60.9, 37.4 (d, J = 3.5 Hz), 11.5; 31 31P NMR (162 MHz, D2O): δ 41.7 (d, J = 27.8 Hz), -10.2 (d, J = 27.8 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N2O 10 P2S [M-H] - , calculated: 416.9928, found: 416.9926; retention time: 8.22 min (method 3). Example 44 Preparation of 5'-O-adenosine triammonium diphosphate-β-thioate (Compound 52)

[0357] Compound 52 was obtained from the protected adenosine monophosphate compound 32 (152 mg, 0.2 mmol), (+)-Ψ* reagent (128 mg, 0.3 mmol), and 4-(hydroxymethyl)phenyl benzoate (114 mg, 0.5 mmol) according to a slightly modified general procedure A. The synthesis of 4-(hydroxymethyl)phenyl benzoate is disclosed in Yang et al., Angew. Chem. Int. Ed. [Angewandte Chemie International Edition] 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, from 0:100 to 40:60) to give 42 mg of the title compound (yield = 43%) after lyophilization. Physical state: white amorphous solid. Compound 52 is characterized by 11H NMR (600 MHz, D2O): δ 8.55 (s, 1H), 8.19 (s, 1H), 6.11 (d, J = 5.5 Hz, 1H), 4.68 - 4.66 (m, 1H), 4.40 - 4.38 (m, 1H), 4.30 (ddd, J = 11.5, 6.0, 2.9 Hz, 1H), 4.23 (dt, J = 11.5, 3.4 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 155.3, 152.5, 148.9, 140.0, 118.4, 86.9, 83.9 (d, J = 8.8 Hz), 74.4, 70.2, 64.7 (d, J = 4.6 Hz); 31 31P NMR (162 MHz, D2O): δ 33.5 (d, J = 30.6 Hz), -11.5 (d, J = 30.6 Hz); HRMS (ESI-TOF) m / z: C 10 H 14 N5O9P2S [M - H] - , calculated: 441.9987, found: 441.9981; retention time: 3.34 min (method 1). Example 45 Preparation of 5'-O-azidothymidine triammonium (R)-triphosphate-α-thioester (Compound (R P )-53)

[0358] According to General Procedure B, Compound (R P )-53 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 30:70) to give 67 mg of Compound (R P )-53 after lyophilization (yield = 57%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-53 is characterized by 1 1H NMR (600 MHz, D2O): δ 7.80 (s, 1H), 6.29 (t, J = 6.9 Hz, 1H), 4.64 (dt, J = 6.7, 3.4 Hz, 1H), 4.34 - 4.25 (m, 3H), 2.54 - 2.45 (m, 2H), 1.96 (s, 3H); 1313C NMR (150 MHz, D2O): δ 166.6, 151.7, 137.3, 111.9, 84.8, 82.9 (d, J = 9.8 Hz), 66.1 (d, J = 6.1 Hz), 61.1, 36.3, 11.7; 31 31P NMR (162 MHz, D2O): δ 42.3 (d, J = 27.7 Hz), -9.1 (d, J = 19.7 Hz), -24.2 (dd, J = 27.7, 19.7 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 12 P3S [M-H] - , calculated value: 521.9651, measured value: 521.9650; retention time: 11.27 min (Method 1). Example 46 Preparation of 5'-O-azidothymidine triammonium (S)-triphosphate-α-thioester (Compound (S P )-53)

[0359] According to General Procedure B, Compound (S P )-53 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 30:70) to obtain 70 mg of Compound (S P )-53 (yield = 59%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-53 is characterized by 1 1H NMR (600 MHz, D2O): δ 7.78 (s, 1H), 6.28 (t, J = 6.9 Hz, 1H), 4.61 (dt, J = 7.1, 3.7 Hz, 1H), 4.34 - 4.23 (m, 3H), 2.55 - 2.45 (m, 2H), 1.96 (s, 3H); 13 13C NMR (150 MHz, D2O): δ 166.6, 151.7, 137.3, 111.8, 84.9, 82.8 (d, J = 9.8 Hz), 65.7 (d, J = 6.4 Hz), 60.8, 36.2, 11.7; 31PNMR (162 MHz, D2O): δ 43.3 (d, J = 26.8 Hz), -9.1 (d, J = 20.0 Hz), -23.6 (dd, J = 26.8, 20.0 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 12 P3S [M-H] - , calculated value: 521.9651, measured value: 521.9650; retention time: 10.43 min (program: method 1). Example 47 Preparation of 5'-O-Thymidine Triammonium (R)-Triphosphate-α-Thioester (Compound (R P )-54)

[0360] According to the general procedure B, compound (R P )-54 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 30:70) to obtain 54 mg of compound (R P )-54 (yield = 48%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-54 is characterized by 1 1H NMR (600 MHz, D2O): δ 7.80 (s, 1H), 6.35 (t, J = 6.9 Hz, 1H), 4.70 (dt, J = 6.5, 3.4 Hz, 1H), 4.30 - 4.26 (m, 2H), 4.23 - 4.20 (m, 1H), 2.41 (ddd, J = 13.9, 7.5, 6.9 Hz, 1H), 2.35 (ddd, J = 13.9, 6.9, 3.6 Hz, 1H), 1.96 (s, 3H); 13 13C NMR (150 MHz, D2O): δ 166.6, 151.8, 137.4, 111.8, 85.3 (d, J = 9.6 Hz), 84.9, 70.8, 65.7 (d, J = 6.2 Hz), 38.5, 11.7; 31P NMR (162 MHz, D2O): δ 42.9 (d, J = 28.2 Hz), -8.2 (d, J = 20.7 Hz), -23.5 (dd, J = 28.2, 20.7 Hz); HRMS (ESI-TOF) m / z: C 10 H 16 N2O 13 P3S [M-H] - , calculated value: 496.9586, measured value: 496.9590; retention time: 5.87 min (Method 1). Example 48 Preparation of 5'-O-Thymidine Triammonium (S)-Triphosphate-α-Thioester (Compound (S P )-54)

[0361] According to General Procedure B, Compound (S P )-54 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 30:70) to give 52 mg of Compound (S P )-54 (yield = 46%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-54 is characterized by 1 H NMR (600 MHz, D2O): δ 7.77 (s, 1H), 6.36 (t, J = 6.9 Hz, 1H), 4.67 (dt, J = 6.2, 3.2 Hz, 1H), 4.29 (ddd, J = 11.9, 7.6, 4.0 Hz, 2H), 4.25 - 4.19 (m, 2H), 2.41 (dt, J = 13.9, 6.9 Hz, 1H), 2.35 (ddd, J = 13.9, 6.9, 3.2 Hz, 1H), 1.96 (s, 3H); 13 C NMR (150 MHz, D2O): δ 166.6, 151.8, 137.4, 111.8, 85.3 (d, J = 9.6 Hz), 85.0, 70.9, 65.6 (d, J = 6.7 Hz), 38.4, 11.7; 31³¹P NMR (162 MHz, D₂O): δ 42.6 (d, J = 26.7 Hz), -7.1 (d, J = 20.0 Hz), -23.3 (dd, J = 26.7, 20.0 Hz); HRMS (ESI-TOF) m / z: C 10 H 16 N₂O 13 P₃S [M-H] - , calculated: 496.9586, found: 496.9590; retention time: 4.83 min (method 1). Example 49 Preparation of 5'-O-uridine triammonium (R)-triphosphate-α-thioester (Compound (R P )-55)

[0362] Following general procedure B, Compound (R P )-55 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 30:70) to give 53 mg of the title compound after lyophilization (yield = 47%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-55 was characterized by 1 ¹H NMR (600 MHz, D₂O): δ 8.04 (d, J = 8.1 Hz, 1H), 5.99 (d, J = 5.1 Hz, 1H), 5.97 (d, J = 8.1 Hz, 1H), 4.46 - 4.43 (m, 1H), 4.41 (t, J = 5.1 Hz, 1H), 4.34 - 4.27 (m, 3H); 13 ¹³C NMR (150 MHz, D₂O): δ 166.2, 151.8, 141.9, 102.6, 88.2, 83.2 (d, J = 9.7 Hz), 73.8, 69.6, 65.3 (d, J = 5.9 Hz); 31 ³¹P NMR (162 MHz, D₂O): δ 43.1 (d, J = 27.7 Hz), -8.9 (d, J = 20.0 Hz), -23.6 (dd, J = 27.7, 20.0 Hz); HRMS (ESI-TOF) m / z: C₉H 14 N₂O 14 P₃S [M-H] -, Calculated value: 498.9379, measured value: 498.9383; retention time: 3.67 min (Method 1). Example 50 5'-O-Uridine triammonium (S)-triphosphate-α-thioester (Compound (S P )-55) Preparation

[0363] According to General Procedure B, from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* reagent (112 mg, 0.26 mmol) and the protected uridine compound 11-1 (222 mg, 0.4 mmol) to obtain compound (S P )-55. Deprotection was carried out at room temperature. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1M NH4HCO3 / water, from 0:100 to 30:70) to obtain 52 mg of compound (S P )-55 (yield = 46%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-55 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.10 (d, J = 8.1 Hz, 1H), 6.00 (d, J = 5.1 Hz, 1H), 5.98 (d, J = 8.1 Hz, 1H), 4.45 - 4.43 (m, 1H), 4.44 (t, J = 5.1 Hz, 1H), 4.33 - 4.27 (m, 3H); 13 13C NMR (150 MHz, D2O): δ 166.3, 151.8, 142.1, 102.6, 88.3, 83.2 (d, J = 9.5 Hz), 73.8, 69.6, 64.8 (d, J = 6.6 Hz); 31 31P NMR (162 MHz, D2O): δ 43.4 (d, J = 27.1 Hz), -8.7 (d, J = 19.9 Hz), -23.5 (dd, J = 27.1, 19.9 Hz); HRMS (ESI-TOF) m / z: C9H 14 N2O 14 P3S [M - H] - , Calculated value: 498.9379, measured value: 498.9383; retention time: 2.91 min (Method 1).

[0364] Figure 2 The LC trace of compound 55 is described. Example 51 Preparation of 5'-O-deoxyadenosine triammonium (R)-triphosphate-α-thioester (Compound (R P )-56)

[0365] According to General Procedure B, Compound (R P )-56 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 40:60) to give 54 mg of Compound (R P )-56 after lyophilization (yield = 47%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-56 was characterized by 1 H NMR (600 MHz, D2O): δ 8.51 (s, 1H), 8.17 (s, 1H), 6.46 (t, J = 6.7 Hz, 1H), 4.34 - 4.30 (m, 1H), 4.27 (ddd, J = 10.9, 7.2, 3.5 Hz, 1H), 4.22 (ddd, J = 10.9, 6.5, 3.6 Hz, 1H), 2.82 (dt, J = 13.6, 6.7 Hz, 1H), 2.60 (ddd, J = 13.6, 6.7, 3.8 Hz, 1H); 13 C NMR (150 MHz, D2O): δ 155.3, 152.5, 148.5, 140.0, 118.4, 85.6 (d, J = 9.5 Hz), 83.6, 71.0, 65.6 (d, J = 6.2 Hz), 39.1; 31 P NMR (162 MHz, D2O): δ 43.2 (d, J = 27.8 Hz), -8.5 (d, J = 20.2 Hz), -23.5 (dd, J = 27.8, 20.2 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 11 P3S [M - H] - , calculated: 505.9702, found: 505.9688; retention time: 6.63 min (Method 1). Example 52 Preparation of 5'-O-deoxyadenosine triammonium (S)-triphosphate-α-thioester (Compound (S P )-56)

[0366] According to General Procedure B, compound (S P )-56 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 40:60) to give 59 mg of the title compound after lyophilization (yield = 51%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-56 was characterized by 1 1H NMR (600 MHz, D2O): δ 8.50 (s, 1H), 8.11 (s, 1H), 6.42 (t, J = 6.7 Hz, 1H), 4.32 - 4.24 (m, 2H), 4.21 - 4.17 (m, 1H), 2.80 (dt, J = 13.6, 6.7 Hz, 1H), 2.59 (ddd, J = 13.6, 6.7, 3.8 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 155.2, 152.3, 148.4, 140.0, 118.3, 85.6 (d, J = 9.6 Hz), 83.6, 71.0, 65.5 (d, J = 6.4 Hz), 39.0; 31 31P NMR (162 MHz, D2O): δ 43.4 (d, J = 27.4 Hz), -7.4 (d, J = 20.7 Hz), -23.2 (dd, J = 27.4, 20.7 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 11 P3S [M - H] - , calculated: 505.9702, found: 505.9688; retention time: 6.05 min (Method 1). Example 53 Preparation of 5'-O-adenosine triammonium (R)-α-thiotriphosphate (Compound (R P )-57)

[0367] According to General Procedure B, compound (R P )-57 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 40:60) to give 59 mg of compound (R P )-57 after lyophilization (yield = 50%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-57 was characterized by 1 1H NMR (600 MHz, D2O): δ 8.58 (s, 1H), 8.21 (s, 1H), 6.12 (d, J = 5.9 Hz, 1H), 4.62 (dd, J = 5.1, 3.6 Hz, 1H), 4.43 - 4.41 (m, 1H), 4.34 (ddd, J = 10.4, 7.5, 2.7 Hz, 1H), 4.28 (ddd, J = 11.9, 6.1, 2.7 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 155.5, 152.7, 149.0, 140.0, 118.5, 86.7, 83.9 (d, J = 9.7 Hz), 74.3, 70.4, 65.5 (d, J = 5.8 Hz); 31 31P NMR (162 MHz, D2O): δ 43.4 (d, J = 27.6 Hz), -10.9 (d, J = 19.6 Hz), -24.1 (dd, J = 27.6, 19.6 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 12 P3S [M - H] - , calculated: 521.9651, found: 521.9662; retention time: 5.85 min (Method 1). Example 54 Preparation of 5'-O-adenosine triammonium (S)-α-thiotriphosphate (Compound (S P )-57)

[0368] According to General Procedure B, compound (S P )-57 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 40:60) to give 61 mg of compound (S P )-57 (yield = 52%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-57 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.63 (s, 1H), 8.18 (s, 1H), 6.12 (d, J = 5.7 Hz, 1H), 4.62 - 4.59 (m, 1H), 4.43 - 4.40 (m, 1H), 4.35 (ddd, J = 11.0, 7.7, 3.0 Hz, 1H), 4.28 (ddd, J = 11.7, 5.5, 3.0 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 155.4, 152.6, 148.9, 140.1, 118.4, 86.8, 83.8 (d, J = 9.5 Hz), 74.3, 70.4, 65.1 (d, J = 6.5 Hz); 31 31P NMR (162 MHz, D2O): δ 43.4 (d, J = 26.9 Hz), -8.5 (d, J = 20.1 Hz), -23.5 (dd, J = 26.9, 20.1 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 12 P3S [M - H] - , calculated: 521.9651, found: 521.9662; retention time: 4.52 min (Method 1). Example 55 Preparation of 5'-O-deoxycytidine triammonium (R)-α-thiotriphosphate (Compound (R P )-58)

[0369] Compound (R P )-58 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* reagent (112 mg, 0.26 mmol), and the protected deoxycytidine compound 12-1 (174 mg, 0.4 mmol) according to the slightly modified general procedure B. 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, from 0:100 to 40:60) to give 40 mg of compound (R P )-58 (yield = 36%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-58 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.02 (d, J = 7.6 Hz, 1H), 6.33 (t, J = 6.7 Hz, 1H), 6.14 (d, J = 7.6 Hz, 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 13C NMR (150 MHz, D2O): δ 166.0, 157.4, 141.8, 96.5, 85.8, 85.3 (d, J = 9.6 Hz), 70.4, 65.4 (d, J = 6.0 Hz), 39.3; 31 31P NMR (162 MHz, D2O) δ 43.1 (d, J = 27.4 Hz), -8.1 (d, J = 20.4 Hz), -23.4 (dd, J = 27.4, 20.4 Hz); HRMS (ESI-TOF) m / z: C9H 15 N3O 12 P3S [M - H] - , calculated: 481.9589, found: 481.9575; retention time: 7.58 min (method 2). Example 56 Preparation of 5'-O-deoxycytidine triammonium (S)-α-thiotriphosphate (Compound (S P )-58)

[0370] According to the slightly modified general procedure B, compound (S P )-58 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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 work-up was neutralized with 10% aqueous AcOH and purified by ion-exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60) to give 41 mg of compound (S P )-58 (yield = 37%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-58 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.07 (d, J = 7.6 Hz, 1H), 6.34 (t, J = 6.6 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 4.64 (dt, J = 6.8, 3.6 Hz, 1H), 4.31 - 4.21 (m, 3H), 2.42 (ddd, J = 14.1, 6.6, 4.0 Hz, 1H), 2.33 (dt, J = 14.1, 6.6, 1H); 13 13C NMR (150 MHz, D2O): δ 165.7, 156.9, 142.1, 96.5, 86.0, 85.4 (d, J = 9.7 Hz), 70.7, 65.4 (d, J = 6.3 Hz), 39.4; 31 31P NMR (162 MHz, D2O): δ 43.4 (d, J = 27.1 Hz), -9.9 (d, J = 19.9 Hz), -23.9 (dd, J = 27.1, 19.9 Hz); HRMS (ESI-TOF) m / z: C9H 15 N3O 12 P3S [M - H] - , calculated: 481.9589, found: 481.9575; retention time: 6.66 min (method 2). Example 57 Preparation of 5'-O-cytidine trisodium (R)-α-thiotriphosphate (compound (R P )-59)

[0371] Compound (R P )-59 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), the (+)-Ψ* reagent (112 mg, 0.26 mmol), and the protected cytidine compound 15-1 (222 mg, 0.4 mmol) according to a slightly modified general procedure B. The coupling step was carried out using 8.0 equivalents of DBU. Deprotection was carried out at 40 °C. The crude product after work-up was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica gel (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone to give 45 mg of compound (R P )-59 after drying under high vacuum. (Yield = 38%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-59 was characterized by 1 1H NMR (600 MHz, D2O): δ 8.06 (d, J = 7.6 Hz, 1H), 6.15 (d, J = 7.6 Hz, 1H), 6.01 (d, J = 4.3 Hz, 1H), 4.47 (t, J = 5.2 Hz, 1H), 4.36 (t, J = 4.7 Hz, 1H), 4.36 - 4.32 (m, 2H), 4.31 - 4.28 (m, 1H); 13 13C NMR (150 MHz, D2O): δ 166.2, 157.8, 141.7, 96.6, 89.0, 82.6 (d, J = 9.7 Hz), 74.2, 69.1, 64.9 (d, J = 5.6 Hz); 31 31P NMR (162 MHz, D2O): δ 42.8 (d, J = 27.9 Hz), -5.8 (d, J = 20.2 Hz), -22.5 (dd, J = 27.9, 20.2 Hz); HRMS (ESI-TOF) m / z: C9H 15 N3O 13 P3S [M - H] - , calculated: 497.9538, found: 497.9533; retention time: 6.81 min (method 2). Example 58 Preparation of 5'-O-cytidine trisodium (S)-triphosphate-α-thioester (Compound (S P )-59)

[0372] According to the slightly modified general procedure B, compound (S P )-59 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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 work-up was neutralized with 10% aqueous AcOH and purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica gel (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone and dried under high vacuum to give 47 mg of compound (S P )-59 (yield = 40%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-59 is characterized by 1 H NMR (600 MHz, D2O): δ 8.13 (d, J = 7.6 Hz, 1H), 6.17 (d, J = 7.6 Hz, 1H), 6.02 (d, J = 4.3 Hz, 1H), 4.45 (t, J = 5.2 Hz, 1H), 4.37 - 4.32 (m, 3H), 4.30 (dt, J = 5.2, 2.5 Hz, 1H); 13 C NMR (150 MHz, D2O): δ 166.2, 157.8, 141.9, 96.7, 89.1, 82.7 (d, J = 9.7 Hz), 74.3, 69.2, 64.4 (d, J = 5.9 Hz); 31 P NMR (162 MHz, D2O): δ 43.2 (d, J = 27.1 Hz), -8.7 (d, J = 19.3 Hz), -23.5 (dd, J = 27.1, 19.3 Hz); HRMS (ESI-TOF) m / z: C9H 15 N3O 13 P3S [M - H] - , calculated: 497.9538, found: 497.9533; retention time: 5.79 min (method 2). Example 59 5'-O-deoxyguanosine trisodium (R)-α-thiotriphosphate (compound (R P) Preparation of

[0373] Compound (R P )-60 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* reagent (112 mg, 0.26 mmol), and the protected deoxyguanosine compound 18-1 (148 mg, 0.4 mmol) according to a slightly modified general procedure B. 8.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to about 3 mL, neutralized with 10% aqueous AcOH, and directly purified by ion-exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60), followed by reverse-phase chromatography on C18-silica (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone and dried under high vacuum to give 35 mg of compound (R P )-60 (yield = 29%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-60 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.16 (s, 1H), 6.32 (t, J = 6.8 Hz, 1H), 4.31 - 4.28 (m, 1H), 4.27 - 4.23 (m, 2H), 2.83 (dt, J = 13.7, 6.8 Hz, 1H), 2.51 (ddd, J = 13.7, 6.8, 3.4 Hz, 1H); 13 13C NMR (150 MHz, D2O) δ 159.0, 153.8, 151.4, 137.8, 116.1, 85.6 (d, J = 9.6 Hz), 83.6, 71.2, 65.7 (d, J = 6.1 Hz), 38.6; 31 31P NMR (162 MHz, D2O) δ 43.3 (d, J = 27.3 Hz), -5.2 (d, J = 19.2 Hz), -21.7 (dd, J = 27.3, 19.2 Hz); HRMS (ESI-TOF) m / z: C10H15N5O11P3S [M - H] - , calculated: 505.9702, found: 505.9688; retention time: 9.78 min (method 2). Example 60 Preparation of 5’-O-deoxyguanosine trisodium (S)-triphosphate-α-thioester (Compound (S P )-60)

[0374] Compound (S P )-60 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* reagent (112 mg, 0.26 mmol) and the protected deoxyguanosine compound 18-1 (148 mg, 0.4 mmol) according to a slightly modified General Procedure B. 8.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to about 3 mL, neutralized with 10% aqueous AcOH and purified directly by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone to give 39 mg of compound (S P )-60 (yield = 32%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-60 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.18 (s, 1H), 6.30 (t, J = 6.8 Hz, 1H), 4.83 - 4.79 (m, 1H), 4.31 - 4.21 (m, 3H), 2.81 (dt, J = 13.7, 6.8 Hz, 1H), 2.52 (ddd, J = 13.7, 6.8, 3.7 Hz, 1H); 13 13C NMR (150 MHz, D2O) δ 159.1, 153.9, 151.3, 137.9, 116.2, 85.6 (d, J = 9.4 Hz), 83.6, 71.1, 65.6 (d, J = 6.3 Hz), 38.6; 31 31P NMR (162 MHz, D2O) δ 43.4 (d, J = 27.5 Hz), -5.8 (d, J = 20.1 Hz), -22.4 (dd, J = 27.5, 20.1 Hz); HRMS (ESI-TOF) m / z: C10H15N5O11P3S [M-H] - , calculated: 505.9702, found: 505.9688; retention time: 9.17 min (Method 2). Example 61 Preparation of 5'-O-guanosine trisodium (R)-triphosphate-α-thioester (Compound (R P )-61)

[0375] Compound (R P )-61 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* reagent (112 mg, 0.26 mmol) and the protected guanosine compound 17-1 (196 mg, 0.4 mmol) according to the generally applicable procedure B with minor modifications. 8.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to approximately 3 mL, neutralized with 10% aqueous AcOH and purified directly by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone to give 39 mg of compound (R P )-61 (yield = 31%, d.r. > 20:1) after drying under high vacuum. Physical state: white amorphous solid. Compound (R P )-61 was characterized by 1 H NMR (600 MHz, D2O): δ 8.20 (s, 1H), 5.93 (d, J = 5.9 Hz, 1H), 4.64 - 4.62 (m, 1H), 4.41 - 4.37 (m, 1H), 4.33 (ddd, J = 10.8, 7.9, 2.7 Hz, 1H), 4.27 (ddd, J = 10.8, 5.9, 3.0 Hz, 1H); 13 C NMR (150 MHz, D2O) δ 159.1, 154.0, 151.7, 137.7, 116.1, 86.6, 83.7 (d, J = 9.3 Hz), 73.8, 70.3, 65.5 (d, J = 5.4 Hz); 31 P NMR (162 MHz, D2O) δ 43.0 (d, J = 28.6 Hz), -5.8 (d, J = 19.9 Hz), -22.4 (dd, J = 28.6, 19.9 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 13 P3S [M-H]- , Calculated value: 537.9600, Measured value: 537.9592; Retention time: 8.92 min (Method 2). Example 62 Preparation of 5'-O-guanosine trisodium (S)-triphosphate-α-thioester (Compound (S P )-61)

[0376] According to the slightly modified General Procedure B, Compound (S P )-61 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* reagent (112 mg, 0.26 mmol) and the protected guanosine compound 17-1 (196 mg, 0.4 mmol). 8.0 equivalents of DBU were used for the coupling step. Deprotection was carried out at 40 °C. After extraction, the aqueous phase was concentrated to about 3 mL, neutralized with 10% aqueous AcOH and directly purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica gel (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone to give 41 mg of Compound (S P )-61 (yield = 33%, d.r. > 20:1). Physical state: White amorphous solid. Compound (S P )-61 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.26 (s, 1H), 5.93 (d, J = 6.0 Hz, 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, 3.0 Hz, 1H), 4.27 (dt, J = 10.8, 4.7 Hz, 1H); 13 13C NMR (150 MHz, D2O): δ 159.1, 154.0, 151.7, 137.9, 116.2, 86.7, 83.8 (d, J = 9.3 Hz), 73.7, 70.4, 65.1 (d, J = 6.7 Hz); 31P NMR (162 MHz, D2O): δ 43.2 (d, J = 27.1 Hz), -5.8 (d, J = 20.0 Hz), -22.4 (dd, J = 27.1, 20.0 Hz); HRMS (ESI-TOF) m / z: C 10 H 15 N5O 13 P3S [M-H] - , calculated: 537.9600, found: 537.9592; retention time: 7.61 min (method 2). Example 63 Preparation of 5'-O-2-thiouridine trisodium (R)-triphosphate-α-thioester (Compound (R P )-62)

[0377] According to General Procedure B, Compound (R P )-62 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* reagent (112 mg, 0.26 mmol), and a regioisomeric mixture of the 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, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica gel (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone and dried under high vacuum to give 35 mg of Compound (R P )-62 (yield = 29%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-62 is characterized by 1 H NMR (600 MHz, D2O): δ 8.23 (d, J = 8.0 Hz, 1H), 6.69 (s, 1H), 6.25 (d, J = 8.0 Hz, 1H), 4.48 - 4.40 (m, 3H), 4.39 - 4.31 (m, 2H); 13 C NMR (150 MHz, D2O): δ 176.5, 164.5, 142.2, 106.8, 93.1, 82.7 (d, J = 9.7 Hz), 74.7, 68.3, 64.4 (d, J = 5.7 Hz); 31P NMR (162 MHz, D2O): δ 42.6 (d, J = 28.3 Hz), -6.0 (d, J = 20.3 Hz), -22.7 (dd, J = 28.3, 20.3 Hz); HRMS (ESI-TOF) m / z: C9H 14 N2O 13 P3S2[M - H] - , calculated value: 514.9150, found value: 514.9168; retention time: 5.44 min (Method 1). Example 64 Preparation of 5’-O-2-thiouridine trisodium (S)-triphosphate-α-thioester (Compound (S P )-62)

[0378] According to General Procedure B, Compound (S P )-62 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* reagent (112 mg, 0.26 mmol), and the regioisomer mixture of the protected 2-thiouridine derivatives Compound 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, from 0:100 to 40:60), followed by reverse phase chromatography on C18-silica gel (1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized. The obtained solid was redissolved in the minimum amount of water, and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone and dried under high vacuum to give 33 mg of Compound (S P )-62 (yield = 27%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-62 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.34 (d, J = 8.1 Hz, 1H), 6.67 (s, 1H), 6.28 (d, J = 8.1 Hz, 1H), 4.48 - 4.43 (m, 2H), 4.42 - 4.38 (m, 2H), 4.35 - 4.31 (m, 1H); 13 13C NMR (150 MHz, D2O): δ 176.2, 163.6, 142.5, 107.0, 93.2, 82.8 (d, J = 9.4 Hz), 74.7, 68.2, 63.7 (d, J = 6.8 Hz); 31³¹P NMR (162 MHz, D₂O): δ 42.9 (d, J = 27.1 Hz), -6.2 (d, J = 20.0 Hz), -22.6 (dd, J = 27.1, 20.0 Hz); HRMS (ESI-TOF) m / z: C₉H 14 N₂O 13 P₃S₂[M - H] - , calculated value: 514.9150, found value: 514.9168; retention time: 4.20 min (Method 1). Example 65 Preparation of 3'-O-Thymidine Triammonium (R)-Triphosphate-α-Thioester (Compound (R P )-63)

[0379] According to General Procedure B, Compound (R P )-63 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 20:80) to give 57 mg of Compound (R P )-63 after lyophilization (yield = 51%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-63 was characterized by 1 ¹H NMR (600 MHz, D₂O) δ 7.69 (s, 1H), 6.34 (t, J = 6.9 Hz, 1H), 5.09 (td, J = 6.9, 3.3 Hz, 1H), 4.31 (q, J = 3.7 Hz, 1H), 3.90 - 3.84 (m, 2H), 2.63 (ddd, J = 14.3, 6.9, 3.3 Hz, 1H), 2.48 (dt, J = 14.3, 6.9 Hz, 1H), 1.90 (s, 3H); 13 ¹³C NMR (150 MHz, D₂O) δ 166.5, 151.7, 137.6, 111.5, 85.5 (d, J = 5.8 Hz), 85.0, 75.6 (d, J = 6.0 Hz), 61.0, 37.5 (d, J = 3.9 Hz), 11.5; 31³¹P NMR (162 MHz, D₂O) δ 42.8 (d, J = 26.2 Hz), -10.0 (d, J = 19.3 Hz), -23.8 (dd, J = 26.2, 19.3 Hz); HRMS (ESI-TOF) m / z: C₁₀H₁₆N₂O₁₃P₃S [M-H] - , calculated value: 496.9551, found value: 496.9603; retention time: 8.45 min (method 3). Example 66 Preparation of 3'-O-Thymidine Triammonium (S)-Triphosphate-α-Thioester (Compound (S P )-63)

[0380] According to General Procedure B, Compound (S P )-63 was obtained from the diphosphate precursor compound 20-1 (169 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 20:80) to give 60 mg of Compound (S P )-63 (yield = 53%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-63 is characterized by 1 ¹H NMR (600 MHz, D₂O): δ 7.69 (d, J = 1.1 Hz, 1H), 6.36 (dd, J = 7.1, 6.4 Hz, 1H), 5.10 (ddt, J = 10.0, 6.4, 3.1 Hz, 1H), 4.29 (q, J = 3.6 Hz, 1H), 3.91 - 3.85 (m, 2H), 2.66 (ddd, J = 14.4, 6.4, 3.1 Hz, 1H), 2.47 (dt, J = 14.4, 7.1 Hz, 1H), 1.90 (d, J = 1.1 Hz, 3H); 13 ¹³C NMR (150 MHz, D₂O) δ 166.5, 151.7, 137.7, 111.5, 85.7 (d, J = 6.3 Hz), 85.0, 75.7 (d, J = 6.0 Hz), 61.0, 37.4 (d, J = 3.7 Hz), 11.5; 3131P NMR (162 MHz, D2O) δ 42.7 (d, J = 26.2 Hz), -7.8 (d, J = 20.1 Hz), -23.3 (dd, J = 26.2, 20.1 Hz); HRMS (ESI-TOF) m / z: C10H16N2O13P3S [M-H] - , calculated value: 496.9551, measured value: 496.9603; retention time: 8.81 min (Method 3). Example 67 31P 1 , 31P 2 -bis-(5'-O-uridine) diammonium (R)-diphosphate-1-thioate (Compound (R P )-64) Preparation

[0381] According to General Procedure C, from the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (+)-Ψ* reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol), Compound (R P )-64 was obtained. Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 20:80) to give 57 mg of Compound (R P )-64 after lyophilization (yield = 42%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-64 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.03 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 8.2 Hz, 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 13C NMR (150 MHz, 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 = 9.4 Hz), 82.6 (d, J = 9.7 Hz), 73.38, 73.35, 69.31, 69.29, 64.54 (d, J = 5.9 Hz), 64.53 (d, J = 6.5 Hz); 31P NMR (162 MHz, D2O) δ 43.3 (d, J = 27.7 Hz), -12.1 (d, J = 27.7 Hz); HRMS (ESI-TOF) m / z: C18H23N4O16P2S [M-H] - , Calculated value: 645.0310, Measured value: 645.0323; Retention time: 4.85 min (Method 1). Example 68 P 1 , P 2 -Bis-(5'-O-uridine) diammonium (S)-diphosphate-1-thioate (Compound (S P )-64) Preparation

[0382] According to General Procedure C, from the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (-)-Ψ* reagent (128 mg, 0.3 mmol) and the protected uridine compound 11-1 (278 mg, 0.5 mmol), Compound (S P )-64 was obtained. Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 20:80) to obtain 54 mg of Compound (S P )-64 (yield = 40%, d.r. > 20:1) after lyophilization. Physical state: White amorphous solid. Compound (S P )-64 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.01 (dd, J = 8.1, 1.4 Hz, 1H), 7.94 (dd, J = 8.1, 1.4 Hz, 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 13C NMR (150 MHz, D2O): δ 165.63, 165.62, 151.2, 141.4, 141.3, 102.3, 102.2, 87.83, 87.77, 82.7 (d, J = 9.5 Hz), 82.6 (d, J = 10.0 Hz), 73.4, 73.3, 69.29, 69.28, 64.7 (d, J = 6.0 Hz), 64.5 (d, J = 5.6 Hz). 31³¹P NMR (162 MHz, D₂O) δ 43.1 (d, J = 26.3 Hz), -12.1 (d, J = 26.3 Hz); HRMS (ESI-TOF) m / z: C₁₈H₂₃N₄O₁₆P₂S [M-H] - , calculated: 645.0310, found: 645.0323; retention time: 3.53 min (method 1). Example 69 ³¹P 1 -(5'-O-Adenosine)-³¹P 2 -(5'-O-Uridine) Diammonium (R)-Diphosphate-2-Thioate (Compound (R P )-65) Preparation

[0383] According to General Procedure C, Compound (R P )-65 was obtained from the protected adenosine monophosphate compound 32 (152 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 20:80) to give 77 mg of Compound (R P )-65 (yield = 55%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-65 is characterized by 1 ¹H NMR (600 MHz, D₂O): δ 8.45 (s, 1H), 8.19 (s, 1H), 7.79 (d, J = 8.1 Hz, 1H), 6.08 (d, J = 5.8 Hz, 1H), 5.84 (d, J = 4.8 Hz, 1H), 5.71 (d, J = 8.1 Hz, 1H), 4.79 - 4.76 (m, 1H), 4.54 (dd, J = 5.0, 3.7 Hz, 1H), 4.40 - 4.37 (m, 1H), 4.35 (ddd, J = 11.4, 4.6, 2.8 Hz, 1H), 4.31 - 4.18 (m, 6H); 1313C NMR (150 MHz, 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 = 9.4 Hz), 83.0 (d, J = 9.7 Hz), 74.2, 73.9, 70.4, 69.7, 65.3 (d, J = 5.5 Hz), 64.8 (d, J = 6.5 Hz); 31 31P NMR (162 MHz, D2O): δ 43.5 (d, J = 27.7 Hz), -12.0 (d, J = 27.7 Hz); HRMS (ESI-TOF) m / z: C19H24N7O14P2S [M-H] - , calculated: 668.0582, found: 668.0587; retention time: 7.82 min (method 1). Example 70 P 1 -(5'-O-Adenosine)-P 2 -(5'-O-Uridine) Diammonium (S)-Diphosphate-2-Thioate (Compound (S P )-65) Preparation

[0384] According to general procedure C, compound (S P )-65 was obtained from the protected adenosine monophosphate compound 32 (152 mg, 0.2 mmol), (-)-Ψ* 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, from 0:100 to 20:80) to give 90 mg of compound (S P )-65 after lyophilization (yield = 64%, d.r. > 20:1).

[0385] Preparation scale: According to general procedure C, compound (S * )-65 was obtained from the protected adenosine monophosphate compound 32 (1.52 g, 2.0 mmol), (-)-Ψ P ) 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, from 0:100 to 20:80) to give 1.01 g of compound (S P)-65 (Yield = 72%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-65 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.43 (s, 1H), 8.14 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 6.06 (d, J = 5.9 Hz, 1H), 5.82 (d, J = 5.1 Hz, 1H), 5.67 (d, J = 8.1 Hz, 1H), 4.56 (dd, J = 5.0, 3.6 Hz, 1H), 4.39 - 4.37 (m, 1H), 4.31 - 4.28 (m, 1H), 4.28 - 4.20 (m, 6H); 13 13C NMR (150 MHz, 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 = 9.6 Hz), 83.0 (d, J = 10.1 Hz), 74.2, 74.0, 70.5, 69.7, 65.30 (d, J = 5.1 Hz), 64.26 (d, J = 5.4 Hz); 31 31P NMR (162 MHz, D2O): δ 43.2 (d, J = 25.8 Hz), -12.0 (d, J = 25.8 Hz); HRMS (ESI-TOF) m / z: C19H24N7O14P2S [M-H] - , calculated: 668.0582, found: 668.0587; retention time: 5.20 min (Method 1). Example 71 P 1 -(5'-O-Adenosine)-P 2 -(5'-O-Uridine) Diammonium (R)-Diphosphate-1-Thioate (Compound (R P )-66) Preparation

[0386] According to the general procedure C, compound (R P )-66 was obtained from the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (+)-Ψ* 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, from 0:100 to 20:80) to give 72 mg of compound (R P)-66 (yield = 51%, d.r. > 20:1). Physical state: white amorphous solid. Compound (R P )-66 is characterized by 1 1H NMR (600 MHz, D2O): δ 8.56 (s, 1H), 8.16 (s, 1H), 7.69 (d, J = 8.1 Hz, 1H), 6.08 (d, J = 5.8 Hz, 1H), 5.83 (d, J = 5.0 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 4.53 (dd, J = 5.0, 3.6 Hz, 1H), 4.42 - 4.39 (m, 1H), 4.33 (ddd, J = 11.6, 4.1, 2.4 Hz, 1H), 4.31 - 4.22 (m, 5H), 4.17 (ddd, J = 11.6, 5.3, 2.6 Hz, 1H); 13 13C NMR (150 MHz, 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 = 9.6 Hz), 83.1 (d, J = 9.4 Hz), 74.4, 74.0, 70.6, 69.6, 65.4 (d, J = 6.4 Hz), 65.0 (d, J = 5.2 Hz); 31 31P NMR (162 MHz, D2O) δ 43.5 (d, J = 27.3 Hz), -12.0 (d, J = 27.3 Hz); HRMS (ESI-TOF) m / z: C 19 H 24 N7O 14 P2S [M-H] - , calculated: 668.0582, found: 668.0557; retention time: 7.11 min (Method 1). Example 72 P 1 -(5'-O-Adenosine)-P 2 -(5'-O-Uridine) Diammonium (S)-Diphosphate-1-Thioate (Compound (S P )-66) Preparation

[0387] According to General Procedure C, from the protected uridine monophosphate compound 34 (127 mg, 0.2 mmol), (-)-Ψ* reagent (128 mg, 0.3 mmol) and the protected adenosine compound 14-1 (342 mg, 0.5 mmol), compound (S P)-66. Deprotection was carried out at 40 °C. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 20:80) to give 79 mg of compound (S P )-66 (yield = 56%, d.r. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (S P )-66 is characterized by 1 1H NMR (600 MHz, D2O) δ 8.51 (s, 1H), 8.16 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 6.07 (d, J = 5.9 Hz, 1H), 5.82 (d, J = 5.1 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 4.79 - 4.76 (m, 1H), 4.54 (dd, J = 4.9, 3.3 Hz, 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 13C NMR (150 MHz, 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 = 10.0 Hz), 83.1 (d, J = 9.6 Hz), 74.3, 73.9, 70.6, 69.6, 65.7 (d, J = 5.9 Hz), 64.9 (d, J = 5.2 Hz); 31 31P NMR (162 MHz, D2O) δ 43.1 (d, J = 26.1 Hz), -12.1 (d, J = 26.1 Hz); HRMS (ESI-TOF) m / z: C 19 H 24 N7O 14 P2S [M - H] - , calculated: 668.0582, found: 668.0557; retention time: 5.45 min (method 1). Example 73 P 1 -(5'-O-7-methylguanosine)-P 2 -(5'-O-guanosine) disodium (R)-triphosphate-1-thioate (compound (R P )-67) Preparation

[0388] A flame-dried round-bottom flask equipped with a stir bar was charged with the 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, molecular sieve (1.0 g) and the (+)-Ψ* reagent (1.08 g, 2.5 mmol, 2.5 equiv.) were added and the reaction was stirred at room temperature for 1 h. Thereafter, the protected 7-methylguanosine compound 36 (0.90 g, 2.0 mmol, 2.0 equiv.) was 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. After completion of the reaction, the resulting mixture was filtered and the solid residue was washed with approximately 2 mL of DMSO. The filtrate was partitioned between 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 pellets were washed twice with acetone. The solid residues from each centrifuge tube were redissolved in the minimum amount of water, combined and purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, from 0:100 to 40:60). The fractions containing the product were combined and the solvent was evaporated in vacuo (temperature ≤40 °C). The remaining solid was co-evaporated with water three times to remove residual buffer. The residue was redissolved in 40 mL of an 80% aqueous AcOH solution and the resulting solution was stirred at 35 °C for 16 h. Subsequently, the volatiles were removed under reduced pressure and the crude product was redissolved in a mixture of MeOH / H2O / Et3N (20:5:1) (100 mL) 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 product was purified by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / aqueous MeCN solution, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized three times to remove the remaining buffer. The solid obtained was redissolved in the minimum amount of water and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone to give 370 mg of compound (R P )-67 (yield = 43%, d.r. >20:1). Physical state: white amorphous solid. Compound (R P )-67 was characterized by 11H NMR (600 MHz, D2O): δ 8.00 (s, 1H), 5.86 (d, J = 2.2 Hz, 1H), 5.78 (d, J = 6.1 Hz, 1H), 4.60 (t, J = 5.6 Hz, 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 13C NMR (150 MHz, 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 = 8.9 Hz), 82.9 (d, J = 9.7 Hz), 74.7, 74.1, 70.4, 68.5, 65.4 (d, J = 5.4 Hz), 63.9 (d, J = 6.5 Hz), 36.0; 31 31P NMR (162 MHz, D2O): δ 42.8 (d, J = 26.0 Hz), -11.6 (d, J = 20.1 Hz), -24.0 (dd, J = 26.0, 20.1 Hz); HRMS (ESI-TOF) m / z: C21H28N10O17P3S [M - 2H] - , calculated value: 817.0573, found value: 817.0567; retention time: 9.63 min (method 2). Example 74 P 1 -(5'-O-7-methylguanosine)-P 2 -(5'-O-guanosine) disodium (S)-triphosphate-1-thioate (Compound (S P )-67) Preparation

[0389] Compound (S P )-67 was obtained using (-)-Ψ* reagent according to a procedure similar to that of (R P )-67. Purified by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5), followed by precipitation as the sodium salt and drying under high vacuum to give 353 mg of compound (S P )-67 (yield = 41%, d.r. > 20:1). Physical state: white amorphous solid. Compound (S P )-67 is characterized by 11H NMR (600 MHz, D2O): δ 8.01 (s, 1H), 5.88 (d, J = 3.0 Hz, 1H), 5.79 (d, J = 5.9 Hz, 1H), 4.66 (t, J = 5.5 Hz, 1H), 4.52 - 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 13C NMR (150 MHz, 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 = 8.6 Hz), 83.3 (d, J = 9.7 Hz), 74.9, 73.8, 70.3, 68.9, 65.4 (d, J = 5.0 Hz), 64.4 (d, J = 5.7 Hz), 35.9; 31 31P NMR (162 MHz, D2O): δ 43.3 (d, J = 25.5 Hz), -11.5 (d, J = 17.2 Hz), -24.1 (dd, J = 25.5, 27.2 Hz); HRMS (ESI - TOF) m / z: C21H28N10O17P3S [M - 2H] - , calculated value: 817.0573, found value: 817.0567; retention time: 8.58 min (method 2). Example 75 Acyclovir triammonium (R)-diphosphate-α-thioester (Compound (R P )-68) Preparation

[0390] According to the general procedure A with slight modification, from the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (+)-Ψ* reagent (85 mg, 0.2 mmol) and acyclovir (112 mg, 0.5 mmol) and using anhydrous DMF (2.0 mL) as the solvent to obtain Compound (R P )-68. 5.0 equivalents of DBU was used for the coupling step. The crude product was purified by ion exchange chromatography on DEAE Sephadex (1M NH4HCO3 / water, from 0:100 to 25:75) to obtain 42 mg of Compound (R P )-68 (yield = 47%, e.e. > 20:1) after lyophilization. Physical state: white amorphous solid. Compound (R P )-68 is characterized by 11H NMR (600 MHz, D2O): δ 7.91 (s, 1H), 5.50 (s, 2H), 4.11 - 4.04 (m, 2H), 3.77 (t, J = 4.5 Hz, 2H); 13 13C NMR (150 MHz, D2O): δ 158.4, 153.5, 151.1, 139.5, 115.5, 72.2, 67.7 (d, J = 8.8 Hz), 64.4 (d, J = 5.9 Hz); 31 31P NMR (162 MHz, D2O): δ 41.4 (d, J = 29.3 Hz), -6.8 (d, J = 29.3 Hz); HRMS (ESI-TOF) m / z: C8H12N5O8P2S [M - H] - , calculated: 399.9887, found: 399.9879; [α] D 25 = +10.6 (c 1.01, DMSO) (measured as the triethylammonium salt); retention time: 7.75 min (method 2). Example 76 Preparation of Acyclovir Triammonium (S)-diphosphate-α-thioester (Compound (S P )-68)

[0391] Compound (S P )-68 was obtained according to a slightly modified general procedure A from the monophosphate precursor compound 7-1 (92 mg, 0.3 mmol), (-)-Ψ* reagent (85 mg, 0.2 mmol), and acyclovir (112 mg, 0.5 mmol) using anhydrous DMF (2.0 mL) as the solvent. 5.0 equivalents of DBU were used for the coupling step. The crude product was purified by ion-exchange chromatography on DEAE Sephadex (1 M NH4HCO3 / water, from 0:100 to 25:75) to give 46 mg of compound (S P )-68 (yield = 51%, e.e. > 20:1) after lyophilization. All characterization data were the same as those of compound (R P )-68 except for the optical rotation. [α] D 25 = -11.0 (c 0.98, DMSO) (measured as the triethylammonium salt); retention time: 7.76 min (method 2). Example 77 Preparation of Compound 72

[0392] Charge a round-bottom flask equipped with a stir bar with 2'-O-methyladenosine (Compound 69) (1.55 g, 5.5 mmol, 1.0 equiv), and then add anhydrous DMF (55 mL). Sequentially add imidazole (0.75 g, 11.0 mmol, 2.0 equiv) and tert-butyldimethylchlorosilane (1.0 g, 6.6 mmol, 1.2 equiv) and stir the reaction mixture at room temperature overnight. Subsequently, quench the reaction by adding concentrated aqueous NaHCO3 and extract with EtOAc. Wash the organic phase with water, brine, dry over Na2SO4, filter, and concentrate under reduced pressure to afford crude Compound 70, which is used in the next step without any further purification.

[0393] Redissolve the crude Compound 70 in anhydrous DCM (30 mL), and then add O Reagent (3.47 g, 8.3 mmol, 1.5 equiv). O The synthesis of the Reagent is disclosed in Huang et al., Science, 2021, 373, 1265 - 1270. Add freshly dried molecular sieves (3.0 g) and stir the mixture for 5 min. Subsequently, cool the reaction mixture to 0 °C, then sequentially add DIPEA (0.10 mL, 0.6 mmol, 0.1 equiv) and DBU (1.30 mL, 8.8 mmol, 1.6 equiv) and stir the reaction under an argon atmosphere for 30 min. Thereafter, dilute the mixture with EtOAc and filter. Wash the filtrate sequentially with 10% aqueous KH2PO4 and brine, dry over Na2SO4, filter, and concentrate under reduced pressure to afford crude Compound 71, which is used in the next step without any further purification.

[0394] The crude compound 71 was redissolved in anhydrous DMF (25 mL), and then the protected guanosine compound 35 (3.58 g, 11.0 mmol, 2.0 equiv) was added. The synthesis of the protected guanosine compound 35 is disclosed in Jo 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. Subsequently, a 1.0 M solution of TBAF in THF (22.0 mL, 22.0 mmol, 4.0 equiv) was added and the reaction was stirred for an additional 4 h at room temperature. Thereafter, MeOH (25 mL) and Et3N (5.0 mL) were added successively, and the reaction mixture was stirred overnight at 50 °C. 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 diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was suspended in water and the filtration step was repeated again. The filtrate was concentrated under reduced pressure, and the crude product was purified by reverse-phase C18-silica chromatography (1 M TEAA / MeCN aqueous solution; from 100:0 to 80:20) and lyophilized to afford 2.40 g of compound 72 (a 2:1 mixture of diastereomers). (Yield in 5 steps = 48%). Physical state: white amorphous solid.

[0395] Compound 72 is characterized by 11H NMR (600 MHz, D2O): δ 8.21 (s, 1H; major + minor), 8.05 (s, 1H; minor), 8.04 (s, 1H; major), 7.87 (s, 1H; minor); 7.85 (s, 1H; major), 6.20 (s, 1H; minor), 6.16 (s, 1H; major), 6.09 (s, 1H; major), 6.06 (s, 1H; minor), 5.89 (d, J = 6.6 Hz, 1H; minor), 5.86 (d, J = 6.3 Hz, 1H; major), 5.50 (d, J = 6.3 Hz, 1H; minor), 5.46 (d, J = 6.9 Hz, 1H; major), 5.29 (dd, J = 6.1, 3.1 Hz, 1H; minor), 5.19 (dd, J = 6.9, 3.1 Hz, 1H; major), 4.66 - 4.62 (m, 1H; major), 4.54 - 4.50 (m, 1H; minor), 4.36 - 4.30 (m, 1H; major + minor), 4.20 - 4.11 (m, 2H; major + minor), 4.06 - 4.03 (m, 1H; minor), 4.00 - 3.96 (m, 1H; major), 3.66 - 3.59 (m, 1H; major + minor), 3.57 - 3.48 (m, 1H; major + minor), 3.46 (s, 3H; major), 3.38 (s, 3H; minor), 3.29 (s, 3H; major + minor), 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 = 7.3 Hz, 12H; nBu4N + ); 1313C NMR (150 MHz, D2O) δ 159.2 (minor), 159.1 (major), 154.9 (major + minor), 154.1 (minor), 154.0 (major), 151.81 (minor), 151.79 (major), 150.4 (minor), 150.3 (major), 147.6 (major + minor), 140.0 (major + minor), 137.1 (major), 137.0 (minor), 118.5 (major + minor), 118.1 (major), 117.0 (minor), 116.0 (major), 115.9 (minor), 89.8 (major), 88.5 (minor), 86.19 (major), 86.15 (minor), 85.9 (d, J = 9.5 Hz; major), 84.7 (major + minor), 84.4 (d, J = 9.4 Hz; minor), 83.5 (major), 82.4 (minor), 81.24 (d, J = 5.5 Hz; major), 81.21 (d, J = 4.7 Hz; minor), 80.6 (major), 80.1 (minor), 72.7 (d, J = 5.5 Hz; major + minor), 65.4 (d, J = 5.1 Hz; major), 65.1 (d, J = 4.9 Hz; minor), 60.6 (major + minor), 57.6 (br; nBu4N + ), 57.3 (major + minor), 52.0 (major), 51.0 (minor), 22.6 (nBu4N + ), 18.6 (nBu4N + ), 12.3 (nBu4N + ); 31 31P NMR (162 MHz, D2O) δ -1.1; HRMS (ESI-TOF) m / z: C 23 H 28 N 10 O 12 P [M - H] - , calculated: 667.1631, found: 667.1621. Example 78 Preparation of Compound 73

[0396] The dinucleoside 72 (2.40 g, 2.6 mmol, 1.0 equiv) was dried by co-evaporation with anhydrous MeCN and the substrate was dissolved in anhydrous MeCN (30 mL). Subsequently, Ψ O reagent (2.22 g, 5.2 mmol, 2.0 equiv) and molecular sieves (3.0 g) were added and the resulting suspension was stirred for 5 min. Ψ OThe synthesis of the reagent was disclosed 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 min. Thereafter, deionized water (3.0 mL) and DBU (2.32 mL, 15.6 mmol, 6.0 equiv) were added successively and the reaction was stirred for an additional 15 min. The mixture was diluted with water, filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The residue was resuspended in water and the filtration step was repeated again. The filtrate was concentrated under reduced pressure and the crude product was purified by reverse-phase C18-silica chromatography (1 M TEAA / MeCN aqueous solution; from 100:0 to 85:15) and lyophilized to afford 1.56 g of compound 73 (a 2:1 mixture of diastereomers). (Yield = 63%). Physical state: white amorphous solid.

[0397] Compound 73 is characterized by 1 H NMR (600 MHz, D2O): δ 8.39 (s, 1H; minor), 8.38 (s, 1H; major), 8.15 (s, 1H; major + minor), 7.90 (s, 1H; major + minor), 6.21 (s, 1H; minor), 6.18 (d, J = 2.3 Hz, 1H; major), 6.10 (s, 1H; major), 6.08 (d, J = 2.3 Hz, 1H; minor), 5.98 (d, J = 7.1 Hz, minor), 5.97 (d, J = 6.8 Hz; major), 5.47 (dd, J = 6.2, 2.3 Hz, 1H; minor), 5.43 (dd, J = 7.0, 2.3 Hz, 1H; major), 5.31 (dd, J = 6.2, 3.6 Hz, 1H; minor), 5.21 (dd, J = 7.0, 3.5 Hz, 1H; major), 4.91 - 4.86 (m, 1H; major + minor), 4.66 (q, J = 4.4 Hz, 1H; major), 4.54 (q, J = 4.2 Hz, 1H; minor), 4.37 - 4.31 (m, 1H; major + minor), 4.25 - 4.15 (m, 3H; major + minor), 4.00 - 3.92 (m, 1H; major + minor), 3.86 - 3.81 (m, 1H; minor), 3.80 - 3.75 (m, 1H; major), 3.47 (s, 3H; major), 3.37 (s, 3H; minor), 3.35 (s, 3H; minor), 3.34 (s, 3H; major), 3.18 (q, J = 7.3 Hz, 12H; Et3NH + ), 1.26 (t, J = 7.3 Hz, 18H; Et3NH + ); 13CNMR(150MHz, D2O): δ 158.1 (major), 158.0 (minor), 154.5 (major + minor), 153.1 (minor), 153.0 (major), 151.8 (major + minor), 150.5 (minor), 150.3 (major), 148.3 (major + minor), 139.2 (major + minor), 137.6 (major), 137.5 (minor), 118.1 (major), 117.9 (major + minor), 116.9 (minor), 115.9 (major), 115.8 (minor), 89.9 (major), 88.6 (minor), 85.8 (d, J = 9.5 Hz; major), 84.6 (major + minor), 84.3 (d, J = 9.4 Hz; minor), 83.6 (major), 82.8 (d, J = 9.1 Hz; major + minor), 82.4 (minor), 82.1 (d, J = 5.5 Hz; major), 82.0 (d, J = 5.5 Hz; minor), 80.5 (major), 80.0 (minor), 72.71 (d, J = 5.7 Hz; major), 72.66 (d, J = 5.8 Hz; minor), 65.4 (d, J = 5.1 Hz; major), 65.1 (d, J = 5.2 Hz; minor), 63.7 (d, J = 4.7 Hz; major + minor), 57.2 (major + minor), 52.0 (major), 50.9 (minor), 46.2 (Et3NH + ), 7.7 (Et3NH + ); 31 P NMR(162MHz, D2O) δ 0.1, -1.2; HRMS(ESI-TOF) m / z: C 23 H 29 N 10 O 15 P2[M - H] - , calculated: 747.1294, found: 747.1287. Example 79 Preparation of Compound 75

[0398] Compound 73 (1.56 g, 1.6 mmol, 1.0 eq) was dried by co-evaporation with anhydrous DMF and dissolved in anhydrous DMF (20 mL). i-Pr2NP(OFm)2 (1.27 g, 2.4 mmol, 1.5 eq) was added to the resulting solution, followed by 5-phenyl-1-H-tetrazole (0.36 g, 2.4 mmol, 1.5 eq), and the reaction was stirred at room temperature for 1 h. Subsequently, tert-butyl hydroperoxide (5.5 M in decane; 0.86 mL, 4.8 mmol, 3.0 eq) was added and the mixture was stirred for an additional 1 h. The reaction was quenched by the addition of Et2O (250 mL), and the resulting suspension was sonicated for 10 min. The solvent was decanted and the remaining oily residue was redissolved in DCM (10 mL). The volatile components were evaporated under reduced pressure, and the residue was co-evaporated successively with MeCN and DCM to afford 2.72 g of crude compound 74 (65% wt. purity as determined by quantitative 31 31P NMR) (NMR yield = 91%).

[0399] Compound 74 was used in the subsequent step without any further purification.

[0400] Compound 74 was characterized by 31 31P NMR (162 MHz, DMSO-d6): δ -1.9, -12.2 (d, J = 20.9 Hz), -12.6 (d, J = 20.9 Hz); HRMS (ESI-TOF) m / z: C 51 H 50 N 10 O 18 P3 [M-H] - , calcd: 1183.2523, found: 1183.2497. P 1 -{5’-O-[2’-O-methyladenosyl-(3’,5’)-guanosine]}-P 2 -(5’-O-7-methylguanosine) (R)-trisodium 3-thiotriphosphate (Compound (R P )-75)

[0401] 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 eq; 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 eq) were added and the mixture was stirred for 10 min. Subsequently, Molecular sieve (1.0 g) and (+)-Ψ* reagent (1.08 g, 2.5 mmol, 2.5 equiv) and the reaction was stirred at room temperature for 1 h. Thereafter, the protected 7-methylguanosine compound 36 (0.90 g, 2.0 mmol, 2.0 equiv) was 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. After completion of the reaction, the resulting mixture was filtered and the solid residue was washed with ca. 2 mL of DMSO. The filtrate was partitioned between 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 pellets were washed twice with acetone. The solid residues from each centrifuge tube were redissolved in the minimum amount of water, combined and purified by ion-exchange chromatography on DEAE Sephadex (gradient 1 M NH4HCO3 / water, from 0:100 to 40:60). The fractions containing the product were combined and the solvent was evaporated in vacuo (temperature ≤40 °C). The remaining solid was co-evaporated with water 3 times to remove the 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 product was redissolved in a mixture of MeOH / H2O / Et3N (20:5:1) (100 mL) 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 product was purified by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / aqueous MeCN, from 100:0 to 95:5). The fractions containing the product were combined and lyophilized 3 times to remove the remaining buffer. The solid obtained was redissolved in the minimum amount of water and the product was precipitated as the sodium salt from 0.2 M NaClO4 in acetone to give 522 mg of the title compound after drying under high vacuum (yield from 72 = 38%, d.r. >20:1). Physical state: white solid.

[0402] Compound (R P )-75 is characterized by 11H NMR (600 MHz, D2O) δ 9.04 (s, 1H), 8.36 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 6.00 (d, J = 5.5 Hz, 1H), 5.85 - 5.80 (m, 2H), 4.97 - 4.92 (m, 1H), 4.78 - 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 13C NMR (150 MHz, D2O) 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 = 9.1 Hz), 82.42 (d, J = 9.5 Hz), 82.40 (d, J = 9.6 Hz), 81.2 (d, J = 4.2 Hz), 74.3, 72.9, 72.1 (d, J = 4.9 Hz), 69.8, 68.5, 64.60 (d, J = 4.6 Hz), 63.5 (d, J = 6.2 Hz), 57.5, 35.6; 31 31P NMR (162 MHz, D2O) δ 42.9 (d, J = 27.4 Hz), -1.0, -11.9 (d, J = 18.7 Hz), -24.2 (dd, J = 27.4, 18.7 Hz); HRMS (ESI-TOF) m / z: C32H42N15O23P4S [M - 2H] - , calculated: 1160.1254, found: 1160.1228; retention time: 8.43 min (method 1). P 1 -{5’-O-[2’-O-methyladenosyl-(3’,5’)-guanosine]}-P 2 -(5’-O-7-methylguanosine) (S)-trisodium 3-thiotriphosphate (compound (S P )-75)

[0403] According to a procedure similar to that for compound (R P )-75 on a 0.1 mmol scale and using the (-)-Ψ* reagent, compound (S P)-75. Purified by reverse-phase column chromatography on C-18 silica gel (gradient 1 M TEAA / MeCN aqueous solution, from 100:0 to 95:5), followed by precipitation as the sodium salt and drying under high vacuum to give 43 mg of the compound (S P )-75 (yield from 72 = 35%, d.r. > 20:1). Physical state: white solid.

[0404] Compound (S P )-75 is characterized by 1 H NMR (600 MHz, D2O): δ 9.16 (s, 1H), 8.46 (s, 1H), 8.18 (s, 1H), 7.97 (s, 1H), 6.04 (d, J = 4.5 Hz, 1H), 5.88 (s, 1H), 5.81 (d, J = 4.5 Hz, 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 (150 MHz, 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 = 8.8 Hz), 82.3 (br), 81.6 (d, J = 3.5 Hz), 74.4, 73.0, 71.9 (d, J = 4.8 Hz), 69.7, 68.7, 64.6 (d, J = 4.9 Hz), 64.4, 63.8, 57.5, 35.6; 31 P NMR (162 MHz, D2O): δ 43.2 (d, J = 26.6 Hz), -1.0, -11.8 (d, J = 18.8 Hz), -24.3 (dd, J = 26.6, 18.8 Hz); HRMS (ESI-TOF) m / z: C32H42N15O23P4S [M - 2H] - , calculated: 1160.1254, found: 1160.1228; retention time: 7.62 min (method 1).

Claims

1. A compound having the following structure:

2. A method for preparing the compound according to claim 1, the method comprising reacting compound 1: with compound 2 or compound 3: in the presence of an acid.

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

4. The method according to claim 2, wherein, Compound 1 is formed by reacting compound 4: with P2S5 in the presence of a base.

5. The method according to 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. The method according to claim 2, wherein, Compound 2 or compound 3 is formed by reacting compound 5 or compound 6: with hydrogen in the presence of a catalyst respectively.

7. The method according to claim 6, wherein, The catalyst is selected from the group consisting of platinum dioxide, palladium on carbon, platinum on carbon, Lindlar catalyst, Raney nickel, nickel, rhodium supported on alumina, palladium, and platinum.

8. A method for preparing a nucleoside dithiophosphate or a salt thereof, the method comprising: (a) Reacting one of compounds 7, 8, or 9: wherein R 1 , R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, CD3, CF3, a straight-chain or branched C1-C 20 alkyl, a straight-chain or branched C2-C 12 alkenyl, a straight-chain or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 、R 7 、 and R 8 are each independently CD3, CF3, a linear or branched C1-C 20 alkyl, a linear or branched C2-C 12 alkenyl, a linear or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; and where Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with the compound as claimed in claim 1 in the presence of a first base to form a chiral thiophosphoramidite transfer reagent; (b) Reacting the chiral thiophosphoramidite transfer reagent with a protected nucleoside or an unprotected nucleoside in the presence of a second base to form a protected nucleoside dithiophosphate; and (c) Deprotecting the protected nucleoside dithiophosphate to form a nucleoside dithiophosphate.

9. The method according to 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-dimethylpyridine, and imidazole.

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

12. The method according to claim 8, wherein, The nucleoside is selected from the group consisting of compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, and compound 19: wherein R 9 is independently hydrogen, acetyl, branched or straight-chain C2-C 20 alkanoyl, benzoyl, aroyl, acryloyl, or heteroaroyl.

13. The method according to claim 8, wherein, The nucleoside dithiophosphate is selected from the group consisting of: where X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

14. The method according to claim 13, wherein, The nucleoside dithiophosphate is 15. The method according to claim 13, wherein, The nucleoside dithiophosphate is 16. The method according to claim 13, wherein, The nucleoside dithiophosphate is 17. The method according to claim 13, wherein, The nucleoside dithiophosphate is 18. The method according to claim 13, wherein, The nucleoside dithiophosphate is 19. The method according to claim 13, wherein, The nucleoside dithiophosphate is 20. The method according to claim 13, wherein, The nucleoside dithiophosphate is 21. The method according to claim 13, wherein, The nucleoside dithiophosphate is 22. The method according to claim 13, wherein, The nucleoside dithiophosphate is 23. The method according to claim 13, wherein, The nucleoside dithiophosphate is 24. A method for preparing a nucleoside triphosphorothioate or a salt thereof, the method comprising: (a) Reacting compound 20 or compound 21: wherein R 1 , R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, CD3, CF3, a linear or branched C1-C 20 alkyl, a linear or branched C2-C 12 alkenyl, a linear or branched C2-C 12 alkynyl, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; R 6 is CD3, CF3, a linear or branched C1-C 20 alkyl group, a linear or branched C2-C 12 alkenyl group, a linear or branched C2-C 12 alkynyl group, aryl, heteroaryl, heterocycle, or C3-C8 cycloalkyl; and where Z is hydrogen, alkylammonium, dialkylammonium, trialkylammonium, or tetraalkylammonium; with the compound as claimed in claim 1 in the presence of a first base to form a chiral thiophosphorotriester transfer reagent; (b) Reacting the chiral thiophosphorotriester transfer reagent with a protected nucleoside or an unprotected nucleoside in the presence of a second base to form a protected nucleoside trithiophosphate; and (c) Deprotecting the protected nucleoside trithiophosphate to form a nucleoside trithiophosphate.

25. The method according to 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. The method according to 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. The method according to claim 26, wherein, The second base is 1,8-diazabicyclo(5.4.0)undec-7-ene.

28. The method according to claim 24, wherein, The nucleoside is selected from the group consisting of: Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, and Compound 19: wherein R 9 is independently hydrogen, acetyl, branched or straight-chain C2-C 20 alkanoyl, benzoyl, aroyl, acryloyl, or heteroaroyl.

29. The method according to claim 24, wherein, The nucleoside tri-thiophosphate is selected from the group consisting of: wherein X is ammonium, trialkylammonium, lithium, sodium, or potassium; and Y is calcium or magnesium.

30. The method according to claim 29, wherein, The nucleoside triphosphorothioate is 31. The method according to claim 29, wherein, The nucleoside tri-thiophosphate is 32. The method according to claim 29, wherein, The nucleoside tri-thiophosphate is 33. The method according to claim 29, wherein, The nucleoside triphosphorothioate is 34. The method according to claim 29, wherein, The nucleoside tri-thiophosphate is 35. The method according to claim 29, wherein, The nucleoside triphosphorothioate is 36. The method according to claim 29, wherein, The nucleoside triphosphorothioate is 37. The method according to claim 29, wherein, The nucleoside tri-thiophosphate is 38. The method according to claim 29, wherein, The nucleoside triphosphorothioate is 39. The method according to claim 29, wherein, The nucleoside triphosphorothioate is

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