Polyphosphated nucleotide, phosphate partially activated nucleotide for the synthesis thereof and method for synthesis thereof, and method for synthesis

Abstract

Provided are: a polyphosphorylated nucleoside having a structure represented by formula (I), which can be efficiently produced using an inexpensive raw material through a short process and a simple operation; a phosphate-partially-activated nucleotide for use in the synthesis of the polyphosphorylated nucleoside; a method for synthesizing the phosphate-partially-activated nucleotide; and a method for synthesizing a polyphosphorylated nucleoside using the phosphate-partially-activated nucleotide. In the formula, B is a protected or unprotected natural or artificial nucleoside base, R1, R2, R4 and R5 are any one of H, alkyl and the like, and R1, R2, R4 and R5 can be the same or different; x and Y are any one of H, OH, OCH3 and the like; h is any one of integers from 1 to 3; p, n, m, and q are each 0 or an integer, p, n, m, and q are in an arbitrary order, and (p + n + m + q) is an integer from 0 to 5000 (inclusive).

Description

TECHNICAL FIELD

[0001] The present application relates to polyphosphorylated nucleosides, phosphorus moiety-activated nucleotides for synthesis thereof, and a method for synthesis of polyphosphorylated nucleosides using the phosphorus moiety-activated nucleotides, and a method for synthesis of polyphosphorylated nucleosides using the phosphorus moiety-activated nucleotides. BACKGROUND

[0002] In recent years, polyphosphorylated nucleosides have attracted attention for their effectiveness. For example, P 1 , P 4 -Di(uridine 5'-)tetraphosphate (hereinafter referred to as "U2P4") or a salt thereof has a sputum discharge inducing effect and is expected as a therapeutic drug for chronic obstructive pulmonary disease (Patent Literature 1). In addition, U2P4 is marketed as an eye drop therapeutic drug for a chronic disease of the corneal epithelium called dry eye, and the demand is high.

[0003]

[0004] In the production of U2P4, the following steps (1) to (3) are employed in Patent Literature 2. (1) The counter cation of uridine 5'-diphosphate as a starting material is replaced with tributylammonium, (2) carbonyldiimidazole (CDI) is allowed to act on the replacement in DMF to imidazolize one of the hydroxyl groups of the terminal side of the phosphoric acid of uridine 5'-diphosphate, and (3) the imidazolizate is allowed to react with uridine 5'-diphosphate in DMF.

[0005] In Patent Literature 2, as another condition for the production of U2P4, a method is disclosed in which one of the hydroxyl groups of uridine 5'-diphosphate is activated with imidazole, and then, in the presence of a Lewis acid, uridine 5'-diphosphate is allowed to react with the uridine 5'-diphosphate activated with imidazole in water.

[0006] In addition, messenger RNA (mRNA) has a triphosphorylated nucleoside structure called a capping structure at the 5' end thereof. An example of the production of a cap structure by a method in which one of the hydroxyl groups of a nucleoside 5'-phosphate is activated with imidazole as described above is disclosed in Patent Literature 3.

[0007] In Patent Literature 4, a method is disclosed in which, in the synthesis of a nucleoside triphosphate, a coupling reaction of a nucleoside 5'-monophosphate and a nucleoside 5'-diphosphate in which one of the hydroxyl groups is activated with imidazole is performed in the presence of a Lewis acid catalyst using DMF as a reaction solvent.

[0008] [Patent Literature]

[0009] [Patent Literature]

[0010] Patent Literature 1: International Publication No. 2008 / 012949

[0011] Patent Literature 2: International Publication No. 2014 / 103704

[0012] Patent Literature 3: International Publication No. 2017 / 053297

[0013] Patent Literature 4: International Publication No. 2022 / 212442 SUMMARY

[0014] PROBLEMS TO BE SOLVED BY THE INVENTION

[0015] Any of the above production methods uses nucleoside 5'-diphosphate as a starting material, but nucleoside 5'-diphosphate is expensive. In addition, in any of the production methods, a condensing agent or an activating agent is used to produce nucleoside triphosphate or nucleoside tetraphosphate, but a multi-step process is required.

[0016] Further, since the reaction efficiency in each step is not necessarily high, a large amount of time is required for purification of a crude product and concentration of a solution obtained by purification. As a result, the yield of the product is low, and the time efficiency and cost performance of production are poor.

[0017] The present application was completed in view of such circumstances, and aims to provide a polyphosphorylated nucleoside that can be produced efficiently by a short process and simple operation using a cheaper starting material, a phosphoric acid moiety-activated nucleotide for synthesis of a polyphosphorylated nucleoside, a synthesis method thereof, and a synthesis method of a polyphosphorylated nucleoside using the phosphoric acid moiety-activated nucleotide.

[0018] MEANS FOR SOLVING THE PROBLEMS

[0019] To solve the above problems, the polyphosphorylated nucleoside, the phosphoric acid moiety-activated nucleotide for synthesis of a polyphosphorylated nucleotide, the synthesis method thereof, and the synthesis method of a polyphosphorylated nucleoside using the phosphoric acid moiety-activated nucleotide of the present application adopt the following means.

[0020] The first aspect of the present application provides a polyphosphorylated nucleoside having a structure represented by Formula (I).

[0021]

[0022] In the formula, B is independently a natural or artificial nucleoside base protected or unprotected with a protecting group;

[0023] R 1 , R 2 is independently H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10H7, 1-pyrenylmethyl, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM) any one of R 1 , R 2 may be the same or different.

[0024] R 3 is independently any one of methyl, ethyl, vinyl derivative, allyl derivative, isopropyl, phenyl derivative, polyfluoroalkyl derivative;

[0025] R 4 , R 5 is independently H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenylmethyl, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM) any one of R 4 , R 5 may be the same or different.

[0026] X, Y are independently any one of H, OH, OCH3, OCH2CH2OCH3, F;

[0027] J is independently any one of O, S, Se, BH;

[0028] Z is any one of OH, NH2, N(CH3)2;

[0029] W is any one of CH2, CH2CH2, CH(CH3);

[0030] V is any one of O, NCH3, NCOR, N-N=NH;

[0031] h is an integer of 1 or more and 3 or less;

[0032] p, n, m, q are independently 0 or an integer, p, n, m, q are in any order, (p+n+m+q) is an integer of 0 or more and 5000 or less.

[0033] In the above first aspect, R 1 , R 2 may be dichloroacetyl, R 3 may be H or a metal salt.

[0034] In the above first aspect, R 1 , R 2 may be phenoxyacetyl, R 3 may be H or a metal salt.

[0035] The second aspect of the present application provides a 5'-diphosphate moiety-activated nucleotide for producing the polyphosphorylated nucleoside of the above first aspect, having a structure represented by the following formula (II).

[0036]

[0037] In the formula,

[0038] B is independently a natural or artificial nucleobase protected or unprotected by a protecting group;

[0039] X, Y, Z are independently OH, OR 6 , SH, SR, BH3, halogen, azole, NR2, NH2;

[0040] R 1 , R 2 are independently an oligonucleotide of 3 to 11-mers having H, OH, halogen, alkyl, hydroxyl protected by an acyl-based protecting group, a carbamoyl-based protecting group, or a silyl-based protecting group, a natural or artificial nucleobase unprotected or protected by a protecting group, R 1 , R 2 may be the same or different,

[0041] R 6 is OBT, OAt, a substituted or unsubstituted vinyl group, or a substituted or unsubstituted allyl group, and the substituted vinyl group can include a cyclic structure.

[0042] The third aspect of the present application is a method for synthesizing the 5'-diphosphate moiety-activated nucleotide of the above second aspect, the method comprising:

[0043]

[0044] (a) a process for preparing a phosphate activator by reacting pyrophosphoryl chloride with a co-catalyst compound in the presence or absence of a catalyst compound in a solvent; and

[0045] (b) a process of reacting the phosphorus acid activator obtained in the process (a) with a nucleoside whose base moiety is protected with a protecting group or is not protected, to produce a 5'-diphosphate partially activated nucleotide (in the formula (II), X = Y = Cl, Z = Cl or O - ).

[0046] The fourth aspect of the present application is the process for producing the 5'-diphosphate partially activated nucleotide according to the second aspect, which comprises:

[0047]

[0048] (a) a process of reacting a polychlorinating agent with a cocatalyst compound in the presence or absence of a catalyst compound, to produce a phosphorus acid activator; and

[0049] (b) a process of reacting the phosphorus acid activator obtained in the process (a) with a nucleoside 5'-monophosphate whose base moiety is protected with a protecting group or is not protected, in a water-hydrophilic organic solvent, to produce a 5'-diphosphate partially activated nucleotide (in the formula, X = Y = azole or heterocyclic compound derivative, Z = azole, heterocyclic compound derivative or O

[0050] The fifth aspect of the present application is the process for producing the 5'-diphosphate partially activated nucleotide according to the second aspect, which comprises:

[0051]

[0052] (a) a process of reacting a polychlorinating agent with a nucleoside 5'-diphosphate whose base moiety is protected with a protecting group or is not protected, in a water-hydrophilic organic solvent, in the presence or absence of a catalyst compound, to produce a nucleoside 5'-diphosphoryl chloride (in the formula, X = Y = Cl) ;

[0053] (b) a process of further adding a cocatalyst compound to the reaction solution after the process (a), to produce a 5'-diphosphate partially activated nucleotide (in the formula, X = Y = azole or heterocyclic compound derivative, Z = azole, heterocyclic compound derivative or O - ).

[0054] In the third to fifth aspects described above, the cocatalyst compound can be imidazole or benzimidazole.

[0055] In the third to fifth aspects described above, the catalyst compound can be an organic catalyst which is HOBT, HOAu, Oxyma (ethyl isonitroso cyanacetic acid ester) or DMT-MM, a Lewis acid such as ferric chloride, aluminum chloride, zinc chloride, copper chloride, magnesium chloride, cesium chloride, cerium triflate or scandium triflate.

[0056] In the fourth or fifth embodiment, the polychlorinating agent may be phosphorus oxychloride, anhydrous dichlorophosphoric acid, phosphorus pentachloride, thionyl chloride or 2-chloro-1,3-dimethylimidazoline chloride.

[0057] A sixth aspect of the present invention provides a method for synthesizing a polyphosphorylated nucleoside, which is the method for synthesizing a polyphosphorylated nucleoside according to the first aspect, comprising the step of condensing the 5'-diphosphate partially activated nucleotides of the third to fifth aspects with the nucleoside 5'-phosphate in the presence of water to obtain the polyphosphorylated nucleoside.

[0058] In the sixth embodiment, the nucleoside 5'-phosphate can be guanosine 5'-phosphate, N7-methylguanosine 5'-phosphate, or a 5'-monophosphorylated nucleic acid selected from RNA, DNA, DNA / RNA chimera, and RNA containing modified nucleosides.

[0059] Effects of the Invention

[0060] According to the method for synthesizing polyphosphorylated nucleosides of the present invention, the target polyphosphorylated nucleosides can be produced by a simple operation using cheaper raw materials. In addition, the target product can be produced efficiently in a short process.

[0061] Furthermore, the 5'-diphosphate partially activated nucleotide used for the synthesis of polyphosphorylated nucleosides according to the present invention can be reacted in a solvent containing only water or an organic solvent containing water, thereby achieving a synthesis method with a low environmental load. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 These are diagrams showing HPLC charts of G2P3 synthesized by the synthesis method of this embodiment before (a) and after (b) purification by anion exchange column chromatography.

[0063] Figure 2 This is a diagram showing an HPLC chart of a crude product after a U2P4 synthesis reaction using uridine 5'-diphosphate as a starting material in Example 1 of this embodiment.

[0064] Figure 3 This is a diagram showing the MS spectrum of the synthesis of U2P4 using uridine 5'-diphosphate as a starting material in Example 2 of this embodiment. The MS spectra of the reaction solution, U2P4, UDP, and uridine monophosphate are shown in order from the top.

[0065] Figure 4 This is an HPLC chart of the crude product obtained after the synthesis reaction in Example 2 of this embodiment.

[0066] Figure 5 This is a diagram showing a preparative LC chromatogram of a crude product obtained after the synthesis reaction in Example 2 of this embodiment.

[0067] Figure 6 is a graph showing an HPLC chart of U2P4 obtained by freeze-drying the Peak4 fraction liquid in Figure 5

[0068] Figure 7 is a graph showing an HPLC chart of the crude product obtained after the reaction in Example 3 of the present embodiment.

[0069] Figure 8 is a graph showing an HPLC chart of the crude product obtained after the synthesis reaction in Example 5 of the present embodiment. DETAILED DESCRIPTION

[0070] Hereinafter, one embodiment of polyphosphorylated nucleosides, phosphomate-activated nucleosides for synthesis of polyphosphorylated nucleosides, and a synthesis method of polyphosphorylated nucleosides using phosphomate-activated nucleosides of the present application will be described.

[0071] The polyphosphorylated nucleosides in the present embodiment have the structure of the following formula (I).

[0072]

[0073] In the formula,

[0074] B is independently a natural or artificial nucleoside base protected with a protecting group or unprotected;

[0075] R 1 , R 2 is independently H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenylmethyl, acetyl, benzoyl, dichloroacetyl, 1-pentanoyl, levulinoyl, phenoxyacetyl, a fatty acid ester having a carbon number of 5 to 22, an alkyl group having a carbon number of 3 to 11 including an alkyne moiety, a biotin ester, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM), R 1 , R 2 may be the same or different.

[0076] R 3 is independently any one of a methyl group, an ethyl group, a vinyl derivative, an allyl derivative, an isopropyl group, a phenyl derivative, a polyfluoroalkyl derivative;

[0077] R​4 , R 5 independently H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenemethyl, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM), R 1 , R 2 may be the same or different;

[0078] R 3 independently any one of methyl, ethyl, vinyl derivative, allyl derivative, isopropyl, phenyl derivative, polyfluoroalkyl derivative;

[0079] X, Y are independently any one of H, OH, OCH3, OCH2CH2OCH3, F;

[0080] J is independently any one of O, S, Se, BH;

[0081] Z is any one of OH, NH2, N(CH3)2;

[0082] W is any one of CH2, CH2CH2, CH(CH3);

[0083] V is any one of O, NCH3, NCOR, N-N=NH;

[0084] h is an integer of 1 or more and 3 or less;

[0085] p, n, m, q are respectively 0 or an integer, p, n, m, q are in any order, (p+n+m+q) is an integer of 0 or more and 5000 or less.

[0086] In the present specification, the nucleoside located at the 5'-most end in Formula (I) is referred to as "left wing", and the nucleoside or nucleotide having a polyphosphate bonded to the 3'-hydroxyl group of the left wing at the 5' end is referred to as "right wing". In the left wing, both the 2'- and 3'-hydroxyl groups of the nucleoside can be acyl-protected.

[0087] As examples of the acyl group, there are acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, aliphatic acid ester having a carbon number of 5 to 22, but not limited to these.

[0088] In the right wing, the sugar possessed by the nucleoside can be all natural five-carbon sugar (ribose or 2' deoxyribose) or can include morpholino nucleoside.

[0089] In the right wing, an oligonucleotide structure having a plurality of nucleotides bound thereto can also be present. In formula (I), the order of p, n, m, and q is different. p, n, m, and q are each 0 or an integer, and (p + n + m + q) is an integer of 0 or more and 5000 or less. (p + n + m + q) is preferably an integer of 0 or more and 200 or less, more preferably an integer of 0 or more and 50 or less, and further preferably an integer of 0 or more and 25 or less.

[0090] B in formula (I) is a natural or artificial nucleoside base. The base moiety can be protected with a protecting group. The base moiety can be unprotected. The protecting group can include a functional group that is labeled. As examples of labeling, a label in a fluorescent functional group, a label in a radioisotope, a label in a stable isotope, a label in biotin, but not limited thereto.

[0091] As examples of the protecting group of the base moiety, acyl-based protecting groups, alkoxy protecting groups, carbamoyl protecting groups, amidine protecting groups, but not limited thereto.

[0092] As examples of the protecting group of the phosphate portion, methyl, ethyl, vinyl derivative, allyl derivative, isopropyl, phenyl derivative, polyfluoroalkyl derivative.

[0093] R at the 2' or 3' position of the nucleoside located in the left wing 1 , R 2 may be H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenylmethyl, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, fatty acid ester having a carbon number of 5 to 22, alkyl having a carbon number of 3 to 11 including an alkyne portion, biotin ester, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM). As examples of the alkyl group, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10H7, 1-pyrenylmethyl, and the like. As examples of acyl groups, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, and the like can be given. Examples of carbamoyl groups include CONHC6H5, CONHC 10 H7, and the like. Further, Boc, Fmoc, and the like, carbonate, allyloxycarbonyl, benzyloxycarbonyl can also be applied. As examples of acetal-type protecting groups, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM), and the like. As examples of silyl-type protecting groups, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tetraisopropylsilyloxy, di-t-butyisilyl, and the like can be given. R 1 , R 2 may be the same or different in one molecule.

[0094] R at the 2' or 3' position of the nucleoside at the 3' end on the right wing 4 , R 5 may be H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenylmethyl, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 H7(NAPOM), and the like. As examples of silyl-type protecting groups, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tetraisopropylsilyloxy, di-t-butyisilyl, and the like can be given. R 10 H7, 1-pyrenylmethyl, and the like. As examples of acyl groups, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinoyl, phenoxyacetyl, and the like can be given. Examples of carbamoyl groups include CONHC6H5, CONHC 10 H7, and the like. Further, Boc, Fmoc, and the like, carbonate, allyloxycarbonyl, benzyloxycarbonyl can also be applied. As examples of acetal-type protecting groups, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10H7 (NAPOM), and the like. As examples of silyl-based protecting groups, mention can be made of trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tetraisopropyl disilyloxy, di-t-butylsilyl, and the like. R 4 , R 5 may be the same or different.

[0095] As examples of known polyphosphorylated nucleosides, mention can be made of dioxazolene (structural formula U2P4) used as a component of eye drops, guanosine (structural formula G) having a methyl group at position 7 on the left side of the triphosphate portion, and the like. 7 G) of mRNA.

[0096] The polyphosphorylated nucleosides of the present embodiment differ from these known substances in that a protecting group is introduced on the 2'-hydroxyl group and / or the 3'-hydroxyl group of the ribose ring on the left wing. The polyphosphorylated nucleosides of the present embodiment, by having such a configuration, can achieve high duplex formation ability and liposolubility. In addition, by protecting the hydroxyl groups, the reaction sites are protected, and thus high reactivity and selectivity can be achieved.

[0097] In synthesizing the polyphosphorylated nucleosides of the present embodiment, as a synthetic intermediate, a nucleoside having an activated 5'-diphosphate portion is used.

[0098]

[0099] In the formula, B is independently a natural or artificial nucleoside base protected or unprotected by a protecting group;

[0100] X, Y, Z are independently OH, OR 6 , SH, SR, BH3, halogen, azole, NR2, NH2;

[0101] R 1 , R 2 are independently 3 to 11-mer oligonucleotides having H, OH, halogen, alkyl, hydroxyl protected by acyl-based protecting groups, carbamoyl-based protecting groups, or silyl-based protecting groups, natural or artificial nucleoside bases unprotected or protected by protecting groups, R 1 , R 2 may be the same or different;

[0102] R 6 is OBT, OAt, substituted or unsubstituted vinyl, or substituted or unsubstituted allyl, and the substituted vinyl can include a cyclic structure.

[0103] The base in the present embodiment includes all bases of naturally occurring nucleosides (hereinafter also referred to as "natural bases"). The most common bases among naturally occurring nucleosides are purines and pyrimidines. As naturally occurring purine rings, adenine, guanine, and N6-methyladenine can be exemplified. As naturally occurring pyrimidines, cytosine, thymine, 5-methylcytosine, and oxaliuracil can be exemplified. As natural nucleosides, adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine, or pseudouridine, 2'-O-methyl or 2'-deoxynucleoside derivatives can be exemplified.

[0104] The base in the present embodiment can be a non-natural base or an artificial base. The non-natural base or the artificial base refers to an artificially synthesized base analog having the same properties as those of a natural base, which is capable of forming an artificial base pair with a base analog that is the counterpart of the base pair (also referred to as "complementary artificial base") in the same manner as a natural base.

[0105] As the synthesis method of the 5'-diphosphate moiety-activated nucleoside, there are three kinds of (1) a synthesis route using a nucleoside as a starting material, (2) a synthesis route using a nucleoside 5'-monophosphate as a starting material, and (3) a synthesis route using a nucleoside 5'-diphosphate as a starting material. Hereinafter, the outline of each synthesis route will be described.

[0106] In the synthesis routes (1) and (2), in the first step, a phosphorylating agent is first activated, and in the next step, it is reacted with a nucleoside or a nucleoside monophosphate.

[0107] (1) Synthesis route using a nucleoside as a starting material

[0108] In the present route, pyrophosphoryl chloride as a phosphorylating agent is reacted with a cocatalyst compound in the presence or absence of a catalyst compound in a solvent to prepare a phosphate activator. Then, the obtained phosphate activator is reacted with a nucleoside protected with a base moiety-protecting group or unprotected in a hydrophilic organic solvent or a water-hydrophilic organic solvent to obtain a target 5'-diphosphate moiety-activated nucleotide (in formula (II), X = Y = Cl, Z = Cl or O - ).

[0109] As the cocatalyst compound used in the synthesis route (1), imidazole or benzimidazole can be exemplified, and benzimidazole is preferred.

[0110] As the catalyst compound used in the synthetic route (1), there can be mentioned organic catalysts such as HOBT, HOAT, HOCU or DMT-MM, Lewis acids such as iron chloride, aluminum chloride, zinc chloride, copper chloride, magnesium chloride, cesium chloride and the like, inorganic or organic salts such as cerium triflate or scandium triflate. The catalyst compound is not essential, and the phosphoric acid activator can be prepared without adding the catalyst compound to the reaction solution. When added, in the present synthetic route (1), a Lewis acid is preferred, and iron chloride is more preferred.

[0111] In the synthetic route (1), it is preferred to use 0.3 to 5.0 equivalents of the catalyst compound, 1.0 to 5.0 equivalents of the co-catalyst compound with respect to one hydroxyl group of the phosphoric acid portion, and 1.0 to 5.0 equivalents of the polychlorinating agent with respect to one hydroxyl group of the phosphoric acid portion, respectively.

[0112] The synthesis in the synthetic route (1) can be carried out in a hydrophilic organic solvent, or a hydrophilic organic solvent containing water. As the hydrophilic organic solvent, trimethyl phosphate is preferred. The reaction temperature is preferably -30°C to 30°C, and more preferably 0°C, in the step of preparing the phosphoric acid activator. It is preferably 10°C to 70°C, and more preferably 50°C, in the step after the addition of the nucleoside.

[0113] As the hydrophilic organic solvent used in the synthesis in the synthetic route (1), there can be mentioned trimethyl phosphate, trimethyl phosphite, acetonitrile, THF, dimethyl carbonate, DMF and the like, with acetonitrile being preferred.

[0114] (2) Synthetic route using nucleoside 5'-monophosphate as a starting material

[0115] In the present route, a polychlorinating agent is reacted with a co-catalyst compound in the presence or absence of a catalyst compound in a solvent to prepare a phosphoric acid activator. Then, the resulting phosphoric acid activator is reacted with a nucleoside 5'-monophosphate having a base moiety protected or unprotected by a protecting group in a hydrophilic organic solvent or a water-hydrophilic organic solvent to obtain a target 5'-diphosphate partially activated nucleotide (in formula (II), X = Y = azole or heterocyclic compound derivative, Z = azole, heterocyclic compound derivative or O - ). As the azole, there can be mentioned imidazoline, benzimidazoline, 1,2,4-triazoline, tetrazoline, pyrazoline and derivatives thereof. As the heterocyclic compound derivative, there can be mentioned pyridinium, 4-N,N'-dimethylaminopyridinium and derivatives thereof.

[0116] As the polychlorinating agent used in the synthetic route (2), phosphorus oxychloride, phosphorus oxychloride anhydride, chlorosulfoxide, or 2-chloro-l,3-dimethylimidazolinium chloride can be given. When anhydrous phosphorus oxychloride is mixed with a Lewis acid as a catalyst compound, an activated substance of diphosphate type is formed, but depending on the reaction conditions, a substance in which only one phosphate is added to the nucleotide monophosphate becomes the main product.

[0117] As the co-catalyst compound used in the synthetic route (2), imidazole or benzimidazole can be given, with imidazole being preferred.

[0118] As the catalyst compound used in the synthetic route (2), an organic catalyst such as HOBT, HOAu, HOCU, or DMT-MM, a Lewis acid such as iron chloride, aluminum chloride, zinc chloride, copper chloride, magnesium chloride, cesium chloride, and the like, an inorganic or organic salt such as cerium triflate or scandium triflate can be given. The catalyst compound is not essential, and the phosphate activated substance can be produced without adding the catalyst compound to the reaction solution. When added, a Lewis acid is preferred in the present synthetic route (2), with iron chloride being more preferred.

[0119] In the synthetic route (2), 0.3 to 5.0 equivalents of the catalyst compound, 1.0 to 5.0 equivalents of the co-catalyst compound with respect to one hydroxyl group of the phosphate portion, and 1.0 to 5.0 equivalents of the polychlorinating agent with respect to one hydroxyl group of the phosphate portion are preferably used.

[0120] The synthesis in the synthetic route (2) can be performed in a hydrophilic organic solvent, a hydrophilic organic solvent containing water, or water. The reaction temperature is preferably 10°C to 30°C in the step of producing the phosphate activated substance, and the target phosphate activated substance can be obtained even at room temperature. In the step after the addition of the nucleotide 5'-monophosphate, the temperature is preferably 0°C to 70°C, and more preferably 25°C.

[0121] As the hydrophilic organic solvent used in the synthesis in the synthetic route (2), acetonitrile, THF, dimethyl carbonate, DMF, and the like can be given, with acetonitrile being preferred.

[0122] (3) In the synthetic route using nucleotide 5'-diphosphate as a starting material, in the present route, a polychlorinating agent is reacted with a nucleotide 5'-diphosphate whose base portion is protected with a protecting group or is unprotected in the presence or absence of a catalyst compound in a solvent, to produce a nucleotide 5'-diphosphochloride (in formula (II), X = Y = Cl). Then, a co-catalyst compound is reacted with the obtained nucleotide 5'-diphosphochloride in a hydrophilic organic solvent, a water-hydrophilic organic solvent, or water, to obtain the target 5'-diphosphate portion-activated nucleotide acid (in formula (II), X = Y = azole or heterocyclic compound derivative, Z = azole, heterocyclic compound derivative, or O -). The pH of the aqueous solution is preferably 1 to 5, more preferably pH 2. As the azole, imidazoline, benzimidazoline, 1,2,4-triazoline, tetrazoline, pyrazoline, and derivatives thereof can be exemplified. As the heterocyclic compound derivative, pyridinium, 4-N,N'-dimethylaminopyridinium, and derivatives thereof can be exemplified.

[0123] As the polychlorinating agent used in the synthetic route (3), phosphorus oxychloride, phosphorus oxydichloride, phosphorus pentachloride, thionyl chloride, or 2-chloro-l,3-dimethylimidazoline chloride, Vilsmeier reagent, preferably 2-chloro-l,3-dimethylimidazolinium chloride can be exemplified. When anhydrous phosphorus oxydichloride is mixed with a Lewis acid as a catalyst compound, an activated substance of the diphosphate type is formed, but depending on the reaction conditions, a substance in which only one phosphate is added to the nucleoside monophosphate becomes the main product. In contrast, 2-chloro-l,3-dimethylimidazoline chloride can be chlorinated without adding a phosphate. Therefore, it can be used when chlorinating a nucleoside diphosphate. In order to promote chlorination, a condensing agent can be used in combination. As the condensing agent, DMT-MM or COMU as a urea onium type condensing agent, DCC, DIC, or EDC as a carbodiimide type condensing agent, Py-BOP as a phosphonium type condensing agent, and the like can be used.

[0124] As the promoter compound used in the synthetic route (3), imidazole or benzimidazole, preferably benzimidazole can be exemplified. Due to the high acidity and steric hindrance of benzimidazole, the reactivity and selectivity in the present synthetic route (3) are improved.

[0125] As the catalyst compound used in the synthetic route (3), HOBT, HOAT, an organic catalyst as Oxyma, iron chloride, aluminum chloride, zinc chloride, copper chloride, magnesium chloride, cesium chloride, and the like Lewis acids, cerium triflate or scandium triflate, and the like inorganic or organic salts can be exemplified. The catalyst compound is not essential, and the phosphate activated substance can be produced without adding the catalyst compound to the reaction solution. When added, in the present synthetic route (3), a Lewis acid is preferred, and iron chloride is more preferred.

[0126] In the synthetic route (3), it is preferred to use 0.3 to 5.0 equivalents of the catalyst compound, 1.0 to 5.0 equivalents of the promoter compound with respect to one hydroxyl group of the phosphate portion, and 1.0 to 5.0 equivalents of the polychlorinating agent with respect to one hydroxyl group of the phosphate portion, respectively.

[0127] The synthesis in the synthetic route (3) can be performed in a hydrophilic organic solvent, a hydrophilic organic solvent containing water, or water. The reaction temperature is preferably -30°C to 30°C, preferably 25°C in the step of producing the phosphate activated substance. Further, in the step after the addition of the promoter compound, it is preferably 10°C to 70°C, more preferably 25°C. The pH of the aqueous solution is preferably 1 to 5, more preferably pH 3.

[0128] As the hydrophilic organic solvent used in the synthesis in the synthetic route (3), acetonitrile, trimethyl phosphate, trimethyl phosphite, THF, dimethyl carbonate, DMF, DMSO, and the like can be mentioned, with acetonitrile being preferred.

[0129] The 5'-diphosphate partially-activated nucleotide of the present embodiment can be confirmed by LC-MS or 31 P-NMR confirms its production.

[0130] The synthesis method of polyphosphorylated nucleosides using 5'-phosphate partially-activated nucleotides of the present embodiment will be described. The 5'-diphosphate partially-activated nucleotide obtained in the above synthetic routes (1) to (3) is condensed with nucleoside 5'-phosphate in the presence of water to obtain a polyphosphorylated nucleoside.

[0131] In the synthesis method of polyphosphorylated nucleosides using 5'-phosphate partially-activated nucleotides of the present embodiment, the nucleoside 5'-phosphate used can be guanosine 5'-phosphate, N7-methylguanosine 5'-phosphate, or 5'-monophosphorylated nucleic acid selected from RNA, DNA, DNA / RNA chimera, and RNA containing modified nucleosides.

[0132] In the synthesis method of the present embodiment, a commercially available inexpensive chlorinating agent or polychlorinating agent can be used to synthesize the phosphate partially-activated nucleoside. Thus, by minimizing the synthetic route using nucleoside or monophosphate as a starting material, the starting material can be obtained at a lower cost compared to the conventional synthesis method.

[0133] In the synthesis method of polyphosphorylated nucleosides of the present embodiment, synthesis is performed using 5'-diphosphate partially-activated nucleotides. The 5'-diphosphate partially-activated nucleotide of the present embodiment can effectively construct polyphosphate when water is used as a solvent for polyphosphate synthesis. Position selectivity, functional group selectivity, and reactivity are high, and side reactions are few.

[0134] In addition, according to the synthesis method of the present embodiment, the number of steps is small compared to the conventional method, and the reaction system is simple, so that the purification step can be made more simple.

[0135] According to the present embodiment, synthesis of 5'-diphosphate partially-activated nucleotides and synthesis of polyphosphorylated nucleosides can be performed in water or a hydrophilic organic solvent containing water. Thus, nucleoside monophosphate or nucleoside diphosphate used as a starting material can be recovered and used again for synthesis.

[0136] According to the synthesis method of polyphosphorylated nucleosides of the present embodiment, one starting material can be converted into an activated diphosphate nucleoside, and polyphosphorylated nucleosides of left-right symmetric structure can be synthesized.

[0137] In addition, according to the synthesis method of polyphosphorylated nucleosides of the present embodiment, a kind of starting material is converted into an activated diphosphorylated nucleotide, which is reacted with a monophosphorylated or diphosphorylated nucleotide or the like. Before forming a symmetrical dimer, by adding an unactivated phosphonucleoside solution (0.1 to 5.0 equivalents, preferably 1.0 equivalent, with respect to the activated diphosphorylated nucleotide) to the activated diphosphorylated nucleotide, a left and right asymmetric polyphosphorylated nucleoside can be synthesized.

[0138] According to the synthesis method of polyphosphorylated nucleosides of the present embodiment, by adding R 1 , R 2 , and any one of biotinylated, the target can be obtained by affinity purification.

[0139] As an example of polyphosphonucleosides, a synthesis method of G2P3 using guanosine as a starting material is described.

[0140]

[0141] Iron chloride (162 mg, 1.0 mmol) was suspended in trimethyl phosphate (2.0 mL). To this solution, pyrophosphoric acid chloride (138 μL, 1.0 mmol) was slowly added dropwise at 0°C, and stirred for 20 minutes. Guanosine (113 mg, 0.4 mmol) was added in small amounts each time, and stirred for 10 minutes. A solution of benzimidazole (307 mg, 2.6 mmol) in acetonitrile (0.8 mL) was added dropwise to the reaction solution at room temperature over 5 minutes. After stirring at 50°C for 10 minutes, 200 μL of deionized water was added dropwise in 10 portions (total 2 mL), and stirred for 10 minutes. A crude product having a sticky consistency in the lower layer was obtained.

[0142] The crude product was diluted with deionized water (50 mL), and purified using an ion exchange column (YMC BioPro IEX SmartSep Q). Elution was performed using A: 0.05 M NH4HCO3, B: 1.0 M NH4HCO3. The obtained fractions were analyzed by UPLC-MS, and the fraction containing G2P3 was recovered. By concentrating and freeze-drying the recovered fraction, white G2P3 was obtained (separation yield 20% to 26%).

[0143] The HPLC chart before purification of G2P3 synthesized by the above method using an anion exchange column chromatography is shown in Figure 1 (a), and the HPLC chart after purification is shown in Figure 1 (b). In HPLC analysis using absorbance at a wavelength of 260 nm as an index, it was confirmed that G2P3 was 99% or more as calculated from the peak area indicating G2P3 and the total area of peaks containing other components. The purification conditions are shown below.

[0144] Column: BioPro SmartSep Q30 5 mL

[0145] Flow rate: 8.0 mL / min

[0146] Injection amount: 20 mg / mL, 0.5 mL injection

[0147] Eluent A: 0.05 M aqueous ammonium bicarbonate

[0148] B: 1.0 M aqueous ammonium bicarbonate

[0149] Gradient (B%): 0%→(60 CV)→15%→(0.1 CV)→100% (5 CV).

[0150] [Example]

[0151] [Example 1: Synthesis of U2P4 using uridine 5'-diphosphate as starting material]

[0152]

[0153] Uridine 5'-diphosphate disodium salt (20 g, 45 mmol) and imidazole (6.0 g, 90 mmol) were dissolved in deionized water (23 ml), 2-chloro-l,3-dimethylimidazolinium chloride (7.5 g, 45 mmol) was added and reacted at 40°C for 1 hour. Further imidazole (6.0 g, 90 mmol) and 2-chloro-l,3-dimethylimidazolinium chloride (7.5 g, 45 mmol) were added and reacted at 40°C for 3 hours. The HPLC profile of the crude product after reaction is shown in Figure 2 The reaction was adjusted to pH 7 with 1.0 M NH4HCO3, diluted with deionized water (500 mL), and purified with an ion exchange column (TOYOPEARL Gigapp Q-650M). Elution was performed using A: 0.05 M NH4HCO3, B: 1.0 M NH4HCO3. The fractions obtained were analyzed by UPLC-MS and the diuridine tetraphosphate (U2P4) was recovered. The recovered U2P4 was dissolved in deionized water (100 ml) and passed through Dowex 50Wx4 Na + The eluted liquid was concentrated, lyophilized, and U2P4 tetrasodium salt was obtained as a white powder (5.55 g, 30% isolated yield).

[0154] [Example 2: Synthesis of U2P4 using uridine 5'-monophosphate as starting material]

[0155]

[0156] Imidazole (2.45 g, 36 mmol) was dissolved in acetonitrile (24 mL). To this solution, phosphorous oxychloride (544 μL, 6 mmol) was added slowly dropwise at room temperature, and stirred for 20 minutes, resulting in the precipitation of white crystals. Uridine monophosphate disodium salt (2.21 g, 6 mmol) was added in small amounts each time, and stirred for 10 minutes. The reaction solution was separated into two layers. After the reaction solution was left to stand at 50°C for 10 minutes, deionized water was added dropwise 10 times (total 2 mL) of 200 μL, and stirred for 10 minutes. N-methyl-imidazole (1.9 mL, 24 mmol) was added dropwise, and reacted at 50°C for 2 to 15 hours. Then, the reaction solution was returned to room temperature, and the upper layer was removed by decantation, to obtain the lower layer of the crude product having a viscous nature.

[0157] The obtained crude product was diluted with deionized water (50 mL), and purified using an ion exchange column (TOYOPEARL Gigapp Q-650M). Elution was performed using A: 0.05 M NH4HCO3, B: 1.0 M NH4HCO3. The obtained fractions were analyzed by UPLC-MS, and a fraction containing diuridine tetraphosphate (U2P4) was recovered. The eluted liquid was concentrated, and freeze-dried to obtain U2P4 as a white solid (0.47 g, 17.8% of the separation yield, 98% purity).

[0158] The LCMS spectrum of the liquid extracted from the reaction solution in the reaction is shown in Figure 2 . According to the MS spectrum of the reaction solution, in addition to the starting material, uridine monophosphate (UMP), and the target substance, U2P4, a peak corresponding to the molecular weight of U2P3, which is a substance in which two uridines are bound by three phosphoric acids, and IntD, which is a substance in which two imidazoles are bound to uridine 5'-diphosphate, were confirmed. One of the structures considered for IntD is shown below. By 31 P-NMR measurement, it was also possible to confirm the presence of IntD (δ 7, 89 ppm, not shown) in the system.

[0159]

[0160] IntD is an active intermediate, and is converted to U2P3 or U2P4.

[0161] The MS spectrum of the synthesis intermediate in the synthesis of U2P4 is shown in Figure 3 .

[0162] Figure 4 The HPLC chart of the crude product obtained after the synthesis reaction in Example 2 is shown in FIG. 1.

[0163] Figure 5 The preparative LC chromatogram of the crude product obtained after the synthesis reaction in Example 2 is shown in FIG. 2.

[0164] Figure 6 HPLC profile of U2P4 obtained by freeze-drying of the Peak4 fraction in Figure 5

[0165] [Example 3: Synthesis of G2P3 using guanosine 5'-monophosphate as starting material]

[0166]

[0167] Benzimidazole (18.1 g, 153 mmol) was suspended in acetonitrile (10.3 mL). To this solution, phosphoryl chloride (2.1 mL, 23.5 mmol) was slowly added at room temperature over 3 minutes, and after stirring for 5 minutes, a previously prepared feed-catalyst solution of guanosine 5'-monophosphate sodium salt (10 g, 24 mmol) and ferric chloride (3.8 g, 23.5 mmol) dissolved in hydrochloric acid (23.5 mL) at pH 2.0 was added dropwise, and the reaction solution was stirred for 30 minutes. Thereafter, the reaction solution was returned to room temperature, and the pH was adjusted to 8.0 using saturated aqueous carbonic acid, to obtain a crude product solution.

[0168] The obtained crude product solution was diluted to 100 mL with deionized water, and purified using a (YMC Corp. BioPro IEX SmartSep Q). In the elution, A liquid: 0.05 M NH4HCO3, B liquid: 1.0 M NH4HCO3 were used, and the obtained fractions were analyzed by UPLC-MS, and fractions containing G2P3 were recovered. The obtained fractions were analyzed by UPLC-MS, and fractions containing guanosine triphosphate (G2P3) were recovered. The eluted liquid was concentrated, freeze-dried, and white G2P3 (7.1 g, separation yield 39.0%, purity 28%) was obtained. Figure 7

[0169] [Example 4: Synthesis of cap analog using guanosine derivative as starting material]

[0170]

[0171] Ferric chloride (162 mg, 1.0 mmol) was suspended in acetonitrile (2.0 mL). To this solution, phosphorous oxychloride (138 μL, 1.0 mmol) was slowly added dropwise at 0°C, and stirred for 20 minutes. 2', 3'-Diphenoxyacetyl-N7-methylguanosine (215 mg, 0.4 mmol) was added in small amounts each time, and stirred for 10 minutes. To the reaction solution, a solution of benzimidazole (307 mg, 2.6 mmol) in acetonitrile (0.8 mL) was added dropwise. After stirring at room temperature for 30 minutes, a solution of 5'-monophosphorylated rAomeG tetrabutylammonium salt (563 mg, 0.4 mmol) in acetonitrile was added dropwise, and stirred for 10 minutes.​​

[0172] Then, the reaction solution was returned to room temperature, and the pH was adjusted to 12 with 1.0 N aqueous sodium hydroxide solution to obtain a crude product solution.

[0173] The crude product solution was diluted with deionized water (10 mL) and purified with an ion exchange column (YMC BioPro IEX SmartSep Q). Elution was performed using A: 0.05 M NH4HCO3, B: 1.0 M NH4HCO3. The obtained fractions were analyzed by UPLC-MS, and the target cap analog was recovered. The eluted solution was concentrated and lyophilized to obtain the target product as a white solid ((100 mg, separation yield 18.8%, 98% purity).

[0174] [Example 5: Synthesis of cap analog using guanosine 5'-monophosphate as a starting material]

[0175]

[0176] Benzimidazole (307 mg, 2.6 mmol) was suspended in acetonitrile (2.0 mL). To this solution, 2-chloro-l,3-dimethylimidazolinium chloride (203 mg, 1.2 mmol) was slowly added at room temperature over 3 minutes, and after stirring for 5 minutes, a previously prepared raw material-catalyst solution in which 5'-diphosphorylated N7-methylguanosine sodium salt (215 mg, 0.4 mmol) and ferric chloride (162 mg, 1.0 mmol) were dissolved in hydrochloric acid (23.5 mL) at pH 3.0 was added dropwise at room temperature, and the reaction solution was stirred at room temperature for 30 minutes. Next, a solution of 5'-monophosphorylated rAomeG tetrabutylammonium salt (563 mg, 0.4 mmol) in acetonitrile (10 mL) was added dropwise, and stirring was performed for 30 minutes to obtain a crude product solution.

[0177] The crude product was diluted with 20% acetonitrile aqueous solution (10 mL) and purified with an ion exchange column (YMC BioPro IEX SmartSep Q). Elution was performed using A: 0.05 M NH4HCO3, B: 1.0 M NH4HCO3. The obtained fractions were analyzed by UPLC-MS, and the target cap analog was recovered. The eluted solution was concentrated and lyophilized to obtain the target product as a white solid (398 mg, separation yield 56.0%, 98% purity). Figure 8 The HPLC chart of the crude product obtained after the reaction is shown in FIG. 6.

[0178] According to the method for synthesizing polyphosphorylated nucleosides of the present embodiment, the target polyphosphorylated nucleosides can be manufactured by a simple operation using a more inexpensive raw material. In addition, the target products can be manufactured in a short process with high efficiency.

[0179] The 5'-diphosphate moiety-activated nucleotide for the synthesis of polyphosphorylated nucleosides according to the present embodiment can be reacted in only water, or an organic solvent containing water as a solvent. Thereby, a synthesis method with a small environmental load can be achieved.

Claims

1. A polyphosphorylated nucleoside having a structure represented by formula (I), Where, B is independently a natural or artificial nucleoside base which is protected or unprotected by a protecting group; R 1 、R 2 are independently H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenemethyl, acetyl, benzoyl, dichloroacetyl, 1-pentanoyl, levulinyl, phenoxyacetyl, fatty acid esters of 5 to 22 carbon atoms, alkyl groups of 3 to 11 carbon atoms containing an alkynyl moiety, biotin esters, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 Any of H7(NAPOM), R 1 、R 2 It can be the same or different; R 3 are independently any one of methyl, ethyl, vinyl derivatives, allyl derivatives, isopropyl, phenyl derivatives, and polyfluoroalkyl derivatives; R 4 、R 5 are independently H, OCH2CH=CH2, CH2C6H5, CH2C6H4-p-OCH3, 2-CH2C 10 H7, 1-pyrenemethyl, acetyl, benzoyl, dichloroacetyl, 1-pentenoyl, levulinyl, phenoxyacetyl, CONHC6H5, CONHC 10 H7, Boc, Fmoc, allyloxycarbonyl, benzyloxycarbonyl, TOM, Pivom, CEM, CH2OCH2C6H4-p-OCH3(PMBOM), CH2O-2-CH2C 10 Any of H7(NAPOM), R 4 、R 5 It can be the same or different; X and Y are independently any one of H, OH, OCH3, OCH2CH2OCH3, and F; J is independently any one of O, S, Se, and BH; Z is any one of OH, NH2, and NHCH3; W is any one of CH2, CH2CH2, and CH(CH3); V is any one of O, NCH3, NCOR, and NN=NH; h is an integer of 1 to 3; p, n, m, and q are each 0 or an integer, p, n, m, and q are in any order, and (p+n+m+q) is an integer from 1 to 22.

2. The polyphosphorylated nucleoside according to claim 1, wherein The R 1 、R 2 is dichloroacetyl, said R 3 is H or metal ion.

3. The polyphosphorylated nucleoside according to claim 1, wherein The R 1 、R 2 is a phenoxyacetyl group, wherein R 3 is H or metal ion.

4. A 5'-diphosphate partially activated nucleotide having a structure represented by the following formula (II), which is used to produce the polyphosphorylated nucleoside having a structure represented by the formula (I) according to claim 1, Where, B is independently a natural or artificial nucleoside base which is protected or unprotected by a protecting group; X, Y, and Z are independently OH, OR 6 , SH, SR, BH3, halogen, azoles, NR2, NH2; R 1 、R 2 R is independently a 3- to 11-mer oligonucleotide having H, OH, halogen, alkyl, hydroxyl protected by an acyl protecting group, a carbamoyl protecting group or a silyl protecting group, or a natural or artificial nucleoside base that is unprotected or protected by a protecting group, 1 、R 2 Can be the same or different, R 6 is OBT, OAt, a substituted or unsubstituted vinyl group, or a substituted or unsubstituted allyl group, and the substituted vinyl group may contain a cyclic structure.

5. A method for synthesizing the 5'-diphosphate partially activated nucleotide having the structure of formula (II) according to claim 4, the method comprising: (a) a step of reacting pyrophosphoryl chloride with a co-catalyst compound in a solvent in the presence or absence of a catalyst compound to prepare an activated phosphoric acid; and (b) reacting the phosphoric acid activated product obtained in step (a) with a nucleoside whose base moiety is protected or unprotected to prepare a nucleoside 5'-diphosphoryl chloride (where X=Y=Cl, Z=Cl or O) - ) process.

6. A method for synthesizing the 5'-diphosphate partially activated nucleotide having the structure of formula (II) according to claim 4, the method comprising: (a) a step of reacting a polychlorinating agent with a co-catalyst compound in the presence or absence of a catalyst compound to prepare an activated phosphoric acid; and (b) reacting the phosphate activated product obtained in step (a) with a nucleoside 5'-monophosphate whose base moiety is protected or unprotected by a protecting group in a water-hydrophilic organic solvent to prepare a 5'-diphosphate partially activated nucleotide (wherein X=Y=azole or heterocyclic compound derivative, Z=azole, heterocyclic compound derivative or O) - ) process.

7. A method for synthesizing the 5'-diphosphate partially activated nucleotide having the structure of formula (II) according to claim 4, the method comprising: (a) a step of reacting a nucleoside 5'-diphosphoryl chloride (wherein X=Y=Cl in the formula (II)) with a polychlorinating agent in a water-hydrophilic organic solvent in the presence or absence of a catalyst compound, wherein the nucleoside 5'-diphosphate whose base moiety is protected by a protecting agent or not is reacted with a polychlorinating agent; and (b) further adding a co-catalyst compound to the reaction solution after step (a) to obtain a 5'-diphosphate partially activated nucleotide (wherein X=Y=azole or heterocyclic compound derivative, Z=azole, heterocyclic compound derivative or O) - ) process.

8. The synthesis method according to any one of claims 5 to 7, wherein The co-catalyst compound is imidazole or benzimidazole.

9. The synthesis method according to any one of claims 5 to 7, wherein The catalyst compound is an organic catalyst such as HOBT, HOAu, HOCU or DMT-MM, ferric chloride, aluminum chloride, zinc chloride, copper chloride, magnesium chloride, cesium chloride, cerium trifluoromethanesulfonate or scandium trifluoromethanesulfonate.

10. The synthesis method according to claim 6 or 7, wherein The polychlorination agent is phosphorus oxychloride, dichlorophosphoric anhydride, thionyl chloride, or 2-chloro-1,3-dimethylimidazoline chloride.

11. A method for synthesizing a polyphosphorylated nucleoside, which is a method for synthesizing a polyphosphorylated nucleoside having a structure of formula (I) according to claim 1, the method comprising: A step of condensing the 5'-diphosphate partially activated nucleotide according to any one of claims 5 to 7 with nucleoside 5'-phosphate in the presence of water to obtain a polyphosphated nucleoside.

12. The synthesis method according to claim 11, wherein The nucleoside 5'-phosphate is selected from guanosine 5'-phosphate, N7-methylguanosine 5'-phosphate, or a 5'-monophosphorylated nucleic acid of RNA, DNA, DNA / RNA chimera, or RNA containing modified nucleosides.

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