RU2708971C1 - Method for producing a salt of α-d-ribofuranoso-1-phosphate or α-d-2-deoxyribofuranoso-1-phosphate - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology and pharmaceutical chemistry. Disclosed is a method of producing a salt α-D-ribofuranose-1-phosphate and salt α-2-D-deoxyribofuranoso-1-phosphate, selected from a barium, magnesium, calcium, sodium or dicyclohexylamine salt. Enzymatic phosphorolysis of 7-methylguanosine hydroiodide salt or 7-methyl-2'-deoxyguanosine hydroiodide salt is performed in the presence of inorganic phosphate or dicyclohexylamine hydrophosphate, and pNP from Enterobacter or E. coli. Obtained α-D-ribofuranose-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate is reacted with a water-ammonia solution of Ba²⁺, Mg²⁺ or Ca²⁺ acetate to obtain corresponding salt. To obtain sodium salt of dicyclohexylamine salt α-D-ribofuranose-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate is dissolved in water and ion-exchange resin Dowex-50 in Na⁺ form is added to solution.

EFFECT: invention provides stereo-specificity of the reaction, complete conversion of the starting compounds and avoids the use of large excess reagents, yield of the desired compounds is 74–96 %.

1 cl, 4 dwg, 10 ex

Description

The technical field to which the invention relates.

The invention relates to the field of biotechnology (the use of enzymes to produce natural metabolites of nucleosides and nucleotides) and pharmaceutical chemistry (the use of enzymes to obtain drugs based on nucleosides).

Currently, a number of drugs based on modified nucleosides are used in clinical practice. These compounds have a wide spectrum of biological activity: antitumor (Cladribine, Fludarabine, Pentostatin, Nelarabin, Vidaza, Decitabine, etc.), antiviral (hepatitis C, herpes, HIV, etc.) (Ribavirin, Vidarabin, Zidovudine, Lamivudin, etc.) ( WB Parker. Enzymology of Purine and Pyrimidine Antimetabolites Used in the Treatment of Cancer. Chem. Rev. 109, 2880-2893, 2009; E De Clercq, G. Li. Clinical Microbiology Reviews, Approved antiviral drugs over the past 50 years, 29 , 695-747, 2016) (Fig. 1). The use of known drugs in therapeutic practice and the search for biologically active substances in a number of analogues of natural nucleosides are closely related to the development of new methods for the synthesis of nucleosides and their analogues. In recent years, the so-called "green" chemistry has been actively developing, the purpose of which is to replace traditional chemical synthesis with various catalytic methods without the use of toxic organic solvents. When producing natural nucleosides and medicinal compounds of a nucleoside nature (cladribine, 5-fluorodeoxyuridine, fludarabine, nelarabin, vidarabine, etc.), enzymatic synthesis is an alternative to multistage chemical synthesis.

In particular, the stage of chemical glycosylation of heterocyclic bases is replaced by an enzymatic one, which proceeds under the influence of nucleoside phosphorylases. Nucleoside phosphorylases in vivo catalyze the condensation of pentose-1-phosphates and heterocyclic bases with the formation of nucleosides and the release of phosphoric acid residues and the reverse reaction - phosphorolysis of nucleosides in the presence of phosphoric acid residues with the formation of heterocyclic bases and pentose-1-phosphates. The equilibrium in this reversible reaction is usually biased towards nucleoside formation. The reaction proceeds selectively, regio-and stereospecific. A wide variety of compounds - possible substrates - provides wide opportunities for the use of nucleoside phosphorylases in the preparation of various biologically active derivatives of nucleosides (I.A. Mikhailopulo, A.I. Miroshnikov, Biologically important nucleosides: modern trends in biotechnology and application. Mendeleev Commun., 2011, 21, 57-68). The development and application of enzymatic and chemical-enzymatic synthesis of nucleosides with the participation of nucleoside phosphorylases is of industrial importance, since it not only makes the production process more environmentally friendly, but also allows to obtain target compounds of high purity with high yields.

Currently, with enzymatic glycosylation of a heterocyclic base, pentose-1-phosphate is obtained directly in the reaction medium from the corresponding nucleoside - a sugar residue donor (transglycosylation reaction). In the transglycosylation reaction, the key point is the shift in the equilibrium in the coupled nucleoside decomposition-formation reactions towards the formation of the target compounds. The use of pentose-1-phosphate in the form of an individual reagent avoids the conjugate reversible reaction, which simplifies the composition of the reaction mixture and, accordingly, the isolation of the target compounds, and also makes it possible to vary the ratio of the starting reagents, shifting the equilibrium of the glycosylation reaction in the desired direction. Currently, pentose-1-phosphates are difficult to access and have a high cost, which affects the cost of the target products. In light of the foregoing, the development and optimization of methods for the synthesis of various pentose-1-phosphates as universal starting compounds for the synthesis of a wide range of natural nucleosides and nucleoside drug compounds is of particular importance.

The level of technology.

Nucleoside analogues are widely used in clinical practice, among them antitumor, antiviral, antiparasitic drugs. For use in medicine, the content of side compounds in the drug substance is of particular importance, which means that high requirements are imposed on the methods of stero- and regio-selectivity for nucleoside synthesis methods for pharmaceutical use.

There are two main methods for producing nucleoside analogues. The first method is based on the modification of natural compounds (M.S. Drenichev, V.E. Oslovsky, S.N. Mikhailov. Cytokinin Nucleosides - Natural compounds with a unique spectrum of biological activities. Current Topics in Medicinal Chemistry, 16, 2562-2576, 2016). In the second, first derivatives of heterocyclic bases and monosaccharide derivatives are obtained, and then nucleoside synthesis is carried out (S.N. Mikhailov, Yu.P. Lysov, G.I. Yakovlev. Use of functionally competent analogues of nucleosides and nucleotides to study enzyme-substrate interactions. Molecular Biology, 33, 393-407, 1999). In this method, the key step in the synthesis is the creation of an N-glycosidic bond.

Enzymatic synthesis methods substantially supplement chemical ones and, in some cases, have undoubted advantages (I.A. Mikhailopulo, A.I. Miroshnikov, Biologically important nucleosides: modern trends in biotechnology and application. Mendeleev Commun., 2011, 21.57-68). An analysis of published data shows that enzymatic methods for creating a glycosidic bond can compete with chemical ones for the preparation of pharmaceutically important nucleosides. To obtain nucleosides, in particular, a glycosylation reaction involving nucleoside phosphorylases (NF) is used: enzymes that catalyze the condensation of pentose-1-phosphates and heterocyclic bases with the formation of nucleosides and the release of phosphoric acid residues and the reverse reaction, nucleoside phosphorolysis in the presence of phosphoric acid residues to form a heterocycle and pentose-1-phosphates:

Pentose-1-phosphate + Base ↔ Nucleoside + Pi.

Nucleoside phosphorylases play a key role in the metabolism of nucleosides. These enzymes are found in almost all organisms, while the differences in their primary structure are relatively small. The most specific enzyme is thymidine phosphorylase (TF) (EC 2.4.2.4), the substrates of which are thymidine and 2'-deoxyuridine. Uridine phosphorylase (UV) (EC 2.4.2.3) carries out the phosphorolysis of uridine, thymidine and 2'-deoxyuridine. The substrates of purinucleoside phosphorylase (PNF) (EC 2.4.2.1) are the purine nucleosides of both the ribo and 2'-deoxyribo series (MJ Pugmire, SE Ealick, Structural analyses reveal two distinct families of nucleoside phosphorylases. Biochemical Journal, 361, 1- 25, 2002). It has been established that in the reverse phosphorolysis of the glycosylation reaction in the case of ribose-1-phosphate and α-D-2-deoxyribose-1-phosphate substrates, only the α-anomers of pentafuranose-1-phosphates act correspondingly, the corresponding β-anomers are not substrates (N. Komatsu, I. Ikeda, Synthesis of 2-deoxy-betα-D-ribose 1-phosphate, NMR comparison and its enzymatic activity for structural elucidation of synthetic alpha-isomer. Nucleosides Nucleotides Nucleic Acids, 22, 1685-1686, 2003).

Currently, in the enzymatic synthesis of nucleoside analogues, the most commonly used reaction is transglycosylation (transfer of a carbohydrate residue from one heterocyclic base to another). In this case, the pentafuranose residue obtained by phosphorolysis of a commercially available or synthesized nucleoside acts as a substrate in the glycosylation reaction. Thus, the synthesis proceeds in two stages. At the first stage, NFs catalyze the cleavage of the N-glycosidic bond in the presence of phosphate to form a heterocyclic base (B1) and pentafuranose-1-phosphate (Fig. 2, stage 1). The process uses enzymes: thymidine phosphorylase, uridine phosphorylase and purine nucleoside phosphorylase. In the next stage (Fig. 2, stage 2), the formation of the target nucleoside occurs due to the transfer of the carbohydrate residue to the heterocyclic base (B2).

Analysis of the transglycosylation reaction showed that the yields of the desired nucleosides depend on the ratio of the equilibrium constants of nucleoside phosphorolysis reactions (K _p1 / K _p2 ). The maximum yield can be achieved if the equilibrium of stage 1 is strongly shifted towards the formation of pentafuranose-1-phosphate, and the equilibrium of stage 2 is shifted towards the target nucleoside (Nuc-2).

In the reversible enzymatic glycosylation reaction of the heterocyclic base with pentafuranose-1-phosphate in the presence of PNP, unlike transglycosylation, there is no conjugation of several equilibrium reactions, and the equilibrium of enzymatic glycosylation is biased towards nucleoside formation (Goldberg RN, Tewari YB, Bhat TN, "Thermodynamics of Enzy of Enzy of Enzy -Catalyzed Reactions - a Database for Quantitative Biochemistry ", Bioinformatics 2004, 20, 2874-2877). Therefore, increasing the yields of this reaction can be achieved without using large excesses of components (which reduces the cost of synthesis and simplifies the isolation of target compounds). From this point of view, pentafuranoso-1-phosphates are optimal substrates to achieve the maximum yield of the target nucleoside, and the development of an effective and convenient method for their synthesis is of particular importance.

The methods of chemical and enzymatic synthesis of pentose-1-phosphates have been the goal of various research groups for many decades.

The following methods are used to obtain these compounds chemically:

1. Direct condensation of the corresponding protected derivative of D-ribofuranosyl chloride with o-H ₃ PO ₄ . A mixture of α and β anomers is obtained with a total yield of 20% (for the entire sequence of steps, based on natural sugar) (DL MacDonald, HG Fletcher. 2-Deoxy-D-ribose. VIII. Synthesis of the Anomeric 2-Deoxy-D -ribofuranose 1-Phosphates. J. Am. Chem. Soc. 84 (7), 1262-1265, 1962)

2. The condensation reaction of the corresponding protected derivative of D-ribofuranosyl chloride with protected derivatives of H ₃ PO ₄ . The resulting mixture of anomers can be separated, the yield of the α-anomer is 21%. (for the entire sequence of steps, based on natural sugar) (GM Tener, RS Wright, HG Khorana. Phosphorylated Sugars. III. Syntheses of of α-D-Ribofuranose 1-Phosphate. J. Org. Chem, 59, 690-692, 1994)

3. Stereoselective chemical synthesis. A group of Japanese researchers proposed a new method for producing derivatives of 2-deoxyribose-1-phosphate. This method allows to obtain 2-deoxyribose-1-phosphate in the form of a mixture of β- and α-anomers (with a significant, up to 98.5: 1.5 predominance of the α-anomer). The total yield for the entire sequence of stages, based on natural sugar, is 66-69%. The sequence of transformations includes 4 stages, one of which is crystallization with the predominant formation of the α-isomer of 2-deoxyribose-1-phosphate. (N. Komatsu, N. Awano. First stereoselective synthesis of 2-deoxy-α-D-ribofuranosyl-1-phosphate: novel application of crystallization-induced asymmetric transformation. J. Org. Chem, 67, 5419-5421, 2002) .

Enzymatic methods for the preparation of pentose-1-phosphates provide stereoselective production of α-anomers. Currently include:

1. Phosphorolysis of the corresponding natural and synthesized nucleosides:

- phosphorolysis of thymidine in the presence of TF. Yield 12-37%. (M. Friedkin, D. Roberts The Enzymatic Synthesis of Nucleosides II. Thymidine and Related Pyrimidine Nucleosides. J. Biol. Chem. 207, 257-266.1954; IV Fateev, MI Kharitonova, KV Antonov, ID Konstantinova, VN Stepanenko, RS Esipov, F. Seela, KW Temburnikar, KL Seley-Radtke, VA Stepchenko, YA Sokolov, AI Miroshnikov, IA Mikhailopulo. Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleos Chemistry-A European Journal, 21, 13401-13419, 2015).

- phosphorolysis of uridine in the presence of UV. Yield 22-31%. (T. Sakai, T. Tochikura, K. Ogata. Metabolisms of Nucleosides in Bacteria. Part III. Nucleoside-N-ribosyl Group Transfer Reaction in Bacteria. Mechanism of Nucleoside-N-ribosyl Group Transfer Reaction in Corynebacterium sepedonicum. Agr. Biolol Chem., 30 (3), 245-253, 1966; IV Fateev, MI Kharitonova, KV Antonov, ID Konstantinova, VN Stepanenko, RS Esipov, F. Seela, KW Temburnikar, KL Seley-Radtke, VA Stepchenko, YA Sokolov , AI Miroshnikov, IA Mikhailopulo. Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90 (Ala Mutant in the Synthesis of Base-Modified Nucleosides. Chemistry-A European Journal, 21, 13401-13419, 2015).

- phosphorolysis of inosine in the presence of NF, xanthine oxidase and catalase. Yield 12% (N.M. Kalckar. The Enzymatic Synthesis Of Purine Ribosides. J. Biol. Chem. 167, 477-486, 1947).

- phosphorolysis of guanosine in the presence of NF. Yield 11%. (M. Friedkin, N.M. Kalckar Desoxyribose-1-Phosphate: I. The Phosphorolysis and Resynthesis of Purine Desoxyribose Nucleoside. J. Biol. Chem. 184: 437-448, 1950; M. Friedkin. Deoxyribose-1-Phosphate II. The Isolation of Crystalline Desoxyribose-1-Phosphate. J. Biol. Chem. 184, 449-460, 1950)

All these chemical synthesis methods allow to obtain pentose-1-phosphates in the form of a mixture of β- and α-anomers. In the first two cases, this mixture can be separated, but the yields of the target α-anomer suitable for use in the enzymatic glycosylation reaction are about 20%. The third method is limited to the synthesis of analogues of 2-deoxyribose-1-phosphates, but gives sufficiently high yields due to the conversion of one isomer to another during crystallization (the yield of α-D-2-deoxyribofuranose-1-phosphate, based on natural sugar, reaches 66-69 %).

These enzymatic synthesis methods are more environmentally friendly, provide regio- and stereo-selectivity of the synthesis, but the maximum yields of α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate, which so far have been obtained by these methods do not exceed 37%. This is because the equilibrium during enzymatic phosphorolysis is shifted towards nucleoside formation. Increased yields can be achieved using excess starting materials. But the use of large excesses significantly increases the cost of synthesis, complicates the composition of the reaction mixture and significantly complicates the isolation of the target compounds.

Disclosure of the invention.

The essence of the invention is the use of hydroiodic salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine as substrates for the production of α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate, respectively, in the form of sodium salts , barium, magnesium, calcium and dicyclohexylamine during enzymatic phosphorolysis with the participation of purine nucleoside phosphorylase PNP.

The invention solves the problem of producing α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate, based on the available 7-methylguanosine (7-Me-Guo) and 7-methyl-2'-deoxyguanosine (7 -Me-dGuo) respectively. The closest prototypes are the publications cited above (N. Komatsu, N. Awano. First stereoselective synthesis of 2-deoxy-α-D-ribofuranosyl-1-phosphate: novel application of crystallization-induced asymmetric transformation. J. Org. Chem, 67, 5419-5421, 2002; IV Fateev, MI Kharitonova, KV Antonov, ID Konstantinova, VN Stepanenko, RS Esipov, F. Seela, KW Temburnikar, KL Seley-Radtke, VA Stepchenko, YA Sokolov, AI Miroshnikov, IA Mikhailopulo. Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90 (Ala Mutant in the Synthesis of Base-Modified Nucleosides. Chemistry-A European Journal, 21, 13401-13419, 2015).

In a known method (Fateev et al., Chem. Eur. J. 2015, 21, 13401-13419) α-D-ribofuranoso-1-phosphate is obtained by phosphorolysis of uridine within 3 days, and α-D-2-deoxyribofuranoso -1-phosphate by phospholysis of thymidine for 12 hours. The reactions are balanced and carried out at a temperature of 50 ° C, which leads to a decrease in product yield to 31% and 37%, respectively. Replacing uridine or thymidine with 7-methylguanosine or 7-methyl-2'-deoxyguanosine allows almost complete phosphorolysis of these substrates under milder conditions (20 ° C) for 5 hours, which can significantly increase the yield of products (93-94%) .

For the synthesis, 7-Me-Guo and 7-Me-dGuo are used in the form of hydroiodic salts, readily soluble in water and aqueous buffer solutions (JW Jones, RK Robins, Purine nucleosides. III. Methylation studies of certain naturally occurring purine nucleosides. J. Am. Chem. Soc, 1963, 85, 193-201) and 7-Me-dGuo (MS Drenichev, CS Alexeev, NN Kurochkin, and S. Mikhailov, Use of Nucleoside Phosphorylases for the Preparation of Purine and Pyrimidine 2'-Deoxynucleosides , Adv. Synth. Catal. 360, 305-312, 2018) ..

The 7-methyl guanosine derivative is quite stable in aqueous solutions over a wide range of temperatures and pH (t≤ ± 90 ° C; 2≤pN≤6). When heated to 90 ° C in an acidic medium (pH≤2), the TV-glycosidic bond is cleaved, and in the main medium (pH≥7), the imidazole ring opens (M. Lahti, N. Santa, E. Darzynkiewicz, N. Lonneberg pH Independent depurination of 7-alkyl guanosine and their 5'-monophosphates. Acta Chemica Scandinavica, 1990, 44, 636-638). 7-Methyl-2'-deoxyguanosine is significantly less stable in aqueous solutions compared to 7-methylguanosine.

7-Me-Guo and 7-Me-dGuo irreversibly undergo phosphorolysis in the presence of PNP with a yield close to quantitative (Fig. 3) (E. Kulikowska, A. Bzowska, J. Wierzchowski, D. Shugar. Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine. Biochimica et Biophysica Acta, 1986, 874, 355-363). The reaction is carried out at pH values close to optimal for PNP activity, as well as for the stability of the starting and target compounds (to obtain maximum yields, it is recommended to carry out the reaction at pH 7.5). For synthesis, PNP can be used from various bacterial sources. For example, from E. coli and Enterobacter. Both enzymes are effective in the phosphorolysis reaction. However, PNP from Enterobacter is more active, the reaction proceeds faster, which minimizes the fraction of nonspecific hydrolysis products and the products of the opening of the imidazole ring of the starting nucleoside.

The irreversibility of phospholysis of 7-Me-Guo and 7-Me-dGuo allows to obtain α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate with yields close to quantitative according to HPLC and up to 74-96 % after isolation (table 1). Irreversibility also avoids the use of large excess reagents. For the synthesis according to the proposed method, a small excess (1.05-1.1 equiv.) Of the phosphate group donor reagent is used with respect to the initial nucleoside. The use of small excesses reduces the cost of synthesis and greatly simplifies the isolation of the target compounds (according to the proposed method, α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate are isolated by crystallization or by precipitation).

Potassium / sodium dihydroorthophosphates (in the production of barium, magnesium and calcium salts) or dicyclohexylamine hydrophosphate (synthesis in the form of cyclohexylamine and sodium salts) are used as a phosphate group donor in the reaction. Cyclohexylamine salts crystallize from the water - n-butanol system. Sodium salts are prepared by reacting cyclohexylamine salts with an ion exchange resin (Dowex 50 Na ⁺ ). Barium, magnesium, calcium and sodium salts of α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate are precipitated from an aqueous-ethanol solution.

Enzymatic reactions are monitored by TLC and HPLC (Fig. 4). The presence of the target compounds is confirmed by TLC with thermal detection (burnout of spots containing ribofuranose and 2-deoxy ribofuranose by heating TLC plates to 400 ° C). At time to (Fig. 4A), the signal of the starting nucleoside (7-Me-Guo or 7-Me-dGuo) is present on the HPLC. With HPLC on Luna® 5u NH2, 7-methyl-guanosine and 7-methyl-2'-deoxyguanosine give signals with RT of 2.5 and 3.3 min, respectively (acetonitrile / water gradient of 1-8% in 6 min, for more details, see The implementation of the invention). During the reaction, the appearance and increase of the signal of 7-methylguanine is observed (RT 6.2 min, the same conditions).

Thus, our method allows us to obtain α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate in the form of sodium, barium, magnesium, calcium, and cyclohexylamine salts, based on the hydroiodic salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine, respectively. The advantages of this method are: the use of easily accessible salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine, regio- and stereo-specificity of the proceeding reaction, the completeness of the conversion of the starting compounds (7-methylguanosine and 7-methyl-2'-deoxyguanosine undergo phosphorolysis irreversibly) and high yield of target compounds.

Brief description of figures and tables

FIG. 1. Cladribine, fludarabine, nonlarabin and vidarabine are pharmaceutically important nucleosides. The figure shows the structure of drugs of the nucleoside nature of cladribine, fludarabine, nonlarabine and vidarabine, widely used in medical practice;

FIG. 2. Reactions of enzymatic phosphorolysis of nucleosides and transglycosylation. NP ₁ , NP ₂ are nucleoside phosphorylases, B ₁ and B ₂ are heterocyclic bases, R-H or OH. The figure shows the reaction patterns of phosphorolysis of nucleosides and glycosylation of ribose-1-phosphates under the action of nucleoside phosphorylases. As successive steps, these reactions are a transglycosylation process (transfer of a pentafuranose fragment from one heterocyclic base to another);

FIG. 3. Obtaining α-D-ribose-1-phosphate and α-D-2-deoxyribose-1-phosphate by phospholysis of 7-methylguanosine and 7-methyl-2'-deoxyguanosine (in the form of hydroiodides) in the presence of purine nucleoside phosphorylases. Reagents and conditions. PNF E. Coli / Enterobacter, 5 h, 74-96%. The figure illustrates the preparation of α-D-ribose-1-phosphate and α-D-2-deoxyribose-1-phosphate from 7-methyl-guanosine and 7-methyl-2'-deoxyguanosine as a substrate in the presence of nucleoside phosphorylases by the phosphorolysis reaction;

FIG. 4. Enzymatic synthesis of α-D-2-deoxyribose-1-phosphate by phosphorolysis of 7-methyl-2'-deoxyguanosine in the presence of PNP (HPLC analysis of the mixture on a Luna® NH ₂ sorbent). The figure illustrates an example of HPLC analysis of the progress of the 7-methyl-2'-deoxyguanosine phosphorolysis reaction. A - before adding PNP (time t ₀ ), B - 5 hours after the start of the reaction (time t - 5 h). 1 - 7-methyl-2'-deoxyguanosine, 2 - 7-methylguanine.

Tab. 1. The yields of α-D-ribofuranose-1-phosphate and α-D-2-deoxyribofuranose-1-phosphate in the form of salts of barium, magnesium, calcium, sodium and cyclohexylamine, obtained by the reaction of enzymatic phosphorolysis. The table describes the yields of α-D-ribofuranoso-1-phosphate and α-D-2-deoxyribofuranoso-1-phosphate in the form of barium, magnesium, calcium, sodium, and cyclohexylamine salts obtained by the phosphoryolysis of 7-methylguanosine and 7-methyl -2'-deoxyguanosine, respectively.

The implementation of the invention

каталожный номер 00G-4378-EO, производитель Phenomenex (США)), оснащенной защитной предколонкой стандарта ЕС (4,0×3,0 мм, диаметр частиц 5 мкм, сорбент Luna® NH₂ каталожный номер AJ0-4302, производитель Phenomenex (США)) в линейном градиенте ацетонитрила от 1 до 8% за 8 минут в деионизированной воде, скорость потока - 1 мл/мин, УФ-детекцию проводят при длине волны 283 нм. Масс-спектры высокого разрешения (HRMS) регистрируют на приборе Bruker Daltonics micrOTOF-Q II с электрораспылительной ионизацией (ESI). Измерения проводят в режиме отрицательно заряженных ионов. Напряжение на капилляре: 4700 V; диапазон сканирования масс m/z 50-3000; калибровка внешняя (Electrospray Calibrant Solution, Fluka); давление на распылителе: 0.4 бар; скорость потока: 3 мкл/мин; газ-распылитель: азот (4 л/мин); температура интерфейса: 200°С. Образцы подаются в распылительную камеру масс-спектрометра после жидкостного хроматографа Agilent 1260, оснащенного колонкой Agilent Poroshell 120 ЕС-С18 (3,0×50 мм; 2,7 мкм); скорость потока 0,2 мл/мин; в ВЭЖХ-хроматограф образцы вещества подаются из раствора ацетонитрил-вода 1:1 (5 мкл) и элюируются в линейном градиенте концентраций ацетонитрила (50→100%). Ионообменную хроматографию выполняют на смоле Dowex 50W-X8 (100-200 mesh) "Bio Rad". Очистку растворителей проводят по стандартным методикам. Для проведения фосфоролиза используют ферментные препараты пуриннуклеозидфофорилазы E.coli (10,6 ед.акт./мл, 0,1 мг/мл) и пуриннуклеозидфофорилазы Enterobacter (20,8 ед.акт./мл, 3,2 мг/мл).The structure of the claimed compounds is confirmed by NMR and Mass spectroscopy. NMR spectra were recorded on a Bruker AMX 300 instrument (Germany). Chemical shifts (δ) in ¹ H-NMR are given in parts per million (ppm) and measured relative to the residual signal of the solvent: D ₂ O - 4.79 ppm. The values of the constants of spin - spin interaction (J) are measured in hertz (Hz). When describing ¹ H-NMR spectra, the following abbreviations are used: c — singlet, broadened — broadened singlet, d — doublet, b — broadened doublet, t — triplet, m — multiplet. Thin layer chromatography was performed on Sorbfil PTCX-AF-A-UV plates (Sorbpolymer CJSC) with UV detection (λ = 254 nm) and thermal detection (heating TLC plates to 400 ° C). An HPLC analysis of phosphorolysis is carried out on a 4.6 × 250 mm chromatographic column (particle diameter 5 μm, Luna® NH2 sorbent, pore diameter

catalog number 00G-4378-EO, manufacturer Phenomenex (USA), equipped with a protective pre-column of EU standard (4.0 × 3.0 mm, particle diameter 5 μm, sorbent Luna® NH ₂ catalog number AJ0-4302, manufacturer Phenomenex (USA )) in a linear gradient of acetonitrile from 1 to 8% in 8 minutes in deionized water, a flow rate of 1 ml / min, UV detection is carried out at a wavelength of 283 nm. High resolution mass spectra (HRMS) are recorded on a Bruker Daltonics micrOTOF-Q II electrospray ionization (ESI) instrument. The measurements are carried out in the mode of negatively charged ions. Capillary voltage: 4700 V; mass scanning range m / z 50-3000; external calibration (Electrospray Calibrant Solution, Fluka); spray pressure: 0.4 bar; flow rate: 3 μl / min; atomizer gas: nitrogen (4 l / min); interface temperature: 200 ° C. Samples are fed into the spray chamber of the mass spectrometer after an Agilent 1260 liquid chromatograph equipped with an Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm; 2.7 μm); flow rate 0.2 ml / min; samples of the substance are fed to the HPLC chromatograph from a 1: 1 acetonitrile-water solution (5 μl) and eluted in a linear gradient of acetonitrile concentrations (50 → 100%). Ion exchange chromatography is performed on a Dowex 50W-X8 (100-200 mesh) Bio Rad resin. Purification of solvents is carried out according to standard methods. For phosphorolysis, enzyme preparations of E. coli purine nucleoside phosphorylase (10.6 units / ml, 0.1 mg / ml) and Enterobacter purine nucleoside phosphorylases (20.8 units / ml, 3.2 mg / ml) are used.

Example 1. Synthesis of barium salt of α-D-ribofuranoso-1-phosphate.

7-Methylguanosine hydroiodide (200 mg, 0.470 mmol) was dissolved in 90 ml of Tris-HCl buffer solution (50 mM; pH 7.5). To the resulting solution, 9.87 ml of potassium dihydrogen phosphate buffer solution (50 mM, pH 7.5; 0.494 mmol) was added. The reaction is initiated by adding 120 μl of E. coli PNP solution with an activity of 10.6 units / ml (or 40 μl Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 98%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer is separated, aqueous ammonia solution of barium acetate (Ba (OAc) ₂ 126.2 mg, 0.494 mmol; water 2 ml; 25% aqueous ammonia solution 0.9 ml) is added. The resulting solution was left for 20 hours at 4 ° C. The precipitate formed is filtered through Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is evaporated in vacuo to a volume of 20 ml (the bath temperature should not exceed 35 ° C, when precipitating in a concentrated solution, it is necessary to re-filter). Ethyl alcohol was added to the resulting concentrated aqueous solution with stirring until a steady turbidity appeared. Leave at 4 ° C for 20 hours. The precipitate was separated by centrifugation, washed with ethanol (10 ml), diethyl ether (10 ml), dried in a vacuum desiccator over P ₂ O ₅ . Yield 161 mg (94%) as an amorphous powder. R _ƒ 0.16 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR (300 MHz, D ₂ O): 5.68 udd (1H, J _1.2 = 4.9, J _HP = 4.4, H1), 4.28 m (1H, H4), 4, 17 dd (1H, J _2.3 = 3.1, H2), 4.08 dd (1H, J _3.4 = 5.8, H3), 3.75 dd (1H, J _{5a, 4} = 3, 2, J _{5a, 5b} = -11.6, H5a), 3.63 dd (1H, J _{5b, 4} = 4.6, H5b). ¹³ C NMR (300 MHz, D ₂ O): 97.47 (C1), 85.28 (C4), 71.65 (C2), 70.08 (C3), 61.85 (C5). ³¹ P NMR (300 MHz, D ₂ O): 1.85. Mass spectrum: calculated for C ₅ H ₁₀ O ₈ P ^- 229.0119, found 229.0103.

Example 2. Synthesis of barium salt of α-D-2-deoxyribofuranoso-1-phosphate. 7-Methyl-2'-deoxyguanosine hydroiodide (300 mg, 0.733 mmol) was dissolved in 140 ml of Tris-HCl buffer solution (50 mM; pH 7.5). To the resulting solution was added 15.4 ml of potassium dihydrogen phosphate buffer solution (50 mM, pH 7.5; 0.770). The reaction is initiated by adding 150 μl of E. coli PNP solution with an activity of 10.6 units / ml (or 60 μl Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methyl-2'-deoxyguanosine according to HPLC proceeds by 96%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 150 ml). The aqueous layer is separated, aqueous ammonia solution of barium acetate (Ba (OAc) ₂ 173.6 mg, 0.770 mmol; water 3 ml; 25% aqueous ammonia solution 1.2 ml) is added. The resulting solution was left for 20 hours at 4 ° C. The precipitate formed is filtered through Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is evaporated in vacuo to a volume of 30 ml (the temperature of the bath should not exceed 35 ° C, when precipitating in a concentrated solution, it is necessary to re-filter). Ethyl alcohol was added to the resulting concentrated aqueous solution with stirring until a steady turbidity appeared. Leave at 4 ° C for 20 hours. The precipitate was separated by centrifugation, washed with ethanol (10 ml), diethyl ether (10 ml), dried in vacuo to dryness. Dried in a vacuum desiccator over P ₂ About ₅ . Yield 255 mg (93%) as an amorphous powder. R _ƒ 0.24 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR (300 MHz, D ₂ O): 5.84 ddd (1H, J _1.2a = 6.6, J _1.2b = 1.4, J _HP = 6.1, H1), 4.28 m (2H, H3, H4), 3.79 dd (1H, J _{5a, 4} = 3.3, J _{5a, 5b} = -12.2, H5a), 3.65 dd (1H, J _{5b, 4} = 5.5, H5'b), 2.44 ddd (1H, J _{2b, 3} = 1H, J _{2a, 3} = 6.6, J _{2a, 2b} = -14.3, H2a), 2.14 ddd (1H, J _{2b, 3} = 2.5, H2b). ¹³ C NMR (300 MHz, D ₂ O): 102.20 (C1), 88.72 (C4), 73.96 (C3), 64.52 (C5), 44.42 (C2). ³¹ P NMR (300 MHz, D ₂ O): 1.80.

Example 3. The synthesis of magnesium salts of α-D-ribofuranoso-1-phosphate.

7-Methylguanosine hydroiodide (200 mg, 0.470 mmol) was dissolved in 90 ml of Tris-HCl buffer solution (50 mM; pH 7.5). To the resulting solution, 9.87 ml of potassium dihydrogen phosphate buffer solution (50 mM, pH 7.5; 0.494 mmol) was added. The reaction is initiated by adding 120 μl of E. coli PNP solution with an activity of 10.6 units / ml (or 40 μl Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 98%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer was separated, aqueous ammonia solution of magnesium acetate (Mg (OAc) ₂ × 4 H ₂ O 105.9 mg, 0.494 mmol; water 2 ml; 25% aqueous ammonia solution 0.9 ml) was added to it. The resulting solution was left for 20 hours at 4 ° C. The precipitate formed is filtered through Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is evaporated in vacuo to a volume of 20 ml (the bath temperature should not exceed 35 ° C, when precipitating in a concentrated solution, it is necessary to re-filter). Ethyl alcohol was added to the resulting concentrated aqueous solution with stirring until a steady turbidity appeared. Leave at 4 ° C for 20 hours. The precipitate was separated by centrifugation, washed with ethanol (10 ml), diethyl ether (10 ml), dried in a vacuum desiccator over P ₂ O ₅ . Yield 111 mg (93%) as an amorphous powder. R / 0.16 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR, ¹³ C NMR and ³¹ P NMR are similar to the spectra of the barium salt of α-D-ribofuranoso-1-phosphate.

Example 4. Synthesis of magnesium salt of α-D-2-deoxyribofuranoso-1-phosphate.

7-Methyl-2'-deoxyguanosine hydroiodide (300 mg, 0.733 mmol) was dissolved in 140 ml of Tris-HCl buffer solution (50 mM; pH 7.5). To the resulting solution was added 15.4 ml of potassium dihydrogen phosphate buffer solution (50 mM, pH 7.5; 0.770 mmol). The reaction is initiated by adding 150 μl of E. coli PNP solution with an activity of 10.6 units / ml (or 60 μl Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methyl-2'-deoxyguanosine according to HPLC proceeds by 96%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 150 ml). The aqueous layer is separated, aqueous ammonia solution of magnesium acetate (Mg (OAc) ₂ × 4 H ₂ O 165.1 mg, 0.770 mmol; water 3 ml; 25% aqueous ammonia solution 1.2 ml) is added. The resulting solution was left for 20 hours at 4 ° C. The precipitate formed is filtered through Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is evaporated in vacuo to a volume of 30 ml (the temperature of the bath should not exceed 35 ° C, when precipitating in a concentrated solution, it is necessary to re-filter). Ethyl alcohol was added to the resulting concentrated aqueous solution with stirring until a steady turbidity appeared. Leave at 4 ° C for 20 hours. The precipitate was separated by centrifugation, washed with ethanol (10 ml), diethyl ether (10 ml), dried in vacuo to dryness. Dried in a vacuum desiccator over P ₂ About ₅ . Yield 161 mg (93%) as an amorphous powder. R _ƒ 0.24 (isopropyl alcohol-ammonia-water, 70:10:20, v / v). ¹ H NMR, ¹³ C NMR and ³¹ P NMR are similar to the spectra of a-0-2-deoxyribofuranoso-1-phosphate barium salt.

Example 5. The synthesis of calcium salts of α-D-ribofuranoso-1-phosphate.

7-Methylguanosine hydroiodide (200 mg, 0.470 mmol) was dissolved in 90 ml of Tris-HCl buffer solution (50 mM; pH 7.5). To the resulting solution, 9.87 ml of potassium dihydrogen phosphate buffer solution (50 mM, pH 7.5; 0.494 mmol) was added. The reaction is initiated by adding 120 μl of E. coli PNP solution with an activity of 10.6 units / ml (or 40 μl Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 98%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer was separated, aqueous ammonia solution of calcium acetate (Ca (OAc) ₂ (anhydrous) 78.1 mg, 0.494 mmol; water 2 ml; 25% aqueous ammonia solution 0.9 ml) was added to it. The resulting solution was left for 20 hours at 4 ° C. The precipitate formed is filtered through Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is evaporated in vacuo to a volume of 20 ml (the bath temperature should not exceed 35 ° C, when precipitating in a concentrated solution, it is necessary to re-filter). Ethyl alcohol was added to the resulting concentrated aqueous solution with stirring until a steady turbidity appeared. Leave at 4 ° C for 20 hours. The precipitate was separated by centrifugation, washed with ethyl alcohol (10 ml), diethyl ether (10 ml), dried in a vacuum desiccator over P2O5. Yield 119 mg (94%) as an amorphous powder. R _ƒ 0.16 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR, ¹³ C NMR and ³¹ P NMR are similar to the spectra of the barium salt of α-D-ribofuranoso-1-phosphate.

Example 6. Synthesis of calcium salt of α-D-2-deoxyribofuranoso-1-phosphate.

7-Methyl-2'-deoxyguanosine hydroiodide (300 mg, 0.733 mmol) was dissolved in 140 ml of Tris-HCl buffer solution (50 mM; pH 7.5). To the resulting solution was added 15.4 ml of potassium dihydrogen phosphate buffer solution (50 mM, pH 7.5; 0.770 mmol). The reaction is initiated by adding 150 μl of E. coli PNP solution with an activity of 10.6 units / ml (or 60 μl Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methyl-2'-deoxyguanosine according to HPLC proceeds by 97%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 150 ml). The aqueous layer is separated, aqueous ammonia solution of calcium acetate (Ca (OAc) ₂ (anhydrous) 121.8 mg, 0.770 mmol; water 3 ml; 25% aqueous ammonia solution 1.2 ml) is added. The resulting solution was left for 20 hours at 4 ° C. The precipitate formed is filtered through Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is evaporated in vacuo to a volume of 30 ml (the temperature of the bath should not exceed 35 ° C, when precipitating in a concentrated solution, it is necessary to re-filter). Ethyl alcohol was added to the resulting concentrated aqueous solution with stirring until a steady turbidity appeared. Leave at 4 ° C for 20 hours. The precipitate was separated by centrifugation, washed with ethanol (10 ml), diethyl ether (10 ml), dried in vacuo to dryness. Dried in a vacuum desiccator over P ₂ About ₅ . Yield 169 mg (91%) as an amorphous powder. R _ƒ 0.24 (isopropyl alcohol-ammonia-water, 70:10:20, v / v). ¹ H NMR, ¹³ C NMR and ³¹ P NMR are similar to the spectra of the barium salt of α-D-2-deoxyribofuranoso-1-phosphate.

Example 7. Synthesis of cyclohexylamine salt of α-D-ribofuranoso-1-phosphate.

7-Methylguanosine hydroiodide (300 mg, 0.706 mmol) and dicyclohexylamine hydrophosphate (230 mg, 0.777 mmol) are dissolved in 100 ml of H ₂ O. The reaction is initiated by adding 120 μl of a E. coli PNP solution with an activity of 10.6 units / ml (or 40 μl of Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 97%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer is separated, evaporated in vacuo to a volume of 2 ml (bath temperature should not exceed 35 ° C, if precipitation occurs in a concentrated solution, it is necessary to re-filter). To the resulting concentrated aqueous solution was added n-butyl alcohol (30 ml), shaken vigorously until a single-phase system formed. Leave at 4 ° C for 60 hours. A crystalline precipitate formed. The precipitate is filtered off, washed with ethyl alcohol (10 ml), diethyl ether (10 ml), dried in vacuo to dryness. Dried in a vacuum desiccator over P ₂ About ₅ . Yield 248 mg (87%) as a crystalline precipitate. R _ƒ 0.21 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR (300 MHz, D ₂ O): 5.64 dd (1H, J _1.2 = 3.9, J _HP = 6.2, H1), 4.24 m (1H, J _4.3 = 4.1, H4), 4.12 dd (1H, J _2.3 = 6.1, H2), 4.07 dd (1H, H3), 3.81 dd (1H, J _{5a, 4} = 3, 3, J _{5a, 5b} = -12.4, H5a), 3.68 dd (1H, J _{5b, 4} = 4.7, H5'b), 3.18 m (2H, H cyclohexylamine residues), 2, 02 m (4H, H cyclohexylamine residues), 1.83 m (4H, H cyclohexylamine residues), 1.68 (2H, H cyclohexylamine residues), 1.37 m (10H, H cyclohexylamine residues). ¹³ C NMR (300 MHz, D ₂ O): 100.10 (C1), 86.90 (C4), 74.36 (C2), 72.62 (C3), 64.27 (C5), 53.10 (C cyclohexylamine residues), 33.09 (C cyclohexylamine residues), 27.04 (C cyclohexylamine residues), 26.54 (C cyclohexylamine residues). ³¹ P NMR (300 MHz, D ₂ O): 1.92. The ¹ H- ³¹ P signals are correlated using NMVS experiments (heteronuclear spin correlation with distant atoms). Signals are observed indicating a correlation of the P atom with the protons H1 (high intensity) and H2 (lower intensity).

Example 8. Synthesis of cyclohexylamine salt of α-D-2-deoxyribofuranoso-1-phosphate.

7-Methyl-2'-deoxyguanosine hydroiodide (300 mg, 0.733 mmol) and dicyclohexylamine hydrophosphate (239 mg, 0.806 mmol) are dissolved in 100 ml of H ₂ O. The reaction is initiated by adding 120 μl of an E. coli PNF solution with an activity of 10.6 units .act. / ml (or 40 μl of Enterobacter PNP solution with an activity of 20.8 u.act./ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 98%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer is separated, evaporated in vacuo to a volume of 2 ml (bath temperature should not exceed 35 ° ↓ С, if precipitation occurs in a concentrated solution, it is necessary to re-filter). To the resulting concentrated aqueous solution was added n-butyl alcohol (30 ml), shaken vigorously until a single-phase system formed. Leave at 4 ° C for 60 hours. Precipitation falls. The precipitate is filtered off, washed with ethyl alcohol (10 ml), diethyl ether (10 ml), dried in vacuo to dryness. Dry in a vacuum desiccator over P ₂ O ₅ . Yield 224 mg (74%) as a powder. R _ƒ 0.26 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR (300 MHz, D ₂ O): 5.84 ddd (1H, J _1.2a = 6.4, J _1.2b = 1.3, J _HP = 5.9, H1), 4.29 m (2H, H3, H4), 3.79 dd (1H, J _{5a, 4} = 3.4, J _{5a, 5b} = -12.2, H5a), 3.53 dd (1H, J _{5b, 4} = 5.2, H5'b), 3.23 m (2H, H cyclohexylamine residues), 2.44 ddd (1H, J _{2a, 3} = 6.4, J _{2a, 2b} = -14.1, H2a), 2.15 ddd (1H, J _{2b, 3} = 2.6, H2b), 2.05 m (4H, H cyclohexylamine residues), 1.88 m (4H, H cyclohexylamine residues), 1.72 m (2H, H cyclohexylamine residues), 1.59 m (2H, H cyclohexylamine residues), 1.38 m (10H, H cyclohexylamine residues). ¹³ C NMR (300 MHz, D ₂ O): 99.63 (C1), 86.07 (C4), 71.21 (C3), 61.69 (C5), 50.39 (C cyclohexylamine residues), 41 72 (C2), 30.39 (C cyclohexylamine residues), 24.33 (C cyclohexylamine residues), 23.83 (C cyclohexylamine residues). ³¹ P NMR (300 MHz, D ₂ O): 1.67.

Example 9. The synthesis of the sodium salt of α-D-ribofuranoso-1-phosphate.

7-Methylguanosine hydroiodide (300 mg, 0.706 mmol) and dicyclohexylamine hydrophosphate (251 mg, 0.847 mmol) were dissolved in 100 ml of H ₂ O. The reaction was initiated by adding 120 μl of an E. coli PNP solution with an activity of 10.6 units / ml (or 40 μl of Enterobacter PNP solution with an activity of 20.8 units / ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 97%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer is separated, evaporated in vacuo to a volume of 2 ml (bath temperature should not exceed 35 ° C, if precipitation occurs in a concentrated solution, it is necessary to re-filter). To the resulting concentrated aqueous solution was added n-butyl alcohol (30 ml), shaken vigorously until a single-phase system formed. Leave at 4 ° C for 60 hours. The precipitate formed is filtered off, washed with ethyl alcohol (10 ml), diethyl ether (10 ml), and dried in vacuo to dryness. The obtained cyclohexylamine salt is dissolved in 10 ml of H ₂ O, 1 ml of Dowex-50 (Na + form) is added to the solution, stirred for 30 min at 20 ° C. The resin is filtered off, washed with water (3 ml). The filtrate was evaporated to dryness in vacuo. Dry in a desiccator over P ₂ O ₅ . Yield 186 mg (96%) as a foam. R _ƒ 0.17 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR (300 MHz, D ₂ O): 5.65 dd (1H, J _1.2 = 3.9, J _HP = 6.4, H1), 4.24 ddd (1H, J _4.5a = 3.3, J _4.5b = 4.7, J _4.3 = 4.1, H4), 4.12 ddd (1H, J _2.3 = 6.2, J _{2, P} = 0.9, H2), 4.07 dd (1H, H3), 3.80 dd (1H, J _{5a, 5b} = -12.4, H5a), 3.67 dd (1H, H5'b). ¹³ C NMR (300 MHz, D ₂ O): 102.31 (C1), 88.14 (C4), 72.64 (C2), 70.26 (C3), 63.33 (C5). ³¹ P NMR (300 MHz, D ₂ O): 1.93.

Example 10. The synthesis of the sodium salt of α-D-2-deoxyribofuranose-1-phosphate.

7-Methyl-2'-deoxyguanosine hydroiodide (300 mg, 0.733 mmol) and dicyclohexylamine hydrophosphate (260 mg, 0.880 mmol) are dissolved in 100 ml of H ₂ O. The reaction is initiated by adding 120 μl of a E. coli PNP solution with an activity of 10.6 units .act. / ml (or 40 μl of Enterobacter PNP solution with an activity of 20.8 u.act./ml), stirred, left at 20 ° C for 5 hours. The conversion of the starting 7-methylguanosine according to HPLC proceeds by 98%. The reaction mixture was cooled in an ice bath, kept at 0 ° C for 30 minutes, then the precipitate of 7-methylguanine was filtered through a Whatman nitrocellulose filter (0.2 μ, 25 mm). The clarified supernatant is extracted with an equal volume of n-butyl alcohol (2 × 100 ml). The aqueous layer is separated, evaporated in vacuo to a volume of 2 ml (bath temperature should not exceed 35 ° C, if precipitation occurs in a concentrated solution, it is necessary to re-filter). To the resulting concentrated aqueous solution was added n-butyl alcohol (30 ml), shaken vigorously until a single-phase system formed. Leave at 4 ° C for 60 hours. Precipitation falls. The precipitate is filtered off, washed with ethyl alcohol (10 ml), diethyl ether (10 ml), dried in vacuo to dryness. The obtained cyclohexylamine salt is dissolved in 10 ml of H ₂ O, 1 ml of Dowex-50 (Na + form) is added to the solution, and stirred for 30 min at 20 ° C. The resin is filtered off, washed with water (3 ml). The filtrate was evaporated to dryness in vacuo. Dry in a desiccator over P ₂ O ₅ . Yield 182 mg (96%) as a foam. R _ƒ 0.24 (isopropyl alcohol - ammonia - water, 70:10:20, v / v). ¹ H NMR (300 MHz, D ₂ O): 5.79 ddd (1H, J _1.2a = 5.2, J _1.2b = 1.4, J _HP = 6.0, H1), 4.25 m (2H, H3, H4), 3.75 dd (1H, J _{5a, 4} = 3.5, J _{5a, 5b} = -12.2, H5a), 3.65 dd (1H, J _{5b, 4} = 5.1, H5'b), 2.39 DDD (1H, J _{2a, 3} = 7.3, J _{2a, 2b} = -14.4, H2a), 2.11 ddd (1H, J _{2b, 3} = 2.5, H2b). ¹³ C NMR (300 MHz, D ₂ O): 98.77 (C1), 87.10 (C4), 70.98 (C3), 63.06 (C5), 40.78 (C2). ³¹ P NMR (300 MHz, D ₂ O): 1.71.

Claims (11)

A method of obtaining a salt of pentose-1-phosphate structure

selected from barium, magnesium, calcium, sodium or dicyclohexylamine salt of α-D-ribofuranoso-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate during enzymatic phosphorolysis:

where X = I; R = H, OH; Pi - inorganic phosphate or dicyclohexylamine hydrophosphate

PNP - purinucleoside phosphorylase from Enterobacter or E. coli; Cation ²⁺ = Ba ²⁺ , Mg ²⁺ , Ca ²⁺ , 2Na ⁺ , dicyclohexylamine

including: - phosphorolysis of the hydroiodic salt of 7-methylguanosine or the hydroiodic salt of 7-methyl-2'-deoxyguanosine in the presence of Pi selected from inorganic phosphate or dicyclohexylamine hydrophosphate

and PNP from Enterobacter or E. coli to produce α-D-ribofuranoso-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate and - the interaction of the obtained phosphate with an aqueous ammonia solution of Ba ²⁺ acetate, Mg ²⁺ or Ca ²⁺ to obtain the corresponding salt, moreover - to obtain the dicyclohexylamine salt of α-D-ribofuranoso-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate, enzymatic phosphorolysis of the hydroiodic salts of 7-methylguanosine or 7-methyl-2'-deoxyguanosine in the presence of dicyclohexylamine hydrophosphate

and PNP from Enterobacter or E. coli, - to obtain the sodium salt of α-D-ribofuranoso-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate, the dicyclohexylamine salt of α-D-ribofuranoso-1-phosphate or α-D-2-deoxyribofuranoso-1-phosphate is dissolved Dowex-50 ion exchange resin in Na ⁺ form is added to the water and to the solution.

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