Methods and compositions for the enzyme-catalyzed synthesis of nucleosides containing protecting groups

Abstract

The present invention provides a method and composition for the enzyme-catalyzed synthesis of nucleosides containing protecting groups. The method includes using pyrimidine nucleoside phosphorylase or uridine phosphorylase and purine nucleoside phosphorylase to catalyze the synthesis of nucleosides containing protecting groups from a substrate, the substrate including a substrate nucleoside, a substrate base, and a substrate phosphate, the substrate base containing a protecting group, the pyrimidine nucleoside phosphorylase including proteins having 80% or more identity to proteins P and NP shown in SEQ ID NO: 1, the uridine phosphorylase including proteins having 80% or more identity to protein UP shown in SEQ ID NO: 2, and the purine nucleoside phosphorylase including proteins having 80% or more identity to protein PNP shown in SEQ ID NO: 3, 7, or 8. This method solves the problem in the prior art that there is no biosynthetic method for producing nucleosides containing protecting groups, and is suitable for the field of enzyme catalysis.

Description

[Technical Field]

[0001] The present invention relates to the field of enzyme catalysis, and in particular to methods and compositions for the enzyme-catalyzed synthesis of nucleosides containing protecting groups.

[0002] This application is based on and claims priority from a Chinese application bearing application number 202211296608.4 and filed on October 21, 2022, the disclosure of which is hereby incorporated by reference in its entirety. [Background technology]

[0003] Nucleic acid-based drugs, such as antisense oligonucleotides and DNA aptamers, can be used to treat eye diseases and other disorders, making them a hot topic in drug development in recent years. Along with this drug development and research, the demand for precursor nucleoside monomers for synthesis in the pharmaceutical market is also increasing. Currently, nucleoside monomers are primarily produced by chemical synthesis. Because nucleosides contain multiple functional groups, such as amino and hydroxyl groups, efficient DNA synthesis often requires selective protection of specific functional groups on the bases, such as the 5' position of 2'-deoxyribose. Furthermore, to obtain a higher-purity product, these protecting groups must be easily removed under relatively mild conditions.

[0004] Commonly used protecting groups include benzoyl, isobutyryl, and acetyl groups. For example, adenine can be protected with a benzoyl group, guanine with an isobutyryl group, and cytosine with a benzoyl or acetyl group. There are few reports on the synthesis of base-protected nucleosides, and they are mainly synthesized by chemical methods. Patent WO 0075154 A2 discloses a chemical method for preparing base-protected nucleosides, including N6-benzoyl-2'-deoxyadenosine, N2-isobutyryl-2'-deoxyguanosine, and N4-benzoyl-2'-deoxycytidine. Patent US 2003162957 A1 discloses a chemical method for preparing N2-isobutyryl-2'-deoxyguanosine. Currently, there are no reports on the biosynthetic production of nucleosides containing protecting groups. Summary of the Invention [Problem to be solved by the invention]

[0005] The primary object of the present invention is to provide a method and composition for the enzyme-catalyzed synthesis of nucleosides containing protecting groups, to overcome the problem in the prior art of the lack of a biosynthetic method for producing nucleosides with protecting groups. [Means for solving the problem]

[0006] In order to achieve the above object, according to a first aspect of the present invention, 1. A method for the enzyme-catalyzed synthesis of nucleosides containing protecting groups, comprising: The method includes the step of synthesizing a nucleoside containing a protecting group by catalyzing a substrate using pyrimidine nucleoside phosphorylase or uridine phosphorylase and a purine nucleoside phosphorylase, the substrate includes a substrate nucleoside, a substrate base, and a substrate phosphate, the substrate base including a protecting group; Pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to PyNP and having the same function, and PyNP is a protein represented by SEQ ID NO: 1; Uridine phosphorylase includes UP or a protein having 80% or more homology to UP and having the same function, UP being the protein represented by SEQ ID NO: 2; Purine nucleoside phosphorylase includes PNP or a protein having 80% or more homology to PNP and having the same function, and PNP includes the protein shown in SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:8.

[0007] Furthermore, the protecting group is located on the base structure of the nucleoside, and preferably the method comprises the steps of generating a pentose phosphate and a free base from a substrate nucleoside and a substrate phosphate under the catalysis of a pyrimidine nucleoside phosphorylase or a uridine phosphorylase, and substituting the phosphate group in the pentose phosphate with the substrate base bearing the protecting group under the catalysis of a purine nucleoside phosphorylase to obtain a nucleoside containing the protecting group.

[0008] Furthermore, the substrate nucleoside is a nucleoside represented by Formula I, and preferably, the substrate nucleoside comprises thymidine, 2'-deoxyuridine, uridine, or urisyl arabinoside; and the substrate base is a base represented by Formula II, Formula III, or Formula IV, and X, Y, and Z are each independently: [ka] [ka] or [ka] is selected from. [ka] wherein R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.

[0009] Preferably, the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine.

[0010] Furthermore, one or more of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, and uridine phosphorylase may be a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0011] Furthermore, the catalytic time of the enzyme catalyst is 4 to 20 hours, the catalytic temperature of the enzyme catalyst is preferably 50 to 70°C, more preferably 60°C, and the concentration of the substrate nucleoside is preferably 20 to 400 mM, the concentration of the substrate base is preferably 10 to 200 mM, and the concentration of the substrate phosphate is preferably 1 to 100 mM.

[0012] Additionally, nucleosides containing protecting groups include N6-benzoyladenosine, N2-isobutyrylguanosine, N4-acetylcytidine, N6-benzoyl-2'-deoxyadenosine, N2-isobutyryl-2'-deoxyguanosine, N4-acetyl-2'-deoxycytidine, N6-benzoylarabinosyladenosine, or N2-isobutyrylarabinosylguanosine.

[0013] In order to achieve the above object, according to a second aspect of the present invention, there is provided a composition comprising any of the enzymes pyrimidine nucleoside phosphorylase or uridine phosphorylase, and purine nucleoside phosphorylase, wherein the pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to PyNP and having the same function, PyNP is the protein represented by SEQ ID NO: 1, the uridine phosphorylase includes UP or a protein having 80% or more homology to UP and having the same function, UP is the protein represented by SEQ ID NO: 2, and the purine nucleoside phosphorylase is PNP or a protein having 80% or more homology to PNP and having the same function, PNP is the protein represented by SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 8.

[0014] Furthermore, one or more of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, and uridine phosphorylase may be a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0015] Furthermore, the composition further comprises a substrate nucleoside and a substrate base, wherein the substrate comprises a substrate nucleoside and a substrate base, and the substrate base comprises a protecting group; the substrate nucleoside is a nucleoside represented by Formula I, preferably the substrate nucleoside comprises thymidine, 2'-deoxyuridine, uridine, or uricil arabinoside; preferably the substrate base is a base represented by Formula II, Formula III, or Formula IV; and X, Y, and Z are each independently: [ka] [ka] or [ka] Preferably, the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine, and preferably, the concentration of the substrate nucleoside is 20 to 400 mM, the concentration of the substrate base is 10 to 200 mM, and the concentration of the substrate phosphate is 1 to 100 mM. [ka] wherein R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3. [Effects of the Invention]

[0016] According to the technical solution of the present invention, a nucleoside containing a protecting group can be synthesized by a biosynthetic method using a purine nucleoside phosphorylase and a pyrimidine nucleoside phosphorylase or a uridine phosphorylase, with a substrate nucleoside and a substrate base containing a protecting group as substrates for enzyme catalysis. [Brief explanation of the drawings]

[0017] The drawings that form a part of this application are intended to provide a further understanding of the invention, and the illustrative embodiments of the invention and their descriptions are intended to illustrate the invention and are not intended to unduly limit the invention. [Figure 1] 1 shows an HPLC chromatogram of N6-benzoyladenosine synthesized using uridine as a substrate according to Example 2 of the present invention. [Figure 2] 1 shows an HPLC chromatogram of N2-isobutyrylguanosine synthesized using uridine as a substrate according to Example 3 of the present invention. [Figure 3] 1 shows an HPLC chromatogram of N4-acetylcytidine synthesized using uridine as a substrate according to Example 4 of the present invention. [Figure 4] 1 shows an HPLC chromatogram of N6-benzoyl-2'-deoxyadenosine synthesized using 2'-deoxyuridine as a substrate according to Example 5 of the present invention. [Figure 5]1 shows an HPLC chromatogram of N2-isobutyryl-2'-deoxyguanosine synthesized using 2'-deoxyuridine as a substrate according to Example 6 of the present invention. [Figure 6] 1 shows an HPLC chromatogram of N4-acetyl-2'-deoxycytidine synthesized using 2'-deoxyuridine as a substrate according to Example 7 of the present invention. [Figure 7] 1 shows an HPLC chromatogram of N6-benzoyl-2′-deoxyadenosine synthesized using thymidine as a substrate according to Example 8 of the present invention. [Figure 8] 1 shows an HPLC chromatogram of N2-isobutyryl-2'-deoxyguanosine synthesized using thymidine as a substrate according to Example 9 of the present invention. [Figure 9] 1 shows an HPLC chromatogram of N6-benzoyl-2′-deoxyadenosine synthesized using thymidine as a substrate according to Example 10 of the present invention. [Figure 10] 1 shows an HPLC chromatogram of N4-acetyl-2′-deoxycytidine synthesized using thymidine as a substrate in Example 11 of the present invention. [Figure 11] 1 shows an HPLC chromatogram of N6-benzoylarabinosyladenosine synthesized using uricil arabinoside as a substrate according to Example 12 of the present invention. [Figure 12] 1 shows an HPLC chromatogram of N2-isobutyrylarabinosylguanosine synthesized using urisylarabinoside as a substrate according to Example 13 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] The embodiments and features of the present invention may be combined with each other as long as they are not inconsistent. The present invention will be described in detail below in combination with the embodiments with reference to the drawings.

[0019] As mentioned in the background art, the production of nucleosides containing protecting groups (nucleosides with protected bases) in the prior art is basically carried out by chemical synthesis, which often involves problems such as harsh reaction conditions and the production of many by-products and contaminants. Therefore, in this application, the inventors have attempted to develop an enzyme-catalyzed synthesis method and composition for nucleosides containing protecting groups, and to produce nucleosides containing protecting groups using biosynthesis. Therefore, a series of protection measures have been proposed in this application.

[0020] In a first exemplary embodiment of the present application, there is provided a method for enzyme-catalyzed synthesis of a nucleoside containing a protecting group, the method utilizing a purine nucleoside phosphorylase and any of the following enzymes: pyrimidine nucleoside phosphorylase or uridine phosphorylase to catalyze the synthesis of a nucleoside containing a protecting group from a substrate, the substrate including a substrate nucleoside, a substrate base, and a substrate phosphate, wherein the substrate base includes a protecting group, the pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology to PyNP and having the same function, and PyNP is selected from the group consisting of PyNP, ... Uridine phosphorylase includes UP or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology to UP and having the same function, and UP is the protein represented by SEQ ID NO: 2. Purine nucleoside phosphorylase includes PNP or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology to PNP and having the same function, and PNP includes the protein represented by SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 8.

[0021] The PNPs include the protein derived from Geobacillus thermoglucosidasius and represented by SEQ ID NO: 3, the protein derived from Thermus thermophilus and represented by SEQ ID NO: 7, and the protein derived from Deinococcus geothermalis and represented by SEQ ID NO: 8. All three of the purine nucleoside phosphorylases can catalyze the substitution of a phosphate group on a pentose phosphate with a substrate base bearing a protecting group to yield a nucleoside containing a protecting group.

[0022] The pyrimidine nucleoside phosphorylase used in this application is derived from Thermus thermophilus, is a protein represented by SEQ ID NO: 1, and is designated as PyNP, and the uridine phosphorylase is derived from Trypanosoma cruzi, is a protein represented by SEQ ID NO: 2, and is designated as UP. In the above method, a nucleoside containing a protecting group can be produced using a substrate nucleoside and a substrate base containing a protecting group as starting materials, using a purine nucleoside phosphorylase and a pyrimidine nucleoside phosphorylase or a uridine phosphorylase.

[0023] The nucleoside containing the protecting group is a nucleoside containing a protecting group on the base structure, and the protecting group is derived from a protecting group on the substrate base. The protecting group may be a protecting group commonly used in the prior art to protect a base. The method is a one-step method, allowing for one-step production in a single vessel, facilitating industrial scale-up production and reducing production costs. Furthermore, since the method is an enzyme-catalyzed method, the reaction conditions are milder and more controllable than chemical synthesis methods, there is less generation of other impurities, fewer by-products are produced, purification is easier, and the production process is economical and environmentally friendly.

[0024] The reaction scheme of the above method is as follows, and the reaction is carried out using thymidine as an example of a substrate nucleoside. [ka]

[0025] First, under the catalysis of the NP1 enzyme (i.e., pyrimidine nucleoside phosphorylase or uridine phosphorylase), the phosphate group (Pi) of the substrate thymidine replaces the base moiety of the substrate nucleoside and links to the pentose to form pentose phosphate and thymine. Next, under the catalysis of the NP2 enzyme (i.e., purine nucleoside phosphorylase), the phosphate group on the pentose phosphate is replaced with the substrate base to yield a nucleoside containing a protecting group.

[0026] As used herein, the abbreviations for amino acid residues are as follows: alanine (Ala, A), asparagine (Asn, N), aspartic acid (Asp, D), arginine (Arg, R), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (Leu, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V).

[0027] The rule for substitutions and replacements is that, in general, amino acids with similar properties will have similar effects when replaced. For example, conservative amino acid substitutions may occur in the above-mentioned homologous proteins. "Conservative amino acid substitutions" include, but are not limited to, the following: Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Vai, Lie, Leu) are substituted with other hydrophobic amino acids. Hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced with other hydrophobic amino acids with bulky side chains. Amino acids with positively charged side chains (Arg, His, Lys) are substituted with other amino acids with positively charged side chains. Amino acids with polar but uncharged side chains (SenThr, Asn, Gin) are substituted with other amino acids with polar but uncharged side chains.

[0028] Those skilled in the art can also make conservative amino acid substitutions according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0029] In one preferred embodiment, the protecting group is located on the base structure of the nucleoside. Preferably, when two enzymes, pyrimidine nucleoside phosphorylase (PyNP) or uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP), are used in combination, the method includes the steps of generating a pentose phosphate and a free base from a substrate nucleoside and a substrate phosphate under the catalysis of the pyrimidine nucleoside phosphorylase or uridine phosphorylase, and then substituting the phosphate group in the pentose phosphate with a substrate base having a protecting group under the catalysis of the purine nucleoside phosphorylase to obtain a nucleoside containing a protecting group. The substrate phosphate is not limited to a specific type and includes, but is not limited to, phosphates commonly used in the prior art, including one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate, or mixtures commonly used in the prior art, such as commercially available phosphate buffer (PB) or phosphate buffer solution (PBS). Here, the phosphate buffer includes a buffer containing sodium dihydrogen phosphate and disodium hydrogen phosphate at a predetermined concentration, and the phosphate buffer includes a buffer containing salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride.

[0030] In one preferred embodiment, the substrate nucleoside is a nucleoside of Formula I, preferably including, but not limited to, thymidine, 2'-deoxyuridine, uridine, or urisyl arabinoside. [ka] wherein R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.

[0031] Formula I represents compounds having chiral moieties, including purified chiral compounds, and mixtures containing different chiral structures (including, but not limited to, racemates). When R1 is -OH or -F, R1 is a chiral substituent. In the Haworth projection shown in Formula I, when R1 at the C2 position faces the same direction as the -OH at the adjacent C3 position (the substituent faces down), the pentose of the substrate nucleoside has a ribose configuration, i.e., the corresponding substituent is Ribo-OH or Ribo-F. When R1 at the C2 position faces the opposite direction to the -OH at the adjacent C3 position (the substituent faces up), the pentose of the substrate nucleoside has an arabinose configuration, i.e., the corresponding substituent is arabino-OH or arabino-F. The above chiral substrate nucleosides can be converted to nucleosides containing protecting groups by the above method without changing the configuration of the pentose structure. Also, when the substrate nucleoside is a mixture of different configurations, any of the substrates of different configurations can be converted to give the corresponding nucleoside containing a protecting group.

[0032] In one preferred embodiment, the substrate base is a base of Formula II, Formula III, or Formula IV, and X, Y, and Z are each independently: [ka] [ka] or [ka] The above substituents are protecting groups, and any of them can be attached to the base structure of Formula II, Formula III, or Formula IV to protect the binding site. [ka]

[0033] Preferably, the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine.

[0034] Using the above substrate nucleosides and substrate bases, the above method allows for flexible combination of the above substrate bases and substrate nucleosides to produce nucleosides containing protecting groups, including, but not limited to, N6-benzoyladenosine, N2-isobutyrylguanosine, N4-acetylcytidine, N6-benzoyl 2-deoxyadenosine, N2-isobutyryl 2-deoxyguanosine, N4-acetyl 2-deoxycytidine, N6-benzoylarabinosyladenosine, or N2-isobutyrylarabinosylguanosine, etc. Any protecting groups are located on the base structure of the nucleoside, not on the pentose structure.

[0035] In one preferred embodiment, one or more of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0036] In catalytic reactions, the above three types of enzymes exist in various forms, such as purified proteins, crude enzyme solutions, or immobilized enzymes, and all of them can catalyze the synthesis of nucleosides having protecting groups. Genes expressing PyNP and / or UP and / or PNP are cloned into host cells, protein expression is induced, and the host cells are then disrupted to obtain a crude enzyme solution containing the target protein. Crude enzyme solutions are easy to prepare, have high catalytic activity, and can reduce production costs for catalytic reactions.

[0037] In one preferred embodiment, the catalytic time of the enzyme catalyst is 4 to 20 hours, and the catalytic temperature of the enzyme catalyst is preferably 50 to 70°C, more preferably 60°C.

[0038] In one preferred embodiment, the concentration of the substrate nucleoside is 20 to 400 mM, and the concentration of the substrate base is 10 to 200 mM.

[0039] The above method is amenable to scale-up reactions, and in the reaction system, the concentration of the substrate nucleoside includes, but is not limited to, 20, 30, 50, 100, 200, 300, or 400 mM, with the maximum concentration being 400 mM. The concentration of the substrate base includes, but is not limited to, 10, 20, 30, 50, 100, 150, or 200 mM, with the maximum concentration being 200 mM, thereby enabling large-scale production of nucleosides containing protecting groups.

[0040] At the appropriate catalytic temperature and time, this enzyme-catalyzed reaction can be completed, and the conversion rate of the substrate base, i.e., the reaction yield, is relatively high. Because the reaction can be completed using only one-step catalysis without the need to add enzymes or other reagents during the reaction, it is suitable for industrial scale-up production. The reaction conditions are mild and easy to control, reducing production equipment costs, energy costs, and risks.

[0041] In one preferred embodiment, the nucleoside containing a protecting group comprises N6-benzoyladenosine, N2-isobutyrylguanosine, N4-acetylcytidine, N6-benzoyl-2'-deoxyadenosine, N2-isobutyryl-2'-deoxyguanosine, N4-acetyl-2'-deoxycytidine, N6-benzoylarabinosyladenosine, or N2-isobutyrylarabinosylguanosine.

[0042] In a second exemplary embodiment of the present application, the present invention includes any of the following enzymes: pyrimidine nucleoside phosphorylase or uridine phosphorylase, and a purine nucleoside phosphorylase, wherein the pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology to PyNP and having the same function, and PyNP is the protein represented by SEQ ID NO: 1; and the uridine phosphorylase includes UP or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology to UP and having the same function, and UP is the protein represented by SEQ ID NO: The present invention provides a composition in which the purine nucleoside phosphorylase is a protein represented by SEQ ID NO:2, and the purine nucleoside phosphorylase includes PNP or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology to PNP and having the same function, and the PNP includes a protein represented by SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:8.

[0043] In one preferred embodiment, one or more of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0044] In one preferred embodiment, the composition further comprises a substrate nucleoside and a substrate base; the substrate includes a substrate nucleoside and a substrate base, the substrate base including a protecting group; The substrate nucleoside is a nucleoside of Formula I. [ka] wherein R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.

[0045] Formula I represents compounds having chiral moieties, including purified chiral compounds, and mixtures containing different chiral structures (including, but not limited to, racemates). When R1 is -OH or -F, R1 is a chiral substituent. In the Haworth projection shown in Formula I, when R1 at the C2 position faces the same direction as the -OH at the adjacent C3 position (the substituent faces down), the pentose of the substrate nucleoside has a ribose configuration, i.e., the corresponding substituent is Ribo-OH or Ribo-F. When R1 at the C2 position faces the opposite direction from the -OH at the adjacent C3 position (the substituent faces up), the pentose of the substrate nucleoside has an arabinose configuration, i.e., the corresponding substituent is arabino-OH or arabino-F. The above compositions include pure chiral compounds and mixtures of compounds with different chiralities (including, but not limited to, racemates). Preferably, the substrate nucleoside comprises thymidine, 2'-deoxyuridine, uridine, or urisyl arabinoside.

[0046] Preferably, the substrate base is a base of formula II, formula III, or formula IV, and X, Y, and Z are each independently: [ka] [ka] or [ka] is selected from. [ka]

[0047] The above substituents are protecting groups and can all be attached to the base structure of Formula II, Formula III, or Formula IV to protect the binding site.

[0048] Preferably, the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine.

[0049] Preferably, the concentration of the substrate nucleoside is 20 to 400 mM, and the concentration of the substrate base is 10 to 200 mM.

[0050] The concentration of the substrate nucleoside may be, but is not limited to, 20, 30, 50, 100, 200, 300, or 400 mM, with a maximum concentration of 400 mM. The concentration of the substrate base may be, but is not limited to, 10, 20, 30, 50, 100, 150, or 200 mM, with a maximum concentration of 200 mM.

[0051] The pyrimidine nucleoside phosphorylase PyNP is derived from Thermus thermophilus and is a protein designated SEQ ID NO: 1. The uracil pyrimidine nucleoside phosphorylase UP is derived from Trypanosoma cruzi and is a protein designated SEQ ID NO: 2. The purine nucleoside phosphorylase PNP includes a protein designated SEQ ID NO: 3 derived from Geobacillus thermoglucosidasius, a protein designated SEQ ID NO: 7 derived from Thermus thermophilus, or a protein designated SEQ ID NO: 8 derived from Deinococcus geothermalis. The composition can catalyze the reaction of a substrate base and a substrate nucleoside to produce a nucleoside containing a protecting group. The enzymes in the composition may each be independently selected from forms such as purified proteins, crude enzyme solutions, or immobilized enzymes, any of which can perform catalytic activity.

[0052] The beneficial effects of the present invention will be further explained below with reference to specific examples.

[0053] Example 1 1. Strain Construction The pyrimidine nucleoside phosphorylase PyNP used was derived from Thermus thermophilus, the uridine phosphorylase UP was derived from Trypanosoma cruzi, and the purine nucleoside phosphorylase PNP was derived from Geobacillus thermoglucosidasius. The protein sequences of the three nucleoside phosphorylases obtained from NCBI are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 (NCBI protein sequence number: EFG53380.1), respectively. The DNA sequences encoding the three enzymes obtained by codon optimization are SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. These were cloned into the expression vector pET28a(+). The resulting plasmid was transformed into E. coli BL21(DE3) host competent, and a single clonal strain was obtained. [ka] [ka] [ka] [ka] [ka] [ka]

[0054] 2. Protein Expression E. coli strains expressing PyNP, UP, and PNP were inoculated into test tubes and cultured at 37°C for 16 hours. After that, they were inoculated into 2 L shake flasks containing 500 mL of Luria-Bertani (LB) medium at a 1% inoculum size and incubated at 37°C for 16 hours. 600The culture was continued until the affinity reached 0.6, and then 0.1 M isopropyl-β-D(-)-thiogalactopyranoside was added to induce protein expression, followed by 18 hours of culture at 20°C. After completion of the culture, the bacterial solution was centrifuged at 7000 rpm for 10 minutes, and the cells were collected for use.

[0055] 3. Preparation of enzyme solution 0.1 g of slime was weighed, 1 mL of pH 7.5 potassium phosphate buffer was added, and the mixture was shaken to homogenize. The bacterial suspension was then disrupted for 5 minutes using an ultrasonic disrupter at 30% power. In all of Examples 2 to 13, catalytic reactions were carried out using enzyme solutions prepared using the above three proteins.

[0056] 4. HPLC detection method Column: Atlantis T3 Column, 4.6 mm x 150 mm, Mobile phase: Water / acetonitrile, Flow rate: 1 ml / min, Temperature: 40°C, UV detector, Detection wavelength: 254 nm, Detection time: 15 min

[0057] Example 2 Synthesis of N6-benzoyladenosine using uridine as a substrate A reaction mixture containing 20 mM uridine, 10 mM N6-benzoyladenine, 4.88 mg of PyNP enzyme solution produced from slime, and 2.39 mg of PNP enzyme solution produced from slime was added to a 2 mM phosphate buffer (pH 7.5) to make a total volume of 1 mL. The mixture was reacted at 60°C for 4 hours, then diluted with an equal volume of DMSO and subjected to HPLC detection as shown in Figure 1. The conversion rate of uridine was 51.03% (i.e., 51.03% of uridine was converted to uracil and pentose phosphate), and the conversion rate of N6-benzoyladenine was 68.20% (i.e., 68.20% of N6-benzoyladenine was bound to pentose phosphate to produce N6-benzoyladenosine). [ka]

[0058] Example 3 Synthesis of N2-isobutyrylguanosine using uridine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.5) was mixed with 20 mM uridine, 10 mM N2-isobutyrylguanine, 4.88 mg of PyNP enzyme solution produced from slime, and 2.21 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 19 hours, and then diluted with an equal volume of DMSO. The mixture was then subjected to HPLC detection as shown in Figure 2. The conversion rate of uridine was 62.16%, and the conversion rate of N2-isobutyrylguanine was 36.51%. [ka]

[0059] Example 4 Synthesis of N4-acetylcytidine using uridine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.0) was mixed with 20 mM uridine, 10 mM N4-acetylcytosine, 4.88 mg of PyNP enzyme solution produced from slime, and 1.53 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 18 hours, and then an equal volume of DMSO was added to dilute the mixture to a certain extent. The mixture was then subjected to HPLC detection as shown in Figure 3. The conversion rate of uridine was 27.35%, and the conversion rate of N4-acetylcytosine was 15.67%. [ka]

[0060] Example 5 Synthesis of N6-benzoyl-2'-deoxyadenosine using 2'-deoxyuridine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.5) was mixed with 20 mM 2'-deoxyuridine, 10 mM N6-benzoyladenine, 4.56 mg of PyNP enzyme solution produced from slime, and 2.39 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 4 hours, and then diluted with an equal volume of DMSO. The mixture was then subjected to HPLC detection as shown in Figure 4. The conversion rate of 2'-deoxyuridine was 45.23%, and the conversion rate of N6-benzoyladenine was 47.83%. [ka]

[0061] Example 6 Synthesis of N2-isobutyryl-2'-deoxyguanosine using 2'-deoxyuridine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.5) was mixed with 20 mM 2'-deoxyuridine, 10 mM N2-isobutyrylguanine, 4.56 mg of PyNP enzyme solution produced from slime, and 2.21 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 19 hours, and then diluted with an equal volume of DMSO. The mixture was then subjected to HPLC detection as shown in Figure 5. The conversion rate of 2'-deoxyuridine was 50.59%, and the conversion rate of N2-isobutyrylguanine was 35.52%. [ka]

[0062] Example 7 Synthesis of N4-acetyl-2'-deoxycytidine using 2'-deoxyuridine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.0) was mixed with 20 mM 2'-deoxyuridine, 10 mM N4-acetylcytosine, 4.56 mg of PyNP enzyme solution produced from slime, and 1.53 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 18 hours, and then diluted with an equal volume of DMSO. The mixture was then subjected to HPLC detection as shown in Figure 6. The conversion rate of 2'-deoxyuridine was 56.21%, and the conversion rate of N4-acetylcytosine was 9.71%. [ka]

[0063] Example 8 Synthesis of N6-benzoyl-2'-deoxyadenosine using thymidine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.5), 40 mM thymidine, 10 mM N6-benzoyladenine, 0.97 mg of PyNP enzyme solution produced from slime, and 0.24 mg of PNP enzyme solution produced from slime were added to 20% dimethylformamide (DMF) to make a total volume of 1 mL. The reaction was allowed to proceed at 60 °C for 4 hours, after which an equal volume of DMSO was added to dilute the mixture. Then, as shown in Figure 7, the mixture was subjected to HPLC detection with 0.1% trifluoroacetic acid (TFA) added to the mobile phase. The conversion rate of thymidine was 22.08%, and the conversion rate of N6-benzoyladenine was 85.7%. [ka]

[0064] Example 9 Synthesis of N2-isobutyryl-2'-deoxyguanosine using thymidine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.5), 60 mM thymidine, 20 mM N2-isobutyrylguanine, 13.57 mg of PyNP enzyme solution produced from slime, and 35.33 mg of PNP enzyme solution produced from slime, and 15% dimethyl sulfoxide (DMSO) were added to make a total volume of 1 mL. The reaction was allowed to proceed at 60 °C for 19 hours, after which an equal volume of DMSO was added to dilute the mixture to a certain extent and subjected to HPLC detection as shown in Figure 8. As a result, the conversion rate of thymidine was 29.98%, and the conversion rate of N2-isobutyrylguanine was 62.49%. [ka]

[0065] Example 10 Scale-up reaction for synthesizing N6-benzoyl-2'-deoxyadenosine using thymidine as a substrate A reaction mixture containing 2 mM phosphate buffer (pH 7.5) was mixed with 400 mM thymidine, 200 mM N6-benzoyladenine, 9.68 g of PyNP enzyme solution produced from slime, and 4.78 g of PNP enzyme solution produced from slime to make a total volume of 100 mL. The mixture was reacted at 60 °C for 16 h, then diluted with an equal volume of DMSO and subjected to HPLC analysis as shown in Figure 9. In scale-up reactions, increasing the ratio of substrate base compared to bench-scale results can increase the yield of purified product. The conversion of thymidine was 43.12%, and the conversion of N6-benzoyladenine was 50.05%. [ka]

[0066] Example 11 Reaction for synthesizing N4-acetyl-2'-deoxycytidine using thymidine as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.0) was added with 400 mM thymidine, 100 mM N4-acetylcytosine, 9.04 mg of PyNP enzyme solution produced from slime, and 76.56 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The reaction was allowed to proceed at 60 °C for 18 hours, after which an equal volume of DMSO was added to dilute the mixture to a certain extent and subjected to HPLC detection as shown in Figure 10. As a result, the conversion rate of thymidine was 30.52%, and the conversion rate of N4-acetylcytosine was 63.70%. [ka]

[0067] Example 12 Synthesis of N6-benzoylarabinosyladenosine using uricil arabinoside as a substrate A reaction mixture containing 2 mM phosphate buffer (pH 7.5) was mixed with 40 mM uricil arabinoside (1-beta-D-arabinofuranosyluracil), 20 mM N6-benzoyladenine, 9.76 mg of UP enzyme solution produced from slime, and 4.78 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 16 hours, and then diluted with an equal volume of DMSO. The mixture was then subjected to HPLC detection as shown in Figure 11. The conversion of uricil arabinoside was 20.73%, and the conversion of N6-benzoyladenine was 12.24%. [ka]

[0068] Example 13 Synthesis of N2-isobutyrylarabinosylguanosine using urisyl arabinoside as a substrate A reaction system containing 2 mM phosphate buffer (pH 7.5) was mixed with 40 mM urisyl arabinoside (1-Beta-D-arabinofuranosyluracil), 20 mM N2-isobutyrylguanine, 9.76 mg of UP enzyme solution produced from slime, and 4.42 mg of PNP enzyme solution produced from slime to make a total volume of 1 mL. The mixture was reacted at 60°C for 16 hours, and then diluted with an equal volume of DMSO. The mixture was then subjected to HPLC detection as shown in Figure 12. The conversion rate of urisyl arabinoside was 14.87%, and the conversion rate of N2-isobutyrylguanine was 2.00%. [ka]

[0069] Example 14: Production of N6-benzoyl-2'-deoxyadenosine involving PNP from other sources A reaction mixture containing 400 mM thymidine, 200 mM N6-benzoyladenine, 9.68 g of PyNP enzyme solution produced from the slime, and 4.78 g of PNP enzyme solution produced from the slime were added to 2 mM phosphate buffer (pH 7.5) to a total volume of 1 mL. The mixture was incubated at 60°C for 16 hours, then diluted with an equal volume of DMSO and subjected to HPLC detection. PNP was derived from Thermus thermophilus and Deinococcus geothermalis, respectively. The protein sequence of PNP from Thermus thermophilus is SEQ ID NO:7, and the corresponding N6-benzoyladenine conversion was 48.79%. The protein sequence of PNP derived from Deinococcus geothermalis is SEQ ID NO:8, and the corresponding conversion rate of N6-benzoyladenine was 46.51%, which is close to the conversion rate of 48.84% for PNP derived from Geobacillus thermoglucosidasius mentioned above. [ka] [ka]

[0070] As is apparent from the above description, the above-described embodiments of the present invention achieve the following technical effects: By using the above-described purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase or uridine phosphorylase to carry out enzymatic catalysis using a substrate nucleoside and a substrate base containing a protecting group as substrates, a nucleoside containing a protecting group can be synthesized by a biosynthetic method, and the production can be scaled up, allowing for the mass production of a nucleoside containing a protecting group under mild reaction conditions in a short time.

[0071] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various modifications and variations to the present invention. Any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. 1. A method for the enzyme-catalyzed synthesis of nucleosides containing protecting groups, comprising: The method includes a step of synthesizing a nucleoside containing the protecting group by catalyzing a substrate using pyrimidine nucleoside phosphorylase or uridine phosphorylase and a purine nucleoside phosphorylase, the substrate comprises a substrate nucleoside, a substrate base, and a substrate phosphate, the substrate base comprising the protecting group; The pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to the PyNP and having the same function, and the PyNP is a protein represented by SEQ ID NO: 1; The uridine phosphorylase includes UP or a protein having 80% or more homology to the UP and having the same function, wherein the UP is a protein represented by SEQ ID NO: 2; The purine nucleoside phosphorylase comprises PNP or a protein having 80% or more homology to the PNP and having the same function, and the PNP comprises a protein represented by SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO:

8.

2. the protecting group is located on the base structure of the nucleoside; Preferably, the method comprises: generating a pentose phosphate and a free base from the substrate nucleoside and the substrate phosphate under the catalysis of the pyrimidine nucleoside phosphorylase or uridine phosphorylase; and substituting a phosphate group in the pentose phosphate with a substrate base having a protecting group under the catalysis of the purine nucleoside phosphorylase to obtain a nucleoside containing the protecting group.

3. The substrate nucleoside is a nucleoside of formula I, 2. The method of claim 1, wherein the substrate nucleoside preferably comprises thymidine, 2'-deoxyuridine, uridine, or urisyl arabinoside. 【Chemistry 1】 (R １ is selected from —H, —OH, or —F, and R ２ is -H or -CH ３ is selected from.)

4. The substrate base is a base represented by Formula II, Formula III, or Formula IV, and X, Y, and Z are each independently: 【Chemistry 2】 【Transformation 3】 or 【Chemistry 4】 is selected from Preferably, the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine; 2. The method of claim 1, wherein the substrate phosphate preferably comprises one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate. 【Transformation 5】

5. The method according to claim 1, wherein one or more of the purine nucleoside phosphorylase, the pyrimidine nucleoside phosphorylase, or the uridine phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

6. The catalytic time of the enzyme catalyst is 4 to 20 hours, Preferably, the catalytic temperature of the enzyme catalyst is 50 to 70°C, more preferably 60°C; The method of claim 1, wherein the concentration of the substrate nucleoside is preferably 20 to 400 mM, the concentration of the substrate base is preferably 10 to 200 mM, and the concentration of the substrate phosphate is preferably 1 to 100 mM.

7. 2. The method of claim 1, wherein the nucleoside containing a protecting group comprises N6-benzoyladenosine, N2-isobutyrylguanosine, N4-acetylcytidine, N6-benzoyl-2'-deoxyadenosine, N2-isobutyryl-2'-deoxyguanosine, N4-acetyl-2'-deoxycytidine, N6-benzoylarabinosyladenosine, or N2-isobutyrylarabinosylguanosine.

8. any one of the following enzymes: pyrimidine nucleoside phosphorylase or uridine phosphorylase, and purine nucleoside phosphorylase; The pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to the PyNP and having the same function, and the PyNP is a protein represented by SEQ ID NO: 1; The uridine phosphorylase includes UP or a protein having 80% or more homology to the UP and having the same function, wherein the UP is a protein represented by SEQ ID NO: 2; The purine nucleoside phosphorylase comprises PNP or a protein having 80% or more homology to the PNP and having the same function, and the PNP comprises a protein represented by SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO:

8.

9. The composition according to claim 8, wherein one or more of the purine nucleoside phosphorylase, the pyrimidine nucleoside phosphorylase, or the uridine phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

10. further comprising a substrate nucleoside, a substrate base, and a substrate phosphate; the substrate base includes a protecting group; The substrate nucleoside is a nucleoside of formula I, Preferably, the substrate nucleoside comprises thymidine, 2'-deoxyuridine, uridine, or urisyl arabinoside; Preferably, the substrate base is a base represented by Formula II, Formula III, or Formula IV, and X, Y, and Z are each independently: 【Transformation 6】 【Transformation 7】 or 【Transformation 8】 is selected from Preferably, the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine; Preferably, the substrate phosphate comprises one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate; The composition according to claim 8 or 9, wherein the concentration of the substrate nucleoside is preferably 20 to 400 mM, the concentration of the substrate base is preferably 10 to 200 mM, and the concentration of the substrate phosphate is preferably 1 to 100 mM. 【Chemistry 9】 (R １ is selected from —H, —OH, or —F; R ２ is -H or -CH ３ is selected from.) 【Chemistry 10】

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