Enzyme-catalyzed method and composition for the 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 more specifically, to an enzyme-catalyzed synthesis method and composition of nucleosides containing protecting groups.

[0002] This application is based on a Chinese application with an application number of 202211296608.4 and a filing date of October 21, 2022 in China, and claims these priorities. The disclosure content of the Chinese application is further incorporated into this application as a whole.

Background Art

[0003] Nucleic acid drugs such as antisense oligonucleotides and DNA aptamers can be used for the treatment of eye diseases and other diseases, and have become one of the focus areas of new drug development in recent years. With the development and research of this drug, the demand for synthetic precursor nucleoside monomers in the drug market is also increasing. Currently, nucleoside monomers are mainly produced by chemical synthesis. Since nucleosides have multiple functional groups such as amino groups and hydroxy groups, in order to efficiently synthesize DNA, it is often necessary to selectively carry out this reaction at specific sites such as the 5'-position of 2'-deoxyribose to protect specific functional groups on the base. Furthermore, in order to obtain higher purity products, these protecting groups need to 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 either 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 WO0075154A2 discloses a chemical method for producing base-protected nucleosides, including N6-benzoyl-2'-deoxyadenosine, N2-isobutyryl-2'-deoxyguanosine, and N4-benzoyl-2'-deoxycytidine. Patent US2003162957A1 discloses a chemical method for producing N2-isobutyryl-2'-deoxyguanosine. Currently, there are no reports on the biosynthesis and production of nucleosides containing protecting groups. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The main objective of the present invention is to provide an enzyme-catalyzed synthesis method and composition for nucleosides containing protecting groups, in order to solve the problem of the prior art that there is no biosynthetic method for producing nucleosides containing protecting groups. [Means for solving the problem]

[0006] To achieve the above objective, according to the first aspect of the present invention, An enzyme-catalyzed method for synthesizing nucleosides containing protecting groups, The process includes the step of catalyzing a substrate to synthesize a nucleoside containing a protecting group using pyrimidine nucleoside phosphorylase or uridine phosphorylase and purine nucleoside phosphorylase, The substrate comprises a substrate nucleoside, a substrate base, and a substrate phosphate, with the substrate base containing a protecting group. Pyrimidine nucleoside phosphorylase includes PyNP, or a protein having more than 80% homology to PyNP and having the same function, where PyNP is the protein indicated by SEQ ID NO:1. Uridine phosphorylase includes UP, or a protein having 80% or more homology to UP and having the same function, where UP is the protein indicated by SEQ ID NO:2. The present invention provides an enzyme-catalyzed synthesis method in which purine nucleoside phosphorylase contains PNP, or a protein having 80% or more homology to PNP and having the same function, and the PNP contains a protein represented by 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 pyrimidine nucleoside phosphorylase or uridine phosphorylase, and replacing the phosphate group in the pentose phosphate with a substrate base having a protecting group under the catalysis of purine nucleoside phosphorylase to obtain a nucleoside containing a protecting group.

[0008] Furthermore, the substrate nucleoside is a nucleoside represented by formula I, preferably the substrate nucleoside includes thymidine, 2'-deoxyuridine, uridine, or urisylarabinoside, and furthermore, the substrate base is a base represented by formula II, formula III, or formula IV, where X, Y, and Z are each independently [ka] [ka] or [ka] Selected from. [ka] (R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.)

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

[0010] Furthermore, one or more of the following are present: purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase, in the form of purified protein, crude enzyme solution, or immobilized enzyme.

[0011] Furthermore, the catalysis time of the enzyme catalyst is 4 to 20 hours, preferably the catalysis temperature of the enzyme catalyst is 50 to 70°C, more preferably 60°C, 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.

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

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

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

[0015] Furthermore, the composition further comprises a substrate nucleoside and a substrate base, the substrate comprises a substrate nucleoside and a substrate base, the substrate base contains a protecting group, the substrate nucleoside is the nucleoside represented by Formula I, preferably, the substrate nucleoside comprises thymidine, 2'-deoxyuridine, uridine, or uracil arabinoside, preferably, the substrate base is the base represented by Formula II, Formula III, or Formula IV, and X, Y, and Z are each independently

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Advantages of the Invention

[0016] According to the technical solution of the present invention, by using purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase or uridine phosphorylase, enzymatic catalysis is carried out with a substrate nucleoside and a substrate base containing a protecting group as substrates, and a nucleoside containing a protecting group can be synthesized by a biosynthesis method.

Brief Description of the Drawings

[0017] The drawings constituting a part of this application are for providing a further understanding of the present invention. The exemplary embodiments and their descriptions of the present invention are for explaining the present invention and do not unduly limit the present invention. [Figure 1] The HPLC chromatogram of N6-benzoyladenosine synthesized with uridine according to Example 2 of the present invention as a substrate is shown. [Figure 2] The HPLC chromatogram of N2-isobutyrylguanosine synthesized with uridine according to Example 3 of the present invention as a substrate is shown. [Figure 3] The HPLC chromatogram of N4-acetylcytidine synthesized with uridine according to Example 4 of the present invention as a substrate is shown. [Figure 4] The HPLC chromatogram of N6-benzoyl-2'-deoxyadenosine synthesized with 2'-deoxyuridine according to Example 5 of the present invention as a substrate is shown. [Figure 5]The HPLC chromatogram of N2-isobutyryl-2'-deoxyguanosine synthesized using 2'-deoxyuridine as a substrate according to Example 6 of the present invention is shown. [Figure 6] The HPLC chromatogram of N4-acetyl-2'-deoxycytidine synthesized using 2'-deoxyuridine as a substrate according to Example 7 of the present invention is shown. [Figure 7] The HPLC chromatogram of N6-benzoyl-2'-deoxyadenosine synthesized using thymidine as a substrate according to Example 8 of the present invention is shown. [Figure 8] The HPLC chromatogram of N2-isobutyryl-2'-deoxyguanosine synthesized using thymidine as a substrate according to Example 9 of the present invention is shown. [Figure 9] The HPLC chromatogram of N6-benzoyl-2'-deoxyadenosine synthesized using thymidine as a substrate according to Example 10 of the present invention is shown. [Figure 10] The HPLC chromatogram of N4-acetyl-2'-deoxycytidine synthesized using thymidine as a substrate according to Example 11 of the present invention is shown. [Figure 11] The HPLC chromatogram of N6-benzoylarabinosyladenosine synthesized using uricylarabinoside according to Example 12 of the present invention is shown. [Figure 12] The HPLC chromatogram of N2-isobutyrylarabinosylguanosine synthesized using urisylarabinoside according to Example 13 of the present invention is shown. [Modes for carrying out the invention]

[0018] The embodiments and features of the embodiments described herein may be combined with each other, provided they do not contradict each other. The present invention will be described in detail below with reference to the drawings, in combination with the embodiments.

[0019] As described in the background information, 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 a large amount of by-products and contaminants. Therefore, in this application, the inventors attempt to develop an enzyme-catalyzed synthesis method and composition for nucleosides containing protecting groups, and produce nucleosides containing protecting groups using a biosynthetic method. Accordingly, the present invention proposes a series of protection methods.

[0020] A representative first embodiment of the present application provides an enzyme-catalyzed method for synthesizing nucleosides containing a protecting group, which uses purine nucleoside phosphorylase and one of the following enzymes: pyrimidine nucleoside phosphorylase or uridine phosphorylase to catalyze a substrate and synthesize a nucleoside containing a protecting group, wherein the substrate comprises a substrate nucleoside, a substrate base, and a substrate phosphate, the substrate base containing a protecting group, the pyrimidine nucleoside phosphorylase comprises 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 SEQ ID The protein indicated by NO:1 is uridine phosphorylase, which includes UP, or proteins that have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology with UP and have the same function, with UP being the protein indicated by SEQ ID NO:2. The purine nucleoside phosphorylase includes PNP, or proteins that have 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology with PNP and have the same function, with PNP being the protein indicated by SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:8.

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

[0022] The pyrimidine nucleoside phosphorylase used in this invention is a protein derived from Thermus thermophilus, indicated by SEQ ID NO:1, and named PyNP, while the uridine phosphorylase is a protein derived from Trypanosoma cruzi, indicated by SEQ ID NO:2, and named 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 raw materials, utilizing purine nucleoside phosphorylase and either pyrimidine nucleoside phosphorylase or uridine phosphorylase.

[0023] The nucleoside containing the above protecting group is a nucleoside in which the protecting group is located on the base structure, and the protecting group originates from the protecting group on the substrate base. The above protecting group may be a protecting group commonly used in the prior art to protect a base. The above method is a one-step method, allowing for one-step production in the same container, facilitating industrial scale-up production and reducing production costs. Furthermore, because the above method is an enzyme-catalyzed method, the reaction conditions are milder and more controllable compared to chemical synthesis methods, other impurities are less likely to be generated, by-products are few and purification is easy, and the production process is economical and environmentally friendly.

[0024] The reaction scheme for 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 is substituted for the base portion of the substrate nucleoside and linked to the pentose, forming 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 substituted for the substrate base (Base), yielding a nucleoside containing a protecting group.

[0026] When used in this specification, 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 (lie, 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 general rule for substitutions and replacements is that if amino acids have similar properties, the effect of the substitution will be similar. For example, conservative amino acid substitutions can occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include, but are not limited to, the following: Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Vai, lie, Leu) can be substituted with other hydrophobic amino acids. Hydrophobic amino acids with coarse side chains (Phe, Tyr, Trp) are substituted with other hydrophobic amino acids with coarse side chains. Amino acids with positively charged side chains (Arg, His, Lys) can be substituted with other amino acids that also have positively charged side chains. Amino acids with polar but uncharged side chains (SenThr, Asn, Gin) can be substituted with other amino acids that also have polar but uncharged side chains.

[0028] Those skilled in the art can also perform 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, and preferably, the above method uses a combination of two enzymes, pyrimidine nucleoside phosphorylase (PyNP) or uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP), comprising the steps of generating pentose phosphate and a free base from a substrate nucleoside and substrate phosphate under the catalyst of pyrimidine nucleoside phosphorylase or uridine phosphorylase, and substituting the phosphate group in the pentose phosphate with a substrate base having a protecting group under the catalyst of purine nucleoside phosphorylase to obtain a nucleoside containing a protecting group. The substrate phosphate is not limited to any particular type and includes, but is not limited to, phosphates commonly used in the prior art, and such phosphates include, but are not limited to, one or more of monohydrogen phosphate, dihydrogen phosphate, monopotassium phosphate, or potassium dihydrogen phosphate, or mixtures such as commercially available phosphate buffers (PB) or phosphate buffers (PBS) commonly used in the prior art. Here, the phosphate buffer contains a buffer consisting of sodium dihydrogen phosphate and disodium hydrogen phosphate at predetermined concentrations. The phosphate buffer contains a buffer consisting of 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 represented by formula I, and preferably includes, but is not limited to, thymidine, 2'-deoxyuridine, uridine, or urisylarabinoside. [ka] (R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.)

[0031] Formula I represents compounds having a chiral moiety, 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, if R1 at position C2 is facing the same direction as the adjacent -OH at position C3 (the substituent is facing downwards), the pentose of this substrate nucleoside will be in a ribose configuration, i.e., the corresponding substituent is Ribo-OH or Ribo-F. If R1 at position C2 is facing the opposite direction from the adjacent -OH at position C3 (the substituent is facing upwards), the pentose of this substrate nucleoside will be in an arabinose configuration, i.e., the corresponding substituent is arabino-OH or arabino-F. The above chiral substrate nucleosides can be produced by the above method to nucleosides containing protecting groups without changing the stereochemistry of the pentose structure. Furthermore, if the substrate nucleosides are a mixture of different stereochemistrys, each substrate with a different stereochemistry can be converted to obtain the corresponding nucleoside containing a protecting group.

[0032] In one preferred embodiment, the substrate base is a base represented by formula II, formula III, or formula IV, where X, Y, and Z are each independently, [ka] [ka] or [ka] The substituents are selected from protecting groups commonly used in conventional technologies, and each of them is a protecting group that can be bonded to a base structure represented by formula II, formula III, or formula IV to protect the binding site. [ka]

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

[0034] Using the above-mentioned substrate nucleosides and substrate bases, the above-mentioned substrate bases and substrate nucleosides can be flexibly combined by the above-mentioned method 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. In all cases, the protecting group is located on the base structure of the nucleoside, not on the pentose structure.

[0035] In one preferred embodiment, one or more of purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase are purified proteins, crude enzyme solutions, or immobilized enzymes.

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

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

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

[0039] The above method allows for scale-up reactions, and the reaction system may contain substrate nucleoside concentrations of 20, 30, 50, 100, 200, 300, or 400 mM, with a maximum concentration of 400 mM. The substrate base concentrations may contain 10, 20, 30, 50, 100, 150, or 200 mM, with a maximum concentration of 200 mM, but not limited thereto, thereby enabling the large-scale production of nucleosides containing protecting groups.

[0040] At the appropriate catalyst temperature and catalysis time described above, this enzyme-catalyzed reaction can be completed, and the conversion rate of the substrate base, i.e., the reaction yield, is relatively high. Since the reaction can be completed with a single-step catalyst without the need to add enzymes or other reagents during the reaction, it is suitable for use in industrial scale-up production. The reaction conditions are mild and easy to control, which can reduce production equipment costs, energy costs, and risks.

[0041] In one preferred embodiment, the protecting group-containing nucleoside includes 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 representative second embodiment of the present application, the enzyme comprises one of the following: pyrimidine nucleoside phosphorylase or uridine phosphorylase, and purine nucleoside phosphorylase, wherein the pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology with PyNP and having the same function, where PyNP is the protein indicated by SEQ ID NO:1, and the uridine phosphorylase comprises UP, or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology with UP and having the same function, where UP is the protein indicated by SEQ ID NO:1. The present invention provides a composition comprising a protein represented by NO:2, purine nucleoside phosphorylase PNP, or a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% or more homology with PNP and having the same function, wherein PNP comprises 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 purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase are purified proteins, crude enzyme solutions, or immobilized enzymes.

[0044] In one preferred embodiment, the composition further comprises a substrate nucleoside and a substrate base, The substrate comprises a substrate nucleoside and a substrate base, and the substrate base contains a protecting group. The substrate nucleoside is the nucleoside shown in formula I. [ka] (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, and in the Haworth projection formula shown in Formula I, if R1 at position C2 is facing the same direction as the adjacent -OH at position C3 (the substituent is facing downwards), the pentose of this substrate nucleoside will be in a ribose configuration, i.e., the corresponding substituent is Ribo-OH or Ribo-F. If R1 at position C2 is facing the opposite direction from the adjacent -OH at position C3 (the substituent is facing upwards), the pentose of this substrate nucleoside will be in 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 having different chiral properties (including, but not limited to, racemates). Preferably, the substrate nucleoside includes thymidine, 2'-deoxyuridine, uridine, or urisylarabinoside.

[0046] Preferably, the substrate base is a base represented by formula II, formula III, or formula IV, where X, Y, and Z are each independently, [ka] [ka] or [ka] Selected from. [ka]

[0047] The substituents described above are protecting groups and can all be bonded to the base structure represented by formula II, formula III, or formula IV to protect the binding site.

[0048] Preferably, the substrate base includes 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 substrate nucleoside concentrations include, but are not limited to, 20, 30, 50, 100, 200, 300, or 400 mM, with a maximum concentration of 400 mM. The substrate base concentrations include, but are 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 indicated by SEQ ID NO:1. The uracilpyrimidine nucleoside phosphorylase UP is derived from Trypanosoma cruzi and is a protein indicated by SEQ ID NO:2. The purine nucleoside phosphorylase PNP includes a protein derived from Geobacillus thermoglucosidasius, indicated by SEQ ID NO:3, or a protein derived from Thermus thermophilus, indicated by SEQ ID NO:7, or a protein derived from Deinococcus geothermalis, indicated by SEQ ID NO:8. The above compositions can catalyze the reaction between a substrate base and a substrate nucleoside to produce a nucleoside containing a protecting group. The enzymes in the composition may be independently selected from forms such as purified protein, crude enzyme solution, or immobilized enzyme, and any of these can perform catalytic activity.

[0052] The beneficial effects of this application will be further explained below with reference to specific examples.

[0053] Example 1 1. Construction of bacterial strains The pyrimidine nucleoside phosphorylase PyNP used is derived from Thermus thermophilus, the uridine phosphorylase UP is derived from Trypanosoma cruzi, and the purine nucleoside phosphorylase PNP is 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 are cloned onto the expression vector pET28a(+). The obtained plasmid was transformed into E. coli BL21(DE3) host competent cells to obtain a single clonal strain. [ka] [ka] [ka] [ka] [ka] [ka]

[0054] 2. Protein expression E. coli strains expressing PyNP, UP, and PNP were inoculated into test tubes and incubated at 37°C for 16 hours. Then, they were inoculated at a 1% dose into a 2 L shaking flask containing 500 mL of Luria-Bertani (LB) medium and incubated at 37°C. 600The cells were cultured until the ratio reached 0.6, and 0.1 M isopropyl-β-D(-)-thiogalactopyranoside was added to induce protein expression. The cells were then cultured at 20°C for 18 hours. The cultured bacterial suspension was centrifuged at 7000 rpm for 10 minutes, and the cells were collected for use.

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

[0056] 4. HPLC detection method Column: Atlantis T3 Column, 4.6 mm × 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 To a reaction system containing 2 mM phosphate buffer (pH 7.5), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 4 hours, and then the same volume of DMSO was added to dilute to a certain factor. The solution was then subjected to HPLC detection as shown in Figure 1. As a result, 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 To a reaction system containing 2 mM phosphate buffer (pH 7.5), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 19 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then subjected to HPLC detection as shown in Figure 2. As a result, 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 To a reaction system containing 2 mM phosphate buffer (pH 7.0), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 18 hours, and then the same volume of DMSO was added to dilute the solution to a certain factor. The solution was then subjected to HPLC detection as shown in Figure 3. As a result, 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 To a reaction system containing 2 mM phosphate buffer (pH 7.5), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 4 hours, and then the same volume of DMSO was added to dilute the mixture to a certain factor. The mixture was then subjected to HPLC detection as shown in Figure 4. As a result, 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 To a reaction system containing 2 mM phosphate buffer (pH 7.5), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 19 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then subjected to HPLC detection as shown in Figure 5. As a result, 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 To a reaction system containing 2 mM phosphate buffer (pH 7.0), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 18 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then subjected to HPLC detection as shown in Figure 6. As a result, 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 To 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, 0.24 mg of PNP enzyme solution produced from slime, and 20% dimethylformamide (DMF) were added to a total volume of 1 mL. The reaction was carried out at 60°C for 4 hours, and then diluted by a certain factor with the same volume of DMSO. As shown in Figure 7, the mixture was subjected to HPLC detection with 0.1% trifluoroacetic acid (TFA) added to the mobile phase. As a result, 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 To 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, 35.33 mg of PNP enzyme solution produced from slime, and 15% dimethyl sulfoxide (DMSO) were added to a total volume of 1 mL. The reaction was carried out at 60°C for 19 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then 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. To a reaction system containing 2 mM phosphate buffer (pH 7.5), 400 mM thymidine, 200 mM N6-benzoyladenine, 9.68 g of PyNP enzyme solution prepared from slime, and 4.78 g of PNP enzyme solution prepared from slime were added to a total volume of 100 mL. The reaction was carried out at 60°C for 16 hours, and then the same volume of DMSO was added to dilute by a certain factor. The solution was then subjected to HPLC detection as shown in Figure 9. In the scale-up reaction, the yield of the purified product can be increased by increasing the proportion of substrate bases compared to the bench scale. As a result, the conversion rate of thymidine was 43.12%, and the conversion rate of N6-benzoyladenine was 50.05%. [ka]

[0066] Example 11: Reaction to synthesize N4-acetyl-2'-deoxycytidine using thymidine as a substrate. To a reaction system containing 2 mM phosphate buffer (pH 7.0), 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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 18 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then 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 uricylarabinoside as a substrate To a reaction system containing 2 mM phosphate buffer (pH 7.5), 40 mM uricylarabinoside (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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 16 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then subjected to HPLC detection as shown in Figure 11. As a result, the conversion rate of uricylarabinoside was 20.73%, and the conversion rate of N6-benzoyladenine was 12.24%. [ka]

[0068] Example 13 Synthesis of N2-isobutyrylarabinosylguanosine using uricylarabinoside as a substrate To a reaction system containing 2 mM phosphate buffer (pH 7.5), 40 mM uricylarabinoside (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 were added to a total volume of 1 mL. The reaction was carried out at 60°C for 16 hours, and then diluted by a certain factor with the same volume of DMSO. The solution was then subjected to HPLC detection as shown in Figure 12. As a result, the conversion rate of uricylarabinoside was 14.87%, and the conversion rate of N2-isobutyrylguanine was 2.00%. [ka]

[0069] Example 14: Production of N6-benzoyl-2'-deoxyadenosine involving PNPs from other sources To a reaction system containing 2 mM phosphate buffer (pH 7.5), 400 mM thymidine, 200 mM N6-benzoyladenine, 9.68 g of PyNP enzyme solution prepared from slime, and 4.78 g of PNP enzyme solution prepared from slime were added to a total volume of 1 mL. The reaction was carried out at 60°C for 16 hours, and then diluted by a certain factor with the same volume of DMSO before being subjected to HPLC detection. Here, the PNPs were derived from Thermus thermophilus and Deinococcus geothermalis, respectively. The protein sequence of the PNP derived from Thermus thermophilus is SEQ ID NO: 7, and the conversion rate of the corresponding N6-benzoyladenine is 48.79%. The protein sequence of PNP derived from Deinococcus geothermalis is SEQ ID NO:8, and the conversion rate of the corresponding N6-benzoyladenine is 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 clear from the above description, the above-mentioned embodiments of the present invention achieve the following technical effects. By using the above-mentioned purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase or uridine phosphorylase, a substrate nucleoside and a substrate base containing a protecting group can be used as substrates for enzymatic catalysis, and a nucleoside containing a protecting group can be synthesized by biosynthesis. Furthermore, scale-up production is possible, and a large quantity of nucleosides containing a protecting group can be produced in a short time and under mild reaction conditions.

[0071] The above description represents only preferred embodiments of the present invention and does not limit it, and various modifications and changes to the present invention are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An enzyme-catalyzed method for synthesizing nucleosides containing protecting groups, The process includes the step of using pyrimidine nucleoside phosphorylase or uridine phosphorylase and purine nucleoside phosphorylase to catalyze a substrate and synthesize a nucleoside containing the protecting group, The substrate comprises a substrate nucleoside, a substrate base, and a substrate phosphate, wherein the substrate base contains the protecting group. The pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having 90% or more identity with PyNP and having the same function, wherein PyNP is the protein indicated by SEQ ID NO:

1. The uridine phosphorylase comprises UP, or a protein having 90% or more identity with UP and having the same function, wherein UP is the protein indicated by SEQ ID NO:

2. The purine nucleoside phosphorylase comprises PNP, or a protein having 90% or more identity with the PNP and having the same function, wherein the PNP comprises a protein represented by SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO:

8. The substrate nucleoside is a nucleoside represented by formula I, The substrate nucleoside includes thymidine, 2'-deoxyuridine, uridine, or uricylarabinoside. The substrate base is a base represented by formula II, formula III, or formula IV, where X, Y, and Z are each independently of each other. 【Chemistry 1】 【Chemistry 2】 or 【Transformation 3】 Selected from, The enzyme-catalyzed synthesis method is characterized in that the substrate base includes N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine. 【Chemistry 4】 (R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.) 【Transformation 5】

2. The protecting group is located on the base structure of the nucleoside, The aforementioned method, A step of generating 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, The method according to claim 1, comprising the step of 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 method according to claim 1, characterized in that the substrate phosphate comprises one or more of monohydrogen phosphate, dihydrogen dihydrogen phosphate, monohydrogen potassium phosphate, or dihydrogen potassium phosphate.

4. The method according to claim 1, characterized in that one or more of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase are purified proteins, crude enzyme solutions, or immobilized enzymes.

5. The catalytic time of the enzyme catalyst is 4 to 20 hours. The catalytic temperature of the enzyme catalyst is 50 to 70°C. The method according to claim 1, characterized in that 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.

6. The method according to claim 1, characterized in that the nucleoside containing the 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.

7. A composition used for the enzymatic catalyst synthesis of nucleosides containing a protecting group, The following enzymes: pyrimidine nucleoside phosphorylase or uridine phosphorylase, and purine nucleoside phosphorylase, The pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having 90% or more identity with PyNP and having the same function, wherein PyNP is the protein indicated by SEQ ID NO:

1. The uridine phosphorylase comprises UP, or a protein having 90% or more identity with UP and having the same function, wherein UP is the protein indicated by SEQ ID NO:

2. The purine nucleoside phosphorylase comprises PNP, or a protein having 90% or more identity with the PNP and having the same function, wherein the PNP comprises a protein represented by SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO:

8. The compound further comprises a substrate nucleoside and a substrate base, The aforementioned substrate base contains a protecting group, The substrate nucleoside is a nucleoside represented by formula I, The substrate nucleoside includes thymidine, 2'-deoxyuridine, uridine, or uricylarabinoside. The substrate base is a base represented by formula II, formula III, or formula IV, where X, Y, and Z are each independently of each other. 【Transformation 6】 【Transformation 7】 or 【Transformation 8】 Selected from, The composition is characterized in that the substrate base comprises N6-benzoyladenine, N2-isobutyrylguanine, or N4-acetylcytosine. 【Chemistry 9】 (R1 is selected from -H, -OH, or -F, and R2 is selected from -H or -CH3.) 【Chemistry 10】

8. The composition according to claim 7, characterized in that one or more of the purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, or uridine phosphorylase are purified protein, crude enzyme solution, or immobilized enzyme.

9. The composition according to claim 7 or 8, further comprising a substrate phosphate.

10. The substrate phosphate comprises one or more of monohydrogen phosphate, dihydrogen dihydrogen phosphate, monohydrogen potassium phosphate, or dihydrogen potassium phosphate, The composition according to claim 9, characterized in that 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.

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