Method for preparing purine nucleosides

Abstract

A method for producing a purine nucleoside is provided. The method comprises the step of producing a purine nucleoside by catalyzing a uridine substrate and a substrate base with pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase; wherein the uridine substrate comprises uridine, 2'-deoxyuridine, or 2'-amino-2'-deoxyuridine; and the substrate base comprises a purine compound, wherein R1 on the purine compound comprises -NH2, -OCH3, or -Cl, and R2 comprises -NH2, -F, or -Cl. This can solve the problem of low yield when producing nucleoside compounds according to the prior art and is applicable to the field of enzyme catalysis.

Description

Technology Field

[0001] The present application claims priority based on a Chinese application with CN application number 202311820673.7 and a filing date of December 27, 2023, and all of the disclosed contents of the said CN application are incorporated into the present application by reference.

[0002] The present invention relates to the field of enzyme catalysts, and specifically to a method for producing purine nucleosides. Background Technology

[0003] Nucleoside analogs serve as active ingredients in pharmaceuticals, such as drug intermediates and receptor agonists, and can exhibit advantages in terms of antiviral and antitumor properties, and are used in the treatment of various diseases. For example, fludarabine is the most preferred drug for the treatment of chronic lymphocytic leukemia and low-grade malignant lymphoma. Currently, nucleoside analogs are mainly synthesized by chemical methods, but the synthesis process requires the use of special chemical catalysts, protection and modification of various functional groups, and multiple complex steps. Generally, this results in disadvantages such as low yield, low selectivity, and environmental contamination.

[0004] In recent years, the synthesis of nucleoside analogs using enzymatic methods has characteristics such as mild conditions and excellent selectivity. In patent application WO9412514A1, 2-fluoroadenosine, a key intermediate of fludarabine, can be synthesized using guanosine as a substrate through a multi-step reaction, but the process requires the use of highly toxic hydrofluoric acid, the reaction pathway is long, the yield is low, and the industrial application of the said pathway is limited. Prior art literature

[0005] WO9412514A1 The problem to be solved

[0006] The main objective of the present invention is to provide a method for producing purine nucleosides to solve the problem of low yield when producing nucleoside compounds using the enzyme synthesis method of the prior art. means of solving the problem

[0007] To realize the above objective, according to one aspect of the present invention, a method for producing a purine nucleoside is provided, said method comprising the step of producing a purine nucleoside by catalyzing a uridine substrate and a substrate base with pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase;

[0008] The uridine substrate is a nucleoside represented by formula I, where R includes -OH, -H, or -NH2;

[0009]

[0010] The substrate base includes a purine compound represented by Formula II, R1 includes -NH2, -OCH3 or -Cl, and R2 includes -NH2, -F or -Cl.

[0011]

[0012] Additionally, R of the uridine substrate is -OH or -H, and at least one of R1 or R2 of the substrate base is a halogen; the substrate base comprises one or a plurality of 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine or 2-amino-6-chloropurine.

[0013] Additionally, R of the uridine substrate is -NH2, and the substrate base comprises one or more of 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine, or 6-methoxyguanine.

[0014] Additionally, the pyrimidine nucleoside phosphorylase comprises a protein having pyrimidine nucleoside phosphate activity having 70% or more homology with the amino acid sequence represented by SEQ ID NO. 1 and / or SEQ ID NO. 2, or the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 2; and the purine nucleoside phosphorylase comprises a protein having purine nucleoside phosphorylation activity having 70% or more homology with the amino acid sequence represented by SEQ ID NO. 3.

[0015] Additionally, R of the uridine substrate is -OH, and the pyrimidine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 2; preferably, R of the uridine substrate is -H or -NH2, and the pyrimidine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 1 and / or SEQ ID NO. 2.

[0016] Additionally, R of the uridine substrate is -H, -NH2 or -OH, and the purine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 3.

[0017] Additionally, the purine nucleoside is 2-fluoroadenosine, 2-chloroadenosine, 2,6-dichloroadenosine, 6-chloro-2-fluoroadenosine, 6-chloroguanosine, 2'-fluorodeoxyadenosine, 2'-chlorodeoxyadenosine, 2,6-dichloropurine-2'-deoxynucleoside, 6-chloro-2-fluoropurine-2'-deoxynucleoside, 2-fluorodeoxyadenosine, 2-chlorodeoxyadenosine, 2,2'-diamino-2'-deoxyadenosine, 2-fluoro-2'-aminodeoxyadenosine, 2-chloro-2'-aminodeoxyadenosine, 2,6-dichloro-2'-aminodeoxyadenosine, 6-chloroguanine-2'-aminodeoxyadenosine or It contains 6-methoxy-2'-aminodeoxyguanosine.

[0018] Additionally, in the above manufacturing method, the concentration of the uridine substrate is 1 to 5000 mM, and the concentration of the substrate base is 1 to 5000 mM.

[0019] Additionally, the reaction time of the above manufacturing method is 1 to 18 hours; preferably, the reaction temperature of the above manufacturing method is 40 to 80°C. Effects of the invention

[0020] According to the technical solution of the present invention, various purine nucleosides can be produced by catalyzing a uridine substrate of a specific structure and a substrate base through a one-step reaction with pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase, and since large-scale production can be realized, they can be synthesized efficiently within a short reaction time and the yield of purine nucleosides is improved. Brief explanation of the drawing

[0021] The drawings constituting part of this application are used to provide a more detailed understanding of the invention, and exemplary embodiments and descriptions of the invention are used to interpret the invention and do not constitute an undue limitation to the invention. The drawings are as follows. Figure 1 shows a schematic diagram of the reaction of a manufacturing method according to an embodiment of the present invention. Figure 2 shows the HPLC detection result of 2,6-dichloroadenosine according to Example 2 of the present invention. Figure 3 shows the HPLC detection result of 6-chloro-2-fluoroadenosine according to Example 2 of the present invention. Figure 4 shows the HPLC detection result of 6-chloroguanosine according to Example 2 of the present invention. Figure 5 shows the HPLC detection result of 2,6-dichloropurine-2'-deoxynucleoside according to Example 4 of the present invention. Figure 6 shows the HPLC detection result of 6-chloro-2-fluoropurine-2'-deoxynucleoside according to Example 4 of the present invention. Figure 7 shows the HPLC detection result of 2,6-dichloro-2'-aminodeoxyadenosine according to Example 5 of the present invention. Figure 8 shows the HPLC detection result of 6-chloroguanine-2'-aminodeoxyadenosine according to Example 5 of the present invention. Figure 9 shows the HPLC detection result of 6-methoxy-2'-aminodeoxyguanosine according to Example 5 of the present invention. Specific details for implementing the invention

[0022] It should be noted that, unless contradictory, the embodiments and features of the embodiments of this application may be combined with one another. Hereinafter, the present invention will be described in detail by combining the embodiments.

[0023] As mentioned in the background art, all prior art methods for synthesizing purine nucleosides have problems such as high cost, long consumption time, and low yield, and are disadvantageous for the manufacture and subsequent utilization of purine nucleosides and their analogs. Accordingly, the inventors of this application catalyze uridine substrates and substrate bases with pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase, and catalyze through a one-step method to produce purine nucleosides. In this reaction, due to the characteristics of the substrates and bases, the reaction time is short and the synthesis efficiency is high, so the product obtained has a high yield and can be produced on a large scale for industrial production. Based on this, the present application has provided the technical solution that it seeks to protect.

[0024] In the first typical embodiment of the present application, a method for producing a purine nucleoside is provided, said method comprising the step of producing a purine nucleoside by catalyzing a uridine substrate and a substrate base with pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase; wherein the uridine substrate is a nucleoside represented by formula I, and R comprises -OH, -H or -NH2, correspondingly, that is, the uridine substrate is uridine, 2'-deoxyuridine or 2'-amino-2'-deoxyuridine;

[0025]

[0026] The substrate base includes a purine compound represented by Formula II, R1 includes -NH2, -OCH3 or -Cl, and R2 includes -NH2, -F or -Cl.

[0027]

[0028] Nucleoside phosphorylase catalyzes the reversible phosphorylation of glycosidic bonds of nucleosides or deoxynucleosides to release a base and produce ribose-1-phosphate or deoxyribose-1-phosphate, and if other bases are present, binds to them to form new nucleosides. This application describes a method using pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase via an enzyme-catalyzed method to convert uridine, 2'-deoxyuridine, and 2'-amino-2'-deoxyuridine into various purine nucleosides, requiring only a single-step reaction. Compared to the problems associated with conventional chemical synthesis methods, such as multiple steps and environmental pollution, the manufacturing method of this application is simple, low-cost, and environmentally friendly. It allows for the synthesis of purine nucleosides through a single-step reaction, features a short reaction time and high synthesis efficiency, and holds significant meaning and favorable application prospects for the industrial synthetic production of nucleoside analogs.

[0029] The pyrimidine nucleoside phosphorylase used in this application is E. coli ( Escherichia coli It is a protein derived from ) and represented by SEQ ID NO. 1, and such pyrimidine nucleoside phosphorylase is named TP; or Geobacillus stearothermophilus ( Geobacillus stearothermophilus It is a protein derived from ) and represented by SEQ ID No. 2, and such pyrimidine nucleoside phosphorylase is named PyNP. Purine nucleoside phosphorylase is Geobacillus thermoglucosidius ( Geobacillus thermoglucosidasius It is a protein derived from ) and represented by SEQ ID No. 3, and such a purine nucleoside phosphorylase is named PNP. The reaction schematic of the above manufacturing method is as shown in FIG. 1.

[0030] In the above enzyme catalytic method, reaction A of FIG. 1 is performed under the catalyst of TP or PyNP, cleaving the CN bond between the pyrimidine group and the ribose group of the substrate nucleoside and attaching a phosphate group to the nucleoside group to form a phosphate ribose intermediate. The phosphate ribose intermediate then performs reaction B of FIG. 1 under the action of PNP, causing the C-1 atom of the ribose group and the N-9 atom of the purine substrate base to form a CN bond, thereby producing a purine nucleoside compound. In reactions A and B, there is no need to add additional coenzymes such as NADPH, and the reaction can be completed using only the substrate and protein, making the reaction simple. Furthermore, since it does not need to be performed in living cells, the reaction cost is significantly reduced and efficiency is greatly improved. The above enzyme catalytic method, performed with this combination of two proteins, can complete the catalytic reaction within a short catalytic time, has a high substrate conversion rate, is suitable for amplification reactions, and enables large-scale industrial production.

[0031] Prior art discloses the catalyzing of similar reactions using similar pyrimidine nucleoside phosphorylases and purine nucleoside phosphorylases to generate new nucleosides using nucleosides and bases as substrates. However, in the field of enzyme catalysis, substrates of different structures significantly affect enzyme catalytic efficiency, and different substituents on substrates generally have a significant impact on the spatial structure and chemical properties of the substrates. It is difficult to obtain insights from the prior art regarding which method should be used to implement similar reactions for different types of substrates, and it is more difficult to predict the effects resulting from catalyzing with different enzymes.

[0032] In a preferred embodiment, R of the uridine substrate is -OH or -H, and at least one of R1 or R2 of the substrate base is a halogen; the substrate base comprises one or a plurality of 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine or 2-amino-6-chloropurine.

[0033] In a preferred embodiment, R of the uridine substrate is -NH2, and the substrate base comprises one or more of 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine, or 6-methoxyguanine.

[0034] When synthesizing halogen-containing nucleoside products, using the manufacturing method of the prior art consumes large amounts of toxic halides, increasing operational requirements for the manufacturing process and polluting the environment. In this application, a non-toxic substrate base having a halogen substituent is used, and a purine nucleoside product containing a halogen can be synthesized by undergoing only a one-step reaction. This process is simple to operate, prevents the use of toxic halides, is safe to operate, and is environmentally friendly.

[0035] In a preferred embodiment, the pyrimidine nucleoside phosphorylase comprises a protein having pyrimidine nucleoside phosphorylation activity having homology of 70% or more (including, but not limited to, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9%) with the amino acid sequence represented by SEQ ID NO. 1 and / or SEQ ID NO. 2. The purine nucleoside phosphorylase comprises a protein having purine nucleoside phosphorylation activity having homology of 70% or more (including, but not limited to, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9%) with the amino acid sequence represented by SEQ ID NO. 3.

[0036] The identity of the present application refers to “homology” between amino acid sequences or nucleic acid sequences, that is, the sum of the ratios of amino acid residues or nucleotides of the same type in the amino acid sequence or nucleic acid sequence. The identity of the amino acid sequence or nucleic acid sequence can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0037] The active site, active pocket, active mechanism, protein structure, etc. of a protein having homology of 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, even 99.9% or more) and having the same function are all highly likely to be identical to the protein provided by the above sequence.

[0038] As used in the text, the abbreviations for amino acid residues are alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 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 (Val; V).

[0039] Rules such as substitution and replacement generally indicate that if amino acids possess similar characteristics, they will exhibit similar effects after substitution. For example, in the aforementioned homologous proteins, conservative amino acid substitutions may occur. “Conservative amino acid substitution” is

[0040] When a hydrophobic amino acid (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) is substituted by another hydrophobic amino acid;

[0041] When a hydrophobic amino acid with a bulky side chain (Phe, Tyr, Trp) is substituted by another hydrophobic amino acid with a bulky side chain;

[0042] When an amino acid having a positively charged side chain (Arg, His, Lys) is substituted by an amino acid having a different positively charged side chain;

[0043] This includes, but is not limited to, cases where an amino acid (Ser, Thr, Asn, Gln) having a polar and uncharged side chain is substituted by an amino acid having a different polar and uncharged side chain.

[0044] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to a person skilled in the art, such as the “blosum62 rating matrix” of the prior art.

[0045] 서열번호 1:

[0046] MFLAQEIIRKKRDGHALSDEEIRFFINGIRDNTISEGQIAALAMTIFFHDMTMPERVSLTMAMRDSGTVLDWKSLHLNGPIVDKHSTGGVGDVTSLMLGPMVAACGGYIPMISGRGLGHTGGTLDKLESIPGFDIFPDDNRFREIIKDVGVAIIGQTSSLAPADKRFYATRDITATVDSIPLITASILAKKLAEGLDALVMDVKVGSGAFMPTYELSEALAEAIVGVANGAGVRTTALLTDMNQVLASSAGNAVEVREAVQFLTGEYRNPRLFDVTMALCVEMLISGKLAKDDAEARAKLQAVLDNGKAAEVFGRMVAAQKGPTDFVENYAKYLPTAMLTKAVYADTEGFVSEMDTRALGMAVVAMGGGRRQASDTIDYSVGFTDMARLGDQVDGQRPLAVIHAKDENNWQEAAKAVKAAIKLADKAPESTPTVYRRISE.

[0047] 서열번호 2:

[0048] MRMVDLIEKKRDGHALTKEEIQFIIEGYTKGDIPDYQMSALAMAIFFRGMNEEETAELTMAMVHSGDTIDLSRIEGIKVDKHSTGGVGDTTTLVLGPLVASVGVPVAKMSGRGLGHTGGTIDKLESVPGFHVEITNDEFIDLVNKNKIAVVGQSGNLTPADKKLYALRDVTATVNSIPLIASSIMSKKIAAGADAIVLDVKTGVGAFMKDLNDAKALAKAMVDIGNRVGRKTMAIISDMSQPLGYAIGNALEVKEAIDTLKGEGPEDFQELCLVLGSHMVYLAEKASSLEEARHMLEKAMKDGSALQTFKTFLAAQGGDASVVDDPSKLPQAKYIIELEAKEDGYVSEIVADAVGTAAMWLGAGRATKESTIDLAVGLVLRKKVGDAVKKGESLVTIYSNREQVDDVKQKLYENIRISATPVQAPTLIYDKIS.

[0049] Sequence No. 3:

[0050] MSIHIEAKQQEIAEKILLPGDPLRAQYIAETFLEGATCYNRVRGMLGFTGTYKGHRISVQGTGMGVPSISIYVNELIQSYHVQTLIRVGTCGAIQKDVNVRDVILAMSASTDSNMNRL TFRGRDYAPTANFALLRTAYEVGAEKGLPLKVGSVFTADMFYNDEPDWETWARYGVLAVEMETAALYTLAAKFGRKALSVLTVSDHILTGEETTAQERQTTFNDMIEVALETAIRVE.

[0051] In a preferred embodiment, R of the uridine substrate is -OH, and the pyrimidine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 2; preferably, R of the uridine substrate is -H or -NH2, and the pyrimidine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 1 and / or SEQ ID NO. 2.

[0052] In a preferred embodiment, R of the uridine substrate is -OH, -H, or -NH2, and the purine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 3; preferably, R of the uridine substrate is -OH, and the purine nucleoside phosphorylase is the amino acid sequence represented by SEQ ID NO. 3.

[0053] Since the active centers of different enzymes may differ for the binding sites of different substrates, when the R of the uridine substrate is -H or -NH2, the pyrimidine nucleoside phosphorylase may be TP or PyNP, and both can bind to these two substrates and cause a catalytic reaction.

[0054] In the case of a catalytic reaction using TP, PyNP, and PNP, the three proteins may exist in various forms, such as purified proteins, coenzyme solutions, or immobilized enzymes, and all can catalyze purine nucleoside synthesis. A gene capable of expressing the protein can be cloned into a host cell, protein expression can be induced, and then the host cell can be lysed to obtain a coenzyme solution containing the target protein. The preparation of the coenzyme solution is simple, it possesses excellent catalytic ability, and can reduce the production cost of the catalytic reaction.

[0055] In a preferred embodiment, the purine nucleoside is 2-fluoroadenosine, 2-chloroadenosine, 2,6-dichloroadenosine, 6-chloro-2-fluoroadenosine, 6-chloroguanosine, 2-fluorodeoxyadenosine, 2-chlorodeoxyadenosine, 2,6-dichloropurine-2'-deoxynucleoside, 6-chloro-2-fluoropurine-2'-deoxynucleoside, 2'-fluorodeoxyadenosine, 2'-chlorodeoxyadenosine, 2,2'-diamino-2'-deoxyadenosine, 2-fluoro-2'-aminodeoxyadenosine, 2-chloro-2'-aminodeoxyadenosine, 2,6-dichloro-2'-aminodeoxyadenosine, 6-chloroguanine-2'-aminodeoxyadenosine or It contains 6-methoxy-2'-aminodeoxyguanosine.

[0056] Since different substrate nucleosides and different substrate bases have different biochemical properties, when TP, PyNP, and PNP are used in the synthesis, various purine nucleosides can be produced by performing enzyme-catalyzed reactions on all of the different substrate nucleosides and substrate bases.

[0057] In a preferred embodiment, the concentration of the uridine substrate in the above manufacturing method is 1 to 5000 mM, and the concentration of the substrate base is 1 to 5000 mM.

[0058] The concentration of the uridine substrate is 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 320, 400, 500, 600, 800, 1000, 15000, 2000, 2500, 3000, 4000, or 5000 mM, and the concentration of the substrate base is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Includes, but is not limited to, 17, 18, 19, 20, 50, 80, 100, 150, 200, 300, 400, 500, 600, 800, 1000, 15000, 2000, 2500, 3000, 4000, or 5000 mM.

[0059] In a preferred embodiment, the catalytic time of the enzyme catalyst is 1 to 18 hours.

[0060] The enzyme catalytic time includes, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18h.

[0061] In a preferred embodiment, the catalyst temperature of the enzyme catalyst is 40 to 80°C.

[0062] The catalyst temperature above includes, but is not limited to, 40, 50, 60, 70, or 80°C.

[0063] The enzyme-catalyzed reaction can be completed within the appropriate catalyst temperature and catalyst time, and the conversion rate of the substrate base, i.e., the reaction yield, is high. There is no need to add additional enzymes or other reagents during the reaction, and the reaction can be completed by catalytically catalyzing in a single step, making it suitable for applications in large-scale industrial production.

[0064] The beneficial effects of the present application will be explained in more detail below by combining specific embodiments.

[0065] Example 1

[0066] 1. Establishment of strain

[0067] The pyrimidine nucleoside phosphorylase used in this application is E. coli ( Escherichia coli It is a protein derived from ) and represented by SEQ ID NO. 1, and such pyrimidine nucleoside phosphorylase is named TP; or Geobacillus stearothermophilus ( Geobacillus stearothermophilus It is a protein derived from ) and represented by SEQ ID No. 2, and such pyrimidine nucleoside phosphorylase is named PyNP. Purine nucleoside phosphorylase is Geobacillus thermoglucosidius ( Geobacillus thermoglucosidasius It is a protein derived from ) and represented by SEQ ID NO. 3, and such a purine nucleoside phosphorylase is named PNP. In the enzyme-catalyzed method above, by efficiently synthesizing purine nucleosides through a one-step method using nucleoside phosphorylases of two different origins, production costs can be reduced and it can be applied to products related to large-scale industrial production. The substrate includes a substrate nucleoside and a substrate base, and may be a natural nucleoside or base structure, or may be an artificially modified similar structure.

[0068] After codon optimization, DNA sequences encoding the three enzymes mentioned above were obtained, and the DNA sequence encoding TP is SEQ ID NO. 4; the DNA sequence encoding PyNP is SEQ ID NO. 5; and the DNA sequence encoding PNP is SEQ ID NO. 6. Each of these was cloned into the expression vector pET28a(+). The obtained plasmids were transformed into E. coli BL21(DE3) host competencies to obtain monoclonal strains.

[0069] Sequence No. 4:

[0070]

[0071] Sequence No. 5:

[0072]

[0073] Sequence No. 6:

[0074] .

[0075] 2. Protein Expression

[0076] E. coli strains expressing TP, PyNP, and PNP were each inoculated into test tubes and cultured at 37°C for 16 hours; subsequently, a 1% inoculum was transferred to a 2L shaking flask containing 500mL of Luria-Bertani medium, and the OD was set at 37°C. 600The culture was carried out until 0.6 was reached, and protein expression was induced by adding isopropyl-beta-D-thiogalactopyranoside at a final concentration of 0.1 M, followed by incubation at 20°C for 18 hours. The cultured suspension was centrifuged at 7000 rpm for 10 minutes, and the cells were collected for later use.

[0077] 3. Preparation of enzyme solution

[0078] Weigh 0.1g of bacterial sludge, add 1mL of potassium phosphate buffer solution with a pH of 7.5, and mix uniformly by shaking. Then, crush the turbid bacterial solution using an ultrasonic grinder at 30% power and for 5 minutes.

[0079] 4. HPLC Detection Method

[0080] The chromatography column is an Atlantis T3 Column, 4.6 mm × 150 mm, the mobile phase is methanol containing 0.1 v / v% trifluoroacetic acid (TFA), the flow rate is 1 mL / min, the column temperature is 40°C, and a UV detector is used, with a detection wavelength of 254 nm and a detection time of 15 minutes.

[0081] Example 2 Synthesis of Purine Nucleoside Using Uridine as Substrate

[0082] A reaction system was prepared in 1 mL of 2 mM phosphate buffer at pH 7.5, containing 20 mM uridine, 10 mM purine analog, a PyNP enzyme solution prepared with 4.8 mg of bacterial sludge, and a PNP enzyme solution prepared with 1.9 mg of bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was finished, 1 mL of DMSO was added and HPLC detection was performed. The detection results for the products 2,6-dichloroadenosine, 2,6-dichloroadenosine, and 6-chloroguanosine are shown in Figures 2, 3, and 4. The conversion rate results are shown in Table 1, and the reaction conversion rate of 2,6-dichloropurine can reach 80% or more.

[0083] substrate base product Purine conversion rate (%) 2,6-Dichloropurine 2,6-Dichloroadenosine +++++ 6-chloro-2-fluoropurine 6-chloro-2-fluoroadenosine +++++ 2-amino-6-chloropurine 6-Chloroguanosine +

[0084] For reference, + indicates a conversion rate of 5–20%, and +++++ indicates a conversion rate of 70–90%. The structural formulas of 2,6-dichloroadenosine, 6-chloro-2-fluoroadenosine, and 6-chloroguanosine are as follows.

[0085] A reaction system was prepared in 1 mL of 2 mM phosphate buffer at pH 7.5, containing 2 mM uridine, 1 mM purine analog, a PyNP enzyme solution prepared with 0.1 mg of bacterial sludge, and a PNP enzyme solution prepared with 0.08 mg of bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was completed, 1 mL of DMSO was added, and HPLC detection was performed. The conversion rate results are shown in Table 2, and the reaction conversion rate of 2-fluoroadenine can reach over 90%.

[0086] substrate base product Purine conversion rate 2-fluoroadenine 2-Fluoradenosine ++++++ 2-chloroadenine 2-Chloroadenosine +++++

[0087] For reference, +++++ indicates a conversion rate of 70–90%, and ++++++ indicates a conversion rate exceeding 90%. The structural formulas of 2-fluoroadenosine and 2-chloroadenosine are as follows.

[0088] Example 3 Amplification reaction using uridine as a substrate

[0089] A reaction system was prepared in 1 mL of 2 mM phosphate buffer at pH 7.5, containing 320 mM uridine, 200 mM purine analog (2-fluoroadenine or 2-chloroadenine), a PyNP enzyme solution prepared with 19.5 mg of bacterial sludge, and a PNP enzyme solution prepared with 15.3 mg of bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was completed, 1 mL of DMSO was added, and HPLC detection was performed. According to the conversion rate results, the reaction conversion rate of 2-fluoroadenine exceeded 80%, and the reaction conversion rate of 2-chloroadenine exceeded 80%.

[0090] Example 4 Synthesis of Purine Nucleoside Using 2'-Deoxyuridine as a Substrate

[0091] A reaction system was prepared in 1 mL of 2 mM phosphate buffer at pH 7.5, containing 20 mM 2'-deoxyuridine, 10 mM purine analog, 4.6 mg of TP or PyNP enzyme solution prepared from bacterial sludge, and 1.9 mg of PNP enzyme solution prepared from bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was completed, 1 mL of DMSO was added, and HPLC detection was performed. The detection results for the products, 2,6-dichloropurine-2'-deoxynucleoside and 6-chloro-2-fluoropurine-2'-deoxynucleoside, are shown in Figures 5 and 6. The conversion rate results are shown in Table 3, and the reaction conversion rate of 2,6-dichloropurine can reach 50% or more.

[0092] substrate base product Purine conversion rate 2,6-Dichloropurine 2,6-Dichloropurine-2'-Deoxynucleoside ++++ 6-chloro-2-fluoropurine 6-chloro-2-fluoropurine-2'-deoxynucleoside +++

[0093] For reference, +++ indicates a conversion rate of 30–50% (excluding the end value of 50%), and ++++ indicates a conversion rate of 50–70%. The structural formulas of 2,6-dichloropurine-2'-deoxynucleoside and 6-chloro-2-fluoropurine-2'-deoxynucleoside are as follows.

[0094] A reaction system was prepared in 1 mL of 2 mM phosphate buffer at pH 7.5, containing 4 mM 2'-deoxyuridine, 1 mM purine analog, 0.9 mg of TP or PyNP enzyme solution prepared from bacterial sludge, and 0.15 mg of PNP enzyme solution prepared from bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was completed, 1 mL of DMSO was added, and HPLC detection was performed. The conversion rate results are shown in Table 4, and the reaction conversion rate of 2-fluoroadenine can reach more than 60%.

[0095] substrate base product Purine conversion rate 2-fluoroadenine 2'-Fluorodeoxyadenosine ++++ 2-chloroadenine 2'-Chlorodeoxyadenosine +++

[0096] For reference, +++ indicates a conversion rate of 30–50% (excluding the end value of 50%), and ++++ indicates a conversion rate of 50–70%. The structural formulas of 2-fluorodeoxyadenosine and 2-chlorodeoxyadenosine are as follows.

[0097] Example 5 Synthesis of Purine Nucleoside Using 2'-Amino-2'-Deoxyuridine as a Substrate

[0098] A reaction system was prepared in 1 mL of phosphate buffer at pH 7.5 with 2 mM, containing 40 mM of 2'-amino-2'-deoxyuridine, 20 mM of a purine analog, a TP or PyNP enzyme solution prepared with 9.8 mg of bacterial sludge, and a PNP enzyme solution prepared with 3.8 mg of bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was finished, 1 mL of DMSO was added and HPLC detection was performed, and the detection results of the products, 2,6-dichloro-2'-aminodeoxyadenosine, 6-chloroguanine-2'-aminodeoxyadenosine, and 6-methoxy-2'-aminodeoxyguanosine, are shown in Figs. 7, 8, and 9. The conversion rate results are shown in Table 5, and the reaction conversion rate of 2,6-dichloropurine can reach 80% or more.

[0099] substrate base product Purine conversion rate 2,6-Dichloropurine 2,6-Dichloro-2'-aminodeoxyadenosine +++++ 2-amino-6-chloropurine 6-Chloroguanine-2'-aminodeoxyadenosine + 6-Methoxyguanine 6-Methoxy-2'-Aminodeoxyguanosine +++

[0100] For reference, + indicates a conversion rate of 5–20%, +++ indicates a conversion rate of 30–50%, and +++++ indicates a conversion rate of 70–90%. The chemical formulas of 2,6-dichloro-2'-aminodeoxyadenosine, 6-chloroguanine-2'-aminodeoxyadenosine, and 6-methoxy-2'-aminodeoxyguanosine are as follows.

[0101] A reaction system was prepared in 1 mL of 2 mM phosphate buffer at pH 7.5, containing 2 mM 2'-amino-2'-deoxyuridine, 1 mM purine analog, 2.5 mg of TP or PyNP enzyme solution prepared from bacterial sludge, and 0.1 mg of PNP enzyme solution prepared from bacterial sludge, and reacted at 60°C for 18 hours. After the reaction was completed, 1 mL of DMSO was added, and HPLC detection was performed. The conversion rate results are shown in Table 6, and the reaction conversion rate of 2-aminopurine can reach over 95%.

[0102] substrate base product Purine conversion rate 2-aminoadenine 2,2'-Diamino-2'-Deoxyadenosine ++++++ 2-fluoroadenine 2-fluoro-2'-aminodeoxyadenosine ++++ 2-chloroadenine 2-chloro-2'-aminodeoxyadenosine ++++

[0103] For reference, ++++ indicates a conversion rate of 50–70%, and ++++++ indicates a conversion rate exceeding 90%. The chemical formulas of 2,2'-diamino-2'-deoxyadenosine, 2-fluoro-2'-aminodeoxyadenosine, and 2-chloro-2'-aminodeoxyadenosine are as follows.

[0104] When the substrate base is 6-chloro-2-fluoropurine, the purine conversion rate can hardly be detected.

[0105] As can be seen from the description above, the above embodiment according to the present invention achieves the following technical effects. By using the enzyme catalytic method, purine nucleoside products can be synthesized within a short catalytic time using uridine nucleosides such as uridine, 2'-deoxyuridine or 2'-amino-2'-deoxyuridine, and substrate bases such as 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine, 2-amino-6-chloropurine, 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine or 6-methoxyguanine as substrates, and can be produced on a large scale, and purine nucleosides of a specific structure can be efficiently synthesized within a short reaction time. Purine nucleosides of this structure are difficult to produce using biocatalytic methods in the prior art and can only be produced using chemical synthesis methods, which involves complex steps, long processing times, poor selectivity, and environmental pollution caused by the use of organic reagents. According to the technical solution of the present application, purine nucleosides of a specific structure can be produced in a single-step reaction, which is simple in steps, uses fewer reagents, is environmentally friendly and efficient, and can be applied to large-scale industrial production.

[0106] The above is merely a preferred embodiment of the present invention and is not intended to limit the invention; those skilled in the art may make various changes and variations to the present invention. Any modification, equivalent substitution, improvement, etc. made within the spirit and principles of the present invention shall all be included within the scope of protection of the present invention.

Claims

Claim 1 A method for preparing a purine nucleoside comprises the step of preparing the purine nucleoside by catalyzing a uridine substrate and a substrate base with pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase; wherein the uridine substrate is a nucleoside represented by Formula I, and R comprises -OH, -H, or -NH2; The above substrate base includes a purine compound represented by Formula II, and A manufacturing method characterized in that R1 comprises -NH2, -OCH3 or -Cl, and R2 comprises -NH2, -F or -Cl. Claim 2 A method for manufacturing according to claim 1, wherein R of the uridine substrate is -OH or -H, and at least one of R1 or R2 of the substrate base is a halogen; and wherein the substrate base comprises one or more of 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine, or 2-amino-6-chloropurine. Claim 3 A method for manufacturing according to claim 1, wherein R of the uridine substrate is -NH2, and the substrate base comprises one or more of 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine, or 6-methoxyguanine. Claim 4 A method for manufacturing according to claim 1, wherein the pyrimidine nucleoside phosphorylase comprises a protein having pyrimidine nucleoside phosphate activity having 70% or more homology with the amino acid sequence represented by SEQ ID NO. 1 and / or SEQ ID NO. 2, or the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 2; and the purine nucleoside phosphorylase comprises a protein having purine nucleoside phosphorylation activity having 70% or more homology with the amino acid sequence represented by SEQ ID NO.

3. Claim 5 A method of preparation according to claim 2, characterized in that R of the uridine substrate is -OH, and the amino acid sequence of the pyrimidine nucleoside phosphorylase is SEQ ID NO.

2. Claim 6 A method of preparation according to claim 2, characterized in that R of the uridine substrate is -H or -NH2, and the amino acid sequence of the pyrimidine nucleoside phosphorylase is represented by SEQ ID NO. 1 and / or SEQ ID NO.

2. Claim 7 A method of preparation according to claim 2, characterized in that R of the uridine substrate is -H, -NH2 or -OH, and the purine nucleoside phosphorylase is an amino acid sequence represented by SEQ ID NO.

3. Claim 8 In claim 1, the purine nucleoside is 2-fluoroadenosine, 2-chloroadenosine, 2,6-dichloroadenosine, 6-chloro-2-fluoroadenosine, 6-chloroguanosine, 2-fluorodeoxyadenosine, 2-chlorodeoxyadenosine, 2,6-dichloropurine-2'-deoxynucleoside, 6-chloro-2-fluoropurine-2'-deoxynucleoside, 2'-fluorodeoxyadenosine, 2'-chlorodeoxyadenosine, 2,2'-diamino-2'-deoxyadenosine, 2-fluoro-2'-aminodeoxyadenosine, 2-chloro-2'-aminodeoxyadenosine, 2,6-dichloro-2'-aminodeoxyadenosine, A method of preparation characterized by comprising one or more of 6-chloroguanine-2'-aminodeoxyadenosine or 6-methoxy-2'-aminodeoxyguanosine. Claim 9 A manufacturing method according to claim 1, characterized in that the concentration of the uridine substrate is 1 to 5000 mM and the concentration of the substrate base is 1 to 5000 mM. Claim 10 A manufacturing method according to claim 1, characterized in that the reaction time of the manufacturing method is 1 to 18 hours. Claim 11 A manufacturing method according to claim 1, characterized in that the reaction temperature of the manufacturing method is 40 to 80℃.

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