Method for preparing purine nucleoside

Abstract

Provided is a method for preparing purine nucleoside. The preparation method comprises catalyzing a uridine substrate and a substrate base by using a pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase, and preparing and obtaining purine nucleoside. The uridine substrate comprises uridine, 2'-deoxyuridine or 2'-amino-2'-deoxyuridine, and the substrate base comprises a purine compound, wherein on the purine compound, R1 comprises -NH 2,-OCH 3 or -Cl, and R2 comprises -NH 2, -F or -Cl. The preparation method can solve the problem of low yield in the preparation of nucleoside compounds in the prior art, and is suitable for the field of enzymatic catalysis.

Description

A method for preparing purine nucleoside

[0001] This application is based on the Chinese application with CN application number 202311820673.7 and application date December 27, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of enzyme catalysis, and in particular to a method for preparing purine nucleosides. Background Art

[0003] Nucleoside analogs, as active pharmaceutical ingredients such as drug intermediates and receptor agonists, offer advantages in antiviral and anti-tumor treatments, and are used to treat a wide range of diseases. For example, fludarabine is the drug of choice for the treatment of chronic lymphocytic leukemia and low-grade lymphoma. Currently, nucleoside analogs are primarily synthesized using chemical methods, but these processes require the use of specialized chemical catalysts, the protection and modification of various functional groups, and involve multiple, complex steps. These processes often suffer from low yields, poor selectivity, and environmental pollution.

[0004] In recent years, the enzymatic synthesis of nucleosides has been developed with the advantages of mild conditions and good selectivity. Patent application WO9412514Al uses guanosine as a substrate and synthesizes 2-fluoroadenosine, a key intermediate of fludarabine, through multiple steps. However, the process requires the use of highly toxic hydrogen fluoride, and the reaction route is long and the yield is low, which limits the industrial application of this route.

[0005] Summary of the Invention

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

[0007] To achieve the above object, according to one aspect of the present invention, a method for preparing purine nucleosides is provided, the method comprising: using pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase to catalyze uridine substrates and substrate bases to prepare purine nucleosides;

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

[0009] The substrate base includes a purine compound represented by formula II, R1 includes -NH2, -OCH3 or -Cl, and R2 includes -NH2, -F or -Cl;

[0010] Furthermore, 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 includes one or more of 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine or 2-amino-6-chloropurine.

[0011] Furthermore, R of the uridine substrate is -NH2, and the substrate base includes one or more of 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine or 6-methoxyguanine.

[0012] Furthermore, the pyrimidine nucleoside phosphorylase includes the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or a protein having more than 70% homology with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having pyrimidine nucleoside phosphorylation activity; the purine nucleoside phosphorylase includes the amino acid sequence shown in SEQ ID NO: 3; or a protein having more than 70% homology with the amino acid sequence shown in SEQ ID NO: 3 and having purine nucleoside phosphorylation activity.

[0013] Furthermore, R of the uridine substrate is -OH, and the pyrimidine nucleoside phosphorylase is the amino acid sequence shown in SEQ ID NO: 2; preferably, R of the uridine substrate is -H or -NH2, and the pyrimidine nucleoside phosphorylase is the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0014] Furthermore, R of the uridine substrate is -H, -NH2 or -OH, and the purine nucleoside phosphorylase has an amino acid sequence shown in SEQ ID NO: 3.

[0015] Further, the purine nucleoside includes 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 6-oxymethyl-2'-aminodeoxyguanosine.

[0016] Furthermore, in the above preparation method, the concentration of the uridine substrate is 1 to 5000 mM, and the concentration of the substrate base is 1 to 5000 mM.

[0017] Furthermore, the reaction time of the above preparation method is 1 to 18 hours; preferably, the reaction temperature of the preparation method is 40 to 80°C.

[0018] By applying the technical solution of the present invention and utilizing pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase, a uridine substrate of a specific structure and a substrate base can be catalyzed to generate a variety of purine nucleosides through a one-step reaction, and scaled-up production can be achieved, thereby achieving efficient synthesis in a shorter reaction time and increasing the yield of purine nucleosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0020] FIG1 shows a reaction schematic diagram of a preparation method according to an embodiment of the present invention.

[0021] FIG2 shows a graph showing the HPLC detection results of 2,6-dichloroadenosine according to Example 2 of the present invention.

[0022] FIG3 shows a graph showing the HPLC detection results of 6-chloro-2-fluoroadenosine according to Example 2 of the present invention.

[0023] FIG4 shows a graph showing the HPLC detection results of 6-chloroguanosine according to Example 2 of the present invention.

[0024] FIG5 shows a graph showing the HPLC detection results of 2,6-dichloropurine-2′-deoxynucleoside according to Example 4 of the present invention.

[0025] FIG6 shows a graph showing the HPLC detection results of 6-chloro-2-fluoropurine-2′-deoxynucleoside according to Example 4 of the present invention.

[0026] FIG7 shows a graph showing the HPLC detection results of 2,6-dichloro-2′-aminodeoxyadenosine according to Example 5 of the present invention.

[0027] FIG8 shows a graph showing the HPLC detection results of 6-chloroguanine-2′-aminodeoxyadenosine according to Example 5 of the present invention.

[0028] FIG9 shows a graph showing the HPLC detection results of 6-oxymethyl-2′-aminodeoxyguanosine according to Example 5 of the present invention. DETAILED DESCRIPTION

[0029] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0030] As mentioned in the background art, existing methods for synthesizing purine nucleosides all suffer from high costs, long time consumption, and low yields, which are not conducive to the preparation and subsequent utilization of purine nucleosides and their analogs. Therefore, in this application, the inventors have attempted to use pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase to catalyze the reaction of uridine substrates and substrate bases to obtain purine nucleosides through a one-step catalytic method. In this reaction, due to the characteristics of the substrates and bases, the reaction time is short and the synthesis efficiency is high, resulting in a high product yield and suitable for industrial scale-up production. On this basis, the protection scheme of this application is proposed.

[0031] In a first typical embodiment of the present application, a method for preparing a purine nucleoside is provided, the method comprising: using pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase to catalyze a uridine substrate and a substrate base to prepare a purine nucleoside; the uridine substrate is a nucleoside represented by formula I, R includes -OH, -H or -NH2, and correspondingly, the uridine substrate is uridine, 2'-deoxyuridine or 2'-amino-2'-deoxyuridine;

[0032] The substrate base includes a purine compound represented by formula II, R1 includes -NH2, -OCH3 or -Cl, and R2 includes -NH2, -F or -Cl;

[0033] Nucleoside phosphorylase can catalyze the reversible phosphorylation reaction of the glycosidic bond of nucleoside or deoxynucleoside, release base and generate ribose-1-phosphate or deoxyribose-1-phosphate, and when another base exists, it combines with it to form a new nucleoside. The present application utilizes pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase by enzyme catalysis method, and can convert uridine, 2'-deoxyuridine and 2'-amino-2'-deoxyuridine into a variety of different purine nucleosides, and only needs to carry out one-step reaction. Compared with the problems such as the many steps and environmental pollution of current chemical synthesis methods, the preparation method in the present application is simple in process, low in cost and environmentally friendly, and purine nucleosides can be synthesized by only one-step reaction, with short reaction time and high synthesis efficiency, which is of great significance and good application prospect to the production of industrialized synthetic nucleoside analogs.

[0034] The pyrimidine nucleoside phosphorylase used in this application is derived from Escherichia coli and is represented by the protein shown in SEQ ID NO: 1. This pyrimidine nucleoside phosphorylase is named TP; or it is derived from Geobacillus stearothermophilus and is represented by the protein shown in SEQ ID NO: 2. This pyrimidine nucleoside phosphorylase is named PyNP. The purine nucleoside phosphorylase is derived from Geobacillus thermoglucosidasius and is represented by the protein shown in SEQ ID NO: 3. This purine nucleoside phosphorylase is named PNP. The reaction diagram of the above preparation method is shown in Figure 1.

[0035] In this enzymatic catalysis method, the A reaction in Figure 1 is carried out under the catalysis of TP or PyNP, the CN bond between the pyrimidine group and the ribose group of the substrate nucleoside is disconnected, and the phosphate group is combined on the nucleoside group to form a phosphate ribose intermediate. Under the action of PNP, the phosphate ribose intermediate is subjected to the B reaction in Figure 1, so that the C-1 atom of the ribose group forms a CN bond with the N-9 atom of the purine substrate base to generate a purine nucleoside compound. In the above-mentioned A and B reactions, there is no need to add coenzymes such as NADPH. Only substrate and protein are used to complete the reaction. The reaction is simple and does not need to be carried out in living cells, which greatly reduces reaction cost and efficiency. The enzymatic catalysis method carried out by this two-protein combination can complete the catalytic reaction within a shorter catalytic time, and the conversion rate of the substrate is high. It is also applicable in the amplified reaction and can be industrially produced on a large scale.

[0036] Although the prior art discloses the use of similar pyrimidine nucleoside phosphorylases and purine nucleoside phosphorylases to catalyze similar reactions, using nucleosides and bases as substrates to catalyze the generation of new nucleosides, in the field of enzyme catalysis, substrates of different structures have a significant impact on the efficiency of enzyme catalysis, and different substituents on substrates generally have a significant impact on the spatial structure, chemical properties, etc. of the substrate. Based on the prior art, it is difficult to obtain insights into what methods are needed to achieve similar reactions for different types of substrates, and it is even more difficult to predict the effects of catalysis by different enzymes.

[0037] 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 includes one or more of 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine or 2-amino-6-chloropurine.

[0038] In a preferred embodiment, R of the uridine substrate is -NH2, and the substrate base includes one or more of 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine or 6-methoxyguanine.

[0039] When synthesizing halogen-containing nucleoside products, using existing preparation methods consumes a large amount of toxic halides, resulting in increased operational requirements for the preparation process and environmental pollution. In contrast, in the present application, a substrate base with a non-toxic halogen substituent is utilized to synthesize halogen-containing purine nucleoside products in a single step. This process is simple to operate, avoids the use of toxic halides, and is safe and environmentally friendly.

[0040] In a preferred embodiment, the pyrimidine nucleoside phosphorylase comprises the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, or a protein having 70% or more (including but not limited to 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9%) homology to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and having pyrimidine nucleoside phosphorylation activity. The purine nucleoside phosphorylase comprises the amino acid sequence of SEQ ID NO: 3, or a protein having 70% or more (including but not limited to 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9%) homology to the amino acid sequence of SEQ ID NO: 3 and having purine nucleoside phosphorylation activity.

[0041] As used herein, homology refers to the "homology" between amino acid sequences or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides in an amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0042] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (for example, 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, or even 99.9% or more) homology and the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the above sequences.

[0043] As used herein, amino acid residues are abbreviated as follows: 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).

[0044] Substitution and replacement rules generally state that amino acids with similar properties will have similar effects after substitution. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0045] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0046] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0047] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0048] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0049] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0050] In a preferred embodiment, R of the uridine substrate is -OH, and the pyrimidine nucleoside phosphorylase has the amino acid sequence shown in SEQ ID NO: 2; preferably, R of the uridine substrate is -H or -NH2, and the pyrimidine nucleoside phosphorylase has the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0051] In a preferred embodiment, R of the uridine substrate is -OH, -H or -NH2, and the purine nucleoside phosphorylase has the amino acid sequence shown in SEQ ID NO: 3; preferably, R of the uridine substrate is -OH, and the purine nucleoside phosphorylase has the amino acid sequence shown in SEQ ID NO: 3.

[0052] The active centers of different enzymes may have different binding sites for different substrates. Therefore, when the R of the uridine substrate is -H or -NH2, the pyrimidine nucleoside phosphorylase can be TP or PyNP, and both can bind to these two substrates to catalyze the reaction.

[0053] Utilizing the catalytic reactions of TP, PyNP, and PNP, these three proteins can be present in various forms, including purified proteins, crude enzyme solutions, or immobilized enzymes, all of which are capable of catalyzing the synthesis of purine nucleosides. Genes expressing these proteins are cloned into host cells, and after inducing protein expression, the host cells are disrupted to obtain a crude enzyme solution containing the target protein. This crude enzyme solution is simple to prepare, exhibits excellent catalytic activity, and can reduce the production cost of the catalytic reaction.

[0054] In a preferred embodiment, the purine nucleoside includes 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 6-oxymethyl-2'-aminodeoxyguanosine.

[0055] Different substrate nucleosides and different substrate bases have different biochemical properties. Therefore, during the synthesis and preparation, TP, PyNP and PNP can all be used to perform enzyme-catalyzed reactions on the above-mentioned different substrate nucleosides and substrate bases, thereby preparing the above-mentioned various purine nucleosides.

[0056] In a preferred embodiment, the concentration of the uridine substrate in the preparation method is 1-5000 mM, and the concentration of the substrate base is 1-5000 mM.

[0057] The concentration of the uridine substrate includes but is not limited to 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. 000 mM and the concentration of the substrate base includes but is not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 80, 100, 150, 200, 300, 400, 500, 600, 800, 1000, 15000, 2000, 2500, 3000, 4000 or 5000 mM.

[0058] In a preferred embodiment, the catalytic time of the enzyme catalysis is 1 to 18 hours.

[0059] The above enzyme catalysis 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 18 hours.

[0060] In a preferred embodiment, the catalytic temperature of the enzyme catalysis is 40-80°C.

[0061] The above-mentioned catalytic temperature includes but is not limited to 40, 50, 60, 70 or 80°C.

[0062] The enzyme-catalyzed reaction is completed within the aforementioned suitable catalytic temperature and catalytic time, with a high substrate base conversion rate, i.e., reaction yield. No additional enzyme or other reagents are required during the reaction, and the reaction can be completed in a single step, making it suitable for industrial scale-up production.

[0063] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0064] Example 1

[0065] 1. Strain Construction

[0066] The pyrimidine nucleoside phosphorylase used in this application is derived from Escherichia coli and is the protein shown in SEQ ID NO: 1. This pyrimidine nucleoside phosphorylase is named TP; or it is derived from Geobacillus stearothermophilus and is the protein shown in SEQ ID NO: 2. This pyrimidine nucleoside phosphorylase is named PyNP. The purine nucleoside phosphorylase is derived from Geobacillus thermoglucosidasius and is the protein shown in SEQ ID NO: 3. This purine nucleoside phosphorylase is named PNP. In the above-mentioned enzymatic method, two nucleoside phosphatases from different sources are used to efficiently synthesize purine nucleosides in one step, which can reduce production costs and be applied to large-scale industrial production of related products. The above-mentioned substrate includes a substrate nucleoside and a substrate base, which can be a natural nucleoside or base structure or an artificially modified similar structure.

[0067] After codon optimization, DNA sequences encoding the three enzymes were obtained: SEQ ID NO: 4 for TP, SEQ ID NO: 5 for PyNP, and SEQ ID NO: 6 for PNP. These sequences were cloned into the expression vector pET28a(+). The resulting plasmids were transformed into competent Escherichia coli BL21(DE3) hosts to generate monoclonal strains.

[0068] 2. Protein Expression

[0069] The E. coli strains expressing TP, PyNP, and PNP were inoculated into test tubes, cultured at 37°C for 16 h, and then inoculated into 2 L shake flasks containing 500 mL Luria-Bertani medium at a 1% inoculum size. The cells were cultured at 37°C until the OD 600 When the pH value was 0.6, 0.1 M isopropyl-β-D-thiogalactopyranoside was added to induce protein expression, and the cells were cultured at 20°C for 18 h. The bacterial culture was centrifuged at 7000 rpm for 10 min to collect the cells for later use.

[0070] 3. Enzyme solution preparation

[0071] Weigh 0.1 g of bacterial sludge, add 1 mL of potassium phosphate buffer with a pH of 7.5, shake and mix, and then use an ultrasonic disruptor to break the bacterial suspension at a power of 30% for 5 min.

[0072] 4. HPLC detection method

[0073] The chromatographic column was Atlantis T3 Column, 4.6 mm x 150 mm, the mobile phase was methanol containing 0.1 v / v% trifluoroacetic acid (TFA), the flow rate was 1 mL / min, the column temperature was 40°C, the detection wavelength was 254 nm, and the detection time was 15 min.

[0074] Example 2 Synthesis of Purine Nucleosides Using Uridine as Substrate

[0075] A 1 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 20 mM uridine, 10 mM purine analog, 4.8 mg of PyNP enzyme solution prepared from bacterial sludge, and 1.9 mg of PNP enzyme solution prepared from bacterial sludge. The reaction was incubated at 60°C for 18 hours. After the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. The results of the products 2,6-dichloroadenosine, 2,6-dichloroadenosine, and 6-chloroguanosine are shown in Figures 2, 3, and 4. The conversion results are shown in Table 1, where the reaction conversion rate of 2,6-dichloropurine reached over 80%.

[0076] Table 1 Note: + represents a conversion rate of 5-20%, and +++++ represents a conversion rate of 70-90%. The structural formulas of 2,6-dichloroadenosine, 6-chloro-2-fluoroadenosine, and 6-chloroguanosine are shown below:

[0077] A 1 mL reaction system containing 2 mM uridine, 1 mM purine analog, 0.1 mg of PyNP enzyme solution prepared from bacterial sludge, and 0.08 mg of PNP enzyme solution prepared from bacterial sludge was prepared in 2 mM phosphate buffer (pH 7.5). The reaction was incubated at 60°C for 18 hours. After completion of the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. The conversion results are shown in Table 2, showing that the conversion rate of 2-fluoroadenine reached over 90%.

[0078] Table 2 Note: +++++ represents a conversion rate of 70-90%, and ++++++ represents a conversion rate >90%. The structural formulas of 2-fluoroadenosine and 2-chloroadenosine are shown below:

[0079] Example 3 Amplification reaction using uridine as substrate

[0080] A 1 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 320 mM uridine, 200 mM purine analog (2-fluoroadenine or 2-chloroadenine), 19.5 mg of PyNP enzyme solution prepared from bacterial sludge, and 15.3 mg of PNP enzyme solution prepared from bacterial sludge. The reaction was incubated at 60°C for 18 hours. After completion of the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. Conversion results showed that the reaction conversion rates of 2-fluoroadenine and 2-chloroadenine were greater than 80%.

[0081] Example 4: Synthesis of Purine Nucleosides Using 2'-Deoxyuridine as a Substrate

[0082] A 1 mL reaction system was prepared in 2 mM phosphate buffer (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. The reaction was incubated at 60°C for 18 h. After the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. The results of the products 2,6-dichloropurine-2'-deoxynucleoside and 6-chloro-2-fluoropurine-2'-deoxynucleoside are shown in Figures 5 and 6. The conversion results are shown in Table 3, where the reaction conversion rate of 2,6-dichloropurine reached over 50%.

[0083] Table 3 Note: +++ represents a conversion rate of 30-50% (excluding the 50% endpoint), and ++++ represents a conversion rate of 50-70%. The structural formulas of 2,6-dichloropurine-2'-deoxynucleoside and 6-chloro-2-fluoropurine-2'-deoxynucleoside are shown below:

[0084] A 1 mL reaction system was prepared in 2 mM phosphate buffer (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. The reaction was incubated at 60°C for 18 hours. After completion of the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. The conversion results are shown in Table 4, showing that the conversion rate of 2-fluoroadenine reached over 60%.

[0085] Table 4 Note: +++ represents a conversion rate of 30-50% (excluding the 50% endpoint), and ++++ represents a conversion rate of 50-70%. The structural formulas of 2-fluorodeoxyadenosine and 2-chlorodeoxyadenosine are shown below:

[0086] Example 5: Synthesis of Purine Nucleosides Using 2'-Amino-2'-Deoxyuridine as a Substrate

[0087] A 1 mL reaction system was prepared in 2 mM phosphate buffer (pH 7.5) containing 40 mM 2'-amino-2'-deoxyuridine, 20 mM purine analog, 9.8 mg of TP or PyNP enzyme solution prepared from bacterial sludge, and 3.8 mg of PNP enzyme solution prepared from bacterial sludge. The reaction was incubated at 60°C for 18 h. After completion of the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. The detection results of the products 2,6-dichloro-2'-aminodeoxyadenosine, 6-chloroguanine-2'-aminodeoxyadenosine, and 6-oxymethyl-2'-aminodeoxyguanosine are shown in Figures 7, 8, and 9. The conversion results are shown in Table 5, where the conversion rate of 2,6-dichloropurine reached over 80%.

[0088] Table 5 Note: + represents a conversion rate of 5-20%, +++ represents a conversion rate of 30-50%, and +++++ represents a conversion rate of 70-90%. The chemistry of 2,6-dichloro-2'-aminodeoxyadenosine, 6-chloroguanine-2'-aminodeoxyadenosine, and 6-oxymethyl-2'-aminodeoxyguanosine is shown below:

[0089] A 1 mL reaction system was prepared in 2 mM phosphate buffer (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. The reaction was allowed to proceed at 60°C for 18 h. After completion of the reaction, 1 mL of DMSO was added, and HPLC analysis was performed. The conversion results are shown in Table 6, showing that the conversion rate of 2-aminopurine reached over 95%.

[0090] Table 6 Note: ++++ represents a conversion rate of 50-70%, and ++++++ represents a conversion rate >90%. The chemical formulas of 2,2'-diamino-2'-deoxyadenosine, 2-fluoro-2'-aminodeoxyadenosine, and 2-chloro-2'-aminodeoxyadenosine are as follows;

[0091] When the substrate base was 6-chloro-2-fluoropurine, the purine conversion rate was almost undetectable.

[0092] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: using the above enzymatic catalysis method, it is possible to synthesize purine nucleoside products in a relatively 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 it is possible to scale up production and efficiently synthesize purine nucleosides of specific structures in a relatively short reaction time. Purine nucleosides of this type of structure are difficult to prepare by biocatalysis in the prior art and can only be prepared by chemical synthesis, which has complex steps, is time-consuming and has poor selectivity, and the use of organic reagents is prone to environmental pollution. The technical solution of the present application can be used to prepare purine nucleosides with specific structures in a one-step reaction, with simple steps, few reagents, environmental protection and high efficiency, and can be applied to industrial scale-up production.

[0093] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a purine nucleoside, characterized in that, The preparation method includes: using pyrimidine nucleoside phosphorylase and purine nucleoside phosphorylase to catalyze uridine substrates and substrate bases to prepare the purine nucleoside; The uridine substrate is a nucleoside represented by Formula I, where R includes -OH, -H or -NH2; The substrate base includes a purine compound represented by Formula II, R1 includes -NH2, -OCH3 or -Cl, and R2 includes -NH2, -F or -Cl; 2. The preparation method according to claim 1, characterized in that, 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 includes one or more of 2-chloroadenine, 2-fluoroadenine, 2,6-dichloropurine, 6-chloro-2-fluoropurine, or 2-amino-6-chloropurine.

3. The preparation method according to claim 1, characterized in that, R of the uridine substrate is -NH2, and the substrate base includes one or more of 2-aminoadenine, 2-fluoroadenine, 2-chloroadenine, 2,6-dichloropurine, 2-amino-6-chloropurine, or 6-methoxyguanine.

4. The preparation method according to claim 1, characterized in that, The pyrimidine nucleoside phosphorylase includes the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2; or a protein having more than 70% homology with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having pyrimidine nucleoside phosphorylating activity; The purine nucleoside phosphorylase includes the amino acid sequence shown in SEQ ID NO: 3; or a protein having more than 70% homology with the amino acid sequence shown in SEQ ID NO: 3 and having purine nucleoside phosphorylating activity.

5. The preparation method 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.

6. The preparation method 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 shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

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

3.

8. The preparation method according to claim 1, wherein, The purine nucleoside includes one or more of 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 6-oxymethyl-2'-aminodeoxyguanosine.

9. The preparation method according to claim 1, characterized in that, In the preparation method, the concentration of the uridine substrate is 1 - 5000 mM, and the concentration of the substrate base is 1 - 5000 mM.

10. The preparation method according to claim 1, characterized in that, The reaction time of the preparation method is 1 - 18 h.

11. According to the preparation method described in claim 1, wherein, The reaction temperature of the preparation method is 40 - 80 °C.

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