One-pot biosynthesis method for arabinonucleosides and compositions

Abstract

The present invention provides a one-pot method for biosynthesizing arabinonucleosides and a composition thereof. The one-pot method for biosynthesizing arabinonucleosides comprises the step of directly producing arabinonucleosides by mixing a substrate, uridine phosphorylase or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase together to biosynthesize the arabinonucleoside. The uridine phosphorylase includes UP shown in SEQ ID NO: 1, and the pyrimidine nucleoside phosphorylase includes P shown in SEQ ID NO: 2. y The purine nucleoside phosphorylase contains PNP as shown in SEQ ID NO: 3, and the substrate contains spongouridine and a substrate base. This method solves the problem of complicated operations involved in the prior art two-step biosynthetic method for producing arabinonucleosides, and is applicable to the field of enzyme catalysis.

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application bearing Chinese patent application number 202211296616.9 and filed on October 21, 2022, the disclosure of which is incorporated herein in its entirety.

[0002] The present invention relates to the field of enzyme catalysis, and in particular to a one-pot method and composition for the biosynthesis of arabinonucleosides. [Background technology]

[0003] Arabinonucleosides are nucleosides that replace deoxyribose with arabinose. When incorporated into DNA, they inhibit DNA replication, thereby affecting cell division and proliferation. Therefore, they can be used as antiviral and antitumor drugs to treat herpes, tumors, AIDS, hepatitis B, and other related diseases. Currently, arabinonucleosides are primarily synthesized by chemical methods (CN1042939C, CN107556356A, CN1128270A, CN103467468A, CN107892707A) and biological methods (CN106929553A, JPH10286097A, CN105237602A). Chemical synthesis, in particular, involves complex processes, high costs, harsh reaction conditions, and the need for heavy metals and organic solvents, potentially posing health risks and environmental pollution. Biological methods use enzymes expressed by microorganisms as catalysts to convert substrates into related products under mild conditions, so the operational steps are simple, the reaction conditions are relatively mild, and there is no need to use heavy metals or organic reagents that are harmful to humans and the environment.

[0004] Patent CN106929553A discloses a two-step method for synthesizing vidarabine, in which uridine phosphorylase is used to convert spongouridine into arabinose-1-phosphate, which is then separated, and adenosine phosphorylase is then used to synthesize vidarabine from arabinose-1-phosphate and adenine, with a conversion rate of more than 90%. Wei Xiaokun and his colleagues reported a two-step synthesis of arabinoguanosine. First, Enterobacter aerogenes, capable of expressing purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, was used to synthesize 2,6-diaminopurine arabinoside from spongouridine and 2,6-diaminopurine. Second, adenosine deaminase from Aspergillus oryzae was used to deaminate 2,6-diaminopurine arabinoside, producing arabinoguanosine. When the substrate concentration was less than 10 mM, the conversion rate of 2,6-diaminopurine arabinoside was 80%, but when the substrate concentration was higher than 10 mM, the conversion rate dropped significantly to less than 50%. Summary of the Invention [Problem to be solved by the invention]

[0005] The main object of the present invention is to provide a one-pot method and composition for the biosynthesis of arabinonucleosides to solve the problem of complicated operations in the prior art two-step biosynthetic method for producing arabinonucleosides. [Means for solving the problem]

[0006] In order to achieve the above object, according to a first aspect of the present invention, there is provided a one-pot method for biosynthesizing an arabinonucleoside, the method comprising the step of co-mixing a substrate, uridine phosphorylase or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase to directly produce an arabinonucleoside by a one-pot method, wherein the uridine phosphorylase includes UP or a protein having 80% or more homology to UP and having the same function, UP being the protein represented by SEQ ID NO: 1; the pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to PyNP and having the same function, PyNP being the protein represented by SEQ ID NO: 2; the purine nucleoside phosphorylase includes PNP or a protein having 80% or more homology to PNP and having the same function, PNP being the protein represented by SEQ ID NO: 3; and the substrate includes spongolidine, a substrate base, and a substrate phosphate.

[0007] The method further includes the steps of: generating arabinose-1-phosphate and a free base from spongolidine and a substrate phosphate under the catalysis of uridine phosphorylase or pyrimidine nucleoside phosphorylase; and substituting the phosphate group of the substrate base from arabinose-1-phosphate under the catalysis of purine nucleoside phosphorylase to obtain an arabinonucleoside.

[0008] Further, the substrate base is a base represented by formula I, wherein R1 is selected from -NH2, ═O, -OMe, or -Cl, and R2 is selected from -H, -NH2, -Cl, or -F; preferably, the substrate base comprises 2,6-diaminopurine, 2-amino-6-methoxypurine, 2-chloroadenine, or 2-fluoroadenine. [ka]

[0009] Furthermore, one or more of the purine nucleoside phosphorylase, uridine phosphorylase, or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0010] Furthermore, the catalytic time for biosynthesis by the one-pot method is 4 to 20 hours, the catalytic temperature for biosynthesis by the one-pot method is preferably 50 to 80°C, more preferably 60 to 70°C, and the concentration of spongouridine is preferably 2 to 320 mM, the concentration of the substrate base is preferably 1 to 200 mM, and the concentration of the substrate phosphate is preferably 1 to 100 mM.

[0011] Further, the arabinonucleoside includes 2,6-diaminopurine arabinoside, nelarabine, 2-chloroadenine arabinoside, or fludarabine, and the substrate phosphate includes one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate, or a phosphate buffer, or a phosphate buffer.

[0012] In order to achieve the above object, according to a second aspect of the present invention, there is provided a composition comprising uridine phosphorylase or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase, wherein the uridine phosphorylase comprises UP or a protein having 80% or more homology to UP and having the same function, UP is the protein shown in SEQ ID NO: 1, the pyrimidine nucleoside phosphorylase comprises PyNP or a protein having 80% or more homology to PyNP and having the same function, PyNP is the protein shown in SEQ ID NO: 2, the purine nucleoside phosphorylase comprises PNP or a protein having 80% or more homology to PNP and having the same function, PNP is the protein shown in SEQ ID NO: 3, Furthermore, one or more of the purine nucleoside phosphorylase, uridine phosphorylase, or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0013] Additionally, the composition further comprises spongouridine, a substrate base, and a substrate phosphate, preferably the substrate phosphate comprises one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate, or a phosphate buffer, or a phosphate buffer.

[0014] Furthermore, the substrate base is a base represented by formula I, R1 is selected from -NH2, =O, -OMe, or -Cl, and R2 is selected from -H, -NH2, -Cl, or -F; preferably, the substrate base comprises 2,6-diaminopurine, 2-amino-6-methoxypurine, 2-chloroadenine, or 2-fluoroadenine; preferably, the concentration of spongouridine is 2-320 mM, the concentration of the substrate base is 1-200 M, and the concentration of the substrate phosphate is 1-100 mM. [ka]

[0015] By applying the technical solution of the present invention, it is possible to produce the target product, arabinonucleoside, by one-pot biosynthesis using spongouridine and a substrate base as substrates, using purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase or uridine phosphorylase. Since there is no need for operations such as replenishing materials or purifying and then re-adding intermediate products during the process, the operation is simpler than that of the two-stage production method, and is advantageous for industrial scale-up production. [Brief explanation of the drawings]

[0016] The drawings in the specification that form a part of this application are intended to provide a further understanding of the present invention, and the schematic examples of the present invention and the description thereof are intended to help understand the present invention and are not intended to unduly limit the present invention. [Figure 1]1 shows an HPLC spectrum of the synthesis of 2,6-diaminopurine arabinoside using spongouridine and 2,6-aminopurine as substrates according to Example 2 of the present invention. [Figure 2] 1 shows an HPLC spectrum of a scaled-up synthesis of 2,6-diaminopurine arabinoside using spongouridine and 2,6-aminopurine as substrates according to Example 3 of the present invention. [Figure 3] 1 shows an HPLC spectrum of the synthesis of nelarabine using spongouridine and 2-amino-6-methoxypurine as substrates according to Example 4 of the present invention. [Figure 4] 1 shows an HPLC spectrum of a scaled-up synthesis of nelarabine using spongouridine and 2-amino-6-methoxypurine as substrates according to Example 5 of the present invention. [Figure 5] 1 shows an HPLC spectrum of the synthesis of 2-chloroadenine arabinoside using spongouridine and 2-chloroadenine as substrates according to Example 6 of the present invention. [Figure 6] 1 shows an HPLC spectrum of the synthesis of fludarabine using spongouridine and 2-fluoroadenine as substrates according to Example 7 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described in detail below with reference to the following examples.

[0018] As mentioned in the background art, in the prior art, the biosynthesis of vidarabine is mainly a two-step method, which is complicated and time-consuming, making it difficult to realize industrial mass production. Therefore, in this application, the inventors attempt to explore a one-pot biosynthetic method for preparing arabinonucleosides, and therefore propose a series of protection schemes in this application.

[0019] In a first exemplary embodiment of the present application, a one-pot method for biosynthesizing an arabinonucleoside is provided, the method comprising the step of co-mixing a substrate, a uridine phosphorylase or a pyrimidine nucleoside phosphorylase, and a purine nucleoside phosphorylase to directly produce an arabinonucleoside in a one-pot manner, wherein the uridine phosphorylase is UP or a protein having 80% or more homology to UP and the same function, the UP being the protein represented by SEQ ID NO: 1, and the pyrimidine nucleoside phosphorylase is Py The term "purine nucleoside phosphorylase" refers to a protein having 80% or more homology with PNP or PyNP and having the same function, where PyNP is the protein shown in SEQ ID NO: 2; the term "purine nucleoside phosphorylase" refers to a protein having 80% or more homology with PNP and having the same function, where PNP is the protein shown in SEQ ID NO: 3; the substrate includes spongouridine and a substrate base, and the substrate may further include a substrate phosphate to provide a phosphate group required for the reaction; and the term "arabinonucleoside" refers to a nucleoside consisting of an arabinose structure and a base structure.

[0020] The uridine phosphorylase used in this application is derived from Trypanosoma cruzi and is the protein shown in SEQ ID NO: 1, designated UP. The pyrimidine nucleoside phosphorylase is derived from Thermus thermophiles and is the protein shown in SEQ ID NO: 2, designated PyNP. The purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus and is the protein shown in SEQ ID NO: 3, designated PNP. Using purine nucleoside phosphorylase and any one of the enzymes uridine phosphorylase and pyrimidine nucleoside phosphorylase, spongolidine and a substrate base can be biosynthesized in a one-pot manner using spongolidine as substrates. This eliminates the need to purify and convert intermediate products, and allows the desired product, an arabinonucleoside, to be directly obtained. The above-mentioned uridine phosphorylase, pyrimidine nucleoside phosphorylase, or purine nucleoside phosphorylase includes a protein having 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% or more homology with UP, PyNP, or PNP and having the same function (i.e., the same catalytic function).

[0021] As used herein, 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 (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). Regarding rules for substitution and replacement, generally, amino acids with similar properties have similar effects after replacement. For example, conservative amino acid replacements may occur in the above-mentioned homologous proteins. "Conservative amino acid replacements" include, but are not limited to, the following:

[0022] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are substituted for other hydrophobic amino acids.

[0023] Hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced with other hydrophobic amino acids with bulky side chains.

[0024] Amino acids with positively charged side chains (Arg, His, Lys) are substituted with other amino acids with positively charged side chains.

[0025] Amino acids with polar but uncharged side chains (Ser, Thr, Asn, Gln) are substituted with other amino acids with polar but uncharged side chains.

[0026] Those skilled in the art can also perform conservative substitutions for amino acids according to amino acid substitution rules familiar to those skilled in the art, such as the conventional "blosum62 scoring matrix."

[0027] In one preferred embodiment, the method includes the steps of: generating arabinose-1-phosphate and a free base from spongolidine and a substrate phosphate under the catalysis of a pyrimidine nucleoside phosphorylase or a uridine phosphorylase; and substituting the phosphate group of the arabinose-1-phosphate for the substrate base under the catalysis of a purine nucleoside phosphorylase to obtain an arabinonucleoside.

[0028] The reaction pathway of the above method is as follows: [ka]

[0029] First, the substrate spongolidine is ligated to the arabinose structure under the catalysis of uridine phosphorylase or pyrimidine nucleoside phosphorylase, with the phosphate group from the substrate phosphate substituting for the base moiety of spongolidine to form phosphorylated arabinose and uracil. Then, under the catalysis of purine nucleoside phosphorylase, the phosphate group of phosphorylated arabinose is substituted for the substrate base to yield an arabinonucleoside.

[0030] In the prior art, a two-step process is typically used to synthesize arabinonucleosides, which affects the yield of the one-pot process. In contrast, in the present application, enzymes with similar functions are screened to obtain the above enzyme combination. Using this enzyme combination, arabinonucleosides can be produced by a one-pot process, thereby improving the substrate conversion rate and the yield of the desired product, arabinonucleoside. While the conversion rate of the above one-pot biosynthetic method is comparable to that of the two-step process in the prior art, the reaction procedures are simpler, and there is no need to separate and purify the intermediate product, facilitating industrial mass production and reducing the time and cost required for the reaction.

[0031] In one preferred embodiment, the substrate base is a base of formula I, wherein R1 is selected from -NH2, ═O, -OMe, or -Cl, and R2 is selected from -H, -NH2, -Cl, or -F, and preferably, the substrate base comprises 2,6-diaminopurine (i.e., 2-aminoadenine), 2-amino-6-methoxypurine, 2-chloroadenine, or 2-fluoroadenine. [ka]

[0032] In one preferred embodiment, one or more of the purine nucleoside phosphorylase, uridine phosphorylase, or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0033] In the catalytic reaction, the above three types of enzymes may exist in various forms, such as purified proteins, crude enzyme solutions, or immobilized enzymes, and all of them can catalyze the synthesis of nucleosides containing protecting groups. Genes expressing PyNP and / or UP and / or PNP 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. Crude enzyme solutions are easy to produce, have good catalytic activity, and can reduce the cost of catalytic production.

[0034] In one preferred embodiment, the catalytic time of the enzyme catalyst is 4 to 20 hours, and the catalytic temperature of the enzyme catalyst is preferably 50 to 80°C, more preferably 60 to 70°C. Preferably, the spongouridine concentration is 2 to 320 mM, the substrate base concentration is 1 to 200 mM, and the substrate phosphate concentration is 1 to 100 mM. The substrate phosphate is not limited to a specific type and includes, but is not limited to, phosphates commonly used in the prior art, such as one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate, or mixtures commonly found in the prior art, such as commercially available phosphate buffer (PB) or phosphate buffer solution (PBS). Here, the phosphate buffer includes a buffer containing sodium dihydrogen phosphate and disodium hydrogen phosphate at a certain concentration. The phosphate buffer includes a buffer containing disodium hydrogen phosphate, potassium dihydrogen phosphate, and salts such as sodium chloride and potassium chloride.

[0035] The above method allows for a scale-up reaction, and in the reaction system, the concentration of spongouridine can be 2 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, or even 320 mM or more, and the concentration of the substrate base can be 1 mM, 2 mM, 10 mM, 20 mM, 50 mM, 100 mM, or even 200 mM or more, thereby enabling large-scale production of arabinonucleosides.

[0036] At the appropriate catalytic temperature and time, the enzyme-catalyzed reaction can be completed, and the conversion rate of the substrate base, i.e., the reaction yield, is high. There is no need to replenish the enzyme or other reagents during the reaction, and there is no need to separate and purify the intermediate product before subsequent catalysis. The reaction can be completed using only the catalyst in a one-pot process, making it suitable for industrial scale-up production. The reaction conditions are mild and easy to control, reducing production equipment costs, energy costs, and hazards.

[0037] In one preferred embodiment, the arabinonucleoside includes, but is not limited to, 2,6-diaminopurine arabinoside, nelarabine (9-(β-D-arabinofuranosyl)-6-methoxy-9H-purin-2-amine), 2-chloroadenine arabinoside, or fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenosine).

[0038] In a second exemplary embodiment of the present application, a composition is provided, the composition comprising uridine phosphorylase, or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase, wherein the uridine phosphorylase comprises UP, or a protein having 80% or more homology to UP and having the same function, and UP is the protein set forth in SEQ ID NO: 1, the pyrimidine nucleoside phosphorylase comprises PyNP, or a protein having 80% or more homology to PyNP and having the same function, and PyNP is the protein set forth in SEQ ID NO: 2, and the purine nucleoside phosphorylase comprises PNP, or a protein having 80% or more homology to PNP and having the same function, and PNP is the protein set forth in SEQ ID NO: 3.

[0039] In one preferred embodiment, one or more of the purine nucleoside phosphorylase, uridine phosphorylase, or pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

[0040] In one preferred embodiment, the composition further comprises spongouridine, a substrate base, and a substrate phosphate, which is not limited to a particular type and includes, but is not limited to, phosphates commonly used in the prior art, including, but not limited to, one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, or mixtures commonly used in the prior art, such as, but not limited to, phosphate buffer.

[0041] In one preferred embodiment, the substrate base is a base of formula I, wherein R1 is selected from -NH2, ═O, -OMe, or -Cl, and R2 is selected from -H, -NH2, -Cl, or -F, and preferably, the substrate base comprises 2,6-aminopurine, 2-amino-6-methoxypurine, 2-chloroadenine, or 2-fluoroadenine. [ka]

[0042] In one preferred embodiment, the concentration of spongouridine is 2 to 320 mM, the concentration of the substrate base is 1 to 200 mM, and the concentration of the substrate phosphate is 1 to 100 mM.

[0043] The concentration of spongouridine includes 2 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, and can even reach 320 mM or more, and the concentration of substrate base includes 1 mM, 2 mM, 10 mM, 20 mM, 50 mM, 100 mM, and can even reach 200 mM or more.

[0044] The uridine phosphorylase is derived from Trypanosoma cruzi and is the protein represented by SEQ ID NO: 1, designated UP. The pyrimidine nucleoside phosphorylase is derived from Thermus thermophiles and is the protein represented by SEQ ID NO: 2, designated PyNP. The purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus and is the protein represented by SEQ ID NO: 3, designated PNP. The composition can catalyze the reaction of a substrate base with spongouridine to produce an arabinonucleoside. The enzymes in the composition may be independently selected from purified proteins, crude enzyme solutions, immobilized enzymes, or other forms, any of which can perform catalytic activity.

[0045] The above-mentioned composition may also be prepared in the form of a product such as a kit.

[0046] The beneficial effects of the present invention will be further explained below with reference to specific examples.

[0047] Example 1 1. Strain Construction The uridine phosphorylase used in this application is derived from Trypanosoma cruzi (designated UP, its amino acid sequence is shown in SEQ ID NO:1). The pyrimidine nucleoside phosphorylase is derived from Thermus thermophiles (designated PyNP, its amino acid sequence is shown in SEQ ID NO:2). The purine nucleoside phosphorylase is derived from Geobacillus stearothermophilus (designated PNP, its amino acid sequence is shown in SEQ ID NO:3). DNA sequences encoding the three enzymes were obtained by codon optimization. The DNA sequence encoding UP 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. These were each cloned into the expression vector pET28a(+). The resulting plasmids were transformed into competent host cells of Escherichia coli BL21(DE3) to obtain monoclonal strains.

[0048] SEQ ID NO:1: TIFF2025534488000007.tif35164 SEQ ID NO:2: TIFF2025534488000008.tif46164 SEQ ID NO:3: TIFF2025534488000009.tif25164 SEQ ID NO:4: TIFF2025534488000010.tif62164 SEQ ID NO:5: TIFF2025534488000011.tif75164 SEQ ID NO: 6: TIFF2025534488000012.tif461652.Protein expression The E. coli strains expressing UP, PyNP, and PNP were inoculated into test tubes, respectively, and cultured at 37°C for 16 hours. After that, a 1% inoculum was inoculated into a 2-L shake flask containing 500 mL of Luria-Bertani medium, and the OD was measured at 37°C. 600After culturing until the β-D-thiogalactopyranoside concentration reached 0.6, 0.1 M isopropyl-β-D-thiogalactopyranoside was added to induce protein expression, and the culture was continued at 20°C for 18 hours. After the culture was completed, the bacterial solution was centrifuged at 7000 rpm for 10 minutes, and the bacterial cells were collected and prepared for use.

[0049] 3. Preparation of enzyme solution 0.1 g of bacterial sludge was weighed, 1 mL of potassium phosphate buffer solution with a pH of 7.5 was added, and the mixture was mixed uniformly by shaking. The bacterial suspension was disrupted using an ultrasonic disrupter at 30% power for 5 minutes.

[0050] 4. Detection Method HPLC detection method: Chromatography column: Agilent Eclipseplus C18, 4.6 × 100 mm, 3.5 μm, mobile phase: water and acetonitrile, flow rate: 1.5 mL / min, temperature: 40 °C, UV: 254 nm.

[0051] Example 2: Synthesis of 2,6-Diaminopurine Arabinoside (2,6-Diamino-9-(beta-D-arabinofuranosyl)purine) A 1 mL reaction mixture was prepared in 2 mM phosphate buffer (pH 7.5) containing 80 mM spongouridine (1-beta-D-arabinofuranosyluracil), 20 mM 2,6-aminopurine, UP enzyme solution prepared with 4.88 mg of bacterial sludge, and PNP enzyme solution prepared with 6.01 mg of bacterial sludge. After 16 hours at 60°C, the conversion of 2,6-aminopurine was 98.78%, as shown in Figure 1. [ka]

[0052] Example 3 Scale-up reaction for the synthesis of 2,6-diaminopurine arabinoside A 1 mL reaction mixture was prepared in 2 mM phosphate buffer (pH 7.5) containing 320 mM spongouridine (1-beta-D-arabinofuranosyluracil), 200 mM 2,6-aminopurine, UP enzyme solution prepared with 19.54 mg of bacterial sludge, and PNP enzyme solution prepared with 60.06 mg of bacterial sludge. After 6 hours of reaction at 60°C, the conversion of 2,6-aminopurine was 89.74%. After 16 hours of reaction, the conversion of 2,6-aminopurine was 96.74%, and the yield of 2,6-diaminopurine arabinoside was 54.61 g / L, as shown in Figure 2.

[0053] Example 4 Synthesis of Nelarabine (9-(β-D-arabinofuranosyl)-6-methoxy-9H-purin-2-amine) A 1 mL reaction mixture was prepared in 2 mM phosphate buffer (pH 7.5) containing 4 mM spongouridine (1-beta-D-arabinofuranosyluracil), 1 mM 2-amino-6-methoxypurine, UP enzyme solution prepared with 0.24 mg of bacterial sludge, and PNP enzyme solution prepared with 0.33 mg of bacterial sludge. After 6 hours at 70°C, the conversion of 2-amino-6-methoxypurine was 98.48%, as shown in Figure 3. [ka]

[0054] Example 5 Scale-up reaction of nelarabine (9-(β-D-arabinofuranosyl)-6-methoxy-9H-purin-2-amine) A 1 mL reaction mixture was prepared in 2 mM phosphate buffer (pH 7.5) containing 320 mM spongouridine (1-beta-D-arabinofuranosyluracil), 80 mM 2-amino-6-methoxypurine, UP enzyme solution prepared with 19.54 mg of bacterial sludge, and PNP enzyme solution prepared with 26.42 mg of bacterial sludge. After 16 hours of reaction at 70°C, the conversion of 6-methoxypurine was 74.82%. After 40 hours of reaction, the conversion of 2-amino-6-methoxypurine was 94.96%. As shown in Figure 4, the yield of nelarabine was 22.58 g / L.

[0055] Example 6 Synthesis of 2-chloroadenine arabinoside A 1 mL reaction mixture was prepared in 2 mM phosphate buffer (pH 7.5), containing 2 mM spongouridine (1-beta-D-arabinofuranosyluracil), 1 mM 2-chloroadenine, 0.12 mg of PyNP enzyme solution prepared with bacterial sludge, and an appropriate amount of PNP enzyme solution prepared with bacterial sludge. The reaction was carried out at 70°C for 20 hours. As shown in Figure 5, the conversion rate of 2-chloroadenine was 93.97%. [ka]

[0056] Example 7 Synthesis of Fludarabine (9-β-D-arabinofuranose-2-fluoroadenosine) A 1 mL reaction mixture was prepared in 2 mM phosphate buffer (pH 7.5), containing 2 mM spongouridine (1-beta-D-arabinofuranosyluracil), 1 mM purine analog 2-fluoroadenine, 0.12 mg of PyNP enzyme solution prepared with bacterial sludge, and an appropriate amount of PNP enzyme solution prepared with bacterial sludge. The reaction was carried out at 70°C for 20 hours. As shown in Figure 6, the conversion rate of 2-fluoroadenine was 92.51%. [ka]

[0057] As can be seen from the above description, the above-described embodiments of the present invention achieve the following technical advantages: In the present application, the above-described purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase or uridine phosphorylase are used to produce the target product, arabinonucleoside, through one-pot biosynthesis using spongouridine and a substrate base as substrates, without the need for intermediate steps such as replenishing materials or purifying and then re-adding intermediate products. Compared with the two-step biosynthesis method of the prior art, the operation is simpler and the reaction time is shorter. Compared with the one-pot biosynthesis method of the prior art, the reaction rate is faster, the substrate conversion rate is higher, and high substrate concentrations can be tolerated, allowing industrial mass production within a short reaction time. Compared with chemical synthesis, the reaction conditions are milder, the process steps are simpler, the cost is lower, and it is environmentally friendly.

[0058] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various modifications and changes to the present invention. Any amendments, equivalent replacements, improvements, etc. made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A one-pot method for biosynthesis of arabinonucleosides, comprising: The method comprises: The method comprises the step of directly producing the arabinonucleoside by a one-pot method by co-mixing a substrate, uridine phosphorylase or pyrimidine nucleoside phosphorylase, and purine nucleoside phosphorylase to synthesize the arabinonucleoside; The uridine phosphorylase includes UP or a protein having 80% or more homology to the UP and having the same function, and the UP is a protein represented by SEQ ID NO: 1; The pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to the PyNP and having the same function, and the PyNP is a protein represented by SEQ ID NO: 2; The purine nucleoside phosphorylase includes PNP or a protein having 80% or more homology to the PNP and having the same function, and the PNP is a protein represented by SEQ ID NO: 3; A one-pot method for biosynthesizing an arabinonucleoside, wherein the substrate comprises spongouridine, a substrate base, and a substrate phosphate.

2. The method comprises: generating arabinose-1-phosphate and a free base from the spongolidine and the substrate phosphate under the catalysis of the uridine phosphorylase or the pyrimidine nucleoside phosphorylase; and substituting the phosphate group of the arabinose-1-phosphate with the substrate base under the catalysis of the purine nucleoside phosphorylase to obtain the arabinonucleoside.

3. The substrate base is a base of formula I, R １ is -NH ２ , ═O, —OMe, or —Cl; R ２ is -H, -NH ２ , —Cl, or —F; 2. The method of claim 1, wherein the substrate base preferably comprises 2,6-diaminopurine, 2-amino-6-methoxypurine, 2-chloroadenine, or 2-fluoroadenine. 【Chemical 1】

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

5. The catalytic time for biosynthesis by the one-pot method is 4 hours to 20 hours; Preferably, the catalytic temperature for the one-pot biosynthesis is 50°C to 80°C, more preferably 60°C to 70°C; The method according to claim 1, characterized in that the concentration of the spongouridine is preferably 2 mM to 320 mM, the concentration of the substrate base is preferably 1 mM to 200 mM, and the concentration of the substrate phosphate is preferably 1 mM to 100 mM.

6. the arabinonucleoside comprises 2,6-diaminopurine arabinoside, nelarabine, 2-chloroadenine arabinoside, or fludarabine; 2. The method of claim 1, wherein the substrate phosphate preferably comprises one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate.

7. 1. A composition comprising: the composition comprises a uridine phosphorylase or a pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase; The uridine phosphorylase includes UP or a protein having 80% or more homology to the UP and having the same function, wherein the UP is a protein represented by SEQ ID NO: 1; The pyrimidine nucleoside phosphorylase includes PyNP or a protein having 80% or more homology to the PyNP and having the same function, and the PyNP is a protein represented by SEQ ID NO: 2; The composition, characterized in that the purine nucleoside phosphorylase comprises PNP or a protein having 80% or more homology to the PNP and having the same function, and the PNP is the protein shown in SEQ ID NO:

3.

8. 8. The composition of claim 7, wherein one or more of the purine nucleoside phosphorylase, the uridine phosphorylase, or the pyrimidine nucleoside phosphorylase is a purified protein, a crude enzyme solution, or an immobilized enzyme.

9. 8. The composition of claim 7, further comprising spongouridine, a substrate base, and a substrate phosphate.

10. The substrate base is a base of formula I, R １ is -NH ２ , ═O, —OMe, or —Cl; R ２ is -H, -NH ２ , —Cl, or —F; Preferably, the substrate base comprises 2,6-diaminopurine, 2-amino-6-methoxypurine, 2-chloroadenine, or 2-fluoroadenine; Preferably, the substrate phosphate comprises one or more of sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium monohydrogen phosphate, or potassium dihydrogen phosphate; The composition according to claim 9, characterized in that the concentration of the spongouridine is preferably 2 mM to 320 mM, the concentration of the substrate base is preferably 1 mM to 200 mM, and the concentration of the substrate phosphate is preferably 1 mM to 100 mM. 【Chemistry 2】

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