A process for the preparation of 2,6-dichloropurine nucleosides and enzymes

Abstract

The application discloses a preparation method and an enzyme of 2,6-dichloropurine nucleoside. The application discloses an enzyme, and the amino acid sequence of the enzyme is shown as SEQ ID NO:1 in a sequence table. The application further discloses a preparation method of 2,6-dichloropurine nucleoside, which comprises the following steps: in a buffer, a compound shown as formula (I) and 2,6-dichloropurine are reacted to prepare 2,6-dichloropurine nucleoside in the presence of an enzyme, and the amino acid sequence of the enzyme is shown as SEQ ID NO:1 in the sequence table. When the method is used for preparing 2,6-dichloropurine nucleoside, the operation is simple, the cost is low, the pollution is small, and a protective group does not need to be introduced.

Description

Technical Field

[0001] This invention relates to a method for preparing 2,6-dichloropurine nucleoside and an enzyme, belonging to the field of biotechnology. Background Technology

[0002] Nucleoside analogues are an important class of anticancer chemotherapeutic agents and drugs for the specific treatment of viral infections. They include various purine and pyrimidine nucleoside derivatives and are mainly antimetabolites. They inhibit the metabolic pathways essential for the survival and replication of tumor cells and viruses by interfering with the synthesis of purines, pyrimidines, purine nucleotides, and pyrimidine nucleotides required for the synthesis of DNA and RNA in tumor cells and viruses. They also produce cytotoxicity by targeting intracellular enzymes and nucleic acids.

[0003] 2,6-Hydropurine nucleosides, as important structural units in the nucleoside series, yield diverse and active antiviral compounds after derivatization, but their synthetic methods are not widely reported in the literature. Existing synthetic methods mainly include: 1) reacting tetraacetylribose, 2,6-dichloropurine, and tin tetrachloride as a catalyst to generate 2,3,5-triacetylnucleoside, followed by reaction with ammonia-methanol to generate 2,6-dichloropurine nucleoside. This method has a yield of only about 62% and uses tin tetrachloride as a catalyst, resulting in significant pollution and high cost; 2) reacting tetraacetylribose and 2,6-dichloropurine under the catalysis of trifluoromethanesulfonic acid to form 2,3,5-triacetyl-2,6,-dichloropurine nucleoside, followed by reaction with ammonia-methanol to generate 2,6-dichloropurine nucleoside. This reaction uses trifluoromethanesulfonic acid as a catalyst, which is expensive, increasing production costs. Furthermore, the large amount of this catalyst required makes cost control difficult and hinders industrial production. Summary of the Invention

[0004] The technical problem this invention aims to solve is that existing methods for synthesizing 2,6-dichloropurine nucleosides are costly, polluting, and require the introduction of protecting groups. Therefore, this invention provides a method and enzyme for preparing 2,6-dichloropurine nucleosides. Using this method, the preparation of 2,6-dichloropurine nucleosides is simple, low-cost, produces less pollution, and does not require the introduction of protecting groups.

[0005] The present invention provides an enzyme, wherein the amino acid sequence of the enzyme is shown as SEQ ID NO:1 in the sequence listing.

[0006] This invention provides a method for preparing 2,6-dichloropurine nucleoside, comprising: in a buffer solution, reacting the compound shown in formula (I) and 2,6-dichloropurine in the presence of an enzyme to obtain 2,6-dichloropurine nucleoside via the following reaction.

[0007]

[0008] Wherein, the R group is a base, and the amino acid sequence of the enzyme is shown in SEQ ID NO:1 in the sequence listing.

[0009] In one embodiment, the enzyme is a ribotransferase.

[0010] In one embodiment, the buffer solution is a phosphate buffer; preferably a potassium phosphate buffer.

[0011] In one particular protocol, the buffer concentration is 50 mM.

[0012] In one embodiment, the buffer solution has a pH of 7.0.

[0013] In one embodiment, the volume ratio of the buffer solution to 2,6-dichloropurine is 0.03 L / g.

[0014] In one embodiment, when the base is purine, the carbon atom at position 2 of the ribose in the compound of formula (I) is connected to the nitrogen atom at position 9 of the purine; when the base is pyrimidine, the carbon atom at position 2 of the ribose in the compound of formula (I) is connected to the nitrogen atom at position 1 of the pyrimidine.

[0015] In one embodiment, the compound represented by formula (I) is adenosine, uridine, guanosine, cytidine, or inosine; preferably adenosine, uridine, or guanosine; more preferably adenosine or uridine; and even more preferably uridine.

[0016] In one embodiment, the reaction is carried out at a temperature of 20–80°C, for example, 20°C or 80°C; preferably, the reaction is carried out at a temperature of 30–70°C, for example, 30°C or 70°C; more preferably, the reaction is carried out at a temperature of 40–70°C, for example, 40°C or 70°C; and even more preferably, the reaction is carried out at a temperature of 50–60°C, for example, 50°C or 60°C.

[0017] In one embodiment, the molar ratio of the compound represented by formula (I) to 2,6-dichloropurine is 1:1 to 3:1; preferably 1.5:1 to 3:1; more preferably 1.5:1.

[0018] In one embodiment, the mass ratio of 2,6-dichloropurine to enzyme is 1:0.1 to 1:1; preferably 1:0.1 to 1:0.4; more preferably 1:0.1.

[0019] In one embodiment, the reaction time is 4 to 40 hours, preferably 20 to 40 hours; more preferably 40 hours.

[0020] In one embodiment, the buffer solution is a 50 mM, pH 7.0 potassium phosphate buffer; the volume ratio of the buffer solution to 2,6-dichloropurine is 0.03 L / g; the mass ratio of 2,6-dichloropurine to ribotransferase is 1:0.1 to 1:0.4; the compound represented by formula (I) is uridine; the molar ratio of the compound represented by formula (I) to 2,6-dichloropurine is 1.5:1; and the reaction is carried out at a temperature of 60°C.

[0021] In one embodiment, the buffer solution is a 50 mM potassium phosphate buffer solution with a pH of 7.0; the volume ratio of the buffer solution to 2,6-dichloropurine is 0.03 L / g; the mass ratio of 2,6-dichloropurine to ribotransferase is 1:0.1; the compound represented by formula (I) is uridine; the molar ratio of the compound represented by formula (I) to 2,6-dichloropurine is 1.5:1; and the reaction is carried out at a temperature of 60°C.

[0022] In one embodiment, the reaction may further include cooling the reaction solution to crystallize after the reaction is complete, thereby precipitating 2,6-dichloropurine nucleoside to obtain 2,6-dichloropurine nucleoside.

[0023] The term "base" is a commonly used term in the biological field, referring to purines and pyrimidines and their derivatives. Bases include, but are not limited to, adenine, guanine, uracil, cytosine, and hypoxanthine.

[0024] The present invention also provides the application of an enzyme in the preparation of 2,6-dichloropurine nucleoside, wherein the amino acid sequence of the enzyme is shown in SEQ ID NO:1 in the sequence listing.

[0025] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0026] The reagents and raw materials used in this invention are all commercially available.

[0027] The positive and progressive effects of this invention are that the method is simple to operate, low in cost, produces less pollution, and does not require the introduction of protecting groups. Detailed Implementation

[0028] The invention will be further described in detail below with reference to specific embodiments. The embodiments are explanations of the invention. The invention is not limited to the following embodiments. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but as long as they do not depart from the basic idea of ​​the invention, they are all within the scope of the invention.

[0029] In the following implementation examples, unless otherwise specified, all reagents and consumables can be purchased commercially, as shown in the table below:

[0030]

[0031] The lyophilized ribotransferase powder used in the following examples is prepared as follows:

[0032] A recombinant *E. coli* strain carrying 2'-deoxyribosyltransferase from *Bacillus psychrosaccharolyticus* (its amino acid sequence is shown in SEQ ID NO:1 in the sequence listing) was inoculated into a fermentation medium. After inducing protein expression fermentation, the fermentation broth was centrifuged to collect bacterial sludge (4000 rpm, 30 min, 4℃). The bacterial cells were washed with 30 ml of 0.9% NaCl solution and centrifuged again to collect bacterial sludge (4000 rpm, 30 min, 4℃). The bacterial precipitate was retained, and 20 ml of Tris-HCl (50 mM) solution was added. The mixture was vortexed and ultrasonically disrupted (500 W, 30 min, on for 2 s, off for 5 s). The supernatant was centrifuged and collected (12000 rpm, 30 min). The supernatant was pre-frozen at -80℃ for 2 h and then freeze-dried in a freeze dryer for 20 h to obtain freeze-dried enzyme powder.

[0033] The fermentation conditions were as follows: a single colony was picked from an LB solid plate containing kanamycin (final concentration 30 μg / ml) and placed into 10 ml of LB liquid medium containing kanamycin (final concentration 30 μg / ml). The seed culture was incubated overnight at 37°C and 300 rpm. 6.25 ml of the seed culture was inoculated into 250 ml of TB medium and incubated at 37°C and 300 rpm for 2–3 hours. Then, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was incubated at 25°C and 450 rpm for 2–3 hours. The LB solid medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder. The LB liquid medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The TB liquid medium contains: 12 g / L peptone, 24 g / L yeast extract, 4 g / L glycerol, 2.31 g / L KH2PO4 and 16.43 g / L K2HPO4·3H2O; the induced protein expression fermentation is carried out using an inducer, which is isopropyl thiogalactoside (IPTG) or lactose.

[0034] 2,6-Dichloropurine standard and 2,6-Dichloropurine nucleoside standard were purchased from Shanghai Linghai Pharmaceutical Technology Co., Ltd. and Shanghai Maclean Biochemical Technology Co., Ltd., respectively.

[0035] 2,6-Dichloropurine standard was detected by HPLC. The HPLC detection conditions were: Waters X-Bridge C18 (150 mm × 4.6 mm × 3.5 μm), flow rate 1.3 ml / min, mobile phase A: 10 mM NH4OAc in water: ACN = 95:5 (v / v), mobile phase B: 10 mM NH4OAc in water: ACN = 5:95 (v / v), wavelength: 254 nm, detection temperature: 35 ℃. The HPLC result was a single peak with a retention time of 3.259 min.

[0036] The 2,6-dichloropurine nucleoside standard was detected by HPLC. The HPLC detection conditions were: Waters X-Bridge C18 (150 mm × 4.6 mm × 3.5 μm), flow rate 1.3 ml / min, mobile phase A: 10 mM NH4OAc in water: ACN = 95:5 (v / v), mobile phase B: 10 mM NH4OAc in water: ACN = 5:95 (v / v), wavelength: 254 nm, detection temperature: 35 ℃. The HPLC result was a single peak with a retention time of 3.059 min.

[0037] Example 1: Effect of different buffer solutions on reaction conversion rate

[0038] A) Use 4 ml of different buffer solutions (pH 7.0, 50 mM) as solvents; the raw material feed amounts are: 133 mg of 2,6-dichloropurine, 339 mg of uridine, and 133 mg of lyophilized ribotransferase powder. Place the reaction mixture in a shaker at 60°C and react at 1000 rpm for 20 h.

[0039] B) Place the reaction solution in 100℃ for 10 min, then centrifuge 1 mL of sample (12000 rpm, 2 min). Take 200 μL of the supernatant, add 1 mL of pH 7.0 sodium phosphate buffer (50 mM), mix thoroughly, pass the sample through a membrane, and analyze the composition of the reaction mixture using high performance liquid chromatography (HPLC). The HPLC detection conditions are: Waters X-Bridge C18 (150 mm × 4.6 mm × 3.5 μm), flow rate 1.3 mL / min, mobile phase A: 10 mM NH4OAc in water: ACN = 95:5 (v / v), mobile phase B: 10 mM NH4OAc in water: ACN = 5:95 (v / v), wavelength: 254 nm, detection temperature: 35℃; determine the reaction conversion rate.

[0040] The reaction conversion rate was measured under different buffer conditions, and the specific results are shown in Table 1.

[0041] Table 1: Reaction conversion rates for different buffer solutions

[0042] Sodium phosphate buffer 30％ Potassium phosphate buffer 52.3％ physiological saline 21.4％ Triethanolamine 18.7％ HEPES 15.3％

[0043] The results of the above comparison of examples show that the reaction conversion rate is the highest, at 53.2%, when the buffer solution is potassium phosphate buffer.

[0044] The HPLC results of the reaction solution showed two peaks with retention times of 3.046 min and 3.259 min, respectively.

[0045] Example 2: Effect of different ribose donors on reaction conversion rate

[0046] A) Use 4 ml of potassium phosphate buffer (pH 7.0, 50 mM) as the solvent; the raw materials are: 133 mg of 2,6-dichloropurine, 1.05 × 10⁻³ mol of ribose donor, and 133 mg of lyophilized ribotransferase powder. Place the reaction mixture in a shaker at 60 °C and react at 1000 rpm for 20 h.

[0047] B) Place the reaction solution in 100°C and heat-treat for 10 min. Centrifuge 1 mL of the sample (12000 rpm, 2 min). Take 200 μL of the supernatant and add 1 mL of pH 7.0 potassium phosphate buffer (50 mM). Mix thoroughly and pass the sample through a membrane. Analyze the composition of the reaction mixture using high-performance liquid chromatography (HPLC) under the conditions described in Example 1. Determine the reaction conversion rate.

[0048] The reaction conversion rate was determined under different ribose donor conditions, and the specific results are shown in Table 2.

[0049] Table 2: Conversion rates of riboglycosylation reactions for different ribose donors

[0050]

[0051] The results of the above examples show that the reaction conversion rate is the highest, at 45.1%, when the ribose donor is uridine.

[0052] The HPLC results of the reaction solution showed two peaks with retention times of 3.046 min and 3.259 min, respectively.

[0053] Example 3: Effect of different reaction temperatures on reaction conversion rate

[0054] A) Use 4 ml of potassium phosphate buffer (pH 7.0, 50 mM) as the solvent; the raw materials are: 133 mg of 2,6-dichloropurine, 339 mg of uridine, and 133 mg of lyophilized ribotransferase powder. Place the reaction mixture in shakers at different temperatures and react at 1000 rpm for 20 h.

[0055] B) Place the reaction solution in 100°C and heat-treat for 10 min. Centrifuge 1 mL of the sample (12000 rpm, 2 min). Take 200 μL of the supernatant and add 1 mL of pH 7.0 potassium phosphate buffer (50 mM). Mix thoroughly and pass the sample through a membrane. Analyze the composition of the reaction mixture using high-performance liquid chromatography (HPLC) under the conditions described in Example 1. Determine the reaction conversion rate.

[0056] The reaction conversion rate was measured under different temperature conditions, and the specific results are shown in Table 3.

[0057] Table 3: Conversion rate of ribose transfer reaction under different temperature conditions

[0058] 20℃ 11.2％ 30℃ 21.2％ 40℃ 38.5％ 50℃ 46.3％ 60℃ 53.8％ 70℃ 31.2％ 80℃ 8.3％

[0059] The results of the above comparison of examples show that the highest conversion rate, 53.8%, is achieved at a reaction temperature of 60°C.

[0060] The HPLC results of the reaction solution showed two peaks with retention times of 3.046 min and 3.259 min, respectively.

[0061] Example 4: Effect of the molar ratio of ribose donor to 2,6-dichloropurine on reaction conversion

[0062] A) Use 4 ml of potassium phosphate buffer (pH 7.0, 50 mM) as the solvent; the raw materials are: 133 mg of 2,6-dichloropurine, different masses of uridine (molar ratio of uridine to 2,6-dichloropurine is 1:1 to 3:1), and 133 mg of lyophilized ribotransferase powder. Place the reaction mixture in a shaker at 60°C and react at 1000 rpm for 20 h.

[0063] B) Place the reaction solution in 100°C and heat-treat for 10 min. Centrifuge 1 mL of the sample (12000 rpm, 2 min). Take 200 μL of the supernatant and add 1 mL of pH 7.0 potassium phosphate buffer (50 mM). Mix thoroughly and pass the sample through a membrane. Analyze the composition of the reaction mixture using high-performance liquid chromatography (HPLC) under the conditions described in Example 1. Determine the reaction conversion rate.

[0064] The reaction conversion rates under different molar ratios were measured, and the specific results are shown in Table 4.

[0065] Table 4: Conversion rate of ribose transfer reaction under different molar ratios of uridine to 2,6-dichloropurine

[0066] 1：1 26.3％ 1.5：1 52.7％ 2：1 46.3％ 2.5：1 43.4％ 3：1 36.3％

[0067] The results of the above examples show that the highest conversion rate (52.7%) was achieved when the molar ratio of ribose donor (uridine) to 2,6-dichloropurine was 1.5:1. As the concentration of ribose donor (uridine) continued to increase, the conversion rate gradually remained constant.

[0068] The HPLC results of the reaction solution showed two peaks with retention times of 3.046 min and 3.259 min, respectively.

[0069] Example 5: Effect of the mass ratio of 2,6-dichloropurine to ribotransferase on the reaction conversion rate

[0070] A) Use 4 ml of potassium phosphate buffer (pH 7.0, 50 mM) as the solvent; the raw materials are: 133 mg of 2,6-dichloropurine and different masses of lyophilized ribotransferase powder (the mass ratio of 2,6-dichloropurine to ribotransferase is 1:0.1 to 1:1), and 256 mg of uridine. Place the reaction mixture in a shaker at 60 °C and react at 1000 rpm for 40 h.

[0071] B) Place the reaction solution in 100°C and heat-treat for 10 min. Centrifuge 1 mL of the sample (12000 rpm, 2 min). Take 200 μL of the supernatant and add 1 mL of pH 7.0 potassium phosphate buffer (50 mM). Mix thoroughly and pass the sample through a membrane. Analyze the composition of the reaction mixture using high-performance liquid chromatography (HPLC) under the conditions described in Example 1. Determine the reaction conversion rate.

[0072] The reaction conversion rates under different mass ratios were measured, and the specific results are shown in Table 5.

[0073] Table 5: Conversion rate of ribotransferase under different mass ratios of 2,6-dichloropurine to ribotransferase

[0074] 1：0.1 54.8％ 1：0.2 53.9％ 1：0.3 54.6％ 1：0.4 54.3％ 1：0.5 51.7％ 1：0.6 52.7％ 1：0.7 52.0％ 1：0.8 51.4％ 1：0.9 51.7％ 1：1 53.3％

[0075] The results of the above examples show that the highest conversion rate (54.8%) was achieved when the mass ratio of 2,6-dichloropurine to ribotransferase was 1:0.1. As the mass of ribotransferase increased, the conversion rate remained constant.

[0076] The HPLC results of the reaction solution showed two peaks with retention times of 3.046 min and 3.259 min, respectively. SEQUENCE LISTING <110> Shanghai Wuquan Pharmaceutical R&D Co., Ltd. Shanghai Hequan Pharmaceutical Co., Ltd. Changzhou Hequan Pharmaceutical Co., Ltd. <120> A method for preparing 2,6-dichloropurine nucleoside and an enzyme <130> P21018256C <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> BP11 <400> 1 Met Ala Lys Ile Tyr Leu Ala Ser Pro Phe Phe Asn Glu Glu Gln Leu 1 5 10 15 Lys His Val Ser Lys Ala Glu Gln Val Leu Arg Asp Leu Gly His Thr 20 25 30 Val Phe Ser Pro Arg Glu Asn Gln Leu Pro Glu Val Glu Phe Gly Ser 35 40 45 Phe Glu Trp Arg Thr Phe Val Phe Lys Asn Asp Leu Glu His Ile Lys 50 55 60 Trp Ala Asp Ile Thr Phe Gly Ile Ile Gly Asp Asn Tyr Asp Asp Thr 65 70 75 80 Gly Thr Ala Trp Glu Leu Gly Ala Ser Tyr Ile Leu Gly Lys Pro Val 85 90 95 Met Leu Phe Ser Pro Thr Gly Glu Ile Ile Asn Leu Met Ile Thr Asp 100 105 110 Ser Leu His Ala Tyr Phe Glu Asp Trp Asn Asp Val Glu Asn Tyr Asp 115 120 125 Phe Ala Thr Leu Pro Ile Lys Pro Tyr Leu Lys Ala Val Lys 130 135 140

Claims

1. A method for preparing 2,6-dichloropurine nucleoside, comprising the following steps: in a buffer solution, reacting the compound shown in formula (I) and 2,6-dichloropurine in the presence of an enzyme to obtain 2,6-dichloropurine nucleoside via the following reaction. in, The R group is a base, characterized in that the amino acid sequence of the enzyme is as shown in SEQ ID NO:1 in the sequence listing, the compound shown in formula (I) is uridine or guanosine, the buffer is phosphate buffer, the molar ratio of the compound shown in formula (I) to 2,6-dichloropurine is 1:1 to 3:1, and the reaction is carried out at a temperature of 40 to 70°C.

2. The preparation method according to claim 1, characterized in that, The preparation method satisfies one or more of the following conditions: a) The volume ratio of the buffer solution to 2,6-dichloropurine is 0.03 L / g; b) The concentration of the buffer solution is 50 mM; c) The pH of the buffer solution is 7.0; d) When the base is purine, the carbon atom at position 2 of the ribose in the compound shown in formula (I) is connected to the nitrogen atom at position 9 of the purine; when the base is pyrimidine, the carbon atom at position 2 of the ribose in the compound shown in formula (I) is connected to the nitrogen atom at position 1 of the pyrimidine. e) The mass ratio of 2,6-dichloropurine to the enzyme is 1:0.1 to 1:1; f) The reaction time is 4 to 40 hours.

3. The preparation method according to claim 2, characterized in that, The preparation method satisfies one or more of the following conditions: g) The buffer solution is a potassium phosphate buffer solution; h) The molar ratio of the compound shown in formula (I) to 2,6-dichloropurine is 1.5:1 to 3:1; i) The mass ratio of 2,6-dichloropurine to enzyme is 1:0.1 to 1:0.4; j) The reaction time is 20-40 hours.

4. The preparation method according to claim 3, characterized in that, The preparation method satisfies one or more of the following conditions: k) The reaction is carried out at a temperature of 50°C or 60°C; l) The molar ratio of the compound shown in formula (I) to 2,6-dichloropurine is 1.5:1; The mass ratio of 2,6-dichloropurine to enzyme in m) is 1:0.1; The reaction time for n) is 40 hours.

5. The preparation method according to claim 1, characterized in that, The buffer solution is a 50 mM, pH 7.0 potassium phosphate buffer; the volume ratio of the buffer solution to 2,6-dichloropurine is 0.03 L / g; the mass ratio of 2,6-dichloropurine to ribotransferase is 1:0.1 to 1:0.4; the compound represented by formula (I) is uridine; the molar ratio of the compound represented by formula (I) to 2,6-dichloropurine is 1.5:1; the reaction is carried out at a temperature of 60°C.

6. The preparation method according to claim 1, characterized in that, The buffer solution is a 50 mM, pH 7.0 potassium phosphate buffer; the volume ratio of the buffer solution to 2,6-dichloropurine is 0.03 L / g; the mass ratio of 2,6-dichloropurine to ribotransferase is 1:0.1; the compound represented by formula (I) is uridine; the molar ratio of the compound represented by formula (I) to 2,6-dichloropurine is 1.5:1; the reaction is carried out at a temperature of 60°C.

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