Method for synthesizing 2-amino-6-chloroguanine based on amide hydrolase

Abstract

The invention discloses a method for synthesizing 2-amino-6-clobird purine based on amide hydrolase, which specifically comprises the following steps: adding the amide hydrolase into a 2-formamido-6-clobird purine aqueous solution, and reacting for 3-5 hours to obtain the 2-amino-6-clobird purine, and the amide hydrolase is self-prepared recombinant amide hydrolase. The 2-amino-6-chlorobird purine prepared by the method provided by the invention is high in purity, low in guanine impurity content, short in reaction time consumption and low in generated wastewater amount. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis of pharmaceutical intermediates, and particularly relates to a method for synthesizing 2-amino-6-chloroguanine based on amidohydrolase. Background Art

[0002] 2-Amino-6-chloroguanine is an important pharmaceutical intermediate. It is an important intermediate for the synthesis of purine antiviral drugs such as famciclovir, abacavir, and penciclovir. It can also be used in the synthesis of anti-cancer and antihypertensive drugs.

[0003] Patent document CN107312003A discloses a method for synthesizing high-purity 2-amino-6-chloroguanine, using guanine as a raw material, stirring and reacting with ViIsmeier reagent at 150-160°C in an organic solvent for 1-2 hours to obtain a chloro intermediate 2-formamido-6-chloroguanine, and the chloro intermediate is hydrolyzed under the action of an alkaline aqueous solution, and separated and purified to obtain 2-amino-6-chloroguanine. Among them, ViIsmeier reagent is obtained by stirring and reacting bis(trichloromethyl) carbonate with N,N-disubstituted amide at 0-10°C for 1 hour. The 2-amino-6-chloroguanine prepared by the method has a purity of more than 99.0% as determined by HPLC, and in the disclosed specific embodiment, the purity is as high as 99.6%.

[0004] Patent document CN114437071A discloses a preparation process of purine derivatives, comprising the following steps: (1) chlorination: add dichloroethane, guanine and dimethylformamide to a reaction kettle, heat to 45-80°C, slowly drop phosphorus oxychloride, keep warm for 2-4h, then heat to 60-90°C and stir for 8-15h, cool to 15°C-45°C; separate the solid and liquid in the chlorination reaction liquid to obtain a chloride intermediate and a mother liquor; (2) hydrolysis: add water to a reaction kettle containing the chloride intermediate, cool to 10-30°C, drop liquid alkali, control the pH of the solution in the reaction kettle to 3.0-5.0, heat to 55-75°C, stir for 4-8h, add acid to adjust the pH to 0-1.0, react for 2-5h, centrifuge and obtain a crude purine derivative 2-amino-6-chloropurine. The weight yield of 2-amino-6-chloropurine prepared according to the method can reach 100%.

[0005] Patent document CN114031620A discloses a method for producing high-purity 2-amino-6-chloroguanine, which comprises: (1) using guanine, DMF, and a chlorination agent to carry out a chlorination reaction; (2) quenching the chlorination reaction; (3) acid hydrolysis to obtain 2-formamide-6-chloroguanine; (4) alkaline hydrolysis to obtain a crude 2-amino-6-chloroguanine; (5) refining using DMF to obtain a product with a purity of >99% and a guanine content of <0.2%.

[0006] It can be understood from the prior art that the conventional synthesis method of 2-amino-6-chloroguanine is to use guanine as the starting material, and guanine first reacts with N,N-dimethylformamide under the action of POCI3 to generate 2-dimethylaminomethylimino-6-chloroguanine, and then hydrolyzes under acidic conditions to obtain the chlorinated intermediate 2-formamido-6-chloroguanine, and then hydrolyzes it to obtain 2-amino-6-chloroguanine through alkaline hydrolysis. The total yield of this method is about 55%, the operation is relatively cumbersome, the amount of wastewater is high, and the three wastes are seriously polluted. In addition, the inventors found that the impurity guanine will be produced during the alkaline hydrolysis process, and it is difficult to remove, resulting in a high level of guanine impurities in the finished product.

[0007] Based on the above deficiencies in the prior art, the present invention provides a method for synthesizing 2-amino-6-chloroguanine, which uses amidohydrolase to hydrolyze the chlorinated intermediate 2-formamido-6-chloroguanine. Compared with directly using alkaline hydrolysis in the prior art, the product guanine prepared by the method provided by the present invention has a low impurity content, a short reaction time, and a small amount of wastewater generated during the preparation process. Summary of the invention

[0008] One object of the present invention is to provide a method for synthesizing 2-amino-6-chloroguanine, wherein the method uses the recombinant amide hydrolase prepared by the present invention to act on the chloro intermediate 2-formamido-6-chloroguanine to prepare 2-amino-6-chloroguanine; another object of the present invention is to provide a use of a recombinant amide hydrolase in the preparation of 2-amino-6-chloroguanine; and a further object of the present invention is to provide a recombinant amide hydrolase and a preparation method thereof.

[0009] In a first aspect, the present invention provides a method for synthesizing 2-amino-6-chloroguanine, characterized in that the method comprises: adding amide hydrolase to an aqueous solution of 2-formamido-6-chloroguanine, reacting for 3-5 hours, and obtaining 2-amino-6-chloroguanine.

[0010] The reaction process is as follows:

[0011]

[0012] Preferably, the synthesis method of 2-amino-6-chloroguanine comprises the following steps:

[0013] (1) dissolving 2-formamido-6-chloroguanine in water at room temperature to form an aqueous solution;

[0014] (2) Raise the temperature to 37°C and adjust the pH to 7.0 using alkali;

[0015] (3) adding amide hydrolase and keeping the reaction warm for 3-5 hours;

[0016] (4) Filter by suction to obtain 2-amino-6-chloroguanine.

[0017] The 2-formamido-6-chloroguanine in the method can be prepared according to any method disclosed in the prior art.

[0018] Preferably, in a specific embodiment of the present invention, the 2-formamido-6-chloroguanine is prepared by the following method: using guanine as a raw material, reacting with dichloroethane and N,N-dimethylformamide under the action of phosphorus oxychloride to generate 2-dimethylaminomethylimino-6-chloroguanine, and then hydrolyzing under acidic conditions to obtain 2-formamido-6-chloroguanine.

[0019] The reaction process is as follows:

[0020]

[0021] The amide hydrolase described in the present invention can be any amide hydrolase that can be purchased commercially. In a preferred embodiment of the present invention, the amide hydrolase is a recombinant amide hydrolase prepared by the technicians of the present invention.

[0022] The sequence of the recombinant amidohydrolase is selected from one of the following amino acid sequences:

[0023] a) consisting of the amino acid sequence of SEQ ID NO: 1;

[0024] b) a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ ID NO: 1;

[0025] c) a sequence that differs from SEQ ID NO: 1 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid.

[0026] The amino acid sequence shown in SEQ ID NO: 1:

[0027] MQPNDITFFQRFQDDILAGRKTITIRDESESHFKTGDVLRVGRFEDDGY FCTIEVTATSTVTLDTLTEKHAEQENMTLTELKKVIADIYPGQTQFYVIEFKC L.

[0028] In a specific embodiment of the present invention, the recombinant amidohydrolase is prepared by the following method:

[0029] The nucleotide sequence shown in SEQ ID NO.2 was connected to a plasmid to obtain a recombinant plasmid, which was then transformed into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium, and the recombinant bacterium was cultured to express the recombinant amide hydrolase.

[0030] The nucleotide sequence shown in SEQ ID NO:2:

[0031] ATGCAGCCAAACGACATCACTTTTTTTCAACGTTTCCAGGATGACATTCTGGCTGGGCGTAAAACCATCACCATCCGCGACGAGTCTGAATCGCACTTCAAAACGGGTGATGTGCTTCGTGTCGGACGTTTTGAAGATGACGGTTATTTTTGCACG ATTGAAGTCACCGCAACCTCAACCGTAACGCTGGATACGCTGACTGAAAAACATGCAGAGCAGGAAAATATGACCCTGACTGAACTGAAAAAAGTCATTGCTGACATCTATCCCGGTCAGACACAATTTTATGTGATTGAATTTAAATGTCTTTAA.

[0032] Further preferably, the preparation method of the recombinant amidohydrolase is as follows:

[0033] (1) The nucleotide sequence shown in SEQ ID NO.2 was digested with pET-28a(+) plasmid and then ligated, and the ligation product was transformed into BL21(DE3) to obtain recombinant bacteria;

[0034] (2) overnight culture, screening positive clones, and obtaining the recombinant expression plasmid pET-28a(+)-amidase;

[0035] (3) Extract the correct recombinant expression plasmid identified by sequencing and inoculate it into LB, screen the successfully constructed transformants using kanamycin resistance, and induce with IPTG to obtain the recombinant protein;

[0036] (4) Separating and purifying the recombinant protein to obtain recombinant amide hydrolase.

[0037] In a second aspect, the present invention provides a recombinant amide hydrolase, characterized in that the sequence of the recombinant amide hydrolase is selected from one of the following amino acid sequences:

[0038] a) consisting of the amino acid sequence of SEQ ID NO: 1;

[0039] b) a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ ID NO: 1;

[0040] c) a sequence that differs from SEQ ID NO: 1 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid.

[0041] In a third aspect, the present invention provides a method for preparing a recombinant amide hydrolase, comprising connecting the nucleotide sequence shown in SEQ ID NO.2 to a plasmid to obtain a recombinant plasmid, then transforming it into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium, and culturing the recombinant bacterium to express the amide hydrolase.

[0042] Preferably, the preparation method of the amidohydrolase is as follows:

[0043] (1) The nucleotide sequence shown in SEQ ID NO.2 was digested with pET-28a(+) plasmid and then ligated, and the ligation product was transformed into BL21(DE3) to obtain recombinant bacteria;

[0044] (2) overnight culture, screening positive clones, and obtaining the recombinant expression plasmid pET-28a(+)-amidase;

[0045] (3) Extract the correct recombinant expression plasmid identified by sequencing and inoculate it into LB, screen the successfully constructed transformants using kanamycin resistance, and induce with IPTG to obtain the recombinant protein;

[0046] (4) Separating and purifying the recombinant protein to obtain amide hydrolase.

[0047] In a fourth aspect, the present invention provides a use of a recombinant amidohydrolase in the preparation of 2-amino-6-chloroguanine.

[0048] Preferably, the substrate of the recombinant amidohydrolase is 2-formamido-6-chloroguanine.

[0049] The method for preparing 2-formamido-6-chloroguanine based on recombinant amido hydrolase provided by the present invention has the following technical effects: 1. Compared with conventional alkaline hydrolysis, the product obtained by hydrolysis using the recombinant amido hydrolase provided by the present invention has high purity, which can reach 99.7% without refining, and the guanine content is lower than 0.2%, which is a significant improvement over the prior art; 2. Compared with conventional alkaline hydrolysis, the method for preparing 2-formamido-6-chloroguanine by hydrolysis using the recombinant amido hydrolase provided by the present invention has a mild reaction and is suitable for large-scale industrial production; 3. Compared with commercially available enzymes with the same effect, the recombinant amido hydrolase provided by the present invention has higher reaction activity. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Preparation Example 1 Preparation of amide hydrolase

[0052] S1: The amidohydrolase gene (SEQ ID NO: 1) and the pET-28a(+) plasmid were digested by conventional methods in the art and then ligated, and the ligation product was transformed into BL21(DE3) to obtain a recombinant bacterium;

[0053] S2: overnight culture, select positive clones for identification, and name the correctly identified recombinant expression plasmid vector: pET-28a(+)-amidase;

[0054] S3: Inoculate the above recombinant bacteria into 50 mL of LB containing 50 mg / L kanamycin, culture at 37°C, 220 rpm for 6-8 h, then inoculate 100 mL of LB at a 2% inoculum and culture at 37°C. When OD600 reaches 0.6-0.8, add IPTG with a final concentration of 0.2 mM, and induce at 18°C, 220 rpm for 16-18 h; collect the cells by centrifugation, discard the supernatant, add an appropriate amount of phosphate buffer to resuspend the cells to OD600 of 100, add the resuspended cells to a high-pressure homogenizer for crushing at a pressure of 700-800 bar until the bacterial solution becomes clear, collect the crushed cells, and centrifuge to obtain the supernatant to obtain the crude enzyme solution of amide hydrolase.

[0055] The amino acid sequence of the amidohydrolase prepared by the method is shown in SEQ ID NO:2.

[0056] Preparation Example 2 Preparation of 2-Formamido-6-chloroguanine

[0057] Add 200gDMF, 200g dichloroethane to the reaction bottle, then slowly drop 200g phosphorus oxychloride, heat to 40-50℃, start to add 50g guanine, the feeding time is about 95-115 minutes, and the temperature is controlled at ≤85℃ when the feeding is completed. After the feeding is completed, continue stirring for 45 minutes, then heat to 90-94℃, reflux, stir and react for 5 hours, the reaction is completed, cool to 40±5℃, discharge, and put the feed liquid into 2L ice water that has been pre-cooled to 0-10℃ in batches for hydrolysis. After the hydrolysis is completed, stand and separate, collect the aqueous phase, neutralize with powdered sodium carbonate at 0-10℃ to the feed liquid pH = 3, heat to 70℃, keep warm for 3h, cool to room temperature, filter and wash to obtain 2-formamido-6-chloroguanine wet product.

[0058] Preparation of 2-amino-6-chloroguanine

[0059] Example 1

[0060] The wet product of 2-formamido-6-chloroguanine obtained in Preparation Example 2 was added into 200 g of water, and the temperature was raised to 37°C. The pH value of the solution was adjusted to 7.0 using 5% sodium hydroxide. The amide hydrolase obtained in Preparation Example 1 was added, and the reaction was kept warm for 3 hours. After the reaction was completed, the filter cake was obtained, which was 2-amino-6-chloroguanine. The yield was 85%, the purity was 99.7%, and the guanine content in the product was 0.2%.

[0061] Comparative Example 1

[0062] Amidohydrolase (recombinant Edwardsiella peptide deformylase) was purchased from Wuhan Huamei Bioengineering Co., Ltd., with the product number CSB-YP017707EJO. The amino acid sequence of the enzyme disclosed in the specification is as follows:

[0063] MAVLQVLHFPDERLRTIAKPVHEVTPEIQRIVDDMFETMYAEEGIGLAATQVDIHQRIIVIDVSENRDQRLVLINPELLQHDGEAGIEEGCLSVPEQRALVTRAENIKVRALDRDGKSFELETDGLLAICIQHEMDHLMGKLFIDYLSPLKRQRIRQKLEKLYKQQARAVN

[0064] The method of use is the same as that of Example 1. The wet 2-formamido-6-chloroguanine obtained in Preparation Example 2 is added into 200 g of water, the temperature is raised to 37°C, the pH value of the solution is adjusted to 7.0 using 5% sodium hydroxide, 1 ml of the recombinant Edwardsiella peptide deformylase is added, the temperature is kept for 6 hours, and after the reaction is completed, the filter cake is 2-amino-6-chloroguanine, the yield is 83%, the purity is 97%, and the guanine content in the product is 0.5%.

[0065] The present technicians calculated the enzyme activity based on the concentration of 2-amino-6-chloroguanine obtained by measurement, and the enzyme activity is defined as the amount of product generated per millimole per minute per ml of enzyme solution. The enzyme activity data of the amidohydrolase provided by the present invention and the commercially available amidohydrolase (recombinant Edwardsiella peptide deformylase) are shown in the following table.

[0066] Table 1 Enzyme activity data

[0067] Amide hydrolase of the present invention Recombinant Edwardsiella peptide deformylase Enzyme activity (mM / min / ml) 1.66 0.83 Enzyme activity ratio 100％ 50％

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synthesizing 2-amino-6-chloroguanine, characterized in that: The method comprises the following steps: adding amide hydrolase to a 2-formamido-6-chloroguanine aqueous solution, reacting for 3-5 hours, and obtaining 2-amino-6-chloroguanine.

2. The synthesis method according to claim 1, characterized in that The synthetic method of 2-amino-6-chloroguanine comprises the following steps: (1) dissolving 2-formamido-6-chloroguanine in water at room temperature to form an aqueous solution; (2) Raise the temperature to 37°C and adjust the pH to 7.0 using alkali; (3) adding amide hydrolase and keeping the reaction warm for 3-5 hours; (4) Filter by suction to obtain 2-amino-6-chloroguanine.

3. The synthesis method according to claim 1 or 2, characterized in that The 2-formamido-6-chloroguanine is prepared by the following method: guanine is used as a raw material, reacted with dichloroethane and N,N-dimethylformamide under the action of phosphorus oxychloride to generate 2-dimethylaminomethylimino-6-chloroguanine, and then hydrolyzed under acidic conditions to obtain 2-formamido-6-chloroguanine.

4. The synthesis method according to claim 1, characterized in that The amide hydrolase is a recombinant amide hydrolase, and the sequence of the recombinant amide hydrolase is selected from one of the following amino acid sequences: a) consisting of the amino acid sequence of SEQ ID NO: 1; b) a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ ID NO: 1; c) a sequence that differs from SEQ ID NO: 1 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid.

5. The synthesis method according to claim 4, characterized in that The recombinant amidohydrolase is prepared by the following method: The nucleotide sequence shown in SEQ ID NO.2 was connected to a plasmid to obtain a recombinant plasmid, which was then transformed into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium, and the recombinant bacterium was cultured to express the recombinant amide hydrolase.

6. The synthesis method according to claim 5, characterized in that The preparation method of the recombinant amidohydrolase is as follows: (1) The nucleotide sequence shown in SEQ ID NO.2 was digested with pET-28a(+) plasmid and then ligated, and the ligation product was transformed into BL21(DE3) to obtain recombinant bacteria; (2) overnight culture, screening positive clones, and obtaining the recombinant expression plasmid pET-28a(+)-amidase; (3) Extract the correct recombinant expression plasmid identified by sequencing and inoculate it into LB, screen the successfully constructed transformants using kanamycin resistance, and induce with IPTG to obtain the recombinant protein; (4) Separating and purifying the recombinant protein to obtain recombinant amide hydrolase.

7. A recombinant amidohydrolase, characterized in that The sequence of the recombinant amidohydrolase is selected from one of the following amino acid sequences: a) consisting of the amino acid sequence of SEQ ID NO: 1; b) a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequence of SEQ ID NO: 1; c) a sequence that differs from SEQ ID NO: 1 in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid.

8. A method for preparing the recombinant amide hydrolase according to claim 7, comprising connecting the nucleotide sequence shown in SEQ ID NO.2 to a plasmid to obtain a recombinant plasmid, then transforming it into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium, and culturing the recombinant bacterium to express the amide hydrolase.

9. The preparation method according to claim 8, characterized in that: The preparation method of the amidohydrolase is as follows: (1) The nucleotide sequence shown in SEQ ID NO.2 was digested with pET-28a(+) plasmid and then ligated, and the ligation product was transformed into BL21(DE3) to obtain recombinant bacteria; (2) overnight culture, screening positive clones, and obtaining the recombinant expression plasmid pET-28a(+)-amidase; (3) Extract the correct recombinant expression plasmid identified by sequencing and inoculate it into LB, screen the successfully constructed transformants using kanamycin resistance, and induce with IPTG to obtain the recombinant protein; (4) Separating and purifying the recombinant protein to obtain amide hydrolase.

10. Use of the recombinant amidohydrolase according to claim 7 in the preparation of 2-amino-6-chloroguanine.

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