Omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate

Abstract

The invention discloses an omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate. The omega-transaminase mutant is obtained by performing single mutation on the56 position or the 134 position of an amino acid sequence as shown in a SEQ ID No.2; the 56 phenylalanine is substituted by valine, histidine or tyrosine; and the 134 threonine is substituted by glycine. The omega-transaminase mutant has relatively high enzyme activity which is higher than 300U / g and is at least 2.7 times that of a wild type since the omega-transaminase mutant is subjected to site-specific mutagenesis by acquiring certain sites possibly influencing the catalytic activity of the omega-transaminase mutant through molecular docking, homologous modeling and other modes, can be used for efficiently catalyzing precursor ketone 1-piperidine-4-(2,4,5-trifluorophenyl)-1,3-dibutanone of the sitagliptin intermediate to synthesize the sitagliptin intermediate (R)-3-amino-1-piperidine-4-(2,4,5-trifluorophenyl)-1-butanone, and has a conversion rate of 85 percent.

Description

Technical field [0001] The present invention relates to the technical field of biochemical industry, in particular to an ω-transaminase mutant and its application in the preparation of sitagliptin intermediates, in particular to the preparation of sitagliptin intermediate 1-piperidine-4-( 2,4,5-trifluorophenyl)-1,3-dibutyl ketone. Background technique [0002] Sitagliptin was developed and developed by Merck and Codexis. It is the first dipeptidyl peptidase-4 (DPP-4) inhibitor approved by the FDA for the treatment of type 2 diabetes (October 2016) ), which is also the active ingredient of Genovie (Sitagliptin Hydrochloride Tablets, JANUVIA), an oral treatment for type II diabetes. Sitagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor. It inhibits its activity by binding the amide moiety to the active part of DPP-4, prolongs the half-life of Incretin, and makes GLP in plasma -1 and GIP activity increases while only slightly increasing their content, but it will not cause sid...

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Problems solved by technology

In this synthesis method, asymmetric hydrogenation catalyzed by ruthenium is required, the catalyst is expensive, the total yield is only 52%, and high-pressure hydrogen is used in the process, and the stereoselectivity is not high

[0006] The international patent application WO2007050485 discloses the synthesis method of the sitagliptin intermediate of Merck, which uses a chiral germanium catalyst to asymmetric hydrogenate enamines to synthesize chiral amines, with a yield of 84% and an ee value of 94%. However, this method requires the use of expensive germanium chiral catalysts, and it is difficult to remove and recover

[0007] Chinese patent application document CN102838511A discloses the production method of sitagliptin intermediate of Zhejiang Haixiang Pharmaceutical Co., Ltd., using Grignard reagent to perform nucleophilic substitution of chiral epichlorohydrin, and then using cyanide to perform substitution hydrolysis to synthesize beta-hydroxy acid. The total yield of this method is only 40%, and highly toxic cyanide is used, so the application is limited

[0008] Chinese patent application document CN102485718A discloses the route of Zhejiang Haixiang Pharmaceutical Co., Ltd. for the synthesis of sitagliptin intermediates, which is synthesized by using methionine as a chiral source, but the yield is only 14%.

[0009] Chinese patent application document CN103014081A discloses that Suzhou Hanzyme Company uses transaminase to transaminate 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester into (R)-3-amino- 4-(2,4,5-trifluorophenyl)-butyric acid methyl ester, but did not disclose the sequence and cloning method of the specific transaminase

However, there are still some ω-transaminases with low enzymatic activity and low conversion rate for specific substrates, especially when the substrate contains large groups such as benzene rings.

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