A transaminase mutant and application thereof in preparation of sitagliptin intermediates

Patent metadata

Field	Value
Publication number	US-2022396816-A1
Application number	US-202117778627-A
Country	US
Kind code	A1
Filing date	Jan 21, 2021
Priority date	Oct 26, 2020
Publication date	Dec 15, 2022
Grant date	—

Abstract

The present invention discloses a transaminase mutant and application thereof in preparation of sitagliptin intermediates, the transaminase mutant is obtained by substitution of tyrosine with proline at position 74, substitution of glutamic acid with aspartic acid at position 228, substitution of leucine with alanine at position 254 and substitution of methionine with threonine at position 290 of the amino acid sequence shown in SEQ ID NO: 2. The present invention uses wet cells or a purified transaminase as a biocatalyst and a sitagliptin precursor ketone or a prochiral carbonyl compound as a substrate to prepare a sitagliptin intermediate or a sitagliptin ester intermediate; the total yield of the method reaches about 82%, and e.e. value of the product reaches 99%.

First claim

1 . A transaminase mutant, wherein the transaminase mutant is obtained by substitution of tyrosine with proline at position 74, substitution of glutamic acid with aspartic acid at position 228, substitution of leucine with alanine at position 254 and substitution of methionine with threonine at position 290 of the amino acid sequence shown in SEQ ID NO: 2.

2 . An encoding gene of the transaminase mutant as claimed in claim 1 , wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 3.

3 . A recombinant genetically engineered strain transformed by the encoding gene of the transaminase mutant as claimed in claim 2 .

4 . An application of the transaminase mutant as claimed in claim 1 in synthesizing a sitagliptin intermediate with a sitagliptin precursor ketone by biocatalysis.

5 . The application as claimed in claim 4 , wherein the application is carried out as follows: wet cells or a purified transaminase as a biocatalyst, [1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione as a substrate, dimethyl sulfoxide as a cosolvent, pyridoxal phosphate as a coenzyme, isopropyl amine as a cosubstrate, and a pH 8-9 triethanolamine buffer as a reaction medium are used to construct a reaction system, a biocatalytic reaction is carried out at 30-45° C. and 100-250 r/min, after the reaction is completed, the reaction solution is subjected to separation and purification to obtain (R)-3-amino-1-(1-piperidinyl)-4-(2,4,5-trifluorophenyl)-1-butanone; in which, the wet wells are obtained by fermentation culture of the recombinant genetically engineered bacteria containing the encoding gene of the transaminase mutant, and the purified transaminase is obtained by subjecting the wet cells to ultrasonication and then extraction.

6 . The application as claimed in claim 5 , wherein in the reaction system, the amount of the wet cells is 10˜100 g/L, the amount of the purified transaminase is 0.01-1.0 g/L, the final concentration of the substrate is 2˜50 g/L, the final concentration of dimethyl sulfoxide is 10-40% (v/v), the final concentration of pyridoxal phosphate is 0.5 g/L, and the final concentration of isopropyl amine is 10 g/L.

7 . An application of the transaminase mutant as claimed in claim 1 in synthesizing a sitagliptin ester intermediate with a prochiral carbonyl compound by biocatalysis, wherein the prochiral carbonyl compound is one selected from the group consisting of the following compounds: 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester, 3-carbonyl-4-(2,4,5-Trifluorophenyl)-butyric acid propyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid isopropyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid ethyl ester, 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid isobutyl ester and 3-carbonyl-4-(2,4,5-trifluorophenyl)-butyric acid benzyl ester.

8 . The application as claimed in claim 7 , wherein the application is carried out as follows: wet cells as a biocatalyst, the prochiral carbonyl compound as a substrate, dimethyl sulfoxide as a cosolvent, pyridoxal phosphate as a coenzyme, isopropyl amine as a cosubstrate, and a pH 8-9 triethanolamine buffer as a reaction medium are used to construct a reaction system, a biocatalytic reaction is carried out at 25-35° C. and 100-250 r/min, after the reaction is completed, the reaction solution is subjected to separation and purification to obtain the sitagliptin ester intermediate; in which, the wet wells are obtained by fermentation culture of the recombinant genetically engineered bacteria containing the encoding gene of the transaminase mutant.

9 . The application as claimed in claim 8 , wherein in the reaction system, the amount of the wet cells is 10˜100 g/L, the final concentration of the substrate is 2˜60 g/L, the final concentration of dimethyl sulfoxide is 10-40% (v/v), the final concentration of pyridoxal phosphate is 0.5 g/L, and the final concentration of isopropyl amine is 10 g/L.

10 . The application as claimed in claim 5 , wherein the wet cells are prepared as follows: the recombinant Escherichia coli strain containing the encoding gene of the transaminase mutant is inoculated into LB liquid medium containing 50 μg/ml kanamycin, cultured at 37° C. and 200 rpm for 12 hours, the resulting inoculum is inoculated into fresh LB liquid medium containing 50 μg/ml kanamycin with 1% incubating volume and cultured at 37° C. and 150 rpm; when OD600 of the cells reaches 0.6-0.8, IPTG is added with the final concentration of 0.1 mM, and the bacteria solution is subjected to induction culture at 28° C. for 12 hours; the resulting solution is subjected to centrifugation at 4° C. and 5000 rpm for 20 min, the resulting supernatant is discarded and sediment is collected, thereby obtaining the wet cells.

Assignees

• Zhejiang Yongtai Technology Co Ltd
• Univ Zhejiang Technology
• Zhejiang yongtai pharmaceutical co ltd

Inventors

• Liu Zhiqiang
• Cheng Feng
• Zhang Xiaojian
• Jia Dongxu
• Zheng Yuguo
• He Renbao
• Jin Yizhong
• Shao Hongming
• Lin Jiaohua
• Zhang Feng

Classifications

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12P17/182 Primary

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title