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(R)-omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate

A technology of mutants and transaminases, which is applied in the application field of preparing sitagliptin intermediate 3-amino-1--4-butan-1-one, can solve the problem of lack of competitive advantage, unfavorable large-scale application, Low substrate concentration and other issues, to achieve high stereoselectivity, high substrate tolerance, high enzyme activity

Active Publication Date: 2020-08-14
ZHEJIANG UNIV OF TECH +2
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  • Abstract
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Problems solved by technology

Chinese patent CN108586346A uses 1-(piperazin-1-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione as a substrate, catalyzed by transaminase (as shown in formula I ), hydrolysis, amino protection, condensation, deprotection, the substrate concentration catalyzed by transaminase is only 367mM, the conversion rate is 99.9%, and the substrate concentration is low, which is not conducive to large-scale industrial application
[0007] In the case that the current asymmetric synthesis of sitagliptin technology is monopolized by Merck and Codexis, the substrate concentration of other technical routes for the preparation of sitagliptin by (R)-ω-TA still does not have a competitive advantage

Method used

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  • (R)-omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate
  • (R)-omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate
  • (R)-omega-transaminase mutant and application thereof in preparation of sitagliptin intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Screening, determination of stereoselectivity and precise determination of enzyme activity of novel ω-TA

[0031] 1. Enzyme source and gene synthesis

[0032] Using the amino acid sequence of the commercial enzyme (R)-ω-TA 117 as a template, three ω-TA wild-type enzyme strains were obtained from the NCBI database by gene mining, namely Capronia epimyces CBS 606.96TA (CeTA for short, GenBank number XP_007730450.1) , Mycolicibacterium litorale TA (abbreviated as MlTA, GenBank number AQT79271) and Exophila xenobiotica TA (abbreviated as ExTA, GenBank number XP_013320890).

[0033] The homology between the above three enzymes and (R)-ω-TA 117 is 44.86%, 51.94% and 42.41%, respectively. Codon optimization was carried out according to the codon preference of E.coli, and three strains of enzymes were synthesized by the method of whole gene synthesis, 6×His-tag tags were added at the end of the nucleic acid sequence, and restriction sites Xho I and Nco I were added at ...

Embodiment 2

[0058] Example 2 Construction and Screening of CeTA Single Point Mutants

[0059] 1. Mutant construction

[0060] The novel (R)-omega-TA screened is subjected to single-point mutation, and mutation primers are designed according to the nucleotide sequence of CeTA (as shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1). Rapid PCR technology, using the recombinant vector pET28b / CeTA as a template, introduces a single mutation to the 77th position of the amino acid sequence of CeTA, and the primers are:

[0061] Forward primer: GACGACCACATCTCT NNK CTGGAAAAATCTTGC (the underline is the mutant base, as shown in SEQ ID NO.4);

[0062] Reverse primer: GCAAGATTTTTCCAG MNN AGAGATGTGGTCGTC (the underline is the mutant base, as shown in SEQ ID NO.5)

[0063] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μ...

Embodiment 3

[0079] Example 3 Construction and Screening of CeTA Two-site Mutant

[0080] Single mutant CeTA constructed according to Example 2 1 Sequence design of mutation primers for site-directed mutagenesis, using rapid PCR technology to recombinant vector pET28b / CeTA 1 as template, for CeTA 1 A single mutation is introduced at position 181 of the amino acid sequence, and the primers are:

[0081] Forward primer: CCGACCGTTAAAAAC NNK CAGTGGGGTGACCTG (the underline is the mutated base, as shown in SEQ ID NO.6);

[0082] Reverse primer: CAGGTCACCCCCACTG MNN GTTTTTAACGGTCGG (the underline is the mutated base, as shown in SEQ ID NO.7).

[0083] The PCR reaction system is the same as the "construction of mutants" in Part 1 of Example 2.

[0084] The PCR amplification conditions were: 95°C for 3min; (95°C for 15s, 50°C for 15s, 63°C for 6.5min) 30 cycles; 72°C for 5min.

[0085] The PCR product was transformed into E.coli BL21(DE3) competent cells, and a single clone was picked in L...

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Abstract

The invention discloses a (R)-omega-transaminase mutant and an application thereof in preparation of a sitagliptin intermediate. The mutant is obtained through multipoint mutation of arginine at the 77th site, leucine at the 181st site, arginine at the 130th site, tyrosine at the 139th site and threonine at the 273th site of an amino acid sequence shown as SEQ ID NO.1. According to the invention,a novel (R)-omega-TA recombinase is screened through a gene mining technology; molecular modification is carried out through a protein engineering technology; an (R)-omega-TA mutant catalyst with highenzyme activity, high substrate tolerance and high stereoselectivity is obtained; and the mutant can be used for asymmetric catalytic synthesis of the sitagliptin intermediate (R)-3-amino-1-(pyrrolidine-1-yl)-4-(2, 4, 5-trifluorophenyl) butan-1-one by taking a precursor ketone analogue 1-(pyrrolidine-1-yl)-4-(2, 4, 5-trifluorophenyl)-1, 3-butanedione as a substrate, and has higher conversion rate.

Description

technical field [0001] The present invention relates to the field of biochemical technology, in particular to a (R)-ω-transaminase mutant and its application in the preparation of sitagliptin intermediates, especially in the preparation of sitagliptin intermediates (R)-3 -Application of amino-1-(pyrrolidin-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one. Background technique [0002] Sitagliptin, English name sitagliptin, full name (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo [4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one, a dipeptidyl group researched and developed by Merck and Codexis Peptidase-4 (DPP-4) inhibitors, which can control blood sugar levels by protecting endogenous incretins and enhancing their effects, are a therapeutic drug with great potential for type Ⅱ diabetes. [0003] The preparation of sitagliptin is mainly an asymmetric synthesis method, usually using transaminase as a biocatalyst for biotransformation. Transaminase (...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/10C12R1/19
CPCC12N9/1096C12N15/70C12P17/10C12Y206/01
Inventor 柳志强彭晨贾东旭李军良程峰张晓健郑裕国何人宝金逸中林娇华
Owner ZHEJIANG UNIV OF TECH
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