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Transaminase mutant and applications of transaminase mutant in Sitagliptin synthesis

A mutant and transaminase technology, applied in the fields of genetic engineering and enzyme catalysis, can solve the problems of expensive reagents, chiral catalysts and chiral auxiliary agents, etc., and achieve the effect of improving enzyme activity

Active Publication Date: 2020-12-18
SHANXI WEIQIDA PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent WO2004087650A2 uses a chiral phosphorous ruthenium catalyst to carry out asymmetric catalytic hydrogenation of sitagliptin precursor ketone to construct a chiral secondary alcohol and then convert it into a chiral secondary amine, but the reagents used in this route are relatively expensive
Patent WO2004085378A1 reported the new Sitagliptin synthesis method of Merck & Co., which won the US President's Green Chemistry Award. The key step is to use a chiral rhodium catalyst to asymmetric hydrogenate enamines to construct a chiral center, which has a simple route. , short steps, high conversion rate and optical purity, but the chiral catalysts and chiral auxiliary agents used are more expensive

Method used

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  • Transaminase mutant and applications of transaminase mutant in Sitagliptin synthesis
  • Transaminase mutant and applications of transaminase mutant in Sitagliptin synthesis
  • Transaminase mutant and applications of transaminase mutant in Sitagliptin synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The screening of embodiment 1 wild-type transaminase

[0069] 1. Enzyme mining and whole gene synthesis

[0070] Using bioinformatics analysis technology, six strains of (R)-ω-transaminase with potential catalytic ability of aromatic ring and its derivatives were excavated from NCBI database: OQE25192.1 (Penicillium steckii), XP_015409010.1 (Aspergillus nomiae), KAF4246444.1 (Aspergillus fumigatiaffinis), XP_013276042.1 (Rhinocladiella mackenziei), XP_023089462.1 (Aspergillus oryzae), XP_020123495.1 (Talaromyces atroroseus), sequence differences are as follows figure 1 shown.

[0071] The nucleic acid sequence was optimized according to the codon preference of Escherichia coli, and after the gene was synthesized, it was cloned into the NcoI and XhoI sites of the plasmid pET22b, and a His tag was added to the C-terminus of the amino acid sequence. Plasmids pET-PSATA(OQE25192.1), pET-ANATA(XP_015409010.1), pET-AFATA(KAF4246444.1), pET-PMATA(XP_013276042.1), pET-AOATA(XP...

Embodiment 2

[0086] Embodiment 2 Transaminase AFATA key site mutation and screening

[0087] 1. Construction of mutants

[0088] Using bioinformatics technology to analyze AFATA, it is determined that T57, R77, K179, E212, I237, T238 and T273 in the amino acid sequence are located in the substrate-cofactor binding region, G50, F51, H53, G54, L56, T57, K84, E115, I117, W147, L181, D185, T187, F191, D205 are dimer polypeptide binding interfaces, and these sites play key roles in the structure and function of the enzyme.

[0089] Since the sitagliptin precursor ketone contains both 1,2,4-triazolo[4,3-a]pyrazine and 2,4,5-trifluorophenyl structures, it reduces transaminase and substrate binding sites resistance. R77, K179, and E212 are related to enzyme catalysis and cofactor binding, and G50 and G54 are relatively small in steric hindrance and will not be changed. The remaining 16 sites were divided into three groups for combined mutations. The first group of mutations: F51V, T57AFG, I117A...

Embodiment 3

[0163] Example 3 The second round of mutation and screening of AFATA key sites

[0164] 1. The second round of mutant construction

[0165] Referring to the method of Example 2, based on the amino acid sequence shown in SEQ ID NO.3, the histidine at position 53 is mutated to leucine (H53L); or the threonine at position 57 is mutated to glycine (T57G) ; or mutation of 84th lysine to histidine (K84H); or mutation of 117th isoleucine to alanine (I117A); or mutation of 191st phenylalanine to alanine (F191A) ; or the 205th aspartic acid mutation to serine (D205S); or the 273th threonine mutation to serine (T273S).

[0166] For the H53L mutation, design forward and reverse primers:

[0167] Forward primer H53L-51: 5'-TGGATGAAGGCTTTATGCTTGGCGATGCGACC-3'

[0168] Reverse primer H53L-31: 5'-GGTCGCATCGCCAAGCATAAAGCCTTCATCCA-3'

[0169] For the T57G mutation, design forward and reverse primers:

[0170] Forward primer T57G-51: 5'-ATGCATGGCGATGCGGGCTATGATGTGACCAC-3'

[0171] Reverse...

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Abstract

The invention discloses a mutant of transaminase SEQ ID NO: 1. The mutant has significantly enhanced enzyme activity and high stereoselectivity; the mutant can efficiently catalyze (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one to synthesize the Sitagliptin, and the e.e. value of products is greater than 99.95%, so thatthe mutant has industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme catalysis, and in particular relates to a transaminase mutant and its application in the synthesis of sitagliptin. Background technique [0002] Sitagliptin, also known as sitagliptin, its full name is 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5 ,6,7,8-tetrahydro-3-trifluoromethyl-1,2,4-triazolo[4,3-a]pyrazine is the first dipeptide developed by Merck DPP-4 inhibitors, and their phosphorylated hydrates are currently type II diabetes drugs that occupy a huge market share. [0003] In the synthetic route of sitagliptin, the introduction of chiral (R)-amino group is one of the key steps. At present, methods including chiral source introduction, selective reduction, chiral induction and kinetic resolution have been developed. Among them, (2Z)-4-oxo-4-[3-(trifluoromethyl)-5,6- Dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7-(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2- Keton...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/70C12N1/21C12P17/18C12R1/19
CPCC12N9/1096C12Y206/01C12P17/182
Inventor 李春刚王金刚韦炎龙李树有彭艾琳秦一
Owner SHANXI WEIQIDA PHARMA IND
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