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CN-121991908-A - Application of alcohol dehydrogenase mutant in synthesis of axial chiral biaryl dimethanol

CN121991908ACN 121991908 ACN121991908 ACN 121991908ACN-121991908-A

Abstract

The invention relates to the technical fields of bioengineering and biocatalysis, in particular to an alcohol dehydrogenase mutant and application thereof. The invention discloses an alcohol dehydrogenase mutant CpAR-M5 (F85A/T125S/F192L/E209A/I213F), and the corresponding amino acid sequence is shown in SEQ ID No. 3. The method can be applied to dynamic kinetic resolution asymmetric reduction of biaryl dimethanol compounds to prepare S-configuration axial chiral biaryl dimethanol compounds, the yield can be up to 95%, the stereoselectivity can be up to 99% ee, and a green, efficient and high-stereoselectivity synthesis strategy is provided for the subsequent synthesis of the biaryl dimethanol compounds.

Inventors

  • HU YUJING
  • Peng Zhuoting
  • CHEN JIE
  • GAO XIAOLONG
  • FANG ZHENG
  • GUO KAI

Assignees

  • 南京工业大学

Dates

Publication Date
20260508
Application Date
20260330

Claims (10)

  1. 1. The alcohol dehydrogenase mutant is characterized in that the amino acid sequence of the alcohol dehydrogenase mutant CpAR-M5 is obtained by mutating phenylalanine at 85 th position to alanine, threonine at 125 th position to serine, phenylalanine at 192 th position to leucine, glutamic acid at 209 th position to alanine and isoleucine at 213 th position of wild type alcohol dehydrogenase CpAR.
  2. 2. The alcohol dehydrogenase mutant according to claim 1, wherein the wild-type alcohol dehydrogenase CpAR is derived from Monilia parapsilosis (STRAIN CDC/ATCC MYA-4646), the amino acid sequence of which is shown in SEQ ID No.2, and the corresponding nucleotide sequence encoding the wild-type alcohol dehydrogenase CpAR2 is shown in SEQ ID No. 1.
  3. 3. The alcohol dehydrogenase mutant CpAR according to claim 1, wherein the amino acid sequence of the alcohol dehydrogenase mutant is obtained by mutating phenylalanine at position 85, threonine at position 125, phenylalanine at position 192, glutamic acid at position 209, isoleucine at position 213 of wild-type alcohol dehydrogenase CpAR to alanine, and phenylalanine, and the amino acid sequence is shown in SEQ ID No. 3.
  4. 4. The alcohol dehydrogenase mutant of claim 3, wherein the nucleotide sequence corresponding to the alcohol dehydrogenase mutant is shown in SEQ ID NO. 4.
  5. 5. A recombinant expression vector comprising the nucleotide sequence of the alcohol dehydrogenase mutant according to claim 4.
  6. 6. A recombinant strain comprising the recombinant expression vector of claim 5 or the nucleotide sequence of the alcohol dehydrogenase mutant of claim 5.
  7. 7. Use of the alcohol dehydrogenase mutant according to claim 1 for the catalytic synthesis of axial chiral biaryl dimethanol compounds.
  8. 8. The method according to claim 7, wherein the catalytic synthesis is carried out using biaryl aldehyde as a substrate, wherein the biaryl aldehyde has the structural formula: the axial chiral biaryl dimethanol is an S-configuration biaryl compound, and has the structural formula:
  9. 9. The use according to claim 7, wherein the catalytic synthesis is an axial chiral biaryl dimethanol compound synthesized by using alcohol dehydrogenase mutant catalytic dynamic kinetic resolution of carbonyl reduction using biaryl aldehyde as a substrate, dimethyl sulfoxide as a cosolvent, isopropanol, and NADP + as a reduction circulation system.
  10. 10. The method according to claim 9, wherein the catalytic synthesis is carried out under the reaction conditions of an initial reaction concentration of aldehyde of 10mM, an initial reaction addition amount of alcohol dehydrogenase crude enzyme solution of 0.2 g/mL, a volume fraction of dimethyl sulfoxide of 2%, a volume fraction of isopropanol of 3%, a pH of 7.0, 50mM Tris-HCl buffer solution, and NADP + of 0.16 mM, and a reaction temperature of 30 ℃ and a rotation speed of 1000 rpm of 3 h.

Description

Application of alcohol dehydrogenase mutant in synthesis of axial chiral biaryl dimethanol Technical Field The invention belongs to the technical fields of bioengineering and biocatalysis, and particularly relates to an alcohol dehydrogenase mutant and application thereof as a biocatalyst in synthesizing an axial chiral biaryl compound with high stereoselectivity. Background The axial chiral biaryl dimethanol is an important chiral structural unit, widely exists in medicine molecules, chiral catalysts and functional materials, and has important application value in the fields of medicinal chemistry, asymmetric catalysis, material science and the like. Therefore, the realization of efficient and high stereoselective synthesis of the axial chiral biaryl dimethanol compound has important scientific significance and application value. In recent years, dynamic Kinetic Resolution (DKR) strategies have received great attention to achieving high enantioselective construction of axial chiral biaryl compounds under the action of chiral catalysts by taking advantage of the property that bridged intermediates, which are unstable in configuration, can be rapidly racemized. However, existing research focused mainly on five-or six-membered bridged intermediate systems, achieving this goal in larger ring transient bridged bodies, including seven-membered bridged intermediate systems, still face significant challenges. This is because the racemic half-life of the seven-membered structure is long on the relevant reaction time scale, and generally ranges from minutes to hours, thus placing extremely stringent demands on the stereocontrol ability of chiral catalysis. Therefore, there is an urgent need for an intermediate with compact structure, wide application range and universality, and a high-efficiency catalytic system based on the intermediate is established to realize high stereoselective synthesis of axial chiral biaryl compounds. The present invention reports the first biocatalysis-based method to achieve axially selective Dynamic Kinetic Resolution (DKR) carbonyl reduction via a transient seven-membered bridged intermediate to build a series of axially chiral biaryl dimethanol. In this method, the engineered alcohol dehydrogenase can catalyze the axial selective reduction of biaryl aldehydes, achieve the stereoselective synthesis of axial chiral biaryl dimethanol compounds, and exhibit good catalytic activity and high stereoselectivity. Disclosure of Invention The invention aims to solve the technical problem of filling the blank of the existing catalytic system for realizing synthesis of axial chiral biaryl dimethanol, and providing a biocatalysis method for realizing stereoselective synthesis of the compound. The invention also solves the technical problem of providing an alcohol dehydrogenase mutant for realizing high-stereoselective biocatalytic conversion of biaryl aldehyde substrates to chiral biaryl dimethanol compounds with specific configurations. In order to solve the technical problems, the invention adopts the following technical scheme: An alcohol dehydrogenase mutant CpAR, wherein the amino acid sequence of the alcohol dehydrogenase mutant CpAR2-M5 is obtained by mutating phenylalanine at 85 th position, threonine at 125 th position, phenylalanine at 192 th position, glutamic acid at 209 th position and isoleucine at 213 th position of wild type alcohol dehydrogenase CpAR into alanine. Wherein the wild-type alcohol dehydrogenase CpAR is derived from Monilia parapsilosis (STRAIN CDC/ATCC MYA-4646), the amino acid sequence of which is shown as SEQ ID NO.2, and the corresponding nucleotide sequence for encoding the wild-type alcohol dehydrogenase CpAR2 is shown as SEQ ID NO. 1. Specifically, the alcohol dehydrogenase mutant CpAR is obtained by mutating 85 th phenylalanine of wild-type alcohol dehydrogenase CpAR into alanine, 125 th threonine into serine, 192 rd phenylalanine into leucine, 209 th glutamic acid into alanine and 213 th isoleucine into phenylalanine, and the amino acid sequence is shown as SEQ ID NO. 3. The nucleotide sequence corresponding to the coded alcohol dehydrogenase mutant is shown as SEQ ID NO. 4. In some embodiments of the invention, the alcohol dehydrogenase mutant is alcohol dehydrogenase mutant CpAR2-M5. Specifically, the amino acid sequence of the alcohol dehydrogenase mutant CpAR-M5 is shown as SEQ ID NO.3, and the corresponding nucleotide sequence is shown as SEQ ID NO. 4. Specifically, the alcohol dehydrogenase mutant CpAR uses a gene expression plasmid derived from Monilia parapsilosis (STRAIN CDC/ATCC MYA-4646) as a template, constructs a mutation library based on structure-oriented protein engineering, and combines a screening method to obtain a good mutant with enhanced or inverted stereoselectivity. And finally obtaining the alcohol dehydrogenase mutant CpAR-M5 through iteration of screening and dominant mutation sites. The alcohol dehydrogenase mutant CpAR-M5 can be used