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Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through multienzyme coupling

A technology of tetrahydroisoquinoline and formic acid, applied in fermentation and other directions, can solve the problem of only 50% theoretical yield, and achieve the effects of mild reaction conditions, high yield and good selectivity

Active Publication Date: 2020-06-09
ZHEJIANG UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The stereoselectivity of the product obtained by this method is high, and the technology is relatively simple, but there is still the problem that the maximum theoretical yield only has 50% (chemoenzymatic synthesis of optically pure (S)-1,2,3,4-tetrahydroquinoline-3 -Research on carboxylic acid[J].Modern Chemical Industry,2003,23(12):23-5.)

Method used

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  • Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through multienzyme coupling
  • Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through multienzyme coupling
  • Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through multienzyme coupling

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1 Genetic Engineering Bacteria Strain Construction

[0077] 1.1 Screening of D-amino acid oxidase and construction of genetically engineered bacteria expressing D-amino acid oxidase

[0078] According to different substrate specificities, D-amino acid oxidases from microorganisms can be divided into two categories: 1) amino acids (such as D-alanine) with a small side chain group preference for substrates, such as Fusarium oxysporum (Fusarium oxysporum)-derived D-amino acid oxidase; 2) preference for amino acids with larger substrate side chain groups (such as D-phenylalanine), such as Trigonopsis variabilis-derived D-amino acid oxidase ( POLLEGIONI L, MOLLAG, SACCHI S, et al. Properties and applications of microbial D-amino acid oxidases: current state and perspectives [J]. Appl Microbiol Biotechnol, 2008, 78(1): 1-16.). The amino acid sequences of these two D-amino acid oxidases were used for BLASTp analysis in the National Center for Biotechnology Informat...

Embodiment 2

[0121] 2.1 Culture of microorganisms

[0122] Composition of liquid LB medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use. If it is solid LB medium, add 15g / L agar.

[0123] The engineered bacteria containing the D-amino acid oxidase gene were inoculated in 5 mL of liquid LB (containing 50 μg / ml kanamycin) medium, and cultured with shaking at 200 rpm for about 8 hours at 37°C. Inoculate in 100mL liquid LB (containing 50μg / ml kanamycin) culture medium according to 1% (V / V) inoculum size, OD 600 After reaching 0.6-0.8, add the inducer isopropylthiogalactoside (initial concentration: 0.1 mM), and induce for 15 hours at 18°C. After the cultivation, pour the culture solution into a 100mL centrifuge tube and centrifuge at 4000rpm for 10min, discard the supernatant, collect the bacterial cells, wash the cells twice with 50mM phosphate buffer (pH 8.0), and store them in a -80°C ult...

Embodiment 3

[0126] Example 3 Preparation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO-PpdpkA multi-enzyme coupling

[0127] According to the method of Example 2, the crude enzyme solution of D-amino acid oxidase derived from Fusarium solani (Fusarium solani) M-0718 and the crude enzyme of pipecolic acid reductase from Pseudomonas putida (Pseudomonas putida) KT2440 were prepared respectively. solution and glucose dehydrogenase crude enzyme solution of Bacillus subtilis (Bacillus subtilis) 168.

[0128] Weigh 0.16g of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid into a 100ml reaction bottle, add 10ml of phosphate buffer (50mM, pH=8.0) Adjust the pH of the solution to 8.0. Add 20ml FsDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, therefore, no additional FAD needs to be added in the crude enzyme solution reaction system), 5ml PpdpkA crude enzyme solution, 5ml glucose dehydrogenase crude enzyme solution, 20mg o...

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Abstract

The invention discloses a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through multienzyme coupling. The method comprises the following steps: by taking raceme of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or raceme of 1,2,3,4-tetrahydroisoquinoline-3-formate as a substrate, enabling (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the substrate to react to generate imidic acid shown in a formula (II) under the catalysis action of oxidative dehydrogenase; and converting the imidic acid shown in the formula (II) into the (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the presence of piperidine acid reductase and a coenzyme capable of providing hydride ions. The method is gentle in reaction condition, good in stereoselectivity, high in reaction efficiency, high in conversion rate, and the like.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through multi-enzyme coupling. Background technique [0002] (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) is an important pharmaceutical intermediate and is widely used in Synthesis of various organic small molecule drugs and peptide-based drugs. For example, (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is an important component of the antihypertensive drug quinapril (Diversity-oriented synthesis of medicinally important 1,2,3,4 -tetrahydroisoquinoline-3-carboxylic acid(Tic) derivatives and higher analogs[J].Org Biomol Chem,2014,12(45):9054-91.). In addition, (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid can be used to synthesize small-molecule antagonists containing a tetrahydroisoquinoline core, acting on the ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12
CPCC12P17/12
Inventor 吴坚平居述云杨立荣施俊巍钱明心
Owner ZHEJIANG UNIV
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