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Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through chemical enzyme process

A technology of tetrahydroisoquinoline and chemical enzymatic method, applied in the field of biocatalysis, can solve problems such as the theoretical yield of only 50%, and achieve the effects of high reaction efficiency and yield, mild reaction conditions and improved yield

Active Publication Date: 2020-06-09
TONGLI BIOMEDICAL +1
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  • Summary
  • 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 chemical enzyme process
  • Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through chemical enzyme process
  • Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through chemical enzyme process

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Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0056] Example 1 Screening of D-amino acid oxidase and construction of genetically engineered bacteria expressing D-amino acid oxidase

[0057] 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 preference for substrate side chain groups, 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 to perform BLASTp analysis in the National Center for Biotechnology Information (NCBI) database (https: / / www.ncbi.nlm.nih.gov / ), and the sequ...

Embodiment 2

[0062] 2.1 Culture of microorganisms

[0063] 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.

[0064] 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 with 1% (V / V) inoculum size, OD 600 After reaching 0.6-0.8, add the inducer isopropylthiogalactopyranoside (final 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 ultra-l...

Embodiment 3

[0070] Example 3 FsDAAO-NH 3 ·BH 3 Preparation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

[0071] Preparation of substrate solution: Prepare 5 g / L racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solution with 50 mM phosphate buffer solution (pH=8.0) and adjust the pH of the solution with 30% ammonia water value to 8.0.

[0072] Add 24mL substrate solution, 6mL FsDAAO crude enzyme solution (the crude enzyme solution already contains enough coenzyme FAD, therefore, no additional FAD needs to be added in the crude enzyme solution reaction system), 12mg catalase lyophilized to 100mL reactor powder and 0.4gNH 3 ·BH 3 . Immediately after mixing, a sample was taken as "0 hour". The reaction system was placed in a constant temperature water bath at 30° C., stirred by magnetic force, and reacted for 24 hours before sampling. The content of two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample was detected by high performance ...

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Abstract

The invention discloses a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid through a chemical enzyme process. The method comprises the following steps: with 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid as a substrate, stereoselectively catalyzing an R-type isomer by utilizing D-amino acid oxidase; carrying out oxidative dehydrogenation to generate a corresponding imidic acid, wherein an S-type isomer which is not catalyzed is kept in a reaction system; reacting the imidic acid with an imidic acid reducing agent to generate a racemic substrate; and stereoselectivelycatalyzing the R-type isomer in the racemic substrate under the action of D-amino acid oxidase to prepare the S-type isomer. According to the method, the reaction yield can reach 80.6% or above, an ee value is larger than 99%, and the method has the advantages of mild reaction conditions, high stereoselectivity, high reaction efficiency, high yield, relatively simple process 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 by chemical enzymatic method. 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 che...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P17/12
CPCC12P41/002C12P41/001C12P17/12C12P41/00C12P13/04C07D217/16Y02P20/55
Inventor 吴坚平居述云杨立荣施俊巍钱明心
Owner TONGLI BIOMEDICAL
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