Method for preparing (s)-1,2,3,4-tetrahydroisoquinoline-1 carboxylic acid and derivatives thereof

Patent metadata

Field	Value
Publication number	US-12480148-B2
Application number	US-201917268455-A
Country	US
Kind code	B2
Filing date	Apr 23, 2019
Priority date	Aug 16, 2018
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

Disclosed is a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof, comprising: taking a racemate of a compound represented by Formula (I) or a racemate of a salt of the compound represented by Formula (I) as a substrate, and making a R-isomer of the compound represented by Formula (I) in the substrate react under the catalysis of oxidative dehydrogenase to generate imino acid represented by formula (II); and converting the imino acid represented by Formula (II) into an S-isomer of the compound represented by Formula (I) in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydrogen anions. The process has mild reaction conditions, strong stereoselectivity, high reaction efficiency, and high conversion rate.

First claim

The invention claimed is:

1 . A method for preparing an S-isomer of a compound represented by Formula (I),

wherein:
in Formula (I), R 1 and R 2 are independently selected from the group consisting of (i) hydrogen, (ii) C 1 -C 6 alkyl, and (iii) C 1 -C 6 alkoxy, and
the method comprises the steps of:
taking a racemate mixture of a compound represented by Formula (I) or salts thereof as a mixture, and
making a R-isomer of the compound represented by Formula (I) in the substrate react under the catalysis of an oxidative dehydrogenase to generate an imino acid represented by Formula (II);

wherein the oxidative dehydrogenase is a D-amino acid oxidase which has an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4;
and
converting the imino acid represented by Formula (II) into an S-isomer of the compound represented by the Formula (I) in the presence of a pipecolic acid reductase and a coenzyme capable of supplying hydrogen anions;

and wherein:
the pipecolic acid reductase has an amino acid sequence as shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8; and
the coenzyme capable of supplying hydrogen anions is NADH, NADPH, or a combination thereof.

2 . The method of claim 1 , wherein:
in the Formula (I), R 1 and R 2 are independently selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, methoxy, or ethoxy; and the salt is an ammonium salt.

3 . The method according to claim 1 , wherein an S-isomer of the compound represented by Formula (I) is (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or (S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid.

4 . The method according to claim 1 , wherein the use form of the D-amino acid oxidase is unorganized D-amino acid oxidase, crude enzyme containing unorganized D-amino acid oxidase, pure D-amino acid oxidase, immobilized D-amino acid oxidase, or cells intracellularly expressing D-amino acid oxidase.

5 . The method according to claim 4 , wherein:
the cell is an engineering bacteria expressing D-amino acid oxidase and containing an expression vector pET-28a(+), and a host cell of the engineering bacteria is E. coli BL21(DE3); and wherein, the D-amino acid oxidase gene is connected to the expression vector pET-28a(+).

6 . The method according to claim 5 , wherein the use form of the pipecolic acid reductase is an unorganized pipecolic acid reductase, a crude enzyme containing unorganized pipecolic acid reductase, a pure pipecolic acid reductase, an immobilized pipecolic acid reductase, or cells intracellularly expressing pipecolic acid reductase.

7 . The method according to claim 6 , wherein:
the cell is an engineering bacteria expressing pipecolic acid reductase and containing an expression vector pET-28a(+), and a host cell of the engineering bacteria is E. coli BL21(DE3); and wherein the pipecolic acid reductase is connected to the expression vector pET-28a(+).

8 . The method according to claim 1 , wherein:
(i) the reaction to generate imino acid is also carried out in the presence of the coenzyme flavin adenine dinucleotide (FAD), (ii) the reaction to generate imino acid is also carried out in the presence of the enzyme catalase, (iii) the reaction to generate imino acid is also carried out at a setting temperature and in the presence of oxygen, wherein the setting temperature is from 20° C. to 70° C., inclusive, or (iv) combinations thereof.

9 . The method according to claim 1 , wherein the implementation process of the method comprises:
first building a reaction system, then, controlling the reaction system to react at a setting temperature in an aerobic environment, in which the reaction system comprises the substrate, the oxidative dehydrogenation, the pipecolic acid reductase, the coenzyme, a coenzyme regeneration system and water, and wherein:
the reaction system selectively comprises a pH buffer and/or pH regulator,
the coenzyme comprises NAD + and/or NADH, or,
the coenzyme comprises NADP + and/or NADPH.

10 . The method according to claim 9 , wherein (i) the coenzyme regeneration system comprises a coenzyme regeneration enzyme and a coenzyme regeneration substrate, (ii) the reaction system further comprises the enzyme catalase, (iii) the reaction system further comprises flavin adenine dinucleotide, or (iv) combinations thereof.

11 . The method according to claim 10 , wherein the coenzyme regeneration enzyme is glucose dehydrogenase, and the coenzyme regeneration substrate is glucose; or the coenzyme regeneration enzyme is alcohol dehydrogenase, and the coenzyme regeneration substrate is isopropanol.

12 . The method according to claim 11 , wherein the glucose dehydrogenase is derived from Bacillus subtilis 168; and/or, the alcohol dehydrogenase is derived from Lactobscillus kefir DSM20587.

13 . The method according to claim 10 , wherein the catalase is bovine liver catalase lyophilized powder.

14 . The method according to claim 10 , wherein a ratio of enzyme activities of the catalase and the oxidative dehydrogenase is (100 to 400):1.

Assignees

• Tongli Biomedical Co Ltd
• Univ Zhejiang

Inventors

• Wu Jianping
• Ju Shuyun
• Shi Junwei
• Yang Lirong
• Qian Mingxin

Classifications

• C12P41/00

  Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C07D217/26 Primary

  Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen · CPC title

• C07B2200/07

  Optical isomers · CPC title

• C12R2001/645

  Fungi {; Processes using fungi} · CPC title