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Biocatalysis preparation method of chiral tetrahydro-2-naphthol compound

A biocatalysis and tetrahydronaphthalene technology, applied in the field of medicine, can solve the problems of harsh reaction conditions, many reaction steps, low optical purity and the like, and achieve the effects of low cost, short reaction steps and high stereoselectivity

Pending Publication Date: 2022-01-28
默沃智造(上海)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the prior art, the synthesis of the chiral intermediate (R)-5,7-difluoro-1,2,3,4-tetrahydronaphthalene-2-ol requires many reaction steps, complicated operations and harsh reaction conditions , low yield, and / or low optical purity, the invention provides a biocatalytic method for (R)-5,7-difluoro-1,2,3,4-tetralin-2-ol The preparation method is to directly obtain (R)-5,7-difluoro-1,2,3,4-tetralone by one-step reduction reaction of the raw material 5,7-difluoro-2-tetralone under mild conditions Hydronaphthalene-2-ol, while the yield is significantly improved, the product has high optical purity, and the ee value can reach 99%

Method used

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  • Biocatalysis preparation method of chiral tetrahydro-2-naphthol compound
  • Biocatalysis preparation method of chiral tetrahydro-2-naphthol compound
  • Biocatalysis preparation method of chiral tetrahydro-2-naphthol compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0159] Example 1: Preliminary Sieve of Carbonyl Rebutase

[0160] In this example, there is no preliminary screening of no less than 10 carbonyl reductase, wherein

[0161] ADH comes from Corynebacterium Sp.st-10, and its genbank serial number is AB190261;

[0162] LSADH comes from Leifsonia Sp.strain S749, its GenBank serial number is AB213459;

[0163] ARQR comes from Agrobacterium Radiobacter ECU 2556, its genbank serial number is WP_015918093.1;

[0164] Fusc2 comes from Fusidioides Strain ATCC 14700, and its genbank serial number is QBB00668.1;

[0165] LK comes from Lactobacillus Kefir, its genbank serial number is WP_054768785.1;

[0166] BD originated from Brevundimonas Diminuta 470-4, and its GenBank serial number is eky29933.1;

[0167] CK20 is derived from Chryseobac-Terium Sp.ca49, and its GenBank serial number is AHC30858.1;

[0168] Fusc1 comes from Fusidioides Strain ATCC 14700, and its genbank serial number is QBB00667.1;

[0169] DHCR comes from Debaryomyces Hanse...

Embodiment 2

[0177] Example 2: Construction of Carbonyl Reprotor Enzyme Engineering

[0178] The carbonyl reductase (LSADH) gene from Leifsonia Sp.strain S749 was brought to a PET28A (+) vector, transferred to E. coli DH5α sensitic cells, kanamycin resistant plate culture. After challenging the positive transformant single colonies and extracts the plasmid sequence, the recombinant plasmid is extracted, and then the host E. coli BL21 (DE3), plate culture, sequential colonies, lb culture, and obtain a recombinant gene engineering of carbonyl reductase (LSADH). bacteria.

Embodiment 3

[0179] Example 3: Construction and screening of carbonyl reduced enzyme mutant libraries

[0180] With wild-type carbonyl reductase LSADH as a template, using a random dot mutant kit ( Iisite-Directed Mutagenesiis Kit is erroneous PCR, or mutated wild-type carbonyl reductase gene LASDH by oriented evolution to obtain a plasmid library comprising an evolutionary carbonyl reductase gene. The constructed plasmid library was transferred to E. coli BL21 (DE3) (item number: Kang as century CW0809S), in an oven at 37 ° C, cultured overnight in LB solid medium containing 50 μg / ml kanamycin. Single colonies were selected to a 96-well plate containing 400 μl of LB liquid medium (containing 50 μg / ml kanamycin), 37 ° C, 200 rpm overnight culture, and obtained seed fluid. 10 μl of seed fluid was then transferred to a 96 deep hole plate containing 400 μl of fermentation medium (containing 50 μg / ml kanamycin) to the OD600 value> 0.8 at 37 ° C. Then, an ultra-concentration of 1 mm isopropy...

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Abstract

The invention discloses a biocatalysis preparation method of a chiral tetrahydro-2-naphthol compound. The biocatalysis preparation method comprises the following steps: (a) with 5,7-difluoro-2-tetralone as a substrate, carrying out asymmetric reduction reaction in the presence of coenzyme under the catalysis of carbonyl reductase in a liquid reaction system to form (R)-5,7-difluoro-1,2,3,4-tetrahydro-2-naphthol; and (b) optionally separating (R)-5,7-difluoro-1,2,3,4-tetrahydro-2-naphthol from the reaction system after the reaction in the step (a). The invention also discloses carbonyl reductase prepared by using the method, a coding gene of the carbonyl reductase, a vector and a bioengineering strain. The method has the advantages of short steps, mild conditions, remarkably improved yield and high optical purity of the product.

Description

Technical field [0001] The invention belongs to the field of medical technology, and more particularly to a biological catalytic preparation method of a chiral tetrahydronaphthalene-2-alcohol compound. Background technique [0002] Hydrochloride is an effective selective dopamine β-hydrolyzate inhibitor. In the existing technique, the synthesis of the inner and her hydrochloride is high, the cost is high, and the main reason is that the chiral intermediate is difficult. [0003] WO 2009 / 021055A1 discloses a chemical synthesis method of internal commitment. The method includes preparation of yellow-green suspension in order to prepare a yellow-green suspension in order to prepare a yellow-green suspension in order to prepare a yellow-green suspension in order to prepare a yellow-green suspension. Cooling to -65 ° C, adding raw material 5,7-difluoro-2-tetrahydronone, and after completion of the reaction to obtain a chiral intermediate (R) -5,7-difluoro -1,2,3,4-tetrahydronaphthal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12N9/04C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12P7/22C12N9/0006C12Y101/01184C12N15/70
Inventor 倪国伟
Owner 默沃智造(上海)生物技术有限公司
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