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Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof

A technology of tetrahydroisoquinoline and dihydroisoquinoline, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of selectivity to be improved

Active Publication Date: 2021-12-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2018, the Helen C.Hailes research group used NCS enzymes to asymmetrically synthesize tricyclic nacresine and its analogues, but the selectivity of this method needs to be improved
Qu et al. used imine reductase to asymmetrically synthesize tetrahydroisoquinoline and its analogues, but their work can only synthesize tetrahydroisoquinoline cores with two rings

Method used

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  • Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof
  • Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof
  • Synthesis method of chiral fused ring tetrahydroisoquinoline alkaloid and analogue thereof

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Experimental program
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Effect test

Embodiment 1

[0012] Embodiment 1: Construction and cultivation of genetically engineered bacteria

[0013]The specific construction and cultivation method of the genetically engineered bacteria producing imine reductase is as follows: the amino acid sequence SEQ ID NO: 1 derived from Myxococcusfulvus is gene-synthesized, constructed into a pET series vector, and expressed heterologously in the host bacteria Escherichia coli . Thaw the strains stored at -80°C, streak on the plate, and culture overnight in a constant temperature incubator at 37°C. Select a single colony on the plate and inoculate it into 20mL LB medium containing the corresponding antibiotic, cultivate it for about 12 hours as a seed solution, inoculate it into 700mL LB medium containing the corresponding antibiotic according to the inoculation amount of 1%, and inoculate it on a shaker at 37°C and 200rpm Grow to OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mmol / L to induce at 25°C for 12 hours, and collect ...

Embodiment 2

[0014] Embodiment 2: IRED1 catalyzes the preparation of optically pure ( R)-8,9-Dimethoxy-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline

[0015] 10 mM 8,9-dimethoxy-2,3,5,6-tetrahydro-1H-pyrrolo[2,1-a]isoquinoline-4-ammonium chloride (dissolved in 10% DMSO), 30mM Glucose, 4U / mL GDH, 0.5mg / mL NADP + , 2.50g of IRED1 genetically engineered bacteria, the buffer solution is 100mL of phosphate buffer solution with pH 7.5, reacted at 25°C and 220rpm for 10h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the extraction was confirmed by thin-layer chromatography. The organic solvent was removed by rotary evaporation to obtain the crude product. The conversion rate of the substrate and the yield of the product were detected by gas phase, and the ee value of the product detected by liquid phase HPLC was 99%. The conversion rate was 87.5%, the yield was 68.1%, and the configuration of the product was (R).

Embodiment 3

[0016] Example 3: IRED1 catalyzes the preparation of optically pure (R)-9,10-Dimethoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline

[0017] 30 mM 9,10-dimethoxy-1,2,3,4,6,7-hexahydropyrido[2,1-a]isoquinoline-5-ammonium chloride (dissolved in 10% DMSO) , 90mM Glucose, 12U / mL GDH, 0.5mg / mL NADP + , 2.50g of IRED1 genetically engineered bacteria, the buffer solution is 100mL of phosphate buffer solution with pH 7.5, reacted at 25°C and 220rpm for 10h. After the reaction, the protein was removed by centrifugation, the supernatant was extracted with ethyl acetate, and the extraction was confirmed by thin-layer chromatography. The organic solvent was removed by rotary evaporation to obtain the crude product. The conversion rate of the substrate and the yield of the product were detected by gas phase, and the ee value of the product detected by liquid phase HPLC was 99%. The conversion was 83.3%, the yield was 74.1%, and the configuration of the product was (R).

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Abstract

The invention provides an imine reductase and application of the imine reductase in preparation of an optical pure chiral fused ring tetrahydroisoquinoline compound through asymmetric reduction; and particularly, the imine reductase is an oxidoreductase derived from Myxococcus fulvus. The imine reductase can be used for realizing the chiral reduction of 10-500 mM of a fused ring dihydroisoquinoline substrate under a mild reaction condition. The imine reductase provided by the invention is wide in substrate spectrum and has relatively high activity on fused ring dihydroisoquinoline.

Description

technical field [0001] The invention relates to a new method for synthesizing a class of condensed-ring tetrahydroisoquinoline alkaloids and their analogues, belonging to the field of biochemical industry. Specifically, it relates to a new method for preparing optically pure chiral condensed ring tetrahydroisoquinoline compounds by enzyme-catalyzed asymmetric reduction. Background technique [0002] Tetrahydroisoquinoline alkaloids are widely distributed in nature and are important chiral amine compounds. The alkaloids with tetrahydroisoquinoline as the structural core have anti-tumor, anti-pathogenic microorganisms, anti-inflammation and central nervous system System regulation and other biological activities (Le V H, Inai M, Williams R M, et al. Ecteinascidins. A review of the chemistry, biology and clinical utility of potent tetrahydroisoquinoline antititumor antibiotics [J]. Natural Product Reports, 2015, 32(2): 328- 347; Johnson JL, Nair D S, Pillai S M, et al. Dissymm...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P17/18C12R1/19
CPCC12P41/002C12P17/182C12P17/18Y02P20/55
Inventor 姚培圆杨林松李键煚徐泽菲陈曦冯进辉吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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