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A method for preparing chiral compounds containing hydroxyl groups

A chiral compound and compound technology, applied in the biological field, can solve the problems of high price, impossibility and instability of reduced coenzyme

Active Publication Date: 2021-02-19
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, the reduced coenzyme is expensive and very unstable, so it is impossible to prepare (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol as a raw material
On the other hand, the cost of using coenzyme II is also much higher than that of coenzyme I

Method used

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  • A method for preparing chiral compounds containing hydroxyl groups
  • A method for preparing chiral compounds containing hydroxyl groups
  • A method for preparing chiral compounds containing hydroxyl groups

Examples

Experimental program
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Effect test

preparation example Construction

[0066] (1) The method for preparing (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol provided by the present invention has high conversion rate, does not need to use coenzyme II, and has low preparation cost;

[0067] (2) The present invention adopts the method of biocatalysis, which is environmentally friendly, has mild conditions and stable process;

[0068] (3) Using the reaction system constructed by the co-expression strain of the present invention, under optimized enzyme expression conditions and optimized reaction conditions, the conversion rate can reach more than 90%, and the product is single, which is very easy to purify.

Embodiment 1

[0070] Embodiment 1 Construction of glucose dehydrogenase and alcohol dehydrogenase recombinant strain

[0071] Alcohol dehydrogenase gene ADH derived from Lactobacillus caucasus was codon optimized and artificially synthesized. Using the same method, the codon optimization of the glucose dehydrogenase (GDH) gene derived from Bacillus was carried out and the gene was artificially synthesized. The genes ADH and GDH were connected with pET-28a respectively, and each was introduced into E.coli BL21(DE3) to obtain recombinant bacteria E.coli BL21-ADH and E.coli BL21-GDH. The recombinant bacteria were tested for expression, and the products ADH and GDH were verified by SDS-PAGE. Finally, a series of optimizations were carried out on its expression conditions.

[0072] Experimental Materials

[0073] 1 experimental equipment

[0074] Table 1-1 Experimental Instruments

[0075]

[0076]

[0077] 2 experimental reagents

[0078] Table 1-2 Experimental Reagents

[0079] ...

Embodiment 2

[0221] Example 2 Co-expression of glucose dehydrogenase and alcohol dehydrogenase

[0222] Lactobacillus caucasus contains alcohol dehydrogenase, which can catalyze the reduction of 2,6-dichloro-3-fluoroacetophenone to (S)-1-(2,6-dichloro-3-fluorophenyl ) ethanol, but the process of this asymmetric reduction reaction requires the reduced coenzyme NADPH to provide reducing hydrogen. Therefore, when using the recombinant Escherichia coli cloned with the alcohol dehydrogenase gene to carry out the reduction reaction of 2,6-dichloro-3-fluoroacetophenone, it is necessary to add expensive NADPH or provide a high-efficiency coenzyme regeneration system. Glucose dehydrogenase can catalyze glucose to gluconic acid, and at the same time convert NAD(P) + Reduction to NAD(P)H. Using genetic engineering means, the key enzyme GDH in coenzyme regeneration and alcohol dehydrogenase ADH are coupled and expressed, and the endogenous coenzyme regeneration is also provided while catalyzing in a...

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Abstract

The invention provides a method for preparing a chiral compound containing a hydroxyl group. The method uses alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) to perform a reduction reaction on a substrate, thereby preparing a chiral compound containing a hydroxyl group. method of active compounds. The method of the invention has high conversion rate, does not need to use coenzyme II, and has low preparation cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for coupling and catalyzing alcohol dehydrogenase and glucose dehydrogenase to prepare a chiral compound containing a hydroxyl group. Background technique [0002] Crizotinib was developed by Pfizer for the treatment of anaplastic lymphoma kinase (ALK)-positive locally advanced and metastatic non-small cell lung cancer (NSCLC), and was approved by the FDA in 2011. In the synthetic technique of crizotinib, the synthetic precursor is (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol, which can be prepared from 2,6-dichloro-3-fluorobenzene Ethanone is prepared by chiral catalytic reduction of alcohol dehydrogenase (ADH). [0003] Chiral reduction of keto groups catalyzed by oxidoreductases is an important method to obtain chiral compounds containing hydroxyl groups. Among them, the chiral reduction products of acetophenone-related compounds are chiral intermed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/22C12P13/02
CPCC12P7/22C12P13/02
Inventor 邵雷陈代杰刘丽勤黄军海张骏梁
Owner SHANGHAI INST OF PHARMA IND CO LTD