Ketoreductase and method for catalytic preparation of (S)-1-(2-chlorophenyl)ethanol by ketoreductase

A technology for catalytic preparation and reductase, applied in oxidoreductase, fermentation and other directions, can solve the problems of low substrate concentration, low conversion rate, hindering industrial production, etc., achieve good catalytic performance, and simplify the production process and cost.

Active Publication Date: 2018-08-17
SYNCOZYMES SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] In view of the prior art, the biocatalytic process of reducing o-chloroacetophenone to obtain (S)-1-(2-chlorophenyl)ethanol mostly has low substrate concentration, low conversion rate, and the process does not have practical value for industrial production. Problems that hinder the process of enzyme-catalyzed preparation of (S)-1-(2-chlorophenyl)ethanol from industrial production

Method used

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  • Ketoreductase and method for catalytic preparation of (S)-1-(2-chlorophenyl)ethanol by ketoreductase
  • Ketoreductase and method for catalytic preparation of (S)-1-(2-chlorophenyl)ethanol by ketoreductase
  • Ketoreductase and method for catalytic preparation of (S)-1-(2-chlorophenyl)ethanol by ketoreductase

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1 Preparation of ketoreductase

[0066] Inoculate the genetically engineered bacteria (vector pET21a, host cell E.Coli BL21(DE3)) containing the coding gene (SEQ ID No.1) of ketoreductase into 5 mL of ampicillin-containing LB test tube medium for activation culture (37° C. Cultivate for 12h), transfer the activated culture to 400mL LB liquid medium containing ampicillin according to 1% inoculum size, culture at 37°C to OD to 0.6-0.8, add IPTG (final concentration 0.1mM) and induce culture at 25°C for 16h. Collect the cells by centrifugation to obtain ketone reductase cells, resuspend the cells in 40 mL of distilled water, and ultrasonically break in an ice-water bath for 15 minutes, collect the supernatant by centrifugation, pre-freeze at -20°C, freeze-dry in vacuum for 48 hours, and crush to obtain recombinant ketones Reductase Enzyme Powder.

Embodiment 2

[0067] Example 2 Preparation of gram-grade (S)-1-(2-chlorophenyl)ethanol

[0068] Add isopropanol (5mL) and substrate o-chloroacetophenone (1.5g) into the reaction vessel, stir well and then add enzyme powder 0.1g, coenzyme NADP + Finally, dilute the volume of 1 mg to 10 mL with pure water, stir the reaction under magnetic force at 30°C, and monitor the progress of the reaction by TLC. After 6 hours, the reaction was completed, the reaction solution was filtered with diatomaceous earth, the liquid phase was extracted three times with the organic phase, the organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain the product. The conventional HPLC profile of the substrate before the reaction solution starts to react is as follows: figure 1 Shown, the chiral HPLC spectrogram of substrate is as attached figure 2 Shown; The 1-(2-chlorophenyl) ethanol chiral HPLC of racemate is as attached image 3 Shown; The chiral HPLC...

Embodiment 3

[0069] Example 3 Preparation of gram-level (S)-1-(2-chlorophenyl)ethanol

[0070] Add isopropanol (6.4mL) and substrate o-chloroacetophenone (2.8g) into the reaction vessel, stir well and add 0.26g of ketoreductase cells, coenzyme NADP + 1.2mg, finally dilute to 10mL with pure water, stir the reaction with magnetic force at 29°C, and detect the progress of the reaction by TLC at the same time. After 6 hours of reaction, diatomaceous earth was filtered, the liquid phase was extracted three times with the organic phase, the organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain the product. Chiral HPLC of the reaction solution such as Figure 12 , The routine HPLC spectrogram detection of reaction solution is as attached Figure 6 , with Figure 12 and 6 The detection of the ee value and conversion rate of the product shows that: the substrate conversion rate=99.5%, and the ee value of the S-type product=99.9%.

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Abstract

The invention belongs to the field of application of genetic engineering technologies to preparation of pharmaceutical intermediates, relates to enzymatic preparation of chiral alcohols and provides ketoreductase and a method for catalytic preparation of (S)-1-(2-chlorophenyl)ethanol by the ketoreductase. According to the method, o-chloroacetophenone is converted into (S)-1-(2-chlorophenyl)ethanolunder the catalytic action of ketoreductase; by application the ketoreductase in a specific amino acid sequence to catalytic preparation of (S)-1-(2-chlorophenyl)ethanol, the substrate conversion rate reaches 99%, ee value of a preparation product is not lower than 99.5%, the maximum reaction substrate concentration is 280g / L, and an industrial scale-up production value is achieved. In addition,by the reaction, a substrate can be completely and efficiently converted into a target product, the prepared product is simple in separation and purification and low in aftertreatment cost, and a whole process is high in environmental friendliness and high in atom utilization rate.

Description

technical field [0001] The invention relates to a preparation method of a pharmaceutical intermediate, in particular to a ketoreductase and a method for preparing (S)-1-(2-chlorophenyl)ethanol by catalysis. Background technique [0002] (S)-1-(2-chlorophenyl)ethanol English name (S)-1-(2-chlorophenyl)ethanol, corresponding CAS number: 131864-71-6, molecular structure formula is: [0003] [0004] The molecule has active groups such as halogenated phenyl groups and chiral hydroxyl groups, and is widely used as an important chiral building block for the synthesis of medicines, pesticides and other fine chemicals. For example, the tumor drug molecule CAS#929095-18-1 of the GlaxoSmithKline GSK-461364A project; the epilepsy drug CAS#112856-44-7 of the Schwabe ADD-137022 project; the cannabinoid receptor 2 drug CAS#1438465-84- All 9 molecules were prepared using (S)-1-(2-chlorophenyl)ethanol as the key chiral intermediate. The molecular structural formula of each drug is: ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P7/22
CPCC12N9/0006C12P7/22
Inventor 竺伟包蕾胡集铖
Owner SYNCOZYMES SHANGHAI
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