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Ketoreductase and application thereof in preparation of (S)-2-chloro-1-(3,4-difluorophenyl) ethanol

A difluorophenyl and reductase technology, applied in the field of biological enzymes, can solve the problems of reduced conversion rate, low conversion rate, and reduced application value

Active Publication Date: 2019-03-05
SYNCOZYMES SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] This technology prepares (S)-2-chloro-1-(3,4-difluorophenyl)ethanol (Formula III) through ketoreductase, but it can be seen from the specific examples given in patent CN105671099 A that with The substrate concentration increases from 100g / L to 150g / L, and the conversion rate of the substrate decreases from 99.6% to 89%, which shows that the enzyme provided by the invention CN 105671099 A can convert its substrate under the reaction conditions of high substrate concentration rate will drop suddenly, although the patent claims that the concentration of the substrate is 1-300g / L in the claims, those skilled in the art can easily know the data variation trend according to the specific examples of the patent, and the patent provides The enzyme has the disadvantage of low conversion rate at high substrate concentration, and its effective and practical substrate catalytic concentration for industrial production will be limited below 150g / L
This limits the application of the patent CN 105671099 A technical solution in industrial production
[0023] However, the maximum complete catalytic concentration of the enzyme provided by the patent CN 106047828 A can reach 200g / L, which is not a very high reaction substrate concentration, and there is still a certain distance from industrialization, and the reaction time under the substrate concentration of 200g / L Above 20h, this further reduces the application value of the enzyme provided in the patent

Method used

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  • Ketoreductase and application thereof in preparation of (S)-2-chloro-1-(3,4-difluorophenyl) ethanol
  • Ketoreductase and application thereof in preparation of (S)-2-chloro-1-(3,4-difluorophenyl) ethanol
  • Ketoreductase and application thereof in preparation of (S)-2-chloro-1-(3,4-difluorophenyl) ethanol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation of ketoreductase

[0068] Inoculate the genetically engineered bacteria (vector pET21a, host cell E.Coli BL21(DE3)) containing the coding gene of ketoreductase (SEQ ID No.1) into 5 mL of ampicillin-containing LB test tube medium for activation culture (37°C Culture for 12 hours), transfer the activated culture to 400mL LB liquid medium containing ampicillin according to 1% inoculation amount, 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 ketoreductase cells, resuspend the cells in 40mL phosphate buffer (10mM, pH 7.5), and ultrasonically disrupt them in an ice-water bath for 15min, collect the supernatant by centrifugation, pre-freeze at -20°C and then vacuum freeze-dry Crush after 48 hours to obtain recombinant ketoreductase enzyme powder.

Embodiment 2

[0069] Example 2 Preparation of gram-grade (S)-2-chloro-1-(3,4-difluorophenyl)ethanol

[0070] Add isopropanol (4mL) and substrate 2-chloro-1-(3,4-difluorophenyl)ethanone (1.5g) into the reaction vessel, stir well and add 0.5g of ketoreductase cells, coenzyme NADP+ (0.3mg), and finally use phosphate buffer to make up to 10mL, stir the reaction under magnetic force at 25°C, and use NaOH (0.5M) to control the reaction pH at about 6.9, and TLC to detect the reaction progress. After the reaction is completed, diatomite is filtered, the liquid phase is extracted three times with the organic phase, the organic phases are combined, dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain the product. HPLC detection conversion rate and product ee value are attached figure 1 , 2 Shown: Substrate conversion rate = 99.7%, S-type product ee value = 99.8%.

Embodiment 3

[0071] Example 3 Preparation of gram-grade (S)-2-chloro-1-(3,4-difluorophenyl)ethanol

[0072] Add isopropanol (4.5mL) and substrate 2-chloro-1-(3,4-difluorophenyl)ethanone (2.0g) into the reaction vessel, stir well and add 0.75g of ketoreductase cells, coenzyme NADP+ (0.7mg), and finally use phosphate buffer to make up to 10mL, magnetically stir the reaction at 45.5°C, and use NaOH (0.5M) to control the reaction pH at about 6.8, and TLC to detect the reaction progress. After the reaction is completed, diatomite is filtered, the liquid phase is extracted three times with the organic phase, the organic phases are combined, dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain the product. HPLC detection conversion rate as attached image 3 , the product ee value is attached figure 2 : Substrate conversion rate = 99.3%, S-type product ee value = 99.8%.

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Abstract

The invention relates to preparation of chiral alcohol by an enzymic method, belongs to the field of preparation of a medical intermediate by a gene engineering technology, and provides ketoreductaseand application thereof in preparation of (S)-2-chloro-1-(3,4-difluorophenyl) ethanol. Ketoreductase of which the source is different from that of ketoreductase in the prior art is provided. The concentration of a substrate 2-chloro-1-(3,4-difluorophenyl) ethanone which can be converted by the ketoreductase reaches 350 g / L or above, moreover, ee value of a product (S)-2-chloro-1-(3,4- difluorophenyl) ethanol can reach 99% or above, and meanwhile, the conversion rate of the substrate is 99% or above. According to the technical scheme, by the ketoreductase which is from the new source, the problem that the substrate concentration of (S)-2-chloro-1-(3,4-difluorophenyl) ethanol prepared by enzymatic hydrolysis in industrial application is not high is solved, and the conversion rate of the high-concentration (S)-2-chloro-1-(3,4-difluorophenyl) ethanol and the ee value of the product are increased.

Description

technical field [0001] The invention relates to a biological enzyme, in particular to a ketoreductase and its application in the preparation of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol. Background technique [0002] Ticagrelor is a platelet aggregation inhibitor, and its clinical efficacy and safety have been verified and supported by the Platelet Inhibition and Patient Outcome Study (PLATO study) and its multiple subgroup studies. The PLATO study also showed that ticagrelor was significantly more effective than clopidogrel, so it was listed as the first-line recommendation by many domestic and foreign guidelines. [0003] The synthetic route of ticagrelor is as formula I: [0004] [0005] Wherein, the compound shown in formula II is as the key intermediate for preparing ticagrelor, because it possesses unique chiral center, the improvement of its preparation method has always been an important work in the field of ticagrelor intermediate synthesis technology. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12P7/22
CPCC12N9/0006C12P7/22
Inventor 竺伟包蕾胡集铖
Owner SYNCOZYMES SHANGHAI
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