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Biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl

A technology for ethyl hydroxybutyrate and biological preparation, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of high dosage of enzymes and cofactors, poor safety, cumbersome operation, etc. Simple operation, high reaction efficiency and mild reaction conditions

Inactive Publication Date: 2013-02-13
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese invention patent applications, such as application numbers 200810124754.2, 201010213724.6, 201110225388.1, etc., disclose several methods for producing (S)-4-chloro-3-hydroxybutyrate ethyl by ketoreductase, but these methods use relatively high amounts of enzymes and cofactors , and need to add glucose as hydrogen donor
Although the substrate concentration can reach 30%, a large amount of organic solvent must be added to form a two-phase system, and the substrate needs to be fed in batches, which is cumbersome to operate and not safe

Method used

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  • Biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1 detects the HPLC / MS method of biotransformation reaction

[0021] Sample treatment: Take 50 μL of reaction solution at different time points, add 950 μL of methanol, dissolve, filter with 0.45 μm microporous membrane, and inject for detection. The injection volume is 1 μL. Chromatographic conditions: the chromatographic column is SB-C18 (2.1×50 mm, 3.5 μm), and the mobile phase is A water (0.1% HCOOH)-B acetonitrile. Flow rate 0.3mL / min, 0-3min 30%B. Mass spectrometry conditions: dry gas flow rate 12L / min, sheath gas pressure 40PSI, dry gas temperature 350°C, capillary voltage 3500V, detection mode is positive ion mode, detected ions 165.1, 167.1, 169.1. (S)-Ethyl 4-chloro-3-hydroxybutyrate has a retention time of 1.1 min, and ethyl 4-chloro-3-oxobutyrate has a retention time of 1.6 min.

Embodiment 2

[0022] Example 2 Gas Chromatographic Method for Detecting Biotransformation Reactions

[0023] Sample treatment: Take 200 μL of the reaction solution at different time points, add an equal volume of ethyl acetate to extract twice, take the organic phase and filter it with a 0.45 μm microporous membrane, and inject a sample for detection, the injection volume is 1 μL. Chromatographic conditions: CP-chirasil-dex CB 25*0.32mm*0.25μm column, carrier gas: nitrogen, inlet temperature: 180°C, detector temperature: 280°C, split ratio: 30:1, temperature program: The initial temperature is 70°C, the temperature is raised to 110°C at 10°C / min, and kept for 6 minutes, then the temperature is raised to 200°C at 40°C / min, and kept for 1.5 minutes. The retention time of ethyl (S)-4-chloro-3-hydroxybutyrate is 7.6min, and the retention time of ethyl 4-chloro-3-oxobutyrate is 3.7min.

Embodiment 3

[0024] Example 3 Preparation of recombinant ketoreductase

[0025] Inoculate a single colony of recombinant Escherichia coli containing the ketoreductase gene from a glycerol tube or transformation plate into 4 mL of liquid LB medium containing ampicillin resistance and activate overnight (37° C., 200 rpm). Transfer 100mL liquid LB medium containing ampicillin resistance from the overnight culture at 1 / 100 inoculum size, culture at 37°C, 200rpm shaking until OD 600 When the value reaches 0.6-0.8, add IPTG and continue culturing overnight at 30°C. The cells were collected by centrifugation, and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0). The cell suspension was ultrasonically disrupted in an ice bath for 10 minutes, centrifuged, the supernatant was pre-frozen overnight, and freeze-dried for 24h-48h to obtain ketoreductase KRED in the form of freeze-dried powder.

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Abstract

The invention relates to a biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl, which uses 4-chloro-3-carbonyl ethyl butyrate as a substrate and enables the substrate to be subjected to asymmetric reduction reaction in the presence of a biological catalyst, a cofactor and a hydrogen donor to produce the (S)-4-chloro-3-hydroxybutyrate ethyl, wherein the biological catalyst is recombinant ketone reductase and the hydrogen donor is isopropyl alcohol; the asymmetric reduction reaction is carried out in a hybrid system of aqueous buffer solution and toluene with pH of 7.0-9.0, and the volume ratio of the aqueous buffer solution to the toluene is 4-20:1. The method provided by the invention solves the problems of requirement of additional coenzyme system, too much added organic solvent, low efficiency of catalyst and high cost in the traditional biological preparation method. The method provided by the invention has the advantages of mild reaction condition, high reaction efficiency and simple operation, and the concentration of the substrate can be increased to 40%, thus improving the preparation efficiency of (S)-4-chloro-3-hydroxybutyrate ethyl and reducing the reaction cost greatly.

Description

technical field [0001] The invention relates to a biological preparation method of ethyl (S)-4-chloro-3-hydroxybutyrate. Background technique [0002] Ethyl (S)-4-chloro-3-hydroxybutyrate is a key chiral intermediate for the preparation of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors such as statins. Statins are currently the best-selling cholesterol-lowering and blood-lipid-lowering drugs in the world, and they are also the class of drugs with the highest sales. Therefore, ethyl (S)-4-chloro-3-hydroxybutyrate, which is a key chiral intermediate of statins, has a high demand and a wide range of applications. [0003] At present, the method of producing ethyl 4-chloro-3-carbonylbutyrate by reduction method can be divided into chemical method and biological method, among which the biological method has the characteristics of mild reaction conditions, strong stereospecificity and high conversion rate. been widely studied and applied. Chinese invention patent appl...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12N9/02C12R1/19
Inventor 鞠鑫唐圆圆
Owner ENZYMEWORKS
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