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(S)-4-chloride-3-ethyl 3-hydroxybutyrate biological preparation method

The technology of ethyl hydroxybutyrate and ethyl carbonyl butyrate is applied in the field of biological preparation of ethyl-4-chloro-3-hydroxybutyrate, can solve the problems of expensive cofactors, complicated operation and the like, achieves simple operation, Improves the effect of low substrate concentrations

Active Publication Date: 2012-07-25
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 the cofactors used in these methods are all expensive NADP, and requires the addition of glucose as a 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

Method used

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  • (S)-4-chloride-3-ethyl 3-hydroxybutyrate biological preparation method

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Experimental program
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Effect test

Embodiment 1

[0022] Take four 20ml three-necked flasks, add 2.37ml phosphate buffer solution (100mM, pH 6.5) to each, and add 1.33ml of substrate 4-chloro-3-carbonyl butyrate ethyl ester, isopropanol 1.3ml, ketone Reductase KRED (commercially purchased) 1020.067g, in three flasks wherein, add respectively the polyethylene glycol 400 of 10%, 30% and 50% of reaction system total volume (10mL), finally add phosphate in each flask The buffer solution was added to 10 ml, and the reaction was stirred at 200 rpm at 30° C., and the conversion rate of the reaction was monitored by gas chromatography. The reaction conversion rate in each flask varies with time as shown in figure 1 shown. From figure 1 Visible, when polyethylene glycol add-on is 50%, the conversion rate of reaction is maximum.

Embodiment 2

[0024] Take four 20ml three-neck flasks, add 2.37ml phosphate buffer solution (100mM, pH 6.5) respectively, and add substrate 4-chloro-3-carbonyl butyric acid ethyl ester 1.33ml, isopropanol 1.3ml respectively successively, Ketoreductase KRED102 (commercially purchased) 0.067g and polyethylene glycol 4005ml, also add the anhydrous calcium chloride of 0.1mM, 1mM and 5mM respectively in three flasks wherein, at 30 ℃, 200rpm stirring reaction, utilize The conversion of the reaction was monitored by gas chromatography. The reaction conversion rate in each flask varies with the reaction time as shown in figure 2 shown. From figure 2 It can be seen that when the amount of calcium ions added is 1mM, the conversion rate of the reaction is the largest.

Embodiment 3

[0026] Add 2.37ml of phosphate buffered saline solution (100mM, pH 6.5) into a 20mL three-necked flask, add substrate 1.33ml of ethyl 4-chloro-3-carbonylbutyrate, 1.30ml of isopropanol, ketoreductase KRED102 (commercial Purchase) 0.02g, polyethylene glycol 4005ml, anhydrous calcium chloride 1mM, under 30 ℃, 200rpm stirring reaction, utilize gas chromatography to monitor the conversion rate of reaction. After 24 hours the conversion reached 85.7%.

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Abstract

The invention relates to an (S)-chloride-3-ethyl 3-hydroxybutyrate biological preparation method, which includes: utilizing 4-chloride-3-carbonyl ethyl butyrate as substrate so that the substrate can be asymmetrically reduce to generation (S)-4-chloride-3-ethyl 3-hydroxybutyrate under the existence of biocatalyst and hydrogen donor, wherein the biocatalyst is ketoreductase KRED102, the hydrogen donor is isopropanol, the asymmetric reduction is carried in water-phase buffer solution with pH (potential of hydrogen) value of 5.0-9.0, and polyethylene glycol 400 as dispersant and divalent metal ions as enzymatic activity intensifier are added into the reaction system. By adding auxiliaries low in cost and wide in sources, the (S)-chloride-3-ethyl 3-hydroxybutyrate biological preparation method overcomes the defects of low substrate concentration and long enzyme reaction time and the like during conventional enzyme catalyzing production effectively, is simple in operation and particularly applicable to popularization and application in the pharmaceutical industry.

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 world's best-selling cholesterol-lowering and blood-lipid-lowering drugs. 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. Among the reductases selected in the biological process, the NAD-dependent type and the one tha...

Claims

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Application Information

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IPC IPC(8): C12P7/62
Inventor 陶军华鞠鑫唐圆圆李斌
Owner ENZYMEWORKS
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