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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: 2013-11-27
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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  • (S)-4-chloride-3-ethyl 3-hydroxybutyrate biological preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Take four 20ml three-necked flasks, add 2.37ml of phosphate buffer solution (100mM, pH 6.5) to each, and add the substrate 1.33ml of ethyl 4-chloro-3-carbonylbutyrate, 1.3ml of isopropanol, ketone Reductase KRED (commercially purchased) 1020.067g, 10%, 30% and 50% polyethylene glycol 400 of the total volume of the reaction system (10mL) were added to three flasks, and finally phosphate was added to each flask The buffer solution was added to 10 ml, and the reaction was stirred at 30° C. and 200 rpm, and the conversion rate of the reaction was monitored by gas chromatography. The change of reaction conversion rate in each flask with time is shown in the figure figure 1 shown. from figure 1 It can be seen that when the amount of polyethylene glycol added is 50%, the conversion rate of the reaction is the largest.

Embodiment 2

[0024] Take four 20ml three-necked flasks, add 2.37ml phosphate buffer solution (100mM, pH 6.5) respectively, and add 1.33ml of ethyl 4-chloro-3-carbonyl butyrate and 1.3ml of isopropanol to each of them in turn. 0.067 g of ketoreductase KRED102 (commercially purchased) and 4005 ml of polyethylene glycol were added to three flasks of which 0.1 mM, 1 mM and 5 mM of anhydrous calcium chloride were respectively added, and the reaction was stirred at 30 ° C and 200 rpm, using The conversion of the reaction was monitored by gas chromatography. The reaction conversion rate in each flask as a function of reaction time is shown in Fig. figure 2 shown. from figure 2 It can be seen that the reaction conversion rate is the highest when the calcium ion addition amount is 1 mM.

Embodiment 3

[0026] In a 20mL three-necked flask, 2.37ml of phosphate buffer solution (100mM, pH 6.5) was added, followed by the substrate 1.33ml of ethyl 4-chloro-3-carbonylbutyrate, 1.30ml of isopropanol, ketoreductase KRED102 (commercial purchased) 0.02g, polyethylene glycol 4005ml, anhydrous calcium chloride 1mM, the reaction was stirred at 30° C. and 200rpm, and the conversion rate of the reaction was monitored by gas chromatography. The conversion reached 85.7% after 24 hours.

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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 (S)-4-chloro-3-hydroxybutyric acid ethyl ester. Background technique [0002] (S)-ethyl 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 hypolipidemic drugs in the world. Therefore, (S)-4-chloro-3-hydroxybutyric acid ethyl ester, 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 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 best ones are NAD-dependent a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/62
Inventor 陶军华鞠鑫唐圆圆李斌
Owner ENZYMEWORKS