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A method for enzymatically preparing (s)-3-hydroxyl-4-chlorobutyric acid ethyl ester

A technology of ethyl chlorobutyrate and ethyl acetate, which is applied in the field of biological preparation of ethyl-3-hydroxy-4-chlorobutyrate, can solve the problems of difficult treatment of three wastes, high cost of reduction, high risk, etc. Low, high product yield, high substrate conversion effect

Active Publication Date: 2015-12-02
SUZHOU CANIMBLE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of asymmetric reduction involving rare metals is too high; the reduction of sodium borohydride or borane is highly dangerous in industrialization, and the three wastes are difficult to deal with
[0005] China has already applied for similar invention patents such as application numbers 200810124754.2, 201010213724.6, 201110225388.1, 201210069347.2, etc., which disclose several methods for preparing (S)-4-chloro-3-hydroxybutyric acid ethyl ester by ketoreductase, but these methods all use Expensive coenzyme factors NAD or NADP, and need to add glucose or isopropanol as hydrogen donors, and must add organic solvents to form a two-phase system, and need to add substrates in batches, the operation is cumbersome

Method used

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  • A method for enzymatically preparing (s)-3-hydroxyl-4-chlorobutyric acid ethyl ester
  • A method for enzymatically preparing (s)-3-hydroxyl-4-chlorobutyric acid ethyl ester
  • A method for enzymatically preparing (s)-3-hydroxyl-4-chlorobutyric acid ethyl ester

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Pour 80L of potassium phosphate buffer solution with pH=7.0 into the 200L reactor, turn on the stirring and start heating, and set the reaction temperature at 37°C. Simultaneous preparation of MgCl 2 Aqueous solution, weigh 19.0g (0.2mol) MgCl 2 Dissolve in deionized water and make up to 2L volume. After the temperature in the reactor is stabilized at 37°C, the prepared 2LMgCl 2 Pour the aqueous solution into the reaction kettle at one time, continue to stir for 10 minutes, then add the weighed ethyl 4-chloroacetoacetate (16.5Kg) into the reaction kettle at one time, and then accurately measure 10L of ketoreductase enzyme Liquid (60U / ml) was added to the reaction system, and the reaction start time was recorded. During the whole reaction process, pay close attention to the temperature and pH value of the reaction system, and pay attention to follow-up detection (HPLC). After the conversion of the substrate is complete, the reaction stops, and it is directly extracte...

Embodiment 2

[0045] Pour 80L of potassium phosphate buffer solution with pH=7.0 into the 200L reactor, turn on the stirring and start heating, and set the reaction temperature at 37°C. Simultaneous preparation of CaCl 2 Aqueous solution, weigh 22.3g (0.2mol) CaCl 2 Dissolve in deionized water and make up to 2L volume. After the temperature in the reactor is stabilized at 37°C, the prepared 2LCaCl 2Pour the aqueous solution into the reaction kettle at one time, continue to stir for 10 minutes, then add the weighed ethyl 4-chloroacetoacetate (16.5Kg) into the reaction kettle at one time, and then accurately measure 10L of ketoreductase enzyme Liquid (65U / ml) was added to the reaction system, and the reaction start time was recorded. During the whole reaction process, pay close attention to the temperature and pH value of the reaction system, and pay attention to follow-up detection (HPLC). After the conversion of the substrate is complete, the reaction stops, and it is directly extracted...

Embodiment 3

[0048] Pour 80L of potassium phosphate buffer solution with pH=7.0 into the 200L reactor, turn on the stirring and start heating, and set the reaction temperature at 37°C. Simultaneous preparation of ZnCl 2 Aqueous solution, weigh 27.2g (0.2mol) ZnCl 2 Dissolve in deionized water and make up to 2L volume. After the temperature in the reactor is stabilized at 37°C, the prepared 2LZnCl 2 Pour the aqueous solution into the reaction kettle at one time, continue to stir for 10 minutes, then add the weighed ethyl 4-chloroacetoacetate (16.5Kg) into the reaction kettle at one time, and then accurately measure 10L of ketoreductase enzyme Liquid (62U / ml) was added to the reaction system, and the reaction start time was recorded. During the whole reaction process, pay close attention to the temperature and pH value of the reaction system, and pay attention to follow-up detection (HPLC). After the conversion of the substrate is complete, the reaction stops, and it is directly extracte...

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Abstract

The invention relates to an enzymatic method for preparing (S)-3-hydroxy-4-chlorobutyric acid ethyl ester. 4-chloroacetoacetic acid ethyl ester is taken as a substrate, in the presence of ketoreductase, an asymmetric reduction reaction is carried out on the substrate so as to generate (S)-4-chlorine-3-hydroxy ethyl butyrate, particularly, the ketoreductase is endoenzyme and is also intact cell enzyme, the reaction is carried out in a water phase buffer solution with the pH of 6.5-7.5 at 35-38 DEG C, wherein coenzyme factors are not used and organic solvents are not added. Compared with the existing biological preparation method, the method is lower in cost and easier in operation as both the expensive coenzyme factors and the organic solvents are not used, the transformation rate of the substrate is high, and the product yield is high, so that the method is more applicable to industrial production.

Description

technical field [0001] The invention relates to a biological preparation method of ethyl (S)-3-hydroxy-4-chlorobutyrate. Background technique [0002] (S)-3-Hydroxy-4-chlorobutyric acid ethyl ester is an essential key intermediate for the preparation of (S)-oxiracetam, and its structure is as follows: [0003] [0004] (S)-Oxiracetam is a nootropic drug that improves thinking, memory and learning. Oxiracetam is clinically used for memory and intellectual impairment caused by mild to moderate vascular dementia, senile dementia and traumatic brain injury. At present, racemic oxiracetam is used clinically, and a large amount of data at home and abroad show that the drug effect of (S)-oxiracetam is obviously better than that of (R)-oxiracetam. A large number of experiments have proved that compared with racemic oxiracetam, (S)-oxiracetam can significantly improve the learning and memory impairment of mice caused by scopolamine. However, there is no (S)-oxiracetam on the ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/62
Inventor 时春娟杨盟陆超
Owner SUZHOU CANIMBLE BIOTECH