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Method for preparing (R)-o-chloromandelic acid through enzyme and application of enzyme

A technology of o-chloromandelic acid and enzymatic preparation, applied in the field of preparation of pharmaceutical intermediate-o-chloromandelic acid, can solve the problems such as low purity of o-chloromandelic acid, high cost and complicated preparation method, and achieves low price, Increase the effect of correct folding and simple process

Inactive Publication Date: 2016-02-24
WUHAN WUYAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current preparation method is more complicated, the cost is high, and the purity of the obtained (R)-o-chloromandelic acid is low, which cannot directly meet the requirements of pharmaceutical intermediates.

Method used

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  • Method for preparing (R)-o-chloromandelic acid through enzyme and application of enzyme
  • Method for preparing (R)-o-chloromandelic acid through enzyme and application of enzyme
  • Method for preparing (R)-o-chloromandelic acid through enzyme and application of enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 prepares the E.coli engineering strain that has recombined nitrilase gene

[0050] Prepare the E.coli engineering strain capable of expressing nitrilase by genetic engineering: the nitrilase gene and the fluorescent protein gene are recombined by an enzyme-free cloning method, and the process is as follows: image 3 As shown, enzyme-free cloning methods such as figure 1 shown. Then add DH5α Escherichia coli competent cells, carry out routine transformation coating plate, pick a single colony for PCR verification, inoculate the correct recombined colony in a small bacterial bottle and culture it overnight at 37°C and 200rpm on a shaker. The bacteria in the shake flask were collected, and the recombinant plasmid was obtained by extracting with a plasmid mini-extraction kit. The extracted plasmids were transformed into RosettaBlue (DE3) competent cells, and the single colonies obtained were respectively recorded as strain 1 # ~Strain 6 # , as a strain for ...

Embodiment 2

[0054] Embodiment 2 prepares SOB culture medium

[0055] The component content and preparation method of each liter of SOB medium are as follows: add 20 g of tryptone, 5 g of yeast extract, and 0.5 g of NaCl in 950 ml of deionized water, shake the container to completely dissolve the solute, add 10 ml of 250 mmol / L KCl Solution (dissolve 1.86g KCl in 100ml deionized water to make 250mmol / l KCl solution), adjust the pH value to 7.0 with 5mol / L KOH, adjust the volume to 1L with deionized water, and sterilize by autoclaving at 121°C for 30min. Before using this solution, add 5ml of sterilized 2mol / L MgCl 2 The solution is the SOB medium.

Embodiment 3

[0056] Embodiment 3 shake flask fermentation culture of recombinant strain

[0057] The bacterial strain 1 that embodiment 1 obtains # Inoculate into SOB liquid culture medium containing 50mg / ml kanamycin, shake the flask on a shaking table at 37°C and 200rpm, and when the OD600 of the bacteria reaches 0.5, move to a shaking table at 30°C overnight, and wait for the growth of the bacteria to Take it out at the end of the plateau phase, and collect resting cells by centrifugation (6000rpm, 5min) at 4°C, and record it as resting cells 1 # , the mass of wet bacteria reaches 4g / L.

[0058] strain 2 # , strain 3 # The same fermentation and culture operation as the same, the obtained resting cells were recorded as resting cells 2 # , resting cells 3 # . resting cell 1 # ~ resting cell 3 # The wet thalline mass is shown in Table 2.

[0059] Table 2

[0060] resting cell number

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Abstract

The invention discloses a method for preparing medical intermediate (R)-o-chloromandelic acid through enzyme. A large amount of nitrilase expressed E. coli is obtained with genetic engineering and biological fermentation methods and produces (R)-o-chloromandelic acid directionally through hydrolysis of o-chloromandelic mandelonitrile racemate. According to the method, the cost of raw materials is low, the process is simple, the reaction condition is mild, the energy consumption is low, few reaction steps are adopted, no conventional toxic reagent participates in a reaction, the method is environment-friendly and pollution-free, the purity and the ee value of prepared (R)-o-chloromandelic acid are both higher than 98%, and the quality requirement of a drug intermediate is met.

Description

technical field [0001] The application belongs to the field of biochemistry or medicinal chemistry, and in particular relates to a preparation method of a pharmaceutical intermediate (R)-o-chloromandelic acid. Background technique [0002] (R)-o-chloromandelic acid is a white crystalline powder with the chemical formula C 8 h 7 ClO 3 , the structural formula is [0003] [0004] The molecular weight is 186.59, and the melting point is 86-92°C. It is an important pharmaceutical intermediate and a raw material for the synthesis of the drug clopidogrel. Clopidogrel is a platelet aggregation inhibitor, which is widely used clinically to prevent and treat cardiovascular and cerebrovascular diseases caused by high platelet aggregation, such as myocardial infarction, stroke, arteriosclerosis, etc. Compared with other platelet inhibitors, it has a stronger effect, It has the advantages of good tolerance and small side effects. Clopidogrel was launched in the United States an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N9/78C12R1/19
CPCC12P7/42C12N9/78C12Y305/05001
Inventor 皮金红尹冬赵涛涛杨诗宏夏静平韩瑞陈晨马立新张琦
Owner WUHAN WUYAO PHARMA
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