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Biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol

A technology of fluorophenyl and fluoroacetophenone, which is applied in the field of biopharmaceuticals, can solve the problems of unfavorable extraction efficiency and operation process, complicated reaction system and high production cost, and achieves convenient industrial production, simple operation process and high ee value. Effect

Inactive Publication Date: 2016-10-26
SYNCOZYMES SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, two kinds of oxidoreductase enzyme powder and exogenous coenzyme NADP are used, the reaction system is complicated and the production cost is high
In addition, when the enzyme powder is used as the catalyst, the emulsification of the post-treatment process is serious, which has a negative impact on the extraction efficiency and the operation process.

Method used

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  • Biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Construction of co-expression recombinant bacteria

[0028] The fully synthetic KRED182 gene was used as a template for PCR amplification, and restriction sites were introduced at both ends (forward primer was introduced into NcoI restriction site, reverse primer was introduced into HindIII restriction site), and NcoI and HindIII were used for its enzymes. cut and recovered the KRED gene fragment. At the same time, the plasmid of the pRSF-Duet vector was extracted and digested with NcoI and HindIII, and the digested vector fragment was recovered. The KRED gene fragment and the vector fragment were ligated using T4 ligase, transformed into E. coli BL21 (DE3), coated on a Kan-resistant plate, and cultured in a 37°C incubator. After the transformants were grown, several single clones were picked for colony PCR verification, and the positive single clone was selected for subsequent experiments, and the strain was named KRED-1 bacteria. Then, the KRED bacterial p...

Embodiment 2

[0029] Example 2 Preparation of free cells and immobilized whole cells of co-expressed recombinant bacteria

[0030] Co-expressed free cells can be obtained by fermentation and culture. The fermentation medium uses 2YT medium, which contains 16g / L peptone, 10g / L yeast powder, 5g / L NaCl, and 2g / L glycerol. The specific steps are as follows: firstly inoculate the strain into a small shaker flask (250ml capacity) containing 50mL of culture medium, cultivate the strain at 37°C for 12h on a shaker to activate the strain, and then inoculate the activated bacteria into a small shaker with a capacity of 250ml. In a 400mL medium shake flask (capacity 1L, with baffle), the inoculum volume is 8ml, incubate at 37°C on a shaker until the OD600nm reaches 0.6-1.0, add IPTG to a final concentration of 0.1mM, and shake at 25°C for induction After 20 h of expression, the co-expressing cells were collected by centrifugation, which were called free cells.

[0031] Weigh 300g of polyvinyl alcohol...

Embodiment 3

[0032] Example 3 Preparation of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol by transformation of co-expressed free cells

[0033] 100mM phosphate buffer (10mL, pH=6.0), glucose (3.0g) and substrate 2,6-dichloro-3-fluoroacetophenone (3.0g) were added to a 25mL reaction vessel, and the volume was adjusted to 15mL , co-expressed free cells (750mg) after stirring evenly, magnetic stirring reaction at 35 ℃, Na 2 CO 3(20%, w / v) pH of the reaction was controlled at about 6.0, and the reaction progress was detected by TLC. After the reaction, the cells were removed by centrifugation, extracted three times with equal volumes of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried under reduced pressure to obtain the product (S)-1-(2,6-dichloro-3-fluorobenzene) base) ethanol 2.84g, the molar yield of the product is 94%, and the ee value is greater than 99.9%. HPLC detects the conversion rate and the ee value of the product, the conversio...

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Abstract

The invention discloses a biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol. The method comprises the following steps: proportionally mixing co-expressed whole cells, 2,6-dichloro-3-fluoroacetophenone, glucose and a buffer solution, reacting at 30-40 DEG C under the pH value of 5-8, and carrying out after-treatment to obtain the target product, wherein the co-expressed whole cells are gene engineering bacteria containing carbonyl reductase and glucose dehydrogenase. Compared with the prior art, the method disclosed by the invention does not need any exogenous coenzyme, and the catalyst can be easily separated from the reaction solution, so that the technique is greatly simplified and the production cost is lowered. The product has higher yield and ee value, and thus, has favorable industrial application value.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol. Background technique [0002] Crizotinib is an ATP-competitive, multi-targeted protein kinase inhibitor developed by Pfizer that inhibits ALK / MET / ROS. The drug has been proven in cancer patients with abnormal ALK, ROS and MET kinase activity It has a significant clinical effect on the human body. In 2011, crizotinib was approved by the U.S. Food and Drug Administration (FDA) for marketing. and metastatic non-small cell lung cancer. Its structural formula is as follows: [0003] [0004] (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol is a key intermediate in the synthesis of crizotinib, and its preparation methods have been extensively studied at home and abroad. The current preparation methods include chemical synthesis and Biotransformation. The chemical synthesis method mainly uses a chiral cata...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12N11/10C12N11/08C12N1/21C12N1/19C12R1/19C12R1/84
CPCC12P7/22C12N9/0006C12N11/08C12N11/10C12Y101/01184C12Y101/9901
Inventor 竺伟高新星胡集铖吴会
Owner SYNCOZYMES SHANGHAI
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