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Method for preparing darunavir intermediate through biological catalysis

A technology of darunavir and biocatalysis, which is applied in the field of biocatalytic preparation of darunavir intermediates, can solve the problems of low enzyme activity and low catalytic reaction efficiency, and achieve high alcohol dehydrogenase activity and high number of coenzyme cycles , The effect of low discharge of three wastes

Active Publication Date: 2020-09-22
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of low enzyme activity in the preparation process of (2S,3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol (structure II) in the prior art. The technical problem of low catalytic reaction efficiency

Method used

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  • Method for preparing darunavir intermediate through biological catalysis
  • Method for preparing darunavir intermediate through biological catalysis
  • Method for preparing darunavir intermediate through biological catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli. Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base , to get splicing primers.

[0022] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.

[0023] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl wxya 2 o

[0024] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the f...

Embodiment 2

[0025] Synthesis of embodiment 2 control protein CKSm gene sequence

[0026] According to the sequence shown in SEQ ID NO.7, Nanjing GenScript Biology was entrusted to carry out the whole gene synthesis of the coding sequence of the protein, and cloned it into pET30a to obtain the control protein expression plasmid NYK-CKSm. Its corresponding amino acid sequence is SEQID NO.8.

Embodiment 3

[0027] Embodiment 3 shake flask expression test

[0028] Pick a single colony of Escherichia coli containing the expression vector and inoculate it in 10ml of high-pressure sterilized medium: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, potassium dihydrogen phosphate 3.4g / L, Ammonium Chloride 2.68g / L, Sodium Sulfate 0.71g / L, Magnesium Sulfate Heptahydrate 0.493g / L, Ferric Chloride Hexahydrate 0.027g / L, Glycerin 5g / L, Glucose 0.8g / L, Add Calcium Namycin to 50mg / L. Cultivate overnight at 30°C, 250rpm. The next day, take a 1L Erlenmeyer flask and insert it into 100ml of high-pressure sterilized medium at an inoculation ratio of 1:100: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, phosphoric acid Potassium dihydrogen 3.4g / L, Ammonium chloride 2.68g / L, Sodium sulfate 0.71g / L, Magnesium sulfate heptahydrate 0.493g / L, Ferric chloride hexahydrate 0.027g / L, Glycerin 5g / L, Glucose 0.3g / L, add kanamycin to 50mg / L. Cultivate at ...

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Abstract

The embodiment of the invention discloses a method for preparing a darunavir intermediate through biological catalysis, and belongs to the technical field of a biological catalysis method and an application. 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol can be converted into (2S, 3S)-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol through a ketoreductase mutant; the ketoreductase mutant is derived from wild type ketoreductase of Starmella magnoliae, has higher alcohol dehydrogenase activity compared with a wild type sequence, has 90% or above similarity with SEQ ID NO.8and has one or more of the following characteristics: I54E, S85A and K182V; and the sequence of the ketoreductase mutant is shown as SEQ ID NO. 4. Alcohols are used for coenzyme circulation, the substrate concentration is up to 110 g / L, the ratio of the substrate dosage to the NADP dosage is up to 1100: 1, the number of times of coenzyme circulation is high, and the downstream application range iseffectively widened.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis methods and applications, and relates to a method for preparing a darunavir intermediate by biocatalysis, in particular to a method for preparing (2S,3S)-1-chloro-3-tert-butoxycarbonyl by biocatalysis Amino-4-phenyl-2-butanol method. Background technique [0002] (2S,3S)-1-Chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol is used as a key intermediate for the preparation of darunavir. Currently, the main production processes include chemical synthesis and biological methods Two, the biological method can be obtained by the corresponding ketoreductase or microbial whole cell biotransformation of 3S-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol. [0003] The chemical method reacts with di-tert-butyl dicarbonate in methanol, and uses L-phenylalanine as the starting material to obtain N-Boc-L-phenylalanine, and then passes through hydrogen chloride after diazotization to obtain keton...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/02C12N9/04C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12P13/02C12N9/0006C12N15/70C12Y101/01184C12N2800/22
Inventor 丁雪峰王乾李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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