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Biological preparation of key intermediate of thiamphenicol and florfenicol

A technology of intermediates and substrates, applied in the field of medicine, can solve the problems of severe environmental pressure, serious water pollution, solid waste pollution, etc.

Active Publication Date: 2019-01-15
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The weak point of existing production route has three points: (1) copper sulfate is used in a large amount in the reaction, and the color of copper sulfate seriously pollutes the water body, and the copper sulfide formed by copper sulfate and hydrogen sulfide produces solid waste pollution, so the use of copper salt brings Severe environmental protection pressure has come; (2) The theoretical yield of traditional splitting is 50%; (3) The resolving agent needs to be recycled and applied mechanically
The weak point of this route is to split 50% of theoretical yield, secondly use two equivalents of methyl sulfone benzaldehyde, increase raw material cost
Although the design of route 2 is reasonable and ingenious, no suitable metal ligand was screened, and the configuration of the hydroxyl group in the transfer hydrogenation reduction product is not required. It needs to go through a tedious three-step reaction of activation, inversion, and hydrolysis.

Method used

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  • Biological preparation of key intermediate of thiamphenicol and florfenicol
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  • Biological preparation of key intermediate of thiamphenicol and florfenicol

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preparation example Construction

[0163] The preparation of the enzyme reduction substrate compound X can refer to the method described in Tetrahedron.2016, 72:1787-1793.

[0164] method

[0165] 1. Enzyme preparation method

[0166] Through conventional techniques in the field, the above-mentioned glucose dehydrogenase for coenzyme regeneration and the target gene were constructed on the same plasmid pET28a(+) vector, and then introduced into the expression host Escherichia coli, and the bacterium containing the target dual enzyme was obtained by inducing expression. Bacterial cells can be obtained directly by centrifugation, or they can be broken to obtain crude enzyme liquid, and the crude enzyme powder is used for subsequent biotransformation reactions.

[0167] 2. Method for preparing compound Y by biocatalytic reduction of compound X

[0168] The invention provides a method for preparing compound Y by catalyzing reduction of compound X by carbonyl reductase. The reaction formula is as follows:

[016...

Embodiment 1

[0179] Embodiment 1, the construction of carbonyl reductase engineering bacteria

[0180] The EA carbonyl reductase target gene and the glucose dehydrogenase target gene were entrusted to a commercial company to carry out the whole gene synthesis, cloned into the pET28a(+) vector, transformed into Escherichia coli DH5α competent cells, cultured on the plate, and picked a single colony of positive transformants After extracting and sequencing the plasmid, the recombinant plasmid was extracted, introduced into BL21 (DE3) strain, cultured in LB, and genetically engineered bacteria capable of inducing the expression of recombinant carbonyl reductase and alcohol dehydrogenase were obtained.

Embodiment 2

[0181] Embodiment 2, the preparation of recombinant carbonyl reductase, glucose dehydrogenase

[0182] Inoculate the genetically engineered bacterium stored in glycerol in the previous step into the LB liquid medium containing kanamycin, cultivate for 13 hours at 37°C and 220rpm to obtain the seed medium, and inoculate the seed culture medium in a proportion of 1.5%. On the liquid medium containing 50ug / ml kanamycin resistance, then cultivate to OD at 37℃, 220rmp 600 If the value is >2.0, add lactose with a final concentration of 1.0%, cool down to 25°C, continue to cultivate for 3 hours, add lactose with a final concentration of 0.5%, and cultivate for 20 hours, put it in a tank, and centrifuge to obtain bacteria, which is ready for biotransformation.

[0183] The fermentation formula is as follows:

[0184] raw material

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Abstract

The invention provides a biological preparation method of a key intermediate of thiamphenicol and florfenicol. Specifically, the method of the present invention comprises the following steps: (a) in aliquid reaction system, a reaction represented by formula A is carried out in the presence of a coenzyme and catalyzed by a carbonyl reductase, using compound X as a substrate, thereby forming compound Y; and (b) optionally separating the compound Y from the reaction system after the reaction of the previous step. The invention also provides a reaction system comprising: (i) an aqueous solvent; (ii) a substrate, said substrate being the compound X; (iii) coenzyme; (iv) carbonyl reductase; (v) a co-substrates; and (vi) enzymes for coenzyme regeneration.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a preparation method of key chiral intermediates of aminoalcohol antibacterial drugs thiamphenicol and florfenicol. Background technique [0002] Thiamphenicol antibiotics include thiamphenicol (thiamphenicol, 1) and its fluoride, florfenicol (florfenicol, 2). They are chemically similar to the obsolete antibiotic chloramphenicol (chloramphenicol, 3), and both belong to the β-aminoalcohol antibiotics [1] . [0003] The chemical structure of amino alcohol drugs is as follows: [0004] [0005] Thiamphenicol was synthesized for the first time in 1952. It has the characteristics of strong antibacterial activity, fast absorption, long-lasting drug effect and low toxicity. It is mainly used clinically for typhoid fever, dysentery, respiratory tract infection, urinary tract infection, hepatobiliary infection, intestinal Infection, surgical infection, brucellosis and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C07C315/04C07C317/32
CPCC12P13/00C12P13/001C07C315/04C07C317/32
Inventor 张福利陈少欣倪国伟邹杰汤佳伟谭支敏郭翔
Owner SHANGHAI INST OF PHARMA IND
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