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Enzymic preparation method of luliconazole intermediate

A technology for enzymatic preparation of azole intermediates, which is applied in the field of enzymatic preparation of luliconazole intermediates, can solve the problems of low chiral ee value and cannot meet industrial production, and achieves good optical purity, green environmental protection, low pollution effect

Inactive Publication Date: 2018-07-17
ZHEJIANG OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Also visible in this document is the biotransformation of (S)-2-chloro-1-(2,4-dichlorophenyl)ethanol, but the chiral ee value is not high (ee value 90), so it cannot meet the requirements of industrial production need

Method used

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  • Enzymic preparation method of luliconazole intermediate
  • Enzymic preparation method of luliconazole intermediate
  • Enzymic preparation method of luliconazole intermediate

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Experimental program
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Embodiment 1

[0031] (1) Synthesis of the original gene of carbonyl reductase: according to the original gene sequence of carbonyl reductase LKADH, codon optimization was carried out on the original gene sequence according to the Escherichia coli codon analysis table, and the target gene was fully synthesized. The two ends were respectively cut with NdeI and XhoI sites and connected to pET26b(+) to obtain the recombinant expression vector pET26b-LKADH. The optimized LKADH original gene sequence is shown in SEQ NO.1, and its encoded amino acid sequence is shown in SEQ NO.2.

[0032] (2) Construction of carbonyl reductase mutants:

[0033] (3) Expression of carbonyl reductase: The recombinant expression vectors pET26b-LKADH and pET26b-LKADHM were transformed into expression strain BL21(DE3) by heat shock transformation method. Pick a single colony, insert the single colony into 5 mL of LB culture medium containing kanamycin, culture at 37°C overnight with shaking, take 1 mL of the bacterial ...

Embodiment 2

[0039]a) Synthesis of ketoreductase gene:

[0040] According to the codon preference of Escherichia coli, the ketoreductase gene KRED (SEQ ID NO.1) was synthesized from the whole gene, and Shanghai Jierui Biotechnology Co., Ltd. was entrusted to carry out the full synthesis of the gene. and XhoI restriction sites and ligated to pET26b(+) to obtain the recombinant expression vector pET26-KRED.

[0041] b) Transform the recombinant expression vector into Escherichia coli: Prepare Escherichia coli BL21 (DE3) competent cells, transform the recombinant expression vector pET26-KRED into BL21 (DE3) competent cells by calcium chloride method, and spread on LB plates containing 50 μg / mL of kanamycin resistance were incubated overnight at 37°C. Pick a single colony, extract the plasmid after a small amount of amplification, then perform 1.5% agarose gel electrophoresis, and observe it under ultraviolet light. The recombinant E. coli that contains the plasmid band is the recombinant E. ...

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Abstract

An enzymic preparation method of a luliconazole intermediat is characterized in that 2-chlorine-1-(2, 4-dichlorophenyl) acetone is used as a substrate and is subjected to asymmetric reduction in the existence of ketoreductase, a cofactor and a hydrogen donor to obtain (S)-2-chlorine-1-(2, 4-dichlorophenyl) ethyl alcohol. The method comprises the following steps: (1) preparing ketoreductase, namely, inoculating single recombinant escherichia coli containing ketoreductase gene into liquid LB culture medium with kanamycin resistance; culturing under temperature of 37 DEG C, and staying overweight; inoculating the activated culture material into the kanamycin containing liquid LB culture medium; oscillating and culturing under the temperature of 37 DEG C until OD600 is 0.6-0.8; adding IPTG with final concentration of 0.1M; performing induced culture for 8-10h under the temperature of 25 DEG C; centrifuging; collecting thallus; and ultrasonically breaking walls to obtain the recombinant ketoreductase; and (2) preparing (S)-2-chlorine-1-(2, 4-dichlorophenyl) ethyl alcohol, namely, adding the substrate 2-chlorine-1-(2, 4-dichlorophenyl) acetone to a buffering solution, and adding certainamount of cofactor NAD(P) and isopropanol; adding the recombinant ketoreductase; reacting by biologically catalyzing under the temperature of 30 DEG C; and adding ethyl acetate for extracting after the reaction is finished, thus obtaining the (S)-2-chlorine-1-(2, 4-dichlorophenyl) ethyl alcohol.

Description

technical field [0001] The invention relates to an enzymatic preparation method of a luliconazole intermediate, in particular to a method for asymmetrically catalyzing the production of a luliconazole intermediate (S)-2-chloro-1-(2,4-dichlorobenzene) by recombinant ketoreductase The method of base) ethanol belongs to the field of biopharmaceutical and biochemical technology. Background technique [0002] Luliconazole is an imidazole antifungal drug developed by Nippon Pesticide Co., Ltd., which inhibits the synthesis of ergosterol by inhibiting lanosterol demethylase, and reduces the accumulation of fungal ergosterol and relative lanosterol. This drug is suitable for fungal treatment , including tinea corporis, candidiasis and tinea versicolor. Compared with previous antifungal topical drugs, the biggest advantage of luliconazole is high skin retention rate, short drug cycle (half that of general drugs), good curative effect and not easy to relapse, so it has great competit...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12N9/04C12R1/19
CPCC12N9/0006C12P7/22C12Y101/01002
Inventor 唐云平郑佳文余芳苗杨最素丁国芳
Owner ZHEJIANG OCEAN UNIV
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