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Biological preparation method of 3-amino-4-(2,4,5-trifluorophenyl)methyl butyrate

A technology of trifluorophenyl and methyl butyrate, applied in the fields of biopharmaceuticals and green chemistry, to achieve the effects of less by-products, simple and convenient operation, and high optical activity

Inactive Publication Date: 2013-04-03
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no transaminases have been used to convert 3-carbonyl-4-(2,4,5-trifluorophenyl)butyrate methyl ester to 3-amino-4-(2,4,5-trifluorophenyl) A report on methyl fluorophenyl)butyrate

Method used

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  • Biological preparation method of 3-amino-4-(2,4,5-trifluorophenyl)methyl butyrate

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Experimental program
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Effect test

Embodiment 1

[0024] Single colonies from glycerol tubes or transformation plates were inoculated into 4 mL of liquid LB medium containing kanamycin resistance for overnight activation (37°C, 200 rpm), and the inoculum of 1 / 100 (v / v) was transferred to 100mL of liquid LB medium containing kanamycin resistance, 37°C, 200rpm shaking culture until the OD600 value reaches 0.6-0.8, add IPTG and continue culturing at 30°C overnight, centrifuge to collect cells, wash with 10mL phosphate buffer (2mM, pH7 .0) Suspend the cells, place the cell suspension in an ice bath for 10 minutes by ultrasonication, then centrifuge, pre-freeze the supernatant overnight to a temperature of -10~-25°C, and freeze-dry for 24h-48h.

Embodiment 2

[0026] In a 20mL three-necked flask, add 3.85mL of 0.1M pH 7.0 phosphate buffer solution, 100mg of substrate, 900μL of isopropylamine, 0.25mL of DMSO, 1mg of pyridoxal phosphate (PLP), 50mg of transaminase, at 30°C, 200rpm stirring paddle Stirring, under the condition of 0.01MPa nitrogen purging, reacted for 24h, and the conversion rate was detected by HPLC>95%. Add hydrochloric acid to adjust the pH to 2-3, filter with celite, add an equal volume of ethyl acetate to extract twice, and rotary evaporate to obtain 90 mg of the product with a purity of >95% and an optical purity of >99%.

Embodiment 3

[0028] In the reactor, add 3.85L of 0.1M pH 7.0 phosphate buffer solution, 100g of substrate, 900mL of isopropylamine, 0.25L of DMSO, 1g of pyridoxal phosphate (PLP), 50g of transaminase, at 30°C, mechanical stirring paddle Stirring, under the condition of 0.01MPa nitrogen purging, reacted for 24h, and the conversion rate was detected by HPLC>95%. Add hydrochloric acid to adjust the pH to 2-3, filter with diatomaceous earth, add an equal volume of ethyl acetate to extract twice, and rotary evaporate to obtain about 90 g of the product, with a purity of >95% and an optical purity of >99%.

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Abstract

The invention relates to a biological preparation method of 3-amino-4-(2,4,5-trifluorophenyl)methyl butyrate. According to the biological preparation method, 3-carbonyl-4-(2,4,5-trifluorophenyl)methyl butyrate is used as a substrate; under the condition that a biocatalyst, a cofactor, an amino donor and a cosolvent exist, the substrate performs the reaction to generate a target product 3-amino-4-(2,4,5-trifluorophenyl)methyl butyrate; the biocatalyst is recombination aminopherase; and the reaction is carried out in water-phase buffer solution with the pH of 6.0 to 9.0. The biological preparation method realizes that 3-carbonyl-4-(2,4,5-trifluorophenyl)methyl butyrate is converted into 3-amino-4-(2,4,5-trifluorophenyl)methyl butyrate by adopting the recombination aminopherase for the first time; compared with a conventional chemical method, the biological preparation method has low energy consumption, is simple and convenient to operate, generates a small number of by-products; and moreover, the product has high optical activity.

Description

technical field [0001] The invention belongs to the fields of biological pharmacy and green chemistry, and in particular relates to a biological preparation method of methyl 3-amino-4-(2,4,5-trifluorophenyl)butyrate. Background technique [0002] Biocatalysis has the obvious characteristics of green, high efficiency and economy. The selective conversion of ketone groups to amino groups by transaminases is one of the most important biotransformation reactions. [0003] Transaminase (amino transferase) is a class of enzymes used to synthesize chiral ammonia or amino acids. Wherein β-amino acid substances are widely used in drug synthesis, can be used to synthesize a series of dipeptidase-IV inhibitors, and are important anti-tumor, anti-infection and drug for treating metabolic disorders (Pharmacol.2004,404 :239, BMC Endocr. Disord. 2008, 8:14). Among the two main options for the preparation of β-amino acids by biotransformation, compared to the enzymatic resolution method ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N9/10
Inventor 乐庸堂鞠鑫
Owner ENZYMEWORKS
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