Biological preparation method of (R)-3-amino-4-(2,4,5-trifluorophenyl) tert-butyl butyrate

A trifluorophenyl, biological preparation technology, applied in the field of biopharmaceuticals and green chemistry, can solve the problems of residual protein risk, high separation cost, poor stability, etc., and achieve no protein residue, high optical activity, simple and convenient operation. Effect

Active Publication Date: 2016-12-07
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The use of transaminases to produce chiral amines has the characteristics of low cost, high efficiency and environmental protection. Patent US 20120329108 and others reported the substrate 3-carbonyl-1-(3-(trifluoromethyl)-5,6-dihydro-[1, Transamination reaction of 2,4]triazol[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one, patent CN 201210555608.1 reported the transamination reaction of 3-carbonyl-4-(2,4,5-trifluorophenyl) methyl butyrate, but so far, no transaminase has been used to convert 3-carbonyl-4-( A report on the conversion of tert-butyl 2,4,5-trifluorophenyl)butyrate (1) to tert-butyl 3-amino-4-(2,4,5-trifluorophenyl)butyrate
[0004] The enzyme conversion process reported in the patent US 20120329108 is used in the last step of the synthesis of raw materials, and the separation cost is high due to the risk of residual protein
CN 201210555608 reported using methyl ester or ethyl ester as substrate, but its stability is poor

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] In a 20mL three-neck flask, add 3.2mL of 0.1M pH 9.5 triethanolamine-hydrochloric acid buffer solution, 500mg of substrate, 900μL of isopropylamine, 0.4mL of Tween 60, 0.4mL of DMSO, 5mg of pyridoxal phosphate (PLP), transaminase EW1101 (Suzhou Hanzyme Biotechnology Co., Ltd.) 50mg, at 30°C, 200rpm paddle stirring, 0.01MPa nitrogen purging, dropwise added 4M isopropylamine to control the pH at 9.5, reacted for 24h, and the conversion rate was 95% by HPLC. 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 90 mg of the product with a purity of 98% and an optical purity of >99%.

Embodiment 2

[0023] In the reactor, 3.2L of 0.1M pH 9.5 triethanolamine-hydrochloric acid buffer solution, 500g of substrate, 900mL of isopropylamine, 0.4L of Tween 60, 0.4mL of DMSO, 5g of pyridoxal phosphate (PLP), transaminase EW1101 ( Suzhou Hanzyme Biotechnology Co., Ltd.) 50g, at 30°C, under the conditions of mechanical stirring and 0.01MPa nitrogen purge, add 4M isopropylamine dropwise to control the pH at 9.5, react for 24h, and the conversion rate by HPLC is 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 90 g of the product with a purity of 98% and an optical purity of >99%.

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Abstract

The invention discloses a biological preparation method of (R)-3-amino-4-(2,4,5-trifluorophenyl) tert-butyl butyrate. According to the method, a 3-carbonyl-4-(2,4,5-trifluorophenyl) tert-butyl butyrate is used as a substrate; under the existence of transaminase, amino donors, auxiliary factors and cosolvents, a reaction is taken to produce a target product of (R)-3-amino-4-(2,4,5-trifluorophenyl) tert-butyl butyrate. The reaction is performed in a water phase buffer solution with the pH being 6.0 to 10.0. The transaminase is used for converting the 3-carbonyl-4-(2,4,5-trifluorophenyl) tert-butyl butyrate into the (R)-3-amino-4-(2,4,5-trifluorophenyl) tert-butyl butyrate for the first time. Compared with the prior art, the process has the advantages that the substrate stability is good; the enzymatic conversion rate is high; the operation is simple and convenient; the subsequent product does not have protein residue; a better green and economical performance is realized; the optical activity of the product is high.

Description

technical field [0001] The invention relates to the technical fields of biopharmaceuticals and green chemistry, in particular to a biological preparation method of (R)-3-amino-4-(2,4,5-trifluorophenyl) tert-butyl butyrate. Background technique [0002] Chiral drug 3-amino-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazol[4,3-a]pyrazine-7(8H)- Base)-4-(2,4,5-trifluorophenyl)butan-1-one is the drug Sitagliptin (trade name JANUVIA) ​​of Merck Company for the treatment of diabetes. Its chiral amino group can be provided by the chiral building block (R)-3-carbonyl-4-(2,4,5-trifluorophenyl)butyric acid tert-butyl ester (2), therefore, compound 2 can be used as the Important chiral precursor of gliptin. [0003] The use of transaminases to produce chiral amines has the characteristics of low cost, high efficiency and environmental protection. Patent US 20120329108 and others reported the substrate 3-carbonyl-1-(3-(trifluoromethyl)-5,6-dihydro-[1, Transamination reaction of 2,4]t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 陈车翘张涛
Owner ENZYMEWORKS
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