Chemo-enzymatic Synthesis Of Chiral Intermediates Of Pregabalin

Pregabalin is the 3-isobutyl substituent of GABA. It is used as an adjuvant therapy for antineuralgic and anti-Epilepsy; besides, pregabalin also is effective on neuropathic pain patients with sleep disorder treatment. (3S)-Cyano-2-ca-rboxyethyl-5-methylhexanoic acid is an important chiral intermediate of pregab-alin.A process is reported for stereoselectively hydrolyzing one enantiomer of r-ac-3-cyano-2-carboxyethyl-5-methylhexanoic acid ethyl ester for (S)-3-cyano-5-m ethylhexanoic acid ethyl ester. Our route begins with the Knoevenagel condensation of isovaleraldehyde and diethyl malonate, followed by the addition reaction with Sodium cyanide to give the key intermediate 3-cyano-2-carboxyethyl-5-methylhexanoic acid ethyl ester. (3S)-Cyano-2-carboxyethyl-5-methylhexanoic acid was prepared from Compound 3-cyano-2-carboxyethyl -5-methylhexanoic acid ethyl ester through biocatalyzed resolutions including microbial-catalyzed and li-pase-catealyzed asymmetric hydrolysis.Firstly, compared with the current synthetic method, the key intermediate 3 -cyano-2-carboxyethyl-5-methylhexanoic acid ethyl ester was prepared from Isovaleraldehyde. After synthetic method was improved and optimized, the yields of the first and second reaction were increased from 78.0 % to 88.5% and from 90.0% to 99.0% respectively. At the same time, to establish the GC method to analyze conversion rate, yield and optical purity of (3S)-cyano-2-carboxy ethyl -5-methylhexanoic acid, we synthesis rac-3-cyano-5-methylhexanoic acid e-thyl ester.Secondly, the method of lipase-catalyzed asymmetric hydrolysis of racemic of 2-carboxyethyl -3-cyano-5-methylhexanoic acid ethyl ester was established. More than thirty kinds of lipases were screened and lipase 23# (Lipoprime 50 T) was found to be the optimum enzyme. The catalytic conditions were also optimized. The optimum concentration of 2-carboxyethyl -3-cyano-5-methylhexa-noic acid ethyl ester was 150mmol/L, and the optimum cultivating temperature was 40℃. Then the hydrolase was applied to the hydrolyzation of the rac-2- carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester, the percentage of (3S)-cyano-2-carboxyethyl-5-methylhexanoic acid ethyl ester in the products was over 99% and the conversion rate was 40%.Finally, in order to screen a large number of bacteria with hydrolytic ability from the soil samples, the high-throughput screening method was establishe-d by using the thin-layer chromatography with petroleum ether-ethanol (90:10) as the expand agent. From the soil samples, we screened 16 strains with selective hydrolysis, and then selected these 16 strains. At last we got 14 # bacteri-a where the e.e._p% value was about 87.9% and the conversion rate was 39.9...