Preparation Of The Chiral Intermediate Of Pregabalin By Microbial Transformation

Pregabalin, (S)-3-aminomethyl-5-methylhexanioc acid, is the 3-isobutyl substituent of GABA and a chiral drug that can be used as an adjuvant therapy for antineuralgic and anti-Epilepsy. The applications of pregabalin have grown markedly in recent years and are likely to expand in the coming years. Therefore, the studies on the preparation methods of optically active pregabalin and screening of biocatalysts are very important both for the knowledge on biocatalytic mechanism and its application.(S)-2-carboxyethyl-3-Cyano-5-methylhexanoic acid ethylester ((S)-CCMAE) is an important chiral intermediate of pregabalin. In this paper, firstly, using racemic CCMAE as the substrate, Pseudomonas sp. CGMCC No.4184 was isolated which can enantioselectively hydrolyze (R)-CCMAE. By optimization of the fermentation medium and the reaction conditions, the enzyme activity increased from 15.92 U/L to 33.43 U/L. Secondly, the resolution conditions by resting cells of Pseudomonas CGMCC No.4184 was investigated, and the results showed that the optimal temperature was 30-35℃and pH 7.5-8.5. After optimization, (S)-CCMAE were successfully prepared with ees of 99.8%. This method was different from all reportorial biotransformation process. Tween-80 and TritonX-100 were found to increase the enantioselectivity (E value) and enzyme activitiy. Finally, Sodium alginate-PVA-CaCl2 method was used to prepare the immobilized cells of Pseudomonas CGMCC No.4184. By optimization of the parameters and the reaction conditions, (S)-CCMAE (ees over 99%) were prepared and the immobilized cells have got good operational stability.In this study, the isolated hydrolase that has the high R-selectivity may be a noval enzyme. In order to improve the catalysis performance and exploit the application of this R-enantioselective hydrolyse, the study on gene cloning-expression and the enzymology characters would be carried out soon.