Study On Preparation Of Chiral Pharmaceutical Intermediates By Asymmetric Reduction Of Carbonyl Compounds With Yeast Cells

With the progress in pharmacology of chiral Pharmaceuticals and the strict regulation in the application of new drug in recent years, the research and development of chiral Pharmaceuticals has become the new direction internationally. Hence, how to synthesize chiral compounds is the key in commercial production of chiral Pharmaceuticals. Many researchers are applying themselves to develop new processes for chiral compound synthesis with high conversion ratio and high stereo-selectivity, in which, biotransformation is attracted interests because of mild reaction condition and high stereo-selectivity.Chlorine substituted acetophenones are important precursors for the synthesis of chiral pharmaceuticals. It has reported that microbials can be used to reduce various carbonyl compounds into chiral alcohols. In this work, the reduction reactions of chlorine substituted acetophenones into chlorine substituted phenyl ethanol catalyzed by yeast cells will be studied in detail.Because no commercial chiral or even racemic chlorine substituted phenyl ethanol is . available in commercial market, the racemic chlorine substituted phenyl ethanols must be synthesized as standards before other research works. The chemical synthesis were performed for racemic 2'-chloro-l-phenyl-ethanol, 3'-chloro-l-phenyl-ethanol and 4'-chloro-l-phenyl-ethanol from corresponding chlorine substituted acetophenones. After separation and purification, the purities of these products were higher than 99%. In the meantime, the analytic method of two enantiomers of racemic chlorine substituted phenyl ethanol must be developed. After optimization of gas chromatographic conditions, the two peaks of enantiomers were well separated by a chiral column, which made the following research works easy.From eleven yeast strains kept in this laboratory, four strains were found the ability to reduce chloroacetophenone into chlorophenyl ethanol with chiral selectivity, including two strains of Saccharomyces cerevisiae, P2 and B5, as well as two strains of Candida sp., Candida pseudotropicalis 104 and C. utilis 1257. Among theses strains, S. cerevisiae B5 showed the best reduction capability with high productivity and stereo-selectivity. Andamong substrates including 2'-chloroacetophenone, 3'-chloroacetophenone, 4'-chloroacetophenone and 3-chloropropiophenone, the highest productivity of the reduced product with S. cerevisiae B5 was observed for 2'-chloroacetophenone.The reduction of 2'-chloroacetophenone to R-2'-chloro-l-phenyl-ethanol by S. cerevisiae B5 was further studied in detail. The results indicated that the enantiometric excess of (R)-2'-chloro-l-phenyl-ethanol was 100% in all the studied conditions. The effects of co-substrates, involving methanol, ethanol, 2-propanol, n-butanol, glucose, glycerol and lactic acid, on both productivity and enantiometric selectivity were evaluated. The experimental data showed that the best co-substrate was ethanol and the optimal ethanol concentration was 5%. The function of ethanol addition is to make the regeneration of NADH by alcohol hydrogenase to supplement the consumption of NADH in substrate reduction. The reaction conditions for the reduction of 2'-chloroacetophenone to R-2'-chloro-l-phenyl-ethanol by 5. cerevisiae B5 were optimized, and the highest productivity was achieved when the bioconversion was taken place at pH8.0 and 25℃ for 24 hrs. Also the effects of substrate concentration, yeast cell amount, the culture and bioconversion conditions on the productivity were examined. The substrate inhibition was observed, and the increase of yeast cell amount was able to increase the productivity. The aerobic cultivation and aerobic bioconversion were favorable for high productivity. In order to alleviate the substrate inhibition, an operation strategy of fed-batch addition of substrate was adopted and the increasing in productivity was observed. Furthermore, the repeated use of S. cerevisiae B5 cells for bioconversion was tested and after 15 cycles, the yeast ceils still remained certain ability to reduce...