Asymmetric Reduction Of Tert-butyl (S)-6-chloro-5-hydroxy-3-oxo-hexanoate With Yeast

Optically active β,δ-dihydroxy esters have been widely used in the synthesis of natural products, polyol fragments, and chiral drugs. Tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate is a chiral building block for HMG-CoA reductase inhibitor, which is a drug for coronary heart disease(CHD).First, because the substrate tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate called for the further experiment is synthesized from (S)-4-chloro-3-hydroxybutanoate ethyl ester, so among the seven yeast strains investigated, Candida pseudotropicalis exhibited the highest activity and stereo-selectivity to COBE. The initial COBE concentration was 22 g/L, Candida pseudotropicalis cell concentration was 100 g/L. The reaction was carried out in the buffer(pH 6.0, 180 rpm) for 8h at 30 °C, in which 10 g/L glucose as the co-substrate. The product yield was 94.1% and the e.e.p value>99%. Obvious inhibition was found in this reaction. When the substrate concentration exceeded 22 g/L, the reaction initial rate decreased significantly. So we chose 22 g/L as the substrate concentration from economic aspect.Second, among the seven yeast strains investigated, Rhodotorula gracilis AS 2.499 exhibited the highest activity and stereo-selectivity to tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate. The stereo-selectivity of the reaction was improved with the thermal treatment. The initial tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate concentration was 1 g/L, Rhodotorula gracilis AS 2.499 cell concentration was 100 g/L. The cells was pretreated at 50℃ with heat for 30 min and the reaction was carried out in the buffer(pH 6.0, 180 rpm) for 48h at 30 ℃, in which 10 g/L glucose as the co-substrate. The product yield was 68.3% and the d.e. value was 95.1%. Obvious inhibition was found in this reaction. When the substrate concentration exceeded 2.5 g/L, the reaction initial rate decreased significantly and the yield and d.e. value was 52.9% and 80.7%. So we chose 1 g/L as the substrate concentration from economic aspect.Finally, the factors which affected the Rhodotorula gracilis AS 2.499's reduction ability were investigated through the mono-factor experiment. The best medium was formed by glucose 50 g/L, peptone 7.5 g/L, yeast extract 7.5 g/L, (NH₄)H₂PO₄ 4 g/L, K₂HPO₄ 1 g/L, KH₂PO₄2 g/L, MgSO4 0.2 g/L. The reactions were carried out with the cells grown form the optimized medium and the original medium, the reactioninitial rate was 44.78 and 26.24 umol/hL. The initial rate increased about 70.7% at the optimized conditions, and the yield could reach 65.3% form 76.1%.