Asymmetric Reduction Of Ethyl 2-oxo-4-phenylbutyrate Catalyzed By Baker's Yeast

Ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-EHPB) is a versatile key intermediate for the synthesis of a variety of angiotensin converting enzymes (ACE) inhibitors such as Cilazapril, Benazepril, and Enalapril etc. Special attention has been paid to the production of (R)-EHPB by the biosynthesis method. In this study, we systematically studied microbial asymmetric reduction of EOPB to optically active (R)-EHPB catalyzed by baker's yeast (Saccharomyces cerevisiae). However, there are still some disadvantages as follow in this process: a relatively low enantiomeric excess value of (R)-EHPB and high biomass: substrate ratio; the severe substrate and product inhibition to the enzymes activity; the toxicity of alpha–phenacyl chloride (α–PC) and organic solvents to the yeast; the reproduction of the coenzyme hardly achieved in an organic solvent, et al.The asymmetric reduction of EOPB catalyzed by baker's yeast in aqueous phase to synthesize EHPB was investigated in detail. The enantiomeric excess value (e.e.) of product (S)-HPBE reached up to 80%. The reaction process and the mechanism of the production of the byproduct PPN were also explored in order to inhibit the side reaction and improve the enantioselectivity of the reduction.In order to improve the enantioselectivity of the asymmetric reduction of EOPB to produce (R)-EHPB, different organic solvents were employed as reaction media in place of water. The enantioselectivity of this reduction could be reversed by using different organic solvents. The enantioselectivity of the asymmetric reduction of EOPB to synthesize (R)-EHPB catalyzed with baker's yeast in diethyl ether can be improved by the introduction ofα-PC. We designed different strategies ofα–PC addition in order to overcome the toxicity ofα-PC to the yeast. The catalytic activity of yeast and the enantioselectivity of the reduction were improved with special pretreatment process of yeast (Strategy 4). The effect ofα-PC on selective inhibition of S-enzymes within yeast cells was investigated.Α-PC as a good alkylating agent can easily react with those residues belong to the active site of the enzyme in yeast cells because of its reactive chloromethyl group binding to its carbonyl group, which might be the major reason of the enhanced enantioselectivity of enzymatic reaction. The change of catalytic behavior of baker's yeast after the chemical pretreatment, which might be caused by an interaction between the yeast andα-PC in ethyl ether, was studied via spectrum analysis (UV and FS). In addition, under the optimum pretreatment conditions, the conversion of EOPB, the yield of EHPB and the e.e. of (R)-EHPB reached 96%,90% and 92% from 6.8%, 60.8% and 62.7%, respectively.The water/organic biphasic system was adopted to overcome the inhibition of a high substrate/product concentration. The baker's yeast showed the best catalytic activity and enantioselectivity in the water/benzene biphasic system. When EOPB concentration was 20 mmol L-1, 95.4% of EOPB conversion, 83.2% of EHPB yield and 97.0% of e.e. (R)-EHPB was obtained by using the chemical-pretreated yeast, respectively, under the following appropriate reaction conditions: 30 oC, Vaq/Vben = 20:40, pH 8.0 phosphate buffer, 1.5% (v/v) of ethanol as the co-substrate. In ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction was shifted towards (R)-side from the (S)-side in [BMIM][PF₆] (27.7%, e.e. (S)), and e.e. (R)-EHPB was increased from 6.6% to 82.5% with the addition of ethanol (1%, v/v) as a co-substrate. The effect of the use of [BMIM][PF₆] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. That is little affected in organic solvent-water biphasic systems. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF₆] concentrations occurs in either organic solvent-water biphasic systems or benzene.The quarternary ammonium salts ionic liquids modified with poly-(ethylene glycol) chain was synthesized for the first time. The solubility of these ionic liquids in different organic solvents was investigated under the different temperature. It was found that these ionic liquids possess the thermoregulated function of"mono-phase under low temperature, biphase under high temperature". A novel concept of thermoregulated ionic liquid biphasic biocatalysis (TILBB) based on the thermoregulated function of ionic liquids for separating the catalyst from the reaction mixure was proposed. The asymmetric reduction of EOPB catalyzed by baker's yeast was investigated in thermoregulated ionic liquid biphasic system composed of IL2 and ethylene glycol dimethyl ether. The enantioselectivity of the reduction in in thermoregulated ionic liquid biphasic system was increased by 25%-30% compared with that in ethylene glycol dimethyl ether. The yeast separated from the ionic liquid can be recycled.