Asymmetric Reduction Of Prochiral Carbonyl To Chiral Alcohol With Baker's Yeast In Aqueous Phase

Single enantiomer pharmaceuticals, agrochemicals and other fine chemicals are more and more important to people's living and industrial and agricultural processes. So to synthesis the key chiral building blocks for these single enantiomer chemical products has become one of the most important and hot fields in organic synthesis. Biocatalysis has become one of the most promising and effective methods in the asymmetric synthesis for its outstanding stereochemical specificity. The asymmetric reduction of prochiral P-oxo ester, aromatic ketone and aliphatic ketone to its corresponding chiral alcohol by yeast cells were studied respectively in this work when ethyl 4-chloro-3-oxobutyrate, acetophenone and 2-octanone were as the model substrates. These chiral alcohols, the products, are the key chiral building blocks for many chiral pharmaceuticals, agrochemicals and other fine chemicals.At first, the asymmetric reduction of P-oxo ester by yeast cells in aqueous phase was investigated when ethyl 4-chloro-3-oxobutyrate (COBE) was chosen as the model substrate. Experiments indicated the product is mainly S (or D)-form ethyl 4-chloro-3-oxobutyrate (CHBE). The yield of CHBE was attractive for asymmetric biocatalysis. It could reach as high as 85%. But the e.e. of S-CHBE was just 85%. The effects of reaction conditions, such as substrate concentration, inhibition of product, reaction pH and temperature, co-substrate and cell culture condition, on the reaction were investigated. The experiments show there were inhibition effects at high COBE and CHBE concentration. And it was found that pH 7-8, 35 ℃ of reaction temperature, glucose as the co-substrate and cells cultured in aerobiotic condition were favorable to this asymmetric reduction reaction.It was found that the stereoselectivity of the asymmetric reduction of COBE by yeast cells was not meeting the practical application requires. So controlling and improving the stereoselectivity of this reaction were studied. The primary technique was pretreatment the yeast cells with enzyme inhibitors, such as heating, allyl alcohol and allyl bromide, for a period before being used in the asymmetric reduction of COBE. It was found that the stereoselectivity of this reaction to produce S-CHBE could be remarkably improved by pretreatment with heating and allyl alcohol. And the e.e. of S-CHBE could reach to 97% and 95% respectively at the optimized pretreatment conditions. On the contrary, pretreating with allyl bromide could turn the stereoselectivity of this asymmetric reduction reaction from mainly producingS-CHBE to R-CHBE. And the e.e. of R-CHBE could reach as high as 98% when the appropriate pretreatment condition was applied. To study the mechanism of this stereochemistry control, the influences of pretreatment to the yeast crude enzymes were investigated. The results indicated the reason of control the stereoselectivity by pretreatment was that the pretreatment inhibited some enzymes in the yeast cell which could reduce COBE. Also, the effect of pretreatment on the yeast cells activity was investigated. The experiments showed the pretreatment didn't impact on the cell activity when the concentration of inhibitor was small or the temperature of heating was low.It was investigated to improve the stereoselectivity and initial substrate concentration of COBE by introducing resin slowly releasing substrate and adsorbing product. The experiments indicated the initial substrate concentration and stereoselectivity could be remarkably increased when AMBERLITE? XAD1600 macropore adsorbing resin was used. Even when the initial substrate concentration increased to 126 mmol/L, the yield and stereoselectivity were satisfactory with introducing this technique. A mathematic model was set up for this complex process. Comparing with the experiment data, this model could excellently simulate to the experiment data.The yeast cells asymmetric reduction of aromatic ketone was also study in this work when the acetophenone (ACP) was as the model substrate. It could be seen that the yeast cells could a...