【作者】 杨忠华；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 博士

【摘要】 手性医药、农药以及其它精细化学品,在日常生活和工农业生产中的重要作用越来越受到人们的重视。因而合成这些手性产品的关键手性砌块也成了当前合成界研究的热点。生物催化因其高效和高度的立体选择性,成为手性合成最重要的方法之一。本文以4-氯乙酰乙酸乙酯、苯乙酮和2-辛酮分别为β-羰基酯、芳香酮和脂肪酮的模型底物,研究了利用活性酵母细胞催化这三类前手性酮不对称还原合成相应手性醇的反应特性。所得到的手性醇是许多手性医药和其它手性精细化学品的关键手性砌块。首先,以4-氯乙酰乙酸乙酯(COBE)为β-羰基酯的模型底物,考察了酵母细胞在水相体系中催化其不对称还原的反应特性。实验发现,酵母细胞在水相体系中能催化该底物的不对称还原,生成以S(或D)-型为主的4-氯-3-羟基丁酸乙酯(CHBE),反应活性很高,产物CHBE的得率可达到85%左右。但反应的立体选择性不是很高,S-CHBE的e.e.值最高只能达到85%左右。同时发现在水相体系中反应,存在一定程度的副反应。实验考察了底物浓度、产物的抑制、反应体系的pH、反应温度以及辅助底物等反应条件和菌体培养条件对酵母细胞不对称还原COBE的影响。发现高浓度底物和产物对反应具有抑制作用,pH7和35℃左右的反应温度有利于反应的进行,以葡萄糖作辅助底物和好氧条件下培养的菌体可以获得较好的反应结果。其次,由于酵母细胞不对称还原COBE的立体选择性达不到实际应用的要求,因此研究了利用酶抑制剂预处理酵母细胞来提高反应的立体选择性。实验发现利用烯丙基化合物和热对酵母细胞进行预处理,可以控制和提高其还原COBE的立体选择性。热和烯丙基醇预处理可以提高S-型产物的e.e.值,通过优化预处理条件可以使热、烯丙基醇预处理后S-CHBE的e.e.值分别达到97%和95%。而烯丙基溴预处理可以使反应立体选择性从通常的生成S-型产物过量转变为R-型产物过量,优化处理条件后能使R-CHBE的e.e.值达到98%。实验进一步考察了预处理对酵母粗酶液中还原COBE酶活和酵母细胞活性的影响,发现预处理控制和提高反应立体选择性的原因是预处理时抑制了胞内一部分催化COBE还原生成另一对映异构体产物的酶的活性。实验发现低强度的预处理对酵母的细胞活性影响不大。第三,活性细胞的催化过程通常具有时-空产率低的不足,这主要是反应底物的初始浓度不高造成的。为了促进活性酵母细胞催化COBE不对称还原反应的时-空产率和反应立体选择性,对利用引入树脂缓释底物和原位吸附产物来提高浙江大学博士学位论文反应底物的初始浓度和反应的立体选择性进行了研究。实验发现使用AMBERLITE⑧xADI 600大孔吸附树脂,可以显著地提高反应底物的初始浓度和反应立体选择性。引入吸附树脂后反应底物的初始浓度即使达到126 mmol几后,反应的立体选择性还可以保持在90%以上。对引入树脂的反应过程进行了分析,得出了描述整个过程的数学方程。利用实验数据对方程参数进行了拟合,发现所得到的方程能很好地模拟整个过程。 第四,为了对利用活性酵母细胞不对称还原芳香酮有所认识,本文以苯乙酮(ACP)为模型底物研究了酵母细胞催化前手性芳香酮不对称还原的反应特性。发现酵母细胞在水相体系中能催化ACP的不对称还原生成以S一型为主的a一苯乙醇(P EA),反应的立体选择性很高,S一PEA的e.e.值可达99%左右。考察了各种反应条件对反应的影响。发现反应条件对反应立体选择性影响不大,但对产物的得率有较大影响,反应体系的pH为7一8、温度30℃左右、酵母细胞用量0.2 g/mL左右能获得较好的产物得率,高浓度的底物和产物对细胞具有较高的毒性,同时发现酵母细胞能选择性地氧化S一PEA为ACP。研究了通过添加树脂来提高反应底物的初始浓度。发现杭州争光树脂厂的ZG3大孔吸附树脂可以很好地促进反应底物初始浓度的提高,使产物浓度达到12.4~o1几。 最后,以2一辛酮(2一Octanone)为模型底物研究了酵母细胞不对称还原前手性脂肪酮的反应特性。发现在水相体系中酵母细胞催化2一辛酮的不对称还原,生成以S一2一辛醇为主的产物。反应的立体选择性很高,S一型产物的e.e.值达95%以上。考察了各种反应条件对反应的影响,发现较高的底物和产物浓度对反应具有抑制作用,pH7.5、反应温度3小36℃以及以葡萄糖为辅助底物对反应较为有利。关键词:不对称还原,手性合成,手性醇,酵母,生物还原,4一氯乙酞乙酸乙酷, S一4一氯一3一经基丁酸乙醋,R一4一氯一3一轻基丁酸乙酷,苯乙酮,S一a一苯乙醇, 2一辛酮,S一2一辛醇更多还原

【Abstract】 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更多还原