【作者】 石玉刚；

【导师】 方云；

【作者基本信息】 江南大学 ， 应用化学， 2009， 博士

【摘要】 α–羟基酯易被转化为其它功能基团而被作为天然产物及手性药物合成中重要的“手性砌块”,（R）-2-羟基-4-苯基丁酸乙酯（（R）-EHPB）是其中一种重要的手性物质,被广泛应用于普利系列血管紧张肽转化酶（ACE,全称angiotensin converting enzyme）抑制剂的合成工业。生物法合成（R）-EHPB因其自身的优点而备受关注,本论文利用廉价易得的面包酵母作为生物催化剂,直接催化2-氧代-4-苯基丁酸乙酯（EOPB）不对称还原合成（R）-EHPB。目前,此过程中仍存在如产物立体选择性不高,明显的底物及产物抑制作用,化学添加剂的毒性,缺少能源供体（糖类等难溶于有机溶剂）难以实现辅酶的原位再生等诸多问题。水相中面包酵母催化还原EOPB,产物以（S）-EHPB为主,e.e. （S）为70-80%。通过研究反应进程,分析了副产物苯丙醇的生成机理,并提出了可行的抑制副反应的方法。有机溶剂中面包酵母催化不对称还原EOPB反应的研究表明,选择合适的有机溶剂作为反应介质,有利于提高酵母催化EOPB合成（R）-EHPB的反应立体选择性。经筛选,市售桃花牌酵母在乙醚单相体系中催化EOPB的反应效果最佳。最适酵母浓度、初始加水量和反应温度分别为50 g L-1,30 g L-1和30 oC。向反应体系中直接添加氯代苯乙酮（α-PC）,可有效地提高酵母催化EOPB（5 mmol L-1）合成（R）-EHPB的反应立体选择性,但α-PC长时间与酵母接触也存在毒性影响,降低酵母催化活性,减小EHPB产率。为此设计了4种α-PC添加策略,发现策略4 （Strategy 4）中,经α-PC预处理操作后的面包酵母既提高立体选择性,又改善了催化活性。面包酵母经α-PC （8 g L-1）预处理仅4 h后,催化还原EOPB转化率、EHPB产率及e.e. （R）-EHPB分别由86.8%,60.8%和62.7% （未使用α-PC）提高至95.9%,90.2%和92.0% （Strategy 4）。使用结构如α-PC分子中含被羰基活化的卤甲基的化学添加剂预处理酵母后,反应的立体选择性均有所提高,说明起作用的关键官能团可能是α-PC分子中的活泼氯甲基。利用紫外及荧光光谱观察酵母预处理过程中α-PC对酵母醇脱氢酶的影响,经α-PC处理之后的面包酵母样品的醇脱氢酶吸收峰的峰位和强度均发生变化,但经苯乙酮预处理的酵母样品的紫外吸收光谱曲线较空白样品未发生变化。激发波长在295 nm时的荧光光谱也得出相似结果。这些实验结果表明,α-PC的活泼氯甲基确实与酵母醇脱氢酶发生了某种不可逆相互作用,较大地改变了酵母的催化行为。利用液液双相体系作为反应介质,克服了单相体系中的底物及产物抑制、底物浓度偏低、有机溶剂的毒性及无法实现辅酶原位再生等不利影响。在水/苯双相体系中面包酵母（经过预处理）表现出较好的催化活性及立体选择性,底物浓度较有机单相体系提高了近四倍（20 mmol L-1）,在合适的反应条件下,即Vaq/Vben = 20: 40,磷酸盐缓冲液pH 8.0,30 oC,添加乙醇（1.5%, v/v）辅底物等,EOPB转化率、EHPB产率及e.e. （R）-EHPB分别达到95.4%、83.2%和97.0%。在[BMIM][PF₆]/水（10:1, v/v）双相体系中,面包酵母催化EOPB反应产物立体选择性由微水离子液体[BMIM][PF₆]单相体系中的S构型转变为R构型。添加辅底物乙醇1% （v/v）,e.e. （R）-EHPB由6.42%提高至82.5%。使用3.2.2节Strategy 4中的经α-PC预处理的面包酵母催化还原EOPB,反应立体选择性达到89.4% （e.e. （R））,且EHPB产率增至56.4%。尽管[BMIM][PF₆]粘度大,极性较高,但面包酵母在离子液体[BMIM][PF₆]/水（10:1, v/v）双相体系中仍具有较好的催化立体选择性,说明[BMIM][PF₆]/水双相体系中的酵母催化反应具有进一步研究价值。首次设计并主要合成了两类结构如下所示的离子液体。测定了不同乙氧基链长的上述离子液体在有机溶剂中的溶剂性能,找到了具有“室温均相,高温分相”的温控离子液体/有机溶剂液液双相体系。在由含乙氧基链的季胺盐型离子液体IL2、乙二醇二甲醚组成的温控离子液体双相体系中,可以进行酵母不对称催化还原EOPB反应,在合适的反应温度下反应于均相体系中进行,反应结束后,适当升温,离子液体析出体系呈两相,含生物催化剂的离子液体相与含产物的有机溶剂相简单相分离后可循环使用。实验结果表明,该体系中酵母催化EOPB反应的立体选择性较单独使用乙二醇二甲醚提高了25%?30%,通过简单分离后,酵母仍具有催化活性。更多还原

【Abstract】 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.更多还原