| Nonaqueous enzymology is a hot research topic in chemistry and biology since 1980s. Compared with aqueous media, enzymatic reaction in organic solvents has several advantages, such as the ability of catalyzing new reactions, higher solution of non-polar substrates, easy isolation of enzyme and product, and the recycle of enzyme. One serious obstacle of enzymatic reactions in conventional organic media is the substantial reduction in reaction rate. Therefore, the study of enzymatic reactions and methods to improve enzymatic activity in organic media is of interest to synthetic chemists in academia and industry, and also to biotechnologists.A Lipase B from Candida antarctica (CALB)/acetamide-catalyzed Michael addition of less-active ketones and aromatic nitroolefins was developed, which is particularly interesting because neither CALB nor acetamide can independently catalyze the reaction to any appreciable extent. This co-catalyst system was applicable to the Michael additions of cyclic and acyclic ketones to a series of aromatic and heteroaromatic nitroolefins. Hydrogen bonds between acetamide and cyclohexanone for the observed activation were confirmed by experimental facts, and new mechanistic insights into CALB/acetamide co-catalysis were presented. The influence of amide to CALB-catalyzed acylation was also investigated, and NMR was used to explore the binding mode of amide and CALB.Formamide, one of so-called ’water mimics’, and water, were used and compared as additives in accelerating asymmetric aldol reaction catalyzed by lipase from porcine pancreas(PPL). When compared with water, formamide displayed different effect and was more likely to act as a co-catalyst to activate substrates rather than a’water mimic’to’loosen up’ enzymes in anhydrous solvents. The hydrogen bond between cyclohexanone and formamide was detected through NMR, and isotopic effect showed that hydrogen bond, in which water and formamide acted as donors, was critical to this promotion for enzymatic promiscuous aldol reaction. Moreover, formamide displayed its advantage in the two-step aldol/dehydration reaction, because water was one of co-products influencing the reaction equilibrium.A novel lipase-catalyzed cascade reaction for the synthesis of spirooxazino derivatives was discovered. Intermediates, isotope labeling and directed evolution of CALB were used to probe the possible mechanism of multi-component reaction of aromatic aldehyde, cyclohexanone, nitroolefin and acetamide. When aldol intermediates were used as substrates, reaction conditions involving hydrolases, enzyme loading, the structure of amide and substrate ratios were optimized, and a serious of spirooxazino derivatives were obtained. All of these novel spirooxazinos had strong fluorescence emission ability and high quantum yields. Chiral spirooxazinos could also be obtained through a multi-enzymatic cascade process, combining PPL-catalyzed asymmetric aldol reaction and CALB-catalyzed multi-component reaction.A novel enzymatic, promiscuous protocol of D-aminoacylase (DA)-catalyzed double Michael addition was developed, for the synthesis of (hetero)spiro[5.5] undecane derivatives in moderate yields. Controlled experiments involving catalyst-free, BSA and inhibited DA verified the role of active site. The catalytic activity of DA was obviously promoted by a small quantity of water. It is notable that almost only the cis isomers were obtained through this biocatalytic methodology in all the cases according to their 1 and 13C NMR spectra.In this thesis,73 compounds were synthesized including 13 Michael adducts,1 Mannich reaction product,18 aldol reaction products,1 aldol/elimination reaction product,8 nitroalkenes, and 15 racemic spirooxzines,10 chiral spirooxzines,2 penta-1,4-dien-3-ones and 5 spiro[5.5]undecanes. These compounds were characterized by 1H NMR,13C NMR, FTIR, ESI-MS and HRMS. |
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