| Enzyme catalytic promiscuity, in another word, a single active site of a given enzyme can catalyze different chemical reactions of natural or non-natural substrates, has received widespread attention as more and more catalytic promiscuities of existing enzymes have been discovered. Actually, all enzymes are thought to have evolved as a result of promiscuous activities in the primitive, ancient enzymes. The relatively few preliminary ancestral generalist enzymes acted on multiple substrates to offer a wider range of metabolic capabilities. The increased catalytic specificity and selectivity are thought to be a result of ramification and evolution. Therefore, enzyme catalytic promiscuity is a key factor in the evolution of new enzyme functions. In addition, promiscuous activity does not normally affect an organism if the promiscuous reaction does not affect the rate of the natural activity or if the substrate for the promiscuous reaction is not native to the enzyme. Thus, there is no selective pressure to remove the promiscuous reaction. Catalytically promiscuous behavior is often hidden behind a native catalytic reaction and only visible under non-natural conditions. Further more, investigations of enzyme catalytic promiscuity not only gain fundamental knowledge about enzyme/substrate interactions and the evolution of new enzymes but help to understand metabolic approach for the biosynthesis of secondary metabolites better. Enzyme catalytic promiscuity could expand the application of biocatalysts and provide a useful method in organic synthesis.For their simple processing requirements, high selectivity and mild reaction conditions, enzymes have been widely used in modern organic synthesis. For the past few years, a new frontier named biocatalytic promiscuity has emerged and largely extended the application of enzymes. Biocatalytic promiscuity, in another word, is the the ability of enzyme to catalyze synthetic reactions, which may vary from its natural role. This kind of biocatalvsts has drawn a lot of attention from chemists for their specialities such as environmentally friendly, economically feasible, simple processing requirements, broad substrate specificity in a wide spectrum of biocatalyzed processes and high stereoselectivity. As one of the most rapidly developing subfields in enzymology, biocatalytic promiscuity not only enriches the existing catalysts, but also provides novel and practical synthetic path ways, in the meantime, it also gives a new thinking and road to green chemistry.The research of biocatalytic promiscuity has developed rapidly and received some achievement recently. The increasing number of enzymes especially hydrolases has been increasingly exploited for asymmetric synthetic transformations. The reaction types refer to asymmetric aldol reactions, C-C Michael additions, asymmetric synthesis of a-aminonitrile amides and the preparation of chiral epoxides and so on. However, according to the current reports, there are still limitations about the enzymatic activity and enantioselectivity, especially the enzymatic reaction mechanisms. Therefore, it’s worth further researching and exploring these problems, meanwhile it is also a challenging and attractive study.In this thesis, the enzyme-catalyzed asymmetric Mannich reaction has been studied. The Mannich reaction, atom-economic and a powerful synthetic method, is an important carbon-carbon bond forming reaction in organic synthesis for the preparation of β-amino carbonyl compounds which are significant intermediates in pharmaceuticals and natural products.In the second chapter of this thesis, the direct asymmetric Mannich reaction catalyzed by protease type XIV from Streptomyces griseus (SGP) was introduced. The SGP-catalyzed three-component Mannich reaction between aromatic aldehydes, aromatic amines and ketones was described. The reaction conditions, including organic solvents, water content, temperature, the molar ratio of substrates and pH, were optimized. We found that the optimized reaction conditions for the model reaction are as followed:acetonitrile is used as solvent, water/water+acetonitrile=0.1(v/v), the molar ratio of4-nitrobenzaldehyde/cyclohexanone is1:15, and the reaction temperature is30℃. After that, the scope of the reaction was tested. For most of the substrates, satisfactory yields and stereoselectivity were obtained. Yields of up to92%with enantioselectivities of up to88%ee (syn-isomer) and diastereoselectivities of up to92:8(syn:anti) were achieved under the optimized conditions.In the third chapter, asymmetric Mannich reaction catalyzed by XY-enzyme A was reported. Asymmetric Mannich reaction catalyzed by XY-enzyme A between aromatic aldehydes, aromatic amines and ketones was described. In this part, the reaction conditions, including organic solvents, water content, temperature, the molar ratio of substrates and pH, were optimized. According to the results, we know that, besides solvent, water content and pH have great effect on the stereoselectivity of XY-enzyme A. We found that the optimized reaction conditions for the model reaction are as followed: acetonitrile is used as solvent, buffer/buffer+acetonitrile=0.15(v/v, pH=8.06), the molar ratio of4-nitrobenzaldehyde/cyclohexanone is1:10, and the reaction temperature is30℃. Then the scope of the reaction was tested. For most of the substrates, satisfactory yields and stereoselectivity were obtained. Yields of up to82%with enantioselectivities of up to89%ee (syn-isomer) and diastereoselectivities of up to90:10(syn:anti) were achieved under the optimized conditions.In the fourth chapter of this thesis, asymmetric Mannich reaction catalyzed by XY-enzyme B was described. Three-component Mannich reaction between aromatic aldehydes, aromatic amines and ketones catalyzed XY-enzyme B was discussed. The reaction conditions, including organic solvents, water content, temperature, the molar ratio of substrates and pH, were optimized. Besides solvent, water content and pH have great effect on the stereoselectivity of XY-enzyme B. The optimized reaction conditions for the model reaction are as followed:isopropanol is used as solvent, buffer/buffer+acetonitrile=0.05(v/v, pH=7.53). the molar ratio of4-nitrobenzaldehyde/cyclohexanone is1:10, and the reaction temperature is30℃. Yields of up to94%with enantioselectivities of up to76%ee (syn-isomer) and diastereoselectivities of up to87:13(syn:anti) were achieved under the optimized conditions. |
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