| With the emergence and development of green chemistry, as well as strengthening the awareness of environmental protection, enzymatic organic reactions, because of its high efficiency, high selectivity, mild reaction conditions, post-processing simple and environmentally friendly, have attracted much more attention by chemists by and biologists. Until now, there about3,000enzymes have been identified and with the development of protein engineering and genetic engineering techniques, the artificial modification of natural enzymes, or synthetic non-natural enzymes become a reality, which in turn promote the use and development of the enzyme in the fields of organic chemistry, especially the high stereoselectivity of the enzymatic organic reactions has become an important means of chiral drug synthesis. Until today, biocatalysis and biotransformation have been achieved its industrialization, large-scale in many areas. The enzyme catalyzes organic reactions as one of important directions for the development of green chemistry, which have now become a hot topic and focus on the chemical research field.Traditional theory holds that the enzyme only can keep their high reaction catalytic activity and enantioselectivity for their natural substrate of chemical reaction in natural aqueous environment. This theory greatly limits the use and development of enzymes in organic chemistry. Until the early1980s, Klibanov and Zaks found that enzymes not only could work in organic solvents containing little or no water, but also acquire remarkable properties such as enhanced stability, altered substrate specificities,’molecular memory’, and the ability to catalyze unusual reactions which are impossible in aqueous media. This feature is called enzymatic promiscuity. Hult and Berglund defined three types of enzymatic promiscuity: condition promiscuity (enzymatic activity in various reaction conditions different from their natural ones), For example, some enzymes were turned out to be able to catalyze the reactions in organic solvents, or in a variety of temperatures, pH and so on; Second, substrate promiscuity (enzymes with a broad substrate specificity); Third, catalytic promiscuity (based on the ability of a single enzyme active site to catalyze several chemical transformations). Until now, most of the research focused on the enzyme third promiscuity:catalytic promiscuity.In recent years, the research of enzyme promiscuity has developed rapidly and received some achievement. The growing number of enzymes especially hydrolases is being increasingly exploited for asymmetric synthetic transformations and enantiopure Pharmaceuticals. During the recent years, many enzymes have become widely used for synthetic transformations such as Aldol reaction, Michael reaction, Domino reaction, Henry reaction and so on. However, there are still limitations about the enzyme catalytic activity and enantioselectivity, especially the enzymatic reaction mechanisms. Therefore, it’s still worth us to further research and exploration these problems. The enzyme promiscuity greatly extended the application of enzyme catalysis in organic chemistry and provides a new pathway of green chemistry. It will be a research area with challenge and opportunity. Therefore, this thesis mainly discussed the application of enzyme catalytic promiscuity on Knoevenagel, Michael and Aldol reactions.The Knoevenagel condensation is one of the most important methods for carbon-carbon double bond formation in synthetic chemistry. The Knoevenagel reaction has been widely employed in organic synthesis to prepare coumarins and their derivatives, which are important intermediates in the synthesis of cosmetics, perfumes and pharmaceuticals. The classical Knoevenagel condensation has been carried out in the presence of bases, Lewis acids, amino acids, Ionic Liquid, Microwave and so on. However, there were only a few reports about enzyme catalyzed Knoevenagel reaction. We found that alkaline proteinase from Bacillus licheniformis No2709could catalyze the Knoevenagel reaction of p-methoxy cinnamaldehyde and acetylacetone. The influence of reaction conditions including solvents, water content, loading of catalyst and temperature were systematically investigated. Under the optimal reaction conditions, we investigated the scope of the enzyme-catalyzed Knoevenagel condensation. The results indicated that when the reaction was carried out in the5%of water content in DMSO/H2O system, the molar ration of aldehyde to ketone was1:1.2, enzyme loading was70mg/ml, the alkaline proteinase catalyzed Knoevenagel reaction affording knoevenagel adducts in moderate to good yields with E/Z selectivities up to>99:1.The Michael addition is one of the important C-C bond-forming reactions. In recent years, there are many reports about enzymatic promiscuity Michael reaction. Warfarin, as one of the most effective anticoagulants, has been introduced for clinical use for more than half a century. Until recently, the mainly strategy to obtain enantioenriched warfarin was developed catalyzed by the organocatalysts such as primary amine, diamine and secondary amine amide. However, biocatalytic preparation of warfarin is keeping unexploited. Herein, this thesis first report the PPL (lipase from porcine pancreas) catalyzed Michael addition of4-hydroxycoumarin and α,β-unsaturated enones to prepare Warfarin and derivatives in organic medium in the presence of small amount water. The results indicated that when the reaction was carried out in the10%of water content in DMSO/H2O system, the molar ration of4-hydroxycoumarin to benzylideneacetone was1:5, enzyme loading was50mg, the PPL catalyzed Michael reaction affording michael adducts in excellent yields (up to95%) with enantioselectivities up to28%ee (S).The asymmetric aldol reaction is one of the most useful methods for carbon-carbon bond formation in organic synthesis. It is also a useful approach for the preparation of pharmaceuticals, fine chemicals and natural products. Therefore, the chapters4-5of this thesis mainly discussed the application of AUAP (acidic protease from Aspergillus usamii No537) and PPL II (Lipase from pig pancreas type II, PPL II) catalytic promiscuity on Aldol reaction. The acid protease is produced by Aspergillus usamii No537, which can hydrolyze protein rapidly under acidic conditions and be widely used in leather, wool, feed and brewing industry. PPL II is one kind of important hydrolases. The molecular weight is about45000-50000daltons and its main role is to break down triglycerides into fatty acids and glycerol. PPL II is widely used in the food, leather, and cosmetics and applied in many fields of medicine. AUAP and PPL are first discovered to display high catalytic activity and enantioselectivity for catalyzing direct asymmetric aldol reactions between aromatic aldehydes and cyclic ketones in organic medium in the presence of water respectively. In our initial studies, the aldol reaction of cyclohexanone and4-nitrobenzaldehyde was used as a model transformation. The influence of some parameters including solvents, water contents, temperature, pH, the molar ratio of substrates and enzyme concentration were systematically investigated. Under the optimal reaction conditions, we further studied the generality of the enzyme-catalyzed direct asymmetric aldol reaction and obtained satisfactory results. When the reaction was carried out in the10%of water content in MeCN/H2O system, the molar ration of aldehyde to ketone was1:5, enzyme loading was100mg/ml, the AUAP catalyzed aldol reaction affording aldol adducts in excellent diastereoselectivities (up to97:3, anti/syn) with high enantioselectivities (up to88%ee, anti). When the reaction was carried out in phosphate buffer (pH=5.41)/CH3CN (v/v=1:9), the molar ration of aldehyde to ketone was1:20, enzyme loading was100mg/ml, PPL II catalyzed Aldol reaction affording the corresponding aldol products with excellent chemical yields (up to98%), and good diastereoselectivity (up to87:13dr, anti/syn) and high enantioselectivity (up to94%ee, anti). |
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