Promiscuous Proteases In The Catalysis Of Asymmetric MBH Reaction, Asymmetric Michael Reaction And Friedel-Crafts Reaction

Enzymes as an efficient catalyst have attracted significant attention due to their environmentally benign quality and mild reaction conditions. In recent years, an increasing number of enzymes have been shown to have promiscuous behavior. In particular, hydrolases are considered one of the most useful enzymes due to its high stability, wide range of substrate scope, and high catalytic efficiency. The investigation of enzyme promiscuity not only broadens our knowledge of biocatalysis but also expand the range of applications of enzymes. Several significant works of catalytic promiscuity of hydrolase have been reported, including aldol reactions, Michael reactions, Mannich reactions, Markovnikov additions, and Henry reactions.The Morita-Baylis-Hillman reaction(MBH reaction) is one of the most important carbon-carbon bond-forming reactions and has drawn considerable attentions in the past decades. Herein we report pepsin from porcine gastric mucosa, a hydrolase, can effectively catalyze the direct asymmetric MBH reactions in combination with DABCO to afford desirable products using 2-cyclohexen-1-one and various substituted aromatic aldehydes. The effects of the reaction medium, temperature, and other variables on the enzymatic MBH reaction were researched in detail, and the catalytic specificity of pepsin was proved by control experiments. In the optimal reaction conditions, we obtained 15 examples, the desired adducts were obtained the yield up to 77% and ee values up to 38%. Although the yields and stereoselectivities were not as high as desired, to the best of our knowledge, we report the first use of pepsin and DABCO together to catalyze the MBH reaction. This method also expanded the application of pepsin in organic synthesis.Many natural products and medicines contain 3,3-substituted oxindole structure with biologically active. Thus, the methods of synthesis such substances have drawn great significance. In the third chapter, we chose papain as a catalyst to synthesis the class of chiral 3,3-substituted oxindole compounds by asymmetric Michael reaction between 3-methyl-oxindole and benzalacetone successfully. We optimized solvent, water content, substrates molar ratio, the amount of enzyme loading, temperature and other reaction conditions. With the optimal conditions in hand, substrates scope was tested, and 12 substrates were obtained. Finally, we obtained the corresponding products yield up to 93%, 68/32 dr, and 86% ee. Although the adaptive response of the substrates in general, the papain-catalyzed Michael reaction still provide a green alternative synthetic route for 3,3-substituted oxindole derivatives. It also broadens the papain catalyzed hydrolysis of versatility in organic synthesis asymmetric carbon-carbon bond formation application.In the fourth chapter of this thesis, we found that α-chymotrypsin can catalyze commercially available isatins and indoles as a green bio-catalyst. The Friedel-Crafts reaction in different solvents obtained different products. After optimization of the reaction conditions such as water content, buffer pH, molar ratio and temperature in 1,2-dichoroethane, 17 substrates for mono-substituted products were achived, the yield up to 97%. Then the reaction conditions in MeOH were optimized, after the substrates scope, we got 25 dual-substituted products, the yield up to 99%. As an example of bio-catalytic process, the discovery expands the α-chymotrypsin applications in catalysis of chemical conversion. Due to the economic and sustainable development of simple and easy operation of the catalytic process, the method has the potential to become a practical method for the synthesis of 3-hydroxy-oxindole and bis-indolyl-oxindole-2one derivatives.Through a series of studies with protease catalytic organic reactions, we found a protease capable of catalyzing different reactions of multifunctional catalytic activity which expanded pepsin, papain and α-chymotrypsin application in organic synthesis. The possible mechanisms of the papain-catalyzed Michael reactions and α-chymotrypsin catalyzed Friedel-Crafts reaction were proposed. The study also shown a feasible green synthesis pathway for the asymmetric MBH reaction and 3,3-substituted oxindole substances.