Promiscuity Of Protease:Enzyme Catalyzed Asymmetric Aldol Reaction And Vinylogous Michael Addition

Enzyme can also catalyze chemical conversion of unnatural substrates which we call this function enzymatic promiscuity. They are usually hidden behind the natural catalytic transformation. Enzymatic promiscuity not only provides more catalysts for organic synthesis, but also laid the foundation for further research to enzymes. In this work, the enzymatic promiscuity was applied in aldol and vinylous Michael reaction.α-Chymotrypsin from bovine pancreas（BPC） is a kind of digestive enzymes that can hydrolyze proteins. Its active group contains a serine residue, belonging to the serine protease. Its active site consists mainly of simultaneous action of Asp-102, His-57 and Ser-195 three amino acid residues that they form the famous catalytic triad. We firstly used BPC to catalyze asymmetric aldol reaction between 4-cyanobenzaldehyde and cyclohexanone. After exploring the effect of solvent, the amount of water, temperature and the amount of enzyme etc., the scope of substrates were subsequently tested under optimized conditions. In order to prove that the reaction indeed happened in the active site of BPC, we did a series of control experiments by using metal ion Cu²⁺ and Ag+, urea, and the specific inhibitor of the active s ite-Gu HC l, PMSF, DEPC and DCC. Also, we used trypsin from bovine pancreas, recombinant chymotrypsin, trypsin from porcine pancreas to catalyze the aldol reaction. The result is indeed verified the aldol reaction indeed occurred in the active site of α- chymotrypsin. On this basis, we propose a possible mechanism.Chiral tertiary alcohol structure in many natural products and biologically active synthetic molecules exists widely. However, due to steric hinder of this structure, there are some difficulties in the synthesis. The catalysts reported for the synthesis of such structure are metal and ligands as well as some small molecules. As a green and sustainable biocatalyst, protease of aspergillus melleus（AMP） was used as a catalyst to synthesis of chiral tertiary alcohols structure successfully through aldol reaction between α,β-unsaturated γ-keto ester and cyclohexhanone in the absence of additional solvent. After screening temperature, the amount of water, molar ratio, the amount of enzyme, we then explored the scope of substrates under optimal conditions. Aromatic ring containing different substituents of unsaturated keto ester can achieve the structure of the chiral tertiary alcohols. Heterocyclic pyridine can tolerate this chiral tertiary alcohols structure. Acyclic acetone, four- membered ring, five- membered ring or six-membered ring and pyran ring ketone can be used as a substrate. After expansion of the substrates, the best results were obtained with yield of up to 90%, diastereoselectivity of up to 93/7 and enantioselectivity of up to70%.Water is green, cheap, and abundant solvent. Water as solvent in organic reaction is clean, safe, and non-polluting. Pepsin is mainly produced in digestive system of human and many other animals, and it can hydrolyze proteins into smaller peptide. Aspartic acid-32 and aspartic acid-215 are in ative site of pepsin. In this thesis, we used water as the solvent and pepsin as catalyst to the vinylogous Michael addition of angelica lactone and N-phenyl maleimide. After screening the molar ratio, enzyme amount, temperature and time, extension of the substrates were done under the optimized conditions, and a series of γ,γ- disubstituted butenolide lactone compounds were obtained. Total of 12 compounds were synthesized, yield up to 98%, diastereoselectivity of up to 99/1. Then we did some control experiments using metal ion Ag+, as well as against its active site specific inhibitors CDI and DCC. We verify the reaction may occur on pepsin activity sites, and we also proposed a possible mechanism.