Biocatalytic Promiscuity: Enzyme Catalyzed Direct Asymmetric Aldol Reaction

Enzyme catalysis has attracted much attention because of the intrinsic advantages such as high stereoselectivity, mild reaction conditions and rapid reaction rate. Recently, the study of enzymatic synthesis becomes the frontier fields of scientific research. However, there is only limited number of enzymes exist in nature, and enzymes only feature limited substrate specificity. Therefore, discovery of novel unnatural activities of existing enzymes to widen the applicability of existing enzymes is significant. Recently, some elegant works on enzyme catalysis in organic solvents have been reported. Some protease and lipase were found had the promiscuity ability to catalyze Michael addition, Henry reaction and Mannich reaction. It greatly extended the application of enzyme catalysis and the exploration of enzymatic promiscuities provides a new pathway for green chemistry. It will be a research area full of challenge and opportunity. Aldol reaction is one of the most important C-C bond forming reactions in organic synthesis. However, there are only limited enzymes except aldolases that can catalyze aldol reaction. Therefore, it is necessary for us to study the promiscuity of enzyme to catalyze aldol reaction.In this thesis, we report three promiscuous enzyme nuclease P1, alkaline protease and chymopapain which have the ability to catalyze direct asymmetric aldol reaction. The aldol reaction between aromatic aldehydes and cyclic ketones catalyzed by the three kinds of enzyme are mainly investigated. In order to further study the mechanism and verify the effect of these enzymes to aldol reaction, we performed some control experiments including the inhibition of the catalytic activity and completely denatured enzymes.Asymmetric direct aldol reaction of 4-cyanobenzaldehyde and cyclohexanone catalyzed by three enzymes was first carried out as a model experiment to find out the optimum reaction conditions such as the solvent, the ratio of substrates, the enzyme loading, the water content, the temperature and the pH value. The results indicate that the optimum solvent is different for different enzymes in aldol reaction and the optimum water content is different for one enzyme in different solvents. Generally, the aldol reaction catalyzed by these three enzymes give the best yield in water-miscible hydrophilic solvent DMSO, but the stereoselectivity is low. Chymopapain catalyzed direct asymmetric aldol reaction in CH3CN obtain high stereoselectivity but low yields. Nuclease P1 has a high stereoselectivity and enantioselectivity under solvent-free conditions. We choose 13 aldehydes and 4 ketones and we obtain 43 known aldol products under the optimun conditions. The 43 known aldol products were verified by 1H NMR,13C NMR and chiral HPLCNuclease P1 (EC 3.1.30.1) from Penicillium citrinum belongs to a family of zinc-dependent endonucleases consisting of 270 amino acid residues with two disulfide bonds which cleaves single-stranded RNA and DNA into 5-mononucleotide. The unnatural ability of nuclease p1 was first discovered to catalyze direct asymmetric aldol reactions between aromatic aldehydes and cyclic ketones under solvent-free conditions. The excellent enantioselectivities of up to 99% ee and high diastereoselectivities of up to> 99:1 (anti/syn) were achieved.A new function of BLAP (alkaline protease from Bacillus licheniformis) was first discovered to catalyze direct aldol reactions between aromatic aldehydes and cyclic ketones in organic medium in the presence of water. The products were obtained in yields of 28-92% with 22-99% ee.Chymopapain, a cysteine proteinase isolated from the latex of the unripe fruits of Carica papaya, displays a promiscuous activity to catalyze the direct asymmetric aldol reactions of aromatic and heteroaromatic aldehydes with cyclic and acyclic ketones in CH3CN in the presence of a phosphate buffer (pH=4.91, Vbuffer/VCH3CN=0.12).