Research On Hydrolases Catalytic C-N Michael Addition In Organic Solvents

Enzymatic synthesis in organic media has brought about an exponential increase in interest in the field of organic synthesis. Moonlighting functions and catalytic promiscuity of enzymes add new dimensions to our understanding of protein structure/function relationships and the evolution of new functions from existing protein scaffolds. These 'extra talents' of enzymes open a new area and widen the application of enzyme in organic synthesis.This thesis, for the first time, focuses on the hydrolase-catalyzed Michael addition of imidazole and its 1H-derivatives, uracil and its 1H-derivatives, 1H-derivatives of cytosine, adenine, 6-benzyl adenine and N~2-acryl guanine to acrylates and methacrylates. In the absence of enzyme catalysts, no additive product was obtained. Using the denatured enzymes and bovine serum albumin (BSA) as catalysts, no additive product was detected ether. These results showed that hydrolases can catalyze C-N Michael addition. In addition, we also discussed the mechanism of hydrolases catalytic Michael addition preliminarily.Using imidazoles, pyridines, purines, amine, alcohol, thiol and C-nucleophiles as Michael donors, 43 compounds were synthesized and characterized by ~1H NMR, ~13C NMR, FTIR and ESI-MS.16 compounds have been confirmed to be new compounds.In these Michael reactions, we examined the influence factor including the enzymes, solvents, structure of donors and acceptors. Initial studies were carried out using imidazole and methyl acrylate as a model system. In 9 commercial hydrolases, Amano Lipase M catalyzed the reaction most effectively. More hydrophilic solvents like N, N-dimethylfomamide (DMF), pyridine, dioxane and acetone were observed to cause lower conversion. On the other hand, hydrophobic solvents such as n-hexane, cyclohexane, isooctane, and isopropyl ether preserve the catalytic activity. As a result, non-polar solvents exhibited the higher conversion yields in the Michael addition of imidazole and methyl acrylate. Structure of reactant affects the reaction in three ways: (a) the length of alcohol component. We have found that the longer alcohol chain, the lower yield. Transform of N-7 to N-9 were observed in Michael addition of adenine and 6-benzyladenine, and when the acceptor had a longer alcohol chain, the transform was slower. (b) The steric effect of side chain in the acceptor. When the acceptor has a same alcohol chain, yield of methacrylates were lower than acrylates even the reactions of the former were carried out in the more effective solvent. (c) The electron donating or withdrawing groups in the donor. Withoutthe influence of solubility, effect of donor structure can be attributed to the electron donating or withdrawing group the donor has.Hydrolases can also catalyze the Michael addition of C-S, C-0 and C-C. In summary, hydrolases have high catalytic activity of hetero-Michael addition especially of C-N and C-S addition. Results of quantum mechanic calculate showed that catalytic function of C-N Michael addition is the behavior of active site in hydrolases and when acrylates acted as substrates of hydrolase, Michael addition can be achieved more easily than transesterification.