Acylases-catalyzed Michael Addition Of N-heterocycle Derivatives

Enzymes had been more and more widely used in organic chemistry. Enzymaticsynthesis in organic media has brought about an exponential increase in interest in the fieldof organic synthesis and provides a large number of alternatve methods for organicsynthises. This thesis focuses on acylases-catalyzed Michael additionof N-heterocycles inorganic media and enzyme-catalyzed cascade acylation/Michael addition.In this thesis, we researched acylases-catalyzed Michael addition of C-N, C-S andC-O bond. Under the catalysis of three promiscuous acylases, including D-Aminoacylasefrom Escherichia coli, Acylase "Amano" from Aspergillus oryzae and immobilizedPenicillin G Acylase from Escherichia coli, the result show that two zinc-active-siteacylases D-Aminoacylase and "Amano" acylases have extremely high Michael activities.Some control experiments demonstrated that the Michael addition was catalyzed by theactive sites of acylases. Based on the acylase mechanism and chemical additionmechanism, we proposed a feasible mechanism for the promiscuous acylase catalyzedMaichael addition and desighned some experiments to preliminarily prove it.The acylase-catalyzed aza-Michael addition had been detailedly discussed. Theinfluence factor including solvents, tempatures, enzyme amounts, substrate concetraionsamd structure of donors and acceptors.had been systematically examined. The best reactiontemperature is between 45-50℃; the reaction activity increases as the polarity of solventdecreases, and DMSO is the best media for hardly dissolved substrates. The enzymaticreactivity decreases as the chain of the alcoholand the steric effect of substituted group inacceptor increased. An electron-rejecting or sterically hindering effect of the substitutedgroup decreased the addition reactivity of N-heterocycle. The electron cloud on theaddition N also influences the addition result greatly.The enzyme-catalyzed cascade acylation/Michael addition was studied. A singleacylase-catalyzed cascade acylation/Michael addition was first attempted and nosatisfactorily result was obtained. Then, the acylation of monosaccharide andα,β-unsaturated vinyl ester catalyzed by proteases and the acylase-catalyzed Michaeladdition of pyridines toα,β-unsaturated sugar ester were investigated to afford 12sugar-containing N-substituted pyridines derivatives. Furthermore, Alkaline protease fromBacillus subtilis and D-Aminoacylase catalyzed-tandem acylation/Michael addition and one-pot reaction of pyridines, glucose andα,β-unsaturated vinyl ester was studied.Compared the reactions of independently acylation, Michael addition and cascade reactionto afford sugar-containing N-substituted imidazoles derivatives, the yield of cascadereaction is lower than independently methods.Using imidazoles, pyridines, purines, amine, alcohol and thiol as Michael donors, 44compounds were synthesized and characterized by ¹H NMR, ¹³C NMR, FTIR andESI-MS. 12 sugar-containing N-substituted pyridines derivatives have been confirmed to benew compounds.