Acylases-catalyzed Markovnikov Addition And Synethsis Of N-heterocycle Derivatives

The study of enzymatic synthesis has attracted much atteintion because of its various advantages, such as the high selectivity, mild condition and rapid reaction rate. Although addition reaction is among the most fundamental types of reactions in organic synthesis, there were only scarce reports about enzymes which are able to catalyze general addition reactions. The recent progress of catalytic promiscuity has greatly extended the application of enzyme catalysis and some lipases and proteases were developed to catalyzing Michael addition and aldol addition. Thus exploration of enzymes with new activities becomes particularly fascinating and remains a great challengeIn this thesis, we report the unprecedented acylases-catalyzed Markovnikov-type addition. Under the catalysis of three promiscuous acylases. including D-Aminoacylase from Escherichia coli, Acylase "Amano" from Aspergillus oryzae and immobilized Penicillin G Acylase from Escherichia coli, a number of N-heterocycles, including some pentacyclic N-heterocycles, pyrimidines and purines, were successfully added to a series of vinyl esters in moderate to excellent yields to prepare 34 N-heterocycle derivatives, among which 33 were new compounds. Some control experiments demonstrated that the Markovnikov addition was catalyzed by the active sites of acylases. Based on the acylase mechanism and chemical addition mechanism, we proposed a feasible mechanism for the promiscuous acylase catalyzed Markovnikov addition and desighned some experiments to preliminarily prove it.Reaction factors such as temperature, solvent and enzyme source were systematically examined. The best reaction temperature is between 45～50℃; the reaction activity increases as the polarity of solvent decreases, and DMSO is the best media for hardly dissolved substrates; 14 acylases, lipases and proteases were examined for the addition activity and the result revealed that acylases possess high Markovnikov addition activities, among which D-aminoacylase was the best catalyst.The influence of the structure of vinyl ester and N-heterocycle was also evaluated. The enzymatic reactivity decreases as the chain of the vinyl ester increased; divinyl dicarboxylates reacted faster than monoacid vinyl esters; the Markovnikov addition of vinyl benzoate is rather slow. An electron-rejecting or sterically hindering effect of the substituted group decreased the addition reactivity of N-heterocycle. The electron cloud on the addition N also influences the addition result g reatly. Generally, the enzymatic reactivity decreases in the order of imidazole, purine and pyrimidine, giving different regioselectivities.