Studies On The Catalytic Promiscuity Of Esterase/Lipase In Carbon-Carbon Bond Formation And Heterocycle Compounds Preparation

Enzymatic catalysis was one of the most important fields in enzyme engineering. In recent years, enzyme catalytic promiscuity has attracted significant attention from the research community due to its potential application in organic synthesis and great significance for the realization of "green chemistry"Esterase BioH was found to catalyze the Aldol reaction, Nitro-Aldol reaction, Knoevenagel condensation reaction and Baylis-Hillman reaction in organic media. Aldol reaction of aromatic aldehydes with ketones, Nitro-Aldol reaction of aromatic aldehydes with nitroalkanes, Knoevenagel condensation reaction of aromatic aldehydes with acetylacetone and Baylis-Hillman reaction of aromatic aldehydes with α,β-unsaturated carbonyl compounds were explored. Enzyme source, reaction media, enzyme concentration were systematically investigated to evaluate the effect of different reaction conditions on the product yield. Control experiments demonstrated that esterase BioH was responsible for the above-mentationed reactions. In addition, site-directed mutagenesis studies were performed on the hydrolysis catalytic triad of BioH. The two mutants "BioH esterase Ser82Ala" and "BioH esterase His235Ala" showed catalytic activities similar to that of the wide-type, indicating an active site other than this catalytic triad.Amano lipase DF was found to catalyze the Knoevenagel condensation/Michael addition cascade reactions of aromatic aldehydes and1,3-cyclic diketones to form a series of2,2’-arylmethylene dicyclohexane-1,3-dione derivatives. The influences of reaction conditions including enzyme source, enzyme concentration and temperature were systematically investigated. Control experiments demonstrated that Amano lipase DF was responsible for the enzymatic Knoevenagel condensation/Michael addition cascade reactions and a possible mechanism was proposed.Esterase BioH was found to catalyze the Michael addition/cyclization cascade reactions of substituted benzalacetones with1,3-cyclic diketones in organic media to form a series of3,4-dihydropyran derivatives. The influences of reaction conditions including enzyme source, enzyme concentration and temperature were systematically investigated. Control experiments demonstrated that esterase BioH was responsible for the enzymatic Michael addition/cyclization cascade reactions. In addition, the two mutants "BioH esterase Ser82Ala" and "BioH esterase His235Ala" showed catalytic activities similar to that of the wide-type, indicating an active site other than this catalytic triad.Amano lipase AS was found to catalyze multi-component reactions of aromatic aldehydes with malononitrile and β-naphthol in organic media to form a series of naphthopyran derivatives. The influences of reaction conditions including enzyme source, reaction media, enzyme concentration and temperature were systematically investigated. Control experiments demonstrated that Amano lipase AS was responsible for the multi-component reactions and a possible mechanism was proposed.In this thesis,60compounds were synthesized including6Aldol reaction products,5Nitro-Aldol reaction products,7Knoevenagel condensation products,9Baylis-Hillman adducts,132,2’-arylmethylene dicyclohexane-1,3-dione derivatives,123,4-dihydropyran derivatives and8naphthopyran derivatives. These compounds were characterized by1H NMR,13C NMR and EI-MS.