Rational Regulation Of Lipase-catalyzed Organic Synthesis Reactions

Lipase has been widely used for organic synthesis reactions because of its high catalytic activity, excellent selectivity and substrate diversity. However, enzymes are susceptible to the external environment, and the reaction types are diversified as well as their substrates are mutable. Therefore, the catalytic activity and enantioselectivity of lipase for organic synthesis reactions are not as good as those for natural reactions. This has largely limited their wider industrial application. Three ways could be adopted to regulate the catalytic activity of enzymes, including protein engineering, substrate engineering and medium engineering. In this thesis, lipase-catalyzed hydrolysis reaction, aminolysis reaction, MBH reaction and Henry reaction were regulated by medium engineering and substrate engineering, the following is the results obtained in this work:(S)-NEMPA with high optical purity could be prepared by the hydrolysis of(R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester((R, S)-NEMPA-ME) catalyzed by lipase PCL, but the reaction activity was not high. We adopted Macrocyclic tetraamines(MTs) as the additive for this lipase-catalyzed hydrolysis reaction, and the catalytic activity and enantioselectivity of lipase PCL were significantly improved. Meantime, we found that the regulation of MTs to the activity and selectivity of lipase PCL was related to the structure of the lipase. The selected optimal additive was MT # 20, and the optimal concentration was 9.6 mmol/L. When compared with the reactions without MTs, MT# 20 could improve the reaction rate about 11times(from 0.5 μmol/h to 5.6 μmol/h).The traditonal catalysts for Morita–Baylis–Hillman(MBH) reaction were organic bases, few biocatalysts have been reported to catalyze the MBH reaction and reaction yield were rather low. In this study, lipase was used to catalyze MBH reaction, but lipase-catalyzed reaction could not be realized. Lipase Novozym 435 catalyzed MBH reaction was realized for the first time by using amide as additive. Reaction conditions were optimized, under the optimal conditions, yield could reach 43.4% at 37℃ for 2days,it is higher than than the references. Besides, Novozym 435/amide were used to catalyze Aza-MBH reaction, the enzyme-catalyzed Aza-MBH reaction was realized for the first time.Deep eutectic solvents(DESs), similar to ILs, are inexpensive and simple to be prepared. DESs was introduced to enzyme-catalyzed Henry reaction to promote the reaction. We reported the first example of biocatalytic Henry reaction in DESs-based medium. Addition of 30 vol % water to DESs further improved the lipase activity. A final yield of 92.2 % for lipase AS-catalyzed Henry reaction was achieved in only 4 h, it was improved by approximately 3-fold when compared with that in pure water and pure DESs. Lipase conformation in DESs- H2 O system was studied by fluorescence spectroscopy. The results showed that lipase conformation would become more flexible, which was beneficial to the interaction between substrate and lipase and thus improved lipase activity. Furthermore, the methodology was also extended to Aza-Henry reaction. Enzyme-catalyzed Aza-Henry reaction was realized for the first time and the reaction yield could reach to 38.7 %.Methoxymethyl chloride(MOMCl) was first introduced as a protective and esterified reagent to obtain MOM-protected caffeic acid MOM ester in this study, and we successfully realized the lipase-catalyzed aminolysis reaction between caffeic acid eaters and chiral amines. Reaction conditions were optimized and e.e.p reached 85.1 %. Methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-butyl ester and MOM ester of four kinds of phenolic acid(cinnamic acid, 3, 4-dimethoxycinnamic acid, MOM-protected ferulaic acid and MOM-protected caffeic acid) were synthesized for verifying the superiority of MOM ester. Then the 24 kinds of esters were used as acyl donor respectively under the same reaction condition. The results showed that reaction rate of MOM ester was superior to the other esters with no loss of enantioselectivity. The reaction rate increased by 1.3-3.3 fold and enantiomeric excess of product(e.e.p) could reach 99 %.In lipase catalyzed hydrolysis reaction, aminolysis reaction and C-C addition reaction, the catalytic activity and enantioselectivity of lipase were improved significantly by substrate and medium engineering. In conclusion, our study provided new thought for rational regulation of the enzyme-catalyzed organic synthesis reactions.