Aldol Reaction And Knoevenel-michael Cascade Reaction Catalyzed By Pel And Others:Optimization Of Reaction Conditions And Related Mechanisms

While traditional chemical synthesis methods bring millions of chemicals and bring convenience to people’s lives,the pollution they produce also brings a heavy blow to the environment.The concept of sustainable development of green chemistry came into being.In order to realize green chemistry and develop green and efficient organic synthesis tools,two novel green synthesis strategies and their intrinsic catalytic mechanisms were investigated in this paper.Enzymes,as green and mild catalysts,have received great attention in fine chemical synthesis.In recent years,studies on the catalytic multifunctionality of enzymes（more and more catalytic multifunctionality of lipases are reported,such as PPL,CAL-B,RML,MML,etc.）have expanded the application scope of enzymes catalyzed in organic synthesis.In order to expand the application of biological enzymes in organic synthesis and enrich the methodology of organic synthesis,we have studied the Aldol reaction of 2-nitrobenzaldehyde and acetone catalyzed by Penicillium expansum lipase（PEL）in deep co-crystal solvent,and found for the first time the versatility of Penicillium expansum lipase in catalyzing the Aldol reaction,its Aldol reaction product was synthesized and prepared,and by setting BSA as a control experiment,non-specific catalysis was excluded,and the special spatial conformation of the enzyme and the specific catalysis of PEL were proved.The specific sites of PEL are essential in catalyzing the Aldol reaction.In order to achieve higher catalytic efficiency,the effects of ketone aldehyde ratio,enzyme loading,water content and the ratio of choline chloride to glycerol in deep eutectic solvent on the catalytic activity of pel were systematically investigated.The optimal yield was 42%.We also described the aldol reaction of aromatic aldehydes and acyclic ketones catalyzed by pel,which was carried out in low to medium yields in a wide range of substrates.In order to explore the mechanism of PEL-catalyzed reaction of 2-nitrobenzaldehyde and acetone,mutants of PEL-S132A、PEL-H241A and PEL-S132A/H241A were constructed by alanine scanning,and it was verified that it lost the main activity of PEL-catalyzed ester hydrolysis.When the main active sites S132 and H241A were mutated,respectively,the reaction catalyzed by the mutants obtained a yield of 36%and 41%,which was indistinguishable from the wild type.But when both were mutated,a yield of 89%was obtained,which was 2.2 times that of the wild type.In order to achieve higher catalytic efficiency,the effects of ketone-aldehyde ratio,enzyme loading,water content,and the ratio of choline chloride to glycerol in the deep co-crystal solvent on the catalytic activity of the PEL-S132A/H241A mutant were systematically investigated.The best yield of 89%was obtained.In addition,we also developed for the first time an environmentally friendly three-component Knoevenagel-Michael cascade reaction of aromatic aldehydes,amines,and active methylene compounds to synthesize a series ofβ-aminoacrylate derivatives.（1）The condition of ammonium sulfate in the catalysis of 4-nitrobenzaldehyde and methyl cyanoacetate was optimized and the Knoevenagel-Michael cascade reaction yielded an optimal yield of 92%of the reaction product.（2）Under the optimal reaction conditions,the versatility of the reaction was discussed by using benzaldehyde substrates modified with different groups.Among them,p-nitrobenzaldehyde,m-nitrobenzaldehyde,o-nitrobenzaldehyde and p-cyanobenzaldehyde can react with methyl cyanoacetate to generate the correspondingβ-amino acrylate,and obtain a yield of 93%,75%,88%and 23%,respectively.On this basis,we also explored the scalability of various primary amine salts as catalysts（as well as one of the substrates）,such as methylamine hydrochloride,ethylamine hydrochloride,benzylamine hydrochloride and histamine hydrochloride.The extensibility of salts as catalysts catalyzed nitrobenzaldehyde and methyl cyanoacetate,ethyl cyanoacetate,and tert-butyl cyanoacetate at different positions,respectively,and the corresponding products（41%-91%yields）were obtained.These findings lay the foundation for the post-synthetic modification of biologically active compounds in medicinal chemistry and the pharmaceutical industry.（3）By controlling the experimental setup and reactions involving multiple substrates,we explored and clarified the mechanism of amines（or ammonium salts）catalyzing the Knoevenagel-Michael cascade reaction:primary amines（or ammonium salts）first catalyzed aromatic aldehydes by Knoevenagel condensation decarboxylation with reactive methylene compounds to yield alpha-cyanoacrylates.The intermediate productα-cyanoacrylate and primary amine（or ammonium salt）continue to undergo Michael addition reaction.Among them,there are two keys for the further conversion ofα-cyanoacrylate intoβ-aminoacrylate:one is that the substituent on the benzene ring of benzaldehyde must be a strong electron withdrawing group,such as-NO2 or-CN.Although the halogen group is also a strong electron withdrawing group,its electron withdrawing ability is weaker than that of-NO₂and-CN,so the reaction stops at the Knoevenagel condensation reaction and cannot continue the Michael addition;the second is another substrate acrylate.Theα-substituent must also be a strong electron withdrawing group-CN.（4）The Knoevenagel-Michael cascade reaction catalyzed by primary amines（or ammonium salts）still has a robust performance at the gram level（the yield of the reaction is 87%）,which is beneficial to the subsequent industrial production and application.This new green synthesis method can also be applied to synthesize intermediates of isothiazolopyrimidines,a class of potent VEGFR₂/KDR inhibitors,and obtain good yields（72%and 89%）,thus providing a green、low-cost、efficient synthesis protocol of some important pharmaceutical intermediates.