Study On The Coupling Reaction Between Nitro Compounds And Diazo Compounds Catalyzed By Metalloenzymes

Due to the strong electron-withdrawing inductive effect of the nitro group,nitroalkanes exhibit mutual isomerization similar to keto-enol tautomerism,namely nitroso-pseudo acid form,and the pseudo acid form can also be regarded as alkenol-type.The presence of alkenol-type endows nitro compounds with the property of accepting free radicals,which can be used to develop a new type of reaction,namely coupling reaction with diazo compounds.Cytochrome P450 exhibits some characteristics of a metal-organic catalyst due to the presence of porphyrin in its structure,such as catalyzing the synthesis of C-C bonds.Small molecule vitamin B₁₂has a structure similar to the auxiliary porphyrin of P450 and can catalyze similar reactions.Therefore,this study investigated the reaction of diazo compounds and nitro compounds under the catalysis of P450 and vitamin B₁₂,which generatedβ-amino acid precursors,opening up a new pathway for the synthesis ofβ-amino acids.1.The mutant variants of P450,P411_HF,P411_CHA,P411_APA,and P411_CHF,were successfully cloned and expressed,and their correctness was verified by sequence alignment and SDS-PAGE experiments.2.We studied the coupling reactions of nitro substrates and diazo substrates catalyzed by different metal enzymes mutants.28 compounds were prepared through chemical methods,and P450 mutants,including P411_HF,P411_CHA,P411_APA,P411_CHF,as well as P411_CHAmutants L437A,L437G,L437I,myoglobin,and Az1 mutant of hydroxyphenylpyruvate dioxygenase（HPPD）,were used to catalyze the coupling reactions of different nitro substrates and diazo substrates.However,no catalytic activity was observed through preliminary screening using thin-layer chromatography and secondary screening using high-performance liquid chromatography.3.The coupling reaction of nitro compounds and diazo compounds was studied using vitamin B₁₂as a catalyst in the presence of the reducing agent Zn.The results showed that the reaction proceeded smoothly at 50℃.The reaction conditions,such as solvent,p H,equivalent of diazo compound,catalyst amount,temperature,and time,were optimized.The highest yield of the reaction was 37.93%.The substrate scope of the B₁₂catalytic system was also investigated,and it was found that various types of diazo substrates could react well with nitro cyclohexane.4.A possible mechanism for the coupling reaction of nitro compounds and diazo compounds catalyzed by vitamin B₁₂was proposed and preliminarily verified.The mechanism of the reaction was explored through a D₂O substitution experiment,and it was found that the H protons in the product came from water,confirming the reliability of the proposed reaction mechanism.