Synthesis Of Fluorinated Compounds Catalyzed By P450

Fluorination is one of the five reactions that chemists dream of.More than 20%of commercial drugs contain fluorine.Although fluorinated compounds are increasingly important in drug development,it is still a very challenging work to directly convert C-H bond to C-F bond in general or specific positions.Compared with chemical synthesis,enzyme catalysis is more efficient.Cytochrome P450 monooxygenase is a natural catalyst that can directly insert oxygen into C-H bond.Scientists found that P450 mutant can catalyze a variety of unnatural reactions.In this study,P411_CHA,a mutant of P450,will catalyze the fluorination of C-H bond to C-F bond.P411_CHA enzyme was successfully cloned and expressed,and the plasmid containing P411_CHA gene was transformed into E.coli.The expression was induced by IPTG and 5-ALA.The protein was soluble and the molecular weight was about 116 k Da by SDS-PAGE gel electrophoresis.The concentration of heme protein in cell lysate was determined by pyridine hemoglobin method..A series of standard fluorine products were prepared.P411_CHA was used for enzyme catalyzed reaction with different substrates.The fluorine sources were NFSI and selectfluor.Three substrates,ethyl benzene,isobutyl benzene andβ-Bromophenylethane,it is preliminarily proved that P411_CHA has the ability to catalyze fluorination reaction.In order to further improve the catalytic activity of P411_CHA,site directed mutagenesis was carried out.The three-dimensional protein structure was constructed by P411_CHA enzyme（PDB:5ucw）.Through computer simulation,it was found that Leu437 may be the entrance of substrate into P411_CHA heme active center,and Leu437 was mutated into amino acid with smaller molecular weight;Connecting the substrate ethyl benzene to P411_CHA molecule,it was found that the distance between Val328 amino acid and substrate was 4.0（?）,which was adjacent to heme cofactor,and the saturation mutation of Val328 amino acid was carried out.The mutant was used for enzyme catalytic reaction,but the catalytic activity of the enzyme was not significantly improved.Based on the comprehensive analysis of the experimental results,it is preliminarily determined that P411_CHA has the ability to catalyze fluorination reaction,which provides a new idea and method for the occurrence of fluorination reaction.This is also the first time that cytochrome P450 mutant is used to catalyze the fluorination of compounds,which also opens up a new way for biocatalytic fluorination in the future.