Cytochrome P450 Catalyzes C-H Bond Amidation

Over the last decade research on cytochrome P450 has demonstrated its ability to simultaneously undergo oxidation while acting as a catalyst in organic synthesis reactions.This alludes to the possibility that there are many unknown functions of cytochrome P450 waiting to be defined.In recent studies,cytochrome P450 has been elucidated for its mutant catalysis ability.In this research the catalyzed P450 mutant C-H bond amidation was studied,with an aim to:1.Determine the catalytic activity of the obtained P450 mutant-P411CHA,according to the gene sequence,using plasmid pET-41a(+)as the vector.Mutant P411CHA protein was successfully expressed,and the cells required for the catalytic reaction were obtained.The whole-cell catalytic substrates 4-ethylanisole and 4-ethyltoluene are respectively reacted with p-toluenesulfonyl azide to amidate the C-H bonds of the secondary carbon atoms on the ethyl groups of the two compounds.Product N-[1-(4-Methoxy-phenyl)-ethyl]-4-methy 1-benzenesulfonamide and 4-Methyl-N-(1-p-tolyl-ethyl)-benzenesulfonamide were obtained,and compared with the standard products of the two target products synthesized by HPLC.The results showed that the catalytically active P450 mutant P411CHA was successfully obtained,which were used in subsequent reactions.2.The mutant P411CHA protein was used to catalyze the amidation reaction of the C-H bond.P411CHA whole cells were added to the synthesized substrate C:N-(2,2-Dimethyl-propionyloxy)-3-pyridin-3-ylpropionamide in order to generate the target product delta-lactam.However after a series of attempts,no product of the amidation reaction was obtained.As a next resort its catalytic potential was tested on subsequent compounds such as cinnamic acid,phenylpropionic acid,methyl cinnamate and methyl phenylpropionate.Among the group,P411CHA exhibited catalytic activity on methyl cinnamate and methyl phenylpropionate,contemporaneously exhibiting esterase activity.3.Obtain a new mutant P411APA.According to the gene sequence,the plasmid pET-22b(+)is used as the vector to express P411CHA,but the unique PeIB fragment on the vector will cause the foreign expression of the protein,which will affect the subsequent use of whole-cell catalytic reactions.Therefore,using a homologous recombination method,replacing the plasmid PZE as a vector,the water-soluble protein was finally successfully obtained and the mutant P411CHA was verified to catalyze the amination reaction of 1-phenyl-1-butene and the nitrogen source PivONH3+TfO-to produce 4-phenyl-3-butene-2-amine.The experiment also uncovered that P411CHA can also recognize this substrate and have the same reaction,successfully expanding the substrate spectrum of this mutant.