Selective aliphatic and aromatic carbon-hydrogen bond activation catalysed by mutants of cytochrome p450cam

The substrate range of the haem monooxygenase cytochrome P450_cam (CYP101) has been broadened by site-directed mutagenesis. The hydroxylation selectivity of five mutants at the 96 position towards a range of substrates has been used to investigate P450_cam -substrate molecular recognition. The substrates contained aromatic and activated and unactivated aliphatic C---H bonds, as well as reactive functional groups. Diphenylmethane, diphenylether, diphenylamine, and 1,1-di-phenylethylene were all hydroxylated regiospecifically at the para position, with no attack at the amine or the olefinic double bond. With benzylcyclohexane the activated benzylic and tertiary C---H bonds were not attacked, and the reactions catalysed by the Y96G and Y96A mutants were highly diastereoselective, with 4-trans-benzylcyclohexanol constituting 90% of the products. 1-Phenyl-1-cyclohexylethylene was oxidised predominantly at the 4-position of the cyclohexane ring without attack at the olefinic double bond, and approximately equal amounts of cis- and trans-4-phenylethenylcyclohexanol were formed. These results show that P450_cam can be engineered to oxidise C---H bonds without attacking more reactive functional groups.