SELECTIVE ALIPHATIC AND AROMATIC CARBON-HYDROGEN BOND ACTIVATION CATALYZED BY MUTANTS OF CYTOCHROME P450(CAM)

The substrate range of the haem monooxygenase cytochrome P450(cam) (CY P101) has been broadened by site-directed mutagenesis. The hydroxylati on selectivity of five mutants at the 96 position towards a range of s ubstrates has been used to investigate P450(cam)-substrate molecular r ecognition. The substrates contained aromatic and activated and unacti vated aliphatic C-H bonds, as well as reactive functional groups. Diph enylmethane, diphenylether, diphenylamine, and 1,1-diphenylethylene we re all hydroxylated regiospecifically at the para position, with no at tack at the amine or the olefinic double bond. With benzylcyclohexane the activated benzylic and tertiary C-H bonds were not attacked, and t he reactions catalysed by the Y96G and Y96A mutants were highly diaste reoselective, 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-pheny lethenylcyclohexanol were formed. These results show that P450(cam) ca n be engineered to oxidise C-H bonds without attacking more reactive f unctional groups.