Developing a Bacterial P450 as a General and Regioselective Catalyst for C-H Bond Oxidation.

Abstract

The biosynthetic macrolide P450 monooxygenase PikCD50N-RhFRED has been employed as a biocatalyst for the stereo- and regioselective oxidation of C–H bonds. Previous results from collaborative work between the Montgomery, Podust and Sherman groups reported employing PikCD50N-RhFRED for the moderately regioselective hydroxylation of several desosamine linked carbocyclic rings, proving PikC to be capable of oxidizing unnatural substrates. The N,N-dimethylamino group in desosamine anchors the substrates in the active site and enables hydroxylation. This thesis describes the expansion the substrate scope of the PikCD50N-RhFRED hydroxylation of unnatural substrates, by probing structural modifications in the aglycone and replacing desosamine with anchoring groups that are easier to synthesize and cleave from the substrates to develop this enzymatic reaction as a synthetic tool for the selective oxidation of C–H bonds. Through substrate engineering, optimized the regioselectivity and stereoselectivity of the oxidation and insights into the factors site-selectivity of PikC catalyzed hydroxylation were gained. A benzylic N,N-dimethylamino anchoring group effectively controlled the regioselectivity of the reaction of 10-deoxymethynolide derivatives while unnatural anchors had little effect on the regioselectivity of the enzymatic oxidation of simple carbocycles. The degree of structural complexity necessary for selective oxidation for 12-membered ring macrolides was probed by synthesis of variety of 12-membered macrolide analogues that more closely resemble the endogenous substrates from PikC. We found it is possible to modestly control selectivity by the choice of anchor, in systems with moderate functionality. Finally, PikCD50N-RhFRED was employed as a bio-catalyst for the selective hydroxylation of small-molecules by coupling of alcohols with larger linkers containing the dimethylamino anchor.