Development of Biocatalytic Strategies for the Directed Oxidation of Small Molecule and Macrocyclic Substrates

Abstract

The cytochrome P450 enzyme PikC has been explored as a robust biocatalyst for late-stage directed C-H hydroxylation reactions in organic synthesis. A collaborative effort between the Sherman, Houk, Podust, and Montgomery labs has resulted in the engineering of a highly active triple mutant fusion protein, PikCD50ND176QE246A-RhFRED. The unique mode of substrate binding to PikC allows for a substrate engineering approach to be employed whereby removable auxiliaries, termed anchors, can render compounds as suitable substrates for PikC oxidations. Previous studies had illustrated that unnatural substrates can be designed to enable the oxidation of inert C-H bonds. However, the ability to tune and reverse the regioselectivity of oxidations was not possible in prior efforts. This dissertation describes the application of a substrate engineering approach for site-selective oxidation of small molecules with a triply mutated form of the PikC biocatalyst using synthetic anchoring groups. Oxidations performed using this approach are highly site- and diastereoselective, and the selectivities seen using PikC are orthogonal to those obtained using transition metal based approaches. Application of anchoring group technology is further streamlined though the development of triazole-based anchors. With this class of directing groups, greater anchoring group structural diversity can be sampled in a high throughput approach. This technology was applied to the site-selective oxidation of unnatural 11- and 12-membered macrocycles synthesized via regiodivergent nickel-catalyzed macrocyclization. In this study, the first example of anchoring group-promoted differential site selectivity is demonstrated and the results are rationalized through computational investigations.