Thesis (Ph. D.)--University of Rochester. Department of Chemistry, 2015.
The late stage oxidation of C(sp3)-H bonds is an attractive reaction for both the late stage diversification of lead compounds as well as the synthesis of natural and synthetic organic compounds. The high bond strength of C(sp3)-H bonds, the large numbers of C(sp3)-H bonds in complex molecules, and the increased reactivity of products as compared to starting materials makes C(sp3)-H bonds challenging targets. The class of cytochromes P450 (P450s) naturally perform the selective monooxygenation of complex carbon scaffolds in the biosynthesis of natural products. The implementation of P450s in organic synthesis is hampered by the difficulty in engineering these proteins for highly regio- and stereoselective oxidations of non-native substrates. Herein we report the design and application of P450s for the late stage functionalization of natural products. First, we applied the recently developed P450 ‘fingerprinting’ method to aid in developing P450 based oxidation catalysts for the oxidation of anti-leukemic sesquiterpene lactone parthenolide. Highly regio- and stereoselective P450 catalysts were developed and applied to the chemoenzymatic synthesis of a novel class of parthenolide derivatives with increased anti-leukemic activity as compared to parthenolide. Two new ‘hotspots’ of the parthenolide scaffold were thus identified for further development, highlighting the use of P450s for the late stage development of drug like compounds. Next, we explored the use of unnatural amino acids (UAAs) to modify the activity and regioselectivity of P450 based oxidation catalysts. A set of structurally diverse UAAs were incorporated into P450s and found to yield viable oxidation catalysts with regioselectivities that differed largely from the parent enzyme. Additionally, para-amino-Phe was shown to have a general activity enhancing effect upon incorporation into P450s leading to the isolation of a variant with the highest activity toward a complex molecule reported to date. These effects could not be reproduced by incorporation of any of the 20 natural amino acids showing that UAA mutagenesis is a useful complementary method to tune the activity and regioselectivity of P450s.
Recently P450s were shown to catalyze intramolecular C-H amination and, looking to expand upon the substrate scope, we chose to explore the reactivity of carbonazidates. Carbonazidates have long been a desirable substrate for C-H amination due to their high atom economy and P450s were found to be uniquely capable of activating this class of substrates. Oxazolidinones were formed via the P450 catalyzed C-H amination of benzylic and allylic C-H bonds. This reactivity was applied to the intramolecular C-H amination of two monoterpene substrates, thus showing that P450s could catalyze the C-H amination of more complex structures. Finally, the mechanism of P450 catalyzed C-H amination was explored using kinetic isotope effect experiments and radical rearrangement probe substrates showing that the C-H amination step likely proceeds through a hydrogen atom abstraction radical rebound type mechanism.
A large limitation to the synthetic use of P450s for C-H amination is the low catalyst activity and large amount of reduction side product (carbamate or sulfonamide) that is formed during the reaction. The proposed mechanism for P450 catalyzed C-H amination was used to direct the engineering of P450 based catalysts. These P450 variants were found to produce less of the undesired reduction products and have greatly increased C-H amination activities, leading to a catalysts with the highest reported C-H amination activity reported to date. These studies further the applicability of P450s to the late stage amination of natural products.
