Evolution and Characterization of Carbene Transferases for Cyclopropanation and Carbon–Silicon Bond Formation

Citation

Lewis, Russell DeRieux (2019) Evolution and Characterization of Carbene Transferases for Cyclopropanation and Carbon–Silicon Bond Formation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/RMEX-Q134. https://resolver.caltech.edu/CaltechTHESIS:06052019-172451535

Abstract

Heme proteins have recently been demonstrated to catalyze cyclopropanation reactions via a putative carbene transfer mechanism. Carbene transfer reactions are not known to occur in natural biological systems, but are highly useful synthetic reactions. There is growing interest in developing new "carbene transferases" that bring new chemical reactions into the realm of biology, and growing interest in engineering these enzymes for use in organic synthesis. Additionally, the mechanistic details of iron porphyrin-catalyzed carbene transfer reactions are largely unknown, especially with regards to how the enzyme environment influences the outcome of a carbene transfer reaction. This thesis details both the engineering of carbene transferases with novel catalytic capabilities and investigations into how these enzymes catalyze carbene transfer reactions. Chapter 1 introduces heme protein-catalyzed carbene transfer reactions and describes the directed evolution of new enzymes that allow access to a range of useful cyclopropane products. Chapter 2 describes the evolution of an enzyme that performs carbene transfer to silicon–hydrogen bonds, resulting in a highly efficient and selective carbon–silicon bond-forming enzyme, the first of its kind. Chapter 3 focuses on the characterization of a key reactive intermediate, the iron-porphyrin carbene, in the active site of the evolved carbon–silicon bond-forming enzyme. This study provides an explanation of the remarkable enantioselectivity of the enzyme and provides a foundation from which to investigate the enzyme reaction mechanism. The mechanism of carbon– silicon bond formation is elucidated in Chapter 4, and the is then used to explain how the enzyme achieves chemoselectivity, which in turn guides the evolution of enzyme variants with altered chemoselectivity. Finally, two off-cycle catalytic pathways that cause inactivation of the carbene transferase are characterized, and methods to prevent and/or circumvent inactivation are investigated (Chapter 5). Overall, the work presented here expands the repertoire of enzyme-catalyzed reactions and facilitates the continuing development of new carbene transferases by developing our mechanistic understanding of this novel class of enzymes.

Thesis Files