Expanding the Catalytic Repertoire of Hemeproteins as Carbene Transferases to Access Diverse Molecular Structures

Abstract

The efficiency, selectivity, and sustainability benefits offered by enzymes are enticing chemists to consider biocatalytic transformations to complement or even supplant more traditional synthetic routes. Increasing demands for efficient and versatile synthetic methods combined with powerful new discovery and engineering tools have prompted innovations in biocatalysis, especially the development of new enzymes for precise transformations. The past decade has witnessed impressive expansion of the catalytic repertoire of enzymes to include new and useful transformations not known (or relevant) in the biological world. The continuing discovery and improvement of these new enzyme activities is opening a floodgate that connects the chemistry of the biological world to that invented by humans over the last 100 years. This thesis describes a new set of enzymes, derived from a cytochrome P450 monooxygenase and a cytochrome c electron-transfer protein, which are able to function as carbene transferases to construct diverse molecular structures, including strained carbocycles and lactone derivatives. Chapter 1 illustrates different approaches researchers have utilized to explore and develop new catalytic machineries of diverse enzymes. These efforts have identified new genetically-encoded biocatalysts that can be tuned and diversified through directed evolution. Chapter 2 presents the discovery of P450 variants that catalyze the formation of highly strained carbocycles, bicyclobutanes and cyclopropenes, via carbene addition to carbon‒carbon triple bonds. The intrinsic strain energies of these small rigid carbocycles allow them to have broad applications in different fields, but also create challenges for their construction. Using a diazo substrate as the carbene precursor, the enzyme variants optimized by directed evolution could act on structurally diverse alkynes (aromatic or aliphatic, terminal or internal) with high efficiency and selectivity, providing an effective route to an array of chiral strained structures. The carbene transferase activity is then extended to the assembly of various lactone structures, a fundamental class of organic moieties with applications in fields varying from synthetic chemistry, to materials science, to medicinal chemistry. Chapter 3 details a strategy using lactone-based carbenes, for the transfer to different functionalities, enabling rapid access to a broad range of α-substituted and spiro-lactones with unprecedented efficiencies and selectivities. A different approach based on intramolecular carbene C–H insertion is outlined in Chapter 4, which allows for the synthesis of lactones in a higher order of structural diversity. Directed evolution of a P450 variant identified a lineage of potent variants, capable of assembling lactones in different sizes (5- to 7-membered) and also with sophisticated three-dimensional structures based on fused, spiro and bridged rings. Computational tools were employed to understand the reaction mechanisms and to explain some mutational effect. In sum, the thesis work lays out how protein engineering integrated with chemical rationalization enables the expansion of the chemical space accessible to native hemeproteins, especially in building diverse molecular structures.