Metalloenzyme Discovery and Pathway Engineering to Functionalizethe Inert C-H bonds of Amino Acids

Princeton University, Princeton NJ

Investigators

Abstract

Metalloenzymes serve in a myriad of critical biological processes. Notably, their ability to natively functionalize inert C–H bonds with high regio- and stereoselectivity has garnered significant attention, as the direct functionalization of chemical feedstock through C–H bond functionalization would serve as a streamlined strategy for accessing desired compounds. This would circumvent many of the circuitous steps required in synthetic chemistry (e.g. protection and deprotection of substrates and products), thereby also addressing some of the chemical challenges associated with sustainability and green chemistry. Because the selective functionalization of inactivated C–H bonds remains an outstanding challenge, the mechanisms that contribute to the unique catalytic properties of metalloenzymes have been studied intensely, with the hope that the catalytic prowess of metalloenzymes could be recapitulated in synthetic systems. However, the advances in molecular biology, biochemistry, and protein engineering have facilitated a paradigm shift. While historically metalloenzymes were limited to catalyzing their native reactions, certain enzyme families, such as such as P450s, are now used to modify non-native substrates on the industrial scale. The early success in utilizing metalloenzymes demonstrates their potential in addressing the current challenges with selectivity and sustainability in synthetic chemistry and demonstrates the need to expand their scope in catalysis. Amino acids are valuable precursors to accessing a wide array of chemically diverse, stereochemically-rich compounds (e.g. therapeutic peptides, alkaloids, penicillins, polyketides, nonribosomal peptides). Given the large number of aliphatic C–H bonds and multiple reactive functional groups present in amino acids, the selective functionalization of these bonds is challenging. Studying and developing new ways to functionalize amino acids using metalloenzymes therefore could provide new avenues to vastly expand the chemical space available to construct new molecules and/or derivatize existing ones. The work described in this proposal aims to study how to functionalize amino acids using metalloenzymes. The first specific aim will focus on the discovery of new reactions catalyzed by understudied metalloenzyme families. Enzyme discovery will be streamlined by finding sequences that colocalize with genes annotated to work on free amino acids, thus limiting the potential substrate scope to canonical amino acids. The second specific aim will focus on engineering reaction pathways by incorporating the chemistry non-heme Fe halogenases. The Chang lab has discovered halogenases that halogenate the aliphatic positions of free L-lysine and L-ornithine. Functionalization of these amino acids provides an avenue for generating valuable functionalized N-heterocycles that could be used for drug discovery and/or modifying existing drug scaffolds. Altogether, the proposed work will contribute to expanding the utility of metalloenzymes to address the challenges associated with installing highly desirable chemical motifs selectively.