Expanding the Chemical Space of Nitrene Transferases: Biocatalytic Construction of C–N Bonds

Citation

Qin, Ziyang (2026) Expanding the Chemical Space of Nitrene Transferases: Biocatalytic Construction of C–N Bonds. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5pv2-nr39. https://resolver.caltech.edu/CaltechTHESIS:07052025-063244748

Abstract

The construction of carbon–nitrogen (C–N) bonds is pivotal to the development of pharmaceuticals, agrochemicals, and advanced materials. While traditional synthetic methods offer robust and efficient solutions, biocatalysis has emerged as a powerful, sustainable alternative for constructing these bonds with precise stereochemical control. Yet, the enzymatic repertoire for C–N bond formation remains limited compared to its chemical counterpart. By leveraging directed evolution and enzyme promiscuity, we have traversed beyond natural enzymatic functions and explored the new-to-nature catalytic landscape of enzymes.

This thesis outlines a suite of strategies that expand the enzymatic toolbox for stereoselective C–N bond construction in two main avenues: the functionalization of more challenging C–H bonds and the development of novel nitrene species. Chapter I reviews contemporary chemical and biocatalytic approaches, highlighting how directed evolution can transcend native enzymatic functions to unlock new reactivities.

Chapter II presents engineered serine-ligated cytochrome P411 variants capable of catalyzing enantioselective propargylic C(sp3)–H amination, granting streamlined access to chiral propargylamines. Chapter III addresses the long-standing challenge of differentiating minimally distinct alkyl groups, methyl and ethyl substituents, by evolving P411 enzymes that perform enantiospecific functionalization of tertiary C–H bonds to construct methyl-ethyl stereocenters with high selectivity.

Chapter IV expands the scope of enzymatic nitrene transfer, enabling intramolecular alkyl and aryl nitrene C–H insertions to synthesize chiral pyrrolidines and methyl indolines. Subsequent biocatalytic derivatization of these products affords complex molecules bearing multiple chiral centers, showcasing the potential of biocatalysis to rapidly build molecular complexity. Chapter V introduces engineered protoglobins capable of mediating intermolecular methylnitrene transfer using a simple and stable N-methyl hydroxylamine recursor. This process uniquely achieves the stereoconvergent conversion of both (Z)- and (E)-silyl enol ether isomers, affording enantiopure N-methyl-a-aminoketones, which defies conventional limitations of enzyme specificity.

Together, these efforts address critical gaps in biocatalytic C–N bond formation, establishing broadly applicable platforms for sustainable and stereoselective synthesis of N-containing molecules. The strategies developed herein not only deepen our understanding of heme-dependent enzymes but also lay the foundation for future innovations in biocatalysis and synthetic biology.