Engineering Heme Proteins for C(sp³)–H Primary Amination

Citation

Gao, Shilong (2024) Engineering Heme Proteins for C(sp³)–H Primary Amination. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gsmr-b827. https://resolver.caltech.edu/CaltechTHESIS:08242023-170135027

Abstract

Primary amine is one of the most prevalent moieties in synthetic intermediates and pharmaceutical compounds. The preparation of aliphatic primary amines via C−H functionalization would provide direct access to the nitrogen-containing compounds from hydrocarbon substrates. While the enzymatic oxyfunctionalization of C–H bonds is well established, the analogous strategy for nitrogen incorporation is unknown in Nature. Likewise, a synthetic method for selective primary amination of aliphatic C–H bonds remains elusive. Combining chemical intuition and inspiration from Nature, chemists and protein engineers have created new heme-containing enzymes for the C(sp³)–H primary amination via directed evolution. This thesis describes some of the efforts in the continued pursuit of these new-to-nature reactions. Chapter I discusses directed evolution in the context of biocatalysis, the strategies for introducing new-to-nature chemistry in enzymes, the discovery of nitrene transferases from the cytochrome P450 monooxygenase, and finally, the development of C(sp³)–H primary aminases. Chapter II details the discovery and engineering of serine-ligated cytochrome P411 enzymes that catalyze the first primary amination of C(sp³)–H bonds with excellent selectivity, affording a broad scope of enantioenriched primary amines. Chapter III demonstrates that these new-to-nature nitrene transferases were engineered to aminate and amidate unactivated, unbiased C(sp³)–H bonds with unprecedented selectivity. In Chapter IV, engineered protoglobins are shown to utilize hydroxylamine (NH₂OH) for nitrene transfer reactions, including benzylic C–H primary amination and styrene aminohydroxylation. Overall, these new-to-nature reactions can be considered the nitrogen analogs to the C–H oxidation chemistry performed by monooxygenases and peroxygenases. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds. Finally, this work hints at the possible future discovery of natural enzymes that use hydroxylamine precursors for amination chemistry.

Thesis Files