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


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


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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biocatalysis;C-H Functionalization;Nitrene Chemistry;Protein Engineering
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Stoltz, Brian M. (chair)
  • Hadt, Ryan G.
  • Gray, Harry B.
  • Arnold, Frances Hamilton
Defense Date:21 August 2023
Funding AgencyGrant Number
Joseph J. Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Department of Energy (DOE)DE-SC0021141
G. Harold & Leila Y. Mathers FoundationMF-2111-02065
Projects:Enzymatic primary amination of benzylic and allylic C(sp³)–H bonds. J. Am. Chem. Soc. 2020, 142(23), 10279–10283, Enzymatic nitrogen insertion into unactivated C–H bonds. J. Am. Chem. Soc. 2022, 144(41), 19097–19105, Enzymatic Nitrogen Incorporation Using Hydroxylamine.
Record Number:CaltechTHESIS:08242023-170135027
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter II adapted for Chapter III
Gao, Shilong0000-0003-2808-6283
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:16165
Deposited By: Shilong Gao
Deposited On:29 Aug 2023 00:07
Last Modified:08 Nov 2023 00:11

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