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Enzymatic Nitrogen Insertion into Unactivated C-H Bonds

Athavale, Soumitra V. and Gao, Shilong and Das, Anuvab and Chandra Mallojjala, Sharath and Alfonzo, Edwin and Long, Yueming and Hirschi, Jennifer S. and Arnold, Frances H. (2022) Enzymatic Nitrogen Insertion into Unactivated C-H Bonds. Journal of the American Chemical Society, 144 (41). pp. 19097-19105. ISSN 0002-7863. PMCID PMC9612832. doi:10.1021/jacs.2c08285. https://resolver.caltech.edu/CaltechAUTHORS:20221024-125854800.29

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Abstract

Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp³)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.2c08285DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc9612832/PubMed CentralArticle
ORCID:
AuthorORCID
Athavale, Soumitra V.0000-0001-5620-6997
Gao, Shilong0000-0003-2808-6283
Das, Anuvab0000-0002-9344-4414
Chandra Mallojjala, Sharath0000-0003-0446-792X
Alfonzo, Edwin0000-0002-4112-8751
Hirschi, Jennifer S.0000-0002-3470-0561
Arnold, Frances H.0000-0002-4027-364X
Additional Information:This research was supported by the Department of Energy (DE-SC0021141) to F.H.A. and by a Ruth Kirschstein NIH Postdoctoral Fellowship (F32GM143799) to E.A. Financial support from the XSEDE Science Gateway Program (under the NSF Grant Numbers ACI-1548562, CHE180061, and CHE210031) (J.S.H. and S.C.M.) and the National Institutes of Health under R15 GM142103 (J.S.H.) is acknowledged.
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)DE-SC0021141
NIH Postdoctoral FellowshipF32GM143799
NSFOAC-1548562
NSFCHE-180061
NSFCHE-210031
NIHR15 GM142103
Issue or Number:41
PubMed Central ID:PMC9612832
DOI:10.1021/jacs.2c08285
Record Number:CaltechAUTHORS:20221024-125854800.29
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20221024-125854800.29
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:117558
Collection:CaltechAUTHORS
Deposited By: Research Services Depository
Deposited On:01 Nov 2022 17:03
Last Modified:01 Nov 2022 17:03

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