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Published March 29, 2023 | Supplemental Material + Submitted
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Enzymatic Nitrogen Insertion into Unactivated C–H Bonds


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 pre-oxidized 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 towards 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.

Additional Information

The content is available under CC BY NC ND 4.0 License. We thank Dr. Zhen Liu for assistance with CalB reactions, Dr. Michael K. Takashe for assistance with collecting X-ray single crystal data, Ziyang Qin for assistance with synthesis and Dr. Mona Shahgholi for HRMS analysis. This research is supported by the Department of Energy (DOE250078) 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 number ACI-1548562, CHE180061 and CHE210031) (J.S.H. and M.S.C.) and the National Institutes of Health under R15 GM142103 (J.S.H.) is acknowledged.

Attached Files

Submitted - enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf

Supplemental Material - si-enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf



Additional details

August 20, 2023
August 20, 2023