Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume -, Issue -, Pages -Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.2c08285
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Funding
- Department of Energy [DE-SC0021141]
- Ruth Kirschstein NIH Postdoctoral Fellowship [F32GM143799]
- XSEDE Science Gateway Program [ACI-1548562, CHE180061, CHE210031]
- National Institutes of Health [R15 GM142103]
- U.S. Department of Energy (DOE) [DE-SC0021141] Funding Source: U.S. Department of Energy (DOE)
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In this study, 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 evolved enzymes showed high promiscuity and activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects supported a stepwise radical pathway. In-enzyme molecular dynamics simulations revealed a predominantly hydrophobic pocket with favorable interactions with the substrate.
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.
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