Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

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.

Mechanistic Insights into the Ene-Reductase-Catalyzed Promiscuous Reduction of Oximes to Amines

“…Promiscuous and new-to-nature enzymatic activities recently obtained increased attention since they allow expansion of the repertoire of biocatalytic transformations. − Although wild-type microorganisms and cell preparations have been reported to transform oximes to the corresponding carbonyl compounds, − pyrazines, or hydroxylamines/amines, − the reduction of an oxime moiety using a defined enzyme has only been reported very recently as a promiscuous activity . Thereby, α-oximo β-keto esters 1 were reduced by ene-reductases from the old yellow enzyme family (EREDs − ) to the presumed corresponding α-amino intermediate 2 (Scheme ), which spontaneously dimerized and oxidized, leading finally to the pyrazine product 3 .…”

MicroED structure of a protoglobin reactive carbene intermediate

“…[5] Heme enzymes are particularly interesting due to their ability to form and transfer reactive carbene and nitrene intermediates to effect transformations not known in biology, and sometimes not even known in chemical catalysis. [6][7][8] Although many new-to-nature heme enzymes have been described, with a wide diversity in their synthetic products, the structural rationale behind these advancements is still missing. Describing the short-lived reactive intermediates of these reactions is of great interest for development of future biocatalysts, but this has proven very challenging.…”

Nitrene transfer reaction with hydroxylamine derivatives

“…95 More importantly, directed evolution of nitrene transferases has unveiled enzymes that can selectively introduce nitrogen at unactivated Csp 3 -H bonds under mild conditions (Scheme 47d). 96…”