Directed Evolution of an Iron(II)‐ and α‐Ketoglutarate‐Dependent Dioxygenase for Site‐Selective Azidation of Unactivated Aliphatic C−H Bonds**

Abstract

FeII‐ and α‐ketoglutarate‐dependent halogenases and oxygenases can catalyze site‐selective functionalization of C−H bonds via a variety of C−X bond forming reactions, but achieving high chemoselectivity for functionalization using non‐native functional groups remains rare. The current study shows that directed evolution can be used to engineer variants of the dioxygenase SadX that address this challenge. Site‐selective azidation of succinylated amino acids and a succinylated amine was achieved as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C−H functionalization with other non‐native functional groups.

Non-Native Site-Selective Enzyme Catalysis

“…204 Site-selective azidation catalyzed by the engineered Fe(II)/KG dependent oxygenase SadX (Scheme 50) has also been combined with both reduction and click chemistry to enable access to amines and triazoles. 351 Given the importance of halogenation in a wide range of transformations, particularly transition metal catalyzed cross-coupling, it is perhaps not surprising that enzymatic halogenation has been paired with a variety of reactions to enable site-selective formation of carbon-carbon and carbon heteroatom bonds. Early efforts towads this end focused on using wild type FDHs to access natural product derivatives with halide substitution that enabled subsequent Suzuki-Miura coupling to access biaryl derivatives (Scheme 61B).…”

“…243,350 SadX was also used as the starting point in a directed evolution effort to evolve site-and chemo-selective azidation biocatalysts. 351 Over four rounds of evolution, the chemoselectivity increased 7.5-fold over the parent enzyme and total yield of the azidated product increased 6.7-fold. In keeping with the need for precise substrate positioning relative to the reactive iron center, the substrate scope for individual mutants from this lineage was narrow.…”

“…Site-selective azidation by engineered SadX variants. 351 Heme-containing proteins have also been used for site-selective C-H functionalization via C-N bond formation. For example, a BM3 variant (P411BM3-CIS-T438S) containing key mutations that reduce monooxygenation activity (T268A) and alter the primary coordination sphere of the heme cofactor (C400S) displayed robust nitrene transfer activity.…”

Overview of Amino Acid Modifications by Iron- and α-Ketoglutarate-Dependent Enzymes

“…Building upon Boal’s discovery of halogenase activity in SadA D157G, Lewis and co-workers developed a directed evolution approach to evolve a biocatalyst for the non-natural C–H azidation reaction on AA-derived substrates (Figure D) . Prior to this work, Matthews et al demonstrated the possibility of performing C–H azidation and nitration with SyrB2 through the use of the appropriate coordinating anions (i.e., N 3 – or NO 2 – ) in the reaction.…”

Hot off the Press

“…Directed evolution of the iron( ii )- and α-ketoglutarate-dependent dioxygenase SadX has generated a biocatalyst capable of selective C–H functionalisation of amino acids. 29 Several variants were shown to perform regioselective azidation of succinylated amino acids (Scheme 1) and succinylated amines with high conversions.…”