Angew Chem Int Ed Engl. 2026 Jun 9:e2116720. doi: 10.1002/anie.2116720. Online ahead of print.

ABSTRACT

Efficient integration of non-natural catalytic motifs into protein scaffolds remains a central challenge in artificial enzyme design. Here we report aniline adenine dinucleotide (AnAD), a NAD⁺-type synthetic cofactor in which the native nicotinamide is replaced by a catalytically active aniline unit. When incorporated into diverse NAD(P)⁺-binding proteins, AnAD introduces an iminium catalysis mechanism that enables tandem Friedel-Crafts alkylation-enantioselective protonation reactions. Screening a panel of natural protein scaffolds revealed broad intrinsic compatibility, while protein engineering further enhanced activity and stereoselectivity, affording artificial enzymes with good substrate generality and tunable enantioselectivity. Mechanistic studies showed that cooperation between AnAD and protein microenvironments, particularly dynamic active-site loop motions, governs both reactivity and enantioselectivity. These results demonstrate the generality and robustness of reprogramming ubiquitous NAD(P)⁺-binding proteins for new-to-nature biotransformations using NAD⁺-type synthetic cofactors.

PMID:42261918 | DOI:10.1002/anie.2116720