Harnessing Enzymatic Diazotization for Chemoenzymatic N–N Bond Formation and Diversification

Diazo compounds are highly versatile intermediates in synthetic chemistry, yet their selective biocatalytic formation remains rare. In nature, diazotases catalyze the ATP-dependent formation of diazo functionalities in the biosynthesis of natural products. However, the broader synthetic potential and substrate scope of these enzymes remains unexplored. Here we demonstrate that the ATP-dependent diazotase CmaA6 can be utilized beyond its native biosynthetic context as a platform for enzymatic diazotization of diverse aromatic amines. CmaA6 exhibits broad promiscuity toward 42 electronically and sterically diverse aromatic amines, enabling the generation of reactive diazo/diazonium intermediates under mild aqueous conditions. These enzymatically-generated intermediates were captured through Japp–Klingemann coupling to afford 34 structurally diverse hydrazone products. Diazenylation with active methylene compounds afforded six additional products, including the preparative-scale synthesis of a hydrazone derivative in 45 four diazonium intermediates underwent azo-coupling, thereby further increasing the accessible product scope from the CmaA6catalyzed diazotization. This work establishes diazotases as versatile tools for selective N–N bond formation and downstream chemical diversification, opening new opportunities for integrating enzymatic diazotization into sustainable cascade processes.