Introducing Azomethine Imines to Chemical Biology: Bioorthogonal Reaction with Isonitriles

Azomethine imines are valuable substrates for chemical synthesis in organic solvents that often require anhydrous conditions. Here, we introduce C,N-cyclic-N′-acyl azomethine imines (AMIs) to bioorthogonal reactions in an aqueous environment. These AMIs are stable under physiological conditions and react rapidly (k2 = 0.1–250 M–1 s–1, depending on pH) and chemoselectively with isonitriles in the presence of biological nucleophiles, including thiols. Live-cell imaging of cell-surface-bound isonitriles underlines the biocompatibility of the AMI–isonitrile ligation, and simultaneous one-pot triple-protein labeling demonstrates its orthogonality to commonly used bioorthogonal reactions, such as the SPAAC and iEDDA ligations.