Reactivity-Guided Detection Strategies for Isocyanide Natural Products Reveal Acid-Induced Hazimycin Artifacts

Isocyanides are unusual functional groups with important roles in synthetic chemistry, chemical biology, and natural product bioactivity, but their detection in complex biological samples remains challenging. Here, we developed complementary HPLC-MS- and fluorescence-based strategies for identifying isocyanide-containing compounds. Label-free MS/MS analysis revealed diagnostic features for selected isocyanides, including [CN]- reporter ions and HCN neutral losses, while deliberate mild acid treatment converted isocyanides to formamides that could be detected through [CH2NO]- reporter ions and characteristic neutral losses. To overcome the limitations of these compound-dependent fragmentation patterns, we established additional workflows based on degradation-guided molecular networking, offline tetrazine labeling, fluorogenic tetrazine-coumarin probes, and postcolumn derivatization. Clofentezine labeling generated diagnostic dichlorinated products, while fluorogenic probes enabled simple visual detection under UV light. Postcolumn derivatization proved particularly useful, as tetrazine infusion generated coeluting isocyanide-tetrazine product pairs and copper infusion exploited the chalkophore properties of isocyanides to produce diagnostic copper complexes with characteristic mass shifts and isotope patterns. Finally, degradation studies of hazimycin revealed acid-induced rearrangement and deformylation chemistry beyond simple isocyanide hydration, leading to the reassignment of previously reported hazimycin derivatives as likely isolation artifacts. Together, this work provides a practical toolbox for isocyanide detection and highlights the need to avoid acidic conditions during their isolation and analysis.