ABSTRACT Natural isonitriles are promising lead compounds for medicinal chemistry. Their identification is, however, challenging due to the hydrolytic lability of the isonitrile group. Here, we use the azomethine imine (AMI)–isonitrile ligation in a reactivity‐based screening protocol for the chemoselective derivatization of natural isonitriles. The herein developed AMI probe rapidly reacts with isonitriles—primary, secondary, tertiary, as well as aromatic—to stable conjugates. A dibromide mass tag enables the detection of low‐abundance isonitriles, even in complex biological matrices. The ligation establishes a new stereogenic center, thereby allowing facile distinction between achiral and chiral isonitriles by the formation of racemates or diastereoisomers, respectively. In addition, a unique reactivity of isonitriles bearing an α‐COOH group was unraveled. This AMI probe enabled the detection of known bacterially produced isonitriles and the identification of a novel fungal isonitrile.
Biedermann et al. (2026) studied this question.