Reinvestigation of an O ‐Salicylaldehyde Ester Functional Group in Aqueous Buffer and Discovery of a Coumarin Scaffold Probe for Selective N‐Terminal Cysteine Labeling

Many intracellular proteins are metabolically unstable, and their half‐life was known to be controlled by the “N‐end rule,” that is, the N‐terminal residue controlled protein stability. To visualize or measure the cellular stability of a protein, depending on the N‐terminal residues, attention is being paid to the development of selective labeling methods for individual N‐terminal amino acids. However, there are only a limited number of functional groups available for specific N‐terminal amino acid labeling in a biological environment. Herein, we report a re‐examination of salicylaldehyde ester for selective N‐terminal residue tagging. Salicylaldehyde ester has been used for chemical ligation to N‐terminal serine or threonine under pyridine/acetic acid conditions. Inspired by previous selective serine/threonine labeling, N‐terminal labeling of salicylaldehyde ester in aqueous buffer has been examined by using boron‐dipyrromethene (BODIPY), rhodamine, and coumarin probes. Surprisingly, the selectivity not only significantly differed, depending on the fluorophore incorporated in salicylaldehyde, but was also perturbed by the addition of a small fraction of phosphate‐buffered saline. In particular, the coumarin‐based salicylaldehyde ester probe showed notable selectivity against N‐terminal cysteine under aqueous buffer conditions. This result reveals the serendipitous discovery of a new N‐terminal cysteine labeling strategy.