Reinvestigation of 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 so-called "N-end rule" that is N-terminal residue controlling for protein stability. To visualize or measure the cellular stability of a protein depending on the N-terminal residues, it is getting attention to develop selective labeling methods for individual N-terminal amino acid. However, there are only a few limited functional groups available for specific N-terminal amino acid labeling in a biological environment. Here, we report re-examination of salicylaldehyde ester for selective N-terminal residue tagging. Salicylaldehyde ester has been utilized for chemical ligation to N-terminal serine or threonine in pyridine-acetic acid condition. Inspired by previous selective serine/threonine labeling, we examined N-terminus labeling of salicylaldehyde ester in aqueous buffer condition using BODIPY, rhodamine, and coumarin probes. Surprisingly, the selectivity was not only significantly differ depending on the fluorophore incorporated in salicylaldehyde, but also perturbed by the addition of a small fraction of PBS buffer solution. In particular, coumarin-based salicylaldehyde ester probe showed notable selectivity against N-terminal cysteine in aqueous buffer condition. This result presents the serendipitous discovery of a new N-terminal cysteine labeling strategy.