Excited-State Intramolecular Proton Transfer Enables Self-Reporting Fluorescent Photoaffinity Labeling in Live Cells

Photoaffinity labeling (PAL) has long served as the central strategy for capturing transient biomolecular interactions in living cells. However, conventional photo-cross-linking tags rely on highly reactive intermediates that react indiscriminately with solvents and generate heterogeneous cross-linked products. Here, we report an excited-state intramolecular proton transfer (ESIPT) chemistry that addresses these limitations within a single scaffold. Upon photoactivation, 2-(2'-hydroxyphenyl)benzimidazole (HBI) undergoes picosecond enol-to-keto tautomerization to deliver a transient Michael-acceptor electrophile that reversibly returns to the ground state in the absence of a proximal nucleophile, intrinsically preserving spatial fidelity without solvent quenching. A systematic heptapeptide (X-LHAPTD) scan establishes chemoselective cysteine capture, while the bright keto-band emission of HBI reports the labeling event in situ. We validate the platform across two case studies: proximity-directed capture of a formylglycine-generating enzyme (FGE)/substrate pair, and de novo interactome profiling of aggregation-prone tau K18-P301L in HEK-Tau-BiFC reporter cells, which revealed eight cellular effectors of tau transmission, including four newly identified ones. ESIPT photochemistry thereby provides a minimally perturbing, self-reporting PAL platform for mapping intracellular interaction networks in their native environment.