Selective
labeling of the protein of interest (POI) in genetically
unmodified live cells is crucial for understanding protein functions
and kinetics in their natural habitat. In particular, spatiotemporally
controlled installation of the labels on a POI under light control
without affecting their original activity is in high demand but is
a tremendous challenge. Here, we describe a novel ligand-directed
photoclick strategy for spatiotemporally controlled labeling of endogenous
proteins in live cells. It was realized with a designer labeling reagent
skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl
and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released
and underwent a proximity coupling reaction with nucleophilic amino
acid residues on the POI in live cells. With fluorescein as a marker,
this photoclick strategy enables time-resolved labeling of carbonic
anhydrase subtypes localized either on the cell membrane or in the
cytoplasm and a discriminable visualization of their metabolic kinetics.
Given the versatility underlined by facilely tethering other functional
entities (e.g., biotin, a peptide short chain) via acylation or (in
cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry
opens up enormous opportunities for discovering dynamic functions
and mechanistic interrogation of endogenous proteins in live cells.