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Schematic representation of in situ probe formation with a pentafluorophenyl (PFP) ester-functionalized trifunctional reagent (43). An amine-containing ligand reacts with the PFP ester to introduce a cysteine reactive chloroacetamide and an alkyne bioorthogonal tag. The resulting probe is used without further purification. Probe labeling is visualized via a copper-catalyzed click reaction with a reporter group.
