Selective Two‐Step Labeling of Proteins with an Off/On Fluorescent Probe

We present a novel design strategy for off/on fluorescent probes suitable for selective two‐step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1 –Ni 2+ , which targets a cysteine‐modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)‐Ni 2+ as the His‐tag recognition site and a 2,4‐dinitrophenyl ether moiety, which quenches the probe’s fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4‐dinitrophenyl ether (donor‐excited PeT; d‐PeT) and also has reactivity with cysteine. His‐tag recognition by the NTA–Ni 2+ moiety is followed by removal of the 2,4‐dinitrophenyl ether quencher by proximity‐enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His‐tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20‐fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light‐excitable fluorescent probe for a His‐based peptide tag. Further, we successfully visualized CysHis 6 ‐peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His 6 Cys‐tagged enhanced blue fluorescent protein (EBFP), but not His 6 ‐tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.