Design and Synthesis of Fluorescent Probe for Polyhistidine Tag Using Macrocyclic Nickel(II) Complex and Fluorescein Conjugate

We report a newly designed polyhistidine tag (His-tag) targeting fluorescent probe, NiL degrees DCF, which we synthesized based on the fluorophore displacement mechanism. A macrocyclic nickel(II) complex (NiL degrees) was employed as a novel binding site for a His-tag motif, and we chose dichlorofluorescein (DCF) (=2',7'-dichloro-3',6'-dihydroxyspiro[iso-benzofuran-1(3H),9'-(9H)xanthen]-3-one) as the fluorophore. A hypochromic shift of NiL degrees DCF from the metal-unbound form (L degrees DCF) in the absorption spectrum suggested that the phenolic oxygen atom of DCF interacted directly with the NiL complex, resulting in efficient fluorescence quenching (Phi = 0.084) in a neutral aqueous solution. When a model peptide having a hexahistidine sequence (H6Y1: YHHHHHH) was added to the solution of NiL degrees DCF, a significant fluorescence enhancement in the emission (Phi = 0.60) was observed. The stoichiometry of the ternary complex between NiL degrees DCF and H6YI was 1:1. The fluorescence intensity increased as the concentration of H6Y1 increased, and the dissociation constant (K-d) was determined to be 24 +/- 1 mu M, consistent with that for the Ni-NTA complex and His(6)-fused proteins (K-d = 1-20 mu M). These results indicate that macrocyclic NiL degrees can serve as a novel binding site for the polyhistidine sequence and that NiL degrees DCF would be applicable to a switchable fluorescent probe for such His-tagged proteins.