Design and Synthesis of Fluorescent Probe for Polyhistidine Tag Using Macrocyclic Nickel(II) Complex and Fluorescein Conjugate
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We report a newly designed polyhistidine tag (His-tag) targeting fluorescent probe, NiL<sup>O</sup>DCF, which we synthesized based on the fluorophore displacement mechanism. A macrocyclic nickel(II) complex (NiL<sup>O</sup>) was employed as a novel binding site for a His-tag motif, and we chose dichlorofluorescein (DCF) (=2′,7′-dichloro-3′,6′-dihydroxyspiro[isobenzofuran-1(3<i>H</i>),9′-(9<i>H</i>)xanthen]-3-one) as the fluorophore. A hypochromic shift of NiL<sup>O</sup>DCF from the metal-unbound form (L<sup>O</sup>DCF) in the absorption spectrum suggested that the phenolic oxygen atom of DCF interacted directly with the NiL<sup>O</sup> complex, resulting in efficient fluorescence quenching (Φ = 0.084) in a neutral aqueous solution. When a model peptide having a hexahistidine sequence (H6Y1: YHHHHHH) was added to the solution of NiL<sup>O</sup>DCF, a significant fluorescence enhancement in the emission (Φ = 0.60) was observed. The stoichiometry of the ternary complex between NiL<sup>O</sup>DCF and H6Y1 was 1:1. The fluorescence intensity increased as the concentration of H6Y1 increased, and the dissociation constant (<i>K</i><sub>d</sub>) was determined to be 24 ± 1 µM, consistent with that for the Ni–NTA complex and His<sub>6</sub>-fused proteins (<i>K</i><sub>d</sub> = 1–20 µM). These results indicate that macrocyclic NiL<sup>O</sup> can serve as a novel binding site for the polyhistidine sequence and that NiL<sup>O</sup>DCF would be applicable to a switchable fluorescent probe for such His-tagged proteins.
- Bulletin of the Chemical Society of Japan
Bulletin of the Chemical Society of Japan 84(4), 386-394, 2011
The Chemical Society of Japan