Solution and Fluorescence Properties of Symmetric Dipicolylamine-Containing Dichlorofluorescein-Based Zn²⁺ Sensors

Abstract

The mechanism by which dipicolylamine (DPA) chelate-appended fluorophores respond to zinc was investigated by the synthesis and study of five new analogs of the 2′,7′-dichlorofluorescein-based Zn 2+ sensor Zinpyr-1 (ZP1). With the use of absorption and emission spectroscopy in combination with potentiometric titrations, a detailed molecular picture has emerged of the Zn 2+ and H + binding properties of the ZP1 family of sensors. The two separate N 3 O donor atom sets on ZP1 each converge to form binding pockets in which all four heteroatoms participate in coordination to either Zn 2+ or protons. The position of the pyridyl group nitrogen atom, 2-pyridyl or 4-pyridyl, has a large impact on the fluorescence response of the dyes to protons despite relatively small changes in pK a values. The fluorescence quenching effects of such multifunctional electron-donating units are often taken as a whole. Despite the structural complexity of ZP1, however, we provide evidence that the pyridyl arms of the DPA appendages participate in the quenching process, in addition to the contribution from the tertiary nitrogen amine atom. Potentiometric titrations reveal ZP1 dissociation constants (K d ) for Zn 2+ of 0.04 pM and 1.2 nM for binding to the first and second binding pockets of the ligand, respectively, the second of which correlates with the value observed by fluorescence titration. This result demonstrates that both binding pockets of this symmetric, ditopic sensor need to be occupied in order for full fluorescence turn-on to be achieved. These results have significant implications for the design and implementation of fluorescent sensors for studies of mobile zinc ions in biology.

Two-photon imaging of Zn 2+ dynamics in mossy fiber boutons of adult hippocampal slices

“…The binding affinities associated with the first observable Zn 2+ -binding event are also identical within the limits of error of each other. The fluorescence responses likely correspond to the formation of 2:1 Zn 2+ -ligand complexes, given that metallation of both metal-binding domains was required for the emissive response in previously described ditopic Zn 2+ sensors (31,32). In these systems, the 1:1 Zn 2+ -ligand complex was too spectroscopically similar to the metal-free probe to allow observation by a simple fluorimetric titration.…”

“…The metal-ligand stoichiometry of Zn A -ZAP4 and Zn B -ZAP4 cannot be assigned with certainty, because these species form only when a large excess of Zn 2+ is present. Owing to its strong resemblance to ZP1 and other ditopic Zn 2+ -responsive probes, we speculate that the Zn A -ZAP4 species is most likely a 2:1 Zn 2+ -ligand complex, the formation of which is preceded by that of a 1:1 complex that is spectroscopically similar to metal-free ZAP4 (31,32). The absorption intensity of the Zn B -ZAP4 species was greater than that of either ZAP4 or Zn A -ZAP4; it is possible that additional equivalents of Zn 2+ increase absorption by triggering the dissociation of ZAP4 aggregates.…”

“…The presence of carboxylic acid on the bottom ring does not appear to affect the synthetic reactions, for the yields of the probes are both ∼60%. The isolation requires no chromatography, and ZAP4 can be crystallized from MeOH and its X-ray structure has been determined, making it the fourth metal-free Zinpyr probe to be structurally characterized to date (31). As with the previously crystallized probes and the Zn 2 -ZP1 complex, the bottom carboxylate is locked into the lactone isomer (11,31).…”

“…The isolation requires no chromatography, and ZAP4 can be crystallized from MeOH and its X-ray structure has been determined, making it the fourth metal-free Zinpyr probe to be structurally characterized to date (31). As with the previously crystallized probes and the Zn 2 -ZP1 complex, the bottom carboxylate is locked into the lactone isomer (11,31). Whether this isomer is the predominant species in solution or whether the lactone formation is a prerequisite for crystal growth is unclear.…”

Cell-Trappable Quinoline-Derivatized Fluoresceins for Selective and Reversible Biological Zn(II) Detection

“…1, QZ2, 25 displays particularly interesting sensing characteristics by comparison to the first generation Zinpyr (or ZP) family of probes. 22,23,[28][29][30][31][35][36][37][38][39] For example, QZ2 has a much lower affinity for Zn(II) (K d1 ~ mid-micromolar) than ZP1 (K d1 ~ picomolar), and can therefore detect higher concentrations of mobile Zn(II) reversibly and without probe saturation.…”