QZ1 and QZ2:  Rapid, Reversible Quinoline-Derivatized Fluoresceins for Sensing Biological Zn(II)

Abstract

QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with 42-and 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K d values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k on values of (3-4) × 10 6 M -1 s -1 , compared to (6-8) × 10 5 M -1 s -1 for select ZP (Zinpyr) dyes, at 4.3 °C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k off values of 150 and 160 s -1 , over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn (II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo. (S5-S9); plots of k obs versus [Zn(II)] (S10-S14); Eyring plots (S15-S19); relative fluorescence emission of the Zn(II)-bound sensors (S20); and both confocal and two-photon microscopy images of biological samples (S21-S23); and experimental details for the representative kinetic analyses. This material is available free of charge via the Internet at

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

“…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 K d values for the 2:1 Zn 2+ -ligand complexes of ZAP4 (23 μM) and 6-CO 2 H-ZAP4 (22 μM) are similar in magnitude to those measured for the QZ family (18).…”

“…The equilibrium constant measured from the forward and backward rates agrees well with that obtained from singular value decomposition analysis of the titrations. The rate constant associated with the dissociation is greater than those measured for ZP1 probes, but less than those measured for QZ probes, as is the rate of Zn 2+ release (18).…”

“…The rapid metal ion release renders the probe less susceptible to metal contamination, much like the QZ and Zinspy probes (18,19) and, to a lesser extent, the Me 4 ZP1 probe (15). The ZAP4 sensor is capable of detecting Zn 2+ in the presence of an equimolar concentration of Fe 2+ .…”

Tracing of intracellular zinc(II) fluorescence flux to monitor cell apoptosis by using FluoZin‐3AM

“…It has been reported that some zinc(II) probes such as ZS5 and QZ1 respond to exogenously added zinc(II) more quickly in neurons than in other normal cell types, such as HeLa and COS-7. 23 Less than 1 min of incubation was sufficient to observe full fluorescence enhancement in hippocampal neurons following treatment with 50 mM zinc(II)/pyrithione, whereas 5 min was required to observe complete turn-on in HeLa cells. 24 Therefore, we questioned whether FluoZin-3AM would respond to intracellular zinc(II) on a faster time scale, a feature that is necessary for monitoring biological zinc(II) flux, in normal cell type.…”

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

“…23,27,33,34 Symmetrical sensors tend to have lower extinction coefficients than their asymmetrical counterparts, thereby lowering fluorescence emission in the metal-free state and increasing the effective dynamic range. 23,25 One of the symmetrical probes illustrated in Fig. 1, QZ2, 25 displays particularly interesting sensing characteristics by comparison to the first generation Zinpyr (or ZP) family of probes.…”