Abstract
Fast ion-chelate dissociation rates and weak ion-chelate affinities are desired kinetic and thermodynamic features for imaging probes to allow reversible binding and to prevent deviation from basal ionic levels. Nevertheless, such properties often result in poor readouts upon ion binding, frequently result in low ion specificity, and do not allow the detection of a wide range of concentrations. Herein, we show the design, synthesis, characterization, and implementation of a Zn2+-probe developed for MRI that possesses reversible Zn2+-binding properties with a rapid dissociation rate (koff = 845 ± 35 s–1) for the detection of a wide range of biologically relevant concentrations. Benefiting from the implementation of chemical exchange saturation transfer (CEST), which is here applied in the 19F-MRI framework in an approach termed ion CEST (iCEST), we demonstrate the ability to map labile Zn2+ with spectrally resolved specificity and with no interference from competitive cations. Relying on fast koff rates for enhanced signal amplification, the use of iCEST allowed the designed fluorinated chelate to experience weak Zn2+-binding affinity (Kd at the mM range), but without compromising high cationic specificity, which is demonstrated here for mapping the distribution of labile Zn2+ in the hippocampal tissue of a live mouse. This strategy for accelerating ion-chelate koff rates for the enhancement of MRI signal amplifications without affecting ion specificity could open new avenues for the design of additional probes for other metal ions beyond zinc.
Highlights
IntroductionOf the cations with a biological role, Zn2+ has garnered much interest due to (i) its involvement, as a tightly bound Zn2+, in the determination of the structure and activity of essential proteins[1] and (ii) its role, as mobile Zn2+, in different secretion pathways of specific tissue.[2−5] Labile Zn2+ was found in relatively large pools in the prostate’s epithelial cells,[6] in the pancreatic β-cells,[7] and in the hippocampal mossy fibers,[8] and its distribution in these tissues was characterized using wellestablished Zn2+-specific fluorescent imaging probes.[9,10] Extensively designed, these probes provide diverse affinity capabilities for Zn2+ to cover a wide range of cation concentrations, which varies between sub-nanomolar and millimolar in different tissues.[11−13] Such variability in Zn2+ affinities was obtained through either replacing the commonly used dipicolyl amine amine (DPA)[14] binding motif with other binding moieties (e.g., thioether,[15] pyrrole,[16] thiophen,[17] quinoline,[18] or pyrazine19) or by reducing the rigidity of the Zn2+-sensors.[20]
The chemical shift offset (Δω) between two exchanging pools of spins is at the core of any designed chemical exchange saturation transfer (CEST) agent[49] and for 19F-CEST-based studies.[45,50−54] This is mainly due to the fact that a larger Δω results in a reduced direct saturation effect, allowing the use of strong saturation powers for an enhanced CEST effect
As a first step in our design, three putative fluorinated derivatives of dipicolyl amine amine (DPA) were synthesized based on the common use of a DPA backbone in both fluorescent-7,8,14 and MRI-22,32responsive probes developed for imaging labile Zn2+ under physiological conditions
Summary
Of the cations with a biological role, Zn2+ has garnered much interest due to (i) its involvement, as a tightly bound Zn2+, in the determination of the structure and activity of essential proteins[1] and (ii) its role, as mobile Zn2+, in different secretion pathways of specific tissue.[2−5] Labile Zn2+ was found in relatively large pools in the prostate’s epithelial cells,[6] in the pancreatic β-cells,[7] and in the hippocampal mossy fibers,[8] and its distribution in these tissues was characterized using wellestablished Zn2+-specific fluorescent imaging probes.[9,10] Extensively designed, these probes provide diverse affinity capabilities for Zn2+ to cover a wide range of cation concentrations, which varies between sub-nanomolar and millimolar in different tissues.[11−13] Such variability in Zn2+ affinities was obtained through either replacing the commonly used dipicolyl amine amine (DPA)[14] binding motif with other binding moieties (e.g., thioether,[15] pyrrole,[16] thiophen,[17] quinoline,[18] or pyrazine19) or by reducing the rigidity of the Zn2+-sensors.[20]. The advances in the design and implementation of MRIresponsive sensors have led to the development of sensors for spatially mapping the distribution of metal ions noninvasively from the deep tissues of live subjects,[22−29] overcoming one of the major limitations of fluorescent-based probes. In addition to cell-penetrable formulations designed to image regions of rich pools of labile zinc in the brain of live animals,[22] other formulations were used to map cell-secreted Zn2+ from both pancreatic[23] and prostate[24] tissues upon glucose stimulation, which showed, in real time, longitudinal modulation in the labile Zn2+ pools in live intact subjects
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