Fast Ion-Chelate Dissociation Rate for In Vivo MRI of Labile Zinc with Frequency-Specific Encodability

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.

images. For the details of X-ray data collection and refinement, see section K.

B. Chemical Synthesis procedure
Compound 1: 6-fluoropicolinaldhyde (166.40 mg, 1.33 mmol) was added to a solution of 2aminoethan-1-ol (32.5 mg, 532.05 μmol) in 1,2-dichloroethane (DCE) (8 mL) and stirred at room temperature for 30 min followed by the addition of sodium triacetoxyborohydride (338 mg, 1.59 mmol). The reaction mixture was then stirred at room temperature for an additional 1 h before being heated to 80 o C for 12 h.
Completion of the reaction was confirmed by thin layer chromatography (TLC) and the solvent was evaporated under reduced pressure. The crude product was dissolved in CHCl3, washed with a saturated solution of NaHCO3 and extracted with CHCl3 (2´20 mL), then the combined organic phase was dried over Na2SO4.
Following filtration, the organic phase was concentrated and purified using silica
Completion of the reaction was confirmed by TLC and the solvent was evaporated.
The crude product was dissolved in CHCl3, washed with a saturated solution of NaHCO3 and extracted with CHCl3 (2´20 mL), and the combined organic phase was dried over Na2SO4. Following filtration, the organic phase was concentrated and purified using silica gel column chromatography (

D. Bloch-McConnell data fitting
To estimate the exchange rate (kex) between Zn 2+ -bound and free 19 F-chelate, multi B1 CEST experiments were perfumed with saturation powers ranging from 5 Hz to 150 Hz. The obtained data (z-spectra) were fitted using the Bloch-McConnell

E. Dissociation constant (Kd) measurements by 19 F NMR
Since the exchange between Zn 2+ -bound and free 5 is slow in the NMR timescale, the Kd values can be evaluated from the ratio between the integrals of the two NMR signals obtained for the free and bound forms ( Figure S2). Thus, the dissociation  Table S1 and representative example in Figure S2) were recorded. Then, the 19 F-NMR signals of Zn 2+ -bound ( • !" ) S9 and free 5 were integrated and the obtained values were used for Kd calculations.
In order to measure a true equilibrium constant, the concentrations were varied in order to obtain 20-80% of the bound form to determine the Kd value (Kd=5.5±0.6 ´10 -3 M). See, Table S1 and Figure S2. After 14 days, a single crystal suitable for X-ray crystallography analysis was obtained.
Compound 5 + Zn Complex: Both Compound 5 (5 mg) and Zn(ClO4)2 . 6H2O (6 mg) were dissolved in 50 µL MeOH separately. The obtained solutions were sonicated until they became clear, mixed at room temperature, and left for slow evaporation.
After five days, a single crystal suitable for X-ray crystallography analysis was obtained.

G. Cell Viability
Cell Titer-Blue assay: Chinese hamster ovary (CHO) cells (12 x 10 3 cells/mL) were cultured in 96-well microplates with Dulbecco's Modified Eagle Medium (DMEM) for 16 hr at 37 o C and 5% CO2. Then, cells were washed with fresh cell medium S10 and treated with Compound 5, which was dissolved in PBS over a range of concentrations (as noted in the graph). After 3.5 hr of incubation, the incubating medium was removed and cells were washed with PBS. Then, fresh DMEM medium (80 µL) was added and followed by the addition of a 20 µL of CellTiter-Blue® reagent (Promega) after which the fluorescence of each well (λex/λem of 573nm/584nm) was recorded using a dedicated plate reader. For each concentration of compound 5, the average fluorescence value was calculated from six biological replicates. Cells treated with 50% DMSO were used as a positive control.

H. MRI experiments Phantom Studies:
Phantom experiments were performed on a 9.4 T wide-bore MR scanner (Bruker matrix size=32 × 32; NA=400; and a saturation pulse B1= 2 µT for 1.5 s. Four data sets were acquired with the parameters mentioned above with two at which B1 was applied "on resonance" (Δω =+3.2 ppm) and another two at which B1 was applied "off resonance" (Δω =-3.2 ppm). These data sets were acquired in alternate order and were averaged out separately. To obtain the 19 F-iCEST MRI contrast, averaged "on resonance" images were subtracted from averaged "off resonance" images. One set of experiments was applied on a group of mice (N=7) implanted S12 with a cannula at the CA3 region where Δω was applied at either +18 ppm or -18 ppm and that data was processed in a similar way to the described above.