Highly Potent MRI Contrast Agent Displaying Outstanding Sensitivity to Zinc Ions

Abstract Zinc ions play an important role in numerous crucial biological processes in the human body. The ability to image the function of Zn2+ would be a significant asset to biomedical research for monitoring various physiopathologies dependent on its fate. To this end, we developed a novel Gd3+ chelate that can selectively recognize Zn2+ over other abundant endogenous metal ions and alter its paramagnetic properties. More specifically, this lanthanide chelate displayed an extraordinary increase in longitudinal relaxivity (r 1) of over 400 % upon interaction with Zn2+ at 7 T and 25 °C, which is the greatest r 1 enhancement observed for any of the metal ion‐responsive Gd‐based complexes at high magnetic field. A “turn‐on” mechanism responsible for these massive changes was confirmed through NMR and luminescence lifetime studies on a 13C‐labeled Eu3+ analogue. This molecular platform represents a new momentum in developing highly suitable magnetic resonance imaging contrast agents for functional molecular imaging studies of Zn2+.

Compound 2 (0.275 g, 0.24 mmol) was dissolved in TFA/CH 2 Cl 2 (2 mL, v/v 80/20) and the solution was stirred for 6 hours at room temperature. The reaction mixture was evaporated to dryness and purified by HPLC using MeCN/H 2 O as the eluent, affording 0.182 g (76%) of compound L as a light yellow oil. 1

1,2-13 C-tert-Butyl bromoacetate (3)
Concentrated H 2 SO 4 (0.19 mL) was added slowly to a vigorously stirred suspension of MgSO 4 (1.300 g, 10.80 mmol) in CH 2 Cl 2 (7 mL) The mixture was stirred for 20 minutes, after which the isotopically labeled 1,2-13 C-bromoacetic acid (0.507 g, 3.60 mmol) was added, followed by addition of tBuOH (1.5 mL). The mixture was stirred for 15 h at room temperature. The insoluble matter was removed by vacuum filtration. The filtrate was transferred to a separatory funnel and washed with water (10 mL) and saturated sodium bicarbonate (10 mL). The aqueous layer was extracted with CH 2 Cl 2 (3× 3 mL). The combined organic layers were washed with brine and dried over anhydrous MgSO 4 . The solvent was evaporated under reduced pressure to afford 3 as a light-yellow liquid (0.341 g, 48%).

NMR measurements
Relaxometric titrations: Proton longitudinal relaxometric titrations with Zn 2+ were performed at 7 T, pH 7.4 (50 mM HEPES buffer) and 25 °C, using using inversion recovery (T 1 ) and Car-Purcell-Meiboom-Gill (T 2 ) pulse sequences. A ZnCl 2 solution of known concentration was added stepwise to the GdL solution (starting concentration 1.0 or 3.0 mM Gd 3+ ), and measurements of T i (i=1, 2) were performed after each addition of the analyte. The longitudinal and transverse relaxivities were calculated from Eq. S1 where T i,obs is the measured T i , T i,d is the diamagnetic contribution of the solvent, and [Gd] is the actual Gd 3+ concentration at each point of the titration.

ITC experiments
The experiments were carried out by placing GdL (50 μM) in the reaction cell and ZnCl 2 (300 μM) in the syringe. All data were recorded in HEPES or PBS (50 mM) at pH 7.4.

Luminescence lifetime experiments
Luminescence lifetime measurements were performed with TbL (1 mM) or EuL* (5 mM) in D 2 O and H 2 O with and without Zn 2+ (2 equiv.) at 25 °C (50 mM HEPES, pH 7.4). The Ln 3+ ion was directly excited (λ ex = 283 and 395 nm for Tb 3+ and Eu 3+ , respectively) and the emission intensity (λ max = 546 and 615 nm for Tb 3+ and Eu 3+ , respectively) was recorded. The excitation and emission slits were set at 10 nm. In total, three independent measurements each with 15 scans were performed to obtain the data set. The obtained curves were fitted with a first order exponential decay with an r 2 > 0.99. The resulting q value, which denotes the hydration number of coordinated water molecules, was then calculated using the equation Eq. S2 for EuL* and Eq. S3 for TbL. [3] (Eu) = 1.