First, a family of contrast agents composed of (1) a Gd-binding moiety, (2) a zinc complexing unit, and (3) linker was developed. Due to a major health problem leading to severe disabilities of the main applicant, only physical chemistry was performed and the synthesis was achieved by other group members.
Several contrast agents with different Gd-binding unit (different charge and structure), and different zinc-binding moieties were studied. Their affinity for HSA was determined by competition measurements followed by luminescence and relaxometric titrations. These affinities were compared to those of the Zn-binding site alone. We could show that :
(1) the highest affinity site in HSA was site II (vs site I)
(2) in the presence of Zn2+, the affinity for HSA is doubled
(3) Relaxometric and luminescent measurements are consistent
(4) The interaction with HSA occurs through the Zn2+ binding moiety
(5) the presence of the Gd3+ complex is necessary to obtain a difference of affinity whether Zn2+ is present or not.
These structural understanding give new insights into the development of efficient Gd3+ complex binding to HSA.
By modifying the Gd3+-binding unit, we could design a thermodynamically stable and kinetically inert complex that shows no toxicity and could be used in vivo. Interestingly at the imaging field (9.4 T), in vitro measurements show a slight relaxivity decrease upon Zn2+ binding. However, in vivo measurements evidenced a contrast increase in the presence of Zn2+, which could be related to Gd3+ accumulation in Zn2+-rich tissues. This highlights the importance of the biodistribution of such contrast agents.
Further structural changes include the modification of the length of the linker between the Gd3+ binding site and the Zn2+ binding site. By decresing the alkyl chain from 4 to 1 carbon atom, we could design a contrast agent that changes its hydration number upon Zn2+ complexation. Using a combination of NMR, luminescence, potentiometric, and relaxivity experiments, completed with DFT calculations, we demonstrated that incorporating a short linker between the Zn2+ sensing unit and the Gd3+ complex leads to unique behavior of the system in the absence of Zn2+, ie an equilibrium between an « open » bishydrated form of the complex, and a « closed » non-hydrated complex. This equilibrium is governed by the protonation of the tertiary amine of the dipycolylamine moiety. A significant increase in efficacy of the system is observed upon Zn2+ binding, and importantly, the complex is highly selective for Zn2+ relative to other physiological cations. A comprehensive structural study reliably determines the microscopic parameters at the origin of the Zn2+ response, primarily an increase in the number of water molecules directly coordinated to Gd3+ upon Zn2+ binding. Crucially, the system maintains a strong response to Zn2+ binding in the presence of Human Serum Albumin, highlighting its potential for biological applications.
Finally, in a more fundamental study, and in order to diversify the entries for Gd3+ ligand, we investigate the coordination properties of an imidazothiadiazole for Ln3+ ions. The thermodynamic stability of the Gd3+ complex was determined by a combination of potentiometric and photophysical measurements. The kinetic inertness was assessed in highly acidic media. The solution structure of the Ln3+ complex was unambiguously determined by a set of photophysical measurements and 1H, 13C, 89Y NMR data in combination with DFT calculations, which proved coordination of the heterocycle to Ln3+. The ability of the imidazothiadiazole moiety to sensitize Tb3+ luminescence was investigated. Finally, the relaxation properties were investigated by recording 1H nuclear magnetic relaxation dispersion (NMRD) profiles and 17O measurements. The water exchange rate is similar to that of GdDOTA as the less negative charge of the ligand is compensated for by the presence of a bulky heterocycle. Relaxivity is constant over a large range of pH values, demonstrating the favorable properties of the complex for imaging purposes.
