Structural and Thermodynamics Studies on Polyaminophosphonate Ligands for Uranyl Decorporation.

The development of actinide decorporation agents with high complexation affinity, high tissue specificity, and low biological toxicity is of vital importance for the sustained and healthy development of nuclear energy. After accidental actinide intake, sequestration by chelation therapy to reduce acute damage is considered as the most effective method. In this work, a series of bis- and tetra-phosphonated pyridine ligands have been designed, synthesized, and characterized for uranyl (UO22+) decorporation. Owing to the absorption of the ligand and the luminescence of the uranyl ion, UV-vis spectroscopy and time-resolved laser-induced fluorescence spectroscopy (TRLFS) were used to probe in situ complexation and structure variation of the complexes formed by the ligands with uranyl. Density functional theory (DFT) calculations and X-ray absorption fine structure (XAFS) spectroscopy on uranyl-ligand complexes revealed the coordination geometry around the uranyl center at pH 3 and 7.4. High affinity constants (log K ∼17) toward the uranyl ion were determined by displacement titration. A preliminary in vitro chelation study proves that bis-phosphonated pyridine ligands can remove uranium from calmodulin (CaM) at a low dose and in the short term, which supports further uranyl decorporation applications of these ligands.

Is hydroxypyridonate 3,4,3-LI(1,2-HOPO) a good competitor of fetuin for uranyl metabolism?

Uranium is widespread in the environment, resulting both from natural occurrences and anthropogenic activities. Its toxicity is mainly chemical rather than radiological. In the blood it is transported as uranyl UO22+ cation and forms complexes with small ligands like carbonates and with some proteins. From there it reaches the skeleton, its main target organ for accumulation. Fetuin is a serum protein involved in biomineralization processes, and it was demonstrated to be the main UO22+-binder in vitro. Fetuin's life cycle ends in bone. It is thus suspected to be a key protagonist of U accumulation in this organ. Up to now, there has been no effective treatment for the removal of U from the body and studies devoted to the interactions involving chelating agents with both UO22+ and its protein targets are lacking. The present work aims at studying the potential role of 3,4,3-LI(1,2-HOPO) as a promising chelating agent in competition with fetuin. The apparent affinity constant of 3,4,3-LI(1,2-HOPO) was first determined, giving evidence for its very high affinity similar to that of fetuin. Chromatography experiments, aimed at identifying the complexes formed and quantifying their UO22+ content, and spectroscopic structural investigations (XAS) were carried out, demonstrating that 3,4,3-LI(1,2-HOPO) inhibits/limits the formation of fetuin-uranyl complexes under stoichiometric conditions. But surprisingly, possible ternary complexes stable enough to remain present after the chromatographic process were identified under sub-stoichiometric conditions of HOPO versus fetuin. These results contribute to the understanding of the mechanisms accounting for U residual accumulation despite chelation therapy after internal contamination.

Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo.

Osteopontin: a uranium phosphorylated binding-site characterization.

Herein, we describe the structural investigation of one possible uranyl binding site inside a nonstructured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phosphopeptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U LIII -edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO2 (2+) /peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein.