Nonmacrocyclic Luminescent Lanthanide Complexes Stable in Biological Media

The synthesis of ligand LpH(8), based on a 2,6-bispyrazolyl-pyridine scaffold functionalized by iminobismethylene-phosphonate functions, is described and its pK values were determined by a combination of pH-spectrophotometric titrations and potentiometry. The interaction of L-p with Tb3+ was investigated in water (0.01 M TRIS/HCl pH = 7.0) by means of UV-vis and fluorescence titration experiments and evidenced the formation of at least three species with 1:1; 1:2, and 2:1 M-L ratios, the 1:1 complex appearing as particularly stable under these conditions (log K-cond > 8). Na-4[LnL(p)H] complexes (Ln = Eu and Tb) were prepared and characterized by elemental analysis, IR spectroscopy, and electrospray mass spectrometry. Their photophysical properties were investigated in aqueous solutions, revealing an excellent shielding of the Ln cations from the solvent environment (no water molecules in the first coordination sphere), very long luminescence lifetimes (tau(H2O) = 1.50 and 3.28 ms, respectively, for Eu and Tb) and reasonable luminescent quantum yields (phi(H2O) = 2.4 and 37%, respectively, for Eu and Tb). Using fetal bovine serum as a model for biological media showed the Tb complex to remain luminescent in these conditions. The structure of the europium complex was studied by means of density functional theory (DFT) modeling, confirming the wrapping of the ligand around the cation, and the very good shielding of the coordinated Ln cation. The conditional stability constant for the formation of the Tb complex with L-p was determined by competition experiments with EDTA and monitored by fluorescence spectroscopy (log KT(bLp)cond = 14.1 +/- 0.3, 0.01 M TRIS/HCl, pH = 7.4) and was used to determine the thermodynamic constant (log K-TbLp = 20.4 +/- 0.4). A systematic comparison with ligand L-c, in which phosphonate functions are replaced by carboxylate ones, is made throughout the study, highlighting the large interest of the introduction of phosphonate moieties to obtain biologically stable luminescent lanthanide complexes.