Spectroscopic analysis of ligand binding to lanthanide-macrocycle platforms

A high-affinity, binary Eu3+ receptor site consisting of 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) was constructed with the goal of improving the detection of dipicolinic acid (DPA), a major component of bacterial spores. Ternary Eu(DO2A)(DPA)(-) complex solutions (1.0 mu M crystallographically characterized TBA.Eu(DO2A)-(DPA)) were titrated with EuCl3 (1.0 nM-1.0 mM); increased Eu3+ concentration resulted in a shift in equilibrium population from Eu(DO2A)(DPA)(-) to Eu(DO2A)(+) and Eu(DPA)(+), which was monitored via the ligand field sensitive D-5(0) -> F-7(3) transition (lambda(em) = 670-700 nm) using luminescence spectroscopy. A best fit of luminescence intensity titration data to a two-state thermodynamic model yielded the competition equilibrium constant (K-c), which in conjunction with independent measurement of the Eu(DPA)(+) formation constant (K-a) allowed calculation of the ternary complex formation constant (K-a'). With this binding affinity by competition (BAC) assay, we determined that K-a' = 1011.21 M-1, which is similar to 1 order of magnitude greater than the formation of Eu(DPA)(+). In general, the BAC assay can be employed to determine ligand binding constants of systems where the lanthanide platform (usually a binary complex) is stable and the ligand bound versus unbound states can be spectroscopically distinguished.