DMNP as an Effective Cage for Ln³⁺: A Spectroscopic and Thermodynamic Study

Dimethoxynitrophenyl-EDTA (DMNP) is a popular calcium cage that is frequently used to investigate the role of Ca²⁺ in signaling processes in vivo. Lanthanides have been used in Ca²⁺ biomimetics due to similarities in coordination properties of Ln³⁺ and Ca²⁺ that may enable fluorescence and NMR studies of functional and structural properties of Ca²⁺ binding proteins. In this study, we show that Tb³⁺, Eu³⁺, and Nd³⁺ bind strongly to DMNP in a 1:1 ratio. Isothermal titration calorimetric measurements of Ca²⁺ displacement by Ln³⁺ in DMNP provide the equilibrium binding constants for Ln³⁺DMNP complexation with association constants, K₁₁ = (1.2 ± 0.7) × 10¹² M^-1 for Eu³⁺, (2.5 ± 1.7) × 10¹² M^-1 for Nd³⁺, and (2.8 ± 0.8) × 10¹² M^-1 for Tb³⁺. The kinetics and thermodynamics of Ca²⁺, Mg²⁺, and Tb³⁺ release from DMNP were characterized using photothermal beam deflection (PBD). Ligand release from the DMNP cage was rapid and occurred within 10 μs upon cage photofragmentation and was associated with similar reaction volume and enthalpy changes that can be attributed to the photoreleased ion solvation. In the case of Ca²⁺DMNP photodissociation at subsaturating Ca²⁺ concentrations, we observed a slower phase with a lifetime of 300 μs that we attribute to Ca²⁺ rebinding to unphotolyzed DMNP. These results demonstrate that DMNP can serve as an effective photolabile cage for oxophilic Ln³⁺ that has similar coordination properties to Ca²⁺ and Mg²⁺.