Structural and thermodynamic analysis of self-association of phenanthridine dyes in aqueous solution by 1H NMR spectroscopy

Self-association of phenanthridine dyes (ethidium monoazide EMB and ethidium diazide EDC) in aqueous solution was studied by one- and two-dimensional H-1 NMR (500 MHz). 2D-TOCSY and 2D-ROESY experiments were used for signal assignment of the dye protons. The concentration and temperature dependences of the chemical shifts of the nonexchangeable protons of EMB and EDC in aqueous solution have been measured. The experimental results were analyzed based oil the infinitely dimensional noncooperative and cooperative models of self-association of molecules. The cooperativity parameter, the equilibrium constants, and the enthalpies and entropies of self-association of the dyes were calculated along with the limiting chemical shifts of EMB and EDC protons in the associates. The cooperativity parameter sigma approximate to 1 indicates that association of phenanthridine dye molecules in aqueous solution is not a cooperative process. The presence of azido groups in the phenanthridine chromophore diminishes the equilibrium constant and the enthalpy of formation of dye aggregates ill aqueous solution. The most plausible structures of EMB and EDC dimers in aqueous solution were derived front the induced proton chemical shifts of the dyes. In the dimer complexes of EMB and EDC, the distances between the planes of the aromatic chromophores are longer than those in the dimer of ethidium bromide due to electrostatic repulsion of the dipole azido groups in the phenanthridine chromophores of the dyes.