Fluorescence-based nitric oxide sensing by Cu(II) complexes that can be trapped in living cells

A series of symmetrical, fluorescein-derived ligands appended with two derivatized 2-methyl-8-aminoquinolines were prepared and spectroscopically characterized. The ligands FL2, FL2E, and FL2A were designed to improve the dynamic range of previously described asymmetric systems, and the copper complex Cu(2)(FL2E) was constructed as a trappable NO probe that is hydrolyzed intracellularly to form Cu(2)(FL2A). The ligands themselves are only weakly emissive, and the completely quenched Cu(II) complexes, generated in situ by combining each ligand with 2 equiv of CuCl(2), were investigated as fluorescent probes for nitric oxide. Upon introduction of excess NO under anaerobic conditions to buffered solutions of Cu(2)(FL2), Cu(2)(FL2E), and Cu(2)(FL2A), the fluorescence increased by factors of 23 +/- 3, 17 +/- 2, and 27 +/- 3, respectively. The corresponding rate constants for fluorescence turn-on were determined to be 0.4 +/- 0.2, 0.35 +/- 0.05, and 0.6 +/- 0.1 min(-1). The probes are highly specific for NO over other biologically relevant reactive oxygen and nitrogen species, as well as Zn(II), the metal ion for which similar probes were designed to detect.