Direct Nitric Oxide Detection in Aqueous Solution by Copper(II) Fluorescein Complexes

A series of FLn (n = 1−5) ligands, where FLn is a fluorescein modified with a functionalized 8-aminoquinoline group as a copper-binding moiety, were synthesized, and the chemical and photophysical properties of the free ligands and their copper complexes were investigated. UV−visible spectroscopy revealed a 1:1 binding stoichiometry for the Cu(II) complexes of FL1, FL3, and FL5 in pH 7.0 buffered aqueous solutions. The reactions of FL2 or FL4 with CuCl2, however, appear to produce a mixture of 1:1 and 1:2 complexes, as suggested by Job's plots. These binding modes were modeled by the synthesis and X-ray crystal structure determination of Cu(II) complexes of 2-[(quinolin-8-ylamino)methyl]phenol (modL), employed as a surrogate of the FLn ligand family. Two kinds of crystals, [Cu(modL)2](BF4)2 and [Cu2(modL‘)2(CH3OH)](BF4)2 (modL‘ = 2-[(quinolin-8-ylamino)methyl]phenolate), were obtained. The structures suggest that one oxygen and two nitrogen atoms of the FLn ligands most likely bind to Cu(II). Introduction of nitric oxide (NO) to pH 7.0 buffered aqueous solutions of Cu(FLn) (1 μM CuCl2 and 1 μM FLn) at 37 °C induces an increase in fluorescence. The fluorescence response of Cu(FLn) to NO is direct and specific, which is a significant improvement over commercially available small molecule-based probes that are capable of detecting NO only indirectly. The NO-triggered fluorescence increase of Cu(FL5) occurs by reduction of Cu(II) to Cu(I) with concomitant dissociation of the N-nitrosated fluorophore ligand from copper. Spectroscopic and product analyses of the reaction of the FL5 copper complex with NO indicated that the N-nitrosated fluorescein ligand (FL5−NO) is the species responsible for fluorescence turn-on. Density functional theory (DFT) calculations of FL5 versus FL5−NO reveal how N-nitrosation of the fluorophore ligand brings about the fluorescence increase. The copper-based probes described in the present work form the basis for real-time detection of nitric oxide production in living cells.