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

A series of FL<i><sub>n</sub></i> (<i>n</i> = 1−5) ligands, where FL<i><sub>n</sub></i> 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 FL<sub>1</sub>, FL<sub>3</sub>, and FL<sub>5</sub> in pH 7.0 buffered aqueous solutions. The reactions of FL<sub>2</sub> or FL<sub>4</sub> with CuCl<sub>2</sub>, 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 FL<i><sub>n</sub></i> ligand family. Two kinds of crystals, [Cu(modL)<sub>2</sub>](BF<sub>4</sub>)<sub>2</sub> and [Cu<sub>2</sub>(modL‘)<sub>2</sub>(CH<sub>3</sub>OH)](BF<sub>4</sub>)<sub>2</sub> (modL‘ = 2-[(quinolin-8-ylamino)methyl]phenolate), were obtained. The structures suggest that one oxygen and two nitrogen atoms of the FL<i><sub>n</sub></i> ligands most likely bind to Cu(II). Introduction of nitric oxide (NO) to pH 7.0 buffered aqueous solutions of Cu(FL<i><sub>n</sub></i>) (1 μM CuCl<sub>2</sub> and 1 μM FL<i><sub>n</sub></i>) at 37 °C induces an increase in fluorescence. The fluorescence response of Cu(FL<i><sub>n</sub></i>) 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(FL<sub>5</sub>) occurs by reduction of Cu(II) to Cu(I) with concomitant dissociation of the <i>N</i>-nitrosated fluorophore ligand from copper. Spectroscopic and product analyses of the reaction of the FL<sub>5</sub> copper complex with NO indicated that the <i>N</i>-nitrosated fluorescein ligand (FL<sub>5</sub>−NO) is the species responsible for fluorescence turn-on. Density functional theory (DFT) calculations of FL<sub>5</sub> versus FL<sub>5</sub>−NO reveal how <i>N</i>-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.