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J Am Chem Soc. 2006 Nov 8;128(44):14364-73.

Direct nitric oxide detection in aqueous solution by copper(II) fluorescein complexes.

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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.


A series of FL(n) (n = 1-5) ligands, where FL(n) 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(1), FL(3), and FL(5) in pH 7.0 buffered aqueous solutions. The reactions of FL(2) or FL(4) with CuCl(2), 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(n) ligand family. Two kinds of crystals, [Cu(modL)(2)](BF(4))(2) and [Cu(2)(modL')(2)(CH(3)OH)](BF(4))(2) (modL' = 2-[(quinolin-8-ylamino)methyl]phenolate), were obtained. The structures suggest that one oxygen and two nitrogen atoms of the FL(n) ligands most likely bind to Cu(II). Introduction of nitric oxide (NO) to pH 7.0 buffered aqueous solutions of Cu(FL(n)) (1 microM CuCl(2) and 1 microM FL(n)) at 37 degrees C induces an increase in fluorescence. The fluorescence response of Cu(FL(n)) 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(5)) 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 FL(5) copper complex with NO indicated that the N-nitrosated fluorescein ligand (FL(5)-NO) is the species responsible for fluorescence turn-on. Density functional theory (DFT) calculations of FL(5) versus FL(5)-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.

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