Synthesis and spectral properties of polymethine-cyanine dye–nitroxide radical hybrid compounds for use as fluorescence probes to monitor reducing species and radicals

Various hybrid compounds comprised of two types of nitroxide radicals and either a pentamethine (Cy5) or trimethine cyanine (Cy3) were synthesized. The nitroxide radicals were linked either via an ester-bond to one or two N-alkyl carboxyl-terminated groups of Cy5, or via two amido-bonds (aminocarbonyl or carbonylamino group) to the 5-position of the indolenine moieties of Cy5 and Cy3. Changes in fluorescence and ESR intensities of the hybrid compounds were measured before and after addition of Na ascorbate in PBS (pH 7.0) to reduce the radicals. Among the hybrid compounds synthesized, those that linked the nitroxide radicals via an aminocarbonyl residue at the 5-position of the indolenine moieties on Cy5 and Cy3 exhibited a 1.8- and 5.1-fold increase in fluorescence intensity with the reduction of the nitroxide segment by the addition of Na ascorbate, respectively. In contrast, fluorescence intensity was not enhanced in the other hybrid compounds. Thus, the hybrid compounds which exhibited an increase in fluorescent intensity with radical reduction can be used in the quantitative measurement of reducing species such as Fe2+ and ascorbic acid, and hydroxyl radicals. Because these hybrid compounds have the advantage of fluorescing at longer wavelengths—661 (Cy5) or 568 (Cy3) nm, respectively, they can be used to measure radical-reducing species or radicals either in solution or in vivo.