THE USE OF FLUOROPHORE-CONTAINING SPIN TRAPS AS POTENTIAL PROBES TO LOCALIZE FREE-RADICALS IN CELLS WITH FLUORESCENCE IMAGING METHODS

Central to the study of free radical processes is the ability to ident ify and localize their cellular site of formation. Under the best of e xperimental conditions, spin trapping/ESR spectroscopy can only charac terize intracellular production of specific free radicals and confocal microscopy can only localize the site of their formation. In this art icle, we report on the development of a fluorophore-containing nitrone , nyl)-2-pyrrolin-1-yl]phenyl]-N-(tert-butyl)nitrone sodium salt (4).( 3) This nitrone (4) reacts with alpha-hydroxyethyl radical with a seco nd order rate constant of 1.7 x 10(5) M(-1) s(-1) to give a characteri stic ESR spectrum. However, we were unable to decrease the fluorescenc e emission, due in part to the small concentration of nitroxide genera ted from the reaction of alpha-hydroxyethyl radical with nitrone (4). Using the fluorophore-containing nitroxide (7) as a model, we found th at only 12% of the nitroxide needs to be reduced to give an almost 400 % increase in the fluorescent emission of (7). Our findings suggest ne w approaches to the development of various fluorophore-containing nitr ones that can both characterize specific free radicals and localize th eir site of intracellular formation.