Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species

We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical ((OH)-O-.) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (-OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O-2(.-)) by using HPF or APF alone, but -OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluoreseein, and for the first time we could visualize -OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and -OCl in many biological and chemical applications.