Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo.

The development of fluorescent probes for superoxide and the hydroxyl radical is a central problem in the field of chemical biology. Superoxide and the hydroxyl radical play a significant role in a variety of inflammatory diseases, and probes that can detect these reactive oxygen species (ROS) have tremendous potential as medical diagnostics and research tools. Fluorescent sensors for superoxide and the hydroxyl radical, such as dihydroethidium (DHE), have been developed; however, they have had limited applicability because of their spontaneous autoxidation, rapid photobleaching, low emission wavelengths, and multiple reaction products with ROS. New chemical probes for superoxide and the hydroxyl radical are therefore greatly needed. Herein, we present a new family of fluorescent ROS sensors, termed the hydrocyanines, which can be synthesized in one step from the commercially available cyanine dyes and can detect superoxide and the hydroxyl radical in living cells, tissue samples, and for the first time in vivo. We anticipate widespread interest in the hydrocyanines given their physical/ chemical characteristics and ease of synthesis. The synthesis of and mechanism by which hydrocyanines image ROS are shown in Figure 1a. The hydrocyanines are synthesized by reducing the iminium cations of the cyanine dyes with NaBH4. Hydrocyanines detect ROS through fluorescent imaging; they are weakly fluorescent because of their disrupted p conjugation; however, oxidation with either superoxide or the hydroxyl radical dramatically increases their fluorescence by regenerating their extended p conjugation. Figure 1b demonstrates the proof of principle of this methodology. Hydro-IR-676 has minimal fluorescence; however, oxidation with superoxide causes a 100-fold increase in its fluorescence intensity. The cyanine dyes comprise a family of approximately 40 dyes, and the reduction methodology presented in Figure 1a was used to synthesize several new fluorescent ROS sensors, which have the physical and chemical properties needed to detect intracellular and extracellular ROS, both in vitro and in vivo. For example, five new ROS sensors, termed hydro-Cy3, hydro-Cy5, hydroCy7, hydro-IR-783, and hydro-ICG, were synthesized by reduction with NaBH4 (Table 1). These molecules have negligible fluorescence, as a result of reduction of their iminium cations; however, after reaction with either superoxide or the hydroxyl radical, they fluoresce at 560, 660, 760, 800, and 830 nm, respectively (see Table 1 and the Supporting Information for details). Hydro-Cy3, hydro-Cy5, hydro-IR676, and hydro-Cy7 have the physical properties needed to detect intracellular ROS, in that they are initially membranepermeable molecules; however, oxidation with intracellular ROS converts them into charged and membrane-impermeable molecules, which should accumulate within cells that are overproducing ROS. In contrast, hydro-ICG and hydro-IRFigure 1. a) Synthesis of hydrocyanines by a one-step reduction with NaBH4. Reaction with superoxide or the hydroxyl radical oxidizes the hydrocyanines to produce the fluorescent cyanine dyes. b) Hydro-IR676 has negligible fluorescence emission; however, oxidation with superoxide causes a 100-fold increase in fluorescence (lex=675 nm, lem=693 nm).