Hydropropidine: A novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide

Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr+), the reduction product of propidium iodide, for detecting extracellular superoxide (O2
• −). HPr+ is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O2
• −. We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr+ and allow for exclusive detection of extracellular O2
• −. Our results indicate that O2
• − reacts with HPr+ (k=1.2×104 M−1 s−1) to form exclusively 2-hydroxypropidium (2-OH-Pr2+) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O2
• − (Zhao et al., Free Radic. Biol. Med.34:1359–1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O2
• − with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O2
• − and HPr+ is slightly higher than that of HE and O2
• − and is closer to that of Mito-HE and O2
• −. Similar to HE, HPr+ undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fenton's reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr2+) and the dimeric products (e.g., Pr2+-Pr2+). In contrast to HE, there was very little intracellular uptake of HPr+. We conclude that HPr+ is a useful probe for detecting O2
• − and other one-electron oxidizing species in an extracellular milieu.

Graphical AbstractFigure optionsDownload high-quality image (206 K)Download as PowerPoint slideHighlights
► Hydropropidine is a cell-impermeable analog of hydroethidine.
► Positive charge on the nitrogen impedes cellular uptake of hydropropidine.
► Extracellular O2
• – reacts with the probe to form a specific hydroxylated product.
► Extracellular one-electron oxidants react with this probe to form dimeric products.