Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide

Hydroethidine (HE) and hydropropidine ($$\hbox {HPr}^{+}$$ HPr + ) are fluorogenic probes used for the detection of the intra- and extracellular superoxide radical anion ($$\hbox {O}_{ {2}}^{\bullet -}$$ O 2 ∙ - ). In this study, we provide evidence that HE and $$\hbox {HPr}^{+}$$ HPr + react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with $$\hbox {O}_{ {2}}^{\bullet -}$$ O 2 ∙ - , leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between $$\hbox {O}_{ {2}}^{\bullet -}$$ O 2 ∙ - and the radical cations of the probes. We also synthesized N-methylated analogs of $$\hbox {HPr}^{+}$$ HPr + and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and $$\hbox {HPr}^{+}$$ HPr + may be used as a scaffold for the design of a new generation of probes for intra- and extracellular superoxide.