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

Abstract

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.

Health position paper and redox perspectives - Disease burden by transportation noise

“…The most used probe for the in vivo and in vitro detection of superoxide radical anion (O 2 •– ) is dihydroethidium (DHE, also known as hydroethidine, HE) [ 409 , 410 ]. This probe forms an O 2 •– -specific red fluorescent product, 2-hydroxyethidium (2-OH-E + ).…”

“…Derivatives of DHE targeted to mitochondria (MitoSOX Red, MitoNeoD) or the extracellular space (hydropropidine) have been also reported [ [414] , [415] , [416] ]. The chemistry of those probes resembles the chemistry of DHE, and therefore the same recommendations regarding the detection of the O 2 •– -specific product apply [ 410 , 411 , 417 , 418 ].…”

Practical Aspects in the Study of Biological Photosensitization Including Reaction Mechanisms and Product Analyses: A Do's and Don'ts Guide†

“…Hydroethidine (HE) also known as dihydroethidium is a specific chemiluminescent probe for the detection of O 2 •− giving rise through oxidation to red 2hydroxyethidium (2-OH-E+) as a red fluorescent product (133). However this compounds has to be separated by HPLC from other fluorescent compounds such as ethidium to allow its selective detection (134,135).…”

Luminescent lanthanide complexes for reactive oxygen species biosensing and possible application in Alzheimer’s diseases

“…2). First oxidation of HE by HO 2 ● yields hydroethidine radical cation HE +● , which then reacts either with (close to diffusion‐controlled, k = (7.4 ± 0.1) × 10 8 L·mol −1 ·s −1 [51], thus competing for with superoxide dismutase in vivo [52]) to specifically yield 2‐HO‐E + or with another intracellular oxidant to yield E + . The main drawbacks are that HE‐based probes are poorly selective for , are susceptible to auto‐oxidation when illuminated and need the use of chromatography‐based separation for specifically detecting 2‐HO‐E + , because both 2‐HO‐E + and E + are usually produced [53].…”