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

Radosław Michalski (1) , David Thiebaut (2) , Bartosz Michałowski (1) , Mehmet M Ayhan (3) , Micael Hardy (2) , Olivier Ouari (2) , Michał Rostkowski (1) , Renata Smulik-Izydorczyk (1) , Angelika Artelska (1) , Andrzej Marcinek (1) , Jacek Zielonka (4) , Balaraman Kalyanaraman (4) , Adam Sikora (1)

1 Institute of Applied Radiation Chemistry [Łódź University of Technology]
2 ICR - Institut de Chimie Radicalaire
3 Department of Chemistry, Gebze Technical University, P.K.:141, 41400 Gebze, Kocaeli, Turkey
4 Department of Biophysics and §Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States

Hydroethidine (HE) and hydropropidine (HPr +) are fluorogenic probes used for the detection of the intra-and extracellular superoxide radical anion (O •− 2). In this study, we provide evidence that HE and 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 O •− 2 , leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between O •− 2 and the radical cations of the probes. We also synthesized N-methylated analogs of 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 HPr + may be used as a scaffold for the design of a new generation of probes for intra-and extracellular superoxide.

Mots clés: Chemical biology; Fluorescent probes; Physical chemistry; Small molecules

Domaines: Chimie

Radosław Michalski, David Thiebaut, Bartosz Michałowski, Mehmet M Ayhan, Micael Hardy, et al.. Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide. Scientific Reports, 2020, 10, ⟨10.1038/s41598-020-75373-2⟩. ⟨hal-03244541⟩