Synthesis and characterization of a 5-membered ring cyclic hydroxylamine coupled to triphenylphosphonium to detect mitochondrial superoxide by EPR spectrometry

Abstract

As mitochondrial superoxide is becoming an attractive metabolic and pharmacological target, there is an important need for developing analytical tools able to detect superoxide with high sensitivity and specificity. Among EPR-based methods, it has been recently reported that cyclic hydroxylamines offer a high sensitivity to measure superoxide production. Here, we report the synthesis and evaluation of mitoCPH, in which a 5-membered ring hydroxylamine was coupled to a triphenylphosphonium moiety to allow mitochondrial accumulation. MitoCPH efficiently reacted with superoxide with a bimolecular rate constant of 1.5 × 10⁴ M⁻¹ s⁻¹. We assessed the ability of this compound to detect superoxide in PBS buffer, lysates, and in paraquat-stimulated cells. We compared its performance with CMH, a nontargeted 5-membered ring hydroxylamine, and mitoTEMPO-H, a classically used 6-membered ring hydroxylamine targeted to mitochondria. MitoCPH presented a higher sensitivity for superoxide anion detection than commonly used mitoTEMPO-H, both in buffer and in cell lysates. While we have described the ability of mitoCPH to detect superoxide in different cellular media, we cannot exclude other potential contributors to the nitroxide production from this probe. Therefore, mitoCPH should be considered as a mitochondria-targeted probe and its use as selective superoxide probe should be used cautiously.

Keywords:

• Cyclic hydroxylamine
• EPR
• ESR
• mitochondria
• paraquat
• superoxide

Acknowledgments

The authors thank Soumia El Aakchioui, Maximilien Richald, and Sébastien Clergue for their help with chemistry protocols, Laure Elens for help in statistical analysis and Thibaut Vazeille for the preparation of cells and lysates.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Samantha Scheinok is a Televie PhD fellow. Pierre Sonveaux and Raphaël Robiette are Senior Research Associates of the Belgian Fonds National de la Recherche Scientifique (FRS – FNRS). The work was supported by an Action de Recherche Concertée from the Communauté Française de Belgique (ARC 14/19-058) and by FRS – FNRS grant CDR J.0209.16. This research used the EPR facilities of the technological platform “Nuclear and Electron Spin Technologies” of the Louvain Drug Research Institute.