The use of cyclic nitroxide radicals as HNO scavengers

Reduction of cyclic stable nitroxides (RNO) by HNO to the respective hydroxylamines (RNO-H) has been demonstrated using EPR spectrometry. HNO shows low reactivity toward piperidine, pyrrolidine and nitronyl nitroxides with rate constants below 1.4 × 105 M− 1 s− 1 at pH 7.0, despite the high driving force for these reactions. The rate constants can be predicted assuming that the reactions take place via a concerted proton–electron transfer pathway and significantly low self-exchange rate constants for HNO/NO and RNO-H/RNO. NO does not react with piperidine and pyrrolidine nitroxides, but does add to HNO forming the highly oxidizing and moderately reducing hyponitrite radicals. In this work, the radicals are produced by pulse radiolysis and the rate constants of their reactions with 2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and 3-carbamoyl-PROXYL have been determined at pH 6.8 to be (2.4 ± 0.2) × 106, (9.8 ± 0.2) × 105, (5.9 ± 0.5) × 105 M− 1 s− 1, respectively. This low reactivity implies that NO competes efficiently with these nitroxides for the hyponitrite radical. The ability of TEMPOL and 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO) to oxidize HNO and their different reactivity toward NO are used to quantify HNO formed via acetohydroxamic acid oxidation. The extent of TEMPOL or C-PTIO reduction was similar to the yield of HNO formed upon oxidation by OH under anoxia, but not by the metmyoglobin and H2O2 reaction system where both nitroxides catalytically facilitate H2O2 depletion and nitrite accumulation. In this system the conversion of C-PTIO into 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (C-PTI) is a minor reaction, which does not provide any mechanistic insight.

Reactivity of HNO and N2O2− towards nitroxides is surprisingly low for these strongly driven reactions. Nitroxides catalytically facilitate H2O2 decomposition and nitrite formation in the presence of heme proteins and hydroxamic acid, and should not be used as HNO or NO scavengers in this system.Figure optionsDownload as PowerPoint slideHighlights
► The reactivity of HNO and N2O2− toward nitroxides (RNO) is surprisingly low.
► Low reactivity of HNO is due to low self-exchange rate constants of HNO and RNO-H.
► Reduction of N2O2− by RNO proceeds via an inner-sphere electron-transfer mechanism.
► RNO catalytically facilitate H2O2 depletion in the presence of MetMb and aceto-HX.
► RNO should not be used as HNO or NO scavenger in the presence of MetMb and H2O2.