Reaction of cyclic nitroxides with nitrogen dioxide: The intermediacy of the oxoammonium cations

Piperidine and pyrrolidine nitroxides, such as 2,2,6,6-tetramethylpiperidinoxyl (TPO) and 3-carbamoylproxyl (3-CP), respectively, are cell-permeable stable radicals, which effectively protect cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The kinetics and mechanism of their reactions with ^.OH, superoxide, and carbon-centered radicals have been extensively studied, but not with ^.NO₂, although the latter is a key intermediate in cellular nitrosative stress. In this research, ^.NO₂ was generated by pulse radiolysis, and its reactions with TPO, 4-OH-TPO, 4-oxo-TPO, and 3-CP were studied by fast kinetic spectroscopy, either directly or by using ferrocyanide or 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate), which effectively scavenge the product of this reaction, the oxoammonium cation. The rate constants for the reactions of ^.NO₂ with these nitroxides were determined to be (7-8) × 10⁸ M^-1 s^-1, independent of the pH over the range 3.9-10.2. These are among the highest rate constants measured for ^.NO₂ and are close to that of the reaction of ^.NO₂ with ^.NO, that is, 1.1 × 10⁹ M^-1 s^-1. The hydroxylamines TPO-H and 4-OH-TPO-H are less reactive toward ^.NO₂, and an upper limit for the rate constant for these reactions was estimated to be 1 × 105 M^-1 s^-1. The kinetics results demonstrate that the reaction of nitroxides with ^.NO₂ proceeds via an inner-sphere electron-transfer mechanism to form the respective oxoammonium cation, which is reduced back to the nitroxide through the oxidation of nitrite to ^.NO₂. Hence, the nitroxide slows down the decomposition of ^.NO₂ into nitrite and nitrate and could serve as a reservoir of ^.NO₂ unless the respective oxoammonium is rapidly scavenged by other reductant. This mechanism can contribute toward the protective effect of nitroxides against reactive nitrogen-derived species, although the oxoammonium cations themselves might oxidize essential cellular targets if they are not scavenged by common biological reductants, such as thiols.