Kinetics and mechanism of hydroxyl radical and OH-adduct radical reactions with nitroxides and with their hydroxylamines

Stable nitroxide radicals are potent antioxidants and are among the most effective non-thiol radioprotectants, although they react with hydroxyl radicals more slowly than typical phenolic antioxidants or thiols. Surprisingly, the reduced forms of cyclic nitroxides, cyclic hydroxylamines, are better reductants yet have no radioprotective activity. To clarify the reason for this difference, we studied the kinetics and mechanisms of the reactions of nitroxides and their hydroxylamines with ·OH radicals and with OH-adducts by using pulse radiolysis, fluorimetric determination of phenolic radiation products, and electron paramagnetic resonance spectrometric determination of nitroxide concentrations following radiolysis. Competition kinetics with phenylalanine as a reference compound in pulse radiolysis experiments yielded rate constants of (4.5 ± 0.4) × 10⁹ M^-1 s^-1 for the reaction of ·OH radical with 2,2,6,6-tetramethylpiperidine-N-oxyl (TPO), 4-hydroxy-TPO (4-OH-TPO), and 4-oxo-TPO (4-O-TPO), (3.0 ± 0.3) × 10⁹ M^-1 s^-1 for deuterated 4-O-TPO, and (1.0 ± 0.1) × 10⁹ M^-1 s^-1 for the hydroxylamine 4-OH-TPO-H. The kinetic isotope effect suggests the occurrence of both ·OH addition to the aminoxyl moiety of 4-O-TPO and H-atom abstraction from the 2- or 6-methyl groups or from the 3- and 5-methylene positions. This conclusion was further supported by final product analysis, which demonstrated that ·OH partially oxidizes 4-O-TPO to the corresponding oxoammonium cation. The rate constants for the reactions of the nitroxides with the OH-adducts of phenylalanine and terephthalate have been determined to be near 4 × 10⁶ M^-1 s^-1, whereas the hydroxylamine reacted at least 50 times slower, if at all. These findings indicate that the reactivity toward ·OH does not explain the differences between the radioprotective activities of nitroxides and hydroxylamines. Instead, the radioprotective activity of nitroxides, but not of hydroxylamines, can be partially attributed to their ability to detoxify OH-derived secondary radicals.