Superoxide Radical Anion Adduct of 5,5-Dimethyl-1-pyrroline <i>N</i>-Oxide (DMPO). 3. Effect of Mildly Acidic pH on the Thermodynamics and Kinetics of Adduct Formation

The nitrone, 5,5-dimethylpyrroline <i>N</i>-oxide (DMPO), is a commonly used spin trap for the detection of superoxide radical anion (O<sub>2</sub><sup>•-</sup>) using electron paramagnetic resonance spectroscopy. This work investigates the reactivity of DMPO to O<sub>2</sub><sup>•-</sup> in mildly acidic pH (5.0−7.0). Mild acidity is characteristic of acidosis and has been observed in hypoxic systems, e.g., ischemic organs and cancer cells. Although the established p<i>K</i><sub>a</sub> for O<sub>2</sub><sup>•-</sup> is 4.8, the p<i>K</i><sub>a</sub> for DMPO is unknown. The p<i>K</i><sub>a</sub> of the conjugate acid of DMPO was determined to be 6.0 using potentiometric, spectrophotometric, <sup>1</sup>H and <sup>13</sup>C NMR, and computational methods. <sup>1</sup>H and <sup>13</sup>C NMR were employed to investigate the site of protonation. An alternative mechanism for the spin trapping of O<sub>2</sub><sup>•-</sup> in mildly acidic pH was proposed, which involves protonation of the oxygen to form the <i>N</i>-hydroxy imino cation and subsequent addition of O<sub>2</sub><sup>•-</sup>. The exoergicity of O<sub>2</sub><sup>•-</sup> addition to protonated DMPO was rationalized using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory.