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

The nitrone, 5,5-dimethylpyrroline N-oxide (DMPO), is a commonly used spin trap for the detection of superoxide radical anion (O₂^•-) using electron paramagnetic resonance spectroscopy. This work investigates the reactivity of DMPO to O₂^•- 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 pK_a for O₂^•- is 4.8, the pK_a for DMPO is unknown. The pK_a of the conjugate acid of DMPO was determined to be 6.0 using potentiometric, spectrophotometric, ¹H and ¹³C NMR, and computational methods. ¹H and ¹³C NMR were employed to investigate the site of protonation. An alternative mechanism for the spin trapping of O₂^•- in mildly acidic pH was proposed, which involves protonation of the oxygen to form the N-hydroxy imino cation and subsequent addition of O₂^•-. The exoergicity of O₂^•- addition to protonated DMPO was rationalized using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory.