Novel DMPO-Derived ¹³C-Labeled Spin Traps Yield Identifiable Stable Nitroxides

The nitrone 5,5-dimethyl-l-pyrroline N-oxide (DMPO) is the most common spin trap used for studying free radicals, yet its spin adducts are rapidly and irreversibly destroyed by cells. A methyl substitution at the 2-position of DMPO results in the nitrone 2,5,5-trimethyl-l-pyrroline N-oxide (M₃PO). Radical addition to M₃PO is expected to produce stable spin adducts; however, they have almost the same N hyperfine splitting (hfs), and, in the absence of a β-hydrogen, different adducts are not distinguishable. To overcome this limitation, the synthesis of M₃PO labeled with ¹³C at the nitronyl (C₂) or the 2-methyl (α or β to the aminoxyl group in the spin adduct, respectively) has been undertaken. [α-¹³C]M₃PO was synthesized from [2-¹³C]acetone in a three-step pathway while [β-^I3C]M₃PO was obtained from DMPO and [¹³C]iodomethane. For M₃PO, the nuclear magnetic moment of ¹³C replaces that of the β-hydrogen of DMPO and provides the additional hfs necessary for spin adduct identification. Primary radicals, such as ∗CH₃, ∗CO₂- and ∗OH were generated radiolytically, sonolytically, or enzymatically, trapped by [¹³C]M₃PO, and gave rise to nitroxide spin adducts which were identified and their magnetic parameters determined. The [¹³C]M₃PO spin adducts were far more stable than those of DMPO. Moreover, they were less susceptible to cellular-induced destruction. However, the superoxide adduct of M₃PO was unstable and did not persist.