NOVEL DMPO-DERIVED C-13-LABELED SPIN TRAPS YIELD IDENTIFIABLE STABLE NITROXIDES

The nitrone 5,5-dimethyl-1-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-1-pyrroline N-oxide (M(3)PO). Radical addition to M(3)PO is expected to produce stable spin adducts; however, they have almost the same N hyperfine splitting (hfs), and, in the absence of a beta-hydrogen, different adducts are not distinguishable. To overcome this limitation, the synthesis of M(3)PO labeled with C-13 at the nitronyl (C-2) or the 2-methyl (alpha or beta to the aminoxyl group in the spin abduct, respectively) has been undertaken. [alpha-C-13]M(3)PO was synthesized from [2-C-13]acetone in a three-step pathway while [beta-C-13]M(3)PO was obtained from DMPO and [C-13]iodomethane. For M(3)PO, the nuclear magnetic moment of C-13 replaces that of the beta-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 [C-13]M(3)PO, and gave rise to nitroxide spin adducts which were identified and their magnetic parameters determined. The [C-13]M(3)PO spin adducts were far more stable than those of DMPO. Moreover, they were less susceptible to cellular-induced destruction. However, the superoxide adduct of M(3)PO was unstable and did not persist.