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 r apidly and irreversibly destroyed by cells. A methyl substitution at t he 2-position of DMPO results in the nitrone 2,5,5-trimethyl-1-pyrroli ne 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 s plitting (hfs), and, in the absence of a beta-hydrogen, different addu cts are not distinguishable. To overcome this limitation, the synthesi s of M(3)PO labeled with C-13 at the nitronyl (C-2) or the 2-methyl (a lpha or beta to the aminoxyl group in the spin abduct, respectively) h as been undertaken. [alpha-C-13]M(3)PO was synthesized from [2-C-13]ac etone in a three-step pathway while [beta-C-13]M(3)PO was obtained fro m 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 ad ditional hfs necessary for spin adduct identification. Primary radical s, such as CH3, *CO2- and *OH were generated radiolytically, sonolyti cally, or enzymatically, trapped by [C-13]M(3)PO, and gave rise to nit roxide spin adducts which were identified and their magnetic parameter s determined. The [C-13]M(3)PO spin adducts were far more stable than those of DMPO. Moreover, they were less susceptible to cellular-induce d destruction. However, the superoxide adduct of M(3)PO was unstable a nd did not persist.