Site-Specific Detection of Free Radicals in Membranes Using an Amphiphilic Spin Trap

The detection of free radicals and related species has attracted considerable attention in recent years due to their critical roles in physiological and pathological processes. Among the various methods for the detection of free radicals, electron spin resonance coupled with spin-trapping technique has been an effective approach for the characterization and quantification of free radicals due to its high specificity. In this study, we designed and synthesized a novel amphiphilic spin trap, 2-(diethoxyphosphoryl)-2-heptadecanyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPHdPO), from 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide with a long hydrocarbon chain at the C-5 position of the pyrroline ring, providing the amphiphilic character. The free-radical-trapping ability of DEPHdPO was evaluated by capturing hydroxyl radicals (^·OH), superoxide anions (\( {\text{O}}_{2}^{ \cdot - } \)), and carbon-centered free radicals in a model membrane prepared from sodium dodecyl sulfate (SDS). The results indicate that the hydrophobic hydrocarbon chain of DEPHdPO can be inserted into the inner core of SDS micelles, and the hydrophilic nitronyl functional moiety is located on the surface layer. Thus, various free radicals, including ^·OH radicals, \( {\text{O}}_{2}^{ \cdot - } \) anions, and carbon-centered radicals could be site-specifically detected near the membrane surface. Moreover, DEPHdPO could be successfully located on the surface of thylakoid membranes, and the nearby photo-initiated \( {\text{O}}_{2}^{ \cdot - } \) anions could be trapped site-specifically.