Quantitative 31P NMR detection of oxygen-centered and carbon-centered radical species Original Research Article

Quantitative 31P NMR spin trapping techniques can be used as effective tools for the detection and quantification of many free radical species. Free radicals react with a nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using 31P NMR in the presence of phosphorus containing internal standards. Initially, the 31P NMR signals for the radical adducts of oxygen-centered (radical dotOH, O2radical dot−) and carbon-centered (radical dotCH3, radical dotCH2OH, CH2radical dotCH2OH) radicals were assigned. Subsequently, the quantitative reliability of the developed technique was demonstrated under a variety of experimental conditions. The 31P NMR chemical shifts for the hydroxyl and superoxide reaction adducts with DIPPMPO were found to be 25.3, 16.9, and 17.1 ppm (in phosphate buffer), respectively. The 31P NMR chemical shifts for radical dotCH3, radical dotCH2OH, radical dotCH(OH)CH3, and radical dotC(O)CH3 spin adducts were 23.1, 22.6, 27.3, and 30.2 ppm, respectively. Overall, this effort forms the foundations for a targeted understanding of the nature, identity, and mechanisms of radical activity in a variety of biomolecular processes.