Planta Med 2008; 74 - SL35
DOI: 10.1055/s-0028-1083915

Development of quantitative 31P NMR methods for the trapping of phenoxy & ketyl free radicals

Z Luca 1, DS Argyropoulos 2
  • 1Dipartimento di Scienze dell'Ambiente e del Territorio, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, 20125, Milano, Italy
  • 2Organic Chemistry of Wood Components Laboratory, Department of Forest Biomaterials Science & Engineering North Carolina State University, NC 123321, USA

Recent work in our laboratory [1] has allowed the development of Quantitative 31P-NMR spin trapping techniques. These methods have been demonstrated to be effective tools for the detection and absolute quantification of many Oxygen and Carbon centered free radical species. Our methods rests on the fact that free radicals react with the nitroxide phosphorous 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 standard. This powerful system was applied to better understand the mechanism of enzymatic oxidation of phenolic and non-phenolic model compounds resembling various lignin biosynthetic pathways. The mechanisms involved consists of H-abstraction reaction involving radical species, such as phenoxy and ketyl radical, which are very difficult intermediates to be detected and quantified with traditional EPR. Our methodology has been shown capable of being able to do just that. The 31P-NMR signals for the radical adducts of phenoxy radicals (PhO·) and ketyl radicals (PhC·CH3OH) were assigned and found to be 25.2 and 29.3 ppm respectively. Moreover to confirm their presence and identity, their mass spectra were also determined. Subsequently, the Spin Trap system was applied to the oxidation of phenols and apocinols in the presence of Peroxidases and 1-Hydroxybenzotriazole (HBT) as mediator. These efforts demonstrate the efficacy of our methodologies that rests on the fact that a variety of radical species can now be readily detected and quantified using quantitative 31P-NMR spin trapping techniques.

References: 1. Argyropoulos, D.S. et al. (2006) Bioorganic & Medicinal Chemistry 15:4017–4028.