Detection of ketyl radicals using ~(31)P NMR spin trapping

摘要

Our recent work has allowed the development of ~(31)P NMR spin trapping techniques for the detection and, at times, absolute quantification of many oxygen- and carbon-centered free radical species. These methods are based on the ability of the nitrone phosphorus compound, 5-diisopropoxy-phosphoryl-5- methyl-1-pyrroline-N-oxide (DIPPMPO), to react with free radical species and form stable radical adducts, which are suitably detected and accurately quantified using ~(31)P NMR. Our continuing efforts have now been focused on the application of this powerful system for the trapping of ketyl radicals, which are very difficult intermediates to be detected and quantified with traditional techniques (i.e., EPR). Ketyl radicals were initially produced using photochemical reactions of acetophenone, whose excited triplet state is able to abstract hydrogen from an H donor. As such, the ~(31)P NMR signals for the radical adducts of the DIPPMPO spin trap with the ketyl radicals were assigned. Furthermore, in an effort to confirm the structure of these adducts, their mass spectra and fragmentation patterns were carefully examined under Gas Chromatogra- phy-Mass Spectrometry (GC-MS) conditions. Subsequently, the DIPPMPO spin trapping system was applied to the oxidation of 1-(3,4-dimethoxyphenyl)ethanol in the presence of horseradish peroxidase (HRP), hydrogen peroxide, and 1-hydroxybenzotriazole (HBT) as the electron carrier (mediator). Our work confirmed that the mechanism consists of a hydrogen abstraction reaction from the α position, involving the ketyl radical: during the oxidation, the hydroxyl, hydroperoxyl, and ketyl radical intermediates were all detected. These efforts demonstrate the efficacy of our methodology that provides for the first time a facile means for the detection of the otherwise elusive ketyl radical species, with important implications in biology, chemistry, and biochemistry.