ESR characterization of a novel spin-trapping agent, 15N-labeled N-tert-butyl-alpha-phenylnitrone, as a nitric oxide donor

We previously found that one of the pharmacological effects of N-tert-butyl-α-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15 N to identify its corresponding 15 NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15 N-PBN, the starting material, ammonium- 15 N chloride, was converted to 2-amino- 15 N-2-methylpropane, oxidized to 2-methyl-2-nitropropane- 15 N, and finally reacted with benzaldehyde to give 15 N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD) 2 , as a trapping agent to measure the NO levels of 15 N-PBN or 14 N-PBN in vitro, the peak intensity of 15 NO[Fe(MGD) 2 ] was over 50% stronger than that of 14 NO[Fe(MGD) 2 ], and that 15 NO and 14 NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15 NO derived from 15 N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15 NO from PBN and 14 NO generated from cells. These results suggested that 15 N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.