ESR Characterization of a Novel Spin-trapping Agent, ¹⁵N-Labeled N-tert-Butyl-α-phenylnitrone, as a Nitric Oxide Donor

Abstract

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 ¹⁵N to identify its corresponding ¹⁵NO in vivo. The properties were examined with an ESR spectrometer. To synthesize ¹⁵N-PBN, the starting material, ammonium-¹⁵N chloride, was converted to 2-amino-¹⁵N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-¹⁵N, and finally reacted with benzaldehyde to give ¹⁵N-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 ¹⁵N-PBN or ¹⁴N-PBN in vitro, the peak intensity of ¹⁵NO[Fe(MGD)₂] was over 50% stronger than that of ¹⁴NO[Fe(MGD)₂], and that ¹⁵NO and ¹⁴NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of ¹⁵NO derived from ¹⁵N-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 ¹⁵NO from PBN and ¹⁴NO generated from cells. These results suggested that ¹⁵N-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.

• nitric oxide
• N-tert-butyl-α-phenylnitrone (PBN)
• ESR
• oxidative stress
• spin trap