Novel glutathione-linked nitrones as dual free radical probes

The detection and identification of transient radicals in biological systems is of importance for the understanding of their roles in a variety of biological processes. Electron paramagnetic resonance spectroscopy coupled with the use of the spin trapping technique has been an indispensable tool for this application owing to its high specificity. In this study, we developed a general method using dual function free radical probes (GS-PBN and its phosphorylated analogue GS-PPN) for the simultaneous determination of transient radicals and the microenvironment where the corresponding spin adducts are situated. This conception was initially proved by high spectral sensitivity of the p-ClPh ^• spin adduct of GS-PBN towards its rotational motion in the glycerol-water system. Results showed that a relatively bulky glutathionyl group in the spin adduct plays an important role in its high sensitivity to the molecular motion. This was further verified by high sensitivity of the p-ClPh^• spin adduct of the newly synthesized probe GS-PPN to its molecular motion. Unlike GS-PBN, GS-PPN can be used to detect O ₂^•- generated in the enzymatic system and PSII membranes of chloroplasts. Based on the relationship between the τ_C values of the superoxide spin adduct and the medium viscosity, the local environment of the adduct in the PSII membranes was determined to be similar to that of the aqueous solution containing ∼15% glycerol (η ≈ 1.33 p).