Reaction of Chromium(V) with the EPR Spin Traps 5,5-Dimethylpyrroline N-Oxide and Phenyl-N-tert-butylnitrone Resulting in Direct Oxidation

Experiments were performed to investigate potential biologically relevant oxidation reactions of a model Cr(V) complex, sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (Na[Cr(ehba)₂O]), with the spin traps 5,5-dimethylpyrroline N-oxide (DMPO) and phenyl-N-tert-butylnitrone (PEN). Electron paramagnetic resonance spectroscopy (EPR) has shown that, under physiological conditions, 100 mM HEPES, pH = 7.4, the reaction of Cr(V) with DMPO resulted in the formation of the hydroxyl radical adduct of DMPO, DMPO/*OH. In the presence of 1.0 M ETOH, this reaction did not display the characteristic α-alcohol adduct of DMPO, implying that DMPO/*OH formation arises from a direct oxidative mechanism and not from a discrete diffusible radical such as the hydroxyl radical, *OH. Upon addition of hydrogen peroxide to Cr(V) and DMPO, a significant increase of the DMPO/*OH adduct was observed, which can be ascribed to the formation of a diffusible radical, *OH. Under acidic conditions, 100 mM acetic acid/sodium acetate, pH = 3.0-6.0, reaction of Cr(V) with DMPO or PEN yielded no EPR-detectable radicals, but led to decreased stability of Cr(V). Product analysis of the reaction of Cr(V) with PBN by HPLC showed cleavage of the imine moiety of the nitrone to form benzaldehyde. Experiments using ¹⁷O-and ¹⁸O-labeled water coupled with IR and EPR spectroscopy were used to examine exchange of the oxo group on Cr(V) and thus to postulate a mechanism for the different pH-dependent reactions. The results suggest that, at physiological pH, nitrone coordination of Cr(TV) induces nucleophilic attack of water at the β-position of DMPO resulting in homolytic bond cleavage and radical formation. At lower pH's, an even electron mechanism predominates and is best accounted for by an oxo-atom transfer from Cr(V) to the spin trap resulting in C=N oxidative cleavage but no radical formation. These same mechanisms may be responsible for the reactions observed between Cr(V) species, generated by the in vivo reduction of Cr(VT), and DNA under various conditions and may be important in Cr(VI) carcinogenesis.