Figure 4.

U0126 directly reacts with hydroxyl radicals produced by Fenton’s reaction and horseradish peroxidase (HRP). (a) ¹H NMR and mass spectrometry show that U0126 does not react with H₂O₂ directly. Addition of Fe(II) results in the appearance of downfield peaks in NMR and new peaks of m/z at 362 and 378 in mass spectra. (b) Mixing U0126, H₂O₂, and Fe(II) in vitro (Fenton reaction) shows bright orange precipitation within 2 min. Control experiments show that mixing U0126 with Fe(II) sulfate results in a faint yellowish color after 3 h, while mixing H₂O₂ and Fe(II) does not induce any color change. (c) Mixing U0126, H₂O₂, and horseradish peroxidase in vitro produces an orange-brown precipitate within seconds. (d) Lipophilicity assay of the reaction products shows that the intense orange-brown compound in the lipophilic phase (n-octanol) as compared to the aqueous phase in the bottom layer. (e) Fluorescence emission spectra of the crude reaction mixture in the n-octanol phase and in the water phase.