Iron-ascorbate complex formation and redox behavior in Tris buffer under aerobic and anaerobic conditions: relevance...

Iron-ascorbate (Fe-Asc) is a clinically approved drug for iron-deficiency anemia (IDA), yet its formation and redox behavior under varying conditions remain poorly understood. Herein, we report the first comprehensive examination of Fe-Asc formation and redox reactivity in Tris buffer as a function of stoichiometry, pH, and oxygen availability. Oxidation of Fe(II) in air-saturated buffer accelerates with pH, yielding multiple species, primarily di- and tri-nuclear clusters. Notably, FeSO₄ with AscH produces a distinct purple complex (λ_max = 510 nm), whose assembly depends critically on O₂, 1:3 stoichiometry of Fe: AscH, and buffer pH 7.5, whereas no such species forms anaerobically. ESI-MS suggests trinuclear clusters, whose stability and redox dynamics are monitored by time-resolved absorbance. The purple color disappears with dithionite or H₂O₂, but dithionite-treated samples regain color upon air exposure, demonstrating redox reversibility. Electrochemically, FeSO₄ shows cathodic (-0.32 V) and anodic (-0.06 V) peaks vs. Ag/AgCl, but the cathodic current decreases markedly in presence of AscH, reflecting Fe-Asc interactions. EPR spectra of FeSO₄ and FeSO₄/AscH are similar, with g = 9.43, 4.37 (high-spin Fe(III), S = 5/2) and g = 1.92 (S = ½, [Fe(III)-Fe(II)] coupling). These results suggest the formation of mixed valence trinuclear clusters with a dynamic ligand environment.