Copper(I)-phenolate complexes as models of the reduced active site of galactose oxidase: Synthesis, characterization, and O₂ reactivity

The Cu(I)-phenolate complexes ¹LCu and ²LCu and the Cu(I)-phenol complex [H²LCu(CNC₆H₃Me₂)] BArF₄ were prepared and structurally characterized by X-ray crystallography, where ¹L^- and ²L^- are ligands comprised of a 2,4-di-tert-butylphenolate linked to 1-isopropyl-1,5-diazacyclooctane or 1,4-diisopropyl-1,4,7-triazacyclononane, respectively. The reduced galactose oxidase (GAO) structural models ¹LCu and ²LCu were found to be highly reactive with O₂, and through combined stopped-flow kinetic and EPR, UV-vis, and resonance Raman spectroscopic studies of the oxygenation of ²LCu at low temperature, new intermediates relevant to those postulated for the active site oxidation step of the GAO catalytic cycle were identified. The oxygenation was shown by kinetics experiments to proceed via initial binding of O₂ to yield a green, unusually thermodynamically stable 1:1 adduct, ²LCu(O₂). Symmetric (η²) binding of a superoxo ligand was indicated by oxygen-isotope-sensitive features in resonance Raman spectra obtained in batch experiments; peaks at v(¹⁶O₂) = 1120 cm^-1, v(¹⁸O¹⁶O) = 1093 cm^-1, and v(¹⁸O₂) = 1058 cm^-1 were assigned as O-O stretching vibrations. These data represent the first experimental evidence for such superoxide coordination in complexes of tetradentate tripodal ligands and provide new precedent for how O₂ may bind at the reduced GAO active site. The 1: 1 Cu/O₂ adduct subsequently evolves into a metastable purple species that is only observable under conditions of substoichiometric O₂. The kinetics of formation of this transient species are second order overall (rate = k₂^′[²LCu(O₂)] [²L1Cu]). It exhibits an absorption band with λ_max = 565 nm (ε = 17,900 M^-1 cm^-1) and multiple oxygen-isotope-sensitive v(Cu-O) and v(O-O) features in the respective regions 500-550 cm^-1 and 700-850 cm^-1 in Raman spectra, with excitation-wavelength-dependent intensities that correlate with the 565 nm absorption feature. On the basis of the combined data available, the presence of multiple isomeric peroxodicopper species in the transient purple solution is postulated.