Observation of oxygenated intermediates in functional mimics of aminophenol dioxygenase

Abstract

Aminophenol dioxygenases (APDO) are mononuclear nonheme iron enzymes that utilize dioxygen (O₂) to catalyze the conversion of o-aminophenols to 2-picolinic acid derivatives in metabolic pathways. This study describes the synthesis and O₂ reactivity of two synthetic models of substrate-bound APDO: [Fe^II(Tp^Me2)(^tBu2APH)] (1) and [Fe^II(Tp^Me2)(^tBuAPH)] (2), where Tp^Me2 = hydrotris(3,5-dimethylpyrazole-1-yl)borate, ^tBu2APH = 4,6-di-tert-butyl-2-aminophenolate, and ^tBuAPH₂ = 4-tert-butyl-2-aminophenolate. Both Fe(II) complexes behave as functional APDO mimics, as exposure to O₂ results in oxidative CC bond cleavage of the o-aminophenolate ligand. The ring-cleaved products undergo spontaneous cyclization to give substituted 2-picolinic acids, as verified by ¹H NMR spectroscopy, mass spectrometry, and X-ray crystallography. Reaction of the APDO models with O₂ at low temperature reveals multiple intermediates, which were probed with UV–vis absorption, electron paramagnetic resonance (EPR), Mössbauer (MB), and resonance Raman (rRaman) spectroscopies. The most stable intermediate at −70 °C in THF exhibits multiple isotopically-sensitive features in rRaman samples prepared with ¹⁶O₂ and ¹⁸O₂, confirming incorporation of O₂-derived atom(s) into its molecular structure. Insights into the geometric structures, electronic properties, and spectroscopic features of the observed intermediates were obtained from density functional theory (DFT) calculations. Although functional APDO models have been previously reported, this is the first time that an oxygenated ligand-based radical has been detected and spectroscopically characterized in the ring-cleaving mechanism of a relevant synthetic system.