Detection of singlet oxygen by EPR: The instability of the nitroxyl radicals

Abstract

The use of spin traps and redox probes coupled with electron paramagnetic resonance (EPR) is a method frequently applied in the evaluation of the efficiency of photosensitizers and photocatalysts in phototherapeutic and photocatalytic processes that involve reactive oxygen species. In this way, the method helps to clarify the mechanism behind photo-induced reactions. Hydroxy-TEMP is a very specific redox probe for selectively identifying and quantifying singlet oxygen (¹O₂). In this work, the kinetics of radical generated by the oxidation products of the Hydroxy-TEMP redox probe was analyzed from EPR spectra in aqueous solutions of several water-soluble porphyrins ([H₂T4MPyP](OTs)₄, Na₄[H₂T4SPP], [H₂T2MPyP](OTs)₄, [ZnT4MyPyP](OTs)₄, [MnT4MyPyP](OTs)₅, H₂T4CPP, and [H₂T4TriMAPP](OTs)4) under white light illumination. Different factors such as the concentration of the redox probe, pH of the medium, and photostability of the porphyrins were evaluated. A systematic study was carried out to reveal the factors associated with stable radical degradation (TEMPOL) by illumination in the visible spectral region in systems containing photosensitizer (porphyrin) and redox probe (Hydroxy-TEMP). With the aid of EPR and gas chromatography coupled with mass spectroscopy (GC-MS) techniques, the mechanism of the radical degradation and the photobleaching of porphyrins were investigated. After successive interactions with the porphyrin in its excited state, in alkaline aqueous solution (pH > 10), the free radical TEMPOL is transformed into TEMPONE until the final diamagnetic product Phorone. A protocol was elaborated to identify and quantify the generation of ¹O₂ by Hydroxy-TEMP reliably, to avoid possible errors in the interpretation of efficiency of photosensitizers.

Keywords: Degradation; Hydroxy-TEMP; Nitroxyl radical; Oxidation; Photodynamic therapy (PDT); Porphyrins; Redox probe; Singlet oxygen; Spin trapping.