EPR Study of KO2 as a Source of Superoxide and •BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H2S and Se-Derivatives

Superoxide radical anion (O2•−) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO2 as a source of O2•− and the electron paramagnetic resonance (EPR) spin trapping 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) technique for the preparation of •BMPO-OOH and/or •BMPO-OH radicals in water solution without DMSO. The method distinguishes the interactions of various compounds with •BMPO-OOH and/or •BMPO-OH radicals over time. Here, we show that the addition of a buffered BMPO-HCl mixture to powdered KO2 formed relatively stable •BMPO-OOH and •BMPO-OH radicals and H2O2, where the •BMPO-OOH/OH ratio depended on the pH. At a final pH of ~6.5–8.0, the concentration of •BMPO-OOH radicals was ≥20 times higher than that of •BMPO-OH, whereas at pH 9.0–10.0, the •BMPO-OH radicals prevailed. The •BMPO-OOH/OH radicals effectively cleaved the plasmid DNA. H2S decreased the concentration of •BMPO-OOH/OH radicals, whereas the selenium derivatives 1-methyl-4-(3-(phenylselanyl) propyl) piperazine and 1-methyl-4-(4-(phenylselanyl) butyl) piperazine increased the proportion of •BMPO-OH over the •BMPO-OOH radicals. In conclusion, the presented approach of using KO2 as a source of O2•−/H2O2 and EPR spin trap BMPO for the preparation of •BMPO-OOH/OH radicals in a physiological solution could be useful to study the biological effects of radicals and their interactions with compounds.

Lawsone Derivatives as Efficient Photopolymerizable Initiators for Free-Radical, Cationic Photopolymerizations, and Thiol—Ene Reactions

Two new photopolymerizable vinyl (2-(allyloxy) 1,4-naphthoquinone, HNQA) and epoxy (2-(oxiran-2yl methoxy) 1,4-naphthoquinone, HNQE) photoinitiators derived from lawsone were designed in this paper. These new photoinitiators can be used as one-component photoinitiating systems for the free-radical photopolymerization of acrylate bio-based monomer without the addition of any co-initiators. As highlighted by the electron paramagnetic resonance (EPR) spin-trapping results, the formation of carbon-centered radicals from an intermolecular H abstraction reaction was evidenced and can act as initiating species. Interestingly, the introduction of iodonium salt (Iod) used as a co-initiator has led to (1) the cationic photopolymerization of epoxy monomer with high final conversions and (2) an increase of the rates of free-radical polymerization of the acrylate bio-based monomer; we also demonstrated the concomitant thiol–ene reaction and cationic photopolymerizations of a limonene 1,2 epoxide/thiol blend mixture with the HNQA/Iod photoinitiating system.

Hydroxyl Radical Generation by the H2O2/CuII/Phenanthroline System under Both Neutral and Alkaline Conditions: An EPR/Spin-Trapping Investigation

The copper–phenanthroline complex CuI(Phen)2 was the first artificial nuclease studied in biology. The mechanism responsible for this activity involves CuII(Phen)2 and H2O2. Even if H2O2/Cu systems have been extensively studied in biology and oxidative chemistry, most of these studies were carried out at physiological pH only, and little information is available on the generation of radicals by the H2O2/CuII-Phen system. In the context of paper pulp bleaching to improve the bleaching ability of H2O2, this system has been investigated, mostly at alkaline pH, and more recently at near-neutral pH in the case of dyed cellulosic fibers. Hence, this paper aims at studying the production of radicals with the H2O2/CuII-Phen system at near-neutral and alkaline pHs. Using the EPR/spin-trapping method, HO• formation was monitored to understand the mechanisms involved. DMPO was used as a spin-trap to form DMPO–OH in the presence of HO•, and two HO• scavengers were compared to identify the origin of the observed DMPO–OH adduct, as nucleophilic addition of water onto DMPO leads to the same adduct. H2O2 decomposition was enhanced by the addition of CuII–Phen (and only slightly by addition of CuSO4), reaching a level similar to the Fenton reagent at near-neutral pH. This evidences the role of Phen, which improves the effect of CuII by tuning the electronic structure and structural properties of the corresponding CuII complexes.

Azacalixphyrins as an Innovative Alternative for the Free-Radical Photopolymerization under Visible and NIR Irradiation without the Need of Co-Initiators

Azacalixphyrins are unique aromatic macrocycles featuring strong absorption from the visible to the near-infrared (NIR) spectral ranges. This work demonstrates through EPR spin-trapping experiments that the N-alkyl tetrasubstituted azacalixphyrin (ACP)...

•BMPO-OOH Spin-Adduct as a Model for Study of Decomposition of Organic Hydroperoxides and the Effects of Sulfide/Selenite Derivatives. An EPR Spin-Trapping Approach

Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O2•−) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O2•− produces relatively stable •BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on •BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of •BMPO-OH/•BMPO-OOH—which is proportional to the transformation/decomposition of •BMPO-OOH—was evaluated. The order of potency of the studied compounds to alter •BMPO-OOH concentration estimated from the time-dependent •BMPO-OH/•BMPO-OOH ratio was as follows: Na2S4 > Na2S4/SeO32− > H2S/SeO32− > Na2S2 ~Na2S2/SeO32− ~H2S > SeO32− ~SeO42− ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of •BMPO-OH/•BMPO-OOH could be useful to study the impact of compounds to influence the transformation of •BMPO-OOH.

Photoreactivity of Bis-retinoid A2E Complexed with a Model Protein in Selected Model Systems

The bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is formed as a byproduct of visual cycle in retinal pigment epithelium (RPE). It contributes to golden-yellow fluorescence of the age pigment lipofuscin, which accumulates in RPE. Lipofuscin can generate a variety of reactive oxygen species (ROS) upon blue-light excitation. Although in model systems photoreactivity of A2E has been determined to be low, this bis-retinoid exhibited significant phototoxicity in RPE cells in vitro. Although the mechanism of A2E-mediated phototoxicity remains mostly unknown, we hypothesize that formation of A2E-adducts with different biomolecules may play an important role. In this study, we investigated the photochemical reactivity of A2E and its complex with bovine serum albumin (BSA) using UV–Vis absorption and emission spectroscopy, EPR-spin trapping, EPR-oximetry, time-resolved singlet oxygen phosphorescence, and the fluorogenic CBA probe. Our data show that A2E after complexation with this model protein photogenerated an increased level of ROS, particularly singlet oxygen. We also demonstrated the ability of A2E to oxidize BSA upon excitation with blue light in aqueous model systems. The data suggest that pyridinium bis-retinoid could oxidatively modify cellular proteins under physiological conditions.

Redox Interactions of Vitamin C and Iron: Inhibition of the Pro-Oxidant Activity by Deferiprone

Ascorbic acid (AscH2) is one of the most important vitamins found in the human diet, with many biological functions including antioxidant, chelating, and coenzyme activities. Ascorbic acid is also widely used in medical practice especially for increasing iron absorption and as an adjuvant therapeutic in iron chelation therapy, but its mode of action and implications in iron metabolism and toxicity are not yet clear. In this study, we used UV–Vis spectrophotometry, NMR spectroscopy, and EPR spin trapping spectroscopy to investigate the antioxidant/pro-oxidant effects of ascorbic acid in reactions involving iron and the iron chelator deferiprone (L1). The experiments were carried out in a weak acidic (pH from 3 to 5) and neutral (pH 7.4) medium. Ascorbic acid exhibits predominantly pro-oxidant activity by reducing Fe3+ to Fe2+, followed by the formation of dehydroascorbic acid. As a result, ascorbic acid accelerates the redox cycle Fe3+ ↔ Fe2+ in the Fenton reaction, which leads to a significant increase in the yield of toxic hydroxyl radicals. The analysis of the experimental data suggests that despite a much lower stability constant of the iron–ascorbate complex compared to the FeL13 complex, ascorbic acid at high concentrations is able to substitute L1 in the FeL13 chelate complex resulting in the formation of mixed L12AscFe complex. This mixed chelate complex is redox stable at neutral pH = 7.4, but decomposes at pH = 4–5 during several minutes at sub-millimolar concentrations of ascorbic acid. The proposed mechanisms play a significant role in understanding the mechanism of action, pharmacological, therapeutic, and toxic effects of the interaction of ascorbic acid, iron, and L1.