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  • br Experimental EPR spectra of probes a f were

    2024-07-10


    Experimental EPR spectra of probes 1a–f were recorded on a Bruker EMX-1572 operating at X-band (9.0–9.9 GHz), at 21 ± 1 °C. The EPR parameters were the same in all experiments: microwave power, 1 mW; modulation amplitude, 5 G; time constant, 10.24 ms; and conversion time, 40.96 ms. Reduced glutathione, l-ascorbic acid, (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic Budesonide sale (Trolox), p-cresol, α-tocopherol (vitamin E), 3,5-di-tert-4-butylhydroxytoluene (BHT) and reduced Triton X-100 were purchased from Sigma-Aldrich. Probes 1a–f were obtained as described in (Aliaga et al., 2016). The determination of the relative antioxidant effectiveness of glutathione dicarboxylate (2) and of p-cresol (5) vis-á-vis the radical probes 1a–f followed the same protocol described previously for the other antioxidants: l-ascorbate (3), trolox (4), α-tocopherol (6) and BHT (7) (Aliaga et al., 2016). Control solutions of probes 1a–f were prepared by adding 10 μl of a methanolic solution of the probe (1 mM) to a phosphate-buffered micellar solution (pH 7) of reduced Triton-X100 (11.0 wt%, 20 mM). The resulting solutions had a final volume of 200 μl and a probe concentration of 50 μM. Test solutions of probes 1a–f were prepared by adding 10 μl of a methanolic solution of the probe (1 mM) to a phosphate-buffered micellar solution (pH 7) of reduced Triton-X100 (11.0 wt%, 20 mM), containing 10 mM of AO. The resulting solutions had a final volume of 200 μl, a probe concentration of 50 μM and an AO concentration of 10 mM.
    Results and discussion Molecular modeling studies have shown that the amphiphobic probes 1a–f orient themselves differently in the Stern layer of a neutral micelle, and that this orientation is responsible for the different locations of their reacting nitroxyl fragment in this heterogeneous microenvironment. Fig. 1 is a schematic representation of these different orientations and locations, depending on the size of the 4-alkanoyl chain (Aliaga et al., 2016). The extreme members of the series, the most hydrophilic 1a and the most hydrophobic probe 1f, have their nitroxyl fragment located in a less hydrophobic environment than probe 1d, of intermediate hydrophobicity. This result is in agreement with the paradoxical observation that a probe with intermediate hydrophobicity shows the highest or the lowest reactivity vis-à-vis a given antioxidant. If the antioxidant resides in the hydrophobic micellar core, its proximity with this probe will increase its antioxidant effectiveness, if compared with the extreme members of the series. If it is located in the aqueous phase, the opposite situation will be observed, and its AO effectiveness towards the probe of intermediate hydrophobicity will be smaller than towards either 1a or 1 f. Thus, a plot of the relative antioxidant effectiveness as a function of the 4-alkanoyl chain-size will give rise to two different patterns, a concave or a convex curve, depending on the location of the antioxidant in a hydrophobic or a hydrophilic pseudo-phase, as shown schematically in Fig. 2. The above protocol was applied to six antioxidants of different hydrophobicities: glutathione (2), ascorbate (3), Trolox anion (4), p-cresol (5), α-tocopherol (vitamin E) (6) and BHT (7). Their structures are given in Scheme 1. They are depicted as neutral or anionic molecules, depending on their pKa’s and their ionization state in neutral aqueous solutions (pH = 7). Table 1 lists logP values of AOs 2–7, which may be used to quantify their hydrophobicities. Plots of the relative antioxidant effectiveness of each of these antioxidants towards radical probes 1, as a function of the increasing hydrophobic chain-length R of 1 are shown in Fig. 3. Inspection of Fig. 3 reveals a progressive shift from a convex to a concave plot, as the antioxidant hydrophobicity increases. Typically hydrophilic AOs, like glutathione dicarboxylate or the ascorbate anion, yield convex plots, while hydrophobic AOs, like α -tocopherol or BHT, exhibit concave curves, in agreement with the general criteria depicted in Fig. 2. Besides assuming a convex or concave form, the observed cut-off curves also vary in their degree of convexity, from highly convex (Fig. 3A and C) or concave (Fig. 3E and F) patterns, to rather shallow curves, as in Fig. 3B and D. Such a variation reflects the selectivity of the probe family vis-à-vis the antioxidant quencher. The cut-off effect is very pronounced for a highly hydrophilic AO like glutathione dicarboxylate (Fig. 3A) or a highly hydrophobic AO like BHT (Fig. 3F), being relatively less selective compared to p-cresol (Fig. 3D), with an intermediate hydrophobicity.