Theoretical and Experimental Studies of the Spin Trapping of Inorganic Radicals by 5,5-Dimethyl-1-pyrroline <i>N</i>-Oxide (DMPO). 3. Sulfur Dioxide, Sulfite, and Sulfate Radical Anions

Radical forms of sulfur dioxide (SO₂), sulfite (SO₃^2–), sulfate (SO₄^2–), and their conjugate acids are known to be generated in vivo through various chemical and biochemical pathways. Oxides of sulfur are environmentally pervasive compounds and are associated with a number of health problems. There is growing evidence that their toxicity may be mediated by their radical forms. Electron paramagnetic resonance (EPR) spin trapping using the commonly used spin trap, 5,5-dimethyl-1-pyrroline <i>N</i>-oxide (DMPO), has been employed in the detection of SO₃^•– and SO₄^•–. The thermochemistries of SO₂^•–, SO₃^•–, SO₄^•–, and their respective conjugate acids addition to DMPO were predicted using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level. No spin adduct was observed for SO₂^•– by EPR, but an S-centered adduct was observed for SO₃^•–and an O-centered adduct for SO₄^•–. Determination of adducts as S- or O-centered was made via comparison based on qualitative trends of experimental hfcc’s with theoretical values. The thermodynamics of the nonradical addition of SO₃^2– and HSO₃^– to DMPO followed by conversion to the corresponding radical adduct via the Forrester–Hepburn mechanism was also calculated. Adduct acidities and decomposition pathways were investigated as well, including an EPR experiment using H₂¹⁷O to determine the site of hydrolysis of O-centered adducts. The mode of radical addition to DMPO is predicted to be governed by several factors, including spin population density, and geometries stabilized by hydrogen bonds. The thermodynamic data supports evidence for the radical addition pathway over the nucleophilic addition mechanism

Reactivities of Superoxide and Hydroperoxyl Radicals with Disubstituted Cyclic Nitrones: A DFT Study

The unique ability of nitrone spin traps to detect and characterize transient free radicals by electron paramagnetic resonance (EPR) spectroscopy has fueled the development of new spin traps with improved properties. Among a variety of free radicals in chemical and biological systems, superoxide radical anion (O₂^•–) plays a critical role as a precursor to other more oxidizing species such as hydroxyl radical (HO^•), peroxynitrite (ONOO^–), and hypochlorous acid (HOCl), and therefore the direct detection of O₂^•– is important. To overcome the limitations of conventional cyclic nitrones, that is, poor reactivity with O₂^•–, instability of the O₂^•– adduct, and poor cellular target specificity, synthesis of disubstituted nitrones has become attractive. Disubstituted nitrones offer advantages over the monosubstituted ones because they allow bifunctionalization of spin traps, therefore accommodating all the desired spin trap properties in one molecular design. However, because of the high number of possible disubstituted analogues as candidate, a systematic computational study is needed to find leads for the optimal spin trap design for biconjugation. In this paper, calculation of the energetics of O₂^•– and HO₂^• adduct formation from various disubstituted nitrones at PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level of theory was performed to determine the most favorable disubstituted nitrones for this reaction. In addition, our results provided general trends of radical reactivity that is dependent upon but not exclusive to the charge densities of nitronyl-C, the position of substituents including stereoselectivities, and the presence of intramolecular H-bonding interaction. Unusually high exoergic Δ<i>G</i>_298K,aq’s for O₂^•– and HO₂^• adduct formation were predicted for (3<i>S</i>,5<i>S</i>)-5-methyl-3,5-bis(methylcarbamoyl)-1-pyrroline <i>N</i>-oxide (<b>11</b>-<i>cis</i>) and (4<i>S</i>,5<i>S</i>)-5-dimethoxyphosphoryl-5-methyl-4-ethoxycarbonyl-1-pyrroline <i>N</i>-oxide (<b>29</b>-<i>trans</i>) with Δ<i>G</i>_298K,aq = −3.3 and −9.4 kcal/mol, respectively, which are the most exoergic Δ<i>G</i>_298K,aq observed thus far for any nitrone at the level of theory employed in this study

Guest Inclusion in Cucurbiturils Studied by ESR and DFT: The Case of Nitroxide Radicals and Spin Adducts of DMPO and MNP

We present an ESR and DFT study of the interaction of cucurbiturils CB[6], CB[7], and CB[8] with di-<i>tert</i>-butyl nitroxide ((CH₃)₃C)₂NO (DTBN) and with spin adducts of 5,5-dimethyl-1-pyrroline-<i>N</i>-oxide (DMPO) and 2-methyl-2-nitrosopropane (MNP). The primary goal was to understand the structural parameters that determine the inclusion mechanism in the CBs using DTBN, a nitroxide with great sensitivity to the local environment. In addition, we focused on the interactions with CBs of the spin adducts DMPO/OH and MNP/CH₂COOH generated in aqueous CH₃COOH. A <i>range</i> of interactions between DTBN and CBs was identified for pH 3.2, 7, and 10. No complexation of DTBN with CB[6] was deduced in this pH range. The interaction between DTBN and CB[7] is evident at all pH values: “in” and “out” nitroxides, with ¹⁴N hyperfine splitting, <i>a</i>_N, values of 15.5 and 17.1 G, respectively, were detected by ESR. Interaction of DTBN with CB[8] was also detected for all pH values, and the only species had <i>a</i>_N = 16.4 G, a result that can be rationalized by an “in” nitroxide in a less hydrophobic environment compared to CB[7]. Computational studies indicated that the DTBN complex with CB[7] is thermodynamically favored compared to that in CB[8]; the orientations of the NO group are parallel to the CB[7] plane and perpendicular to the CB[8] plane (pointing toward the annulus). Addition of sodium ions led to the ESR detection of a three-component complex between CB[7], DTBN, and the cations; the ternary complex was not detected for CB[8]. The DMPO/OH spin adduct was stabilized in the presence of CB[7], but the effect on <i>a</i>_N was negligible, indicating that the N–O group is located <i>outside</i> the CB cavity. Computational studies indicated more favorable energetics of complexation for DMPO/OH in CB[7] compared to DTBN. An increase of <i>a</i>_N was detected in the presence of CB[7] for the MNP/CH₂COOH adduct generated in CH₃COOH, a result that was assigned to the generation of the three-component radical between the spin adduct, sodium cations, and CB[7]

Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin Trapping Studies

Arsenic is one of the most environmentally significant pollutants and a great global health concern. Although a growing body of evidence suggests that reactive oxygen species (ROS) mediate the mechanism of arsenic toxicity, the exact mechanism remains elusive. In this study, we examine the capacity of trivalent arsenic species arsenous acid (iAs^III), monomethylarsonous acid (MMA^III), and dimethylarsinous acid (DMA^III) to generate ROS through a theoretical analysis of their structures, redox properties, and their reactivities to various ROS using a density functional theory (DFT) approach at the B3LYP/6-31+G**//B3LYP/6-31G* level of theory and by employing electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-<i>N</i>-oxide (DMPO) as a spin trap. Results show that the oxidized forms (As^IV) are structurally more stable compared to the reduced forms (As^II) that impart elongated As–O bonds leading to the formation of As^III and hydroxide anion. Enthalpies of one-electron reduction and oxidation indicate that increasing the degree of methylation makes it harder for As^III to be reduced but easier to be oxidized. The order of increasing favorability for arsenical activation by ROS is O₂ < O₂^•– < HO^•, and the oxidation of DMA^III to DMA^V is highly exoergic in multiple redox pathways with concomitant generation of radicals. This is followed by MMA^III and by iAs^III being the least favorable. Spin trapping studies showed a higher propensity for methylated arsenicals to generate radicals than iAs^III upon treatment with H₂O₂. However, in the presence of Fe^II,III, all showed radical generation where MMA^III gave predominantly C-centered adducts, while acidified iAs ^III and DMA^III gave primarily HO-adducts, and their formation was affected in the presence of SOD suggesting a As^III–OO/OOH radical intermediate. Therefore, our results suggest a basis for the increased redox activity of methylated arsenicals that can be applied to the observed trends in arsenic methylation and toxicity in biological systems

Kinetics and Mechanism of Ultrasonic Activation of Persulfate: An in Situ EPR Spin Trapping Study

Ultrasound (US) was shown to activate persulfate (PS) providing an alternative activation method to base or heat as an in situ chemical oxidation (ISCO) method. The kinetics and mechanism of ultrasonic activation of PS were examined in aqueous solution using an in situ electron paramagnetic resonance (EPR) spin trapping technique and radical trapping with probe compounds. Using the spin trap, 5,5-dimethyl-1-pyrroline-<i>N</i>-oxide (DMPO), hydroxyl radical (^•OH) and sulfate radical anion (SO₄^•–) were measured from ultrasonic activation of persulfate (US-PS). The yield of ^•OH was up to 1 order of magnitude greater than that of SO₄^•–. The comparatively high ^•OH yield was attributed to the hydrolysis of SO₄^•– in the warm interfacial region of cavitation bubbles formed from US. Using steady-state approximations, the dissociation rate of PS in cavitating bubble systems was determined to be 3 orders of magnitude greater than control experiments without sonication at ambient temperature. From calculations of the interfacial volume surrounding cavitation bubbles and using the Arrhenius equation, an effective mean temperature of 340 K at the bubble–water interface was estimated. Comparative studies using the probe compounds <i>tert</i>-butyl alcohol and nitrobenzene verified the bubble–water interface as the location for PS activation by high temperature with ^•OH contributing a minor role in activating PS to SO₄^•–. The mechanisms unveiled in this study provide a basis for optimizing US-PS as an ISCO technology

Reactive Nitrogen Species Reactivities with Nitrones: Theoretical and Experimental Studies

Reactive nitrogen species (RNS) such as nitrogen dioxide (^•NO₂), peroxynitrite (ONOO^–), and nitrosoperoxycarbonate (ONOOCO₂^–) are among the most damaging species present in biological systems due to their ability to cause modification of key biomolecular systems through oxidation, nitrosylation, and nitration. Nitrone spin traps are known to react with free radicals and nonradicals via electrophilic and nucleophilic addition reactions and have been employed as reagents to detect radicals using electron paramagnetic resonance (EPR) spectroscopy and as pharmacological agents against oxidative stress-mediated injury. This study examines the reactivity of cyclic nitrones such as 5,5-dimethylpyrroline <i>N</i>-oxide (DMPO) with ^•NO₂, ONOO^–, ONOOCO₂^–, SNAP, and SIN-1 using EPR. The thermochemistries of nitrone reactivity with RNS and isotropic hfsc's of the addition products were also calculated at the PCM­(water)/B3LYP/6-31+G**//B3LYP/6-31G* level of theory with and without explicit water molecules to rationalize the nature of the observed EPR spectra. Spin trapping of other RNS such as azide (^•N₃), nitrogen trioxide (^•NO₃), amino (^•NH₂) radicals and nitroxyl (HNO) were also theoretically and experimentally investigated by EPR spin trapping and mass spectrometry. This study also shows that other spin traps such as 5-carbamoyl-5-methyl-pyrroline <i>N</i>-oxide, 5-ethoxycarbonyl-5-methyl-pyrroline <i>N</i>-oxide, and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline <i>N</i>-oxide can react with radical and nonradical RNS, thus making spin traps suitable probes as well as antioxidants against RNS-mediated oxidative damage

Synthesis of Tris-hydroxymethyl-Based Nitrone Derivatives with Highly Reactive Nitronyl Carbon

A novel series of α-phenyl-<i>N</i>-<i>tert</i>-butyl nitrone derivatives, bearing a hydrophobic chain on the aromatic ring and three hydroxyl functions on the <i>tert</i>-butyl group, was synthesized through a short and convenient synthetic route based on a one-pot reduction/condensation of tris­(hydroxymethyl)­nitromethane with a benzaldehyde derivative. Because of the presence of hydroxyl functions on the <i>tert</i>-butyl group, an intramolecular Forrester–Hepburn reaction leading to the formation of an oxazolidine-<i>N</i>-oxyl compound was observed by electron paramagnetic resonance (EPR). The mechanism of cyclization was further studied by computational methods showing that intramolecular hydrogen bonding and high positive charge on the nitronyl carbon could facilitate the nucleophilic addition of a hydroxyl group onto the nitronyl carbon. At high nitrone concentrations, a second paramagnetic species, very likely formed by intermolecular nucleophilic addition of two nitrone molecules, was also observed but to a lesser extent. In addition, theoretical data confirmed that the intramolecular reaction is much more favored than the intermolecular one. These nitrones were also found to efficiently trap carbon-centered radicals, but complex spectra were observed due to the presence of oxazolidine-<i>N</i>-oxyl derivatives

Thiol-Dependent Reduction of the Triester and Triamide Derivatives of Finland Trityl Radical Triggers O₂‑Dependent Superoxide Production

Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV–vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O₂) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O₂ by the trityl carbanions was shown to be superoxide radical (O₂^•–) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (<i>k</i>) depend on the types of both trityl radicals and thiols with the order of <i>k</i>_ET‑03/Cys (0.336 M^–1 s^–1) > <i>k</i>_ET‑03/GSH (0.070 M^–1 s^–1) > <i>k</i>_AT‑03/Cys (0.032 M^–1 s^–1) > <i>k</i>_AT‑03/GSH (0.027 M^–1 s^–1). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the p<i>K</i>_a of the thiols and is further reflected by the rate of O₂^•– production and consumptions of O₂ and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes

Reactivities of Substituted α‑Phenyl‑<i>N</i>-<i>tert</i>-butyl Nitrones

In this work, a series of α-phenyl-<i>N</i>-<i>tert</i>-butyl nitrones bearing one, two, or three substituents on the <i>tert</i>-butyl group was synthesized. Cyclic voltammetry (CV) was used to investigate their electrochemical properties and showed a more pronounced substituent effect for oxidation than for reduction. Rate constants of superoxide radical (O₂^•–) reactions with nitrones were determined using a UV–vis stopped-flow method, and phenyl radical (Ph^•) trapping rate constants were measured by EPR spectroscopy. The effect of <i>N</i>-<i>tert</i>-butyl substitution on the charge density and electron density localization of the nitronyl carbon as well as on the free energies of nitrone reactivity with O₂^•– and HO₂^• were computationally rationalized at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory. Theoretical and experimental data showed that the rates of the reaction correlate with the nitronyl carbon charge density, suggesting a nucleophilic nature of O₂^•– and Ph^• addition to the nitronyl carbon atom. Finally, the substituent effect was investigated in cell cultures exposed to hydrogen peroxide and a correlation between the cell viability and the oxidation potential of the nitrones was observed. Through a combination of computational methodologies and experimental methods, new insights into the reactivity of free radicals with nitrone derivatives have been proposed

Synthesis and Characterization of PEGylated Trityl Radicals: Effect of PEGylation on Physicochemical Properties

Tetrathiatriarylmethyl (TAM, trityl) radicals have attracted considerable attention as spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging owing to their sharp EPR singlet signals and high biostability. However, their <i>in vivo</i> applications were limited by the short blood circulation lifetimes and strong binding with albumins. Our previous results showed that PEGylation is a feasible method to overcome the issues facing <i>in vivo</i> applications of TAM radicals. In the present study, we synthesized a series of new PEGylated TAM radicals (TTP1, TPP2, TNP1, TNP2, d-TNP1, and d-TNP3) containing various lengths and numbers of mPEG chains. Our results found that the pattern of PEGylation exerts an important effect on physicochemical properties of the resulting TAM radicals. Dendritic PEGylated TAM radicals, TNP1 and TNP2, have higher water solubility and lower susceptibility for self-aggregation than their linear analogues TPP1 and TPP2. Furthermore, dendritic PEGylated TAM radicals exhibit extremely high stability toward various biological oxidoreductants as well as in rat whole blood, liver homogenate, and following <i>in vivo</i> intravenous administration in mice. Importantly, the deuterated derivatives, especially d-TNP3, exhibit excellent properties including the sharp and O₂-sensitive EPR singlet signal, good biocompatibility, and prolonged kinetics with half-life time of ≥10 h in mice. These PEGylated TAM radicals should be suitable for a wide range of applications in <i>in vivo</i> EPR spectroscopy and imaging