Theoretical and experimental studies of the spin trapping of inorganic radicals by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 1. Carbon dioxide radical anion

The carbon dioxide radical anion (CO2*-) is known to be generated in vivo through various chemical and biochemical pathways. Electron paramagnetic resonance (EPR) spin trapping with the commonly used spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been employed in the detection of CO2*-. The thermodynamics of CO2*- addition to DMPO was predicted using density functional theory (DFT) at the B3LYP/6-31++G**//B3LYP/6-31G* and B3LYP/6-311+G* levels with the polarizable continuum model (PCM) to simulate the effect of the bulk dielectric effect of water on the calculated energetics. Three possible products of CO2*- addition to DMPO were predicted: (1) a carboxylate adduct, (2) pyrroline-alcohol and (3) DMPO-OH. Experimentally, UV photolysis of H2O2 in the presence of sodium formate (NaHCO2) and DMPO gave an EPR spectrum characteristic of a C-centered carboxylate adduct and is consistent with the theoretically derived hyperfine coupling constants (hfcc). The pKa of the carboxylate adduct was estimated computationally to be 6.4. The mode of CO2*- addition to DMPO is predicted to be governed predominantly by the spin (density) population on the radical, whereas electrostatic effects are not the dominant factor for the formation of the persistent adduct. The thermodynamic behavior of CO2*- in the aqueous phase is predicted to be similar to that of mercapto radical (*SH), indicating that formation of CO2*- in biological systems may have an important role in the initiation of oxidative damage in cells.     

Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 3. Effect of mildly acidic pH on the thermodynamics and kinetics of adduct formation

The nitrone, 5,5-dimethylpyrroline N-oxide (DMPO), is a commonly used spin trap for the detection of superoxide radical anion (O2*-) using electron paramagnetic resonance spectroscopy. This work investigates the reactivity of DMPO to O2*- in mildly acidic pH (5.0-7.0). Mild acidity is characteristic of acidosis and has been observed in hypoxic systems, e.g., ischemic organs and cancer cells. Although the established pKa for O2*- is 4.8, the pKa for DMPO is unknown. The pKa of the conjugate acid of DMPO was determined to be 6.0 using potentiometric, spectrophotometric, 1H and 13C NMR, and computational methods. 1H and 13C NMR were employed to investigate the site of protonation. An alternative mechanism for the spin trapping of O2*- in mildly acidic pH was proposed, which involves protonation of the oxygen to form the N-hydroxy imino cation and subsequent addition of O2*-. The exoergicity of O2*- addition to protonated DMPO was rationalized using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory.     

Reactivity of superoxide radical anion and hydroperoxyl radical with alpha-phenyl-N-tert-butylnitrone (PBN) derivatives

Nitrones have exhibited pharmacological activity against radical-mediated pathophysiological conditions and as analytical reagents for the identification of transient radical species by electron paramagnetic resonance (EPR) spectroscopy. In this work, competitive spin trapping, stopped-flow kinetics, and density functional theory (DFT) were employed to assess and predict the reactivity of O(2)(*-) and HO(2)(*) with various para-substituted alpha-phenyl-N-tert-butylnitrone (PBN) spin traps. Rate constants of O(2)(*-) trapping by nitrones were determined using competitive UV-vis stopped-flow method with phenol red (PR) as probe, while HO(2)(*) trapping rate constants were calculated using competition kinetics with 5,5-dimethylpyrroline N-oxide (DMPO) by employing EPR spectroscopy. The effects of the para substitution on the charge density of the nitronyl-carbon and on the free energies of nitrone reactivity with O(2)(*-) and HO(2)(*) were computationally rationalized at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level of theory. Theoretical and experimental data show that the rate of O(2)(*-) addition to PBN derivatives is not affected by the polar effect of the substituents. However, the reactivity of HO(2)(*) follows the Hammett equation and is increased as the substituent becomes more electron withdrawing. This supports the conclusion that the nature of HO(2)(*) addition to PBN derivatives is electrophilic, while the addition of O(2)(*-) to PBN-type compounds is only weakly electrophilic.     

Theoretical study of the spin trapping of hydroxyl radical by cyclic nitrones: a density functional theory approach

The hydroxyl radical (*OH) is an important mediator of biological oxidative stress, and this has stimulated interest in its detection. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) and its alkoxycarbonyl and alkoxyphosphoryl analogues have been employed as spin traps for electron paramagnetic resonance (EPR) spectroscopic radical detection. Energies of optimized geometries of nitrones and their corresponding *OH adducts were calculated using density functional theory (DFT) at the B3LYP/6-31+G//B3LYP/6-31G level. Calculations predict that the trans adduct formation is favored in alkoxycarbonyl nitrones, while cis adducts with intramolecular H-bonding is favored for alkoxyphosphoryl nitrones. Addition of *OH to a phosphoryl-substituted nitrone is more exoergic than the carbonylated nitrones. Charge and spin densities on the nitrone spin traps were correlated with their rates of addition with *OH, and results show that the charge density on the nitronyl C, the site of *OH addition, is more positive in phosphorylated nitrones compared to DMPO and the alkoxycarbonyl nitrones. The dihedral angle between the beta-H and nitroxyl O bonds is smaller in phosphorylated nitrones, and that aspect appears to account for the longer half-lives of the spin adducts compared to those in DMPO and alkoxycarbonyl nitrones. Structures of nitrones with trifluoromethyl-, trifluoromethylcarbonyl-, methylsulfonyl-, trifluoromethylsulfonyl-, amido-, spiropentyl-, and spiroester substituents were optimized and their energies compared. Amido and spiroester nitrones were predicted to be the most suitable nitrones for spin trapping of *OH due to the similarity of their thermodynamic and electronic properties to those of alkoxyphosphoryl nitrones. Moreover, dimethoxyphosphoryl substitution at C-5 was found to be the most efficient substitution site for spin trapping of *OH, and their spin adducts are predicted to be the most stable of all of the isomeric forms.     

Theoretical and experimental studies of the spin trapping of inorganic radicals by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 3. Sulfur dioxide, sulfite, and sulfate radical anions

Radical forms of sulfur dioxide (SO(2)), sulfite (SO(3)(2-)), sulfate (SO(4)(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 N-oxide (DMPO), has been employed in the detection of SO(3)(?-) and SO(4)(?-). The thermochemistries of SO(2)(?-), SO(3)(?-), SO(4)(?-), 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 SO(2)(?-) by EPR, but an S-centered adduct was observed for SO(3)(?-)and an O-centered adduct for SO(4)(?-). 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 SO(3)(2-) and HSO(3)(-) 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 H(2)(17)O 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.     

The fidelity of spin trapping with DMPO in biological systems

Unlike direct ESR, spin trap methodology depends on the absolute fidelity of the spin trap reaction. Two alternative reactions of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) leading to radical adduct artifacts have been discovered and investigated: inverted spin trapping and the Forrester-Hepburn nucleophilic mechanism. These two alternate pathways to radical adducts are a combination of one-electron oxidation and nucleophilic addition, in either order. In biological systems, serious artifacts have been reported due to the Forrester-Hepburn mechanism, which is initiated by the addition of a nucleophile to DMPO. It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) can react with DMPO via a nonradical, nucleophilic reaction, and it has been further proposed that DMPO/(?)SO(3)(-) formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical ((?)SO(3)(-)). The one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase (HRP)/hydrogen peroxide (H(2)O(2)) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the absence of DMPO, the initial rate of (bi)sulfite-dependent oxygen and H(2)O(2) consumption was determined to be half of the initial rate of DMPO/(?)SO(3)(-) radical adduct formation as determined by ESR, demonstrating that, under our experimental conditions, DMPO exclusively forms the radical adduct by trapping the (?)SO(3)(-).     

Reactive oxygen species scavenging ability of a new compound derived from weathered coal

The scavenging activity of three fulvic acids (named XWCS-1, XWCS-4, and XWCS-8 according to time taken for ozonolysis) obtained by ozonolysis of humic acid extracted from Xinjiang (China) weathered coal and a fulvic acid (named XWCFA) extracted from the same coal towards reactive oxygen species such as superoxide radical (O(2)(.)(-)) and hydroxyl radical ((.)OH) was investigated with an electron spin resonance (ESR)-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. O(2)(.)(-) was generated with a hypoxanthine-xanthine oxidase system. (.)OH was generated by three different methods; (i) FeSO(4)-hydrogen peroxide (H(2)O(2)) system, (ii) Cu(en)(2)-H(2)O(2) system, and (iii) UVB photolysis of H(2)O(2). At physiological pH, XWCS-1 had the greatest O(2)(.)(-) scavenging activity, followed by XWCS-4, XWCS-8 and XWCFA. XWCFA had the greatest ?OH scavenging activity among the four fulvic acids, whereas XWCS-1 and XWCS-4 enhanced the production of (.)OH from a metal-catalyzed hydroxyl radical generating system, suggesting that these molecules act as prooxidants in the presence of metal ion.     

Comparative DFT study of the spin trapping of methyl, mercapto, hydroperoxy, superoxide, and nitric oxide radicals by various substituted cyclic nitrones

The thermodynamics of the spin trapping of various cyclic nitrones with biologically relevant radicals such as methyl, mercapto, hydroperoxy, superoxide anion, and nitric oxide was investigated using computational methods. A density functional theory (DFT) approach was employed in this study at the B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level. The order of increasing favorability for Delta G(rxn) (kcal/mol) of the radical reaction with various nitrones, in general, follows a trend similar to their respective experimental reduction potentials as well as their experimental second-order rate constants in aqueous solution: NO (14.57) < O2*- (-7.51) < *O2H (-13.92) < *SH (-16.55) < *CH3 (-32.17) < *OH (-43.66). The same qualitative trend is predicted upon considering the effect of solvation using the polarizable continuum model (PCM): i.e., NO (14.12) < O2*- (9.95) < *O2H (-6.95) < *SH (-13.57) < *CH3 (-32.88) < *OH (-38.91). All radical reactions with these nitrones are exoergic, except for NO (and O2*- in the aqueous phase), which is endoergic, and the free energy of activation (Delta G) for the NO additions ranges from 17.7 to 20.3 kcal/mol. This study also predicts the favorable formation of certain adducts that exhibit intramolecular H-bonding interactions, nucleophilic addition, or H-atom transfer reactions. The spin density on the nitronyl N of the superoxide adducts reveals conformational dependences. The failure of nitrones to trap NO at normal conditions was theoretically rationalized due to the endoergic reaction parameters.     

Theoretical study on the reaction mechanism of (CH3)3CO(.) radical with NO

The reaction mechanism of (CH3)3CO(.) radical with NO is theoretically investigated at the B3LYP/6-31G* level. The results show that the reaction is multi-channel in the single state and triplet state. The potential energy surfaces of reaction paths in the single state are lower than that in the triple state. The balance reaction: (CH3)3CONO←→ (CH3)3CO(.)+NO, whose potential energy surface is the lowest in all the reaction paths, makes the probability of measuring (CH3)3CO(.) radical increase. So NO may be considered as a stabilizing reagent for the (CH3)3CO(.)radical.     

Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline n-oxide (DMPO). 1. The thermodynamics of formation and its acidity

The nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been the most widely used spin trap for the detection of transient free radicals in chemical, biological, and biomedical research using electron paramagnetic resonance (EPR) spectroscopy. A density functional theory (DFT) approach was used to predict the thermodynamics of formation of the superoxide anion/hydroperoxyl radical (O2*-/*O2H) adduct of DMPO as well as its pK(a) in aqueous systems. At the B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level, we predicted (in the gas phase and with a polarizable continuum model (PCM) for water) three conformational minima for both the DMPO-O2- and DMPO-O2H adducts. Using DFT and the PCM solvation method, the pK(a) of DMPO-O2H was predicted to be 14.9 +/- 0.5. On the basis of free energy considerations, the formation of DMPO-O2H at neutral pH proceeds via initial addition of O2*- to DMPO to form the DMPO-O2- adduct and then subsequent protonation by water (or other acidic sources) to form DMPO-O2H. Under acidic conditions, the addition of *O2H to DMPO is predicted to be more exoergic than the addition of O2*- and is consistent with available experimental kinetic data.     

Rate constants of hydroperoxyl radical addition to cyclic nitrones: a DFT study

Nitrones are potential synthetic antioxidants against the reduction of radical-mediated oxidative damage in cells and as analytical reagents for the identification of HO2* and other such transient species. In this work, the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) and PCM/mPW1K/6-31+G(d,p) density functional theory (DFT) methods were employed to predict the reactivity of HO2* with various functionalized nitrones as spin traps. The calculated second-order rate constants and free energies of reaction at both levels of theory were in the range of 100-103 M-1 s-1 and 1 to -12 kcal mol-1, respectively, and the rate constants for some nitrones are on the same order of magnitude as those observed experimentally. The trend in HO2* reactivity to nitrones could not be explained solely on the basis of the relationship of the theoretical positive charge densities on the nitronyl-C, with their respective ionization potentials, electron affinities, rate constants, or free energies of reaction. However, various modes of intramolecular H-bonding interaction were observed at the transition state (TS) structures of HO2* addition to nitrones. The presence of intramolecular H-bonding interactions in the transition states were predicted and may play a significant role toward a facile addition of HO2* to nitrones. In general, HO2* addition to ethoxycarbonyl- and spirolactam-substituted nitrones, as well as those nitrones without electron-withdrawing substituents, such as 5,5-dimethyl-pyrroline N-oxide (DMPO) and 5-spirocyclopentyl-pyrroline N-oxide (CPPO), are most preferred compared to the methylcarbamoyl-substituted nitrones. This study suggests that the use of specific spin traps for efficient trapping of HO2* could pave the way toward improved radical detection and antioxidant protection.     

Photohydroxylation of 1,4-benzoquinone in aqueous solution revisited

In water, photolysis of 1,4-benzoquinone, Q gives rise to equal amounts of 2-hydroxy-1,4-benzoquinone HOQ and hydroquinone QH(2) which are formed with a quantum yield of Phi=0.42, independent of pH and Q concentration. By contrast, the rate of decay of the triplet (lambda(max)=282 and approximately 410 nm) which is the precursor of these products increases nonlinearly (k=(2-->3.8) x 10(6) s(-1)) with increasing Q concentration ((0.2-->10) mM). The free-radical yield detected by laser flash photolysis after the decay of the triplet also increases with increasing Q concentration but follows a different functional form. These observations are explained by a rapid equilibrium of a monomeric triplet Q* and an exciplex Q(2)* (K=5500+/-1000 M(-1)). While Q* adds water and subsequent enolizes into 1,2,4-trihydroxybenzene Ph(OH)(3), Q(2)* decays by electron transfer and water addition yielding benzosemiquinone (.)QH and (.)OH adduct radicals (.)QOH. The latter enolizes to the 2-hydroxy-1,4-semiquinone radical (.)Q(OH)H within the time scale of the triplet decay and is subsequently rapidly (microsecond time scale) oxidized by Q to HOQ with the concomitant formation of (.)QH. On the post-millisecond time scale, that is, when (.)QH has decayed, Ph(OH)(3) is oxidized by Q yielding HOQ and QH(2) as followed by laser flash photolysis with diode array detection. The rate of this pH- and Q concentration-dependent reaction was independently determined by stopped-flow. This shows that there are two pathways to photohydroxylation; a free-radical pathway at high and a non-radical one at low Q concentration. In agreement with this, the yield of Ph(OH)(3) is most pronounced at low Q concentration. In the presence of phosphate buffer, Q* reacts with H(2)PO(4) (-) giving rise to an adduct which is subsequently oxidized by Q to 2-phosphato-1,4-benzoquinone QP. The current view that (.)OH is an intermediate in the photohydroxylation of Q has been overturned. This view had been based on the observation of the (.)OH adduct of DMPO when Q is photolyzed in the presence of this spin trap. It is now shown that Q*/Q(2)* oxidizes DMPO (k approximately 1 x 10(8) M(-1) s(-1)) to its radical cation which subsequently reacts with water. Q*/Q(2)* react with alcohols by H abstraction (rates in units of M(-1) s(-1)): methanol (4.2 x 10(7)), ethanol (6.7 x 10(7)), 2-propanol (13 x 10(7)) and tertiary butyl alcohol ( approximately 0.2 x 10(7)). DMSO (2.7 x 10(9)) and O(2) ( approximately 2 x 10(9)) act as physical quenchers.     

自由基HO2与C2H2反应势能面的理论研究

应用量子化学从头计算和密度泛函理论(DFT)对HO2+C2H2反应体系的反应机理进行了研究.在B3LYP/6-311G**和CCSD(T)/6-311G**水平上计算了HO2+ C2H2反应的二重态反应势能面.计算结果表明,主要反应方式为自由基HO2的H原子和C2H2分子中的C原子结合,经过一系列异构化,最后分解得到主要产物P1 (CH2O+ HCO).此反应是放热反应,化学反应热为-321.99 kJ·mol-1.次要产物为P2 (CO2 +CH3),也是放热反应.     

在光动力光疗中类卟啉稀土配合物光敏作用原初反应的特征: 产生自由基I型机制的ESR研究

本文用ESR方法研究了类卟啉稀土配合物[(CO2H-APPC)Gd]Cl2的光敏反应。用4-hydro-tetramethylpiperidine-N-oxide radical(4-hydro-TEMPO)作探针, 通过对其消自旋的作用, 证实[(CO2H-APPC)Gd]Cl2光敏反应中有阳离子自由基[(CO2H-APPC)Gd]^+产生, 加入还原剂可促使[(CO2H-APPC)Gd]^+生成。经由5, 5-Dimethyl-1-pyrrolineN-oxide(DMPO)对超氧阴离子(O2^-)和羟基自由基(.OH)的自旋捕捉及对该自旋加合物[DMPO-O2^-]和[DMPO-OH]的ESR测定, 证实有O2^-和.OH产生, 并用SOD清除O2^-和甲酸钠清除.OH的实验, 进一步证实O2^-和.OH的产生。上述结果说明[(CO2H-APPC)Gd]Cl2光敏反应存在着产生[(CO2H-APPC)Gd]^+和活性氧自由基的I型机制。     

Reactions of hydrated electron with various radicals: spin factor in diffusion-controlled reactions

The reactions of hydrated electron (eaq-) with various radicals have been studied in pulse radiolysis experiments. These radicals are hydroxyl radical (*OH), sulfite radical anion (*SO3-), carbonate radical anion (CO3*-), carbon dioxide radical anion (*CO2-), azidyl radical (*N3), dibromine radical anion (Br2*-), diiodine radical anion (I2*-), 2-hydroxy-2-propyl radical (*C(CH3)2OH), 2-hydroxy-2-methyl-1-propyl radical ((*CH2)(CH3)2COH), hydroxycyclohexadienyl radical (*C6H6OH), phenoxyl radical (C6H5O*), p-methylphenoxyl radical (p-(H3C)C6H4O*), p-benzosemiquinone radical anion (p-OC6H4O*-), and phenylthiyl radical (C6H5S*). The kinetics of eaq- was followed in the presence of the counter radicals in transient optical absorption measurements. The rate constants of the eaq- reactions with radicals have been determined over a temperature range of 5-75 degrees C from the kinetic analysis of systems of multiple second-order reactions. The observed high rate constants for all the eaq- + radical reactions have been analyzed with the Smoluchowski equation. This analysis suggests that many of the eaq- + radical reactions are diffusion-controlled with a spin factor of 1/4, while other reactions with *OH, *N3, Br2*-, I2*-, and C6H5S* have spin factors significantly larger than 1/4. Spin dynamics for the eaq-/radical pairs is discussed to explain the different spin factors. The reactions with *OH, *N3, Br2*-, and I2*- have also been found to have apparent activation energies less than that for diffusion control, and it is suggested that the spin factors for these reactions decrease with increasing temperature. Such a decrease in spin factor may reflect a changing competition between spin relaxation/conversion and diffusive escape from the radical pairs.     

Quantum chemical analysis of the unfolding of a penta-glycyl 3(10)-helix initiated by HO(●), HO2(●), and O2(-●)

In this study, the thermodynamic functions of hydrogen abstraction from the C(α) and amide nitrogen of Gly(3) in a homo-pentapeptide (N-Ac-GGGGG-NH(2); G5) by HO(●), HO(2)(●), and O(2)(-●) were computed using the Becke three-parameter Lee-Yang-Parr (B3LYP) density functional. The thermodynamic functions, standard enthalpy (ΔH°), Gibbs free energy (ΔG°), and entropy (ΔS°), of these reactions were computed with G5 in the 3(10)-helical (G5(Hel)) and fully-extended (G5(Ext)) conformations at the B3LYP/6-31G(d) and B3LYP/6-311+G(d,p) levels of theory, both in the gas phase and using the conductor-like polarizable continuum model implicit water model. H abstraction is more favorable at the C(α) than at the amide nitrogen. The secondary structure of G5 affects the bond dissociation energy of the H-C(α), but has a negligible effect on the dissociation energy of the H-N bond. The HO(●) radical is the strongest hydrogen abstractor, followed by HO(2)(●), and finally O(2)(-●). The secondary structure elements, such as H-bonds in the 3(10)-helix, protect the peptide from radical attack by disabling the potential electron delocalization at the C(α), which is possible when G5 is in the extended conformation. The unfolding of the peptide radicals is more favorable than the unfolding of G5(Hel); however, only the HO(●) can initiate the unfolding of G5(Hel) and the formation of G5(Ext)(●). These results are relevant to peptides that are prone to undergoing transitions from helical structures to β-sheets in the cellular condition known as "oxidative stress" and the results are discussed in this context.     

Spin trapping by 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO): theoretical and experimental studies

The nitrone 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO) was synthesized and characterized. Spin trapping of various radicals by AMPO was demonstrated for the first time by electron paramagnetic resonance (EPR) spectroscopy. The resulting spin adducts for each of these radicals gave unique spectral profiles. The hyperfine splitting constants for the superoxide adduct are as follows: isomer I (80%), a(nitronyl)(-)(N) = 13.0 G and a(beta)(-)(H) = 10.8 G; isomer II (20%), a(nitronyl)(-)(N) = 13.1 G, a(beta)(-)(H) = 12.5 G, and a(gamma)(-)(H) = 1.75 G. The half-life of the AMPO-O(2)H was about 8 min, similar to that observed for EMPO but significantly shorter than that of the DEPMPO-O(2)H with t(1/2) approximately 16 min. However, the spectral profile of AMPO-O(2)H at high S/N ratio is distinguishable from the spectrum of the (*)OH adduct. Theoretical analyses using density functional theory calculations at the B3LYP/6-31+G//B3LYP/6-31G level were performed on AMPO and its corresponding superoxide adduct. Calculations predicted the presence of intramolecular H-bonding in both AMPO and its superoxide adduct. The H-bonding interaction was further confirmed by an X-ray structure of AMPO, and of the novel and analogous amido nitrone 2-amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH(2)-AMPO). The thermodynamic quantities for superoxide radical trapping by various nitrones have been found to predict favorable formation of certain isomers. The measured partition coefficient in an n-octanol/buffer system of AMPO was similar to those of DMPO and DEPMPO. This study demonstrates the suitability of the AMPO nitrone for use as a spin trap to study radical production in aqueous systems.     

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 (O(2)(?-)) 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 O(2)(?-) is important. To overcome the limitations of conventional cyclic nitrones, that is, poor reactivity with O(2)(?-), instability of the O(2)(?-) 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 O(2)(?-) and HO(2)(?) 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 ΔG(298K,aq)'s for O(2)(?-) and HO(2)(?) adduct formation were predicted for (3S,5S)-5-methyl-3,5-bis(methylcarbamoyl)-1-pyrroline N-oxide (11-cis) and (4S,5S)-5-dimethoxyphosphoryl-5-methyl-4-ethoxycarbonyl-1-pyrroline N-oxide (29-trans) with ΔG(298K,aq) = -3.3 and -9.4 kcal/mol, respectively, which are the most exoergic ΔG(298K,aq) observed thus far for any nitrone at the level of theory employed in this study.     

Spin trapping by 5,5-dimethylpyrroline-N-oxide in Fenton media in the presence of Nafion perfluorinated membranes: limitations and potential

Spin trapping by 5,5-dimethylpyrroline-N-oxide (DMPO) was used for the detection of radicals in Fenton media in the presence and absence of Nafion perfluorinated ionomers. For ethanol as solvent, the same types of spin adducts were detected in the presence or absence of Nafion. Solvent-derived adducts, DMPO/*OC2H5 and DMPO/*CH(OH)CH3, were identified, and their presence was rationalized by Fe(III)-catalyzed nucleophilic addition of ethanol to the spin trap and hydrogen abstraction by *OH radicals; oxygen radical adducts, DMPO/*O2(-) and DMPO/*OOH, were also detected. In Fenton media with methanol as solvent (and no Nafion), the DMPO/*O2(-) adduct dominated immediately after sample preparation, and a mixture consisting of DMPO/*OCH3, DMPO/*CH3, DMPO/*O2(-), and DMPO/*OOH adducts was detected after 30 min. In the presence of Nafion, only the adduct DMPO/*OH was detected. For water as solvent, only the DMPO/*OH adduct was detected, in both the absence and the presence of Nafion. The full hyperfine tensor components of this adduct were determined in Fenton media in the presence of Nafion with water and methanol as solvents. In Nafion/water exposed to the Fenton reagent at 358 K for 3 h, a DMPO adduct of a carbon-centered radical was also identified and assigned to a Nafion-derived fragment; its exact nature is under investigation. Variations of the 14N and Hbeta hyperfine splittings of a given adduct with the local polarity were key to the identification of some DMPO adducts, in particular DMPO/*O2(-). Both *OOH and O2*- adducts, with different 14N and Hbeta splittings, were detected simultaneously in some samples, for the first time in the spin trapping literature. Comparison with the results of a direct electron spin resonance study of Nafion exposed to the Fenton reagent indicated that spin trapping by DMPO can provide complementary information on the type of radicals present during Nafion degradation. The spin trapping approach described in this paper is limited, however, to systems that do not contain organic solvents.     

正十六烷光催化降解的羟自由基测定及其反应速率常数

以5,5-二甲基-1-吡咯啉-N-氧化物(DMPO)为自旋捕集剂,采用电子顺磁共振(EPR)方法,在光照的TiO₂磷酸缓冲水溶液(pH=7.4)中检测到羟自由基的自旋加合物(DMPO-OH),其强度随光照时间增加而加大.在1min时达到稳态,此时DMPO-OH的产生和猝灭达到平衡.根据已知的羟自由基(HO·)与DMPO结合的速率常数k0,推导出纳米级TiO₂光催化生成羟自由基氧化正十六烷(n-C_{16H34)的速率常数k=5.0×10¹¹mol^-1·L·s^-1.}