Phenoxy radical detection using 31P NMR spin trapping

Recent work in our laboratory has allowed the development of quantitative ³¹P NMR spin trapping techniques. These methods have been demonstrated to be effective tools for the detection and absolute quantification of many oxygen‐ and carbon‐centered free radical species. Our methods rest on the fact that a free radical reacts with the nitroxide phosphorus compound, 5‐diisopropoxy‐phosphoryl‐5‐methyl‐1‐pyrroline‐N‐oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using ³¹P NMR in the presence of phosphorus containing internal standards. This system was applied for the detection of phenoxy radicals, as an alternative to traditional EPR techniques. More specifically, the phenoxy radicals were produced via the oxidation of different phenols by K₃Fe(CN)₆. The ³¹P NMR signals for the radical adducts of phenoxy radicals (PhO·) were assigned and found to be located at 25.2 ppm. Subsequently, this spin trapping system was applied to the oxidation of various phenols in the presence of peroxidases and 1‐hydroxybenzotriazole (HBT) as a mediator: the 2,4,6‐trichlorophenol and 2,4,6‐tri‐tert‐butylphenol were oxidized and only phenoxy radical adducts were detected, whereas during the oxidation of 2,4‐dimethylphenol and isoeugenol, other adducts were detected and related to radical delocalization. These preliminary efforts demonstrate the efficacy of our methodologies, so that a variety of radical species can now be readily detected and quantified using quantitative ³¹P NMR spin trapping techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

NMR Spin Trapping: Insight into the Hidden Life of Free Radical Adducts

In our original work (V. Khramtsov, L.J. Berliner and T.L. Clanton, Magn. Reson. Med. 42:228??34, 1999), a ³¹P nuclear magnetic resonance spin trapping (ST NMR) approach aimed to overcome comparatively short lifetimes of the paramagnetic adducts by detecting stable diamagnetic adducts of the degradation of phosphorous-containing nitrone was proposed. High stability of the NMR adducts of the nitrone spin traps with C-centered radicals and the ability to track their origin from paramagnetic adducts make ST NMR a valuable tool for the studies of these radicals in chemical and biological systems. Complementary ST NMR and electron paramagnetic resonance studies of the nitrone adducts derived from the addition of O- and S-centered radicals and nucleophiles revealed new insights into the pathways of the paramagnetic and diamagnetic adduct formation and degradation. In particular, use of ST NMR allowed for observation of reversible nucleophilic addition to the nitrones. On one hand, nucleophilic addition may result in a possible spin trapping artifact via the Forrester–Hepburn mechanism; whereas, on the other hand, the reverse reaction provides the key step in the “recycling??mechanism that is important for the antioxidant actions of the nitrones.     

Development of fluorinated,NMR-active spin traps for studies of free radical chemistry

Five fluorinated analogs of the spin trap phenyl-t-butyl nitrone (PBN) have been synthesized and evaluated for use as NMR-active traps. The introduction of the fluorine substituent allows selective observation of chemical reactions involving the spin traps. Although the paramagnetic adducts themselves are not directly observable by this approach as a consequence of extensive broadening, the reduced forms (hydroxylamines) can be readily observed. NMR studies of the trapping of the phenyl radical produced from the thermal decomposition of phenylazotriphenyl methane have been carried out. The observation of fluorinated benzaldehydes in these studies reflects the formation and subsequent degradation of oxygen-centered radicals under some conditions. Relative trapping efficiencies for the phenyl radical in the series 2-F, 4-F, 2,6-difluoro, 2-CF₃, and 4-CF₃ substituted PBN analogs have been determined based on an analysis of the ¹⁹F NMR resonance intensities of the reduced phenyl radical adducts. The relatively large proton hyperfine coupling constants observed for 2,6-difluoro and 2-CF₃ PBN analogs allow direct observation by ESR of adduct formation in solutions containing both PBN and either of these analogs. The introduction of fluorine substituents into the trap has only a small effect on trapping efficiency.     

Alkylperoxyl spin adducts of pyrroline‐N‐oxide spin traps: Experimental and theoretical CASSCF study of the unimolecular decomposition in organic solvent,potential applications in water

Spin trapping coupled with electron paramagnetic resonance spectroscopy is the most direct method for detection of very low concentrations of free radicals, and it has been intensively used to detect superoxide or alkylperoxyl radicals in biological systems, using cyclic nitrones as spin traps. The half‐life time of the ensuing spin adducts depends dramatically on the spin trap structure; however, their mechanism of decay has never been definitely established. We investigated at the MRMP2/CASSCF (MultiReference second‐order Møller‐Plesset perturbation theory/Complete Active Set Self Consistent Field) level of theory the mechanism of decay of methylperoxyl and tert‐butylperoxyl spin adducts formed with various cyclic nitrones. We showed that no transition state can be located for the O─O homolytic cleavage, which yields an intermediate biradical with the following sequence ^?O─N─C─O^?. Then, homolytic cleavage of the N─C bond yields a nitrosoaldehyde, through an early transition state with a very low activation energy. For each nitrone used as spin trap, electrospray ionization mass spectrometry analysis of the spin trapping mixture allowed to detect the presence of the expected nitrosoaldehyde. We generated tert‐butylperoxyl spin adducts in toluene, and we found a good correlation between their half‐life time and the bond dissociation energy of their peroxidic O─O bond. The theoretical method was then extended to hydroperoxyl spin adducts in water and provided promising results.     

Time‐resolved resonance Raman and density functional theory study of the photochemistry of 4‐benzoylpyridine in acetonitrile and 2‐propanol

A nanosecond time‐resolved resonance Raman (ns‐TR³) spectroscopic investigation of the intermolecular hydrogen‐abstraction reaction of the triplet state of 4‐benzoylpyridine (4‐BPy) in 2‐propanol solvent is reported. The TR³ results reveal a rapid hydrogen abstraction (<10 ns) by the 4‐BPy triplet state (nπ*) with the 2‐propanol solvent, leading to formation of a 4‐BPy ketyl radical and an associated dimethyl ketyl radical partner from the solvent. The recombination of these two radical species occurs with a time constant about 200 ns to produce a para‐N‐LAT (light absorbing transient). The structure, major spectral features, and identification of the ketyl radical and the para‐N‐LAT coupling complex have been determined and confirmed by comparison of the TR³ results with results from density functional theory (DFT) calculations. A reaction pathway for the photolysis of 4‐BPy in 2‐propanol deduced from the TR³ results is also presented. The electron‐withdrawing effect of the heterocyclic nitrogen for 4‐BPy on the triplet state makes it have a significantly higher chemical reactivity for the hydrogen abstraction with 2‐propanol compared to the previously reported corresponding benzophenone triplet reaction under similar reaction conditions. In addition, the 4‐BPy ketyl radical reacts with the dimethyl ketyl radical to attach at the para‐N atom position of the pyridine ring to form a cross‐coupling product such as 2‐[4‐(hydroxy‐phenyl‐methylene)‐4h‐pyridin‐1‐yl]‐propan‐2‐ol instead of attacking at the para‐C atom position as was observed for the corresponding benzophenone reaction reported in an earlier study. Copyright © 2008 John Wiley & Sons, Ltd.     

Pulse-shift measurement: a direct observation of radical pair dynamics in a micelle

The hydrogen abstraction reactions of benzophenone in a micellar solution in the absence and presence of 1,4-cyclohexadiene are compared with time-resolved electron spin resonance (ESR) and optically detected (OD) ESR. “Pulse shift” measurement by OD-ESR, which observes the effect of a resonant microwave pulse at different delay times after laser excitation, reveals that the lifetime of the radical pair becomes much shorter in the presence of 1,4-cyclohexadiene. This explains the change of chemically induced dynamic electron polarization from spin-correlated radical pair polarization in the absence of 1,4-cyclohexadiene to conventional, E^*/A polarization in its presence. The rate constants determined by this technique indicate that the escaping rate of cyclohexadienyl radical is ten times larger than those of alkyl and benzophenone ketyl radicals.     

某些含氮有机物光催化氧化产生的自由基中间体

本文用自旋捕捉技术与ESR相结合的方法研究了某些含氮有机物TiO₂光催化降解的过程中形成的活泼中间体.选择的化合物中有丁胺、戊胺、庚胺、六氢吡啶、吡啶、2-甲基吡啶、2,6-二甲基吡啶,这些分子中的氮原子分别作为初级脂肪胺、饱和环中的二级胺以及芳环中的三级胺出现,实验中观察到N-中心自由基及C-中心自由基与自旋捕捉剂加合物的ESR信号,表明这些自由基参与这些化合物光降解的初始过程,对深入了解其光降机理是有意义的.     

Involvement of triplet state in the photodissociation of hydrogen peroxide: experimental evidence from time-resolved EPR study

The dissociation of photoexcited hydrogen peroxide to generate a pair of hydroxyl radicals is generally believed to take place in a repulsive electronic singlet state. The results presented here, based on time-resolved EPR experiments on the spin polarisation pattern of the acetone ketyl radical (CH₃)₂C^?OH, generated on photodissociation of H₂O₂ in 2-propanol with a 248?nm laser light, strongly indicate significant involvement of a repulsive triplet state of excited hydrogen peroxide.     

Electron spin polarisation in the photoreduction of xanthone in alcohol: effect of concentration and temperature

Time-resolved EPR studies of the hydrogen abstraction reaction of photoexcited xanthone in 2-propanol were carried out as a function of the concentration of xanthone and the sample temperature. The temperature was varied from 22°C to about ?30°C, and the concentration from about 0.2 to 4.0?mM. At low temperature or concentration, the observed spectra of the xanthone ketyl radical and the propan-2-olyl radical could be simulated as a superposition of a hyperfine-independent component due to the emissive triplet mechanism and a hyperfine-dependent component due to the S–T₀ radical pair mechanism. However, with an increase in the concentration of xanthone, the relative contribution of TM decreases, and, concomitantly, the net absorptive component of only the xanthone ketyl radical increases. As the spin polarisation mechanisms do not predict any concentration dependence, this unusual behaviour is explained by invoking the enhancement of the spin–lattice relaxation rates due to Heisenberg spin exchange occurring at high local concentrations of the radicals. The net absorptive signal is attributed to thermally equilibrated radicals. The observed temperature dependence of the spin polarisation behaviour is similarly explained. The origin of the net absorptive signal in the TREPR spectra of the acetone?2-propanol system is also attributed to thermally equilibrated radicals. The self-quenching mechanism of xanthone is proposed to be an electron-transfer reaction from an excited xanthone molecule to another xanthone in the ground state.     

二苯甲酮与醚、酚的光化夺氢反应中产生的活泼自由基的ESR研究

本文用具立体阻碍的自旋捕捉剂2,4,6,-三-特丁基亚硝基苯(TBN)与ESR相结合的方法,研究了七种醚及四种酚与二苯甲酮光化夺氢反应中产生的活泼自由基。从ESR谱的超精细结构确证,处于激发态的二苯甲酮是夺取醚中接于氧的a-c上的氢; ?₂CO+ROR→?₂?OH+R'?HOR 而且只是其中的R'?HOR可与TBN形成(Ⅰ)型及(Ⅱ)型两种自由基加合物: 对于苯酚衍生物而言,二苯甲酮却是夺取酚羟基中的质子而形成R-O-?,它与TBN也形成(Ⅰ)型及(Ⅱ)型两种自由基加合物。     

二苯甲酮与烃的光化夺氢反应中产生的活泼自由基的ESR研究

本文用具立体阻碍的自旋捕捉剂2,4,6-三-特丁基亚硝基苯(TBN)与ESR相结合的方法,研究了十种烷烃及四种芳烃与二苯甲酮光化夺氢反应中产生的活泼自由基。结果表明,R₃R₂R₁Ċ和TBN形成烷氧苯胺自由基加合物。R-ĊH-R'与TBN形成烷氧苯胺自由基加合物和氮氧自由基加合物。TBN可用于区分有、无空间位阻的烷烃,芳烃自由基。自旋加合物的ESR波谱可表明自由基中未偶电子所在碳原子附近的有效质子数目。     

卷烟烟气中活性自由基的自旋捕获ESR研究

卷烟烟气中的瞬态自由基对人体健康是相当有害的，而检测活性自由基最常用的方法是自旋捕获方法. 本研究中使用高效新型捕捉剂DEPMPO能够直接在水相中捕捉到烷基自由基与羟基自由基，并未发现烷氧自由基加合物. 考虑到DEPMPO对活性自由基的捕捉能力和加合物ESR谱图解析特征性都优于PBN与DMPO，且在有机溶剂中溶解较多的氧分子. 因此认为以往文献中所捕获的烷氧自由基来源于烷基自由基氧化后的次级自由基产物.     

Detection of free radicals produced by a pulsed electrohydraulic discharge using electron spin resonance

The detection of free radicals such as hydroxyl radical and hydrogen radical for plasma in solution induced by a pulsed electrohydraulic discharge are successfully performed using electron spin resonance measurement. The plasma reactor is a barrier-type and consists of a stainless needle high-voltage (+35 to 65 kV) electrode partially immersed in the solution and a Pyrex glass solution container around which an aluminum film grounded electrode is wrapped. Streamers are induced in the solution. After adding iron (II) sulfate or radical trapping agent before the plasma application, the spectrum for radicals is clearly detected. A reactive dye solution is dramatically decolorized.     

Proton Exchange Effects in Laser Photolystic P-Benzosemiquinone Spin Polarized Radicals

The TRESR spectra of benzosemiquinone radical and the ethylene glycoi ketyl radical formed from laser photolysis of P-benzoquinone in ethylene glycoi or ethylene glycol/water systems are presented. The photon exchange between p-benzosemiquinone radicals PBQH⁺ and their protonated forms PBQH⁺ is studied by adding H₂SO₄ to the solution. The experimental results show that different hyperfine lines for PBQH⁺ have different time-dependence which depend upon the fraction of the overall number of nuclear spin states, and that the lines with smaller fraction are decay fast.     

Simulation of EPR Spectra as a Tool for Interpreting the Degradation Pathway of Hyaluronan

Electron paramagnetic resonance (EPR) spin trapping is one of the choice techniques for identifying free radicals and is often used in the study of biological systems. However, its sensitivity can result in a typical complicated EPR spectrum. The accurate simulation of these systems is essential for correct identification of the radical species, whenever more than one species contributes to the spectrum. Programs implementing the linear combination of single simulations allow the interpretation of EPR spectra without modifying experimental conditions. In this study, this approach was used to investigate the influence of the ferrous ion and the role of oxygen, as well on the formation of transient radical species, in the whole mechanism of hyaluronan degradation. Degradation was carried out under different environmental conditions (air, O₂, Ar, N₂, N₂ + CO₂) and EPR spin trapping studies were performed. The advantages of the simulation of multiple species EPR spectra were applied to the obtained results and some aspects of hyaluronan degradation mechanism were elucidated. The depolymerization reaction pathway has been defined according to two possible subsequent steps: the first is consistent with formation of an amidyl radical that induces a series of strand scissions, which stabilize at two different levels of molecular weight. The second step occurs when the molecular weight is lower than before and two different adducts are generated.     

EPR spectroscopy of fullerene adducts

EPR spectroscopy has been used to identify and characterize the paramagnetic adducts of small free radicals with C₆₀ through measurements of the hyperfine interactions of protons and¹³C nuclei. The initially formed mono-radical adducts (RC₆₀) have unpaired spin density localized near the point of attachment of the radical. Generally, they are in thermal equilibrium with their diamagnetic dimer, and have a surprisingly large barrier to internal rotation about the new bond. The tri- and penta-radical adducts have electronic structures similar to those of allyl and cyclopentadienyl radicals, respectively.NRCC No. 35262     

醛类的光分解与光氧化初级过程的ESR研究 取代苯甲醛的研究

本文采用自由基捕捉技术与ESR相结合的方法,研究了14种芳香醛的紫外光解及光氧化的初过程,结果表明:1.10种取代芳香醛的光解中主要活性自由基为芳酰基ϕĊO。2.光氧化初级过程(即引发过程)与光解所产生之自由基相同。但光氧化中自由基积累速度比光解为快,因此肯定由于氧参与了自由基的引发,从而加速其进程。3.有羟基取代的芳香醛,未检出自由基。     

ESR-spin trapping study on the sonochemistry of liquids in the presence of oxygen. Evidence for the superoxide radical anion formation

The sonolysis of water and some organic liquids such as ethylene glycol, methanol and chloroform in the presence of oxygen, at 20 and 475 kHz ultrasound frequencies has been investigated by the ESR-spin trapping technique. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), 3,3,5,5-tetramethylpyrroline-N-oxide (TMPO) and N-tert-butyl-alpha-phenyl nitrone (PBN) were able to trap superoxide radical anion, generated as the result of the sonication of the organic media. The addition of superoxide dismutase (SOD) resulted in a dramatic decrease of the ESR signal intensity of the superoxide radical adduct. In addition, the thermolysis of the liquids under ultrasound was shown by ESR detection of the spin adducts of the radicals formed by homolytic fragmentation. Occasionally, the nature of the detected spin adduct was dependent on the sonication time or on the frequency of the ultrasonic radiation. Experiments carried out in the presence of 2-methyl-2-nitrosopropane (MNP) resulted in the detection of radicals originating from thermal decomposition of the spin trap, showing its lability under ultrasonic radiation.     

Radical reorientation dynamics studied by positive-muon avoided level crossing resonance

Spin polarised positive muons substituted for a hydrogen nucleus in organic radicals are used as spin labels to sense reorientational motion of the radicals in orienting environments. The effect of uniaxial motion on the spectrum of the muonated cyclohexadienyl radical in a benzene single crystal and of its hexamethyl derivative in polycrystalline hexamethylbenzene is demonstrated. Isotopic spectra for cyclohexadienyl radicals adsorbed on SiO₂ are explained by translational diffusion around the 7 nm grain. The potential of the technique to study transient radical intermediates of processes in heterogeneous catalysis is discussed.     

Free radical scavenging activities of white and red wines: An EPR spin trapping study

Free radical scavenging activities of wine samples were investigated by means of electron paramagnetic resonance (EPR) spin trapping. 1,1-Diphenyl-2-picrylhydrazyl served as a source of free radicals, OH radicals were produced by the Fenton system, and in situ thermally generated SO₄⁻ radicals from K₂S₂O₈ and carbon-centered radicals from azo-compounds were also used. 5,5-Dimethylpyrroline-N-oxide (DMPO) was used as a spin trapping agent. Three white and four red wine samples from the Slovak wine regions, along with 12% (v/v) aqueous ethanol solution as a reference sample, were compared. In general, the lowest scavenger activity was found in the reference sample, a higher one in the white, and a considerably higher one in the red wines, except when the Fenton system was used as the radical source. Here, unexpectedly, unlike in the other radical sources, the wine samples increased the yield of DMPO-adducts by about 5 times, compared to the reference sample. This is probably due to an additional activation of the Fenton system by wine components. Higher concentrations of Mn²⁺ ions as determined by EPR spectroscopy corresponded to an increased scavenging activity of wines.