Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay

The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R=H(2)N(HN)C-C(CH(3))(2)). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern-Volmer's plot) for each spin trap showed good linearity, and their slopes, k (SB)/k (ST), were estimated to be 87.7±2.3, 267±15, 228±9, and 213±16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k (SB)/k (ST) values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay.     

Synthesis and spin-trapping behavior of 5-ChEPMPO, a cholesteryl ester analogue of the spin trap DEPMPO

[structure: see text] 5-(Cholesteryloxyethoxyphosphoryl)-5-methylpyrroline N-oxide (5-ChEPMPO), a DEPMPO analogue bearing a cholesterol group on the phosphorus atom, has been prepared and used to trap peroxyl-, alkoxyl-, thiyl-, and carbon-centered radicals in organic solvent. The important steric hindrance in 5-ChEPMPO does not affect the properties of 5-ChEPMPO in comparison to DEPMPO for the spin trapping of an enantiopure linoleic acid hydroperoxide. The 5-ChEPMPO-OOL spin adduct was observed by ESR and confirmed by ESI-MS/MS experiments. The relaxation terms of the 5-ChEPMPO-lipid peroxyl spin adduct were compared with those of other peroxyl spin adducts, and it was shown that the cholesteryl group has only a weak influence on the exchange rate between adduct conformers.     

Singlet Oxygen–mediated Hydroxyl Radical Production in the Presence of Phenols: Whether DMPO–·OH Formation Really Indicates Production of ·OH?¶

The reaction of singlet oxygen (1O2) generated by ultraviolet-A (UVA)-visible light (lambda > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-*OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO-*OH decreased in the presence of either a *OH scavenger or a quencher of *O2 and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both 1O2- and DMPO-mediated formation of free *OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that 1O2, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO-*OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of *OH. These observations indicate that 1O2 reacts first with DMPO, and the resulting DMPO-1O2 intermediate is immediately decomposed/reduced to give *OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of 1O2 to *OH. Furthermore, DEPMPO did not cause the spin-trapping agent-mediated generation of *OH like DMPO did.     

Synthesis and Spin‐Trapping Properties of a Trifluoromethyl Analogue of DMPO: 5‐Methyl‐5‐trifluoromethyl‐1‐pyrroline N‐Oxide (5‐TFDMPO)

The 5‐diethoxyphosphonyl‐5‐methyl‐1‐pyrroline N‐oxide superoxide spin adduct (DEPMPO?OOH) is much more persistent (about 15 times) than the 5,5‐dimethyl‐1‐pyrroline N‐oxide superoxide spin adduct (DMPO?OOH). The diethoxyphosphonyl group is bulkier than the methyl group and its electron‐withdrawing effect is much stronger. These two factors could play a role in explaining the different half‐lifetimes of DMPO?OOH and DEPMPO?OOH. The trifluoromethyl and the diethoxyphosphonyl groups show similar electron‐withdrawing effects but have different sizes. We have thus synthesized and studied 5‐methyl‐5‐trifluoromethyl‐1‐pyrroline N‐oxide (5‐TFDMPO), a new trifluoromethyl analogue of DMPO, to compare its spin‐trapping performance with those of DMPO and DEPMPO. 5‐TFDMPO was prepared in a five‐step sequence by means of the Zn/AcOH reductive cyclization of 5,5,5‐trifluoro‐4‐methyl‐4‐nitropentanal, and the geometry of the molecule was estimated by using DFT calculations. The spin‐trapping properties were investigated both in toluene and in aqueous buffer solutions for oxygen‐, sulfur‐, and carbon‐centered radicals. All the spin adducts exhibit slightly different fluorine hyperfine coupling constants, thereby suggesting a hindered rotation of the trifluoromethyl group, which was confirmed by variable‐temperature EPR studies and DFT calculations. In phosphate buffer at pH 7.4, the half‐life of 5‐TFDMPO?OOH is about three times shorter than for DEPMPO?OOH and five times longer than for DMPO?OOH. Our results suggest that the stabilization of the superoxide adducts comes from a delicate balance between steric, electronic, and hydrogen‐bonding effects that involve the β group, the hydroperoxyl moiety, and the nitroxide.     

Synthesis,crystal structure,and ESR study of a novel phosphorylated lipophilic spin trap

In this article, the synthesis of a novel alpha-phosphorus-containing spin trap DEPPEPO [2-(diethoxyphosphoryl)-2-phenethyl-3,4-dihydro-2H-pyrrole-1-oxide] and the evaluation of its ability to spin-trap radicals, especially superoxide and hydroxyl radicals, are described. Single crystal X-ray structure analysis reveals that there exist a lot of intramolecular nonbonded attractive interactions in the molecule. The phenethyl group is located away from the diethoxyphosphoryl group and the nitronyl plane, and only one face of the nitronyl plane is sterically hindered by the oxygen attached to the phosphorus with a double bond. The latter feature is responsible for the stereoselection of the free radical additions on the nitronyl moiety. The ability of DEPPEPO to trap the active superoxide anion radical generated in the HX/XO system and the stability of their spin adduct were investigated with that for DEPMPO. The half-life of DEPPEPO is about 13.4 min, and as a result, the DEPPEPO seems to be a promising lipophilic spin trap, perhaps in both in vitro and in vivo ESR investigation. Because DEPPEPO is a solid compound, it is quite easy to purify by recrystallization and to store the compound even at room temperature. In addition, an obvious increase in lipophilicity for DEPPEPO was found as a contribution of 2-substituted phenethyl.     

Evidence of an electron-transfer mechanism in the peroxynitrite-mediated oxidation of 4-alkylphenols and tyrosine

The mechanism of interaction of the peroxynitrite with some 4-alkylphenols and tyrosine was mainly studied by means of ESR spectroscopy and product analysis. The radical intermediates, detected as spin adducts to the 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), were identified as carbon-centered radicals to the benzene ring. The reaction seems to proceed via an electron transfer process (ET), most likely mediated by a NOx derivative, leading to the intermediacy of a phenoxyl-type radical as proved by the detection of the corresponding Pummerer-type ketone. No evidence of the formation of hydroxyl radicals, due to the homolytic cleavage of the peroxynitrite at physiological pH was obtained, even though DEPMPO hydroxyl spin adducts were detected: the latter most likely arises from the direct attack of the spin trap by the oxidant species. The possible involvement of HCO(3)(-)/CO(2), i.e., the formation of the nitrosoperoxycarbonate, ONOOCO(2)(*)(-), was also investigated.     

可见光响应光触媒的测试研究

在液-固相光催化反应体系,采用自旋捕捉-电子自旋共振（ESR）技术,进行可见光响应光触媒的测试研究。在光催化反应体系中加入自由基捕捉剂5-二乙氧基磷酰基-5-甲基-1-吡咯啉-氮氧化物（DEPMP0）,测试不同的光辐射条件下溶液的ESR波谱,测试跟踪光催化反应过程中自由基的动态。本法简便、准确、快捷,可用于光触媒性能的测试以及光催化反应机理的研究。     

Nitric oxide release from the unimolecular decomposition of the superoxide radical anion adduct of cyclic nitrones in aqueous medium

Nitrones such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO) have become the spin-traps of choice for the detection of transient radical species in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy. The mechanism of decomposition of the superoxide radical anion (O2(.-)) adducts of DMPO, DEPMPO and EMPO in aqueous solutions was investigated. Our findings suggest that nitric oxide (NO) was formed during the decomposition of the O2(.-) adduct as detected by EPR spin trapping using Fe(II)N-methyl-d-glucamine dithiocarbamate (MGD). Nitric oxide release was observed from the O2(.-) adduct formed from hypoxanthine-xanthine oxidase, PMA-activated human neutrophils, and DMSO solution of KO2. Nitric oxide formation was not observed from the independently generated hydroxyl radical adduct. Formation of nitric oxide was also indirectly detected as nitrite (NO2(.-)) utilizing the Griess assay. Nitrite concentration increases with increasing O2(.-) concentration at constant DMPO concentration, while NO2(.-) formation is suppressed at anaerobic conditions. Moreover, large excess of DMPO also inhibits NO2(.-) formation which can be attributed to the oxidation of DMPO to hydroxamic acid nitroxide (DMPO-X) by nitrogen dioxide (NO2), a precursor to NO2(.-). Product analysis was also conducted to further elucidate the mechanism of adduct decay using gas chromatography-mass spectrometry (GC-MS) technique.     

Preparation and use as spin trapping agents of new ester-nitrones

The synthesis of two new nitrones, N-benzylidene-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPN) and N-[(1-oxidopyridin-1-ium-4-yl)methylidene]-1-ethoxycarbonyl-1- methylethylamine N-oxide (EPPyON), is described. Measurement of their n-octanol-phosphate buffer partition coefficient permitted evaluation of their lipophilicity. Their capacity to act as spin trapping agents was investigated in aqueous media. Although these nitrones were unsuitable for detecting hydroxyl radical, they efficiently trapped various carbon- and oxygen-centred radicals, including superoxide, in aqueous media. The half-lives of their superoxide adducts were determined at pH 5.8 and 7.2. At neutral pH, the superoxide spin adduct of DEEPN was found to be as persistent as that of 5-diethoxyphosphoryl-5-methyl-3,4-dihydropyrrole N-oxide (DEPMPO). Consequently, DEEPN was believed to be an efficient trap for superoxide detection in aqueous media.     

Deuterated analogues of the free radical trap DEPMPO: synthesis and EPR studies

Three analogues of 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO, 1) labelled with two (1-d2), five (1-d5) or seven (1-d7)2H were synthesized and used to trap the tert-butylperoxyl radical. The EPR spectra of 1-d2-OOBu(t) and 1-d7-OOBu(t) spin adducts exhibited more straightforward patterns and better signal to noise ratio than those obtained with 1 or 1-d5. The use of the easily available 1-d2 as spin trap could help significantly the analysis of the EPR signals when the signal of either superoxide or alkylperoxyl spin adduct is superimposed with the signals of other spin adducts.     

DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water

Peroxidation is an important process both in chemistry and biology, and peroxyl radicals play a crucial role in various pathological situations involving lipid and protein peroxidation. A few secondary and tertiary peroxyl radicals can be detected directly by Electron Spin Resonance (ESR). However, primary and secondary alkylperoxyl radicals have extremely short lifetimes and their direct observation is impossible in biological samples. DMPO has been used to trap alkylperoxyl radicals generated in biological systems and the characterization of DMPO-alkylperoxyl spin adducts has been claimed by different authors. However, it was then clearly shown that all the assignments made previously to DMPO-OOR adducts were actually due to DMPO-OR adducts. We have investigated the potential of DEPMPO to characterize the formation of alkylperoxyl radicals in biological milieu. Various DEPMPO-OOR (R = Me, primary or secondary alkyl group) spin adducts were unambiguously characterized and the formation of DEPMPO-OOCH(3) was clearly established during the reaction of tert-butylhydroperoxide with chloroperoxidase and cytochrome c.     

2-甲基取代对吡咯啉-N-氧化物类自旋捕捉剂与超氧阴离子自由基加合物稳定性的影响

20世纪60年代末期出现的自旋捕捉技术已在多个学科领域中得到了普遍应用,特别是近年来该技术的生物体系活性氧自由基检测中获得了广泛的应用,极大地推动了生物自由基领域的研究。     

Detection of hydrogen atom adduct of spin-trap DEPMPO. The relevance for studies of biological systems

We proposed EPR spectroscopy using spin-trap DEPMPO as a novel method for the detection of a hydrogen atom (*H) produced by chemical and biological systems. In complex EPR spectra of DEPMPO adducts in biological systems, spectral lines of unknown origin have been observed. We have assumed (Baci?, G.; Mojovi?, M. Ann. N. Y. Acad. Sci. 2005, 1048, 230-243) that those lines represent the spectrum of a hydrogen atom (*H) adduct i.e., DEPMPO/H. An electrochemical system known to produce only *H radicals was used here in order to obtain a separate spectrum of the DEPMPO/H adduct. An acquired spectrum as well as a computer spectral simulation of the DEPMPO/H adduct showed considerable resemblance with additional lines in the EPR spectra of DEPMPO adducts in biological systems-plant plasma membranes and cell walls. This shows that such a radical is produced by plants as well as that DEPMPO is suitable for detection in both electrochemical and biological systems.     

Synthesis and structure of 5,5-diethoxycarbonyl-1-pyrroline N-oxide (DECPO). Application to superoxide radical trapping

DECPO, a new analogue of EMPO was synthesized through a two-step synthetic pathway. Its structure and its application to trap superoxide were investigated. The ESR detection of the DECPO-OOH spin adduct is easy even at low concentration of superoxide. In comparison with DEPMPO, the trapping of superoxide with DECPO is faster and the detection of DECPO-OOH can be performed using a very low nitrone concentration (0.5 mM).     

Characterization of a new methylated Beta-cyclodextrin with a low degree of substitution by electrospray ionization mass spectrometry and liquid chromatography/mass spectrometry

A new methylated beta-cyclodextrin (Me-beta-CD) with a low degree of substitution (DS) was characterized by electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography coupled with ESI-MS (LC/ESI-MS). For ESI-MS analyses, the composition of the infused sample solution was optimized in order to obtain only singly charged ammoniated CDs without fragmentation. The DS value (i.e. the number of methyl groups per glucopyranose unit) was found to be 0.7, which was in accordance with the values previously obtained by other methods. The LC/ESI-MS analysis, derived from a method using evaporative light scattering detection, allowed the study of the substitution isomers of each derivative and appears to be an easy and rapid tool for the accurate characterization of Me-beta-CD mixtures.     

Synthesis of the cis diastereoisomer of 5-diethoxyphosphoryl-5-methyl-3-phenyl-1-pyrroline N-oxide (DEPMPPOc) and ESR study of its superoxide spin adduct

The cis and trans diastereoisomers of 5-diethoxyphosphoryl-5-methyl-3-phenyl-1-pyrroline N-oxide (DEPMPPO), the C(3)-phenyl analogue of DEPMPO, were prepared in three steps from phenylacetaldehyde and used in ESR-spin trapping of various carbon-, oxygen- and sulfur-centred radicals. In the case of the cis-isomer, the presence of the phenyl group cancels the alternating line width phenomenon observed for the DEPMPO-OOR (R = H, Bu^t) spin adducts. The ESR spectra of the DEPMPPOc-OOR spin adducts exhibit more straightforward patterns and are more easily assignable.     

DEPMPO and lipophilic analogues: synthesis and EPR studies

DEPMPO (5-diethylphosphono-5-methyl-1-pyrroline N-oxide) is now recognised as a major trap for the applications of spin-trapping techniques in biological milieu. We report herein a new synthetic route for DEPMPO ending with the reductive cyclisation of the phosphorylated γ-nitroaldehyde 8. In comparison with the usual synthesis of DEPMPO, involving the oxidation of the parent pyrrolidine 3, the new approach avoids the formation of over oxidation by-products thus making easier the purification of DEPMPO. Lipophilic analogues of DEPMPO have been prepared through addition of various Y₂P(O)H on 2-methyl-1-pyrroline, followed by oxidation of the ensuing pyrrolidines. These new nitrones were used to trap different radicals in water and organic solvents.     

An EPR study on wastewater disinfection by peracetic acid,hydrogen peroxide and UV irradiation

EPR spectroscopy was applied to obtain qualitative and quantitative information on the radicals produced in disinfection processes of wastewater for agricultural reuse. The DEPMPO spin trap was employed to detect hydroxyl and carbon-centered short living radicals in two different peracetic acid solutions and a hydrogen peroxide solution used for water disinfection either in the absence or in the presence of UV-C irradiation. Moreover, three different kinds of water (wastewater, demineralized water, distilled water) were analysed in order to assess the contribution of Fenton reactions to the radical production. The spectroscopic results were discussed in relation to the efficiency of the different oxidizing agents and UV irradiation in wastewater disinfection evaluated as Escherichia Coli, Faecal and Total Coliforms inactivation.     

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.