Radical Intermediates and Antioxidants: An Esr Study of Radicals Formed on Carnosic Acid in the Presence of Oxidized Lipids

Carnosic acid, an antioxidant extracted from rosemary, is shown to produce radicals when in contact with oxidized methyl oleate in the absence of air above 50°C. Two radical species are formed: the first one, stable up to -110°C, is an hydroxy-phenoxy radical whose ESR spectrum was analyzed by studying its temperature dependence and its sensitivity to deuterium/proton exchange. The second species was observed above 110°C, its ESR spectrum was identical to the spectrum obtained when carnosol, another antioxidant extracted from rosemary, was heated at the same temperature in the presence of oxidized lipid. This observation is probably due to the transformation of carnosic acid into carnosol; the analysis of the corresponding ESR spectrum suggests the formation of a keto phenoxy radical exhibiting a great delocalization of the unpaired electron.     

Electron spin resonance study of melanin treated with reducing agents.

The electron spin resonances (ESR) of several native and modified melanins have been determined. Melanins isolated from black wool and synthesized from 3,4-dihydroxyl-L-phenylalanine (L-DOPA) and tyrosine all show similar ESR signals. Modification of the isolated melanins by treatment with reducing agents causes some lightening in color and slight changes in the ESR spectra. Lithium and liquid ammonia (Birch) reduction applied to melanins from wool and L-DOPA gave very different results, as reflected by ESR spectra, but in both cases the changes were much greater than those produced by other treatments. In general, reductive treatments in nonaqueous media in the presence of metals increase the free radical content and line width, whereas treatment in aqueous media resulted in decreased free radical content. These observations are consistent with a melanin pigment which is an irregular polymer and has unpaired electrons localized on different but similar monomer units.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Characterization of the high-resolution ESR spectra of superoxide radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Analysis of conformational exchange

It has been previously reported that the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) can form stable radical adducts with superoxide radical. However, the presence of diastereomers of DEPMPO radical adducts and the appearance of superhyperfine structure complicates the interpretation of the ESR spectra. It has been suggested that the superhyperfine structure in the ESR spectrum of DEPMPO/^?OOH is a result of conformational exchange between conformers. The analysis of the temperature dependence of the ESR spectrum of DEPMPO/^?OOH and of its structural analog DMPO/^?OOH have demonstrated that both ESR spectra contain exchange effects resulting from conversion between two conformers. Computer simulation calculates a conformer lifetime on the order of 0.1?μs for DMPO/^?OOH at room temperature. However, temperature dependence of the ESR spectrum of DEPMPO/^?OOH suggests that superhyperfine structure does not depend on the conformational exchange. We have now found that the six-line ESR spectrum with superhyperfine structure should be assigned to a DEPMPO-superoxide-derived decomposition product. Therefore, ESR spectra previously assigned to DEPMPO/^?OOH contain not only the two diastereomers of DEPMPO/^?OOH but also the decomposition product, and these spectra should be simulated as a combination of four species: two conformers of the first diastereomer, one conformer of the second diastereomer and the superoxide-derived decomposition product. The presence of four species has been supported by the temperature dependence of the ESR spectra, nucleophilic synthesis of radical adducts, and isotopic substitution experiments. It is clear that to correctly interpret DEPMPO spin trapping of superoxide radicals, one must carefully consider formation of secondary radical adducts.     

Photolysis of N-methyl and N-acetyl substituted amino acids

Summary Experiments on the production of free radicals in aqueous solutions of N-methyl and N-acetyl substituted amino acids by UV light are reported. The ESR spectra observed at 77 K show that the bond of the CH_3–group of these substances is ruptured to a great deal compared with other bond ruptures producing paramagnetic centres. But in N-methyl substituted amino acids the methyl radical is less predominant than in N-acetyl substituted ones. At temperatures higher than 200 K the ESR spectra of all substances studied in our experiments are similar. It is supposed that a radical is formed with an unpaired spin near an oxygen atom.A part of this paper was reported at a Congress in Freiburg i. Br. (Germany) organized by the Deutsche Gesellschaft für Biophysik, October 2–4, 1974.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Characterization of the high-resolution ESR spectra of superoxide radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Analysis of conformational exchange

It has been previously reported that the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) can form stable radical adducts with superoxide radical. However, the presence of diastereomers of DEPMPO radical adducts and the appearance of superhyperfine structure complicates the interpretation of the ESR spectra. It has been suggested that the superhyperfine structure in the ESR spectrum of DEPMPO/^⋅OOH is a result of conformational exchange between conformers. The analysis of the temperature dependence of the ESR spectrum of DEPMPO/^⋅OOH and of its structural analog DMPO/^⋅OOH have demonstrated that both ESR spectra contain exchange effects resulting from conversion between two conformers. Computer simulation calculates a conformer lifetime on the order of 0.1 μs for DMPO/^⋅OOH at room temperature. However, temperature dependence of the ESR spectrum of DEPMPO/^⋅OOH suggests that superhyperfine structure does not depend on the conformational exchange. We have now found that the six-line ESR spectrum with superhyperfine structure should be assigned to a DEPMPO-superoxide-derived decomposition product. Therefore, ESR spectra previously assigned to DEPMPO/^⋅OOH contain not only the two diastereomers of DEPMPO/^⋅OOH but also the decomposition product, and these spectra should be simulated as a combination of four species: two conformers of the first diastereomer, one conformer of the second diastereomer and the superoxide-derived decomposition product. The presence of four species has been supported by the temperature dependence of the ESR spectra, nucleophilic synthesis of radical adducts, and isotopic substitution experiments. It is clear that to correctly interpret DEPMPO spin trapping of superoxide radicals, one must carefully consider formation of secondary radical adducts.     

Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites

The oxidation of the phenacetin metabolites p-phenetidine and acetaminophen by peroxidases was investigated. Free radical intermediates from both metabolites were detected using fast-flow ESR spectroscopy. Oxidation of acetaminophen with either lactoperoxidase and hydrogen peroxide or horseradish peroxidase and hydrogen peroxide resulted in the formation of the N-acetyl-4-aminophenoxyl free radical. Totally resolved spectra were obtained and completely analyzed. The radical concentration was dependent on the square root of the enzyme concentration, indicating second-order decay of the radical, as is consistent with its dimerization or disproportionation. The horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of p-phenetidine (4-ethoxyaniline) at pH 7.5-8.5 resulted in the one-electron oxidation products, the 4-ethoxyaniline cation free radical. The ESR spectra were well resolved and could be unambiguously assigned. Again, the enzyme dependence of the radical concentration indicated a second-order decay. The ESR spectrum of the conjugate base of the 4-ethoxyaniline cation radical, the neutral 4-ethoxyphenazyl free radical, was obtained at pH 11-12 by the oxidation of p-phenetidine with potassium permanganate.     

Characterization of the structure and reactions of free radicals from serotonin and related indoles

The ESR spectra of the free radicals formed by the autoxidation of serotonin, 5-hydroxyindole, and 5-hydroxytryptophan in 1 N NaOH are presented. The analysis of the hyperfine splitting constants in H2O and D2O characterize these free radicals as semiquinone-imines, the one-electron oxidation product of the corresponding indole. At alkaline pH, autoxidation of these compounds ultimately leads to solid precipitate and unresolved ESR spectra characteristic of polymeric material. The reduction of cytochrome c at pH 7.4 by a wide variety of indoles correlates with the amplitude of the ESR signal in 1 N NaOH, as do other processes thought to be related to 5-hydroxyindole free radical formation. Relative to the rate of cytochrome c reduction, neither serotonin nor the serotonin free radical appears to react with oxygen to form superoxide. In the presence of NAD(P)H, the serotonin radical most probably oxidizes NAD(P)H to form the NAD(P). radical. The NAD(P). radical then reacts with oxygen to form superoxide, which ultimately reduces cytochrome c.     

E.S.R. Spin Trapping Analysis of Gamma Induced Radicals in Sucrose

Radicals induced by gamma-irradiation in sucrose, in the solid state at different temperatures and in aqueous solution, have been studied by the spin-trapping method. Electron spin resonance (ESR) combined with high performance liquid chromatography (HPLC) then spectra analysis with a simulation program (VOYONS), revealed seven main radical species. Their nitrogen and hydrogen splitting constants were compared with those obtained from fructose and glucose units. Assignments of chemical structures are discussed for three radical species.     

High resolution proton nuclear magnetic resonance spectroscopy of lactoperoxidase

The first high resolution proton nuclear magnetic resonance spectra are reported for the native ferric and ferric cyano complexes of bovine lactoperoxidase. The spectrum of the native species exhibits broad heme signals in a far downfield region characteristic of the high-spin ferric state. The low-spin cyano complex yields a proton nuclear magnetic resonance spectrum with signals as far as 68.5 ppm downfield and as far as -28 ppm upfield of the tetramethylsilane reference. These peak positions are anomalous with respect to those seen only as far as 35 ppm downfield in other cyano hemoprotein complexes. An extreme asymmetry in the unpaired spin delocalization pattern of the iron porphyrin is suggested. The unusual proton nuclear magnetic resonance properties parallel distinctive optical spectral properties and the exceptional resistance to heme displacement from the enzyme. Lactoperoxidase utilized in these studies was isolated from raw milk and purified by an improved, rapid chromatographic procedure.     

Structure and redox properties of radicals derived from one-electron oxidised methylxanthines

The repair of the one electron oxidised form of a xanthine derivative by another xanthine derivative was studied by reacting aqueous binary mixtures of xanthine derivatives with sulphate radical anion (SO(4)( -)) and following the concentration of both compounds as a function of time. The relative behaviour observed enabled the establishment of a qualitative order of antioxidant capacities for the several xanthines studied in acidic and neutral media. The same order was confirmed quantitatively by measuring the reduction potentials of the xanthines by cyclic voltammetry. Theoretical DFT calculations were used to calculate the relative stabilities of the tautomers of each xanthine neutral radical. It was also demonstrated that the deprotonation of a xanthine radical cation never occurs from N1, unless no other possibility is available. At high pH values, it was possible to obtain the ESR spectra of the radical anions derived from 1-methylxanthine, 3-methylxanthine and xanthosine. The theoretical calculations also enabled the assignment of the ESR hyperfine coupling constants of the spectra of these radical anions. The coupling constants calculated are in good agreement with the experimental values.     

The structure of the guanine cation: ESR/ENDOR of cyclic guanosine monophosphate single crystals after X irradiation at 10 K.

Following X irradiation of 3',5'-cyclic guanosine monophosphate single crystals at 10 K, several free radicals were trapped and detected by ESR/ENDOR/FSE spectroscopy. The two dominant species both have unpaired spin located on the guanine base. One is the product of net hydrogen atom loss from the exocyclic amino group. The spectroscopic characteristics of this resonance leave this assignment unambiguous. The experimental conditions make it likely that this species was formed by deprotonation of the guanine base cation. The nature of the other species is more uncertain. However, the evidence is consistent with the assignment that it is a net OH adduct to the C4 position of the base. Several species in which the unpaired spin was located on the sugar-phosphate region of the molecule were also observed. The mechanisms for the decay of the primary radicals, also leading to the well-known C8 hydrogen addition radical of the guanine base, are described and discussed.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

Movement of methylphenazyl free radicals in polar and nonpolar liquids.

A protonated, charged free radical of methylphenazine methosulfate (PMS) was generated at carbon electrodes in a buffered aqueous medium. This radical diffused from the aqueous phase into nonpolar organic solvents, where it was stable for extended periods. The electron spin resonance (ESR) spectrum of the free radical species in the nonpolar solvent was significantly different from that of the aqueous species. This difference was attributed to the loss of electric charge through deprotonation at the solution interface, followed by solvation of the uncharged species in the organic phase. ESR spectra are presented for PMS free radicals in polar and nonpolar liquid phases, along with electrochemical results and conclusions regarding the mechanisms of movement and toxicity of phenazyl free radicals in biological systems.     

Characterization of the high resolution ESR spectra of the methoxyl radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)

Spin-trapping investigators are largely limited by the instability of the radical adducts. Spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms very stable alkoxyl radical adducts. However, the presence of two chiral centers in the DEPMPO alkoxyl radical adduct results in two diastereomers with distinctive ESR spectra, which complicates the interpretation of the ESR spectra. We have analyzed the high resolution ESR spectra of the DEPMPO/_•OCH₃ radical adduct. DEPMPO/_•OCH₃ has been synthesized by the nucleophilic addition of alcohols to DEPMPO. The electron spin resonance (ESR) spectrum of DEPMPO/_•OCH₃ in oxygen-free methanol solution reveals superhyperfine structure with hyperfine coupling constants as small as 0.3 G. In order to simplify the analysis of the electron spin resonance (ESR) spectrum, we synthesized the DEPMPO/_•OCD₃ radical adduct. Computer simulation of the DEPMPO/_•OCD₃ ESR spectrum revealed two diastereomers. Hyperfine coupling constants of γ-protons and ₁₇O from the -OCH₃ group were also determined. ESR spectra of DEPMPO/_•OCH₃ in phosphate buffer have also been characterized. The presence of specific hyperfine couplings from the -OCH₃ group can be used for the unambiguous identification of the DEPMPO/_•OCH₃ radical adducts. We suggest that the analysis of high resolution ESR spectra can be used for the unambiguous characterization of DEPMPO radical adducts.     

Capacity of ascorbyl palmitate to produce the ascorbyl radical in vitro: an electron spin resonance investigation

This study aims to compare the electron spin resonance (ESR) spectra emitted by human blood loaded with either ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbic acid (AA), or with AA. Whole blood of a healthy male individual was equilibrated with equimolar concentrations of AP and AA of 200, 400, and 800 micromol/l. The intensity of the ESR signal, expressed as the peak-to-peak amplitude, reflects the amount of unpaired spins that are created due to the reducing action of AA and is proportional, in relative terms, to the amount of the ascorbyl radical formed. We found that the blood with AP emitted an ESR signal whose singlet shape, width, and location precisely correlate with the known characteristics of the ascorbyl radical in vitro. The signal magnitude increased linearly with increasing concentrations of AP and was similar to that of AA. We conclude that AP is biologically active, as it generates the ascorbyl radical, an action that also underlies the scavenging process by ascorbic acid. To this end, ascorbyl-6-palmitate might have potential advantages, due to its ability to penetrate biomembranes and to act at the lipid-related molecular target sites.     

Dosimetric and kinetic investigations of γ-irradiated sodium tartrate dihydrate

Effects of gamma radiation on solid sodium tartrate dihydrate (NaTA) were studied using electron spin resonance (ESR) spectroscopy. One main singlet located at g = 2.0034 and many weak lines located at low and high magnetic field sides were found in the irradiated samples. Dosimetric and kinetic features of the radical species responsible for the experimental ESR spectra were explored through the variations in the signal intensities with respect to applied microwave power, temperature and storage time. Activation energies of the involved radical species were also determined using data derived from annealing studies.     

A spectroscopic characterization of a phenolic natural mediator in the laccase biocatalytic reaction

Multi-frequency ESR combined with NALDI-TOF MS has been used for the characterization of 3,5-dimethoxy-4-hydroxyacetophenone radical intermediate and by-products formed during the Coriolopsis gallica laccase catalytic reaction. A stable radical species is formed and an intense and well-structured ESR spectrum was detected and fully characterized at S-, X- and W-bands. The presence of by-products generated as the result of by-reactions has been investigated and analyzed through NALDI-TOF MS, performing the experiments versus time. The superior radical stability of such phenoxy radical, due to steric hindrance in ortho to the phenol group and the great delocalization of the unpaired electron on the acetyl substituent, makes acetosyringone particularly interesting for biotechnological applications. This represents a good example for the development of new stable laccase mediator molecules.     

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.