Superoxide radical anion adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 2. The thermodynamics of decay and EPR spectral properties

The formation of the superoxide radical anion (O2*-) adduct of the nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as detected by electron paramagnetic resonance (EPR) spectroscopy is one of the most common techniques for O2*- detection in chemical and biological systems. However, the nature of DMPO-O2H has confounded spin-trapping investigators over the years, since there has been no independently synthesized DMPO-O2H to date. A density functional theory (DFT) approach was used to predict the isotropic hyperfine coupling constants arising from the N, beta-H, and gamma-H nuclei of DMPO-O2H using explicit interactions with water molecules as well as via a bulk dielectric effect employing the polarizable continuum model (PCM). Theoretical calculation on the thermodynamics of DMPO-O2H decay shows favorable intramolecular rearrangement to form a nitrosoaldehyde and a hydroxyl radical as products, consistent with experimental observations. Some pathways for the bimolecular decomposition of DMPO-O2H and DMPO-OH have also been computed.     

DMPO-OH radical formation from 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in hot water.

When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70 degrees C for 30 min, formation of DMPO-OH was observed by ESR. This DMPO-OH radical formation was suppressed under an argon atmosphere. When water was replaced with ultra-pure water for ICP-MS experiments, DMPO-OH radical formation was also diminished. Under an argon atmosphere in ultra-pure water, the intensity of the DMPO-OH signal decreased to about 1/20 of that observed under aerobic conditions with regular purified water. The addition of hydroxyl radical scavengers such as mannitol did not affect the formation of DMPO-OH, but the signal turned faint in the presence of EDTA. We suggest that DMPO reacted with dissolved oxygen to form DMPO-OH.     

Influence of conformation on the EPR spectrum of 5,5-dimethyl-1-hydroperoxy-1-pyrrolidinyloxyl: a spin trapped adduct of superoxide

Spin trapping, a technique used to characterize short-lived free radicals, consists of using a nitrone or nitroso compound to "trap" an unstable free radical as a long-lived aminoxyl that can be characterized by EPR spectroscopy. The resultant aminoxyl exhibits hyperfine splitting constants that are dependent on the spin trap and the free radical. Such is the case with 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) and 2,2-dimethyl-5-hydroperoxy-1-pyrrodinyloxyl (DMPO-OOH) whose hyperfine splitting constants, A(N) = A(H) = 14.9 G and A(N) = 14.3 G, A(H)(beta) = 11.7 G, and A(H)(gamma) = 1.25 G, respectively, have been used to demonstrate the generation of HO(*) and O(2)(*)(-). However, to date, the source of the apparent A(H)(gamma) hyperfine splitting in DMPO-OOH is not known. We consider three possible explanations to account for the unique EPR spectrum of DMPO-OOH. The first is that the gamma-splitting arises from one of the hydrogen atoms at either carbon 3 or carbon 4 of DMPO-OOH. The second is that the gamma-splitting originates from the hydrogen atom of DMPO-OOH. The third is that the conformational properties of DMPO-R change upon going from DMPO-OH to DMPO-OOH. Experimental and theoretical chemical approaches as well as EPR spectral modeling were used to investigate which of these hypotheses may explain the asymmetric EPR spectrum of DMPO-OOH. From these studies it is shown that the 12-line EPR spectrum of DMPO-OOH results not from any proximal hydrogen, but from additional conformers of DMPO-OOH. Thus, the 1.25 G hyperfine splitting, which has been assigned as a gamma-splitting, is actually from two individual EPR spectra associated with different conformers of DMPO-OOH.     

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.     

Theoretical and experimental studies of the spin trapping of inorganic radicals by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 2. Carbonate radical anion

Previous studies have shown that the enzyme-mediated generation of carbonate radical anion (CO(3)(.-)) may play an important role in the initiation of oxidative damage in cells. This study explored the thermodynamics of CO(3)(.-) addition to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) using density functional theory at the B3LYP/6-31+G(**)//B3LYP/6-31G* and B3LYP/6-311+G* levels with the polarizable continuum model to simulate the effect of the bulk dielectric effect of water on the calculated energetics. Theoretical data reveal that the addition of CO(3)(.-) to DMPO yields an O-centered radical adduct (DMPO-OCO2) as governed by the spin (density) population on the CO(3)(.-). Electron paramagnetic resonance spin trapping with the commonly used spin trap, DMPO, has been employed in the detection of CO(3)(.-). UV photolysis of H(2)O(2) and DMPO in the presence of sodium carbonate (Na(2)CO(3)) or sodium bicarbonate (NaHCO(3)) gave two species (i.e., DMPO-OCO(2) and DMPO-OH) in which the former has hyperfine splitting constant values of a(N) = 14.32 G, a(beta)-Eta = 10.68 G, and a(gamma-H) = 1.37 G and with a shorter half-life compared to DMPO-OH. The origin of the DMPO-OH formed was experimentally confirmed using isotopically enriched H(2)(17)O(2) that indicates direct addition of HO(.) to DMPO. Theoretical studies on other possible pathways for the formation of DMPO-OH from DMPO-OCO(2) in aqueous solution and in the absence of free HO(.) such as in the case of enzymatically generated CO(3)(.-), show that the preferred pathway is via nucleophilc substitution of the carbonate moiety by H(2)O or HO(-). Nitrite formation has been observed as the end product of CO(3)(.-) trapping by DMPO and is partly dependent on the basicity of solution. The thermodynamic behavior of CO(3)(.-) in the aqueous phase is predicted to be similar to that of the hydroperoxyl (HO(2)(.)) radical.     

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). 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.     

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.     

Calculated hyperfine coupling constants for 5,5-dimethyl-1-pyrroline N-oxide radical products in water and benzene

The optimized structures of some radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were computed by different methods on ESR spectra. As trapped radicals, H, N₃, NH₂, CH₃, CCl₃, OOH in water and F, OH, CF₃, CH₂OH, OC₂H₅ in benzene solutions were used. The calculated isotropic hyperfine coupling constants of all the trapped radicals were compared with the corresponding experimental data. The hyperfine coupling constant due to the β proton of the nitroxide radical was seen to be consist with the McConnel’s relation α_β = B ₀ + B ₁cos²θ and, to be effected with the opposite spin density of oxygen nucleus bonded to the nitrogen. It was concluded that in hyperfine calculations the DFT(B3PW91)/LanL2DZ level is superior computational quantum model relative to the used other level. Also, the study has been enriched by the computational of the optimized geometrical parameters, the hyper conjugative interaction energies, the atomic charges and spin densities for all the radical adducts.     

Synthesis of new functionalized 5,5-dimethyl-1-pyrroline 1-oxides and their investigation as spin traps

The reactions of 5,5-dimethyl-3-oxo-1-pyrroline 1-oxide (3-oxo-DMPO, 1) with NH₂OH and N₂H₄ afforded oxime (2a) and hydrazone (2b), respectively. The reaction products were studied as spin traps for the short-lived radicals HO^·, Ph^·, PhCO₂ ^·, NC(Me₂)C^·, and NC(Me₂)CO^·. The nitroxides generated in the reactions of the above-mentioned short-lived radicals with nitrones 1 and 2a,b were characterized by ESR spectroscopy. Of these nitrones, oxime 2a is the most effective radical trap.     

Distinguishing the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical quenching effect from the hydroxyl radical scavenging effect in the ESR spin-trapping method

The spin-trapping method has been widely accepted to measure the hydroxyl radical scavenging activity in electron spin resonance (ESR) spectroscopy, however, the disappearance of the trapped signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical may lead to an erroneous conclusion, as the decreased signal must result from the rapid quenching of the DMPO-OH formed. The DMPO-OH quenching activity of a compound can be measured by tracing the signal decay resulting from its addition after the cessation of DMPO-OH formation. It was found that Fe(II) ions could quench the DMPO-OH radical quite rapidly, and phosphate ions could enhance the DMPO-OH radical quenching activity of the Fe(II) ions.     

Esters of 5-carboxyl-5-methyl-1-pyrroline N-oxide: a family of spin traps for superoxide

Apparent rate constants, at acidic pH and neutral pH for the reaction of a family of ester-containing 5-carboxyl-5-methyl-1-pyrroline N-oxides with superoxide (O2*-) were estimated, using ferricytochrome c as a competitive inhibitor. It was of interest to note that the rate constants were similar among the different nitrones and not that significantly different from that found for 5-(diethoxyphosphoryl)-5-dimethyl-1-pyrroline N-oxide. At acidic pH, the rate constant for spin trapping O2*- was 3-fold greater than that at physiological pH. Subsequent experiments determined the half-life of aminoxyls, derived from the reaction of these nitrones with O2*-. The EPR spectra were modeled by using a global analysis method. The results clearly demonstrated that EPR spectra of all the aminoxyls were inconsistent with a model that included a single gamma-hydrogen splitting. A better interpretation modeled them as two diastereomers with identical nitrogen splittings and slightly different beta-hydrogen splittings. Detailed line width analyses slightly favored an equal line width-unequal population ratio for the two diastereomers.     

An HPLC and EPR investigation on the stability of DMPO and DMPO spin adducts in vivo

Application of the spin trapping technique in intact animals requires an understanding of the stability and distribution of the spin traps and their spin adducts in vivo. We studied the stability of DMPO in vivo in mice using HPLC and the stability of spin adducts of DMPO by EPR in plasma, whole blood, peritoneal fluid, and homogenized heart tissue of the rat. At 15 minutes after intraperitoneal injection DMPO had similar concentrations in the liver, heart, and blood of the mice and 40% remained in the organs 2 hours after the injection. In contrast, the spin adduct DMPO-OH was short lived, with a half-life of 3.0 minutes in plasma, and was not detectable 1 minute after formation in whole blood and homogenized heart tissue. The carbon centered spin adduct DMPO-CH(OH)CH₃ was more stable, having half-lives of 16, 11, 3.6, and 0.79 minutes in plasma, peritoneal fluid, whole blood, and homogenized heart tissue, respectively. The spin adduct DMPO-SO₃ was sufficiently stable for the adduct to be observed directly from living mice.     

Combined ESR and thermodynamic studies of the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO): hindered rotation around the O-O bond evidenced by two-dimensional simulation of temperature-dependent spectra

Experiments were performed to elucidate the origin of the superhyperfine structure and line width alternation (LWA) seen in the ESR spectrum of the major diastereoisomer (1) of DEPMPO-OOH, the remarkably persistent superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). Using selectively deuterated DEPMPO derivatives, we demonstrated that the superhyperfine pattern can be unambiguously attributed to long-range couplings. The recording in pyridine of highly resolved spectra in a wide temperature range, combined with two-dimensional simulation, allowed us to characterize an inverted LWA in 1 and revealed a uniform line broadening in the spectrum of the minor DEPMPO-OOH diastereoisomer (2), with both effects originating from a chemical exchange between conformers. When the individual spectra of 1 presenting LWA in the fast-exchange regime were simulated, four equally good fits were obtained and this ambiguity could be resolved by using a two-dimensional simulation technique. The thermodynamic and kinetic constants of this exchange were consistent with a rotation around the O-O bond. We propose that line broadening effects in 1 and 2 result from this O-O rotation concerted with the pseudo-rotation of the pyrrolidine ring.     

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).     

Synthesis and spin trapping properties of 1,1-dimethyl-3-(trifluoromethyl)-1H-isoindole N-oxide

We have achieved an efficient synthesis of 1,1-dimethyl-3-(trifluoromethyl)-1H-isoindole N-oxide (2) in 39% yield by seven steps from 2-bromobenzoic acid (3). This compound serves as a spin trap reagent, giving a strong and stable ESR signal of the radical adduct of 2 in the presence of i-amyloxy radical generated by UV photolysis of i-amyl nitrite.     

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.     

NMR and EPR studies of the reaction of nucleophilic addition of (bi)sulfite to the nitrone spin trap DMPO

The reaction of the nitrone spin trap 5,5‐dimethylpyrroline‐N‐oxide (DMPO) with sodium (bi)sulfite in aqueous solutions was investigated using NMR and EPR techniques. Reversible nucleophilic addition of (bi)sulfite anions to the double bond of DMPO was observed, resulting in the formation of the hydroxylamine derivative 1‐hydroxy‐5,5‐dimethylpyrrolidine‐2‐sulfonic acid, with characteristic ¹H and ¹³C NMR spectra. The reaction mechanism was suggested and corresponding equilibrium constants determined. The mild oxidation of the hydroxylamine results in the formation of an EPR‐detected spectrum identical with that for the DMPO adduct with sulfur trioxide anion radical. The latter demonstrates that a non‐radical addition reaction of (bi)sulfite with DMPO may contribute to the EPR detection of SO₃^?? radical. This possibility must be taken into account in spin trapping analysis of sulfite radical. Copyright © 2003 John Wiley & Sons, Ltd.