Reactivities of substituted α-phenyl-N-tert-butyl nitrones.

In this work, a series of α-phenyl-N-tert-butyl nitrones bearing one, two, or three substituents on the tert-butyl group was synthesized. Cyclic voltammetry (CV) was used to investigate their electrochemical properties and showed a more pronounced substituent effect for oxidation than for reduction. Rate constants of superoxide radical (O2(•-)) reactions with nitrones were determined using a UV-vis stopped-flow method, and phenyl radical (Ph(•)) trapping rate constants were measured by EPR spectroscopy. The effect of N-tert-butyl substitution on the charge density and electron density localization of the nitronyl carbon as well as on the free energies of nitrone reactivity with O2(•-) and HO2(•) were computationally rationalized at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level of theory. Theoretical and experimental data showed that the rates of the reaction correlate with the nitronyl carbon charge density, suggesting a nucleophilic nature of O2(•-) and Ph(•) addition to the nitronyl carbon atom. Finally, the substituent effect was investigated in cell cultures exposed to hydrogen peroxide and a correlation between the cell viability and the oxidation potential of the nitrones was observed. Through a combination of computational methodologies and experimental methods, new insights into the reactivity of free radicals with nitrone derivatives have been proposed.

5-Hydroxy-2,2,6,6-tetramethyl-4-(2-methylprop-1-en-yl)cyclohex-4-ene-1,3-dione, a novel cheletropic trap for nitric oxide EPR detection.

The title compound behaves as an efficient cheletropic trap for both NO and NO(2) radicals in the presence of oxygen, yielding EPR observable nitroxide and alkoxynitroxide, respectively.

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: a combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study.

Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione (GSH) and 2-methyl 2-nitroso propane (MNP) in an oxidative medium, to evaluate the reactivity of this tripeptide as a nucleophile toward a nitroso compound. Depending on the experimental conditions, different radical species could be detected by EPR, which allowed some structural assumptions. These samples were then submitted to electrospray ionization, in both positive and negative ion modes, for structural elucidation in tandem mass spectrometry. Although the primary nitroxide products could not be detected in MS, structurally related compounds such as hydroxylamine and O-methyl hydroxylamine could be fully characterized. In the absence of light, a S-adduct was formed via a Forrester-Hepburn reaction, that is, a nucleophile addition of MNP onto the thiol function in reduced glutathione to yield a hydroxylamine intermediate, further oxidized into nitroxide. In contrast, irradiating the reaction medium with visible light could allow an inverted spin trapping reaction to take place, involving the oxidation of both MNP and GSH before the nucleophilic addition of the sulfenic acid function onto the nitrogen of MNP, yielding a so-called O-adduct. It was also found that dilution of the reaction medium with methanol for the purpose of electrospray ionization could allow nitroxides to be indirectly observed either as hydroxylamine or O-methyl hydroxylamine species.

Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate.

Spin trapping using a nitrone and electron paramagnetic resonance (EPR) spectroscopy is commonly employed in the identification of transient radicals in chemical and biological systems. There has also been a growing interest in the pharmacological activity of nitrones, and there is, therefore, a pressing need to develop nitrones with improved spin trapping properties and controlled delivery in cellular systems. The beta-cyclodextrin (beta-CD)-cyclic nitrone conjugate, 5-N-beta-cyclodextrin-carboxamide-5-methyl-1-pyrroline N-oxide (CDNMPO) was synthesized and characterized. 1-D and 2-D NMR show two stereoisomeric forms (i.e., 5S- and 5R-) for CDNMPO. Spin trapping using CDNMPO shows distinctive EPR spectra for superoxide radical anion (O2(*-)) compared to other biologically relevant free radicals. Kinetic analysis of O2(*-) adduct formation and decay using singular value decomposition and pseudoinverse deconvolution methods gave an average bimolecular rate constant of k = 58 +/- 1 M(-1) s(-1) and a maximum half-life of t(1/2) = 27.5 min at pH 7.0. Molecular modeling was used to rationalize the long-range coupling between the nitrone and the beta-CD, as well as the stability of the O2(*-) adducts. This study demonstrates how a computational approach can aid in the design of spin traps with a relatively high rate of reactivity to O2(*-), and how beta-CD can improve adduct stability via intramolecular interaction with the O2(*-) adduct.

Synthesis and spin-trapping properties of a new spirolactonyl nitrone.

Spin trapping using electron paramagnetic resonance (EPR) spectroscopy is commonly employed for the identification of transient radicals in chemical and biological systems. A spirolactonyl-nitrone with rigid H-bond acceptor, 7-oxa-1-azaspiro[4.4]non-1-en-6-one 1-oxide, CPCOMPO, has been synthesized and characterized, and its spin-trapping properties were investigated. The rate of formation of CPCOMPO-O2H was determined using competition kinetics between the superoxide/hydroperoxyl radical (O2*-/HO2*) trapping by CPCOMPO and the spontaneous dismutation of this radical in aqueous media. The rate constant of 60 M-1.s-1 is the highest rate constant thus far observed at neutral pH for any nitrones using the kinetic method employed. Decay kinetics were also experimentally investigated for CPCOMPO-O2H. The effect of rigid H-bond acceptor on the stability of the CPCOMPO-O2H were computationally rationalized and compared to that of EMPO-O2H, which has flexible H-bond acceptor, and results show the need of a "loose" H-bond acceptor for improved adduct stability.

Reactivity of superoxide radical anion with cyclic nitrones: role of intramolecular H-bond and electrostatic effects.

Limitations exist among the commonly used cyclic nitrone spin traps for biological free radical detection using electron paramagnetic resonance (EPR) spectroscopy. The design of new spin traps for biological free radical detection and identification using EPR spectroscopy has been a major challenge due to the lack of systematic and rational approaches to their design. In this work, density functional theory (DFT) calculations and stopped-flow kinetics were employed to predict the reactivity of functionalized spin traps with superoxide radical anion (O2*-). Functional groups provide versatility and can potentially improve spin-trap reactivity, adduct stability, and target specificity. The effect of functional group substitution at the C-5 position of pyrroline N-oxides on spin-trap reactivity toward O2*- was computationally rationalized at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) and PCM/mPW1K/6-31+G(d,p) levels of theory. Calculated free energies and rate constants for the reactivity of O2*- with model nitrones were found to correlate with the experimentally obtained rate constants using stopped-flow and EPR spectroscopic methods. New insights into the nucleophilic nature of O2*- addition to nitrones as well as the role of intramolecular hydrogen bonding of O2*- in facilitating this reaction are discussed. This study shows that using an N-monoalkylsubstituted amide or an ester as attached groups on the nitrone can be ideal in molecular tethering for improved spin-trapping properties and could pave the way for improved in vivo radical detection at the site of superoxide formation.

Persilylated phosphoranyl radicals: the first persistent noncyclic phosphoranyl radicals.

Persistent noncyclic phosphoranyl radicals have been prepared and observed by electron paramagnetic resonance (EPR) for the first time. They were obtained by UV-photolysis of a solution containing a bis(trialkylsilyl) peroxide (R = Me, Et) and a tris(trialkylsilyl) phosphite (R = Me, Et, iPr). EPR parameters (a(P) approximately 100 mT) are typical of phosphoranyl radicals exhibiting a trigonal-bipyramidal structure, with the odd electron in an equatorial site. Analysis of the pseudo-first-order decay shows that these phosphoranyl radicals decay by S(H)2 homolytic substitution on the bis(trialkylsilyl) peroxide and by loss of a trialkylsilyloxyl radical (alpha-scission reaction). Both the S(H)2 and alpha-scission reactions depend on the steric bulk of the alkyl groups, that is, the bulkier the alkyl group, the slower the S(H)2 and alpha-scission reactions.

Spin trapping of superoxide by diester-nitrones.

The nitrone N-[(1-oxidopyridin-1-ium-4-yl)-methylidene]-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPyON) was synthesized and used as a spin trapping agent. The kinetic aspects of the superoxide detection by this new spin trap and by two other diester-nitrones, i.e. 2,2-diethoxycarbonyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPO) and N-benzylidene-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPN), were examined by determining the rate constants for the trapping reaction and for the spin adduct decay at pH 7.2. Comparing the results obtained to those given by analogous monoester-nitrones showed that both the spin trapping and the adduct decay reactions were faster in the presence of a second ester group in the cyclic nitrone series, while the superoxide trapping capacities of linear diester-nitrones were found to be dramatically weak. It follows from this study that DEPO and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) are superior when it comes to superoxide detection. Below 0.005 mol dm(-3), DEPO is to date the only nitrone capable of clearly detecting superoxide, while EMPO should be preferred at higher spin trap concentration.

Evidence of overestimation of rate constants for the superoxide trapping by nitrones in aqueous media.

Since major disagreements exist regarding the kinetics of superoxide trapping by nitrones, the underlying theory of one of the most popular method used in these studies was reinvestigated. It involves a competition between the nitrone of interest and a superoxide scavenger, and implies that the superoxide spontaneous dismutation, the spin adduct decay, and the consumption of the competitor during the course of the experiments are negligible events. Evidences of the importance of these three unduly neglected reactions are given, and errors connected to their omission are estimated. Hence this Stern-Volmer-like method should be avoided in the determination of rate constants for the trapping of superoxide by nitrones.

A new kinetic approach to the evaluation of rate constants for the spin trapping of superoxide/hydroperoxyl radical by nitrones in aqueous media.

A new kinetic approach to the evaluation of rate constants for the spin trapping of superoxide/hydroperoxyl radical by nitrones in buffered media is described. This method is based on a competition between the superoxide trapping by the nitrone and the spontaneous dismutation of this radical in aqueous media. EPR spectra are recorded as a function of time at various nitrone concentrations, and kinetic curves are obtained after treatment of these spectra using both singular value decomposition and pseudo-inverse deconvolution methods. Modelling these curves permits the determination of the rate constants k(T) and k(D) for the superoxide trapping and the adduct decay reactions, respectively. Kinetics parameters thus obtained with six nitrones, namely the 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (EMPO), the 5-diethoxyphosphoryl-5-methyl-3,4-dihydro-5H-pyrrole N-oxide (DEPMPO), the 5,5-dimethyl-3,4-dihydro-5H-pyrrole N-oxide (DMPO), the 1,3,5-tri[(N-(1-diethylphosphono)-1-methylethyl)-N-oxy-aldimine]benzene (TN), the N-benzylidene-1-ethoxycarbonyl-1-methylethylamine N-oxide (EPPN), and the N-[(1-oxidopyridin-1-ium-4-yl)methylidene]-1-ethoxycarbonyl-1-methylethylamine N-oxide (EPPyON), indicate that cyclic nitrones trapped superoxide faster than the linear ones. However, the low k(T) values obtained for compounds show that there is still a need for new molecules with better spin trapping capacities.

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.

N-2-(2-ethoxycarbonyl-propyl) alpha-phenylnitrone: an efficacious lipophilic spin trap for superoxide detection.

The spin trap N-2-(2-ethoxycarbonyl-propyl) alpha-phenylnitrone, EPPN 1, synthesized by methods previously described, has been purified by recrystallization. A measure of its octanol-phosphate buffer partition coefficient (P(oct) = 29.8) indicated that EPPN was quite lipophilic, yet it could be easily solubilized in water up to 60 mmol L(-1). Although this nitrone was unsuitable for detecting hydroxyl radical, it efficiently trapped several carbon-centered radicals as well as superoxide in aqueous media, without yielding any artifactual signals. Kinetic studies of the superoxide adduct decay gave rate constants k(D) of 2 x 10(-3) and 2.1 x 10(-3) s(-1) at pH 5.6 and pH 7, respectively. EPPN can be considered as an easily prepared and highly pure spin trap, allowing efficient detection of superoxide in aqueous environments.