Béatrice Tuccio

A combined spin trapping/EPR/mass spectrometry approach to study the formation of a cyclic peroxide by dienolic precursor autoxidation

The spontaneous addition of air oxygen to a dienolic compound, yielding a cyclic peroxide, was followed by spin trapping (ST) combined with EPR spectroscopy and mass spectrometry (MS). Using two different nitrones, the ST/EPR study allowed the detection of the spin adduct of a radical intermediate, and the radical centre in the addend was identified after similar experiments performed with two different (13)C-labelled analogues of the substrate. The media were also submitted to electrospray ionisation, in both positive and negative modes, for structural characterisation of the spin adducts by tandem mass spectrometry. This allowed the structure of the hydroxylamine derivatives of the nitroxides formed to be identified. Following these results, a mechanism pathway was proposed for this autoxidation.

β-Phosphorylated α-phenyl-N-tert-butylnitrone (PBN) analogues: a new series of spin traps for oxyl radicals

The two β-phosphorylated nitrones, N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide 3(PPN) and 1-diethoxyphosphoryl-1-methyl-N-[(1-oxidopyridin-1-ium-4-yl)methylidene]ethylamine N-oxide 4 (4-PyOPN), which are the phosphorylated analogues of α-phenyl-N-tert-butylnitrone (PBN) and α-(1-oxidopyridin-1-ium-4-yl)-N-tert-butylnitrone (4-PyOBN), respectively, have been prepared and their ability to trap oxygen-centred radicals has been investigated.

PPN-type nitrones: preparation and use of a new series of β-phosphorylated spin-trapping agents†

The synthesis of six members of a new series of β-phosphorylated nitrones derived from α-phenyl-N-tert-butylnitrone (PBN)‡ is described. A new method, based on HPLC, is used to evaluate their n-octanol–phosphate buffer partition coefficient. As is the case for other PBN-type traps, the lipophilicity of these nitrones is found to be greatly dependent on the structure of their aromatic moiety. The capacity of these compounds to act as spin trapping agents is surveyed by using them to trap various free radicals in organic or aqueous media, and the EPR parameters of the different spin adducts obtained are reported herein.

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.

Effect of two new PBN‐derived phosphorylated nitrones against postischaemic ventricular dysrhythmias

Spin traps might exert antioxidant cardioprotective effects during myocardial ischaemia–reperfusion where free radicals are thought to be responsible for the occurrence of reperfusion injury. The aim of our study was to investigate the effects of two new α‐phenyl N‐tert‐butylnitrone (PBN)‐derived β‐phosphorylated nitrones: 2‐N‐oxy‐N‐[benzylidène amino] diéthyl propyl‐2‐phosphate (PPN) and 1‐diethoxyphosphoryl‐1‐methyl‐N‐[(1‐oxido‐pyridin‐1‐ium‐4‐yl) methylidene] ethylamine N‐oxide (4‐PyOPN) compared with PBN on (1) the evolution of cardiovascular parameters and (2) the postischaemic recovery. Anaesthetized rats were injected with 120 μmol/kg of the nitrones or 14 μmol/kg of amiodarone, used as a reference antidysrhythmic drug. Ischaemia was induced in vivo through ligation of the left anterior descending coronary artery for 5 min followed by 15 min of reperfusion after release. Cardiovascular parameters and occurrence of ventricular premature beats (VPB), ventricular tachycardia (VT) and fibrillation (VF) were recorded throughout the experiment. Under nonischaemic conditions, none of the three spin traps was shown to modify cardiovascular parameters during the 25‐min measurement period. Solvent‐treated (NaCl 0.9%) animals challenged with ischaemia–reperfusion exhibited 39 ± 10 VPB, 156 ± 39 s of VT and 60% mortality caused by sustained VF. Nitrones improved slightly postischaemic recovery, reducing the occurrence of VF and mortality to 33% whereas amiodarone injection totally suppressed rhythm disturbances and mortality. Our study has shown only limited antidysrhythmic cardioprotective effects of PBN‐derived β‐phosphorylated nitrones during reperfusion after a regional myocardial ischaemia but also minor antioxidant properties of these spin trapping agents.

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.

Characteristics and use as spin trapping agent of a β-phosphorylated nitroso compound, DEPNP

2-(Diethylphosphonate)-nitrosopropane (DEPNP), prepared by oxidation of the corresponding aminophosphonate, was found to essentially exist as monomer in both water and organic solvents. The mechanisms of its degradation under 80°C heating or visible light exposure were studied by EPR spectroscopy: its decomposition gave rise to paramagnetic by-products, which have been identified as DEPNP / ·C(CH3)2[P(O)(OC2H5)2] and DEPNP / ·P(O)(OC2H5)2 spin adducts. Despite this drawback, DEPNP was successfully used as spin trapping agents to scavenge various carbon — and phosphorus-centred free radicals both in aqueous and organic media, giving rise to intense EPR spectra characteristic of the species trapped.

Electrochemical characterisation of β-phosphorylated nitrone spin traps

Oxidation and reduction potentials have been determined for a series of thirteen β-phosphorylated nitrones in water and in acetonitrile. The effect of structural modifications on potential window is briefly discussed.

Spin-trapping of free radicals by PBN-type β-phosphorylated nitrones in the presence of SDS micelles†

Three PBN-type β-phosphorylated nitrones, PPN 1, 4-PyOPN 2, and 4-ClPPN 3, have been used to trap two carbon centred free radicals, CH3˙ and CO2˙–, in a water–SDS biphasic system. The location of the traps and their adducts has been found by EPR spectroscopy. 4-PyOPN is found to remain in water, while 4-ClPPN is preferentially sequestered into the micellar structure, and PPN partitioned between the two phases. With the three nitrones, the CO2˙– spin adduct always remains in the bulk aqueous phase. In contrast, the three CH3˙ spin adducts seem to be present in both phases. However, 4-PyOPN-CH3 is essentially located in water, and 4-ClPPN–CH3 in the micelles, while PPN–CH3 is clearly shown to partition between the aqueous environment and the micellar interior. For all these spin adducts, the phosporus hyperfine splitting constant, aP, is found to be a good indicator of aminoxyl behaviour in the presence of micelles. For the various sodium dodecyl sulfate concentrations, the PPN–CH3 affinity for the micellar phase was evaluated from the average aP value.

Combining EPR and ESI-MS/MS to study the reactivity of alkylthiols and cysteine towards 2-methyl-2-nitrosopropane (MNP)

An approach combining EPR spectroscopy and electrospray ionisation mass spectrometry was employed to study the reactivity of two alkylthiols and of cysteine towards 2-methyl-2-nitrosopropane (MNP) in the presence of H2O2. Depending on the experimental conditions, various nitroxides could be EPR-detected, while their diamagnetic derivatives were structurally characterised after tandem mass spectrometry experiments. Sample dilution in methanol before electrospray ionisation was also found to generate methyl-hydroxylamine derivatives and could thus constitute a simple derivatisation process to stabilise hardly MS-detectable radical species. Upon white light irradiation, the three thiols tested led to alkoxynitroxides according to an inverted spin trapping process. On the other hand, only the reduced form of cysteine was found to react with MNP in the dark, yielding an alkylthionitroxide adduct via a Forrester–Hepburn mechanism. Besides a better understanding of the reactivity of thiols towards nitroso compounds, the results obtained underlined the particular behaviour of cysteine, suggesting that the use of a molecule as simple as MNP could allow a nitroxide moiety to be linked to a cysteinyl residue.