Biomedical subjects

Hakim Karoui

Publications and source records attributed to Hakim Karoui.

7 recordsLinked to original sources

PubMed2006

Nitroxide bound beta-cyclodextrin: is there an inclusion complex?

236CDTIPNO, a derivative of the persistent free radical TIPNO (1,1-dimethylethyl 2-methyl-1-phenylpropyl nitroxide) covalently bound to a permethylated-beta-cyclodextrin, was prepared. Self-association of 236CDTIPNO in water was proved by solvent- and competition-dependent EPR spectroscopy experiments with 2,6-di-O-Me-beta-cyclodextrin (DIMEB) and permethylated-beta-cyclodextrin (TRIMEB) as external hosts competing for accommodation of the TIPNO moiety. Temperature-dependent EPR spectra were simulated with a novel two-dimensional (field-temperature) EPR simulation program that afforded a full determination of the thermodynamic parameters characterizing the rate constants of the self-inclusion reaction derived from Arrhenius and Eyring models. This method allows separating the line broadening effects due to relaxation from a chemical exchange, even if only the fast exchange regime is accessible experimentally. The activation parameters for the forward and backward steps were consistent with an equilibrium between a nonassociated form and a weakly associated form, with activation free enthalpies for each reaction of around 34 kJ.mol(-)(1).

Journal Article↗

PubMed2006

Inclusion complexes of EMPO derivatives with 2,6-di-O-methyl-beta-cyclodextrin: synthesis, NMR and EPR investigations for enhanced superoxide detection.

The free radical trapping properties of eight 5-alkoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO) type nitrones and those of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were evaluated for trapping of superoxide anion radicals in the presence of 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CD). (1)H-NMR titrations were performed to determine both stoichiometries and binding constants for the diamagnetic nitrone-DM-beta-CD equilibria. EPR titrations were then performed and analyzed using a two-dimensional EPR simulation program affording 1 : 1 and 1 : 2 stoichiometries for the nitroxide spin adducts with DM-beta-CD and the associated binding constants after spin trapping. The nitroxide spin adducts associate more strongly with DM-beta-CD than the nitrones. The ability of the nitrones to trap superoxide, the enhancement of the EPR signal intensity and the supramolecular protection by DM-beta-CD against sodium L-ascorbate reduction were evaluated.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Inclusion complexes of PBN-type nitrone spin traps and their superoxide spin adducts with cyclodextrin derivatives: parallel determination of the association constants by NMR titrations and 2D-EPR simulations.

(1)H NMR and electron paramagnetic resonance (EPR) titrations were used to determine the association constants of the complexes of alpha-phenyl-N-tert-butylnitrone (PBN) analogues and their superoxide spin adducts, respectively, with methylated beta-cyclodextrins. A 1:1 stoichiometry for the nitrones with randomly methylated beta-cyclodextrin and 2,6-di-O-methyl-beta-cyclodextrin and 1:1 and 1:2 stoichiometries for the corresponding cyclodextrin-nitroxide complexes were observed. After the superoxide radical spin trapping reaction, EPR titrations afforded the association constants of the corresponding cyclodextrin-nitroxide complexes. Two-dimensional EPR simulations indicated a bimodal inclusion of the nitroxide free radical spin adducts into the cyclodextrins. For all the nitrone-cyclodextrin and nitroxide-cyclodextrin complexes, the association constants were always higher for the nitroxide complexes than for the nitrone complexes. A cooperative system concerning the complexation of the nitroxide spin adduct with a cyclodextrin was evidenced by EPR titrations. The efficiency of the cyclodextrin inclusion technique to trap superoxide and to resist bioreduction by sodium l-ascorbate was also investigated.

Cyclodextrins↗

PubMed2005

Assignment of the EPR spectrum of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) superoxide spin adduct.

[structure: see text] Spin trapping consists of using a nitrone or a nitroso compound to "trap" an unstable free radical as a long-lived nitroxide that can be characterized by electron paramagnetic resonance (EPR) spectroscopy. The formation of DMPO-OOH, the spin adduct resulting from trapping superoxide (O(2)(*)(-)) with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been exploited to detect the generation of superoxide in a wide variety of biological and chemical systems. The 12-line EPR spectrum of DMPO-OOH has been either reported or mentioned in more than a thousand papers. It has been interpreted as resulting from the following couplings: A(N) approximately 1.42 mT, A(H)beta approximately 1.134 mT, and A(H)gamma(1H) approximately 0.125 mT. However, the DMPO-OOH EPR spectrum has an asymmetry that cannot be reproduced when the spectrum is calculated considering a single species. Recently, it was proposed that the 0.125 mT splitting was misassigned and actually results from the superimposition of two individual EPR spectra associated with different conformers of DMPO-OOH. We have prepared 5,5-dimethyl-[3,3-(2)H(2)]-1-pyrroline N-oxide (DMPO-d(2)), and we showed that the EPR spectrum of the corresponding superoxide spin adduct is composed of only six lines, in agreement with the assignment of the 0.125 mT splitting to a gamma-splitting from a hydrogen atom bonded to carbon 3 of DMPO. This result was supported by DFT calculations including water solvation, and the asymmetry of the DMPO-OOH EPR spectrum was nicely reproduced assuming a chemical exchange between two conformers.

Journal Article↗

PubMed2003

Bicarbonate-dependent peroxidase activity of human Cu,Zn-superoxide dismutase induces covalent aggregation of protein: intermediacy of tryptophan-derived oxidation products.

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3-. HCO3--dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion (CO3.). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3--dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3-did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3.-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3., forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3-/H2O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3--dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed.

Amyotrophic Lateral Sclerosis↗

PubMed2002

Spin trapping of superoxide in the presence of beta-cyclodextrins.

The trapping of superoxide anion with DMPO and DEPMPO has been carried out in the presence of a methylated beta-cyclodextrin, Me-beta-CD; inclusion of the spin adducts in the cavity of Me-beta-CD resulted in a seven-fold increase of their half-life (t1/2 = 96 min for DEPMPO-superoxide spin adduct) and in their protection towards glutathione peroxidase (Gpx) and also ascrobate anion in the case of DEPMPO.

Cyclodextrins↗

PubMed1999

Synthesis of a New Spin Trap: 2-(Diethoxyphosphoryl)-2-phenyl-3,4-dihydro-2H-pyrrole 1-Oxide.

Recently, the synthesis of a new phosphorylated nitrone, 2-(diethoxyphosphoryl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (DEPMPO) (2) was described. The presence of the phosphorylated group strongly stabilized the DEPMPO-superoxide spin adduct. To understand the role of the diethoxyphosphoryl group in this stabilization, a new phosphorylated nitrone, 2-(diethoxyphosphoryl)-2-phenyl-3,4-dihydro-2H-pyrrole 1-oxide (DEPPPO) (7), was prepared through a four-step synthetic pathway, and its ability to trap free radicals was investigated. Data obtained from spin trapping experiments of a wide variety of free radicals generated in situ showed the formation of two diastereoisomers spin adducts with different phosphorus and hydrogen coupling constants. Superoxide trapping by DEPPPO gave a persistent nitroxide spin adduct, and its half-time life was measured and compared to that of the DEPMPO analogue.

Journal Article↗