Hakim Karoui

Properties of dinitroxides for use in dynamic nuclear polarization (DNP)

We have investigated the properties of a series of dinitroxides as polarizing agents for SS NMR/DNP applications. Our results clearly establish that an orthogonal relative orientation of electron g tensors is a crucial requirement to obtain high enhancement DNP factors. Moreover, the ratio of the proton Larmor frequency over the e(-)-e(-) dipolar coupling (omega(H)/omega(D)) influences the efficiency of the cross effect (CE) mechanism, thus the Ree distance between the unpaired electrons must be adapted to omega(H).

Spin trapping of superoxide in the presence of β-cyclodextrins

The trapping of superoxide anion with DMPO and DEPMPO has been carried out in the presence of a methylated β-cyclodextrin, Me-β-CD; inclusion of the spin adducts in the cavity of Me-β-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 ascorbate anion in the case of DEPMPO.

Improving the Trapping of Superoxide Radical with a β-Cyclodextrin– 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) Conjugate

During the last decade, the detection of superoxide radical in biological systems continued to receive increasing attention. The superoxide radical is positioned upstream in many free radical cascades leading to reactive oxygen species (ROS). These species are assumed to act as mediators in various physiological and pathological processes, such as signal transduction, aging, inflammatoryand age-related diseases. The search for reliable tools to study ROS in biological systems is important not only for unraveling their complex contributions but also for the development of diagnostic and therapeutic applications. In recent years, hydroethidine derivatives and other fluorescent probes have been developed successfully as ROS probes. However, the complexity of the dye chemistry in biological systems could lead potentially to misinterpretations concerning the ROS implication, and so there is a need to develop complementary techniques. In the late 70s, the coupled spin trapping/EPR technique (ST/EPR) has been developed for the detection and characterization of free radicals. This technique is based on the trapping of free radicals by diamagnetic spin traps leading to the formation of persistent spin adducts exhibiting EPR patterns characteristic of the radical trapped. A large number of studies using the ST/EPR technique has been devoted to the characterization of superoxide generated by various chemical and biological systems. Until the late 90s, almost all these studies reported the use of the wellknown 5,5-dimethyl-pyrroline-N-oxide (DMPO) as spin trap. However, in biological systems, the short half-lifetime ( 1 min) of the DMPO–superoxide spin adduct and its spontaneous decomposition to the DMPO–hydroxyl spin adduct severely limited the characterization of superoxide by ST/EPR. In the meantime, spin traps such as 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) and (Mito-DEPMPO), which exhibit improved performances compared with those of DMPO, have been developed. However, in biological systems the characterization of superoxide by ST/EPR is still hampered by the readily reduction of the spin adducts to EPR silent compounds by bioreductants and the relatively low trapping rate of superoxide radical (from 0.1 to 85 mol 1 L s , depending of the spin trap) compared with its disproportionation rate (3 10 mol 1 L s 1 at pH 7.3) and its reaction with SOD enzymes (5 10 mol 1 L s ). A few years ago, we showed that the addition of six equivalents of b-cyclodextrin derivatives during spin-trapping experiments resulted in an improved superoxide radical detection, due to the increase of the half-life time of the corresponding spin adduct and to its partial protection to reduction by ascorbate and glutathione/glutathione peroxidase. These results were attributed to the selective inclusion of the superoxide spin adduct (Kspinadduct > Knitrone) preventing further interactions with its immediate surrounding. Aiming at expanding these features to biological systems, we and others recently turned our attention to cyclodextrin functionalized nitrone spin traps. In the recent [a] Dr. M. Hardy, Dr. D. Bardelang, Dr. H. Karoui, Dr. J.-P. Finet, Dr. O. Ouari, Prof. P. Tordo UMR 6264, Laboratoire Chimie Provence, Equipe SREP Universit s d’Aix-Marseille 1, 2, 3 et CNRS Avenue Escadrille Normandie Niemen 13397 Marseille Cedex 20 (France) Fax: (+33) 491-288-758 E-mail : paul.tordo@univ-provence.fr olivier.ouari@univ-provence.fr [b] Prof. A. Rockenbauer Chemical Research Center, Institute for Structural Chemistry 1525 Budapest, PO Box 17 (Hungary) [c] Dr. L. Jicsinszky Cyclolab Ltd, 1525 Budapest, PO Box 435 (Hungary) [d] R. Rosas Spectropole, Universit Paul C zanne Centre Scientifique de Saint-J r me 13397 Marseille cedex 20 (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901342.

Inclusion complexes of EMPO derivatives with 2,6-di-O-methyl-β-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.

BDPA-Nitroxide Biradicals Tailored for Efficient Dynamic Nuclear Polarization Enhanced Solid-State NMR at Magnetic Fields up to 21.1 T

Dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance (NMR) has developed into an invaluable tool for the investigation of a wide range of materials. However, the sensitivity gain achieved with many polarizing agents suffers from an unfavorable field and magic angle spinning (MAS) frequency dependence. We present a series of new hybrid biradicals, soluble in organic solvents, that consist of an isotropic narrow electron paramagnetic resonance line radical, α,γ-bisdiphenylene-β-phenylallyl (BDPA), tethered to a broad line nitroxide. By tuning the distance between the two electrons and the substituents at the nitroxide moiety, correlations between the electron-electron interactions and the electron spin relaxation times on one hand and the DNP enhancement factors on the other hand are established. The best radical in this series has a short methylene linker and bears bulky phenyl spirocyclohexyl ligands. In a 1.3 mm prototype DNP probe, it yields enhancements of up to 185 at 18.8 T (800 MHz 1H resonance frequency) and 40 kHz MAS. We show that this radical gives enhancement factors of over 60 in 3.2 mm sapphire rotors at both 18.8 and 21.1 T (900 MHz 1H resonance frequency), the highest magnetic field available today for DNP. The effect of the rotor size and of the microwave irradiation inside the MAS rotor is discussed. Finally, we demonstrate the potential of this new series of polarizing agents by recording high field 27Al and 29Si DNP surface enhanced NMR spectra of amorphous aluminosilicates and 17O NMR on silica nanoparticles.