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Liu Y.,Biomedical Imaging Center | Song Y.,Biomedical Imaging Center | Rockenbauer A.,Institute of Structural Chemistry | Sun J.,Biomedical Imaging Center | And 4 more authors.
Journal of Organic Chemistry | Year: 2011

Measurement of thiol concentrations is of great importance for characterizing their critical role in normal metabolism and disease. Low-frequency electron paramagnetic resonance (EPR) spectroscopy and imaging, coupled with the use of exogenous paramagnetic probes, have been indispensable techniques for the in vivo measurement of various physiological parameters owing to the specificity, noninvasiveness and good depth of magnetic field penetration in animal tissues. However, in vivo detection of thiol levels by EPR spectroscopy and imaging is limited due to the need for improved probes. We report the first synthesis of trityl radical-conjugated disulfide biradicals (TSSN and TSST) as paramagnetic thiol probes. The use of trityl radicals in the construction of these biradicals greatly facilitates thiol measurement by EPR spectroscopy since trityls have extraordinary stability in living tissues with a single narrow EPR line that enables high sensitivity and resolution for in vivo EPR spectroscopy and imaging. Both biradicals exhibit broad characteristic EPR spectra at room temperature because of their intramolecular spin-spin interaction. Reaction of these biradicals with thiol compounds such as glutathione (GSH) and cysteine results in the formation of trityl monoradicals which exhibit high spectral sensitivity to oxygen. The moderately slow reaction between the biradicals and GSH (k2 ∼ 0.3 M-1 s -1 for TSSN and 0.2 M-1 s-1 for TSST) allows for in vivo measurement of GSH concentration without altering the redox environment in biological systems. The GSH concentration in rat liver was determined to be 3.49 ± 0.14 mM by TSSN and 3.67 ± 0.24 mM by TSST, consistent with the value (3.71 ± 0.09 mM) determined by the Ellmans reagent. Thus, these trityl-based thiol probes exhibit unique properties enabling measurement of thiols in biological systems and should be of great value for monitoring redox metabolism. © 2011 American Chemical Society. Source

Liu Y.,Ohio State University | Villamena F.A.,Ohio State University | Song Y.,Ohio State University | Sun J.,Ohio State University | And 2 more authors.
Journal of Organic Chemistry | Year: 2010

Simultaneous evaluation of redox status and oxygenation in biological systems is of great importance for the understanding of biological functions. Electron paramagnetic resonance (EPR) spectroscopy coupled with the use of the nitroxide radicals have been an indispensable technique for this application but are still limited by low oxygen sensitivity and low EPR resolution in part due to the moderately broad EPR triplet and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl and its isotopically labeled 15N analogue TNN15 were synthesized and characterized. Both biradicals exhibited much stronger spin-spin interaction with J > 400 G compared with that of the previous synthesized trityl-nitroxide biradicals TN1 (∼160 G) and TN2 (∼52 G) with longer linker chain length. The enhanced stability of TNN14 was evaluated using ascorbate as reductant, and the effect of different types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the biradicals by ascorbate share the same oxygen sensitivity. Of note is that the 15N-labeled TNN15-H with an EPR doublet exhibits improved EPR signal amplitude as compared with that of TNN14-H with an EPR triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the trityl-nitroxide biradicals. © 2010 American Chemical Society. Source

Liu Y.,Ohio State University | Villamena F.A.,Ohio State University | Rockenbauer A.,Institute of Structural Chemistry | Zweier J.L.,Ohio State University
Chemical Communications | Year: 2010

Novel trityl-nitroxide biradicals were synthesized and exhibited enhanced sensitivity and stability for rapid and simultaneous measurement of redox status and oxygenation by electron paramagnetic resonance spectroscopy. © 2010 The Royal Society of Chemistry. Source

Durand G.,University of Strasbourg | Choteau F.,University of Strasbourg | Prosak R.A.,Ohio State University | Rockenbauer A.,Institute of Structural Chemistry | And 2 more authors.
New Journal of Chemistry | Year: 2010

Due to the dual property of synthetic nitroxide compounds to either act as probe or antioxidant, efforts toward their selective targeting using specific ligands have been extensively explored. Herein, we report the synthesis of novel amphiphilic nitroxides in which the nitroxyl group is grafted onto an amphiphilic carrier comprising a lactobionamide polar group, a non-polar alkyl chain and an amino acid as scaffold. Piperidine and pyrrolidine nitroxides such as 4-amino-TEMPO (4-AT) and 3-carboxyproxyl (3-CP), respectively, were grafted onto the amphiphilic carriers. To further investigate the effect of the nature of the chain on the physical-chemical and biological properties of nitroxides, hydrogenated or perfluorinated alkyl chains were used. The self-aggregation properties in aqueous media of these surfactant-like nitroxides were confirmed by dynamic light scattering (DLS) as well as electron paramagnetic resonance (EPR) spectroscopy, and were correlated with their respective lipophilicity. The effect of the carrier groups on the electrochemical property of nitroxides was investigated using cyclic voltammetry, and the rates of reduction using ascorbate as reducing agent were measured. Finally, their cytoprotective property against toxic concentrations of hydrogen peroxide using bovine aortic endothelial cells was also investigated. © 2010 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique. Source

Kim S.-U.,Ohio State University | Liu Y.,Ohio State University | Nash K.M.,Ohio State University | Zweier J.L.,Ohio State University | And 2 more authors.
Journal of the American Chemical Society | Year: 2010

Nitrone spin traps have been employed as probes for the identification of transient radical species in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy and have exhibited pharmacological activity against oxidative-stress-mediated diseases. Since superoxide radical anion (O2•-) is a major precursor to most reactive oxygen species and calix[4]pyrroles have been shown to exhibit high affinity to anions, a cyclic nitrone conjugate of calix[4]pyrrole (CalixMPO) was designed, synthesized, and characterized. Computational studies at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level suggest a pendant-type linkage between the calix[4]pyrrole and the nitrone to be the most efficient design for spin trapping of O2•-, giving exoergic reaction enthalpies (ΔH298K,aq) and free energies (ΔG298K,aq) of -16.9 and -2.1 kcal/mol, respectively. 1H NMR study revealed solvent-dependent conformational changes in CalixMPO leading to changes in the electronic properties of the nitronyl group upon H-bonding with the pyrrole groups as also confirmed by calculations. CalixMPO spin trapping of O 2•- exhibited robust EPR spectra. Kinetic analysis of O 2•- adduct formation and decay in polar aprotic solvents using UV-vis stopped-flow and EPR methods gave a larger trapping rate constant for CalixMPO and a longer half-life for its O2•- adduct compared to the commonly used nitrones. The unusually high reactivity of CalixMPO with O2•- was rationalized to be due to the synergy between the α-effect and electrostatic effect by the calix[4]pyrrole moiety on O 2•- and the nitrone, respectively. This work demonstrates for the first time the application of an anion receptor for the detection of one of the most important radical intermediates in biological and chemical systems (i.e., O2•-). © 2010 American Chemical Society. Source

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