I. A. Grigor'ev

EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines.

Abstract Superoxide (O2•−) has been implicated in the pathogenesis of many human diseases, but detection of the O2•− radicals in biological systems is limited due to inefficiency of O2•− spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O2•− in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O2•−, producing stable nitroxides and allowing site-specific O2•− detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O2•−. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O2•− both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O2•− stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O2•−. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O2•− radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

Synthesis of the tetraethyl substituted pH-sensitive nitroxides of imidazole series with enhanced stability towards reduction

The synthesis of 2,2,5,5-tetraethylimidazole nitroxides from 3-ethylpent-2-ene is described. The newly synthesized nitroxides, namely 4-methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazol-1-yloxy (1), 3,4-dimethyl-2,2,5,5-tetraethylperhydroimidazol-1-yloxy (2) and 2,2,5,5-tetraethyl-4-pyrrolidin-1-yl-2,5-dihydro-1H-imidazol-1-oxyl (3), were found to be pH sensitive spin probes, with pK values of 1.2, 4.95 and 7.4, respectively. The most important finding was the fact that these new nitroxides were 20-30 times more stable in the presence of ascorbate and had significantly longer halflifes in rat blood as compared to 2,2,5,5-tetramethyl analogs. The latter observation provides a unique advantage for the application of tetraethyl substituted imidazole nitroxides as functional EPR probes.

Cytotoxicity of spin trapping compounds

Spin trapping compounds are used frequently to detect free radicals released by cells. Their cytotoxicity has to be considered in order to prevent perturbations of normal cell growth and viability. Eleven spin traps (eight nitrones and three nitroso traps) have been tested for their effects on bovine aortic endothelial cells (toxicity range, 50% survival rate). The lowest cytotoxicity was found for 5,5‐dimethylpyrroline‐1‐oxide and 2,2,4‐trimethyl‐2H‐imidazole‐1‐oxide whereas nitrosobenzene and 2‐methyl‐2‐nitrosopropane exerted the strongest cytotoxic effects. In addition, three nitronyl nitroxides were tested. Their cytotoxicity was found to be dependent on substitution, and the toxic concentration of a lipophilic derivative was found to be more than two orders lower as compared to a hydrophilic derivative. The results of this study indicate that most spin traps can be used in cell cultures at customary (i.e. millimolar) concentrations; caution is recommended when nitroso spin traps are applied to cells.

In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors

Approach for in vivo real‐time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L‐band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony‐stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony‐stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony‐stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double‐resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med, 2011.

Nitroxides with two pK values—useful spin probes for pH monitoring within a broad range

A series of 4-dialkylamino-2,5-dihydroimidazole nitroxides with pyridine-4-yl, 4-dimethylaminophenyl or 4-hydroxyphenyl groups in position 2 of the imidazole ring were prepared using the reaction of RMgBr with corresponding 5-dialkylamino-4,4-dimethyl-4H-imidazole 3-oxides. The EPR spectra of the nitroxides were shown to be pH-sensitive due to consecutive protonation of the amidino moiety and the basic group(s) at position 2 of the imidazole ring. The 5,5-dimethyl-4-(dimethylamino)-2-ethyl-2-pyridine-4-yl-2,5-dihydro-1H-imidazol-1-oxyl showed a monotonic increase in the isotropic nitrogen hyperfine (hfi) coupling constant alpha(N) of 1 .4 G over a pH range from 2 to 6.5. Such a broad range of pH-sensitivity could be useful for many biophysical and biomedical applications, including pH-monitoring in the stomach.

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts.

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.

In Vitro and In Vivo Measurement of pH and Thiols by EPR-Based Techniques

In vitro and in vivo measurements of pH and thiols provide critical information on physiology and pathophysiology of living organisms, particularly related to oxidative stress. Stable nitroxides of imidazoline and imidazolidine types provide the unique possibility of measuring local values of pH and glutathione content in various biological systems, including in vivo studies. The basis for these applications is the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in the electron paramagnetic resonance (EPR) spectra of these probes, measured by low-frequency EPR techniques. The applications of some newly developed pH and SH probes in model systems of pharmacological interest, biological fluids, tissues, and cells as well as in vivo studies in isolated hearts and in the gut of living animals are discussed.

Superoxide-Mediated Reduction of the Nitroxide Group Can Prevent Detection of Nitric Oxide by Nitronyl Nitroxides

Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated .NO chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without detectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN concentration. Model experiments using hypoxanthine/xanthine oxidase in aqueous solution and KO2 in dimethylsulfoxide as sources of O2.- revealed that there is a rapid reduction of nitronyl nitroxides by superoxide. The second order rate constant for the reaction of the water soluble NN with O2.- was determined to be 8.8 x 10(5) M-1s-1, which is more than two orders of magnitude higher than the value reported previously for reaction with .NO (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the presence of glutathione, ascorbic acid or rabbit liver microsomes. Incorporation of both nitronyl and imino nitroxides into liposomes strongly decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposomal encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.

In vivo detection of a pH‐sensitive nitroxide in the rat stomach by low‐field ESR‐based techniques

A study was made of the in vivo detectability of a pH‐sensitive, imidazolidine spin probe, and the efficacy of low‐frequency electron spin resonance (ESR)‐based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally‐detected ESR (LODESR) and field‐cycled dynamic nuclear polarization (FC‐DNP) for in vitro and in vivo measurements, and radiofrequency (RF)‐ and X‐band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC‐DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer‐term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC‐DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system. Magn Reson Med 49:558–567, 2003.

Route to stable nitroxides with alkoxy groups at α- carbon - the derivatives of 2- and 3-imidazolines

Abstract Oxidation of 1-hydroxy-5,5-dimethyl-3-imidazoline-3-oxides containing one or two hydrogen atoms in position 2 of the heterocycle with lead dioxide in alcohol leads to stable nitroxides with alkoxy groups at α-carbon, the derivatives of 2- and 3-imidazolines, via the intermediate formation of 4H-imidazol-1,3-dioxides.