Tetrathiatriarylmethyl radicals are ideal spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of affordable large-scale syntheses. We report the large-scale synthesis of the Finland trityl radical using an improved addition protocol of the aryl lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivo applications.

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 000:000–000, 2011.

M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47(phox-/-) mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.

Biological applications of stable nitroxyl radicals, NR, include their use as contrast agents for magnetic resonance imaging, spin labels, superoxide dismutase mimics, and antioxidants. The rapid reduction of NR in biological samples into hydroxylamines, HA, significantly limits their application. In its turn, reoxidation of HA back to the NR has been used for detection of reactive oxygen species, ROS. In this work comparative studies of the reduction of pyrrolidine, imidazoline and imidazolidine NR by ascorbate were performed taking advantage of recently synthesized tetraethyl substituted NR with much higher stability towards reduction both in vitro and in vivo. Surprisingly, these NR kept 10-50% of initial intensity of electron paramagnetic resonance signal for about 1 h in the presence of hundred fold excess of ascorbate. To explain this data, reoxidation of the corresponding HA by ascorbate radical and dehydroascorbic acid back to the NR was proposed. This hypothesis was supported by direct measurement of the NR appearance from the HA upon ascorbate radical generation by ascorbate oxidase, or in the presence of the dehydroascorbic acid. The reversible reaction between NR and ascorbate was observed for the various types of the NR, and the rate constants for direct and reverse reactions were determined. The equilibrium constants for oneelectron reduction of the tetraethyl substituted NR by ascorbate were found to be in the range from 2.65×10 −6 to 10 −5 which is significantly lower than corresponding values for the tetramethyl substituted NR (less or about 10 −4 ). This explains an establishment of EPR-detectable quasiequilibrium level of tetraethyl substituted NR in the presence of excess of ascorbate. The redox reactions of the NR-HA couple in ascorbate containing medium was found to be significantly affected by glutathione, GSH. This effect was attributed to the reduction of ascorbate radical by GSH, and the rate constant of this reaction was found to be equal to 10 M −1 s −1 . In summary, the data provide new insight into the redox chemistry of NR and HA, and significantly affect interpretation and strategy of their use as redox-and ROS-sensitive probes, or as antioxidants.