Andrey A. Bobko

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.

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.

Synthesis and Characterization of Amino Derivatives of Persistent Trityl Radicals as Dual Function pH and Oxygen Paramagnetic Probes

Triarylmethyl radicals, TAMs, are useful soluble paramagnetic probes for EPR spectroscopic and imaging applications because of their extraordinary stability in living tissues, narrow line width, high analytical resolution at micromolar concentrations and enhanced sensitivity to molecular oxygen. Recently we proposed the concept of dual function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure (J. Am. Chem. Soc. 2007, 129 (23), 7240-7241). In this paper we report the synthesis of TAM derivatives containing amino groups. The synthesized TAMs combine stability with oxygen and pH sensitivity, in the range of pH from 6.8 to 9.0. To decrease the number of spectral components and improve probe solubility at physiological pH, asymmetric TAM derivatives containing both carboxyl and amino functions were synthesized. The presence of nitrogen and hydrogen atoms in direct proximity to protonatable amino groups resulted in strong pH-induced changes to the corresponding hyperfine splittings, Delta hfs approximately (300-1000) mG, comparable to the values of hfs themselves. Large pH-dependent line shifts of individual spectral components, with narrow linewidths of (160-280) mG, allow for easy discrimination between the pH effect and the observed oxygen-dependent line broadening of about (6 +/- 0.5) mG per % oxygen. The synthesized TAM derivatives represent the first dual function pH and oxygen paramagnetic probes with reasonably valuable properties for biomedical research.

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.

NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols

BackgroundOvarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied.MethodsNCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies.ResultsCells treated with NCX-4040 (25 μM) showed a significant reduction of cell viability (A2780 WT, 34.9 ± 8.7%; A2780 cDDP, 41.7 ± 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 ± 11.8% versus NCX-4040+cisplatin, 26.4 ± 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 ± 4.4% versus NCX-4040+cisplatin, 56.4 ± 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression.ConclusionThe results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.

Trityl-based EPR probe with enhanced sensitivity to oxygen

An asymmetric derivative of the triarylmethyl radical, TAM-H, containing one aldehyde and two carboxyl groups, was synthesized. The electron paramagnetic resonance (EPR) spectrum of TAM-H is characterized by a doublet of narrow lines with a linewidth of 105 mG under anoxic conditions and hyperfine interaction constant of 245 mG. The partial overlap of the components of the doublet results in enhanced sensitivity of the spectral amplitudes ratio to oxygen compared with oxygen-induced linewidth broadening of a single line. Application of the TAM-H probe allows for EPR measurements in an extended range of oxygen pressures from atmospheric to 1 mm Hg, whereas the EPR spectrum linewidth of the popular TAM-based oxygen sensor Oxo63 is practically insensitive to oxygen partial pressures below 20 mm Hg. Enhanced sensitivity of the TAM-H probe relative to Oxo63 was demonstrated in the detection of oxygen consumption by Met-1 cancer cells. The TAM-H probe allowed prolonged measurements of oxygen depletion during the hypoxia stage and down to true anoxia (<or=1.5 mm Hg).

Phosphonated Trityl Probes for Concurrent in Vivo Tissue Oxygen and pH Monitoring Using Electron Paramagnetic Resonance-Based Techniques

Previously we proposed the concept of dual function pH and oxygen paramagnetic probes based on the incorporation of ionizable groups into the structure of persistent triarylmethyl radicals, TAMs (J. Am. Chem. Soc.2007, 129, 7240-7241). In this paper, we synthesized an asymmetric monophosphonated TAM probe with the simplest doublet hfs pattern ideally suited for dual function electron paramagnetic resonance (EPR)-based applications. An extraordinary low line width of the synthesized deuterated derivative, p1TAM-D (ΔHpp ≤ 50 mG, Lorentz line width, ≤20 mG) results in high sensitivity to pO2 due to oxygen-induced line broadening (ΔLW/ΔpO2 ≈ 0.5 mG/mmHg or ≈400 mG/mM); accuracy of pO2 measurement, ≈1 mmHg). The presence of a phosphono group in the p1TAM-D structure provides pH sensitivity to its EPR spectra in the physiological range of pH from 5.9 to 8.2 with the ratio of signal intensities of protonated and deprotonated states being a reliable pH marker (accuracy of pH measurements, ± 0.05). The independent character of pH and [O2] effects on the EPR spectra of p1TAM-D provides dual functionality to this probe. The L-band EPR studies performed in breast tumor-bearing mice show a significant difference in extracellular pH and pO2 between tumor and normal mammary gland tissues, as well as the effect of animal breathing with 100% O2 on tissue oxygenation. The developed dual function phosphonated p1TAM-D probe provides a unique tool for in vivo concurrent tissue oxygen and pH monitoring.

Design of liposome-based pH sensitive nanoSPIN probes: nano-sized particles with incorporated nitroxides

Liposome-based nanoSized Particles with Incorporated Nitroxides, or nanoSPINs, were designed for EPR applications as pH probes in biological systems. Phospholipid membrane of the liposomes with incorporated gramicidin A showed selective permeability to a small analyte, H(+), while protecting entrapped sensing nitroxide from biological reductants. An application of the pH-sensitive nanoSPIN in an ischemia model in rat heart homogenate allows for monitoring ischemia-induced acidosis while protecting encapsulated nitroxide against bioreduction.

In Vivo Proton–Electron Double-Resonance Imaging of Extracellular Tumor pH Using an Advanced Nitroxide Probe

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

Dual-function pH and oxygen phosphonated trityl probe.

Triarylmethyl radicals (TAMs) are used as persistent paramagnetic probes for electron paramagnetic resonance (EPR) spectroscopic and imaging applications and as hyperpolarizing and contrast agents for magnetic resonance imaging (MRI) and proton-electron double-resonance imaging (PEDRI). Recently we proposed the concept of dual-function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure ( J. Am. Chem. Soc. 2007 , 129 , 7240 - 7241 ). In this paper we report the synthesis of a deuterated derivative of phosphonated trityl radical, pTAM. The presence of phosphono substitutes in the structure of TAM provides pH sensitivity of its EPR spectrum in the physiological range from 6 to 8, the phosphorus hyperfine splitting acting as a convenient and highly sensitive pH marker (spectral sensitivity, 3Δa(P)/ΔpH ≈ 0.5 G/pH unit; accuracy of pH measurements, ±0.05). In addition, substitution of 36 methyl protons with deuterons significantly decreased the individual line width of pTAM down to 40 mG and, as consequence, provided high sensitivity of the line-width broadening to pO(2) (ΔH/ΔpO(2) ≈ 0.4 mG/mmHg; accuracy of pO(2) measurements, ≈1 mmHg). The independent character of pH and [O(2)] effects on the EPR spectra of pTAM provides dual functionality to this probe, allowing extraction of both parameters from a single EPR spectrum.