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.

A new low-field EPR approach for noninvasive measurements of myocardial oxygen tension and tissue acidity was developed. The approach was applied to monitor myocardial pO2 and pH in a model of global no-flow ischemia (30 min) and reperfusion (I/R) in isolated perfused rat hearts. The myocardial oxygen measurements were performed using deuterated Finland trityl radical probe. A rapid decrease of myocardial pO2 from 160 mmHg to about 2 ± 1 mmHg was observed within the first minute of ischemia followed by incomplete restoration of pO2 to 50 mmHg during 30 minutes of reperfusion. The lower oxygen concentration after ischemia was attributed to the 50% reduction in coronary flow after ischemia as a consequence of myocardial I/R damage. Myocardial pH measurements using a specially designed imidazoline pH-sensitive nitroxide showed severe myocardial acidification to pH 6.25 during 30 minutes of ischemia. Preconditioning of the hearts with two 5 minute periods of ischemia significantly reduced the acidification of myocardial tissue during sustained ischemia. Noninvasive EPR monitoring of myocardial oxygenation and pH may provide important insights into the mechanisms of I/R injury and a background for development of new therapeutic approaches.

A sterically hindered bis-spirocyclic C(2)-symmetric chiral pyrrolidine-type nitroxide has been successfully synthesized starting from an l-tartaric derived nitrone. Starting from a pyrrolidine flanked by two methylene groups, complete quaternization of the two α-carbon atoms has been accomplished through iteration of completely regio- and stereoselective intramolecular cycloaddition reactions and organometallic additions to key nitrone intermediates, formed in turn by oxidation procedures. This method appears to be very useful for building up bulky spirocyclic moieties adjacent to a nitroxide group and provides an important supplementation to traditional methods of nitroxide synthesis. The synthesized chiral nitroxide showed a very high stability to reduction with ascorbate (k ≈ 8 × 10(-3) M(-1) s(-1)).

Triarylmethyl radicals, TAMs, are used as persistent paramagnetic probes for EPR spectroscopic and imaging applications and as hyperpolarizing and contrast agents for 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 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 of pH from 6 to 8, the phosphorus hyperfine splitting being convenient and highly sensitive pH marker (spectral sensitivity, 3ΔaP/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 linewidth of pTAM down to 40 mG and, as consequence, provided high sensitivity of the linewidth broadening to pO2 (ΔH/ΔpO2≈0.4 mG/mmHg; accuracy of pO2 measurements, ≈1 mmHg). The independent character of pH and [O2] effects on the EPR spectra of pTAM provides dual functionality to this probe allowing for an extraction of both parameters from a single EPR spectrum.

No abstract