Ilirian Dhimitruka

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.

Synthesis and Characterization of Ester-Derivatized Tetrathiatriarylmethyl Radicals as Intracellular Oxygen Probes

Electron paramagnetic resonance (EPR) spectroscopy using paramagnetic probes has been employed as an important tool for the accurate determination of oxygen (O2) concentrations in biological systems. However, paramagnetic probes are still limited by their intracellular penetrability. Various esterified trityl derivatives were synthesized and characterized, and an X-ray structure of one of the triyl radicals was determined. The ester-derivatized trityls exhibited higher sensitivity to O2 concentration compared to the trityl tricarboxylate CT-03. Cyclic voltammetry was also carried out to assess the susceptibility of the trityl radicals to oxidation and reduction. Among all of the ester-derivatized trityls studied, facile hydrolysis of the acetoxymethoxy esters to the respective carboxylate was observed using porcine liver esterase. This study demonstrates that cellular permeability of the trityl radicals can be achieved by varying the type and number of ester groups. Therefore, ester-derivatized trityl radicals show great potential as intracellular EPR oximetry probes and imaging agents.

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.

Synthesis, structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthesized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated. The compounds are potential candidates for targeted spin probes in EPR imaging.

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.

Interstitial Inorganic Phosphate as a Tumor Microenvironment Marker for Tumor Progression

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the “growth rate hypothesis” may underline its potential role in tumorigenesis and tumor progression.

New spectral-spatial imaging algorithm for full EPR spectra of multiline nitroxides and pH sensitive trityl radicals

An algorithm is derived and demonstrated that reconstructs an EPR spectral-spatial image from projections with arbitrarily selected gradients. This approach permits imaging wide spectra without the use of the very large sweep widths and gradients that would be required for spectral-spatial imaging with filtered back projection reconstruction. Each projection is defined as the sum of contributions at the set of locations in the object. At each location gradients shift the spectra in the magnetic field domain, which is equivalent to a phase change in the Fourier-conjugate frequency domain. This permits solution of the problem in the frequency domain. The method was demonstrated for 2D images of phantoms consisting of (i) two tubes containing (14)N and (15)N nitroxide and (ii) two tubes containing a pH sensitive trityl radical at pH 7.0 and 7.2. In each case spectral slices through the image agree well with the full spectra obtained in the absence of gradient.

Fourier Transform EPR Spectroscopy of Trityl Radicals for Multifunctional Assessment of Chemical Microenvironment

Pulse techniques in electron paramagnetic resonance (EPR) allow for a reduction in measurement times and increase in sensitivity but require the synthesis of paramagnetic probes with long relaxation times. Here it is shown that the recently synthesized phosphonated trityl radical possesses long relaxation times that are sensitive to probe the microenvironment, such as oxygenation and acidity of an aqueous solution. In principle, application of Fourier transform EPR (FT-EPR) spectroscopy makes it possible to acquire the entire EPR spectrum of the trityl probe and assess these microenvironmental parameters within a few microseconds. The performed analysis of the FT-EPR spectra takes into consideration oxygen-, proton-, buffer-, and concentration-induced contributions to the spectral shape, therefore enabling quantitative and discriminative assessment of pH, pO2, and concentrations of the probe and inorganic phosphate.

Electron paramagnetic resonance monitoring of ischemia-induced myocardial oxygen depletion and acidosis in isolated rat hearts using soluble paramagnetic probes.

A new low‐field electron paramagnetic resonance 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 in isolated perfused rat hearts. The myocardial oxygen measurements were performed using deuterated Finland trityl radical probe. A rapid decrease in 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 min of reperfusion. The lower oxygen concentration after ischemia was attributed to the 50% reduction in coronary flow after ischemia as a consequence of myocardial ischemia and reperfusion damage. Myocardial pH measurements using a specially designed imidazoline pH‐sensitive nitroxide showed severe myocardial acidification to pH 6.25 during 30 min of ischemia. Preconditioning of the hearts with two 5‐min periods of ischemia significantly reduced the acidification of myocardial tissue during sustained ischemia. Noninvasive electron paramagnetic resonance monitoring of myocardial oxygenation and pH may provide important insights into the mechanisms of ischemia and reperfusion injury and a background for development of new therapeutic approaches. Magn Reson Med, 2012.