Denis A. Komarov

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.

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase.

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-dl-alanine (DOPA) has been studied. The ability of DOPA to react with has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and being equal to (3.4±0.6)×105 M−1 s−1. The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with

Synthesis of a chiral C2-symmetric sterically hindered pyrrolidine nitroxide radical via combined iterative nucleophilic additions and intramolecular 1,3-dipolar cycloadditions to cyclic nitrones.

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)).

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.

Functional electron paramagnetic resonance imaging of ischemic rat heart: Monitoring of tissue oxygenation and pH

Electron paramagnetic resonance (EPR) imaging in the spectral‐spatial domain with application of soluble paramagnetic probes provides an opportunity for spatially resolved functional measurements of living objects. The purpose of this study was to develop EPR methods for visualization of oxygenation and acidosis of ischemic myocardium.

Effect of Sterical Shielding on the Redox Properties of Imidazoline and Imidazolidine Nitroxides.

The oxidant properties of the series of 2,2,5,5-tetraalkyl imidazoline and imidazolidine nitroxides were investigated. An increase in the number of bulky alkyl substituents leads to a decrease in the rate of reduction with ascorbate, which makes the electrochemical reduction potential more negative and shifts the equilibrium in the mixture of nitroxide and reference hydroxylamine (3-carboxy-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-1-(15)N) toward the starting compounds. The effect of structural factors on these reactions was analyzed by means of multiple regression using the Fujita steric constant Es and the inductive Hammett constant σI. Satisfactory statistical outputs were obtained in all of the biparameter correlations, denoting that the oxidant properties of the nitroxides are determined by steric and electronic effects of the substituents. The data imply that bulky substituents can stabilize nitroxide and/or destabilize hydroxylamine.

Fast backprojection-based reconstruction of spectral-spatial EPR images from projections with the constant sweep of a magnetic field

In this paper, we introduce a procedure for the reconstruction of spectral-spatial EPR images using projections acquired with the constant sweep of a magnetic field. The application of a constant field-sweep and a predetermined data sampling rate simplifies the requirements for EPR imaging instrumentation and facilitates the backprojection-based reconstruction of spectral-spatial images. The proposed approach was applied to the reconstruction of a four-dimensional numerical phantom and to actual spectral-spatial EPR measurements. Image reconstruction using projections with a constant field-sweep was three times faster than the conventional approach with the application of a pseudo-angle and a scan range that depends on the applied field gradient. Spectral-spatial EPR imaging with a constant field-sweep for data acquisition only slightly reduces the signal-to-noise ratio or functional resolution of the resultant images and can be applied together with any common backprojection-based reconstruction algorithm.

A 750-MHz Electronically Tunable Resonator Using Microstrip Line Couplers for Electron Paramagnetic Resonance Imaging of a Mouse Tumor-Bearing Leg

Objective: The purpose of this work was to develop an electronically tunable resonator operating at 750 MHz for continuous-wave electron paramagnetic resonance (CW-EPR) imaging of a mouse tumor-bearing leg. Methods: The resonator had a multi-coil parallel-gap structure with a sample space of 16 mm in diameter and 20 mm in length. Microstrip line couplers were used in conjunction with varactor diodes to enable resonance frequency adjustment and to reduce the nonlinear effects of the varactor diodes. The resonator was modeled by the finite-element method and a microwave circuit simulation was performed to clarify its radiofrequency characteristics. Results : A tunable resonator was evaluated in terms of its resonance frequency, tunable frequency band, and conversion efficiency of the RF magnetic field. The developed resonator provided a tunable frequency band of 4 MHz at a central frequency of 747 MHz and a conversion efficiency of 34 μT/W1/2. To demonstrate the application of this tunable resonator to EPR imaging, three-dimensional EPR images of a sample solution and a mouse tumor-bearing leg were obtained. Conclusion: The developed tunable resonator satisfied our initial requirements for in vivo EPR imaging and may be able to be further improved using the present finite-element and circuit models if any problems arise during future practical applications. Significance: This work may help to promote EPR imaging of tumor-bearing mice in cancer-related studies.