Triarylmethyl Radicals Research Articles

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.

Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution.

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.

First Combined in Vivo X-Ray Tomography and High-Resolution Molecular Electron Paramagnetic Resonance (EPR) Imaging of the Mouse Knee Joint Taking into Account the Disappearance Kinetics of the EPR Probe

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 μm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.

A phosphonated triarylmethyl radical as a probe for measurement of pH by EPR

A new water soluble phosphonated tetrathiatriarylmethyl radical has been synthesized and its application for pH measurement in a physiological range by EPR is reported.

Three‐dimensional EPR/NMR image coregistration using a MATLAB‐based software

AbstractThe coregistration of electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) images in three‐dimensional (3D) data space typically involves a process of visual inspection and manual alignment of the images. This is due primarily to the use of separate scanners, which can result in a number of potential differences between the two image types, such as resolution, field‐of‐view, and image orientation. In this article, we describe recently developed methodology and software that enables automatic coregistration of EPR and NMR acquired images without the need for manual alignment. The technique requires the use of fiducial markers during both EPR and NMR image acquisition, which contain a solution of free radical spin probes, and allow adjustment of the positions and the fields‐of‐view of the measured images. In this work we present results for EPR/NMR image coregistration using an isoflurane‐anesthetized mouse, injected with triarylmethyl radical (Oxo63) spin probes. The same spin probes were also used inside the fiducial markers and in plastic tubes used for phantom measurements. Using our MATLAB‐based software, we were able to successfully coregister EPR/NMR images in approximately 60 s. © 2011 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 39B: 180–190, 2011

Frequency bandwidth extension by use of multiple Zeeman field offsets for electron spin-echo EPR oxygen imaging of large objects

Electron spin-echo (ESE) oxygen imaging is a new and evolving electron paramagnetic resonance (EPR) imaging (EPRI) modality that is useful for physiological in vivo applications, such as EPR oxygen imaging (EPROI), with potential application to imaging of multicentimeter objects as large as human tumors. A present limitation on the size of the object to be imaged at a given resolution is the frequency bandwidth of the system, since the location is encoded as a frequency offset in ESE imaging. The authors' aim in this study was to demonstrate the object size advantage of the multioffset bandwidth extension technique. The multiple-stepped Zeeman field offset (or simply multi-B) technique was used for imaging of an 8.5-cm-long phantom containing a narrow single line triaryl methyl compound (trityl) solution at the 250 MHz imaging frequency. The image is compared to a standard single-field ESE image of the same phantom. For the phantom used in this study, transverse relaxation (T(2e)) electron spin-echo (ESE) images from multi-B acquisition are more uniform, contain less prominent artifacts, and have a better signal to noise ratio (SNR) compared to single-field T(2e) images. The multi-B method is suitable for imaging of samples whose physical size restricts the applicability of the conventional single-field ESE imaging technique.

Use of the Frank sequence in pulsed EPR

The Frank polyphase sequence has been applied to pulsed EPR of triarylmethyl radicals at 256 MHz (9.1 mT magnetic field), using 256 phase pulses. In EPR, as in NMR, use of a Frank sequence of phase steps permits pulsed FID signal acquisition with very low power microwave/RF pulses (ca. 1.5 mW in the application reported here) relative to standard pulsed EPR. A 0.2 mM aqueous solution of a triarylmethyl radical was studied using a 16 mm diameter cross-loop resonator to isolate the EPR signal detection system from the incident pulses.

Resolution-recovery for EPR imaging of free radical molecules in mice

A method of post-processing to enhance the image resolution of the distribution of free radical molecules obtained with continuous-wave electron paramagnetic resonance (CW-EPR) imaging is reported. The low spatial resolution of EPR imaging, which has created difficulties in biomedical applications, was overcome by the method of resolution-recovery for EPR imaging. High spatial resolution images for the distribution of free radical molecules with a very short relaxation time were obtained with this method. The method's two-step postprocessing consists of conventional deconvolution and filtered back-projection and a process of iterative deconvolution. The resolution-recovery method was demonstrated with three-dimensional (3D) imaging of stable nitroxyl radicals in mouse head. In phantom experiments with a solution of triarylmethyl (TAM) radicals, the spatial resolution was improved by a factor of 7 with the resolution-recovery method.

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 (≤ 1.5 mm Hg).

Esterified trityl radicals as intracellular oxygen probes

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O 2 concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O 2 concentrations and the effects of menadione and KCN on the rates of O 2 consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O 2 concentration and consumption.

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.

Slice-selective images of free radicals in mice with modulated field gradient electron paramagnetic resonance (EPR) imaging

Continuous wave (CW) electron paramagnetic resonance (EPR) imaging can be used to obtain slice-selective images of free radicals without measuring three-dimensional (3D) projection data. A method that incorporated a modulated magnetic field gradient (MFG) was combined with polar field gradients to select a slice in the subject noninvasively. The slice-selective in vivo EPR imaging of triarylmethyl radicals in the heads of live mice is reported. 3D surface-rendered images were successfully obtained from slice-selective images. In the experiment in mice, a slice thickness of 1.8 mm was achieved.

Trityl Radicals as Persistent Dual Function pH and Oxygen Probes for in Vivo Electron Paramagnetic Resonance Spectroscopy and Imaging: Concept and Experiment

Triarylmethyl radicals, TAMs, have been the popular choice for electron paramagnetic resonance (EPR) spectroscopy and imaging applications because of their extraordinary stability in cells and tissues, narrow linewidths resulting in high analytical resolution at μM concentrations, and enhanced sensitivity to O2. In this Communication we propose a general approach and provide experimental data allowing for improvement of the functionality of TAM radicals by developing pH sensitive derivatives. The magnetic resonance parameters, both g-factor and hydrogen hyperfine splitting, of several TAM radicals containing different number of carboxyl groups and hydrogen atoms attached to the aryl substituents were found to be pH dependent. This pH effect was measured using X-band and Q-band EPR spectroscopy and is related to reversible protonation of carboxyl groups. Taking into account that both oxygen and pH play key roles in cellular metabolism and homeostasis, dual function pH and oxygen probes based on TAM radical...

Neutral Organic Mixed-Valence Compounds: Synthesis and All-Optical Evaluation of Electron-Transfer Parameters

In this paper we present the synthesis as well as a detailed study of the electrochemical and photophysical properties of a series of neutral organic mixed-valence (MV) compounds, 1-7, in which different amine donor centers are connected to perchlorinated triarylmethyl radical units by various spacers. We show that this new class of compounds are excellent model systems for the investigation of electron transfer due to their uncharged character and, consequently, their excellent solubility, particularly in nonpolar solvents. A detailed band shape analysis of the intervalence charge-transfer (IV-CT) bands in the context of Jortner's theory allowed the electron-transfer parameters (inner vibrational reorganization energy lambdav, outer solvent reorganization energy lambdao, and the difference in the free energy between the diabatic ground and excited states, DeltaG degrees degrees , as well as the averaged molecular vibrational mode v) to be extracted independently. In this way we were able to analyze the solvatochromic behavior of the IV-CT bands by evaluating the contribution of each parameter. By comparison of different compounds, we were also able to assign specific molecular moieties to changes in vv. For this class of molecules, we also demonstrate that the adiabatic dipole moment difference Deltamicroab and, consequently, the electronic coupling V12 can be evaluated directly from the absorption spectra by a new variant of the solvatochromic method. Furthermore, an investigation of the electrochemistry of compounds 1-7 by cyclic voltammetry as well as spectroelectrochemistry shows that, not only in the neutral MV compounds but also in their oxidized forms, a charge transfer can be optically induced but with exchanged donor-acceptor functionalities of the redox centers.

Fast‐response VHF pulsed 2 KW power amplifiers

AbstractA pulsed power amplifier operating at a nominal 250 MHz with 2 kW (+63 dBm) output was designed to meet the specifications for an application in a pulsed EPR (electron paramagnetic resonance) spectrometer and was built by Tomco Technologies of Adelaide, South Australia. Key design features were pulse fidelity for short (e.g., 40 ns) RF pulses, output noise blanking after the pulse, and output power linear with input power from very low levels up to 2 kW output. A summary of the specifications, design, and performance of the amplifier is presented. The 2 kW RF amplifier tested has approximately 12 ns rise times and 15 ns fall times, permitting the formation of approximately 40 ns RF pulses. It has negligible amplitude droop over microsecond‐long pulses and negligible phase change over microsecond long pulses. There was observable but small phase change during the rise and fall times of the pulse. Even for input pulses as short as 16 ns, the output pulse peaks at more than half of the rated power output. Application to measuring the free induction decays of triaryl methyl radicals in aqueous solution was demonstrated. © 2006 Wiley Periodicals, Inc. Concepts Magn Reson 29B: 185–190, 2006.

Reactivity of Molecular Oxygen with Ethoxycarbonyl Derivatives of Tetrathiatriarylmethyl Radicals

Tetrathiatriarylmethyl (TAM) radicals are commonly used as oximetry probes for electron paramagnetic resonance imaging applications. In this study, the electronic properties and the thermodynamic preferences for O2 addition to various TAM-type triarylmethyl (trityl) radicals were theoretically investigated. The radicals' stability in the presence of O2 and biological milieu was also experimentally assessed using EPR spectroscopy. Results show that H substitution on the aromatic ring affects the trityl radical's stability (tricarboxylate salt 1-CO2Na > triester 1-CO2Et > diester 2-CO2Et > monoester 3-CO2Et) and may lead to substitution reactions in cellular systems. We propose that this degradation process involves an arylperoxyl radical that can further decompose to alcohol or quinone products. This study demonstrates how computational chemistry can be used as a tool to rationalize radical stability in the redox environment of biological systems and aid in the future design of more biostable trityl radicals.

Electron paramagnetic resonance imaging of tumor hypoxia: Enhanced spatial and temporal resolution for in vivo pO2 determination

The time-domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen-dependent linewidth (LW) to generate pO2 maps with minimal errors. A methodology for noninvasive pO2 imaging based on the application of TD-EPR using oxygen-induced LW broadening of a triarylmethyl (TAM)-based radical is presented. The decay of pixel intensities in an image is used to estimate T2*, which is inversely proportional to pO2. Factors affecting T2* in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line-broadening. Suitable experimental and image-processing parameters were obtained to produce pO2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor-bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of approximately 1.0 mm and a temporal resolution of approximately 9 s for 2D pO2 mapping, with a reliable oxygen resolution of approximately 1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution.

Multifrequency EPR of four triarylmethyl radicals

Continuous-wave spectra at W-band of four triarylmethyl (trityl) radicals at 100 K in 1∶1 water-glycerol exhibit rhombic electron paramagnetic resonance spectra. The rigid-lattice line widths at W-band are only 3 to 5 times larger than at X-band or S-band, and fluid-solution line widths are much narrower than those for rigid lattice, which indicates that unresolved anisotropic nuclear hyperfine couplings make significant contributions to the rigid-lattice line widths. Spin-flip lines are observed in glassy-solution spectra at X-band and S-band, but not at W-band or 250 MHz. At 100 KTm is dominated by spin diffusion of solvent protons and is independent of microwave frequency. Between about 130 and 170 K, 1/Tm for trityl-CH3 is enhanced by rotation of the methyl groups at a rate comparable to inequivalences in the hyperfine interaction. Motional averaging of anisotropic interactions enhances spin echo dephasing between about 200 and 300 K. The temperature dependence of 1/T1 is similar for the four radicals and is consistent with assignment of the Raman process and a local mode as the dominant relaxation processes. The similarity inT1 values at W-band and X-band supports this assignment.

Impact of resonator on direct-detected rapid-scan EPR at 9.8 GHz

Rapid-scan electron paramagnetic resonance spectra at 9.8 GHz were obtained on a Bruker E580 spectrometer. Spectra of lithium phthalocyanine (LiPc) needles (T1=8 μs,T2=3.4 μs) and of a 0.2 mM aqueous solution of Nycomed triarylmethyl (trityl-CD3) radical (T1=11.5 μs,T2=8 μs) were recorded at scan rates between 3.4·103 and 7.5·105 G/s at the center of sinusoidal scans. Signals for LiPc were obtained with a split-ring resonator, a rectangular resonator and a dielectric resonator. At faster scan rates the small bandwidth of the high-Q dielectric resonator filters, out high-frequency components of the rapid-scan signals. Field inhomogeneities induced by the rapidly changing magnetic field increase with scan rate and are greater with the dielectric and split-ring resonantors than with the rectangular resonator. Data for trityl-CD3 were recorded with the rectangular resonator. The extended trityl sample, about 3 mm long, shows larger effects of magnetic field inhomogeneities than the small LiPc crystals.

The solution conformation of triarylmethyl radicals

Hyperfine coupling tensors to 1H, 2H, and natural abundance 13C were measured using X-band pulsed electron nuclear double resonance (ENDOR) spectroscopy for two triarylmethyl (trityl) radicals used in electron paramagnetic resonance imaging and oximetry: methyl tris(8-carboxy-2,2,6,6-tetramethyl-benzo[1,2 d:4,5- d′]bis(1,3)dithiol-4-yl) and methyl tris(8-carboxy-2,2,6,6-tetramethyl(- d 3)-benzo[1,2 d:4,5- d′]bis(1,3)dithiol-4-yl). Quantum chemical calculations using density functional theory predict a structure that reproduces the experimentally determined hyperfine tensors. The radicals are propeller-shaped with the three aryl rings nearly mutually orthogonal. The central carbon atom carrying most of the unpaired electron spin density is surrounded by the sulfur atoms in the radical and is completely shielded from solvent. This structure explains features of the electron spin relaxation of these radicals and suggests ways in which the radicals can be chemically modified to improve their characteristics for imaging and oximetry.