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In rapid scan EPR the rapidly-changing magnetic field induces a background signal that may be larger than the EPR signal. A method has been developed to correct for that background signal by acquiring two sets of data, denoted as scan 1 and scan 2. In scan 2 the external field B 0 is reversed and the data acquisition trigger is offset by one half cycle of the scan field relative to the settings used in scan 1. For data acquired with a cross-loop resonator subtraction of scan 2 from scan 1 cancels the background and enhances the EPR signal. Experiments were performed at an EPR frequency of about 258 MHz, which is in the range that is commonly used for in vivo imaging. Samples include nitroxide radicals, a trityl radical, a dinitroxide, and a nitroxide in the presence of a magnetic field gradient. This method has the advantage that no assumption is made about the shape of the background signal, and it provides an approach to automating the background correction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

X-band electron paramagnetic resonance spectra of immobilized nitroxides were obtained by rapid scan at 293 K. Scan widths were 155 G with 13.4 kHz scan frequency for (14)N-perdeuterated tempone and for T4 lysozyme doubly spin labeled with an iodoacetamide spirocyclohexyl nitroxide and 100 G with 20.9 kHz scan frequency for (15)N-perdeuterated tempone. These wide scans were made possible by modifications to our rapid-scan driver, scan coils made of Litz wire, and the placement of highly conducting aluminum plates on the poles of a Bruker 10″ magnet to reduce resistive losses in the magnet pole faces. For the same data acquisition time, the signal-to-noise for the rapid-scan absorption spectra was about an order of magnitude higher than for continuous wave first-derivative spectra recorded with modulation amplitudes that do not broaden the lineshapes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bimolecular collision rate constants of a model solute are measured in water at T = 259-303 K, a range encompassing both normal and supercooled water. A stable, spherical nitroxide spin probe, perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl, is studied using electron paramagnetic resonance spectroscopy (EPR), taking advantage of the fact that the rotational correlation time, τ(R), the mean time between successive spin exchanges within a cage, τ(RE), and the long-time-averaged spin exchange rate constants, K(ex), of the same solute molecule may be measured independently. Thus, long- and short-time translational diffusion behavior may be inferred from K(ex) and τ(RE), respectively. In order to measure K(ex), the effects of dipole-dipole interactions (DD) on the EPR spectra must be separated, yielding as a bonus the DD broadening rate constants that are related to the dephasing rate constant due to DD, W(dd). We find that both K(ex) and W(dd) behave hydrodynamically; that is to say they vary monotonically with T/η or η/T, respectively, where η is the shear viscosity, as predicted by the Stokes-Einstein equation. The same is true of the self-diffusion of water. In contrast, τ(RE) does not follow hydrodynamic behavior, varying rather as a linear function of the density reaching a maximum at 276 ± 2 K near where water displays a maximum density.

We show that at low concentrations (≤5 mM) TEMPONE radicals in liquid toluene exhibit higher DNP efficiency than in water. In spite of reduced coupling factors, the improved DNP performance in toluene results from favourable saturation and leakage factors, as determined by pulse electron-electron double resonance (ELDOR) and NMR relaxation, respectively. The extracted coupling factors at 0.35 Tesla support theoretical predictions of the Overhauser mechanism.

Picosecond rotational correlation times of perdeuterated tempone (PDT) are found in alkane and aromatic liquids by directly using the spectral width of the central electron paramagnetic resonance line. This is done by mathematically eliminating the nonsecular spectral density from the spectral parameter equations, thereby removing the need to assume a particular form for it. This is preferable to fitting a constant correction factor to the spectral density, because such a factor does not fit well in the low picosecond range. The electron-nuclear spin dipolar interaction between the probe and solvent is shown to be negligible for the very rapid rotation of PDT in these liquids at the temperatures of the study. The rotational correlation times obtained with the proposed method generally agree to within experimental uncertainty with those determined by using the traditional parameters. Using the middle line width offers greater precision and smoother trends. Previous work with the central line width is discussed, and past discrepancies are explained as possibly resulting from residual inhomogeneous broadening. The rotational correlation time almost forms a common curve across all of the solvents when plotted with respect to isothermal compressibility, which shows the high dependence of rotation on liquid free volume.

Electron spin relaxation times of perdeuterated tempone (PDT) 1 and of a nitronyl nitroxide (2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) 2 in aqueous solution at room temperature were measured by 2-pulse electron spin echo (T(2)) or 3-pulse inversion recovery (T(1)) in the frequency range of 250 MHz to 34 GHz. At 9 GHz values of T(1) measured by long-pulse saturation recovery were in good agreement with values determined by inversion recovery. Below 9 GHz for 1 and below 1.5 GHz for 2,T(1)~T(2), as expected in the fast tumbling regime. At higher frequencies T(2) was shorter than T(1) due to incomplete motional averaging of g and A anisotropy. The frequency dependence of 1/T(1) is modeled as the sum of spin rotation, modulation of g and A-anisotropy, and a thermally-activated process that has maximum contribution at about 1.5 GHz. The spin lattice relaxation times for the nitronyl nitroxide were longer than for PDT by a factor of about 2 at 34 GHz, decreasing to about a factor of 1.5 at 250 MHz. The rotational correlation times, τ(R) are calculated to be 9 ps for 1 and about 25 ps for 2. The longer spin lattice relaxation times for 2 than for 1 at 9 and 34 GHz are due predominantly to smaller contributions from spin rotation that arise from slower tumbling. The smaller nitrogen hyperfine couplings for the nitronyl 2 than for 1 decrease the contribution to relaxation due to modulation of A anisotropy. However, at lower frequencies the slower tumbling of 2 results in a larger value of ωτ(R) (ω is the resonance frequency) and larger values of the spectral density function, which enhances the contribution from modulation of anisotropic interactions for 2 to a greater extent than for 1., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The work in part 6 of this series (J. Phys. Chem. A 2009, 113, 4930), addressing the task of separating the effects of Heisenberg spin exchange (HSE) and dipole-dipole interactions (DD) on electron paramagnetic resonance (EPR) spectra of nitroxide spin probes in solution, is extended experimentally and theoretically. Comprehensive measurements of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (pDT) in squalane, a viscous alkane, paying special attention to lower temperatures and lower concentrations, were carried out in an attempt to focus on DD, the lesser understood of the two interactions. Theoretically, the analysis has been extended to include the recent comprehensive treatment by Salikhov (Appl. Magn. Reson. 2010, 38, 237). In dilute solutions, both interactions (1) introduce a dispersion component, (2) broaden the lines, and (3) shift the lines. DD introduces a dispersion component proportional to the concentration and of opposite sign to that of HSE. Equations relating the EPR spectral parameters to the rate constants due to HSE and DD have been derived. By employing nonlinear least-squares fitting of theoretical spectra to a simple analytical function and the proposed equations, the contributions of the two interactions to items 1-3 may be quantified and compared with the same parameters obtained by fitting experimental spectra. This comparison supports the theory in its broad predictions; however, at low temperatures, the DD contribution to the experimental dispersion amplitude does not increase linearly with concentration. We are unable to deduce whether this discrepancy is due to inadequate analysis of the experimental data or an incomplete theory. A new key aspect of the more comprehensive theory is that there is enough information in the experimental spectra to find items 1-3 due to both interactions; however, in principle, appeal must be made to a model of molecular diffusion to separate the two. The permanent diffusion model is used to illustrate the separation in this work. In practice, because the effects of DD are dominated by HSE, negligible error is incurred by using the model-independent extreme DD limit of the spectral density functions, which means that DD and HSE may be separated without appealing to a particular model., (© 2012 American Chemical Society)

Carotenoids, natural pigments widely distributed in algae and plants, have a conjugated double bond system. Their excitation energies are correlated with conjugation length. We hypothesized that carotenoids whose energy states are above the singlet excited state of oxygen (singlet oxygen) would possess photosensitizing properties. Here, we demonstrated that human skin melanoma (A375) cells are damaged through the photo-excitation of several carotenoids (neoxanthin, fucoxanthin and siphonaxanthin). In contrast, photo-excitation of carotenoids that possess energy states below that of singlet oxygen, such as β-carotene, lutein, loroxanthin and violaxanthin, did not enhance cell death. Production of reactive oxygen species (ROS) by photo-excited fucoxanthin or neoxanthin was confirmed using a reporter assay for ROS production with HeLa Hyper cells, which express a fluorescent indicator protein for intracellular ROS. Fucoxanthin and neoxanthin also showed high cellular penetration and retention. Electron spin resonance spectra using 2,2,6,6-tetramethil-4-piperidone as a singlet oxygen trapping agent demonstrated that singlet oxygen was produced via energy transfer from photo-excited fucoxanthin to oxygen molecules. These results suggest that carotenoids such as fucoxanthin, which are capable of singlet oxygen production through photo-excitation and show good penetration and retention in target cells, are useful as photosensitizers in photodynamic therapy for skin disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To develop an estimation method of gadolinium magnetic resonance imaging (MRI) contrast agents, the effect of concentration of Gd compounds on the ESR spectrum of nitroxyl radical was examined. A solution of either 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) was mixed with a solution of Gd compound and the ESR spectrum was recorded. Increased concentration of gadolinium-diethylenetriamine pentaacetic acid chelate (Gd-DTPA), an MRI contrast agent, increased the peak-to-peak line widths of ESR spectra of the nitroxyl radicals, in accordance with a decrease of their signal heights. A linear relationship was observed between concentration of Gd-DTPA and line width of ESR signal, up to approximately 50 mmol/L Gd-DTPA, with a high correlation coefficient. Response of TEMPONE was 1.4-times higher than that of TEMPOL as evaluated from the slopes of the lines. The response was slightly different among Gd compounds; the slopes of calibration curves for acua[N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]glycinato(3-)]gadolinium hydrate (Gd-DTPA-BMA) (6.22 μT·L/mmol) and gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelate (Gd-DOTA) (6.62 μT·L/mmol) were steeper than the slope for Gd-DTPA (5.45 μT·L/mmol), whereas the slope for gadolinium chloride (4.94 μT·L/mmol) was less steep than that for Gd-DTPA. This method is simple to apply. The results indicate that this method is useful for rough estimation of the concentration of Gd contrast agents if calibration is carried out with each standard compound. It was also found that the plot of the reciprocal square root of signal height against concentrations of contrast agents could be useful for the estimation if a constant volume of sample solution is taken and measured at the same position in the ESR cavity every time.

We propose the use of the pulse electron double resonance (ELDOR) method to determine the effective saturation factor of nitroxide radicals for dynamic nuclear polarization (DNP) experiments in liquids. The obtained values for the nitroxide radical TEMPONE-D,(15)N at different concentrations are rationalized in terms of spin relaxation and are shown to fulfil the Overhauser theory.

EPR linewidth measurements of 2,N-(4-n-butyl benzilidene) 4-amino 2,2,6,6-tetramethyl piperidine 1-oxide (BBTMPO) in toluene at 1 GHz (L-Band), 4 GHz (S-Band), 9 GHz (X-Band) and 34 GHz (Q-Band) microwave frequencies indicate the presence of a distribution of relaxation times. The empirical response parameter introduced by Cole-Davidson for the analysis of dielectric relaxation in liquids has been used for the analysis of EPR relaxation data in the L-Band and S-Band frequency regions. The Cole-Davidson parameter can assume values in the range 0 < β ≤ 1. When β = 1, one obtains the Debye-type spectral density. The calculated linewidth data at 1 GHz and 4 GHz agree with a Cole-Davidson parameter of 0.7 for the spherocone shaped BBMTPO solute. β < 1 at the L- and S-bands suggests the presence of an asymmetrical distribution of relaxation times associated with different modes of relaxation mechanisms or internal molecular motions. This study shows EPR experiments at low microwave frequencies are more sensitive to the shape of the correlation function. Differences between this study and an earlier study [6] on perdeuterated 2,2,6,6-tetramethly-4-piperidone N oxide (PD-Tempone) in toluene are attributed to the size of and absence of deuteration in the BBTMPO probe., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H(2)O(2)) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H(2)O(2) were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H(2)O(2). Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.

This paper describes a novel experiment on nitroxide radical spin labels using a multiarm EPR W-band bridge with a loop-gap resonator (LGR). We demonstrate EPR spectroscopy of spin labels by linear sweep of the microwave frequency across the spectrum. The high bandwidth of the LGR, about 1 GHz between 3 dB points of the microwave resonance, makes this new experiment possible. A frequency-tunable yttrium iron garnet (YIG) oscillator provides sweep rates as high as 1.8x10(5) GHz/s, which corresponds to 6.3 kT/s in magnetic field-sweep units over a 44 MHz range. Two experimental domains were identified. In the first, linear frequency sweep rates were relatively slow, and pure absorption and pure dispersion spectra were obtained. This appears to be a practical mode of operation at the present level of technological development. The main advantage is the elimination of sinusoidal magnetic field modulation. In the second mode, the frequency is swept rapidly across a portion of the spectrum, and then the frequency sweep is stopped for a readout period; FID signals from a swept line oscillate at a frequency that is the difference between the spectral position of the line in frequency units and the readout position. If there is more than one line, oscillations are superimposed. The sweep rates using the YIG oscillator were too slow, and the portion of the spectrum too narrow to achieve the full EPR equivalent of Fourier transform (FT) NMR. The paper discusses technical advances required to reach this goal. The hypothesis that trapezoidal frequency sweep is an enabling technology for FT EPR is supported by this study., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

A DNP set-up is described where a liquid sample is hyperpolarized by the electron-nucleus Overhauser effect in a field of 0.34 T and transferred to a field of 14.09 T for NMR detection. In contrast to a previous set-up, using two dedicated magnets for polarization and detection, a dedicated ferroshim system was inserted into the bore of a 14.09 T shielded cryomagnet to provide a homogeneous low-field region in the stray field above the magnetic center. After polarization in the low-field the sample is transferred to the high-field magnetic center within 40 ms by a pneumatic shuttle system. In our set-up a standard high-resolution inverse (1)H/(13)C selective probe was used for NMR detection and a homebuilt EPR cavity, operating in the TM(110) mode was used for polarisation. First experimental data are presented. We observed a maximum proton Overhauser enhancement of up to epsilon(HF) = -3.7 in the high-field position for a 5 mM 4-Oxo-TEMPO-D,(15)N (TEMPONE)/H(2)O sample. While this reproduces the DNP enhancement observed also in the old set-up, with the new set-up we observe enhancement on larger molecules that were impossible to enhance in the old set-up. Therefore, we can demonstrate for the first time Overhauser enhanced high resolution proton spectra of glucose and 2,2-dimethyl-2-silapentane-5-sulfonic acid sodium salt (DSS) in D(2)O, where the high resolution spectrum was acquired in the high-field position after polarizing the sample in the low-field.

Dynamic nuclear polarization is emerging as a potential tool to increase the sensitivity of NMR aiming at the detection of macromolecules in liquid solution. One possibility for such an experimental design is to perform the polarization step between electrons and nuclei at low magnetic fields and then transfer the sample to a higher field for NMR detection. In this case, an independent optimization of the polarizer and detection set ups is required. In the present paper we describe the optimization of a polarizer set up at 15 MHz (1)H NMR/9.7 GHz EPR frequencies based on commercial hardware. The sample consists of the nitroxide radical TEMPONE-D,(15)N in water, for which the dimensions were systematically decreased to fit the homogeneous B(1) region of a dielectric ENDOR resonator. With an available B(1) microwave field up to 13 G we observe a maximum DNP enhancement of -170 at room temperature by irradiating on either one of the EPR lines. The DNP enhancement was saturated at all polarizer concentrations. Pulsed ELDOR experiments revealed that the saturation level of the two hyperfine lines is such that the DNP enhancements are well consistent with the coupling factors derived from NMRD data. By raising the polarizing field and frequencies 10-fold, i.e. to 140 MHz (1)H/94 GHz EPR, we reach an enhancement of -43 at microwave field strengths (B(1) approximately 5 G). The results are discussed in view of an application for a DNP spectrometer.

Water (1)H relaxation rate measurements of (15)N-(2)H-TEMPONE solutions at temperatures ranging from 298 to 328 K have been performed as a function of magnetic field from 0.00023 to 9.4 T, corresponding to (1)H Larmor frequencies of 0.01 to 400 MHz. The relaxation profiles were analyzed according to the full theory for dipolar and contact relaxation, and used to estimate the coupling factor responsible for observed solution DNP effects. The experimental DNP enhancement at (1)H Larmor frequency of 15 MHz obtained by saturating one of the lines of the (15)N doublet is only ca. 20% lower than the limiting value predicted from the relaxation data, indicating that the experimental DNP setup is nearly optimal, the residual discrepancy arising from incomplete saturation of the other line.

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.

EPR spectra of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (PDT) are studied as functions of molar concentration, c, and temperature, T, in water and 70 wt % glycerol in water. The increase of the intrinsic line width averaged over the three hyperfine lines, B(tot), varies linearly with c with zero intercept in both solvents at all temperatures; therefore dB(tot)/dc is independent of c. The spin exchange induced dispersion, from which the spin exchange frequency, omega(e), may be computed, increases linearly with B(tot), passing through the origin in water and in 70% glycerol at high temperatures; however, at low temperatures, where dipolar interactions broaden the spectra, linearity does not prevail until B(tot) > 1 G due to a contribution of dipolar interactions to the dispersion. The broadening constant due to spin exchange, dB(e)/dc, is found from the slope of the linear region, permitting a computation of the dipolar constant, dB(dip)/dc = dB(tot)/dc - dB(e)/dc. Thus, the separation of concentration broadening into spin exchange and dipolar contributions is effected without having to appeal to some supposed temperature dependence of the two interactions. The fractional broadening by spin exchange, Omega(T), is near unity at high temperatures in both solvents, decreasing to zero in 70% glycerol at 273 K. Omega(T) is a continuous function of the inverse rotational correlation time of PDT but is discontinuous as a function of T/eta where eta is the shear viscosity. Omega(T) = 0.5, where spin exchange and dipolar interactions contribute equally to the line width occurs at T/eta = 20 +/- 1 K/cP in 70% glycerol. Hydrodynamic predictions of dB(e)/dc via the Stokes-Einstein (SE) equation are remarkably accurate in 70% glycerol comparable with the results in a series of alkanes. In water, dB(e)/dc is linear with T/eta with zero intercept as required by the SE; however, with slope a factor of 0.73 smaller. dB(dip)/dc is reasonably predicted by the SE only at very small values of eta/T very quickly following an approximately logarithmic dependence rather that the linear prediction. Values of dB(dip)/dc approach a plateau above eta/T = 0.20 cP/K that is about one-half the solid state limit. Line shifts due to spin exchange are not yet useful to deduce values of Omega(T) due to a lack of knowledge of the time between re-encounters; however, they may be used to verify the values determined from line broadening and spin exchange induced dispersion. Some effects at low temperatures in 70% glycerol suggest that the effects of dipolar interaction are inadequately described by the widely accepted theory.

Bimolecular collisions between perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-l-oxyl molecules in three alkanes have been studied by measuring the electron paramagnetic resonance (EPR) spectral changes induced by spin exchange. We define an "encounter" to be a first-time collision followed by a series of re-encounters prior to the diffusing pair's escaping each other's presence. The present work stems from a recent proposal [B. L. Bales et al., J. Phys. Chem. A 107, 9086 (2003)] that an unexpected linear dependence of the spin-exchange-induced EPR line shifts on spin-exchange frequency can be explained by re-encounters of the same probe pair during one encounter. By employing nonlinear least-squares fitting, full use of the information available from the spectral changes allows us to study encounters and re-encounters separately. The encounter rate constants appear to be dominated by hydrodynamic forces, forming a common curve for hexane, decane, and hexadecane when plotted against T/eta, where eta is the shear viscosity. Unexpectedly, encounters are not dependent on the ratio mu = a/a(s), where a and a(s) are the van der Waals radii of the nitroxide probe and the solvent, respectively. It is argued that the near coincidence of the resulting encounter rate constant with the hydrodynamic prediction is likely due to a near cancellation of terms in the general diffusion coefficient. Thus, the semblance of hydrodynamic behavior is coincidental rather than intrinsic. In contrast, the mean times between re-encounters do depend on the relative sizes of probe and solvent. For hexane at lower temperatures, the Stokes-Einstein equation apparently describes re-encounters well; however, at higher temperatures and for decane and hexadecane, departures from the hydrodynamic prediction become larger as mu becomes smaller. This is in qualitative agreement with the theory of microscopic diffusion of Hynes et al. [J. Chem. Phys. 70, 1456 (1979)]. These departures are well correlated with the free volume available in the solvent; thus, the mean times between re-encounters form a common curve when plotted versus the free volume. Because free volume is manifested macroscopically by the isothermal compressibility, it is expected and observed that the re-encounter rate also forms a common curve across all three solvents when plotted with respect to compressibility. The existence of a common curve for alkanes raises the prospect of using EPR to determine the compressibility of substances such as fossil fuels and biological membranes.

A comparative study of tissue redox-status imaging using commonly used redox sensitive nitroxides has been carried out using electron paramagnetic resonance imaging (EPRI), Overhauser magnetic resonance imaging (OMRI) and conventional T(1)-weighted magnetic resonance imaging, MRI. Imaging studies using phantoms of different nitroxides at different concentration levels showed that EPRI and OMRI sensitivities were found to be linearly dependent on line width of nitroxides up to 2 mM, and the enhancement in MRI intensity was linear up to 5 mM. The sensitivity and resolution of EPRI and OMRI images depended significantly on the line width of the nitroxides whereas the MRI images were almost independent of EPR line width. Reduction of the paramagnetic 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3CP) by ascorbic acid (AsA) to the diamagnetic by hydroxylamine was monitored from a sequence of temporal images, acquired using the three imaging modalities. The decay rates determined by all the three modalities were found to be similar. However the results suggest that T(1)-weighted MRI can monitor the redox status, in addition to providing detailed anatomical structure in a short time. Therefore, a combination of MRI with nitroxides as metabolically responsive contrast agents can be a useful technique for the in vivo imaging probing tissue redox status.

We have developed a general electron paramagnetic resonance (EPR) method to measure electrostatic potential at spin labels on proteins to millivolt accuracy. Electrostatic potential is fundamental to energy-transducing proteins like myosin, because molecular energy storage and retrieval is primarily electrostatic. Quantitative analysis of protein electrostatics demands a site-specific spectroscopic method sensitive to millivolt changes. Previous electrostatic potential studies on macromolecules fell short in sensitivity, accuracy and/or specificity. Our approach uses fast-relaxing charged and neutral paramagnetic relaxation agents (PRAs) to increase nitroxide spin label relaxation rate solely through collisional spin exchange. These PRAs were calibrated in experiments on small nitroxides of known structure and charge to account for differences in their relaxation efficiency. Nitroxide longitudinal (R(1)) and transverse (R(2)) relaxation rates were separated by applying lineshape analysis to progressive saturation spectra. The ratio of measured R(1) increases for each pair of charged and neutral PRAs measures the shift in local PRA concentration due to electrostatic potential. Voltage at the spin label is then calculated using the Boltzmann equation. Measured voltages for two small charged nitroxides agree with Debye-Hückel calculations. Voltage for spin-labeled myosin fragment S1 also agrees with calculation based on the pK shift of the reacted cysteine.

Two-dimensional (2D) Fourier transform ESR techniques, such as 2D-ELDOR, have considerably improved the resolution of ESR in studies of molecular dynamics in complex fluids such as liquid crystals and membrane vesicles and in spin labeled polymers and peptides. A well-developed theory based on the stochastic Liouville equation (SLE) has been successfully employed to analyze these experiments. However, one fundamental assumption has been utilized to simplify the complex analysis, viz. the pulses have been treated as ideal non-selective ones, which therefore provide uniform irradiation of the whole spectrum. In actual experiments, the pulses are of finite width causing deviations from the theoretical predictions, a problem that is exacerbated by experiments performed at higher frequencies. In the present paper we provide a method to deal with the full SLE including the explicit role of the molecular dynamics, the spin Hamiltonian and the radiation field during the pulse. The computations are rendered more manageable by utilizing the Trotter formula, which is adapted to handle this SLE in what we call a "Split Super-Operator" method. Examples are given for different motional regimes, which show how 2D-ELDOR spectra are affected by the finite pulse widths. The theory shows good agreement with 2D-ELDOR experiments performed as a function of pulse width.

High-level ab initio g and A tensor components have been calculated for PD-tempone and tempo-palmitate (TP) radical spin probes dissolved in n-pentyl and n-hexyl cyanobiphenyl liquid crystals. Solvent effects have been included in the proposed approach by means of the polarizable continuum model, allowing for solvent anisotropy. An in-depth analysis of the electronic structure of probes was performed to choose a suitable model for TP and make the calculations more accessible. Computed magnetic tensor components have been compared with corresponding values measured in the rigid limit. The quality of the results suggests the use of quantum-mechanical data to determine the order parameter of the nematic from experimental electron-spin resonance measurements.

Intensity of EPR spectra of stable organic free radicals, nitroxides, is decreasing with time if the radicals are dissolved in beer. The process is determined by a chemical reaction of nitroxide reduction by components naturally present in beer. Kinetics can be described as a simple irreversible first order with respect to both nitroxide and one reducing agent. Effective concentration of the reducing agent and the corresponding reaction rate constant has been determined. It is demonstrated that the nitroxide reduction is sensitive to the presence of solvent-accessible SH groups of proteins present in beer. It is proposed that quantitative analysis of reduction kinetics of small water-soluble nitroxide radicals such as TEMPO and TEMPOL can be used to assess the reducing power of beer. The effect of accelerated aging of beer achieved at elevated temperatures on nitroxide reduction kinetics is demonstrated.

Objective: To study the alkaloids from Armeniaca mume Sieb., Methods: The alkaloids were extracted with chloroform from 80% alcoholic extract and concentrated. Then they were transferred into aqueous solution and absorbed by macroporous cation exchange resin. The concentrated total eluent was extracted with chloroform. The alkaloids were purified by recrystallization with petroleum ether and sublimation. Then they were determined by Fourier Transform Infrared spectroscopy, Mass spectrometry and Nuclear magnetic resonance etc methods., Result: Two alkaloids were separated and their structures were elucidated as 2,2,6,6-tetramethyl-4-piperidone and tert-butylurea on the basis of spectral evidence., Conclusion: They are new alkaloids from A. mume.

Low-energy visible light (LEVL) has previously been found to modulate various processes in different biological systems. One explanation for the stimulatory effect of LEVL is light-induced reactive oxygen species formation. In the present study, both sperm and skin cells were illuminated with LEVL and were found to generate singlet oxygen (1O2). The detection of 1O2 was performed using a trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy. In addition, we have shown that, together with O2 generation, LEVL illumination increases the reductive capacity of the cells, which explains the difficulties encountered in 1O2 detection. The potential of visible light to change the cellular redox state may explain the recently observed biostimulative effects exerted by LEVL.

We describe herein a novel metabolic fate of the 2,2,6,6-tetramethyl-piperidine (2,2,6,6-TMPi) moiety to a ring-contracted 2,2-dimethyl pyrrolidine (2,2-DMPy) in human liver microsomal incubations. The existence of this pathway was demonstrated for three compounds (I-III) of varied structures suggesting that this may be a general biotransformation reaction for the 2,2,6,6-TMPi moiety. The 2,2-DMPy metabolites formed in incubations of the three compounds with human liver microsomes were characterized by online high performance liquid chromatography coupled to a high resolution hybrid quadrupole-time-of-flight mass spectrometer. Suggested elemental composition obtained from accurate mass measurements of the molecular ions and fragment ions of the metabolites clearly indicated the loss of a mass equivalent to C(3)H(6) from the parent 2,2,6,6-TMPi functionality. Additional accurate tandem mass spectrometry data indicated that one of the original two gem-dimethyl groups was intact in the metabolite structure. Proof of a ring-contracted 2,2-DMPy structure was obtained using (1)H-NMR experiments on a metabolite purified from liver microsomal incubations, which showed only two geminal methyl groups, instead of four in the parent compound. Two-dimensional correlation spectroscopy and decoupling experiments established aliphatic protons arranged in a pyrrolidine ring pattern. The fact that the formation of 2,2-DMPy metabolites in human liver microsomes was NADPH-dependent suggested that this novel metabolic reaction was catalyzed by the cytochrome P450 (P450) enzyme(s). Immunoinhibition studies in human liver microsomal incubations using anti-P450 monoclonal antibodies and experiments with insect cell microsomes containing individually expressed recombinant human P450 isozymes indicated that multiple P450 isozymes were capable of catalyzing this novel metabolic transformation.

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).

EPR linewidth measurements of PD-Tempone in toluene at 1 (L-Band), 4 (S-Band), 9 (X-Band) and 34 GHz (Q-Band) microwave frequencies indicate the presence of a distribution of relaxation times. The empirical response parameter introduced by Cole-Davidson for the analysis of dielectric relaxation in liquids has been used for the analysis of EPR relaxation data in the low frequency region. The Cole-Davidson parameter can assume values in the range 0 < beta < or = 1. When beta = 1, one obtains the Debye-type spectral density. The calculated linewidth data at 1 GHz agrees with a Cole-Davidson distribution function with a width parameter 0.83 +/- 0.04 for a spherical solute. Beta < 1 at L-band suggests the presence of an asymmetrical distribution of relaxation times associated with different modes of relaxation mechanisms or internal molecular motions. This study shows EPR experiments at low microwave frequencies are more sensitive to the shape of the correlation function.

The EPR spectra of nitroxide spin labels have been simulated as a function of microwave field, H1, taking into account both magnetic field modulation and molecular rotation. It is found that the saturation of the second integral, S, of the first harmonic in-phase absorption spectrum is approximated by that predicted for slow-passage conditions, that is, S approximately H1/1 + PH21, in all cases. This result is independent of the degree of inhomogeneous broadening. In general, the fitting parameter, P, depends not only on the T1 and T2 relaxation times, but also on the rate of molecular reorientation and on the modulation frequency. Calibrations for determining the relaxation times are established from the simulations. For a given modulation frequency and molecular reorientation rate, the parameter obtained by fitting the saturation curves is given by 1/P = a + 1/gamma2eT1 . Teff2, where Teff2 is the effective T2. For molecular reorientation frequencies in the range 2 x 10(7)-2 x 10(8) s-1, Teff2 is dominated by the molecular dynamics and is only weakly dependent on the intrinsic T02, allowing a direct estimation of T1. For reorientation frequencies outside this range, the (T1T2) product may be determined from the calibrations. The method is applied to determining relaxation times for spin labels undergoing different rates of rotational reorientation in a variety of environments, including those of biological relevance, and is verified experimentally by the relaxation rate enhancements induced by paramagnetic ions., (Copyright 1998 Academic Press.)

The stable free radical 4-oxo-2,2,6,6-tetramethylpiperidinooxy (2,2,6,6-tetramethylpiperidone-N-oxyl, TEMPONE) in nitrous oxide-saturated aqueous formate solution has been shown to be a quantitative dosimeter for in situ radiolysis electron spin resonance (ESR) studies. The bleach of the TEMPONE ESR signal is proportional to the absorbed dose in steady-state radiolysis/field-modulation and in pulse radiolysis/time-resolved studies. The G value for consumption of TEMPONE in nitrogen-deoxygenated solution is 5.3 (molecules per 100 eV), while the value in nitrous oxide-saturated solution is 6.0. Measurement of the radiolytically produced yield of carbon dioxide shows that the product of reaction of carbon dioxide radical anion with TEMPONE gives the corresponding hydroxylamine. The stability of the integrated intensity of the TEMPONE ESR bleach has been evaluated in the presence and absence of oxygen and found to be stable for hours. The ESR linewidth of TEMPONE solutions irradiated under anoxic conditions was constant in the postirradiation period. However, when oxygen was added postirradiation, the oxygen-broadened ESR linewidth decreased slowly toward the width observed in anoxic media, indicating consumption of dioxygen by, with concomitant production of, diamagnetic species in solution.

The production of singlet oxygen by H2O2 disproportionation and via the oxidation of H2O2 by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H2O2 and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe2+ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of 1O2 with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O2.-/HO2. and .OH has been also investigated. It has been found that both O2.-/HO2. and .OH radicals formed in a phosphate buffer solution (pH 7.4, 37 degrees C), respectively by a xanthine-oxidase/hypoxanthine system and via H2O2 UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.

Sonodynamic therapy is a promising new modality for cancer treatment based on the synergistic effect on tumor cell killing by combination of a drug (typically a photosensitizer) and ultrasound. The mechanism of sonodynamic action was suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. In this work we studied the aqueous sonochemical reactions of the gallium-porphyrin derivative ATX-70, one of the most active sonodynamic agents found, using 50 kHz ultrasound. The experiments were carried out in the presence of 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TMP), which reacts with singlet oxygen or .OH radicals to give the EPR-detectable nitroxide 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TMP-NO). Recently it has been suggested that the enhancement of TMP-NO yields in the presence of aqueous solutions of ATX-70 exposed to ultrasound was evidence for the formation of singlet oxygen in the system. Our results show that the surfactant cetyltrimethylammonium bromide (CTAB) can mimic the ATX-70-induced increase in the TMP-NO signal, but it fails to reproduce the behavior of ATX-70 in D2O: while the yields of TMP-NO in the presence of ATX-70 increase in D2O, the opposite effect was found with the surfactant CTAB. However, our data show that the increased TMP-NO yields in D2O are paralleled by an increased concentration of ATX-70 dimer, a form that is inactive in the photochemical generation of singlet oxygen. Our finding that the ATX-70-dependent enhancement of the TMP-NO signal was highest at approximately 20% O2, in both N2/O2 and argon/O2 mixtures, and decreased with increasing oxygen concentration is not compatible with the singlet oxygen mechanism. Finally, our results on the temperature dependence of the ATX-70-induced formation of TMP-NO are not consistent with the photochemical excitation of ATX-70 by sonoluminescent light: the ATX-70-dependent enhancement of TMP-NO signal increased with temperature in the range 10-25 degrees C, while the intensity of sonoluminescence of aqueous solutions both in multiple-bubble fields and in single-bubble experiments is known to decrease with increasing temperature.

The production of 2,2,6,6-tetramethyl-4-piperidone-N-oxyl by reaction of 2,2,6,6-tetramethyl-4-piperidone (TMPone) with ultrasonically generated active species in oxygenated solutions of hematoporphyrin (Hp) was studied by electron spin resonance spectroscopy. The nitroxide production rate in air-saturated TMPone solutions in phosphate-buffered saline of pH 9.0 was significantly higher in the presence of Hp than in its absence. The enhancement of nitroxide production by Hp was significantly inhibited in the presence of sodium azide or histidine in the solution. The production rate with Hp was doubled by substitution of deuterium oxide, while the rate without Hp increased only modestly. These results suggest that a substantial amount of active oxygen can be generated by ultrasound in aqueous solutions of Hp. Since the production rate was not reduced by mannitol and no nitroxide was produced in nitrogen-saturated solutions, it appears that hydroxyl radicals do not account for a major portion of the active oxygen species which reacted with TMPone to yield a nitroxide.

In this paper we propose theoretical models for the conformations of triacetonamine and protonated triacetonamine (Vincubine, an anticancer chemotherapeutic agent) developed by quantum and molecular mechanics techniques. We discuss the theoretical factors which are involved in the stabilization of the conformations calculated by the MNDO, MM2 and COPEANE methods and show the relative percent abundance of each molecular shape. Graphic representations of the conformers are depicted.