A Multifrequency (X-, Q-, and W-band) EPR and DFT Study of a Photopolymerizable Dental Resin

The free radicals generated during the polymerization process of Z100 (3 M ESPE) dental resin were examined by electron paramagnetic resonance (EPR) in X-, Q- and W-bands. Experimental generation and spectra simulations were associated with density functional theory (DFT) calculations to determine the molecular structure and explain the EPR spectrum formation. It was assumed that the EPR spectrum was formed by the sum of two different types of radicals: “propagating” and allylic. The spectra simulations and DFT calculations showed good agreement, indicating that the proposed model fully explained the nine lines of the EPR spectrum in X-band and showed that the spectrum formation is the sum of “9 + 5” lines, rather than the “5 + 4” lines predicted early. Simulations in Q- and W-bands showed very close correlation and were essential to support the proposed model.     

Multifrequency (X-band to W-band) CW EPR of the organic free radical in petroleum asphaltene

Petroleum of Arabian and Colombian origin was studied by electron paramagnetic resonance (EPR) spectroscopy at X- (9 GHz), Q- (34 GHz) and W-bands (94 GHz). The experiments were performed at room temperature (about 300 K) and at 77 K (W-band only). The asymmetry in the lines corresponding to free radicals was observed more intensely in the W-band spectra. The values of the line width ΔH in the spectra increased linearly with the microwave frequency utilized in the EPR experiments. A mathematical simulation of the free radical signal for the EPR spectra in three bands with a set of parameters corresponding to a single species was attempted, but this was not exactly coincident with the experimental signals, suggesting that the hyperfine interaction of the unpaired electron with its neighborhood corresponds to more than one species of radical in the molecular structure of the petroleum asphaltene.     

An in situ electrochemical cell for Q- and W-band EPR spectroscopy

A simple design for an in situ, three-electrode spectroelectrochemical cell is reported that can be used in commercial Q- and W-band (ca. 34 and 94 GHz, respectively) electron paramagnetic resonance (EPR) spectrometers, using standard sample tubing (1.0 and 0.5 mm inner diameter, respectively) and within variable temperature cryostat systems. The use of the cell is demonstrated by the in situ generation of organic free radicals (quinones and diimines) in fluid and frozen media, transition metal ion radical anions, and on the enzyme nitric oxide synthase reductase domain (NOSrd), in which a pair of flavin radicals are generated.     

“Unusual” lines observed in low-frequency cw ENDOR of photoexcited triplet state molecules: the primary donor triplet in photosynthetic reaction centers as an example

The origin of frequently observed “negative” (opposite phase) ENDOR lines in the low-frequency region of triplet state ENDOR spectra is explained in terms of microwave hole burning and RF modulation phenomena. From this, a new method of detecting burnt side holes in EPR spectra is derived which is based on cw ENDOR instrumentation. The method uses the modulation satellites that are induced by a longitudinal RF field component and appear around any EPR line, including burnt holes (“negative” lines). The longitudinal RF field was generated by a coil oriented parallel to the external field, but a longitudinal component of the RF field also exists in most conventional ENDOR spectrometers because of slight misalignments of the ENDOR coil generating the transversal RF field. The lines it induces in the low-frequency part of ENDOR spectra are generally considered as artifacts. It is shown, however, that RF induced modulation satellites provide valuable information concerning the lines distant from the spectral position in the EPR spectrum chosen for ENDOR observation. This allows one to record the pattern of side holes burnt by microwave saturation through forbidden transitions that carries information about ENDOR frequencies comparable to what can be extracted from ESEEM experiments. Such comparability is demonstrated for examples of nitrogen ENDOR of photoexcited triplet states of the primary donor in photosynthetic reaction centers and related compounds.     

Absolute signs of hyperfine coupling constants as determined by pulse ENDOR of polarized radical pairs

A novel method that allows the determination of absolute signs of hyperfine coupling constants in polarized radical pair (RP) pulse electron-nuclear double resonance (ENDOR) spectra is presented, The variable mixing time (VMT) ENDOR method used here leads to a separation of ENDOR transitions originating from different electron spin manifolds by employing their dependence on the time-dependent parameters of the pulse sequence. The simple kinetic model of the RP VMT ENDOR experiment shows very good agreement with the experiments performed on the P ₇₀₀ ^.+ A ₁ ^.- RP in photosystem I. This method relies on the selective excitation of absorptive or emissive lines of one radical in the RP EPR spectrum and therefore requires high spectral resolution. This condition was fulfilled for the system studied at the low-field edge of the RP EPR spectrum obtained at Q-band. The method presented here has a very high sensitivity and does not require any equipment additional to the one used for RP pulse ENDOR. The VMT ENDOR method offers the possibility for selective suppression of signals from different electron spin manifolds.     

Electron spin relaxation times and internal motions of radicals in the solid state investigated by ENDOR and pulsed EPR

The angular dependent ENDOR spectra of the radical formed by ψ-irradiated single crystal of 4-methyl-2,6-di-t-butylphenol have been studied and the full hyperfine tensors of all the t-butyl and the ring protons have been obtained atT=190 K. We found six different tensors for the t-butyl protons. This result shows that the t-butyl groups are slowly rotating on the ENDOR time scale, whereas each methyl group rotates fastly. The dynamical parameters of the motions have been determined by pulsed EPR experiments. The longitudinal relaxation and the phase memory times have been measured in the temperature range 130–290 K. Three different kinds of motions have been detected and the resulting values of the dynamical parameters have been compared with those obtained for the undamaged molecule by previous NMR studies.     

Pulse EPR, ENDOR, and ELDOR Study of Anionic Flavin Radicals in Na+-Translocating NADH:Quinone Oxidoreductase

The Na⁺-translocating nicotinamide adenine dinucleotide (NADH):quinine oxidoreductase (Na⁺–NQR) is a component of respiratory chain of various bacteria and it generates a redox-driven transmembrane electrochemical Na⁺ potential. It contains four different flavin prosthetic groups, including two flavin mononucleotide (FMN) residues covalently bound to the subunits NqrB and NqrC. Na⁺–NQR from Vibrio harveyi was poised at different redox potentials to prepare two samples, containing either both FMN_NqrB and FMN_NqrC or only FMN_NqrB in a paramagnetic state. These two samples were comparatively studied using pulse electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron-electron double resonance (ELDOR) spectroscopy. The echo-detected EPR spectra and electron spin relaxation properties were very similar for flavin radicals in both samples. The splitting of the outer peaks in the proton ENDOR spectra, assigned to the C(8α) methyl protons, allows to identify both radicals as anionic flavosemiquinones. The mean interspin distance of 20.7 Å between these radicals was determined by pulse ELDOR experiment, which allows to estimate the edge-to-edge distance (r _e) between these flavin centers as: 11.7 Å < r _e < 20.7 Å. The direct electron transfer between FMN_NqrB and FMN_NqrC during the physiological turnover of the Na⁺–NQR complex is suggested.     

Schonland ambiguity in the electron nuclear double resonance analysis of hyperfine interactions: principles and practice

For the analysis of the angular dependence of electron paramagnetic resonance (EPR) spectra of low-symmetry centres with S=1/2 in three independent planes, it is well-established-but often overlooked-that an ambiguity may arise in the best-fit g<--> tensor result. We investigate here whether a corresponding ambiguity also arises when determining the hyperfine coupling (HFC) A<--> tensor for nuclei with I=1/2 from angular dependent electron nuclear double resonance (ENDOR) measurements. It is shown via a perturbation treatment that for each set of M(S) ENDOR branches two best-fit A<--> tensors can be derived, but in general only one unique solution simultaneously fits both. The ambiguity thus only arises when experimental data of only one M(S) multiplet are used in analysis or in certain limiting cases. It is important to realise that the ambiguity occurs in the ENDOR frequencies and therefore the other best-fit result for an ENDOR determined A<--> tensor depends on various details of the ENDOR experiment: the M(S) state of the fitted transitions, the microwave frequency (or static magnetic field) in the ENDOR measurements and the rotation planes in which data have been collected. The results are of particular importance in the identification of radicals based on comparison of theoretical predictions of HFCs with published literature data. A procedure for obtaining the other best-fit result for an ENDOR determined A<--> tensor is outlined.     

Middle macroradicals and their influence on the strength of oriented polymers

The macroradicals formed through the detachment of hydrogen atoms in amorphous-crystalline polymers are studied using electron-nuclear double resonance (ENDOR) spectroscopy. The ¹H ENDOR spectra are analyzed, and the hyperfine interaction constants of the nearest neighbor and remote protons are measured. The conformational structure of radicals in poly(ethylene) (PE), poly(propylene) (PP), and poly(caproamide) (PCA) is determined. The ENDOR and EPR spectra of fibers subjected to tensile loading are recorded. It is revealed that torsional strain arises in the radicals with a nonplanar structure. The results of mechanical testing of irradiated oriented polymers demonstrate that the weakening of β bonds in the radicals has an effect on the macroscopic strength.     

High-frequency EPR and ENDOR: Time-domain spectroscopy of ribonucleotide reductase

This paper discusses time-domain electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) experiments aimed at elucidating the enzymatic mechanism of ribonucleotide reductase (RNR), the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. The article begins with a discussion of the current state of the art of instrumentation for high-frequency EPR and ENDOR and some suggestions as to future developments. We then provide an introduction to the chemistry of RNR and a discussion of the high-field EPR and ENDOR spectra of the tyrosyl radical (Y^?) in the R2 subunit of class I RNR. Finally, we describe two examples illustrating the use of high-frequency EPR and ENDOR to elucidate the enzymatic mechanism of RNR. EPR and ENDOR have played an important role for these studies since the mechanism involves several different radical intermediates. These intermediates are all present in low concentrations relative to the Y^? concentration and they possess similarg-values. Spectral overlap, therefore, has been a problem with X-band EPR. At high frequencies the spectra are resolved to the point that individual powder lineshapes are discernible. In addition, we describe our approach, on the basis of differential relaxation, to suppress the spectrum of the dominant Y^?. High-frequency EPR and ENDOR therefore has permitted us to determine the structure of several radical intermediates which in turn have contributed to the understanding of the enzymatic mechanism of RNR.     

Fast right-angle spinning EPR on organic radicals: Resolution enhancement and angle determination

Fast right-angle sample spinning (RAS) with rotation frequencies up to 17 kHz at temperatures down to 205 K is applied to electron paramagnetic resonance (EPR) experiments on organic radicals. Echo-detected RAS EPR provides substantial resolution enhancements for the range of anisotropies between 10 and 100 MHz which is not accessible with either magic-angle sample spinning EPR or anisotropy-resolved EPR on the basis of slow rotation. The larger reorientation angles in experiments with fast spinning cause strong phase shifts of the echo, which manifest themselves as regions with negative intensity in the spectrum. These phase shifts and thus the lineshape in echo-detected RAS EPR depend significantly on the relative orientation of theg and hyperfine tensor. For the determination of anisotropies in poorly resolved spectra of organic radicals in disordered solids, we introduce the two-dimensional fixed-angle rotation experiment as an alternative to anisotropy-resolved EPR.     

EPR study of gamma-irradiated 2-Bromo-4′-methoxyacetophenone single crystals

The gamma-irradiated single crystals of 2-Bromo-4′-methoxyaceto-phenone (2B4MA) were investigated using electron paramagnetic resonance (EPR) technique. Density-functional theory calculations were employed to investigate and identify the radicals that have been assumed to be formed upon irradiation of 2B4MA single crystals. The EPR spectra of 2B4MA were recorded at different orientations in the magnetic field at room temperature. Taking into account the chemical structure and experimental spectra of irradiated single crystal of 2B4MA, it was assumed that at least two different radicals were produced in the sample. Following this assumption, six possible radicals were modeled and EPR parameters were calculated by using the DFT, B3LYP/6-311+G(d), for the modeled radicals individually. The calculated hyperfine coupling constants and g-tensors were used as initial values for simulation studies. The three crystallographic axes on the simulated spectra were well matched with experimental spectra for the two modeled radicals. Thus, we identified the R1 type radical and R4 type radical as paramagnetic species produced in gamma-irradiated 2B4MA.     

EPR Studies of DOPA–Melanin Complexes with Fe(III)

Paramagnetic centers in 3,4-dihydroxyphenylalanine–melanin and its complexes with Fe(III) were examined by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic centers of melanin play an important role in detoxification of environment and they reveal high activity in binding of metal ions. Two different signals were observed in EPR spectra: lines of o-semiquinone free radicals and lines of paramagnetic Fe(III). Amplitudes of EPR lines of both free radicals and iron ions decrease with increasing Fe(III) content in melanin–metal ion complexes. Free radical concentrations in the melanin samples, g-factors, amplitudes and line widths of EPR spectra were determined. It was stated that fast spin–lattice relaxation processes exist in both free radical system and paramagnetic iron ions in melanin complexes.     

Spin-label oximetry at Q- and W-band

Spin-lattice relaxation times (T?s) of small water-soluble spin-labels in the aqueous phase as well as lipid-type spin-labels in membranes increase when the microwave frequency increases from 2 to 35 GHz (Hyde, et al., J. Phys. Chem. B 108 (2004) 9524-9529). The T?s measured at W-band (94 GHz) for the water-soluble spin-labels CTPO and TEMPONE (Froncisz, et al., J. Magn. Reson. 193 (2008) 297-304) are, however, shorter than when measured at Q-band (35 GHz). In this paper, the decreasing trends at W-band have been confirmed for commonly used lipid-type spin-labels in model membranes. It is concluded that the longest values of T? will generally be found at Q-band, noting that long values are advantageous for measurement of bimolecular collisions with oxygen. The contribution of dissolved molecular oxygen to the relaxation rate was found to be independent of microwave frequency up to 94 GHz for lipid-type spin-labels in membranes. This contribution is expressed in terms of the oxygen transport parameter W=T??1(Air)-T??1(N?), which is a function of both concentration and translational diffusion of oxygen in the local environment of a spin-label. The new capabilities in measurement of the oxygen transport parameter using saturation-recovery (SR) EPR at Q- and W-band have been demonstrated in saturated (DMPC) and unsaturated (POPC) lipid bilayer membranes with the use of stearic acid (n-SASL) and phosphatidylcholine (n-PC) spin-labels, and compared with results obtained earlier at X-band. SR EPR spin-label oximetry at Q- and W-band has the potential to be a powerful tool for studying samples of small volume, ~30 nL. These benefits, together with other factors such as a higher resonator efficiency parameter and a new technique for canceling free induction decay signals, are discussed.     

Electron nuclear quadruple resonance for assignment of overlapping spectra

Multiple resonance methods are important tools in EPR for revealing the network of hyperfine levels of free radicals and paramagnetic centers. The variations of electron nuclear double resonance (ENDOR) or electron spin-echo envelope modulation (ESEEM) techniques help to correlate nuclear frequencies with each other. These methods have limited utility when there is extensive overlap or suspected overlap in the EPR spectrum between different species or different orientations. In the ENDOR spectrum, overlap and second-order shifts of lines also leads to ambiguity in assignment and interpretation. A new electron nuclear multiple resonance method is presented here that is based on population transfer ENDOR. It is a quadruple resonance method that correlates ENDOR lines and reveals the network of hyperfine levels in samples with unoriented paramagnetic species and in samples with overlapping EPR or ENDOR lines.     

Methyl dynamics studied by ENDOR spectroscopy: a new method

A new method for the investigation of methyl group rotation dynamics in free radicals in solids is described, based on the temperature dependence of the methyl protons ENDOR enhancement. The method is shown to work for hindering barriers of any height, and for both radicals trapped in single crystals and in powdered samples. As specific examples, the Arrhenius parameters for the methyl rotation in free radicals produced by γ-irradiation of 1-alanine and 4-methyl-2, 6 di-t-butyl-phenol are determined.     

EPR studies of15N- and2H-substituted pH-sensitive spin probes of imidazoline and imidazolidine types

The isotopically substituted analogs of pH-sensitive imidazoline and imidazolidine radicals have been synthesized and investigated with electron paramagnetic resonance (EPR) spectroscopy. The introduction of²H and¹⁵N into the structure of the radical is a useful approach to enhance the information obtained from spin-labeling experiments. The spectra of the radicals have been analyzed with 9.8 (X-band) and 130 GHz (D-band) EPR spectroscopy. The substitution of¹H for²H leads to significant narrowing of Gaussian line width, while the substitution of¹⁴N for¹⁵N in the nitroxyl fragment decreases both the number of spectral lines and Lorentzian line width. These effects result in a significant increase in the peak intensities up to 5–7 times for X-band EPR spectra of one of the imidazoline radicals (R4). The increase in spectral resolution allowed us to reveal the hyperfine interaction splitting with the attached proton (0.36 G) in the protonated form of the radical R4. The influence of proton exchange of the radicals with phosphate and acetate buffers on their EPR spectra has been studied in H₂O and D₂O. The corresponding rate constants of the proton exchange have been calculated from fitting of the simulated EPR spectra line shapes to experimental spectra. The data obtained demonstrated the advantages of the isotopically substituted spin pH probes in spectral resolution and sensitivity which can be an important factor particularly for applications in vivo where the fundamental sensitivity is much lower. The sensitivity of EPR spectra of these spin probes to the buffer capacity could be of practical importance taking into account the biological relevance of monitoring this parameter in some pathological states.     

ENDOR evidence for the quantum exchange of the methylene protons

ENDOR spectroscopy was used for studying methylene proton couplings in the 6-yl (or H-addition) radical in a single crystal of 1-methyluracil at 4.2K. The two methylene protons were magnetically equivalent in all crystal (radical) orientations in the magnetic field, indicating proton quantum exchange. The two protons compose magnetically single entity with spin I=1. The observed small ENDOR line splittings of about 0.60MHz agree well with the theoretical expectations for such a system.     

EPR and density functional studies on 3-pyridylmethaniminoxy radical:1H hyperfine couplings as a structural criterion for iminoxyls derived from pyridinealdoximes

3-pyridylmethaniminoxyl has been generated by γ-irradiation of the parent oxime in the solid state and by incorporation of 3-pyridinealdoxime into the channels of thermally activated pentasil zeolite ZSM-5. The radical formed by both methods exhibits an electron paramagnetic resonance (EPR) spectrum with a characteristic powder pattern. The assignment of the hyperfine structure of the anisotropic spectra to the couplings with¹⁴N and¹H nuclei of Z geometrical isomer of 3-pyridylmethaniminoxy radical has been aided significantly by comparison with the coupling parameters calculated with the density functional theory (DFT). It has been found that 3- and 4-pyridylmethaniminoxyls generated in the oxime matrices, in contrast to the radicals derived from 2-pyridinealdoxime, have a possibility to isomerize. The EPR spectra of γ-irradiated 3- and 4-pyridinealdoximes show a distinct temperature dependence of particular isomer contributions which have been correlated with the relative energies of the isomers derived from DFT calculations. Hybrid density functional methods have been also used to determine the structure of transition states for isomerization (via inversion at nitrogen) and rotation (of pyridyl ring) as well as to predict the appropriate energy barriers.     

ENDOR studies of alkyl substituted p-Benzosemiquinones in reversed micelles and in 2-propanol

Various substituted p-benzosemiquinone radical anions, inter alia ubisemiquinone and derivatives, have been investigated in 2-propanol and in reversed micelles by EPR and ENDOR spectroscopy. Unsymmetrical semiquinones, with respect to the oxygen atoms, experience remarkable hyperfine shifts depending on the medium. This effect even allows differentiation between stereoisomers. Immobilization of the semiquinone molecules at the water-surfactant interface in reversed micelles gives rise to pronounced asymmetric linewidth effects. In the case of 2-cyclohexyl-3-methyl-1,4-benzosemiquinones, mixtures of two species (conformers) have been observed.