Determination of the g-, hyperfine coupling- and zero-field splitting tensors in EPR and ENDOR using extended Matlab codes.

The analysis of single crystal electron magnetic resonance (EMR) data has traditionally been performed using software in programming languages that are difficult to update, are not easily available, or are obsolete. By using a modern script-language with tools for the analysis and graphical display of the data, three MatLab® codes were prepared to compute the g, zero-field splitting (zfs) and hyperfine coupling (hfc) tensors from roadmaps obtained by EPR or ENDOR measurements in three crystal planes. Schonland's original method was used to compute the g- and hfc -tensors by a least-squares fit to the experimental data in each plane. The modifications required for the analysis of the zfs of radical pairs with S = 1 were accounted for. A non-linear fit was employed in a second code to obtain the hfc -tensor from EPR measurements, taking the nuclear Zeeman interaction of an I = ½ nucleus into account. A previously developed method to calculate the g- and hfc -tensors by a simultaneous linear fit to all data was used in the third code. The validity of the methods was examined by comparison with results obtained experimentally, and by roadmaps computed by exact diagonalization. The probable errors were estimated using functions for regression analysis available in MatLab. The software will be published at https://doi.org/10.17632/ps24sw95gz.1, Input and output examples presented in this work can also be downloaded from https://old.liu.se/simarc/downloads?l=en.

Tannic Acid Radicals in the Presence of Alkali Metal Salts and Their Impact on the Formation of Silicate-Phenolic Networks.

Polyphenolic molecules have become attractive building blocks for bioinspired materials due to their adhesive characteristics, capacity to complex ions, redox chemistry, and biocompatibility. For the formation of tannic acid (TA) surface modifications based on silicate-phenolic networks, a high ionic strength is required. In this study, we investigated the effects of NaCl, KCl, and LiCl on the formation of TA coatings and compared it to the coating formation of pyrogallol (PG) using a quartz-crystal microbalance. We found that the substitution of NaCl with KCl inhibited the TA coating formation through the high affinity of K+ to phenolic groups resulting in complexation of TA. Assessment of the radical formation of TA by electron paramagnetic resonance spectroscopy showed that LiCl resulted in hydrolysis of TA forming gallic acid radicals. Further, we found evidence for interactions of LiCl with the Siaq crosslinker. In contrast, the coating formation of PG was only little affected by the substitution of NaCl with LiCl or KCl. Our results demonstrate the interaction potential between alkali metal salts and phenolic compounds and highlight their importance in the continuous deposition of silicate-phenolic networks. These findings can be taken as guidance for future biomedical applications of silicate-phenolic networks involving monovalent ions.

In Quest of the Alanine R3 Radical: Multivariate EPR Spectral Analyses of X-Irradiated Alanine in the Solid State.

The amino acid l-α-alanine is the most commonly used material for solid-state electron paramagnetic resonance (EPR) dosimetry, due to the formation of highly stable radicals upon irradiation, with yields proportional to the radiation dose. Two major alanine radical components designated R1 and R2 have previously been uniquely characterized from EPR and electron-nuclear double resonance (ENDOR) studies as well as from quantum chemical calculations. There is also convincing experimental evidence of a third minor radical component R3, and a tentative radical structure has been suggested, even though no well-defined spectral signature has been observed experimentally. In the present study, temperature dependent EPR spectra of X-ray irradiated polycrystalline alanine were analyzed using five multivariate methods in further attempts to understand the composite nature of the alanine dosimeter EPR spectrum. Principal component analysis (PCA), maximum likelihood common factor analysis (MLCFA), independent component analysis (ICA), self-modeling mixture analysis (SMA), and multivariate curve resolution (MCR) were used to extract pure radical spectra and their fractional contributions from the experimental EPR spectra. All methods yielded spectral estimates resembling the established R1 spectrum. Furthermore, SMA and MCR consistently predicted both the established R2 spectrum and the shape of the R3 spectrum. The predicted shape of the R3 spectrum corresponded well with the proposed tentative spectrum derived from spectrum simulations. Thus, results from two independent multivariate data analysis techniques strongly support the previous evidence that three radicals are indeed present in irradiated alanine samples.

New Evidence for Hydroxyalkyl Radicals and Light- and Thermally Induced Trapped Electron Reactions in Rhamnose.

Radical formation and trapping of radicals in X-irradiated crystals of rhamnose at 6 K were investigated using electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques, complemented with periodic density functional theory (DFT) calculations. The two major radical species at 6 K were the O4-centered alkoxy radical and the intermolecularly trapped electron (IMTE), previously also detected by other authors. The current experimental results provided hyperfine coupling constants for these two species in good agreement with the previous data, thus providing a consistency check that improves their credibility. In addition to the O4-centered alkoxy radical and the IMTE, the C3-centered and C5-centered hydroxyalkyl radicals are the most prominent primary species at 6 K. The C3-centered radical appears in two slightly different conformations at 6 K, designated C and D. The C5-centered radical exhibits a coupling to a methyl group with tunneling rotation at 6 K, and analysis of one of the rotational substates (A) of the spin system yielded an understanding of the structure of this radical. Visible light bleaching of the IMTE at 6 K led to the C3-centered radical C, and thermal annealing above 6 K resulted in a conversion of the C to the D conformation. In addition, thermal annealing releases the IMTE, apparently resulting in the formation of the C2-centered radical. It is possible that the thermal decay of the IMTE also contributes to a small part of the C3-centered radical (D) population at 85 K. There are several other products trapped in rhamnose crystals directly after irradiation at 6 K, among which are resonance lines due to the C2 H-abstraction product. However, these other products are minority species and were not fully characterized in the current work.

Lithium Formate for EPR Dosimetry (2): Secondary Radicals in X-Irradiated Crystals.

The secondary radiation-induced radicals in lithium formate monohydrate were studied using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques complemented with periodic density functional theory (DFT) calculations. Single crystals of lithium formate monohydrate were X irradiated at 77 K and at room temperature. The main radicals present after irradiation at 77 K are the CO(2)(•-) radical (R1), the recently identified protonated electron-gain product, HCOOH(•-) (R2) (Krivokapic et al., Radiat Res 2014: 181:503-11), and a different geometrical conformation of this latter radical, a species that, up until now, has remained unidentified (R3). The successful quantum chemical modeling of R3 confirmed its structure and also provided a possible mechanism for its formation. After irradiation at 295 K, the crystals were investigated both shortly after irradiation and after storage for eight months at room temperature in ambient environments. After long-term storage the CO(2)(•-) radical had significantly decayed and the EPR spectra were dominated by two minority radicals. Both of these radicals are most likely formate-centered π-radicals, and based on the observed EPR parameters (g- and hyperfine coupling tensors) tentative candidates are the CO(•-) radical and the dimer formed by the CO(2)(•-) radical and a neighboring formate molecule yielding the radical (-)O(2)C·O·(•)CH·O(-).

Direct radiation effects to the amino acid side chain: EMR and periodic DFT of X-irradiated L-asparagine at 6 K.

Radical formation in single crystals of L-asparagine monohydrate following X-irradiation at 6 K has been investigated at 6 K and at elevated temperatures using various electron magnetic resonance (EMR) techniques such as electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) spectroscopy. Molecular structures of the three free radicals stable at 6 K were assessed by detailed analysis of the experimental data and density functional theory (DFT) calculations in a periodic approach. Radical LI is assumed to result from one-electron reduction at the amide functional group in the asparagine side chain followed by protonation at the amide carbonyl oxygen by proton transfer from a neighboring molecule across a hydrogen bond. Radical LII is assigned to a one-electron reduction of the carboxyl group in the amino acid backbone, followed by proton transfer across a hydrogen bond between a carboxylic oxygen and a neighboring asparagine molecule. Radical LIII is suggested to be formed by a net CO2 abstraction from an initial one-electron oxidized amino acid backbone. For the DFT modeling of LIII at 6 K, it was chosen to include the CO2 group stably embedded in the crystalline lattice. The assignments made are discussed in relation to previous work on L-asparagine. The relevance of these results to possible charge transfer processes in protein:DNA complexes is discussed.

Automated generation of radical species in crystalline carbohydrate using ab initio MD simulations.

As the chemical structures of radiation damaged molecules may differ greatly from their undamaged counterparts, investigation and description of radiation damaged structures is commonly biased by the researcher. Radical formation from ionizing radiation in crystalline α-l-rhamnose monohydrate has been investigated using a new method where the selection of radical structures is unbiased by the researcher. The method is based on using ab initio molecular dynamics (MD) studies to investigate how ionization damage can form, change and move. Diversity in the radical production is gained by using different points on the potential energy surface of the intact crystal as starting points for the ionizations and letting the initial velocities of the nuclei after ionization be generated randomly. 160 ab initio MD runs produced 12 unique radical structures for investigation. Out of these, 7 of the potential products have never previously been discussed, and 3 products are found to match with radicals previously observed by electron magnetic resonance experiments.

Lithium formate for EPR dosimetry: radiation-induced radical trapping at low temperatures.

Radiation-induced primary radicals in lithium formate. A material used in EPR dosimetry have been studied using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-Induced EPR (EIE) techniques. In this study, single crystals were X irradiated at 6-8 K and radical formation at these and higher temperatures were investigated. Periodic density functional theory calculations were used to assist in assigning the radical structures. Mainly two radicals are present at 6 K, the well-known CO2(•-) radical and a protonated electron-gain product. Hyperfine coupling tensors for proton and lithium interactions were obtained for these two radicals and show that the latter radical exists in four conformations with various degrees of bending at the radical center. Pairs of CO2(•-) radicals were also observed and the tensor for the electron-electron dipolar coupling was determined for the strongest coupled pair, which exhibited the largest spectral intensity. Upon warming, both the radical pairs and the reduction product decay, the latter apparently by a transient species. Above 200 K the EPR spectrum was mainly due to the CO2(•-) (mono) radicals, which were previously characterized as the dominant species present at room temperature and which account for the dosimetric EPR signal.

Solved? The reductive radiation chemistry of alanine.

The structural changes throughout the entire reductive radiation-induced pathway of l-α-alanine are solved on an atomistic level with the aid of periodic DFT and nudged elastic band (NEB) simulations. This yields unprecedented information on the conformational changes taking place, including the protonation state of the carboxyl group in the "unstable" and "stable" alanine radicals and the internal transformation converting these two radical variants at temperatures above 220 K. The structures of all stable radicals were verified by calculating EPR properties and comparing those with experimental data. The variation of the energy throughout the full radiochemical process provides crucial insight into the reason why these structural changes and rearrangements occur. Starting from electron capture, the excess electron quickly localizes on the carbon of a carboxyl group, which pyramidalizes and receives a proton from the amino group of a neighboring alanine molecule, forming a first stable radical species (up to 150 K). In the temperature interval 150-220 K, this radical deaminates and deprotonates at the carboxyl group, the detached amino group undergoes inversion and its methyl group sustains an internal rotation. This yields the so-called "unstable alanine radical". Above 220 K, triggered by the attachment of an additional proton on the detached amino group, the radical then undergoes an internal rotation in the reverse direction, giving rise to the "stable alanine radical", which is the final stage in the reductive radiation-induced decay of alanine.

Structural specificity of alkoxy radical formation in crystalline carbohydrates.

A DFT study of radiation induced alkoxy radical formation in crystalline α-l-rhamnose has been performed to better understand the processes leading to selective radical formation in carbohydrates upon exposure to ionizing radiation at low temperatures. The apparent specificity of radiation damage to carbohydrates is of great interest for understanding radiation damage processes in the ribose backbone of the DNA molecule. Alkoxy radicals are formed by deprotonation from hydroxyl groups in oxidized sugar molecules. In α-l-rhamnose only one alkoxy radical is observed experimentally even though there are four possible sites for alkoxy radical formation. In the present work, the origin of this apparently specific action of radiation damage is investigated by computationally examining all four possible deprotonation reactions from oxygen in the oxidized molecule. All calculations are performed in a periodic approach and include estimates of the energy barriers for the deprotonation reactions using the Nudged Elastic Band (NEB) method. One of the four possible radical sites is ruled out due to the lack of a suitable proton acceptor. For the other three possible sites, the reaction paths and energy profiles from primary cationic radicals to stable, neutral alkoxy radicals are compared. It is found that deprotonation from one site (corresponding to the experimentally observed radical) differs from the others in that the reaction path is less energy demanding. Hence, it is suggested that the alkoxy radical formation is not necessarily site specific, but that the observed radical is formed in much greater abundance than the others due to the different energetics of the processes and reaction products.

Identification of primary free radicals in trehalose dihydrate single crystals X-irradiated at 10 K.

Primary free radical formation in trehalose dihydrate single crystals X-irradiated at 10 K was investigated at the same temperature using X-band Electron Paramagnetic Resonance (EPR), Electron Nuclear Double Resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques. The ENDOR results allowed the unambiguous determination of six proton hyperfine coupling (HFC) tensors. Using the EIE technique, these HF interactions were assigned to three different radicals, labeled R1, R2 and R3. The anisotropy of the EPR and EIE spectra indicated that R1 and R2 are alkyl radicals (i.e. carbon-centered) and R3 is an alkoxy radical (i.e. oxygen-centered). The EPR data also revealed the presence of an additional alkoxy radical species, labeled R4. Molecular modeling using periodic Density Functional Theory (DFT) calculations for simulating experimental data suggests that R1 and R2 are the hydrogen-abstracted alkyl species centered at C5' and C5, respectively, while the alkoxy radicals R3 and R4 have the unpaired electron localized mainly at O2 and O4'. Interestingly, the DFT study on R4 demonstrates that the trapping of a transferred proton can significantly influence the conformation of a deprotonated cation. Comparison of these results with those obtained from sucrose single crystals X-irradiated at 10 K indicates that the carbon situated next to the ring oxygen and connected to the CH(2)OH hydroxymethyl group is a better radical trapping site than other positions.

The energy dependence of lithium formate and alanine EPR dosimeters for medium energy x rays.

PURPOSE: To perform a systematic investigation of the energy dependence of alanine and lilthium formate EPR dosimeters for medium energy x rays. METHODS: Lithium formate and alanine EPR dosimeters were exposed to eight different x-ray beam qualities, with nominal potentials ranging from 50 to 200 kV. Following ionometry based on standards of absorbed dose to water, the dosimeters were given two different doses of approximately 3 and 6 Gy for each radiation quality, with three dosimeters for each dose. A reference series was also irradiated to three different dose levels at a 60Co unit. The dose to water energy response, that is, the dosimeter reading per absorbed dose to water relative to that for 60Co gamma-rays, was estimated for each beam quality. In addition, the energy response was calculated by Monte Carlo simulations and compared to the experimental energy response. RESULTS: The experimental energy response estimates ranged from 0.89 to 0.94 and from 0.68 to 0.90 for lithium formate and alanine, respectively. The uncertainties in the experimental energy response estimates were typically 3%. The relative effectiveness, that is, the ratio of the experimental energy response to that following Monte Carlo simulations was, on average, 0.96 and 0.94 for lithium formate and alanine, respectively. CONCLUSIONS: This work shows that lithium formate dosimeters are less dependent on x-ray energy than alanine. Furthermore, as the relative effectiveness for both lithium formate and alanine were systematically less than unity, the yield of radiation-induced radicals is decreased following x-irradiation compared to irradiation with 60Co y-rays.

Radical formation in lithium formate EPR dosimeters after irradiation with protons and nitrogen ions.

Radical formation in polycrystalline lithium formate monohydrate after irradiation with gamma rays, protons and nitrogen ions at room temperature was studied by continuous-wave electron paramagnetic resonance (EPR) spectroscopy. The linear energy transfer (LET) of the various radiation beams was 0.2, 0.7-3.9 and 110-164 keV/microm for gamma rays, protons and nitrogen ions, respectively. Doses between 5 and 20 Gy were given. The EPR reading (the area under the EPR absorption resonance) increased linearly with dose for all types of radiation. As the LET increased, the relative effectiveness (the EPR reading per dose relative to that for gamma rays) decreased, while the EPR line width increased. Track structure theory and modeling of detector effectiveness predicted the dosimeter response observed after proton and nitrogen-ion irradiation. A semi-empirical line broadening model including dipolar spin-spin interactions was developed that explained the dependence of the line width on LET. The findings indicate that the local radical density in lithium formate is increased after high-LET irradiation.

On the identity of the radiation-induced stable alanine radical.

Using periodic DFT calculations, it is concluded that the stable radiation-induced alanine radical most probably is the result of reductive deamination and protonation of the detached amino group, yielding an NH(4)(+) ammonium ion and a negatively charged radical.

Radicals in 5-methylcytosine induced by ionizing radiation. Electron magnetic resonance for structural and mechanistic analyses.

Single crystals of 5-methylcytosine hemihydrate and 5-methylcytosine hydrochloride were X-irradiated and studied at 10 K and at higher temperatures using X- and K-band EPR, ENDOR and EIE spectroscopy. In the hemihydrate crystals, four radicals were identified at 10 K, one of them being the recently reported N1-deprotonated one-electron oxidation product (Krivokapic et al., J. Phys. Chem. A 113, 9633-9640, 2009). The other radicals were the 3alphaH radical and the C6 and C5 H-addition radicals (the 5-yl and 6-yl radicals, respectively). After irradiation at 295 K, only the 3alphaH and the 5-yl radicals were observed. In the hydrochloride crystals, at least seven different radicals were present after irradiation at 10 K. These were the N1-deprotonated one-electron oxidation product, the 3alphaH radical, three different one-electron reduction products, and the 5- and 6-yl radicals. DFT calculations were used to assist in assigning the observed couplings. The 3alphaH and 5-yl radicals were dominant after thermal annealing to room temperature. In neither crystal system did the N1-deprotonated oxidation product transform into the 3alphaH radical upon warming. The radical yield was significantly greater after irradiation at 300 K compared to that after irradiation at 10 K followed by warming to 300 K and was also considerably greater in the hydrochloride crystals than in the hemihydrate crystals.

Dosimetry of stereotactic radiosurgery using lithium formate EPR dosimeters.

Small lithium formate EPR (electron paramagnetic resonance) dosimeters (diameter 3 mm, height 2 mm) were produced and employed for 2D dosimetry of stereotactic radiosurgery (SRS). An anthropomorphic head phantom with an in-house made insert holding 45 lithium formate dosimeters was used. A spherical target was outlined centrally in planning CT images of the head and an SRS dose plan with three arcs was made using the iPlan planning system. Beam collimation was achieved with the BrainLAB m3 micro-MLC. The minimum target dose was 15 Gy. The planned dose distribution was compared to measurements. For dosimetry, a dosimeter calibration series was generated with doses from 1 to 20 Gy. At the treatment unit, three replicate measurement series were performed. The measurements gave on average 2.2% lower dose at the plateau of the dose distribution compared to the dose plan. Larger differences were seen in the penumbra, where the dose plan underestimated the dose gradients. By repeated measurements, the systematic and random error in the SRS delivery was estimated to less than 1 mm. In conclusion, the planning system produced an intracranial dose distribution with tolerable accuracy. Furthermore, small lithium formate EPR dosimeters were useful for measuring SRS dose distributions.

Oxidation and reduction products of X irradiation at 10 K in sucrose single crystals: radical identification by EPR, ENDOR, and DFT.

Electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) measurements on sucrose single crystals at 10 K after in situ X irradiation at this temperature reveal the presence of at least nine different radical species. Nine proton hyperfine coupling tensors were determined from ENDOR angular variations and assigned to six of these species (R1-R6) using EIE. Spectral simulations indicate that four of those (R1-R3 and R6) dominate the EPR absorption. Assisted by periodic density functional theory (DFT) calculations, R1 and R2 are identified as H-abstracted C1- and C5-centered radicals, R3 is tentatively assigned to an H-abstracted C6-centered radical, and R6 is identified as an alkoxy radical where the abstracted hydroxy proton has migrated to a neighboring OH group via intermolecular proton transfer. The latter radical had been characterized and identified in a previous study, but the present DFT calculations provide additional insight into its conformation and particular properties. This study provides the first step in unraveling the formation mechanism of the stable sucrose radicals detected after room-temperature irradiation and contributes to the understanding of the initial stages of radiation damage to solid-state carbohydrates.

Relaxation-time determination from continuous-microwave saturation of EPR spectra.

Based on the theories of Portis and of Castner 50 years ago, different continuous-wave measurement procedures for analyzing the microwave saturation power dependence of inhomogeneously broadened EPR lines were developed. Although these procedures have been refined, they still use only a few selected points on the saturation curve. A non-linear least-squares procedure for analyzing the microwave-power dependence of inhomogeneously broadened lines using all data points on a saturation curve has been developed. This procedure provides a simple alternative method to obtain magnetic relaxation data when the more direct pulse-saturation techniques are not available or are less suitable. The latter includes applications of quantitative EPR such as dosimetry. Then microwave saturation data should be obtained under conditions similar to those used in the quantitative measurements, which are usually made on first derivative spectra recorded using continuous-wave spectrometers. Selected applications to benchmark literature data and within the field of EPR dosimetry are discussed. The results obtained illustrate that relaxation times comparable to those yielded by various pulse-saturation EPR techniques can be obtained. It appears as a systematic feature that, whenever the pulse EPR data are fitted using bi-exponential functions, the shortest relaxation times obtained are those that correspond best to those measured using the current continuous-wave saturation method.

Primary oxidation products of 5-methylcytosine: methyl dynamics and environmental influences.

The primary oxidation product in X-irradiated single crystals of 5-methylcytosine hemihydrate and 5-methylcytosine hydrochloride has been studied at 10 K, using electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) spectroscopies. The radical is characterized by large couplings to the methyl protons and appears to be deprotonated at N1 in both crystal systems. In the hydrochloride crystal the methyl group is completely frozen at 10 K, whereas in the hemihydrate crystal it undergoes tunneling rotation. For the hemihydrate crystal, four ENDOR lines associated with transitions within the A and E rotational states were followed in three planes of rotation. Large ENDOR shifts as measured by saturation of the high- and low-field parts of the EPR spectrum indicate that the rotation is rather slow. Sidebands due to mixing of A and E rotational states are expected for slow rotation and were observed in both the EPR and the EIE spectra. The ENDOR shifts and the sideband frequencies indicate a tunneling splitting between 40 and 60 MHz. Estimates of the barrier to rotation in both crystalline systems were calculated using cluster and single-molecule density functional theory methods, and the results are consistent with those obtained by analysis of the experimental results.

ENDOR and HYSCORE analysis and DFT-assisted identification of the third major stable radical in sucrose single crystals X-irradiated at room temperature.

Recently, the chemical structure of two of the three major stable radicals (T2 and T3) produced in sucrose single crystals by X-irradiation at room temperature was identified by comparing Density Functional Theory (DFT) calculations of Electron Magnetic Resonance parameters with experimental results [H. De Cooman, E. Pauwels, H. Vrielinck, E. Sagstuen, F. Callens and M. Waroquier, J. Phys. Chem. B, 2008, 112, 7298-7307]. Ambiguities concerning an unusual proton hyperfine coupling (HFC) tensor prevented the identification of the third major stable radical (T1). In the present work, experimental results of continuous wave Electron Nuclear Double Resonance experiments on sucrose single crystals and Hyperfine Sublevel Correlation Spectroscopy experiments on sucrose powder are presented that lift these remaining ambiguities. Using the final set of experimental HFC tensors and employing advanced DFT calculations, the chemical structure of the T1 radical is established: an allylic-type radical with approximately half of the spin density localised on the C2' carbon of the fructose unit, involving glycosidic bond cleavage at the fructose side and a concerted formation of a carbonyl group at the C1' carbon. The electronic structure of the T1 radical is discussed in more detail by means of additional DFT calculations, yielding a better understanding of the peculiar properties of the unusual proton HFC tensor mentioned above.