Determination of the g Tensors for the Dominant Stable Radicals in X-Irradiated beta-D-Fructose Single Crystals

In spite of recent successful identifications of radicals produced after X-ray irradiation at 10 and 77 K in beta-D-fructose, the structure of the two stable radicals dominating the electron paramagnetic resonance (EPR) spectrum after room temperature irradiation is still unclear. Based on the agreement between proton hyperfine (HF) tensors obtained in electron nuclear double resonance (ENDOR) experiments and the results of single molecule density functional calculations, a model for these radicals, involving OH abstraction at the C2 ring position, had previously been proposed, but this assignment could not be confirmed when the radical was embedded in a crystal environment. In this paper, we therefore provide additional experimental information for these radicals. First, their g tensors are determined from angular dependent ENDOR-induced EPR experiments. The relatively large anisotropy of these tensors is indicative of delocalization of the unpaired electron onto a neighboring oxygen atom. Second, EPR spectra of fructose powders, selectively enriched in C-13 on various ring positions, are presented, demonstrating that the HF interaction with the carbon atom C3 is larger than with the C2. Combining the g tensor, proton and C-13 HF data, we conclude that the structure of the stable radicals differs strongly from that of intact molecules and that further advanced quantum chemical modeling will be required to fully identify them

Combined Electron Magnetic Resonance and Density Functional Theory Study of Thermally Induced Free Radical Reactions in Fructose and Trehalose Single Crystals

Both as models for studying the effects of radiation on the DNA sugar unit and for applications in dosimetry, radiation-induced defects in sugars have in the past few decades been intensively studied with electron magnetic resonance (EMR) techniques, often with considerable success. However, irradiation generally gives rise to a large variety of free radicals, resulting in strongly composite Electron Paramagnetic Resonance (EPR) spectra. This complexity makes studying them quite a challenge. Despite considerable efforts, little is still known about the identity of the radicals and even less about the radical formation and transformation processes and mechanisms. At room temperature (RT) the primary radiation products, which may be stabilized upon low temperature (LT) irradiation, transform into stable radicals via multistep reaction mechanisms. While the species formed at LT are expected to be formed by simple processes, the molecular structure and geometry of the stable radicals may differ considerably from that of the intact molecule even in the solid state (crystals). Studying the intermediate radicals in the reactions occurring after LT irradiation helps elucidating the formation and identity of the stable radicals. The structural identification of these radicals is in most cases the result of a combination of EPR, Electron Nuclear Double Resonance (ENDOR) and ENDOR Induced EPR (EIE) experiments and advanced quantum chemistry calculations based on Density Functional Theory (DFT). In the present study a summary is given of the experimental EMR results obtained so far on radiation-induced radicals at different temperatures in fructose and trehalose single crystals and powders. “In situ” X-irradiation at LT (10 K) without annealing, leads to spectra strongly different from those observed after RT irradiation and offers the possibility to study and characterize the primary radiation products [1]. Performing EMR measurements on samples irradiated and/or annealed at various temperatures between LT (10 K or 77 K) and RT allows us to study the intermediate products, and such studies therefore have the potential to devise mechanistic links between the primary radicals and the stable radicals. In the present work, our own measurements are compared with results reported in the EMR literature. An outline at future experimental (EMR) and theoretical (DFT) research will also be given

Electron magnetic resonance study of primary free radicals in trehalose single crystals

Radiation-induced radicals in sugars have recently gained considerable interest with respect to both fundamental and applied research. A number of studies are available that focus on the dosimetric characteristics of sugar systems. Other studies, like ours, aim to understand the identity and the structural properties of the involved radicals, and the radical reactions in which the primary or secondary products can be linked to the stable radicals. Furthermore, carbohydrates represent extremely suitable model systems in which primary radiation-induced events may be studied. For instance, the detection of a trapped electron center in organic solids was first made in carbohydrate single crystals1,2 and the nature of alkoxy radicals is readily investigated in these systems 3. We present here experimental results obtained for low temperature radiation-induced radicals in trehalose single crystals. After 10 K in situ X-irradiation of trehalose single crystals four dominant radicals are present. Two radical species, labeled R1 and R2 are characterized by anisotropic g factors typical for the alkoxy type radicals. The R1 EPR spectrum is a broad singlet for most orientations, indicating only small proton hyperfine couplings. The direction of the maximum g value (gmax) indicates that O4’ is the most likely site for the unpaired electron. R2 mainly exhibits a quartet EPR spectrum and the gmax direction favours O2 as the site of the unpaired electron. The other two radicals, R3 and R4, are characterized by a rather isotropic g tensor typical of alkyl radicals. R3 is characterized by a rather isotropic triplet in EPR which suggests two almost equivalent β proton hyperfine interactions. It is obtained by a net hydrogen abstraction from the C3’ position. The second alkyl radical has most likely the unpaired electron localized at the C2 position. A major purpose of this study is to compare the obtained results with the results reported by De Cooman et al4 for 10 K X-irradiated sucrose single crystals. Trehalose and sucrose have a close structural similarity: they both are disaccharides composed of two units linked by a glycosidic oxygen bridge between their two anomeric carbon atoms, C1 and C1’. The study of very similar products may lead to a better general understanding of the radiation chemistry of carbohydrates

Direct-effect radiation chemistry of solid-state carbohydrates using EMR and DFT

To contribute to a mechanistic understanding of radical reaction pathways in the sugar-phosphate backbone of DNA, we are investigating primary radicals induced by X-rays, as well as their transformation into stable radicals or diamagnetic products, in crystalline sugar and sugar derivatives. Radicals are identified and characterized mainly via the hyperfine interactions of the electron spin with protons in the molecular environment. These interactions are determined experimentally with electron magnetic resonance (EMR) techniques and compared to theoretical ab initio calculations based on density functional theory in a periodic approach. Different stages of the radiation-induced processes are investigated by irradiating in situ at various temperatures and controlled annealing experiments. Here, results obtained in single crystals of the dipotassium salt of glucose 1-phosphate (K2G1P) and the disaccharides sucrose and trehalose are presented. The dominant radical in K2G1P after irradiation at 77 K exhibits a broken phospho-ester bond and is chemically identical to one of the major stable sucrose radicals, the latter all being characterized by a broken glycosidic bond. This suggests that the ester bond is radiation sensitive and that the phosphate group is not essential for the reaction pathway leading to this scission. Surprisingly, however, no evidence for glycosidic bond scission has so far been observed in trehalose. Rather, a simple H-abstraction alkyl radical is remarkably stable in this system. In all three compounds, dominant radicals are formed with one or several concerted carbonyl group formations. Extended studies are necessary to establish how and to which extent structural or geometrical factors determine the radiation chemistry, but certain general principles are starting to emerge

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 suggest 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 CH2OH hydroxymethyl group is a better radical trapping site than other positions

ENDOR in field-frequency space: orientation, species and quantum state selection

Due to its specific detection method, via saturation of the EPR spectrum at a certain magnetic field position, ENDOR measurements are highly selective. Already in the late sixties Rist and Hyde recognized the possibilities of obtaining angular dependent information from powder specimens by recording the field-dependence of the ENDOR spectrum, by grace of the orientation selection principle.1 However, also for single crystal samples it makes sense to record ENDOR spectra in the two-dimensional field-frequency space (FF-ENDOR). Next to orientation selectivity – when several symmetry-related orientations of the same paramagnetic species are simultaneously detected in the EPR spectrum – such measurements feature species selectivity and, for systems with S > ½ and/or I > ½, also quantum state selectivity. The former facilitates the interpretation of multi- composite EPR spectra, as illustrated in the figure below. Quantum state selectivity offers possibilities of determining the relative signs of spin Hamiltonian parameters, e.g. zero-field splitting and hyperfine, or hyperfine and quadrupole principal values. All these effects will be illustrated through recent examples in or research of radiation-induced radicals in sugars and high-spin transition ion or rare-earth doped fluoride crystals

Electron Magnetic Resonance study of the Structure and Thermal Stability of Radiation-Induced Free Radicals in Trehalose Single Crystals

Radiation-induced radicals in sugars have recently gained considerable interest with respect to both fundamental and applied research. A number of studies are available that focus on the dosimetric characteristics of sugar systems. Other studies, like ours, aim to understand the identity and the structural properties of the involved radicals, and the radical reactions in which the primary or secondary products can be linked to the stable radicals. We present here experimental results obtained on radiation-induced radicals in trehalose single crystals. A major purpose of this study is to check if the cleavage of the glycosidic bond and carbonyl formation are common radiation-induced processes produced by irradiation in disaccharides. Recently, the chemical structures of three dominant radicals obtained after room temperature (RT) irradiation in sucrose single crystals, were identified by De Cooman et al.1-3 All three radicals have a broken glycosidic bond and a carbonyl group. Trehalose was selected as the object of this study because of its close structural similarity with sucrose : it is a disaccharide composed of two [...]-D-glucosyl units linked by a glycosidic oxygen bridge between their two anomeric carbon atoms, C1 and C1’. After RT irradiation of trehalose single crystals three dominant radicals are present. One radical species is characterized by a rather isotropic triplet due to the interaction of the unpaired electron with two almost equivalent protons in [...] positions. The other two radical species exhibit only proton hyperfine couplings smaller than 20 MHz and therefore are characterized by a broad EPR singlet. In addition to these radicals, two other less dominant species characterized by a doublet structure are present. Storing the irradiated trehalose crystal at RT for three months or heating it to 40° for three days, changes the EPR spectrum completely, the dominant species now being characterized by a doublet of doublets

Electron magnetic resonance study of the structure and thermal stability of radiation-induced radicals in fructose and trehalose

Both as models for studying the effects of radiation on the DNA sugar unit and for applications in dosimetry, radiation-induced defects in sugars have in the past few decades been intensively studied with electron magnetic resonance (EMR) techniques, often with considerable success. However, irradiation generally gives rise to a large variety of free radicals, resulting in strongly composite Electron Paramagnetic Resonance (EPR) spectra. This complexity makes studying them quite a challenge and despite considerable efforts, little is still known about the identity of the radicals and even less about the radical formation and transformation processes and mechanisms. At room temperature (RT) the primary radiation products, which may be stabilized upon low temperature (LT) irradiation, transform into stable radicals via multiple step reaction mechanisms. While the species formed at LT are expected to be formed by simple processes, the molecular structure and geometry of the stable radicals may differ considerably from that of the intact molecule even in the solid state (crystals). Studying the intermediate radicals in the reactions occurring after LT irradiation helps elucidating the formation and identity of the stable radicals. The structural identification of these radicals is in most cases the result of a combination of EPR, Electron Nuclear Double Resonance (ENDOR) and ENDOR Induced EPR (EIE) experiments and advanced quantum chemistry calculations based on Density Functional Theory (DFT). In the present doctoral study a summary is given of the experimental EMR results obtained so far on radiation-induced radicals at different temperatures in fructose and trehalose single crystals and powders. Performing EMR measurements on samples irradiated and/or annealed at various temperatures between LT (10 K or 77 K) and RT allows us to study the intermediate products, and such studies therefore have the potential to devise mechanistic links between the primary radicals and the stable radicals