Lithium Formate for EPR Dosimetry: Radiation-Induced Radical Trapping at Low Temperatures Author(s): André Krivokapić, Siv G. Aalbergsjø, Hendrik De Cooman, Eli Olaug Hole, William H. Nelson, and Einar Sagstuen Source: Radiation Research, 181(5):503-511. 2014. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR13582.1 URL: http://www.bioone.org/doi/full/10.1667/RR13582.1

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RADIATION RESEARCH

181, 503–511 (2014)

0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13582.1

Lithium Formate for EPR Dosimetry: Radiation-Induced Radical Trapping at Low Temperatures Andre´ Krivokapic´,a,1 Siv G. Aalbergsjø,a Hendrik De Cooman,b Eli Olaug Hole,a William H. Nelsonc,2 and Einar Sagstuena a

Department of Physics, University of Oslo, N-0316 Oslo, Norway; b Department of Solid State Sciences, Ghent University, Krijgslaan 281-S1, B9000 Ghent, Belgium; and c Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303

as a sensitive radiation dosimeter in electron paramagnetic resonance (EPR) spectroscopy (1–7). The radiation sensitivity of LiFo is 56 times higher than that of the wellestablished EPR-dosimeter alanine and displays a linear doseresponse relationship at least between 0.2 and 1,000 Gy (2). In addition to being more tissue equivalent than alanine, these properties may make LiFo more suitable for the lower doses used in radiation therapy, although there are still some issues that remain to be solved regarding environmental influences (air humidity, temperature, light) on the stability of the radicals formed (5, 8). For LiFo to become established as a dosimeter it is also essential to understand its radiation physics and chemistry. In general, radiation produces various primary species, which may transform into (more) stable species along multistep pathways. Therefore, of particular interest are the chemical and electronic structures of the initially formed radicals as well as the more deeply trapped end products. It was previously shown that the dominant radiation product formed in LiFo at room temperature (accounting for the  dosimetric EPR signal) is the well-known CO2 – radical (3, 5). It is formed by a net dehydrogenation of the formate ion and is characterized by a large electron spin density on carbon. This radical exhibits a plethora of small hyperfine couplings to nearby hydrogen and lithium nuclei, and at least one more radical species is present at room temperature, however, little is known about its structure. In this report, the radical structures of the shallowly trapped species, which are stable only at very low temperatures, have been investigated in detail. Single crystals of LiFo have been X irradiated and studied at 68 K, using EPR, electron nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) spectroscopic techniques. Quantum chemical calculations with periodic boundary conditions were performed to assist in the identification of the structures.

Krivokapic´, A., Aalbergsjø, S. G., De Cooman, H., Hole, E. O., Nelson, W. H. and Sagstuen, E. Lithium Formate for EPR Dosimetry: Radiation-Induced Radical Trapping at Low Temperatures. Radiat. Res. 181, 503–511 (2014).

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 68 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. Ó 2014 by Radiation Research Society

INTRODUCTION

In the last decade, lithium formate monohydrate, HCOO– Liþ H2O (LiFo), has attracted considerable attention for use Editor’s note. The online version of this article (DOI: 10.1667/ RR13446.1) contains supplementary information that is available to all authorized users. 1 Address for correspondence: Department of Physics, University of Oslo, P.O. Box 1048 Blindern, N-0316 Oslo, Norway; e-mail: [email protected]. 2 Prof. William H. Nelson passed away prior to the completion of this work.

METHODS Single crystals of lithium formate monohydrate (HCOO– Liþ H2O) were grown from aqueous solutions by slow evaporation at 20–508C. 503

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The crystals are orthorhombic with space group Pna21 and four asymmetric units in the unit cell (9). The lengths of the crystal axes ˚ , b ¼ 6.4906 A ˚ and c ¼ 4.8523 A ˚ . The formate ions are: a ¼ 9.9844 A are connected by a network of hydrogen bonds, as shown in Fig. 1. There is only one hydrogen bond between one of the formate oxygens and water, whereas short-range electrostatic interactions connect lithium ions with both of the formate oxygens as well as with neighboring water molecules. The crystal structure is layered with only weak interactions between the layers. The experimental procedures for K- and X-band (24 and 9.8 GHz, respectively) EPR, ENDOR and EIE spectroscopy were as previously described (10, 11). The crystals, situated inside the EPR cavity vacuum shroud at approximately 6 K, were X irradiated (65 kV/45 mA) using a tungsten-anode X-ray tube to doses of about 40 kGy and studied at this and higher temperatures. The orthogonal abc crystal axis reference system was used for the hyperfine coupling tensor analysis. The spectroscopic data for the crystals were collected by rotating the crystals around the b and c crystal axes in addition to a skewed axis defined by the polar angles h ¼ 908, / ¼ 578, which resolved the Schonland ambiguity (12, 13). The polar angles are defined with c as the polar axis and a as the azimuthal angle reference axis. Additional studies, including partially deuterated samples for which the (easily exchangeable) water hydrogens are substituted by deuterium but the formate hydrogens are not, were performed at 8 K and higher temperatures using an X-band (9.5 GHz) Bruker Elexsys 560 spectrometer. For these purposes, the crystals were X irradiated (60 kV/40 mA, chromium-anode X-ray tube) at 8 K to doses in the range of 36 kGy. However, proton NMR studies (at 500 MHz) of the deuterated samples actually used in the experiments revealed that about 30% of the water protons in the crystals had not been exchanged. Potential radical structures were investigated with density functional theory (DFT) calculations with periodic boundary conditions using the CP2K program (14). The crystallographic unit cell was doubled in the b and c directions, and was chosen as the periodic unit to prevent interactions between the periodic images of the radical. This periodic unit contained 16 LiFo and water units, 128 atoms in total. The experimentally determined atomic coordinates (9) were taken as the starting point for the calculations. The Gaussian and augmented plane wave (GAPW) approach (15, 16) was used along with the BLYP functional (17, 18) and a 6311G** (19) basis set which was modified so that all p and d orbitals of the lithium ions were removed. The initial plane wave cutoff was  set to 280 Ry whereas the final geometries of the CO2 – and  HCOOH – radicals were reoptimized using a 400 Ry cutoff. It should be noted that lithium is present in the crystal structure in the form of Li þ ions and the removal of the p and d orbitals did not result in any  significant change in the calculated EPR parameters for the CO2 – radical.  The CO2 – radical was modeled by removing the hydrogen atom of one of the formate ions in the crystal structure and setting the total charge and spin multiplicity of the periodic unit to 0 and 2, respectively. The geometry was then optimized without any  constraints. The HCOOH – radical was modeled by transferring the proton from the neighboring water molecule hydrogen bonded to the formate. The total charge and spin multiplicity of the periodic cell was 1 and 2, respectively. Constrained geometry optimizations were then performed in two steps. The first step was to constrain the oxygen– proton bond length to prevent back transfer of the proton. Subsequently, the HCOO torsional angle was constrained to 308 to bend the molecular structure and stabilize the radical. The constrained geometry optimizations were performed as restraints with a force constant of 0.5. Finally, the resulting structure was optimized without any constraints. Further separation of charge and spin in the system was obtained by multiple proton shuffling along the hydrogen bonds of the water chain in the crystal (see Fig. 1) towards the already deprotonated OH–. This was achieved by performing a constrained

FIG. 1. The bonding scheme in single crystals of LiFo, viewed down the b axis. Hydrogen bonds and electrostatic interactions are marked with dotted lines and the numbers indicate the OH and ˚ . The atoms are symbolized by spheres as OLi distances in A follows: C ¼ black, O ¼ red, H ¼ grey, Li ¼ blue.

geometry optimization where the OH bonds of the next two water ˚ . Again the final structure molecules in the cell were restrained to 1.5 A was freely optimized.

RESULTS AND DISCUSSION

After irradiation at 68 K, at least two different radical species were observed. These were identified from the  experiments and from the DFT calculations as the CO2 – – radical and the protonated anion HCOOH radical. In  addition, pairs of CO2 – radicals were observed and characterized. The EPR and EIE spectra indicated the possible presence of other species in minor amounts, but due to a lack of ENDOR signals their structures could not be established. Figure 2 shows single crystal K-band EPR spectra (35 mT field sweeps) of LiFo with the magnetic field along each of the three crystallographic axes. The lines from the two radicals overlap in Fig. 2 (see also Fig. 5 and see Supplementary Fig. S1; http://dx.doi.org/10.1667/ RR13582.1.S1). Shown in Fig. 3 is a K-band ENDOR spectrum taken off the central (high-intensity) part of the b-axis EPR spectrum of Fig. 2, along with corresponding EIE spectra from the main ENDOR lines (indicated by Roman numerals). At this particular orientation ENDOR lines I–III did not give strong EIE signals and are not shown. In general, however, EIE spectra from ENDOR lines III were almost identical to those for lines IV. From the angular variation of the ENDOR spectra it was possible to determine seven hyperfine coupling tensors that could be associated with the two major radicals using EIE.

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505



Radical R1, the CO2 – Radical

FIG. 2. 1 derivative K-band EPR spectra of single crystals of lithium formate monohydrate, X irradiated and observed at 6 K. The magnetic field is aligned along the three crystallographic axes a, b and c, as indicated. The asterisk indicates lines from the radical pairs. In the bottom spectrum these lines occur outside the magnetic field range (see Fig. 4). The spectra are normalized to a common microwave frequency.

Coupling tensors I and II in Table 1 are both due to coupling with lithium nuclei as evidenced by the ENDOR lines being centered around the free lithium frequency (indicated in Fig. 3). They exhibit axial symmetry, which is typical for couplings between an unpaired electron and distant nuclei for which the point (magnetic) dipole approximation is valid, and are very similar to two of the  corresponding tensors for the CO2 – radical (R1) that were determined previously after irradiation at room temperature (3). It is therefore concluded that tensors I and II are due to  the CO2 – radical, formed and stabilized already at 6 K. This radical is most likely formed by deprotonation of the oxidized formate ion, with most (;80%) of the electron  spin localized on carbon. The CO2 – radical is by far the dominant radical species present after irradiation at room temperature, and in a previous article (3) several lithium and weak proton couplings were reported. In the current study, the ENDOR data allowed reliable tensor analysis for only the two strongest Li couplings (see Table 1) and in addition, some other weak-coupling ENDOR lines were observed. The hyperfine couplings to neighboring nuclei of the  CO2 – radical were also determined using periodic DFT calculations as described in the Methods section. The results for the two above mentioned lithium couplings and the radical g tensor are included in Table 1, together with the corresponding experimental results from the previous room temperature study (3). A complete set of previously obtained hyperfine coupling tensors (3) together with corresponding calculated counterparts are shown in Supplementary Table S1 (http://dx.doi.org/10.1667/RR13582. 1) Also shown in Table 1 are the angular deviations

FIG. 3. Panel A: K-band ENDOR spectrum acquired from the low-field line of the central part of the EPR b-axis spectrum (Fig. 2) of a single crystal of lithium formate, X irradiated and measured at 6 K. Lines from the couplings in Table 1 are indicated, as well as the free proton and free lithium frequencies. Panel B: The EPR b-axis spectrum (adapted from Fig. 2) from which the ENDOR was obtained, together with the EIE spectra obtained from ENDOR lines IV–VII in the top panel. The spectra are normalized to a common microwave frequency.

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FIG. 4. 1 derivative X-band EPR spectra of partially deuterated single crystals of lithium formate, X irradiated and observed at 8 K. The magnetic field is aligned along the three crystallographic axes a, b and c, as indicated. The asterisks indicate lines from the radical pairs. These particular lines have been amplified by a factor of 10 in the two bottom spectra for illustration purposes. The spectra are normalized to a common microwave frequency.

between the experimentally obtained (at 6 and 295 K) eigenvectors and those obtained by the DFT calculations. The deviations of all of the eigenvectors are smaller for the room temperature tensors, possibly because of more precise  data as the CO2 – radical was the main focus in that study, although a temperature effect cannot be ruled out. Regardless, the eigenvector deviations for the maximum dipolar principal value are small at either temperature. Whereas the calculated isotropic coupling values are somewhat larger than the experimental ones, the anisotropic principal values are nearly identical. This good agreement demonstrates the reliability of using periodic DFT calculations for assigning radical structures in this system. 

The CO2 – Radical Pairs

The outermost lines [indicated by asterisks (*)] in the EPR spectra in Fig. 2 were assigned to radical pairs because of their large splitting and characteristic anisotropy. They  are presumably due to CO2 – radicals as they exhibit the same g-anisotropy as the monoradicals. In addition, ENDOR spectra taken off these EPR lines gave only weak lines close to the free lithium and free proton frequencies,  which is also in accordance with CO2 – radicals. Figure 4 shows X-band EPR axis spectra (60 mT field sweeps) of partially deuterated crystals X irradiated and measured at 8 K. The radical pair EPR lines were clearly observed when rotating the crystals around the three crystallographic axes and from these experiments the tensor for the electron spin dipolar coupling (with principal values 2D, D – 3E, D þ 3E, where D and E are the zero-field splitting constants) (20–22) was obtained and is included in Table 1.

FIG. 5. 1 derivative X-band EPR spectra of a single crystal of lithium formate monohydrate with the magnetic field along the crystallographic b axis, X irradiated at 8 K and warmed to the temperatures indicated and measured at these temperatures. The dashed lines indicate new lines growing in.

For some orientations at or near the b and c axes each of the outer-wing EPR lines appeared to be split into doublets and triplets, with line separations up to 1.1 mT. Upon warming to around 100 K (see below and Supplementary Fig. S1; http://dx.doi.org/10.1667/RR13582.1.S1) these lines converged into single, more intense lines. This  suggests that at the lowest temperatures, the CO2 – pairs are frozen in slightly different conformations resulting in slightly different intermolecular distances and that furthermore, when warmed the radicals merge into the energetically most favorable conformation by geometric relaxation. The radical pairs started decaying at temperatures above 120 K. The dipolar coupling tensor should be traceless and the small deviation from this, as apparent in Table 1, is probably due to the presence of several groups of radical pairs and their different conformations, making exact determinations of corresponding high- and low-field resonance positions difficult. The eigenvector of the maximum principal value is almost parallel to the crystallographic c axis. A simplified expression relating the interaction energy D ¼ DZZ/2 ¼ 26.7 mT to the separation distance R between them, l0 3 ð1Þ gl R3 ; 4p 2 B where g is the radical g-factor and lB is the Bohr-magneton, D=glB ¼

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TABLE 1  Hyperfine Coupling Tensors for Two Lithium Couplings of the CO2 – Radical in Single Crystals of Lithium Formate Monohydrate Experimentally Determined after X Irradiation at 6 K (Current Study, Tensors I and II) and 295 K [Tensors I (Room Temperature) and II (Room Temperature) (3)] and Calculated using Periodic DFT [Current Study, Tensors I (P) and II (P)] Eigenvectors Tensor

Isotropic value

I

9.8 (1)

II

6.6 (1)

Anisotropic values

a

Experimental, 6 K, current study (in MHz) 3.0 (1) –0.712 (3) –1.2 (1) –0.667 (10) –1.8 (1) –0.219 (3) 2.8 (1) 0.715 (1) –1.3 (1) 0.069 (1) –1.5 (1) 0.696 (10)

b 0.021 0.291 –0.956 0.699 –0.102 –0.708

(14) (59) (12) (149) (144) (25)

Experimental, room temperature, previous study (3). Hyperfine couplings in MHz 9.70 (1) 2.99 (1) –0.693 (1) 0.089 (4) –1.30 (1) –0.686 (2) 0.225 (16) –1.69 (1) –0.222 (1) –0.970 (2) 6.85 (1) 2.78 (1) 0.763 (1) 0.637 (17) –1.31 (1) 0.447 (1) –0.400 (25) –1.47 (1) 0.467 (2) –0.659 (22) 2.0032 –0.043 0.998 2.0008 0.894 0.060 1.9965 –0.445 0.026

I (room temperature) II (room temperature) G

Periodic DFT calculations, current study. Hyperfine couplings in MHz 13.15 2.72 –0.6984 0.1004 –1.06 –0.7084 0.0438 –1.65 –0.1017 –0.9940 9.44 2.60 0.7915 0.6002 –1.20 0.4741 –0.4842 –1.40 0.3857 –0.6366 2.0046 –0.1682 0.9857 2.0014 0.8370 0.1463 1.9956 –0.5206 –0.0833

I(P) II(P) g

d (deg)

c –0.702 0.685 0.193 –0.022 –0.992 0.121

(38) (18) (47) (15) (21) (207)

4.6 14.6 15.3 10.6 35.7 36.9

–0.715 0.692 0.095 0.108 –0.800 0.590 –0.050 0.443 0.895

(12) (4) (11) (16) (15) (30)

1.7 10.5 10.6 3.0 6.3 6.5 7.6 7.8 8.1

–0.7086 0.7045 –0.0405 0.1151 –0.7354 0.6678 –0.0064 0.5272 0.8497



D

Experimental, 8 K, current study. Electronelectron dipolar coupling tensor of CO2 – radical pairs (in mT)a –0.6 (1) –53.4 (1) 0.005 (3) –0.077 (1) 0.997 (5) 24.3 (1) 0.999 (3) 0.015 (5) –0.004 (1) 29.1 (1) 0.014 (1) –0.997 (2) –0.077 (3)

Notes. d denotes the angular deviation from the corresponding DFT-calculated eigenvectors. Also included are the experimental and calculated g-tensor, as well as the experimentally determined dipolar coupling tensor of the radical pair. The numbers in parenthesis denote the uncertainty in the last digits. a The zero-field splitting constants D and E are 26.7 and 0.8 mT, respectively. The apparently nonzero isotropic value is a nonphysical result originating from the unconstrained fitting procedure.

˚ , which is gives an effective interspin distance R ¼ 4.71 A ˚ close to the c-axis length of 4.85 A (9). Weaker lines from other radical pairs, most likely also  composed of CO2 – radicals, were also present but could not be analyzed. The dipolar couplings of these pairs exhibited different anisotropies. The maximum observed splitting from the strongest coupled of these pairs was around 50 mT (;308 from the b axis in the bc plane). 

Radical R2, the HCOOH – Radical (in Four Conformations)

The central part of the EPR spectra in Figs. 2 and 4 is dominated by several doublet structures giving rise to ENDOR lines IIIVII and the corresponding EIE spectra shown in Fig. 3. The hyperfine coupling tensors calculated

from these ENDOR lines are shown in Table 2 and are all due to proton interactions. The width of the (doublet) EIE spectra from ENDOR lines III–VI at several orientations always matched the corresponding ENDOR frequencies. The tensors III–VI have very similar anisotropic principal values, indicating that they may be due to the same radical in different geometrical conformations, each of them providing one of the couplings III–VI. From the width and shape of the EIE spectra, coupling VII appears to be connected to coupling IV (possibly also to coupling III). When disregarding major chemical and structural changes to the molecules in the lattice after irradiation at this low temperature (6 K), there are not many possible radical candidates that can give rise to these couplings. The  anisotropic tensors are typical for p-radical .C –H a-

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TABLE 2 Experimental and DFT Calculated Hyperfine Coupling Tensors for Radical R2 in Lithium Formate, X Irradiated and Observed at 6 K Eigenvectors Tensor

Isotropic value (MHz)

III

46.5 (1)

IV

53.9 (1)

V

75.6 (1)

VI

84.3 (1)

VII

–5.0 (1)

P (CH)

69.9

P (OH)

–3.1

Crystallographic directions:

Anisotropic values (MHz) 28.5 –0.9 –27.4 28.7 –0.9 –27.5 28.5 –2.2 –26.2 28.0 –2.1 –25.7 14.1 –8.5 –5.6

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

a –0.753 –0.276 –0.597 –0.702 –0.390 –0.596 –0.635 –0.582 –0.508 –0.587 –0.662 –0.466 0.800 0.261 0.541

Periodic cell DFT calculations 28.6 –0.577 –4.4 –0.627 –24.1 –0.523 14.5 0.751 –8.9 0.072 –5.6 0.657 CH ? to O–C–O

–0.807 –0.259

b (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (11) (11) (11)

–0.392 0.917 0.071 –0.551 0.827 0.108 –0.646 0.761 –0.065 –0.728 0.683 –0.052 0.119 0.814 –0.569

d (deg)

c (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (10) (6) (15)

0.528 0.288 –0.799 0.451 0.404 –0.796 0.424 0.287 –0.859 0.353 0.308 –0.883 0.588 –0.519 –0.620

–0.715 0.698 –0.048 0.202 0.921 –0.332

0.395 0.347 –0.851 0.629 –0.382 –0.677

–0.268 0.961

0.527 0.093

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (2) (2) (3)

22.6 24.1 8.6 12.3 15.9 10.3 5.3 5.3 1.1 3.0 2.9 3.9 5.9 14.8 15.4

Notes. The numbers in parenthesis denote the uncertainty in the last digit(s). d denotes the angular deviation from the DFT-calculated principal directions. P (CH) and P (OH) indicate proton couplings from the carbon bonded and oxygen bonded protons of the modeled structure.

proton couplings (23) indicating that the majority of the spin density is located at the carbon atom of the formate moiety. Of the four tensors III–VI in Table 2, the eigenvectors of tensor III deviate the least from those expected based on the crystallographic data; the eigenvector of the largest (positive) anisotropic principal value differs only 7.98 from the crystallographic C–H direction while the eigenvector of the intermediate principal value differs 11.68 from the perpendicular to the O–C–O plane. The corresponding eigenvectors for the other tensors IV–VI exhibit increasingly larger deviations, about 318 for tensor VI. The third eigenvector of these tensors [expected to be along the in plane external bisector of the O–C –O fragment (23)] does not change to the same degree nor in the same consistent manner. By using the Gordy-Bernhard relation (24) for the maximum anisotropic coupling, ad ¼ Qd  qp, where Qd ¼ 38.7 MHz and qp is the spin density on C, the four tensors give values for qp between 0.72 and 0.74.  In contrast to planar p-orbital .C –H a-couplings, however, the isotropic parts of the coupling tensors III–VI are all positive. Furthermore, the isotropic couplings increase the more the eigenvectors of the dipolar couplings deviate from the crystallographic directions expected for a  planar .C –H coupling. This strongly suggests that the radical is bent (rather than being reoriented rigidly as a

whole), which results in rotation of the C–H bond approximately in the original O–C–O bisecting plane. This would produce more sp3 bonding character at the central carbon atom of the radical and therefore bring positive electron spin density to the proton which in turn increases the isotropic coupling value from its normal negative value in the planar conformation to progressively more positive coupling as the bending becomes greater. The general behaviors described here for both the anisotropic and isotropic parts of the hyperfine interactions have been discussed in detail by Erling and Nelson (25). Tensor VII in Table 2 is also due to a proton coupling, as evidenced by the ENDOR lines being centered around the free proton frequency, mp. EIE spectra from ENDOR lines belonging to this weak coupling gave doublets with a splitting matching the ENDOR frequencies of coupling IV at these same orientations. Additional weak (proton) coupling ENDOR lines gave EIE spectra similar to those of couplings V and VI (EIE spectra not shown). However, the tensors of these weak couplings could not be determined. It thus seems that couplings III–VI are all accompanied by weaker, mutually similar couplings, one of them being coupling VII. The splitting from these couplings was not resolved in the EIE spectra and only contributed to the line width. The hyperfine coupling tensor principal

RADICALS IN LOW-TEMPERATURE IRRADIATED LITHIUM FORMATE

values of VII are somewhat unusual (assuming that the correct sign of the principal values has been chosen). Whereas the anisotropic tensor is typical of a b-proton coupling, the isotropic coupling value is negative. However, similar proton coupling principal values have previously been observed experimentally and predicted by periodic DFT calculations for radicals in irradiated single crystals of sucrose and glucose phosphate (26, 27). It seems probable that coupling VII may be due to a proton added to one of the formate oxygens. Considering the overall charge balance, this would indicate that all four radicals are structurally (but not geometrically) similar protonated one-electron reduced formate ions. Based on the crystal structure, the most likely oxygen site for protonation would be the one that is hydrogen-bonded to the crystalline water (see Fig. 1). Periodic DFT calculations were performed on this structure (Structure 1) as described in the Methods section. The resulting proton coupling tensors, P(CH) and P(OH), for the radical being protonated through the hydrogen-bond are included in Table 2. The anisotropic (dipolar) principal values of tensor P(CH) are remarkably similar to those of tensors III–VI. Also shown in Table 2 are the deviations between the eigenvectors for the experimental tensors III–VI and those for the calculated tensor P(CH). The deviations are smallest for tensors V and VI, while the calculated isotropic coupling value is closest to that of tensor V. Similarly, the calculated coupling tensor for the added proton at the hydrogen-bonded oxygen, P(OH) in Table 2, has principal values almost identical to those of the corresponding experimental tensor VII. The eigenvector of the largest anisotropic value of P(OH) deviates only 68 from the corresponding eigenvector of tensor VII. The overall excellent agreement between the calculated and experimentally obtained couplings strongly supports the radical model where protonation takes place across the hydrogen bond and it can be concluded that radical R2 is most probably the one-electron reduction product protonated on oxygen, as shown in Structure 1.

Apparently, this radical exists in at least four different conformations at the lowest temperatures. The calculated electron spin distribution leaves ca. 9% on surrounding atoms in the lattice, while the spin density on carbon is 67%, in agreement with the experimental results discussed

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for tensors III–VI. The remaining spin is mainly distributed on the oxygens. Although the current results cannot determine whether the radical is formed by (i) the formate trapping an electron followed by proton transfer from water, or by (ii) hydrogenation, presumably from reduced water, mechanism (i) is expected to energetically be the most feasible reaction. The ENDOR line corresponding to coupling VII was also observed in X-band spectra of the incompletely deuterated crystals. However, due to the presence of both water deuterons and water protons (see the Methods section) in these crystals, the results could not unequivocally determine whether or not coupling VII is due to an exchangeable proton. An alternative to proton transfer from water is protonation at the other non-hydrogen bonded oxygen of the reduced formate, or hydrogen atom addition at this oxygen of an intact formate possibly resulting from a neighboring C–H dissociation (28). Preliminary periodic DFT calculations for such radicals, however, did not yield structures with EPR properties compatible with those in Table 2 as two tensors were obtained with isotropic coupling values of about 70 MHz and this type of structure was not pursued further. Warming Experiments

Figure 5 shows X-band EPR spectra of a nondeuterated LiFo crystal X irradiated at 8 K and warmed to various temperatures with the magnetic field along the b axis. At 50 K and above, new lines are appearing in the outer part of the central portion of the spectra, while lines associated with radical R2 decay. This may indicate a transformation of this radical. The spectra were acquired at the indicated temperatures and with increasing microwave power so that they are not quantitatively directly comparable. Warming and re-cooling another irradiated crystal to 6 K displayed the same spectral changes (see Supplementary Fig. S1; http://dx.doi.org/10.1667/RR13582.1.S1), showing that the changes observed at the various temperatures are irreversible. At 80 K and with the magnetic field along the a axis, a new ENDOR line appeared at 50.4 MHz (X-band). This line gave an EIE spectrum whose spectral width (ca. 4.6 mT) corresponded to the new EPR lines at this orientation. The spectral pattern appeared to be a triplet or possibly, and perhaps more likely judging from the EPR spectrum, an unresolved quartet, where the middle two lines nearly overlap. No further ENDOR measurements were possible at these elevated temperatures, and the structure of the new radical was not further pursued in the current work but will be investigated further in ongoing work in our laboratories. Radical R2 started decaying at 50 K in the nondeuterated crystals and appeared to have disappeared completely at 180 K (see Fig. 5 and Supplementary Fig. S1; http://dx.doi.org/ 10.1667/RR13582.1.S1). In partially deuterated crystals the transformation occurred at a slightly elevated temperature; the decay started at 80 K and R2 was still present at 200 K.

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This isotope effect might point to proton (deuteron) transfer across the hydrogen bond network playing a role in the transformation and decay of R2. In crystalline LiFo the water molecules form a continuous hydrogen bonded chain in the c axis direction (see Fig. 1). Thus if R2 is formed by proton transfer from water, its formation may be followed by proton transfer to the remaining OH– through the water chain. The radical structure of R2 was calculated (by DFT) after two such proton transfer steps had occurred along the c chain and a stable optimized structure with nearly identical coupling tensors as those (calculated) in Table 2 was obtained. This suggests that long-range proton transfer in this water network, which in effect removes the charge (OH–) further away from the radical, may be a feasible way of trapping the radicals (29–31). The new lines growing in concomitantly with the decay of R2 were no longer observable around 180 K. Thus, neither of these two radical species are candidates for the minority room temperature radical [denoted radical II in the previous study (3)]. After warming to room temperature the EPR spectra are qualitatively identical to spectra recorded after irradiation at room temperature. Judging by the high radiation sensitivity of LiFo (2), irradiation at room temperature results in a much larger radical yield than after irradiation at 6 K. This is consistent with radical R1 being formed by deprotonation of the oxidized formate (3). Deprotonation at carbon is more likely at room temperature due to thermally excited C–H vibrational states and separates the charge from the spin which in turn makes the radical less susceptible to immediate recombination (32). CONCLUSIONS

After X irradiation of LiFo crystals at 6 K two radicals are  formed. One is the CO2 – radical (R1) which is observed both as monoradicals and as radical pairs. Only the monoradicals survive warming to room temperature. The  other radical (R2) is the product HCOOH – formed by protonation at one of the oxygens of the one-electron reduced formate, where the proton transfer takes place across the hydrogen bond. This radical is observed in four conformations. Upon warming from 6 K, radical R2 partly transforms into a new species (hitherto unidentified), which along with R2, subsequently decays apparently without any successors. Therefore, it appears that neither of these relatively shallowly trapped radicals are candidates or precursors for the minority radicals present at room temperature, and hence they will not influence the dosimetric properties of LiFo. ACKNOWLEDGMENTS Prof. Freddy Callens at the Department of Solid State Sciences, Ghent University, is acknowledged for his support to Dr. Hendrik De Cooman. Dr. Ewald Pauwels, scientific coordinator of the UGent High Performance

Computing infrastructure, is acknowledged for expert advice on periodical DFT calculations. Mr. Dirk Petersen at the Department of Chemistry, University of Oslo, is acknowledged for recording the NMR spectra. The calculations were performed on the Titan and Abel clusters owned by the University of Oslo and the Norwegian Metacenter for Computational Science (NOTUR). A grant for computing resources at NOTUR from the Research Council of Norway is acknowledged. Received: October 18, 2013; accepted: December 16, 2013; published online: April 10, 2014

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