Q-band EPR and ENDOR of low temperature X-irradiated beta-D-fructose single crystals

Beta-D-fructose single crystals were in situ X-irradiated at 80 K and measured using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques at Q-band (34 GHz) microwave frequencies. The measurements revealed the presence of at least four carbon-centered radicals stable at 80 K. By means of ENDOR angular variations in the three principal crystallographic planes, six proton hyperfine coupling tensors could be determined and were assigned to four different radicals by the aid of EIE. Two of the radicals exhibit only beta-proton hyperfine couplings and reveal almost identical EIE spectra. For the other two radicals, the major hyperfine splitting originates from a single alpha-proton hyperfine coupling and their EIE spectra were also quite similar. The similarity of the EIE spectra and hyperfine tensors led to the assumption that there are only two essentially different radical structures. The radical exhibiting only beta-proton hyperfine couplings was assigned to a C3 centered radical arising from H3 abstraction and the other radical suggested to be an open-ring species with a disrupted C2-C3 bond and a double C2-O2 bond. A possible formation mechanism for the latter open-ring radical is presented. By means of cluster density functional theory (DFT) calculations, the structures of the two radicals were determined and a fairly good agreement between the calculated and experimental hyperfine tensors was found.     

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.     

Radiation products at 77 K in trehalose single crystals: EMR and DFT analysis

The radicals obtained in trehalose dihydrate single crystals after 77 K X-irradiation have been investigated at the same temperature using X-band electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) techniques. Five proton hyperfine coupling tensors were unambiguously determined from the ENDOR measurements and assigned to three carbon-centered radical species (T1, T1*, and T2) based on the EIE spectra. EPR angular variations revealed the presence of four additional alkoxy radical species (T3 to T6) and allowed determination of their g tensors. Using periodic density functional theory (DFT) calculations, T1/T1*, T2, and T3 were identified as H-loss species centered at C4, C1', and O2', respectively. The T4 radical is proposed to have the unpaired electron at O4, but considerable discrepancies between experimental and calculated HFC values indicate it is not simply the (net) H-loss species. No suitable models were found for T5 and T6. These exhibit a markedly larger g anisotropy than T3 and T4, which were not reproduced by any of our DFT calculations.     

Electron magnetic resonance and density functional theory study of room temperature X-irradiated β-D-fructose single crystals

Stable free radical formation in fructose single crystals X-irradiated at room temperature was investigated using Q-band electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR induced EPR (EIE) techniques. ENDOR angular variations in the three main crystallographic planes allowed an unambiguous determination of 12 proton HFC tensors. From the EIE studies, these hyperfine interactions were assigned to six different radical species, labeled F1-F6. Two of the radicals (F1 and F2) were studied previously by Vanhaelewyn et al. [Vanhaelewyn, G. C. A. M.; Pauwels, E.; Callens, F. J.; Waroquier, M.; Sagstuen, E.; Matthys, P. J. Phys. Chem. A 2006, 110, 2147.] and Tarpan et al. [Tarpan, M. A.; Vrielinck, H.; De Cooman, H.; Callens, F. J. J. Phys. Chem. A 2009, 113, 7994.]. The other four radicals are reported here for the first time and periodic density functional theory (DFT) calculations were used to aid their structural identification. For the radical F3 a C3 carbon centered radical with a carbonyl group at the C4 position is proposed. The close similarity in HFC tensors suggests that F4 and F5 originate from the same type of radical stabilized in two slightly different conformations. For these radicals a C2 carbon centered radical model with a carbonyl group situated at the C3 position is proposed. A rather exotic C2 centered radical model is proposed for F6.     

Study of rhamnose radicals in the solid state adopting a density functional theory cluster approach

A theoretical study is performed on the radiation-induced radicals in crystalline alpha-l-rhamnose, using density functional theory (DFT) calculations. Irrespective of earlier structural assignments, a host of possible radical models is examined in search for a structure that accurately reproduces experimental electron paramagnetic resonance (EPR) properties. A cluster approach is followed, incorporating all hydrogen bond interactions between radical and crystalline environment. Hyperfine coupling tensors as well as g tensors are determined and a comparison is made with available experimental data. Three carbon-centered hydroxyalkyl radicals are validated, in accordance with experimental suggestions for their structure. The occurrence of a carbon-centered oxygen anion radical for one of the radical species is rejected on theoretical grounds, and instead an altered hydroxyalkyl structure is suggested. Our cluster calculations are able to determine g and hyperfine tensors for the oxygen-centered alkoxy radical in rhamnose, in accordance with one of the two measurements for this species. For all radical models, quantitative agreement with experimental hyperfine tensors is obtained by performing full cluster DFT calculations. The inclusion of the molecular environment for the determination of this EPR property proved to be essential.     

Radiation-induced radicals in glucose-1-phosphate. II. DFT analysis of structures and possible formation mechanisms

Four radiation-induced carbon-centered radicals in dipotassium glucose-1-phosphate dihydrate single crystals are examined with DFT methods, consistently relying on a periodic computational scheme. Starting from a set of plausible radical models, EPR hyperfine coupling tensors are calculated for optimized structures and compared with data obtained from EPR/ENDOR measurements, which are described in part I of this work. In this way, an independent structural identification is made of all the radicals that were observed in the experiments (R1-R4) and tentative reaction schemes are proposed. Also, the first strong evidence for conformational freedom in sugar radicals is established: two species are found to have the same chemical composition but different conformations and consequently different hyperfine coupling tensors. Analysis of the calculated energies for all model compounds suggests that the radiation chemistry of sugars, in general, is kinetically and not necessarily thermodynamically controlled.     

An EPR, ENDOR and EIE study of gamma-irradiated poly (lactide-co-glycolide) polymers

Gamma radiation of poly (lactide-co-glycolide) raw polymers and processed microspheres under vacuum and at 77 K results in the formation of a series of free radicals. The resulting powder electron paramagnetic resonance (EPR) spectrum contains a distribution of several different radicals, depending on the annealing temperature, and is therefore difficult to interpret. By utilising the selectivity of the electron nuclear DOuble resonance (ENDOR) and associated ENDOR induced EPR (EIE) techniques, a more direct approach for the deconvolution of the EPR spectrum can be achieved. Using this approach, the radiolytically induced CH3 *CHC(O)R- chain scission radical was identified at 120 K by simulation of the EIE spectrum. At elevated temperatures (250 K), this radical decays considerably and the more stable radicals -O*CHC(O)-, CH3 *C(OR)C(O)- and CH3 *C(OH)C(O)- predominate. This work demonstrates the utility of the EIE approach to supplement and aid the interpretation of powder EPR spectra of radicals in a polymer matrix.     

EPR, ENDOR, and DFT study of free radicals in L-lysine·HCl·2H2O single crystals X-irradiated at 298 K

With K-band EPR (Electron Paramagnetic Resonance), ENDOR (Electron-Nuclear DOuble Resonance), and EIE (ENDOR-induced EPR) techniques, three free radicals (RI-RIII) in L-lysine hydrochloride dihydrate single crystals X-irradiated at 298 K were detected at 298 K, and six radicals (R1, R1', R2-R5) were detected if the temperature was lowered to 66 K from 298 K. R1 and RI dominated the central portion of the EPR at 66 and 298 K, respectively, and were identified as main chain deamination radicals, (-)OOC?H(CH(2))(4)(NH(3))(+). R1' was identified as a main chain deamination radical with the different configuration from R1 at 66 K, and it probably formed during cooling the temperature from 298 to 66 K. The configurations of R1, R1', and RI were analyzed with their coupling tensors. R2 and R3 each contain one α- and four β-proton couplings and have very similar EIEs at three crystallographic axes. The two-layer ONIOM calculations (at B3LYP/6-31G(d,p):PM3) support that R2 and R3 are from different radicals: dehydrogenation at C4, (-)OOCCH(NH(3))(+)CH(2)?H(CH(2))(2)(NH(3))(+), and dehydrogenation at C5, (-)OOCCH(NH(3))(+)(CH(2))(2)?HCH(2)(NH(3))(+), respectively. The comparisons of the coupling tensors indicated that R2 (66 K) is the same radical as RII (298 K), and R3 is the same as RIII. Thus, RII and RIII also are the radicals of C4 and C5 dehydrogenation. R4 and R5 are minority radicals and were observed only when temperature was lowered to 66 K. R4 and R5 were only tentatively assigned as the side chain deamination radical, (-)OOCCH (NH(3))(+)(CH(2))(3)?H(2), and the radical dehydrogenation at C3, (-)OOCCH(NH(3))(+)?H(CH(2))(3)(NH(3))(+), respectively, although the evidence was indirect. From simulation of the EPR (B//a, 66 K), the concentrations of R1, R1', and R2-R5 were estimated as: R1, 50%; R1', 11%; R2, 14%; R3, 16%; R4, 6%; R5, 3%.     

NH3 and O2 interaction with tetrahedral Ti3+ ions isomorphously substituted in the framework of TiAlPO-5. A combined pulse EPR, pulse ENDOR, UV-Vis and FT-IR study

Continuous Wave (CW), pulse Electron Paramagnetic Resonance (EPR) and pulse Electron Nuclear Double Resonance (ENDOR) spectroscopies, in conjunction with UV-Vis and Infrared (IR) spectroscopies, are used to investigate the chemical reactivity of tetrahedrally coordinated Ti(3+) ions isomorphously substituted in the framework of AlPO-5 towards NH(3) and O(2). The coordination of ammonia to Ti(3+) centres is followed in detail by complementary vibrational and electron magnetic resonance techniques. In particular HYSCORE spectra allow identifying the coordination of two ammonia molecules to Ti(3+) centres resolving the full hyperfine and quadrupole (14)N coupling tensors. The reactivity of the reduced TiAlPO sample towards molecular oxygen is detailed by means of CW-EPR and pulse ENDOR spectroscopy. (17)O(2) is employed, allowing to establish the formation of a "side-on" η(2) O(2)(-)-Ti(4+) electrostatic complex. Pulse ENDOR spectra provide detailed information on the local environment of the formed superoxide radical anion which acts as a paramagnetic probe, providing evidence for Ti-O-Ti oligomeric species.     

Decomposition study of the electron paramagnetic resonance spectrum of irradiated alanine

Recent Electron Paramagnetic Resonance (EPR) studies on alanine powders as a function of irradiation dose and temperature on the one hand and single crystal Electron Nuclear DOuble Resonance (ENDOR) studies on the other hand, showed the presence of at least three radicals contributing to the total alanine EPR spectrum. The latter spectrum obtained after irradiation at room temperature (RT), is dominated by the well-known stable-alanine-radical (SAR) CH3C*HCOO-, also denoted R1. Appropriate heating of irradiated alanine causes the relative contribution of R1 to decrease, resulting in a spectrum mainly caused by the H-abstraction radical CH3C*(NH3)COO-, denoted R2. Although the EPR spectrum of these two radicals could be satisfactorily simulated, their influence on dose reconstruction has not been reported yet. Therefore, a detailed Maximum Likelihood Common Factor Analysis (MLCFA) study has been performed on EPR spectra from polycrystalline alanine samples, after irradiation and heat treatments. Conclusions concerning the number of contributing radicals and their influence on the RT irradiated alanine EPR spectrum will be made.     

EPR and ENDOR study of radiation-induced radical formation in purines: sodium inosine crystals X-irradiated at 10 K

X-irradiated single crystals of sodium inosine (Na(+)*Inosine(-)*2.5H(2)O), in which the hypoxanthine base is present as the N1-deprotonated anion, were investigated using K-band (24 GHz) electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and ENDOR induced EPR (EIE) techniques at 10 K. At least five different radicals were present immediately after irradiation at 10 K. R1, which decayed upon warming the crystals to 50 K, was identified as the electron-loss product of the parent N1-deprotonated hypoxanthine base. Hyperfine couplings to HC8 and HC2 were fully characterized with ENDOR spectroscopy, and the identification was supported by DFT calculations. R2, which also decayed on warming to 50 K, exhibited nearly equal couplings to HC2 and HC8. Taken in combination with an extensive set of DFT calculations, the experimental results indicate that R2 is the (doubly negative) product of electron-gain by the initially anionic N1-deprotonated hypoxanthine parent. R3, which exhibited hyperfine coupling only to HC8 could not be identified. R4, which persisted on annealing to 260 K, exhibited one large alpha-proton hyperfine coupling which was fully characterized by ENDOR. Based on DFT calculations and the experimental data, R4 was identified as the product of net H-abstraction from C5'. The remaining HC5' was the source of the measured alpha-proton coupling. R5, present at low temperature and the only observable radical after warming the crystals to room temperature, was identified as the C8-H addition radical. The alpha-coupling to HC2 and beta-couplings to the pair of C8 methlyene protons were fully characterized by ENDOR.     

Radiation-induced radicals in alpha-D-glucose: Comparing DFT cluster calculations with magnetic resonance experiments

Using density functional theory (DFT) calculations, an enhanced theoretical examination was made of the radiation-induced radicals in alpha-d-glucose. For the carbon-centred radicals in this sugar, the effect of the model space on the radical geometry as well as on the calculated radical hyperfine coupling tensors was examined. The findings were compared with previously published tensors, as determined by electron paramagnetic resonance (EPR) experiments and single molecule DFT calculations. A cluster approach was adopted, in which intermolecular interactions (predominantly hydrogen bonds) between the radical species and its environment were explicitly incorporated. This substantially improved the correspondence with experimental findings in comparison with single molecule calculations of an earlier examination. In a direct comparison between both computational methods for the glucose radicals, it was shown that the extent of the model space plays an important part in the determination of the radical geometry. Furthermore, the model space also has an impact on the calculated hyperfine coupling tensors. Full cluster EPR calculations, in which the paramagnetic properties are calculated for the entire model space of the cluster, give an excellent agreement with the experimental EPR measurements.     

Model Calculations of Radiation-Induced Damage in 1-Methyluracil:9-Ethyladenine

Detailed EPR and ENDOR experiments on the cocrystalline complex of 1-methyluracil:9-Ethyladenine (MUEA) have revealed that the major radiation-induced products observed at 10 K on MU are: MUEA1, a radical formed by net hydrogen abstraction from the N1-CH₃ methyl group, MUEA2, the MU radical anion, and MUEA3, the C5 H-addition radical. The following four products were observed on the adenine moiety at 10 K, MUEA4, the N3 protonated adenine anion, MUEA5, the native adenine cation, MUEA6, the amino deprotonated adenine cation, and MUEA7, the C8 H-addition radical formed by net H-addition to C8 of the adenine base. The geometries, energetics, and hyperfine properties of all possible radicals of MU and EA, the native anions and cations, as well as radicals formed via net hydrogen atom abstraction (deprotonated cations) or addition (protonated anions) were investigated theoretically. All systems were optimized using the hybrid Hartree–Fock–density functional theory functional B3LYP, in conjunction with the 6-31G(d,p) basis set of Pople and co-workers. Calculations of the anisotropic hyperfine couplings for all the radicals observed in MUEA are presented and are shown to compare favorably with the experimentally measured hyperfine couplings. The calculated ionizations potentials indicate that EA would be the preferred oxidation site. In MUEA, both the adenine cation and its N4-deprotonated derivative were observed. The calculated electron affinities indicate that MU would be the preferred reduction site. In MUEA radical, MUEA2 is a uracil reduction product, however the protonation state of this radical could not be determined experimentally. Calculations suggest that MUEA2 is actually the C4=O protonated anion.     

Structure of radicals from X-irradiated guanine derivatives: an experimental and computational study of sodium guanosine dihydrate single crystals

In sodium guanosine dihydrate single crystals, the guanine moiety is deprotonated at N1 due to growth from high-pH (>12) solutions. Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) studies of crystals X-irradiated at 10 K detected evidence for three radical forms. Radical R1, characterized by two proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the N1-deprotonated guanine unit. R1 exhibited an unusually distorted structure leading to net positive isotropic components of the hydrogen alpha-couplings. Radical R2, characterized by one proton and one nitrogen hyperfine coupling, was identified as the primary electron-loss product. This product is equivalent to that of deprotonation at N1 by the guanine cation and represents the first ENDOR characterization of that product. Radical R3, characterized by a single hydrogen hyperfine coupling, was identified as the product of net dehydrogenation at C1' of the ribose moiety. The identification of radicals R1-R3 was supported by density functional theory (DFT) calculations on several possible structures using the B3LYP/6-311G(2df,p)//6-31G(d,p) approach. Radical R4, detected after warming the crystals to room temperature, was identified as the well-known product of net hydrogenation of C8 of the (N1-deprotonated) guanine component. Radical R1, evidently formed by protonation of the primary electron addition product, was present as roughly 60% of the total radicals detected at 10 K. Radical R2 was present as roughly 27% of the total yield, and the concentration of R3 contributed the remaining 13%. R3 is evidently the product of one-electron oxidation followed by deprotonation; thus, the balance of oxidation and reduction products is approximately equal within experimental uncertainty.     

Structure of radicals from X-irradiated guanine derivatives. 2. An experimental and computational study of 9-ethylguanine single crystals

An EPR (electron paramagnetic resonance) and ENDOR (electron-nuclear double resonance) study of 9-ethylguanine crystals X-irradiated at 10 K detected evidence for three radical forms. Radical R1, characterized by three proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the guanine unit. R1, which evidently formed by protonation of the primary electron addition product, exhibited an unusually distorted structure leading to net positive isotropic components of the alpha-coupling to the hydrogen attached to C8 of the guanine unit. Radical R2, characterized by two nitrogen and three proton hyperfine couplings, was identified as the primary electron loss product, *G+. Distinguishing between *G+ and its N1-deprotonated product is difficult because their couplings are so similar, and density functional theory (DFT) calculations were indispensable for doing so. The results for R2 provide the most complete ENDOR characterization of *G+ presented so far. Radical R3 exhibited a narrow EPR pattern but could not be identified. The identification of radicals R1 and R2 was supported by DFT calculations using the B3LYP/6-311+G(2df,p)//6-31+G(d,p) approach. Radical R4, detected after irradiation of the crystals at room temperature, was identified as the well-known product of net hydrogenation at C8 of the guanine component. Spectra from the room temperature irradiation contained evidence for R5, an additional radical that could not be identified. Radical concentrations from the low temperature irradiation were estimated as follows: R1, 20%; R2, 65%; R3, 15%.     

Radiation-induced radicals in glucose-1-phosphate. I. Electron paramagnetic resonance and electron nuclear double resonance analysis of in situ X-irradiated single crystals at 77 K

Electron magnetic resonance analysis of radiation-induced defects in dipotassium glucose-1-phosphate dihydrate single crystals in situ X-irradiated and measured at 77 K shows that at least seven different carbon-centered radical species are trapped. Four of these (R1-R4) can be fully or partly characterized in terms of proton hyperfine coupling tensors. The dominant radical (R2) is identified as a C1-centered species, assumedly formed by a scission of the sugar-phosphate junction and the concerted formation of a carbonyl group at the neighboring C2 carbon. This structure is chemically identical to a radical recently identified in irradiated sucrose single crystals. Radical species R1 and R4 most likely are C3- and C6-centered species, respectively, both formed by a net hydrogen abstraction. R3 is suggested to be chemically similar to but geometrically different from R4. Knowledge of the identity of the sugar radicals present at 77 K provides a first step in elucidating the formation mechanism of the phosphoryl radicals previously detected after X-irradiation at 280 K. In paper II, the chemical identity, precise conformation, and possible formation mechanisms of these radical species are investigated by means of DFT calculations and elementary insight into the radiation chemistry of sugar and sugar derivatives is obtained.     

Free radical products in X-irradiated Rochelle salt: low temperature ENDOR and DFT studies

Single crystals of Rochelle salt, [⁻OOC-CHOH-CHOH-COO⁻, Na⁺, K⁺]·4H₂O, X-irradiated at 10 K, have been examined using EPR, ENDOR and EIE spectroscopic techniques to characterize the radiation induced radicals stable at that temperature and their reactions upon warming. The one-electron gain product was observed and from the hyperfine interaction with a β-proton it was unambiguously centered at the C₄ position of the tartrate moiety. An additional nearly isotropic hyperfine structure of about 21 MHz was tentatively assigned to interaction with a sodium ion exhibiting a close contact to O₃ in the crystal. Evidence was obtained that the one-electron reduced radical had become protonated at one of the C₄ bonded carboxyl oxygens, most probably O₄. No evidence for the corresponding C₁-centered reduction product was found. Two resonance lines (R2, A1) were shown by EIE to belong to a species formed by decarboxylation at C₃, a secondary oxidation product. Two other resonance lines (K1, K2) were assigned to two varieties of another decarboxylation radical, centered at C₂, distinguished by differences in the potassium ion coordination. Furthermore, one other resonance line (A2) was tentatively ascribed to a third decarboxylation radical, centered at the opposite end of the tartrate moiety. The precursor of these products, that is, the one-electron loss product, was not observed after X-irradiation at 10 K. Thermally induced free radical reactions followed by EPR in the temperature range of 12-119 K indicate that a water molecule or a hydroxyl ion is eliminated from the one-electron reduction product radical and that a C₃-centered radical is formed. The reduction and oxidation reaction pathways of hydroxy acid derivatives are discussed.     

Free radical conformations and conversions in X-irradiated single crystals of L-cysteic acid by electron magnetic resonance and density functional theory studies

Single crystals of L-cysteic acid monohydrate were X-irradiated and studied at 295 K using EPR, ENDOR, and EIE techniques. Three spectroscopically different radicals were observed. These were a deamination radical reduction product (R1), and two oxidation products formed by hydrogen abstraction (radicals R2, R3). R2 and R3 were shown to exhibit the same chemical structure while exhibiting very different geometrical conformations. Cluster DFT calculations at the 6-31G(d,p) level of theory supported the experimental observations for radicals R1 and R2. It was not possible to simulate the R3 radical in any attempted cluster; hence, for this purpose a single molecule approach was used. The precursor radicals for R1, R2, and R3, identified in the low-temperature work on L-cysteic acid monohydrate by Box and Budzinski, were also investigated using DFT calculations. The experimentally determined EPR parameters for the low-temperature decarboxylated cation could only be reproduced correctly within the cluster when the carboxyl group remained in the proximity of the radical. Only one of the two observed low-temperature carboxyl anions (stable at 4 and 48 K) could be successfully simulated by the DFT calculations. Evidence is presented in support of the conclusions that the carboxyl reduction product already is protonated at 4 K and that the irreversible conversion between the two reduction products is brought forward by an umbrella-type inversion of the carboxyl group.     

EPR and ENDOR study of radiation-induced radical formation in purines: hypoxanthine hydrochloride monohydrate crystals X-irradiated at 10 K

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) study of hypoxanthine.HCl.H(2)O crystals irradiated at low temperatures (10 K) identified three radical species. In these crystals, the parent molecules exist in a cationic form with a proton at N7. R1 was the product of net hydrogen addition to N3 and exhibited alpha-proton hyperfine couplings to HC2, HN1, HC8, and HN3. The coupling to HC2 has an isotropic component smaller than usual, evidently an indication that the bonds to C2 are nonplanar. R2 was the product of net hydrogen loss from N7, equivalent to the one-electron oxidation product of neutral hypoxanthine, and exhibited alpha-proton hyperfine couplings to HC2 and HC8. Both couplings are characteristic of planar bonding arrangements at the centers of spin. R3 was provisionally identified as the product of net hydrogen addition to O6 and exhibited hyperfine alpha-proton couplings to HC8 and NH1. To identify the set of radicals, the experiments employed four crystal types: normal, deuterated only at NH positions, deuterated at HC8 and NH positions, and deuterated at HC8 only. The low-temperature data also showed clear evidence for H/D isotope effects in formation and/or stabilization of all radicals. To aid and support the identifications, the experimental results were compared to DFT calculations performed on a variety of radical structures plausible for the parent molecule and molecular packing within the crystal.     

Single crystals of L-O-serine phosphate X-irradiated at low temperatures: EPR, ENDOR, EIE, and DFT studies

Single crystals of the phosphorylated amino acid L-O-serine phosphate were X-irradiated and studied at 10 K and at 77 K using EPR, ENDOR, and EIE techniques. Two radicals, R1(10 K) and R1(77 K), were detected and characterized as two different geometrical conformations of the protonated reduction product >CH-C(OH)(2). R1(10 K) is only observed after irradiation at 10 K, and upon heating to 40 K, R1(10 K) transforms rapidly and irreversibly into R1(77 K). The transition from R1(10 K) to R1(77 K) strongly increases the isotropic hyperfine coupling of the C-CH(beta) coupling (Delta = 32 MHz) and the major C-OH(beta) coupling (Delta = 47 MHz), in sharp contrast to the their much reduced anisotropic hyperfine couplings after the transition. An umbrella-like inversion of the carboxylic acid center, accompanied by minor geometrical adjustments, explains the changes of these observed isotropic and anisotropic couplings. DFT calculations were done on the reduced and protonated L-O-serine phosphate radical at the B3LYP/6-311+G(2df,p)//B3LYP/6-31+G(d) level of theory in order to support the experimental observations. Two different conformations of the anion radical, related by an inversion at the carboxylic center, could be found within the single molecule partial energy-optimization scheme. These two conformations reproduce the experimental hyperfine couplings from radicals R1(10 K) and R1(77 K). A third radical, radical R2, was observed experimentally at both 10 and 77 K and was shown to be due to the decarboxylated L-O-serine phosphate oxidation product, a conclusion fully supported from the DFT calculations. Upon thermal annealing from 77 to 295 K, radicals R1(77 K) and R2 disappeared and all three previously observed room-temperature radicals could be observed. No phosphate-centered radicals could be observed at any temperatures, indicating that the phosphate-ester bond break for one of the room-temperature radicals does not occur by dissociative electron capture at the phosphate group.