2016
The Journal of Physical Chemistty, Vol. 82, No. 18, 1978
W. P. Unruh, T. Gedayloo, and J. D. Zimbrick
ENDOR Measurements of Proton Tunneling in 5-Thymyl Radicals in Acidic Glasses Wesley P. Unruh,+ Teymoor Gedayloo,' and John
D. Zlmbrlck"
Departments of Physics and Radiation Biophyslcs, Universlty of Kansas, Lawrence, Kansas 66045, and Department of Physics, California State Polytechnic University, San Luis Obispo, California 9340 1 (Received March 14, 1977; Revised Manuscript Received June 12, 1978) Publication costs assisted by the University of Kansas and the Natlonal Institute of General Medical Sciences
Radicals formed by hydrogen atom attack on thymine (5-thymylradicals) in acidic glasses at low temperatures have been investigated by EPR and ENDOR spectroscopy, and comparisons made with the same radicals formed in single crystal thymidine. In both environmentsthe 77 K EPR spectrum consists of the conventional eight-line 5-thymyl radical (5T.) resonance. At -40 K, the EPR spectrum of 5T. changes as additional lines appear due to proton tunneling of the three CB-methylprotons. EPR and ENDOR measurements at 4.2 and 1.2 K show no temperature dependence in this additional structure. Measurements of the frequency shifts of the methyl proton ENDOR frequencies were used to determine the proton tunneling frequencies, by application of the tunneling theory in the literature. Our results show the tunneling of the (&-methylprotons to be -880 MHz in the acid glass matrix, and that it is reduced to -100 MHz in the single crystal environment. No temperature dependence was observed in the range from 4.2 to 1.2 K. A search for EPR side bands due to the tunneling frequency was made in the glass matrix, but none were detected.
I. Introduction In recent years, many EPR studies have been reported on the nature and identity of free radicals produced in DNA and its constituent molecules by ionizing radiation. Detailed summaries of the principal results can be found in recent review papers on the ~ubject.l-~ Electron nuclear double resonance (ENDOR)4is a technique which can be combined with EPR to provide unique spectral information for cases in which EPR spectra are complex, and spectral lines are inhomogeneously broadened and unresolved. ENDOR spectroscopy measures the resonance frequencies of those nuclei interacting relatively strongly with the unpaired spin responsible for the EPR spectrum. This is accomplished by saturating an EPR transition and determining the radiofrequency at which it is partially unsaturated by resonance of the nuclei in its environment. ENDOR has been used to study a variety of organic free radicals in liquid, glass, polycrystalline, and single-crystal phases.6-1s Some of these studies are particularly concerned with the application of ENDOR to the investigation of methyl proton tunneling13-16in several types of free radicals, including 5T- in single-crystalthymidine.17 Thus far, no reports of ENDOR studies on free radicals in DNA or its constituent bases in low temperature glasses have appeared in the literature. In a preliminary reportlg we presented the results of an initial study on the EPR and ENDOR spectra of 5,6-dihydrothymine-5-~1(5T.) radicals in acid glass at 77 and 4.2 K. The EPR spectrum at 4.2 K appeared to be composed of many more lines than were present at 77 K, but we were unable to present conclusive ENDOR spectra. Here we report the results of an EPR and ENDOR study at 77,4.2 and 1.2 K on the 5T- radical formed in acid glass, and comparisons with 5-thymyl radicals formed in single-crystal thymidine. This kind of study allows a direct determination of matrix effects (ordered nonaqueous vs. nonordered aqueous) on the paramagnetic properties of 5T- and provides information which may be important in interpreting EPR and ENDOR data on 5T. in DNA, as suggested by Box et al.17 +Departmentof Physics, University of Kansas. Department of Physics, California State Polytechnic University. * Department of Radiation Biophysics, University of Kansas. Author to whom correspondence should be addressed. 0022-3654/78/2082-2010$01 .OO/O
Freedz0 has shown that the EPR structure of free radicals trapped near a methyl group is modified by splittings between combined spin and torsional oscillator states, and that one can expect to see additional temperature-dependent EPR structure due to rotation of the CH, between its three equivalent positions around the bond axis of the carbon. This phenomenon has been extensively investigated by Clough and his collaborators, who have used a variety of techniques to measure the tunneling frequencies of methyl groups in several different environments. Of particular interest to us is their calculation of the effect of this motion on the ENDOR spectrum of the methyl protons in the fragment .C-CH3. These calculations are based upon a reprehentation of the torsional splittings of methyl proton spin states in terms of an effective exchange interaction between the methyl protons. Such an approach13-16allows one to use perturbation calculations to obtain ENDOR frequencies both for the case where the tunneling frequencies are higher than the hyperfine interaction frequency, and lower than the hyperfine interaction frequency of the proton-free radical interaction. Recently Box et a1.I' have calculated the ENDOR frequencies to be expected for the specific case of the 5T. radical in a single-crystal matrix, including in the treatment the methylene protons as well. Their calculated EPR and ENDOR frequencies are obtained from a perturbation expansion which is valid for the case in which the tunneling barrier is larger than the hyperfine energy. Except for the more complex notation and EPR level structure (due to the specific inclusion of the methylene protons in the calculation), these calculations are comparable to those done earlier by Clough and Poldy14 and essentially the same approximations and representation of rotational barrier potentials are used in both treatments. We prefer to interpret our results with the theory of Clough and Poldy14because it deals with only the effects of rotating methyl protons, and can be easily generalized to cases where additional static interactions are present (and thus is not restricted to the specific consideration of thymidine). This treatment is also of importance because it shows that the signs of the ENDOR frequency shifts can be used to determine unambiguously whether the tunneling frequencies are higher than or lower than the hy0 1978 American Chemical Society
5-Thymyl Radicals in Acidic Glasses
perfine interaction frequencies of the methyl protons.16 Qualitatively, one expects to see two quite distinctive characteristics in the ENDOR response of rapidly tunneling methyl p r ~ t o n s : ~(a) ~ Jthe ~ ENDOR frequencies obtained from the lowest and highest EPR line will be shifted by more than just the corresponding proton resonance frequency shift due to the magnetic field shift, and (b) ENDOR responses obtained from the second EPR lines at each end of the spectrum are at quite a different frequency because they involve spatial functions of quite different symmetry. Other distinctive spectral shifts are seen, as well, and would be quite useful in the investigation of single crystal spectra. These effects must be averaged over all possible angles 6' when one interprets the ENDOR spectra obtained from acid glass samples. Thus the ENDOR responses have the characteristic shape of powder spectra. Fortunately, the hyperfine tensors for both the methyl protons and the methylene protons are dominated by the contact interaction, making them nearly isotropic and leading to ENDOR spectra which are only a few megaHertz wide. 11. Materials and Methods Reagent-grade H,S04 and polycrystalline thymine from Sigma Chemical Co. were used without further purification. Solutions were prepared from singly distilled water which was further purified by all-glass triple distillation over alkaline permanganate and acid dichromate. Solutions which were 120 mM thymine and 9 M H2S04 were pipetted into 2-mm diameter A1 foil molds and immediately quenched in liquid nitrogen to form clear glass cylinders, after which the A1 foils were removed. Under these conditions, no observable chemical reactions occurred between the acid solution and the foils. The glasses were irradiated at 77 K with 6oCoy rays in an Atomic Energy of Canada, Ltd. Model 200 Gamma Cell which provided a dose rate of 43.3 krd/h as calibrated by ferrous sulfate dosimetry. After irradiation, the samples were annealed to a temperature of 171 K and held at that temperature for 2 min in a variable temperature dewar before being recooled to 77 K. This annealing procedure has been shown21to result in (a) mobilization of hydrogen atoms (Ha) which have been produced by irradiation and trapped in the matrix at 77 K; (b) reaction of the H. with thymine molecules in the matrix to form 5-thymyl radicals; and (c) recombination of SO4-. radical anions so that their EPR spectrum does not interfere with that of the 5-thymyl radical spectrum. The annealed samples were stored in darkness at 77 K until they were needed for analysis. In the case of these acid glass samples, approximately 8-mm long fragments were cut from an annealed sample and were mounted in polyfoam inside the ENDOR cavity. This entire operation was carried out under liquid nitrogen. The cavity was then attached to the waveguide (also under liquid nitrogen) and placed in the experimental dewar without appreciable warmup. Single crystals of thymidine were grown from saturated aqueous solution. Polycrystalline thymidine was dissolved in triply distilled water at 50 "C and allowed to crystallize in a beaker while the solution cooled at a rate of -20 OC/h. The resulting tetragonal crystals were needlelike (typically 1 X 1 X 6 mm), with the c axis parallel to the long dimension. Irradiation at 300 K produced essentially no 5T. radicals, whereas irradiation at 77 K did produce these radicals, but at a much lower yield than for the acid glass samples. In contrast, the 5T. radicals, once formed at 77 K, were quite stable at 300 K. Samples stored (after radical formation) at 300 K in the dark showed no de-
The Journal of Physical Chemistry, Vol. 82, No. 18, 1978 2017
tectable signal decrease after several months. There is some evidence that the 5T. radicals can be bleached by W light, but this aspect of their structure was not pursued. Crystals used in this study were irradiated a t 77 K in the 6oCogamma cell facility of the Solid State Division, Oak Ridge National Laboratory. The total dose was 42 X lo6 rd, accumulated at a rate of -6 X lo6 rd/h. After removal from the gamma cell and warming to 300 K, they were red in color, and maintained both this color and their EPR signals indefinitely. Both the single crystal and acid glass samples showed appreciable microwave loss, even at 4.2 K, and especially with the single crystal samples size had to be restricted so that the microwave Q of the ENDOR cavity was not degraded. The EPR-ENDOR spectrometer used in this study is described e l ~ e w h e r e . ~The ~ , ~frequency ~ sweep of the radiofrequency generator which provides the nuclear resonance field is controlled by a signal averager, and the resulting signals are stored digitally. Fast transient ENDOR is obtained by sweeping the radiofrequency rapidly and directly averaging the output of the superheterodyne EPR spectrometer (without demodulation of a steady-state low frequency signal). This method minimizes the effects of nuclear relaxation, since one detects only initial electronic populations of the various hyperfine level pairs rather than the steady-state populations obtained while circulating spins through all the levels. This arrangement allows great flexibility in the frequency range and sweep rates used in the experiment, and also provides the opportunity to subtract the responses seen on a particular EPR hyperfine line from those seen on the adjacent lines in order to find those unique to a particular EPR line. The microwave frequency was -9.4 GHz.
111. Results A. EPR Spectra. A typical set of EPR spectra of an irradiated annealed glass at 77 K is shown in Figures 1A-C. These three spectra show the changes which occur as the microwave power and field modulation amplitude are increased. It can be seen in Figure 1A that the spectrum taken at very low power and modulation amplitude consists principally of the well-knownNeight-line spectrum of total width -140 G and splitting -20 G attributed to 5T.. In addition, a series of weak resonances between the lines of the 5T. spectrum can be seen. These weak resonances are detectable only at extremely low microwave power ( H J and modulation amplitude (H,)settings. Figures 1B and 1C show the range of Hl and H, values within which the weak resonance disappears from the spectrum. The values of H, and H, for the spectrum of Figure 1C in which the weak resonance has disappeared completely are comparatively low (Hl= 50 pW, H, = 8 G). They are typical of those used in previous published studies on 5T. radicals and may explain why the weak resonances are not visible in published spectra of 5T. radicals. Figure 2 presents an absorption spectrum at 77 K (spectrum A) and an absorption spectrum at 1.2 K (spectrum B) taken from the same glass sample. These spectra show marked changes in the number of spectral lines and in the relative intensities of these lines as a function of temperature. The additional splittings which develop at low temperatures are characteristic of the effects of methyl group rotational splittings of the EPR levels.13J4J7Because the g tensor of the 5T. radical is isotropic the EPR spectra look essentially identical in the glass and in single crystals (as can be seen by comparing Figure 2 with the spectrum shown in ref 17). A c-axis first de-
2018
The Journal of Physical Chemistry, Vol. 82, No. 18, 1978
W. P. Unruh, T. Gedayloo, and J. D. Zimbrlck
A 6 7 8
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Flgure 1. First derivative EPR spectra of 5-thymyl radicals in acidic glass at 77 K ('OCo y dose = 0.6 Mrd, Y = 9.2 GHz): spectrum A, microwave power = 0.05pW, H, = 0.5 G; spectrum B, microwave power = 0.05 mW, H, = 0.5 G; spectrum C, microwave power = 0.05 mW, H, = 8 G.
Figure 2. Variation of EPR spectra of 5-thymyi radicals with sample y dose = 0.6 Mrd, Y = 9.2 GHz): spectrum A, integral temperature absorption at 77 K, microwave power 0.1 mW; spectrum B, integral absorption at 1.2 K, microwave power N 1 nW.
rivative EPR spectrum obtained from one of our single crystal samples is shown in Figure 3, where it is compared with the first derivative spectrum of the glass sample used to obtain the spectra in Figure 2. There is clearly another four-line radical overlapping the center of the 5T. spectrum in both cases; however the work reported here involves only the outer lines of the spectrum which are attributable to 5T.. For reference purposes the EPR lines on either end are numbered to agree with the nomenclature used in ref 25 in describing the methyl proton splittings. Both the glass and single-crystal samples could be cycled in temperature between 77 and 1.2 K without any detectable permanent alteration to either spectrum. The additional structure due to the methyl proton rotation
Flgure 3. Comparison of first derivative EPR spectra of 5-thymyl radicals in glassy and single crystal matrices: spectrum A, acidic glass at 4.2 K; spectrum B, single-crystal c-axis spectrum at 4.2 K. The transition numbering corresponds to the convention used in ref 25. Line positions shown in Figure 8 are included for comparison.
disappears rather suddenly as the temperature is raised, in agreement with observations made in a different radical system.14 An approximate measurement of this transition temperature was made in the single-crystal case (the slowest tunneling frequency) by recording EPR derivative shapes as the sample warmed from 4.2 to 77 K. We observed essentially complete disappearance of the tunneling structure within a 10 K range centered at -30 K. In the temperature range from 4.2 to 1.2 K the only spectral changes seen are due to small changes in line width and spin-lattice relaxation times, in both the glass and single-crystal samples. The total EPR splitting in the glass at low temperatures is 140 G, with a splitting of 10 G between adjacent extrema1 lines. Although there is appreciable overlap, the spectrum can be seen to be the result of the characteristic seven-line tunneling methyl spectrum13*14 split further by the 1:2:1 spectrum of the two very nearly equivalent methylene protons. Since the total methyl proton splitting is thus -60 G, this leaves an average methylene splitting of -40 G, in agreement with estimates obtained from the high-temperature eight-line composite spectrum of 5thymyl radicals.24These results are, of course, quite close to those obtained from the single-crystal spectrum. It is interesting to compare the low-power 77 K spectrum (Figure 1A) with the low-temperature spectrum in Figure 3A. The residual structure seen at 77 K matches quite well the line splittings seen at low temperatures. It may possibly be due to a small number of radicals which have local environments in which the rotational barriers are so high that full rotational averaging is not achieved even at 77 K. B. ENDOR Spectra. Strong ENDOR signals were observed from each of the EPR lines of 5T.in acid glass throughout the temperature range from 4.2 to 1.2 K. No ENDOR signals were detected at 77 K. Since the EPR of 5T. is isotropic, the ENDOR signals consist of a powder average over all orientations, and are broadened. Figures 4A and 4B show survey traces taken from 10 to 100 MHz on the extreme low-field EPR line and its adjacent neighbor, respectively. Clear differences are seen in the responses below 80 MHz. No other high-frequency responses are present in any spectra taken on any of the nominally resolved EPR lines. Ordinarily, one expects to see proton ENDOR responses occurring in pairs. If the relaxation mechanisms among the various saturated spin levels are reasonable, one sees
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The Journal of Physical Chemistry, Vol. 82, No, 18, 1978 2019
5-Thymyl Radicals in Acidic Glasses vn
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Figure 4. ENDOR signals from 5-thymyl radicals in acid glass at 1.2 K: spectrum A, taken from the extreme low field EPR line, (1) of Figure 3; spectrum B, taken on the adjacent EPR line, (2) of Figure 3; Y, is the proton resonance frequency at the field used (-3300 G). (The rapid sweep rate used to achieve good signalhoise disturbs the spin populations in a way which leads to slightly altered relative line intensities. Undlstorted ENDOR line shapes are to be found in succeeding figures.)
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MHz Flgure 6. Subtracted ENDOR responses of 5-thymyl radicals in acid glass at 1.2 K, for the full frequency range from 1 to 75 MHz: spectrum A is the difference spectrum obtalned from subtractlng ENDOR responses from EPR lines 1 and 2; spectrum B is obtained from subtracting ENDOR responses from EPR lines 8 and 7. Y, is the proton resonance frequency in each case. The arrows locate the corresponding narrow transitions obtained from the methyl protons in the c-axis single crystal ENDOR spectrum.
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MHz Figure 5. ENDOR responses between 20 and 90 MHz from 5-thymyl radicals in acid glass: spectrum A was taken on EPR line (1) of Figure 3; spectrum B on line (2); spectrum C is the difference (A - B) and shows just the ENDOR transitions at 42 and 67 MHz due to the tunneling methyl protons.
the nuclear transitions between states coupled to both the M , = + 1 / 2 and M, = -1/2 electronic levels. For hyperfine interactions, A , smaller than the free proton nuclear resonance frequency, v,, one should see (in first order) two lines at Y, f A / 2 . For large hyperfine interactions ( A / 2 > v,), the lines should be found at A / 2 f Y,. This latter case is the one which applies to the ENDOR responses to be expected from both the methylene and methyl protons in 5T. radicals. In addition, one generally sees a "distant ENDOR" response at v, due to protons essentially uncoupled from the radical spin, and this frequency provides a valuable check on the field calibration, and on the field shifts between the various EPR lines. The intensities of these various ENDOR responses, which are dependent on the various possible electronic and nuclear relaxation mechanisms, are often unexpectedly low, or even absent. In practice, one often finds the high frequency (Ma= -l/z) ENDOR transitions to be much more intense than those at low frequency ( M , = +1/2), especially if the lower line is below 15 MHz. This can be seen in the glass spectra discussed below. In Figure 5 one sees ENDOR responses obtained from the extremal EPR lines between 20 and 90 MHz. Traces A, B, and C, respectively, are obtained from the low-field EPR line (l),its neighbor ( 2 ) , and the difference between the two responses (A - B). Traces A and B show two broad peaks at -40-50 and -70-80 MHz common to both EPR
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MHz Flgure 7. ENDOR spectrum between 20 and 90 MHz taken on lines 2 and 7 (Figure 3) of the EPR spectrum of 5-thymyl radicals in acid glass at 1.2 K. The broad methylene proton response is common to both spectra, as is the single methyl proton response at -68 MHz. The arrows locate the corresponding narrow transitions obtained from the methylene protons in the c-axis single crystal ENDOR spectrum.
lines. There is an additional peak at -67 MHz in B. Both broad peaks completely subtract out in trace C. This difference spectrum clearly shows that the extremal line gives a transition at -45 MHz and its neighbor yields a different transition (inverted because of the subtraction) at -67 MHz. The ENDOR responses to be expected from the methylene protons are common to all EPR lines, and the two broad responses which subtract out are those due to just the two methylene protons at the C6position in the molecule. The entire frequency range from 1to 75 MHz is shown in the two subtracted traces of Figure 6. These traces contain only the methyl proton responses and the residual free nuclear response Y, (the narrow line at -14 MHz). Note that these traces show the -45 MHz peaks to be shifted with respect to each other by an amount different from the shift of Y, resulting from the two different magnetic fields used (AH 140 G). This is especially apparent in the weaker M , = +l/z responses seen near 15 MHz, for which the shifts are in the opposite direction. Figure 7 shows just the ENDOR responses obtained on lines 2 and 7. The two broad methylene responses are seen,
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The Journal of Physical Chemistry, Vol. 82, No. 18, 1978
with the additional methyl response at -68 MHz. Notice that the approximate rule that v = A / 2 f u, implies A / 2 = 30 MHz for the methyl protons and A / 2 60 MHz for the methylene protons in the glass matrix. Single-crystal ENDOR spectra were taken for comparison purposes, with the applied field oriented along the c axis of the crystalline thymidine. The transitions seen were quite narrow, and gave hyperfine interaction constants in good agreement with those obtained by Box et al.17 from their 35-GHz data. For comparison purposes, we have included stick diagrams in Figures 6 and 7 showing where these single-crystal responses were seen for the c-axis orientation. These comparisons show that both the methyl proton and methylene proton hyperfine interactions are essentially unchanged in the glass and are reasonably isotropic, as is to be expected from the agreement between the single crystal and glass EPR spectra. Moreover, the same methyl transitions are seen in the glass as in the single crystal. In addition most importantly, the relative shifts of the 4045-MHz methyl proton ENDOR lines are quite different in the two cases, implying that the rotational shifts of the ENDOR frequencies are different in the glass. Because the frequency shifts are seen to be different in the two cases, it is of interest to see whether any temperature dependence in the ENDOR line positions can be detected. Both single crystal and acid glass samples were measured for a selection of temperatures from 4.2 to 1.2 K. No frequency shifts were detected for any of the transitions seen. This is especially clear in the case of the single crystal spectra, in which shifts as small as Av = 10 kHz could have been seen. Surveys through the EPR lines, which are individually about 8-G wide, show no change in ENDOR shape as one shifts the field; only the intensities of the broad powder pattern change as one scans across an individual hyperfine line, as is expected for an isotropic g value. Surveys through the EPR structure show that all unshifted lines have the same shape on all EPR lines, and subtract out exactly from line to line. Half the methyl proton lines are shifted and half are not, but the two types share some common relaxation paths. Thus we have an internal check on line shape distortion effects. Since the unshifted set show no extra shifts in the powder spectra, and do show precisely the correct field-dependent Zeeman shifts, all the extra observed shifts are due to the tunneling. These extra shifts are exactly correlated with those transitions known to shift from the single-crystal results. The methylene protons provide a further check, since they have comparable width, anisotropy, and relaxation times. No evidence is seen for unusual effects and unexpected shifts in their spectra, and they subtract out precisely along with the unshifted methyl lines. Finally, no changes are seen in ENDOR line shapes as one varies the temperature between 4.2 and 1.2 K, although the relaxation times vary quite significantly over this range of temperatures. These checks, as well as the agreement between axial powder averages and the observed line shapes, show that the observed ENDOR spectra are free of spurious distortion due to relaxation effects and can be relied upon to give the true tunneling shifts. IV. Discussion The EPR spectrum of irradiated annealed glasses taken at 77 K consists principally of eight lines which conform closely to the spectral parameters of the well-accepted model of 5T,26radicals. At low temperatures, the 1:3:3:1 component of this spectrum due to three equivalent methyl protons (rapidly tunneling at 77 K) is changed into the
W. P. Unruh, T. Gedayloo, and J. D. Zimbrick
I l l
I l l
METHYL SPLlTT ING
METHYLENE SPLITTING
I I I IIII Figure 8. Low temperature EPR lines from methylene and tunneling methyl protons in acid glass deduced from ENDOR measurements of hyperfine constants at 1.2 K.
nominal seven line spectrum (1:1:1:2:1:1:1) characteristic of a tunneling methyl group. The additional 1:2:1 splitting of the two nearly equivalent methylene protons gives the spectrum shown in Figure 8, which agrees well with the EPR spectrum observed in this and previous work.17 The residual splittings seen in the low-power spectrum of Figure 1A raise the possibility that in the glass a few radicals retain their tunneling splitting even at 77 K, due to a local environment which produces high rotational barriers. Although the theoretical description needed for the interpretation of this ENDOR data has been presented earlier,l*l' it will be useful to summarize the main results which have applicability to this ENDOR data. The spin-Hamiltonian describing the coupled methyl proton-radical levels between which both EPR and ENDOR transitions are induced can be written ad3
This expression ignores the small effects of dipole-dipole coupling between adjacent methyl protons, and treats the effects of the torsional splittings of the EPR states by means of an exchange constant J, related to the tunneling frequency as 3 J = ut. In the slow tunneling limit, J
