INT . J . RADIAT . BIOL .,
1978,
VOL .
33,
NO .
2, 1 2 1-137
Radical formation in single crystals of hypoxanthine-HCI-HO, inosine, and the disodium salt of 5'-inosine-monophosphate HERBERT ZEHNER, ERIC WESTHOF, WOLFGANG FLOSSMANN and ADOLF MULLER Institut fur Biophysik and Physikalische Biochemie der Universitat Regensburg, D-8400 Regensburg, Germany
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(Received 15 August 1977 ; accepted 26 September 1977)
Radical formation in single crystals of hypoxanthine • HCl • H 2 O, inosine and Nae -5'-IMP • (7 . 5 H2O) by X-irradiation has been studied using electronspin-resonance spectroscopy at 9 . 5 and 35 GHz. In all crystals both H-addition radicals at position C2 and C8 of the purine ring are found . The coupling constants of these two radicals are different and depend strongly on the protonation state of the base. INDO-calculations indicate that the C8-radical is protonated at 06 . In Nae -5'-IMP OH-addition radicals at position C2 of the purine ring are formed . Electron adduct radicals are found in the neutral and the N7protonated base after X-irradiation at 77 K . In Nae-5'-IMP no electron adduct is formed but a radical which probably is the cation. In hypoxanthine • HCl H 2 O a radical could be observed after X-irradiation at 77 K, which results from addition of a Cl - to the nitrogen N1 .
1 . Introduction In two previous papers (Zehner, Flossmann and Westhof 1976, Zehner, Westhof, Flossman and Muller 1977), we have studied the formation of H-addition radicals in several single crystals of adenine derivatives . We have shown that, in most of the crystals analysed, radicals resulting from H-addition at two different positions are present : carbon C2 of the pyrimidine part and carbon C8 of the purine part . The e .s .r .-parameters of both radicals are different and depend on the protonation state of the molecule . With molecular orbital calculations at the INDO level, it is possible to reproduce the trends observed in the spin-density distributions of the different radicals in dependence on the protonation state . The stability of the radicals depends on the crystalline environment (Zehner et al . 1977, Westhof, Flossman, Zehner and Muller 1977 b) . In this work, we present the results of e .s .r . studies on X-irradiated single crystals of the purine base hypoxanthine, its nucleoside, and its nucleotide . As in the case of adenine derivatives, the H-addition may occur at two positions : C2 or C8 . 0
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122
H. Zehner et al .
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It will be shown that X-irradiation produces both kinds of H-addition radicals and that their e .s .r . parameters again depend on the protonation state of the molecule . In single crystals of Nae -5'-IMP under special conditions, OHaddition radicals at C2 are produced . X-irradiation at 77 K leads in single crystals of hypoxanthine and inosine to radicals which are identified as electron adduct radicals on the basis of INDO calculations . A further radical, which results from Cl- addition to the nitrogen N1 of the purine base, is identified in single crystals of hypoxanthine • HC1 • H 20 .
2.
Materials and methods Single crystals of hypoxanthine • HCl • H 2 O were grown by slow evaporation at room temperature of a 2N . HCl solution saturated at 40°C . The crystals are monoclinic with space group P21 Ic (Sletten and Jensen 1969) . They present a pronounced cleavage plane, which is parallel to the (102)-plane (Sletten and Jensen 1969) . For the e .s .r. measurements, an orthogonal coordinate system a*, b, c* was chosen, where a* and b are in the cleavage plane and c* perpendicular to the cleavage plane ; b is the crystallographic symmetry axis . Single crystals of inosine were grown by slow cooling of aqueous solutions saturated at 50°C . There are three crystalline forms of inosine : two anhydrous ones with one orthorombic (Subramanian, Madden and Bugg 1973) and the other one monoclinic (Munns and Tollin 1970) and one hydrated form which is also monoclinic (Thewalt, Bugg and Marsh 1970) . The crystals we have analysed belong to the anhydrous orthorhombic form . They were of poor quality and either twinned or warped . It was not therefore possible to define an orthogonal coordinate system for the e .s .r . measurements . Single crystals of Nae -5'-IMP . (7 . 5 H2 O) were grown by evaporation of an aqueous solution at 40 ° C . The crystals are orthorhombic with Z= 8 and space group C2221 (Nagashima and litaka 1968) . The crystallographic axes were used for the e .s .r . measurements . All three kinds of crystals were prepared at four different deuteration stages : full protonated, easily-exchangeable protons deuterated, the proton bonded to C8 deuterated, and full deuterated, where the easily-exchangeable and the proton bonded to C8 are deuterated . The procedure for the deuteration, the irradiation, and the e .s .r . measurements were as described previously (Zehner, Flossmann and Westhof 1976) .
3 . Results 3 .1 . The H-addition radicals 3 .1 .1 . Hypoxanthine • HCl • H2 O Immediately after X-irradiation at 300 K, one observes the spectrum at the top left of figure 1 . Warming the crystal for 3 hours at 100 ° C yields the spectrum at the top right of figure 1 . The structure of the extreme lines changes from a triplet to a quartet . This indicates that the outer triplet obtained immediately after X-irradiation results from overlapping of the lines of two different radicals with similar /3-proton splitting . The spectrum at the bottom left is obtained with a crystal where the C8-proton is deuterated . A comparison with the upper spectra shows that the most extreme lines disappear and the middle lines of the
123
Radical formation in hypoxanthine derivatives
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20 G
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r' C8 HH
C8 HD C8 HH
Figure 1 . E .s .r . spectra of single crystals of hypoxanthine • HCl • H20 X-irradiated at room temperature . The magnetic field is parallel to the purine plane and roughly parallel to the C8-H bond . Top left : immediately after X-irradiation . Top right : after warming the crystal for 3 hours at 100°C . Bottom left : C8 deuterated crystal immediately after X-irradiation . Bottom right : Crystal grown from DCl after warming for 3 hours at 100°C .
triplet remain . Some supplementary lines also appear on either side of the central main line . The outer lines, with a total splitting of 90 G, must therefore belong to the radical resulting from H-addition at position C2 . These outer lines disappear upon heating, confirming the conclusion reached at above . Accordingly, the inner lines are those of the C8-HD addition radical . In the top right spectrum of figure 1, which is attributed to the C8-HH addition radical, two supplementary couplings are observed . These present the typical anisotropy of a-protons and disappear with crystals of hypoxanthine DCl • D2 0 . Thus, they arise from the proton bonded to N7 and N9, respectively (the molecule is protonated at N7 in crystals of hypoxanthine . HCl • H2O) . The e .s .r . parameters are contained in table 1 . The methylene protons of the C2-addition radical are probably non-equivalent with values of 42 and 47 G . The maximal values of the a-protons bonded to N7 and N9 of the C8-addition radical are similar to those observed for the C8-addition radical in adenine 2HC1 (Zehner et al . 1977) . X-irradiation at 77 K and subsequent warming to 300 K yields a small amount of both H-addition radicals . However, the amount of C2-addition radical is larger than immediately after X-irradiation at room temperature . It was not possible to observe any transformation of radicals . Illumination with light (N 2 -laser at A=337 nm or high-pressure mercury lamp) at 77 or 300 K of single crystals of hypoxanthine . HCl • H 2 O also produces both kinds of H-addition radical . Doping of the crystals with proflavine does not increase the yield of H-addition radicals . However, a further singlet is observed in the middle of the spectrum .
H . Zehner et al .
124
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Radical formation in hypoxanthine derivatives
125
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3 .1 .2 . Inosine Figure 2 shows spectra of single crystals of inosine after X-irradiation at 300 K . The top spectrum is obtained with a C8-deuterated crystal . Therefore, the outer lines with a total splitting of 70 G belong to the C2-addition radical . Because of the larger line-width, the lines of the C8-HD addition radical are not resolved . In the lower spectrum, gained with a fully protonated crystal, additional lines are seen in the outer parts of the spectrum . This deuteration effect reveals the presence of C8-addition radicals . The methylene proton splittings of the C8-addition radical are much larger than those of the C2-
C2-HH
C2-HH C8-HH
Figure 2 . E.s .r . spectra of single crystals of inosine X-irradiated at room temperature . The magnetic field is parallel to the purine ring of the molecules . Observation frequency is 35 GHz . Top : C8-deuterated crystal . Bottom : protonated crystal .
addition radical . In the case of the non-protonated adenine molecule, the reverse situation was found (Zehner et al. 1976, Zehner et al . 1977). Because of the relative orientation of the bases in the crystal we could not extract more e.s .r . parameters than those given in table 1 . Both H-addition radicals are stable in crystals of inosine under illumination with light or heating to 120•C . 3 .1 .3 . Nat-5'-IMP - ( 7. 5 H2 O) Spectra obtained with single crystals of the disodium salt of ionsine monophosphate X-irradiated at 300 K are shown in figure 3 . The main radical produced is characterized by a triplet of doublets . The triplet splitting is
1 26
H . Zehner et al .
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35 . 0 G and the doublet splitting is not present in C8-deuterated crystals (bottom left). The other weak lines belong to another site of the same radical, where hyperfine interaction with nitrogen is visible . In the lower spectrum at the left, the lines typical of the C8-HD radical are not observed . Thus, only the C2-addition radical is produced . The e .s .r . parameters are contained in table 1 . The C2-addition radical is stable against illumination with light or warming to 80 • C . We have not analysed the other lines present in the spectrum . It should be added that these crystals contain several water molecules and that a very large amount of H-addition instead of D-addition radicals is formed even in carefully deuterated crystals .
y C2 HH
Figure 3 . E.s .r . spectra of single crystals of Nat-5'-IMP - (7 . 5 H2O) . The magnetic field is parallel to the (bc)-plane 30• from c. Top : protonated crystals . Bottom : C8-deuterated crystals . Left : X-irradiated at room temperature . Right : X-irradiated at 77 K and warmed up to room temperature . Stick diagrams indicate line positions only for one site except spectra bottom left where stick diagrams for the two magnetically inequivalent molecules could be seen .
Irradiation at 77 K produces no H-addition radicals. After annealing to 300 K, one observes the spectra at the right of figure 3 . The spectra are very similar to those at the left . However, some new outer lines can be seen, which disappear on deuteration of the C8-proton . Assuming that these lines arise from the C8-addition radical, a methylene proton coupling of 44 G is obtained which compares well with that observed in inosine crystals . After warming, the radical concentration is only 10 per cent of the initial radical concentration at 77 K . Therefore, no radical transformation could be observed . After photobleaching with A > 600 nm at 77 K and subsequent warming to 300 K of a crystal X-irradiated at 77 K, one does not observe H-addition radicals . Some link between the low-temperature radicals and the H-addition radicals is therefore possible .
Radical formation in hypoxanthine derivatives
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H. Zehner et al .
3.2 . INDO calculations on the H-addition radicals Table 2 contains the results of INDO calculations on several H-addition radicals of hypoxanthine and guanine . The experimental results for guanine are presented in table 1 . Concerning the experimental methylene proton coupling constants, the main conclusions are that, in the non-protonated form of hypoxanthine, the fl-proton couplings of the C2-addition radical are smaller than those of the C8-addition radical and that in the N7-protonated form they are very similar . The first experimental conclusion is well reproduced by INDO. However, the N7-protonated form yields very different fl-proton couplings for both H-addition radicals in contrast with the second conclusion . In order to reproduce the experimental trend, a supplementary protonation at 06 is necessary . This supplementary protonation at 06 seems to be also necessary in the case of guanine derivatives . In this case, the methylene proton couplings are nearly identical in both the protonated and the non-protonated form (table 1) . This experimental result can only be reproduced if the C8addition radical of the N7-protonated guanine molecule is further protonated at 06 . The other small couplings, as well as the nitrogen couplings, neither discredit nor confirm this supplementary protonation . However, the unexpectedly large coupling observed for the proton bonded to N7 and N9 in hypoxanthine and guanine is better reproduced in the doubly-protonated form of the C8-addition radical . In the case of neutral and N1-protonated adenine derivatives, a supplementary coupling from the proton bonded to C8 was observed for the C2addition radical . In agreement with the calculations, such a coupling has only been observed in the neutral form of hypoxanthine .
3.3 . The OH-addition radical After warming to 300 K a crystal of Na,-5'-IMP X-irradiated at 77 K, lines appear in the middle of the spectrum, which are not present immediately after X-irradiation at room temperature . The orientation-dependence of these lines and the effect of deuteration on them indicate that they present the same hyperfine interactions as the C2-HH addition radical but with the coupling of one methylene proton less . Thus, instead of two equivalent fl-protons of 35 G, only one fl-proton of 20 G is observed . The effect of the C8-deuteration is shown in figure 3 : the same small coupling originating from H(C8) present in C2-HH radical is present in the doublet structure of the new lines . Figure 4 also shows that the nitrogen coupling is the same for both radicals . We attribute these lines to the radical resulting from OH-addition at position C2 . For pyrimidines, it was found experimentally that the fl-proton of OHaddition radicals is smaller than in H-addition radicals (Westhof, Flossman and Miiller 1977 a) . INDO calculations have also presented this trend . The formation of OH-addition radicals in purine-derivative single crystals has not yet been reported . However, OH-addition radicals at position C8 have been detected, after X-irradiation of frozen aqueous solutions of adenine and guanine derivatives (Gregoli, Olast and Bertinchamps 1974) . In table 3, the experimental and theoretical couplings for OH-addition radicals at position C2 or C8 of the purine ring in adenine, guanine, and hypoxanthine are given . It should be
Radical formation in hypoxanthine derivatives
1 29
i
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Figure 4 . E .s .r . spectra of single crystals of Naz -5'-IMP • (7 .5 H20), X-irradiated at 77 K and warmed to room temperature . The magnetic field is parallel to the c-axis . Top : fully protonated crystal . Bottom : C8-deuterated crystal . Stick diagrams indicate line positions of C2-HH and C2-HOH radicals . The small amount of C8-HH and C8-HD radicals could not be seen in this orientation . remarked that, in pyrimidine and purine crystals, OH-addition was found at the less frequent position of addition (i .e . C6 and C2, respectively) and that, in both cases, some relationship with an observed cation radical was apparent .
3 .4. The base radical anions In figure 5 are presented the spectra obtained after X-irradiation at 77 K of single crystals of fully protonated hypoxanthine . HCI • H20 (top) and of crystals where the C8-proton has been exchanged for a deutron (bottom) . The spectra at the left were taken at 9 . 5 GHz and those at the right at 35 GHz . The influence of the frequency of observation indicates the presence of two radicals : a doublet and a quartet . The C8-deuteration replaces the doublet by a singlet and removes the large splitting of the quartet, which appears as a singlet because of overlapping lines . Since crystals of hypoxanthine . DO . D2 0 yield identical spectra, the small splitting should arise from H(C2) . The gfactor of the quartet spectrum is more anisotropic than that of the doublet spectrum . The maximal splitting of the doublet spectrum is obtained with the magnetic field perpendicular to the C8-H bond in the molecular plane and is equal to 17 . 0 G .
130
H . Zehner et al .
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Radical formation in hypoxanthine derivatives
1 31
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Figure 5 . E .s .r . spectra of single crystals of hypoxanthine • HCl • H20 X-irradiated and observed at low microwave power at 77 K . The magnetic field is parallel to the purine plane and roughly normal to the C8-H bond . Top : protonated crystals . Bottom : C8-deuterated crystals . Left : frequency of observation 9 . 5 GHz . Right : frequency of observation 35 GHz .
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Figure 6 . E .s .r . spectra of single crystals of inosine (left) and Nae-5'-IMP (right) Xirradiated and observed at 77 K . Top : protonated crystals. Bottom :C8-deuterated crystals . The orientation of the magnetic field is the same as in figure 2 for the inosine spectra . For the Nae -5'-IMP spectra the magnetic field is parallel to the b-axis .
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132
H . Zehner et al .
Crystals of inosine irradiated at 77 K do not yield well-resolved spectra . The two spectra at the left of figure 6 show that the C8-deuteration has no effect . Identical spectra are also obtained with crystals grown from heavy water . Therefore, the doublet splitting present in the spectrum arises from the proton bonded to C2 . This splitting is anisotropic and has the maximum value of 15 . 0 G . At the right of figure 6 are shown spectra gained with crystals of Na e-5'-IMP The effect of C8-deuteration is to replace the quartet by a triplet . Replacement of the easily exchangeable protons by deuterons has no influence on the spectra . Therefore, the spectra arise from hyperfine interaction between the unpaired electron and the proton bonded to C8 and one nitrogen nucleus . The angle of the magnetic field with the p 7 -orbital is about 35° . The maximal nitrogen coupling is about 8 . 5 G . When the magnetic field is parallel to the a-axis (i .e . roughly parallel to the purine ring), spectra taken at 9 . 5 and at 35 GHz present a doublet of 7 . 0 G, which collapses to a singlet after C8-deuteration . In the 35 GHz spectra, an additional broad unresolved structure is visible, which indicates that at least one supplementary radical is present . It therefore appears that, in the two crystals where the hypoxanthine molecule is not protonated, irradiation produces different radicals at 77 K . In all three crystals, the radicals produced after X-irradiation at 77 K are easily photo-bleached by visible light and disappear on warming to 300 K. In the case of Na e-5'-IMP, H-addition radicals and OH-addition radicals appear after annealing to room temperature .
3 .5 . INDO calculations on the ionic-radicals Radicals produced after X-irradiation at low temperatures are usually of ionic nature . Therefore, we have calculated the anion and cation radicals of the neutral and several protonated forms of the hypoxanthine molecule . The results are contained in table 4 . First, consider the radical anions . In the electron adduct of the neutral molecule, the spin density is localized mainly on carbon C2, giving a larger coupling with H(C2) . On the other hand, the electron adduct of the N7-protonated molecule gives a larger coupling with H(C8), since the spin density is mainly localized on C8 . Experimentally, we found two radicals behaving in this manner . In inosine crystals, a radical was characterized by a doublet splitting arising from H(C2), whereas in hypoxanthine HCl - H2 O crystals, a similar radical was characterized by a doublet splitting arising from H(C8) . We therefore consider that these two doublet splittings arise from the electron adduct radical of the neutral and N7-protonated molecule . Two radicals are still to be explained : the quartets observed in hypoxanthine HCI • H2 O and Na e-5'-IMP . We tentatively assign these two different quartets to the cation radicals of the neutral molecule in the case of Na e-5'-IMP and to the cation radical of the N7-protonated molecule in the case of hypoxanthine HCl • H20 . The INDO calculations of table 4 indicate that the cation radical of the neutral molecule should present couplings with H(C8) and with Nl and N3 . We only observe coupling with one nitrogen . This could indicate that we have not calculated the proper structure of the cation radical . The same
Radical formation in hypoxanthine derivatives
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problem occurs with the cation radical of the N7-protonated molecule . It presents large couplings with the nitrogens N1 and N3 . However, it has already been observed that INDO calculation overemphasized nitrogen couplings, thereby reducing the proton couplings (Westhof and Van Rooten 1976) . The calculations indeed yield much too small couplings with the protons H(C2) and H(C8) . We did several calculations on purine-derivative cation radicals without good agreement with experimental couplings . This may reflect some limitations of the INDO method .
3 .6 .
The
N-chloro
radical
At high incident microwave power, the spectra obtained at 77 K with single crystals of hypoxanthine • HCl • H20 X-irradiated at 77 K reveal the presence of another radical characterized by large hyperfine anisotropic couplings and a strongly anisotropic g-factor (see figure 7) . At all orientations, the spectra could be analysed in terms of hyperfine interaction between the unpaired electron and one chlorine nucleus, one nitrogen nucleus, and one hydrogen nucleus .
S
i f V
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Y Yr y
h
Figure 7 . E .s .r. spectra of single crystal of hypoxanthine • HCl • H20 X-irradiated and observed at high microwave power at 77 K . Top : the magnetic field is parallel to the b-axis, Bottom : the magnetic field is parallel to the (a*c*)-plane 50° from a* . Stick diagrams indicate the line positions for the N-chloro radical . The 37 C1 isotope is shown below the spectra, the 35 C1 isotope above the spectra .
Radical formation in hypoxanthine derivatives
135
As can be seen in figure 7, both chlorine isotopes, 35C1 and 37 C1, could be resolved . From the orientation dependence of the spectra, the principal values and direction cosines of the hyperfine tensors could be extracted and are contained in table 5 .
Nucleus
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Principal values
Directions
113 25 25
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H(C2)
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Parallel to C2-H direction in crystal Parallel to p,,-orbital Normal to both directions
g
2 . 001 2 . 027 2 . 023
Parallel Nl-Cl directiohn Parallel p 2 -orbital Normal to both directions
Hyperfine tensors of the N-chloro-radical in hypoxanthine • HCl • H 2 O . All values in Gauss . The g-values are no principal values .
The direction cosines of the maximal principal value of the nitrogen coupling coincides with the direction of the N1-H bond in the undamaged crystal, while those of the chlorine coupling coincide with the N1 . . . Cl - direction. These two directions make an angle of 6 ° with each other . The hydrogen coupling arises from the proton bonded to C2, because deuteration of neither the easily exchangeable protons, nor of H(C8), reduces this coupling . The coupling is slightly anisotropic and not at all typical of an a-proton . Similar spectra have been observed with single crystals of adenine • 2HC1 (Huttermann 1975), and cytosine • HCl (Westhof, Flossmann and Muller 1975) . They were assigned to the radical resulting from Cl - addition to the nitrogen N1 in adenine and N3 in cytosine after removal of the proton bonded to these nitrogens (Westhof et al. 1975) . However, no direct evidence could be presented to exclude addition at the amino group (Westhof et al . 1975) . The observation of a radical having the same hyperfine tensors and behaviour in hypoxanthine • HCl • H 2O crystals definitively proves the addition at position N1 in purine derivatives and N3 in cytosine . In adenine . 2HC1 crystals, no additional hydrogen coupling could be observed, but the line-width is about 8-12 G in these crystals and less than 4 G in hypoxanthine • HCl • H 2 O . 4.
Discussion The H-addition radicals are the main species trapped in single crystals of purine derivatives after X-irradiation at room temperature . These radicals may be formed by two reaction mechanisms, either through direct hydrogen atom addition, or through protonation of an electron adduct (Westhof et al . 1977 b) . We could not ascertain whether one of these reactions leads preferentially to one kind of radicals . However, there is evidence that both reaction
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H. Zehner et al .
mechanisms occur . Indeed, irradiation with light of A > 300 nm of hypoxanthine ' HCl • H20 yields both kinds of H-addition radicals . On the other hand, the presence of ionic radicals at 77 K indicates that ionic reactions are possible. The OH-addition radical observed in Na,-5'-IMP could also be produced through an ionic precursor . In this case, the cation radical and not the anion radical would be involved (Gregoli et al . 1974) . It is interesting to note that in Nae -5'-IMP, no anion radicals were detected but instead most probably cation radicals . The presence of water and not OH-groups from the sugar seems to be necessary to form the OH-addition radical, since such radicals were never observed in single crystals of nucleosides which crystallize without so many water molecules . In table 6, the results are presented concerning the occurence of H-addition radicals in all purine derivatives (except guanine) we have analysed . The crystals can be divided into three groups . The first, and smaller, contains those where only C2-addition radicals were observed . In the second, which is slightly larger, only C8-addition radicals are detected . And finally, in the third group, both kinds of H-addition radical are produced . Neither the substituents, nor the molecules of crystallization or the protonation state seems to be decisive for the production of one kind of radical . In the case of the doubly-protonated adenine molecule, however, the formation of C8-addition radicals is preferred . It appears also that the C2-addition radical is less stable than the C8-addition radical (Zehner et al. 1977) . The local crystal environment seems, therefore, to be the dominating factor controlling the formation and the stability of these H-addition radicals . Crystal
C 2
C 8
Adenine • +H 2 SO 4 • H20 Na2-5'-inosine monophosphate • (7 . 5 H 2 O) Ba-5'-inosine monophosphate • 6 H20t
X X X
-
Adeninet 9-CH 3 -Adenine Nae -5'-Adenosine monophosphatet Purine ribosidef Adenosine • DMSO Adenine • H2SO 4 9-CH3-Adenine • 2HBr
-
X X X X X X X
Adenine • 2HC1 Adenine • HCl • H2O Desoxyadenosine • H20 Adenosine Adenosine • HCl 5'-Adenosine monophosphate • H2 0 Hypoxanthine • HCl • H20 Inosine Na-5'-inosine monophosphate • 8 H20t
X X X X X X X X X
X X X X X X X X X
t Results were gained from powder spectra . Table 6 .
Occurrence of H-addition radicals in crystals of purine derivatives after X-irradiation at room temperature .
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Radical formation in hypoxanthine derivatives
137
La formation de radicaux dans des monocristaux de ('hypoxanthine • HCI • H20, inosine et Na e-5'-IMP • 7,5 H 2 O a ete analysee par resonance paramagnetique electronique (9,5 et 35 GHz) sous 1'effet de rayonnement . Des radicaux de 1'addition d'hydrogene en C2 et en C8 du noyau purique ont ete identifies dans tons les cristaux . Les constantes de couplages de ces deux radicaux sont differentes et dependent fortement de l'etat des protons de la base . Les calculs par la methode INDO indiquent, qu'au radical C8 l'addition d'un proton a lieu en 06 . L'addition du groupement OH en C2 du noyau purique peut etre observee daps la molecule de Na e -5'-IMP . Sous 1'effet du rayonnement X a 77 K l'addition d'electrons pent etre observee daps la base neutralisee et protonis6e en N7 . Aucune addition d'electrons s'est formee dans la molecule de Nae -5'IMP-, cependant un radical etant probablement le cation . Dans la molecule de l'hypoxanthine • HCl • H20 un autre radical a ete trouve sous 1'effet du rayonnement X a 77 K provenant de l'addition d'un Cl- sur l'azote NI du noyau purique . Die Radikalbildung in Einkristallen von Hypoxanthin • HCl • H 2O, Inosin and Nae -5'IMP • 7,5 H20 durch Rontgenbestrahlung wurde mit Hilfe der Elektronenspin-ResonanzSpektroskopie bei 9 . 5 and 35 GHz untersucht. In allen Kristallen wurden H-Anlagerungsradikale an Position C2 and C8 des Purinrings gefunden . Die Kopplungskontanten dieser beiden Radikale sind unterschiedlich and hangen sehr vom Protonierungszustand der Base ab . INDO-Rechnungen deuten an, daf3 das C-8-Radikal an 06 protoniert ist . In Nae -5'-IMP wird OH-Anlagerung an C2 des Purinrings beobachtet . Nach Rontgenbestrahlung bei 77 K findet man in der neutralen and an N7-protonierten Base Elektronenaddukte . In Na e -5'-IMP wird kein Elektronenaddukt gebildet, jedoch ein Radikal, das moglicherweise das Kation ist . In Hypoxanthin • HC1 - H2O kann man nach Rontgenbestrahlung bei 77 K ein weiteres Radikal beobachten, das durch Anlagerung eines Cl - an den Stickstoff N1 des Purinrings entstanden ist .
REFERENCES
ALEXANDER, CH ., and GORDY, W ., 1967, Proc . natn . Acad . Sci. U .S.A ., 58, 1279. GREGOLI, S ., OLAST, M ., and BERTINCHAMPS, A ., 1974, Radiat. Res ., 60, 388 . HUTTERMANN, J ., 1975, Y. magn . Res., 17, 66 . MUNNS, A . R . J ., and TOLLIN, P ., 1970, Acta crystallogr ., B, 26, 1101 . NAGASHIMA, N ., and IITAKE, Y ., 1968, Acta crystallogr. B, 24, 1136 . SLETTEN, J ., and JENSEN, L . M ., 1969, Acta crystallogr. B, 25, 1608 . SUBRAMANIAN, E., MADDEN, J . J ., and BUGG, C . E ., 1973, Biochem . biophys . Res. Commun ., 50,691 . THEWALT, U ., BUGG, C . E ., and MARSH, R . E ., 1970, Acta crystallogr., B, 26, 1089 . WESTHOF, E ., FLOSSMANN, W ., and MULLER, A ., 1975, Int ., . Radiat . Biol., 28, 427 ; 1977 a, Radiat . Res ., 70, 304 . WESTHOF, E ., FLOSSMANN, W ., ZEHNER, H ., and MULLER, A ., 1977 b, Discuss. Faraday Soc., 63 (in the press) . WESTHOF, E ., and VAN ROOTEN, M ., 1976, Z. Naturf. C, 31, 371 . ZEHNER, H ., FLOSSMANN, W., and WESTHOF, E ., 1976, Z. Naturf. C, 31, 225 . ZEHNER, H ., WESTHOF, E ., FLOSSMANN, W ., and MULLER, A ., 1977, Z. Naturf. C, 32, 1 .
R .B .
K
1978,
VOL .
33,
NO .
2, 1 2 1-137
Radical formation in single crystals of hypoxanthine-HCI-HO, inosine, and the disodium salt of 5'-inosine-monophosphate HERBERT ZEHNER, ERIC WESTHOF, WOLFGANG FLOSSMANN and ADOLF MULLER Institut fur Biophysik and Physikalische Biochemie der Universitat Regensburg, D-8400 Regensburg, Germany
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(Received 15 August 1977 ; accepted 26 September 1977)
Radical formation in single crystals of hypoxanthine • HCl • H 2 O, inosine and Nae -5'-IMP • (7 . 5 H2O) by X-irradiation has been studied using electronspin-resonance spectroscopy at 9 . 5 and 35 GHz. In all crystals both H-addition radicals at position C2 and C8 of the purine ring are found . The coupling constants of these two radicals are different and depend strongly on the protonation state of the base. INDO-calculations indicate that the C8-radical is protonated at 06 . In Nae -5'-IMP OH-addition radicals at position C2 of the purine ring are formed . Electron adduct radicals are found in the neutral and the N7protonated base after X-irradiation at 77 K . In Nae-5'-IMP no electron adduct is formed but a radical which probably is the cation. In hypoxanthine • HCl H 2 O a radical could be observed after X-irradiation at 77 K, which results from addition of a Cl - to the nitrogen N1 .
1 . Introduction In two previous papers (Zehner, Flossmann and Westhof 1976, Zehner, Westhof, Flossman and Muller 1977), we have studied the formation of H-addition radicals in several single crystals of adenine derivatives . We have shown that, in most of the crystals analysed, radicals resulting from H-addition at two different positions are present : carbon C2 of the pyrimidine part and carbon C8 of the purine part . The e .s .r .-parameters of both radicals are different and depend on the protonation state of the molecule . With molecular orbital calculations at the INDO level, it is possible to reproduce the trends observed in the spin-density distributions of the different radicals in dependence on the protonation state . The stability of the radicals depends on the crystalline environment (Zehner et al . 1977, Westhof, Flossman, Zehner and Muller 1977 b) . In this work, we present the results of e .s .r . studies on X-irradiated single crystals of the purine base hypoxanthine, its nucleoside, and its nucleotide . As in the case of adenine derivatives, the H-addition may occur at two positions : C2 or C8 . 0
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122
H. Zehner et al .
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It will be shown that X-irradiation produces both kinds of H-addition radicals and that their e .s .r . parameters again depend on the protonation state of the molecule . In single crystals of Nae -5'-IMP under special conditions, OHaddition radicals at C2 are produced . X-irradiation at 77 K leads in single crystals of hypoxanthine and inosine to radicals which are identified as electron adduct radicals on the basis of INDO calculations . A further radical, which results from Cl- addition to the nitrogen N1 of the purine base, is identified in single crystals of hypoxanthine • HC1 • H 20 .
2.
Materials and methods Single crystals of hypoxanthine • HCl • H 2 O were grown by slow evaporation at room temperature of a 2N . HCl solution saturated at 40°C . The crystals are monoclinic with space group P21 Ic (Sletten and Jensen 1969) . They present a pronounced cleavage plane, which is parallel to the (102)-plane (Sletten and Jensen 1969) . For the e .s .r. measurements, an orthogonal coordinate system a*, b, c* was chosen, where a* and b are in the cleavage plane and c* perpendicular to the cleavage plane ; b is the crystallographic symmetry axis . Single crystals of inosine were grown by slow cooling of aqueous solutions saturated at 50°C . There are three crystalline forms of inosine : two anhydrous ones with one orthorombic (Subramanian, Madden and Bugg 1973) and the other one monoclinic (Munns and Tollin 1970) and one hydrated form which is also monoclinic (Thewalt, Bugg and Marsh 1970) . The crystals we have analysed belong to the anhydrous orthorhombic form . They were of poor quality and either twinned or warped . It was not therefore possible to define an orthogonal coordinate system for the e .s .r . measurements . Single crystals of Nae -5'-IMP . (7 . 5 H2 O) were grown by evaporation of an aqueous solution at 40 ° C . The crystals are orthorhombic with Z= 8 and space group C2221 (Nagashima and litaka 1968) . The crystallographic axes were used for the e .s .r . measurements . All three kinds of crystals were prepared at four different deuteration stages : full protonated, easily-exchangeable protons deuterated, the proton bonded to C8 deuterated, and full deuterated, where the easily-exchangeable and the proton bonded to C8 are deuterated . The procedure for the deuteration, the irradiation, and the e .s .r . measurements were as described previously (Zehner, Flossmann and Westhof 1976) .
3 . Results 3 .1 . The H-addition radicals 3 .1 .1 . Hypoxanthine • HCl • H2 O Immediately after X-irradiation at 300 K, one observes the spectrum at the top left of figure 1 . Warming the crystal for 3 hours at 100 ° C yields the spectrum at the top right of figure 1 . The structure of the extreme lines changes from a triplet to a quartet . This indicates that the outer triplet obtained immediately after X-irradiation results from overlapping of the lines of two different radicals with similar /3-proton splitting . The spectrum at the bottom left is obtained with a crystal where the C8-proton is deuterated . A comparison with the upper spectra shows that the most extreme lines disappear and the middle lines of the
123
Radical formation in hypoxanthine derivatives
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r' C8 HH
C8 HD C8 HH
Figure 1 . E .s .r . spectra of single crystals of hypoxanthine • HCl • H20 X-irradiated at room temperature . The magnetic field is parallel to the purine plane and roughly parallel to the C8-H bond . Top left : immediately after X-irradiation . Top right : after warming the crystal for 3 hours at 100°C . Bottom left : C8 deuterated crystal immediately after X-irradiation . Bottom right : Crystal grown from DCl after warming for 3 hours at 100°C .
triplet remain . Some supplementary lines also appear on either side of the central main line . The outer lines, with a total splitting of 90 G, must therefore belong to the radical resulting from H-addition at position C2 . These outer lines disappear upon heating, confirming the conclusion reached at above . Accordingly, the inner lines are those of the C8-HD addition radical . In the top right spectrum of figure 1, which is attributed to the C8-HH addition radical, two supplementary couplings are observed . These present the typical anisotropy of a-protons and disappear with crystals of hypoxanthine DCl • D2 0 . Thus, they arise from the proton bonded to N7 and N9, respectively (the molecule is protonated at N7 in crystals of hypoxanthine . HCl • H2O) . The e .s .r . parameters are contained in table 1 . The methylene protons of the C2-addition radical are probably non-equivalent with values of 42 and 47 G . The maximal values of the a-protons bonded to N7 and N9 of the C8-addition radical are similar to those observed for the C8-addition radical in adenine 2HC1 (Zehner et al . 1977) . X-irradiation at 77 K and subsequent warming to 300 K yields a small amount of both H-addition radicals . However, the amount of C2-addition radical is larger than immediately after X-irradiation at room temperature . It was not possible to observe any transformation of radicals . Illumination with light (N 2 -laser at A=337 nm or high-pressure mercury lamp) at 77 or 300 K of single crystals of hypoxanthine . HCl • H 2 O also produces both kinds of H-addition radical . Doping of the crystals with proflavine does not increase the yield of H-addition radicals . However, a further singlet is observed in the middle of the spectrum .
H . Zehner et al .
124
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Radical formation in hypoxanthine derivatives
125
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3 .1 .2 . Inosine Figure 2 shows spectra of single crystals of inosine after X-irradiation at 300 K . The top spectrum is obtained with a C8-deuterated crystal . Therefore, the outer lines with a total splitting of 70 G belong to the C2-addition radical . Because of the larger line-width, the lines of the C8-HD addition radical are not resolved . In the lower spectrum, gained with a fully protonated crystal, additional lines are seen in the outer parts of the spectrum . This deuteration effect reveals the presence of C8-addition radicals . The methylene proton splittings of the C8-addition radical are much larger than those of the C2-
C2-HH
C2-HH C8-HH
Figure 2 . E.s .r . spectra of single crystals of inosine X-irradiated at room temperature . The magnetic field is parallel to the purine ring of the molecules . Observation frequency is 35 GHz . Top : C8-deuterated crystal . Bottom : protonated crystal .
addition radical . In the case of the non-protonated adenine molecule, the reverse situation was found (Zehner et al. 1976, Zehner et al . 1977). Because of the relative orientation of the bases in the crystal we could not extract more e.s .r . parameters than those given in table 1 . Both H-addition radicals are stable in crystals of inosine under illumination with light or heating to 120•C . 3 .1 .3 . Nat-5'-IMP - ( 7. 5 H2 O) Spectra obtained with single crystals of the disodium salt of ionsine monophosphate X-irradiated at 300 K are shown in figure 3 . The main radical produced is characterized by a triplet of doublets . The triplet splitting is
1 26
H . Zehner et al .
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35 . 0 G and the doublet splitting is not present in C8-deuterated crystals (bottom left). The other weak lines belong to another site of the same radical, where hyperfine interaction with nitrogen is visible . In the lower spectrum at the left, the lines typical of the C8-HD radical are not observed . Thus, only the C2-addition radical is produced . The e .s .r . parameters are contained in table 1 . The C2-addition radical is stable against illumination with light or warming to 80 • C . We have not analysed the other lines present in the spectrum . It should be added that these crystals contain several water molecules and that a very large amount of H-addition instead of D-addition radicals is formed even in carefully deuterated crystals .
y C2 HH
Figure 3 . E.s .r . spectra of single crystals of Nat-5'-IMP - (7 . 5 H2O) . The magnetic field is parallel to the (bc)-plane 30• from c. Top : protonated crystals . Bottom : C8-deuterated crystals . Left : X-irradiated at room temperature . Right : X-irradiated at 77 K and warmed up to room temperature . Stick diagrams indicate line positions only for one site except spectra bottom left where stick diagrams for the two magnetically inequivalent molecules could be seen .
Irradiation at 77 K produces no H-addition radicals. After annealing to 300 K, one observes the spectra at the right of figure 3 . The spectra are very similar to those at the left . However, some new outer lines can be seen, which disappear on deuteration of the C8-proton . Assuming that these lines arise from the C8-addition radical, a methylene proton coupling of 44 G is obtained which compares well with that observed in inosine crystals . After warming, the radical concentration is only 10 per cent of the initial radical concentration at 77 K . Therefore, no radical transformation could be observed . After photobleaching with A > 600 nm at 77 K and subsequent warming to 300 K of a crystal X-irradiated at 77 K, one does not observe H-addition radicals . Some link between the low-temperature radicals and the H-addition radicals is therefore possible .
Radical formation in hypoxanthine derivatives
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3.2 . INDO calculations on the H-addition radicals Table 2 contains the results of INDO calculations on several H-addition radicals of hypoxanthine and guanine . The experimental results for guanine are presented in table 1 . Concerning the experimental methylene proton coupling constants, the main conclusions are that, in the non-protonated form of hypoxanthine, the fl-proton couplings of the C2-addition radical are smaller than those of the C8-addition radical and that in the N7-protonated form they are very similar . The first experimental conclusion is well reproduced by INDO. However, the N7-protonated form yields very different fl-proton couplings for both H-addition radicals in contrast with the second conclusion . In order to reproduce the experimental trend, a supplementary protonation at 06 is necessary . This supplementary protonation at 06 seems to be also necessary in the case of guanine derivatives . In this case, the methylene proton couplings are nearly identical in both the protonated and the non-protonated form (table 1) . This experimental result can only be reproduced if the C8addition radical of the N7-protonated guanine molecule is further protonated at 06 . The other small couplings, as well as the nitrogen couplings, neither discredit nor confirm this supplementary protonation . However, the unexpectedly large coupling observed for the proton bonded to N7 and N9 in hypoxanthine and guanine is better reproduced in the doubly-protonated form of the C8-addition radical . In the case of neutral and N1-protonated adenine derivatives, a supplementary coupling from the proton bonded to C8 was observed for the C2addition radical . In agreement with the calculations, such a coupling has only been observed in the neutral form of hypoxanthine .
3.3 . The OH-addition radical After warming to 300 K a crystal of Na,-5'-IMP X-irradiated at 77 K, lines appear in the middle of the spectrum, which are not present immediately after X-irradiation at room temperature . The orientation-dependence of these lines and the effect of deuteration on them indicate that they present the same hyperfine interactions as the C2-HH addition radical but with the coupling of one methylene proton less . Thus, instead of two equivalent fl-protons of 35 G, only one fl-proton of 20 G is observed . The effect of the C8-deuteration is shown in figure 3 : the same small coupling originating from H(C8) present in C2-HH radical is present in the doublet structure of the new lines . Figure 4 also shows that the nitrogen coupling is the same for both radicals . We attribute these lines to the radical resulting from OH-addition at position C2 . For pyrimidines, it was found experimentally that the fl-proton of OHaddition radicals is smaller than in H-addition radicals (Westhof, Flossman and Miiller 1977 a) . INDO calculations have also presented this trend . The formation of OH-addition radicals in purine-derivative single crystals has not yet been reported . However, OH-addition radicals at position C8 have been detected, after X-irradiation of frozen aqueous solutions of adenine and guanine derivatives (Gregoli, Olast and Bertinchamps 1974) . In table 3, the experimental and theoretical couplings for OH-addition radicals at position C2 or C8 of the purine ring in adenine, guanine, and hypoxanthine are given . It should be
Radical formation in hypoxanthine derivatives
1 29
i
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Figure 4 . E .s .r . spectra of single crystals of Naz -5'-IMP • (7 .5 H20), X-irradiated at 77 K and warmed to room temperature . The magnetic field is parallel to the c-axis . Top : fully protonated crystal . Bottom : C8-deuterated crystal . Stick diagrams indicate line positions of C2-HH and C2-HOH radicals . The small amount of C8-HH and C8-HD radicals could not be seen in this orientation . remarked that, in pyrimidine and purine crystals, OH-addition was found at the less frequent position of addition (i .e . C6 and C2, respectively) and that, in both cases, some relationship with an observed cation radical was apparent .
3 .4. The base radical anions In figure 5 are presented the spectra obtained after X-irradiation at 77 K of single crystals of fully protonated hypoxanthine . HCI • H20 (top) and of crystals where the C8-proton has been exchanged for a deutron (bottom) . The spectra at the left were taken at 9 . 5 GHz and those at the right at 35 GHz . The influence of the frequency of observation indicates the presence of two radicals : a doublet and a quartet . The C8-deuteration replaces the doublet by a singlet and removes the large splitting of the quartet, which appears as a singlet because of overlapping lines . Since crystals of hypoxanthine . DO . D2 0 yield identical spectra, the small splitting should arise from H(C2) . The gfactor of the quartet spectrum is more anisotropic than that of the doublet spectrum . The maximal splitting of the doublet spectrum is obtained with the magnetic field perpendicular to the C8-H bond in the molecular plane and is equal to 17 . 0 G .
130
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Radical formation in hypoxanthine derivatives
1 31
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Figure 5 . E .s .r . spectra of single crystals of hypoxanthine • HCl • H20 X-irradiated and observed at low microwave power at 77 K . The magnetic field is parallel to the purine plane and roughly normal to the C8-H bond . Top : protonated crystals . Bottom : C8-deuterated crystals . Left : frequency of observation 9 . 5 GHz . Right : frequency of observation 35 GHz .
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Figure 6 . E .s .r . spectra of single crystals of inosine (left) and Nae-5'-IMP (right) Xirradiated and observed at 77 K . Top : protonated crystals. Bottom :C8-deuterated crystals . The orientation of the magnetic field is the same as in figure 2 for the inosine spectra . For the Nae -5'-IMP spectra the magnetic field is parallel to the b-axis .
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132
H . Zehner et al .
Crystals of inosine irradiated at 77 K do not yield well-resolved spectra . The two spectra at the left of figure 6 show that the C8-deuteration has no effect . Identical spectra are also obtained with crystals grown from heavy water . Therefore, the doublet splitting present in the spectrum arises from the proton bonded to C2 . This splitting is anisotropic and has the maximum value of 15 . 0 G . At the right of figure 6 are shown spectra gained with crystals of Na e-5'-IMP The effect of C8-deuteration is to replace the quartet by a triplet . Replacement of the easily exchangeable protons by deuterons has no influence on the spectra . Therefore, the spectra arise from hyperfine interaction between the unpaired electron and the proton bonded to C8 and one nitrogen nucleus . The angle of the magnetic field with the p 7 -orbital is about 35° . The maximal nitrogen coupling is about 8 . 5 G . When the magnetic field is parallel to the a-axis (i .e . roughly parallel to the purine ring), spectra taken at 9 . 5 and at 35 GHz present a doublet of 7 . 0 G, which collapses to a singlet after C8-deuteration . In the 35 GHz spectra, an additional broad unresolved structure is visible, which indicates that at least one supplementary radical is present . It therefore appears that, in the two crystals where the hypoxanthine molecule is not protonated, irradiation produces different radicals at 77 K . In all three crystals, the radicals produced after X-irradiation at 77 K are easily photo-bleached by visible light and disappear on warming to 300 K. In the case of Na e-5'-IMP, H-addition radicals and OH-addition radicals appear after annealing to room temperature .
3 .5 . INDO calculations on the ionic-radicals Radicals produced after X-irradiation at low temperatures are usually of ionic nature . Therefore, we have calculated the anion and cation radicals of the neutral and several protonated forms of the hypoxanthine molecule . The results are contained in table 4 . First, consider the radical anions . In the electron adduct of the neutral molecule, the spin density is localized mainly on carbon C2, giving a larger coupling with H(C2) . On the other hand, the electron adduct of the N7-protonated molecule gives a larger coupling with H(C8), since the spin density is mainly localized on C8 . Experimentally, we found two radicals behaving in this manner . In inosine crystals, a radical was characterized by a doublet splitting arising from H(C2), whereas in hypoxanthine HCl - H2 O crystals, a similar radical was characterized by a doublet splitting arising from H(C8) . We therefore consider that these two doublet splittings arise from the electron adduct radical of the neutral and N7-protonated molecule . Two radicals are still to be explained : the quartets observed in hypoxanthine HCI • H2 O and Na e-5'-IMP . We tentatively assign these two different quartets to the cation radicals of the neutral molecule in the case of Na e-5'-IMP and to the cation radical of the N7-protonated molecule in the case of hypoxanthine HCl • H20 . The INDO calculations of table 4 indicate that the cation radical of the neutral molecule should present couplings with H(C8) and with Nl and N3 . We only observe coupling with one nitrogen . This could indicate that we have not calculated the proper structure of the cation radical . The same
Radical formation in hypoxanthine derivatives
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problem occurs with the cation radical of the N7-protonated molecule . It presents large couplings with the nitrogens N1 and N3 . However, it has already been observed that INDO calculation overemphasized nitrogen couplings, thereby reducing the proton couplings (Westhof and Van Rooten 1976) . The calculations indeed yield much too small couplings with the protons H(C2) and H(C8) . We did several calculations on purine-derivative cation radicals without good agreement with experimental couplings . This may reflect some limitations of the INDO method .
3 .6 .
The
N-chloro
radical
At high incident microwave power, the spectra obtained at 77 K with single crystals of hypoxanthine • HCl • H20 X-irradiated at 77 K reveal the presence of another radical characterized by large hyperfine anisotropic couplings and a strongly anisotropic g-factor (see figure 7) . At all orientations, the spectra could be analysed in terms of hyperfine interaction between the unpaired electron and one chlorine nucleus, one nitrogen nucleus, and one hydrogen nucleus .
S
i f V
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Y Yr y
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Figure 7 . E .s .r. spectra of single crystal of hypoxanthine • HCl • H20 X-irradiated and observed at high microwave power at 77 K . Top : the magnetic field is parallel to the b-axis, Bottom : the magnetic field is parallel to the (a*c*)-plane 50° from a* . Stick diagrams indicate the line positions for the N-chloro radical . The 37 C1 isotope is shown below the spectra, the 35 C1 isotope above the spectra .
Radical formation in hypoxanthine derivatives
135
As can be seen in figure 7, both chlorine isotopes, 35C1 and 37 C1, could be resolved . From the orientation dependence of the spectra, the principal values and direction cosines of the hyperfine tensors could be extracted and are contained in table 5 .
Nucleus
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Table 5 .
Principal values
Directions
113 25 25
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71 50 45
Parallel N1-H direction in crystal Parallel p z -orbital Normal to both directions
H(C2)
6 5 9
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g
2 . 001 2 . 027 2 . 023
Parallel Nl-Cl directiohn Parallel p 2 -orbital Normal to both directions
Hyperfine tensors of the N-chloro-radical in hypoxanthine • HCl • H 2 O . All values in Gauss . The g-values are no principal values .
The direction cosines of the maximal principal value of the nitrogen coupling coincides with the direction of the N1-H bond in the undamaged crystal, while those of the chlorine coupling coincide with the N1 . . . Cl - direction. These two directions make an angle of 6 ° with each other . The hydrogen coupling arises from the proton bonded to C2, because deuteration of neither the easily exchangeable protons, nor of H(C8), reduces this coupling . The coupling is slightly anisotropic and not at all typical of an a-proton . Similar spectra have been observed with single crystals of adenine • 2HC1 (Huttermann 1975), and cytosine • HCl (Westhof, Flossmann and Muller 1975) . They were assigned to the radical resulting from Cl - addition to the nitrogen N1 in adenine and N3 in cytosine after removal of the proton bonded to these nitrogens (Westhof et al. 1975) . However, no direct evidence could be presented to exclude addition at the amino group (Westhof et al . 1975) . The observation of a radical having the same hyperfine tensors and behaviour in hypoxanthine • HCl • H 2O crystals definitively proves the addition at position N1 in purine derivatives and N3 in cytosine . In adenine . 2HC1 crystals, no additional hydrogen coupling could be observed, but the line-width is about 8-12 G in these crystals and less than 4 G in hypoxanthine • HCl • H 2 O . 4.
Discussion The H-addition radicals are the main species trapped in single crystals of purine derivatives after X-irradiation at room temperature . These radicals may be formed by two reaction mechanisms, either through direct hydrogen atom addition, or through protonation of an electron adduct (Westhof et al . 1977 b) . We could not ascertain whether one of these reactions leads preferentially to one kind of radicals . However, there is evidence that both reaction
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136
H. Zehner et al .
mechanisms occur . Indeed, irradiation with light of A > 300 nm of hypoxanthine ' HCl • H20 yields both kinds of H-addition radicals . On the other hand, the presence of ionic radicals at 77 K indicates that ionic reactions are possible. The OH-addition radical observed in Na,-5'-IMP could also be produced through an ionic precursor . In this case, the cation radical and not the anion radical would be involved (Gregoli et al . 1974) . It is interesting to note that in Nae -5'-IMP, no anion radicals were detected but instead most probably cation radicals . The presence of water and not OH-groups from the sugar seems to be necessary to form the OH-addition radical, since such radicals were never observed in single crystals of nucleosides which crystallize without so many water molecules . In table 6, the results are presented concerning the occurence of H-addition radicals in all purine derivatives (except guanine) we have analysed . The crystals can be divided into three groups . The first, and smaller, contains those where only C2-addition radicals were observed . In the second, which is slightly larger, only C8-addition radicals are detected . And finally, in the third group, both kinds of H-addition radical are produced . Neither the substituents, nor the molecules of crystallization or the protonation state seems to be decisive for the production of one kind of radical . In the case of the doubly-protonated adenine molecule, however, the formation of C8-addition radicals is preferred . It appears also that the C2-addition radical is less stable than the C8-addition radical (Zehner et al. 1977) . The local crystal environment seems, therefore, to be the dominating factor controlling the formation and the stability of these H-addition radicals . Crystal
C 2
C 8
Adenine • +H 2 SO 4 • H20 Na2-5'-inosine monophosphate • (7 . 5 H 2 O) Ba-5'-inosine monophosphate • 6 H20t
X X X
-
Adeninet 9-CH 3 -Adenine Nae -5'-Adenosine monophosphatet Purine ribosidef Adenosine • DMSO Adenine • H2SO 4 9-CH3-Adenine • 2HBr
-
X X X X X X X
Adenine • 2HC1 Adenine • HCl • H2O Desoxyadenosine • H20 Adenosine Adenosine • HCl 5'-Adenosine monophosphate • H2 0 Hypoxanthine • HCl • H20 Inosine Na-5'-inosine monophosphate • 8 H20t
X X X X X X X X X
X X X X X X X X X
t Results were gained from powder spectra . Table 6 .
Occurrence of H-addition radicals in crystals of purine derivatives after X-irradiation at room temperature .
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Radical formation in hypoxanthine derivatives
137
La formation de radicaux dans des monocristaux de ('hypoxanthine • HCI • H20, inosine et Na e-5'-IMP • 7,5 H 2 O a ete analysee par resonance paramagnetique electronique (9,5 et 35 GHz) sous 1'effet de rayonnement . Des radicaux de 1'addition d'hydrogene en C2 et en C8 du noyau purique ont ete identifies dans tons les cristaux . Les constantes de couplages de ces deux radicaux sont differentes et dependent fortement de l'etat des protons de la base . Les calculs par la methode INDO indiquent, qu'au radical C8 l'addition d'un proton a lieu en 06 . L'addition du groupement OH en C2 du noyau purique peut etre observee daps la molecule de Na e -5'-IMP . Sous 1'effet du rayonnement X a 77 K l'addition d'electrons pent etre observee daps la base neutralisee et protonis6e en N7 . Aucune addition d'electrons s'est formee dans la molecule de Nae -5'IMP-, cependant un radical etant probablement le cation . Dans la molecule de l'hypoxanthine • HCl • H20 un autre radical a ete trouve sous 1'effet du rayonnement X a 77 K provenant de l'addition d'un Cl- sur l'azote NI du noyau purique . Die Radikalbildung in Einkristallen von Hypoxanthin • HCl • H 2O, Inosin and Nae -5'IMP • 7,5 H20 durch Rontgenbestrahlung wurde mit Hilfe der Elektronenspin-ResonanzSpektroskopie bei 9 . 5 and 35 GHz untersucht. In allen Kristallen wurden H-Anlagerungsradikale an Position C2 and C8 des Purinrings gefunden . Die Kopplungskontanten dieser beiden Radikale sind unterschiedlich and hangen sehr vom Protonierungszustand der Base ab . INDO-Rechnungen deuten an, daf3 das C-8-Radikal an 06 protoniert ist . In Nae -5'-IMP wird OH-Anlagerung an C2 des Purinrings beobachtet . Nach Rontgenbestrahlung bei 77 K findet man in der neutralen and an N7-protonierten Base Elektronenaddukte . In Na e -5'-IMP wird kein Elektronenaddukt gebildet, jedoch ein Radikal, das moglicherweise das Kation ist . In Hypoxanthin • HC1 - H2O kann man nach Rontgenbestrahlung bei 77 K ein weiteres Radikal beobachten, das durch Anlagerung eines Cl - an den Stickstoff N1 des Purinrings entstanden ist .
REFERENCES
ALEXANDER, CH ., and GORDY, W ., 1967, Proc . natn . Acad . Sci. U .S.A ., 58, 1279. GREGOLI, S ., OLAST, M ., and BERTINCHAMPS, A ., 1974, Radiat. Res ., 60, 388 . HUTTERMANN, J ., 1975, Y. magn . Res., 17, 66 . MUNNS, A . R . J ., and TOLLIN, P ., 1970, Acta crystallogr ., B, 26, 1101 . NAGASHIMA, N ., and IITAKE, Y ., 1968, Acta crystallogr. B, 24, 1136 . SLETTEN, J ., and JENSEN, L . M ., 1969, Acta crystallogr. B, 25, 1608 . SUBRAMANIAN, E., MADDEN, J . J ., and BUGG, C . E ., 1973, Biochem . biophys . Res. Commun ., 50,691 . THEWALT, U ., BUGG, C . E ., and MARSH, R . E ., 1970, Acta crystallogr., B, 26, 1089 . WESTHOF, E ., FLOSSMANN, W ., and MULLER, A ., 1975, Int ., . Radiat . Biol., 28, 427 ; 1977 a, Radiat . Res ., 70, 304 . WESTHOF, E ., FLOSSMANN, W ., ZEHNER, H ., and MULLER, A ., 1977 b, Discuss. Faraday Soc., 63 (in the press) . WESTHOF, E ., and VAN ROOTEN, M ., 1976, Z. Naturf. C, 31, 371 . ZEHNER, H ., FLOSSMANN, W., and WESTHOF, E ., 1976, Z. Naturf. C, 31, 225 . ZEHNER, H ., WESTHOF, E ., FLOSSMANN, W ., and MULLER, A ., 1977, Z. Naturf. C, 32, 1 .
R .B .
K
