INT . J . RADIAT . BIOL .,
1976,
VOL .
30,
NO.
4, 301-315
Radical formation in salts of pyrimidines III. Cytosine . Hao crystals W. FLOSSMANN, E. WESTHOF and A. MOLLER Institut fur Biophysik and physikalische Biochemie, Universitat Regensburg, D-84 Regensburg, Germany
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(Received 7 June 1976 ; accepted 6 August 1976)
The radicals produced by X-irradiation at 77 K and at 300 K in cytosine monohydrate crystals have been analysed by electron-spin-resonance (e .s .r.) spectroscopy . Three radicals have been identified at 77 K : the anion radical and the cation radical of the cytosine molecule, together with the radical resulting from H-abstraction from the nitrogen N1 . Irradiation at 300 K produces radicals resulting from H-addition at three different positions of the cytosine molecule . These are the C5-addition radical, the C6-addition radical, and the 02-addition radical . The results are compared with those found previously by other authors .
1. Introduction The free radicals produced by ionizing radiation in single crystals of cytosine monohydrate have been analysed repeatedly (Herak and Galogaza 1969, Cook, Elliott and Wyard 1967, Dertinger 1967, Herak, Galogaza and Dulcic 1969). Herak and Galogaza (1969) identified after irradiation at 77 K the cytosine cation radical but were not quite sure about the identification of another radical, which they tentatively assigned to the cytosine anion radical . We have examined single crystals of cytosine . H2 0 after X-irradiation at 77 K and were able to isolate three different radical species. Despite correct identification, the hyperfine tensors for the cation radical given by Herak and Galogaza (1969) had to be corrected substantially, owing to the presence of a third species in the e.s .r . spectra. Our analysis has yielded evidence for the assignment of the anion radical of the cytosine molecule without any doubt . The third species is found to be the radical resulting from hydrogen abstraction from the nitrogen N1 . Such an identification has been previously proposed by Cook et al. (1967) for some lines of the spectra obtained after X-irradiation at 300 K . Further, these authors identified the radical resulting from H-addition at position C5. We show that the lines attributed by Cook et al . (1967) to the radical resulting from H-abstraction from N1 are indeed the result of D-addition at position C5 . A further radical resulting from H-addition to 02 was tentatively assigned by Dertinger (1967) to a small doublet present in the spectra taken at 35 GHz . In two previous papers, we have shown that the radical resulting from H-addition at position C6 is also present (Flossmann, Westhof and Muller 1976) and that the identification of Dertinger (1967) is correct (Westhof, Flossmann and Muller 1975) . In the present paper, the hyperfine tensors of these two radicals are given . In addition to these, two new radicals have been isolated. Because of the total number of different radicals produced with similar hyperfine couplings, the spectra are very complex . Therefore, a whole gamut R.B .
X
302
W. Flossmann et al .
of methods was employed for the separation of the various components . Variation of temperatures was combined with bleaching by light as a standard routine . We should like to emphasize that our analysis rests heavily on results obtained through specific deuteration of the hydrogen bonded to carbon C5 and on the use of two frequencies of observation . Furthermore, the strong dependence of the spectra on the dose of radiation given to the crystal was exploited . Also, crystals doped with copper were investigated . H\N/H
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pg i
N3
~H
5li s
0~ \/C N H I
H Cytosine
2 . Experimental Single crystals of cytosine monohydrate were grown from saturated aqueous solutions by slow evaporation at room temperature . The crystal structure was reported by Jeffrey and Kinoshita (1963) and later refined by McClure and Craven (1973) . The crystals are monoclinic with space group P2 1 /c . There are four molecules in the unit cell . For the e .s .r . measurements, an orthogonal system a*, b, c was chosen . The b-axis is parallel to the pyrimidine ring and nearly perpendicular to the C5-H bond . The other two axes are skew with respect to the molecular plane . Three types of crystal have been analysed . In crystals of type A, the cytosine molecule is fully protonated (cytosine . H 2O) . In crystals of type $, the hydrogen atoms bonded to the nitrogen atoms (amino group and N1) are exchanged with deuterium (cytosine . D2 0) . Finally, in the crystals of type C, the hydrogen atom bonded to C5 has also been replaced by deuterium (H6cytosine . D2 0), leaving only the hydrogen at C6 protonated . The proton at position C5 was exchanged with deuterium in the following way . First, concentrated DC1 solutions of cytosine were warmed to 100°C for some hours . The chlorine ions were then removed by neutralization . This substitution was verified by high-resolution NMR . The experimental procedure and equipment were as described previously (Flossmann, Westhof and Muller 1975) . 3. Results 3 .1 . Irradiation and observation at 77 K The spectra obtained at different orientations with single crystals of cytosine . H 2O, cytosine . D 20, and H6-cytosine . D 2 0 are presented in figures 1, 2, and 3 . The orientation of the magnetic field is for figure 1 in the molecular plane perpendicular to the b-axis, i .e . roughly parallel to the C5-H bond ; for figure 2, it is parallel to the b-axis, i .e . nearly perpendicular to the C5-H bond ; and for figure 3, perpendicular to the molecular plane . The second spectrum of figure 1 is the same as the cne presented in figure 3 of reference (Herak and Galogaza 1969) . The orientation should be, however, perpendicular to the
Radicals in cytosine . H2O
303
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P
t II
t II
V
I 20G
,_
Figure 1 .
E .s.r. spectra of crystals of cytosine . H20 (upper spectrum), cytosine . D,O (middle spectrum), and H6-cytosine . D 2 0 (lower spectrum) X-irradiated and observed at 77 K . The frequency of observation is 9 . 5 GHz. The magnetic field is oriented perpendicular to the b-axis in the molecular plane . The stick diagrams indicate the lines due to the radicals numbered by roman figures on the right .
b-axis and not parallel to it as indicated in the caption of figure 3 of reference (Herak and Galogaza 1969) . A comparison of the three spectra of figure 1 shows that only the doublet of about 20 G located at g=2 . 0028 is independent of the deuteration stage . This coupling therefore arises from the proton bonded to C6, which is the only proton present in crystals of type C . Another radical characterized by a singlet
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I
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II
I II
I II
Figure 2 . E .s .r. spectra of cytosine crystals of type A, B and C after X-irradiation and observation at 77 K . The frequency of observation is 35 GHz . The magnetic field is parallel to the b-axis . The arrows indicate the central lines of Mn++ (MnO in MgO) used as a standard .
at g = 2 . 0050 is also present in crystals of type C . In crystals of type B, where the proton bonded to C5 is not exchanged, a doublet of about 7 G is observed at g=2-0050 instead of a singlet . This doublet therefore arises from the proton bonded to C5 . In the fully-protonated crystals, another coupling splits the doublet to give a 1 : 2 : 1 triplet . Thus, two couplings contribute to the spectra of this second radical : one from the proton bonded to C5 and one from a proton bonded to a nitrogen atom . The spectra of figure 2 were taken at 35 GHz to separate the overlapping lines of these two radicals . The third spectrum of figure 2 (type C) shows a small doublet of 10 G at g = 2 . 0031 and a large singlet at g = 2 . 0039 . In crystals of type B, a doublet of 22 G is found at 2 .0039, and the small doublet is unchanged . The large doublet must now be assigned to a proton bonded to C5
Radicals in cytosine . H20
305
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and the small one to a proton bonded to C6 . Such a behaviour for the two radicals observed is expected from the theoretical anisotropy of an a-proton . Indeed, in figure 1, the magnetic field is approximately parallel to the C5-H bond direction in the molecular plane . Therefore, at this orientation, an a-proton bonded to C5 should give minimal coupling, and one bonded to C6 a much larger one . On the other hand, in figure 2, the magnetic field is perpendicular to the C5-H bond direction, and an a-proton bonded to C5 must have a maximal coupling, whereas one bonded to C6 must have a much smaller coupling . In crystals of type A, another coupling of 5 G splits the lines of the doublet of 22 G.
I
'j
JV
.1
1
20G I
Figure 3 . E .s .r. spectra of cytosine crystals of type A, B and C X-irradiated and observed at 77 K . Same remarks as for figure 1 apply. The magnetic field is perpendicular to the molecular plane .
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From the spectra taken with the magnetic field in the molecular plane, one can conclude that oneradical is characterized exclusively by an (X-proton bonded to C6, and. the other by an a-proton bonded to C5 and one bonded to a nitrogen atom . Figure 3 shows the spectra obtained when the magnetic field is perpendicular to the molecular plane for crystals of type A, B, and C . The first two spectra are similar to those presented in figure 2 of reference (Herak and Galogaza 1969) . These authors interpreted the second spectrum of figure 3 as arising mostly from a radical presenting two non-equivalent nitrogen couplings and one a-proton . However, the third spectrum of figure 3, obtained with crystals of type C, indicates the presence of three radicals . In the middle of the spectrum a doublet of narrow lines with a splitting equal to 15 G is present accompanied by several less intense lines on both sides . The lines of this doublet are present in all three spectra and should therefore arise from the a-proton bonded to C6 . At this orientation, a radical containing spin density in the 2p .,-orbital of a nitrogen atom should present maximal hyperfine coupling with the nitrogen . The numerous and less intense lines are therefore assigned to the radical previously characterized by an a-proton at C5, which should present hyperfine interaction with two non-equivalent nitrogen atoms equal to 13 . 1 G and 4 . 0 G, respectively . With the same nitrogen couplings, the second spectrum of figure 3 can be explained if the coupling of the a-proton bonded to C5 is equal to 11 . 2 G . The broad lines present on each side of the spectrum do not belong to this second radical, for the variation of their spacing following deuteration at C5 is different . These lines are due to a third radical presenting hyperfine coupling with one nitrogen (25 G) and with the a-proton bonded to C5 (13 . 7 G) . Since they assumed that all the lines of the second spectrum of figure 3 belong to the same radical, Herak and Galogaza (1969) obtained wrong hyperfine tensors for the second radical . The presence of a third radical can be deduced from the variable intensity of the lines in the spectra of figures 2 and 3 . This third radical must accordingly possess a similar hyperfine coupling with the proton bonded to C5 as that of the second radical . The spectra presented in figure 4 confirm the fact that we are dealing with three different radical species . The upper spectrum of figure 4 is obtained with single crystals of cytosine . H 2 O doped with copper ions . The relative concentration of the first and second radical is drastically changed after Cu++-doping . This spectrum should be compared with the first spectrum of figure 1 . On the other hand, the second spectrum of figure 4 is obtained with cytosine crystals of type C after irradiation at 77 K followed by illumination with the red light of a He-Ne laser (A=633 nm) . A comparison between this spectrum and the third one of figure 3 clearly shows that the third radical is not so sensitive to light as the first two radicals . Further, the second spectrum of figure 4 confirms that the third radical is characterized by a 1 : 1 : 1 triplet and not a large doublet . One can conclude that, after irradiation of cytosine monohydrate crystals at 77 K, three radicals are produced . The first one presents hyperfine interaction with the proton bonded to C6 ; the second one with the proton bonded to C5, with a proton bonded to a nitrogen, and with two non-equivalent nitrogen atoms ; and finally, the third one has interaction with the proton bonded to C5 and with one nitrogen atom . The principal values and principal directions of the different hyperfine tensors are contained in table 1 .
Radicals in cytosine . H2 O
HEN/H
HEN H
I ,C,,
I C
I
0/C
OAC H ~ H
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Radical I
H \N/H
I
I N /H
/H
I p
II
Radical (I) a-H(C6)
Radical (II) a-H(C5)
Ni
N3
g
Radical (III) N a-H(C5)
C
Radical III
Direction cosines b
Anisotropic
-14 . 5
Isotropic
-7 . 1 -22 .0 -11 . 2
+6 . 3 -8 .6 +2 . 2
-13 . 4 4.0 0.0
Isotropic
25 .0 -13 .7
C
Radical 11
+8 . 5 -8 . 0 -0 . 5
2 .0023 2 . 0040 to 2 .0050
CH
C/ \N/ H
- 6.0 -22 . 5 -15 . 0
13 . 1 0 .0
N~C~
H
O#C\N/C I H
Principal values (G) Total
307
c
0 . 3582 0 . 7713 0 .4670
-0. 9202 0 .4725 0 . 0309
-0 . 1580 -0 . 4264 0 . 8790
0 . 8709 0 .0166 0 .4904
0 . 0242 -0 . 9998 0 . 0150
-0 . 4907 0 . 0079 0 . 8711
parallel 2p .-orbital in the molecular plane parallel 2p -orbital in the molecular plane parallel 2p, -orbital in the molecular plane
parallel 2p .--orbital parallel 2p,,-orbital
Table 1 . Hyperfine tensors for the a-protons of the anion radical (I) and of the cation radical (II) . The maximal couplings of the two non-equivalent nitrogens of radical II are given, as well as its g-tensor . The g-tensor of radical III is similar to that of radical II . The probable errors are estimated to be ± 1 . 0 G for the hyperfine tensors and ± 0 .0002 for the g-tensor . The probable errors of the direction cosines are estimated to be ± 6° . The direction cosines 1, m, n are referred to the a*, b, c axis respectively . The direction cosines for the other site are given by 1, - m, n .
The first radical is clearly the anion of the cytosine molecule, since pyrimidine anion radicals typically present hyperfine interaction with the proton bonded to C6 (Baudet, Berthier and Pullman 1962, Sevilla 1971) . Anion radicals of the cytosine molecule (radical I) have previously been observed in single crystals of cytidine-3'-phosphate (Box, Potter and Budzinski 1975) and of cytosine . HCl
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III
Figure 4 . Top spectrum : E .s .r . spectrum obtained with a single crystal of cytosine . HO doped with copper ions after X-irradiation and observation at 77 K . The magnetic field is oriented perpendicular to the b-axis in the molecular plane . Bottom spectrum : E .s .r. spectrum obtained with a single crystal of H6-cytosine . D S O after X-irradiation and subsequent illumination with a He-Ne laser at 77 K . The temperature of observation is 77 K . The magnetic field is perpendicular to the molecular plane .
(Westhof et al. 1975) . The second radical is the cation radical (radical II), which was correctly identified by Herak and Galogaza (1969) . The coupling of the easily exchangeable proton should arise from the proton bonded to N1 . The occurrence of such a coupling in this second radical indicates that the cation radical is still positively charged . We identify the third radical with the deprotonated cation radical, which is equivalent to the radical resulting from hydrogen abstraction from the nitrogen NI (radical III) . With the help of INDO calculations, we have shown that the thymine cation radical and the radical resulting from hydrogen abstraction from N1 should present the same couplings, except for the supplementary coupling of the substituent at N1 in the cation radicals (Westhof and Van Rooten 1976) . This has been experimentally verified by Sevilla (1976) . In this connection, one should note that the 2p z-spin density distribution in the cytosine cation radical compares well with that in the thymine cation radical : pcs 0 . 20 ; p Nl 0 . 40 (Sevilla 1976) . However, thelarge nitrogen coupling of radical III, 25 G, is closer to that, 28 . 8 G, of the radical resulting from hydrogen abstraction in barbituric acid crystals (Hiittermann, Schmidt and Weymann 1976) . INDO calculations indicate that the barbituric acid cation radical has a much lower nitrogen coupling than the radical resulting from hydrogen abstraction from N1 . In the case of the
Radicals in cytosine . H20
309
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cytosine cation radical, INDO calculations yield Ni11= 7 . 1 G and =15 . 9 G, N311 and H(C5) I8O = -2 . 0 G . As in the case of thymine, the couplings of the nitrogens are calculated too large with the consequence that the coupling due to the proton bonded to C5 is too small . However, the deprotonated cation radical is not easy to calculate, because of convergence problems . This could indicate that the radical resulting from hydrogen abstraction from N1 exists in a tautomeric form different from that of the undamaged molecule . More detailed calculations will be presented elsewhere . On warming a crystal irradiated at 77 K to room temperature most radicals disappear which renders any study of secondary radical reactions impossible . 3 .2 . Irradiation and observation at 300 K Irradiation at room temperature of cytosine monohydrate produces three different hydrogen addition radicals : the C5-addition radical (radical IV), the C6-addition radical (radical V), and the 02-addition radical (radical VI) . H\ N/H
H\N/H
N *L-C,H I I \H O/CAN "C~
I
K\ /H N
I
I
H\ ,Cy
N.
H I H
I
H
H
Radical IV
Radical V
I
N"CyH II
1
HC'C'N'C" I H H Radical VI
The maximal yield of C5-addition radicals is obtained at doses ranging from 2 to 3 Mrad . On the other hand, the 02-addition radical dominates the spectra only at very high doses (-10 M rad) . The concentration of C6-addition radical is always very low after X-irradiation . However, we have shown that subsequent illumination with light of A > 400 nm induces a transformation of C5-addition radicals into C6-addition radicals (Flossmann et al. 1976) . A high concentration of C6-addition radicals can be produced in this way . The C5addition radical was first identified by Cook et al. (1967) . The hyperfine tensor of the a-proton bonded to C6 was later refined (Herak et al . 1969) . Cook et al . (1967) observed that, at microwave power greater than 1 mW, the middle part of the spectrum saturates at a different rate from the outer lines of the spectrum . The central part of the spectrum is, in fact, due to the 02-addition radical, which was first identified by Dertinger (1967) at 35 GHz . 3 .2 .1 . The C5-addition radical (radical IV) This radical is characterized by two equivalent and isotropic #-protons of 37 . 0 G and an a-proton bonded to C6 . We have redetermined the hyperfine tensor of this a-proton and the results are contained in table 2 . Because of the presence of spin density on the nitrogen N1, the line-width of this radical is large and anisotropic . Along the N1-C6 bond, the coupling of-the nitrogen Nl is 2 . 9 G. This coupling is minimal when the magnetic field is perpendicular to the molecular plane. Therefore, the width of the lines of the C5-addition radical is minimal (about 4 . 0 G), and this radical is most easily detected at this orientation . It has already been noticed that the hyperfine coupling of such an a-nitrogen is not maximal when the magnetic field is perpendicular to the
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3 10
Principal values (G) Total
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Radical (IV) a - H(C6)
Radical (V) a-H(C5)
Radical (VI) a - H(C6)
-8 . 1 -30-0 -17 . 7
+10 . 5 -11 .4 +0 . 9
-18 . 6
Isotropic
-8-5 -26 . 5 -15 .0
+8 . 2 -9 . 8 +1 . 7
-16 . 7
Isotropic
-4. 5 -12 . 3 -9 . 1
+4 . 1 -3-7 -0 . 5
-8-6 Radical (VI) g
Anisotropic
Direction cosines a*
b
c
0 . 3582 0 . 7713 0 . 4670
-0 . 9202 0 . 4725 0 . 0309
-0 . 1580 -0-4264 0 . 8790
0 . 8709 0 . 0166 -0 . 4904
0 . 0242 -0 . 9998 -0 . 0150
-0-4907 0 . 0079 -0 . 8711
-0 . 068 0 . 865 -0 . 497
0 . 997 0 . 082 0 . 007
-0 . 046 0 . 495 0 . 868
0 . 863 -0 . 015 -0 . 505
0 . 038 0 . 999 0 . 035
0 . 504 -0 . 049 0 . 862
Isotropic
2 . 0075 2 . 0052 2 . 0026
+0 .0024 +0 .0001 -0 . 0025
2 . 0051
Isotropic
Table 2 . Hyperfine tensors for the a-protons of the C5-addition radical (IV), of the C6-addition radical (V), and of the 02-addition radical (VI) . The g-tensor of radical VI is also given . Remarks as for table 1 apply . molecular plane, but minimal (Flossmann, Huttermann, Muller and Westhof 1973) .
The upper spectrum of figure 5 is obtained with a cytosine . D 2 0 crystal irradiated at 300 K and observed with the magnetic field perpendicular to the molecular plane . As was already noted by Cook et al. (1967), even in deuterated crystals the hydrogen addition to C5 is present (C5-HH radical) . In addition, several other lines, which were not present in cytosine . H20 crystals, dominate the spectrum. The upper spectrum of figure 5 is not exactly the same as the one represented in figure 7 of reference (Cook et al. 1967) . However, at a specific orientation and after a specific dose, all deuterated crystals observed yield the same spectrum as the one of figure 7 in reference (Cook et al. 1967) . Since at this orientation, the magnetic field is perpendicular to the molecular plane, Cook et al. (1967) analysed this spectrum as arising from two nonequivalent nitrogens and one a-proton . It is, however, possible to interpret all these lines at all orientations with one fl-proton of 37 . 0 G, one a-proton bonded to C6, and one isotropic coupling to a deuterium atom (5 . 5 to 5 . 9 G) . This deuterium coupling is precisely what should be expected if a a-proton of 37 . 0 G is replaced by a deuteron (37 . 0 /6 . 51 = 5 . 68) . Therefore, we attribute to these lines the radical resulting from D-addition at position C5 (C5-HD In contrast, Cook et al. (1967) assigned to these lines the radical radical) .
Radicals in cytosine . H2 O
311
MMD)
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~---T .
W(HH)
V(HH)
Figure 5 . E .s .r . spectra of crystals of cytosine . D2 0 X-irradiated and observed at 300 K at 9 . 5 GHz . For the upper spectrum, the magnetic field is perpendicular to the molecular plane . For the two bottom spectra, it is parallel to the b-axis. The last spectrum was obtained after subsequent illumination with light of A > 400 nm . In this spectrum, the lines due to the C6-HH radical with a D-atom at position C5 are indicated by stick diagrams at the bottom .
resulting from H-abstraction from the nitrogen N1 . Further evidence for our assignment is given in the next section . 3 .2 .2 . The C6-addition radical (radical V) In a previous paper (Flossmann et al. 1976), we have shown that, in pyrimidine derivatives, the C5-addition radicals transform into C6-addition radicals on irradiation with light of wavelength A > 400 nm . Immediately after irradiation at room temperature, the lines belonging to this radical are already
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present, albeit with a low intensity, as can be seen from the second spectrum of figure 5 . After irradiation with light, the concentration of the C6-addition radical is high enough to permit an analysis of the hyperfine tensors . The hyperfine tensor of the a-proton bonded to C5 is given in table 2 . The isotropic coupling of the methylene protons is about 50 . 0 G . These fl-protons are, in fact, slightly non-equivalent (49 . 0 and 53 . 0 G) . In contrast to the C5-addition radical, the width of the lines of the C6-addition is minimal in the molecular plane and maximal perpendicular to it . INDO calculations yield some spin density on the nitrogen atom N3 which in the C6-addition radical is not an a-nitrogen as the N1 nitrogen is for the C5-addition radical . With the help of INDO calculations, we have also shown that the C6-addition radical has to be protonated at N3 in order to reproduce the large fl-proton couplings (Flossmann et al. 1976) . The second spectrum of figure 5 shows also that after X-irradiation of cytosine . D 20 crystals, both H- and D-addition radicals to both positions C5 and C6 are produced . On subsequent illumination with light, the concentration of both types of radical V resulting from H- or D-addition to C6 increases, whereas most of the two corresponding C5-radicals disappear . However, additional lines are clearly detected (figure 5, third spectrum) after irradiation with light . These lines are due to the C6-HH radical with a D-atom at position C5 . These observations reinforce our revised assignment of the C5-HD radical for, on irradiation with light, this radical is able to yield either the C6-HD radical or the C6-HH radical with a deuterium at position C5 . 3 .2 .3 . The 02-addition radical (radical VI) The identification and the structure of this radical has already been discussed elsewhere (Westhof et al. 1975) . By comparing spectra taken at 35 GHz of cytosine . H2 0 and cytosine . HC1 crystals, we were able to show that, for the 02-addition radical, the doublet splitting presented originates from the proton bonded to C6 and not from the proton added to 02 (Westhof et al. 1975) . The principal values and principal direction cosines of both the g-tensor and the hyperfine tensor are given in table 2 . The direction cosines with respect to the a*, b, c axis system of the C2-02 bond, as calculated from the crystal structure, are the following : 0 . 908, 0 . 033, 0 .416. The angle between the direction of this bond and that of the maximal principal value of the g-tensor is equal to 6° . A comparison of the direction cosines of the C6-H bond and those of the minimal principal value of the hyperfine tensor would not give such a good agreement ; the two directions make an angle of 23° . This arises from the treatment of the experimental data . We have diagonalized separately the g-tensor and the hyperfine tensor . Such a procedure is incorrect in case of strong anisotropy of the g-tensor and when the principal axes of the g-tensor are different from those of the hyperfine tensor . However, we could not follow the doublet splitting through sufficient orientations to make a correct analysis . Nevertheless, the third spectrum of figure 6 clearly indicates that the hyperfine coupling is due to the proton bonded to C6, since this spectrum has been obtained with a crystal of type C, where H(C6) is the only non-exchanged proton . A supplementary coupling of 3 . 0 G splits the doublet lines when the magnetic field is in the molecular plane and perpendicular to the b-axis . This
Radicals in cytosine . H2 O
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L
L TI
,.
200
313
VI
VI
,
Figure 6 . E .s .r. spectra of cytosine . D,0 crystals after low dose (top) and high dose (middle) of radiation. The magnetic field is parallel to the b-axis . The arrows indicate the lines of Mn++ . The lower spectrum is obtained with a crystal of type C after a high dose of radiation . The orientation of the magnetic field is the same as for the two upper spectra .
coupling disappears in crystals grown from D 20 . One could attribute to this splitting the hydroxyl proton . However, from INDO calculations and from the orientation of maximal coupling, we think that the proton bonded to N1 is responsible. The lines of the doublet splitting become very broad when the magnetic field is perpendicular to the molecular plane . As confirmed by INDO calculations, this is due to hyperfine interaction with the two nitrogens N1 and N3 . The 02-additional radical is already present at low doses (0.4 Mrad) . However, at high doses (10 Mrad), it completely dominates the spectrum.
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This can be observed in the first two spectra of figure 6 . The upper one is obtained after a low dose . The lines due to the C5-HH and C5-HD radicals can be observed as well as those of a large doublet of 65 G . The second spectrum is obtained after a high dose . The C5-addition radical can only be observed at high gain, but the small doublet of the 02-addition radical is prominent . However, two lines separated by 27 . 5 G are also present . These lines do not belong to the 02-addition radical, since they disappear when the H(C5) proton is exchanged with a deuteron (third spectrum of figure 6) . The radicals responsible for the doublet of 27 . 5 G and for the large one of 65 G will be discussed in a following paper . 4.
Conclusions By comparing the formation of H-addition radicals in single crystals of pyrimidine derivatives (Flossmann et al. 1973, Flossmann et al. 1975, Westhof et al. 1975, Flossmann et al. 1976), the following conclusions emerge . H-addition radicals can be the result of direct hydrogen addition, or of a two-step process through protonation of an anionic intermediate radical . We have shown that the first mechanism-the excitation path-is predominant in van der Waalscrystals, and the second one-the ionization path-in strongly hydrogen-bonded crystals containing small polar molecules . Whereas the ionization path could not be detected in the van der Waals crystals formed by the pure bases, in crystals containing molecules of crystallization both paths have been observed . The origin of the hydrogen is either the methyl groups or the hydrogen bonds . In bases non-substituted at N1, hydrogen may be supplied directly by the ring, since radicals resulting from hydrogen abstraction from the nitrogen N1 are observed (Dulcic and Herak 1972, Zehner, Flossmann, Westhof and Muller 1976) . Concerning the sites of attack, we have shown that protonation of the electron-adduct radicals occurs at C6, giving C6-addition radicals, and that the C5-addition radicals result from hydrogenation of the neutral molecule (Westhof and Flossmann 1975, Flossmann et al. 1975, Westhof et al . 1975) . Indeed, until now, C6-addition radicals have been detected only if anion radicals could be stabilized at lower temperatures . On the other hand, C5-addition radicals have been observed in all crystals studied . Furthermore, INDO calculations have shown that C5-addition radicals are neutral and C6-addition ones positively charged in unsubstituted pyrimidines (Flossmann et al . 1976) . Because of its charge, the C6-addition radical is only stabilized in an anionic environment . This explains the absence or small concentration of C6-addition radicals in van der Waals crystals and its increased concentration in crystals containing polar molecules . In all previous crystals studied, the electron adduct radicals were in fact neutral since the bases were already protonated in the undamaged crystals . Upon further protonation at C6, these electron adduct radicals lead to the positively-charged C6-addition radicals . The cytosine monohydrate crystals are interesting, since in them the radicals resulting from electron addition or abstraction remain negatively and positively charged . Next to the competition between the two carbons of the C5-C6 double bond, the oxygen 02 is also a position of hydrogenation in cytosine . In cytosine . H 2O crystals, the concentration of 02-addition radicals at high doses
Radicals in cytosine . H2 0
315
is much higher than that of any other radical . The mechanism of formation of this radical is not yet clear . In both crystals where we could detect it, the oxygen 02 participates in an inter-pyrimidine hydrogen bond . This could be an important condition for its formation, as already noted by Dertinger (1967) . In crystals of 1-CH 3-cytosine . HBr and deoxycytidine . HCl, where the oxygen 02 fails to participate in hydrogen bonding, we could not find the characteristic lines of the 02-addition radical . Further examples will be needed to reach definite conclusions .
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ACKNOWLEDGMENT
This research was supported by grants from the Deutsche Forschungsgemeinschaft . Les radicaux induits par irradiation X a 77 K et a 300 K dans des monocristaux de cytosine . H 2 O ont ete analyses par la methode de resonance paramagnetique electronique (r.p .e .) . A 77 K, trois radicaux ont ete identifies : le radical cationique et le radical anionique de la molecule de cytosine ainsi que le radical produit par l'abstraction d'hydrogene de l'azote N1 . L'irradiation a 300 K produit des radicaux qui resultent de l'addition d'hydrogene sur trois atomes de la molecule de cytosine . Ceux-ci sont le radical d'addition en C5, le radical d'addition en C6, et le radical d'addition en 02 . Ces resultats sont compares a ceux trouves precedemment par d'autres auteurs. Es wurden die Radikale mit Elektronen-Spin -Resonanz-(ESR)-Spektroskopie analysiert, die durch Rontgenstrahlen in Kristallen von Cytosin-Monohydrat bei 77 K and 300 K erzeugt werden . Drei Radikale wurden bei 77 K identifiziert : das AnionRadikal and das Kation-Radikal des Cytosinmolekuls sowie das Radikal, das durch H-Abstraktion von N1-Stickstoff entsteht. Bestrahlung bei 300 K erzeugt Radikale, die durch H-Addition an drei verschiedenen Positionen des Cytosinmolekuls entstehen . Es sind dies das C5-Additionsradikal, das C6-Additionsradikal and das 02-Additionsradikal . Die Ergebnisse werden mit deren anderer Autoren verglichen .
REFERENCES BAUDET, J., BERTHIER, G ., and 264, 762 . Box, H . C ., POTTER, W . R ., and COOK, J . B ., ELLIOTT, J . P ., and DERTINGER, H ., 1967, Z. Naturf.,
PULLMAN, B ., 1962,
C . r . hebd. Seanc. Acad. Sci. Paris,
BUDZINSKI, E . E ., 1975, J. chem . Phys ., 62, 3476 . WYARD, S . J ., 1967, Molec. Phys., 13, 49 . 22b, 1266 . DuLCic, A ., and HERAK, J . N ., 1972, Y. chem. Phys ., 57, 2537 . FLOSSMANN, W ., HUTTERMANN, J ., MOLLER, A ., and WESTHOF, E ., 1973, Z. Naturf., 28c, 523 . FLOSSMANN, W ., WESTHOF, E ., and MOLLER, A., 1975, Int. Y. Radiat. Biol ., 28,105 ; 1976, chem . Phys., 64, 1688 . HERAK, J . N ., and GALOGAZA, V ., 1969, J. chem . Phys., 50, 3101 . HERAx, J . N., GALOGAZA, V., and DuLcIc, A ., 1969, Molec. Phys ., 17, 555 . HUTTERMANN, J ., SCHMIDT, G., and WEYMANN, D ., 1976, Y. Mag. Res., 21, 221 . JEFFREY, G . A., and KINOSHITA, Y ., 1963, Acta crystallogr ., 16, 20. McCLuRE, R . J ., and CRAVEN, B . M ., 1973, Acta crystallogr ., B, 29, 1234. SEVILLA, M . D ., 1971, Y. phys . Chem ., 75, 626 ; 1976 Ibid ., 80, 1898 . WESTHOF, E ., and FLOSSMANN, W ., 1975, J. Am . chem . Soc ., 97, 6622 . WESTHOF, E ., FLOSSMANN, W ., and MOLLER, A., 1975, Int. ,7. Radiat. Biol., 28, 427 . WESTHOF, E ., and VAN RooTEN, M ., 1976, Z. Naturf., 31c, 371 . ZEHNER, H ., FLOSSMANN, W ., WESTHOF, E ., and MOLLER, A ., 1976, Molec . Phys . (in the press) .
Y.
1976,
VOL .
30,
NO.
4, 301-315
Radical formation in salts of pyrimidines III. Cytosine . Hao crystals W. FLOSSMANN, E. WESTHOF and A. MOLLER Institut fur Biophysik and physikalische Biochemie, Universitat Regensburg, D-84 Regensburg, Germany
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(Received 7 June 1976 ; accepted 6 August 1976)
The radicals produced by X-irradiation at 77 K and at 300 K in cytosine monohydrate crystals have been analysed by electron-spin-resonance (e .s .r.) spectroscopy . Three radicals have been identified at 77 K : the anion radical and the cation radical of the cytosine molecule, together with the radical resulting from H-abstraction from the nitrogen N1 . Irradiation at 300 K produces radicals resulting from H-addition at three different positions of the cytosine molecule . These are the C5-addition radical, the C6-addition radical, and the 02-addition radical . The results are compared with those found previously by other authors .
1. Introduction The free radicals produced by ionizing radiation in single crystals of cytosine monohydrate have been analysed repeatedly (Herak and Galogaza 1969, Cook, Elliott and Wyard 1967, Dertinger 1967, Herak, Galogaza and Dulcic 1969). Herak and Galogaza (1969) identified after irradiation at 77 K the cytosine cation radical but were not quite sure about the identification of another radical, which they tentatively assigned to the cytosine anion radical . We have examined single crystals of cytosine . H2 0 after X-irradiation at 77 K and were able to isolate three different radical species. Despite correct identification, the hyperfine tensors for the cation radical given by Herak and Galogaza (1969) had to be corrected substantially, owing to the presence of a third species in the e.s .r . spectra. Our analysis has yielded evidence for the assignment of the anion radical of the cytosine molecule without any doubt . The third species is found to be the radical resulting from hydrogen abstraction from the nitrogen N1 . Such an identification has been previously proposed by Cook et al. (1967) for some lines of the spectra obtained after X-irradiation at 300 K . Further, these authors identified the radical resulting from H-addition at position C5. We show that the lines attributed by Cook et al . (1967) to the radical resulting from H-abstraction from N1 are indeed the result of D-addition at position C5 . A further radical resulting from H-addition to 02 was tentatively assigned by Dertinger (1967) to a small doublet present in the spectra taken at 35 GHz . In two previous papers, we have shown that the radical resulting from H-addition at position C6 is also present (Flossmann, Westhof and Muller 1976) and that the identification of Dertinger (1967) is correct (Westhof, Flossmann and Muller 1975) . In the present paper, the hyperfine tensors of these two radicals are given . In addition to these, two new radicals have been isolated. Because of the total number of different radicals produced with similar hyperfine couplings, the spectra are very complex . Therefore, a whole gamut R.B .
X
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of methods was employed for the separation of the various components . Variation of temperatures was combined with bleaching by light as a standard routine . We should like to emphasize that our analysis rests heavily on results obtained through specific deuteration of the hydrogen bonded to carbon C5 and on the use of two frequencies of observation . Furthermore, the strong dependence of the spectra on the dose of radiation given to the crystal was exploited . Also, crystals doped with copper were investigated . H\N/H
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pg i
N3
~H
5li s
0~ \/C N H I
H Cytosine
2 . Experimental Single crystals of cytosine monohydrate were grown from saturated aqueous solutions by slow evaporation at room temperature . The crystal structure was reported by Jeffrey and Kinoshita (1963) and later refined by McClure and Craven (1973) . The crystals are monoclinic with space group P2 1 /c . There are four molecules in the unit cell . For the e .s .r . measurements, an orthogonal system a*, b, c was chosen . The b-axis is parallel to the pyrimidine ring and nearly perpendicular to the C5-H bond . The other two axes are skew with respect to the molecular plane . Three types of crystal have been analysed . In crystals of type A, the cytosine molecule is fully protonated (cytosine . H 2O) . In crystals of type $, the hydrogen atoms bonded to the nitrogen atoms (amino group and N1) are exchanged with deuterium (cytosine . D2 0) . Finally, in the crystals of type C, the hydrogen atom bonded to C5 has also been replaced by deuterium (H6cytosine . D2 0), leaving only the hydrogen at C6 protonated . The proton at position C5 was exchanged with deuterium in the following way . First, concentrated DC1 solutions of cytosine were warmed to 100°C for some hours . The chlorine ions were then removed by neutralization . This substitution was verified by high-resolution NMR . The experimental procedure and equipment were as described previously (Flossmann, Westhof and Muller 1975) . 3. Results 3 .1 . Irradiation and observation at 77 K The spectra obtained at different orientations with single crystals of cytosine . H 2O, cytosine . D 20, and H6-cytosine . D 2 0 are presented in figures 1, 2, and 3 . The orientation of the magnetic field is for figure 1 in the molecular plane perpendicular to the b-axis, i .e . roughly parallel to the C5-H bond ; for figure 2, it is parallel to the b-axis, i .e . nearly perpendicular to the C5-H bond ; and for figure 3, perpendicular to the molecular plane . The second spectrum of figure 1 is the same as the cne presented in figure 3 of reference (Herak and Galogaza 1969) . The orientation should be, however, perpendicular to the
Radicals in cytosine . H2O
303
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P
t II
t II
V
I 20G
,_
Figure 1 .
E .s.r. spectra of crystals of cytosine . H20 (upper spectrum), cytosine . D,O (middle spectrum), and H6-cytosine . D 2 0 (lower spectrum) X-irradiated and observed at 77 K . The frequency of observation is 9 . 5 GHz. The magnetic field is oriented perpendicular to the b-axis in the molecular plane . The stick diagrams indicate the lines due to the radicals numbered by roman figures on the right .
b-axis and not parallel to it as indicated in the caption of figure 3 of reference (Herak and Galogaza 1969) . A comparison of the three spectra of figure 1 shows that only the doublet of about 20 G located at g=2 . 0028 is independent of the deuteration stage . This coupling therefore arises from the proton bonded to C6, which is the only proton present in crystals of type C . Another radical characterized by a singlet
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I
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II
I II
I II
Figure 2 . E .s .r. spectra of cytosine crystals of type A, B and C after X-irradiation and observation at 77 K . The frequency of observation is 35 GHz . The magnetic field is parallel to the b-axis . The arrows indicate the central lines of Mn++ (MnO in MgO) used as a standard .
at g = 2 . 0050 is also present in crystals of type C . In crystals of type B, where the proton bonded to C5 is not exchanged, a doublet of about 7 G is observed at g=2-0050 instead of a singlet . This doublet therefore arises from the proton bonded to C5 . In the fully-protonated crystals, another coupling splits the doublet to give a 1 : 2 : 1 triplet . Thus, two couplings contribute to the spectra of this second radical : one from the proton bonded to C5 and one from a proton bonded to a nitrogen atom . The spectra of figure 2 were taken at 35 GHz to separate the overlapping lines of these two radicals . The third spectrum of figure 2 (type C) shows a small doublet of 10 G at g = 2 . 0031 and a large singlet at g = 2 . 0039 . In crystals of type B, a doublet of 22 G is found at 2 .0039, and the small doublet is unchanged . The large doublet must now be assigned to a proton bonded to C5
Radicals in cytosine . H20
305
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and the small one to a proton bonded to C6 . Such a behaviour for the two radicals observed is expected from the theoretical anisotropy of an a-proton . Indeed, in figure 1, the magnetic field is approximately parallel to the C5-H bond direction in the molecular plane . Therefore, at this orientation, an a-proton bonded to C5 should give minimal coupling, and one bonded to C6 a much larger one . On the other hand, in figure 2, the magnetic field is perpendicular to the C5-H bond direction, and an a-proton bonded to C5 must have a maximal coupling, whereas one bonded to C6 must have a much smaller coupling . In crystals of type A, another coupling of 5 G splits the lines of the doublet of 22 G.
I
'j
JV
.1
1
20G I
Figure 3 . E .s .r. spectra of cytosine crystals of type A, B and C X-irradiated and observed at 77 K . Same remarks as for figure 1 apply. The magnetic field is perpendicular to the molecular plane .
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From the spectra taken with the magnetic field in the molecular plane, one can conclude that oneradical is characterized exclusively by an (X-proton bonded to C6, and. the other by an a-proton bonded to C5 and one bonded to a nitrogen atom . Figure 3 shows the spectra obtained when the magnetic field is perpendicular to the molecular plane for crystals of type A, B, and C . The first two spectra are similar to those presented in figure 2 of reference (Herak and Galogaza 1969) . These authors interpreted the second spectrum of figure 3 as arising mostly from a radical presenting two non-equivalent nitrogen couplings and one a-proton . However, the third spectrum of figure 3, obtained with crystals of type C, indicates the presence of three radicals . In the middle of the spectrum a doublet of narrow lines with a splitting equal to 15 G is present accompanied by several less intense lines on both sides . The lines of this doublet are present in all three spectra and should therefore arise from the a-proton bonded to C6 . At this orientation, a radical containing spin density in the 2p .,-orbital of a nitrogen atom should present maximal hyperfine coupling with the nitrogen . The numerous and less intense lines are therefore assigned to the radical previously characterized by an a-proton at C5, which should present hyperfine interaction with two non-equivalent nitrogen atoms equal to 13 . 1 G and 4 . 0 G, respectively . With the same nitrogen couplings, the second spectrum of figure 3 can be explained if the coupling of the a-proton bonded to C5 is equal to 11 . 2 G . The broad lines present on each side of the spectrum do not belong to this second radical, for the variation of their spacing following deuteration at C5 is different . These lines are due to a third radical presenting hyperfine coupling with one nitrogen (25 G) and with the a-proton bonded to C5 (13 . 7 G) . Since they assumed that all the lines of the second spectrum of figure 3 belong to the same radical, Herak and Galogaza (1969) obtained wrong hyperfine tensors for the second radical . The presence of a third radical can be deduced from the variable intensity of the lines in the spectra of figures 2 and 3 . This third radical must accordingly possess a similar hyperfine coupling with the proton bonded to C5 as that of the second radical . The spectra presented in figure 4 confirm the fact that we are dealing with three different radical species . The upper spectrum of figure 4 is obtained with single crystals of cytosine . H 2 O doped with copper ions . The relative concentration of the first and second radical is drastically changed after Cu++-doping . This spectrum should be compared with the first spectrum of figure 1 . On the other hand, the second spectrum of figure 4 is obtained with cytosine crystals of type C after irradiation at 77 K followed by illumination with the red light of a He-Ne laser (A=633 nm) . A comparison between this spectrum and the third one of figure 3 clearly shows that the third radical is not so sensitive to light as the first two radicals . Further, the second spectrum of figure 4 confirms that the third radical is characterized by a 1 : 1 : 1 triplet and not a large doublet . One can conclude that, after irradiation of cytosine monohydrate crystals at 77 K, three radicals are produced . The first one presents hyperfine interaction with the proton bonded to C6 ; the second one with the proton bonded to C5, with a proton bonded to a nitrogen, and with two non-equivalent nitrogen atoms ; and finally, the third one has interaction with the proton bonded to C5 and with one nitrogen atom . The principal values and principal directions of the different hyperfine tensors are contained in table 1 .
Radicals in cytosine . H2 O
HEN/H
HEN H
I ,C,,
I C
I
0/C
OAC H ~ H
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Radical I
H \N/H
I
I N /H
/H
I p
II
Radical (I) a-H(C6)
Radical (II) a-H(C5)
Ni
N3
g
Radical (III) N a-H(C5)
C
Radical III
Direction cosines b
Anisotropic
-14 . 5
Isotropic
-7 . 1 -22 .0 -11 . 2
+6 . 3 -8 .6 +2 . 2
-13 . 4 4.0 0.0
Isotropic
25 .0 -13 .7
C
Radical 11
+8 . 5 -8 . 0 -0 . 5
2 .0023 2 . 0040 to 2 .0050
CH
C/ \N/ H
- 6.0 -22 . 5 -15 . 0
13 . 1 0 .0
N~C~
H
O#C\N/C I H
Principal values (G) Total
307
c
0 . 3582 0 . 7713 0 .4670
-0. 9202 0 .4725 0 . 0309
-0 . 1580 -0 . 4264 0 . 8790
0 . 8709 0 .0166 0 .4904
0 . 0242 -0 . 9998 0 . 0150
-0 . 4907 0 . 0079 0 . 8711
parallel 2p .-orbital in the molecular plane parallel 2p -orbital in the molecular plane parallel 2p, -orbital in the molecular plane
parallel 2p .--orbital parallel 2p,,-orbital
Table 1 . Hyperfine tensors for the a-protons of the anion radical (I) and of the cation radical (II) . The maximal couplings of the two non-equivalent nitrogens of radical II are given, as well as its g-tensor . The g-tensor of radical III is similar to that of radical II . The probable errors are estimated to be ± 1 . 0 G for the hyperfine tensors and ± 0 .0002 for the g-tensor . The probable errors of the direction cosines are estimated to be ± 6° . The direction cosines 1, m, n are referred to the a*, b, c axis respectively . The direction cosines for the other site are given by 1, - m, n .
The first radical is clearly the anion of the cytosine molecule, since pyrimidine anion radicals typically present hyperfine interaction with the proton bonded to C6 (Baudet, Berthier and Pullman 1962, Sevilla 1971) . Anion radicals of the cytosine molecule (radical I) have previously been observed in single crystals of cytidine-3'-phosphate (Box, Potter and Budzinski 1975) and of cytosine . HCl
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III
Figure 4 . Top spectrum : E .s .r . spectrum obtained with a single crystal of cytosine . HO doped with copper ions after X-irradiation and observation at 77 K . The magnetic field is oriented perpendicular to the b-axis in the molecular plane . Bottom spectrum : E .s .r. spectrum obtained with a single crystal of H6-cytosine . D S O after X-irradiation and subsequent illumination with a He-Ne laser at 77 K . The temperature of observation is 77 K . The magnetic field is perpendicular to the molecular plane .
(Westhof et al. 1975) . The second radical is the cation radical (radical II), which was correctly identified by Herak and Galogaza (1969) . The coupling of the easily exchangeable proton should arise from the proton bonded to N1 . The occurrence of such a coupling in this second radical indicates that the cation radical is still positively charged . We identify the third radical with the deprotonated cation radical, which is equivalent to the radical resulting from hydrogen abstraction from the nitrogen NI (radical III) . With the help of INDO calculations, we have shown that the thymine cation radical and the radical resulting from hydrogen abstraction from N1 should present the same couplings, except for the supplementary coupling of the substituent at N1 in the cation radicals (Westhof and Van Rooten 1976) . This has been experimentally verified by Sevilla (1976) . In this connection, one should note that the 2p z-spin density distribution in the cytosine cation radical compares well with that in the thymine cation radical : pcs 0 . 20 ; p Nl 0 . 40 (Sevilla 1976) . However, thelarge nitrogen coupling of radical III, 25 G, is closer to that, 28 . 8 G, of the radical resulting from hydrogen abstraction in barbituric acid crystals (Hiittermann, Schmidt and Weymann 1976) . INDO calculations indicate that the barbituric acid cation radical has a much lower nitrogen coupling than the radical resulting from hydrogen abstraction from N1 . In the case of the
Radicals in cytosine . H20
309
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cytosine cation radical, INDO calculations yield Ni11= 7 . 1 G and =15 . 9 G, N311 and H(C5) I8O = -2 . 0 G . As in the case of thymine, the couplings of the nitrogens are calculated too large with the consequence that the coupling due to the proton bonded to C5 is too small . However, the deprotonated cation radical is not easy to calculate, because of convergence problems . This could indicate that the radical resulting from hydrogen abstraction from N1 exists in a tautomeric form different from that of the undamaged molecule . More detailed calculations will be presented elsewhere . On warming a crystal irradiated at 77 K to room temperature most radicals disappear which renders any study of secondary radical reactions impossible . 3 .2 . Irradiation and observation at 300 K Irradiation at room temperature of cytosine monohydrate produces three different hydrogen addition radicals : the C5-addition radical (radical IV), the C6-addition radical (radical V), and the 02-addition radical (radical VI) . H\ N/H
H\N/H
N *L-C,H I I \H O/CAN "C~
I
K\ /H N
I
I
H\ ,Cy
N.
H I H
I
H
H
Radical IV
Radical V
I
N"CyH II
1
HC'C'N'C" I H H Radical VI
The maximal yield of C5-addition radicals is obtained at doses ranging from 2 to 3 Mrad . On the other hand, the 02-addition radical dominates the spectra only at very high doses (-10 M rad) . The concentration of C6-addition radical is always very low after X-irradiation . However, we have shown that subsequent illumination with light of A > 400 nm induces a transformation of C5-addition radicals into C6-addition radicals (Flossmann et al. 1976) . A high concentration of C6-addition radicals can be produced in this way . The C5addition radical was first identified by Cook et al. (1967) . The hyperfine tensor of the a-proton bonded to C6 was later refined (Herak et al . 1969) . Cook et al . (1967) observed that, at microwave power greater than 1 mW, the middle part of the spectrum saturates at a different rate from the outer lines of the spectrum . The central part of the spectrum is, in fact, due to the 02-addition radical, which was first identified by Dertinger (1967) at 35 GHz . 3 .2 .1 . The C5-addition radical (radical IV) This radical is characterized by two equivalent and isotropic #-protons of 37 . 0 G and an a-proton bonded to C6 . We have redetermined the hyperfine tensor of this a-proton and the results are contained in table 2 . Because of the presence of spin density on the nitrogen N1, the line-width of this radical is large and anisotropic . Along the N1-C6 bond, the coupling of-the nitrogen Nl is 2 . 9 G. This coupling is minimal when the magnetic field is perpendicular to the molecular plane. Therefore, the width of the lines of the C5-addition radical is minimal (about 4 . 0 G), and this radical is most easily detected at this orientation . It has already been noticed that the hyperfine coupling of such an a-nitrogen is not maximal when the magnetic field is perpendicular to the
W. Flossmann et al .
3 10
Principal values (G) Total
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Radical (IV) a - H(C6)
Radical (V) a-H(C5)
Radical (VI) a - H(C6)
-8 . 1 -30-0 -17 . 7
+10 . 5 -11 .4 +0 . 9
-18 . 6
Isotropic
-8-5 -26 . 5 -15 .0
+8 . 2 -9 . 8 +1 . 7
-16 . 7
Isotropic
-4. 5 -12 . 3 -9 . 1
+4 . 1 -3-7 -0 . 5
-8-6 Radical (VI) g
Anisotropic
Direction cosines a*
b
c
0 . 3582 0 . 7713 0 . 4670
-0 . 9202 0 . 4725 0 . 0309
-0 . 1580 -0-4264 0 . 8790
0 . 8709 0 . 0166 -0 . 4904
0 . 0242 -0 . 9998 -0 . 0150
-0-4907 0 . 0079 -0 . 8711
-0 . 068 0 . 865 -0 . 497
0 . 997 0 . 082 0 . 007
-0 . 046 0 . 495 0 . 868
0 . 863 -0 . 015 -0 . 505
0 . 038 0 . 999 0 . 035
0 . 504 -0 . 049 0 . 862
Isotropic
2 . 0075 2 . 0052 2 . 0026
+0 .0024 +0 .0001 -0 . 0025
2 . 0051
Isotropic
Table 2 . Hyperfine tensors for the a-protons of the C5-addition radical (IV), of the C6-addition radical (V), and of the 02-addition radical (VI) . The g-tensor of radical VI is also given . Remarks as for table 1 apply . molecular plane, but minimal (Flossmann, Huttermann, Muller and Westhof 1973) .
The upper spectrum of figure 5 is obtained with a cytosine . D 2 0 crystal irradiated at 300 K and observed with the magnetic field perpendicular to the molecular plane . As was already noted by Cook et al. (1967), even in deuterated crystals the hydrogen addition to C5 is present (C5-HH radical) . In addition, several other lines, which were not present in cytosine . H20 crystals, dominate the spectrum. The upper spectrum of figure 5 is not exactly the same as the one represented in figure 7 of reference (Cook et al. 1967) . However, at a specific orientation and after a specific dose, all deuterated crystals observed yield the same spectrum as the one of figure 7 in reference (Cook et al. 1967) . Since at this orientation, the magnetic field is perpendicular to the molecular plane, Cook et al. (1967) analysed this spectrum as arising from two nonequivalent nitrogens and one a-proton . It is, however, possible to interpret all these lines at all orientations with one fl-proton of 37 . 0 G, one a-proton bonded to C6, and one isotropic coupling to a deuterium atom (5 . 5 to 5 . 9 G) . This deuterium coupling is precisely what should be expected if a a-proton of 37 . 0 G is replaced by a deuteron (37 . 0 /6 . 51 = 5 . 68) . Therefore, we attribute to these lines the radical resulting from D-addition at position C5 (C5-HD In contrast, Cook et al. (1967) assigned to these lines the radical radical) .
Radicals in cytosine . H2 O
311
MMD)
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~---T .
W(HH)
V(HH)
Figure 5 . E .s .r . spectra of crystals of cytosine . D2 0 X-irradiated and observed at 300 K at 9 . 5 GHz . For the upper spectrum, the magnetic field is perpendicular to the molecular plane . For the two bottom spectra, it is parallel to the b-axis. The last spectrum was obtained after subsequent illumination with light of A > 400 nm . In this spectrum, the lines due to the C6-HH radical with a D-atom at position C5 are indicated by stick diagrams at the bottom .
resulting from H-abstraction from the nitrogen N1 . Further evidence for our assignment is given in the next section . 3 .2 .2 . The C6-addition radical (radical V) In a previous paper (Flossmann et al. 1976), we have shown that, in pyrimidine derivatives, the C5-addition radicals transform into C6-addition radicals on irradiation with light of wavelength A > 400 nm . Immediately after irradiation at room temperature, the lines belonging to this radical are already
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present, albeit with a low intensity, as can be seen from the second spectrum of figure 5 . After irradiation with light, the concentration of the C6-addition radical is high enough to permit an analysis of the hyperfine tensors . The hyperfine tensor of the a-proton bonded to C5 is given in table 2 . The isotropic coupling of the methylene protons is about 50 . 0 G . These fl-protons are, in fact, slightly non-equivalent (49 . 0 and 53 . 0 G) . In contrast to the C5-addition radical, the width of the lines of the C6-addition is minimal in the molecular plane and maximal perpendicular to it . INDO calculations yield some spin density on the nitrogen atom N3 which in the C6-addition radical is not an a-nitrogen as the N1 nitrogen is for the C5-addition radical . With the help of INDO calculations, we have also shown that the C6-addition radical has to be protonated at N3 in order to reproduce the large fl-proton couplings (Flossmann et al. 1976) . The second spectrum of figure 5 shows also that after X-irradiation of cytosine . D 20 crystals, both H- and D-addition radicals to both positions C5 and C6 are produced . On subsequent illumination with light, the concentration of both types of radical V resulting from H- or D-addition to C6 increases, whereas most of the two corresponding C5-radicals disappear . However, additional lines are clearly detected (figure 5, third spectrum) after irradiation with light . These lines are due to the C6-HH radical with a D-atom at position C5 . These observations reinforce our revised assignment of the C5-HD radical for, on irradiation with light, this radical is able to yield either the C6-HD radical or the C6-HH radical with a deuterium at position C5 . 3 .2 .3 . The 02-addition radical (radical VI) The identification and the structure of this radical has already been discussed elsewhere (Westhof et al. 1975) . By comparing spectra taken at 35 GHz of cytosine . H2 0 and cytosine . HC1 crystals, we were able to show that, for the 02-addition radical, the doublet splitting presented originates from the proton bonded to C6 and not from the proton added to 02 (Westhof et al. 1975) . The principal values and principal direction cosines of both the g-tensor and the hyperfine tensor are given in table 2 . The direction cosines with respect to the a*, b, c axis system of the C2-02 bond, as calculated from the crystal structure, are the following : 0 . 908, 0 . 033, 0 .416. The angle between the direction of this bond and that of the maximal principal value of the g-tensor is equal to 6° . A comparison of the direction cosines of the C6-H bond and those of the minimal principal value of the hyperfine tensor would not give such a good agreement ; the two directions make an angle of 23° . This arises from the treatment of the experimental data . We have diagonalized separately the g-tensor and the hyperfine tensor . Such a procedure is incorrect in case of strong anisotropy of the g-tensor and when the principal axes of the g-tensor are different from those of the hyperfine tensor . However, we could not follow the doublet splitting through sufficient orientations to make a correct analysis . Nevertheless, the third spectrum of figure 6 clearly indicates that the hyperfine coupling is due to the proton bonded to C6, since this spectrum has been obtained with a crystal of type C, where H(C6) is the only non-exchanged proton . A supplementary coupling of 3 . 0 G splits the doublet lines when the magnetic field is in the molecular plane and perpendicular to the b-axis . This
Radicals in cytosine . H2 O
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L
L TI
,.
200
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VI
VI
,
Figure 6 . E .s .r. spectra of cytosine . D,0 crystals after low dose (top) and high dose (middle) of radiation. The magnetic field is parallel to the b-axis . The arrows indicate the lines of Mn++ . The lower spectrum is obtained with a crystal of type C after a high dose of radiation . The orientation of the magnetic field is the same as for the two upper spectra .
coupling disappears in crystals grown from D 20 . One could attribute to this splitting the hydroxyl proton . However, from INDO calculations and from the orientation of maximal coupling, we think that the proton bonded to N1 is responsible. The lines of the doublet splitting become very broad when the magnetic field is perpendicular to the molecular plane . As confirmed by INDO calculations, this is due to hyperfine interaction with the two nitrogens N1 and N3 . The 02-additional radical is already present at low doses (0.4 Mrad) . However, at high doses (10 Mrad), it completely dominates the spectrum.
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This can be observed in the first two spectra of figure 6 . The upper one is obtained after a low dose . The lines due to the C5-HH and C5-HD radicals can be observed as well as those of a large doublet of 65 G . The second spectrum is obtained after a high dose . The C5-addition radical can only be observed at high gain, but the small doublet of the 02-addition radical is prominent . However, two lines separated by 27 . 5 G are also present . These lines do not belong to the 02-addition radical, since they disappear when the H(C5) proton is exchanged with a deuteron (third spectrum of figure 6) . The radicals responsible for the doublet of 27 . 5 G and for the large one of 65 G will be discussed in a following paper . 4.
Conclusions By comparing the formation of H-addition radicals in single crystals of pyrimidine derivatives (Flossmann et al. 1973, Flossmann et al. 1975, Westhof et al. 1975, Flossmann et al. 1976), the following conclusions emerge . H-addition radicals can be the result of direct hydrogen addition, or of a two-step process through protonation of an anionic intermediate radical . We have shown that the first mechanism-the excitation path-is predominant in van der Waalscrystals, and the second one-the ionization path-in strongly hydrogen-bonded crystals containing small polar molecules . Whereas the ionization path could not be detected in the van der Waals crystals formed by the pure bases, in crystals containing molecules of crystallization both paths have been observed . The origin of the hydrogen is either the methyl groups or the hydrogen bonds . In bases non-substituted at N1, hydrogen may be supplied directly by the ring, since radicals resulting from hydrogen abstraction from the nitrogen N1 are observed (Dulcic and Herak 1972, Zehner, Flossmann, Westhof and Muller 1976) . Concerning the sites of attack, we have shown that protonation of the electron-adduct radicals occurs at C6, giving C6-addition radicals, and that the C5-addition radicals result from hydrogenation of the neutral molecule (Westhof and Flossmann 1975, Flossmann et al. 1975, Westhof et al . 1975) . Indeed, until now, C6-addition radicals have been detected only if anion radicals could be stabilized at lower temperatures . On the other hand, C5-addition radicals have been observed in all crystals studied . Furthermore, INDO calculations have shown that C5-addition radicals are neutral and C6-addition ones positively charged in unsubstituted pyrimidines (Flossmann et al . 1976) . Because of its charge, the C6-addition radical is only stabilized in an anionic environment . This explains the absence or small concentration of C6-addition radicals in van der Waals crystals and its increased concentration in crystals containing polar molecules . In all previous crystals studied, the electron adduct radicals were in fact neutral since the bases were already protonated in the undamaged crystals . Upon further protonation at C6, these electron adduct radicals lead to the positively-charged C6-addition radicals . The cytosine monohydrate crystals are interesting, since in them the radicals resulting from electron addition or abstraction remain negatively and positively charged . Next to the competition between the two carbons of the C5-C6 double bond, the oxygen 02 is also a position of hydrogenation in cytosine . In cytosine . H 2O crystals, the concentration of 02-addition radicals at high doses
Radicals in cytosine . H2 0
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is much higher than that of any other radical . The mechanism of formation of this radical is not yet clear . In both crystals where we could detect it, the oxygen 02 participates in an inter-pyrimidine hydrogen bond . This could be an important condition for its formation, as already noted by Dertinger (1967) . In crystals of 1-CH 3-cytosine . HBr and deoxycytidine . HCl, where the oxygen 02 fails to participate in hydrogen bonding, we could not find the characteristic lines of the 02-addition radical . Further examples will be needed to reach definite conclusions .
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ACKNOWLEDGMENT
This research was supported by grants from the Deutsche Forschungsgemeinschaft . Les radicaux induits par irradiation X a 77 K et a 300 K dans des monocristaux de cytosine . H 2 O ont ete analyses par la methode de resonance paramagnetique electronique (r.p .e .) . A 77 K, trois radicaux ont ete identifies : le radical cationique et le radical anionique de la molecule de cytosine ainsi que le radical produit par l'abstraction d'hydrogene de l'azote N1 . L'irradiation a 300 K produit des radicaux qui resultent de l'addition d'hydrogene sur trois atomes de la molecule de cytosine . Ceux-ci sont le radical d'addition en C5, le radical d'addition en C6, et le radical d'addition en 02 . Ces resultats sont compares a ceux trouves precedemment par d'autres auteurs. Es wurden die Radikale mit Elektronen-Spin -Resonanz-(ESR)-Spektroskopie analysiert, die durch Rontgenstrahlen in Kristallen von Cytosin-Monohydrat bei 77 K and 300 K erzeugt werden . Drei Radikale wurden bei 77 K identifiziert : das AnionRadikal and das Kation-Radikal des Cytosinmolekuls sowie das Radikal, das durch H-Abstraktion von N1-Stickstoff entsteht. Bestrahlung bei 300 K erzeugt Radikale, die durch H-Addition an drei verschiedenen Positionen des Cytosinmolekuls entstehen . Es sind dies das C5-Additionsradikal, das C6-Additionsradikal and das 02-Additionsradikal . Die Ergebnisse werden mit deren anderer Autoren verglichen .
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