ESR study of the ionic radical species in an irradiated single crystal of isocytosine

JOURNAL

OF MAGNETIC

RESONANCE

33, 319-329 (1979)

ESR Study of the Ionic Radical Species in an Irradiated Single Crystal of Isocytosine J. N . H E R A K

Faculty of Pharmacy and Biochemistry, University of Zagreb AND

B. RAKVIN AND M. BYTYCI* Rudjer Bogkovid Institute, Zagreb, Croatia, Yugoslavia Received March 2, 1978 The radicals in a single crystal of isocytosine, irradiated with gamma rays at 77 K, were studied with the ESR spectroscopy. Three types of radicals were detected. One of them, whose ESR spectrum exhibits two 14N and two 1H coupling nuclei, is interpreted to be the isocytosine cation radical. The second species, characterized by a doublet structure, is thought to be formed by a proton transfer to the isocytosine anion. The third species could not be analysed in detail. All three species are unstable upon warming or upon illumination with green Ar laser light.

INTRODUCTION

The role that pyrimidines play in biological systems makes these compounds the subject of various investigations. There have been numerous ESR studies of the radicals in the pyrimidine compounds, produced by ionizing irradiation. Many of these studies have been carried out on single crystals, primarily because in such ordered systems the anisotropic components of the ESR parameters that give additional data on the radicals studied can be determined. Ionic radical species have been observed in the pyrimidine bases in several crystalline systems at low temperatures. While anions have been observed in the crystals of cytosine (1, 2), cytosine hydrochloride (3), barbituric acid (4), cytidine 3'-phosphate (5), thymidine (6) and 1-methyl uracil (7), the cations have been found only in the crystal of cytosine monohydrate (1, 2). Isocytosine is a very similar compound to cytosine and the system of hydrogen bonding in this crystal (8) is similar to that in cytosine H20. Therefore one expects that the radicals formed in these two systems will be similar. The present investigation shows that it is really the case. Isocytosine is, besides cytosine, the only pyrimidine system in the crystal form, in which the cation radicals are observed. Another radical species present in isoeytosine is related to the anion. The details of its structure will be discussed. * Permanent Address: Faculty of Science, University of Pri~tina, Pri~tina, Yugoslavia 319 0022-2364/79/020319-11 $02.00/0 Copyright© 1979by AcademicPress,Inc. All rights of reproduction in any form reserved. Printed in Great Britain

320

HERAK, RAKVIN, AND BYTYCI o U

2 I sl NH~z C~/C\H,6)

2 s I° NzH~'~/C~I"I{B)

0 ~"':"z~ / 6c ~H

HH) (A)

H ISOCYTOSINE

(B)

CYTOSINE

FIG. 1. Two isocytosine tautomers, in comparison to cytosine. EXPERIMENTAL Single crystals of isocytosine were grown from aqueous solution either by slow evaporation of a concentrated solution at r o o m temperature or by slow controlled cooling of the solution from about 40°C to 15°C. Partly deuterated crystals were grown by the latter procedure in a sealed vessel from the heavy water solution. In this way the hydrogen atoms bonded to nitrogens and oxygens in the molecules are replaced by deuterium. The crystals are monoclinic with space group P21/n. There are eight molecules in the unit cell, four of them in tautomeric form A and the remaining four in form B (Fig. 1). The external appearance of the crystal varied significantly from harvest to harvest. In most cases very many faces were approximately equally developed, which made the identification of the crystallographic axes and planes and hence the controlled orientation of the crystal in the magnetic field of the spectrometer very difficult. In most cases we found s o m e rather typical species. All our studies were made on the crystals portrayed in Fig. 2(a). The identification of

-'

/

\ _ ..... Ib

(a)

y###\

.. (b)

FIG. 2. External appearance of the single crystalof isocytosine, with crystallographicand reference axes (a) and the relation of the reference axes to the molecules in the crystal (b).

RADICALS FROM ISOCYTOSINE

321

the crystallographic axes was done with the use of the X-ray technique. For the purposes of the E S R analysis the orthogonal coordinate system a*, b and c* was used. The c* axis is chosen to be approximately in the molecular ring planes. Each molecule of type A is paired with a molecule of type B (8). The plane defined by the ring atoms of molecule A differs from the plane defined by the ring atoms of molecule B by 9 °. The least-square-fit plane defined by the atoms of both molecules makes an angle of 60 ° with an identical plane related by the crystallographic symmetry axis. The c* axis represents the intersection of these two planes (Fig. 2(b)). The crystal was irradiated in liquid nitrogen in a 6°Co source at the dose rate of approximately 0.6 M r a d / h to a total dose up to 5 Mrad. The spectra were recorded at 77 K with a Varian E-3 spectrometer. The hyperfine couplings and the g tensor were determined from the data recorded in three mutually perpendicular planes with the use of a first-order perturbation treatment, as described by Farach and Poole (9).

i

PPH

(a)

(b)

. v .U ~

,,lllTl

a 20 GAU55 GAUSS ~

,,

FIG. 3. The first derivativeESR spectra of the isocytosinesingle crystal,irradiated and observed at 77 K, for I-Ilia*:(a) nondeuterated crystal,(b) deuterated crystalimmediatelyafter irradiation and (c) deuterated crystal after a short warming to about 120 K.

322

H E R A K , RAKVIN, A N D BYTYCI

The complexity of the hnit cell structure together with the uncertainty of the crystal orientations in the spectrometer make the ESR analysis rather complex, as will be shown later. ANALYSIS OF T H E S P E C T R A

Figures 3, 4 and 5 show representative ESR spectra. The patterns in Fig. 3 are recorded for the magnetic field in the a* direction, Hlla*. The upper curve represents a spectrum of the undeuterated crystal and the lower two refer to the crystal grown from heavy water. Figure 4 shows the spectra for HI[c* for a nondeuterated and a deuterated crystal, respectively. Figures 3 and 4 demonstrate that in the crystal of isocytosine at least three different types of radicals are induced by ionizing irradiation. The Cation Radical

We will show that the most abundant radical represented by the most intense ESR spectrum is the isocytosine cation radical. In the Hlla* direction the radical gives rise

DPPH

1 (a]

(b)

t t

FIG. 4. The spectra for Hllc* for nondeuterated crystal (a) and for deuterated crystal (b). The arrows under the spectra indicate the "doublet" radical resonance lines.

RADICALS FROM ISOCYTOSINE

323

DPPH

NONDEUTERATED

I

DEUTERATED

I

20 GAUSS

!

FIG. 5. The spectra for the magnetic field in the (a*, b) plane, 30° off the b axis. An additional proton splitting is seen in the nondeuterated crystal.

to a ten-line pattern in a deuterated crystal and a more complex spectrum in a nondeuterated crystal (Fig. 3). In the H]lc* direction the same radical is represented by a well defined doublet in both deuterated and nondeuterated crystals (Fig. 4). The well resolved spectra presented in Figs. 3 and 4 are so well defined that it is obvious that they must originate from a single type of radicals, probably a single tautomer, either A or B. In the further analysis we will show that this is really the case. The hyperfine patterns of the deuterated radicals are caused by the couplings of a 1H and two 14N nuclei in a single radical. The nitrogen couplings are much m o r e orientationdependent than the proton couplings as shown in Fig. 6. F r o m the angular variation of the couplings and g- factor the principal elements of the hyperfine splitting tensors and the g tensor are determined. They are presented in Table 1. The ~4N couplings

HERAK, RAKVIN, AND BYTYCI

324

TABLE 1 PRINCIPAL ELEMENTS OF THE COUPLING TENSORS AND g TENSOR

Principal Elementsa

Directionsb

2.0022 2.0047 2.0037 -17.8 --7.3 --23.7

parallelto 2pz orbital in the ring plane in the ring plane 0.858 :~0.511 :~0.509 +0.462 0.726 :~0.509 0.223 :V0.460 0.859

AN(l)

All= 7.9 A± ~ 0

parallelto 2pz orbital in the ring plane

AN(3)

Alt= 13.9 parallel to 2pz orbital A± ~ 0 in the ring plane

g AH(5~ Cation

g=2.003 "Doublet" Anion AH(6)

-7.0 -3.5 -12.0

parallelto 2pz orbital in the C(6)-H(6 ) direction in the ring plane

The couplings are expressed in gauss (G). b The directions for the AI~(5~coupling tensor elements are expressed by the direction cosines with respect to the a*, b* and c* axes. a

are axial. The maximum values are in the directions perpendicular to the ring planes. The values in the ring plane are essentially equal to zero, as observed quite generally for the nitrogen couplings in the ~r-electronic conjugated radicals. The proton coupling parameters are rather typical for an a proton. The proton coupling in a deuterated crystal may come either from H(5~ or H(6) because these two are the only hydrogen atoms nonexchangeable upon deuteration. The direction defined by the direction cosines 0.462, 0.726 and - 0 . 5 0 9 or - 0 . 4 6 2 , 0.726 and 0.509 (the set of values which gives a better fit is used) makes an angle of 14 ° with the C(s)-H(5> bond direction of molecule A and 9 ° with the C(5)-H(5> bond of molecule B. The same direction makes angles of 52 ° and 50 ° with the C(6)-H(6) directions of molecule A and B, respectively. On the basis of this comparison the coupling of the H(6) proton can be safely ruled out in both tautomers. The same comparison is not sufficient to point out whether we deal with tautomer A or tautomer B. It is the additional coupling in the nondeuterated radical that gives information on the location of the radical. The extra proton coupling in the nondeuterated crystal (see Figs. 3 and 5) comes from the exchangeable hydrogen, either H m in tautomer A or H(3> in tautomer B. The orientations of the N(I)-H(1) and N(3)-H(3) bonds in molecules A and B, respectively, are different enough to be resolvable by the orientation of their proton dipolar coupling term. It is expected that the minimum of the total proton coupling will be close to the direction of the respective N - H bond, and that the maximum

R A D I C A L S F R O M ISOCYTOSINE

325

2.00,~ 2.002 25 O (.9 Z

20

B

...I

fl_

15 0 ¢_)

10

a"

30 °

60 °

b

30 °

60 °

c*

30 °

60 °

Q~

CRYSTAL ORIENTATION FIG. 6. Angular variation of three couplings and the g-factor for the cation radical.

coupling will be in the direction perpendicular to it, and in the ring plane (I0). Unfortunately, the complete tensor elements of the exchangeable proton coupling could not be determined. However, the distinction between the couplings of H~I~in tautomer A and H~3~ in tautomer B comes from the relative magnitudes of the coupling in various crystal orientations. In the HI]a* direction the extra proton coupling is about 3.5 G (Fig. 3). For HI]c*, the magnetic field makes an angle of 19.5 ° with the No~-Ho~ bond of tautomer A and 75 ° with the N(3~-H(3~ bond of tautomer B. Thus, if it is H(I~ of tautomer A that gives rise to the extra coupling, the hyperfine splitting is expected to be smaller, and if it is H~3~of tautomer B the coupling would be larger than that in the a* direction (I0). As judged from Fig. 4, the coupling in the HHc* direction is definitely smaller than that in the HHa* direction. The same analysis for the magnetic field in the (a*, b) plane, 30 ° off the b axis (ring plane) gives additional evidence that the splitting arises from H~I~of tautomer A. In that crystal orientation the magnetic field makes angles of 75 ° and 17 ° with the N - H bonds of tautomers A and B, respectively. The observed coupling of 5 . 0 G completely fits tautomer A. From the above experimental data it is obvious that the unpaired electron spin is delocalized: significant concentration is found on C(5~, N~I~and N(3~ atoms. Since H(I~ is present in the radical, one can safely identify the radical to be the cation of isocytosine A. This radical is completely analogous to the cytosine cation radical (I, 2). Even very similar hyperfine couplings and consequently similar spin distribution are observed.

326

HERAK, RAKVIN, AND BYTYCI

We attempted to correlate the "observed" spin densities with the ones deduced from the INDO MO theory. Unfortunately, INDO calculations failed to converge for the atomic coordinates of the undamaged molecule A (8). However, various MO calculations (11-15) and the observations in various glasses (13, 14, 16) for other pyrimidine cationic radicals agree in the fact that appreciable spin densities are found on the C and possibly N(3) atoms. Thus it is quite generally found that large spin densities on the above mentioned atoms in the pyrimidines represent the cation radicals or the related deprotonated isoelectronic neutral radicals. In the present system it is obviously the cation since the presence of H(I> in the radical has been undoubtly proven.

The Anionic Radicals The spectra in Figs. 3 and 4 demonstrate that besides the cation radical at least two other types of radicals are present. One of them is represented by a doublet in most crystal orientations (indicated by the arrows in Fig. 4). A low relative concentration of the radicals represented by the doublet resonance made it difficult to determine

12.5

.-J 13.. E) O L)

I

,51 5 2.5 12.5

..-~"

3' 0 °

' ° 60

~,

' ° 120

' ° 150

c*

10

7.5 I

!

a¢

!

30

60

c~

!

120

I

150

!

ct*

I

b,

~0 o

~ o

, a.~

, ° 120

, ° 150

b,

10 75

5 2.5

CRYSTAL

ORIENTATION

FIG. 7. The comparison of the measured splittings (circles) and the predicted angular variations of the H(6) coupling of tautomer A (solid lines) and tautomer B (dashed lines).

RADICALS FROM ISOCYTOSINE

327

the coupling- and g-tensor parameters. We found that at somewhat elevated microwave power level (10 mW), in contrast to the cation radical patterns the doublet pattern was essentially unsaturated and the ESR signals of the two radicals were of approximately equal intensity. Thus we were able to measure the doublet splitting at least in several orientations in the three orthogonal planes. The broad resonance lines and the presence of the cation radical lines masked the site splittings and thus we actually measured the "average" splitting of the two magnetically distinct species of type A or of type B or of four distinct species, two of each tautomer. The measured splittings are represented by the circles in Fig. 7. Instead of deducing the coupling tensor elements from these data and then comparing the deduced direction cosines with the directions of various C - H bonds in the crystal, we used the opposite procedure. We assumed that it was an a proton coupling of either H(s~ or H(6~ atoms of tautomer A or tautomer B. By assuming various spin densities on C(5~or C(6~ in both tautomers and by assuming relative magnitudes and directions of the coupling tensor principal elements as predicted by the theory (10), we calculated angular variations of the couplings and compared it with the observations. The H(s~ proton in both tautomers A and B could be safely ruled out. The fit of the expected coupling of H(6~ with the unpaired spin of 0.27 on C(6~ in both tautomers is also shown in Fig. 7. As it is seen, it is impossible to conclude whether the present radical is associated with species A or with species B. The spectroscopic properties of the "doublet" radical may be summarized as follows. The predominant hyperfine coupling comes from H(6) as an a proton. The isotropic component of the coupling is about 7.5 G and consequently the principal elements should be close to the values 3.5, 7 and 12 G. In other pyrimidines such properties are attributed to the anionic, not necessarily negatively charged species (here we use the term "anionic" in a broader sense, for any species having one electron more in the ~r system than the original molecule). More precisely, the observed data are strikingly similar to the values for the cytosine radical formed by the net addition of a hydrogen atom to O(2~ (2, 17). We suggest that in the present case the anion radical protonated at O(4~ is present. The difference between the two lower spectra in Fig. 3 accounts for the third radical. This radical is obviously the first one to disappear upon warming the crystal. Its E S R spectrum is rather broad and unresolved and not enough data could be collected to make any reliable judgement on the radical structure. DISCUSSION It has been proven that the radicals formed by ionizing irradiation of the crystal of isocytosine have much in common with the radicals observed in the crystal of a similar system--cytosine. The existence of two tautomers and the very complex crystal structure of isocytosine make the ESR analysis of the present system much more complex and the results more uncertain, except perhaps the data associated with the cation radical. The mere fact that there are two tautomers of isocytosine imposes the additional question: is the damage randomly distributed between the two tautomers or is one of them preferred? From the present analysis it is obvious that at least the cations are located predominantly or entirely on one species,

328

HERAK, RAKVIN, AND BYTYCI

tautomer A. This means that the cation radical has lower energy when on species A than on species B. Our attempt to prove that by the I N D O M O calculations failed, because the calculation of species A failed to converge. The presence of the positively charged radicals in the crystal suggests that also negative species should be present. They are not necessarily in the radical form. Anion radicals have been demonstrated beyond question in the crystal of cytosine (1, 2, 18). The "doublet" radical detected in the present study is more likely to be neutral, but isoelectronic with the isocytosine anion radical. It is possible that this radical is formed by a transfer of a proton from the hydrogen-bonded neighbouring molecules, thus leaving a negative charge on a diamagnetic neighbour. However, the concentration of the "doublet" radical is too low to compensate all the positive charge of the cations. A part of the negative charge might be associated with the third radical species present in the system. Alternatively, all the negative charge is associated with the third radical. At present neither of these possibilities is supported by the experimental evidence. All the three radicals are very unstable. They disappear at temperatures much below room temperature. The poor stability of the radicals is also demonstrated by illumination with an Ar laser at 77 K: the radical concentration decreases by an order of magnitude within several minutes. This fact supports the idea of the ionic nature of the radicals, including the "doublet" radical. The big difference in stability between the "doublet" radical and the spectroscopically related radical in cytosine seems to support the association of the present radical with a charged diamagnetic species. If it is the case, the doublet radical is formed by a proton transfer from a neighbour molecule to the isocytosine anion. In order to clarify this problem, a use of a more precise E N D O R technique would be of a great help.

ACKNOWLEDGMENT This work is supported by the Research Council of Croatia, Yugoslavia.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

J. N. HERAKAND V. GAI.OGA'~A,J. Chem. Phys. 50, 3101 (1969). W. FLOSSMANN,E. WESTHOF.AND A. MULLER,Int. J. Radiat. Biol. 30, 301 (1976). E. WESTHOF,W. FLOSSMANN.AND A. MULLER,Int. J. Radiat. Biol. 28, 427 (1975). H. C. Box AND E. E. BUDZINSKI,J. Chem. Phys. 59, 1588 (1973). H. C. Box, W. R. POTTER,AND E. E. BUDZINSKI,J. Chem. Phys. 62, 3476 (1975). H. C. Box AND E. E. BUDZtNSKI,J. Chem. Phys. 62,.197 (1975). W. FLOSSMANN.E. WESTHOF.AND A. MIALLER,](nt. J. Radiat. Biol. 28, 105 (1975). B. D. $I--IARMAAND J. F. McCONNELL,Acta Cryst. 19, 797 (1965). H. A. FARACHAND C. P. POOLE. JR.. in "Solving the Spin Hamiltonian for the Electron Spin Resonance of Irradiated Organic Single Crystals", (J. S. Waugh, Ed.), Advances in Magnetic Resonance, Vol. 5, AcademicPress, New York, 1971, pp. 229-303. H. M. McCONNELLAND J. STRATHDEE,Mol. Phys. 2, 129 (1959). J. BAUDET, G. BERTI-IIER,AND B. PULLMAN,Compt. Rend. 254, 762 (1962). M. J. MANTIONEAND B. PULLMAN,Biochem. Biophys. Acta 91, 387 (1964). M. D. SEVILLA,Jr. Phys. Chem. 75, 626 (1971). M. D. SEVILLA.C. VANPAEMELAND C. NICHOLS,J. Phys. Chem. 76, 3571 (1972).

RADICALS FROM ISOCYTOSINE

15. 16, 17. 18.

E. WESTHOF AND M. VAN ROOTEN, Z. Naturforschg. 31e, 371 (1976). M. D. SEVILLA~ J. Phys. Chem. 80, 1898 (1976). J. N. I-IERAK, D. R. LENARD, AND C. A. McDOWELL, J. Magn. Reson. 26, 189 (1977). A. DuL~Id AND J. N. HERAK, Radiat. Res. 71, 75 (1977).

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