ESR study of an X-ray-induced nitroxide radical in 3-hydroxyguanine

JOURNAL

OF MAGNETIC

RESONANCE

67,53 l-538 (1986)

ESR Study of an X-Ray-Induced Nitroxide Radical in 3-Hydroxyguanine’ C. ALEXANDER

JR., AND A. N. M. S. SHAHIDAIN

Department of Physics and Astronomy, University of Alabama, University, Alabama 35486 AND

M. S. JAHAN Department of Physics, Memphis State University, Memphis, Tennessee 38152 Received, November 2 1, 1985 Single crystals of 3-hydroxyguanine have been X irradiated at 77 K and studied by ESR at 300 K. A nitroxide radical formed by abstraction of a hydrogen at N3 is the primary radical species formed by the radiation. A nitrogen hype&e tensor with principal values of 1.7,6.1, and 16 G, and agtensor with principal values of 2.0013,2.0062, and 2.0088 have been determined. These results indicate a spin density of 0.23 on N3. Spectra from pairwise-trapped nitroxide radicals were observed at some orientations. Q 1986 ~eadcmic FTes. Inc.

INTRODUCTION

The radiation chemistry of the DNA base guanine has been studied by many investigators and the radical species stable at room temperature is a hydrogen adduct (1). This H-adduct radical is formed in all DNA bases after room-temperature irradiation. On the other hand, the radical formed at room temperature in some purineN-oxide molecules has been shown to be an acylnitroxide species (2, 3). Nitroxide radicals are considered to be important in many biological reactions (for a review, see Ref. (4)). They are quite persistent and are easily formed by oxidation of biological molecules (5-9). In addition, the -CONHOH group where the nitroxide radical is often formed by H abstraction has been established as a site to which radiation damage is transferred (10). Because of these previous results we thought it would be interesting to study the radiation chemistry of a guanine molecule containing a -NOH group such as 3-hydroxyguanine. This molecule also exhibits oncogenic properties as do some other purine&oxides (I I). EXPERIMENTAL

RESULTS

Powdered samples of 3-hydroxyguanine were obtained from J. C. Parham of the Sloan-Kettering Institute for Cancer Research. These powders were used to grow crystals ’ This research was supported in part by Grant 1 RO 1 CA 202 45-O 1A 1 awarded by the National Cancer Institute. DHEW. 531

0022-2364186 $3.00 Copyright Q 1986 by Academic Press, Inc. All rights of nproduction in any form resewed.

532

ALEXANDER,

SHAHIDAIN,

/\

P

AND JAHAN

A\ 8

(A)

-H

II

II H,N+;

‘; I OH

FIG. 1. (A) The chemical form of the 3kydroxyguanine showing the reference axes, a, b, c, used in this study.

9/

Ii

molecule. (B) The external features of the crystal,

without further purification. Small crystals were obtained by dissolving the samples in dilute HCl at room temperature and then slowly evaporating the mixture at an elevated temperature of 30°C. The chemical form and external features of the crystal are shown in Fig. 1. X irradiation was performed with a unit operating at 40 kV and 30 mA, having an effective energy of 10 keV. An approximate exposure rate was 1.7 X lo5 R/min. Crystals were irradiated at 77 K and ESR spectra were recorded at 300 K on a spectrometer operating at 25 GHz. The modulation frequency used was 32 kHz, the modulation amplitude was approximately 3 G (pp), and the microwave power was approximately 0.5 mW. Figure 2 shows the typical ESR spectra of 3-OHG recorded at 300 K following an X irradiation at 77 K. For these spectra, the crystal was aligned along the a and b reference axes in the ca and bc planes, respectively, and in the ab plane it was aligned along a direction 130” from the a reference axis. The spectra showed three-line hyper-fine patterns in most orientations, which is characteristic of an unpaired electron coupling to a nucleus of spin 1, where the spin density is primarily localized in a p orbital. The singlet observed in certain orientations (such as the strong central line in the bottom spectrum of Fig. 2) is due to the unresolved hyperfme coupling expected when the external magnetic field is perpendicular to the p orbital containing the unpaired density. Doublet splitting of each of the triplet pattern was observed when the crystal was rotated about the a and c axes. This splitting is due to site splitting of two magnetically inequivalent radicals.

NITROXIDE

RADICAL

IN 3-HYDROXYGUANINE

533

9=1.9800

HX 1 30° from a-axis in the

RG. 2. Second-derivative ESR spectra of the irradiated 3-hydroxyguanine crystal at room temperature. The magnetic field was aligned as indicated on the spectra. An absorption line from a C?’ marker with g = 1.9800 is shown by the arrow.

The g value for each ESR spectrum was measured as a function of the rotation angle of the magnetic field with respect to the crystal orientation. From least-squares fits of the g values we obtained elements of squared g tensor by use of the formula g* = k, + kpzos 28 + kssin 28, where g is the experimentally determined g value, 0 is the angle of rotation, and k, , k2 and k3 are related to the elements of the squared g tensor (12). The g tensor was diagonalized to obtain principal values and directions. From these derived principal values and a knowledge of the crystal orientation, g values for different orientations were calculated by use of the formula g = (h - g” - h)“*, where g is the g value, h is the direction of the magnetic field, and g is the g tensor. The g value was calculated for 5” intervals in a particular plane and was compared to the experimental values. The g-value results are shown in Fig. 3; the points represent the experimental data and the solid line represents values calculated from the experimentally determined g tensors. The principal values and direction cosines are given in Table 1. The nuclear hyperhne splittings of each ESR spectrum were also measured as a function of the angle of rotation of the applied magnetic field with respect to the crystal orientation. Data were taken at 5” intervals for 180” rotations of the applied magnetic field for the three planes of rotation. These data were then least-square fitted to obtain

534

ALEXANDER,

SHAHIDAIN,

AND JAHAN

90 60 30 90 60 30 90 60 30

2.000

2.005 g-VALUE

2.010

FIG. 3. Calculated (lines) and observed (points) g-value variations of the ESR absorptions for rotations of the crystal about the a, b, or c axis. Two magnetically inequivalent sites are observed for rotations about the a and c axes.

a squared hyperhne tensor and this tensor was then diagonalized to give principal values and directions. The procedure was analogous to that described for the g-value calculations. Figure 4 shows the experimental points and the calculated tensor fit to the nitrogen hyperfine splitting; the principal values and the direction cosines are given in Table 1.

TABLE 1 Principal Values of the g Tensor and Hyperfine Tensor for the Radical Observed in 3-Hydroxyguanine Coupling constants

Principal values

g1 g2 g3

2.0013 f 0.001” 2.0062 2.0088

Al A2 -43

16.0 + 1.5 G” 1.7 6.1

Direction cosines for molecules 0.828 -0.523 0.202

0.535 0.630 -0.564

0.168 0.575 0.801

0.831 -0.547 0.099

0.556 0.815 -0.162

0.008 0.191 0.982

’ The large errors are due to the poor quality of the crystal and the difficulty of aligning it accurately.

NITROXIDE

RADICAL

IN 3-HYDROXYGUANINE

535

90 60 30 90 60 30 90 60 30

10

0 SPLITTING

20 (GAUSS)

FIG. 4. Calculated (lines) and observed (points) 14N hyperfine splittings for rotations of the crystal about the u, b, or c axis. Two magnetically inequivalent sites are observed for rotation about the a and c axes.

It has been found that for an unpaired electron in the p orbital of a nitrogen atom, the 14N hyperline coupling has a maximum value when the applied magnetic field is parallel to the p orbital direction and a minimum when the applied magnetic field is in a plane which is perpendicular to the p orbital direction. The observed hyperfine splittings in the present work were typical of a r-type nitrogen radical. The perpendicular to the guanine ring is parallel to the axis of the nitrogen p orbitals. The principal values of the 14N hyperline tensor can be expressed in terms of an isotropic component AF and an anisotropic component A,, by the equations All

= PAAF

AL

= /WF-A,J

+ up)

where pn is the spin density in the p orbital of an 14N atom, and A,, and Al are the measured hyperfine splittings when the magnetic field is parallel and perpendicular to the nitrogen p orbital, respectively. Using the hyperfine principal values in Table 1, the above equations, and 17.1 G as the value of the anisotropic coupling for a spin density of unity on the nitrogen atom, AF, A,,, and the nitrogen p orbital spin density for this radical were calculated to be AF = 7.93 G A, = 4.01 G pr = 0.23.

536

ALEXANDER,

SHAHIDAIN, RADICAL

AND JAHAN

MODEL

The large g value variation given in Table 1 is a good indication that the unpaired electron is localized on either a sulfur or an oxygen atom. This is because the spinorbit coupling constant (A) is large for these two atoms (sulfur, X = -382 cm-‘; oxygen, X = 151 cm-‘), compared to this constant for nitrogen or carbon (carbon, X = -28 cm-‘; nitrogen, X = -76 cm-‘) (12). Since there are no sulfur molecules in the 3OHG crystal, it is expected that there is considerable spin density on an oxygen atom. If the spin density is localized in a p orbital on the oxygen, the minimum g-value direction would be parallel to the direction for the maximum hyperfine splitting, and the maximum g-value direction should correspond to the direction of the oxygen bonding orbital. From our data in Table 1, we calculate a value of 9” for the angle between the directions of the minimum g value and the maximum nitrogen hyperhne splitting. The experimental results presented above can be used to predict a radical model for irradiated 3-OHG. The radical is a r-type nitrogen radical with considerable spin density on an oxygen atom. One radical model which is consistent with these data is a nitroxide species, formed when the hydrogen atom is abstracted from the oxygen in the number three position (see inset). This type of radical has been observed in two other hydroxypurine molecules, 3-hydroxyxanthine, and 1-hydroxyxanthine (2, 3).

OH

I 0*

H

I H

The 14N coupling constants and the g-value variation are quite similar to the values found in these other hydroxypurine molecules. For illustrative purposes, ESR results of these three hydroxypurines are tabulated in Table 2. TABLE 2 Principal Values of the .Qand Hvperline Tensors for Three Hvdroxypurine

Radicals

Principal values Coupling constants

1-Hydroxyxanthine

L?l

2.0031

g2 g3

1.0085

Al A2

A3

3-Hydroxyxanthine

3-Hydroxyguanine

2.0099

2.0050 2.0010 2.0105

2.00623 2.00130 2.00879

2.6 G 13.6 0.8

1.2 G 14.2 2.9

1.7 G 15.9 6.1

NITROXIDE

RADICAL

IN 3-HYDROXYGUANINE

537

A McLachlan molecular orbital calculation was carried out for the nitroxide radical model with a X = 1.2 value. The calculated spin densities with values greater than 0.07 were 0.18 on N3 on 0.55 on the attached oxygen, These values are consistent with our experimental g-value variation and hyperfine couplings. In our previous investigations of irradiated purine-N-oxide single crystals, we found strong spectra from pairs of nitroxide radicals after X irradiation at 77 K (2, 3, 13). This pairwise trapping exhibited unusual temperature characteristics because it proved to be stable at room temperature. Compared to our earlier purine-iv-oxide crystals, our 3-OHG crystals were more difficult to grow and were not as well formed. The 3-OHG ESR spectra were consequently weaker and radical pair absorption lines were more difficult to detect. Nevertheless, there were crystal orientations where lines characteristic of pairwise trapping were observed. These lines can be seen in the bottom spectrum of Fig. 2. In this spectrum, the large central line is from the nitroxide monoradical (the 14N splitting is not resolved at this orientation) and the symmetric outer lines are attributed to nitroxide radical pairs. These lines were symmetric about the central maximum and they broaden and move symmetrically away from the center as the crystal is rotated from this orientation. Unfortunately these lines become too weak to be observed as the nitrogen splitting increases, and they can be followed for only a few degrees of rotation. We interpret these spectra as evidence that pairwise trapping of nitroxide radicals also occurs in 3-OHG crystals and that this radical configuration is also stable at room temperature. CRYSTAL

STRUCTURE

CHARACTERISTICS

The crystal structure of 3-hydroxyguanine has not been determined, but some details of the structure can be determined from our ESR data. The direction of the perpendicular to the purine plane is along the direction of the p orbital on the nitrogen atom. A direction of the p orbital can be determined from an average of the directions and of the maximum hyperfine direction and the minimum g-value direction. This calculated direction is given by direction cosines (with respect to the u, b, c axis system) with values of 0.831, 0.556, 0.088. This direction gives the orientation of the purine molecules with respect to the chosen reference axes. The direction of the N3-03 bond of the molecule should be the same as that of the maximum g value which is given by direction cosines with values of 0.202, 0.564, 0.80 1. The ESR data indicate that there are two magnetically inequivalent sites for molecules in the crystal and that these two are equivalent for rotations of the crystal about one axis (called the b axis). These symmetry considerations are consistent with a monoclinic-type crystal structure for the 3-hydroxyguanine crystal. CONCLUSIONS

In contrast to most guanine derivatives, no H-adduct radicals were observed at room temperature after 77 K irradiation. The primary stable radical formed in 3hydroxyguanine is a nitroxide formed by hydrogen abstraction from the oxygen atom bonded to N3. The spin density in the p orbital on N3 was determined to be 0.23. This radical has now been shown to be the primary species formed in three irradiated purine-N-oxide molecules. This result is very interesting in light of the report by Shields

538

ALEXANDER,

SHAHIDAIN,

AND

JAHAN

that the -NOH group of hydroxamic acids is particularly sensitive to ionizing radiation (IO). The present results on 3-OHG and our previous results on 3-OHX and I-OHX (2, 3) (which contain hydroxamic acid groups) show that the irradiation damage to purine-N-oxides is stabilized or trapped at the -NOH site and the spin density is highly localized in that part of this conjugated molecule. These results are also consistent with earlier solution ESR reports that a nitroxide is the primary radical species formed by oxidation of cyclic hydroxamic acids (14). Our results also indicate that room-temperature-stable pair-wise.trapping of nitroxide radicals occurs in 3-OHG crystals as was found in other irradiated puke-N-oxides. REFERENCES

1. W. A. BERNHARD, Adv. Rudiut. Biol. 9, 199 (1981). 2. M. S. JAHAN AND C. ALEXANDER, JR., R&at. Rex 74,25 1 (1978). 3. K. SOGABE AND C. ALEXANDER, JR., Radiat. Res. 97,443 (1984). 4. R. P. MASON, “Reviews in Biological Toxicology” (Hodgson, Bend, Philpot, Eds.), p. 15 1, Elsevier, Amsterdam, 1979. 5. A. R. FORRESTER, M. M. OCILVY, AND R. H. THOMPSON, J. Chem. Sot. C, 108 1 (1970). 6. S. H. BLOBSTEIN, R. W. GRADY, S. R. MESHNICK, AND A. CERAMI, B&hem. Parmacol. 27, 2939

(1979). 7. H. BARTSCH,

J. A. MILLER, AND E. C. MIUER, B&him. Biophys. Acta 273,40 (1972). L. M. SOONG, R. N. WALKER, AND M. STUART, Cancer Res. 36,276l (1976). KIESC, Xenobioticu 1, 553 (1971). SHIELDS, Radiat. Rex 100,418 (1984). SUGIURA, M. N. TELLER, J. C. PARHAM, AND G. B. BROWN, Cancer Res. 30, 184 (1970). CARRINGTON AND A. D. MCLACHLAN, “Introduction to Magnetic Resonance,” Harper & Row, New York, 1967. 13. M. S. JAHAN AND C. ALEXANDER, JR., J. Map. Resort. 43,21 (1981). 14. A. T. BALABAN, I. PAXARA, AND F. CUBAN, J. Magn. Reson. 7,241 (1972).

8. R. 9. M. 10. H. 11. K. 12. A.

A. nOn>,