Identification of a monoradical species from the EPR study of its radical pair in irradiated 3-hydroxyxanthine single crystals

JOURNAL

OF MAGNETIC

RESONANCE

43, 21-27 (1981)

Identification of a Monoradical Species from the EPR Study of Its Radical Pair in Irradiated 3-Hydroxyxanthine Single Crystals* M. SHAH Department

of Physics

and

JAHAN? Astronomy,

AND CHESTER The

University

ALEXANDER, of Alahamu,

JR.

University,

Alabama

35486

Received August 13, 1980 A model for a monoradical species formed in 77-K X-irradiated 3-hydroxyxanthine has been determined from a study of its radical pair EPR spectra. The monoradical and radical pairs were stable at room temperature and were studied at that temperature. The radical was found to be a r-type nitrogen-centered species with hyperfine values of A,, = 9.7 t 1 G and A, approximately 1 G and g-tensor principal values of 2.0093, 2.0081, and 2.0029. The spin density on the nitrogen atom was calculated to be between 0.15 and 0.21, and this was compared to theoretical values from Hiickel molecular orbital calculations. A radical model that is consistent with these data is hydrogen abstraction from N,,,. The average distance between radicals forming the pair is 6.75 8, and the crystalline interplane spacing is approximately 3.7 A.

Ultraviolet-induced radicals trapped in the oncogenic purine N-oxide, 3-hydroxyxanthine (3-OHX), have been studied by several investigators (l-4). In our previous study (5) we observed a stable nitroxide radical, Rl, with structure (A),

d

H A

which was found to be the primary radical species induced when 3-OHX was uv irradiated at 77 or 300 K. In the same study we also observed that radicals were trapped in pairs when 3-OHX was irradiated by X rays or high-energy electrons at 77 K. Two radical pairs, I and II, observed at 300 K were attributed to radicals of type Rl situated in adjacent crystal planes. In this paper we are reporting a new monoradical species, R2, that is trapped in 3-OHX when irradiated by X rays or high-energy electrons at 77 K. Detection and identification of R2 are very difficult * This research was supported by Grant 1 ROl CA 20245-OlAl, awarded by the National Cancer Institute, DHEW. t Present address: Department of Physics, Memphis State University, Memphis, Tennessee 38152. 21

0022-2364/81/040021-07$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

22

JAHANANDALEXANDER

LLLL A’: ?

LLlL Y

$1

LLI b

2

1 G

+ILLI I+

4J 1

LLLLJ

I

AI -102G , An=260

I

A2

dlJ

-IAl

LIL 1

A2

A~=rnG

FIG. 1. Second-derivative EPR spectrum for a 3-hydroxyxanthine crystal irradiated by X rays at 77 K and observed at 300 K. The magnetic field was aligned 50” from the a axis in the ab plane. The line marked by the arrow is one of the three lines due to radical R2 with hyperline splitting A,. The groups of lines separated by A,,, are due to radical pair III with hyperfme splittingAJ2. The dipolar splittings A, and Ai, and hyperfine splittings A,/2 characterize radical pairs I and II.

because, except along a few orientations, the resonance lines of R2 are always superimposed on those due to Rl. Fortunately, we observed a third type (III) of radical pair, which is trapped simultaneously with I and II and which was found to have a smaller hyperfine splitting and a different center of resonance compared to those of I and II. We have been able to determine a g tensor and approximate hyperfine values for the monoradical species R2 by following the resonance lines of radical pair III. We have also determined some crystal structure characteristics from the radical pair data. EXPERIMENTAL

RESULTS

Crystal growing, crystal structure parameters, and irradiation processes were discussed in our previous paper (5). Crystals were irradiated at 77 K and EPR spectra were taken at 300 K. Second derivatives of the absorption lines were recorded on a home-built spectrometer operating at 25 GHz. The modulation frequency used was 32 kHz, the modulation amplitude was approximately 2 G (pp), and the microwave power was approximately 0.5 mW. According to the EPR theory of radical pairs (6) the resonant magnetic field is given by H = H,, + 4 - ; (A;gn’

where

+ A$n”),

RADICAL

TABLE PRINCIPAL

VALUESOFTHE~ AND RADICAL R2 OBSERVEDIN

Coupling constants

23

FROM 3-HYDROXYXANTHINE 1 HYPERFINETENSORSFORTHE 3-HYDROXYXANTHINE

Principal values

Direction cosines in the a, h, c’ system

2.0093 2.0029 2.0081

0.028 0.999 0.003

9.1 + 1G 0-*2G

1.00

0.912 -0.028 -0.234

0.234 -0.003 0.972

0.0 I to a axis

0.0

A = D(3 co? tl - 1) + E sin’ 8 co? 4.

[31

D and E are zero-field splitting constants; 8 and 4 are the azimuthal and equitorial

angles between the vector reff and the direction calculated from

of the magnetic field. The reff is

D = 3/3re;.

[41

The center of the group of resonance lines due to a radical pair, as given by Eq. [2], is the same as that of the isolated monoradicals. The last term of Eq. [l] shows that the hyperfine splitting of the radical pair spectra is the same as that for two coupling monoradicals, each with one-half the hyperfine splitting of the isolated monoradical. The EPR spectra for radical pair III is shown in Fig. 1 for an orientation of the crystal with the external magnetic field 50” from the crystalline a axis in the ab plane. By comparing the spectra for pairs I, II, and III, it can be seen that the

FIG. 2. Experimental (0) and calculated (lines) g-value variations rotation of the crystal in c’a, bc’, and ab planes.

of the EPR absorptions

for

24

JAHANANDALEXANDER

FIG. 3. Angular dependence of the dipolar splitting, A, measured in the ab, bc’, and c’a planes. Circles indicate the observed splittings due to radical pair III and the solid lines represent the theoretical curves calculated from the experimentally determined tensors.

hyperfine splitting (A,) for III is smaller than for I and II (A,), and the center of the spectrum is not the same as the center of the I and II spectra. Also it is evident in Fig. 1 that an extra line (marked by an arrow) is present in the central resonance in addition to the three-line RI nitroxide spectrum (described in our previous report): this line is the only line of the R2 monoradical that can be observed under the stronger Rl spectrum. These facts indicate that the pairs of radicals responsible for the spectra of III are not the same as those that produce the spectra for I and II. The hyperfine pattern with intensity ratios 1:2:3:2:1 is of the same type (but with different hyperfine splittings) as that for I and II and is exactly what is expected for the interaction of two identical nitrogen centered radicals. The g principal values given in Table 1 were obtained by following the g-value variation for III for the three rotation planes and obtaining a g tensor by least-squares fitting to the formula,

RADICAL

FROM

TABLE CHARACTERISTIC Direction

cosines

relative

to axis

N

h

CC

20.543 0.938 t0.001

0.839 TO.543 -0.010

0.010 +0.001 0.999

25

3-HYDROXYXANTHINE

DATA

2 OF RADICAL

Principal values of splitting tensor A(G) 181.5 -61.5 - 129.5

PAIR III

D ((3

E ((3

Effective distance reff L-4)

-90.75

34.0

6.75

g2 = K1 + Kz cos 28 + K3 sin 28,

[51

where 8 is the angle of rotation and K,, Kz, and K3 are related to the elements of a squared g tensor (7). In Fig. 2 the observed g values (points) are compared with those calculated (lines) from the parameters in Table 1. The hyperfine splitting of radical pair III could be measured when both the electron-electron dipolar splitting (A) and the hyperfine splitting were large. This situation occurred for some orientations when the crystal was rotated about the c’ or b axes. There was no observable hyperfine splitting for u-axis rotations. The hyperfine variation was axially symmetric, similar to that of the g-value variation and to the hyperfine splitting of radical Rl. By using the observable hyperfine splittings and symmetry characteristics, we calculated an approximate hyperfine tensor, which is given in Table 1. These EPR parameters in Table 1 characterize the monoradical species R2 responsible for the radical pair III spectra. Since the absorption spectra for the nitroxide radical Rl are much stronger than those for this second monoradical species R2, there are only a few orientations where any lines are observable, and it is only from the radical pair spectra that the characteristics of the R2 radical could be determined. This second monoradical species, R2, is a r-type nitrogen-centered radical with a g value that is almost axially symmetric. The g-value variation is large enough to suggest delocalization of the unpaired electron to oxygen atoms. An approximate spin density pn, on the nitrogen atom, was calculated by using Eqs. [6], [7], shown below, and a value of Ap of 17.1 (8) as the value of the anisotropic coupling for a spin density of unity on the nitrogen atom. A,, A,

= PAAF

+

24-d

[61

= PAAF

-

AP.).

[71

The spin density on the nitrogen atom was calculated to be 0.15 + 0.21. Zero-field splitting parameters for radical pair III were obtained with a leastsquares fitting procedure like that used for the g-tensor principal values. Fig. 3 shows the variation of the dipolar splitting in three rotation planes and the characteristic values for III are given in Table 2. The r eff vector is approximately in the ab plane and its length is 6.75 A. The relative positions of the two R2 monoradicals forming the radical pair III is shown in Fig. 4.

26

JAHAN

AND ALEXANDER

0

-5. -6. -7. -8FIG. 4. A section through the crystal unit cell, perpendicular to c’, in single crystals of 3-hydroxyxanthine. Heavy bars indicate the segments of molecules, N,,,-O,,,, where most of the unpaired spin density is localized. The relf vectors for radical pair III as well as those for I and II are represented by the dark lines. CONCLUSIONS

The R2 radical species is a nitrogen-centered radical with electron delocalization to oxygen atoms. Without a crystal to give a definite assignment of the radical structure, but by eters and Huckel molecular orbital calculations, we can model. Hydrogen abstraction from N,,, would give radical, as shown below:

considerable unpaired structure it is difficult using our EPR paramsuggest a reasonable R2, with structure (B)

Theoretical spin density calculations for this radical by the Huckel method give the values in Table 3. The values of 0.14 on N,,,, 0.12 on OC2), and 0.10 on OC6) would be consistent with our experimental g-value variation and hyperfine coupling. This radical is formed at the same time as the hydrogen abstraction radical

RADICAL

FROM

TABLE THEORETICAL HWKEL Atom N (1) C (2, 0 121 N,, 0 (3, C (41

Spin

27

3-HYDROXYXANTHINE 3

SPIN DENSITIES CALCULATIONS

density 0.04 0.02 0.12 0.14 0.04 0.13

Atom C (51 C 161 0 161 N (71 N t9,

FROM

Spin

density 0.20 0.01 0.10 0.08 0

Rl, but the yield of the R2 species, for the same radiation dose, is less. The R2 species is formed by X rays or high-energy electrons, but it was not observed after uv irradiation. The intensity of the radical pair III spectra was comparable to that of radical pairs I and II. This could indicate that the formation of highly concentrated radical “clusters” is as likely for the R2 species as for the Rl species. An interplane spacing for the molecules in the crystalline structure can be calculated from the length of the reff vector and the angle of this vector to the a axis. This calculation gives a spacing of 3.7 A, which is reasonable for this type of crystal and which is in good agreement with a 3.6-A value calculated from the radical pair data of I and II. REFERENCES I. 2. 3. 4. 5. 6. 7. 8.

G. B. BROWN, M. N. TELLER, I. SMULLYAN, N. T. M. BIRDSALL, T. C. LEE, J. C. PARHAM, AND G. STOHRER, Cancer Res. 33, 1113 (1973). G. B. BROWN, Prop. Nucleic Acid Res. Mol. Biol. 8, 209 (1968). I. PULLMAN, J. C. PARHAM, AND G. B. BROWN, Radiat. Res. 47, 242 (1971). J. C. PARHAM, I. PULLMAN, AND G. B. BROWN, Tetrahedron 29, 3329 (1973). M. S. JAHAN AND C. ALEXANDER, JR., Radiat. Res. 74, 251 (1978). Y. KURITA, J. Chem. Sot. Jpn. 85, 833 (1964). A. CARRINGTON AND A. D. MCLACHLAN, “Introduction to Magnetic Resonance,” Chap. 7, pp. 103- 105, Harper & Row, New York, 1967. J. R. MORTON, Chem. Rev. 64, 453 (1964).