ENDOR

J . Phys. Chem. 1991, 95, 1494-1503

1494

Primary Reduction and Oxidation of Thymine Derivatives. ESRIENDOR of Thymidine and 1-Methylthymine X-Irradiated at 10 K Eli 0. Hole, Einar Sagstuen, Department of Physics, University of Oslo, P.O. Box 1048, Blindern. 0316 Oslo 3, Norway

William H. Nelson,* Department of Physics and Astronomy, Georgia State University, Atlanta. Georgia 30303

and David M. Close Department of Physics, East Tennessee State University, P.O. Box 22060 A, Johnson City, Tennessee 37614 (Received: July 12, 1990)

Single crystals of thymidine and I-methylthymine were X-irradiated at 10 K and studied by using K-band ESR, ENDOR, and FSE spectroscopy. In thymidine, six primary radicals were identified. The major species were the 04-protonated anion (TI), the radical formed by net H addition to the C6 position of the base (TII), the radical formed by net H abstraction from the methyl group (TIII), and the alkoxy radical (TVI). The two minority radicals were one formed by net H addition to the C5 position of the base (TIV) and one in which the unpaired spin was localized on the sugar moiety, thought to be a CI' H abstraction radical (TV). A small, nonexchangeable coupling in radical TI was shown to be due to a coupling to the methyl group. Evidence is presented that the characteristics of this coupling tensor indicates the protonation state of the thymine anion radical. Upon annealing, the anion decayed at about 40 K with no detectable successor. The alkoxy radical decays in the same temperature range. In I-methylthymine, three major radical species were identified. These were the 0 4 protonated anion radical (MTI), the radical formed by net H abstraction from the C5 methyl group (MTII), and the radical formed by net H addition to the C5 position of the base (MTIII). The protonation state of the anion was interpreted from the characteristics of a small coupling with the methyl group. The anion decayed at about 40 K with no detectable successor. The thymin-5-yl radical slowly grew in upon prolonged annealing above 250 K. In both crystals the protonated anion is the reduction product. Comparison of the 0 4 protonated product's magnetic parameters with those of products thought to be thymine anions in other thymine systems in the solid state led to the proposal that all were similarly protonated. Finally, evidence is reviewed for reactions in which thymin-5-yl radicals may arise from the 0 4 protonated anions or other precursors.

Introduction Thymine (structure I, dR replaced with H) is one of the five base constituents of nucleic acids. The first free-radical product identified in dry DNA after exposure to ionizing radiation was the 6-dihydrothymin-5-yl radical, TH2 (structure 11, dR replaced with H) formed by net hydrogen addition to the C6 position of the thymine base.lP2 Radiation-chemical experiments using DNA and pyrimidine derivatives have shown that the 5-yl radical is formed in aqueous solution by protonation at C6 of the primary reduction product, the thymine anion T (structure 111, dR replaced with H).3-5 Other experiments indicate that this process may also occur in the solid state (crystals, frozen aqueous. solut/on, frozen glasses)! However, in crystalline matrices, the T- TH2 conversion has not been followed in detail but has been accepted as a working model.

-

I

I

I

dR

dR

dR

I

I1

I11

One result of this model was the fairly widely accepted scheme for primary oxidation and reduction of DNA by direct Here, electrons are trapped predominantly at thymine whereas oxidation takes place at guanine. A major piece of evidence for this model is the ESR observation of the thymin-5-yl radical at high temperatures following the disappearance of the primary doublet resonance that has been ascribed to the T radical trapped at low temperatures. Recently,.evidence has accumulated indicating that the twocomponent T-/G+ model for the primary radiation response of DNA by direct effects must be modified. In particular, it seems necessary to take into account other sites for the primary damage, as well as alternative mechanisms for their secondary Among many questions to be answered is whether the 5-yl radical is formed from a negatively charged thymine anion precursor in the solid state, or if the pristine reduction product may engage in other reactions. I n a recent short communicationI2 evidence was presented that in a single crystal of thymidine (structure I , dR = deoxyribose), ( I ) the anion was protonated at 0 4 even at 4.2 K and thus was stabilized as a neutral product, and (2) the protonated anion was very unstable and decayed before 40 K with no radical successor.

~

( I ) Ehrenberg, A.; Ehrenberg, L.; Lofroth, G . Nature 1963, 200, 376. (2) Eisinger. J.: Schulman, R. G.h o c . Nail. Acad. Sci. U.S.A. 1%3,50,

694. (3) Ormercd, M.G. Int. J . Radial. Biol. 1965, 9, 291. (4) Holroyd, R. A.; Glass, J. W . Int. J . Radial. Biol. 1971, 14, 445. (5) (a) Das, S.; Deeble, D. J.; Schuchmann, M.N.; von Sonntag, C. I n t . J . Radial. Biol. 1984, 46, 7. (b) Deeble, D. J.: Das, S . ; von Sonntag, C. J. Phys. Chem. 198589, 5784. (c) Novais, H. M.;Steenken, S.J. Am. Chem. SOC.1986. /OR. I . (d) Das, S.: Deeble, D. J.; von Sonntag, C. Z . Naturforsch. 1985, 40c, 292. (6) Bernhard, W. A . Ado. Radial. Biol. 1981, 9, 199.

(7) Graslund. A.; Ehrenberg, A,; Rupprecht, A,; Strom, G . Biochem. Biophys. Acta 1971, 254, 172. (8) Huttermann, J.; Voit, K. I n Electronic Magnetic Resonance in The Solid Slate, Weil, J., Ed., Canadian Society for Chemistry: Ottawa, Canada, 1987; p 267. (9) Symons, M . C. R. J . Chem. Soc., Faraday Trans. 1 1987, 83, I . (IO) (a) Bernhard, W. A.: Patrzalek, A. 2.Radial. Res. 1989, 1 1 7 , 379. (b) Bernhard, W. A . J. Phys. Chem. 1989, 93, 2187. ( I I ) Huttermann, J. Free Radical Res. Commun. 1989, 6 , 2 5 . (12) Sagstuen, E.; Hole, E. 0.;Nelson, W. H.; Close, D. M.J. Phys. Chem. 1989. 93, 5974.

0022-3654/9l/2095-l494$02.50/0 0 1991 American Chemical Society

The Journal o/ Physical Chemistry, Vol. 95, No. 3, 1991 1495

Reduction and Oxidation of Thymine Derivatives

TABLE I: Hyperfine Coupling Parameters for the Protonated Thymine Aniona TI in Crystals of Thymidine X-Irradiated at 10 KbC coupling . 1

principal values -55.7 (3) -30.3 (3) -13.2 (3)

isotropic value -33.1 (2)

42.6 (4) 29.2 (4) 27.6 (3)

eigenvectors

dipolar values -22.6 (3) 3.2 (3) 20.3 (3)

0.732 (5) 0.680 (6) 0.048 (1 5)

33.1 (2)

9.5 (4) -3.9 (4) -5.5 (3)

0.282 (16) 0.190 (142) 0.940 (27)

-11.9 (4) -7.6 (4) 3.2 (3)

-5.4 (2)

-6.5 (4) -2.2 (4) 8.6 (3)

-8.6 (3) -7.9 (2) -3.7 (3)

-6.7 (2)

-1.9 (3) -1.2 (2) 3.1 (3)

(a)

-0.670 (49) 0.707 (45) -0.226 (16) 0.670 (81) 0.195 (301) 0.717 (32)

(b) 0.656 -0.722 0.221 0.806 -0.579 -0.124 -0.640 -0.705 -0.307

(5) (8) (19)

(8) (24) (9) (47) (43) (26) -0.739 (40) 0.076 (270) 0.670 (30)

(C)

0.185 -0.130 -0.974 0.521 -0.793 0.317

(8) (22) (4)(14) (50) (117)

-0.376 (17) -0.061 (36) 0.925 (7) -0.076 (390) 0.978 (40) -0.195 (61)

Calculated Dipolar Principal Values for Coupling 4 -1.9 0.7 12 -0.9 0.166 2.9 0.682

-0.702 0.197 0.685

0.021 0.966 -0.257

Directions from the Crystal Structure 0.7068 0.2008 -0.2363 0.68 13

-0.7068 0.1731 -0.1678 0.6953

0.0297 -0.9642 0.9571 -0.2289

thymine base perpendicular C6-H bond' N3-H bond C 5 4 7 bond

OStructurc IV, d R = deoxyribose. *The coupling parameters are given in megahertz. CNumbersin parentheses represent the standard deviation in the last quoted digit(s). dThe signs of the principal values have been reversed compared to those in the previous publication.'* 'This direction was incorrectly given in ref 12.

In this paper, more complete results on the primary radiation response of thymidine and 1 methylthymine are presented. The main emphasis is on the structure and secondary reactions of the reduction products, with particular attention given to any connection bctween the anions, 0 4 protonated or not, and the 5-yl radicals. Thus, the relation between these two products is discussed, concluding with the suggestion that environmental factors and other reactions are important in determining the formation of the DNA thymine radical. Experimental Section Single crystals of thymidine and 1methylthymine were obtained from aqueous solutions of the commercial chemicals (Sigma Chemical Co.) by slow evaporation from covered vessels in an oven maintained at 35-50 'C. Partially deuterated crystals of each material (with all nitrogen- and oxygen-bonded protons exchanged with deuterons) were prepared in a similar manner from D 2 0 solutions. Single crystals of thymidine are orthorhombic with space group P2,2,2,,13while single crystals of I-methylthymine are monoclinic with space group PZ,/C.'~ For the I-methylthymine crystals, the orthogonal (a*bc) reference system was used. Both types of crystals are anhydrous. The experimental procedures, which involve X-irradiation and measurements at temperatures close to that of liquid helium (a conservative estimate of 10 K is used throughout this work) or at pumped nitrogen temperature (65 K ) have been previously A recent improvement consists of the described in introduction of a Hughes 24-dB low-noise microwave preamplifier in the detection circuit. This permits the operation of the spectrometer at power levels in the nanowatt range. In the case of thymidine, four independent data sets were collected from three planes of rotation, i.e., rotation about ( a ) , (1 3) Young, D.W.;Tollin.

P.;Wilson, H. R. Aero Crysfallogr. 1%9,825,

1423. (14) (a) Hoogsteen, K. Acfa Crysrallogr. 1963, 16, 28. (b) Kvick, A.; Koetzle, T. F.; Thomas, R. J. Chem. Phys. 1974,61, 271 I . (15) (a) Nelson, W.H.; Hole, E. 0.; Sagstuen, E.; Close, D. M. Inr. J . Radiuf. B i d . 1988,54,963. (b) Because of the large number of data points, figures showing the angular dependence of the data are not included. The errors derived from the data are given in the tables for each coupling tensor. The overall rms error of fitting the data was in the range 20-50 kHz. The data files and angular variation plots may be obtained from the authors upon rquest.

(c), and a skew axis located in the ( c a ) plane 68.8O from (c). Similarly, in the case of I-methylthymine, four independent sets of data were collected from three planes of rotation, Le., rotation about ( b ) , (c), and a skew axis defined by the polar angles B = 107.5' and = 121.3°.15bIn this manner the Schonland ambiguityI6 always was solved and unique hyperfine coupling tensors were obtained. Field-swept ENDOR (FSE) techniques" were used to assist in the assignment of different ENDOR lines to specific radicals and to ensure that the coupling tensors derived from the data are all related to the same molecule in the unit cell. Results and Analysis ( A ) Thymidine. Six different free-radical products were identified in thymidine (TdR) single crystals after X-irradiation at about 10 K. Some of these were analyzed in detail in the and will thus previous communication12 or by other only be described briefly. Radical TI: The 04-Protonated Anion. In the previous communication,'* it was clearly documented that a species with structure IV (dR = deoxyribose) was formed and trapped at 10

dk Iv K. The spectroscopic observation of the proton at 0 4 makes this assignment unambiguous. The ability to clearly detect this 0protons presence is the result of its out-of-plane position. This (16) Schonland. D. S. Proc. Phys. Soc. London 1959, A73, 788. (17) Kevan, L.; Kispert, L. D. Electron Spin Double Resonance Spectroscopy; Wiley: New York, 1976. (18) (a) Box, H. C.; Budzinski, E. E. J. Chem. Phys. 1975,62, 197. (b) Box, H.C.; Budzinski, E. E.; Potter, W. R. J. Chem. Phys. 1974,61, 1136. (19) (a) Herak, J. N.; McDowell, C. A. J. Magn. Reson. 1974, 16,434. (b) Herak, J. N.; Velenik, A,; McDowell, C. A. J. Magn. Reson. 1982, 19,

64. (20) (a) Huttermam, J. I n f . J. Radial. Biol. 1970, 17, 249. (b) Flossmann, H.;Zehner, H.; Muller, A. 2. Naturforsch. 1980, C35. 20. (c) Westhof, E.;Flossmann, W.; Zehner, H.; Muller, A. Faraday Discuss. Chem. Soc. 1977. 63, 248.

Hole et al.

1496 The Journal of Physical Chemistry, Vol. 95, No. 3, 1991

TABLE II: Un ired Spin Density Distribution for the Tbyaine Anion‘ T,, the 04-Protonated Anion T,+4’,bJ and the 0 2 , 0 4 Doubly Protonated Anion T,+2’,4’ Calculated by Using the I N W RHF/CI SCF-MO Method‘

g.

radical T, T,+4‘ T,+2’.4’

d -I 0 I

NI

c2

02

0.050 0.113 0.149

0.001 -0.008 -0.004

-0.003 -0.006 -0.002

N3 0.052 0.122 0.055

c4 0.143 0.463 0.480

04

c5 0.098 -0.096 -0.080

0.180 0.062 0.078

@Ta, structure 111, dR replaced with H. ”,+4’, T:,

or T:+2’,

structure IV,dR replaced with H. (The alternative notation is T:.6 depending upon the mechanism of formation. ‘Reference 27. /This is the net charge of the radical.

fact, along with the specific hydrogen bonding arrangement of 0 4 in the crystal, described below, are evidence that the proton was transferred to 0 4 across a hydrogen bridge. In the crystal, the HN3-N3-C4-04 fragments of two thymidine molecules are bridged by 03’ (in the sugar) of two other molecules forming a centrosymmetric hydrogen bonded tetramer. The dihedral angle C5-C4-04-H03’ is 1 56O.I3 After the formation of radical TI the added proton remains in the original out-of-plane hydrogenbond direction, thereby enabling its direct observation. Figure 1 shows ENDOR spectra from normal and partially deuterated crystals of thymidine. This figure demonstrates that the proton added at 0 4 and the proton at N3 are in fact exchangeable. Clearly, the proton at C6 and the proton responsible for the fourth coupling associated with this radical are nonexchangeable. For completeness, all four coupling tensors are reproduced in Table I. Of the four couplings associated with radical TI, the smallest (denoted coupling 4 in Table I ) was previously ascribed to a >C6-HCI’ interaction.I2 However, the almost identical interaction recently observed in 1 -methylthymine (see below) forced reexamination of this assignment. An alternative interpretation arises from the observation that the eigenvector of the numerically smallest principal value is very close to the C5-CH3 bond direction (the deviation is about 3O). Furthermore, the eigenvector for the numerically largest value is close to the pyrimidine ring normal (the deviation is about 6’). These properties are characteristic of a coupling to a P-methyl group that is bonded to the central C atom of an allylic type fragment.2i To reassign coupling 4 in line with the arguments above, three requirements must be met: (1) The signs of the principal elements of the hyperfine coupling tensor must be reversed, which implies that there must be a small negative spin density at C5. (2) The methyl group must rotate freely in order that the ENDOR is a single line. (3) The combined dipolar coupling from the small negative spin density at C5 and large positive spin densities at C4 and C6 must match the experimental dipolar coupling. The following discussion shows that the data meet these requirements. For the rotating methyl model, the magnitude of the spin density at C5 can be calculated from the isotropic part of coupling 4, and the Heller-McConnell relation for

aiso= p*(Bo

+ B2 cos2 0)

(1)

For the case of free rotation, this simplifies to aiso= p”QP where Q1, = 80 M H z . ~With ~ ai, = -6.7 MHz (see Table I), this relation yields p” = -0.08. The dipolar coupling tensor of the rapidly rotating methyl group was calculated by considering the H3,part as one effective H located along the C5-C7 bond direction. For calculating the combined dipolar couplings (with C4, C5, and C6) the procedure developed by McConnell and StrathdeeZ4was adopted and was modified as prescribed by Derbyshire,25aBarfield,25band Wells.2k The actual method of calculation was outlined in a previous report.26 The set of parameters used for the dipolar calculation were as follows: the C6-C5(CH3)-C4 system was assumed to (21) Theisen, H.; Sagstuen, E. J . Cfiem.Pfiys. 1981, 74, 2319. (22) (a) Heller, C.: McConnell, H. M. J. Cfiem.Pfiys. 1960.32. 1535. (b) McConnell, H. M.; Chesnut, D. B. J. Chem. Pfiys. 1958, 28, 107. (23) Fessenden, R. H.; Schuler, R. J . Cfiem. Pfiys. 1963, 39, 2147. (24) McConnell, H. m.; Strathdee, J. Mol. Phys. 1959, 2, 129. (25) (a) Derbyshire, W. Mol. Pfiys. 1962, 5 , 225. (b) Barfield, M. J . Cfiem. Pfiys. 1970, 3836. (c) Wells, J. W. J. Cfiem. Pfiys. 1976, 66, 632. (26) Sagstuen, E.: Awadelkarim, 0.;Lund, A,; Masiakowski, J. J . Cfiem. Pfiys. 1986.85. 3223.

4

40

C6 0.454 0.357 0.331

“The alternative notation is

2 1

45

50

55

$0

65

ENDOR FREQUENCY (MHzI

845

850

855

860

865

870

MAGNETIC FIELD (mT)

Figure 1. ENDOR and FSE spectra from a single crystal of thymidine X-irradiated at I O K. The magnetic field is parallel to ( b ) . (a) 3565-MHz ENDOR from a normal crystal. Lines denoted 1-4 are from the 04-protonated anion, radical TI. (b) Same as (a) but from a partially deuterated crystal. (c) Same as (a) but recorded a t I O K after annealing at 50 K. (d) FSE from the ENDOR lines 1-4 in (a). (e) FSE from the ENDOR lines 1 and 4 in (b).

be planar with regular bonding angles of 1 20°; the bond lengths were taken from the crystal s t r ~ c t u r e and ’ ~ were assumed to be the same as those of the parent molecule; the effective C5-H distance was taken to be 2.0 A; the standard value of 3.25 was used for Slater’s screening constant; the value of 0.45 was used for the spin densities at both C6 and C4 (the C6 value was taken from ref 12, while the C4 value was taken from an INDO calculation for the protonated anion, as is described below). After some trial and error, excellent correspondence between the calculated and experimental dipolar tensors was obtained with a spin density of -0.06 at C5, as is shown in Table I. As an additional test of the rotating methyl model, INDO calculations based on the RHF/CI scheme developed by Oloff and HUttermann2’ were performed for the negatively charged anion as well as for the 06-protonated anion. The main results of these calculations are presented in Table 11. These results show that indeed a small, negative spin density is expected at C5 for the protonated anion (INDO predicts -0.07, in good agreement with the value deduced above). Taken together, the results described above are strong evidence that coupling 4 of Table I can reliably be assigned to the rapidly rotating methyl group at C5. A consequence of this result is that the features of the C5-CH3 coupling may be used as an indicator of the protonation state of the molecule. Specifically, for the negatively charged anion, the INDO calculated spin density at C5 is positive (+0.09),and the spin density at C4 is much less (0.15 vs 0.45). Thus, the geometry of the dipolar tensors should be quite different in the charged molecule than in the protonated one. Radical TII: The Thymidin-5-yl Radical. The well-known spectral features of the 6-dihydrothymidin-5-yl radical (structure (27) Oloff. H.; Huttermann. J. J. Magn. Reson. 1980, 40. 415.

The Journal of Physical Chemistry, Vol. 95, NO. 3, 1991 1497

Reduction and Oxidation of Thymine Derivatives

TABLE 111: Hypertine Coupling Panmeters for the Thymidin-5-y14 (TII) and Thymidin-7-ylb(TIII) Radicals in Crystals of Thymidine X-lmdiated at 10 Kcd

radical TI1

coupling 81

TI1

82

TI11

principal values

isotropic value

123.8 (2) 114.6 (2) 112.5 (2)

117.0 ( I )

118.3 (2) 107.7 ( I ) 105.9 (3)

eigenvectors (b)

(a) 0.557 (8) 0.578 (47) 0.596 (41)

-0.091 (27) 0.756 (44) -0.648 (50)

0.825 (4) -0.307 (30) -0.474 (22)

110.6 ( I )

0.070 (10) 0.971 (13) 0.227 (56)

0.493 (1 1) -0.232 (51) 0.838 (13)

-0.867 (7) -0.054 (25) 0.495 (1 2)

-41.8 (3) -29.7 (3) -14.0 (2)

-28.5 ( I )

0.721 (9) 0.688 (IO) 0.082 (12)

0.680 (IO) -0,725 (IO) 0.108 (20)

0.134 (11) -0.022 (20) -0.990 (2)

(C)

Tlll

a2

-65.4 (3) -42.9 (3) -20.4 (2)

-42.9 (2)

0.684 (6) 0.705 (7) 0.187 (10)

0.669 (6) -0.709 (7) 0.223 (13)

0.230 (6) -0.027 (14) -0.957 (2)

TI11

a3

-70.2 (3) -42.8 (3) -22.2 (4)

-45.1 (2)

0.519 (5) 0.717 (8) 0.464 (9)

0.529 (5) -0.697 (8) 0.485 (9)

-0.671 (3) 0.006 (9) 0.741 (3)

-0.7068 0.1731 0.47 15

0.0297 -0.9642 0.7460

Directions from the Crystal Structure 0.7068 thymine base perpendicular 0.2008 C6-H bond 0.4703

c4-04

OStructure 11, dR = deoxyribose. bStructure V, dR = deoxyribose. 2 MHz) could be observed from this radical. In partially deuterated crystals, the thymidin-5-yl H addition radical was only weakly present while D adducts dominated the ESR spectra. This behavior was observed by other authors as

we11.20b,28Both @-methyleneproton ENDOR lines were observed in the partially deuterated crystals, indicating no positional preference for the D in the methylene group. The presence of HD adducts shows that the protons/H atoms for a major part of the 5-yl radicals formed at 10 K originate from exchangeable (polar) site(s). In the 5-yl radical, the onset of methyl group rotation occurs at about 65 K, with virtually free rotation at 77 K, as is demonstrated in Figure 2. At 90 K, an ENDOR line at 65.1 MHz (along ( c * ) ) appeared. This line corresponds to a hyperfine splitting of 57.2 MHz or 20.4 G, which is identical with the methyl coupling expected for the 5-yl radical.” This line was not present at 50 K, and it disappeared again upon recooling the crystal, as is expected for the reversible transition between free and restricted rotation. A final point is that it was argued that the methyl group rotates freely even at 10 K in radical TI. Thus, the restricted rotation of the methyl group in the 5-yl radical below 77 K must be the result of potential barriers imposed by the structure of that specific radical, Le., the addition of an extra proton at C6. Radical TIII: The 7-yl Hydrogen Abstraction Radical. A resonance characteristic of three inequivalent and nonexchangeable (28) Herak, J. N. J. Chem. Phys. 1970, 52,6440.

1498 The Journal of Physical Chemistry, Vol, 95, No. 3, 1991

Hole et al.

TABLE IV: Hyperfine Coupling Parameters for the Thymidin-Cyl“ (TIV) and the 8-Hb (TV) Radicals in Crystals of Thymidine X-Irradiated at 10 K P v d radical

coupling

TIV

PI

TV

Bl

TV

B2

TXe

principal

isotropic

values 140.2 (2) 130.1 (3) 127.9 (3)

value

eigenvectors (b)

(0)

(C)

132.7 (2)

0.874 (5) 0.093 (50) 0.476 (16)

0.135 (15) 0.896 (36) -0.423 (79)

0.466 (9) -0.434 (63) -0.771 (35)

84.7 (2) 75.7 (3) 73.3 (4)

77.9 (2)

0.789 ( 1 1) 0.568 (20) 0.235 (27)

-0.582 (IO) 0.569 (30) 0.581 (30)

-0.197 (22) 0.597 (37) -0.780 (28)

60.3 (2) 44.4 (2) 43.9 (2)

49.5 ( I )

0.182 ( I O ) 0.232 (250) 0.955 (61)

-0.983 (2) 0.035 (44) 0.179 (13)

-0.008 (19) 0.972 (61) -0.235 (256)

13.6 (3) 11.9 (1) 8.8 (3)

11.4 ( I )

0.846 (33) 0.234 (122) 0.478 (37)

-0.501 (33) 0.046 (47) 0.864 ( 1 9)

0.181 (99) -0.971 (30) 0.157 (84)

“Structure VI, d R = deoxyribose. ”Structure VI1, dR = deoxyribose. cThe splitting parameters are given in megahertz. dNumbers in parentheses represent the standard deviation in the last quoted digit(s). runidentified radical.

a-proton interactions was also present immediately after irradiation at IO K. The ENDOR lines from this resonance are denoted TI11 in the spectrum shown in Figure 3a, and the FSE from these lines are given in Figure 3b. This resonance has been observed in all thymine derivatives studied so fari9q20and is unequivocally assigned to the thymidin-7-yl radical formed by a net hydrogen abstraction from the methyl group (structure V, dR = deoxyribose). In the O

H

0

dk

dk

V

VI

present work the three hyperfine coupling tensors were evaluated and are given in Table 111. The data presented here agree well with the previous ENDOR measurement^.'^^ No other couplings of significant magnitude (>2 MHz) could be associated with this radical in thymidine. Radical TIV: The 6-yl Radical. In Figure 3a is shown an ENDOR spectrum from an X-irradiated crystal of thymidine at IO K. Among the many lines observed is one at 102 MHz (marked TIV) representing an unusually large coupling. It is almost isotropic, indicating that it is due to a proton in @-positionto the unpaired electron. The hyperfine coupling tensor for this resonance line is given in Table IV. The unusually large magnitude of this coupling makes an assignment to an aliphatic free radical (in the sugar moiety) less probable (the isotropic value of the coupling is larger than the standard B2 value of 126 MHz6 used for aliphatic radicals). The FSE spectrum in Figure 3c shows that the @coupling is accompanied by one somewhat smaller coupling. The data indicate that the smaller coupling exhibits considerable anisotropy. Unfortunately, it was not possible to follow this coupling throughout the rotation planes, but sufficient data are available to indicate that this coupling arises from the a-proton at the C6-position of the thymidine base. Experiments with deuterated crystals proved that both couplings are nonexchangeable. These two couplings are like those expected from the thymidin-6-yl radical, formed by net hydrogen addition to the C5position of thc base (structure VI, dR = deoxyribose). Due to the prcsence of the larger methyl group, the added proton is located almost perpendicular to the pyrimidine ring. The HellerMcConnell relation22afor @ couplings (eq 1 ) with Bo = 0, B2 = 168 MHz$ ai, = 132.7 MHz (from Table I I I ) , and 0 = Oo yields a spin density p* of 0.79. This is in line with that estimated by other authors (typically 0.75).20b Furthermore, it can be noted that the eigenvector for the maximum splitting value deviates about IOo from the expected direction calculated by assuming a perpendicular orientation of the C5-H bond with respect to the pyrimidine plane. The nature of the larger coupling taken together

with the angular behavior of the smaller coupling supports the identification of radical TIV as the 6-yl radical of the thymine base. From the ESR spectra, it is clear that this is a minor product in thymidine. It is interesting to note, however, that very similar spectral parameters also were obtained for a relatively more abundant species in 1-methylthymine (see below). Flossmann et al. also observed this product in several other thymine derivatives20b Radical TV: A 2@-H Sugar Radical. In Figure 3a are also indicated two relatively weak ENDOR lines that exhibit only small anisotropy (these lines mhave been marked TV), and in Figure 3d is shown the FSE spectrum associated with these lines. Experiments with deuterated crystals showed that the responsible protons are nonexchangeable. The hyperfine coupling tensors are given in Table IV. It is reasonable to assign these two @-couplings to a radical centered in the sugar moiety of thymidine. At low temperatures a large geometrical reorganization of the radical fragment is not expected. Thus, a first guess of the radical structure may be obtained by correlating the eigenvectors for the maximum 0coupling with the C-H, direction of the crystal s t r ~ c t u r e . ’The ~ best agreement is obtained for a radical formed by H abstraction from Cl’, i.e. the deviation between the eigenvectors for the maximum splitting values and the two CI’-.HC2’ bonds is about 8O and 1 l o . Now, using the Heller-McConnel relation for @protons22a(eq 1) and assuming that the two @-protonsare bonded to the same carbon atom, and two isotropic values in Table 1V yield two siinultaneous equations for p* and 0. Using Bo = 3 MHz, B2 = 126 M H z , ~and the auxiliary condition that the difference between the two dihedral angles should be 120°, the equations yield p* = 0.67, 8, = 18.5O, and O2 = 41.4’. This is reasonably close to the values obtained from the crystallographic data,” 2 1 O and 27O, respectively, assuming that the electron LEO occupies an orbital perpendicular to the N.-OI’-C2’ fragment. Consequently, radical TV is identified as that formed by a net hydrogen abstraction from CI’ of the sugar moiety of thymidine as shown by structure V11.

TJwH~2~ H

OH

VI1

Radical TVI: The Alkoxy Radical. The alkoxy radicals found by Box et al.’” in their previous study of thymidine were also found in these experiments. No reexamination of this radical is presented here. However, a curious characteristic of the alkoxy resonance was a considerable distortion of the line shape, shown in Figure 4a. This occurs at the lowest temperature (=IO K) and even at very low microwave power (=IO0 nW). As shown in Figure 4b, the line shape became normal when the sample was warmed to

The Journal of Physical Chemistry, Vol. 95, No. 3, 1991 1499

Reduction and Oxidation of Thymine Derivatives

TABLE V Hyperfine Coupling Parameters for the Protonated Metbylthymine Anion' MTI in Crystals of 1-Methylthymine X-Irradiated at 10 KbS

coupling 1

2

3

principal values

isotropic value

-61.1 -33.9 -18.4 -12.0 -7.6 0.5 -7.5 -7.0 -2.8

(4) (4) (5)

-37.8 (2)

(4) (4) (3) (3) (4) (4)

dipolar

eigenvectors

(a+)

(b)

values -23.3 (4) 3.9 (4) 19.4 (5)

0.749 (4) 0.503 (12) 0.430 (12)

-0.535 ( 5 ) 0.078 (17) 0.841 (4)

-0.390 (9) 0.860 (8) -0.328 (18)

-6.4 (2)

-5.6 (4) -1.2 (4) 6.9 (3)

-0.830 (25) 0.520 (43) -0.200 (31)

0.330 (22) 0.170 (31) -0.929 (8)

0.449 (44) 0.837 (27) 0.312 (26)

-5.8 (2)

-1.7 (3) -1.2 (4) 3.0 (4)

0.016 (272) 0.561 (46) -0.828 (33)

0.890 (181) -0.385 (414) -0.244 (59)

0.060 0.523 -0.850

0.890 -0,414 -0.192

0.455 (345) 0.733 (229) 0.506 (48)

(C)

Calculated Dipolar Principal Values for Coupling 3 -1.9 -0.9 2.9

0.452 0.745 0.490

Directions from the Crystal Structure thymine base perpendicular 0.4499 0.4257 C6-H bond -0.3590 N3-H bond C 5 4 7 bond

0.5101

0.07 IO 0.8603 -0.8933 -0.8376

0.8905

-0.2800 0.2700 -0.1950

'Structure IV, dR replaced with CH3. *The splitting parameters are given in megahertz. CNumbersin parentheses represent the standard deviation in the last quoted digit(s).

I

I

8415 a515 8615 MAGNETIC FIELD (mT) Figure 4. ESR at 24026.31 MHz from thymidine crystals with the external field along ( c ) following irradiation at IO K to a total dose of 4 kGy. The prominent ESR features are from alkoxy radicals, 7-yl radicals, and 04-protonated anions. Weak features can also be seen from 5-yl radicals. (a) ESR at IO K showing the distorted shape of the alkoxy ESR lincs. (b) ESR at 15 K showing a more normal shape of the alkoxy

ESR lines.

= I 5 K . No similar distortion was observed by Box and Budzinski, even at 4.2 K , in their experiment (see Figure 1 of ref 18a). Aside from their capability of lower temperature, the only other significant difference in apparatus is the detection technique: they used no field modulation, while 50-kHz field modulation was used in the present experiment. Thus the behavior must arise from "passage" effects due to the field modulation rate used in our apparatus. Radical T X An Unidentified Radical. One additional very weak and small @-typehyperfine coupling was observed in both normal and partially deuterated crystals. The coupling tensor is included in Table IV. FSE studies showed that this coupling is associated with a narrow resonance in the center of the ESR spectrum. A further analysis of this resonance proved impossible, but the direction of the eigenvectors indicate another sugar-centered radical. It is interesting to note that this coupling also was observed by Box and co-workers after irradiation of partially deuterated thymidine crystals at 4.2 K (e.g., see the ENDOR line at 42.4 MHz in Figure 1 of ref 18a). In the present work, there are a few other weak couplings in the 40-55-MHz region of which some may bc associated with the same radical, but it was not possible to obtain sufficient data for analyzing them. Dose Effrcts and Radical Transformations. Thymidine crystals were exposcd to X-ray doses in the range 4.5-100 kGy at IO K.

It was observed qualitatively that the concentration of radical TI seemed to reach saturation at doses lower than for radicals TI1 and T I L It was also observed that the concentration of alkoxy radicals18asaturated at doses lower than that of TII. Otherwise, the main effect of the higher doses was an improvement in the signal-to-noise ratio of the spectra. After irradiation at IO K, thymidine crystals were exposed to white light before warming. This treatment induced no changes in the ESR or ENDOR spectra. On the other hand, upon thermal annealing above 40 K, the 04-protonated anion, radical TI, decayed with no detectable successor radical. The disappearance of the resonance lines from this radical is demonstrated in Figure 1. In addition, the alkoxy lines were essentially absent from the ESR after annealing the crystals to ca. 30 K. The onset of methyl group rotation in the 5-yl radicals (TII) occurred at about 60 K and was completed at about 90 K, as shown in Figure 2. In the same temperature interval, the resonance lines from radical TIV, the 6-yI radical, disappeared. Due to the initial low yield of radical TIV, this decay could not be followed, nor could it be established if any successor radical was formed. After these, no other temperature-induced changes were observed in the spectra up to room temperature. ( B ) I-Methylthymine. Three radical species were trapped and identified in crystals of I-methylthymine (MeT) after X-irradiation at 10 K. Some weak lines appeared in the ENDOR spectra at a few orientations indicating the presence of additional minority species. However, the concentration of these was too low for a reliable data analysis. Radical MTI: The 04-Protonated Anion. Figure 5 shows ESR and ENDOR spectra from a crystal of I-methylthymine after X-irradiation at IO K. In Figure 5a,b, several of the ENDOR lines have been denoted I, and the corresponding stick spectra have been indicated in the ESR spectra. FSE and thermal annealing studies (see below) show that these lines must be ascribed to one single radical species yielding a doublet ESR pattern. The hyperfine coupling tensors obtained from the ENDOR data are given in Table V. The similarity with the coupling parameters for the TI-radical in Table I is striking, except for the missing H 0 4 interaction. Particularly notable is that coupling 3 in Table V closely reproduces the coupling ascribed to the CS-CH, coupling in thymidine (coupling 4 in Table I, discussed in detail above). On the basis of the close correspondence between the two data sets (in particular upon the C5-CH, coupling) radical MTI is identified as the thymine base anion, protonated at 0 4 (structure IV, dR replaced with CH,). I n I-methylthymine crystals, the HN3-N3-C4-04 fragment participates in a planar centrosymmetric hydrogen-bonded dimer with a neighboring m o l e c ~ l e . ' ~

1500 The Journal of Physical Chemistry, Vol. 95, No. 3, 1991

Hole et al.

TABLE VI: Hypemne Coupling Parameters for the Metbyltbynin-7-yP (MTII), the Methylthymin-6-ylb(MTIII) Radical, and an Unidentified Species (MX) in Crystals of 1-Methylthymine X-Irradiated at 10 Kcqd

radical MTll

principal values

coupling

-37.4 -27.1 -13.1 -67.7 -43.6 -21.0 -72.5 -44.1 -23.1

“I

MTll

a2

isotropic value -25.9 (2)

(4) (3) (4) (4) (4) (5) (3) (4) (5)

-44.1 (3)

eigenvectors 0.706 (1 1) 0.512 (21) 0.490 ( 1 3) 0.774 (5) 0.496 (1 1) 0.393 (IO) 0.062 (9) 0.518 (12) 0.853 (7)

(b) -0.608 (7) 0.082 (17) 0.790 (6) -0.478 (6) 0.052 (14) 0.877 (3) -0.998 ( I ) 0.053 (9) 0.039 (7)

-0.364 0.855 -0.369 -0.415 0.867 -0.277 0.025 0.854 -0.520

(a*)

(C)

(21) (13) (1 8) (1 1)

(7) (13) (7) (7) (12)

MTll

“3

MTlll

“I

-87.2 (3) -53.2 (4) -26.7 (4)

-55.7 (3)

0.735 (3) 0.507 (6) 0.449 (6)

-0.487 (4) -0.064 (IO) 0.871 (2)

-0.470 (6) 0.860 (3) -0.200 (9)

MTllI

A

143.5 (3) 135.9 (2) 132.6 (3) 58.8 52.0

137.3 ( I )

0.988 (5) 0.127 (38) 0.092 (26) 0.485 0.453 -0.748

0.074 (24) 0.137 (60) -0.988 (9) -0.854 0.060 -0.517

0.139 (38) -0.982 (9) -0.126 (60) -0.189 -0.890 0.415

0.0710 0.8603 0.0760 -0.8376

0.8905 -0.2800 -0.4343 -0.1950

MXC

-46.6 (2)

54.1 51.5

Directions from the Crystal Structure thymine base perpendicular

0.4499 0.4257 0.8970 0.5101

C6-H bond C4-04 bond C5-C7 bond

‘Structure V, dR replaced with CH,. bStructure VI, dR replaced with CHI. CThesplitting parameters are given in megahertz. dNumbers in parentheses represent COH couplings by Gough and Taylor.29 From an analysis of protonated benzosemiquinones they found the isotropic component of the coupling to follow the relation (in megahertz)

aOH= p0(-53.2)

+ ~ “ ~ ( 8 4 . 0COS^ ) e

(2) Applying this to radical MTI with 0 = 90° and p = 0.02 yields aOH = -1.06 MHz. The dipolar coupling tensor for H 0 4 localized in the molecular plane was calculated as previously described.12 Combining the results from this calculation with the isotropic value arrived at above, the net principal values of 6.3, -4,4, and -5.0 MHz for the X 4 4 H coupling tensor were obtained. Lines from

such a small anisotropic coupling are difficult to detect as they fall among the many others arising from weakly coupled protons near the free proton resonance. The spin density at C6 was calculated to be 0.51 from the McConnell relation22busing QcH = -73.4 MHz,I2 while Bernhard’s procedure30 with QrdiP of 38.7 MHz yields 0.50. (The corresponding values from the 04-protonated anion in thymidine12are 0.45 from McConnell’s relation and 0.52 from Bernhard’s relation.) With calculations like those from coupling 4 of radical TI in thymidine, the isotropic part of coupling 3 indicates a CS spin density of -0.07. The dipolar part was calculated in the manner described above for thymidine, and the result is included in Table V. The overall agreement is very good, lending support to the interpretation given below. Radical MTII The 7-yl Hydrogen Abstraction Radical. Three ENDOR lines denoted I1 in Figure 5a,b all yield the same FSE and are thus ascribed to one single radical species. The hyperfine coupling tensors obtained from these ENDOR lines are given in Table VI. The data in Table VI compare very well with those for the TI11 radical in Table 111. This, together with the directions of the eigenvectors relative to the virgin crystal structure, unequivocally pinpoints the 7-yl radical (structure V with dR replaced with CH3) as responsible for this resonance. Radical MTIII: The 6-yl Radical. In I-methylthymine, as in thymidine an ENDOR line varying between 103 and 108 MHz was observed at IO K. The FSE from this line was a four-line pattern showing that a second coupling is associated with the resonance. The corresponding ENDOR line was easily followed throughout the rotation planes. The hyperfine coupling tensors derived from the data are shown in Table VI. The smaller coupling in Table VI is of the a-type with the eigenvector for the minimum principal value deviating only 5.3O from the C6-H bond direction. The eigenvector for the intermediate principal value is close (3.6O deviation) to the pyrimidine normal. With a Q value of -73.4 MHz in the McConnell relation,22bthe spin density at C6 is estimated to be 0.75, while Bernhard’s procedure30 with Qrdipof 38.7 MHz yields the same ~~

(29) Gough, T. E.;Taylor, G . A. Con.J . Chem. 1969, 4 7 , 3717.

(30) Bernhard, W.A. J . Chem. Phys. 1984.81, 5928.

The Journal of Physical Chemistry. Vol. 95, No. 3, I991

Reduction and Oxidation of Thymine Derivatives

1501

TABLE VII: ComDarison of Proton Coupline Constants' from Various Thymine Electron-Addition Products

HC6

methyl

HN3

system Td R

aim

Bdipb

-33.1

0.52

TdR'

-32.8

MeT

-37.8

-22.6 3.2 20.3 -22.7 2.9 19.8 -23.3 3.9 19.4

Tmd

-40.0

-18.8 -2.0 20.4

0.53

oligo(T)c

-36.5

0.53

Tf

-33.04

-21.4 0.8 20.5 na

&6

ais0 -5.4

Bdip

aiw

-6.5 -2.2 8.6

-6.7

0.5 I

0.50

ref this work 18a

-6.4

-5.6 -1.2 6.9

-5.8

this work

20b

-4.0

-0.56

f3.7

na

2.5

1 Oa

5c

'All are in megahertz; Bdi the dipolar components of the hyperfinecouplings, are in the order E,, By, B, from top to bottom. bThe spin densities were calculated from B, as &scribed in ref 30 with QIip= 38.7 MHz. ' 0 4 proton not detected; experiments at 4 K, and with partially deuterated crystals. dTm = thymine monohydrate at 77 K; ESR only was used for the experiments. In 12 M LiCl glass at 77 K. f I n aqueous solution at ca. 300 K.

number. The large @-couplingcorrelates very well with expectations for a proton bonded to C5 in a direction almost perpendicular to the base. Specifically, the eigenvector for the maximum principal element deviates only 2' from that calculated for the orientation indicated. (For this calculation the additional hydrogen was assumed to be directly above C5 by 1 .O A.) However, with the Heller-McConnell relationZZafor P-protons with Bo = 0, B2 = 168 MHz, and 0 = Oo (the same parameters as used in thymidine), p"B2 = 126 MHz, which is smaller than the observed isotropic value of 137.3 MHz. Thus, Bo and B2 are probably slightly underestimated. In conclusion, all data are consistent with a radical formed by net hydrogen addition to the C5 position of the thymine base, structure VI with dR replaced with CH3. Unidentified Radicals. At several orientations, a few additional weak ENDOR resonance lines could be observed. Among these, one was observed for all crystals used, including partially deuterated ones, and it exhibited an angular variation in accord with a CS-CH3 coupling with a significant unpaired spin density on C5 and with the methyl group rapidly rotating. Table VI includes the best-fitting trial tensor used for the simulation of the angular variation of this line in two planes of rotation. The isotropic value of the coupling, 54.1 MHz or 1.93 mT, is remarkably similar to the value of 2.2 mT reported for the C5methyl coupling of the charged cation of 1 ,3-dimethylthymine3Ia and to the values ranging between 1.88 and 2.1 1 mT for the C5-methyl coupling of deprotonated thymine and 1 -methylthymine cation radicals.20b*31b*c This coupling is also like that from the rotating 5-methyl group of 5-yl radicals. However, no ESR evidence was seen for this interaction, nor was any ESR or ENDOR evidence found for the two protons at C5. The available experimental data are insufficient to permit assignment of this coupling. Radical Transformations. Upon annealing crystals of 1 methylthymine X-irradiated at 10 K, radical MTI (the 0 4 protonated anion) decayed at approximately 40 K. This is demonstrated in Figure 5c. Here the ESR and ENDOR spectra shown are those obtained after a short annealing to 50 K followed by recooling to IO K. These spectra were recorded at the same orientation and instrumental settings as those in Figure 5b. The loss of the ESR features and the ENDOR resonance lines assigned to radical MTI is evident. Upon further annealing, no other (31)(a) Rhodes, C.J.; Podmore, I . D.; Symons, M . C. R. J . Chem. Res. M.D. J . Pfiys. Cfiem. 1974, 80, 1898. (c) Sevilla, M . D.;Suryanarayana, D.; Morehouse, K. M . J . Pfiys. Cfiem.1981, Synop. 1988, 120. (b) Sevilla, 85, 1027.

(32)Madden, K. P.; Bernhard, W. A. J . Phys. Cfiem. 1982, 86, 4033.

changes could be observed in the spectra until about 250 K. At this temperature, the thymine C6 H-addition radical (the 5-yl radical) grew in slowly and reached its final concentration after several hours at room temperature.

Discussion The results presented in this paper show clearly that thymine anions protonate at 0 4 in thymidine (TdR) and that the protonation occurs below 10 K. Strong evidence is also presented that the same behavior occurs in I-methylthymine (MeT). These results have a bearing on at least two issues in current work aimed at characterizing the identity and chemical behavior of the initial products of directly ionized DNA. Magnetic Character of T.The first issue is the question of the magnetic character of thymine anions. Several studies of thymine in solids identified products of electron addition10aJ8a-20b but presented no direct evidence for the protonation state. Table VI1 compares the ESR parameters of these electron adducts to the 04-protonated anion (Ta+4') in thymidine and to the nonprotonated anion (p)from thymine in solution. The major coupling in all cases is from the proton at C6. The isotropic value of the coupling varies from 32.8 (TdR) to 40.0 MHz (Tm); however, it is striking to note that the B, value of the dipolar coupling is virtually identical for all cases in the solid state. Bernhard30argued that the dipolar coupling is more reliable than the isotropic coupling asNanindicator of the *-spin density at the LY carbon. It therefore appears that there is little difference in the 2p" spin densities at C6 in those products. From the data presented in Table VII, two conclusions are possible. One is that the properties of T,+4' and T in the solid state are virtually identical. In line with this possibility is that Box and BudzinskiIEafound values for the HC6 coupling in TdR identical with those reported here for the 04-protonated structure. Since their sample temperature was lower than ours (4.2 K vs near I O K), the protonation may have been prevented from occurring. The consequence of this possible conclusion is that distinguishing the "true" thymine anion from the 04-protonated anion with ESR alone will be virtually impossible. This follows since in most situations the added proton will be in the molecular plane and therefore will have very little isotropic coupling. The second possible conclusion is that the electron adducts in all the solid-state studies are protonated at 0 4 . The principal basis for this is the near identity of the B, values of the dipolar couplings and thus the near identity of the C6 spin densities among the products. The fact that Box and Budzinski did not detect the added proton in their study of thymidine does not contradict this

1502 The Journal of Physical Chemistry, Vol. 95, No. 3, 1991

Hole et al.

the 5-yl radicals were not observed to form in MeT until higher conclusion, since this proton is exchangeable and they used temperatures, a mechanism other than direct protonation of T partially deuterated crystals.Iga at C6 must be responsible. We believe the second conclusion to be more reasonable since it is expected (e.g., from INDO calculations, Table 11) that It is reasonable to expect that the differences in the stability successive protonations of the charged anion will reduce the spin and formation probability of protonated anions result from the density at C6. The similar values of the HC6 isotropic coupling state of the specific systems. In aqueous solution the frequent of T,+4' in thymidine and that of T in solutionSCdemonstrate and random encounters between solvent and solute or between the charge dependence of the Q value in McConnell's relation.6*22b solute and solute provide ready availability of protons to add at Thus, while the isotropic couplings of T,+4' (in TdR) and T (in any site, especially ones that participate in hydrogen bonds (e&, 0 4 ) . This also provides ready acceptors for protons in reversing thymine) may be nearly equal, their dipolar couplings should not the 0 4 protonation. In solids at low temperatures (whether be equal and thus should provide the basis for distinguishing the two (unless they are in solution). The spectral differences observed crystals or DNA) the situation is more nearly static. The prowhen thymine electron adducts are formed in acidic g l a s ~ e s ' ~ ~ . ~tonation ~ at 0 4 is thought to occur by transfer of a proton across must then be due to additional protonation, probably at 0 2 . a hydrogen bond in which it participates. However, C6 participates Reactions Proposed for Thymine Derivatives. The second issue in no hydrogen bonds and therefore has no similar supply of of relevance to current work with DNA is the connection between protons. During the irradiation, the electron-loss products easily T or its 04-protonated form and the thymin-5-yl radical observed deprotonate, providing a general supply of protons to the lattice. in DNA. The basic question is whether both forms lead to these It is reasonable to assume that some of these protons would be 5-yl radicals. Associated with this is the additional question of available for protonation qt C6, provided this is not precluded by the potentially faster protonation at 04.34 Therefore, unless whether the thymin-5-yl radicals in DNA form only from direct protonation of thymine anions at C6 (thereby requiring these the irradiation is in progress, the low "proton availability" combined with the slow rate of C6 protonation appears to lead to a anions as precursors): i.e., are there other mechanisms operable in DNA that can also yield 5-yl radicals to thymine? I n the low probability for 5-yl radicals forming by C6 protonation in following discussion, evidence will be given that the ionizationsolids. initiated mechanisms leading to 5-yl radicals in solids of thymine The possibility of direct protonation of the anion at C6 during derivatives at low temperatures are more varied than simply that irradiation is supported by the set of radicals observed in TdR. of anion protonation at C6. Specifically, 5-yl radicals were found along with the OCprotonated The protonation behavior of thymine electron adducts has been anions (and others) after irradiation at 10 K. However, since 6-yl investigated in two recent s t u d i e ~ . ~Deeble ~ . ~ et al. found that radicals were also found, it is possible that a 6-yl to 5-yl transelectron adducts of uracil derivatives in aqueous solution protonate formation (to be discussed below) was marginally active in TdR rapidly, but reversibly, at 0 4 , while they protonate slowly and during low-temperature irradiation. irreversibly at C6.Sb Novais and Steenken corroborated the Evidence for a direct connection between anions and 5-yl C6-protonation behavior in their study.% An important inference radicals was reported by Flossmann et for thymine monofrom this is that 0 4 protonation is innocuous in that it is reversible hydrate (Tm). Specifically, they found that the "anions" were to the T- parent, with the result that the only lasting product of present following irradiation at 77 K and were thus more stable T- is that of the slow C6 protonation. It is notable that C6 than those in TdR and MeT. They also found that illumination protonation was very slow with water as the protonating agent of the crystals with red light (A > 600 nm) caused transformation (5IO3 S - I ) , ~ ~ whereas more rapid reaction requires stronger proof the "anions" into 5-yl radicals, with the added net H being an tonating agents such as phosphate. The rate of this reaction in exchangeable one. In Tm the proton hydrogen bonded to 0 4 (the DNA is uncertain since the protonating agent is expected to be one expected to be transferred following electron capture) is one water, sugar, or another base. of the water protons.3s This same proton is the closest exThe possibility of proton transfer between paired DNA bases changeable one to C6 of a neighboring molecule (e3.6 A). has been considered recently by Steenken3' in terms of pK, difAssuming the "anions" to be protonated at 0 4 (as argued above), ferences. Straightforward application of this procedure to TdR two possible mechanisms can be proposed for the T,+4' to 5-yl in which the anions (pK, = 7) are protonated by ribose H03' (pK, transformation ion Tm. One is that the red light activated reversal i= 12.5) predicts that the protonation should not occur. However, of the 0 4 protonation and also provided sufficient energy for direct a significant concentration of the T,+4' was observed in TdR. This C6 protonation, with the donor being the water nearest C6 of the shows that the factors controlling the protonation/deprotonation resulting p. The second is that the red light "mobilized" the behavior in solids may be significantly different from the correweakly bound H at 0 4 , which then added to C6 of the neighbor. sponding factors in solution as reflected in pK, values. As a The molecular arrangements of TdR and MeT do not have this consequence, care should be taken in applying the pK, difference feature of a neighboring C6 being near the 0 4 - H 0 4 portion of procedure to DNA. the molecule. Anionic products in solid TdR and MeT were observed to have A second possible fate for protons released to the lattice during a behavior different from those in solution. The behavior of MeT irradiation is to become trapped at a site of high negative charge was most clearcut: the (04-protonated) anions decayed at about within a molecule. In hydrogen-bonded solids, the most suitable 50 K with no obvious successor, and the 5-yl radicals formed slowly of these sites are of limited (or no) availability, as they are hyat about 250 K. The easy reversibility of the protonation in drogen-bond acceptors. An alternative site in pyrimidines is the solution is consistent with the marginal stability of the protonated C5=C6 double bond. INDO predicts ~ 0 . 1excess electronic anions in solids (of TdR as well as MeT). However, an important charge at C5 in the neutral parent, and =O,l deficiency at C6; difference is that the decay in solids cannot be simply the reversal proton attack at C5 thereby is favored. Such a protonated parent o 04protonation. I f the reversal occurred, the result would be molecule has increased cross section for electron capture. Thus , the characteristic spectrum of which was not observed. Since the origin of the net H addition at C5 (6-yl radicals) may be the result of H+ addition, followed by electron capture, a mechanism previously proposed by Bernhard for uracL6 (33) Kaalhus, 0.; Johansen, S. A. Ann. N.Y. Aead. Sci. 1973,222, 1973. (34) This provision is significant for two reasons: ( I ) Herak et al.'9b The behavior summarized above for MeT is consistent with a demonstrated that the 5-yl radicals in thymidine have no proton at 0 4 , which mechanism involving proton addition followed by electron capture. argues that doubly protonated products do not form and consequently that Specifically, the major oxidation product is thought to be the result C6-protonation requires reversal of the 04-protonation; (2) a significant part of the potential for protonation am" from the coulombic influence of the net of net H loss from the 5-methyl group (radical MTII). The charge, which is neutralized by the 0 4 protonation. observation in MeT that the, 6-yl radicals resulted from addition (35) Gerdil, R. Acta Crysfallogr. 1961, 14, 333. of a nonexchangeable net H may be taken as evidence that it (36) Zell. 1.; HOttermann, J.; Grilslund, A.; Rupprecht, A,; Kbhnlein, W. originated from the methyl group. I t is important to note that Free Radical Res. Commun. 1989, 6, 105. (37) Steenken, S.Chem. Reu. 1989, 89, 503. the product cannot be the result of an intramolecular proton shift

rl

J . Phys. Chem. 1991, 95, 1503-1507 since it is one of net H addition. Another possibility for. formation of 6-yl radicals is direct H addition to CS,where the H’s arise from the methyl via dissociative superexcitation. However, Das et al. found in aqueous solutions of uracil that direct H addition occurred at both C5 and C6 in the approximate ratio 2:l .Sd In view of this result, and the fact that the initial population in MeT included no significant contribution from 5-yl radicals, !i seems unlikely that the 6-yl radicals were formed by the direct H mechanism. Regardless of how 6-yl radicals form in MeT, they have been shown by Flossmann et a1.*Obto transform irreversibly into the 5-yl form under activation by either white light at 77 K or heat. The irreversibility of this 1,2 hydrogen shift indicates that the 5-yl form is more stable than the 6-yl form in thymine. Therefore, the evidence with thymine derivatives in solids indicates that 5-yl radicals will be the net result of any mechanism producing any combination of 6-yl and 5-yl radicals. Significance for DNA. The basic thymine-related fact from DNA studies that must be accounted for is the formation of thymin-5-yl radicals. In the discussion above, three mechanisms were proposed for producing 5-yl radicals of thymine: (1) direct protonation of T at C6: (2) protonation at C5 followed by electron addition; (3) direct H addition to C5 or C6. It is clear from a variety of studiesSbscthat the 5-yl radicals can be formed by direct protonation of the anion at C6. However, in the solid-state studies discussed above, it is not clear that this mechanism is a significant source of the considerable population of 5-yl radicals found after warming the sample to ca. 300 K. In particular, no direct connection was found between the anions in MeT and the production of 5-yl radicals. Recent results with oriented DNA fibers found that the spectral component assigned to T did not behave as

1503

expected upon incorporation of methyl-deuterated thymine into the DNA.36 This is evidence that the concentration of T is less than previously thought and thus that the thymin-5-yl radicals formed upon warming the DNA may arise from other precursors. The complete set of evidence, in particular the evidence that thymine 5-yl radicals do not require anions as precursors, divides into two parts the basic question of how the 5-yl radicals form in DNA: ( I ) What factors (molecular arrangement, hydrogenbonding mates, water presence and positioning, etc.) control the effectiveness of the possible mechanism? (2) What is the relative importance of these in DNA?

Summary and Conclusions In summary, the discovery of a proton at 0 4 in the anions of TdR raises the possibility that all “anions” of thymine derivatives reported so far in magnetic resonance studies of solids are similarly protonated. This discovery also shows that pK, values alone are insufficient to predict proton transfer across hydrogen bonds in solids. In addition, experimental evidence available at this time supports several plausible mechanisms for formation of thymine 5-yl radicals that do not require direct protonation at C6 of T. The basic result is the proposal that the appearance of the thymine 5-yl radicals in DNA is the result of the variety of possible reactions as affected by the local conditions within DNA. Acknowledgment. This work was supported by N I H Grant CA36810 and by NATO Travel Grant RG 0426/88. Additional travel support was obtained from Norges Allmenvitenskapelige Forskningsrad (NAVF). Discussions with W. A. Bernhard and S. Steenken concerning the nature and behavior of thymine radiochemical products are gratefully acknowledged.

Raman Spectroscopic Study of Hydrogen Bondlng in Aqueous Carboxylic Acid Solutions. 2. Deuterio Analogues in Heavy Watert Naoki Tanaka, Hiromi Kitano,* and Norio Ise Department of Polymer Chemistry, Kyoto University, Kyoto, Japan (Received: July 5, 1990)

Raman spectra of deuterio analogues of acetic acid and propionic acids in heavy water at 25 OC were measured. The carbonyl stretching region of the spectra was resolved into four envelopes by using band-fitting programs according to the assumption of the existenceof monomeric, dimeric, and polymeric acetic acid species formed by hydrogen bonding. Dimerization constants for the monomer-dimer equilibrium, KD, of carboxylic acid-d, were calculated from the normalized band areas. KD’s in heavy water solutions were greater than those in aqueous solutions. The contribution of hydrophobic interaction to the KD was thermodynamically discussed. Furthermore, normal-coordinate analysis of water-associated acetic acid molecules was carried out to clarify the reason for the splitting of C-C stretching vibration of acetic acid-dl heavy water solution.

Introduction It has henfound by various methdsI-4 that the dimerization constant of carboxylic acids tends to increase with increasing- alkyl . chain length. It is concluded that hydrophobic interaction stabilizes the carboxylic acid dimers in aqueous solution. In the previous study,s we investigated the dimerization of carboxylic acids in aqueous solution using Raman spectroscopy. The effects of alkyl chain length and the concentration of carboxylic acids on dimerization constants ( K D )in aqueous solution were investigated by the band resolution of the spectra. In the present study, KD’s of deuterio analogues of acetic acid and propionic acid in heavy water were estimated at various concentrations by the band resolutions of the spectra. The dif‘Presented at the 39th Annual Meeting of the Society of Polymer Science, Japan, at Kyoto International Hall in May, 1990. To whom correspondence should be addressed.

0022-3654/91/2095-l503$02.50/0

ference in KDvalues for normal carboxylic acids and their deuterio analogues were quantitatively discussed in terms of the hydroPhobic interaction.

Experimental Section Materials. Acetic acid and propionic acid were obtained from Nacalai Tesque, Kyoto, Japan, and used after distillation. Acetic acid-dl and acetic-d, acid-d, were guaranteed reagents from Aldrich Chemical Co., Milwaukee, WI, and CEA, Gif-sur-Yvette, France, respectively. Propionic acid-d, was prepared by hydrolysis ( I ) Nash, G. R.; Monk, C. B. J . Chem. Soc. 1957,4274. (2) Suzuki, K.; Taniguchi, Y.;Watanabe. T. J . Phys. Chem. 1973, 77,

1918. (3)Katchalsky, A.; Eisenberg, H.;Lifson, S.J . Am. Chem. Soc. 1951, 73, 5889. (4) Martin, D. L.; Rossotti, F. J . L. Proc. Chem. SOC.London 1959, 60. ( 5 ) Tanaka, N.; Kitano, H.; Ise, N. J . Phys. Chem. 1990, 94. 6290.

0 1991 American Chemical Society