EPR study of a sulfur-centered π radical in γ-irradiated single crystal of 2-thiothymine

Available online at www.sciencedirect.com

Journal of Molecular Structure 876 (2008) 234–239 www.elsevier.com/locate/molstruc

EPR study of a sulfur-centered p radical in c-irradiated single crystal of 2-thiothymine Erim Besˇic´ *, Vjeran Gomzi Department of Biophysics, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇic´a 1, 10002 Zagreb, Croatia Received 7 March 2007; received in revised form 19 June 2007; accepted 20 June 2007 Available online 5 July 2007

Abstract Single crystals of 2-thiothymine (5-methyl-2-thiouracil) were c-irradiated at 77 K and studied using EPR spectroscopy. In addition to previously observed sulfur r radical, another sulfur-centered radical of p type was formed and analyzed at 100 K. The large values and the directions of the g-tensor principal elements undoubtedly confirmed the assignment. The observed isotropic 1:3:3:1 quartet structure of the EPR spectra, attributed to the couplings with methyl group protons, indicates the large delocalization of the spin along the 2-thiothymine molecule. Doublet splitting of the 1:3:3:1 quartets observed in some crystal orientations supports the idea that the pristine radical is an anion, formed by the capture of an electron at S(2) and subsequently neutralized by addition of a proton to S(2). From the crystal structure data it is concluded that the captured proton is the H 0 (3) of neighboring molecule, participating in the formation of the N 0 (3)–S(2) hydrogen bonding between two adjacent 2-thiothymine molecules in the unit cell.  2007 Elsevier B.V. All rights reserved. Keywords: EPR spectroscopy; 2-Thiothymine; Sulfur-centered radicals; Thio-nucleobases

1. Introduction Thio-nucleobases have been used as intrinsic photolabels to probe the structure and organization of RNA molecules in solution and to identify the contacts between nucleic acids and proteins in nucleoprotein complexes [1]. On the contrary to the regular nucleic-acid bases, thioanalogs absorb light at long wavelengths and thus could be selectively photoactivated into the electronic triplet state with high affinity for crosslinking to other nucleic-acid bases and amino acid residues. In such experiments, the most frequently used thioanalog base is 4-thiouracil, naturally occurring in some bacterial tRNAs [2,3]. Some other non-natural thioanalogs, including 2-thiothymine, have also been used for specific targeting of the selected sites in nucleic acids [4,5].

*

Corresponding author. Tel.: +385 1 4818 288. E-mail addresses: [email protected] (E. Besˇic´), [email protected] (V. Gomzi). 0022-2860/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2007.06.033

Thioanalogs of nucleic-acid bases are also known to be good hole traps in the systems of stacked bases exposed to ionizing radiation. In a number of studies it has been shown that these substances, imbedded in small quantities in ordered structures of the nucleic-acid bases, represent preferred sites for localization of the migrating holes [6– 12]. In the single crystals of nucleic acids and their constituents, as well as in the single crystals of 2-thiocytosine [6], 6thioguanine [9] and 2-thiouracil [13], c-irradiated at low temperature (77 K), only radicals of the p type were observed. The exceptions are the radicals formed by the reaction of sulfur-centered base radicals with the Cl ions in some hydrochlorinated matrices [7,8]. Also, radiationinduced free radicals stabilized on thio-nucleobases are always of the cationic origin [6,9,10,13,14]. Irradiated thioanalog of thymine, 2-thiothymine (5methyl-2-thiouracil), exhibits some different properties from regular bases and other thio-nucleobases. Only in the single crystals of 2-thiothymine ionizing radiation induces two types of sulfur-centered radicals, with the char-

E. Besˇic´, V. Gomzi / Journal of Molecular Structure 876 (2008) 234–239

acteristics quite unusual in comparison to the free radicals formed in similar systems. One of the free radicals associated with sulfur atom of 2thiothymine is the cation radical of c-electron configuration, formed by a loss of an electron from 2-thiothymine and subsequent deprotonation at N(3) [14]. Sulfur-centered r radical was the first observed radical of r-electron configuration in all nucleic-acid bases or their thioanalogs in ordered structure. In the present paper, we report the detailed EPR analysis of another sulfur-centered radical, formed in the single crystals of 2-thiothymine, c-irradiated at 77 K and recorded at 100 K. 2. Experimental Anhydrous single crystals of 2-thiothymine (purchased from Aldrich) were grown from saturated aqueous solution by slow evaporation in thermostatic oven at 300 K. The beaker containing the solution was covered with aluminum foil to reduce evaporation. Crystals of good quality were obtained after three weeks, and these were stable for months when exposed to the atmosphere. The obtained anhydrous crystals were of monoclinic structure, with the space group P21/n and the unit cell dimension a = 426 pm, b = 1451 pm, c = 1026 pm, b = 92.27 [15]. There are four molecules in the unit cell, two of them magnetically distinct. For the evaluation of the spectroscopic tensors the spectra were recorded in three mutually perpendicular planes. Since the reference system deviated from the crystallographic system by only 2.27, which is within the experimental error in the alignment of the crystal in the external magnetic field, three mutually perpendicular planes were defined by the axes , and . The crystals were irradiated with gamma rays in a 60 Co source at the temperature of the liquid nitrogen (77 K) to the total dose of 35 kGy. The EPR measurements were performed at 100 K with a Bruker Elexsys E500 X-band spectrometer equipped with the temperature control unit. Following irradiation, crystals were mounted on the sample holders with any of the crystallographic axes perpendicular to the external magnetic field. The spectra were recorded in steps of 10 as the crystals were rotated in three reference planes. 3. Results 3.1. EPR spectra Fig. 1 presents the EPR spectrum of a single crystal of 2thiothymine, c-irradiated at 77 K and recorded at 100 K, for the magnetic field in the direction of the b crystallographic axis. For this orientation all paramagnetic species in the crystal lattice are magnetically equivalent, raising the same EPR spectrum.

331

332

333

235

334

335

336

magnetic field / mT Fig. 1. EPR spectrum of the c-irradiated single crystal of 2-thiothymine. Spectrum was recorded at 100 K for magnetic field in plane bc (H^a), in the direction of the b crystallographic axis, with the microwave frequency of 9.502 GHz and the microwave power of 60 mW. The bars beneath the spectrum represent the position of the resonance lines of sulfur-centered p radical (solid lines) and r radical (dashed lines).

The EPR spectrum of the system exhibits the pattern composed of two groups of lines. First group is represented by a double triplet structure, as it is indicated with the dashed line bars beneath the spectrum. This group of resonances is associated with previously observed sulfur-centered r radical, formed by the radiation-induced loss of an electron from S(2) and subsequent deprotonation at N(3) [14]. The lines of r radical were not fully resolved because of their partial saturation induced by the appliance of extreme high microwave power of 60 mW. Measurements using such high microwave power were necessary since the second group of lines became fully resolved only at this power. The EPR spectrum of this group of lines is represented by the quartet with the intensity ratio 1:3:3:1, as it is depicted with the solid line bars under the spectrum in Fig. 1. The 1:3:3:1 quartet structure of the spectrum is obviously brought about by the coupling of the unpaired electron with three magnetically equivalent protons. The observed value of 1.15 mT of these proton couplings remained unchanged in all orientations of the crystal, in each of the three reference planes. Such isotropic splitting is characteristic for the coupling of the unpaired electron with three b-protons and these are assigned to methyl protons of 2-thiothymine. On the contrary to the observed extremely isotropic proton couplings, the positions of the resonance lines exhibited large angular variation as the sample were rotated in the external magnetic field. This quite anisotropic character, as well as the observed large values of the g-factor, confirmed that the present radical is associated with sulfur atom of 2-thiothymine. In some crystal orientations an additional proton splitting was observed, nicely noticed by the doubling of the resonance lines. Unfortunately, in the most crystal orientations these doublet substructures were not fully resolved, so the coupling tensor of this hyperfine interaction could not be precisely determined.

E. Besˇic´, V. Gomzi / Journal of Molecular Structure 876 (2008) 234–239

236

Saturation of the resonance lines of the radical was observed only at the extremely high microwave power of 80 mW, which is quite unusual for the free radicals formed in similar systems. Upon thermal annealing of the sample it was observed that the radical decay at about 150 K with no obvious successor radical(s). At the temperature of 280 K the resonance lines of well-known 5-yl and 7-yl radicals were detected. The appearance of these radicals was expected since they are always present in irradiated single crystals of thymine and its derivatives at room temperature [16–19].

Table 1 Principal elements of the g-tensor Tensor

Principal values

Direction cosines





g

2.0921 2.0604 2.0044

0.311 0.725 0.609 0.767 0.404

0.912 0.053 0.391 0.122 0.871

0.282 0.687 0.692 0.630 0.281

Ring normal S(2)–C(2) bond direction

g min O

3.2. Evaluation of EPR parameters The g-tensor was determined by solving the spin Hamiltonian which included only the Zeeman interaction of the unpaired electron with the external magnetic field [20]. Fig. 2 shows the angular dependence of the g-factor recorded in the three reference planes. The circles represent the experimentally observed values and the full lines are curves reproduced with the calculated g-tensor parameter values listed in Table 1. As it can be seen, the curves fit well in the experimental data. As seen from the data in Table 1, maximum and minimum principal value of the g-tensor is along the ring plane (deviations 2.9 and 4.5, respectively) and the medium principal value is perpendicular to the ring plane (deviation 5.6). Also, the direction of the maximum principal value is within C(2)–S(2) bond (deviation 5.8). Relative orientation of the g-tensor principal elements along the radical skeleton is shown in Fig. 3. 3.3. Structure of the radical The large values (up to 2.092) and the anisotropic character of the g-tensor, as well as the direction of the g-tensor maximal principal value, undoubtedly confirm that the

2.08

H⊥a

H⊥b

c

ca bc a crystal orientation

H⊥c

g-factor

2.06

2.04

2.02

2.00

b

b

a

Fig. 2. Angular variations of the g-factor recorded in the three reference planes. The circles (o) represent the experimentally observed values and the full lines are curves reproduced with the g-tensor parameter values listed in Table 1.

C

H

5

C

gmed

H g max

C H3

4

N3 C S

6

2 1

N

C

H

H Fig. 3. Structure of the radical and the relative orientations of the gtensors principal elements along the radical skeleton.

present radical is associated with the sulfur atom of 2-thiothymine. The observed isotropic quartet hyperfine splitting of the resonance lines with the intensity ratio 1:3:3:1 is assigned to the coupling of the unpaired electron with the methyl protons. This suggests that significant spin density is located at methyl group of 2-thiothymine. The absence of hyperfine couplings with neighboring N(1) and N(3) is quite surprising, indicating unusual distribution of the electron spin in the radical. Nitrogen hyperfine splitting in organic radicals in irradiated crystals of regular bases and thio-nucleobases is dominant for the direction of magnetic field perpendicular to the ring plane [6–12]. Approaching this orientation by the rotation of the crystal in (a, b) reference plane, broadening and some smaller intensities of the resonance lines were observed, which could be provoked by the unresolved nitrogen splitting. In the orientations for magnetic field being in the ring plane, the lines became sharper and much more intensive, probably due to smaller, also unresolved nitrogen splitting. The large anisotropy and the relative orientations of the g-tensor principal values with the respect to the 2-thiothymine ring confirmed that the radical is of p-electron configuration. Since it was generally found in pyrimidines that the radicals of anionic origin have significant spin density on C(5) and C(7) (radicals of cationic origin have most of the electron spin at C(6) [16,17]), we propose that the radical is anion in origin, primarily formed by the capture of an electron at S(2). The observed doublet splitting of 1:3:3:1 quartets in some crystal orientations supports the idea that the neutralization of negative charge in this p anion is followed by the addition of a proton to S(2). The protonation at S(2) is thought to occur by the transfer of a proton across a hydrogen bond in which it partici-

E. Besˇic´, V. Gomzi / Journal of Molecular Structure 876 (2008) 234–239

pates. From the crystal structure data it is concluded that the captured proton is the H 0 (3) of neighboring 2-thiothymine molecule. The radical is finally stabilized by the formation of the N 0 (3)–S(2) hydrogen bonding between two adjacent 2-thiothymine molecules. The ability to detect coupling with H 0 (3) in some crystal orientations, along with the fact that N 0 (3)–S(2) is hydrogen bonded in an out-of-plane direction, suggests that the trapping site of this proton is out of the ring plane. Proposed structure of the radical is presented in Fig. 3. 3.4. MO calculations In order to support the assignment and understand better the electronic structure of the radical, the MO calculation of a cluster of two neighboring 2-thiothymine molecules has been performed using the B3LYP method [21]. Geometry optimization in the 6-31G(d) basis set was used and the single-point calculations were done with the 6-311++G(2d,p) basis set. For the calculations the Gaussian 98 package of programs was used [22]. In geometry optimization the coordinates from the crystal structure were used as the initial values for all atoms except H 0 (3) which has been transferred to the S(2) for presumed anionic origin of the radical. The best agreement with the spin density distribution expected from experimentally derived results is obtained if the N(1), C(5) and C(6) atoms of the first and N 0 (1), C 0 (5) and C 0 (6) atoms of the second 2-thiothymine molecule have been kept fixed at their crystal coordinates. Calculations were performed for the presumed anionic origin of the radical. The results of the calculations show that most of the spin density is located at C(2)–S(2) bond (71%) and on C(5)–C(7)–H3 fragment (17%), which is qualitatively in good agreement with experimentally observed data. Relative small spin densities found on N(1) and N(3) (9% and 5%, respectively) are also consistent with the features of the EPR spectra in which no nitrogen splitting has been resolved. Calculated distribution of the spin for presumed anionic origin of the radical is shown in Fig. 4. As it can be seen, spin density is localized almost entirely on the radical molecule protonated at S(2). The relative large calculated spin density of about 2% on H 0 (3) (much more than spin densities on the other hydro-

237

gen atoms of the radical molecule, which are less than 0.1%), is consistent with the idea that H 0 (3) participates in the radical structure by the neutralization of the primarily formed negative charge. This supports our presumption that the radical is an anion in origin.

4. Discussion During the studies of the hole transfer in c-irradiated systems of nucleic-acid bases containing small quantities of thioanalogs, radicals associated with the sulfur atom of 2-thiocytosine [6,7], 6-thioguanine [8] and 2-thiouracil [13] have been reported. All of these radicals were formed by the radiation-induced loss of an electron and subsequent deprotonation of the pristine cation radical. The unpaired electron, mainly centered on sulfur, is delocalized in the pyrimidine or purine ring, in analogy to the base radicals in natural nucleic acids and the related model systems [13,23]. The review of the sulfur-centered radicals in the systems of the base thioanalogs is given in Table 2. Only in the single crystals of 2-thiothymine, irradiated and recorded at low temperature, two types of sulfur-centered radicals were observed. Characteristics of these two radicals are different from all other thio-nucleobases and all naturally occurring bases. One of the free radicals associated with sulfur atom of 2-thiothymine is the cation radical of r-electron configuration, formed by the loss of an electron from the p-orbital of sulfur and subsequent deprotonation at N(3) [14]. Electronic rearrangement after the deprotonation changes the ground electronic state of the radical from p to r type. Finally the unpaired electron is shared by r-orbital of C(2)–S(2) bond and non-bonding r-orbital on N(3), formed after deprotonation. This was the first report on a r radical in one of the nucleic-acid bases or their thioanalogs in ordered structure that is not associated with three-electron bond formed by the interaction with electron-donating group, like Cl in some hydrochlorinated matrices [7,8]. Also, neutralization of the positive charge in this radical is achieved by deprotonation at N(3), in contrast to the events observed in similar radicals of related systems, where the proton bound to N(1) Table 2 Types of sulfur-centered radicals in thioanalogs of the nucleic-acid bases System

Crystal matrix

Type

2-Thiocytosine [6,7,9]

Thiocytosine Cytosine Æ H2O Cytosine Æ HCl 5-Methyl-cytosine Æ H2O 5-Methyl-cytosine Æ HCl 2-Thiouracil Guanine HCl Æ H2O Guanine HCl Æ 2H2O GuanineÆ2HCl 2-Thiothymine 2-Thiothymine

p p r (Cl–S–C<) p r (Cl–S–C<) p p p r (Cl–S–C<) r p

5-Methyl-2-thiocytosine [10] 2-Thiouracil [13] 6-Thioguanine [8]

Fig. 4. Calculated spin density distribution in the protonated sulfurcentered p radical.

2-Thiothymine [14] 2-Thiothymine

E. Besˇic´, V. Gomzi / Journal of Molecular Structure 876 (2008) 234–239

238

Fig. 5. The pathway leading to formation of r (a) and p (b) sulfur-centered radical in c-irradiated single crystal of 2-thiothymine.

is eliminated. The formation of r-radical from primarily formed cation radical is depicted in Fig. 5a. The existence of another sulfur-centered radical in irradiated system of 2-thiothymine is reported here. Although the assignment of the spectra to the radical associated with sulfur suggests that most of the spin is located in C(2)–S(2) bond, experimentally observed hyperfine couplings, assigned to methyl protons couplings, indicates that appreciable amount of spin is also on C(5)–C(7)–H3 fragment. For the model of rotating methyl protons, the magnitude of the spin density at C(5) can be calculated from the isotropic part of methyl proton couplings, and the Heller– McConnell relation for b-couplings [24] aiso ¼ qp ðB0 þ B2 cos2 hÞ:

ð1Þ

For the case of free rotation, this simplifies to aiso = qpQb, where Qb = 2.855 mT [25]. With aiso = 1.15 mT, this relation yields qp = 0.4. This result implies that appreciable amount of the spin (40%) is located on C(5)– C(7)–H3 fragment, which quantitatively is not in good agreement with calculated one (17%). Since the methyl group is placed opposite to S(2) in 2-thiothymine molecule, detection of the methyl protons couplings designates the large delocalization of the unpaired electron. The performed MO calculations qualitatively confirmed such unusual distribution of the electron spin. On the basis of the observed experimental data and the experiences achieved in the studies of similar radicals in related systems we concluded that the present radical is an anion in origin, primarily formed by the capture of an electron at S(2). Subsequent stabilization of the primarily formed radiation damage is achieved by the formation of the N 0 (3)–S(2) hydrogen bonding between two adjacent 2-thiothymine molecule with the

H 0 (3) participating in protonation of an anion. Neutralization of the negative charge by similar linkage has been reported in anions formed in irradiated crystals of thymine derivatives [23]. Fig. 5b shows a scheme resuming the pathway leading to formation of neutralized sulfur-centered p-radical after exposition of 2-thiothymine to c-radiation. Anion types of radicals are always present in the single crystals of thymine [19], thymidine [18] and 1-methylthymine [18]. In the later two systems the pristine anion is formed by the capture of an electron at O(4) and the subsequent neutralization of negative charge is accomplished by the protonation at O(4) via transfer of a proton across hydrogen bond in which it participates. In anhydrous thymine single crystals O(4) does not participate in hydrogen bonding. Nevertheless, subsequent to electron trapping a proton still adds to O(4), indicating strong tendency of thymine anions for protonation at this position, probably due to large negative charge on O(4) of the charged anion. The substitution of the oxygen with sulfur at C(2) in 2-thiothymine does not change a tendency of thymine-like systems for formation a pristine anion radical, but obviously affects the place of capturing the radiation-induced electron. Instead at O(4), which is generally observed in all anions of thymine derivatives so far, capture of an electron and subsequent protonation in 2-thiothymine rather takes place at S(2), favoring the N 0 (3)–H 0 (3)–S(2) linkage between two adjacent 2-thiothymine molecules instead expected N 0 (1)– H 0 (1)–O(4). 5. Conclusions The present study demonstrates that irradiated thioanalog of thymine, 2-thiothymine, exhibits some different

E. Besˇic´, V. Gomzi / Journal of Molecular Structure 876 (2008) 234–239

properties from regular bases and other thio-nucleobases. Only in the single crystals of 2-thiothymine, two types of sulfur-centered radicals were observed. One of the free radicals associated with sulfur atom of 2-thiothymine is the previously analyzed cation radical of r-electron configuration. Another sulfur-centered radical was observed when the extremely high microwave power was applied and the detailed EPR analysis of this radical is reported here. It has been shown that the radical is of p-electron configuration and anionic origin, primarily formed by the capture of an electron at S(2). Subsequent neutralization of the negative charge is achieved by the formation of the N 0 (3)–S(2) hydrogen bonding between two adjacent 2-thiothymine molecule with the H 0 (3) participating in protonation of an anion. Acknowledgement This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia. References [1] A. Favre, C. Saintome´, J.L. Fourrey, P. Clivio, P. Laugaˆa, Photochem. Photobiol. B Biol. 42 (1994) 109. [2] C. Saintome, P. Clivio, J.L. Fourrey, A. Woisard, P. Laugaˆa, A. Favre, Tetrahedron 56 (9) (1997) 1197. [3] B. Bartholomew, B.R. Braun, G.A. Kassavetis, E.P. Geiduschek, J. Biol. Chem. 269 (27) (1994) 18090. [4] I.V. Kutyavin, R.L. Rhinehart, E.A. Lukhtanov, V.V. Gorn, R.B. Meyer, H.B. Gamper, Biochemistry 35 (34) (1996) 11170.

239

[5] D. Compagno, J.N. Lampe, C. Bourget, I.V. Kutyavin, L. Yurchenko, E.A. Lukhtanov, V.V. Gorn, H.B. Gamper, J. Biol. Chem. 274 (12) (1999) 8191. [6] K. Sankovic´, D. Krilov, J.N. Herak, J. Mol. Struct. 190 (1988) 277. [7] K. Sankovic´, D. Krilov, J.N. Herak, Radiat. Res. 128 (1991) 119. [8] J.N. Herak, K. Sankovic´, D. Krilov, J. Hu¨ttermann, Radiat. Res. 151 (3) (1999) 319. [9] K. Sankovic´, D. Krilov, T. Pranjic´-Petrovic´, J. Hu¨ttermann, J.N. Herak, Int. J. Radiat. Biol. 70 (1996) 603. [10] J.N. Herak, K. Sankovic´, D. Krilov, M. Jaksˇic´, J. Hu¨ttermann, Radiat. Phys. Chem. 50 (2) (1997) 141. [11] J.N. Herak, K. Sankovic´, E.O. Hole, E. Sagstuen, Phys. Chem. Chem. Phys. 2 (21) (2000) 4971. [12] K. Sankovic´, E. Malinen, Z. Medunic´, E. Sagstuen, J.N. Herak, Phys. Chem. Chem. Phys. 5 (2003) 1665. [13] P. Jørgensen, E. Sagstuen, Radiat. Res. 88 (1) (1981) 29. [14] E. Besˇic´, K. Sankovic´, V. Gomzi, J.N. Herak, Phys. Chem. Chem. Phys. 3 (2001) 2723. ˇ alogovic´, E. Besˇic´, K. Sankovic´, Acta Crystall. C58 [15] D. Matkovic´-C (2002) 568. [16] E.O. Hole, E. Sagstuen, W.H. Nelson, D.M. Close, J. Phys. Chem. 95 (3) (1991) 1494. [17] A. Dulcˇic´, J.N. Herak, Radiat. Res. 47 (1971) 573. [18] J. Hu¨ttermann, Int. J. Radiat. Biol. 17 (3) (1970) 249. [19] E. Sagstuen, E.O. Hole, W.H. Nelson, D.M. Close, J. Phys. Chem. 96 (3) (1992) 1121. [20] F.A. Farach, C.P. Poole Jr., in: J.S. Waugh (Ed.), Solving the spin Hamiltonian for the electron spin resonance of irradiated single crystals, Advances in Magnetic Resonance, vol. 5, Academic Press, New York and London, 1971, p. 229. [21] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (2) (1988) 785. [22] M. Frisch et al., Gaussian 98, Gaussian, Inc, Pittsburgh PA, 2001 (1998). [23] J. Weiland, J. Hu¨ttermann, Int. J. Radiat. Biol. 74 (1998) 341. [24] C. Heller, H.M. McConnell, J. Chem. Phys. 32 (1960) 1535. [25] R.H. Fessenden, R. Schuler, J. Chem. Phys. 39 (1963) 2147.