Conformational and Electronic Properties of the Two Cis (5S,6R) and (5R,6S) Diastereoisomers of 5,6-Dihydroxy-5,6-dihydrothymidine: X-ray and Theoretical Studies Franck Jolibois, † Lucienne Voituriez, † Andre ´ Grand, ‡ and Jean Cadet* ,† De ´ partement de Recherche Fondamentale sur la Matie ` re Condense ´ e/CEA/Grenoble, SESAM/LAN and SESAM/CC, 17, Rue des Martyrs, F-38054 Grenoble Cedex 09, France Received July 21, 1995 X The structure of (+)-cis-(5S,6R)-5,6-dihydroxy-5,6-dihydrothymidine was obtained using X-ray crystallography [space group P2 1 with a ) 10.130(3) Å, b ) 6.434(9) Å, c ) 11.02(5)Å, and  ) 112.646(2)°]. The comparison of the two cis diastereoisomers of thymidine glycol (I, II) showed several structural and conformational differences. The solid state structures appear to be in agreement with the results of 1 H NMR studies which were carried out in aqueous solution. Conformational and electronic properties of the ground state of the molecules I and II were obtained using ab initio LSD-DFT theory. Only slight differences between the crystal structure and the optimized geometry are observed for each of the two oxidized nucleosides. On the other hand, molecules I and II exhibit significant differences in their electronic properties. In particular, the dipole moment of (5S,6R)-thymidine glycol (I) is twice smaller than that of the (5R,6S) diastereoisomer (II). It is noteworthy that these differences in the electronic properties between the two compounds may be related to changes in the rotameric population around the C4′-C5′ bond. The repartition of the electrostatic potential is different in the two compounds. These observations lead to a better understanding of the structural changes when the above lesions are included within a DNA molecule. Introduction Much attention has been devoted in the two past decades to the determination of the mechanisms of radical oxidation of nucleic acids associated with exposure to ionizing radiation (1-5). One of the major radiation- induced nucleobase lesions identified so far within naked (6-10) and cellular DNA (11, 12) is 5,6-dihydroxy-5,6- dihydrothymine. It is noteworthy that the so-called “thymine glycol” has been shown to be generated in cells under other oxidizing conditions including near-UV ir- radiation (12) treatment with hydrogen peroxide (13) and various carcinogens (14, 15). Efforts have also been made to gain insights into the biological role of 5,6-hydroxy- 5,6-dihydrothymine (16). In this respect, the latter oxidized base does not appear to be a premutagenic lesion (17) when assessed in single-stranded DNA by using the transfection assay. It was also found that the coding properties of the diol were retained (18-21) despite the fact that the 5,6-ethylenic bond of the pyrimidine ring is saturated and the pyrimidine ring is no longer planar. Thymine glycol was found to be efficiently bypassed by the Escherichia coli Klenow fragment (19, 22). Similar observation was made in the same sequence context when single- and double-stranded genomes containing a thymine glycol residue were replicated in E. coli strains (23). However, since 5,6-dihydroxy-5,6-dihydrothymine was found to form an efficient hydrogen bond with adenine (24), it was concluded that this important class of oxidative DNA damage cannot be considered as an effective premutagenic lesion. It should be added that the presence of thymine glycol in pSV2 plasmids did not affect the transformation frequency in humans cells (25). Evidence was provided that the “thymine glycol” is a substrate for the N-glycosylase activity of E. coli endo- nuclease III (26-29). In addition, 5,6-dihydroxy-5,6- dihydrothymine was also found to be removed from oxidized DNA through an excision repair process medi- ated by the E. coli UV ABC nuclease complex (30, 31). Efforts were also made to determine the chemical and conformational features of 5,6-dihydroxy-5,6-dihydrothym- ine and related nucleoside derivatives. The absolute configuration of the four cis and trans diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine which may be produced by the reaction of hydroxyl radicals (5) and through the photosensitized formation of a pyrimidine radical cation (32) was assigned (33, 34). The main change in the conformational properties of these four thymidine glycols, which was inferred from a detailed 1 H NMR study in aqueous solutions, deals with the shift in the dynamic equilibrium between the two puckered sugar conformers 2 E (C2′-endo) T 3 E (C3′-endo) toward the 2 E form. This is more pronounced for the (6S) diastereoi- somers (35). The presence of the thymine glycol in a duplex DNA was shown to induce significant distortion in the vicinity of the damage (36). Relevant structural and conformational information regarding both the pyrimidine moiety and the furanose ring of the (-)-cis-(5R,6S)-“thymidine glycol” was inferred from a X-ray crystallographic investigation (37). In addition, it should be added that the X-ray structure of the cis isomer of 5,6-dihydrothymine was also resolved (38). Theoretical study of the structures of the four cis and trans enantiomers of 5,6-dihydroxy-5,6-dihydrothym- ine was carried out using Hartree-Fock ab initio quan- * To whom correspondence should be addressed. Phone: (33)-76- 88-49-87; Fax: (33)-76-88-50-90; E-Mail: cadet@drfmc.ceng.cea.fr. † SESAM/LAN. ‡ SESAM/CC. X Abstract published in Advance ACS Abstracts, December 15, 1995. 298 Chem. Res. Toxicol. 1996, 9, 298-305 0893-228x/96/2709-0298$12.00/0 © 1996 American Chemical Society + +