Synthesis and Biochemical Properties of Cyanuric Acid Nucleoside-Containing DNA Oligomers Didier Gasparutto, † Sandrine Da Cruz, † Anne-Gae ¨lle Bourdat, † Michel Jaquinod, ‡ and Jean Cadet* ,† Laboratoire des Le ´ sions des Acides Nucle ´ iques, Service de Chimie Inorganique et Biologique, De ´ partement de Recherche Fondamentale sur la Matie ` re Condense ´ e, CEA-Grenoble, F-38054 Grenoble Cedex 9, France, and Laboratoire de Spectrome ´ trie de Masse des Prote ´ ines, Institut de Biologie Structurale, F-38027 Grenoble Cedex, France Received November 23, 1998 1-(2-Deoxy--D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers. To investigate the biological and structural significance of dY residue in DNA, the latter modified 2′-deoxynucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using the phosphoramidite approach. For this purpose, a convenient glycosylation method involving 3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) was devised. The anomeric mixture of modified 2′-deoxyribonucleosides (1/2 R/) was resolved by silica gel purification of the 5′-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired -phosphoramidite monomer (5). After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophore- sis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double- stranded DNA fragment as inferred from melting temperature measurements. The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease P 1 , snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment of E. coli polymerase I. Introduction Several studies have established that 8-oxo-7,8-dihy- dro-2′-deoxyguanosine (8-oxodGuo), 1 which may result from hydroxylation of the C8 position of 2′-deoxygua- nosine (dGuo), is the major stable modified nucleoside present in oxidized DNA. The structural and biological features of 8-oxodGuo have been widely studied during the past decade (1-5). Interestingly, 8-oxodGuo is cur- rently used as a biomarker of DNA oxidation induced by both endogenous (cellular metabolism, oxidative stress, etc.) and exogenous (ionizing radiation, photosensitizers, xenobiotics, etc.) agents (6, 7). Several studies have shown that 8-oxodGuo, which exhibits a lower ionization potential than the normal DNA nucleosides, is highly reactive toward oxidizing reagents (8-11). This suggests that the latter nucleoside may also be sensitive to in vivo DNA oxidation. Recently, 8-oxodGuo has been found to be an efficient substrate of photosensitized reactions which give rise to a set of secondary products. Thus, at the nucleoside level, it was shown that the major product of singlet oxygen oxidation (type II mechanism) was the cyanuric acid nucleoside (1)(12, 13). Although the latter model studies provided a large amount of information about the structure and the mechanism of formation of 1, the presence of dY has not been established so far at the DNA level. In this paper, we report the first synthesis of cyanuric acid nucleoside (1) and its site-specific insertion into several oligodeoxyribonucleotides using phosphoramidite chemistry. The modified DNA oligomers were used to determine the hybridization properties of 1 upon substi- tution of the guanine parent base. In addition, informa- tion about biochemical features, including enzymatic cleavage, repair, and mutagenicity, is provided. * To whom the correspondence should be addressed. Tele- phone: (33)-4-76-88-49-87. Fax: (33)-4-76-88-50-90. E-mail: cadet@ drfmc.ceng.cea.fr. † CEA-Grenoble. ‡ Institut de Biologie Structurale. 1 Abbreviations: dY, 1-(2-deoxy--D-erythro-pentofuranosyl)cyanuric acid; 8-oxodGuo, 8-oxo-7,8-dihydro-2′-deoxyguanosine; ODN, oligode- oxyribonucleotide; DMTrCl, 4,4′-dimethoxytrityl chloride; TFA, tri- fluoroacetic acid; CSPDE, calf spleen phosphodiesterase; SVPDE, snake venom phosphodiesterase; FAB-MS, fast atom bombardment mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis; endo III, endonuclease III; Fpg, formamidopyrimidine DNA glycosylase. 630 Chem. Res. Toxicol. 1999, 12, 630-638 10.1021/tx980255e CCC: $18.00 © 1999 American Chemical Society Published on Web 06/25/1999