Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts

Published online 5 August 2015 Nucleic Acids Research, 2015, Vol. 43, No. 16 7717–7730 doi: 10.1093/nar/gkv797 Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts Shuhei Kusano1 , Shogo Ishiyama1 , Sik Lok Lam2,* , Tsukasa Mashima3 , Masato Katahira3 , Kengo Miyamoto4 , Misako Aida4 and Fumi Nagatsugi1,* 1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai-shi, Miyagi 980-8577, Japan, 2 Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China, 3 Institute of Advanced Energy, Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan and 4 Department of Chemistry, Graduate School of Science, Hiroshima University,1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan ABSTRACT DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl2 , NiCl2 , ZnCl2 and MnCl2 . In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion. INTRODUCTION Strategies for preparing crosslinked duplex DNA have attracted attention due to their many applications in a variety of fields, including DNA repair, gene regulation and nanotechnology. DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clin- ical anticancer drugs, such as nitrogen mustards and platinum agents (1,2). Drug resistance in tumor cells through enhanced ICL repair is a major problem in cancer treatment (3,4). Although a number of repair pathways have been implicated in ICL repair, the molecular mechanism remains poorly understood (5,6). Determining the chemical structure of crosslinked duplex DNA could help elucidate the repair mechanism (7). Covalently linked duplex DNA can be prepared by using a variety of crosslinked dinucleotides (8–15). Oligonucleotides (ODNs) containing O6 -guanine-alkyl-O6 -guanine ICL products were used to investigate the repair of DNA ICLs by O6 -alkylguanineDNA alkyltransferase (16,17). Plasmids containing N4 CethylN4 C that mimicked nitrogen mustard ICL, and N3 Tethyl-N3 T or N1 I-ethyl-N3 T ICL that mimicked the nitrosourea ICL structure were used to investigate the repair mechanism in cells (18). In an alternative approach, duplex DNA that contained a reactive moiety in both strands was used to prepare covalently linked duplex DNA (19– 27). ICL duplex DNA has been synthesized by disulfide bond linkage (21,27), click chemistry (25,26) and amide bond formation (22). These strategies produced a variety of ICL duplex DNA structures by adjusting the linker length between the DNA strand and each reactive moiety and these strategies were used to form the DNA nanostructure. However, these methods for preparing ICL duplex DNA could not be used to control gene regulation. Crosslink-forming oligonuleotides (CFOs) bind to the target mRNA to form an irreversible complex, and effectively inhibit translation. Various functional groups have been developed for ICL formation (28) by photoirradiation, including psoralen (29,30), diaziridine (31) and carbazoles (32). In addition, reactive functional groups activated by * To whom correspondence should be addressed. Tel: +81 22 217 5633; Fax: +81 22 217 5633; Email: Correspondence may also be addressed to Sik Lok Lam. Tel: +852 3943 8126; Fax: +852 2603 5057; Email: Present address: Shuhei Kusano, Department of Chemistry, Faculty of Science, Fukuoka University, Nanakuma 8-19-1, Fukuoka 814-0180, Japan.  C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received May 27, 2015; Revised July 24, 2015; Accepted July 28, 2015 7718 Nucleic Acids Research, 2015, Vol. 43, No. 16 MATERIAL AND METHODS Synthesis of the AOVP derivative phosphoramidite 4-amino-2-(2-octylthioethyl)-6-oxo-1-(3 ,5 -O-dibenzyl2 ,4 -dideoxy-D-ribityl)pyrimidine (5). Lithium hydride (109 mg, 13.7 mmol) was added to a suspension of 3 (532 mg, 1.88 mmol) and 4 (995 mg, 2.53 mmol) in dioxane (19 ml). After stirring for 30 min at room temperature, the reaction mixture was heated to 100o C and stirred for 5 days. After cooling to room temperature, saturated NH4 Cl was added to the reaction mixture. The layers were separated and the aqueous phase was extracted with CH2 Cl2 . The organic phase was washed with brine, dried over anhydrous Na2 SO4 , filtered, and concentrated in vacuo to obtain an oil. The residue was purified by column chromatography (CHCl3 /MeOH, 1:0 to 40:1) to afford 5 (366 mg, 34%) as a pale yellow oil; 1 H NMR (400 MHz, CDCl3 ) ␦ 0.879 (t, J = 6.8 Hz, 3H), 1.26–1.34 (m, 10H), 1.55 (quint, J = 8.0 Hz, 2H), 1.77–2.02 (m, 4H), 2.48 (t, J = 8.0 Hz, 2H), 2.84–2.91 (m, 4H), 3.58 (t, J = 2.8 Hz, 2H), 3.73–3.76 (m, 1H), 3.90–3.97 (m, 1H), 4.04–4.44 (m, 1H), 4.48 (d, J = 4.0 Hz, 2H), 4.54 (d, J = 2.8, 2H), 4.62 (brs, 2H), 7.27–7.36 (m, 10H); 13 C NMR (100 MHz, CDCl3 ) ␦ 14.1, 22.6, 28.7, 28.8, 29.2, 29.6, 31.8, 32.5, 33.3, 34.0, 34.6, 39.7, 66.5, 71.2, 73.0, 74.1, 86.0, 127.5, 127.6, 127.7, 127.9, 128.3, 128.4, 138.3, 138.4, 160.0, 160.9, 163.1; HRMS-ESI (m/z): [M+Na]+ calcd for C33 H47 N3 NaO3 S, 588.3230; found, 588.3229. 2-(2-octylthioethyl)-6-oxo-4-phenoxyacetylamino-1-(3 ,5 O-dibenzyl-2 ,4 -dideoxy-D-ribityl) pyrimidine (6). Phenoxyacetyl chloride (0.18 ml, 1.31 mmol) was added to a solution of 11 (366 mg, 0.647 mmol) in pyridine (6.5 ml) at 0o C. After stirring for 1h at 0o C, the reaction mixture was allowed to warm to room temperature. After additional stirring at room temperature for 4h, the reaction mixture was diluted with CH2 Cl2 , washed with saturated NaHCO3 and brine, dried over anhydrous Na2 SO4 , filtered, and concentrated in vacuo. The residue was purified (...truncated)