4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA

6774–6781 Nucleic Acids Research, 2013, Vol. 41, No. 13 doi:10.1093/nar/gkt197 Published online 18 June 2013 4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA Atsushi Nishimoto1, Daichi Jitsuzaki1, Kazumitsu Onizuka1, Yosuke Taniguchi1,2, Fumi Nagatsugi2,3 and Shigeki Sasaki1,2,* 1 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan, 2CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan and 3Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan Received January 29, 2013; Revised February 27, 2013; Accepted March 1, 2013 ABSTRACT The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many crosslinking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson–Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 50 -side of the opposing uridine. In the absence of a 50 -adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research. INTRODUCTION Many chemical entities, of either exogenous or endogenous origins, cause the alkylation of or damage to DNA and RNA; thus, they have a strong impact on biological functions of nucleic acids (1–3). Chemotherapeutic agents, such as mitomycin C, exert their effects by the alkylation of DNA (4). On UV irradiation, psoralen forms DNA adducts with both duplex strands to form an interstrand cross-link. This compound is widely used for therapy (5) and mechanistic studies of processes, such as DNA repair (6). The enhanced inhibition of translation by antisense oligodeoxynucleotides (ODNs) has been demonstrated by the cross-link formation between psoralen and target RNA (7,8). Cross-link formation is also used to maintain 3D nucleic acid structures (9–11). A variety of functional groups have also been developed to enable interstrand cross-linking, including disulfide bonds (12), benzophenone derivatives (13), carbazoles (14), quinone methides (15,16), phenylselenyl derivatives of pyrimidines (17) and furan derivatives (18). To further advance these studies, an efficient cross-linking method is still desired. To address the need for an efficient cross-linking agent, we previously developed a 2-amino-6-vinylpurine derivative (1) based on a hybridization-assisted strategy. This compound exhibited efficient and selective cross-linking to cytosine bases (19–25). The close proximity of the vinyl group to the cytosine base in the hybridized complex contributed to the high reactivity of 1 (Figure 1). An ODN incorporating a sulfide-protected derivative of 2-amino-6-vinylpurine was shown to be useful for inhibiting and modulating intracellular gene expression (26). A characteristic feature of this 2-amino6-vinylpurine is that the vinyl group is directed towards the Watson–Crick base pairing face to react with the 4-amino group of cytosine. Generally, the reactive group for cross-link formation is not located near the Watson– Crick face. The efficient cross-link formation by 1 suggests that partial base pairing and/or shape complementarity can benefit from a proximity effect. Further efforts have continued towards the biological applications and improvement of the 2-amino-6-vinylpurine unit (27–30). In this study, newly designed 4-vinylpyrimidine-2-one nucleoside analogs (T-vinyl 2 and U-vinyl 3) have been *To whom correspondence should be addressed. Tel: +81 92 642 6615; Fax: +81 92 642 6615; Email: ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2013, Vol. 41, No. 13 6775 H 2N R N O O O N N N NH2 N NH2 O N RNA 1 N O O O N O N N N N RNA 2: R=CH3 3: R=H Figure 1. The 2-amino-6-vinylpurine derivative (1) for cross-linking with cytosine and newly designed 4-vinyl substituted pyrimidine derivatives, T-vinyl (2) and U-vinyl (3). shown to exhibit fast and selective cross-link formation with either adenine or uracil bases, depending on the target sequence. Herein, we describe in detail the synthesis of nucleoside analogs 2 and 3, evaluation of their cross-linking reactivity, analysis of the cross-linked products derived from 2 and highlight its potential application for cross-linking in triplex DNA. treated with 0.5 M NaOH solution to generate the vinyl group, thus providing the cross-linking ODNs (3,4 and 5) in good yields. The vinyl group of ODN was hydrated with a half-life of 4 h at 25 C and pH 7.0. RESULTS AND DISCUSSION Evaluation of the cross-linking reaction MATERIALS AND METHODS The 4-vinyl-(1H)-5-methylpyrimidine-2-one derivative (2) was designed for cross-link formation with the 6-amino group of adenine, based on the expectation that the two bases would exhibit shape-complementarity resembling a T–A base pair. A similar derivative lacking the 5-methyl group (3) was also synthesized for comparison. The synthesis of the nucleoside units and their incorporation into ODNs are shown in Scheme 1. The syntheses described herein are an extension of earlier reports from this group concerning the introduction of a vinyl group via Suzuki– Miyaura coupling. The 2,4,6-triisopropylbenzensulfonyl derivative of tert-butyldimethylsilyl (TBDMS)protected thymidine (7) or 20 -deoxyuridine (8) was treated with 2,4,6-trivinylcyclotriboroxane pyridine complex in the presence of Pd(PPh3)4, LiBr and K2CO3 in H2O-dioxane. As the resulting vinylated products (9 and 10) were not sufficiently stable for isolation, they were isolated after protection with octanethiol (11 and 12) (31). They were then converted to the corresponding phosphoramidite precursors (13 and 14) using conventional methods and were (...truncated)