Communication www.rsc.org/chemcomm CHEMCOMM Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine·pyrimidine interruption Dominique Guianvarc’h, a Rachid Benhida, a Jean-Louis Fourrey,* a Rosalie Maurisse b and Jian-Sheng Sun* b a Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse 91198. Gif-sur-Yvette Cédex, France. E-mail: fourrey@icsn.cnrs-gif.fr; Fax: +33-1-69077247; Tel: +33-0169283053 b Laboratoire de Biophysique, UMR 8646 CNRS-Muséum National d’Histoire Naturelle, INSERM U201, 43 rue Cuvier 75231. Paris Cedex 05, France. E-mail: sun@mnhn.fr; Fax: +33-1-69077247; Tel: +33-01407937 Received (in Cambridge, UK) 25th April 2001, Accepted 21st July 2001 First published as an Advance Article on the web 23rd August 2001 Thermal denaturation experiments have established that an oligonucleotide incorporating the artificial nucleobase S, does form a stable triplex with a double stranded DNA which exhibits a pyrimidine interruption within the oligo- purine sequence. For many years, triple helix-forming oligonucleotides (TFOs) have been known to be able to bind in the major groove of oligopyrimidine·oligopurine sequences of double-stranded DNA (dsDNA). TFOs establish specific hydrogen bonds with the oligopurine strand of dsDNA through the formation of T·A3T and C·G3C + base triplets 1 in Hoogsteen (pyrimidine- motif, Fig. 1) or T·A3A and T·A3T, and C·G3G triplets in reverse Hoogsteen configuration (purine- or mixed-motif). 2 Hence, dsDNA sequence recognition by TFOs has potential applications in gene expression modulation and in gene targeting technologies. 3 Unfortunately, these interesting appli- cations must be restricted to long oligopyrimidine·oligopurine sequences only ( > about 15 bp) since any interruption by even a single A·T or G·C base pair strongly destabilizes triple helix formation. 4 Consequently, during the last decade, considerable efforts have been devoted, so far with limited success, to circumvent such a sequence limitation. 5 Two approaches have been undertaken to design and synthesize: (1) new base analogs featuring an extended heterocyclic ring system for achieving specific hydrogen bonds with all hydrogen bond-forming sites available in the major groove side of the inverted A·T and G·C base pairs; 6 (2) nucleobases capable of stabilizing, non- sequence-specifically, triple helix formation at the purine·pyr- imidine interruption sites, either by the attachment of an intercalating agent in conjunction with an appropriate nucleo- base, or by a nucleobase alone. 7 The second strategy (universal base approach) has been more successful than the first (specific base approach). In the literature, a 4-(3-benzamidophenyl)imi- dazole (D 3 ) has been shown to equally stabilize the triplex at both A·T and G·C sites. 6a Subsequent NMR studies showed that the binding mode is intercalation 6b which is consistent with the lack of base pair discrimination. In this work, a new base analog (S) has been synthesized and incorporated into a TFO in an attempt to achieve better triplex stabilization than the D 3 nucleobase at the purine·pyrimidine sites within its cognate dsDNA sequence. Compared to D 3 the S nucleobase consists of two unfused aromatic rings which are linked to 2A-deoxyribose by an acetamide motif instead of a three ring construct attached directly to 2A-deoxyribose (Fig. 2). The synthesis of the required phosphoramidite 5 to serve for the incorporation of S in TFOs is straightforward. Thus, compound 2, readily obtained by catalytic hydrogenation of 2-acetamido- 4-(3-nitrophenyl)thiazole 1, 8 was acylated with 2-(2-deoxy- 5-O-dimethoxytrityldeoxyribosyl)acetic acid 3 9 using 2-chloro- 1-methylpyridinium iodide. Finally, the resulting derivative 4 was phosphitylated in the usual manner to give the desired phosphoramidite 5 (Scheme 1). The capacity of triple helix stabilization of the novel nucleobase S was assessed in a model system where S was incorporated in the middle of a 18-mer TFO (at position Z) 10 and was screened against all four possible base pairs (X·Y = T·A, C·G, A·T or G·C) in the target oligopyrimidine·oligopurine sequence (Table 1). The thermal denaturation experiments 11 indicated that the T m value of the triplex containing an A·T3S triplet (T m = 50 °C) was very close to those of perfect triplexes without any interruption of oligopyrimidine·oligopurine se- quences (T·A3T or C·G3C + , T m = 51 or 50 °C, respectively). It was noted that the use of S base provides a 5–8 °C triplex stabilization as compared to the best base triplet made of natural bases (A·T3S vs. A·T3G; G·C3S vs. G·C3T), respectively. In terms of triplex stability, the novel S nucleobase is at least as good as the previously reported D 3 base. The main difference † Electronic supplementary information (ESI) available: experimental details. See http://www.rsc.org/suppdata/cc/b1/b103743a/ Fig. 1 Canonical T·A3T and C·G3C + base triplets in Hoogsteen configuration (pyrimidine-motif). R = 2A-deoxyribosyl. Fig. 2 Structures of the 2A-deoxynucleosides featuring nucleobases S and D 3 . This journal is © The Royal Society of Chemistry 2001 1814 Chem. Commun., 2001, 1814–1815 DOI: 10.1039/b103743a Published on 23 August 2001. Downloaded by University of Michigan Library on 30/10/2014 17:23:12. View Article Online / Journal Homepage / Table of Contents for this issue