Pyrene-modified PNAs: Stacking interactions and selective excimer emission in PNA2DNA triplexes

Pyrene-modified PNAs: Stacking interactions and selective excimer emission in PNA2DNA triplexes Alex Manicardi*1, Lucia Guidi1, Alice Ghidini1,2 and Roberto Corradini*1 Full Research Paper Open Access Address: 1Department of Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43124, Parma, Italy. Fax: +39 0521 905472; Tel: +39 0521 905410 and 2Present Address: Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Hälsovägen 7, 14183, Huddinge, Sweden Beilstein J. Org. Chem. 2014, 10, 1495–1503. doi:10.3762/bjoc.10.154 Email: Alex Manicardi* - ; Roberto Corradini* This article is part of the Thematic Series "Nucleic acid chemistry". * Corresponding author © 2014 Manicardi et al; licensee Beilstein-Institut. License and terms: see end of document. Received: 11 March 2014 Accepted: 03 June 2014 Published: 02 July 2014 Guest Editor: H.-A. Wagenknecht Keywords: modified nucleobase; nucleic acids; PNA; pyrene excimer; SNP recognition; triplex stabilization Abstract Pyrene derivatives can be incorporated into nucleic acid analogs in order to obtain switchable probes or supramolecular architectures. In this paper, peptide nucleic acids (PNAs) containing 1 to 3 1-pyreneacetic acid units (PNA1–6) with a sequence with prevalence of pyrimidine bases, complementary to cystic fibrosis W1282X point mutation were synthesized. These compounds showed sequence-selective switch-on of pyrene excimer emission in the presence of target DNA, due to PNA2DNA triplex formation, with stability depending on the number and positioning of the pyrene units along the chain. An increase in triplex stability and a very high mismatch-selectivity, derived from combined stacking and base-pairing interactions, were found for PNA2, bearing two distant pyrene units. Introduction Peptide nucleic acid (PNA) probes are very selective in the recognition of DNA and have been used in a large variety of diagnostic methods, easily allowing the detection of point mutations at very low concentrations [1-3]. Poly-pyrimidine PNA can form very stable triplexes of the type PNA/DNA/PNA with poly-purine DNA, via both Watson–Crick and Hoogsteen base pairing (Figure 1). These structures are so stable that dsDNA undergoes displacement of the non-complementary strand [4-7]. However, the formation of triplex structures is limited to homopyrimidine sequences since the presence of one or more purine residues destabilizes these complexes and favour the formation of less stable duplexes [8]. Therefore it would be of great value to adopt strategies for the stabilization of triplex structures even in the presence of non-pyrimidine bases. From the available structural data on these complexes [7], it is possible to envisage that any pair of groups protruding from both thymines methyl groups of a TAT triplet and able to give rise to attractive interactions (Figure 1a) would stabilize the 1495 Beilstein J. Org. Chem. 2014, 10, 1495–1503. triplex. If these groups are aromatic fluorophores, changes in the fluorescence properties can be observed upon interaction with DNA, thus enabling to study the occurring interactions and to produce switching PNA probes. Fluorescent switching probes for DNA detection are very useful tools in diagnostics applications such as real-time PCR and in situ hybridisation [9,10]. Among the possible reporter groups, pyrene has been proposed in several in vitro detection systems, due to the sensitivity of its fluorescence properties to microenvironment and due to its ability to produce stabilizing stacking interactions and to show excimer fluorescence [11-20]. Furthermore, pyrene has been shown to favour self-assembly processes of supramolecular structures [21-28] and interact with carbon nanostructures such as nanotubes [29] or graphene [30], thus allowing to create composite material with special properties. PNA fluorescent probes bearing pyrene units as “universal base” were described [31,32], and recently, pyrrolidinyl-PNA bearing a uracil-linker pyrene unit showed good fluorescence response and mismatch recognition [33]; though terminal pyrene units were shown to stabilize triplexes formed by oligonucleotide probes [34], the effect of single- or multiple pyrene units on PNA in the formation of triplex structures has still to be addressed. We have recently reported the modification of uracil at C5 by hydroxymethylation, followed by substitution with chloride and then with azide, which can be used for click chemistry or as a masked amino group both in a PNA monomer and in PNA oligomers, allowing to produce a variety of modified PNAs from a single precursor [35]. This chemistry introduces a moderate degree of flexibility which can be useful for allowing interactions with other groups to occur within the major groove. In this work we applied this strategy to the synthesis of new mono-, di- and tri-functionalised PNA containing a 1-pyreneacetic acid residue linked to this C5-aminomethyl group (Figure 1b). As a model sequence, we chose a 9-mer (Figure 1c) complementary to a purine-rich tract of DNA which is present in the mutated form of the human cystic fibrosis (CFTR) gene, and which was previously studied in our lab using PNA and modified PNA probes [36,37]; this mutation is characterised by the presence of an adenine instead of guanine, and corresponds to one of the most frequent point mutations connected with cystic fibrosis (M-W1282X). Results and Discussion Synthesis of the PNA strands Two different approaches were followed for the introduction of the pyrene units in the PNA strands. The probe containing only 1 pyrene unit (PNA1, Figure 1c) was synthesized by on-resin modification of 5-azidomethyluracil precursor, as described previously [35], whereas a pyrene-containing modified monomer 1 (Scheme 1), more suitable for automated synthesis, was Figure 1: (a) TAT triplet structure showing Watson–Crick and Hoogsteen base pairing; the binding can be reinforced by the concurrent interaction between two groups protruding from C5 position of thymine into the major groove; (b) pyrene-modified uracil derivative used in PNA monomer in the present study; (c) sequences of PNA and DNA used. T indicates pyrene modified nucleobases; bold letters indicate the position of W1282X point mutation. 1496 Beilstein J. Org. Chem. 2014, 10, 1495–1503. designed for the realization of all the other oligomers (PNA2–6, Figure 1c). For the synthesis of the modified monomer bearing the pyrene moiety, we started from the 5-azidomethyluracil building block 2 previously synthesized by our lab [35]. The azide function was first reduced under Staudinger conditions to the corresponding amine 3, on which 1-pyreneacetic acid was linked using HBTU/DIPEA as condensing agent. The ester 4 was then hydrolyzed to the acid 5, and linked to the Fmoc-protected PNA backbone using EDC/DhBtOH as activating mixture; the PNA monomer 1, was then obtained by ester hydrolysis of 6 under acidic conditions. The PN (...truncated)