Solid phase synthesis of DNA-3 0 -PNA chimeras by using Bhoc/Fmoc PNA monomers Domenica Capasso, a Lorenzo De Napoli, b Giovanni Di Fabio, b Anna Messere, c Daniela Montesarchio, b Carlo Pedone, d Gennaro Piccialli e,p and Michele Saviano d a Istituto di Microbiologia, Seconda Universita Á di Napoli, S. Aniello a Caponapoli, 80138 Napoli, Italy b Dipartimento di Chimica Organica e Biochimica, Universita Á degli Studi di Napoli Federico II, via Cynthia 4, 80126 Napoli, Italy c Dipartimento di Scienze Ambientali, Seconda Universita Á di Napoli, via Vivaldi 43, 81100 Caserta, Italy d Centro di Studio di Biocristallogra®a del CNR, via Mezzocannone 6, 80134 Napoli, Italy e Dipartimento di Chimica delle Sostanze Naturali, Universita Á degli Studi di Napoli `Frederico II', Via D. Montesano 49, I-80131 Napoli, Italy Received 26 June 2001; revised 3 September 2001; accepted 20 September 2001 Abstract ÐOligonucleotides carrying a peptide nucleic acid PNA) tail at the 3 0 -end have been ef®ciently prepared by an on-line automated synthetic protocol exploiting commercially available Bhoc/Fmoc PNA monomers for the assembly of the PNA tract, followed by a deprotection/reprotection of the base protecting groups. The syntheses of the ODN domain in the chimeras have then been performed by standard methods. The hybridization properties of the synthesized chimeras with complementary DNA fragments have been investigated by thermal denaturation experiments. q 2001 Elsevier Science Ltd. All rights reserved. 1. Introduction Peptide nucleic acids PNAs) are DNA mimics in which the deoxyribose-phosphate backbone has been replaced by N-2-aminoethyl)glycine units. 1 They can selectively bind to complementary DNA and RNA by Watson±Crick base pairing 2 and are resistant to enzymatic degradation. 3 For these reasons they are promising candidates for the development of gene therapeutic drugs in antisense and/or antigene approaches. 4 However these potential biochemical applications are limited by poor water solubility and inability to activate RNase H in PNA±RNA hetero- duplexes. 5 These drawbacks could be overcome by using PNA±DNA hybrids chimeras) which possess unaltered binding af®nity towards complementary nucleic acids and are highly water-soluble and resistant to the degradation by exonucleases. 1b,6 A number of papers have recently appeared in the literature dealing with the synthesis of these chimeras; studies on their hybridization properties 1b,7 and behavior as enzymatic substrates 7c,d have been also carried out. The type of the linkage at the DNA±PNA junction and the orientation of the PNA tract N±C vs. C±N) in the chimera seem to be crucial for the effectiveness of the recognition processes. 7a,8 The synthetic approaches so far proposed for C)-PNA-N)- 3 0 -p-DNA-5 0 are based on chemical template-directed ligation 7a or `on line' solid phase strategies. 7c,8,9 In the ®rst approach, the PNA and DNA fragments to be connected are mixed in the presence of complementary DNA or PNA strands as templates and suitable junction promoters imida- zole/EDC) to give the desired chimera. As an alternative to the above solution strategy, many on line solid phase protocols have been described, in which the polymer- supported PNA tract is assembled before the oligodeoxy- nucleotide ODN) chain through classical peptide chemis- try. Then a stable and structurally favorable junction with the ®rst nucleotide unit is obtained via 3 0 -phosphoramidate bond formation, followed by standard phosphoramidite synthesis of the DNA tract. The latter pathways require the usage of ad hoc designed, commercially unavailable PNA monomer building blocks carrying a protecting group for the base amino functions which can be removed in alkaline conditions, and an acidic labile group MMT or DMT) for the transient protection of the 2-ethylamino func- tion. Thus it is possible to follow the same deprotection procedures used in ODN synthesis: acidic treatment by dichloroacetic acid DCA) addition in the elongation steps and basic conditions by conc. ammonia treatment), in the ®nal step for detachment from the support and complete deprotection of the oligomer. In an effort to develop a simpler and more versatile approach to DNA±PNA chimeras, we focused our attention on the possibility of using commercially available PNA Tetrahedron 57 2001) 9481±9486 Pergamon TETRAHEDRON 0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0040-402001)00944-9 Keywords: nucleic acid analogues; peptide analogues; protecting groups; solid-phase synthesis. p Corresponding author. Tel.: 139-081-674127/6; fax: 139-081-674393; e-mail: picciall@cds.unina.it