Synthesis of achiral and chiral peptide nucleic acid PNA) monomers using Mitsunobu reaction Bogdan Falkiewicz, a,p Aleksandra S. Koøodziejczyk, b Bogdan Liberek b and Kazimierz Wis Âniewski c a Faculty of Biotechnology, University of Gdan Âsk and Medical University of Gdan Âsk, Køadki 24, 80-822 Gdan Âsk, Poland b Faculty of Chemistry, University of Gdan Âsk, Sobieskiego 18, 80-952 Gdan Âsk, Poland c Ferring Research Institute, San Diego, CA, USA Received 30 October 2000; revised 25 June 2001; accepted 19 July 2001 Abstract ÐPeptide nucleic acids PNAs) are intensively studied DNA analogues. We elaborated an ef®cient procedure for the synthesis of N-, C-protected pseudodipeptides with a reduced peptide bond and then peptide nucleic acid PNA) monomers, based on the Mitsunobu reaction of N-Boc-b-amino alcohols with N-o-nitrobenzenesulfonyl-protected oNBS-protected) amino acid esters. Using the new pro- cedure, we obtained protected PNA monomer backbones with various amino acid side chains. The pseudodipeptide secondary amine groups were then deprotected by thiolysis, and after appropriate work-up, acylated with thymin-1-ylacetic acid to give the protected monomers. The procedure seems to be of general applicability and allows various modi®cations of PNA structure by using diverse alcohols and amino acid esters. q 2001 Elsevier Science Ltd. All rights reserved. 1. Introduction Peptide or more generally, polyamide) nucleic acids PNAs) are relatively novel DNA analogues which can mimic oligonucleotides forming heteroduplexes with complementary DNA or RNA. 1 In PNAs a polyamide or peptide backbone replaces the phosphodiester pentose back- bone of DNA or RNA. Depending on the manner of the attachment of the nucleobase to the polyamide backbone, two main groups of polyamide nucleic acids can be singled out: 2 - Type I: PNAs containing a polyamide backbone con- sisting of N-aminoalkyl)aminoacid units to whose secondary amine groups nucleobases are attached by an alkylcarbonyl linker. These PNAs may be achiral or chiral as illustrated in Fig. 1) - Type II: PNAs containing a backbone consisting of amino acid residues carrying nucleobases in their side chains, frequently called `chiral PNA' or `cPNA'. The most widely known PNAs, those based on a N-2- aminoethyl)glycine backbone Fig. 1), were designed and synthesised in 1991 by a group of Danish chemists. 1 These PNA molecules ef®ciently and sequence-speci®cally bind to the complementary according to Watson±Crick or Hoogsteen rules) strand of DNA, RNA or PNA oligomers. 3 Complexes of PNA oligomers with natural nucleic acids show high thermal stability and PNAs of this type prove to be better ligands of DNA or RNA than native nucleic acids. 2 PNA oligomers show the ability to displace the pyrimidine strand of homopurine/homopyrimidine dsDNA and to form PNA±DNA duplexes or PNA p DNA±PNA triplexes. 4 Due to their interesting properties, PNAs can serve as e.g. nucleic acid-targeted compounds with antigene and antisense properties or excellent molecular probes, and they already have numerous applications in the ®elds of molecular biology and experimental medicine. 5 Both types of PNAs may also ®nd various interesting applications in chemistry 6 and technology, e.g. as electrochemical bio- sensors, 7 and in optical data storage. 8 The main limitations of the usefulness of PNAs are their poor solubility in physio- logical solutions and low permeability through cellular membranes. The PNA structure is easy to modify and it is probable that synthesis of altered e.g. chiral) 9 monomers would subsequently result in oligomers with improved Tetrahedron 57 2001) 7909±7917 Pergamon TETRAHEDRON 0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0040-402001)00759-1 Figure 1. Unprotected type I chiral PNA monomer. Conventional parts: aminoalkyl a), amino acid b), linker c), and nucleobase B) d). R 1 or R 2 or both) are different from H. Keywords: Mitsunobu reaction; peptide nucleic acids PNA); pseudo- peptides; reduced peptide bond. p Corresponding author. Tel./fax: 148-58-301-2807; e-mail: bogdan.falkiewicz@wp.pl