FULL PAPER DOI: 10.1002/ejoc.200800891 A Practical and Efficient Approach to PNA Monomers Compatible with Fmoc-Mediated Solid-Phase Synthesis Protocols Andrea Porcheddu,* [a] Giampaolo Giacomelli, [a] Ivana Piredda, [a] Mariolino Carta, [a] and Giammario Nieddu [a] Dedicated to the memory of Dr. Charles Mioskowski Keywords: Peptide nucleic acids / Guanine / Protecting groups / Solid-phase synthesis A straightforward synthesis of orthogonally protected PNA monomers is described. Protected aminoethylglycine (Aeg) monomers were efficiently prepared by reductive amination of N-Fmoc-glycinaldehyde with glycine methyl ester and the subsequent acylation of the free amine with N-bis-Boc-pro- tected nucleobase acetic acids. The exocyclic amine group of the nucleobases, including the notoriously difficult-to-protect guanine nucleobase, was protected with a bis-Boc carbamate Introduction Natural oligonucleotides (DNA, RNA) have limited po- tential as therapeutic agents, and main reason is their poor in vivo stability as a result of the susceptibility of the phos- phodiester backbone to undergo enzymatic cleavage by cellular nucleases. To overcome this general problem in the antisense field, there have been significant advances in the discovery and development of analogues bearing structural features that improve the pharmacological properties of natural oligonucleotides. Peptide nucleic acids (PNAs) are DNA/RNA mimics in which the sugar–phosphate back- bone of natural nucleic acid has been replaced by an un- charged pseudopeptide skeleton composed of N-(2-aminoe- thyl)glycine units (Figure 1). [1] The nucleobases A, C, G and T are linked to this achiral skeleton through a two-atom carboxymethyl spacer. PNAs have very flexible structures, but they are still able to bind complementary DNA and RNA strands with high specificity and selectivity. [2] PNA– RNA and PNA–DNA hybrids are more stable than the cor- responding nucleic acid complexes. The increased stability of PNA–DNA and PNA–RNA duplex in comparison to DNA–DNA (RNA) duplex is mainly attributed to the lack of electrostatic repulsion between the two strands. At the [a] Dipartimento di Chimica, Università degli Studi di Sassari, Via Vienna 2, 07100 Sassari, Italy Fax: +39-079-229559 E-mail: anpo@uniss.it Supporting information for this article is available on the WWW under http://www.eurjoc.org or from the author. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2008, 5786–5797 5786 group; this increased the solubility of the nucleobases in the most common organic solvents. The current protocol allows all Aeg monomers to be prepared on both the micro- and macroscale, which avoids or minimizes the use of toxic rea- gents or solvents, and moreover, cheap starting materials are used. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) same time, the change in ionic strength has little effect on the stability of PNA–DNA duplexes. [3] They also possess high chemical and biological stability due to the unnatural amide backbone that is not recognized by nucleases or pro- teases and thus not degraded inside a living cell. [4] These properties of PNAs make these oligomers of significant interest in many disciplines of chemistry, molecular biology and medicine. [5] A ubiquitous requirement in the field of PNAs research [6] is the preparation of monomers for subse- quent oligomerization. A PNA monomer consists of N-pro- tected (2-aminoethyl)glycine to which a protected nucleo- base is attached (Figure 1). These two protecting groups have to be orthogonal, that is, Pg 2 must be stable under the conditions used to remove Pg 1 . Figure 1. General structures of a PNA monomer. In the literature, during these last years several combina- tions of protecting groups were reported for PNA synthe- sis. [7] PNA oligomers can be prepared by following standard solid-phase synthesis protocols for peptides by using, for example, a (methylbenzhydryl)amine polystyrene resin as the solid support. [8] Previous reports on PNA synthesis have focused on Boc/Cbz protection methods (Pg 1 = Boc and