PNAs are relatively novel DNA analogues, intensively studied due to their potential as gene-targeted drugs with antigene and antisense properties. In 1996 we elaborated a new method of synthesis of PNA monomer backbones based on the Mitsunobu reaction with N-tosyl-protected (Tos) amino acid esters as acidic components of the reaction. Since the method used for the Tos group removal requires conditions incompatible with various functional groups, here we modified the procedure by replacing the tosyl group with o-nitrobenzenesulfonyl (o-NBS) group. Using the new procedure we obtained protected PNA monomer backbones with various amino acid side chains. The pseudodipeptide secondary amine groups were then deprotected by thiolysis, and after standard work-up acylated with thymin-1-ylacetic acid, to give the protected monomers. Since the deprotection of the secondary amine group occurs under mild conditions, the procedure is of general applicability and allows various modifications of PNA structure by using diverse beta-amino alcohols and alpha-amino acid esters.