Epimerization of peptide nucleic acids analogs during solid-phase synthesis: optimization of the coupling conditions for increasing the optical purity

Abstract

Peptide nucleic acid (PNA) analogs based on N^α-(thymin-1-ylacetyl)ornithine were previously shown to form triplexes with complementary RNA. In order to obtain optically pure compounds for hybridization experiments, chiral monomers based on D- or L-ornithine, N^δ-Fmoc-N^α-(thymin-1-ylacetyl)ornithine 2 and N^δ-Fmoc-N^α-(uracil-1-ylacetyl)ornithine 3 were synthesized either by a one-step or by a simple three-step procedure starting from N^δ-protected ornithine; the latter procedure led to enantiomerically pure products. Oligomerization of 2 and 3 was carried out either in solution, or by solid-phase peptide synthesis (SPPS) on an MBHA-Rink amide resin. The oligomers turned out to contain large amounts of epimerization products, especially those obtained by SPPS. Therefore, we examined carefully the parameters which may be involved in epimerization: the nature of the coupling reagent, of the base, and the addition mode. Coupling of the monomer L-3 was performed under various conditions. Lower racemization was found to occur when using (7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as coupling agent and 2,4,6-trimethylpyridine (sym-collidine, TMP) as base, without preactivation, leading to a residual 4% of the D-enantiomer. By applying a procedure based on the stepwise addition of the base the D-enantiomer content was reduced to less than 1%. Using this procedure, a decamer of L-3 was synthesized, which was shown to contain less than 2% of the D-ornithine derivative.