Efficient Solution Phase Synthesis of PPII Helix Mimicking Ena/VASP EVH1 Inhibitors from Proline-Derived Modules (ProMs)

In the search for efficient inhibitors for the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain to reduce cell motility in metastatic cancer, we previously developed a toolkit of proline-derived modules (ProMs), which mimic the PPII helix found in the natural −FPPPP− binding motif of EVH1. In this work, we describe the modular assembly of these ProM-based pentapeptidic EVH1 ligands through liquid phase peptide synthesis. We initially used pentafluorophenyl (Pfp) active esters for amide bond formation and built up the growing peptide chain from the C- to the N-terminus. Switching to HATU/DIPEA coupling conditions and changing the directionality of the synthesis from the N- to the C-terminus afforded the target ligands with improved overall yields and purity. Employing a Fmoc-protected (instead of the N-acetylated) phenylalanine derivative as N-terminal building block significantly reduced epimerization. In contrast to the originally used solid phase peptide synthesis (SPPS), the developed solution phase method allowed for a facile alteration of the C-terminal ProM unit and the production of various pentapeptidic ligands in an efficient fashion even on a multigram scale.