Display Selection of a Hybrid Foldamer-Peptide Macrocycle.

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

Differential Peptide Multi-Macrocyclizations at the Surface of a Helical Foldamer Template.

Hybrid sequences comprising a peptide with several Cys residues and an aromatic foldamer helix with several chloroacetamide functions at its surface were synthesized. Such products may in principle form numerous macromulticyclic thioether products by intramolecularly combining all Cys residues and all chloroacetamide functions. However, we show that the reactive sites on the structurally defined helix can be placed at such locations that the peptide selectively stitches itself to form a series of different macrocycles within mostly one preferred product. Reactions were monitored by HPLC and products with two, three or four macrocycles were identified using LC-MS and NMR. The series of selective macrocyclizations define a sort of reaction trail where reaction sites otherwise identical are involved successively because of their precise positioning in space. The trails can be predicted to a large extent based on structural considerations and the assumption that smaller macrocycles form faster.

Conformational interplay in hybrid peptide-helical aromatic foldamer macrocycles.

Macrocyclic peptides are an important class of bioactive substances. When inserting an aromatic foldamer segment in a macrocyclic peptide, the strong folding propensity of the former may influence the conformation and alter the properties of the latter. Such an insertion is relevant because some foldamer-peptide hybrids have recently been shown to be tolerated by the ribosome, prior to forming macrocycles, and can thus be produced using an in vitro translation system. We have investigated the interplay of peptide and foldamer conformations in such hybrid macrocycles. We show that foldamer helical folding always prevails and stands as a viable means to stretch, i.e. unfold, peptides in a solvent dependent manner. Conversely, the peptide systematically has a reciprocal influence and gives rise to strong foldamer helix handedness bias as well as foldamer helix stabilisation. The hybrid macrocycles also show resistance towards proteolytic degradation.

Precise Enzymatic Cleavage Sites for Improved Bioactivity of siRNA Lipo-Polyplexes.

Sequence-defined cationic lipo-oligomers are potent siRNA carriers, forming stable lipo-polyplexes based on both electrostatic and hydrophobic interactions and, after endocytosis and endosomal protonation, facilitating the delivery of siRNA into the cytosol. After completion of the nucleic acid delivery process, carriers should be readily biodegradable to ensure minimum accumulation of amphiphilic molecules that are harmful to lysosomes and other intracellular organelles. Endolysosomal enzymes may degrade a surplus of carrier molecules left over in lysosomes and thereby facilitate the generation and rapid excretion of cleavage products. By solid-phase supported synthesis, a library of sequence-defined lipo-oligomers was generated containing artificial and natural amino acids comprising precise enzymatic cleavage sites. Incorporating either short cleavable l-arginine sequences (RR), noncleavable d-arginine linkers (rr), or varieties of both tailored the degradability of lipo-oligomers, as demonstrated upon incubation with the endolysosomal protease cathepsin B. Cleavage products were identified by MALDI-TOF mass spectrometry. The effect of improved intracellular degradation on cell tolerability was studied by transfecting Huh7-eGFPLuc and DU145-eGFPLuc cells. Positioning of enzymatic cleavage sites between a lipophilic diacyl domain and an ionizable oligocationic siRNA binding unit enabled efficient enzymatic degradation of the carrier and reduced the lytic potential under lysosomal conditions. Highly degradable carriers containing at least one l-arginine dipeptide linker significantly improved the viability of transfected cells without hampering gene silencing activity. Therefore, the precise integration of enzymatic cleavage sites in lipo-oligomers is a promising strategy toward biocompatible nucleic acid carriers.