Stitched α-Helical Peptides via Bis Ring-Closing Metathesis

Abstract

Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.

Synthesis of Cyclic Peptides and Peptidomimetics by Metathesis Reactions

“…Recently, the next generation of hydrocarbon-stapled peptide systems was reported [62] in which two hydrocarbon staples are formed from a single amino acid precursor Àbis-pentenylglycine(2-(amino)-2-(pent-4-enyl)hept-6-enoic acid)À, as shown in Scheme 7, thereby creating a spiro-macrocyclic connectivity pattern of the amino acid side chains which is known to be exceptionally rigidifying the bicyclic peptide. This approach of introducing contiguous hydrocarbon staples along one side of the α-helix has been coined "stitching" which provides peptides superior helix induction, thermal stability, resistance against proteolysis, and cell permeability properties relative to peptide stapling.…”

“…RCM of 36 could lead to the formation of three bis-metathesized peptides, 37-39. Based on model studies it turned out (see Table 1 in [62]) that intraresidue RCM (reaction a; to form cyclononenylglycine) was disfavored as was the undesired reaction b, suggesting that compound 37 would be highly unlikely. Reaction c on the other hand would lead to an S i,i+4 R(8) staple (see Fig.…”

Regulation of protein–protein interactions using stapled peptides

“…These can take the form of multiple individual staples ( Figure 4D) or "stitched" stapled peptides incorporating two alkenyl chains on one α-carbon of the central amino acid (Figure 4E). [51][52][53][54] One staple constraint has been shown to be ineffective for longer peptides; however, two staple constraints can significantly increase the helicity. 54 Stitched α-helical peptides can be thought to be the next generation in terms of hydrocarbon stapling, creating spiro-macrocycles, which are known to be stabilizing.…”

“…[51][52][53][54] One staple constraint has been shown to be ineffective for longer peptides; however, two staple constraints can significantly increase the helicity. 54 Stitched α-helical peptides can be thought to be the next generation in terms of hydrocarbon stapling, creating spiro-macrocycles, which are known to be stabilizing. Stitched peptides have been shown to give increased helicity, protease resistance, and cell permeability compared to the monostapled peptides.…”

“…Stitched peptides have been shown to give increased helicity, protease resistance, and cell permeability compared to the monostapled peptides. 54 The size of the macrocycle formed upon RCM is important to achieve an increased degree of helicity in the constrained α-helical peptide. Different ring sizes are achieved depending on the length of the amino acid side chains used for the RCM.…”