Chemical synthesis of hydrocarbon-stapled peptides for protein interaction research and therapeutic targeting

Abstract

The peptide α-helix represents one of nature's most featured protein shapes and is employed in a diversity of protein architectures, from the cytoskeletal infrastructure to the most intimate contact points between crucial signaling proteins. By installing an all-hydrocarbon crosslink into native sequences, the shape and biological activity of natural peptide α-helices can be recapitulated, yielding a chemical toolbox that can be used both to interrogate the protein interactome and to modulate interaction networks for potential therapeutic benefit. Here, current methodology for synthesizing stabilized α-helices (SAH) corresponding to key protein interaction domains is described. A stepwise approach is taken for the production of crosslinking non-natural amino acids, incorporation of the residues into peptide templates, and application of ruthenium-catalyzed ring-closing metathesis to generate hydrocarbon-stapled peptides. Through facile derivatization and functionalization steps, SAHs can be tailored for a broad range of applications in biochemical, structural, proteomic, cellular, and in vivo studies. Curr. Protoc. Chem. Biol. 3:99-117 © 2011 by John Wiley & Sons, Inc.

Keywords: hydrocarbon stapling; olefin metathesis; peptide; photoreactive; protein interaction; targeting; α‐helix.

Fig. 1

Characteristic features of hydrocarbon-stapled peptides. (A) Circular dichroism demonstrates that hydrocarbon stapling can transform an unfolded peptide into a sturdy α-helix(Bird et al., 2010). (B) Protease resistance of hydrocarbon-stapled peptides. In this example, unmodified, singly-, and doubly-stapled peptides were exposed to chymotrypsin in vitro and the persistence of full-length peptide monitored over time by LC/MS(Bird et al., 2010). (C) Fluorescently-labeled stapled peptides are taken up by cells via the pinosomal pathway and gradually released into the cytosol for distribution to target binding sites, as shown here for a BCL-2 family α-helix (BID SAHB) that tracks to the mitochondria(Walensky et al., 2004). (D) Stapled helices can exhibit enhanced binding affinity for their target protein compared to the corresponding unmodified peptide. In this example, a BCL-2 family α-helix (BIM SAHB) demonstrated improved binding affinity for an anti-apoptotic target (left) and a previously undetected binding interaction with a pro-apoptotic target (right)(Walensky et al., 2006).

Fig. 2

Synthetic steps for generating α,α-disubstituted non-natural amino acids bearing olefin tethers using a BPB-based chiral auxiliary.

Fig. 3

Synthetic steps for the automated production of hydrocarbon-stapled peptides using Fmoc chemistry.

Fig. 4

Production and derivatization of hydrocarbon-stapled peptide α-helices for a diversity of experimental and therapeutic applications.