A hydrogen bond surrogate approach for stabilization of short peptide sequences in α-helical conformation

(Chemical Equation Presented) α-Helices constitute the largest class of protein secondary structures and play a major role in mediating protein-protein interactions. Development of stable mimics of short α-helices would be invaluable for inhibition of protein-protein interactions. This Account describes our efforts in developing a general approach for constraining short peptides in α-helical conformations by a main-chain hydrogen bond surrogate (HBS) strategy. The HBS α-helices feature a carbon-carbon bond derived from a ring-dosing metathesis reaction in place of an N-terminal intramolecular hydrogen bond between the peptide i and i + 4 residues. Our approach is centered on the helix-coil transition theory in peptides, which suggests that the energetically demanding organization of three consecutive amino adds into the helical orientation inherently limits the stability of short α-helices. The HBS method affords preorganized α-turns to overcome this intrinsic nucleation barrier and initiate helix formation. The HBS approach is an attractive strategy for generation of ligands for protein receptors because placement of the cross-link on the inside of the helix does not block solvent-exposed molecular recognition surfaces of the molecule. Our metathesis-based synthetic strategy utilizes standard Fmoc solid phase peptide synthesis methodology, resins, and reagents and provides HBS helices in sufficient amounts for subsequent biophysical and biological analyses. Extensive conformational analysis of HBS α-helices with 2D NMR, circular dichroism spectroscopies and X-ray crystallography confirms the α-helical structure in these compounds. The crystal structure indicates that all i and i + 4 C=O and NH hydrogen-bonding partners fall within distances and angles expected for a fully hydrogen-bonded α-helix. The backbone conformation of HBS α-helix in the crystal structure superimposes with an rms difference of 0.75 Å onto the backbone conformation of a model α-helix. Significantly, the backbone torsion angles for the HBS helix residues fall within the range expected for a canonical α-helix. Thermal and chemical denaturation studies suggest that the HBS approach provides exceptionally stable α-helices from a variety of short sequences, which retain their helical conformation in aqueous buffers at exceptionally high temperatures. The high degree of thermal stability observed for HBS helices is consistent with the theoretical predictions for a nucleated helix. The HBS approach was devised to afford internally constrained helices so that the molecular recognition surface of the helix and its protein binding properties are not compromised by the constraining moiety. Notably, our preliminary studies illustrate that HBS helices can target their expected protein receptors with high affinity.