Peptides that bind macromolecular receptors in an extended conformation can often be converted to mimetics that retain binding but have improved protease resistance and membrane permeability. 1 However, peptides that must fold upon themselves in order to bind a receptor have proven difficult to improve by similar approaches, because of their larger size and the difficulty of mimicking functionality presented on a complex folded molecular surface. One such folded peptide structure that participates widely in biomolecular recognition events is the R-helix. 2, 3 Most peptides that bind their receptors in an R-helical conformation have little helical structure when free in solution. Stabilizing the helical form of such peptides is thus expected to favor receptor binding by virtue of preorganization. Furthermore, the intramolecular hydrogen bonding associated with helix formation reduces the exposure of the polar amide backbone, thereby reducing the barrier to membrane penetration and increasing the resistance to protease cleavage. A number of approaches for covalent helix-stabilization have been reported, 4 but most involve cross-links that are both polar and pharmacologically labile, such as disulfides5 and lactam bridges. 6, 7 An important conceptual advance on this front is the development by Grubbs and co-workers of chemistry for olefinic cross-linking of helices through O-allyl serine residues located on adjacent helical turns, via ruthenium-catalyzed ring closing metathesis (RCM). 8 The particular cross-links analyzed in that study, however, showed no evidence of enhancing helical stability, highlighting the difficulty of this problem from a design standpoint. Here we have taken an alternate metathesis-based approach, namely to screen multiple configurations of all-hydrocarbon crosslinks differing in position of attachment, stereochemistry, and cross-linker length. Where some configurations impart significant helix-stabilization, others actually destabilize the helix. We show that stabilizing an R-helix in this way leads to markedly increased resistance to proteolysis.

The actual structure of cross-links positioned on one face of an R-helix is very dependent upon the stereochemistry at the attachment points (Figure 1). We therefore designed unnatural amino acids 1 having either R or S stereochemistry at the R-carbon and bearing alkyl tethers of various lengths (Figure 1). To avoid the intrinsic helix-destabilizing effect of D-configured amino acids while capitalizing on the helix-stabilizing effect of R, R-disubsti-