A variety of structural motifs have been developed for constraining peptides sequences. These constraining motifs include, but are not limited to, D-amino acids, 2 hydrocarbon-stapled peptides, and heterocyclic amide isosteres. Recent studies in our lab have shown that incorporation of dominant rotors that limit rotational freedom can stabilize two major conformational ensembles, or "wells", in solution. High rotational barriers embedded in these constructs significantly restrict the conformational space a peptide macrocycle can sample in solution and can prevent switching between the two conformational ensembles in solution (Figure ). Although rigidified conformations play an important role in the design of therapeutic agents and chemical probes, conformational heterogeneity can also be an important determinant of the functional behavior of a macrocycle. For example, cyclosporine A relies on a switch between conformations to enter cell membranes. Increasing backbone flexibility has led to the reduction of the koff for stapled peptide macrocycles binding to receptor protein 14-3-3. Conformational selection and multi-step, dynamic association processes are common in protein-protein interactions, and strategies for controlling the conformational state of potential inhibitors are in demand. We have sought out to design a system with increased coverage of macrocycle conformational space. With a focus on the locations where key transannular interactions occur, we hypothesized that small modifications in those regions would engender the most significant effect on overall conformation. We term these interactions "structural pins," which represent hydrogen bonds that stabilize overall macrocycle backbone conformation by impeding local bond rotation. Identification of structural pins should enable a better selection of site modifications in conformation/activity studies. This is because minor structural modifications of a macrocycle can have a significant effect on overall conformation, while the magnitude of modification can be unpredictable and highly dependent on local interactions. Using the dominant rotor systems described previously, the accessible conformational space of a macrocycle can be restricted by increasing the barrier to interconversion about a biaryl linkage 5 (Figure ). To provide a complementary method to the dominant rotor approach that can be used to increase conformational heterogeneity while also influencing the ground state conformation, we considered systems in which the ground state conformation is equipped with a hydrogen bond (a structural pin) that stabilizes overall macrocycle conformation by impeding local bond rotations. For a system in which a single conformational state is stabilized by this construct, modification of the pin would raise the ground state conformational energy such that Comparison of exocyclic and endocyclic structural pins along with protonation and methylation as strategies for disruption of the pin other conformational states become accessible (Figure ). The barrier to interconversion is expected to be reduced (Figure ). The appearance of a second conformation in solution would imply that the barrier to conformational transition is low enough that it can be traversed at room temperature. Similar strategies aimed at ground state destabilization have been described in enzyme design, but have not received attention by the peptide community. Earlier studies have identified that the combination of an endocyclic tertiary amine and an exocyclic amide in a macrocycle resulted in the exocyclic amide forming a hydrogen bond with the third residue N-H while the amine did not form interactions with the macrocycle 13 (Figure ). Investigations into amine/heterocycle combinations (reduced amide/heterocycle, or RAH motifs) revealed that tertiary backbone amines in oxadiazole-containing peptidic macrocycles routinely participate in hydrogen bonding to proximal amide protons and exhibit significantly reduced pKB's 14 (Figure ). We hypothesized that this hydrogen bonding interaction is a structural pin for the entire macrocycle conformation. Reduced amide peptide linkages can induce turns and kinks in linear and cyclic peptides, however they typically interact with the rest of the peptide backbone via a protonated ammonium at physiological pH. This is not the case for the RAH macrocycles. The RAH macrocycles have successfully been used as scaffolds to display the leucine-threonine-aspartate tripeptide motif in the same conformation as it adopts in mucosal addressin cell adhesion molecule-1 (MAdCAM-1). This enabled in vitro inhibition of integrin binding to MAdCAM-1, making these macrocycles biologically relevant scaffolds. At neutral pH these RAH macrocycles adopt single conformations with spectral indications of reduced conformational heterogeneity relative to their homodetic analogs.