preference, similarly to the effect observed in 2. The corresponding amide (6, 8) and N-methylated (7, 9) derivatives were synthesized and characterized in d6-DMSO as previously described for 5. Both amide-containing macrocycles 6 and 8 adopted an analogous conformation to 1, with the same hydrogen bond pattern and turn placement. However, N-Me macrocycles 7 and 9 each adopted distinct conformations. Both 7 and 9 displayed exchange between two conformations, analogous to the behaviour of 5. Although both a to b coupling and MD simulations indicated the fifth residue sidechain did not change conformation upon incorporation of the N-Me, the hydrogen bonding patterns and overall backbone conformation changed significantly. The third and sixth residue amide protons were found to be involved in transannular hydrogen bonds for 7, while MD simulations indicated the threonine side chain was hydrogen bonding with the threonine carbonyl. For 9, the fourth and fifth residues amides were involved in intramolecular hydrogen bonding. Both 7 and 9 represent novel conformational states for RAH macrocycles. The difference between 7 and 9 is striking, considering the similarity between 6 and 8. The only structural difference the fifth residue sidechain. For threonine derivative 7 the side chain is hydrogen bonding to its amide C=O, while for isoleucine compound 9 there is no such interaction. This can account for the conformational difference between the two, however the question then becomes why this interaction does not influence the conformation of 6. b-branched residues are known to exert significant conformational influence on homodetic cyclic peptides. Presumably, when the amine/amide hydrogen bond is present in 6 and restricting the proline y angle this rigidifying effect is transmitted throughout the ring and dominates the conformational preference of the backbone so that the side chain does not affect it (Figure ). The Thr OH---O=C interaction becomes the "structural pin" for 7, but it is not a strong enough interaction to cause conformational rigidity, since two conformers are observed. This highlights the strength of the RAH as a unit to control conformation in peptides, but equally, that removal of this interaction is also useful if one wants to access flexible conformations. This enables the RAH unit to act as conformational "hotspot" that is predictably sensitive to N-methylation. This is in contrast to the unpredictable effects of backbone N-methylation in homodetic peptides. The physicochemical properties of a macrocycle are dependent on its conformational behaviour. We were interested in relating the effects of increasing conformational heterogeneity on lipophilicity. Reverse phase HPLC times are often used as indicators of lipophilicity. Accordingly, we compared the retention time of amide macrocycle 8 and N-methylated macrocycle 9, which differ only by substitution of the amide proton for a methyl group. On a 15 minute gradient from 5% to 95% acetonitrile in water, 8 had a retention time of 5.69 minutes while 9 had a retention time of 5.63 minutes. These values are within the margin of error and suggest that compounds 7 and 8 have essentially the same lipophilicity. This is counterintuitive to conventional medicinal chemistry wisdom, as 9 is lacking a hydrogen bond donor and has an extra lipophilic alkyl group. Nmethylation of macrocycles is expected to increase retention time versus all-NH congeners. Fairlie et al. have predicted in silico that addition of methyl groups to the RAH macrocyclic scaffold can increase retention time if they connect hydrophobic surfaces. While this is contrast with our results for 8 and 9, it is clear that N-methylation that specifically prevents conformationally stabilizing transannular hydrogen bonds does not increase lipophilicity, likely due to an increase in backbone interactions with aqueous solvent. When a macrocyclic scaffold containing a non-canonical Brønsted base is modified in proximity to the base, predictive rules can change due to coupled intramolecular interactions. In our case, conventional strategies to influence permeability by extending hydrophobic surfaces no longer apply to the same extent. This is similar to Lokey et al's finding that replacement of too many backbone amide protons with methyl groups can actually decrease lipophilicity. The ability of Cyclosporin A to permeate membranes while maintaining aqueous solubility and the capacity to engage polar binding is dependent on its conformational heterogeneity. It could be that N-methylation of the oxadiazole/amine macrocycle scaffold provides a tool to for the rational design of such behaviour. The RAH macrocycle scaffold provides an approach to influencing permeability that is complementary to the strategy of extending hydrophobic surfaces that was reported by Fairlie. In summary, we have found that removal of key intramolecular hydrogen bonds that restrict bond rotation in a macrocycle backbone drastically influences the conformation of the entire ring. Introduction of an N-Me group on the second amide adjacent to a basic amine to prevent hydrogen bonding increases conformational heterogeneity and results in the appearance of novel conformations. The decreased rigidity of the backbone allows sidechain-backbone interactions to influence conformation and produces conformations distinct from those previously reported for this class of macrocycles. This offers further support for the strong influence backbone Brønsted base can have on overall macrocycle conformation when they can interact with other functionality, along with acting as a structural pin on the macrocycle that is predictably conformationally sensitive to modification. This is unlike the relatively insignificant effects of N-methylation of backbone amides in a homodetic peptide. The incorporation of the N-Me group did not increase the lipophilicity of the macrocycle although it is adjacent to other hydrophobic surfaces, suggesting that stabilizing effect of intermolecular interactions must be considered when optimizing the lipophilicity of a macrocycle. The finding that overall conformational heterogeneity changes when interactions of the basic amine are modified should be particularly useful for tuning the flexibility of macrocycles to optimizing entropy of protein binding or designing macrocycles capable switching conformations in various environments. It should also provide a tool for displaying peptide binding motifs in a range of complementary conformations to elaborate conformation-activity relationships while designing macrocycle-protein interactions. Additionally, these results provide a conceptual framework for the identification of structural pins in other macrocycles.