The hydrogen bond between the endocyclic amine and the adjacent amide is present in all macrocycles of this structural class and is presumed to play an important role in the conformational stability they display. The RAH macrocycles have a strong tendency to adopt b-turns, regardless of amino acid sequence. Disrupting this conserved hydrogen bond on the overall organization of the macrocycle has enabled us to determine its importance for the overall conformational stabilization of the macrocycle (Figure ). Due to the coupled nature of bond rotations in cyclic compounds, one can imagine that restricting the y torsional angle of the first residue and the f torsional angle of the next residue influences the conformation of the entire macrocycle in a manner complementary to the "dominant rotor" effect described for biaryl-containing peptide macrocycles. Specifically, the strong hydrogen bonding interaction seen in 1 behaves as a "structural pin" that significantly restricts the pyrollidine y torsional angle and the f torsional angle of the second residue (Figure ). By protonation of the backbone amine of 1, the pyrollidine nitrogen is converted from a hydrogen bond acceptor to a hydrogen bond donor, allowing us to investigate the downstream effects of modification of this structural pin on the conformation. Treatment of 1 with aqueous HCl to furnish 2 resulted in significant backbone reorganization (Figure ). To understand the differences in hydrogen bonding between the two macrocycles, variable temperature 1 H NMR experiments were conducted. A chemical shift dependence of less than 4 ppb/K typically indicates that a proton is involved in an intramolecular hydrogen bond. Interestingly, VT 1 H NMR of the HCl salt in DMSO indicated that no amides were involved in intramolecular hydrogen bonding, which is in contrast to 1 that contains two H-bonds. There were also effects on the side chains in the proximal part of the macrocycle. Coupling constants of the phenylalanine b protons moved close to average values while the two resonances moved closer together (Figure ), indicating free rotation and a greater occupancy of the phenylalanine c-space. This result highlights an increase in flexibility in an area of the macrocycle remote from the protonated amine. In our initial reports, pH titrations indicated a low pKB of 1.9 for the amine.