For example, protonation of the lysine residues in the SNase protein results in significant conformational changes. A link between protonation states and macrocycle conformation was also reported by Mügge et al., when they found that cyclic pentapeptides containing a D-aspartate residue would adopt two distinct conformations at different pH values. Based on the protonation state of the aspartate side chain, the macrocycle would either adopt a βII'-turn containing conformation (pH > 5) or a conformation that lacked this turn (pH < 4). However, previous investigations of backbone amine protonation in cyclic pentapeptides have not indicated significant conformational shifts occurring in DMSO. While we were excited that protonation resulted in a new conformational state for 2, the low basicity of the backbone amine means that the new conformational state will not be accessible at biologically relevant pH values. Attempting to mimic the effects of amine protonation with a more stable modification, we moved on to eliminate the possibility of the hydrogen bond interaction via N-methylation of the adjacent amide to relax constraints on the local torsional angles. Nmethylation of internal H-bond donors in peptidic macrocycles is an established method for removing hydrogen bonding interactions in peptides. In small molecule design, methylation can be used to induce a specific conformation via steric interactions. Backbone C-methylation of the 12-membered cyclic clinical candidate lorlatinib locks the conformation of a remote biaryl linkage. The effects of N-methylating peptidic macrocycles are much less predictable. Methylation of heterocycle/peptide macrocycle natural product apratoxin A results in global conformational reorganization, 28 while mono-N methylation of homodetic macrocycles only significantly influences overall conformation if it is employed in combination with a D-residue. N-methylated macrocycle 5 was prepared using previously reported methods and characterized in d6-DMSO. The appearance of two sets of peaks in the 1 H NMR spectrum was indicative of two conformations interconverting on the NMR timescale. This was confirmed by the appearance of EXSY cross-peaks in the ROESY spectrum (Figure ). Peak doubling was observed for the a protons of all residues except for Leu4. Additionally, the amide protons displayed peak doubling. The shifting of the backbone amide protons and a protons remote from the N-methylation is an indication that the conformational effects were being transmitted across the entire ring. We also evaluated the intramolecular hydrogen bonding pattern of the major conformation (5a) using VT 1 H NMR. Chemical shift/temperature coefficients indicated only the Ala6 amide proton was involved in intramolecular hydrogen bonding. NOE-guided MD simulations suggested that it was hydrogen bonding to the Gly4 carbonyl (Figure ). This is significantly different than the secondary structure that was observed for 1. Additionally, the Jab 3 coupling values for residue Leu3 was changed from 4.