Dynamics of (Hetero)aryl Motifs: An Integrative Approach To Study the Conformational Landscape in Macrocycles

Macrocyclic peptides are emerging as a powerful therapeutic modality owing to their potential oral bioavailability and capacity to engage targets long considered undruggable. The introduction of aromatic heterocycles into macrocycles further expands this structural space by imparting distinct conformational preferences. However, accurate determination of the solution-state structures and dynamics of the resulting molecules remains challenging. Here, we integrate complementary isotropic and anisotropic NMR observables with enhanced sampling simulations and density functional theory (DFT) calculations to systematically investigate aryl- and heterobiaryl-containing cyclic peptides in different solvent systems. Our results based on residual dipolar coupling (RDC) measurements reveal conformational dynamics of macrocycles and significantly extend the knowledge gained by conventional NMR analysis based on nuclear Overhauser effects (NOEs) by more faithfully capturing the solution-state ensembles. Our methodology enables conformational analysis in both fast- and slow-exchange regimes on the NMR time scale. Notably, up to three interconverting backbone conformers at the DFT level are required to fully reconcile the experimental data for each ring system. We identify aryl and heterobiaryl motifs as structural elements governing the conformational landscape in our systems, modulating the populations of multiple thermodynamically accessible states characterized by distinct intramolecular hydrogen-bonding networks and unusual backbone geometries. Together, this integrative NMR-computational framework provides a precise and general strategy for resolving complex conformational ensembles of macrocycles, which should inform the objectives for synthetic modification and pave a way for the structure-based rational design of next-generation peptide therapeutics.