Heterophyllin B: Combining Isotropic and Anisotropic NMR for the Conformational Analysis of a Natural Occurring Cyclic Peptide

ABSTRACT Heterophyllin B is a natural occurring cyclic peptide with diverse attributed bioactivities. NMR‐based conformational analysis of cyclic peptides often poses a challenge due to limited isotropic solution‐state NMR data. In this study, we combined isotropic and anisotropic NMR observables including J ‐coupling, NOEs, amide proton temperature coefficients, and residual dipolar couplings (RDCs), which enabled the determination of a minimal conformational ensemble of heterophyllin B in methanol at density functional theory (DFT) accuracy. For conformational sampling of a cyclic peptide with a high degree of conformational freedom, we proposed a computational strategy that combines the Conformer–Rotamer Ensemble Sampling Tool (CREST) with the Commandline Energetic SOrting (CENSO). This combined computational and NMR‐based approach offers a robust framework for the conformational analysis of cyclic peptides.