Isotopic Editing Unveils Ergosterol-Dependent Sugar Orientation Constraints That Stabilize Amphotericin B Channel Assemblies and May Contribute to Their Clustering

High Resolution Image Download MS PowerPoint Slide Amphotericin B (AmB) is the drug of choice for treating systemic fungal infections. Its selective toxicity is thought to arise from preferential binding to ergosterol in fungal membranes, which enables stable ion-permeable pores. The amino sugar mycosamine, attached to the AmB macrolide ring, has been proposed to mediate specific interactions with ergosterol; however, its orientation in membranes remains elusive, mainly because glycosidic-linkage conformations are difficult to analyze in lipid bilayers. Here we apply an isotopic editing strategy─late-stage glycosylation combined with site-selective isotopic editing ( 2 H/ 13 C labeling)─to determine mycosamine orientation in membrane-embedded AmB assemblies. Isotopic labeling required both an edited aglycone and an edited sugar: C19- 2 H was introduced by reducing a 19-keto intermediate obtained via deglycosylation, and 1- 13 C mycosamine was efficiently prepared from 1- 13 C-glucose. Semisynthesis from natural-product-derived feedstocks thus enabled access to 2 H/ 13 C-labeled AmB for solid-state NMR. The results indicated that the addition of ergosterol significantly sharpened the 1′- 13 C signal, restricting the sugar orientation. Intramolecular 13 C{ 2 H} REDOR measurements defined the mycosamine orientation in ergosterol-containing membranes. All-atom molecular dynamics simulations revealed stronger AmB–ergosterol associations, narrower Φ and Ψ dihedral angle distributions of the mycosamine moiety, and more favorable hydrogen-bond interaction energies in AmB–ergosterol systems compared to AmB–cholesterol. Moreover, antiparallel association between channel assemblies exhibited enhanced stability, driven by strong hydrogen bonding between AmB and ergosterol. This clustering behavior was further supported by restricted-motion signatures observed in 2 H NMR of 39,40-hexadeuterated AmB isotopologue obtained via biosynthetic precursor incorporation. Furthermore, a synergistic effect is suggested, in which clustering of the assemblies stabilizes the architecture of the active ion channel. These results provide direct structural insight into ergosterol–mycosamine interactions that organize and stabilize AmB pores.