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

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 (2H/13C labeling)to determine mycosamine orientation in membrane-embedded AmB assemblies. Isotopic labeling required both an edited aglycone and an edited sugar: C19-2H was introduced by reducing a 19-keto intermediate obtained via deglycosylation, and 1-13C mycosamine was efficiently prepared from 1-13C-glucose. Semisynthesis from natural-product-derived feedstocks thus enabled access to 2H/13C-labeled AmB for solid-state NMR. The results indicated that the addition of ergosterol significantly sharpened the 1'-13C signal, restricting the sugar orientation. Intramolecular 13C-{2H} 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 2H 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.