A post-PKS oxidation of the amphotericin B skeleton predicted to be critical for channel formation is not required for potent antifungal activity

Abstract

The clinically vital antimycotic agent amphotericin B represents the archetypal example of a channel-forming small molecule. The leading model for self-assembly of the amphotericin B channel predicts that C(41) carboxylate and the C(3′) ammonium ions form intermolecular salt bridges/hydrogen bonds that are critical for stability. We herein report a flexible degradative synthesis pathway that enables the removal of either or both of these groups from amphotericin B. We further demonstrate with extensive NMR experiments that deleting these groups does not alter the conformation of the polyene macrolide skeleton. As predicted by the leading model, amphotericin B derivatives lacking the mycosamine sugar that contains the C(3′) ammonium ion are completely inactive against Saccharomyces cerevisiae. However, strikingly – and in strong contradiction with the current model – the amphotericin B derivative lacking the C(41) carboxylate is at least equipotent to the natural product. Collectively, these findings demonstrate that the leading model for the mechanism of action of amphotericin B must be significantly revised – either the C(41) carboxylate is not required for channel formation, or channel formation is not required for antifungal activity.

Figure 1

A. A bird's eye view of the current "barrel stave" model for the AmB channel. Salt bridges and/or hydrogen bonds (dashed lines) between oppositely-charged C(41)-carboxylate and C(3')-ammonium ions are predicted to be critical for channel stabilization. B. These two functional groups are installed as post-PKS modifications of the macrolide skeleton.

Figure 2

Superposition of the ground state conformation of the macrolactone skeletons of compounds 1–4 (or their more soluble analogs, see SI for details).

Figure 3

A. Disc diffusion assay with S. cerevesiae (40 µg of compound per disc). Similar results were achieved with C. albicans (SI). B. Broth dilution assays; values represent the average of three experiments.

Scheme 1^a

^a Reagents and conditions (a) (i) Fmoc-succinimide, pyridine, DMF:MeOH 2:1, 23 °C, 12 h; (ii) CSA, THF:MeOH 1:1, 0 °C, 1 h, 90% (two steps); (b) TESOTf, 2,6 lutidine, hexanes, 0 °C, 3 h, 96%; (c) 2-thiopyridyl chloroformate, Et₃N, Et₂O, 0 °C, 30 min, 91%; (d) LiBH₄, Et₂O, 23 °C, 2 h, 88%; (e) I₂, PPh₃, imidazole, THF, 0 °C, 1 h, 78%; (f) DDQ, CaCO₃, THF, 23 °C, 10 min, 67%; (g) (S)-CBS oxazaborolidine, Me₂S·BH₃, CH₂Cl₂, −10 °C, 30 min, 6:1 d.r., 79%; (h) NaBH₄, DMPU, 23 °C, 6 h, 78%; (i) (i) HF/pyridine, THF:pyridine 3:2, 0 °C, 6 h; (ii) AcOH:H₂O:THF 1:1:2, 23 °C, 30 min; HPLC, 38% (two steps); (j) allyl bromide, i-Pr₂NEt, DMF, 23 °C, 8 h, 86%; (k) DDQ, CaCO₃, THF, 23 °C, 20 min, 65%; (l) NaBH₄, THF:MeOH 3:1, 0 °C, 30 min, >20:1 d.r., 77%; (m) HF/pyridine, THF:pyridine 5:3, 0 → 23 °C, 6 h, 56%; (n) CSA, THF:H₂O 2:1, 23 °C, 5h; HPLC, 81%; (o) Pd(PPh₃)₄, thiosalicylic acid, THF, 23 °C, 13 h; HPLC, 50%; (p) HF/pyridine, THF:pyridine 5:3, 0 → 23 °C, 6.5 h, 73%; (q) NaBH₄, DMSO, 23 °C, 8 h, 58%; (r) (i) CSA, THF:H₂O 2:1, 23 °C, 30 min; (ii) piperidine, DMSO:MeOH 4:1, 23 °C, 3 h; HPLC, 56% (two steps).