Amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans for over 50 years with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of the first derivatives of amphotericin that kill yeast but not human cells.

The clinically vital antimycotic agent amphotericin B represents the archetypal example of a channelforming 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.The leading model for the antifungal action of amphotericin B (AmB, 1) involves its selfassembly into a membrane-spanning ion channel. 1 This natural product thus represents a potential prototype for small molecules with the capacity to perform ion channel-like functions in living systems. Efforts to harness this potential and/or improve the notoriously poor therapeutic index of this clinically vital antimycotic 2 would benefit from a molecular-level understanding of this channel activity.Although the evidence that AmB can self-assemble in lipid membranes to form discrete ion conducting channels is strong, 1,3 the molecular architecture of this channel assemblage and its role in antifungal activity remain poorly understood. 4 Despite this, the leading "barrelstave" model 5 is an often cited textbook classic (Fig. 1A). 6 Extensive computer modeling studies predict that this complex is stabilized by a ring of salt bridges 7a and/or hydrogen bonds 7b-c at the channel periphery between oppositely-charged C(41)-carboxylate and C(3′)-

Site-selective functionalizations of complex small molecules can generate targeted derivatives with exceptional step-efficiency, but general strategies for maximizing selectivity in this context are rare. Here we report that site-selectivity can be tuned by simply modifying the electronic nature of the reagents. A Hammett analysis is consistent with linking of this phenomenon to the Hammond postulate: electronic tuning to a more product-like transition state amplifies site-discriminating interactions between a reagent and its substrate. This strategy transformed a minimally site-selective acylation reaction into a highly selective and thus preparatively useful one. Electronic tuning of both an acylpyridinium donor and its carboxylate counterion further promoted site-divergent functionalizations. With these advances, a range of modifications to just one of the many hydroxyl groups appended to the ion channel-forming natural product amphotericin B was achieved. Thus, electronic tuning of reagents represents an effective strategy for discovering and optimizing site-selective functionalization reactions.

Not so complex: A novel iterative cross‐coupling strategy provides access to useful building blocks that enable the simple preparation of complex polyene natural‐product motifs in all possible stereoisomeric forms. The method was used to synthesize the polyene core of vacidin A (see structure).