An amphotericin B-fluorescein conjugate as a powerful probe for biochemical studies of the membrane.

Amphotericin B is an antimycotic agent extracted and isolated from a soil streptomycete and is currently in clinical use against chronic fungal infections. An enormous amount of data on the biological activity of this polyketide has been accumulated, which underlines the potency and effectiveness of the medicament, as well as its importance in membrane research. Even so, after five decades of intensive research the details of the mechanism of action of amphotericin B remain far from completely elucidated. It is generally accepted that amphotericin B induces leakage of electrolytes and small molecules from exposed cells. However, a definitive answer to the question of whether such electrolyte efflux is the primary cause of amphotericin-B-induced cell death remains elusive. Recent attempts at mechanistic inquiry have involved use of the tools of synthesis and metabolic engineering. There is a need for the design and synthesis of amphotericin B probes with which to test the various existing hypotheses. Herein, we describe the use of a readily accessible piperazine linker as a synthetic anchor for the introduction of reporter groups such as fluorescein to amphotericin B. We used the resulting probe to conduct several in vivo and in vitro comparative studies involving mammalian and fungal cells, as well as vesicles (Liposomes). Several of the observations documented herein are of particular significance: 1) the amphotericin–fluorescein conjugate is rapidly internalized in mammalian cells, but no such uptake occurs in fungal cells; 2) in contrast, the conjugate is localized in the fungal membrane, as observed by epifluorescence microscopy; 3) despite the fact that liposome studies show the amphotericin conjugate to lead to rapid K efflux, the compound is not lethal to yeast over a wide range of concentrations; 4) the conjugate was found to be uniformly distributed throughout the membrane in exponentially growing and budding yeast. The various intriguing properties of this probe may facilitate further studies aimed at gaining an understanding of the difference between the behavior of amphotericin towards mammalian cells and that towards fungal cells, and may already provide an insight into the origins of the severe side-effects of the drug. Despite the density of functionality of amphotericin B, it only has two addressable reactive centers that allow selective conjugation: the primary amine function found in the pendant mycosamine moiety and the carboxylic acid group in the amphoteronolide fragment (Scheme 1). Analysis of prior work in this area suggests that conjugates anchored through the mycosamine moiety must contain a linker that preserves a protonatable amine function to allow observation of the fast kinetics of K release in liposomes. Murata and co-workers recently published a study in which a linker was introduced