Organic & Biomolecular Chemistry Dynamic Article Links Cite this: DOI: 10.1039/c0ob00685h www.rsc.org/obc PAPER Synthesis of 6-F-ergosterol and its influence on membrane-permeabilization of amphotericin B and amphidinol 3 Yusuke Kasai, a Nobuaki Matsumori,* a Hiroyuki Ueno, a Kenichi Nonomura, a Shinya Yano, a Murata Michio* a and Tohru Oishi b Received 7th September 2010, Accepted 22nd November 2010 DOI: 10.1039/c0ob00685h Two well-known antifungals, amphotericin B (AmB) and amphodinol 3 (AM3), are thought to exert antifungal activity by forming ion-permeable channels or pores together with sterol molecules. However, detailed molecular recognitions for AmB-sterol and AM3-sterol in lipid bilayers have yet to be determined. Toward 19 F NMR-based investigation of the molecular recognition underlying their potent antifungal activity, we synthesized 6-fluoro-ergosterol in five steps via ring opening of (5a,6a)-epoxide of ergosterol acetate with using novel combination of TiF 4 and n-Bu 4 N + Ph 3 SiF 2 - . Then we evaluated its activity of promoting pore formation of AmB and AM3, and found that pore formation of AmB was barely promoted by 6-F-ergosterol in clear contrast to the dramatic promotion effect of unmodified ergosterol, whereas AM3 activity was markedly enhanced in the presence of 6-F-ergosterol, which was comparable to that of unmodified ergosterol. These results indicate that the introduction of an F atom at C6 position of ergosterol plays an inhibitory role in interacting with AmB, but it is not the case with AM3. Introduction Amphotericin B (AmB, Fig. 1) has been a standard drug for treatment of deep-seated systemic fungal infections for nearly 50 years. 1–3 For lack of better alternatives, as well as the rare occurrence of resistant strains, 4 the clinical importance of AmB has remained unchanged. It is widely accepted that AmB exerts its antifungal activity by forming transmembrane ion-permeable self- assemblies together with ergosterol (Fig. 1), an abundant sterol in fungal membranes. The selective toxicity of AmB is accounted for by its higher affinity to ergosterol than cholesterol (Fig. 1), a component of mammalian membrane. However, detailed molecular recognition between AmB and ergosterol in lipid bilayers has yet to be determined. Similarly, amphidinol 3 (AM3, Fig. 1) also shows membrane permeabilizing activity. 5–7 AM3 was isolated from marine di- noflagellate Amphidinium krebsii as a potent antifungal, hemolytic and cytotoxic substance, 6,8 and then shown to form pores across biological membranes. 5,6 Recently, we have revealed that the pore- formation of AM3 is dramatically promoted in the presence of cholesterol and ergosterol, 7 which is presumably related to the fact revealed by surface plasmon resonance (SPR) experiments that a Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan. E-mail: matsmori@chem.sci.osaka-u.ac.jp, murata@chem.sci.osaka- u.ac.jp; Fax: +81 6 6850-5790 +81 6 6850-5774; Tel: +81 6 6850-5789 +81 6 6850-5774 b Department of Chemistry, Graduate School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan AM3 has much higher affinity to sterol-containing membranes than sterol-free one. 9 As in the case of AmB, however, the mecha- nism of sterol’s promoting AM3 activity is far from understanding. Meanwhile, 19 F NMR has been of interest for many years in investigating biological systems due in large part to the attractive properties of 19 F, which include 100% natural abundance, spin I = 1/2, large magnetogyric ratio, and low background signals in biological samples. In particular, solid-state 19 F NMR has become an important tool for characterizing molecular interactions in lipid bilayers. In the course of our structural studies on the ion-channel formed by AmB and sterol molecules, 10 particularly the molecular recognitions for AmB–AmB 11 and AmB–sterol, 12 we have prepared several fluorinated derivatives of AmB 13,14 and fluorinated cholesterol (Fig. 1), 15 and used them for solid- state NMR measurements. 16 In this study, toward the 19 F NMR investigation of AmB-ergosterol and AM3-ergosterol interactions in membrane, we developed a novel preparation method of a fluorinated derivative of ergosterol, 6-F-ergosterol (Fig. 1). We then assessed its activity of promoting membrane permeabilization by AmB and AM3. Results Preparation of 6-F-ergosterol Synthesis of 6-F-ergosterol is shown in Scheme 1. Although synthesis of 6-F-ergosteryl acetate 4 from ergosteryl acetate 1 was reported by Barrett et al. 17 by treatment with chromyl fluoride (CrO 2 F 2 ) 18 giving fluorohydrin 3 followed by dehydration with This journal is © The Royal Society of Chemistry 2011 Org. Biomol. Chem. Downloaded by Osaka University on 21 January 2011 Published on 11 January 2011 on http://pubs.rsc.org | doi:10.1039/C0OB00685H View Online