Matsumori Nobuaki, Sawada Yuri, Murata Michio
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
J Am Chem Soc. 2006 Sep 13;128(36):11977-84. doi: 10.1021/ja063433w.
Amphotericin B (AmB 1) is known to assemble and form an ion channel across biomembranes. We have recently reported that conformation-restricted derivatives of AmB 2-4 show different ergosterol preferences in ion-channel assays, which suggested that the orientation of the mycosamine strongly affects the sterol selectivity of AmB. The data allowed us to assume that compound 3 showing the highest selectivity would reflect the active conformation of AmB in the channel assembly. In this study, to gain further insight into the active conformation of AmB, we prepared a new intramolecular-bridged derivative 5, where the linker encompassed a hydrophilic glycine moiety. The derivative has almost equivalent ion-channel activity to those of AmB and 3. The antifungal activity of 5 compared with 3 improves significantly, possibly because the increasing hydrophilicity in the linker enhances the penetrability through the fungal cell wall. Conformation of 5 was well converged and very similar to that of 3, thus further supporting the notion that the conformations of these derivatives reproduce the active structure of AmB in the channel complex. Then we used the derivative to probe the mobility of AmB in the membrane by solid-state NMR. To measure dipolar couplings and chemical shift anisotropies, we incorporated [1-(13)C,(15)N]glycine into the linker. The results indicate that 5 is mostly immobilized in ergosterol-containing DMPC bilayers, implying formation of large aggregates of 5. Meanwhile some fraction of 5 remains mobile in sterol-free DMPC bilayers, suggesting promotion of AmB aggregation by ergosterol.
两性霉素B(AmB 1)已知可组装并形成跨越生物膜的离子通道。我们最近报道,AmB 2 - 4的构象受限衍生物在离子通道测定中表现出不同的麦角固醇偏好性,这表明霉胺的取向强烈影响AmB的固醇选择性。这些数据使我们推测,具有最高选择性的化合物3将反映AmB在通道组装中的活性构象。在本研究中,为了进一步深入了解AmB的活性构象,我们制备了一种新的分子内桥连衍生物5,其中连接体包含一个亲水性甘氨酸部分。该衍生物具有与AmB和3几乎相当的离子通道活性。与3相比,5的抗真菌活性显著提高,可能是因为连接体中亲水性的增加增强了其穿过真菌细胞壁的渗透性。5的构象高度收敛且与3非常相似,从而进一步支持了这些衍生物的构象再现了通道复合物中AmB活性结构的观点。然后我们使用该衍生物通过固态核磁共振探测AmB在膜中的流动性。为了测量偶极耦合和化学位移各向异性,我们将[1 - (13)C,(15)N]甘氨酸掺入连接体中。结果表明,5在含麦角固醇的二肉豆蔻酰磷脂酰胆碱(DMPC)双层中大多固定不动,这意味着形成了5的大聚集体。同时,5的一部分在不含固醇的DMPC双层中仍保持可移动性,这表明麦角固醇促进了AmB的聚集。