Abstract
Polytheonamide B was isolated from a marine sponge Theonella swinhoei as the largest non-ribosomal peptide, and was found to have a potent cytotoxicity caused by formation of 06.3-helix structure and a monovalent cation channel in the lipid bilayers (Figure 1). The length of 136.3-helical ion channel formed by the single molecule of polytheonamide is long enough to span the lipid bilayer. These unique features are intriguing as the structural platform for designing novel ion channel molecules. Here, we report the total synthesis and functional study of the artificial ion channel peptide, dansylated polytheonamide mimic (1, Figure 2). The original sequence of polytheonamide B contains various non-proteinogenic amino acids, which require the multistep syntheses. To preserve the ability of forming stable 06.3-helical structure and reduce the synthetic steps in the total synthesis of 1, these amino acids were substituted to commercially available or synthetically more accessible ones, which have the similar amide and hydroxyl groups. At the 44th residue, the alkyne-type side chain was introduced as a handle for functionalization via click chemistry. Non-proteinogenic amino acids were synthesized prior to peptide synthesis (Figure 3). These amino acids were used in the automated solid-phase peptide syntheses to give the two peptide segments (Scheme 1). Subsequently, thioesterification of the N-terminal segment and introduction of the dansyl moiety to the C-terminal segment led to segments 15 and 18, respectively. Ag^+ mediated coupling reaction between 15 and 18 gave the protected peptide 2. Then, the 15 protective groups (Tmb, Tr, t-Bu) were simultaneously removed under the acidic conditions to generate 1. To evaluate functions of the novel artificial peptide, we performed the biological studies. The cytotoxicity assay against p388 mouse leuckemia cells revealed that 1 had a strong cytotoxicity (IC50 = 12 nM). Furthermore, the membrane disruption and ion transport assays using fluorescent probe-encapsulated liposomes indicated that 1 transported alkaline metal cations across the membrane without the membrane disruption (Figure 4). In conclusion, design and total synthesis of 1 was successfully achieved. Synthesized 1 has a strong cytotoxicity and cation channel activity. The simplified structure and automated synthetic route are beneficial for practical syntheses of various derivatives. Our rationally designed peptide structure will be utilized as a novel platform of a channel structure.
