Solid-Phase Total Synthesis and Dual Mechanism of Action of the Channel-Forming 48-mer Peptide Polytheonamide B.

Abstract

Polytheonamide B (1) is a unique peptide natural product because of its extremely complex structure, a channel-forming ability in vitro, and the extremely potent cytotoxicity. The 48-mer sequence of 1 comprises alternating d,l-amino acids and possesses an array of sterically bulky β-tetrasubstituted and hydrogen bond forming residues. These unusual structural features are believed to drive 1 to fold into a 4.5 nm long tube, form a transmembrane ion channel at the plasma membrane, and exert cytotoxicity. Despite its potential biological application, however, multiple substitutions by these unusual residues significantly heightened the synthetic challenges, impeding the solid-phase peptide synthesis (SPPS) of 1. In this study, we first addressed the synthesis problem by extensive optimization of various factors of the SPPS. Adaptation of a new protective group strategy allowed for elongation of a 37-mer peptide on resin, to which an N-terminal 11-mer fragment was condensed. Removal of the 18 protective groups and resin gave rise to 1 in excellent overall yield (4.5%, 76 steps from 17). The SPPS protocol is operationally simple and was proven easily amenable to total synthesis of the fluorescent 48-mer probe 2. Synthetic 1 and 2 were utilized for analysis of their cellular behavior. Reflecting its ion-channel function, the addition of 1 to MCF-7 cells rapidly diminished a potential across the plasma membrane. Furthermore, fluorescence imaging study revealed that 1 and 2 were also internalized into the cells, accumulating in acidic lysosomes and neutralizing the lysosomal pH gradient. These new findings indicated that 1 is capable of exerting two functions upon causing apoptotic cell death of mammalian cells: It induces free cation transport across the plasma as well as lysosomal membranes. The present chemical and biological studies provide valuable information for the design and synthesis of polytheonamide-based molecules with more potent and selective biological activities.