Synthesis of sanguinamide B derivatives and their mechanism of action

Abstract

This thesis describes structure-activity relationship studies of the natural product Sanguinamide B (San B) and elucidates the mechanism of action of several derivatives. San B was discovered by Molinski in 2009. It is a heterocyclic macrocycle containing an oxazole, two thiazoles, two prolines, and three hydrophobic amino acids; valine, alanine, and leucine. The prolines are both trans, trans configuration about the amide bonds. However, no biological activity or mechanism of action were reported. Given San B s structurally interesting features, the heterocyclic moieties and trans rotation around the prolyl amides, we pursued the first total synthesis of San B. We recently published our synthetic efforts of both the natural product and its conformers. My first project investigates the structure-activity relationship of San B analogues in bacteria and colon cancer cell line HCT-116. Our San B derivatives involved substituting the three hydrophobic amino acids with either an N-Me, glycine, an L- or a D- phenylalanine. We conserved the heterocyclic moieties and prolines as they are likely to play key roles in the structure of San B. Our goal was to observe how the conformational changes induced by each substitution affected the biological activity. Using a convergent solution phase synthesis I contributed three derivatives out of twelve in this series. All San B analogues were subjected to conformational analysis using variable temperature and 2D NMR in order to assign the trans or cis configuration of the prolyl amide bonds. We then tested all twelve analogs against gram positive and negative bacteria as well as in cytotoxicity assays using the colon cancer cell line (HCT-116). My second project involved elucidating two active San B analogs mechanism of action. My thesis describes the synthesis of tagged derivatives and their use to find their mechanism of action in pull-down assay. Identification of protein targets pulled down by these tagged compounds provides insight into their mechanism. We identified the protein targets as ribosomal proteins, which are involved in protein biosynthesis within the cell. In order to validate this pull-down result we tested our compounds in an in vitro translation assay. This assay indicates that the pull-down results were valid and San B interferes with translation between mRNA and protein synthesis, which leads to cell death. Finally, we determined the mode of cancer cell death through microscopy, and flow cytometry, where one compound induces apoptosis, while the second induces necrosis. These exciting results show the potential for this class of compounds as anticancer leads, and my work has shown that both compounds act as translational inhibitors, stopping proteins from being synthesized within the cell.