Synthetic and Mechanistic Studies with Biologically Active Macrocyclic Peptides

This thesis describes research concerned with the design, synthesis and biological evaluation of cyclic peptides that target infectious diseases. Chapter 2 describes the design and synthesis of cyclic peptides related to Laspartomycin C. A small library of lipopeptides were synthesized and tested for their activity. In collaboration with the group of Bert Janssen (Utrecht University) high-resolution crystal structures were obtained for two compounds. Interestingly, the results show that a high-ordered crystal arrangement is formed, this higher-ordered arrangement is driven by various amino acids not present in the parent compound but found in the amphomycin/friulimicin class. Chapter 3 builds upon the results described in Chapter 2. To further understand the amino acids that play a key role in the activity of Laspartomycin C three achiral amino acids (Gly) were substituted with the bulkier 2-aminoisobutyric acid (Aib) counterpart. These findings led to the discovery that position eight in Laspartomycin is crucial for activity as it is part of the calcium binding motif. The other two positions were much more amenable to change and further substituted for either a L- or D-Alanine. While overall activity increased when going from Aib to either L- or D-alanine it was found that there was an eight-fold difference in activity between L- and D-alanine at position six (with D-alanine being more active). Position six is always a D-amino acid in the broader CDA family, with Laspartomycin C being the only one that contains a Gly at that position. Chapter 4 reports a fast and convenient synthetic route to obtaining bicyclic peptides that are active against Gram-negative pathogens. Specifically, this synthesis route uses a chemoenzymatic approach to generate large quantities of polymyxin E nona-peptide (PMEN). This PMEN can be covalently linked to the second β-hairpin peptide macrocycle. These peptides bind to the newly reported BamA complex in Gram-negative pathogens and even demonstrate potent activity against MCR-1 pathogens. Chapter 5 focuses on the design and synthesis of β-hairpin peptide macrocycles that can be recognized by the newly discovered bacterial enzyme EarP. EarP transfers a rhamnose to a specific arginine residue in its acceptor protein EF-P. The in vitro rhamnosyltransferase activity of EF-P is abolished when presented with linear substrate peptide (derived from EF-P). However, the enzyme readily glycosylates the same sequence in a cyclized β-hairpin mimic. This was done in collaboration with the group of Marthe Walvoort (University of Groningen). Using detailed NMR studies, it was established that the active peptide substrates all share some degree of β-hairpin formation