Webhofer, Katharina Johanna; (2025) Novel Synthetic Strategies Toward Cadaside and Malacidin Analogues: Probing Structure-Activity Relationships in Calcium-Dependent Antibiotics. Doctoral thesis (Ph.D), UCL (University College London).

Text Katharina Webhofer - PhD Thesis.pdf - Accepted Version Access restricted to UCL open access staff Download (15MB)

Abstract

Antimicrobial resistance continues to outpace discovery and development, keeping pressure on the research of new antibacterial agents. Calcium-dependent antibiotics, with daptomycin as the most prominent representative, remain a credible route forward. Members of the class require Ca2+ for activity, and share common design features (lipid tail, compact macrocycle, and Ca2+-binding motif). Among recent entrants, malacidin A/B and cadaside A/B display promising activity against resistant Gram-positive bacteria, but synthetic routes, particularly of cadasides, have been limited, hindering further research. This thesis addresses that bottleneck by developing an on-resin route to cyclic lipopeptide scaffolds that supports microwave-assisted macrocyclisation and rapid depsi-bond integration. The approach uses standard Fmoc solid-phase peptide synthesis with orthogonal protection to allow facile lipidation and sequence alterations, followed by a concise solution-phase ring-closure under microwave heating. The final synthetic route reduces total synthesis time and facilitates rapid analogue generation. Using this platform, we prepared fifteen simplified analogues spanning the malacidin and cadaside families. Designs tested ring size, backbone edits (e.g., Asp/Glu substitutions), stereochemical inversions, and side-chain modifications, while keeping the overall Ca2+-dependent architecture in view. Biological evaluation combined minimum inhibitory concentration (MIC) testing against Bacillus subtilis 168 with thin-layer-chromatography-based binding/competition assays to explore the mode of action. Two clear trends emerged. First, backbone simplification generally decreases activity 100-fold, while simultaneously abolishing Ca2+-dependence. Second, selected cadaside variants exhibit a previously unexplored precursor preference, with evidence for interaction with C55-P. Interaction of malacidin with lipid II was abolished completely. The data highlights the importance of Asp/Glu editing and stereochemistry in maintaining lipid-precursor recognition and antibacterial effect. In summary, this thesis delivers a practical synthetic route to two challenging CDA scaffolds, and novel insights into structure-activity relationships (SARs). Collectively, the developed synthetic route, SAR data, and proposed next steps establish a tractable path to probe and exploit Ca2+-dependent lipopeptides as drug candidates.

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