title: Biocompatible peptide stapling
Creators: name: Morewood, Richard
Publisher: name: The Australian National University
Year: 2024

description: Peptide stapling involves the linking of two residues within a peptide, constraining its conformation and potentially bestowing advantageous properties upon it. While various stapling techniques are currently available, the objective of this work is to explore the development of novel biocompatible stapling approaches and their application to infectious disease targets. In chapter one, Zika virus (ZIKV) is presented, with a focus on its lifecycle and the current protease inhibitors available. The chapter elucidates peptides, detailing their nature and the processes involved in their synthesis. Several exemplary peptide drugs are discussed to provide context. Additionally, the technique of peptide stapling is introduced, accompanied by an overview of prevalent stapling methodologies. Chapter two presents a novel two-component peptide stapling method that builds on the one-component technique pioneered by the Nitsche group. The approach introduces a two-component strategy, utilising the reaction between the nitriles of 2,6-dicyanopyridine (DCP) and two 1,2-aminothiols present in a peptide. The 1,2-aminothiols can either be incorporated as an N-terminal cysteine or as an unnatural amino acid. This efficient and biocompatible method was used to synthesise multiple stapled ZIKV NS2B-NS3 protease (ZiPro) inhibitors. Chapter three investigates a novel peptide stapling method involving alpha-bromoketones. The approach employs aliphatic linkers endowed with terminal alpha-bromopyruvate esters. During the stapling procedure, these linkers react with two 1,2-aminothiols in the peptide, generating a cyclic ketenamine at each end stapling the peptide. The synthesis of these linkers was easily achieved through Fischer esterification of bromopyruvic acid with aliphatic diols. This allows the production of varying linker lengths, granting chemists the flexibility to determine the optimal geometry for their specific peptide stapling needs. By stapling a peptide designed to inhibit ZiPro, this technique was used in the synthesis of three distinct ZiPro inhibitors with differing linker lengths. Chapter four introduces a photoswitchable peptide stapling method using perfluoroazobenzene. This linker enables direct stapling of azobenzene onto the thiol of cysteine via nucleophilic aromatic substitution. A distinct feature of peptides stapled using this technique is the ability to control their conformation using UV and visible light. This was demonstrated with an aurein 1.2 derivative containing two cysteines, which exhibited varying helicity under different light wavelengths. Additionally, a stapled ZiPro inhibitor was synthesised and evaluated; however, its activity remained consistent regardless of exposure to trans or cis-inducing wavelengths. Chapter five presents co-authored publications, accompanied by a statement of contribution for each. The first co-authored article introduces a one-component peptide stapling technique that utilises the reaction between a 2-cyanoisonicotinamide bearing residue and an N-terminal cysteine for stapling. The second article delves into the screening of substrate-derived peptides and peptidomimetics against the SARS-CoV-2 main protease. The third article highlights a one-component approach to peptide bicyclisation, capitalising on the reaction between a DCP-bearing residue and two 1,2-aminothiols within the peptide. The fourth article showcases an innovative lanthanoid protein tag and its applications in both NMR and electron paramagnetic resonance spectroscopy. The final article details a technique for genetically encoded meta-cyanopyridylalanine into peptides, enabling macrocyclisation through a reaction with an N-terminal cysteine. Chapter six summarises the work presented in chapters two, three, and four. Additionally, the chapter offers prospective avenues for future investigations.