Synthesis and Testing of Lipopeptide and Peptide Probes for Bacterial Lipoprotein Processing Enzymes

Abstract

Bacterial lipoproteins perform essential functions in bacteria and are often crucial for survival, growth and virulence. Bacterial lipoproteins feature a fully conserved N-terminal cysteine bearing a 1,2-diacylglyceryl lipid moiety which anchors the proteins in the cytoplasmic membrane of the bacteria. These lipids are post-translationally installed by the bacterial lipoprotein processing pathway. In Gram-negative bacteria, this pathway consists of a unique series of enzymes - Lgt, LspA and Lnt - with no homologues in mammals and thus inhibition of these enzymes offers opportunities for novel antibiotic discovery. To facilitate high-throughput screening (HTS) for potential inhibitors suitably specific, sensitive and responsive molecular probes are required. In the second chapter, the effect of diacylglyceryl lipid chain length on the rate of enzymatic turnover of a FRET peptide activity probe for LspA is investigated. This builds on previous work within the group which developed a FRET probe for LspA that is suitable for HTS. The synthesis begins with the assembly of a series of Fmoc diacylglyceryl cysteine building blocks. An additional diacylglyceryl cysteine structure is also synthesised in which the FRET quencher is moved from the peptide C-terminus to the lipid chain. These lipidated cysteines are then used in solid-phase peptide synthesis to produce a series of FRET probes for LspA with varying lipid chain lengths and one alternative structure. The probes are reacted with LspA and their rates of turnover by the enzyme are compared. The probe experiencing the highest turnover rate by the enzyme was identified. In Chapter 3 the development of a novel strategy for synthesising lipopeptides using a thiol-Michael addition reaction under aqueous conditions is reported. This method uses dehydroalanine as an orthogonal reactive handle on a model peptide, and lipid-thiol reagents which are reacted with the peptide under basic, aqueous, surfactant-mediated conditions. The synthesis of three different diacylglyceryl lipid thiols is described. The optimal reaction conditions were found using a model reaction and applied to a series of structurally diverse lipids, demonstrating the scope of this reaction. The reaction was also successfully demonstrated on an alternative peptide sequence. This study culminates with the synthesis of a functional FRET probe for LspA via the described method, and the enzyme kinetic parameters are reported for the interaction of this novel probe with the enzyme. Chapter 4 outlines initial efforts to identify a suitable substrate peptide that may be used as an activity probe for Lgt. The synthesis of a fluorescent peptide substrate and initial testing with Lgt was carried out to verify the peptide as a substrate for Lgt. The peptide was modified with a fluorescent N-Me-Abz group and reacted with a corresponding fluorescent lipid substrate bearing a nitrobenzoxadiazole (NBD). These moieties are hypothesised to form a FRET pair when the peptide is lipidated by Lgt. Initial biological testing of a second peptide substrate for Lgt is also described. Synthesis of an inhibitor peptide for Lgt was carried out to enable further substrate testing. Therefore, key preliminary studies were performed which identified two peptides with the potential to be used to assay Lgt activity. Chapter 5 describes the overall conclusions and future work of the projects described in this thesis. Chapter 6 contains the experimental details and compound characterisation for the work described in this thesis. In summary, the work described in this thesis has characterised a series of FRET probes for LspPae which may be used for HTS of potential inhibitors in the future. From this series, a probe with the highest sensitivity was successfully identified. Additionally, the development of a synthetic peptide lipidation methodology has been established, utilising Dha chemistry and bespoke lipid-thiol reagents. Initial testing of potential peptide substrates for Lgt was also carried out with the view to develop a novel activity probe for Lgt in the future. Overall, these results contribute to a better understanding of the enzymes of the bacterial lipoprotein processing pathway and will allow them to be screened for novel inhibitors in the future.