The Synthesis and Biological Evaluation of Trehalose Diesters as Mincle Ligands
Authors:   , Foster Amy Jane 
Published: May 2023

<p>The macrophage inducible C-type lectin (Mincle) is a pattern recognition receptor (PRR) that commonly recognises both pathogen-associated and danger-associated molecular patterns (PAMPs and DAMPs, respectively). Over the past decade, significant efforts have been dedicated to determining the binding partners of Mincle, which has led to the identification of a range of both natural and unnatural Mincle ligands. Mincle agonists such as trehalose dimycolate (TDM) and trehalose dibehenate (TDB), have been shown to activate antigen presenting cells to produce a Mincle-dependent inflammatory response. Further evaluation of Mincle ligands has revealed several useful therapeutic properties such as anti-cancer and adjuvant activity.</p>
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<p>Brartemicin, an aromatic-containing trehalose diester, was proposed as a potential Mincle ligand due to its structural similarity to known trehalose-based Mincle ligands. Preliminary studies employed both theoretical and experimental binding models to demonstrate that Brartemicin could bind bovine Mincle with strong affinity. The first section of this thesis describes an efficient synthetic strategy to prepare Brartemicin, along with a series of rationally designed lipidated Brartemicin analogues. The ability of the synthesised analogues to signal through Mincle was assessed using a range of in vitro assays, which demonstrated that C18- containing Brartemicin analogues are potent Mincle agonists. Additionally, evaluation of the designated lead compound in an in vivo model highlighted the capacity of this ligand to augment the TH1-polarised immune response to a model vaccine antigen.</p>
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<p>The next section of this thesis describes the synthesis and initial screening of a second generation (2G) library of Brartemicin analogues specifically designed to probe the functional requirements for Mincle agonism. More specifically, the 2G-Brartemicin derivatives investigate the importance of lipid position (e.g. ortho- vs. meta- vs. para-substituted), number of lipid chains (e.g. mono- vs. di-substituted), linkage type (e.g. ether-linked vs. nitrogen-linked vs. sulphur-linked vs. carbon-linked), and aromatic position on the lipid chain. The synthesised 2G-Brartemicin analogues were screened for their ability to signal through Mincle using a green fluorescent proteins (GFP)-reporter assay and a bone-marrow derived macrophage (BMDM) assay.</p>
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<p>The final section of this thesis details the synthesis and preliminary biological evaluation of several prodrugs of TDB, which were designed to selectively cleave in regions of the body that contain reduced oxygen content (e.g. tumours). These hypoxia-activated prodrugs of TDB (H-TDBs) make use of the N,N'-dimethylethylenediamine linker system to incorporate several nitroaromatic masking groups at the 4-hydroxyls of trehalose, which are thought to be important for mediating binding to Mincle. The synthetic methodology features a convergent strategy that allows for the late-stage incorporation of different masking groups. Finally, the synthesised H-TDBs were tested for their ability to fragment to give TDB using chemical reduction. </p>