The Synthesis and Biological Evaluation of Ligands for C-Type Lectin Receptors

Abstract

Macrophage inducible C-type lectin (Mincle), expressed on antigen presenting cells (APCs), is an important player in innate immunity due to its capacity to recognise pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), which are involved in pathogen recognition and in tissue homeostasis, respectively. Several ligands can bind Mincle and activate APCs to generate an inflammatory immune response, with prominent examples including the mycobacterial glycolipid, trehalose dimycolate (TDM), and the synthetic analogue thereof, trehalose dibehenate (TDB). Trehalose glycolipids exhibit anti-tumour properties and an ability to switch the phenotype of macrophages from one that is tumour promoting to one that is tumour suppressive. Insomuch, Mincle ligands have potential as adjuvants for both prophylactic and therapeutic (e.g. cancer) vaccines. Maradolipids, which were originally extracted from the nematode Caenorhabditis elegans, are glycolipids containing trehalose with symmetrical or asymmetrical iso-branched and straight chain fatty acids. The incorporation of the iso-branch makes them distinct from linear trehalose diesters (TDEs) and might provide the compounds with enhanced immunomodulatory properties. Chapter 2 of this thesis reports on the synthesis of maradolipids (or iso-TDEs) and linear trehalose diesters (TDEs). The ability of the compounds to activate bone marrow derived macrophages (BMDMs) so as to elicit cytokine and chemokine production is then investigated. Both the maradolipids and TDEs activated macrophages in a Mincle-dependent manner, however the maradolipids led to greater cytokine production and a faster immune response, thus indicating that iso-TDEs these trehalose glycolipids may have superior adjuvant activity. Moreover, in these studies it was demonstrated for the first time that trehalose glycolipids with a chain length of ≥C12 led to a Mincle-dependent immune response and one that is less inflammatory in nature. Chapter 3 of this thesis explores the structure-activity relationship between monoacylglycerides (MAGs) and their ability to signal through Mincle and activate monocytes. To this end a series of linear, iso-branched, and α-branched MAGs with defined stereochemistry were synthesised and their ability to signal through Mincle was assessed using nuclear factor of activated T cell-green fluorescence protein (NFAT-GFP) reporter cells. As the MAGs only activate hMincle, the immunostimulatory response to the MAGs was then determined by stimulating human peripheral blood monocytes with MAGs and then assessing cytokine production. The structure activity relationship (SAR) study determined that the incorporation of longer lipid chains and branching on the lipid backbone enhanced the immunomodulatory activity of MAGs, while the stereochemistry of the glycerol moiety affected cellular activation, with the sn-1 isomers leading to more pronounced immune response compared to the sn-3 isomers. Furthermore, the cytotoxic effect of the synthesised MAGs was explored to determine if there was a correlation between thebcytotoxicity and immune cell activation. Chapter 4 of this thesis details an efficient synthetic route for the synthesis of α-glucosyl diacylglyceride (αGlc-DAG), whereby the target compounds were prepared in 7 steps and in 47% overall yield with excellent α-selectivity. The Mincle agonist activity of the synthesised αGlc-DAGs was then determined using NFAT-GFP-reporter cells, which determined that the C14 acyl chain αGlc-DAG was the most potent murine Mincle and human Mincle agonist compared to the other derivatives. The incorporation of C14 αGlc-DAG into liposomes for Streptococcus pneumoniae in vivo immunisation studies using the capsular polysaccharide vaccine antigen PPV was then carried out. The initial in vivo assay results indicated that C14 αGlc-DAG enhanced immunity against S. pneumoniae infection and thus can potentially participate as a vaccine adjuvant for S. pneumoniae vaccines. Finally, Chapter 5 of this thesis focuses on synthetic strategies for the development of anti-cancer hypoxia-activated TDB (H-TDB) prodrug. The prodrug was designed to selectively cleave in hypoxic regions by undergoing reduction. Here, commercially available 4-aminobenzyl ether was used as a linker between the nitroimidazole trigger and the effector (TDB). The synthesis of 2-nitroimidazole and suitably protected trehalose diester derivatives were achieved in good yields. The chapter mainly discusses different synthetic strategies to effectively connect the trigger, the linker, and the effector domain to achieve the target H-TDB prodrug