Going to any length? Trehalose diesters of various chain lengths have been synthesised in order to determine the effect of lipid length on innate immune recognition, as determined by NO and cytokine production by macrophages. In this work, we show that longer lipids (C(20) -C(26)) are required for macrophage activation, with C(22) giving optimal activity.

The C22 and C26 trehalose monoesters, each containing a single acyl chain, were synthesised in good overall yields and found to activate macrophages in a Mincle-dependent manner. The activities of the monoesters paralleled those of their diester counterparts, and both mono- and diesters could activate the immune response in the absence of priming. This is the first time that trehalose monoesters have been found to activate macrophages, and these studies thus provide an important framework for the rational design of other Mincle agonists.

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The Fráter-Seebach alkylation is a highly efficient means to diastereoselectively introduce α-substituents to chiral β-hydroxy esters, however, the yields of reactions in which longer chain alkyl halides are used can be disappointing. To provide a more robust protocol for the alkylation of β-hydroxy esters, we prepared a variety of long-chain allylic iodides with the view that the greater reactivity of the allylic system would

In this study, the role of lipoteichoic acid (LTA) anchors in the activation of the innate immune response was investigated through the chemical synthesis of a series of LTA derivatives and the determination of their ability to induce NO production in bone marrow-derived macrophages (BMM). To this end, an efficient synthesis of the sn-3-O-(α-D-galactofuranosyl)-1,2-di-O-acylglycerol LTA core was developed, which was then used as a key structure to produce both phosphate and glycerylphosphate-funtionalised LTA anchors, as well as galactofuranosyldiglycerides with different fatty acid chain lengths. With a series of LTA anchors in hand, we then determined the effect of these glycolipids on the innate immune response by exploring their capacity to activate macrophages. Here, we report that several of the LTA-derivatives were able to induce NO production by BMMs. In general, the unnatural (sn-1) core glycolipid anchors showed lower levels of activity than the corresponding natural (sn-3) analogues, and the activity of the glycolipids also appears to be dependent on the length of lipid present, with an optimum lipid length of C20 for the sn-3 derivatives. Interestingly, a triacylated anchor and the 6-O-phosphorylated anchor, showed only modest activity, while the 6-O-glycerophosphorylated derivative was unable to induce NO production. Taken as a whole, our results highlight the subtle effects that glycolipid length can have on the ability to activate BMMs.

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<p>The development of novel therapies for cancer and other diseases is an area of enormous research effort due to the growing need for better patient outcomes. As such, not only is the chemical synthesis of new drugs and adjuvants required, but ways to improve drug delivery also need to be explored. Accordingly, there has been much recent effort towards the synthesis and the biological evaluation of bacterial cell wall components as immunomodulatory compounds. To this end, trehalose glycolipids (TGs), which have been isolated from bacteria of the Mycobacteria family, are of significant interest, due to their anti-tumour and adjuvant activities.  In this thesis, the efficient synthesis of trehalose monoesters (TMEs) was investigated and the ability of these monoesters to activate macrophages via Mincle was studied and compared to the activities of their trehalose diester (TDE) counterparts. In this way, a better understanding of how TG structure influences biological activity was explored. Liposomes containing a representative TG (the C26 TDE) were also synthesised, with the objective being to explore whether TGliposomes could be used as improved drug delivery vehicles. To meet these overall objectives, TGs in solution, as well as TG incorporated into liposomes, were tested for their ability to activate macrophages derived from both C57BL/6 and Mincle-/- mice, whereby the Mincle receptor is a known receptor for TDEs.  In the TME studies, an optimised synthesis of the monoesters was developed. The ability of the TMEs to active macrophages was explored and, for the first time, it was observed that TMEs have similar biological activities to TDEs. In the TGliposome studies, a variety of liposomes containing different concentrations of phosphatidylcholine (PC) as well as the C26 TDE was prepared, so as to explore how differences in these two constituent parts influence the activation of macrophages. From this work, it was observed that increasing concentrations of TG in the liposome and increasing concentrations of liposomes gave increased macrophage activation. A concentration of PC above 200 !M also led to macrophage activation, and non-specific cell death was observed at time points > 48 h (for the wild type macrophages) and at time points ≥ 48 h for the Mincle-/- macrophages. Thus, in the case of the TG liposomes, macrophage activation is independent of Mincle, which was unusual as macrophage activation in the case of the individual TGs was dependent on this receptor.  Taken as a whole, these results pave the way for further investigations into utilising TGs in the treatment of diseases. In particular, this work provided insight into the requirement of TG/Mincle binding for improved TGs as potential adjuvants. Moreover, these studies demonstrated that the incorporation of TGs into liposomes leads to enhanced macrophage activation and therefore, potentially enhanced phagocytosis by these immune cells. Accordingly, TG-liposomes may find future application as drug delivery vehicles, in diseases where macrophages play a prominent role.</p>

The cover picture shows the differential activation of macrophages by synthetic trehalose diesters of various chain lengths. Small-chain analogues (C 4 -C 10 ) show no activity and medium-length analogues (C 18 ) show moderate activity; however, longer derivatives (C 20 -C 26 ) lead to optimal activation of macrophages, as evidenced by NO production and the generation of cytokines such as IL-6 and IL-1b. For more details, see the paper by M. S. M. Timmer, B. L. Stocker et al. on p. 2572 ff.