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Abstract—α,α′-Trehalose 6,6′-glycolipids have long been known for their immunostimulatory properties. The adjuvanticity of α,α′-trehalose 6,6′-glycolipids is mediated by signalling through the macrophage inducible C-type lectin (Mincle) and the induction of an inflammatory response. Herein, we present an aryl-functionalised trehalose glycolipid, AF-2, that leads to the release of cytokines and chemokines, including IL-6, MIP-2 and TNF-α, in a Mincle-dependent manner. Furthermore, plate-coated AF-2 also leads to the Mincle-independent production of IL-1β, which is unprecedented for this class of glycolipid. Upon investigation into the mode of action of plate-coated AF-2, it was observed that the treatment of WT and Mincle−/− bone marrow derived macrophages (BMDM), murine RAW264.7 cells, and human monocytes with AF-2 led to lytic cell death, as evidenced using Sytox Green and lactate dehydrogenase assays, and confocal and scanning electron microscopy. The requirement for functional Gasdermin D and Caspase-1 for IL-1β production and cell death by AF-2 confirmed pyroptosis as the mode of action of AF-2. The inhibition of NLRP3 and K+ efflux reduced AF-2 mediated IL-1β production and cell death, and allowed us to conclude that AF-2 leads to Capase-1 dependent NLRP3 inflammasome-mediated cell death. The unique mode of action of plate-coated AF-2 was surprising and highlights how the physical presentation of Mincle ligands can lead to dramatically different immunological outcomes.

Isoglobotrihexosylceramide (iGb3) is a known NKT cell agonist, however the specific interactions required to trigger NKT cell TCR activation in response to this mammalian glycolipid are not fully understood. Here we report the synthesis of 1,3-β-Gal-LacCer (βG-iGb3) that displays a β-linked terminal sugar. βG-iGb3 activated NKT cells to a similar extent as iGb3 with a terminal α-linkage, indicating that the conformation of the terminal sugar residue of iGb3 is not essential to facilitate NKT cell TCR recognition. In addition, the immunological activity of four recently described iGb3 analogues with modifications to their terminal sugar or lipid backbone were also investigated. These iGb3 analogues all induced NKT cell proliferation, with IL-13 the predominate cytokine detected. This highlights the ability of the NKT cell TCR to accommodate variations in iGb3-based glycolipids and suggests that undiscovered NKT cell ligands may exist within the lacto-series of mammalian glycosphingolipids.

Lipidated derivatives of the natural product brartemicin show much promise as vaccine adjuvants due to their ability to signal through the macrophage inducible C-type lectin (Mincle). We synthesised three lipophilic amide-linked brartemicin derivatives and compared their agonist activity to that of their ester-linked counterparts in vitro. We demonstrate that the brartemicin amide derivatives activate bone-marrow-derived macrophages (BMDMs) in a Mincle-dependent manner, as evidenced by the production of the pro-inflammatory cytokine IL-1β in wildtype but not Mincle-/- cells. The amide derivatives showed activity that was as good as, if not better than, their ester counterparts. Two of the amide derivatives, but none of the ester-derivatives, also led to the production of IL-1β by human-derived monocytes. As the production of IL-1β is a good indicator of vaccine adjuvanticity potential, these findings suggest that amide-linked brartemicin derivatives show particular promise as vaccine adjuvants.

The correct diagnosis of multiple sclerosis (MS) remains challenging due to the complex pathophysiological and clinical characteristics of the disease. Consequently, there has been immense interest in finding a non-invasive diagnostic test for MS. Recent studies found that serum anti-α-D-Glcp-(1→4)-α-D-Glcp (GAGA4) IgM antibodies were upregulated in MS patients, and this finding led to the development of a commercial diagnostic test (gMS® Dx test), although the test has poor selectivity and has not been independently validated. Herein, we developed an enzyme-linked immunosorbent assay (ELISA) to evaluate the use and reliability of several anti-glucose IgM antibodies, including those against GAGA4, as diagnostic biomarkers for MS. In contrast to previous studies, our results show that serum anti-GAGA4 IgM antibody levels are not significantly higher in MS patients, which could potentially explain the poor selectivity of the commercial test.

Many studies have investigated how trehalose glycolipid structures can be modified to improve their Macrophage inducible C-type lectin (Mincle)-mediated adjuvanticity. However, in all instances, the ester-linkage of α,ά-trehalose to the lipid of choice remained. We investigated how changing this ester-linkage to an amide influences Mincle signalling and agonist activity and demonstrated that Mincle tolerates this functional group change. In in vivo vaccination studies in murine and ovine model systems, using OVA or Mannheimia haemolytica and Mycoplasma ovipneumoniae as vaccine antigens, respectively, it was demonstrated that a representative trehalose diamide glycolipid was able to enhance antibody-specific immune responses. Notably, IgG titres against M. ovipneumoniae were significantly greater when using trehalose dibehenamide (A-TDB) compared to trehalose dibehenate (TDB). This is particularly important as infection with M. ovipneumoniae predisposes sheep to pneumonia.

The correct diagnosis of multiple sclerosis (MS) remains challenging due to the complex pathophysiological and clinical characteristics of the disease. Consequently, there has been immense interest in finding a non-invasive diagnostic test for MS. Recent studies found that serum anti-α-d-Glc

A series of glycitylamines, which were prepared in few steps from readily available carbohydrates, were tested for their ability to inhibit tuberculosis growth in an Alamar Blue BCG colourimetric assay. Several derivatives, including (2R,3R)-1-(hexadecylamine)pent-4-ene-2,3-diol, (2R,3R)-1-(hexadecylmethylamino)pent-4-ene-2,3-diol and 5-deoxy-5-hexadecylmethylamino-D-arabinitol, were prepared in good to excellent (44-90%) overall yield and exhibited micromolar (20-41μM) inhibitory activity that was similar to the first line tuberculosis (TB) drug ethambutol (39μM) in the same assay. The ease and low cost of the synthesis of the glycitylamines offers definite advantages for their use as potential TB drugs.

The application of N-glycosyl-N-alkyl-methoxyamine bi-functional linkers for the synthesis of a variety of glycoconjugates is described. The linker contains a specific functional group, such as an amine, azide, thiol, or carboxylic acid, which can be used for conjugation methodologies that include amide ligation, sulfonylation, copper-mediated Huisgen cycloaddition or thiol-maleimide coupling. In this way, glycoconjugates equipped with biotin, a fluorescent reporter, or a protein were efficiently synthesised, thus demonstrating the versatility of this type of oxyamine linker for the construction of glycoconjugate probes.

Isoglobotrihexosylceramide (iGb3, 1) is an immunomodulatory glycolipid that binds to CD1d and is presented to the T-cell receptor (TCR) of invariant natural killer T (iNKT) cells. To investigate how modifications to the lipid tail or terminal sugar residue of iGb3 influence iNKT cell activity, we developed an efficient and divergent synthetic route that provided access to both sugar and lipid iGb3 analogues which utilised a lactosyl 2-azido-sphingosine derivative as a common intermediate. In this way, iGb3 (1) and the unprecedented analogues 6'''-deoxy-iGb3-sphingosine 2, 6'''-deoxy-iGb3-sphinganine 3, C12 N-acyl iGb3 4 and C20:2 N-acyl iGb3 5 were prepared so that key structure-activity relationships can be explored.

A protecting-group-free synthetic strategy for the synthesis of piperidines has been explored. Key in the synthesis is an I2-mediated carbamate annulation, which allows for the cyclization of hydroxy-substituted alkenylamines into piperidines, pyrrolidines, and furans. In this work, four chiral scaffolds were compared and contrasted, and it was observed that with both d-galactose and 2-deoxy-d-galactose as starting materials, the transformations into the piperidines 1-deoxygalactonorjirimycin (DGJ) and 4-epi-fagomine, respectively, could be achieved in few steps and good overall yields. When d-glucose was used as a starting material, only the furan product was formed, whereas the use of 2-deoxy-d-glucose resulted in reduced chemo- and stereoselectivity and the formation of four products. A mechanistic explanation for the formation of each annulation product could be provided, which has improved our understanding of the scope and limitations of the carbamate annulation for piperidine synthesis.

The ability of a series of pyrrolidines to inhibit several glycosidases was investigated. Using Fleet’s ‘mirror-image postulate’, it was proposed that enantiomeric derivatives of 1,4-dideoxy-1,4-imino- d -lyxitol (a known α- d -galactosidase inhibitor) would show inhibitory activity against α- l -fucosidases. Some modest α- l -fucosidase inhibitory activity was observed for selected compounds (particularly an aminomethyl pyrrolidine) and it was proposed that better activity could be obtained by modifying the C-2 side chain of the pyrrolidine core. The d - galacto carbamate scaffold also exhibited somewhat selective, albeit modest, α- l -fucosidase inhibitory activity and may prove to be an interesting scaffold for further development.

The immunomodulatory glycolipid α-galactosylceramide (α-GalCer) binds to CD1d and exhibits potent activity as a ligand for invariant CD1d-restricted natural killer-like T cells (iNKT cells). Structural analogues of α-GalCer have been synthesised to determine which components are required for CD1d presentation and iNKT cell activation, however, to date the importance of the phytosphingosine 4-hydroxyl for iNKT cell activation has been disputed. To clarify this, we synthesised two 4-deoxy α-GalCer analogues (sphinganine and sphingosine) and investigated their ability to activate murine and human iNKT cells. Analysis revealed that the analogues possessed comparable activity to α-GalCer in stimulating murine iNKT cells, but were severely compromised in their ability to stimulate human iNKT cells. Here we determined that species-specific glycolipid activity was due to a lack of recognition of the analogues by the T-cell receptors on human iNKT cells rather than insufficient presentation of the analogues on human CD1d molecules. From these results we suggest that glycolipids developed for potent iNKT cell activity in humans should contain a phytosphingosine base.

An efficient and highly stereoselective synthesis of L-ido-aminoiminosugar 3 has been described. Key in the synthesis is the application of a diastereoselective Strecker reaction and the extension of our carbamate annulation methodology to protected and functionalized alkenylamines. In addition, the identification of the primary iodide as a key intermediate during the carbamate annulation reaction provides insight into the mechanism of this reaction.

New methodology for the protecting-group-free synthesis of primary amines is presented. By optimizing the metal hydride/ammonia mediated reductive amination of aldehydes and hemiacetals, primary amines were selectively prepared with no or minimal formation of the usual secondary and tertiary amine byproduct. The methodology was performed on a range of functionalized aldehyde substrates, including in situ formed aldehydes from a Vasella reaction. These reductive amination conditions provide a valuable synthetic tool for the selective production of primary amines in fewer steps, in good yields, and without the use of protecting groups.

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discovery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone ( 10a ) and 6-amino-7-methylsulfonyl-5,8-quinolinequinone ( 12 ), that exhibited selectivity for leukemic cells over T-cells, a highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone ( 18a ) exhibiting the highest AI activity (0.11 μM), while 6,7-dichloro-5,8-quinolinequinone ( 7a ), 6,7-dichloro-2-methyl-5,8-quinolinequinone ( 7b ), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol ( 19 ) also exhibited good AI activity and specificity. Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolinequinone ( 11 ) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone ( 14 ), exhibited low MICs (1.56–3.13 μg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the functional group substitution of the quinolinequinone core and biological activity were also identified.

As noted inhibitors of glycosidases, iminosugars have enormous therapeutic potential in the treatment of a number of diseases such as cancer, diabetes, and lysosomal storage disorders. Accordingly, much effort has been expended over the past decades in developing novel and efficient methodologies for the synthesis of iminosugars. This microreview highlights recent (post 2005) developments in the synthesis of pyrrolidine-containing iminosugars and includes discussions of methodologies such as ring-closing metathesis, aldol and pericyclic reactions, and protecting-group free strategies.

The protecting-group-free asymmetric synthesis of 1,2,4-trideoxy-1,4-imino-L-xylitol is readily achieved in five steps from 2-deoxy-D-ribose and with an overall yield of 48%. Key in this synthesis is the application of our recently developed Vasella-reductive amination and carbamate annulation methodologies to the synthesis of 2-deoxy-aza-sugars. The carbamate annulation occurred with excellent yield and diastereoselectively (>20:1 d.r.), in favour of the 3,4-cis isomer.

A protecting group free synthesis of 2,3-cis substituted hydroxypyrrolidines is reported. Two novel reaction methodologies allow for the stereoselective formation of cyclic carbamates from olefinic amines, and the formation of primary amines via a Vasella/reductive amination reaction, both performed in aqueous media.

A remarkably efficient synthesis of the biologically important iminosugar 1-deoxygalactonojirimycin (DGJ) is presented. Key to this strategy is the development of a novel carbamate annulation reaction that favours formation of a six-membered carbamate-containing piperidine skeleton over its five-membered counterpart.

Depending on the substrates - which are derived from carbohydrates - the I2-mediated cyclization yields either furan or piperidine derivatives.

New methodology for the protecting-group-free synthesis of primary amines is presented. By optimizing the metal hydride/ammonia mediated reductive amination of aldehydes and hemiacetals, primary amines were selectively prepared with no or minimal formation of the usual secondary and tertiary amine byproduct. The methodology was performed on a range of functionalized aldehyde substrates, including in situ formed aldehydes from a Vasella reaction. These reductive amination conditions provide a valuable synthetic tool for the selective production of primary amines in fewer steps, in good yields, and without the use of protecting groups.

As noted inhibitors of glycosidases, iminosugars have enormous therapeutic potential in the treatment of a number of diseases such as cancer, diabetes, and lysosomal storage disorders. Accordingly, much effort has been expended over the past decades in developing novel and efficient methodologies for the synthesis of iminosugars. This microreview highlights recent (post 2005) developments in the synthesis of pyrrolidine-containing iminosugars and includes discussions of methodologies such as ring-closing metathesis, aldol and pericyclic reactions, and protecting-group free strategies.

A five-step, protecting group free synthesis of 2,3-cis substituted hydroxy-pyrrolidines is presented. Key steps in the synthesis are the chemoselective formation of a primary amine via a Vasella reductive amination using ammonia as the nitrogen source, and the stereoselective formation of a cyclic carbamate from an alkenylamine. Improvement of the reductive amination, by way of the use of alpha-picoline borane as a more environmentally benign reducing agent, is also presented.

The cover picture shows a representation of the synthesis of several iminosugars. Iminosugars are sugar mimetics found widely in nature. They have potential in the treatment of a number of diseases, and accordingly, much effort has been spent in developing efficient routes for their syntheses. Recent developments in the synthesis of pyrrolidine-containing iminosugars are outlines in the Microreview by B. L. Stocker, M. S. M. Timmer et al. on page 1615 ff. The plant in the background is a Ponga (silver fern, cyathea dealbata) endemic to New Zealand. It was chosen to highlight the fact that iminosugars are naturally occurring in many plant species from around the world. The authors thank Sam Win-Mason for the photograph used for the cover picture.

Carbohydrates, in the form of glycoconjugates, have recently been shown to control a wide range of cellular processes. Accordingly, students interested in the study of organic chemistry and biomedical sciences should be exposed to carbohydrate chemistry. To this end, we have developed a sequence of experiments that leads the student from the derivatization of naturally occurring D-glucose through to the formation of simple alkyl and aryl glycosides, and in doing so this experiment provides a comprehensive overview of the fundamentals of glycoside synthesis. These experiments focus on the control of stereochemistry at the anomeric center and NMR spectroscopy for the structural elucidation of products.