Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists.

Invariant natural killer T (iNKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical iNKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of iNKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor−α-GalCer–CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon iNKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human iNKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue.

The synthesis of threitol ceramide, which is a non-glycosidic analogue of the potent CD1d antigen α-galactosyl ceramide, is described. The synthesis of a 14C-labelled threitol ceramide analogue is also presented. This radiolabelled analogue will allow the intracellular trafficking pattern/itinerary of this iNKT-CD1d cell agonist to be studied.(2S,3S,4R)-2-Azido-3,4-O-isopropylidene-1,3,4-octadecanetriolC21H41N3O3Ee = 100%[α]D22=+9.6 (c 1.0, CHCl3)Source of chirality: chiral poolAbsolute configuration: (2S,3S,4R)1-O-[l-Threitol]-2-amino-1,3,4-d-ribo-octadecantriolC22H47NO6Ee = 100%[α]D22=+23 (c 1.0, CHCl3)Source of chirality: chiral poolAbsolute configuration: (2S,3S,4R,2′S,3′S)

An aryl-end-capped dodecayne has been prepared using a four-fold fluoride-mediated dechlorosilylation of a masked dodecayne precursor containing four β-chlorovinylsilane residues that serve as masked alkynes; the unstable dodecayne product has been characterised by UV-vis absorption spectroscopy and ‘matrix-free’ MALDI-TOF mass spectrometry.

In the presence of NaBH(OAc)3, a 1,5-keto-aldehyde, contained within a side-chain of an η4-dienetricarbonyliron complex, undergoes a double reductive amination sequence with a series of primary amines, to provide the corresponding piperidine products in good to excellent yield. The dienetricarbonyliron complex functions as a powerful chiral auxiliary in this cascade process, exerting complete control over the stereoselectivity of the reaction, with the formation of a single diastereoisomeric product. The sense of stereoinduction has been confirmed by X-ray crystallography. Removal of the tricarbonyliron moiety can be effected with CuCl2 to afford the corresponding 2-dienyl-substituted piperidine in excellent yield. Attempted extension of this cyclisation strategy to the corresponding azepane ring system using a 1,6-keto-aldehyde as the cyclisation precursor was unsuccessful; in this case, the reaction stopped after a single reductive amination on the aldehyde to provide an acyclic keto-amine product.

Grubbs' first-generation, Ru metathesis complex 3 catalyses the hydrosilylation of terminal alkynes. The reaction exhibits an interesting selectivity profile that is dependent on the reaction concentration and more importantly on the silane employed.

In the presence of NaBH(OAc)3, a 1,5-keto-aldehyde, contained within a side-chain of an η4-dienetricarbonyliron complex, undergoes a double reductive amination sequence with a series of primary amines, to provide the corresponding piperidine products in good to excellent yield. The dienetricarbonyliron complex functions as a powerful chiral auxiliary in this cascade process, exerting complete control over the stereoselectivity of the reaction, with the formation of a single diastereoisomeric product. The sense of stereoinduction has been confirmed by X-ray crystallography. Removal of the tricarbonyliron moiety can be effected with CuCl2 to afford the corresponding 2-dienyl-substituted piperidine in excellent yield. Attempted extension of this cyclisation strategy to the corresponding azepane ring system using a 1,6-keto-aldehyde as the cyclisation precursor was unsuccessful; in this case, the reaction stopped after a single reductive amination on the aldehyde to provide an acyclic keto-amine product.

An aryl-end-capped dodecayne has been prepared using a four-fold fluoride-mediated dechlorosilylation of a masked dodecayne precursor containing four β-chlorovinylsilane residues that serve as masked alkynes; the unstable dodecayne product has been characterised by UV-vis absorption spectroscopy and ‘matrix-free’ MALDI-TOF mass spectrometry.