Veerapen et al.
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of disaccharides containing α-galactosyl linkages. Hence, 3, 4, 6-tri-O-acetylgalactopyranosyl
chloride (7) was obtained from β-D-galactopyranose pentaacetate and converted to the
corresponding thioglycoside 8 by reacting with thiophenol in the presence of caesium
carbonate. The tethered compound 9 was then synthesised following the procedure described
by Bols. Rearrangement of the silylene 9, catalyzed by N-iodosuccinimide (NIS) in anhydrous
nitromethane at 80 °C, yielded the desired product 10 along with some small amounts of 5
after 2 h. It was observed that by careful monitoring and quenching of the reaction as soon as
compound 9 was consumed, helped in minimizing the regeneration of phytosphingosine
derivative 5 and hence enhanced the yield of the glycosylated product. Methanolysis, followed
by hydrogenation of the azide then afforded compound 2. Finally, N-acylation with the fully
saturated fatty acid, hexacosanoic acid, was achieved via reaction of the corresponding acid
chloride with the free amine 2 in a 1:1 mixture of THF and saturated sodium acetate solution.
Target compound 1 was obtained as a white solid after concentration of the organic phase and
purification of the residue by flash chromatography. The spectroscopic data of the latter were
consistent with the literature.
While our first approach (Scheme 2) is more direct and higher yielding, the alternative route
(Scheme 3) also provides the additional benefit of freeing the hydroxyl group at C-2 on the
sugar residue (compound 10). This allows for selective modification on α-GalCer; such as the
introduction of an additional sugar residue. Specifically, the diglycosyl ceramide, Gal
(α1→2GalCer) 12 has been used to study lysosomal glycolipid processing. Briefly,
galactosidases from lysosomes are responsible for truncating oligoglycosyl ceramides to
monoglycosyl ceramides before they can bind to CD1d and be presented to iNKT cells. Scheme
4 depicts a new strategy for synthesizing disaccharide 11 via NIS/TfOH activation of sugar
donor 4 at -78 °C in anhydrous CH2Cl2 for 3 h. Once more, the directing effect of the bulky
silyl group ensured the formation of the desired α-linkage, as confirmed by the H-1 and C-1
signals in 1H and 13C NMR. Compound 12 was obtained after routine procedures, similar to
those described above and exhibited spectroscopic data consistent with the literature.
We next tested the biological activity of α-GalCer and Gal(α1→2GalCer). Both lipids
stimulated human and mouse iNKT cells in the presence of CD1d-expressing antigen-
presenting cells (APC) (Fig. 1a and b). α-GalCer, but not Gal(α1→2GalCer), stimulated iNKT
cells in an APC-free CD1d-Fc fusion protein plate assay (Fig. 1c). In fix/pulse, pulse/fix
experiments α-GalCer stimulated an iNKT cell response under both conditions, whereas Gal
(α1→2GalCer) resulted in cytokine release only under the pulse/fix condition (Fig. 1d).
Together, these data suggest that α-GalCer and Gal(α1→2GalCer) described here can stimulate
human and mouse iNKT cells. Furthermore, Gal(α1→2GalCer), in contrast to α-GalCer,
required uptake and processing to generate the biologically active monoglycosyl ceramide.
In conclusion, we have demonstrated the versatility of both compounds 4 and 10 as crucial
intermediates in practical and high-yielding syntheses of α-GalCer and other biologically
important derivative, such as Gal(α1→2GalCer).
Acknowledgements
G.S.B. acknowledges support in the form of a Personal Research Chair from Mr. James Badrick, Royal Society
Wolfson Research Merit Award, as a former Lister Institute-Jenner Research Fellow, the Medical Council and The
Wellcome Trust (084923/B/08/7).
References and notes
1. Kronenberg M. Annu. Rev. Immunol. 2005;23:877. [PubMed: 15771592]
2. Brutkiewicz R.R. J. Immunol. 2006;177:769. [PubMed: 16818729]
3. Bendelac R. Rivera M.N. Park S.H. Roark J.H. Annu. Rev. Immunol. 1997;15:535. [PubMed: 9143699]
Published as: BioorgMed Chem Lett. 2009 August 01; 19(15): 4288-4291.