Synthesis of r-Galactosyl Ceramide (KRN7000) and Analogues Thereof via a Common Precursor and Their Preliminary Biological Assessment Mario Michieletti, † Antonio Bracci, † Federica Compostella, ‡ Gennaro De Libero, § Lucia Mori, § Silvia Fallarini, † Grazia Lombardi, † and Luigi Panza* ,† Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, UniVersita ` del Piemonte Orientale “Amedeo AVogadro”, Via BoVio 6, 28100 NoVara, Italy, Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, UniVersita ` di Milano, Via Saldini 50, 20133 Milano, Italy, and Experimental Immunology, Department of Biomedicine, Basel UniVersity Hospital, Hebelstrasse 20, 4031-Basel, Switzerland panza@pharm.unipmn.it ReceiVed September 11, 2008 A new practical synthesis of R-GalCer and of its analogues is presented, opening the chance to easily modify the sphingosine chain. The common precursor is a disaccharide, obtained by coupling tetra-O-benzyl-D-galactose with allyl 2,3-O-isopropylidene-D-lyxofuranoside. Introduction of alkyl chains via Wittig reaction (for R-GalCer and OCH) or via Williamson reaction (for oxa analogues) followed by standard synthetic steps allows one to efficiently obtain such com- pounds. The analogues are able to activate iNKT cells when presented by CD1d expressing cells. R-GalCer (KRN7000) is one of the most powerful activating glycolipids when presented by CD1d proteins expressed on antigen-presenting cells (e.g., monocytes, dendritic cells, and B cells). The stimulation of iNKT cells by engagement of the T-cell receptor initiates a cascade of events leading to the release of different Th1- or Th2-type cytokines. 1 The crystal structure of CD1d alone or complexed with R-GalCer has been described, 2 highlighting the importance of the length of the acyl and the alkyl chains in modulating TCR binding affinity and cytokine bias. 3 Several syntheses of R-GalCer have been reported, based on the use of various galactosyl donors (e.g., phosphates, imidates, halides, thiosugars, etc.) 4 and a sphingosine or a ceramide acceptor, with variable efficiency. 5 Here we describe a novel approach to the synthesis of R-GalCer (11a) and R-GalCer analogues starting from a disaccharide and building the lipidic part on the reducing end, thus avoiding the difficulties of glycosylation reactions on ceramide acceptors and opening an easy access to R-GalCer and various analogues modified on the sphingosine chain. 6 We exploited D-lyxose as precursor, with the suitable stere- ochemistry, of the phytosphingosine moiety. However, differ- ently from previous approaches 7 in which lyxose was converted into phytosphingosine before the glycosylation, we reversed the sequence by previously forming a 5-galactosyl lyxoside and introducing the lipid chain or mimics thereof on the obtained disaccharide. Our synthetic route started from the bromide 1, obtained from commercial tetra-O-benzyl-D-galactose by reaction with oxalyl bromide, which was coupled with the D-lyxose derivative 2 in presence of tri(1-pyrrolidine)phosphine oxide as activating agent 8 (Scheme 1), giving 3 together with traces of its -anomer in 95% yield (R/ >95:5). Careful chromatography gave pure 3 in 84% yield. Acceptor 2 was easily obtained from D- mannofuranose following the procedure of Brimacombe et al. 9 The obtained disaccharide 3 was then deallylated to give the key intermediate 4 in 90% yield. Disaccharide 4 is a versatile compound for the synthesis of either R-GalCer or its alkyl analogues (e.g., OCH) by Wittig olefination or for the synthesis of oxa analogues by Williamson alkylation. † Universita ` del Piemonte Orientale. ‡ Universita ` di Milano. § Basel University Hospital. (1) (a) Tsuji, M. Cell. Mol. Life Sci. 2006, 1889. (b) Wu, D.; Zajonc, D. M.; Fujio, M.; Sullivan, B. A.; Kinjo, Y.; Kronenberg, M.; Wilson, I. A.; Wong, C. H. Proc. Natl. Acad. Sci. U.S.A. 2006, 11, 3972. (c) De Libero, G.; Mori, L. Nat. ReV. Immunol. 2005, 5, 485. (d) Chang, Y. J.; Huang, J. R.; Tsai, Y. C.; Hung, J. T.; Wu, D.; Fujio, M.; Wong, C.-H.; Yu, A. L. Proc. Natl. Acad. Sci. U.S.A. 2007, 25, 10299. (2) Koch, M.; Stronge, V. S.; Shepard, D.; Gadola, S. D.; Mathew, B.; Ritter, G.; Fersht, A. R.; Besra, G. S.; Schmidt, R. R.; Jones, E. Y.; Cerundolo, V. Nat. Immunol. 2005, 8, 819–826. (3) (a) Goff, R. 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