A divergent approach to the synthesis of iGb3 sugar and lipid analogues via a lactosyl 2-azido-sphingosine intermediate.

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.

Enzymatic synthesis of Gb3 and iGb3 ceramides.

Gb3 and iGb3 are physiologically important trihexosylceramides with a terminal alpha-d-Galp-(1-->4)-beta-d-Galp- and alpha-d-Galp-(1-->3)-beta-d-Galp sequence, respectively. In particular iGb3 is attracting considerable attention as it is believed to serve as a ligand for natural killer T cells. Whether or not iGb3 is present in humans and which enzyme might be responsible for its synthesis is at present a matter of lively debate. In the current investigation we evaluated human blood group B galactosyltransferase (GTB) for its ability to catalyze the formation of iGb3 from lactosylceramide and UDP-Galp. GTB is a retaining glycosyltransferase that in vivo catalyzes the transfer of galactose from UDP-Galp donors to OH-3 of Galp on the H-antigen (alpha-l-Fucp-(1-->2)-beta-d-Galp) acceptor forming the blood group B antigen. GTB tolerates modifications in donor and acceptor substrates and its ability to accept lactosides as acceptors makes it a possible candidate for iGb3 production in humans. For comparison iGb3 and Gb3 were also synthesized from the same acceptor using an alpha-(1-->3)- and alpha-(1-->4)-specific galactosyltransferase, respectively. All the enzymes tested catalyzed the desired reactions. Product characterization by NMR analysis clearly differentiated between the alpha-Galp-(1-->3)-Galp and alpha-Galp-(1-->4)-Galp product, with the GTB product being identical to that of the alpha-(1-->3)-GalT-catalyzed reaction. The rate of transfer by GTB however was very low, only 0.001% of the rate obtained with a good substrate, H antigen disaccharide (octyl alpha-l-Fucp-(1-->2)-beta-d-Galp). This is too low to account for the possible formation of the iGb3 structure in humans in vivo.

Alpha anomers of iGb3 and Gb3 stimulate cytokine production by natural killer T cells.

Natural killer T cells (NKT cells) respond to presentation of specific glycolipids with release of a variety of proinflammatory and immunomodulatory cytokines. The repertoire of glycolipid antigens for these cells includes alpha-glycosylceramides, alpha-glycosyldiacylglycerols, and the triglycosylceramide iGb3. Two features of iGb3 set it apart from these other antigens: (i) three sugars are required for stimulation and (ii) the glycosidic bond between ceramide and the proximal sugar is beta in iGb3, whereas it is alpha in other antigens. We have synthesized the alpha versions of iGb3 and Gb3 and demonstrate that they are effective antigens for NKT cells and that they do not require lysosomal processing to the monoglycosylceramides for stimulation. These triglycosylceramides constitute a new class of antigen that stimulates NKT cells comparably to monoglycosylceramides.

Globotriose-functionalized gold nanoparticles as multivalent probes for Shiga-like toxin.

Compared to monovalent carbohydrates, multivalent carbohydrate ligands exhibit significantly enhanced binding affinities to their interacting proteins. Here, we report globotriose (P(k) ligand)-functionalized gold nanoparticle (AuNP) probes for the investigation of multivalent interactions with the B(5) subunit of Shiga-like toxin I (B-Slt). Six P(k)-ligand-encapsulated AuNPs (P(k)-AuNPs) of varying particle size and linker length were synthesized and evaluated for their potential as multivalent affinity probes by using a surface plasmon resonance competition assay. The affinity of these probes for the interacting proteins was greatly affected by nanoparticle size, linker length, and ligand density on nanoparticle surface. For example, the 20-nm 20-P(k)-l-AuNP, which had a relatively long linker showed a >10(8)-fold increase in affinity compared with the mono P(k) ligand. This intrinsic high-affinity AuNP probe specifically captured the recombinant B-Slt from Escherichia coli lysate, and the resulting purity of the B-Slt was >95 %. We also developed a robust P(k)-AuNP-based detection method for Slt-I by combining the technique with silver enhancement.

Synthesis and structure-activity relationship study of isoglobotrihexosylceramide analogues.

Invariant natural killer T (iNKT) cells are innate T lymphocytes that express T cell receptors binding to exogenous and endogenous glycosphingolpid antigens presented by a nonpolymorphic, non-MHC antigen presenting molecule, CD1d. The endogenous glycosphingolipid metabolite, isoglobotrihexosylceramide (iGb3), is the first known natural ligand for both human and mouse iNKT cells, whose activity has been confirmed in a variety of iNKT cell clones generated by different investigators, representing the majority of the iNKT cell population. The signaling pathway mediated by T cell receptor is largely influenced by the structural variation of glycosphingolpid antigens, leading to multiple and varied biological functions of iNKT cells. In order to investigate the structural requirements behind iGb3 triggered iNKT cell activation, the structure-activity relationship (SAR) of iGb3 needs to be characterized. In this study, iGb3 analogues containing 2' '', 3' '', 4' '' and 6' '' deoxy terminal galactose were synthesized for probing the SAR between iGb3 and TCR. The biological assays on the synthetic iGb3 analogues were performed with use of the murine iNKT cell hybridoma DN32.D3. The results showed that the 2' '' and 3' '' hydroxyl groups of terminal galactose play more important roles for the recognition of iGb3 by TCR; while 4' '' and 6' '' hydroxyl groups were not as crucial for this recognition. These studies might help to understand the general structural requirements for natural endogenous ligands recognized by iNKT cells.

Monovalent Gb3-/Gb2-derivatives conjugated with a phosphatidyl residue: a novel class of Shiga toxin-neutralizing agent.

Shiga toxin (Stx) exerts toxic activity by binding to glycosphingolipids, mainly globotriaosyl (Gb(3)) ceramide, on the surface of target cells. The inhibition of toxin-receptor binding is a promising therapeutic approach to prevent Stx-mediated diseases. In this study, we synthesized monovalent Stx-ligands of phosphatidylethanolamine dipalmitoyl-Gb(3) (Gb(3)-PEDP) and galabiosyl (Gb(2))-PEDP and we examined their neutralizing activity against Stx-1 and Stx-2 in vitro. Both Gb(3)-PEDP and Gb(2)-PEDP strongly neutralized the cytotoxicity of Stx-1 and Stx-2. It is likely that the mechanism of neutralization involved formation of liposomes and consequently clustering of sugar units. We propose monovalent Gb(3)-/Gb(2)-derivatives conjugated with phosphatidyl residue as a novel class of Stx-neutralizing agent.

Modification of the ceramide moiety of isoglobotrihexosylceramide on its agonist activity in stimulation of invariant natural killer T cells.

Isoglobotrihexosylceramide (iGb3) is an endogenous antigen of mammalian cells and can stimulate invariant natural killer T (iNKT) cells to evoke autoimmune activities by the release of T helper 1 (Th1) and Th2 cytokines. Th1 cytokines are correlated with the antitumor and antiviral response, while Th2 cytokines are correlated with the amelioration of autoimmune diseases. iGb3 is a very weak agonist compared to the exogenous alpha-galactosylceramide; however, modification of the ceramide moiety has been advocated as one of the approaches to improve its stimulatory activity and to change the bias of release of Th1 and Th2 cytokines. Two analogues of iGb3, 2H-iGb3 and HO-iGb3 with different ceramide moieties, were synthesized. Bioassay results showed that HO-iGb3 was much more effective in stimulating iNKT cells than iGb3 at low concentration. The assay also showed that the CD1d/2H-iGb3 complexes are remarkably efficient in stimulating iNKT cells.

Thio-isoglobotrihexosylceramide, an agonist for activating invariant natural killer T cells.

Thio-isoglobotrihexosylceramide (S-iGb3) might be resistant to alpha-galactosidases in antigen-presenting cells and have a longer retaining time in the lysosome before being loaded to CD1d. The biological assay showed that S-iGb3 demonstrates a much higher increase as a stimulatory ligand toward invariant natural killer T (iNKT) cells as compared to iGb3. [structure: see text].

Chemoenzymatic syntheses of iGb3 and Gb3.

Efficient chemoenzymatic syntheses of iGb3 and Gb3 have been developed. Isoglobotrihexose and globotrihexose were enzymatically synthesized by a three-enzyme system in both solid and solution phases. Then iGb3 and Gb3 were chemically synthesized by coupling of the corresponding trisaccharides with lipid.

Adamantyl globotriaosyl ceramide: a monovalent soluble mimic which inhibits verotoxin binding to its glycolipid receptor.

The globotriaosylceramide (Gb3) verotoxin (VT) interaction is one of several examples of glycolipid receptors where the ceramide (or lipid) free oligosaccharides fail to show the expected binding parameters. We present a novel, yet simple strategy to synthesize monovalent, water soluble glycosphingolipid mimics which retain receptor function. Replacing the fatty acid chain with rigid, three dimensional hydrocarbon frames, such as adamantane, gives a novel class of neohydrocarbon glycoconjugates. Such adamantyl conjugates derived from Gb3 showed significantly enhanced solubility in water compared to natural Gb3. Adamantyl-Gb3 showed a thousand fold enhanced inhibitory activity (IC50 = 1 microM) for VT-Gb3 binding as compared to a lipid free Gb3 oligosaccharide derivative, alphaGal1-4betaGal1-4betaGlc1-O-CH2CH(CH2SO2C 4H9)2 (IC50 > 2 mM). This represents a new approach to the generation of antagonists of glycolipid receptors.

Synthesis and use of novel fluorescent glycosphingolipids for estimating beta-glucosidase activity in vitro in the absence of detergents and subtyping Gaucher disease variants following administration into intact cells.

Two novel fluorescent glycolipids, LRO-glucosylceramide (LRO-GC) and LRO-trihexosylceramide (LRO-THC) were synthesized and utilized for estimating activities of the lysosomal, acid beta-glucosidase in cell extracts and intact skin fibroblasts, derived from normal individuals and patients with Gaucher disease subtypes. The uniqueness of the glycolipids is the fact that a fluorescent probe (lissamine rhodamine) is linked in a sulfonylamide linkage to the sphingosyl residue of the sphingolipid. Thus, the product of enzymatic hydrolysis, lissamine rhodamine sulfonylamido sphingosine (LRO-ceramide) cannot be further hydrolyzed and remains a metabolic end product. A unique property of LRO-GC as a substrate for the lysosomal, acid beta-glucosidase in vitro was the observation that enzymatic hydrolysis occurs in the absence of detergents and that hydrolytic rates are, in fact, reduced in the presence of Triton X-100 and/or sodium taurocholate. Also, both glycolipids penetrated the membrane of intact fibroblasts in the absence of serum and were hydrolyzed in lysosomes of the intact cells. The rates of intracellular hydrolysis decreased with the severity of the Gaucher disease subtypes. Using LRO-THC as substrate, the intracellular ratio of LRO-ceramide to LRO-glucosylceramide was an indicator for the specific GD-subtype.

Total synthesis of globotriaosylceramide (Gb3) and lysoglobotriaosylceramide (lysoGb3).

We have recently reported a highly efficient and stereocontrolled synthesis of globotriaosylceramide (Gb3, 1) in optically pure form. Key to our synthetic strategy was the implementation of the two-stage activation of thioglycosides for formation of the glycosidic bonds and the utilization of (2S, 3S, 4E)-2-azido-3-O-(tert-butyldimethylsilyl)-4-octadecen-1,3-di ol (9) as a sphingosine equivalent. The syntheses of Gb3 (1) and lysoGb3 (2) were achieved by stereocontrolled coupling of 2,3,4,6-tetra-O-benzyl-alpha-D-galactosyl fluoride (15) with phenyl O-(6-O-benzoyl-2,3-di-O-pivaloyl-beta-D-galactopyranosyl)- (1----4)-2,3,6-tri-O-pivaloyl-1-thio-beta-D-glucopyranoside (14) to form the P kappa antigen trisaccharide masked as a phenyl 1-thioglycoside at the reducing end. Thioglycoside 16 was converted into glycosyl fluoride 19, which was coupled to 9 in high yield. The coupled product 20 was converted into the title compounds 1 and 2 in four and three steps, respectively. This article presents the total synthesis of 1 and 2 in full experimental detail.

Total synthesis of ceramide trihexoside accumulating with Fabry's disease.

We report the synthesis of natural ceramide trihexoside, viz. gal (alpha-1 leads to 4) gal (beta-1 leads to 4) gluc (beta-1 leads to 1) ceramide (XIII). It involves the Koenigs-Knorr reaction of the bromide II with the aglucon XIX, and of the chloride VII with the D-enantiomer of the ceramide ester XII. A positional isomer of XIII was obtained as a by-product. A novel technique in the Koenings-Knorr reaction is described.