A Diversity-Oriented Strategy for Chemoenzymatic Synthesis of Glycosphingolipids and Related Derivatives.

A diversity-oriented strategy combining enzymatic glycan assembly and on-site lipid remodeling via chemoselective cross-metathesis and N-acylation was developed for glycosphingolipid (GSL) synthesis starting from a common, simple glycoside. The strategy was verified with a series of natural GSLs and GSL derivatives and showed several advantages. Most notably, it enabled two-way diversification of the glycan and lipid, including introduction of designed molecular tags, to provide functionalized GSLs useful for biological studies and applications.

Synthesis of the Non Reducing End Oligosaccharides of Glycosphingolipids from Ascaris suum.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcß1→OR (1), Galß1→3Galα1→3GalNAcß1→OR (2), Galß1→6Galα1→3GalNAcß1→OR (3), Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (4) and GlcNAcß1→6Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1-4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-ß-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

Synthetic Strategies for Modified Glycosphingolipids and Their Design as Probes.

The plasma membrane of cells contains a diverse array of lipids that provide important structural and biological features. Glycolipids are typically a minor component of the cell membrane and consist primarily of glycosphingolipids (GSLs). GSLs in vertebrates contain a multifarious assortment of glycan headgroups, which can be important to biological functions based on lipid-lipid and lipid-protein interactions. The design of probes to study these complex targets requires advanced synthetic methodologies. In this Review, we will discuss recent advances in chemical and chemoenzymatic synthesis of GSLs in conjunction with the use of these approaches to design new probes. Examples using either chemical or enzymatic semisynthesis methods starting from isolated GSLs will also be reviewed. Focusing primarily on vertebrate glycolipids, we will highlight examples of radionuclide, fluorophore, photoresponsive, and bioorthogonal tagged GSL probes.

Generation of anti-oligosaccharide antibodies that recognize mammalian glycoproteins by immunization with a novel artificial glycosphingolipid.

Here we report a new method for the efficient generation of antibodies that recognize the fine structures of oligosaccharides on glycoproteins. We found a newly designed artificial glycosphingolipid carrying a very long chain fatty acid to be a strong immunogen in mice, with the serum of immunized mice containing antibodies recognizing the oligosaccharide structure of the immunogen. First, we found that conjugation of a simple ceramide analogue to target oligosaccharides could enhance the immunogenicity of these oligosaccharides in these immunized mice. This effect was confirmed in mice immunized with the artificial glycosphingolipids carrying 6'-Sialyl-LacNAc, 3'-Sialyl-LacNAc and LacNAc. Next, we tried to improve the immunogenic enhancing effect of the ceramide analogue. In a model experiment using 6'-Sialyl-LacNAc oligosaccharide, we manipulated the alkyl chains to several lengths, and found that a longer alkyl chain length of the fatty acid correlated with high immunogenicity. Among these we examined, artificial glycosphingolipids conjugated with a ceramide analogue carrying a very long chain fatty acid (lignoceric acid) showed the strongest immunogenicity. By using the artificial glycosphingolipid containing 6'-Sialy-LacNAc and lignoceric acid, we succeeded in the generation of a new anti-6'-Sialyl-LacNAc antibody that recognizes 6'-Sialyl-LacNAc carrying glycoproteins but does not bind to 6'-Sialyllactose, asialo-glycoporoteins and glycoproteins carrying 3'-Sialyl-LacNAc. These results indicate that the established technology is valuable for the targeted generation of monoclonal antibodies against glycoproteins containing specific oligosaccharide structures.

Recent Advances in the Chemistry of Glycoconjugate Amphiphiles.

Glyconanoparticles essentially result from the (covalent or noncovalent) association of nanometer-scale objects with carbohydrates. Such glyconanoparticles can take many different forms and this mini review will focus only on soft materials (colloids, liposomes, gels etc.) with a special emphasis on glycolipid-derived nanomaterials and the chemistry involved for their synthesis. Also this contribution presents Low Molecular Weight Gels (LMWGs) stabilized by glycoconjugate amphiphiles. Such soft materials are likely to be of interest for different biomedical applications.

Syntheses of Fluorescent Gangliosides for the Studies of Raft Domains.

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes. The functional evaluations of the synthesized fluorescent gangliosides demonstrated the great influence of fluorescent dye on the physical properties of gangliosides in PMs and revealed the fluorescent ganglioside analogs which show similar behaviors to the native gangliosides.

Synthesis and biological evaluation of neoglycosphingolipids.

Various neoglycosphingolipids were efficiently synthesized in a one-step reaction by the coupling of free sugars with an N-alkylaminooxy-functionalized ceramide analogue. The bioactivity studies demonstrated that most of these compounds could upregulate the expression of matrix metalloproteinase-9 (MMP-9, extracellular matrix proteins associated with tumor migration) in murine melanoma B16 cells in a similar manner to the natural ganglioside monosialodihexosylganglioside (GM3), which highlights the potential use of these neoglycosphingolipids as inhibitors of tumor migration.

Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis.

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized ß-D-Galp-(1→6)-ß-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-ß-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-ß-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galß1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

Synthetic Glycosphingolipids for Live-Cell Labeling.

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or-more commonly-to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

Total Synthesis of Marine Glycosphingolipid Vesparioside B.

The first total synthesis of a major component of marine glycolipid vesparioside B ( Scheme 1 , 1, R1 = n-C22H45, R2 = n-C14H29) has been accomplished through a convergent [4 + 3] coupling strategy. Key steps included stereoselective installment of a set of challenging 1,2-cis-glycoside bonds. A 2-quinolinecarbonyl-assisted α-galactosylation and a novel ß-arabinosylation were developed, respectively, to synthesize the α-galactofuranosidic and the ß-arabinopyranosidic linkages. Furthermore, a 4,6-O-benzylidene-controlled α-galactopyranosylation reaction allowed the efficient connection of the left tetrasaccharide donor 2 with the right disaccharide lipid acceptor 3, hence leading to the total synthesis of 1.

Design and synthesis of a novel glycosphingolipid derived from polyhydroxy 2-pyrrolidinone and phytoceramide appended by a 1,​2,​3-​triazole linker.

Synthetic analogs of glycosphingolipids (GSLs) have been demonstrated as potential therapeutic interventions in certain patho-physiological conditions. This article reviews reports of various bioactive synthetic GSLs, emanated from the Bittman laboratory. KRN7000, a synthetic GSL which is a α-galactosylceramide (α-GalCer) is a potent immunomodulatory agent. Bittman et al. reported several modifications of C-glycosides of KRN7000 with an eye towards achieving selective cytokine response during iNKT cell activation. However, GSLs with azasugar variants were not explored which inspired us to derive a polyhydroxy 2-pyrrolidinone azasugar from d-galactose and append to the phytoceramide via a 1,2,3-triazole linker to afford GSL analog 12. This novel GSL analog 12 may be used to explore the immunomodulatory activity, and other biological activities against targets involving iminosugar or azasugar based therapeutics.

Total syntheses of disulphated glycosphingolipid SB1a and the related monosulphated SM1a.

Total syntheses of two natural sulphoglycolipids, disulphated glycosphingolipid SB1a and the structurally related monosulphated SM1a, are described. They have common glycan sequences and ceramide moieties and are associated with human epithelial carcinomas. The syntheses featured efficient glycan assembly and the glucosyl ceramide cassette as a versatile building block. The binding of the synthetic sulphoglycolipids by the carcinoma-specific monoclonal antibody AE3 was investigated using carbohydrate microarray technology.

Synthesis of a new glycosphingolipid, neurosporaside, from Neurospora crassa.

The glycosphingolipid neurosporaside (α-D-Glcp-(1 → 2)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 →)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

Synthesis of N-oxyamide-linked neoglycolipids.

N-Oxyamide-containing compounds have shown improved metabolic stability and interesting secondary structures due to the good hydrogen bond-donating property of N-oxyamide. ß-Glucolipids linked by the N-oxyamide bond have been successfully synthesized as novel mimics of glycoglycerolipids and glycosphingolipids.

Intramolecular nitrogen delivery for the synthesis of C-glycosphingolipids. Application to the C-glycoside of the immunostimulant KRN7000.

The key reaction in this approach to C-glycosphingolipids is the stereoselective iodocyclization of a sugar-linked homoallylic carbonimidothioate. E and Z reaction substrates were assembled in a convergent fashion via an alkene metathesis strategy and exhibited the same alkene facial selectivity in the iodocyclization irrespective of alkene geometry, although the E alkene was found to be less reactive.

Synthesis of neoglycosphingolipid from methoxyamino-functionalized ceramide.

An efficient approach for the synthesis of a methoxyamino-functionalized ceramide was established from phytosphingosine using specific Nß→Nα acyl migration of the octadecanoyl group during the removal of Nα-Fmoc protective group. One step glycoblotting reaction of the ceramide mimic with lactose afforded a neoglycosphingolipid showing potent inhibitory activity against recombinant endoglycoceramidase II from Rhodococcus sp.

Glycosphingolipids: synthesis and functions.

Glycosphingolipids (GSLs) comprise a heterogeneous group of membrane lipids formed by a ceramide backbone covalently linked to a glycan moiety. Hundreds of different glycans can be linked to tens of different ceramide molecules, giving rise to an astonishing variety of structurally different compounds, each of which has the potential for a specific biological function. GSLs have been suggested to modulate membrane-protein function and to contribute to cell-cell communication. Although GSLs are dispensable for cellular life, they are indeed collectively required for the development of multicellular organisms, and are thus considered to be key molecules in 'cell sociology'. Consequently, the GSL make-up of individual cells is highly dynamic and is strictly linked to the cellular developmental and environmental state. In the present review, we discuss some of the available knowledge, open questions and future perspectives relating to the study of GSL biology.

Synthesis of α-O- and α-S-glycosphingolipids related to Sphingomonous cell wall antigens using anomerisation.

Analogues of glycolipids from Spingomonadacaece with O- and S- and SO2-linkages have been prepared using chelation induced anomerisation promoted by TiCl4. Included are examples of the anomerisation of intermediates with O- and S-glycosidic linkages as well as isomerisation of ß-thioglycuronic acids (ß-glycosyl thiols). The ß-O-glucuronide and ß-O-galacturonide precursors were efficiently prepared using benzoylated trichloroacetimidates. ß-Glycosyl thiols were precursors to ß-S-derivatives. Triazole containing mimics of the natural glycolipids were prepared using CuI promoted azide-alkyne cycloaddition reactions in THF. The glycolipid antigens are being evaluated currently for their effects on iNKT cells.

D-Glucosamine-derived synthons for assembly of L-threo-sphingoid bases. Total synthesis of rhizochalinin C.

A five-step transformation of D-glucosamine, commencing with indium-mediated Barbier reaction without isolation of intermediates, into (R,R)-2-aminohex-5-ene-1,3-diol in 45-51% is described. The latter is a useful synthon for assembly of L-threo-sphingoid bases: long-chain aminoalkanols and aminoalkanediols with configurations antipodal to that found in mammalian D-erythro-sphingosine but prevalent among invertebrate-derived sphingolipids. The utility of the method is demonstrated by the first total synthesis of rhizochalinin C, the long-chain, "two-headed" sphingoid base aglycon of the natural product rhizochalin C from the marine sponge Rhizochalina incrustata.

Specific synthesis of neurostatin and gangliosides O-acetylated in the outer sialic acids using a sialate transferase.

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides' outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.