Total synthesis of dansyl and biotin functionalized ganglioside GM3 by chemoenzymatic method

Ganglioside GM3, as well as other gangliosides, offers a variety of modifications in its sialic acid and ceramide moieties GM3 exhibits various types of important biological activities, due to the inability to effectively observe the trafficking of ganglioside GM3, developing sensitive research tools for specific monitoring of GM3 expression and activity is thus desirable. The total synthesis of a dansyl and biotin bifunctionalized fluorescent ganglioside GM3 were reported in this article. From lactose after 13 reaction steps, the compound of 2′ -biotinoylaminoethyl-6-N-dansylamido-6-deoxy-β-D-galatopyranosyl-(1→4)-β-D-glucopy-ranoside was obtained in total yield of 16.2%. Sialylation of dansyl and biotin functionalized lactose by enzymatic method gave dansyl and biotin labeled ganglioside GM3. The fluorescent property of this compound was also investigated.     

Chemical and chemoenzymatic synthesis of S-linked ganglioside analogues and their protein conjugates for use as immunogens

Analogues of the tumor-associated gangliosides GM(3) and GM(2) containing terminal S-linked neuraminic acid residues and an amino terminated, truncated ceramide homologue have been synthesized and conjugated to a protein. The synthesis involved coupling of a S-linked sialyl alpha(2-->3) galactose disaccharide with a glucosyl sphingosine analogue, followed by elaboration and deprotection to give amino-terminated glycosyl ceramide 1. Glycosyltransferase-catalyzed extension of the trisaccharide 1 provided access to the modified GM(2) tetrasaccharide 2 or sulphur-containing GD(3) analogue 30. Owing to their potentially enhanced resistance to endogenous exo-glycoside hydrolases and their inherent non-self character, carbohydrate antigens containing non-reducing terminal thioglycosidic linkages may be more immunogenic than O-linked antigens and may stimulate the production of antibodies capable of recognizing naturally occurring oligosaccharides. Our initial results suggest that in fact these antigens are viable immunogens and furthermore, that immune sera cross reacts with O-gangliosides in the context of a heterologous glycoprotein conjugate.     

Chemoenzymatic synthesis of biotin-appended analogues of gangliosides GM2, GM1, GD1a and GalNAc-GD1a for solid-phase applications and improved ELISA tests

Biotinylated analogues of gangliosides GM2, GM1, GD1a and GalNAc-GD1a were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. The presence of a biotin moiety in the aglycone part of these mimics allows for attachment of these materials onto various streptavidin-coated surfaces. Analysis of the interaction of biotin-appended GM1 with the B subunit of Escherichia coli heat-labile enterotoxin performed in a modified ELISA procedure shows the potential of this compound to replace the natural GM1 in toxin detection.     

Isolation and structure of monomethylated GM3-type ganglioside molecular species from the starfish Luidia maculata

Two monomethylated GM(3)-Type ganglioside molecular species, 1 and 2, have been obtained from the polar lipid fraction of the chloroform/methanol extract of the starfish Luidia maculata. The structures of these gangliosides have been determined on the basis of chemical and spectroscopic evidence as 1-O-[8-O-methyl-(N-acetyl-alpha-D-neuraminosyl)-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranosyl]-ceramide (1) and 1-O-[8-O-methyl-(N-glycolyl-alpha-D-neuraminosyl)-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranosyl]-ceramide (2). The ceramide moieties were composed of heterogeneous unsubstituted fatty acid, 2-hydroxy fatty acid, sphingosine and phytosphingosine units. Compound 1, designated as LMG-3, represents new ganglioside molecular species. Compound 2 was a known ganglioside molecular species.     

Biosynthesis of conjugatable saccharidic moieties of GM2 and GM3 gangliosides by engineered E. coli

Oligosaccharidic moieties of GM(2) and GM(3) gangliosides bearing an allyl or a propargyl aglycon, are efficiently biosynthesized on the gram scale by growing metabolically engineered Escherichia coli cells in the presence of the corresponding lactoside acceptors and sialic acid.     

New Insights on Non-Enzymatic Oxidation of Ganglioside GM1 Using Mass Spectrometry

Gangliosides are acidic glycosphingolipids that are present in cell membranes and lipid raft domains, being particularly abundant in central nervous systems. They participate in modulating cell membrane properties, cell–cell recognition, cell regulation, and signaling. Disturbance in ganglioside metabolism has been correlated with the development of diseases, such as neurodegenerative diseases, and in inflammation. Both conditions are associated with an increased production of reactive oxidation species (ROS) that can induce changes in the structure of biomolecules, including lipids, leading to the loss or modification of their function. Oxidized phospholipids are usually involved in chronic diseases and inflammation. However, knowledge regarding oxidation of gangliosides is scarce. In order to evaluate the effect of ROS in gangliosides, an in vitro biomimetic model system was used to study the susceptibility of GM1 (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer) to undergo oxidative modifications. Oxidation of GM1 under Fenton reaction conditions was monitored using high resolution electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Upon oxidation, GM1 underwent oxidative cleavages in the carbohydrate chain, leading to the formation of other gangliosides GM2 (GalNAcβ1-4Gal(Neu5Acα2-3)1-4Glcβ1Cer), GM3 (Neu5Acα2-3Galβ1-4Glcβ1Cer), asialo-GM1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer), asialo-GM2 (GalNAcβ1-4Galβ1-4Glcβ1Cer), of the small glycolipids lactosylceramide (LacCer), glucosylceramide (GlcCer), and of ceramide (Cer). In addition, oxygenated GM1 and GM2 (as keto and hydroxy derivatives), glycans, oxidized glycans, and oxidized ceramides were also identified. Nonenzymatic oxidation of GM1 under oxidative stress contributes to the generation of other gangliosides that may participate in the imbalance of gangliosides metabolism in vivo, through uncontrolled enzymatic pathways and, consequently, play some role in neurodegenerative processes.

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Synthesis of N-modified ganglioside GM3 derivatives

Ganglioside GM3 and its derivatives have many important biological functions. Using diethyl phosphite protected sialic acid as glycosyl donor and 3,2′,3′,4′-unprotected lactose as glycosyl acceptor, the sialic acid-containing trisaccharide was assembled with excellent anomeric stereoselectivity. The trisaccharide was further coupled with ceramide precursor to yield sphingosine 1. Based on this key intermediate, two different series of N-modified GM3 analogues with modifications on either the nitrogen of sialic acid residue or the nitrogen of ceramide moiety and GM3 itself were synthesized smoothly.     

Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.     

Gangliosides and sulphatide in human cerebrospinal fluid: quantitation with immunoaffinity techniques

A sensitive micromethod involving extraction, purification and thin-layer chromatography (TLC)-enzyme immunostaining was developed for the quantation of gangliosides and sulphatide, as markers for neuronal disorders and myelin disturbances, in individual samples of less than 5 ml of cerebrospinal fluid. The gangliosides of the gangliotetraose series were individually determined with cholera toxin subunit B by TLC-enzyme-linked immunosorbent assay (ELISA) after chromatography and subsequent sialidase hydrolysis to II3NeuAc-GgOse4Cer (GM1). Other gangliosides and sulphatide were determined with specific monoclonal antibodies by TLC-ELISA. The total ganglioside content varied between 100 and 230 nmol/l in ten normal cerebrospinal fluid samples from adults. The major gangliosides were of the gangliotetraose series, represented by GM1, IV3NeuAc,II3NeuAc-GgOse4Cer, (GD1a), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3 (NeuAc)2-GgOse4Cer (GT1b) of which the b-series gangliosides dominated, i.e., GD1b and GT1b.     

Evaluation and evidence of natural gangliosides with two unsaturated bonds in the ceramide structure obtained by a combination of MALDI-MS and NMR spectroscopy

Gangliosides are membrane-associated glycosphingolipids. N-Acetyl GM3 and N-glycolyl GM3 are two tumor-associated antigens expressed in cancer tissues such as melanoma and mammalian cancer. In order to use these antigens in GM3-based vaccines for patients with early stage cancer, the synthetic version is recommended to avoid the risk of animal virus transmission from the source. However, the isolation of natural gangliosides is of comparative value for the structural characterization. The structures of N-acetyl and N-glycolyl GM3 extracted from dog and horse erythrocytes were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance techniques; additionally, the natural N-acetyl ganglioside was compared to a synthetic one. In addition to the main compound with C24:0 fatty acid chain, a minor component with an additional unsaturation in the ceramide chain was detected, in both the dog and the horse gangliosides. This paper shows spectroscopic evidence of the aforementioned compounds.     

Contribution to the total synthesis of caribenolide I

Stereoselective synthesis of C₁₃-C₂₉ fragment of caribenolide I, a potent antitumour macrolide isolated from a marine dinoflagellate Amphidinium sp. is described. The key step relies on a highly diastereoselective C-glycosylation, using a bulky chiral oxazolidin-2-thione, to control the absolute configuration of C₂₁. The C₂₄ and C₂₅ stereogenic centres were controlled by the enantioselective vinylogous Mukayiama aldol reaction, whereas C₁₄ and C₁₆ stereogenic centres were built from a chiral bis-epoxide.     

A liquid chromatography/tandem mass spectrometric approach for the determination of gangliosides GD3 and GM3 in bovine milk and infant formulae

A liquid chromatographic/tandem mass spectrometric method using pneumatically assisted electrospray ionisation (LC/ESI-MS/MS) was developed for the determination of gangliosides GD3 and GM3 in milk and infant formulae. The gangliosides were extracted in a chloroform/methanol/water environment and cleaned up by solid-phase extraction (SPE) on an end-capped C8 sorbent. The gangliosides were detected in negative ion mode after separation on a reversed-phase (RP) C5 analytical column. From the different ganglioside molecular species, product ions at m/z 290 corresponding to an N-acetylneuraminic acid fragment were produced in the collision cell and used in selected reaction monitoring. A standard addition technique was applied for quantification. The relative repeatability standard deviations were less than 5% for GD3 (level 10 mg/L) and 14% for GM3 (level 0.1-0.2 mg/L).     

Enantioselective synthesis of decarestrictine J

An efficient total synthesis of decarestrictine J has been achieved using ring-closing metathesis and Yamaguchi esterification as key steps. The stereogenic centres were generated by means of iterative hydrolytic kinetic resolution (HKR) of racemic epoxides.     

Chip-nanoelectrospray quadrupole time-of-flight tandem mass spectrometry of meningioma gangliosides: a preliminary study

A strategy combining high-performance thin layer chromatography (HPTLC), laser densitometry, and fully automated chip-based nanoelectrospray (nanoESIchip) performed on a NanoMate robot coupled to QTOF-MS was developed, optimized, and for the first time applied for mapping and structural identification of gangliosides (GGs) extracted and purified from a human angioblastic meningioma specimen. While HPTLC pattern indicated only seven fractions migrating as GM3, GM2, GM1, GD3, GD1a (nLD1, LD1), GD1b, GT1b, and possibly GD2, due to the high sensitivity, mass accuracy, and ability to ionize minor species in complex mixtures, nanoESIchip-QTOF MS was able to discover significantly more GG species than ever reported in meningioma. Thirty-four distinct glycosphingolipid components of which five asialo, one GM4, nine GM3, two GM2, two GD3, nine GM1, and six GD1 differing in their ceramide compositions were identified. All structures presented long-chain bases with 18 carbon atoms, while the length of the fatty acid was found to vary from C11 to C25. MS screening results indicated also that the diversity of the expressed GM1 structures is higher than expected in view of the low proportions evidenced by densitometric quantification. Simultaneous fragmentation of meningioma-associated GM1 (d18:1/24:1) and GM1 (d18:1/24:0) by MS/MS using CID confirmed the postulated structures of the ceramide moieties and provided data on the glycan core, which document that for each of the GM1 (d18:1/24:1) and GM1 (d18:1/24:0) forms both GM1a and GM1b isomers are expressed in the investigated meningioma tissue.     

A formal total synthesis of crocacin C

An efficient total synthesis of a potent antifungal and moderate cytotoxic agent crocacin C is described. The synthesis involves the generation of four contiguous stereogenic centres via desymmetrization of a meso bicyclic dihydrofuran using asymmetric hydroboration.     

Antibody synthesis induced by endogenous internal images

In this study, immunization with a vaccine consisting of multiple F(abt’)₂ fragments of affinity-purified antitetanus toxoid antibodies covalently bound to a carrier protein successfully induced antitetanus toxoid antibodies. Further studies showed that this vaccine preparation contained no biologically detectable tetanus antigen. The induced antitetanus antibody (Ab1t’) titer was higher than the titer of antibodies binding control antigens. The immunizing F(abt’)₂ preparation did not elicit a secondary antitetanus response from mice primed with tetanus toxoid and, hence, appeared free of tetanus epitopes. The specificity of Ab1t’ was established by absorption and inhibition with antigen. Immunization with antitetanus F(abt’)₂ (Ab1t’) fragments appears to have elicited naturally occurring autologous antitetanus toxoid antibody (Ab1t’) through an idiotypic pathway. As predicted by network theory, anti-idiotype (Ab2) and antitetanus (Ab1t’) cycled reciprocally. Clonotypic characterization of Ab1t’ using isoelectric focusing and affinity immunoblotting showed increases in Ab1t’ titer to be the result of increased synthesis by limited subsets of antitetanus toxoid B-cell clones and not increased synthesis by multiple clones, as is characteristic of antigen-driven Ab1 responses. Many Ab1 and Ab1t’ clonotypes had identical pIs, suggesting that they either share V region genes or are the product of the same B-cell clones. These findings indicate that immunization with polyclonal multivalent Ab1 preparations can trigger active synthesis of antibodies with the same specificity. The results provide further evidence for naturally occurring idiotypic cascades that could be exploited for studies of catalytic antibodies.     

Stereocontrolled synthesis and alkylation of cyclic beta-amino esters: asymmetric synthesis of a (-)-sparteine surrogate

A convenient method for the stereoselective synthesis of cyclic beta-amino esters from an iodo alphabeta-unsaturated ester and alpha-methylbenzylamine is described. Subsequent enolate generation and alkylation proceeds with complete stereocontrol, with the two stereogenic centres working together. In this way, a functionalised piperidine suitable for alkaloid natural product synthesis was prepared. The usefulness of the methodology is exemplified with the concise synthesis of a (-)-sparteine surrogate.     

A first total synthesis of a hybrid-type ganglioside associated with amyotrophic lateral sclerosis-like disorder

The hybrid ganglioside X1, which was identified in the bovine brain, was synthesized for the first time. Ganglioside X1 is believed to be involved in the development of amyotrophic lateral sclerosis-like disorders in patients with neurological disorders after treatment with bovine brain gangliosides. A convergent approach using two branched glycan units, the GM2-core trisaccharide and the lacto-ganglio tetrasaccharide, efficiently provided the highly branched heptasaccharide part of ganglioside X1, which was conjugated with the ceramide part to produce the protected ganglioside X1. Global deprotection delivered homogenous ganglioside X1, with which serum from the patient was reacted.     

A metal-oxo mediated approach to the synthesis of 21,22-diepi-membrarollin

A synthesis of 21,22-diepi-membrarollin (14) is described, which employed sequential metal-oxo mediated oxidative cyclisations to introduce six of the seven stereogenic centres.     

Total synthesis of (−)-baconipyrone C

A highly stereoselective asymmetric total synthesis of marine polypropionate (−)-baconipyrone C has been achieved. Utilization of desymmetrization technique to create five stereogenic centres, Sharpless epoxidation, Gilman's reaction and resolution of methyl group using enzyme PS-C is the highlight of the synthesis.