A Fucosylated Lactose-Presenting Tetravalent Glycocluster Acting as a Mutual Ligand of Lectins A (PA-IL) and B (PA-IIL)-Synthesis and Interaction Studies.

The Gram-negative bacterium is an important opportunistic human pathogen associated with cystic fibrosis. produces two soluble lectins, the d-galactose-specific lectin PA-IL (LecA) and the l-fucose-specific lectin PA-IIL (LecB), among other virulence factors. These lectins play an important role in the adhesion to host cells and biofilm formation.

Glycan microarrays: new angles and new strategies.

Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on solid surfaces in a spatially discrete pattern, are sensitive and versatile tools for the analysis of glycosylation changes in complex biological samples. Glycoarrays are also suitable for monitoring multiple molecular interactions with biomolecules where sugars are involved, offering a large variety of bioassay options. In this paper we review the most important glycan microarray types currently used with their main applications, and discuss some of the future challenges the technology faces.

Neoglycoproteins as carbohydrate antigens: synthesis, analysis, and polyclonal antibody response.

The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization.

Synthesis of N-glycopeptides applying glycoamino Acid building blocks with a combined fmoc/boc strategy.

Mono-, di- and trisaccharide representing the reducing terminal of the core structure of N-glycans were incorporated into Leu-Lys-Asn-Gly-Gly-Pro hexapeptide that is a partial structure of the Trp-cage mini-protein by linear assembly. These studies provide evidence that the used combination of Fmoc and Boc strategy and mild conditions result in glycopeptides in high purity and reasonable yield.

Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans.

N-Acetyl-D-glucosamine, chito-oligomers and carbon starvation regulated chiA, chiB, and nagA gene expressions in Aspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and the N-acetyl-beta-D-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-related chiA or the autolysis-coupled chiB genes although both of them were down-regulated under diamide stress.

Dominant antibody responses to Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc containing carbohydrate epitopes in Pan troglodytes vaccinated and infected with Schistosoma mansoni.

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcbeta1-4GlcNAc, GalNAcbeta1-4(Fucalpha1-3)GlcNAc, and Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)).

Synthesis of fragments of the glycocalyx glycan of the parasite Schistosoma mansoni.

The chemical synthesis of alpha-L-Fucp-(1 --> 3)-beta-D-GalpNAc-(1 --> 4)-beta-D-GlcpNAc-(1 --> 3)-alpha-D-GalpO(CH2)5NH2, beta-D-GalpNAc-(1 --> 4)-[alpha-L-Fucp-(1 --> 3)-]beta-D-GlcpNAc-(1 --> 3)-alpha-D-GalpO(CH2)5NH2, and alpha-L-Fucp-(1 --> 3)-beta-D-GalpNAc-(1 --> 4)-[alpha-L-Fucp-(1 --> 3)-]beta-D-GlcpNAc-(1 --> 3)-alpha-D-GalpO(CH2)5NH2 is described. These structures represent fucosylated oligosaccharide fragments of the glycocalyx glycan of the cercarial stage of the parasite Schistosoma mansoni, and in protein-conjugated form they are potential diagnostics in the search for antibodies raised against the glycan in the serum of infected humans.

Anomalous Zemplén deacylation reactions of alpha- and beta-D-mannopyranoside derivatives.

Reaction of mono-, di-, and trisaccharide derivatives of methyl beta-D- and octyl beta-D-mannopyranosides bearing ester groups at isolated and non-isolated positions on the same molecule, under Zemplén conditions (catalytic amount of sodium methoxide in methanol) gave partially deacylated compounds, in which the O-acyl groups were retained at isolated sites. In the case of one disaccharide, all the benzoyl groups remained intact at the reducing end, while all the acetyl functions were removable from the nonreducing end. In another case, both isolated ester groups at positions 2 and 4 were retained at the reducing end.

Chemical and chemo-enzymatic synthesis of the alpha-Neu p5Ac-(2-->6)- beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp element that is part of N-linked carbohydrate chains of human lutropin.

In the framework of a project aimed at the elucidation of the nature of the functional importance of the N-glycosylation of the alpha-subunit of the glycoprotein hormones human lutropin and human chorionic gonadotropin, the structural element alpha-Neu p5Ac-(2-->6)-beta-D-GalpNac-(1-->4)- beta-D-GlcpNAc-(1-->2)-alpha-D-Manp, which is part of the carbohydrate chains of human lutropin, has been prepared by chemical and chemo-enzymatic synthesis in the form of its propyl glycoside. Condensation of 4-O- acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-alpha/beta-D-glucopyranosyl trichloroacetimidate with allyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside gave after deacetylation allyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) -(1-->2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranoside. Ethyl 3-O-benzyl-2-deoxy-2-phthalimido-l-thio-beta-D-glucopyranoside was converted into the galacto-derivative ethyl 4,6-di-O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D -galactopyranoside via an oxidation-reduction route, as well as via SN2-type substitution with acetate.

Syntheses of spacer-armed carbohydrate components of the Mycobacterium avium serocomplex serovar 8.

p-Nitrophenyl glycosides of 3-O-Me-beta-D-Glcp-(1-->3)-alpha-L-Rhap, alpha-L-Rhap-(1-->2)-6-deoxy-alpha-L-Talp, and 3-O-Me-beta-D-Glcp-(1-->3)-alpha-L-Rhap-(1-->2)-6-deoxy-alpha-L-++ +Talp have been prepared, related to Mycobacterium avium. Various glycosylation methods have been used for the formation of the interglycosidic linkages. The p-nitrophenyl derivatives were converted into p-isothiocyanatophenyl glycosides, capable of forming neoglycoproteins.

Synthesis of a fucosylated and a non-fucosylated core structure of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-O-(3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-beta-D-glucopyranoside (4) and methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-4-O- (3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-6- O-alpha-L-fucopyranosyl-beta-D-glucopyranoside (5), which represent the invariant hexasaccharide core structure of the xylose-containing glycans of N-glycoproteins and its 6-O- fucosylated derivative. Ethyl 4-O-[3-O-allyl-4-O-benzoyl-6-O-tert-butyldimethylsilyl-2-O- (2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1- thio-beta-D-glucopyranoside (9) was coupled with methyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranoside (11). Desilylation of the resulting tetrasaccharide derivative, followed by condensation with 2,3,4,6- tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7), gave methyl 4-O-(4-O-[3-O-allyl-4- O-benzoyl-6-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-2-O-(2,3,4 -tri-O- acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)- 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside (14).

Chemical synthesis of the pyruvic acetal-containing trisaccharide unit of the species-specific glycopeptidolipid from Mycobacterium avium serovariant 8.

The functionalized, pyruvic acetal-containing haptenic trisaccharide, p-trifluoroacetamidophenyl 6-deoxy-2-O-(3-O-[4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-meth yl- beta-D-glucopyranosyl]-alpha-L-rhamnopyranosyl)-alpha-L-talopyranosid e (19), a component of the glycolipid from Mycobacterium avium serovar 8 was synthesized. For the preparation of the terminal pyruvic acetal-containing unit, benzyl 2-O-benzyl-3-O-methyl-beta-D-glucopyranoside (6) was condensed with methyl 2,2-di(ethylthio)propionate (1) in the presence of SO2Cl2-CF3SO3H catalyst to yield benzyl 2-O-benzyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-methyl-beta -D- glucopyranoside (7S), which was then converted into the suitably substituted glycosyl donor 2-O-acetyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-methyl-alph a-D- glucopyranosyl trichloroacetimidate (11). The disaccharide glycosyl acceptor p-nitrophenyl endo-3,4-O-benzylidene-6-deoxy-2-O-(2,4-di-O-benzyl-alpha-L-rhamnopyrano syl)- alpha-L-talopyranoside (15) was glycosylated with 11 in the presence of trimethyl trifluoromethanesulfonate to furnish the protected trisaccharide p-nitrophenyl 2-O-(3-O-[2-O-acetyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-m ethyl-beta- D-glucopyranosyl]-2,4-di-O-benzyl-alpha-L-rhamnopyranosyl)-endo-3,4- O-benzylidene-6-deoxy-alpha-L-talopyranoside (16).

Synthesis of p-trifluoroacetamidophenyl 6-deoxy-2-O-(3-O-[2-O-methyl-3-O- (2-O-methyl-alpha-D-rhamnopyranosyl)-alpha-L-fucopyranosyl]-alpha-L- rhamnopyranosyl)-alpha-L-talopyranoside: a spacer armed tetrasaccharide glycopeptidolipid antigen of Mycobacterium avium serovar 20.

The synthesis of the title tetrasaccharide glycoside 38 is reported. p-Nitrophenyl endo-3,4-O-benzylidene-6-deoxy-alpha-L-talopyranoside (4), 3-O-acetyl-2,4-di-O-benzyl-alpha-L-rhamnopyranosyl trichloroacetimidate (7), methyl 3-O-acetyl-4-O-benzyl-2-O-methyl-1-thio-beta-L-fucopyranoside (15), 3-O-acetyl-4-O-benzyl-2-O-methyl-alpha-L-fucopyranosyl bromide (16), and ethyl 3-O-acetyl-4-O-benzyl-2-O-methyl-1-thio-alpha-D-rhamnopyranoside (33) were prepared as intermediates. Compound 4 was glycosylated with imidate 7 as well as with methyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-alpha-L-rhamnopyranoside (9), affording the same disaccharide derivative 8.

Synthesis of a selectively protected trisaccharide building block that is part of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of ethyl 4-O-[3-O-allyl-4,6-O-isopropylidene-2-O-(2,3,4-tri-O-acetyl-beta-D- xylopyranosyl)-beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalim ido-1 - thio-beta-D-glucopyranoside (16), a key intermediate in the synthesis of xylose-containing carbohydrate chains from N-glycoproteins. Condensation of ethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D- glucopyranoside (5) with 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide, using silver triflate as a promoter, gave the beta-linked disaccharide derivative 8 (84%). O-Deacetylation of 8 and then isopropylidenation afforded 10, which was converted via oxidation-reduction into ethyl 4-O-(3-O-allyl-4,6-O-isopropylidene-beta-D-mannopyranosyl)-3,6-di-O-benz yl-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (12).

ROESY studies and HSEA calculations on xylose-containing oligosaccharides related to N-glycoproteins.

Conformational studies on beta-D-Xyl-(1----2)-[alpha-D-Man-(1----6)]-beta-D-OMe, beta-D-Xyl-(1----2)-[alpha-D-Man-(1----3)]-beta-D-Man-OMe, and beta-D-Xyl-(1----2)-[alpha-D-Man-(1----3)]-[alpha-D-Man-(1----6)]-beta-D -Man-Ome have been carried out using rotating-frame n.O.e.

Synthesis of four structural elements of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis of the oligosaccharides beta-D-Xylp-(1----2)-beta-D-Manp-OMe (12), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-OMe (17), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)]-beta-D-Manp+ ++-OMe (21), and beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)] [alpha-D-Manp-(1----6)]-beta-D-Manp-OMe (25) is described. Methyl 3-O-benzyl-4,6-O-isopropylidene-beta-D-mannopyranoside (6) was prepared from the corresponding glucoepimer (4) by oxidation, followed by stereoselective reduction. Condensation of 6 with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl bromide in the presence of mercuric cyanide gave a 1:9 mixture of methyl 3-O-benzyl-4,6-O-isopropylidene-2-O-(2,3,4- tri-O-acetyl-alpha- (7a) and -beta-D-xylopyranosyl)-beta-D-mannopyranoside (7), and then 7 was converted into the acetylated disaccharide-glycoside 11.

1H- and 13C-n.m.r. assignments for structural elements of xylose-containing N-linked oligosaccharides, using 1D-and 2D-n.m.r. experiments.

1H-N.m.r.

Synthesis of the tetrasaccharide core region of antigenic lipo-oligosaccharides characteristic of Mycobacterium kansasii.

The oligosaccharide core region, beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-alpha-D-Glcp- 1----1)-alpha-D-Glcp (1), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2',3',4',6'-hexa-O-benzyl-6-O-(1-phenylethyl)-alpha, alpha-trehalose (4). Compound 4 was obtained by LiAlH4-AlCl3-type hydrogenolysis of 2,3,2',3',4',6'-hexa-O-benzyl-4,6-O-(S)-(1-phenylethylidene)-alpha , alpha-trehalose.

Glycosylated trehalose. Synthesis of the oligosaccharides of the glycolipid-type antigens from Mycobacterium smegmatis.

The oligosaccharide components, 3-O-Me-beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-beta-D-Glcp-(1----6)- alpha-D-Glcp-(1----1)-alpha-D-Glcp (1) and beta-D-Glcp-(1----4)-beta-D-Glcp-(1----6)-alpha-D-Glcp-(1----1)-alpha-D- Glcp, of the glycolipid-type antigens isolated from M. smegmatis have been synthesised from 2,3,4,2',3',4',6'-hepta-O-acetyl-alpha,alpha-trehalose and the appropriate glycosyl bromides under Helferich conditions with Hg(CN)2 as the promoter. Condensation of the trisaccharide glycosyl bromide 27 gave an orthoester derivative (28) which could be rearranged, using HgBr2 or boron trifluoride etherate, into the acetylated derivative (29) of 1.

Synthesis of the tetrasaccharide repeating-unit of the lipopolysaccharide isolated from Pseudomonas maltophilia.

The title tetrasaccharide having the structure 3-O-Me-beta-L-Xylp-(1----4)- alpha-L-Rhap-(1----4)-alpha-L-Rhap-(1----2)-L-Rhap was obtained by reaction of the alpha-acetobromo derivative of 4-O-(3-O-methyl-beta-L-xylopyranosyl)-L-rhamnopyranose and benzyl 3,4-di-O-benzyl-2-O-(2,3-O-isopropylidene-alpha-L-rhamnopyranosyl)- alpha-L-rhamnopyranoside, followed by removal of the protecting groups. The synthesised compounds were characterised on the basis of n.m.

Intracellular hydrolysis of EGTA-esters.

It has been established that the hydrolysis of EGTA-acetoxymethylester (AME) by red blood cells is about the tenth of the hydrolysis of acetylthiocholine (Ac-S-Ch). This splitting of AME could be inhibited by about 50% by prostigmine at a concentration of 0.75 X 10(-5) mol/l, while the splitting of Ac-S-Ch was totally inhibited by the same prostigmine concentration.