Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides.

The 2-bromoethyl beta-glycosides of the disaccharide galabiose [Gal(alpha1-4)Gal] and the trisaccharides globotriose [Gal(alpha1-4)Gal(beta1-4)Glc] and 3'-sialyllactose [Neu5Ac(alpha2-3)Gal(beta1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52-95% yields after purification by HPLC. 1H NMR spectroscopy revealed that beta-elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.

Carbohydrate specificity of the Escherichia coli P-pilus papG protein is mediated by its N-terminal part.

The adherence of pyelonephritic Escherichia coli isolates to mammalian host cells is mediated by the P-pili structures on the bacterial surface. The protein constituting the distal part of the pili structure, papG, interacts with glycan receptors on the host cell. Variation in specificity for different glycoconjugates between the isolates, that may reflect variation in host tropism, has been correlated to three different classes of papG. Truncated variants of the class I, II and III papG adhesins were produced as fusion protein in E. coli and analysed for carbohydrate binding. The results showed that both carbohydrate binding and specificity of the papG adhesin resided in a linear part of the N-terminus of the protein.

The use of human milk fucosyltransferase in the synthesis of tumor-associated trimeric X determinants.

We have studied the fucosylation of a chemically synthesized trimer of N-acetyllactosamine [(LacNAc)3-EtPhNHCOCF3] with a fucosyltransferase preparation from normal human milk, which utilizes both type-1 and type-2 structures, whether sialylated or not. When fucose residues were added enzymically to the (LacNAc)3-EtPhNHCOCF3 hexasaccharide, mono-, di-, or trifucosylated oligosaccharide species were formed, containing the Lewisx determinant (Gal beta 1-->4[Fuc alpha 1-->3]Glc-NAc beta 1-->3). With excess GDP-fucose and prolonged reaction times, the trifucosylated product was formed in almost quantitative yield. Kinetic analysis of the fucosylation reaction indicated that there is a significant difference in the rate of transfer of the first, second and third fucose residues onto the acceptor molecule. The location of the fucose residues in the monofucosylated and difucosylated intermediate products was assessed by analyzing the digests obtained after endo-beta-galactosidase treatment by HPLC and reverse-phase chromatography. In addition, the fucosylated (LacNAc)3-EtPhNHCOCF3 structures were characterized by HPLC and were identified by 400-MHz 1H-NMR spectroscopy. There is a highly preferred order in which the fucosyl residues are attached to (LacN-Ac)3-EtPhNHCOCF3. In the major pathway, the first two fucose residues are transferred with equal preference to the medial (GN3) and proximal (GN1) GlcNAc residues, whereas the third fucose is attached to the distal (GN5) GlcNAc residue. These results are of relevance in understanding the role of alpha-3-fucosyltransferase in the biosynthesis of Lewisx-related cell-surface carbohydrate structures, that function as ligands for selectin-type cell-adhesion molecules and may play a role in the invasion and metastasis of several carcinoma.

Synthesis of the trisaccharide, 2-(p-trifluoroacetamidophenyl)ethyl O-alpha-D-galactopyranosyl-(1-4)-O-beta-D-galactopyranosyl-(1-4)-2-acet amido-2-deoxy-beta-D-glucopyranoside, corresponding to the blood group P1 determinant.

The 2-(p-trifluoroacetamidophenyl)ethyl beta-glycoside of the P1 antigen trisaccharide, O-alpha-D-galactopyranosyl-(1-4)-O-beta-D-galactopyranosyl-(1-4)-2- acetamido-2-deoxy-D-glucopyranose, was synthesized. Thioglycoside intermediates were used as building blocks.

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy.

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of an H type 2 and a Y (Le(y)) glycoside from thioglycoside intermediates.

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2- deoxy-4-O-[2-O-(alpha-L-fucopyranosyl)-beta-D-galactopyranosyl]-beta-D- glucopyranoside 1 and the tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-3-O-(alpha-L- fucopyranosyl)-4-O-[2-O-(alpha-L-fucopyranosyl)-beta-D-galactopyranosyl]- beta-D-glucopyranoside 2 were synthesized. Thioglycosides, suitably protected, activated directly with methyl trifluoromethanesulfonate or dimethyl(methylthio)sulfonium tetrafluoroborate or activated after bromine treatment with halophilic reagents, were used as glycosyl donors in the construction of the glycosidic linkages.

Synthesis of Gal beta 1-3GlcNAc and Gal beta 1-3GlcNAc beta-SEt by an enzymatic method comprising the sequential use of beta-galactosidases from bovine testes and Escherichia coli.

Gal beta 1-3GlcNAc (1) and Gal beta 1-3GlcNAc beta-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes beta-galactosidase with lactose as donor and N-acetylglucosamine and GlcNAc beta-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with beta-galactosidase from Escherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to greater than 95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal beta 1-6GlcNAc beta-SEt (3) was synthesized by the transglycosylation reaction using beta-galactosidase from Escherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.

Synthesis of a tri- and a hepta-saccharide which contain alpha-L-fucopyranosyl groups and are part of the complex type of carbohydrate moiety of glycoproteins.

Reaction of a thioglycoside with methyl trifluoromethanesulfonate (methyl triflate) in the presence of a hydroxyl compound is an efficient glycosylation method. Thus, methyl triflate-promoted condensation of ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-3-O-(2,3,4-tri-O-benzyl-a lpha-L- fucopyranosyl)-beta-D-glucopyranoside with benzyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside and with benzyl 2,4-di-O-benzyl-3,6-di-O-(3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl)-alp ha-D- mannopyranoside gave a trisaccharide and a heptasaccharide derivative, respectively. The trisaccharide 1 and the heptasaccharide 2, which are parts of the complex type of glycoproteins, were obtained by removal of the protecting groups and N-acetylation. (formula: see text).

Synthesis of a tetra- and a nona-saccharide which contain alpha-L-fucopyranosyl groups and are part of the complex type of carbohydrate moiety of glycoproteins.

Methyl trifluoromethanesulfonate-promoted condensation of ethyl 6-O-benzyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-3-O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)- beta-D- glucopyranoside with benzyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside and benzyl 2,4-di-O-benzyl-3,6-di-O-(3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl)-alp ha-D- mannopyranoside gave a tetrasaccharide and a nonasaccharide derivative, respectively. The tetrasaccharide 1 and the nonasaccharide 2 were obtained after removal of the protecting groups and N-acetylation. (formula: see text).

Synthesis of a disaccharide component of the capsular polysaccharide antigen of Streptococcus pneumoniae type 1.

Methyl 2-acetamido-4-amino-2,4,6-trideoxy-3-O-(alpha-D-galactopyranosyl-uronic acid)-alpha-D-galactopyranoside has been synthesised. The parent disaccharide is a structural element of the capsular polysaccharide antigen of Streptococcus pneumoniae type 1.

Synthesis of a nona- and an undeca-saccharide that form part of the complex type of carbohydrate moiety of glycoproteins.

Silver trifluoromethanesulfonate-promoted condensation of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-beta-D-glucopyranosyl bromide with benzyl 2,4,-di-O-benzyl-6-O-(3,4-di-O-benzyl-alpha-D-mannopyranosyl)-3-O-(3,6-di-O- benzyl-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside gave nonasaccharide and undecasaccharide derivatives. The nonasaccharide 2 and the undecasaccharide 3 were obtained by removal of the protecting groups followed by N-acetylation. (formula; see text).

Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features.

A series of synthetic oligosaccharides, resembling natural N-acetyllactosamine type glycans, were tested for their ability to inhibit the binding of labeled ligand to the mammalian hepatic lectin on rabbit hepatocytes at 2 degrees C. A dramatic hierarchy of inhibitory potency (tetraantennary greater than triantennary much greater than biantennary much greater than monoantennary) could be demonstrated. The range of concentration required for 50% inhibition of labeled ligand binding extended from approximately 1 mM, for the monoantennary oligosaccharides, to approximately 1 nM for triantennary oligosaccharides, even though the absolute Gal concentration increased only 3-fold. It was found that the number of Gal residues/cluster and their branching mode are major determinants of binding affinity of ligands to the hepatic lectin on the surface of hepatocytes.

Synthesis of oligosaccharides that form parts of the complex type of carbohydrate moieties of glycoproteins. Three glycosides prepared for conjugation to proteins.

Condensation of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-2-deoxy-2- phthalimido-beta-D-glucopyranosyl bromide with p-nitrophenyl 3-O-benzoyl-4,6-di-O-benzylidene-alpha-D-mannopyranoside, p-nitrophenyl 3,6-di-O-benzyl-alpha-D-mannopyranoside and p-nitrophenyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave protected tri- and pentasaccharides. The oligosaccharide glycosides 1, 2 and 3 were obtained after de-protection of these condensation products. These oligosaccharides will, after suitable conversions, be conjugated to proteins for biological studies.