René Roy

Syntheses and some applications of chemically defined multivalent glycoconjugates.

Classical multivalent neoglycoproteins have been widely used to study a great number of carbohydrate-protein interactions. The synthesis of other neoglycoconjugates with various shapes, valencies, and conformations has reached considerable levels of sophistication and holds promise as a new tool for glycobiology and biomedical applications. Within the last few years, advances have been made towards both the syntheses and understanding of the antigenic properties of water-soluble glycopolymers. Some of these glycopolymers are finding applications as inhibitors of microbial adhesins and as carriers for drug delivery to specific cells. Novel dendritic carbohydrate structures are emerging as potent ligands for carbohydrate-binding proteins.

Inhibition of adhesion of type 1 fimbriated Escherichia coli to highly mannosylated ligands.

The inhibitory potencies of a number of mannosides, di‐ and trivalent mannosides, a set of mannose‐terminating dendrimers, and five types of mannose‐bearing neoglycoproteins were determined by using a binding assay that measures the binding of 125I‐labeled, highly mannosylated neoglycoprotein to a type 1 fimbriated Escherichia coli (K12) strain in suspension. The IC50 values (the concentration of inhibitor that causes 50u2009% reduction in the bound 125I‐ligand to E. coli) obtained by this method were much lower than the equivalent values obtained by hemagglutination or in assays that involve microplate immobilization. Two important factors that strongly influence the affinity to E. coli adhesin are: 1) the presence of an α‐oriented aglycon that has a long aliphatic chain or an aromatic group immediately next to the glycosyl oxygen, and 2) the presence of multiple mannosyl residues that can span a distance of 20 nm or longer on a relatively inflexible structure. The two best inhibitors, which are a highly mannosylated neoglycoprotein with the longest linking arm between a mannose and protein amino group and the largest mannosylated dendrimer (fourth generation), exhibited sub‐nM IC50 values.

Amphiphilic p-tert-Butylcalix[4]arene Scaffolds Containing Exposed Carbohydrate Dendrons

The best of both worlds. Amphiphilic hybrid molecules, in which water-exposed glycodendrons are attached to a hydrophobic p-tert-butylcalix[4]arene scaffold, display strong affinities for both carbohydrate-binding proteins and polystyrene surfaces (shown schematically). The molecules can form monolayers useful in bioanalytical devices.

Macromolecular recognition: effect of multivalency in the inhibition of binding of yeast mannan to concanavalin A and pea lectins by mannosylated dendrimers.

The synthesis and binding properties of a new family of high affinity alpha-D-mannopyranoside ligands are described. The synthesis of the new multivalent ligands is based on the scaffolding of multiantennary branches of L-lysine residues having electrophilic N-chloroacetylated end groups as core structures. An alpha-D-mannopyranoside with p-substituted aryl aglycon ending with a thiol group was prepared and covalently attached to each of the branches of the dendritic structures. The resulting glycodendrimers with 2 (12), 4 (14), 8 (16), and 16 (18) mannoside residues were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl and p-nitrophenyl alpha-D-mannopyranosides as standards. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohemagglutinin (Canavalia ensiformis, concanavalin A) and to pea lectin (Pisum sativum) were determined. Analogous mannosylated copolyacrylamides were also prepared for comparison. The IC50 values were also plotted as a function of dendrimer valencies. The inhibitions showed 16-mer 18 to be approximately 600- and 2000-fold more potent than methyl alpha-D-mannopyranoside, and 66- and 1383-fold more potent than p-nitrophenyl alpha-D-mannopyranosides with Con A and pea lectins, respectively. Even when these numbers are expressed relative to single mannopyranoside residues per dendrimers, the relative potencies against the aromatic mannoside are still 4- and 86-fold better against Con A and pea lectins. These results unequivocally indicate that the optimum inhibitory binding properties of the new mannosylated dendrimers vary with both dendrimers and lectin valencies.

Glycodendrimers: novel glycotope isosteres unmasking sugar coding. case study with T-antigen markers from breast cancer MUC1 glycoprotein.

Glycodendrimers are relatively novel synthetic biomacromolecules that are made of biologically relevant carbohydrate ligands constructed at the periphery of a wide range of highly functionalized and repetitive scaffolds having varied molecular weights and structures. They were aimed to fill the gap between glycopolymers, having generally dispersed higher molecular weight, and small glycoclusters, in the study of multivalent carbohydrate protein interactions. In a way, glycodendrimers, with their spheroidal or dendritic (wedge) type structures, were initially designed as bioisosteres of cell surface multiantennary glycans. Taken as a curiosity and elegant molecules at their beginning, they are now considered as potent inhibitors of microbial adhesins. They have also been shown to play some roles in signal transduction and in receptor cross-linking. This brief report will describe advances that have been made toward the syntheses of a range of glycodendrimers bearing the immunodominant T-antigen disaccharide [beta-D-Gal-(1-3)-alpha-D-GalNAc] found on malignant cells of carcinomas, particularly related to breast cancer. This antigen, usually cryptic on healthy tissues, is greatly increased on cancer cells as a result of aberrant glycosylation. It is considered to be an important cancer marker. The high incidence of these carcinomas to invade other tissues such as lymph nodes, lung, and liver by metastasis was one of the arguments raised to generate T-antigen dendrimers that might have the potential to block the receptor sites following surgery. The synthesis of the T-antigen disaccharide will be briefly described, followed by the elaboration of neoglycoproteins and glycopolymers used to raise monoclonal antibodies against the T-antigen and for screening purpose, respectively. Scaffolds made of poly(amidoamine) (PAMAM), poly(propylene imine), N,N-bis(acrylamido)acetic acid, and finally hyperbranched L-lysine were used to construct relatively small glycodendrimers bearing T-antigen moieties. Few glycodendrimers were also linked to fluorescein and biotin probes to generate ligands that can be used to detect T-Ag receptor sites.

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and capillary liquid chromatography-electrospray mass spectrometry (cLC-ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC-ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man8GlcNAc2) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC-PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.

“Active” and “latent” thioglycosyl donors in oligosaccharide synthesis. Application to the synthesis of α-sialosides

Abstract The use of a new “active” and “latent” thioglycosyl donor strategy in glycoside synthesis is described using arylthio α-sialosides of different reactivities.

Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins

Clusters of di-, tri-, and tetra-antennary α-D-mannopyranosides were synthesized in good yields based on the coupling of amine-bearing mono- or trisaccharide {Man α(1→6)[Man α(1→3)]Man} haptens to poly-isocyanate or -isothiocyanate tethering cores. The relative binding properties of the resulting multivalent ligands were determined by turbidimetric and solid phase enzyme-linked lectin assays (ELLA) using plant lectins (phytohemagglutinins) Concanavalin A (Con A) and Pisum sativum (pea lectin) having four and two carbohydrate binding sites, respectively. Rapid and efficient cross-linking between tetravalent Con A and mannopyranosylated clusters were measured by a microtiter plate version of turbidimetric analyses. In inhibition of binding of the lectins to yeast mannan, the best tetravalent monosaccharide (30) and trisaccharide (31) inhibitors were shown to be 140 and 1155 times more potent inhibitors than monomeric methyl α-D-mannopyranoside against pea lectin and Con A, respectively. Compounds 30 and 31 were thus 35 and 289 fold more potent than the reference monosaccharide based on their hapten contents. As a general observation, the ligands bearing the Man α(1→6)[Man α(1→3)]Man trimannoside structures were found to be more potent inhibitors for Con A than the ligands having single mannoside residues, whereas pea lectin could not discriminate between the two types of ligands.

Synthesis of novel dendritic glycosides

Abstract Dendritic thiolated glycosides, with and without a spacer moiety, exhibiting valencies of two, four and eight were synthesized using L-lysine as a divalent core structure via solid-phase chemistry.

Polysialosides scaffolded on p-Tert-butylcalix[4]arene

Abstract p - Tert -butylcalix[4]arene ( 1 ) was transformed into known tetraethyl ester 2 to provide cone-shaped calix[4]arene used for the scaffolding of tetravalent α-sialoside. Ester 2 was transformed into acid chloride 4 which after treatment with mono-Boc-1,4-butanediamine, trifluoroacetolysis and N-chloroacetylation afforded tetraamine 7 . Conjugation of α-thiosialoside 9 by nucleophilic substitution and deprotection furnished water-soluble tetravalent α-thiosialoside 12 which bound strongly to the plant lectin wheat germ agglutinin.