L. M. Likhosherstov

Identifying human milk glycans that inhibit norovirus binding using surface plasmon resonance

Human milk glycans inhibit binding between norovirus and its host glycan receptor; such competitive inhibition by human milk glycans is associated with a reduced risk of infection. The relationship between the presence of specific structural motifs in the human milk glycan and its ability to inhibit binding by specific norovirus strains requires facile, accurate and miniaturized-binding assays. Toward this end, a high-throughput biosensor platform was developed based on surface plasmon resonance imaging (SPRi) of glycan microarrays. The SPRi was validated, and its utility was tested, by measuring binding specificities between defined human milk glycan epitopes and the capsids of two common norovirus strains, VA387 and Norwalk. Human milk oligosaccharide (HMOS)-based neoglycoconjugates, including chemically derived neoglycoproteins and oligosaccharide-glycine derivatives, were used to represent polyvalent glycoconjugates and monovalent oligosaccharides, respectively, in human milk. SPRi binding results established that the glycan motifs that bind norovirus capsids depend upon strain; VA387 capsid interacts with two neoglycoproteins, whereas Norwalk capsid binds to a different set of HMOS motifs in the form of both polyvalent neoglycoproteins and monovalent oligosaccharides. SPRi competitive binding assays further demonstrated that specific norovirus-binding glycans are able to inhibit norovirus capsid binding to their host receptors. A polyvalent neoglycoconjugate with clustered carbohydrate moieties is required for the inhibition of VA387 capsid binding to host receptor glycans, whereas both monovalent oligosaccharides and polyvalent neoglycoconjugates are able to inhibit Norwalk capsid binding to its host receptor. Binding of HMOS and HMOS-based neoglycoconjugates to norovirus capsids depends upon the specific strain characteristics, implying that HMOS and their polyvalent derivatives are potential anti-adhesive agents for norovirus prophylaxis.

Structure of simusan, a new acidic exopolysaccharide from Arthrobacter sp.

Simusan, a major exopolysaccharide produced by an ethanol-utilizing Arthrobacter sp. strain CE-17, contains D-glucose, D-mannose, D-galactose, L-rhamnose, D-glucuronic acid, and pyruvic acid in the ratios approximately 3:2:1:1:1:1 as well as O-acyl groups (presumably (presumably residues of acetic and palmitic acid). On the basis of chemical modifications of the polysaccharide, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and an octa-saccharide fragment, Smith degradation, and 1H and 13C NMR analysis, the following structure of the repeating unit was established: [formula: see text] It is suggested that at least one of the glucose residues and the galactose residue are O-acetylated.

A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins

O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.

A method for reductive cleavage of N-glycosylamide carbohydrate-peptide bond

Abstract A new, mild method for the splitting of N -linked oligosaccharides from glycoproteins includes treatment of glycoproteins (ovomucoid, flavoprotein, ribonuclease B, hemagglutinin, or transferrin) with 2 m LiBH 4 in 25m m LiOH-50m m Li citrate-70% aqueous tert -butyl alcohol (5 h, 45°), followed by hydrolysis of the resulting glycosylamine with aqueous acetic acid. The oligosaccharides formed were easily isolated by gel filtration and cation-exchange chromatography in 60–80% yields. The reaction was accompanied by the intense reductive cleavage of peptide bonds with formation of amino alcohols, but N -deacetylation of hexosamine was completely excluded. The optimal conditions of this reaction were chosen by use of a model glycopeptide, 2-acetamido-4- O -(2-acetamido-2-deoxy-β- d -glucopyranosyl)-1- N -(4- l -aspartoyl)-2-deoxy-β- d -glucopyranosylamine.

2-Acetamido-3,6-anhydro-2-deoxy-D-hexoses: products of the alkaline degradation of 2-acetamido-2-deoxy-D-hexoses

Abstract The formation of 2-acetamido-3,6-anhydro-2-deoxy- D -hexoses during the alkaline degradation of 2-acetamido-2-deoxy- D -hexoses is reported for the first time. Thus, 2-acetamido-3,6-anhydro-2-deoxy- D -glucose and - D -mannose are formed from 2-acetamido-2-deoxy- D -glucose or - D -mannose. The structure of 2-acetamido-3,6-anhydro-2-deoxy- D -mannose has been elucidated, and certain derivatives have been obtained. The 3,6-anhydro derivative formed during the alkaline degradation of 2-acetamido-2-deoxy- D -galactose is different from those formed from the D - gluco and D - manno analogues. This allows identification of a 2-acetamido-2-deoxy- D -hexose that decomposes during the alkaline treatment of glycoproteins in an amino acid analyzer.

Structure of the major exopolysaccharide produced by Azotobacter beijerinckii B-1615

A. beijerinckii strain B-1615 produced two acidic exopolysaccharides in the ratio approximately 9:1. The minor polysaccharide contained mannuronic and guluronic acids in the ratio 2.3:1 and is a bacterial alginate. The major polysaccharide consisted of D-galactose, L-rhamnose, and pyruvic acid in the ratios 2:1:1 and was acetylated. On the basis of methylation analysis, and 1H- and 13C-n.m.r. spectroscopy of the polysaccharide before and after removal of the pyruvic acid residues and O-deacetylation, it was concluded that the major polysaccharide had the structure [formula; see text] with up to 1.5 OAc groups per repeating unit.

Click chemistry in the synthesis of the first glycoconjugates of bacteriochlorin series

A regioselective synthesis of glycoconjugates based on bacteriochlorophyll a and lactose derivatives has been carried out. The conjugation was achieved via 1,3-dipolar cycloaddition of bacteriochlorins containing a terminal triple bond and a lactose azide derivative. The conjugates obtained in this way had one or two disaccharide fragments attached to pyrrol A, the exocyclic imide ring of the tetrapyrrolyc macrocycle, or to both positions. Exhaustive NMR analysis by 1D and 2D NMR experiments (1H-1H COSY, TOCSY, ROESY, 1H-13C HSQC, HMBC, and 1H-15N HMBC) allowed us to determine the structures and configurations of the glycoconjugates obtained. A bioassay of the glycoconjugates using the Hep2 cell line showed that the highest efficiency was observed for the glycosylated bacteriopurpurinimide containing a lactose residue at pyrrole ring A.

Synthesis of N-glycyl-β-glycopyranosylamines, human milk fucooligosaccharide derivatives

The action of ammonium carbamate in aqueous methanol in the presence of NH3 on three-, penta-, hexa-, octa-, and nonasaccharides of human milk and on decasaccharide (N-glycan from human immunoglobulin (IgG)) containing one or two a-l-fucose residues led to the corresponding β-glycopyranosylamines. After their N-acylation upon treatment with N-hydroxysuccinimide ester of N-Boc-glycine or N-Boc-glycine anhydride (Boc is the tert-butyloxycarbonyl) with subsequent removal of the Boc group, N-glycyl-β-glycopyranosylamines were obtained in up to 75% yield.

Mass spectrometry of glycoproteins: O-glycosyl derivatives of L-serine and L-threonine.

Abstract The mass spectra of acetylated O -glycosyl derivatives of serine and threonine methyl esters show characteristic features which allow identification of the amino acid and sugar moieties and the type of linkage between them. Some information concerning the sequence of sugar units and the position of the glycosidic bond in the carbohydrate moiety of disaccharide O -glycosyl derivatives of serine methyl ester is provided also by mass spectrometry.

Synthesis ofN-chioroacetyl-β-glycopyranosylamines, derivatives of monosaccharides and lactose

N-Chloroacetyl-β-glycopyranosylimines were synthesized from various monosaccharide (hexoses, pentoses, deoxysugars, uronic acids, and sugar phosphates) and a disaccharide (lactose) byN-acylation of the corresponding β-glycosylamines with chloroacetic anhydride in DMF In some cases, treatment of rnonosacc harides with NH3 in the presence of (NH4)2CO3 in MeOH or aqueous MeOH was more efficient than the methods previously described, as it gave β-glycosylarnines in higher yields.