V. E. Piskarev

Branching pattern and sequence analysis of underivatized oligosaccharides by combined MS/MS of singly and doubly charged molecular ions in negative-ion electrospray mass spectrometry

We previously reported that sequence and partial linkage information, including chain and blood-group types, of reducing oligosaccharides can be obtained from negative-ion electros-pray CID MS/MS on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without derivatization (Chai, W.; Piskarev, V.; Lawson, A. M. Anal. Chem.2001, 73, 651–657). In contrast to oligonucleotides and peptides, oligosaccharides can form branched structures that result in a greater degree of structural complexity. In the present work we apply negative-ion electrospray CID MS/MS to core-branching pattern analysis using nine 3,6-branched and variously fucosylated oligosaccharides based on hexasaccharide backbones LNH/LNnH as examples. The important features of the method are the combined use of CID MS/MS of singly and doubly charged molecular ions of underivatized oligosaccharides to deduce the branching pattern and to assign the structural details of each of the 3- and 6-branches. These spectra give complimentary structural information. In the spectra of [M -H]−, fragment ions from the 6-linked branch are dominant and those from the 3-linked branch are absent, while fragment ions from both branches occur in the spectra of [M - 2H]2−. This allows the distinction of fragment ions derived from either the 3- or 6-branches. In addition, a unique D2β-3 ion, arising from double D-type cleavage at the 3-linked glycosidic bond of the branched Gal core residue, provides direct evidence of the branching pattern with sequence and partial linkage information being derived from C- and A-type fragmentations, respectively.

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.

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.

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.

Synthesis of mono- and di-α-l-fucosylated 2-acetamido-2-deoxy-N-glycyl-β-d-glucopyranosylamines, spacered fragments of glycans of N-glycoproteins

Abstractα-l-Fucp-(1→3)-d-GlcNAc, α-l-Fucp-(1→6)-[α-l-Fucp-(1→3)]-d-GlcNAc, and β-d-Galp-(1→3)-[α-l-Fucp-(1→4)]-d-GlcNAc were converted into corresponding β-glycopyranosylamines by action of ammonium carbamate in aqueous boric acid, or in aqueous methanol in case of α-l-Fucp-(1→6)-d-GlcNAc. N-Acylation of these fucooligosaccharides with N-Z-glycine N-hydroxysuccinimide ester (Z is benzyloxycarbonyl) followed by hydrogenolytic removal of Z-group afforded corresponding N-glycyl-β-glycopyranosylamines of these fucooligosaccharides; three of them model carbohydrate-peptide region of N-glycoproteins, and the forth is an amino-spacered Lea-antigen.

Synthesis of mono- and di-α-l-fucosylated 2-acetamido-2-deoxy-N-glycyl-β-d-glucopyranosylamines modeling N-glycoprotein carbohydrate-peptide bond region based on 2-acetamido-N-(N-tert-butyloxycarbonylglycyl)-2-deoxy-β-d-glucopyranosylamine

A synthesis of glycyl-spacered N-β-glycosides of diand trisaccharides such as α-l-Fucp-(1→3)-β-d-GlcNAcp-NHCOCH2NH2 (1), α-l-Fucp-(1→6)-[α-{asl}-Fucp-(1→3)]-β-d-GlcNAcp-NHCOCH2NH2 (2), α-l-Fucp-(1→6)-β-d-GlcNAcp-NHCOCH2NH2 ({sn3}), which model the carbohydrate-peptide bond region of fucosylated N-glycoproteins of animals and plants, was accomplished. The synthesis was carried out by the fucosylation of unprotected N-glycoside β-{}scd-GlcNAcp-NHCOCH2NHBoc (Boc is the tert-butyloxycarbonyl) (4), 4,6-O-benzylidene derivative of compound 4, and protected disaccharide 1 (α-l-FucpBn3-(1→3)-β-d-GlcNAcp-NHCOCH2NHBoc (Bn is the benzyl)) with ethyl 2,3,4-tri-O-benzyl-1-thio-β-l-fucoside (in the presence of CuBr2 and an excess of Et4NBr). A possibility of the selective ?-fucosylation of N-glycoside 4 at the primary hydroxy group in the presence of secondary hydroxy groups (in 34% yield) was demonstrated. Amino spacered N-glycosides 1–3 were obtained after the removal of protecting groups.

Synthesis of N-glycyl-β-glycopyranosyl amines, derivatives of natural oligosaccharides — glucose analogs of Lex antigen

Treatment of the natural tri-, tetra-, and pentasaccharides, β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, α-l-Fucp-(1→2)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, and α-l-Fucp-(1→2)-[α-d-GalNAcp-(1→3)]-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, which are glucose analogs of Lex, with ammonium carbamate in aqueous methanol gave the corresponding β-glycopyranosyl amines. After their N-acylation with N-Z-glycine N-hydroxysuccinimidyl ester (Z is benzyloxycarbonyl) with subsequent hydrogenolytic removal of Z-group, corresponding N-glycyl-β-glycopyranosyl amines were obtained in yields up to 70%.

Localization and characterization of the carbohydrate-binding site of the porcine lymphocyte mannan-binding protein.

Mannan-binding proteins found in the liver and serum of several vertebrate species are supposed to play an important role in the intracellular transport of glycoproteins, as well as in several protective reactions including complement activation and elimination of various pathogens. To study these protective functions at molecular level it is necessary to understand the fine oligosaccharide specificity and mutual relation among various forms of these soluble lectins. We have isolated mannan-binding protein as peripheral membrane proteins of porcine lymphocytes. This lectin was purified to homogeneity and shown to possess many properties in common with the well studied rat liver proteins (mol. mass, subunit composition and general organization of the molecule). Binding studies performed with three series of defined oligosaccharides (high mannose, hybrid type, and complex) on native lectin molecules as well as isolated carbohydrate-binding domains revealed distinctive features of this mannan-binding protein, including its impaired ability to bind the oligosaccharide ligand after reduction and decyclization at core N-acetyl-D-glucosamine 1.

A new method for the synthesis of aldehyde-spacered oligosaccharides by oxidation of l -tartaric acid derivative

N-(N-[(2R,3R)-2,3-Dihydroxy-3-carboxypropionyl]glycyl)-β-d-glycopyranosylamines and N-[N-(formylcarbonyl)glycyl]-β-d-glycopyranosylamines, derivatives of lactose, human milk tri-, tetra-, and pentasaccharides, and synthetic di- and trisaccharides, were synthe- sized. N-Glycyl-β-d-glycopyranosylamines of these oligosaccharides were N-acylated with (+)-di-O-acetyl-l-tartaric anhydride, and the derivatives with α-glycol and terminal carboxy groups in the aglycon moiety were obtained after O-deacylation (70—80% yield). The short-term (80 s) selective oxidation of the α-glycol group of the tartaric acid residue in these derivatives with dilute periodic acid gave the target compounds with the terminal aldehyde group in aglycon in ~90% yield.

Synthesis of N -( N -bromoacetylglycyl)-β-D-glycopyranosylamines, derivatives of monoand di-α-L-fucosylated diand trisaccharides

N-(N-Bromoacetylglycyl)-β-glycopyranosylamines of diand trisaccharides, namely, α-LFucp-(1→6)-β-D-GlcNAcp-NHCOCH2NHCOCH2Br, α-L-Fucp-(1→3)-β-D-GlcNAcpNHCOCH2NHCOCH2Br, α-L-Fucp-(1→6)-[α-L-Fucp-(1→3)]-β-D-GlcNAcp-NHCOCH2NHCOCH2Br, α-L-Fucp-(1→4)-[β-D-Galp-(1→3)]-β-D-GlcNAcp-NHCOCH2NHCOCH2Br, and α-L-Fucp-(1→2)-β-D-Galp-(1→4)-β-D-Glcp-NHCOCH2NHCOCH2Br, were synthesized in ~70% yields by treatment of the appropriate N-glycyl-β-glycopyranosylamines with N-bromoacetoxysuccinimide in aqueous DMF. The synthesized diand trisaccharide bromo derivatives are proposed for the obtaining of neoglycoconjugates of physiologically active substances, for instance, of daunorubicin anticancer agent.