First synthesis of the immunodominant beta-galactofuranose-containing tetrasaccharide present in the cell wall of Aspergillus fumigatus

β-Galf-(1→5)-β-Galf-(1→6)-α-Manp-(1→6)-α-Manp, the immunodominant epitope in the cell-wall galactomannan of Aspergillus fumigatus, was synthesized for the first time as its allyl glycoside. The key disaccharide glycosyl donor, 2,3,5,6-tetra-O-benzoyl-β-d-galactofuranosyl-(1→5)-2-O-acetyl-3,6-di-O-benzoyl-β-d-galactofuranosyl trichloroacetimidate (10), was constructed by 5-O-glycosylation of 1,2-O-isopropylidene-3,6-di-O-benzoyl-α-d-galactofuranose (4) with 2,3,5,6-tetra-O-benzoyl-β-d-galactofuranosyl trichloroacetimidate (5), followed by 1,2-O-deacetonation, acetylation, selective 1-O-deacetylation, and trichloroacetimidation. The target tetrasaccharide 16 was obtained by the condensation of allyl 2,3,4-tri-O-benzoyl-α-d-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-d-mannopyranoside (14) as glycosyl acceptor with the disaccharide glycosyl donor 10, followed by deprotection.     

Synthesis of a tetrasaccharide substrate of heparanase

A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely β-d-GlcA(2S)-(1→4)-α-d-GlcN(NS,6S)-(1→4)-β-d-GlcA-(1→4)-α-d-GlcN(NS,6S)-OMe, was synthesized in a convergent manner via coupling of a pair of the disaccharide building blocks as a key step.     

Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-β(1?→?4)-Glc-β(1?→?3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC₅₀ of 270?nM and 2.4?µM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.     

Synthesis of a mannose nonasaccharide existing in the exopolysaccharide of Cryphonectria parasitica

alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)[alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)]-alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->2)]-alpha-D-Manp, existing in the exopolysaccharide of Cryphonectria parasitica was synthesized as its allyl glycoside in a regio- and stereoselective manner.     

Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum lipoferum SR65

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose and d-glucose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays important roles in the growth and development of plants. The interaction between the roots of the plants and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design.     

Synthesis of two heptasaccharide analogues of the lentinan repeating unit

beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (18) and the allyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-beta-D-Glcp-(1-->6)]-alpha-D-Glcp (29) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6-O-benzylidenated mono-13 or disaccharide 8 were used as the acceptor to ensure the beta linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the beta linkage in the synthesis of 29.     

Oxidation and metal-ion affinities of a novel cyclic tetrasaccharide

The cyclic tetrasaccharide, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was oxidized in high yield to a dicarboxylic acid, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->). The parent and oxidized compound were then screened for the ability to form stable complexes with 20 metal cations. Ion-exchange thin-layer chromatography was utilized to survey binding in aqueous and 50% methanolic solutions. The screening identified Pb2+, Fe2+ and Fe3+ as forming strong metal chelates with the oxidized cyclic tetrasaccharide. The stoichiometry of the oxidized cyclic tetrasaccharide and Pb2+ complex was determined to be 1:1 using aqueous gel-permeation chromatography. Perturbations between the free and complexed structure were examined using NMR spectroscopy. Molecular simulations were used to identify a probable structure of oxidized cyclic tetrasaccharide complexed with Pb2+.     

Synthesis of beta-(1-->6)-branched (1-->3)-glucododecaose and -glucopentadecaose with alternate beta- and alpha-bonds in the backbone

Beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)](2-3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp were synthesized as their methoxyphenyl glycosides in a concise way with a trisaccharide as the building block.     

Efficient synthesis of a 3,6-branched mannose hepta- and octasaccharide

Alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp and alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp, were synthesized as their methyl glycosides in a regio- and stereoselective way.     

酶法合成功能性低聚糖

功能性低聚糖具有无毒、无残留、稳定性强等特点,作为新型绿色添加剂被广泛应用在食品、饲料、医药行业。国际市场上10余种低聚糖产品中除大豆低聚糖、棉籽糖外,主要采用酶法制备。用于合成功能性低聚糖的酶包括糖苷酶、糖基转移酶和磷酸化酶。本文综述了功能性低聚糖种类、性质和制备方法,分析了酶法合成低聚糖的优缺点,阐述了磷酸化酶种类、催化特性和低聚糖产物。多酶法合成策略和目标酶的分子改造将是酶法合成功能性低聚糖的发展方向。     

Synthesis of a hexasaccharide fragment of group E streptococci polysaccharide and the tetrasaccharide repeating unit of E. coli O7:K98:H6

Syntheses of a hexasaccharide, the dimer of the repeating unit of the group E streptococci polysaccharide, and a tetrasaccharide, the repeating unit of the E. coli O7:K98:H6, were achieved by constructing alternate alpha-L-(1-->2)- and alpha-L-(1-->3)-linked L-rhamnopyranose backbones and substituting with beta-linked D-glucopyranose side chains for the former, and a D-glucopyranosyluronate branch for the latter, respectively, at O-2 of the L-rhamnose ring.     

Synthesis of the beta anomer of the spacer-equipped tetrasaccharide side chain of the major glycoprotein of the Bacillus anthracis exosporium

The beta glycoside of the tetrasaccharide sequence beta-Ant-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->2)-l-Rhap, whose aglycon allows conjugation to proteins, was synthesized for the first time. A stepwise synthetic approach was applied with thioglycosides as glycosyl donors, and the beta anomer of the compound was obtained equipped with a spacer group whose further transformation allows conjugation to suitable carriers. To synthesize the beta-anthrosyl linkage with high stereoselectivity, a linker-equipped rhamnotriose derivative was glycosylated with ethyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-dideoxy-1-thio-beta-d-glucopyranoside. Further functionalization of the tetrasaccharide thus obtained, followed by deprotection, gave the target substance.     

Synthesis of galactose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose and its lauryl glycoside

Coupling of the trisaccharide acceptor either 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-5-O-acetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (13) or lauryl 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,5-di-O-acetyl-alpha-D-glucopyranoside (15) with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate (12) gave alpha-linked hexasaccharides 14 and 16, respectively, while coupling of either 13 or 15 with trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate 17 did not afford any hexasaccarides. The analogues of the immunomodulator beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-beta-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp (1) was obtained by deprotection of 14 and 16.     

Synthesis of an octamannosyled glycan chain, the key oligosaccharide structure in ER-associated degradation

The high-mannose type decasaccharide (Man(8)GlcNAc(2)), the proposed ligand of ER residing mannosidase-like proteins (MLP), and its monoglycosylated homologue (alpha-Glc(1)Man(8)GlcNAc(2)) were synthesized. The oligosaccharide assembly was performed in a convergent and stereoselective manner, using three oligosaccharide components, a core trisaccharide having a beta-mannoside bond, a liner mannotriose, and a branched mannotetraose.     

A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer

Background

The glycan moieties sialyl-Lewis-X and/or -A (sLe^X/A) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation.

Synthesis of a D-rhamnose branched tetrasaccharide, repeating unit of the O-chain from Pseudomonas syringae pv. Syringae (cerasi) 435

The first synthesis of a d-rhamnose branched tetrasaccharide, corresponding to the repeating unit of the O-chain from Pseudomonas syringae pv. cerasi 435, as methyl glycoside is reported. The approach used is based on the synthesis of an opportune building-block, that is the methyl 3-O-allyl-4-O-benzoyl-alpha-D-rhamnopyranoside, which was then converted into both a glycosyl acceptor and two different protected glycosyl trichloroacetimidate donors. Successive couplings of these three compounds afforded the target oligosaccharide. The reported synthesis is also useful to perform the oligomerization of the repeating unit.     

Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.     

Synthesis of the tetrasaccharide residue of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agelas clathrodes

A tetrasaccharide, alpha-l-Rhap-(1-->3)-beta-d-GalpNAc-(1-->6)-alpha-d-Galp-(1-->2)-alpha-d-Galp, the carbohydrate moiety of clarhamnoside isolated from the marine sponge Agelas clathrodes, was synthesized as its propyl glycoside via a convergent approach. The key steps to the synthetic strategy were the stereoselective construction of the reducing-end disaccharide alpha-d-Galp-(1-->2)-d-Galp (5) and efficient coupling with the terminal disaccharide alpha-l-Rhap-(1-->3)-d-GalpNAc building block, in which the N-phthalimido-protected trifluoroacetimidate 13 was proved to be an effective donor.     

Solution conformation and dynamics of a tetrasaccharide related to the LewisX antigen deduced by NMR relaxation measurements

¹H-NMR cross-relaxation rates and nonselectivelongitudinal relaxation times have been obtained at two magnetic fields (7.0and 11.8 T) and at a variety of temperatures for the branchedtetrasaccharide methyl3-O--N-acetyl-galactosaminyl--galactopyranosyl-(14)[3-O--fucosyl]-glucopyranoside (1), an inhibitor of astrocyte growth. Inaddition, ¹³C-NMR relaxation data have also been recorded atboth fields. The ¹H-NMR relaxation data have been interpretedusing different motional models to obtain proton–proton correlationtimes. The results indicate that the GalNAc and Fuc rings display moreextensive local motion than the two inner Glc and Gal moieties, since thosepresent significantly shorter local correlation times. The¹³C-NMR relaxation parameters have been interpreted in termsof the Lipari–Szabo model-free approach. Thus, order parameters andinternal motion correlation times have been deduced. As obtained for the¹H-NMR relaxation data, the two outer residues possess smallerorder parameters than the two inner rings. Internal correlation times are inthe order of 100 ps. The hydroxymethyl groups have also different behaviour,with the exocyclic carbon on the glucopyranoside unit showing the highestS². Molecular dynamics simulations using a solvated systemhave also been performed and internal motion correlation functions have beendeduced from these calculations. Order parameters and interproton distanceshave been compared to those inferred from the NMR measurements. The obtainedresults are in fair agreement with the experimental data.     

Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification

The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40–120 μm) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 μmol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.