A double regio- and stereoselective glycosylation strategy for the synthesis of N-glycans

A building block approach for biantennary N-linked oligosaccharides from glycoproteins (N-glycans) has been developed. Starting from a core trisaccharide (beta-mannosyl chitobiose) containing a benzylidene-protected beta-mannoside, the attachment of the disaccharide building blocks for the antennae can be performed in a double regio- and stereoselective manner. A short synthesis of a GlcNPhtbeta1,2Man donor was developed. The benzylidene acetal moiety, as a minimal protection of the beta-mannoside, allows selective alpha-glycosylation at OH-3 of the 2,3-diol with GlcNbeta1,2Man trichloroacetimidate donors. Subsequent debenzylidenation leads to a 4,6-diol, which can be selectively extended at OH-6. Overreaction at OH-4 was generally low when phthalimido-protected donors were used. This general strategy represents a modular synthesis of N-glycans and their glycoconjugates.     

Positive-ion ESI mass spectrometry of regioisomeric nonreducing oligosaccharide fatty acid monoesters: in-source fragmentation of sodium adducts

Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na](+). In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1' and C-2' of the alpha-D-Glcp-(1-2)-beta-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry.     

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.     

Degradation of Target Oligosaccharides by Anthraquinone–Lectin Hybrids with Light Switching

Anthraquinone–lectin hybrids were effectively synthesized using water‐soluble anthraquinone derivative 11 with concanavalin A (ConA) and hygrophorus russula lectin (HRL) to give anthraquinone–ConA ( 16 ) and anthraquinone–HRL ( 17 ) hybrids, respectively. These anthraquinone–lectin hybrids effectively and selectively degraded oligosaccharides containing a mannose residue as a non‐reducing terminal sugar, which has affinity for ConA and HRL, under photo‐irradiation with long‐wavelength UV light without additives and under neutral conditions. In addition, anthraquinone–HRL ( 17 ) selectively photo‐degraded only Man(α1,6)Man, which has a high affinity for HRL, among several mannosides by recognition of both the type and glycosidic linkage profile of the sugar in an oligosaccharide.     

Chemoselective synthesis of oligosaccharides of 2-deoxy-2-aminosugars

Along with the application of the S-benzoxazolyl glycosides to the high-yielding synthesis of disaccharides of the 2-amino-2-deoxy series, chemoselective assembly of oligosaccharides containing multiple residues of 2-amino-2-deoxyglycoses is reported. This modified armed-disarmed approach is relying on the observation that 2-N-trichloroethoxycarbonyl derivatives of S-benzoxazolyl glycosides are significantly more reactive than their 2-N-phthaloyl counterparts in MeOTf-promoted glycosylations. This allowed efficient chemoselective synthesis of 1,2-trans-linked oligosaccharides, the disarmed reducing end of which can be activated for immediate second step glycosidation in the presence of a more powerful activator, AgOTf. As a result of this two-step activation, trans-trans-patterned trisaccharides could be assembled in a highly efficient manner. This result differs from the classic armed-disarmed technique, according to which usually cis-trans-patterned oligosaccharides are generated.     

寡糖衍生化及基质辅助激光解吸电离飞行时间质谱分析方法研究

为提高中性寡糖在基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)中的检测灵敏度,建立了以1-(4-氰基苯基)-4-哌啶碳酰肼(CPH)为衍生化试剂对寡糖的标记方法。寡糖的还原端与CPH的酰肼基团反应生成腙,使得寡糖被CPH标记,衍生物以MALDI-TOF质谱进行分析。结果表明:在反应温度95℃,醋酸浓度为0.125%(V/V),CPH过量100倍的条件下,衍生产率可达最大,并且CPH衍生可使中性寡糖在MALDI-TOF质谱中的检测灵敏度提高10倍。本方法简便快速,灵敏度高,适合微量寡糖链的质谱分析。     

A Method of Orthogonal Oligosaccharide Synthesis Leading to a Combinatorial Library Based on Stationary Solid‐Phase Reaction

A new, efficient synthesis of oligosaccharides, which involves solid‐phase reactions without mixing in combination with an orthogonal‐glycosylation strategy, is described. Despite a great deal of biological interest, the combinatorial chemistry of oligosaccharides is an extremely difficult subject. The problems include 1) lengthy synthetic protocols required for the synthesis and 2) the variety of glycosylation conditions necessary for individual reactions. These issues were addressed and solved by using the orthogonal‐coupling protocol and the application of a temperature gradient to provide appropriate conditions for individual reactions. Furthermore, we succeeded in carrying out solid‐phase reactions with neither mechanical mixing nor flow. In this report, the synthesis of a series of trisaccharides, namely, α/β‐L ‐Fuc‐(1→6)‐α/β‐D ‐Gal‐(1→2/3/4/6)‐α/β‐D ‐Glc‐octyl, is reported to demonstrate the eligibility of the synthetic method in combinatorial chemistry.     

Preparation of Building Blocks for Carba‐Oligosaccharides: Some Protected 5a′‐Carba‐D‐hexopyranosyl‐1,5‐anhydro‐2‐deoxy‐D‐arabino‐hex‐1‐enitols,and 5a,5a′‐Dicarba Congeners Thereof

Four configurational types of two protected O‐linked (5a‐carba‐D‐hexopyranosyl)‐D‐glucal and carba‐D‐glucal derivatives were prepared in order to provide versatile synthetic intermediates readily convertible into carba‐oligosaccharides of biological interest. These compounds may also find application as donors for elongation of carba‐oligosaccharide chains having O‐linked carbahexopyranose residues at nonreducing ends.     

Linkage and Branch Analysis of High-Mannose Oligosaccharides Using Closed-Ring Labeling of 8-Aminopyrene-1,3,6-Trisulfonate and P-Aminobenzoic Ethyl Ester and Negative Ion Trap Mass Spectrometry

A strategy based on negative ion electrospray ionization tandem mass spectrometry and closed-ring labeling with both 8-aminopyrene-1,3,6-trisulfonate (APTS) and p-aminobenzoic acid ethyl ester (ABEE) was developed for linkage and branch determination of high-mannose oligosaccharides. X-type cross-ring fragment ions obtained from APTS-labeled oligosaccharides by charge remote fragmentation provided information on linkages near the non-reducing terminus. In contrast, A-type cross-ring fragment ions observed from ABEE-labeled oligosaccharides yielded information on linkages near the reducing terminus. This complementary information provided by APTS- and ABEE-labeled oligosaccharides was utilized to delineate the structures of the high-mannose oligosaccharides. As a demonstration of this approach, the linkages and branches of high-mannose oligosaccharides Man(5)GlcNAc(2), Man(6)GlcNAc(2), Man(8)GlcNAc(2), and Man(9)GlcNAc(2) cleaved from the ribonuclease B were assigned from MS(2) spectra of ABEE- and APTS-labeled derivatives.     

2-Oxabutane as a substitute for internal monomer units of oligosaccharides to create lectin ligands

The synthesis of bioactive oligosaccharides is too tedious to scale up for commercialization. However, structurally simplified glycomimetics are commercializable, if they can be synthesized much more easily than the oligosaccharides while having a comparable bioactivity. In this study, we propose a 2-oxabutane (OB) structure as an imitation of the internal monosaccharide units in oligosaccharides. Two trimannoside and three pentamannoside OB-glycomimics were synthesized in remarkably short steps. Among them, Manα1-OB-2Man 10, a trimannoside mimic, showed eight-fold affinity toward concanavalin A (ConA) relative to methyl mannoside in latex agglutination lectin assay and equilibrium dialysis assay (EDA), while the other mimics showed three- to four-fold affinities. EDA indicated that the bindings between each mimic molecule and a ConA subsite were all in one-to-one stoichiometry and thus these mimics were monovalent ligands, excluding multivalence effect for the high affinities. The strong affinity of 10 could be explained by the occupation of two mannose binding sites of a ConA subsite by its two mannose units. Mimic 10 proved to be even a better ligand for ConA than the natural disaccharide Manα1,2Man, while been much more easy to synthesize, thereby illustrating the potential of the approach here presented.     

3-氨基-9-乙基咔唑衍生化寡糖混合物的高效液相色谱分离及激光解吸电离飞行时间质谱分析

建立了以3-氨基-9-乙基咔唑(AEC)为衍生化试剂对寡糖的标记方法。寡糖的还原端与AEC的伯氨基反应生成烯胺,再被NaBH3CN还原为二级胺,使得寡糖被AEC标记。衍生物通过反相高效液相色谱分离纯化,采用的色谱柱为Waters Symmetry C18柱(3.9 mm×150 mm,5 μm),乙腈和乙酸铵水溶液(pH 4.5)为流动相,梯度洗脱,在254 nm波长处检测,并以基质辅助激光解吸电离飞行时间质谱进行分析。在此衍生化条件和色谱条件下,葡寡糖衍生物分离良好,并且AEC衍生可显著提高葡寡糖的质谱检测灵敏度。该方法适用于寡糖的分离纯化和结构分析,并与生物质谱具有良好的兼容性,表明该方法在微量寡糖链分析方面有广阔的应用前景。     

Conformational analysis of β-1,2-linked mannobiose to mannoheptaose, specific antigen of pathogenic yeast Candida albicans

Candida albicans contains characteristic β-1,2-linked oligomannosyl moieties in the cell wall mannan. Reduction of the reducing termini of these oligosaccharides by NaBH(4) causes a significant downfield shift in the NMR signals for the second and third mannose units and upfield shift of the fourth mannose unit. To show the correlation between the shift in the NMR signals and the conformations of the β-1,2-linked mannooligosaccharides, we performed molecular mechanics calculations. Six energy minima of the β-1,2-linked mannobiose were found in the relaxed map computed using the AMBER force field. Five of the six energy minima could also be generated by a simulated annealing from a 900 K molecular dynamics. Similarly, the solution conformation of the β-1,2-linked mannotriose to mannoheptaose was identified by the high temperature-simulated annealing. In the mannotetraose, the nonreducing terminal mannose unit was located near the reducing terminal one and formed a folded conformation. This result suggests that a mannose unit affects the H-1 chemical shifts of not only the second mannose unit, but also the third and fourth mannose units.     

Prompt chemoenzymatic synthesis of diverse complex-type oligosaccharides and its application to the solid-phase synthesis of a glycopeptide with Asn-linked sialyl-undeca- and asialo-nonasaccharides

We describe herein the preparation of 24 pure asparagine-linked oligosaccharides (Asn-oligosaccharides) from asparagine-linked biantennary complex-type sialylundecasaccharide [(NeuAc-alpha-2,6-Gal-beta-1,4-GlcNAc-beta-1,2-Man-alpha-1,6/1,3-)(2)-Man-beta-1,4-GlcNAc-beta-1,4-GlcNAc-beta-1-asparagine, 2] obtained from egg yolk. Our synthetic strategy aimed at adapting branch specific exo-glycosidases digestion (beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase) of the individual asialo-branch after preparation of monosialyloligosaccharides obtained from 2 by acid hydrolysis of NeuAc. In order to perform branch specific exo-glycosidase digestion, isolation of pure monosialyloligosaccharides obtained was essential. However, isolation of two kinds of monosialyloligosaccharides are difficult by HPLC due to their highly hydrophilic nature. Therefore, we examined chemical protection with hydrophobic protecting (Fmoc and benzyl) groups. These chemical protection enabled us to separate the monosialyloligosaccharides by use of a HPLC column (ODS) on synthetic scales. Using these pure monosialiloligosaccharides enable us to obtain 24 Asn-linked oligosaccharides (100 mg scale) within a few weeks by branch specific exo-glycosidase digestions (alpha-D-neuraminidase, beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase). In addition, solid-phase synthesis of glycopeptide having Asn-linked sialyl-undeca- and asialo-nonasaccharides thus obtained, was also performed on an acid labile HMPA-PEGA resin.     

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.     

Conformational analysis of the disaccharide methyl α‐d‐mannopyranosyl‐(1→3)‐2‐O‐acetyl‐β‐d‐mannopyranoside monohydrate

The crystal structure of methyl α‐d ‐mannopyranosyl‐(1→3)‐2‐O‐acetyl‐β‐d ‐mannopyranoside monohydrate, C₁₅H₂₆O₁₂·H₂O, ( II ), has been determined and the structural parameters for its constituent α‐d ‐mannopyranosyl residue compared with those for methyl α‐d ‐mannopyranoside. Mono‐O‐acetylation appears to promote the crystallization of ( II ), inferred from the difficulty in crystallizing methyl α‐d ‐mannopyranosyl‐(1→3)‐β‐d ‐mannopyranoside despite repeated attempts. The conformational properties of the O‐acetyl side chain in ( II ) are similar to those observed in recent studies of peracetylated mannose‐containing oligosaccharides, having a preferred geometry in which the C2—H2 bond eclipses the C=O bond of the acetyl group. The C2—O2 bond in ( II ) elongates by ～0.02 Å upon O‐acetylation. The phi (?) and psi (ψ) torsion angles that dictate the conformation of the internal O‐glycosidic linkage in ( II ) are similar to those determined recently in aqueous solution by NMR spectroscopy for unacetylated ( II ) using the statistical program MA′AT, with a greater disparity found for ψ (Δ = ～16°) than for ? (Δ = ～6°).     

Switching extended 1,3-diequatorial and bent 1,3-diaxial states of a disubstituted hinge sugar by ligand exchange reactions on Pt(II)

The bent conformation of a trisaccharide containing 2,4-diaminoxylopyranoside, in which both end sugars are presented in 1,3-diaxial orientation, is fixed by chelation of the diamino groups to Pt(II) and unfixed by a ligand exchange reaction with NaCN or thiourea giving its extended conformation.     

Anomeric information obtained from a series of synthetic trisaccharides using energy resolved mass spectra

The majority of structural investigations of oligosaccharides based on mass spectrometry use naturally occurring oligosaccharides, which do not allow extracting any common feature associated with anomeric structures and linkage positions. In order to address the issue to find such characteristics possibly contained in oligosaccharide structure, a synthetic combinatorial trisaccharide library was analyzed. The trisaccharides used in the analysis consisted of L-fucose, D-galactose and D-glucose, in which individual glycosidic linkages existed in either alpha- or beta-anomers. The analysis of energy-resolved mass spectra (ERMS) and the scattered plot analysis of some parameters obtained from ERMS for a series of trisaccharides revealed that lower activation energy was required for the dissociation of alpha-glycosides of these sugars compared to those of the corresponding beta-anomers. It is suggested that this finding may be useful in structural analysis of natural oligosaccharides.     

Conversion of Complex Sialooligosaccharides into Polymeric Conjugates and their Anti-Influenza Virus Inhibitory Potency

ABSTRACT

To investigate the specificity of various influenza virus strains we have prepared polyacrylic type conjugates of undecasaccharide (Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1)₂-3,6Manβ1-4GlcNAcβ1-4GlcNAc (YDS), and trisaccharides 6‵-sialyl-N-acetyllactosamine (6‵SLN), 6‵-sialyllactose (6‵SL), and 3‵-sialyllactose (3‵SL). Free oligosaccharides were transformed to glycosylamine-1-N-glycyl derivatives by sequential action of NH₄HCO₃, chloroacetic anhydride, and aqueous NH₃. The known derivatization protocol has been optimized for these sialooligosaccharides. Coupling of obtained amino-spacered derivatives with poly(4-nitrophenyl acrylate) gave rise to two types of conjugates, namely with polyacrylic acid and polyacrylamide backbones; the conversion proceeded quantitatively and without destruction of the oligosaccharides. The content of oligosaccharides in the conjugates was 10, 20, and 30% mol for 3‵SL, 6‵SL, 6‵SLN, and 2, 5 and 10% mol for YDS. Free oligosaccharides and the glycoconjugates were tested as inhibitors of influenza virus adhesion, and also as blockers of virus infectivity in MDCK cell culture. Biantennary YDS demonstrated similar activity to trisaccharide 6‵SLN both as the free form and neoglycoconjugate.     

Identification of reducing and nonreducing neutral carbohydrates by laser‐enhanced in‐source decay (LEISD) MALDI MS

In this work, laser‐enhanced in‐source decay (LEISD) technique of matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI‐FT‐ICR‐MS) was used to distinguish reducing and nonreducing carbohydrates. Interestingly, easier cleavage of (1 → 2)‐linked glycosidic bonds for nonreducing carbohydrates containing D‐fructofuranosyl units was observed in MALDI‐FT‐ICR‐MS, which was in agreement with the result of theoretical calculation by the software package Gaussian 09. Importantly, no cross‐ring cleavage of fructofuranosyl residues was detected in the LEISD spectra of nonreducing carbohydrates. LEISD method therefore offers an attractive alternative for fast and efficient differentiation of reducing and nonreducing carbohydrates, and the positions of nonreducing monosaccharide residues in a carbohydrate chain could be easily speculated. Copyright © 2013 John Wiley & Sons, Ltd.     

Pd(OAc)2-catalyzed carbonylation of amines

A phosphine-free catalytic system [Pd(OAc)2-Cu(OAc)2-air] induced a substrate-specific carbonylation of amines in boiling toluene under CO gas (1 atm). Symmetrical N,N'-dialkylureas were obtained by the carbonylation of primary amines. N,N,N'-Trialkylureas were selectively formed by addition of a secondary amine to the above reaction vessel. Secondary amines did not give tetraalkylureas. However, dialkylamines with a phenyl group on their alkyl chains, such as N-monoalkylated benzylic amine or phenethylamine derivatives, underwent a direct aromatic carbonylation to afford five- or six-membered benzolactams. In the carbonylation, the chelation effect or steric repulsion between Pd(II) and the meta-substituent in the ortho-palladation and the ring sizes of cyclopalladation products that were formed prior to carbonylation were found to generate good site selectivity and increase the reaction rate. In contrast, carbonylation of omega-arylalkylamines with a hydroxyl group gave neither ureas nor benzolactams but instead produced 1,3-oxazolidinones smoothly. Hydrochlorides of amines also underwent carbonylation to afford the corresponding amides under the conditions used. This procedure made it possible to prepare ureas of amino acid esters and N-alkylcarbamates in practical yields.