Synthesis and characterization of some anomeric pairs of per-O-acetylated aldohexopyranosyl cyanides (per-O-acetylated 2,6-anhydroheptononitriles). On the reaction of per-O-acetylaldohexopyranosyl bromides with mercuric cyanide in nitromethane

The synthesis and characterization of the anomeric pairs of the per-O-acetylaldohexopyranosyl cyanides of D-galactose, L-fucose, D-glucose, and D-mannose, as well as of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl cyanide, are described. Cyanation of the readily available, per-O-acetylaldohexopyranosyl bromides with mercuric cyanide in nitromethane, and subsequent purification, gave the corresponding, crystalline glycosyl cyanides with a high degree of 1,2-trans stereoselectivity. Thus, per-O-acetylated aldohexopyranosyl cyanides of the 1,2-trans configuration were obtained in yields ranging from 20 to 79%, whereas the corresponding 1,2-cis anomers were obtained in yields of less than or equal to 8.4%, the ratios of the 1,2-trans:1,2-cis anomers so prepared being greater than or equal to 8.5:1. The principal by-products of these irreversible, cyanation reactions were the per-O-acetylated 1,2-O-[1-(exo- and endo-cyano)ethylidene]aldohexopyranoses, obtained in yields of up to 40%. The structural assignments of the per-O-acetylaldohexopyranosyl cyanides were unequivocally established by elemental analysis, chemical transformation, vibrational spectroscopy, and 13C- and 1H-nuclear magnetic resonance spectroscopy. Correlations between the physical properties and the anomeric configurations of these C-aldohexopyranosyl compounds are described.     

Studies on the origin of stereoselectivity in the synthesis of 1,2-trans glycofuranosyl azides

The stereoselectivity of the 1,2-trans directed, Lewis acid-catalysed azidation of peracylated furanoses was found to depend on the reactivity of the azide donor (azide nucleophilicity) and the configuration at the anomeric centre relative to the neighbouring 2-O-acyl group. Reactions of 1,2-trans glycosyl esters with highly nucleophilic azide donors, generated from SnCl4 and Me3SiN3, were stereospecific. The results are interpreted in terms of the rapid reaction of the azide species with bicyclic 1,2-acyloxonium (1,2-O-alkyliumdiyl-D-glycofuranose) ions, which were the primarily formed reactive intermediates. When using 1,2-cis glycosyl esters as starting materials the selectivity was reduced (90-94% de); the same is true with 1,2-trans counterparts if less nucleophilic Me3SiN3 in combination with Me3SiOTf catalyst was used. This occurred due to the appearance of the more reactive but less selective oxocarbenium (glycofuranoxonium) ions either as primarily formed reactive intermediates in the former case or after equilibration with acyloxonium ions in the latter case. Protected 1,2-trans beta-D-glycofuranosyl azides with ribo, xylo and 3-deoxy-erythro-pento configurations were best prepared from the corresponding glycosyl esters using 0.05 equivalents of SnCl4, i.e., under anomerization-free conditions. Azidation of methyl glycofuranosides proceeds with inferior (80-90% de) and less predictable selectivity irrespective of the starting anomeric configuration.     

Differentiating the 2,3-diols of glucopyranosides by 4,6-O-benzylidene-protected-1,2-D-glucopyranosylorthoesters strategy

A facile and efficient method to differentiate the 2,3-diols of glucopyranosides based on 1,2-orthoesters strategy was developed. Stable thioglucosides were employed as the starting materials to prepare the corresponding 1,2-orthoesters. When treated with HCl aqueous solution and followed with Et(3)N, differentiation of the 2,3-diols was efficiently achieved along with the generation of a convertible anomeric hydroxyl group. In addition, an easy and practical method based on NOE was proposed to determine whether the 1,2-orthoesters were endo-type or exo-type.     

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.     

A click chemistry approach to glycomimetics: Michael addition of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose to 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose--a convenient route to novel 4-deoxy-(1-->5)-5-C-thiodisaccharides

The base catalyzed conjugate Michael addition of the 1-thiosugar, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose, 1, to a new highly reactive enone 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose, 2, proceeds steroselectively with formation of adduct 3 in 94% yield. Convenient stereoselective reduction of the C-3 keto function of 3 with L-Selectride followed by in situ acetylation produces thiodisaccharide 4 in good 82% yield. Cleavage of the 1,2-O-isopropylidene protecting group with p-toluenesulfonic acid in methanol, followed by de-O-acetylation, produced an inseparable anomeric mixture of methyl 4-deoxy-5-C-(beta-D-glucopyranosyl)-thio-alpha/beta-L-ribo-pyranoside 5 in 72% overall yield. This approach constitutes a new general two-step click chemistry route to the previously unknown class of 4-deoxy-(1-->5)-5-C-thiodisaccharides as stable and biologically important glycomimetics.     

Differentiating the 2,3-diols of glucopyranosides by 4,6-O-benzylidene-protected-1,2-d-glucopyranosylorthoesters strategy

A facile and efficient method to differentiate the 2,3-diols of glucopyranosides based on 1,2-orthoesters strategy was developed. Stable thioglucosides were employed as the starting materials to prepare the corresponding 1,2-orthoesters. When treated with HCl aqueous solution and followed with Et₃N, differentiation of the 2,3-diols was efficiently achieved along with the generation of a convertible anomeric hydroxyl group. In addition, an easy and practical method based on NOE was proposed to determine whether the 1,2-orthoesters were endo-type or exo-type.     

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

The ¹H- and ¹³C-n.m.r. spectra of the anomeric methyl (methyl d-galactosid)uronates, as well as the ¹H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d-galactopyranosiduronates showed good correlation with those of the corresponding anomeric d-galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d-galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d-galactopyranosid)uronates and their corresponding peracetates are in the ⁴C₁(d) conformation. The chemical shifts in the ¹³C-n.m.r. spectra show good correlation with those of the methyl d-galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.     

Stereoselective glycosylations using benzoylated glucosyl halides with inexpensive promoters

Reactions of O-benzoylated glucopyranosyl halide (I, Br), isolated or generated in situ from per-benzoylated glucose (8a) and trimethylsilyl halide, with various alcohols were efficiently promoted by zinc halide (Cl, Br) or N-bromosuccinimide with a catalytic ZnI₂ to give the corresponding 1,2-trans-β-glucosides in good to high yields. When the anomeric halogenation of 8a was carried out in the presence of reactive alcohols, 1,2-cis--glucosides were selectively formed.     

D-Galactose 6-phosphate: anomeric distribution and analysis of its synthesis from D-galactose and polyphosphoric acid

D-Galactose 6-phosphate as synthesized by direct phosphorylation of D-galactose with polyphosphoric acid is contaminated with two of its positional isomers. These were separated from D-galactose 6-phosphate and from each other, and identified as D-galactose 3- and 5-phosphate by enzymic, chromatographic, and mass-spectral analysis. The previous misidentification of these isomers as furanose forms of D-galactose 6-phosphate has led to erroneous reports concerning the anomeric distribution of D-galactose 6-phosphate. The anomeric distribution of D-galactose 6-phosphate in a purified preparation was determined by gas-liquid chromatography and ¹³C-n.m.r. spectroscopy to be 32% α-pyranose, 64% β-pyranose, and no more than 4% furanose anomers.     

Synthesis of anomeric 1,5-anhydrosugars as conformationally locked selective α-mannosidase inhibitors

Anomeric 1,5-anhydrosugar 2 was synthesized from d-glucose derived N-Cbz protected aminodiol 8. The key step involves, acid catalyzed hydrolysis of 1,2-acetonide group in 8 to get hemiacetal that concomitantly undergoes formation of the pyranose ring by attack of C-3 hydroxyethyl group on anomeric C-1, leading to the formation of dioxabicyclo[3.2.1]octane skeleton which on hydrogenolyis gave 2. The glycosidase inhibitory activities of hydroxy- and amino-substituted anomeric 1,5-anhydrosugars 1 and 2, respectively, showed selective inhibition of α-mannosidase. These results were substantiated by molecular docking studies using WHAT IF software and AUTODOCK 4.0 program.     

A novel synthesis of D-galactofuranosyl,D-glucofuranosyl and D-mannofuranosyl 1-phosphates based on remote activation of new and free hexofuranosyl donors

The selective synthesis of 1,2-cis-hexofuranosyl 1-phosphates was readily accomplished according to a procedure based on the 'Remote Activation Concept'. This approach required (i) the preparation of suitable 1,2-trans-hexofuranosyl donors, so that new heterocyclic thiofuranosides were designed and synthesized, (ii) the stereocontrolled phosphorylation of the corresponding unprotected donors and (iii) the simple and fast purification of the resulting anomeric phosphates. This approach showed to be equally efficient in the galactose, glucose and mannose series.     

Determining the solution conformational entropy of O-linked oligosaccharides at quasi-physiological conditions: size-exclusion chromatography and molecular dynamics

The physiological functions of oligosaccharides are influenced by a number of structural parameters such as anomeric configuration, glycosidic linkage, and degree of polymerization. These parameters affect the conformation of the oligosaccharides which, in turn, is responsible for characteristics such as aptameric and enzymatic binding, chiral recognition, and the structural targeting of bacterial and parasitic recognition events. Here, we measure the solution conformational entropy (DeltaS) of two series of oligosaccharides, linear malto- and cellooligosaccharides, using size-exclusion chromatography (SEC). For each series, we have determined DeltaS as a function of degree of polymerization (DP). The choice of oligosaccharides studied also allowed us to compare the influence of anomeric configuration on DeltaS, and to do so as a function of DP. Studies were conducted in water at physiological temperature and pH in order to resemble conditions within the human body. Experimental results were augmented with results from molecular dynamics computer modeling simulations in aqueous solvent. A comparison between experimental and computational data showed how the techniques can complement each other. An example of the latter is the considerable enthalpic contribution to the chromatographic separation of alpha- and gamma-cyclodextrin, which may have gone unnoticed if not for the large discrepancy between the results obtained by the separate techniques.     

alpha-L-iduronate ring conformations in heparin and heparin derivatives. 13-C Nuclear-magnetic-resonance analysis and titration data for variously desulphated and periodate-oxidized heparins.

A heparin derivative that had been O/N-desulphated and re-N-acetylated was investigated by 13C n.m.r. spectroscopy and potentiometric titration. Three forms of uronic acid were observed, tentatively identified as beta-D-glucuronate, and two different forms of alpha-L-iduronate. A comparison of the n.m.r. spectra of heparin, an oligosaccharide (beta-D-glucuronate-2-acetamido-2-deoxy-alpha-D-glucose)n, and heparin that had been subjected to selective oxidation of beta-D-glucuronate, enabled the position of the anomeric carbon of the latter residue to be assigned [delta 102.9 (p.p.m.)]. Periodate oxidation of O/N-desulphated heparin destroyed in addition, approx. 40% of the alpha-L-iduronate content. The remainder of the alpha-L-iduronate residues displayed only one anomeric resonance, at delta 99.7 (p.p.m.). In another preparation, after sequential desulphation of heparin (N-desulphation, re-N-acetylation and O-desulphation) the anomeric resonance of the alpha-L-iduronate residue shifted downfield [from delta99.7 (p.p.m.) to delta 102.3]indicating a change in ring conformation. These data support the interpretation that the unsulphated alpha-L-iduronate residues may adopt two conformations. It was shown that the proportions of alpha-L-iduronate conformers are determined by the sequence of desulphation operations. Also minor components of heparin were assigned.     

Condensation reactions between 2-phenylthio-2,3-dideoxyribose derivatives and nucleic bases.

The effects of phenylthio group at C-2 of 2,3-dideoxyribose were examined for the stereoselectivities in condensation reactions between sugars and pyrimidine bases. In the case of mono-PhS sugar, the 1,2-trans-anomers were mainly obtained in the ratio of 9:1. Condensation reactions with di-PhS-sugar gave the anomeric mixtures of alpha:beta = 2:8.     

Hemicellulose-based multifunctional macroinitiator for single-electron-transfer mediated living radical polymerization

A multifunctional macroinitiator for single-electron-transfer mediated living radical polymerization (SET-LRP) was designed from acetylated galactoglucomannan (AcGGM) by α-bromoisobutyric acid functionalization of the anomeric hydroxyl groups on the heteropolysaccharide backbone. This macroinitiator, with a degree of substitution of 0.15, was used in the SET-LRP of methyl acrylate, catalyzed by Cu(0)/Me(6)-TREN in DMSO, DMF, or DMSO/H(2)O in various concentrations. Kinetic analyses confirm high conversions of up to 99.98% and a living behavior of the SET-LRP process providing high molecular weight hemicelluloses/methyl acrylate hybrid copolymers with a brush-like architecture.     

1H- and 13C-n.m.r.-spectral study of synthetic methyl d-manno-oligosaccharides

¹H- and ¹³C-n.m.r. spectra for 16 synthetic methyl manno-oligosaccharides were recorded, and the signals for the anomeric protons and anomeric carbon atoms in branched manno-pentaosides and -hexaosides were assigned, based on the data for methyl manno-biosides and -triosides. These n.m.r. data identified the branching pattern of high-mannose types of glycans of glycopeptides with those of unambiguously synthesized manno-oligosaccharides, and confirmed the structures proposed for such glycans.     

Sugar-linked biodegradable polymers: Regio-specific ester bonds of glucose hydroxyls in their reaction with maleic anhydride functionalized polystyrene and elucidation of the polymer structures formed

In the development of sugar-linked synthetic polymers as biodegradable polymers, it is imperative to know the variety of polymer structures formed by the reaction of a multi-functional sugar molecule with the functionalized synthetic polymer on which the sugar is to be anchored. Enzymes produced by the microorganisms causing the polymer to biodegrade can be sensitive to the particular type of sugar hydroxyl utilized (such as anomeric, primary, or secondary hydroxyl group) for getting anchored to the polymer. In this paper, we present synthesis of regio-specific ester derivatives of glucose with anhydride, functionalized polymers, i.e., ester formation specifically with the anomeric, primary or secondary hydroxyls of glucose. Characterization of these different esters groups was done using FTIR spectroscopy; each ester peak was further deconvoluted to yield its different components. For this purpose, we studied the reactions of d-glucose, 6-O-trityl glucose, methyl glucoside, 1,2-5,6-diisopropylidene-d-glucose, and 1,2,3,4-tetraacetyl-d-glucose with maleic anhydride functionalized polystyrene (PSMAH). In this study, the primary hydroxyl of glucose was found to be even more reactive than the anomeric hydroxyl. The peaks at 1716, 1725, and 1729–1737 cm⁻¹ were assigned to the ester carbonyl of the anomeric, primary, and secondary hydroxyls of glucose (C2, C3, and C4), respectively. An attempt was made to quantify the extent to which the different polymer structures are formed in a particular reaction by taking ratios of non-variable reference peaks (polystyrene peak at 1493 cm⁻¹) and variable peaks caused by the reaction (the residual anhydride carbonyl at 1780 cm⁻¹).     

Chemical synthesis of a (1→2)-d-glucopyranan

1,2-Anhydro-3,4,6-tri-O-benzyl-α-d-glucopyranose was polymerized with a number of Lewis acids. Phosphorus pentafluoride at ?60° caused polymerization to a product rich in β linkages. Other Lewis acids at higher temperatures gave perbenzylated polysaccharides of lower molecular weight with less stereoselectivity. Debenzylation of the most-regular derivative gave a polysaccharide whose specific rotation was +14.7° and whose ¹³C-n.m.r. spectrum had six absorptions corresponding to those of natural (1→2)-β-d-glucopyranans and additional minor peaks presumably due to some α-anomeric configurations. It was estimated to have ～90% of β linkages.     

A 2D-1H-n.m.r. study of some Shigella flexneri O-polysaccharides

A 1H-n.m.r. study of the O-polysaccharides from different types of Shigella flexneri has been performed. With the aid of 2D-n.m.r. techniques, namely, J-resolved, spin-spin correlation, and NOESY experiments, most of the structural features of these polysaccharides could be deduced. Sequences could generally be obtained from the NOESY experiments. When using a prolonged mixing time in these experiments, cross-peaks due to spin diffusion from one anomeric proton to the anomeric proton of an adjacent residue could be obtained, thereby giving complementary sequence information.     

Dependence of 13C chemical shifts on the spatial interaction of protons,and its application in structural and conformational studies of oligo- and poly-saccharides

Regularities in the variation of chemical shifts and the glycosidation effects in the ¹³C-n.m.r. spectra of disaccharides were found to depend on the configuration at the anomeric centre of the glycosidating pyranose, and the absolute configuration of both pyranoses moieties. These empirical regularities are explained in terms of the spatial proton-proton interactions within the statistically averaged, or preferred, conformation near the glycosidic linkage. The applicability of these effects for the determination of the anomeric and absolute configuration and the sequence of pyranose residues in oligo- and poly-saccharides is discussed. The conformational properties of glycosidic linkages in disaccharides and disaccharide fragments of oligo- and poly-saccharides are compared on the basis of ¹³C-n.m.r. data.