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PubMed1992

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.

Carbohydrate Conformation↗
PubMed1989

High-performance liquid chromatographic identification of disaccharides generated from heparan sulphate isomers using heparitinases.

Specific heparan sulphate-lyases, heparitinases I and II, were used to identify unsaturated disaccharide constituents generated from heterogeneous heparan sulphate isomers. All determinations were made using high-performance liquid chromatography with a column containing a sulphonized styrene-divinylbenzene copolymer. Unsaturated disaccharides generated from variously sulphated heparan sulphate isomers after simultaneous digestion with heparitinases I and II facilitated separation of the individual disaccharides, based on sulphate groups at the specific position of the uronic acid and glucosamine residues. The simultaneous digestion with heparitinases I and II produces unsaturated disaccharides from heparan sulphate isomers with the structure of 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-amino-deoxy-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-aminodeoxy-6-O-sulpho-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-6-O-sulpho-D-glucose, 4-deoxy-2-O-sulpho-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-amino-2-deoxy-6-O-sulpho-D-glucose and 4-deoxy-2-O-sulpho-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-6-O-sulpho-D-glucose.

Chromatography, High Pressure Liquid↗
PubMed2000

Detection and quantification of the sulfated disaccharides in chondroitin sulfate by electrospray tandem mass spectrometry.

A new method of identifying and quantifying the disaccharide building blocks of glycosaminoglycans is introduced. The polysaccharides are subjected to an enzymatic digestion that releases the sulfated disaccharides. The disaccharides are then identified using a combination of electrospray ionization mass spectrometry and tandem mass spectrometry. Quantification of the isomeric disaccharides is also achieved by tandem mass spectrometry, using a recently developed methodology which quantifies mixtures of isomers without the use of chromatography or prior separation. Using mass spectrometry to characterize the components of glycosaminoglycans significantly reduces both sample consumption and analysis time of traditional methods.

Chondroitin ABC Lyase↗
PubMed1989

Structure elucidation methodology for disaccharides based on carbon-13 nuclear magnetic resonance spectrum simulation.

Molecular mechanics techniques are used to derive structural parameters that allow carbon-13 nuclear magnetic resonance spectra of disaccharides to be modeled. On the basis of a collection of 40 disaccharide spectra, four linear models are derived that allow complete spectra to be simulated. On the basis of these 40 compounds, the average difference between simulated and observed chemical shifts is +/- 0.45 ppm. The majority of structural parameters used are based on interatomic distances computed from modeled three-dimensional atomic coordinates. Found particularly useful in modeling the disaccharide spectra are the effects of oxygen electron lone pairs and encodings of the structural environments of hydrogens alpha to the carbon whose chemical shift is being predicted. The computed models are examined, and their predictive ability is assessed. The potential application of the methodology to the structure elucidation of disaccharides is demonstrated.

Carbohydrate Conformation↗
PubMed2004

Glycosaminoglycan disaccharide alters the dimer dissociation constant of the chemokine MIP-1 beta.

Chemokines are immune system proteins that recruit and activate leukocytes to sites of infection. This recruitment is believed to involve the establishment of a chemokine concentration gradient by the binding of chemokines to glycosaminoglycans (GAGs). In previous studies, we elucidated the GAG binding site of the chemokine MIP-1beta and implicated the involvement of the chemokine dimer in GAG binding through residues across the dimer interface. In the present studies, nuclear magnetic resonance spectroscopy was used to investigate the effect of GAG binding on MIP-1beta dimerization. Using several dimerization-impaired variants of MIP-1beta (F13Y, F13L, L34W, and L34K), these studies indicate that the addition of disaccharide to the mutants increases their dimerization affinities. For MIP-1beta F13Y, the presence of the disaccharide increases the chemokine dimerization affinity about 9-fold as evidenced by a decrease in the dimer dissociation constant from 610 to 66 microM. Even more dramatically, the dimerization affinity of MIP-1beta L34W also increases upon addition of disaccharide, with the dimer dissociation constant decreasing from 97 to 6.5 microM. After this effect for the mutants of MIP-1beta was shown, similar experiments were conducted with the CC chemokine RANTES, and it was demonstrated that the presence of disaccharide increases its dimerization affinity by almost 7-fold. These findings provide further evidence of the importance of the dimer in chemokine function and provide the first quantitative investigation of the role of GAGs in the manipulation of the MIP-1beta quaternary structure.

Amino Acid Substitution↗
PubMed2005

Influence of disaccharide structure on prebiotic selectivity in vitro.

To obtain structure-function information of a range of carbohydrates, which are available only in very small quantities, an in vitro fermentation method using 7 mg of carbohydrate, 0.7 mL of basal medium, and 1% (w/v) of fecal bacteria was validated against a pH-controlled batch culture with 150 mL of basal medium and 1.5 g of test carbohydrate. This method was used to determine the influence of different glycosidic linkages and monosaccharide compositions of disaccharides on the selectivity of microbial fermentation. A prebiotic index (PI) was calculated for each disaccharide. Generally, disaccharides with linkages of 1-2, 1-4, and 1-6 generated a high PI score, with kojibiose and sophorose showing the greatest values (21.62 and 18.63, respectively). Apart from 6alpha-mannobiose, mannose-containing disaccharides gave a low PI due to low numbers of bifidobacteria and lactobacilli and an increase in bacteroides. The structure-function information obtained in this study may lead to a predictive understanding of how specific structures are fermented by the human gut microflora.

Bacteria↗
PubMed2005

Computational study of the dynamics of mannose disaccharides free in solution and bound to the potent anti-HIV virucidal protein cyanovirin.

In this paper, we present a computational study of the dynamics of the potent anti-HIV virucidal protein cyanovirin in complex with mannose disaccharides. Recently, it has been experimentally demonstrated that cyanovirin binds mannose oligomers on the surface of glycoprotein gp120. gp120, a protein on the surface of the HIV virus, is key in the process of viral docking and transfer of genetic material into human cells. Cyanovirin prevents the transfer of viral RNA into human cells. In this study, we found that, among all residues that show nuclear Overhauser effects in the solution NMR experiments, residues Glu41 and Arg76 appear to interact with the sugar at the high-affinity binding site through stronger Coulombic interactions. In particular, Arg76 participates in a dynamical mechanism that caps and locks the sugar once it is bound to the protein. We also studied the distribution of glycosidic torsional angles of mannose disaccharides in solution and compared it with those when bound at the high- and low-affinity sites of the protein. Throughout our 20 ns simulations, we find that the sugar bound to the high-affinity site preserves the most favorable conformation in solution while the sugar bound at the low-affinity site does not. The sugar at the low-affinity site can adopt both conformations, but we find it most predominantly on the one that is least probable for the free sugar in solution. We also carried out a detailed study of the interactions between the disaccharides and different amino acids as well as between the disaccharide and the solvent at both binding locations.

Anti-HIV Agents↗
PubMedDate not supplied

[Synthesis of alpha- and beta-glycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone].

The synthesis of thioglycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone was studied using the glycosylation of a series of suitably protected 3-monohydroxy- and 3,4-dihydroxyderivatives of phenyl 2-azido-2-deoxy-1-thio-alpha- and 1-thio-beta-D-galactopyranosides by galactosyl bromide, fluoride, and trichloroacetimidate. In the reaction with the monohydroxylated glycosyl acceptor, the process of intermolecular transfer of thiophenyl group from the glycosyl acceptor onto the cation formed from the molecule of glycosyl donor dominated. When glycosylating 3,4-diol under the same conditions, the product of the thiophenyl group transfer dominated or the undesired (1-->4), rather than (1-->3)-linked, disaccharide product formed. The aglycone transfer was excluded when 4-nitrophenylthio group was substituted for phenylthio group in the galactosyl acceptor molecule. This led to the target disaccharide, 4-nitrophenyl 2-azido-4,5-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-beta-D-galactopyranoside, in 57% yield. This disaccharide product bears nonparticipating azide group in position 2 of galactosamine and can hence be used to form alpha-glycoside bond. 2-Azide group and the aglycone nitro group were simultaneously reduced in this product and then trichloroacetylated, which led to the beta-glycosyl donor, 4-trichloroacetamidophenyl 4,6-O-diacetyl-2-deoxy-3-O-(2,3,4,6-tetra- O-acetyl-beta-D-galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D- galactopyranoside, in 62% yield. The resulting glycosyl donor was used in the synthesis of tetrasaccharide asialo-GM1.

Carbohydrate Conformation↗
PubMed2001

Investigation of disaccharide recognition by molecularly imprinted polymers.

The selectivity of carbohydrate-imprinted polymers for several disaccharides, namely cellobiose, maltose, lactose and gentiobiose, is investigated. An ternary ligand-Cu(II)-carbohydrate complex was formed in alkaline solution and captured afterwards in the polymer. The accessibility of the polymer matrix for disaccharides was investigated by HPLC analysis, refractometry and 1H NMR spectroscopy applying excess of the original template during rebinding experiments under saturation conditions in unbuffered, aqueous solution at neutral pH and 20 degrees C. The selective discrimination of the alpha- and beta-glycosidic linkage of cellobiose and maltose is demonstrated. It is further shown, that the disaccharide-imprinted polymers slightly distinguish between the 1,4-beta- and the 1,6-beta-glycosidic linkage of cellobiose and gentiobiose, while cellobiose and lactose are not selectively recognized. Due to the weak apparent binding constant of the functional Cu(II) monomers with the targeted disaccharides at physiological pH, the recognition process is dominated by the shape of the created imprinted cavity under the applied conditions.

Carbohydrate Sequence↗
PubMed2003

A stereodivergent, two-directional synthesis of stereoisomeric C-linked disaccharide mimetics.

Dipyranones, such as 1,2-bis[(2R,3S,6S)-3-hydroxy-6-methoxy-3-oxo-6H-pyran-2-yl]ethane, were exploited as templates for the synthesis of some novel C-linked disaccharide analogues. Efficient methods, such as stereoselective reduction and dihydroxylation, were developed for two-directional functionalisation of these templates. Peracetylated derivatives of ten stereoisomeric disaccharide analogues [acetic acid 4,5-diacetoxy-6-methoxy-[(3',4',5'-triacetoxy-6'-methoxytetrahydropyran- 2'-yl)ethyl]tetrahydropyran-3-yl esters] were synthesised from a virtual library of 136 compounds; furthermore, an additional eight stereoisomers could have been synthesised simply by using the enantiomeric ligand in the enantioselective step. The ability of (2S,3S,4R,5R,6R)-6-methoxy-2-[2'-((2'R,3'R,4'S, 5'R,6'S)-3',4',5'-trihydroxy-6'-methoxytetrahydropyran-2'-yl) ethyl]tetrahydropyran-3,4,5-triol to bind to the repressor protein, LacI, was estimated to be similar to that of isopropyl-beta-thiogalactoside. The disaccharide mimetics were concluded to be a new and interesting class of C-linked disaccharide mimetics with promising, though largely unstudied, biological activity.

Bacterial Proteins↗
PubMed2000

Disaccharide analysis of skin glycosaminoglycans in atrophoderma of Pasini and Pierini.

There are divergent opinions as to whether atrophoderma of Pasini and Pierini (APP) is a nosologic entity or a primary atrophic morphoea. In this study, we used high performance liquid chromatography to analyse the skin disaccharide contents of glycosaminoglycan (GAG) in two patients with APP and compared the results with those from a typical atrophic morphoea patient. Perilesional uninvolved skin was used as a control in each patient. In the atrophic phase morphoea, both the total amount of disaccharide per skin punch-biopsy and the amount of DeltaDi-4S(DS) - the main disaccharide unit of dermatan sulphate - per mg dry weight were increased. These changes were consistent with sclerotic phase morphoea. In contrast, the total amount of disaccharide per skin punch-biopsy was decreased and the amount of DeltaDi-4S(DS) per mg dry weight was decreased or unchanged in APP. Our results suggest that GAG metabolism in APP may be unique and quite different from that in morphoea.

Adolescent↗
PubMed2002

The binding surface and affinity of monomeric and dimeric chemokine macrophage inflammatory protein 1 beta for various glycosaminoglycan disaccharides.

Chemokines comprise a family of proteins that function in the immune response to recruit leukocytes to sites of infection. This recruitment is believed to be carried out by the establishment of a chemokine gradient by the binding of chemokines to sulfated polysaccharides known as glycosaminoglycans (GAGs) located on the extracellular surface of endothelial cells. In the present studies, multidimensional NMR spectroscopy was used to study the interaction of monomeric and dimeric chemokine macrophage inflammatory protein (MIP)-1 beta variants with a series of differentially sulfated disaccharides. The data define a GAG binding surface, including both basic and uncharged residues such as Arg(18), Asn(23), Val(25), Thr(44), Lys(45), Arg(46), and Ser(47). Dissociation constants determined from these NMR studies consistently show for each disaccharide that dimeric wild type MIP-1 beta binds more tightly than monomeric MIP(9). Furthermore, analysis of the binding surface suggests that participation in the dimer of residues Met(3), Gly(4), and Ser(5) may be responsible for this higher affinity. These studies also indicate that the specificity of MIP-1 beta for particular GAG disaccharides is directly related not only to the degree of disaccharide sulfation but also to the position of the sulfate moiety, with O-sulfation at position 2 of the hexuronic acid unit and position 6 of the D-glucosamine being major determinants for binding.

Carbohydrate Sequence↗
PubMed2003

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.

Amino Acid Sequence↗
PubMed2001

Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis.

Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun. Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography. Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography. Both disaccharides were obtained in gram amounts at 90% or higher purity. Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.

Animals↗
PubMed1986

Absence of a generalized disaccharide effect in adult female rats.

Adult female Sprague-Dawley rats were either prefed ground nonpurified diet, starved 48 h, then refed a purified carbohydrate diet for 72 h or shifted from ground nonpurified diet directly to a purified carbohydrate diet for 72 h. Diets were formulated to contain 65% carbohydrate either as the disaccharides maltose or sucrose or as their respective monosaccharide equivalents glucose and invert sugar (glucose: fructose, 1:1). Alternations in hepatic glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and malic enzyme (ME) activities, relative liver size and food efficiency were determined. Rats starved and refed invert sugar had higher levels of G6PDH and ME than those red glucose, indicating a positive fructose effect. The greatest changes in hepatic enzyme activities were observed in rats consuming diets containing sucrose. Positive fructose and disaccharide effects were obtained with sucrose for all enzymes studied in both dietary shift and starve-refeed studies. No disaccharide effect was observed with maltose. In conclusion, females did not display a generalized disaccharide effect with either dietary shifting or starvation refeeding.

Animals↗
PubMed1994

Linkage position analysis of pyridylamino-disaccharides by HPLC of fluorogenic Smith degradation products.

Oligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA-oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0 degrees C for 15 min or at 4 degrees C for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4 degrees C for 40 h were purified by reversed phase HPLC, and the fraction collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0 degrees C for 15 min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product(s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney alpha-L-fucosidase.

Acetylglucosamine↗
PubMed1994

A new disaccharide Fuc alpha 1-2Man found in human urine.

Human urine collected from healthy individuals was ultrafiltered and the filtrate was gel-filtered. The fraction including disaccharides was pyridylaminated to convert the reducing sugars to fluorescent pyridylamino (PA)-derivatives. A PA-disaccharide consisting of Fuc and Man was purified by gel filtration, reversed-phase HPLC, and size fractionation HPLC. Structural analysis revealed that the disaccharide was Fuc alpha 1-2Man-PA. The disaccharide is considered to be a metabolite of unknown glycoconjugates.

Carbohydrate Sequence↗
PubMed1984

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases.

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.

Aminopyridines↗