Antagonists of the myelin-associated glycoprotein: a new class of tetrasaccharide mimics

The tetrasaccharide substructure 1 of the ganglioside GQ1balpha shows a remarkable affinity for the myelin-associated glycoprotein (MAG). In the search for structurally simplified and pharmacokinetically improved mimics of 1, biphenyl was identified as a feasible replacement for the core disaccharide Galbeta(1-3)GalNAc according to saturation transfer difference (STD) NMR and molecular modeling investigations. Using Suzuki coupling, a convergent synthesis of the mimics was achieved. To optimize the yields of the coupling reactions, the catalytic effects of microwave irradiation and conventional heating were compared. The biological evaluation of mimics 3 and 4 was performed in a competitive target-based assay. It was found that the relative inhibitory potency (rIP) of antagonist 3 was clearly enhanced in comparison to the reference trisaccharide 2, despite the former having a much simpler structure. In addition to the improved synthetic feasibility, an increase of the partition coefficient between octanol and water (logP), and therefore a beneficial change in the pharmacokinetic properties of 3 and 4 was achieved.     

Synthesis of the alpha-D-GlcpA-(1-->3)-alpha-L-Rhap-(1-->2)-L-Rha trisaccharide isolated from the cell wall hydrolyzate of the green alga, Chlorella vulgaris

The title trisaccharide was synthesized from 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl chloride (10), ethyl 2,4-di-O-benzyl-1-thio- (5) and benzyl 3,4-di-O-benzyl-alpha-L-rhamnopyranoside (9). The disaccharide 11 obtained from compounds 5 and 10 was used as the glycosyl donor to glycosylate the rhamnopyranoside derivative 9 having free OH-2 using the NIS-AgOTf-mediated glycosylation methodology. Zemplén deacetylation of the trisaccharide 12 resulted in the 6"-OH derivative (13), which was selectively oxidized with CrO3 to the uronic acid derivative 14. The benzyl groups were removed by catalytic hydrogenolysis to furnish the target trisaccharide (1).     

Enzymatic synthesis and characterization of oligosaccharides structurally related to the repeating unit of Pullulan

A trisaccharide (Glcalpha1-4Glcalpha1-6Glc) and a tetrasaccharide (Glcalpha1-4Glcalpha1-4Glcalpha1-6Glc) the structures of which are related to that of repeating unit of pullulan have been obtained, exploiting the transglycolytic activity of Aspergillus niger cyclodextrin glucanotransferase. Both products were obtained in one-pot reaction using as a donor the alpha-cyclodextrin and as an acceptor the disaccharide isomaltose. The regioselectivity of the reaction was 85% for the tetrasaccharide and 80% for the trisaccharide. The yield of reaction resulted to be 42% for the synthesis of trisaccharide and 25% for that of tetrasaccharide. Purification of products was performed by size exclusion chromatography and by semipreparative reverse phase HPLC after reversible derivatization with 2-aminopyridine. Structural characterization was performed by capillary electrophoresis, ion-spray mass spectrometry, and by 13C-NMR spectroscopy. A comparison of these results with those obtained by using alpha-D-glucosidase, which had been effective for the synthesis of the disaccharide isomaltose, is reported.     

Synthesis of an alpha-linked dimer of the trisaccharide repeating unit of the exopolysaccharide produced by Pediococcus damnosus 2.6

A hexasaccharide, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->2)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->2)]-D-Glcp, the alpha-linked dimer of the trisaccharide repeating unit of the exopolysaccharide produced by Pediococcus damnosus 2.6, was synthesized as its methyl glycoside. Condensation of fully benzoylated alpha-D-glucopyranosyl trichloroacetimidate (1) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (2) selectively furnished (1-->3)-linked disaccharide 3, and subsequent 2-O-acetylation, desulfation, and trichloroacetimidate formation afforded the disaccharide donor 6. Meanwhile, selective 3-O-coupling of methyl 4,6-O-benzylidene-alpha-d-glucopyranoside (8) with 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (7), followed by coupling with 1 gave the trisaccharide 10. Removal of the benzylidene group of 10, benzoylation, and deallylation produced the trisaccharide acceptor 12. Condensation of 12 with 6 yielded a pentasaccharide mixture 13 with beta and alpha isomers in a ratio of 2:1. Removal of the benzylidene group of 13, followed by benzoylation gave the pentasaccharide mixture 14. Selective 2'-deacetylation of the isolated beta-linked 14beta with MeCOCl/MeOH/CH2Cl2 did not give the expected pentasaccharide acceptor, and serious decomposition occurred, indicating a large steric hindrance at C-2'. Alternatively, 2,3-di-O-glycosylation of allyl 4,6-O-benzylidene-beta-D-glucopyranoside (21) with 1 gave 22, then deallylation and trichloroacetimidate formation afforded the trisaccharide donor 24. Condensation of 12 with 24 furnished only the alpha-linked hexasaccharide 25, and its deprotection gave the free hexaoside 27.     

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.     

Synthetic mucin fragments: methyl 3-O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl-beta-D- 3-O-(2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl- beta-D-glucopyranosyl)-beta-D-galactoyranoside. pyranosyl)-beta-D-galactopyranoside

Methyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (5) was obtained crystalline by way of its 3-O-allyl derivative, which was in turn obtained by ring-opening of a presumed 3,4-O-stannylene derivative of methyl beta-D-galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-beta-D-glucopyra no)-[2,1-d]-2-oxazoline in 1,2-dichloroethane in the presence of p-toluenesulfonic acid afforded the disaccharide derivative methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2, 4,6-tri-O-benzyl-beta-D-galactopyranoside (6) Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7, which was acetalated with alpha, alpha-dimethoxytoluene to afford the 4',6'-O-benzylidene acetal (10). Catalytic hydrogenolysis of the benzyl groups of 7 afforded the title disaccharide 8. Glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave the fully protected trisaccharide derivative 12. Systematic removal of the protecting groups of 12 then furnished the title trisaccharide 14. The structures of 5, 8, and 14 were all confirmed by 13C-n.m.r. spectroscopy. The 13C-n.m.r. chemical shifts for methyl alpha- and beta-D-galactopyranoside, and also those of their 3-O-allyl derivatives, are recorded, for the sake of comparison, in conjunction with those of compound 5.     

Synthesis and evaluation of cardiac glycoside mimics as potential anticancer drugs

The cardiac glycoside digitoxin, consisting of a steroid core linked to a labile trisaccharide, has been used for centuries for the treatment of congestive heart failure. The well known pharmacological effect is a result of the ability of cardiac glycosides to inhibit the Na(+), K(+)-ATPase. Within recent years cardiac glycosides have furthermore been suggested to possess valuable anticancer activity. To mimic the labile trisaccharide of digitoxin with a stabile carbohydrate surrogate, we have used sulfur linked ethylene glycol moieties of varying length (mono-, di-, tri- or tetra-ethylene glycol), and furthermore used these linkers as handles for the synthesis of bivalent steroids. The prepared compounds were evaluated for their potencies to inhibit the Na(+), K(+)-ATPase and for their cytotoxic effect on cancerous MCF-7 cells. A clear trend is observed in both inhibition and cytotoxic effect, where the bioactivity decreases as the size increases. The most potent Na(+), K(+)-ATPase inhibitors are the compounds with the shortest ethylene glycol chain (K(app) 0.48 μM) and thiodigitoxigenin (K(app) 0.42 μM), which both are comparable with digitoxigenin (K(app) 0.52 μM). For the cancer cell viability assay the shortest mimics were found to have highest efficacy, with the best ligand having a monoethylene glycol unit (IC(50) 0.24 μM), which was slightly better than digitoxigenin (IC(50) 0.64 μM), while none of the novel cardiac glycoside mimics display an in vitro effect as high as digitoxin (IC(50) 0.02 μM).     

汉麻籽不同萌发时期的生长活性物质非靶向代谢组学研究

为了使汉麻籽在食品工业中发挥其最大价值,找寻汉麻籽在萌发过程中的最佳处理时期十分重要。本研究利用非靶向代谢组学研究方法和液相色谱-质谱(LC-MC)联用技术分析汉麻籽在发芽12及24 h的代谢物数量,结合多元统计分析方法与单变量统计分析,筛选汉麻籽三个时期组间的显著性差异代谢物(未发芽与发芽12 h的汉麻籽,未发芽与发芽24 h的汉麻籽,发芽12 h与发芽24 h的汉麻籽)。结果表明在正、负离子监测模式下,三个时期共发现显著性差异代谢物26个,第一组中16种表现为上调,8种表现为下调;第二组中13种表现为上调,9种表现为下调;第三组中13种表现为上调,8种表现为下调。其中,在汉麻籽发芽12 h时二羟基丙酮(DHA),4-羟基丁酸内酯,4-氨基丁酸,嘧啶等表现为上调;D-脯氨酸,烟酸酯,L-脯氨酸等表现为下调,且其变化倍数远远大于其他成分,影响作用较明显。本研究为萌发汉麻籽在食品开发中的应用提供参考。     

Detection of Kestoses and Kestose-Related Oligosaccharides in Extracts of Festuca arundinacea, Dactylis glomerata L., and Asparagus officinalis L. Root Cultures and Invertase by C and H Nuclear Magnetic Resonance Spectroscopy

A previous study (KL Forsythe, MS Feather [1989] Carbohydr Res 185: 315-319) showed that ¹³C nuclear magnetic resonance spectroscopy can be used to detect and identify mixtures of 1-kestose and neokestose after conversion to the acetate derivatives. In this study, unequivocal assignments are made for the anomeric carbon and proton signals for the above two trisaccharide acetates as well as for 6-kestose hendecaacetate and for nystose tetradecaacetate (a 1-kestose-derived tetrasaccharide). A number of oligosaccharide fractions were isolated from several plant species, converted to the acetates, and nuclear magnetic resonance spectra obtained. Using the above reference data, the following information was obtained. The trisaccharide fraction from Dactylis glomerata L. stem tissue and Asparagus officinalis L. roots contain both 1-kestose and neokestose, and the tetrasaccharide fractions contain three components, one of which is nystose. Penta- and hexasaccharide acetates were also isolated from A. officinalis L. roots and were found to contain, respectively, four and at least five components. All components of both of the above species appear to contain a kestose residue and to be produced by the sequential addition of fructofuranosyl units to these. The trisaccharide fraction from Festuca arundinacea is complex, and contains at least five different components, two of which appear to be 1-kestose and neokestose.     

A facile regio- and stereoselective synthesis of mannose octasaccharide of the N-glycan in human CD2 and mannose hexasaccharide antigenic factor 13b

A highly concise and effective synthesis of the mannose octasaccharide of the N-linked glycan in the adhesion domain of human CD2 was achieved via TMSOTf-promoted selective 6-glycosylation of a trisaccharide 4,6-diol acceptor with a pentasaccharide donor, followed by deprotection. The pentasaccharide was constructed by selective 3,6-diglycosylation of 1,2-O-ethylidene-beta-D-mannopyranose with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, while the trisaccharide was obtained by selective 3-O-glycosylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside with the same disaccharide trichloroacetimidate, followed by debenzylidenation. The mannose hexasaccharide antigenic factor 13b was synthesized by condensation of a trisaccharide donor, 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, with a trisaccharide acceptor, methyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside, followed by deprotection.     

Application of new, high-sensitivity, 1H-13C-n.m.r.-spectral techniques to the study of oligosaccharides

Four n.m.r. methods that are especially useful for characterization of oligosaccharides are applied to the trisaccharide alpha-Neu5Ac-(2----3)-beta-Gal-(1----4)-Glc (1). Three of these are two-dimensional, heteronuclear methods that provide chemical-shift correlation maps having much higher sensitivity than was previously possible, because they rely on indirect observation of 13C via 1H detection. These methods are used to assign, completely, the 1H- and 13C-n.m.r. spectra of both anomers of the trisaccharide. In addition to these two-dimensional methods, a one-dimensional method is used to measure 1H-1H coupling-constants accurately within each sugar ring. The values of the coupling constants thus measured for 1 are evidence that the conformations of the individual sugar rings are not affected by linkage into the trisaccharide.     

Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.     

Alpha-galactose based neoglycopeptides. Inhibition of verotoxin binding to globotriosylceramide

Solution and solid phase strategies for the synthesis of alpha-galactose based neoglycopeptide derivatives 2-13 were developed. Neoglycopeptides generated were tested for the inhibition of verotoxin binding to globotriosylceramide (Gb3) using ELISA. Among all of the compounds tested, only the lipid derivatives of neoglycopeptides, 11, 12 and 13 were found to be inhibitors, IC50 = 2.0 mM (11b and 12c) and 0.2 mM (11c and 13c). All of the inhibitors (11b, 11c, 12c and 13c) have a similar branching of the two alpha-galactosyl units at the N-terminal glycine residue of a short peptide and a lipid moiety attached at the C-terminal site. Both of these factors seem to be crucial for the inhibition. It is interesting to note that the inhibitors have only a portion of the natural trisaccharide ligand. The secondary groups either may contribute in sub-site oriented interactions with the protein receptors or may mimic the internal sugar units of the cell-surface ligand, Gb3.     

Isolation of a trisaccharide containing 2-amino-2-deoxy-D-galacturonic acid from the Bordetella pertussis endotoxin

4-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl-6-O-(2-amino-2-deoxy-alpha-D-galactopyranuronyl)-D-glucopyranose, a branched-chain trisaccharide, was isolated after hydrolysis of Bordetella pertussis endotoxin with 4 M HCl for 1 h at 100 degrees C. The trisaccharide was present in both polysaccharide moieties of the two constituent lipopolysaccharides of this endotoxin. Its structure was established by analysis of the 400-MHz nuclear magnetic resonance spectrum and by chemical and enzymatic degradation.     

Synthesis of the trisaccharide portion of soyasaponin beta g: evaluation of a new glucuronic acid acceptor

The synthesis of the trisaccharide portion of soyasaponin beta g was successfully achieved using a new glucuronic acid acceptor: methyl 1-O-allyl-3,4-di-O-methoxymethyl-beta-D-glucuronate (9). This compound and methyl 1-O-allyl-3,4-di-O-tert-butyldimethylsilyl-beta-D-glucuronate (8) were both prepared from glucuronolactone via a glycal intermediate. The former compound 9 was successfully coupled to ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-beta-D-galactopyranoside (13) in excellent yield. Synthesis of the protected trisaccharide was then completed by the addition of a suitably protected rhamnose derivative to the disaccharide portion. The reactivity of the glucuronic acid derivative 9 was also explored with trichloroacetimidate and fluoride donors.     

New conformational constraints in isotopically (13C) enriched oligosaccharides

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.      

稻螟赤眼蜂诱导滞育条件的优化

应用滞育技术是延长赤眼蜂货架期的可行性措施之一。为实现工厂化生产稻螟赤眼蜂滞育蜂,本研究以在3种光周期(16 L∶8 D、12 L∶12 D和8 L∶16 D)下繁育的稻螟赤眼蜂种群为材料,考察了诱导始期(寄生后发育12 h、24 h、36 h、48 h、60 h和72 h)、诱导温度(10℃和13℃)和诱导历期(14 d、21 d、28 d和35 d)对稻螟赤眼蜂滞育的影响。研究结果表明短光照下繁育的种群更容易进入滞育状态;寄生后发育24 h和12 h为最敏感的诱导始期,随着诱导始期虫龄的增长,滞育效果也随之下降;诱导温度对滞育也有显著影响,其中8 L∶16 D的种群在13℃下诱导的滞育率要显著高于10℃,但其他两个种群在10℃下诱导的滞育率要显著高于13℃;诱导历期的增加可以显著提高稻螟赤眼蜂的滞育率。供试稻螟赤眼蜂种群的较适宜滞育条件为8 L∶16 D下繁育的赤眼蜂种群寄生后发育12 h于13℃下诱导滞育28 d或35 d,以及寄生后发育24 h于10℃或13℃下诱导滞育35 d,滞育率可超过95%。     

Engineering Escherichia coli to produce Bordetella pertussis oligosaccharide with multiple trisaccharide units

The immunogenicity of the pertussis vaccine can be significantly improved by adding Bordetella pertussis oligosaccharide with multiple trisaccharide units. The more trisaccharide units there are, the better the efficiency of the immune response induction. However, natural B. pertussis oligosaccharides usually contain only a single terminal trisaccharide unit. In addition, B. pertussis is pathogenic, and there are potential safety hazards when preparing oligosaccharides from B. pertussis. In this study, Escherichia coli MG1655 was engineered to produce B. pertussis oligosaccharides containing multiple trisaccharide units. Fifty-nine genes relevant to the biosynthesis of the O-antigen and core oligosaccharide of lipopolysaccharide, enterobacterial common antigen, and colanic acid were deleted in MG1655, resulting in strain MDCO020. Then, 25 genes relevant to the biosynthesis of the oligosaccharide antigen in B. pertussis and 3 genes relevant to the repeating trisaccharide unit in Pseudomonas aeruginosa PAO1 were overexpressed in MDCO020, resulting in the recombinant E. coli MDCO020/pWpBpD5. The production of B. pertussis oligosaccharide with multiple trisaccharide units by MDCO020/pWpBpD5 was confirmed by SDS-PAGE and ¹H NMR analyses, and its immune response-stimulating activity was confirmed by using rabbit anti-pertussis serum.     

A Haemophilus influenzae strain associated with Fisher syndrome expresses a novel disialylated ganglioside mimic

The non-typeable Haemophilus influenzae strain DH1 was isolated from a 25 year old male patient with Fisher syndrome, a postinfectious autoimmune condition characterized by the presence of anti-GQ1b IgG antibodies that target and initiate damage to peripheral nerves. DH1 was found to display an alphaNeuAc(2-8)alphaNeuAc(2-3)betaGal branch bound to the tetraheptosyl backbone core of its lipooligosaccharide (LOS). The novel sialylation pattern was found to be dependent on the activity of a bifunctional sialyltransferase, Lic3B, which catalyzes the addition of both the terminal and subterminal sialic acid residues. Patient serum IgGs bind to DH1 LOS, and the reactivity is significantly influenced by the presence of sialylated glycoforms. The display by DH1, of a surface glycan that mimics the terminal trisaccharide portion of disialosyl-containing gangliosides, provides strong evidence for its involvement in the development of Fisher syndrome.     

Conformational analysis of an alpha-galactosyl trisaccharide epitope involved in hyperacute rejection upon xenotransplantation.

alpha-Galactosyl epitopes are carbohydrate structures bearing an alpha-Gal-(1-->3)-Gal terminus (alpha-Gal epitopes). The interaction of these epitopes on the surface of animal cells with anti alpha-Gal antibodies in human serum is believed to be the main cause in antibody-mediated hyperacute rejection in xenotransplantation. In this paper, conformational analysis of an N-linked alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp trisaccharide epitope was conducted in terms of each monosaccharide residue conformation, primary hydroxymethyl group configuration, and interglycosidic conformations. Selective 2D J-delta INEPT experiments have been carried out at three different temperatures to evaluate three-bond, long-range 13C-1H coupling constants for the crucial alpha-(1-->3) linkage. The NMR experimental data were complemented by theoretical calculations. The flexibility and dynamics of the trisaccharide have been studied by Metropolis Monte Carlo simulations. Ensemble-averaged three-bond, long-range 13C-1H coupling constants and nuclear Overhauser effects were in good agreement with the experimental data. The alpha-(1-->3) glycosidic linkage has shown a restricted flexibility as indicated by NMR spectroscopy and molecular modeling.