Fungal beta-N-acetylhexosaminidases with high beta-N-acetylgalactosaminidase activity and their use for synthesis of beta-GalNAc-containing oligosaccharides

About 60 fungal strains were tested for production of extracellular beta-N-acetylhexosaminidases. A unique beta-N-acetylhexosaminidase with the beta-GalNAc-ase/beta-GlcNAc-ase ratio of 2.3-2.8 was found in the culture filtrates of some strains of Penicillium oxalicum. Addition of 20% (w/v) MgSO(4) increased the beta-GalNAc-ase/beta-GlcNAc-ase ratio to the value of 3.35. Cultivation conditions influence this ratio as well. beta-N-Acetylhexosaminidases from P. oxalicum CCF 2430 and Aspergillus oryzae CCF 1066 considerably differing in the GalNAc-ase activity were used for the synthesis of the following structures beta-D-GalpNAc-(1-->4)-D-GlcpNAc, beta-D-GalpNAc-(1-->6)-D-GlcpNAc, beta-D-GalpNAc-(1-->6)-D-GalpNAc, beta-D-GalpNAc-(1-->4)-alpha-D-GlcpNAcOAll and beta-D-GalpNAc-(1-->6)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAcOAll to demonstrate the application of these new enzymes.     

Synthesis of the O-linked pentasaccharide in glycoproteins of Trypanosoma cruzi and selective sialylation by recombinant trans-sialidase

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The synthesis of the pentasaccharide, beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-beta-D-Galp-(1-->6)-[beta-D-Galf-(1-->4)]-D-GlcpNAc and the corresponding alditol, previously isolated by reductive beta-elimination of the mucins, is described. The key step was the 6-O-glycosylation of a easily accessible derivative of beta-D-Galf-(1-->4)-D-GlcpNAc with a beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-D-Galp donor using the trichloroacetimidate method. The beta-linkage was diastereoselectively obtained by the nitrile effect. The pentasaccharide is the major oligosaccharide in the mucins of T. cruzi, G strain and presents two terminal beta-D-Galp residues for possible sialylation by TcTS. A preparative sialylation reaction was performed with its benzyl glycoside and the sialylated product was isolated and characterized. NMR spectroscopic analysis showed that selective monosialylation occurred at the terminal (1-->3) linked galactopyranose.     

Synthesis of the Lewis b hexasaccharide and squarate acid-HSA conjugates thereof with various saccharide loadings

The Lewis b hexasaccharide, alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> 3)-[alpha-L-Fucp-(1 --> 4)]-beta-D-GlcpNAc-(1 --> 3)-beta-D-Galp-(l --> 4)-beta-D-Glcp, has been synthesised using a convergent synthesis. Starting from ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside, a thioglycoside tetrasaccharide donor block, was constructed through two orthogonal glycosylations with glycosyl bromide donors. First, the galactose moiety was introduced using silver triflate as a promoter and then the two fucose residues under halide-assisted conditions. Finally, this tetrasaccharide was linked to a spacer-equipped 3I,4I-diol lactose acceptor in a DMTST-promoted coupling to give, after deprotection, the Lewis b hexasaccharide as its 2-aminoethyl spacer-equipped derivative. This was coupled to human serum albumin (HSA), using the squarate ester methodology, in various saccharide-protein ratios, to give neoglycoconjugates with different saccharide loadings in about 50%) efficiency.     

Conformational studies of blood group A and blood group B oligosaccharides using NMR residual dipolar couplings

The conformations of two synthetic trisaccharides of blood group A and B (alpha-L-Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-alpha-D-Galp and alpha-L-Fucp-(1-->2)-[alpha-D-Galp-(1-->3)]-alpha-D-Galp, respectively) and of a type A tetrasaccharide alditol, Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-beta-D-Galp-(1-->3)-GalNAc-ol, were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.     

Primary structure of four human milk octa-, nona-, and undeca-saccharides established by 1H- and 13C-nuclear magnetic resonance spectroscopy.

The structures of two octasaccharides, one nonasaccharide, and one undecasaccharide, isolated from human milk, have been investigated by 1H- and 13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides are: beta-D-Galp-(1----4)-[alpha-L-Fucp- (1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp+ ++- (1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc; beta-D-GALp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1---- 3)-beta-D - Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----3)-beta -D-Galp- (1----4)-D-Glc; beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1---- 6)-(alpha - L-Fucp-(1----2)-beta-D-Gal-(1----3)-[alpha-L-Fucp-(1----4)]- beta-D-GlcpNAc- (1----3))-beta-D-Galp-(1----4)-D-Glc; and alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3) -beta-D- Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----6)-[alp ha-L- Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)]-beta-D -Galp- (1----4)-D-Glc. The two octasaccharides have been previously isolated from human milk as a mixture, and in a pure form from new-born feces, but the n.m.r. data were not provided. These two octasaccharides display the di-Lewis X and the composite Lewis A-Lewis X antigenic determinant, previously described as neo-antigens of adenocarcinoma cell lines.     

Enzymatic synthesis of blood group A and B trisaccharide analogues

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.     

Synthesis of a novel glycosphingolipid from the millipede, Parafontaria laminata armigera, and the assembly of its carbohydrate moiety into multivalent structures

A novel glycosphingolipid, beta-D-Manp-(1-->4)-[alpha-L-Fucp-(1-->3)]-beta-D-Glcp-(1-->1)-Cer, found in the millipede, Parafontaria laminata armigera, and multivalent derivatives of its carbohydrate moiety were synthesized. As the key step, the target glycolipid (1) was obtained through an inversion reaction at the 2-position of a beta-glucopyranoside residue yielding a beta-mannopyranoside. In addition, the synthesis of fluorescently labeled trimer and tetramer glycoconjugates (2, 3) was achieved by iterative amide bond formation using a monomer unit (24).     

The use of porcine liver (2----3)-alpha-sialyltransferase in the large-scale synthesis of alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc, the epitope of the tumor-associated carbohydrate antigen CA 50.

alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc (2) and, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn+ ++ were prepared on a large scale by the action of beta-D-Galp-(1----3)-D-GalpNAc (2----3)-alpha-sialyltransferase (partially purified from porcine liver) on beta-D-Galp-(1----3)-D-GlcpNAc and beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn, respectively. The trisaccharide 2 is the epitope of the tumor-associated carbohydrate antigen CA 50, highly expressed in human pancreatic adenocarcinoma.     

The structures of oligosaccharides excreted by sheep with swainsonine toxicosis

Eleven oligosaccharides were purified form the urine of sheep with swainsonine toxicosis induced by the feeding of Astragalus lentiginosus. Oligosaccharides were extracted by charcoal adsorption, chromatographed on Bio-Gel P-2, and partially fractionated by preparative-layer chromatography. Separation into individual compounds was completed by semi-preparative high pressure liquid chromatography. Structures were determined by a combination of high pressure liquid chromatography and exo- and endo- glycosidase action, methanolysis followed by gas-liquid chromatography, methylation analysis, and high resolution nuclear magnetic resonance spectroscopy. Two homologous series of oligosaccharides were identified: (a) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----2)-alpha-D-Manp(1----3)-[alpha-D-Manp+ ++-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)-[alpha- D-Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc (minor series); (b) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-alpha-D-Manp-(1----6)-beta-D-Manp -(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp++ +-(1----4)-beta-D-GlcpNAc - (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)- [alpha-D-Manp-(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D- GlcpNAc (major series).     

Hydroxy protons in conformational study of a Lewis b tetrasaccharide derivative in aqueous solution by NMR spectroscopy

The 1H NMR chemical shifts, vicinal coupling constants, temperature coefficients, and exchange rates of the hydroxy protons of a Lewis b tetrasaccharide derivative, alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> 3)[alpha-L-Fucp-(1 --> 4)]-beta-D-GlcpNAc-1-O(CH2)2NHCOCHCH2, have been measured in aqueous solution. The data did not show any evidence for persistent hydrogen bonds participating in the stabilization of the structure. While most of the hydroxy proton signals have chemical shifts similar to those of the corresponding methyl glycosides, four of them, O(3)H, O(4)H, and O(6)H of Galp, and O(2)H of the Fucp linked to GlcpNAc, exhibit large upfield shifts. This shielding effect has been attributed to the orientation of the hydroxy protons toward the amphiphilic region constituted by the hydroxy groups of the Galp residue and mainly the ring and methyl hydrogens of the Fucp unit attached to the GlcpNAc. The close face to face stacking interaction between the Fucp linked to the GlcpNAc and the Galp residues, as well as the steric interaction between the Fucp linked to the Galp and the GlcpNAc are confirmed by the additional inter-residue NOEs of the exchangeable protons in sugar units which are not directly connected.     

Determination of the immunodominant part in the O-antigenic polysaccharide from Escherichia coli O128 by ELISA-inhibition study

The immunodominant part in the O-antigenic polysaccharide from Escherichia coli O128 was immunologically characterized by an enzyme-linked immunosorbent assay (ELISA). The antibody specificity was determined by the inhibitory effects of the methyl glycosides of constituent mono- and oligosaccharides synthesized related to the O-antigenic polysaccharide from E. coli O128. It was found that methyl alpha-L-fucopyranoside was the most effective inhibitor amongst the monosaccharides while the highest antibody specificity was directed towards the trisaccharide with the structure: beta-D-GalpNAc-(1-->6)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-1-->OMe suggesting that the monospecific antibody has the extended combining site.     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [alpha-L-Fucp-(1-2)-beta-D-Galp-(1-4)-D-Glc, Ka= 4.2 x 10(3) M(-1)]. The following antigens were bound with a weaker affinity: H-disaccharide alpha-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Ley alpha-L-Fucp-(1-2)-beta-D-Galp-(1-4)-[alpha-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (Ka 1.1-1.7 x 10(3) M(-1)). The lowest binding was observed for L-fucose (Ka = 2.7 x 10(2) M-1) and trisaccharide Lea, (3-Galp-(1-3)-[a-L-Fucp-(1-4)]-GlcNAc (Ka = 6.4 x 10(2) M(-1)). The Lea, Lea, and Lex pentasaccharides and Leb hexasaccharide were not bound to LABA.     

Structure of a fucoidan from the brown seaweed Fucus serratus L

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1:0.1 and small amounts of xylose and galactose were isolated from the brown seaweed Fucus serratus L. The fucoidan structure was investigated by 1D and 2D 1H and 13C NMR spectroscopy of its desulfated and de-O-acetylated derivatives as well as by methylation analysis of the native and desulfated polysaccharides. A branched structure was suggested for the fucoidan with a backbone of alternating 3- and 4-linked alpha-L-fucopyranose residues, -->3)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->, about half of the 3-linked residues being substituted at C-4 by trifucoside units alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-alpha-L-Fucp-(1-->. Minor chains built up of 4-linked alpha-fucopyranose and beta-xylose residues were also detected, but their location, as well as the position of galactose residues, remained unknown. Sulfate groups were shown to occupy mainly C-2 and sometimes C-4, although 3,4-diglycosylated and some terminal fucose residues may be nonsulfated. Acetate was found to occupy C-4 of 3-linked Fuc and C-3 of 4-linked Fuc in a ratio of about 7:3.     

The use of NMR residual dipolar couplings in aqueous dilute liquid crystalline medium for conformational studies of complex oligosaccharides

C-H dipolar coupling values were measured for a natural-abundance sample of the pentasaccharide beta-D-Galp-(1-->3)-[alpha-L-Fucp-(1-->4)]-beta-D-GlcNAcp-(1 -->3)-beta-D- Galp-(1-->4)-beta-D-Glcp ('lacto-N-fucopentaose 2') (LNF-2), in a 7.5% solution of dimyristoyl phosphatidylcholine-dihexanoyl phosphatidylcholine bicelle liquid crystals oriented in the NMR magnetic field. Interpretation of the dipolar coupling data and NOE confirms the conformational model for the Lewis(a) trisaccharide epitope based on NOE, molecular dynamics simulations, and scalar coupling data and provided new structural information for the remaining residues of the pentasaccharide. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.     

Synthesis and enzymatic evaluation of modified acceptors of recombinant blood group A and B glycosyltransferases

The disaccharide alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> O)-Octyl (1) is an acceptor for the human blood group A and B glycosyltransferases. Seven analogues of 1, containing deoxy, methoxy and arabino modifications of the Fuc residue, were chemically synthesized and kinetically evaluated in radioactive enzymatic assays. Both the enzymes tolerate modification of the 3'-OH on the fucose residue. The 2'-OH was found to be key to the recognition of the acceptors by these enzymes. The arabino derivative was recognized as an acceptor by the A transferase (Km of 200 microM), but not the B transferase and is the first synthetic acceptor capable of distinguishing between the two enzyme activities.     

Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation

Oxazoline mono-, di-, tri- and hexasaccharides, corresponding to the core components of N-linked glycoprotein high mannose glycans, are synthesised as potential glycosyl donors for endohexosaminidase catalysed glycosylation of glycopeptides and glycoprotein remodelling. The crucial beta-D-Manp-(1-->4)-D-GlcpNAc linkage is synthesised via epimerisation of gluco disaccharide substrates by sequential triflation and nucleophilic substitution. Oxazolines are formed directly from the anomeric OPMP protected N-acetyl glucosamine derivatives. Efficient endohexosaminidase catalysed glycosylation of a synthetic beta-D-GlcpNAcAsn glycosyl amino acid is demonstrated with the trisaccharide oxazoline donor.     

Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose,respectively

Two analogues of the Le(x) trisaccharide, alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed beta-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding alpha-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le(x) antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.     

Biochemical characterization of inner sugar chains of acrosome reaction-inducing substance in jelly coat of starfish eggs

The inception of the acrosome reaction (AR) in the starfish Asterias amurensis is perceived to be strongly associated with sulfated polysaccharide chains derived from an extremely large proteoglycan-like molecule called AR-inducing substance (ARIS), in which one of the sugar fragments, named fragment 1 (Fr. 1), was composed of the repeating units of [-->4]-beta-D-Xylp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Fucp-4 (SO3-)-(1-->3)- alpha-L-Fucp-4(SO3-)-(1-->4)-alpha-L-Fucp-(1-->)n. In the current study, this sugar chain is inferred to link to the peptide part by O-glycosidic linkage through a sugar chain with different structural features from Fr. 1. This inner sugar portion of ARIS was isolated as Fr. 2 from the sonicated products of pronase digest of ARIS. Fr. 2, which retains AR-inducing activity to an admirable extent and has an apparent molecular size of 400 kDa, is composed of Gal, Xyl, Fuc, GalNAc, and GlcNAc in a molar ratio of 5:1:5:4:2 with O-sulfate substitutions at Gal-4, Gal-2, Gal-2,3 and Gal-2,4 (disulfated), Fuc-4, and GlcNAc-6. The study of Fr. 2 revealed that the major portion of the inner sugar chain of ARIS is composed of the heptasaccharide units of -->3)-Galp-(1-->3)-Fucp-(1-->3)-Galp-(1-->4)-GalNAcp-(1-->4)-GlcNAcp-6(SO3-)-(1-->6)-Galp-4(SO3-)-(1-->4)-GalNAcp-(1-->. This new structure of inner sugar chains of ARIS is elucidated by using electrospray ionization MS along with tandem mass analysis, sugar composition analysis, and methylation analysis of the sugar fragments obtained by acid-catalyzed resin-based partial hydrolysis of Fr. 2. Furthermore, this study corroborates that the sulfate groups are solely liable to the anionic character of ARIS, which ought to be present in the sugar chains of ARIS for its biological activity.     

Structure and cross-reactivity of the O-antigen of Proteus vulgaris O8.

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O8 lipopolysaccharide followed by gel permeation chromatography. Studies of the polysaccharide by sugar and methylation analyses and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, demonstrated the presence of a tetrasaccharide repeating unit having the following structure: [sequence: see text] The role of an epitope associated with the alpha-L-FucpNAc-(1-->3)-D-GlcpNAc disaccharide in serological cross-reactivity of P. vulgaris O8 is discussed.     

Conformational analysis of two xylose-containing N-glycans in aqueous solution by using 1H NMR ROESY and NOESY spectroscopy in combination with MD simulations

The conformational behavior of the synthetic hexa- and heptasaccharide methyl beta-glycosides alpha-D-Manp-(1 --> 6)-[alpha-D-Manp-(1 --> 3)-][beta-D-Xylp-(1 --> 2)-]beta-D-Manp-(1 --> 4)-beta-D-GlcpNAc-(1 --> 4)-beta-D-GlcpNAc-(1 --> OMe and alpha-D-Manp-(1 --> 6)-[alpha-D-Manp-(1 --> 3)-][beta-D-Xylp-(1 --> 2)-]beta-D-Manp-(1 --> 4)-beta-D-GlcpNAc-(1 --> 4)-[alpha-L-Fucp-(1 --> 6)-]beta-D-GlcpNAc-(1 --> OMe, representing the xylosylated and the xylosylated alpha-(1 --> 6)-fucosylated core structures of N-glycans in alpha(D)-hemocyanin of the snail Helix pomatia, respectively, were investigated by 1H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. 1H and 13C chemical shifts of the oligosaccharides were assigned using 1H-(1)H COSY, TOCSY, and NOESY, and 1H-(13)C HMQC techniques. Experimental 2D 1H cross-peak intensities from one series of NOESY and one series of ROESY experiments of the two oligosaccharides were compared with calculated values derived from MD trajectories using the CROSREL program, yielding information about the conformation of each glycosidic linkage of the methyl glycosides. The flexibility of the linkages was described by generalized order parameters and internal rotation correlation times. Analysis of the data indicated that several conformations are likely to exist for the alpha-D-Man-(1 --> 6)-beta-D-Man, the alpha-L-Fuc-(1 --> 6)-beta-D-GlcNAc, and the alpha-D-Man-(1 --> 3)-beta-D-Man linkage, whereas the beta-D-Xyl-(1 --> 2)-beta-D-Man-(1 --> 4)-beta-D-GlcNAc-(1 --> 4)-beta-D-GlcNAc fragment occurs in one rigid conformation. No significant differences were found between the corresponding structural elements in both methyl glycosides. NOESY and ROESY experiments proved to be suitable for providing the experimental data required, however, due to more overlap within the ROESY spectra, reducing the accuracy of the analysis, NOESY spectral analysis is preferred.