Depicting the MM3 potential energy surfaces of trisaccharides by single contour maps: application to beta-cellotriose and alpha-maltotriose

The adiabatic potential energy surfaces (PES) of two trisaccharides (beta-cellotriose and alpha-maltotriose) were obtained using the MM3 force field. Each PES can be described by a single 3D contour map for which the energy is plotted against the two psi glycosidic angles. Given the usually small variations of the phi glycosidic torsional angle in the low-energy regions of disaccharide maps (at least with MM3), it is valid to leave both phi glycosidic angles to relax in the process of building the conformational map of trisaccharides. The surfaces are those expected from the map of disaccharides containing the same linkages and monosaccharide units (i.e., beta-cellobiose and alpha-maltose), with second-order factors altering the 'symmetry' of both linkages. A large low-energy region appears for beta-cellotriose, comprising four minima in close proximity, with barriers between them below 0.6 kcal/mol. On the other hand, for alpha-maltotriose a main global minimum is observed, with several surrounding local minima. The surfaces obtained agree with single-crystal X-ray data on these trisaccharides and derivatives. A reduction of the linkage flexibilities is observed when passing from the disaccharides to the trisaccharides. Furthermore, the linkage closer to the reducing end appears to be less flexible than the linkage closer to the non-reducing end.     

MM3 potential energy surfaces of trisaccharides. II. Carrageenan models containing 3,6-anhydro-D-galactose

The adiabatic potential energy surfaces (PES) of six trisaccharides-namely 3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp, beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp, and their derivatives sulfated on positions 2 and 4 of the beta-galactose unit-were obtained using the MM3 force field. Each PES was described by a single contour map for which the energy is plotted against the two psi glycosidic angles, given the small variations of the phi glycosidic torsional angle in the low-energy regions of disaccharide maps. In five of the six examples, the surfaces are those expected from the maps of the disaccharidic repeating units of carrageenans, with less important factors altering the additive effect of both linkages. However, when a sulfate group is present on C2 of a beta-galactose reducing end, a new low-energy minimum in a different region is produced, originated in a hydrogen bond between the first and third monosaccharidic moieties of the trisaccharide. The flexibility of the beta-linkages is nearly identical to that in their disaccharide counterparts, while that of the alpha-linkages is slightly reduced, independent of their presence closer or further away from the reducing end. A fair agreement is observed between the x-ray fiber diffraction analysis for a kappa-carrageenan double helix and the surfaces obtained for the trisaccharide analogs of that polymer.     

MM3 potential energy surfaces of alpha-3-linked L-fucobiose and fucotriose and their sulfated counterparts

The adiabatic potential energy surfaces (PES) of alpha-L-Fuc-(1-->3)-alpha-L-Fuc and their counterparts disulfated at 2,2' and 4,4', and tetrasulfated at 2,2',4,4', which are representative of fucoidan structures, were obtained using the mm3 force field, and plotted as contour maps and as 2D graphs representing the energy versus the psi angle. The surfaces of the corresponding trisaccharides were also obtained and represented by a single 3D contour map for which the energy is plotted against the two psi glycosidic angles. For the nonsulfated disaccharide, similar populations of two minima occur. A substantial sulfate effect is observed. Whereas sulfation on both of the 2-positions shift the global minimum to positive psiH angles, sulfation on both of the 4-positions deepen the well at negative psiH values. A similar effect occurred in their galactose counterparts. Sulfation on the 2- and 4-positions carry the additive effect of both groups. The same trend was observed for both linkages present in the trisaccharides, with minor differences. For instance, the 4,4',4" trisulfated compound exhibits a trend by which the glycosidic linkage closer to the nonreducing end appears to be highly flexible, with similar energies in both conformers. Raising the dielectric constant on nonsulfated oligosaccharides was found to give a better agreement with experimental determinations.     

MM3 potential energy surfaces of the 2-linked glucosyl trisaccharides alpha-kojitriose and beta-sophorotriose

The adiabatic potential energy surfaces (PES) of two trisaccharides with 2-linkages (alpha-kojitriose and beta-sophorotriose) were obtained using the MM3 force field, and are represented by a single 3D contour map for which the energy is plotted against the two psi glycosidic angles. In spite of the proximity of the positions where the two monosaccharidic units are linked to the central monosaccharide, an almost independent behavior of both linkages was found for the alpha-linked trisaccharide alpha-kojitriose, i.e., the surfaces are those expected from the maps of the disaccharide containing the same linkage. A slight shift of the position of the global minimum is found to occur, due to a hydrogen bond between the third and first monosaccharide units, which also leads to an increase in flexibility. On the other hand, for the beta-linked trisaccharide beta-sophorotriose, the surface is sharply different from that expected by observation of the disaccharide map. Some of the expected minima cannot appear unless a serious deformation of the phi and/or psi angles is produced. Furthermore, the global minimum corresponds to a combination of different conformations for each of the linkages, whereas another minimum with only slightly higher energy has both glycosidic linkages in a conformation less favored for the disaccharide, though close to that predicted in crystal diffraction studies.     

Disaccharide conformational maps: 3D contours or 2D plots?

The potential energy surfaces of several alpha-(1-->3)- and beta-(1-->4)-linked disaccharides were obtained and plotted in terms of energy versus psi glycosidic angle. These plots were compared to those obtained previously in the way of the usual 3D contour maps, which relate the energy with the two glycosidic angles (phi and psi). Given the usually small variations of the phi angle in the low-energy regions (at least using MM3), both kinds of graphs lead to similar conclusions concerning flexibility measurements by two different methods and assessment of the effects of sulfation and/or hydroxyl group orientation. Only second-order effects were found with some sulfated disaccharides, not changing the general conclusions. The computational efforts required to produce those plots are smaller, and the plots are easier to interpret. Besides, the conversion of a 3D map into a 2D plot leaves the possibility of constructing 3D maps of carbohydrates including a second variable different to phi, e.g., the second psi angle of a trisaccharide or the omega angle of a 6-linked disaccharide.     

A convergent synthesis of trisaccharides with alpha-Neu5Ac-(2 --> 3)-beta-D-gal-(1 --> 4)-beta-D-GlcNAc and alpha-Neu5Ac-(2 --> 3)-beta-D-gal-(1 --> 3)-alpha-D-GalNAc sequences

The syntheses of three trisaccharides: alpha-Neu5Ac-(2 --> 3)-beta-D-Gal-(1 --> 4)-beta-D-GlcNAc --> OMe, alpha-Neu5Ac-(2 --> 3)-beta-D-Gal6SO3Na-(1 --> 4)-beta-D-GlcNAc --> OMe, and alpha-Neu5Ac-(2 --> 3)-beta-D-Gal-(1 --> 3)-alpha-D-GalNAc --> OBn were accomplished by using either methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-beta-D-glycero-D-g alacto-2-nonulopyranoside)onate or methyl (phenyl N-acetyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-beta-D-gl ycero-D-galacto-2-nonulopyranoside)onate as the sialyl donor. The N,N-diacetylamino sialyl donor appears to be more reactive than its parent acetamido sugar when allowed to react with an disaccharide acceptor under the same glycosylation conditions. The trisaccharides, as well as the intermediate products, were fully characterized by 2D DQF 1H-1H COSY and 2D ROESY spectroscopy.     

Conformational analysis of the anomeric forms of kojibiose, nigerose, and maltose using MM3

Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1 → 2)-, (1 → 3)-- and (1 → 4)--linked -glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20° grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.     

Remote control of alpha- or beta-stereoselectivity in (1-->3)-glucosylations in the presence of a C-2 ester capable of neighboring-group participation

In (1-->3)-glucosylation the glycosyl bond originally present in either donor or acceptor is shown to control the stereoselectivity of the forthcoming bond, i.e., the newly formed glycosidic linkage has the opposite anomeric configuration of that of either the donor or acceptor. Therefore, with alpha-(1-->3)-linked disaccharides with nonreducing ends that have the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with an alpha-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with 3-OH free as the acceptor, beta-linked trisaccharides were obtained. Meanwhile, with beta-(1-->3)-linked disaccharides that have nonreducing ends with the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with a beta-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with the 3-OH free as the acceptor, alpha-linked trisaccharides were obtained in spite of the C-2 neighboring group participation.     

Conformational free energy maps for globobiose (alpha-D-Galp-(1-->4)-beta-D-Galp) in implicit and explicit aqueous solution

Four Ramachandran maps of the conformational potential of mean force (PMF) for the galactose disaccharide globobiose (alpha-D-Galp-(1-->4)-beta-D-Galp) were calculated in vacuum, explicit water, with a simple high dielectric constant and a distance-dependent dielectric coefficient, respectively. This simple model of the galactan alpha-(1-->4)-linkage is shown to be conformationally restricted, with only a small range of syn-phi/syn-psi conformations predominating at standard temperature and pressure. This has implications for the preferred conformation and chain dynamics of alpha-galactosides. In addition, comparison of the relevant PMF surfaces reveals the substitution of a high dielectric constant for explicit water solution to be a valid approximation for reproducing the minimum energy conformation of this glycosidic linkage.     

Conformation of the branched O-specific polysaccharide of Shigella dysenteriae type 2: molecular mechanics calculations show a compact helical structure exposing an epitope which potentially mimics galabiose

Conformational analyses of the branched repeating unit of the O-antigenic polysaccharide of Shigella dysenteriae type 2 have been performed with molecular mechanics MM3. A filtered systematic search on the trisaccharide alpha-D-GalNAc-(1-->3)-[alpha-D-GlcNAc-(1-->4)]-alpha-D-GalNAc forming the branch, shows essentially a single favored conformation. Also, the downstream alpha-D-GalNAc-(1-->4)-alpha-D-Glc linkage is sterically constrained. The alpha-D-Glc-(1-->4)-beta-D-Gal moiety, however, forms a more flexible link region between the branch points, and shows a 90 degrees bend similar to what is known for the galabiose moiety occurring in globo-glycolipids. The calculations indicate that consecutive repeating units in their minimum energy conformation arrange in a helical structure with three repeating units per turn. This helix is very compact and appears to be stabilized by hydrophobic interactions involving the N-acetyl groups at the branch points. Random conformational search suggests the existence of another helical structure with four repeating units per turn. It appears possible that the alpha-D-Glc-(1-->4)-beta-D-Gal moiety, which is exposed on the surface of the helical structures, can evade recognition by the immune system of the host by the mimicry of globo structures.     

Potential energy surfaces of carrageenan models: carrabiose,beta-(1 --> 4)-linked D-galactobiose,and their sulfated derivatives

The adiabatic conformational surfaces of several beta-linked disaccharides, which correspond to the repeating structures of carrageenans, were calculated using the MM3 force-field. The studies were carried out on the disaccharide beta-D-Galp-(1 --> 4)-alpha-D-Galp and eight sulfated derivatives, as well as on carrabiose (beta-D-Galp-(1 --> 4)-3,6-An-alpha-D-Galp) and five sulfated derivatives. The presence of 3,6-anhydrogalactose does not change the main features of the maps, although it increases the flexibility of the glycosidic linkage. Sulfation neither produces a striking effect on the map shape, nor a shift on the global minimum, which always remains with psi (theta(C-1'-O-4-C-4C-5)) in trans orientation, and phi (theta(O-5'-C-1'-O-4-C-4)) with a value close to -80 degrees. This effect differs from that occurring on the alpha linkage of equivalent disaccharides, for which the sulfation pattern on the beta-galactose unit shifts the global minima to different positions. A reduction in the flexibility (originated in a deepening of the global minimum well) is observed by sulfation on position 2 of the beta-D-galactose unit, and by sulfation of position 6 of the alpha-D-galactose unit (when the beta-D-galactose unit is 4-sulfated). Within the compounds containing 3,6-anhydrogalactose, the effect of sulfation is even less noticeable. The calculated low-energy regions on carrabiose derivatives agree with X-ray diffraction data on carrageenan fibers and on peracetylated carrabiose dimethyl acetal, and with NOE calculations carried out on kappa-carrabiose.     

Effect of Enzyme Preparation from the Marine Mollusk Littorina kurila on Fucoidan from the Brown Alga Fucus distichus

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units -->3)-alpha-L-Fucp-(2,4-di-SO3(-))-(1-->4)-alpha-L-Fucp-(2SO3(-))-(1-->, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.     

Efficient modelling protocols for oligosaccharides: from vacuum to solvent

The determination of conformational preferences of oligosaccharides is best approached by describing their preferred conformations on potential energy surfaces as a function of the glycosidic linkage φ, ψ torsional angles. For proper molecular mechanics modelling the flexibility of the rotatable pendant groups must also be considered. The so called adiabatic maps partially mimic the flexibility within the 10 dimensional conformational space of the pendant groups of the given disaccharide. These molecular mechanics maps are considered to be the state-of-the art of the φ, ψ potential energy surface of disaccharides recently calculated. The RAMM (RAndom Molecular Mechanics) method was shown to be able to calculate such profiles automatically. Additionally, based on the continuum solvent approach, RAMM allows the calculation of the effects of solvent on conformational energy profiles. Molecular dynamics simulations are also useful tools to study the influence of solvent on conformational behaviour of oligosaccharides. The capability of the RAMM calculational protocol to locate low-energy conformers on the multidimensional potential energy hypersurfaces of disaccharides is illustrated and compared with molecular dynamics simulations with and without inclusion of the solvent. This revised version was published online in August 2006 with corrections to the Cover Date.     

An efficient synthesis of two monosulfated trisaccharides with the Galbeta1,3GlcNacbeta1,3Galbeta-O-allyl backbone

The GlcNAcbeta(1-->3) Gal linked disaccharide 7 was synthesized as key building blocks for the construction of target monosulfated trisaccharides 1 and 2 using oxazoline 3 as glycosyl donor promoted by BF3 x Et2O.     

Morphology of Western larch arabinogalactan

A molecular modeling study has revealed that (1 --> 3)-beta-D-galactan can not only adopt a triple helical structure similar to that of the corresponding glucan but can also accommodate a highly flexible beta-D-Gal-(1 --> 6)-beta-D-Gal disaccharide moiety as a side group 6-linked to every galactosyl unit in the main chain. The resulting triple helix, applicable to Western larch arabinogalactan, can assume quite different morphologies since the side group has access to several allowed conformational states. Some of the preferred modes of association between these helices have been visualized using preliminary X-ray fiber diffraction data.     

Synthesis of neoglycoproteins containing O-methylated trisaccharides related to excretory/secretory antigens of Toxocara larvae

The disaccharides allyl beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 10a and 10b and the trisaccharides allyl 2-O-methyl-alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin. The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly immunogenic O-glycans located at the surface of Toxocara larvae.     

Structure of the O-antigen of the main lipopolysaccharide isolated from Sinorhizobium fredii SMH12

The lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, has been studied. Isolation of lipopolysaccharide by the phenol-water method leads to a mixture of two polysaccharides; polyacrylamide gel electrophoresis indicates that both are possibly lipopolysaccharides. The structures of the O-antigen of the main lipopolysaccharide and its deacetylated form are determined by sugar and methylation analysis, partial hydrolysis, lithium degradation, ESI-MS/MS, and NMR studies. Here we show that the fast-growing S. fredii SMH12 produces a lipopolysaccharide whose O-antigen has a repeating unit consisting of the trisaccharide -->4)-alpha-D-Gal pA-(1-->3)-2-O-Ac-alpha-L-Rha p-(1-->3)-2-O-Ac-alpha-D-Man p-(1-->. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. The tandem mass spectrometry studies rule out both an alternating and a random distribution of methyl groups and suggest the existence of zones in the polysaccharide rich in methyl groups interspersed with zones without methyl groups.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses.     

Defining the carbohydrate specificities of aplysia gonad lectin exhibiting a peculiar D-galacturonic acid affinity

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.     

Saturation transfer difference NMR and computational modeling of a sialoadhesin-sialyl lactose complex

The siglecs are a family of I-type lectins binding to sialic acids on the cell surface. Sialoadhesin (siglec-1) is expressed at much higher levels in inflammatory macrophages and specifically binds to alpha-2,3-sialylated N-acetyl lactosamine residues of glycan chains. The terminal disaccharide alpha-D-Neu5Ac-(2-->3)-beta-D-Gal is thought to be the main epitope recognized by sialoadhesin. To understand the basis of this biological recognition reaction we combined NMR experiments with a molecular modeling study. We employed saturation transfer difference (STD) NMR experiments to characterize the binding epitope of alpha-2,3-sialylated lactose, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 to sialoadhesin at atomic resolution. The experimental results were compared to a computational docking model and to X-ray data of a complex of sialyl lactose and sialoadhesin. The data reveal that sialoadhesin mainly recognizes the N-acetyl neuraminic acid and a small part of the galactose moiety of 1. The crystal structure of a complex of sialoadhesin with sialyl lactose 1 was used as a basis for a modeling study using the FlexiDock algorithm. The model generated was very similar to the original crystal structure. Therefore, the X-ray data were used to predict theoretical STD values utilizing the CORCEMA-STD protocol. The good agreement between experimental and theoretical STD values indicates that a combined modeling/STD NMR approach yields a reliable structural model for the complex of sialoadhesin with alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-D-Glc 1 in aqueous solution.