Hard-sphere Exo-anomeric Research Articles

MMC and LD simulations of alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-OMe. A model for the terminal trisaccharide in glycoprotein precursors.

The conformational flexibility and the dynamics of alpha-D-Glcp-(1-->2)-alpha-D-Glcp(1-->3)-alpha-D-Glcp-OMe (I) has been investigated by Metropolis-Monte Carlo with the HSEA (Hard Sphere Exo-Anomeric) force field and Langevin dynamics simulations employing two different CHARMm (Chemistry at HARvard Molecular Mechanics) force fields, CHEAT95 and PARM22. The conformational space spanned by the molecule is similar for the two former force fields but differ significantly for the latter. Hydrogen bonding between O2" and O4 of the title compound is analysed in comparison to NMR and preliminary results from X-ray powder diffraction studies.

Methyl 2-O-α-D-Mannopyranosyl-β-D-glucopyranoside

The structure of the title compound, C 13 H 24 O 11 , has been determined. The torsion angles of the glycosidic linkage connecting the two sugar residues, φH (H1'-C1'-O2-C2) and ψH (C1'-O2-C2-H2), have values of -62.7 (2)° and -28.6 (2)°, respectively. The conformation in the crystal is similar to that obtained by energy minimization in vacuo using the HSEA (hardsphere exo-anomeric) force field. A chain of seven interresidue hydrogen bonds, involving all possible H-atom donors in the molecule is observed. The chain is terminated by a ring O atom as an acceptor.

A carbohydrate domain common to both sialyl Le(a) and sialyl Le(X) is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1

The specificity of endothelial cell leukocyte adhesion molecule-1, ELAM-1, for binding to a panel of carbohydrate structures was determined by a sensitive cell binding assay with immobilized synthetic glycoconjugates. ELAM-1 cDNA transfectants were found to bind Sialyl Lea (sialylated lacto-N-fucopentaose II) or sialylated Lewis a antigen (NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc), as well as or slightly better than Sialyl Lex (sialylated lacto-N-fucopentaose III) or sialylated Lewis X antigen (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A monoclonal antibody, HECA-452, which has been identified recently as recognizing ELAM-1 ligands in addition to those containing Sialyl Lex, was also found to bind both Sialyl Lex and Sialyl Lea. Hard sphere exo-anomeric (HSEA) calculations were performed on these two hexasaccharides. The conformations indicate that Sialyl Lea and Sialyl Lex show a high degree of similarity in both the nonreducing and reducing termini. As Lea and Lex show much weaker reactivity, the determinants recognized by ELAM-1 and HECA-452 probably involve neuraminic acid and fucose residues which on one face of both Sialyl Lex and Sialyl Lea can be similarly positioned. The finding that Sialyl Lea is a potent ligand for ELAM-1 is important, as circulating Sialyl Lea and Sialyl Lex containing mucins which are elevated in the serum of many cancer patients may block leukocyte interactions with ELAM-1 and may contribute to the pathological immunodepression observed in these patients.

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.

A new force‐field program for the calculation of glycopeptides and its application to a heptacosapeptide‐decasaccharide of immunoglobulin G1

Energetically favored conformations of glycopeptide 1 were calculated using the newly developed force-field program, GEGOP (geometry of glycopeptides). The three-dimensional structure of glycopeptide 1, which is part of the Fc fragment of IgG1, has been calculated. 1 contains 27 amino acid residues from Pro291 to Lys317 and a biantennary decasaccharide N-linked to Asn297. The conformations of the peptide and the carbohydrate parts are shown to be mutually dependent. Single glycosyl residues of 1 exhibit interaction energies of up to -31.8 kJ/mol with the peptide portion. Generally, only a few of the glycosyl residues of the oligosaccharide moiety express significant interaction energies with the peptide part. No easy prediction is possible of glycosyl residues which exhibit favorable interaction energies. However, in all of the calculated structures, the glycosyl residues of the 1-6-linked branches show strong attractive forces for the peptide part. 1-6-glycosidically linked branches can adopt a larger number of conformations than other linkages due to their high flexibility which allows more favorable interactions with proteins. We developed the GEGOP program in order to be able to study the preferred conformations of large glycopeptides. The program is based on the GESA (geometry of saccharides) program and utilizes the HSEA (hard sphere exo anomeric) force field for the carbohydrate part and the ECEPP/2 (empirical conformation energy program for peptides) force field [Némethy, G., Pottle, M. S. & Scheraga, H. A. (1983) J. Phys. Chem. 87, 1883-1887] for the peptide part. The GEGOP program allows the simultaneous relaxation of all rotational degrees of freedom of these glycoconjugates during the energy optimization process. Thus, mutual interactions between glycosyl and amino acid residues can be studied in detail.

An n.m.r. and conformational analysis of the terminal trisaccharide from the serologically active glycolipid of Mycobacterium leprae in different solvents

The 1H- and 13C-n.m.r. spectra of allyl 2- O-[4- O-(3,6-di- O-methyl-β- d-glucopyranosyl)-2,3-di- O-methyl- a- l-rhamnopyranosyl]-3- O-methyl- a- l- rhamnopyranoside ( 3), a glycoside of the terminal trisaccharide found in the phenolic glycolipid I from Mycobacterium leprae, and those of the two component disaccharides, allyl 4- O-(3,6-di- O-methyl-β- d-glucopyranosyl)-2,3-di- O-methyl- a- l-rhamnopyranoside ( 1) and allyl 2- O-(2,3-di- O-methyl- a- l-rhamnopyranosyl)-3- O-methyl- a- l-rhamnopyranoside ( 2) have been assigned completely by 1D and 2D techniques. The preferred conformations, determined by chemical shift and n.O.e. studies, were different in D 2O, CD 3OD, and CDCl 3. The preferred conformation of 3 accorded with the results of hard-sphere exo-anomeric (HSEA) calculations.

The conformation of core oligosaccharides from Escherichia coli and Salmonella typhimurium lipopolysaccharides as predicted by semi-empirical calculations.

The preferred conformation of the hexose and heptose regions of core saccharides from Enterobacteriaceae lipopolysaccharides was calculated. The Hard Sphere Exo Anomeric (HSEA) approach was used and the minimum energy conformation of the Salmonella typhimurium and Escherichia coli R1, R2, R3, R4 and K12 cores calculated. The results indicate that most of the cores are sterically crowded, with small degrees of freedom, and that the hexose and heptose parts form two separate regions. The core structures exhibit a 'front'-side and a 'back'-side, the former being similar for all the structures and the latter being characteristic for each core type.

Conformational analysis of the sialyl alpha(2----3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential.

The conformation is described of the sialyl alpha(2----3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----N)Asn and NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----N)Asn is presented and for each glycosidic linkage, except for the alpha(2----6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the beta(1----4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the alpha(2----3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2----6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(2----6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta structural element. This provides an explanation for the shift effects induced by alpha(2----6) attachment of NeuAc to the N-acetyllactosamine unit.

The conformation of Salmonella O-antigenic polysaccharide chains of serogroups A, B, and D 1 predicted by semi-empirical, hard-sphere (HSEA) calculations

The preferred conformation of the tetrasaccharide repeating units of Salmonella Serogroups A, B, and D 1 have been calculated. The semiempirical calculations used the Hard-Sphere, Exo-Anomeric (HSEA) approach to derive a conformational model, which could be used to assist the interpretation of conformations significantly populated in aqueous solution. The calculated model was extended to include a pentasaccharide repeating unit bearing an α- d-glucopyranose-branch point. The 3,6-dideoxyhexose to d-mannose linkage was shown to possess a steep energy surface with a minimum, which results in good exposure of the dideoxyhexose O-2 and O-4 atoms. Stereochemical changes involving the equatorial or axial disposition of these atoms are the distinguishing structural features of the A, B, and D 1 serogroups. A lipophilic surface involving the 6-deoxy groups of the dideoxy- d-hexose and l-rhamnose residues was identified, and the possible implications of these features in antigenic determinants is discussed. The preferred conformation predicted by the HSEA method correlates with the known antigenic specificities of polysaccharides belonging to these Salmonella serogroups.

Spatial conformation of glycans and glycoproteins.

Ten years ago, we anticipated future results by building the Y-shaped molecular model of a biantennary glycan. Progressively, this structure has been refined and modified thanks to experimental data obtained by using physical methods: X-ray diffraction, electron spin resonance (EPR), nuclear magnetic resonance (NMR) including two-dimensional NMR and one-dimensional 1H-nuclear Overhauser effect (NOE) experiments, neutron scattering and hard-sphere exo-anomeric (HSEA) calculations. So, the concept evolved successively from the Y-, to the T-, the bird- and the "broken wing"-conformation, until the demonstration, that these conformers are interconvertible. The bird-conformation as well as the concept of the mobility of antennae are in a good agreement with the reactivity of lectins, including membrane lectins, by rendering accessible any specific sugar structure, and with the activity of glycosyltransferases by making reachable the substitutable hydroxyl groups even in the case of pentaantennary structures. Along this line, we know now that the tetraantennary glycans adopt an "umbrella conformation" in which the four antennae are disposed parallely to the protein surface and act as protective shields. So could be explained the resistance towards proteases and the weak antigenicity of numerous glycoproteins as well as the peculiar behaviour and resistance of metastatic cancerous cells since it has been recently demonstrated that membrane glycoproteins and fibronectin of this kind of cells are significantly enriched in tri- and tetraantennary glycans.

Further justification for the exo-anomeric effect. Conformational analysis based on nuclear magnetic resonance spectroscopy of oligosaccharides

Hard-sphere (HS) calculations predict three conformers for the branched B human blood group antigenic determinant (α LFuc(1 → 2)[α DGal(1 → 3)]βDGal) and two conformers for the linear tetrasaccharide (αLRha(1 → 2)αLRha(1 → 3)αLRha(1 → 3)βDGlcNAc), which constitutes a part of the repeating unit of the Shigellaflexneri O-antigen, which differ in conformational energy by less than 0.7 and 0.2 kcal/mol, respectively. However, a detailed 1H nmr study of specific interunit deshielding effects, quantitative treatment of spin-lattice relaxation times, and nuclear Overhauser enhancements requires that only one of the conformers thus predicted be, in fact, conformationally preferred in solution. These conformers are those predicted by hard-sphere exo-anomeric (HSEA) calculations.