J. Albert van Kuik

Structural requirements of the fructan-lipid interaction.

Fructans are a group of fructose-based oligo- and polysaccharides. They are proposed to be involved in membrane protection of plants during dehydration. In accordance with this hypothesis, they show an interaction with hydrated lipid model systems. However, the structural requirements for this interaction are not known both with respect to the fructans as to the lipids. To get insight into this matter, the interaction of several inulins and levan with lipids was investigated using a monomolecular lipid system or the MC 540 probe in a bilayer system. MD was used to get conformational information concerning the polysaccharides. It was found that levan-type fructan interacted comparably with model membranes composed of glyco- or phospholipids but showed a preference for lipids with a small headgroup. Furthermore, it was found that there was an inulin chain-length-dependent interaction with lipids. The results also suggested that inulin-type fructan had a more profound interaction with the membrane than levan-type fructan. MD simulations indicated that the favorable conformation for levan is a helix, whereas inulin tends to form random coil structures. This suggests that flexibility is an important determinant for the fructan-lipid interaction.

A 1H NMR database computer program for the analysis of the primary structure of complex carbohydrates

A 1H NMR database computer program has been developed to determine the primary structure of complex carbohydrates. The database contains carbohydrate structures, their corresponding 1H NMR data, and literature references. From an input list of chemical shift values, the program generates an output list of partially or completely matching carbohydrate structures. In order to facilitate the recognition of the matching part of the selected carbohydrate structures, these structures are displayed with the matching structural elements highlighted. This new 1H NMR database, together with the search program described, now provides a fast access to the published 1H NMR data of complex carbohydrates and furnishes easy links to carbohydrate structures. The performance of the program is demonstrated by the analysis of five carbohydrate fractions prepared from a pool of horse serum glycoproteins.

Sulfated N‐linked carbohydrate chains in porcine thyroglobulin

N‐linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide‐alditols was fractionated by high‐voltage paper electrophoresis, the acidic fractions were further separated by high‐performance liquid chromatography on Lichrosorb‐NH2, and analyzed by 500‐MHz 1H‐NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide‐alditols, the following sulfated carbohydrate chains could be identified:

Improved carbohydrate force field for gromos: ring and hydroxymethyl group conformations and exo-anomeric effect

Abstract In this work, improvements of the carbohydrate force field for gromos have been carried out by combined molecular mechanics (MM) and molecular dynamics (MD) calculations. With the original force field, a far too small relative energy (4.5 kJ mol −1 ) between the ‘normal’ chair conformation ( 4 C 1 ) and the ‘inverted chair’ conformation ( 1 C 4 ) of the methyl β- d -glucopyranoside has been observed in vacuum, compared with ab initio and MM3 calculations that predict 16.0–30.0 kJ mol −1 . The ring inversion has been solved by a large increase of the bond-angle force constants involving the oxygen atom of hydroxyl groups. The consequence of such a modification for the relative energy between the two chair conformations is an increase to 13.2 kJ mol −1 . Furthermore using a potential-of-mean-force calculation through umbrella sampling, with explicit solvent molecules, on both methyl β- d -glucopyranoside and methyl β- d -galactopyranoside, it has been found that the rotamer distribution of the hydroxymethyl group does not reproduce accurately NMR data. The hydroxymethyl group conformer distribution has been improved by increasing the torsional barrier around the considered bond (OACS2CS1OS) leading to a closer agreement with experimental rotamer distributions. In addition, an improved dihedral potential has been used to account for the exo-anomeric effect. MM calculations in vacuum on the methyl β- d -fructofuranoside have shown that our force field modifications have only a slight influence on the conformation of the five-membered ring. This was confirmed by MD simulation in aqueous solution.

Additional fucosyl residues on membrane glycoproteins but not a secreted glycoprotein from cystic fibrosis fibroblasts

Glycopeptides derived from peripheral membrane glycoproteins of skin fibroblasts of seven patients with cystic fibrosis (CF) had an increase in fucosyl residues when compared with those of seven age, race and sex matched controls (Pediatr Res 1985;19:368-374). To further define these results, the membrane glycopeptides which bound to immobilized lentil lectin and thereby enriched in fucosyl residues linked alpha 1----6 to N-acetylglucosamine attached to asparagine, were Pronase digested, partially purified and examined by 500-MHz 1H-NMR spectroscopy. The CF derived glycopeptides had two different features when compared to those from Controls (1) an increased number of fucosyl residues linked alpha 1----6 to the N-acetylglucosamine attached to asparagine and (2) fucosyl residues linked alpha 1----3 to a branch N-acetylglucosamine. The glycopeptides from both sources were of the di and triantennary type containing sialic acid linked alpha 2----3 and alpha 2----6 to galactose in an approximate molar ratio of 3:2 and 2:1, from CF and Control, respectively. Glycopeptides derived from a glycoprotein, fibronectin, secreted from CF fibroblasts were also examined by 1H-NMR spectroscopy and showed no evidence of fucosyl residues linked alpha 1----3 to branch N-acetylglucosamine and a lesser percentage of core fucose than found in the peripheral membrane glycopeptides. These results define further the altered fucosylation of the CF peripheral membrane glycoproteins.

Conversion of GalNAcβ(1–4)GlcNAcβ-OMe into GalNAcβ(1–4)-[Fucα(1–3)]GlcNAcβ-OMe using human milk α3/4-fucosyltransferase synthesis of a novel terminal element in glycoprotein glycans

Incubation of GalNAcβ(1–4)GlcNAcβ‐OMe with GDP‐Fuc in the presence of human milk α3/4‐fucosyltransferase resulted in the formation of GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe. Under conditions that led to complete α3‐fucosylation of Galβ(1–4)GlcNAcβ‐OEt, GalNAcβ(1–4)GlcNAcβ‐OMe was fucosylated for more than 85%. For the identification of the isolated fucosylated products one‐ and two‐ dimensional 1H‐NMR spectroscopy was applied. In vacuo molecular dynamics simulations of Galβ(1–4)[Fucα(1–3)]GlcNAcβ‐OEt and GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe using the CHARMm based force field CHEAT, demonstrated only small differences between the conformations of these compounds. This illustrates the minor conformational influence of the substituent at C‐2′, i.e. a hydroxyl function versus a N‐acetyl group.

Synthesis and conjugation of oligosaccharide fragments related to the immunologically reactive part of the circulating anodic antigen of the parasite Schistosoma mansoni

The immunoreactive part of the circulating anodic antigen (CAA) from the parasite Schistosoma mansoni is a threonine-linked polysaccharide consisting of →6)-[β-D-GlcpA-(1→3)]-β-D-GalpNAc-(1→ repeating disaccharides. In the framework of an immunochemical project, as a follow-up of earlier synthesized di- to tetrasaccharide CAA fragments, the synthesis of a spacer-containing pentasaccharide fragment, 3-(2-aminoethylthio)propyl (2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→6)-[(β-D-glucopyranosyluronic acid)-(1→3)]-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→6)-[(β-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-β-D-galactopyranoside, is described. Moreover, 1-O-[3-(2-aminoethylthio)propyl]-N-acetyl-β-D-galactosamine was synthesized. Oxidation steps in the synthesis of tri- to pentasaccharide CAA fragments were performed using pyridinium dichromate and acetic anhydride. TEMPO-catalyzed oxidations were explored in the synthesis of 1-O-[6-aminohexyl]-β-D-glucuronic acid and 3-aminopropyl (2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→6)-[(β-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-β-D-galactopyranoside, affording short reaction times and high yields. All synthesized compounds, including the earlier described 3-(2-aminoethylthio)propyl-spacered di-, tri-, and tetrasaccharide CAA fragments, were conjugated to BSA using squaric diester chemistry with coupling efficiencies in the range of 30–90%. The efficiency decreased when larger oligosaccharides were coupled to BSA. Finally, conformational analyses of the tri- and tetrasaccharide fragments were performed using Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations.

Databases of complex carbohydrates

Abstract Ready access to the wealth of data being amassed on the physical properties of complex carbohydrates is of particular interest to those involved in the study and manipulation of food carbohydrate structure and functionality. This article describes the main databases offering on-line, CD-ROM or diskette retrieval of such information.