Jeremy P. Carver

Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection

Ten characterized sialylated oligosaccharides from bovine fetuin (B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming, Biochemistry, in press; and D. A. Cumming, C. G. Hellerqvist, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and B. Bendiak, Biochemistry, in press) were chromatographed using high-performance anion-exchange chromatography with pulsed amperometric detection. At near neutral pH values, oligosaccharides were separated according to their number of formal negative charges from sialic acid; however, at alkaline pH, the neutral portion of the oligosaccharides enhanced resolution due to oxyanion formation. Specifically, trisialylated triantennary oligosaccharides containing a Gal-beta(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal-beta(1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of alpha(2,6)- and alpha(2,3)-linked sialic acids (alpha(2,6)-linked sialic acid reduced retention time). The relative molar electrochemical responses for di-, tri-, tetra-, and pentasialylated oligosaccharides were found to be similar (4.8 +/- 14% relative to glucose). Coelution studies were performed with each of the characterized oligosaccharides and the mixture of oligosaccharides which were released from fetuin with N-glycanase. The relative proportion of the major classes of sialylated oligosaccharides (bi-, tri-, tetra-, and penta-) varied significantly in bovine fetuin from different sources.

Quantum mechanical and NMR spectroscopy studies on the conformations of the hydroxymethyl and methoxymethyl groups in aldohexosides.

The potential energy surfaces of the hydroxymethyl and methoxymethyl groups in methyl hexopyranosides have been extensively studied, employing quantum mechanical calculations and high resolution NMR data. The structure and energy of the C-5-C-6 rotamers were calculated at the B3LYP level of the density functional theory (DFT). For all, geometry optimizations were carried out for 264 conformers of 16 methyl D-gluco- and methyl D-galactopyranoside derivatives 1-16 at the B3LYP/6-31G** level. For all calculated minima, single-point calculations were performed at the B3LYP/6-311++G** level. Solvent effects were considered using a self-consistent reaction field method. Values of the vicinal coupling constants 3J(H-5-H-6R), 3J(H-5-H-6S), 3J(C-4-H-6R), and 3J(C-4-H-6S) for methyl D-glucopyranosides, methyl D-galactopyranosides and their 6-O-methyl derivatives 9-16 were measured in two solvents, methanol and water. The calculated gg, gt, and tg rotamer populations of the hydroxymethyl and methoxymethyl groups in 9-16 agreed well with experimental data. The results clearly showed that the population of gg, gt, and tg rotamers is sensitive to solvent effects. It was concluded that the preference of rotamers in 1-16 is due to the hydrogen bonding and solvent effects.

Experimental structure determination of oligosaccharides

Abstract There is evidence of significant internal motion in oligosaccharides yet the past year has shown a continuing focus on the representation of oligosaccharides by single three-dimensional structures. Methods are needed to generate a more realistic representation, i.e. an ensemble of structures consistent with the experimental data. New approaches hold out hope for the eventual development of such methods.

Syntheses of model oligosaccharides of biological significance.Synthesis of methyl 3,6-DI-O-(α-d-mannopyranosyl)-α-d-mannopyranoside and the corresponding mannobiosides

Abstract Methyl 2- O -allyl-4,6- O -benzylidene-3- O -(2,3,4,6-tetra- O -acetyl- α - d -mannopyranosyl)- α - d -mannopyranoside( 12 ) was prepared in 90 % yield by Helferich glycosylation of methyl 2- O -allyl-4,6- O -benzylidene- α - d -mannopyranoside ( 9 ) with tetra- O -acetyl- α - d -mannopyranosyl bromide ( 11 ). Removal of the benzylidene group and second Helferich glycosylation with 11 led to methyl 2- O -allyl-3,6-di- O -(2,3,4,6-tetra- O -acetyl- α - d -mannopyranosyl)- α - d -mannopyranoside ( 14 ) which, after deallylation and Zemplen deacetylation, gave the title compound 5. The disaccharides methyl 3- O -( α - d -mannopyranosyl)- α - d -mannopyranoside ( 7 ) and methyl 6- O -( α - d -mannopyranosyl)- α - d -mannopyranoside ( 6 ) have also been synthesized. Complete assignments of the 1 H-n.m.r. spectra of the compounds 5, 6 , and 7 are given.

On the reaction pathways and determination of transition-state structures for retaining α-galactosyltransferases

The catalytic mechanism of retaining glycosyltransferases is not yet completely understood, but one possible mechanism, by analogy with retaining glycosidases, is a double-displacement mechanism via a covalent glycosyl-enzyme intermediate (CGE). We have investigated various reaction pathways for this mechanism using non-empirical quantum mechanical methods. Because a double-displacement mechanism presumes a reaction happening in two steps, we have used predefined reaction coordinates to calculate the potential energy surface describing each step of the mechanism. By investigating several potential candidates to act as a catalytic base, this study attempts to shed some light on the unclear mechanism of the second step of the reaction. All intermediates and transition states on the reaction pathways were characterized using basis sets up to the DFT/B3LYP/6-311++G**//DFT/B3LYP/6-31G* level. Reaction pathways and structural changes were compared with the results previously obtained for inverting glycosyltransferases. The outcome of this study indicates, that among the reaction models investigated, the energetically favorable one is also the most plausible given the existing experimental data. This model requires the presence of only one catalytic acid in the active site with the UDP functioning as a general base in the second step of the reaction. This mechanism is in agreement with both kinetic data in the literature and the description of X-ray structures of retaining glycosyltransferases solved up to today.

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human α1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.

Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides

An analysis has been carried out of the three-dimensional structure of a tetrasaccharide, Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcN Ac beta 1-OCD3, which is a fragment from the high mannose type of N-linked oligosaccharides. Although earlier work had suggested that this fragment might adopt a stable three-dimensional structure, both n.m.r. and conformational energy calculations support the existence of an ensemble of structures. The conformational entropy calculated from the ensemble and the distribution of distances between the terminal Man(alpha 1-3) and GlcN Ac residues, however, suggests that a significant fraction of the ensemble has the two terminal residues in close proximity.

Lectin Receptors and Lectin Resistance in Chinese Hamster Ovary Cells

Chinese hamster ovary (CHO) cells previously selected in a single-step for resistance to one or two different lectins and assigned to individual phenotypic groups on the basis of their unique patterns of lectin resistance, have been examined for their lectin-binding abilites. The lectin-binding parameters of CHO cells were shown to be very complex in a detailes study of the binding of 125I-WGA to wild-type (WT) cells. On the basis of these results, standard assay conditions were established and comparative binding studies between the twenty-two WT and lectin-resistant (LecR) clones were performed. A general correlation of lectin resistance with decreased lectin-binding ability and of lectin sensitivity with increased lectin-binding ability was found, although several exceptions to this trend were observed.

Accessibility of D-Mannopyranoside Glycosylating Synthons by Acetolysis for Preparations of Oligosaccharide Moieties of N-Linked Glycoproteins

Abstract Selective acetolysis of methyl 2, 3, 4, 6-tetra-O-benzyl-α-D-manno-pyranoside (2) allows for easy preparation of 1-acetates of 2, 3,4, 6-tetra-O-benzyl (5), 6-O-acetyl-2, 3, 4, tri-O-benzyl-(6), 4, 6-di-O-acetyl-2,3-di-O-benzyl-(7), 3, 4, 6-tri-O-acetyl-2-O-benzyl-(8), and 2, 4, 6-tri-O-acetyl-3-O-benzyl-D-mannopyranoside (9). 8 and 9 formed are separated by preparative HPLC in 30-60g scale. The time course of previously described acetolyses of 3, 4, 6-tri-O-benzyl- 1, 2-O-(1-methoxyethyidene)-β-D-mannopyranose (3), and methyl 2, 3-dt-O-benzyl-4, 6-O-benzylldene-α-D-mannopyranoside (4) giving 9, 1, 2, 6-tri-O-acetyl-3, 4-di-O-benzyl-(10), and 1, 2-di-O-acetyl-3, 4, 6-tri-O-benzyl-(11) α-D-mannopyranose as well 7 have been studied.

Ab initio molecular orbital calculation of carbohydrate model compounds 4. Flexibility of Ψ-type glycosidic bonds in carbohydrates

Abstract An ab initio study of the conformational behavior of aglycon OC glycosidic bonds and rotameric distribution in O-methylated carbohydrates has been carried out on axial and equatorial 1,2-, 1,3- and 1,4-dimethoxytetrahydropyran as models. The geometry of the conformers about the C(aglycon)O(glycosidic) bond (Ψ-type) in 12 models was determined by gradient optimization at the SCF level using split valence 6-31G ∗ basis sets. The potential of rotation has been calculated using 6-31G ∗ and 631 + G ∗ basis sets. At all levels of theory, both axial and equatorial forms prefer the GT or TG conformers around the CO glycosidic bond over the GG conformer. Exceptions are models of (1 → 2) linkages with the equatorial anomeric methoxyl group ( E2A and E2E ), where the TG conformer is not present. Calculated potential energy profiles show high flexibility within a 1.5 kcal mol −1 low energy region that is 180 ° wide. The glycosidic bond angle ClO i C i depends on the torsion angle Ψ and assumes values in the interval from 115 ° to 125 °.