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Biomedical subjects

D M Whitfield

Publications and source records attributed to D M Whitfield.

17 recordsLinked to original sources
PubMed2001

Chemoenzymatic iterative synthesis of difficult linkages of oligosaccharides on soluble polymeric supports.

[reaction: see text]. A trisaccharide donor containing a cis-Galpalpha(1-->4)Galp linkage was prepared using a synthetic strategy based on chemoenzymatic oligosaccharide synthesis on a soluble polymeric support. Significantly, only retaining glycosyltransferases gave complete reactions, whereas inverting enzymes showed little or no activity with poly(ethylene glycol) (MPEG)-bound lactose as an acceptor. The MPEG-attached trisaccharide was shown to bind to Verotoxin-1 by transfer NOE studies through the Galpalpha(1-->4)Galp portion of the molecule.

Carbohydrate Conformation↗
PubMed2001

The conformational origin of the barrier to the formation of neighboring group assistance in glycosylation reactions: a dynamical density functional theory study.

Static and dynamical Density Functional Theory studies of 2,6-di-O-acetyl-3,4-O-isopropylidene-D-galactopyranosyl cation have shown that this cation can exist in two conformers characterized as (2)S(O) and B(2,5), respectively. The (2)S(O) conformer has the O-2 acyl group equatorial with the carbonyl syn to H-2 and is populated by monocyclic oxocarbenium ions. These conformational features are present in the structurally related glycosyl donor ethyl 2,6-di-O-benzoyl-3,4-O-isopropylidene-beta-D-galactothiopyranoside as determined by X-ray diffraction studies. The B(2,5) conformer has O-2 axial and allows the carbonyl to rotate and close the five-membered ring to form a bicyclic dioxolenium ion. Constraints based on natural internal coordinates were implemented to study this conformational transition. In this way the barrier to interconversion has been determined to be 34 kJ mol(-)(1) with a transition state characterized as (O)S(2) and a pathway involving pseudorotation. Thus, for the first time the structures and energetics of the key ions postulated to be involved in neighboring group assisted glycosylation reactions have been determined.

Journal Article↗
PubMed2001

Immunostimulant oxidized beta-glucan conjugates.

beta-Glucans are polysaccharides that act as nonspecific immune system stimulants. However, many beta-Glucans are sparingly soluble in water. This work describes an oxidative procedure, which solubilizes the beta-Glucan from Saccharomyces cerevisiae and maintains its immunostimulatory properties. Furthermore, the carboxylates at the site of oxidation allow for the conjugation of small molecule immunostimulants. Both the parent oxidized beta-glucan and its conjugates with O-beta-alanyl-5-[6-(N,N'-dimethylamino)purin-9-yl]pentanol stimulate cytotoxic T-lymphocytes (CTLs), B cells and macrophages. In addition, they both stimulate natural killer (NK) cells, a property which the small molecule purine does not possess.

Adjuvants, Immunologic↗
PubMed2000

Polymer-supported and chemoenzymatic synthesis of the Neisseria meningitidis pentasaccharide: a methodological comparison.

Neisseria meningitidis trisaccharide [GlcNAc[(1-->3)Galbeta(1-->4)Glc-R], tetrasaccharide [Galbeta(1-->4)GlcNAcbeta(1--> 3)Galbeta(1-->4)Glc-R], and a pentasaccharide [Neu5Acalpha(2-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)G albeta(1-->4)Glc-SPh] were prepared via conventional chemical synthesis, polymer-supported synthesis, and chemoenzymatic methods, starting from D-lactose. The polymer polyethyleneglycol monomethylether (MPEG) and the linker dioxyxylene (DOX) were used with a lactose-bound acceptor to improve the purification process. Several enzymes (LgtA, GalE-LgtB fusion, and CMP-Neu5Ac synthetase/sialyltransferase fusion) were used for syntheses of these oligosaccharides. Excellent stereo- and regioselectivities as well as high yield (> 90% from Gal(1-->4)Glc-SPh) of the pentasaccharide were obtained. Both of the convenient processes are suitable for efficient preparation of target oligosaccharides.

Antigens, Bacterial↗
PubMed2000

Ready access to sialylated oligosaccharide donors.

[formula: see text] Numerous glycoconjugates contain the disaccharide Neu5Ac alpha (2-->3)DGalp. An efficient way to incorporate this disaccharide into synthetic glycoconjugates is to develop a disaccharide building block. This communication reports a chemoenzymatic route to such a building block which requires as few as four steps. Some examples using more chemical steps are also presented, which increase the flexibility. These disaccharide donors were used to prepare synthetic trisaccharides.

Carbohydrate Conformation↗
PubMed1999

Synthesis of a disialylated hexasaccharide of type VIII group B Streptococcus capsular polysaccharide.

As part of our program to design, develop and prepare protective vaccines against the bacterial pathogens Group B Streptococcus, we report the synthesis of a disialylated hexasaccharide. This hexasaccharide represents a portion of the serotype-specific capsular polysaccharide of Type VIII that has the tetrasaccharide repeat unit [beta-L-Rhap-(1-->4)-beta-D-Glcp-(1-->4)-[alpha-Neu5Ac-(2--> 3)]-beta-D- Galp-(1-->4)]n. A tetrasaccharide corresponding to this repeat unit has been synthesized by us [E. Eichler, H.J. Jennings, D.M. Whitfield, J. Carbohydr. Chem., 16 (1997) 385-411]. Since the protective epitopes are believed to involve several repeat units, methods to extend this tetrasaccharide were examined. This objective requires a glycosylation of the unreactive OH-4 of the beta-L-Rhap, which was accomplished by coupling a D-Galp glycosyl trichloroacetimidate donor with a beta-L-Rhap-(1-->4)-D-Glcp acceptor. Subsequent coupling of this trisaccharide as a donor to an alpha-Neu5Ac-(2-->3)-D-Galp disaccharide acceptor gave a pentasaccharide. The pentasaccharide was deprotected and enzymatically sialylated using an alpha-(2-->3)-sialyltransferase from Campylobacter jejuni to give the title hexasaccahride alpha-Neu5Ac-(2-->3)- beta-D-Galp-(1-->4)-beta-L-Rhap-(1-->4)-beta-D-Glcp-(1-->4)-[alpha -Neu5Ac- (2-->3)]-beta-D-Galp-(1-->O)-(CH2)3N3.

Bacterial Capsules↗
PubMed1998

Polymer-supported tin carbohydrate chemistry.

It was anticipated that stannylation of carbohydrates could be achieved using tin on a polymer-support. Such immobilization simplifies the purification of the carbohydrate because the toxic tin reagent can be removed by filtration. In this case an alkene linker (3-buten-1-ol) was added to chloromethylated 2% cross-linked polystyrene by etherification. Photochemical hydrostannylation with dibutyltinchlorohydride gave a polymer bound trialkyl tin chloride. The Sn-Cl could be hydrolysed with NaOH to yield a resin with terminal Sn-O bonds. Highly regioselective acylation of methyl alpha-D-mannopyranoside (alphaMeMan) to its 3-O-benzoyl derivative was achieved. Traces of the mono 6-O-benzoate and the 3,6-di-O-benzoate were also obtained. Methyl alpha-D-glucopyranoside was also selectively acylated to its 2-O-benzoate but this reaction gave a more complex mixture. The isolated yields (10-30% based on sugar) were disappointingly low. The yields were improved to about 60% with 5% cross-linked resin.

Acylation↗
PubMed1996

Glycosylation reactions--present status future directions.

A short review of the present status of glycosylation reactions is presented. The reactivity of both proven and newer glycosylation methods are briefly discussed. Emphasis is placed on the control of stereochemistry and regiochemistry. As well, the identification and avoidance of side reactions is covered. Polymer-supported synthesis of oligosaccharides is noted as a promising direction for eliminating some of the problems associated with purification. It is suggested that a better understanding of the mechanism of glycosylation reactions is necessary for future improvements to stereoselectivity and regioselectivity. A key advance would be methods for enhancing the reactivity of weakly nucleophilic hydroxyls.

Carbohydrate Sequence↗
PubMed1993

Isoglobotetraosylceramide is a marker for highly metastatic cells in rat mammary adenocarcinomas.

We have previously identified a neutral glycolipid antigen which appears to be a surface antigenic marker for the metastatic subpopulation in the R3230AC rat mammary adenocarcinoma (S.A. Carlsen, M. Barry, and K. Newton, Clin. Exp. Metastasis, 8: 141-151, 1990). In this article we describe the structural characterization of this glycolipid antigen. The sequence of the sugars in the saccharide portion of the molecule was determined by specific glycosidase cleavage and further confirmed by mass spectroscopic analysis. The nature of the linkages between the monosaccharide units was determined by methylation analysis. The final structure was confirmed by NMR analysis and found to be isoglobotetraosylceramide (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4Gle beta 1-O-ceramide). We also present evidence that the cells marked by this antigen have a higher metastatic potential than the cells lacking this glycolipid as measured by the formation of lung colonies after i.v. injection of the cells into the tail vein of the rat. Furthermore, isoglobotetraosylceramide seems to play a direct role in the metastatic process since the blocking of exposed antigen with monoclonal antibodies, or their Fab fragments, results in a highly significant decrease in lung colony formation.

Adenocarcinoma↗
PubMed1992

Characterization and cellular distribution of acidic peptide and oligosaccharide metal-binding compounds from kidneys.

Two low-molecular-mass Ni-binding fractions first isolated from human kidneys [Templeton & Sarkar (1985) Biochem. J. 230, 35-42] are further characterized. Both components are acidic and are readily separated from each other by gel chromatography on Bio-Gel P-2. After equilibration with 63Ni the largest complex constitutes about 30% of the radioactive 63Ni and is an approx. 3.5 kDa peptide and the smallest species comprise short oligosaccharides containing 70% of the radioactivity. Both of these components are found in human, bovine and porcine kidneys as well as in a porcine proximal tubule-like cell line LLC-PK1. There is a small variation in amino acid composition between species. The oligosaccharides are reducing sugars and contain sulphate, glucosamine, glucuronic acid and iduronic acid with two to four overall negative charges. The monosaccharide composition was determined by h.p.l.c. with pulsed amperometric detection of the acid hydrolysates and by gas chromatography. In the LLC-PK1 cell line the acidic peptide is both intracellular and extracellular, whereas the oligosaccharides are only intracellular. The concentration of extracellular peptide, as measured by 63Ni binding, is found to increase after exposure of the cells to low micromolar concentrations of Ni, whereas the oligosaccharide concentrations, also measured by 63Ni binding, remain constant. The oligosaccharide component is decreased by 40% in the presence of NH4Cl, suggesting that is derived from degradation of internalized heparan sulphate.

Aged↗
PubMed1992

Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.

As model compounds for Ni(II)-binding heparin-like compounds isolated from human kidneys (Templeton, D.M. & Sarkar, B. (1985) Biochem. J. 230 35-42.), we investigated two disaccharides--4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-2,5-anhydro- D-mannitol, disodium salt (1a), and 4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-6-O- sulfo-2,5-anhydro-D-mannitol, trisodium salt (1b)--that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (1a) was active whereas the disulfate (1b) was inactive in a high-performance liquid chromatography (HPLC) binding assay with the tracer ions 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(II) binding fractions from a complete pool of nitrous-acid-derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (1a) and the other as a tetrasulfated trisaccharide by high resolution FAB-MS, NMR and HPLC-PAD. Similarly, two synthetic disaccharides-methyl, 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-alpha-D-glucosamine, trisodium salt [IdopA2S(alpha 1,4)GlcNS alpha Me, 2a], and 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-6-O-sulfo- alpha-D-glucosamine, tetrasodium salt [IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b]--were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H NMR titrations of 1a, 1b, 2a, and 2b with Zn (OAc)2 were analyzed to yield 1:1 Zn(II)-binding constants of 472 +/- 59, 698 +/- 120, 8,758 +/- 2,237 and 20,100 +/- 5,598 M-1, respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions.

Binding Sites↗
PubMed1992

Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation.

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn(II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S (alpha 1,4)AnManOH, 1 alpha, IdopA2S (alpha 1,4)AnManOH6S, 1b, IdopA2S-(alpha 1,4)GlcNS alpha Me, 2a, and IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated [(NOE)]s, [T1]s, and [J]s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L-idopyranosiduronate ring and the 4E and 4T3 of the anhydro-D-mannitol ring are evaluated as are rotations about the C5-C6 hydroxymethylene of the AnManOH(6S) or GlcNS (6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH(6S) or GlcNS(6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially "frozen" such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH(6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS(6S) residues or the sulfate oxygens of the 6-sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2 alpha and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(alpha 1,4)GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.

Binding Sites↗
PubMed1991

Diagnostic methods for the determination of iduronic acid in oligosaccharides.

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.

Chondroitin Sulfates↗
PubMed1991

Metal binding to heparin monosaccharides: D-glucosamine-6-sulphate, D-glucuronic acid, and L-iduronic acid.

In order to ascertain which residues in heparin may be responsible for its metal binding capacities we have investigated metal binding to some of its component monosaccharides by 1H and 13C NMR. The diamagnetic Zn ion and the paramagnetic Ni ion were used as probes. 4-Methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulpho-D-glucosamine was used as a model for O-sulphates. Only weak interactions with the sulphate group were found. The 4C1 ring conformation of sodium methyl-beta-D-glucopyranosiduronate was not perturbed by binding to its carboxylate and little evidence exists for chelation. By contrast, the ring conformation of the sodium methyl-alpha-L-idopyranosiduronate is affected by the addition of Zn greater than Pb greater than Cd greater than Ca much greater than K ions. The sodium salt is suggested to be an equilibrium mixture of the 2SO and 1C4 ring conformations. Cation binding to the carboxylate group shifts this equilibrium towards the 1C4 conformation and suggests additional binding to O5 or, less likely, O4. This effect appears to be electrostatic in nature, as excess Na and protonation produce similar shifts. Lead complexation is different from the other ions and suggests some covalent character. The control of the ring conformation of iduronic acid by metal ions may have biological implications for the action of heparin and heparin-like compounds.

Cadmium↗
PubMed1990

Interactions of a molybdenum(VI) oxo-cation with some uronic acids: a 1H- and 13C-nuclear magnetic resonance study.

The interactions of molybdate, [MoO4]2- with some uronic acids in aqueous D2O solution were studied by 1H- and 13C-NMR. Evidence is presented for a specific binding site of an Mo(VI) oxo-cation (Mo2O5(2+] with alpha-,beta-D-glucuronic acid. It is proposed that complexation involves the carboxylate oxygen (O-6b) and O-4 of the hydroxyl oxygen in a pyranose form (4C1), in the pH region 3.5-5.8.

Carbon Isotopes↗