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Benjamin G Davis

Publications and source records attributed to Benjamin G Davis.

At least 37 records · Page 2Linked to original sources
PubMed2005

Lectins: tools for the molecular understanding of the glycocode.

Recent progress in glycobiology has revealed that cell surface oligosaccharides play an essential role in recognition events. More precisely, these saccharides may be complexed by lectins, carbohydrate-binding proteins other than enzymes and antibodies, able to recognise sugars in a highly specific manner. The ubiquity of lectin-carbohydrate interactions opens enormous potential for their exploitation in medicine. Therefore, extraordinary effort is made into the identification of new lectins as well as into the achievement of a deep understanding of their functions and of the precise mechanism of their association with specific ligands. In this review, a summary of the main features of lectins, particularly those found in legumes, will be presented with a focus on the mechanism of carbohydrate-binding. An overview of lectin-carbohydrate interactions will also be given, together with an insight into their energetics. In addition, therapeutic applications of lectins will be discussed.

Carbohydrates↗
PubMed2005

Investigation of the interaction between peanut agglutinin and synthetic glycopolymeric multivalent ligands.

The interaction between synthetic glycoplymers bearing beta-D-galactose side groups and the lectin peanut agglutinin (PNA) was investigated by UV-difference spectroscopy and isothermal titration calorimetry (ITC). UV-difference spectroscopy indicated that the polymer-lectin interaction was stronger than that between PNA and either the corresponding monomer, D-galactose or D-lactose. The thermodynamics of binding (K, DeltaG, DeltaH, DeltaS and n) were determined from ITC data by fitting with a two-site, non-cooperative binding model. It was found that the glycopolymer displayed around a 50 times greater affinity for the lectin than the parent carbohydrate, and around 10 times greater than the monomer, on a valency-corrected basis. Binding was found to be entropically driven, and was accompanied by aggregation and precipitation of protein molecules. Furthermore, interesting differences between polymers prepared either from deacetylated monomers, or by deacetylation of pre-formed polymers, were found.

Galactans↗
PubMed2005

Glyco- and peptidomimetics from three-component Joullié-Ugi coupling show selective antiviral activity.

Chlorination-elimination chemistry coupled with three-component Joullié-Ugi reaction and facile deprotection allowed efficient access to an array of polyhydroxylated pyrrolidines through parallel synthesis that may be considered to be a library of imino (aza) sugars (glycomimetics) and/or dihydroxyprolyl peptides (peptidomimetics). The utility of generating such a library was illustrated by screening against 15 different targets that revealed potent and selective inhibition of the Gaucher's disease glycosyltransferase enzyme glucosylceramide synthase and of primary pathogen model for human hepatitis C virus (HCV) and bovine diarrhoeal virus (BVDV). An observed selectivity for this HCV model over hepatitis B virus and remarkably low toxicity suggest a novel mode of action.

Antiviral Agents↗
PubMed2005

High-throughput mass-spectrometry monitoring for multisubstrate enzymes: determining the kinetic parameters and catalytic activities of glycosyltransferases.

A novel high-throughput screening (HTS) method with electrospray time-of-flight (ESI-TOF) mass spectrometry allows i) rapid and broad screening of multisubstrate enzyme catalytic activity towards a range of donor and acceptor substrates; ii) determination of full multisubstrate kinetic parameters and the binding order of substrates. Two representative glycosyltransferases (GTs, one common, one recently isolated, one O-glycosyltransferase (O-GT), one N-glycosyltransferase (N-GT)) have been used to validate this system: the widely used bovine beta-1,4-galactosyltransferase (EC 2.4.1.22), and the recently isolated Arabidopsis thaliana GT UGT72B1 (EC 2.4.1.-). The GAR (green/amber/red) broad-substrate-specificity screen, which is based on the mass ion abundance of product, provides a fast, high-throughput method for finding potential donors and acceptors from substrate libraries. This was evaluated by using six natural and non-natural donors (alpha-UDP-D-Glucose (UDPGlc), alpha-UDP-N-Acetyl-D-glucosamine (UDPGlcNAc), alpha-UDP-D-5-thioglucose (UDP5SGlc), alpha-GDP-L-fucose (GDPFuc), alpha-GDP-D-mannose (GDPMan), alpha,beta-UDP-D-mannose (UDPMan)) and 32 broad-ranging acceptors (sugars, plant hormones, antibiotics, flavonoids, coumarins, phenylpropanoids and benzoic acids). By using the fast-equilibrium assumption, KM, kcat and KIA were determined for representative substrates, and these values were used to determine substrate binding orders. These screening methods applied to the two very different enzymes revealed some unusual substrate specificities, thus highlighting the utility of broad-ranging substrate screening. For UGT72B1, it was shown that the donor specificity is determined largely by the nucleotide moiety. The method is therefore capable of identifying GT enzymes with usefully broad carbohydrate-transfer ability.

Animals↗
PubMed2005

Developing promiscuous glycosidases for glycoside synthesis: residues W433 and E432 in Sulfolobus solfataricus beta-glycosidase are important glucoside- and galactoside-specificity determinants.

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG.

Alkylation↗
PubMed2004

"Polar patch" proteases as glycopeptiligases.

The strategy of combined site directed mutagenesis and chemical modification with polar prosthetic groups was used to broaden substrate specificity of proteases resulting in the first successful formation of glycopeptides through the use of glucoamino acid acyl donors in yields of up to 90%.

Glycolipids↗
PubMed2004

LEAPT: lectin-directed enzyme-activated prodrug therapy.

Targeted drug delivery to selected sites allows reduced toxicity, enhanced efficiency and interchangeable target potential [Langer, R. (2001) Science 293, 58-59 and Molema, G. & Meijer, D. K. F., eds. (2001) Drug Targeting (Wiley-VCH, Weinheim, Germany)]. We describe a bipartite drug-delivery system that exploits (I) endogenous carbohydrate-to-lectin binding to localize glycosylated enzyme conjugates to specific, predetermined cell types followed by (II) administration of a prodrug activated by that predelivered enzyme at the desired site. The carbohydrate structure of an alpha-L-rhamnopyranosidase enzyme was specifically engineered through enzymatic deglycosylation and chemical reglycosylation. Combined in vivo and in vitro techniques (gamma scintigraphy, microautoradiography and confocal microscopy) determined organ and cellular localization and demonstrated successful activation of alpha-L-rhamnopyranoside prodrug. Ligand competition experiments revealed enhanced, specific localization by endocytosis and a strongly carbohydrate-dependent, 60-fold increase in selectivity toward target cell hepatocytes that generated a >30-fold increase (from 0.02 to 0.66 mg) in protein delivered. Furthermore, glycosylation engineering enhanced the serum-uptake rate and enzyme stability. This created enzyme activity (0.2 units in hepatocytes) for prodrug therapy, the target of which was switched simply by sugar-type alteration. The therapeutic effectiveness of lectin-directed enzyme-activated prodrug therapy was shown through the construction of the prodrug of doxorubicin, Rha-DOX, and its application to reduce tumor burden in a hepatocellular carcinoma (HepG2) disease model.

Animals↗
PubMed2004

Selective electrochemical glycosylation by reactivity tuning.

Electrochemical glycosylation of a selenoglycoside donor proceeds efficiently in an undivided cell in acetonitrile to yield beta-glycosides. Measurement of cyclic voltammograms for a selection of seleno-, thio-, and O-glycosides indicates the dependence of oxidation potential on the anomeric substituent allowing the possibility for the rapid construction of oligosaccharides by selective electrochemical activation utilising variable cell potentials in combination with reactivity tuning of the glycosyl donor. A variety of disaccharides are readily synthesised in high yield, but limitations of the use of selenoglycosides as glycosyl donors for selective glycosylation of thioglycoside acceptors are exposed. The first electrochemical trisaccharide synthesis is described.

Electrochemistry↗
PubMed2004

Glycodendriproteins: a synthetic glycoprotein mimic enzyme with branched sugar-display potently inhibits bacterial aggregation.

The continuing ability of bacteria to resist current antibiotic treatments highlights the need for alternative strategies for inhibiting their pathogenicity. Bacterial attachment is a major factor in infectivity and virulence. This key binding phase of bacteria to any potential host is mediated by adhesin proteins and so these present an attractive therapeutic target for antiinfective blocking strategies. However, the natural ligands to adhesins are large, typically complex molecules that are difficult to mimic with small molecules. We describe here a method that creates precise synthetic mimics of glycoproteins that are designed to bind adhesins. By using protein-degrading enzymes as the basis for these mimics we have created large-molecule protein ligands that inhibit aggregation of pathogenic bacteria at levels greater than a million-fold higher than small-molecule inhibitors of adhesins.

Actinomyces↗
PubMed2003

Precise structure activity relationships in asymmetric catalysis using carbohydrate scaffolds to allow ready fine tuning: dialkylzinc-aldehyde additions.

The ready construction of 24 stereochemically and functionally diverse carbohydrate ligand structures from a core D-glucosamine scaffold has allowed the evaluation of broad ranging structure activity relationships in ligand accelerated zincate additions to aldehydes, with variations in deltadeltaG+/+(R-S) of up to 5650 J mol(-1) that create opposing senses of asymmetric induction and that are consistent with models based on several ligand X-ray structures and molecular mechanics analysis. Factorial analysis of enantioselectivity using key dihedral angles and steric volume on N-2 also highlight the potential for the use of factorial design in ligand construction.

Aldehydes↗
PubMed2003

Highly diastereoselective additions to polyhydroxylated pyrrolidine cyclic imines: ready elaboration of aza-sugar scaffolds to create diverse carbohydrate-processing enzyme probes.

Representative diastereomeric, erythritol and threitol polyhydroxylated pyrrolidine imine scaffolds have been rapidly elaborated to diversely functionalized aza-sugars through highly diastereoselective organometallic (RM) additions (R=Me, Et, allyl, hexenyl, Ph, Bn, pMeO-Bn). The yields for these additions have all been substantially enhanced from previously optimised levels (<58 %) for normal additions using a reverse addition procedure (e.g. R=Ph; 44 % normal mode --> 78 % reverse mode). The high diastereoselectivities (>98 % de for all except R=Me) are consistent with additions that are controlled by the configuration of the C-2 centre adjacent to the azomethine imine carbon and the conformation of the pyrrolidine imine. The high potential of this method was demonstrated by concise syntheses of 1-epi- and 2-epi-desacetylanisomycins. In addition, the late stage addition of hydrophobic substituents, which this imine addition methodology allows, enabled the preparation of novel aza-sugars with enhanced inhibitory potential. This was highlighted by the screening of a representative selection of these "hydrophobically-modified" aza-sugars against a diverse panel of 12 non-mammalian and human carbohydrate-processing enzymes. This identified a novel nanomolar alpha-galactosidase inhibitor (IC(50)=250 nM) and a novel highly selective glucosylceramide synthase inhibitor (IC(50)=52 microM, no alpha-glucosidase inhibition at 1 mM). Furthermore, analysis of the structure-activity relationships of racemic series of inhibitors allowed some validation of Fleet's mirror-image enzyme active site postulate.

Journal Article↗