Peter Critchley

Glycosidase-catalysed oligosaccharide synthesis: preparation of N-acetylchitooligosaccharides using the β-N-acetylhexosaminidase of Aspergillus oryzae

The beta-N-acetylhexosaminidase of Aspergillus oryzae catalyses the formation of 2-acetamido-4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D- glucopyranose (di-N-acetylchitobiose) and 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D- glucopyranose from p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and 2-acetamido-2-deoxy-D-glucopyranose. The ratio of the two disaccharides is time-dependent. The ratio of (1-->4)- to (1-->6)-isomers is a maximum (approximately 9:1) at the point of disappearance of the glycosyl donor. If left to evolve, the ratio changes to 92:8 in favour of the (1-->6)-isomer. Either the (1-->4)- or the (1-->6)-isomer can be isolated by treating the appropriately enriched dissaccharide mixture with the beta-N-acetylhexosaminidase of Jack bean (Canavalia ensiformis) or the beta-N-acetylhexosaminidase of A. oryzae, respectively. Di-N-acetylchitobiose [GlcNAc(beta 1-4)GlcNAc] is an efficient donor of 2-acetamido-2-deoxy-D-glucopyranosyl units in reactions catalysed by the N-acetylhexosaminidase of A. oryzae. Di-N-acetylchitobiose itself acts as acceptor to give tri-N-acetylchitotriose [GlcNAc(beta 1-4)GlcNAc(beta 1-4)GlcNAc]. As the trisaccharide accumulates it, in turn, acts as acceptor giving tetra-N-acetylchitotetraose [GlcNAc(beta 1-4)GlcNAc(beta 1-4)GlcNAc(beta 1-4)GlcNAc]. The product mixture consisting of mono-, di-, tri-, and tetrasaccharides is conveniently separated by charcoal-Celite chromatography.

Enzymatic synthesis of glycosides using the β-galactosidase of Escherichia coli: regio- and stereo-chemical studies

β-Galactosyl transfer from lactose to acceptor alcohols (R)-(–)-butan-2-ol, (RS)-butan-2-ol, (S)-(+)-propane-1,2-diol, (RS)-propane-1,2-diol, (S)-(+)-butane-1,3-diol, (RS)-butane-1,3-diol, propane-1,3-diol, (S)-(+)-isopropylideneglycerol (1,2-O-isopropylidene-sn-glycerol) and (RS)-iso-propylideneglycerol (rac-1,2-O-isopropylideneglycerol) was studied, catalysed by the β-galactosidase (β-D-galactoside galactohydrolase EC 3.2.1.23) of Escherichia coli. Preference for galactosyl transfer to the R-enantiomers of chiral alcohols was observed, although selectivity was not pronounced. Higher selectivity for transfer to the primary hydroxy groups of the primary–secondary diols was observed. The results are interpreted in terms of a proposed active site model for the enzyme.

Biological properties of N-acyl and N-haloacetyl neuraminic acids : Processing by enzymes of sialic acid metabolism, and interaction with influenza virus

Several unnatural N-acyl neuraminic acids (N-propionyl, N-hexanoyl, N-benzoyl, N-trifluoroacetyl, N-chloroacetyl, N-difluoroacetyl) were prepared enzymatically using immobilised sialic acid aldolase. N-Trifluoroacetyl-, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were shown to enhance up to 10-fold the rate of association of influenza virus A to a sialoglycolipid neomembrane by surface plasmon resonance, and were found to act as weak inhibitors (K(iapp) 0.45-2.0 mM) of influenza virus neuraminidase. The N-propionyl, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were found to be substrates for recombinant Escherichia coli CMP sialate synthase, to give the corresponding CMP-N-acyl-neuraminic acids. CMP-N-propionyl neuraminic acid was found not to be a substrate for CMP-N-acetyl neuraminic acid hydroxylase from pig submandibular gland.

Carbohydrate–protein interactions at interfaces: comparison of the binding of Ricinus communis lectin to two series of synthetic glycolipids using surface plasmon resonance studies

Two C-lactosyl lipids and the related C-galactosyl lipids have been synthesised and their binding to RCA120 plant lectin was compared with a second series of thiolactosylethoxyalkanes. The interactions were measured quantitatively in real time by surface plasmon resonance (BIAcore) at a range of concentrations and temperatures from 5 to 30 degrees C. The C-galactosyl lipid (1,3-dimethyl-5-[beta-D-galactopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) bound much more weakly with a K(A) = 8.86 x 10(5) than the corresponding C-lactosyl lipid (1,3-dimethyl-5-[beta-D-galactopyranosyl-(1 --> 4)-beta-D-glucopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) (K(A) = 2.31 x 10(7)). The influence of the linker region of the two different series of lactosyl lipids was clearly demonstrated by the differences in the binding to RCA120 lectin. The changes in kinetic values and in the enthalpic and entropic contribution to the free energy of binding reflected the importance of the linker and the hydrocarbon anchor holding the synthetic glycolipids in the neomembrane.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of dextran and dextrin derivatives.

Application of matrix-assisted laser desorption/ionization (MALDI) to the analysis of dextran and dextrin derivatives, specifically glucose saccharides, by time-of-flight (TOF) mass spectrometry is reported. MALDI-TOF analysis was carried out on alpha-, beta-and gamma-cyclodextrin, two O-methylated beta-cyclodextrins of differing degrees of substitution (DS) and dextrans (a linear glucose saccharide), as pure and doped solutions and as mixtures of two or more of these analytes. Doping was carried out with trace amounts of inorganic salts. The purpose of the analysis of the cyclodextrins was to determine whether they would form inclusion complexes with the various added cations, or whether less specific cation addition/exchange was occurring either prior to desorption or in the gas phase.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A comparison of fragmentation patterns of linear dextran obtained by in-source decay, post-source decay and collision-induced dissociation and the stability of linear and cyclic glucans studied by in-source decay.

In the first part of this study, fragmentation patterns from a range of dextran oligomers (containing 4–20 anhydroglucose units) were compared using three different methods of analysis coupled with matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. Collision-induced dissociation (CID), prompt in-source decay (ISD) and post-source decay (PSD) all caused cleavage of the glycosidic bonds. Both CID and, to a lesser extent, ISD caused further cleavage of pyranose rings of the individual sugar residues. There was very little cleavage of pyranose rings detected in the PSD spectrum. Derivatisation of the reducing end-groups of the oligodextrans with 1-phenyl-3-methyl-5-pyrazolone (PMP) restricted cleavage in the MALDI mass spectrometer to the non-reducing end and also enabled the saccharides to be separated by high-performance liquid chromatography (HPLC) so that a single chain length could be examined as a standard. Maltoheptaose was also used as a standard. In the second part of the study, prompt ISD-MALDI mass spectrometry was used to compare the fragmentation of three oligoglucans, viz. dextran, maltodextrin and gamma cyclodextrin, that have different linkages and different secondary structure. The results showed that the degree of fragmentation correlated with the degree of freedom in the saccharide chains in solution as determined by NMR. Dextran, with the most random conformation, was fragmented most whereas there was little evidence of any fragments, not even glycosidic bond breakage, from cyclodextrin, even when the laser power was increased considerably. The fragmentation pattern of maltodextrin was intermediate. The patterns of fragmentation produced by MALDI mass spectrometry, particularly where standards are available to calibrate the spectrum and the energy of the laser is controlled, can be used to predict the type of linkage present.

Carbohydrate–protein interactions at interfaces: synthesis of thiolactosyl glycolipids and design of a working model for surface plasmon resonance

Thiolactosyl lipids designed for carbohydrate-protein binding studies have been synthesised. One representative was selected for binding studies with a plant lectin RCA120, the agglutinin from Ricinus communis. The interactions were measured quantitatively in real time using a BIAcore surface plasmon resonance instrument. Removal of much of the galactose from the thiolactosyl lipid in situ with beta-galactosidase showed that the lectin binding was highly specific. A dissociation constant KD = 8.77 x 10(-8) M was measured for 1-[2-[2-(2-[beta-D-galactopyranosyl-(1-->4)-1-thio-beta-D -glucopyranosyl]ethoxy)ethoxy]ethoxy]octadecane 30 which is four orders of magnitude greater than that determined for binding to lactose in solution. A concentration of lactose of > 80 mM was required to block the lectin binding to thiolactosyl lipid in a neomembrane.

Biotransformations in carbohydrates synthesis. N-Acetylgalactosaminyl transfer on to methyl N-acetyl-β-D-glucosaminide (methyl 2-acetamido-2-deoxy-β-D-glucopyranoside) and methyl N-acetyl-α-D-glucosaminide (methyl 2-acetamido-2-deoxy-α-D-glucopyranoside) catalysed by a β-N-acetylgalactosaminidase from Aspergillus oryzae

Using a crude N-acetylgalactosaminidas from Aspergillus oryzae, the β-N-acetylgalactosaminyl moiety of p-nitrophenyl N-acetylgalactosaminide was transferred to the C-4 and C-6 hydroxy groups of methyl N-acetyl-β-D-glucosaminide and methyl N-acetyl-α-D-glucosaminide and, for the latter, with high efficiency and selectivity for transfer to the C-4 position.

Biotransformations in carbohydrate synthesis. N-Acetylgalactosaminyl and N-acetylglucosaminyl transfer onto methyl α- and β-glucosides catalysed by the β-N-acetylhexosaminidase from Aspergillus oryzae

Regioselectivity of N-acetylhexosaminyl transfer onto methyl α- and β-D-glucosides catalysed by the N-acetyl hexosaminidase from Aspergillus oryzae is strongly dependent on the configuration at the anomeric centre of the acceptor: with the β-glucoside, 1,3- and 1,4-transfer products are formed, whereas with the α-glucoside, 1,4-and 1,6-transfer products are obtained.

Use of Enzymatic Digestion and Chemical Methylation Followed by Matrix-assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry to Sequence a 60kDa Commercial Fraction of Cellulose Acetate

Cellulose acetate was characterized by using enzyme in both digestion and chemical derivation and acetolysis. The fragments were normalised and compared on an anhydroglucose scale, using mass spectrometry to identify the different sized fragments. It was determined that at least two sub-populations for cellulose acetate existed within the parent. The macroscopic effect of this variation in the degree of acetylation will be a modification of the structural properties of the polymer chains. It was found that through comparison with enzyme-based degradation, an estimation of the acetylation topography of the cellulose acetate fraction could be made. Enzyme degradation produced a number of oligosaccharides of more than 10 glucose units, presumably resistant to enzyme degradation because they contained acetate groups. Chemical hydrolysis gave a random ladder of short sequences of mainly 3–4 glucose units some of which had a high methyl ether content, that were analysed by mass and converted to an anhydroglucose mass scale. This approach could be used to demonstrate differences between large biopolymers of cellulose acetate that previously gave an overall average rather than a specific ladder average.