David H.G. Crout

Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis

Remarakable advances in glycobiology in recent years have stimulated a resurgence of interest in carbohydrate chemistry. The challenge of producing the complex glycosides and oligosaccharides needed for research in glycobiology has led to the development of enzymatic methods that are now firmly established as part of the synthetic repertoire of the carbohydrate chemist.

Immobilization/stabilization on Eupergit C of the β-galactosidase from B. circulans and an α-galactosidase from Aspergillus oryzae.

Two synthetically useful glycosidases, the β-galactosidase from Bacillus circulans and an α-galactosidase from Aspergillus oryzae have been immobilized on Eupergit C. The immobilized enzymes retain high catalytic activity and show increased thermal stability compared with the free enzymes.

Glycosidases—a great synthetic tool

Glycosidases were used to prepare oligosaccharide structures of physiological and medicinal relevance. The study included an extensive screening of crude enzymatic preparations for α- and β-galactosidase, α- and β-mannosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase and α-l-fucosidase activities. The enzymes were assessed with respect to regioselectivity of glycosyl transfer on to carbohydrate acceptors. The purification procedures for individual biocatalysts are described in detail.

Solvent effect on enzyme-catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β-d-glucopyranosides

Abstract Almond β- d -glucosidase was used to catalyze alkyl-β- d -glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β- d -glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.

Stereochemistry of the 4-hydroxyisoleucine from Trigonella foenum-graecum

Abstract The stereochemistry of the 4-hydroxyisoleucine from fenugreek ( Trigonella foenum-graecum ) has been reinvestigated. The absolute configuration was shown to be (2 S ,3 R ,4 S by a combination of chemical, spectroscopic and X-ray crystallographic techniques.

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.

Glycosidase-catalysed synthesis of glycosides by an improved procedure for reverse hydrolysis: application to the chemoenzymatic synthesis of galactopyranosyl-(1→4)-O-α-galactopyranoside derivatives

Abstract β-Galactosidase from Aspergillus oryzae, α-galactosidase from Aspergillus niger, β-mannosidase from Helix pomatia and β-glucosidase from almond were used to synthesise different glycosides by reverse hydrolysis: GlcβO(CH2)6OH 1 was obtained in 61% yield, β-D-Glc-O(CH2)3CH-CH2 2 in 50% yield, β-D-Glc-O(CH2)2Si(Me)3 3 in 11% yield, β-D-Gal-O(CH2)6OH 4 in 48% yield, β-D-Gal-O(CH2)3CH-CH2 5 in 22% yield, α-D-Gal-O(CH2)6OH 6 in 47% yield, α-D-GalO(CH2)3CH-CH2 7 in 37% yield and β-D-ManO(CH2)6OH 8 in 12% yield. Using the appropriate glycosides methyl O-(2,3,4,6-tetra-O-benzyl-α-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzoyl-1-O-α-D-galactopyranoside 11 and 6′-benzoylhexyl-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzoyl-1-O-β-D-galactopyranoside 15 were synthesised. This chemoenzymatic approach avoided at least four chemical steps that would have been necessary in a conventional synthesis.

Immobilisation on polystyrene of diazirine derivatives of mono- and disaccharides: biological activities of modified surfaces.

The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.

Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.

Chemoenzymatic synthesis of ethyl 1-thio-(β-D-galactopyranosyl)-O-β-D-glycopyranosyl disaccharides using the β-galactosidase from Bacillus circulans

Abstract Different ethyl 1-thio-β-D-disaccharides have been synthesised by transgalactosylation using the β-galactosidase from Bacillus circulans as biocatalyst. This β-galactosidase shows mainly a β-1–4 specificity in the galactosyl transfer. Gal-β-(1–4)-O-β-D-GlcSEt 15 was obtained in 36% yield, Gal-β-(1ndash;4)-O-α-D-GlcSEt 19 in 30% yield, Gal-β-(1–4)-O-β-D-GalSEt 17 in 60% yield, Gal-β-(1–4)-O-β-D-GalNAcSEt 20 in 49% yield, Gal-β-(1–4)-O-β-D-Gal-β-(1–4)-O-β-D-GalNAcSEt 21 in 9% yield, Gal-β-(1–6)-O-β-D-GlcSEt 16 in 3% yield and Gal-β-(1–3)-O-β-D-XylSEt 18 in 25% yield.