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.

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.

Trisaccharide synthesis by glycosyl transfer from p-nitrophenyl beta-D-N-acetylgalactosaminide on to disaccharide acceptors catalysed by the beta-N-acetylhexosaminidase from Aspergillus oryzae.

The beta-N-acetylhexosaminidase from Aspergillus oryzae catalysed the transfer of beta-D-N-acetylgalactosaminyl residues from p-nitrophenyl beta-D-N-acetylglucosaminide on to disaccharide acceptors consisting of thioethyl glycosides of alpha-D-Glc-(1-->4)-beta-D-Glc, beta-D-Glc-(1-->4)-beta-D-Glc and beta-D-Glc-(1-->6)-beta-D-Glc. The principle of 'anomeric control' was exemplified by the results which showed that an alpha-linkage between the units of the acceptor favoured exclusively the formation of a new (1-->4)-linkage, whereas the beta-configuration in the acceptor led to a mixture of (1-->4)- and (1-->3)-linked products, as observed for simple glycosides of monosaccharide acceptors. With the thioethyl beta-lactoside as acceptor, beta-D-Gal-(1-->6)-beta-D-Gal-(1-->4)-beta-D-GlcSEt was formed, owing to the action of residual beta-D-galactosidase activity in the N-acetylhexosaminidase on the thioethyl beta-lactoside acting as both donor and acceptor.

Glycosidase-catalysed oligosaccharide synthesis: preparation of N-acetylchitooligosaccharides using the beta-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.

Stereochemistry of the incorporation of valine methyl groups into methylene groups in cephalosporin C.

'Chiral methyl valines', i.e. samples of valine labelled stereospecifically in the methyl groups with 2H and 3H, were incorporated into cephalosporin C by a suspension of washed cells of Cephalosporium acremonium. Analysis by 3H n.m.r. of the cephalosporin C produced showed that the conversion of the 3-pro-S-methyl group of valine into the acetoxymethyl side-chain was a highly stereospecific process. By contrast, conversion of the 3-pro-R-methyl group into the endocyclic methylene group of the dihydrothiazine ring was shown to proceed by a non-stereospecific process.

Metabolism of methyl methacrylate: evidence for metabolism by the valine pathway of catabolism in rat and in man.

1. Evidence to support the suggestion that methyl methacrylate is metabolized by the normal pathway of valine catabolism has been obtained by the administration of methyl [Me-14C]methacrylate to rats. 2. Of the administered dose, 80% was respired as 14CO2 as predicted, and methylmalonic acid, specifically labelled with 14C in a manner consistent with the proposed pathway of metabolism, was excreted (0.22%) dose). 3. Following administration of sodium [Me-2H3]methacrylate to a human subject, [Me-2H3]methylmalonic acid was detected in the urine by g.l.c.-mass spectrometry.

Stereospecificity of the enzymatic dehydrogenation in the biosynthesis of 3-ethylidene-L-azetidine-2-carboxylic acid from isoleucine by Streptomyces cacaoi.

(2RS,3S,4S)-[2-14C,4-3H]Isoleucine and (2S,3S,4R)-[U-14C,4-3H]isoleucine have been prepared by stereospecific syntheses. The addition of these substrates to Streptomyces cacaoi led to the isolation of polyoxins from which 3-ethylidene-L-azetidine-2-carboxylic acid was isolated by hydrolysis. The pro-R hydrogen at C-4 of isoleucine was lost and the pro-S hydrogen was retained in the biosynthesis of 3-ethylidene-L-azetidine-2-carboxylic acid. These results indicate that the enzymatic dehydrogenation of isoleucine to form 3-ethylidene-L-azetidine-2-carboxylic acid involves the antiperiplanar elimination of the hydrogen at C-3 and the pro-R hydrogen at C-4.

Stereochemistry of valine biosynthesis. Configuration of the product of rearrangement of alpha-acetolactate.

When alpha-aceto[1,3,5-13C3]lactate (2-hydroxy-2-methyl-3-oxo[1,3,5-13C3]butanoate) was incubated with a cell-free system prepared from Salmonella typhimurium, the valine produced was labelled in the C-4 pro-S position. This result proves that during the tertiary ketol rearrangement catalysed by the reductoisomerase of the isoleucine-valine pathway, the methyl group transfer is to the re face of the trigonal centre at C-3 of alpha-acetolactate.

Methylmethacrylate metabolism in man. The hydrolysis of methylmethacrylate to methacrylic acid during total hip replacement.

Methylmethacrylate, the monomeric component of the polymethylmethacrylate cement used in orthopedic surgery, has been shown to undergo hydrolysis to methacrylic acid during hip replacement operations. Circulating levels of methacrylic acid were comparable with those of methylmethacrylate. Concentrations of both methylmethacrylate and methacrylic acid normally lay in the range 0--15 micrograms/cc. No correlation could be discerned between changes in the concentrations of methylmethacrylate and methacrylic acid, and changes in blood pressure.

Toxicology of methyl methacrylate: the rate of disappearance of methyl methacrylate in human blood in vitro.

1. The rate of disappearance of methyl methacrylate in blood has been determined using an isotope dilution technique. 2. At a concentration of 10(-4) mol dm(-3), methyl methacrylate disappears with pseudo first order kinetics. 3. The half-life of methyl methacrylate in blood at 37 degrees C lies in the range 20--40 min. 4. The half-life showed no dependence on the age or sex of the blood donor. 5. A major, possibly the only, pathway of metabolism is by hydrolysis to methacrylic acid.