J. Michael Williams

An improved method for the liquid chromatography of the 1-deoxy-1-(2-pyridylamino)alditol derivatives of oligosaccharides and its application to structural studies of the carbohydrate moeities of glycoproteins

Abstract The 1-deoxy-1-(2-pyridylamino)alditols prepared by reductive amination of lactose, 2-acetamido-2-deoxy- d -glucose, and 2,5-anhydro- d -mannose have been characterised, and the efficiency of the reductive amination procedure, especially with 2,5-anhydro- d -mannose and 2-amino-2-deoxy- d -glucose hydrazone as starting materials, has been studied. The latter compound, which is a model for the oligosaccharide hydrazones released from glycoproteins and glycopeptides by hydrazinolysis, was first N-acetylated and the hydrazone group was found to be removed hydrolytically when a cation-exchange resin was used for deionisation. Such loss of the hydrazone group is desirable because the N-acetylated hydrazone was not efficiently derivatised by reductive amination. An amine-modified silica column was used to separate the components of a mixture of the pyridylamino derivatives of oligosaccharides from mono- to dodeca-saccharide in 20 min. A neutral fluorescent by-product, formed in all reductive aminations, was identified as (2-amino-1-pyridyl)cyanoborane and was eluted well before monosaccharide derivatives and thus did not interfere with the analysis.

Nitroaliphatic compounds in Hippocrepis comosa and other legumes in the European flora

Abstract In a survey of 148, mostly European, angiosperm species, 3-nitropropionate was found only in extracts of the legumes, Hippocrepis balearica, H. comosa, Lotus uliginosus and Scorpiurus muricatus . Three separate nitropropanoyl esters of glucose, 6-(3-nitropropanoyl)-αβ- d -glucopyranose, 1,6- di -O-(3- nitropropanoyl -β- d - glucopyranose (cibarian) and 1,2,6- tri -O-(3- nitropropanoyl )-β- d - glucopyranose (karakin) were identified in shoot extracts of H . comosa . Evidence for the presence of the 3-nitropropanoyl ester of 4-hydroxybutanoic acid in extracts of H . comosa is also presented.

Structural analysis of the carbohydrate moieties of glycoproteins by regiospecific degradation and liquid chromatography

Abstract The reaction sequence of hydrazinolysis, nitrosation, and reduction, followed by liquid chromatography (l.c.) has been studied as a method for the routine structural analysis of the asparagine-bound oligosaccharides of glycoproteins. Glycopeptides derived from IgM and ovalbumin by proteolysis were used as test materials. The hydrazinium sulphate-catalysed hydrazinolysis was superior to the longer uncatalysed reaction, in that there was less non-specific degradation and higher degree of N -deacetylation. The nitrosation products were reduced in situ with sodium cyanoborohydride, and the l.c. analysis required 20 min for the fractionation of oligosaccharides up to decasaccharide. The l.c. profile is characteristic of the structure of the carbohydrate unit. The analytical l.c. column may also be used to isolate oligosaccharide fractions in quantities of several hundred micrograms.

A comparison of the results of sequential hydrazinolysis-nitrosation and alkali-mediated cleavage-nitrosation of theO-linked oligosaccharides of gastric mucus glycoproteins

Analysis of the oligosaccharides released from pig gastric mucus glycopolypeptides by hydrazinolysis showed that degradation had occurred. Nitrosation of the products followed by reduction gave a mixture that had a low content of 2,5-anhydro-D-talitol, which implied destruction of much of the terminal reducing 2-amino-2-deoxy-D-galactose. Under the conditions of hydrazinolysis, cellobiose was largely unchanged but laminaribiose gave a complex mixture that probably contained glucose hydrazone (13C-n.m.r. data). In order to avoid degradation, the hydrazinolysis-nitrosation sequence should be applied to the reduced oligosaccharides released on cleavage with alkali.

The search for d-glucose derivatives suitable for the study of natural hydrogen isotope fractionation

Abstract In order to apply Site-specific Natural Isotope Fractionation (SNIF) to chemical, biochemical, and environmental studies of d -glucose, there is a need for readily prepared compounds which give 2 H NMR spectra with all or most signals resolved. Changing substituents of C-1 and/or C-6 of α- d -glucopyranose pentaacetate improved the dispersion of deuterium signals, the best results being achieved with 1,2,3,4-tetra- O -acetyl-6-deoxy-6-thiocyanato-α- d -glucopyranose, for which only the 2 H-2 and 2 H-4 signals were not resolved, and with 2,3,4-tri- O -acetyl-6-bromo-6-deoxy-α- d -glucopyranosyl bromide, for which only the 2 H-6a and 2 H-6b signals were not resolved. Periodate oxidation of methyl 4,6- O -benzylidene-α- d -glucopyranoside and 4,6-dichloro-4,6-dideoxy- d -galactose was also examined as a possible source of useful compounds. Products obtained from the benzylideneglucoside gave inadequate resolution and broad deuterium signals. The oxidation of 4,6-dichloro-4,6-dideoxy- d -galactose was not straightforward. The α anomer was oxidised more rapidly than the β anomer. The oxidation product, 2,4-dichloro-2,4-dideoxy-3- O -formyl- d -threose, was too labile to be isolated pure and its hydrolysis to 2,4-dichloro-2,4-dideoxy- d -threose was accompanied by a slow elimination to form 2,4-dichlorobut-2-enal.

Hydrazinolysis of monosaccharides: a two-step synthesis of chiral pentane-1,2,3-triols from pentoses and observations on the hydrazinolysis of glycoproteins and glycopeptides

Abstract 1,2-Dideoxyalditols, the corresponding 1-alkenes, and 1-deoxyalditols are formed in various proportions from d -glucose, d -mannose, l -arabinose, and d -xylose by the action of refluxing hydrazine. Sequential hydrazinolysis, catalytic hydrogenation, and chromatography afford a route to 1,2-dideoxyalditols. For example, 1,2-dideoxy- l -erythro-pentitol is formed from l -arabinose in 42% yield, and d -xylose is a source of 1,2-dideoxy- d -threo-pentitol (50%). Under the conditions (anhydrous hydrazine at 100° for 30 h in the absence of air) used by Montreuil for the hydrazinolysis of glycoproteins and glycopeptides, no 1,2-dideoxyalditol was formed; degradation was incomplete, there being some aldose hydrazone present. Under Kochetkovs hydrazinolysis conditions (105° for 10 h with hydrazinium sulphate), less degradation occurred and the product from d -galactose was identified as 1-deoxy- d -tagatose hydrazone.

Sugar lactams and lactim ethers, useful precursors of cyclic amidines, from intramolecular nucleophilic displacements

Abstract The cyclisation of the conjugate base of N-aryl-2,3:5,6-di-O-isopropylidene-4-O-methanesulfonyl-D-gulonamides gave the D-allonolactam acetals. Sodium methoxide-promoted cyclisation of 2,3:5,6-di-O-isopropylidene-4-O-methanesulfonyl-D-mannononitrile gave the D-talonolactim ether. Tetrazole formation without isolation of the intermediate azide was illustrated by the conversion of the aforementioned nitrile sulfonate into the D-talonotetrazole. Cyclic amidines were prepared from the lactam and lactim ether derivatives. The D-allono derivatives were also converted into D-ribono-1,4 lactam and amidine derivatives.

Carbohydrate-derived surfactants : synthesis and phase behaviour of methyl 4',6'-Di-O-alkyl-β-lactosides

In order to study the physical properties of lactose-derived surfactants, methyl 4′, 6′-di-O-hexyl-β-lactoside and methyl 4′, 6′-di-O-octyl-β-lactoside were prepared. The 4′, 6′-O-isopropylidene ketal was used as a temporary protecting group in the key intermediate. The 3′, 4′-O-benzylidene acetal was identified as a minor by-product in the preparation of methyl 4′, 6′-O-benzylidene-β-lactoside by acid-catalysed transacetalation. The phase behaviour of these derivatives is reported.

IS SULFATE LOST DURING THE CHEMICAL RELEASE OF OLIGOSACCHARIDES FROM GLYCOPROTEINS

Abstract Model experiments using methyl α-d-galactopyranoside 6-sulfate have implied that the Carlson conditions (1 M sodium borohydride and 0.05 M sodium hydroxide at 45°C) for the release of O-linked oligosaccharides from glycoproteins do not cause significant loss of sulfate ester groups. However the more vigorous conditions used to release N-linked oligosaccharides are more likely to cause considerable sulfate loss from a galactoside 6-sulfate as a result of 3,6-anhydride formation. Experiments with dextran sulfate suggested that sulfate loss due to oxirane formation was also not significant under the Carlson conditions. These results were supported by experiments with [35S]sulfate-labelled human rectal mucus glycoprotein.

Synthesis and lyotropic phase behaviour of methyl 3′,4′-di-O-hexyl- and -di-O-octyl-β-lactoside and partial O-acetylation of methyl 3′,4′-di-O-octyl-β-lactoside

Methyl 3′,4′-O-isopropylidene-β-lactoside was used as an intermediate in the synthesis of the 3′,4′-di-O-hexyl- and 3′,4′-di-O-octyl-β-lactoside. The following seven partially acetylated derivatives were prepared by partial O-acetylation of the dioctyllactoside: 6′- and 6-monoacetate, 2′,6- and 6,6′-diacetate, 2,6,6′-triacetate, and 2,2′,6,6′- and 2,3,6,6′-tetraacetate. The lyotropic phase behaviour of some of these derivatives was studied.