Neil Baggett

A fluorometric assay using 4-methylumbelliferyl α-l-iduronide for the estimation of α-l-iduronidase activity and the detection of Hurler and Scheie syndromes

Abstract Incubation of 4-methylumbelliferyl α-L-iduronide with whole cell homogenates prepared from cultured skin fibroblasts and amniotic cells, and peripheral blood leukocytes gave 4-methylumbelliferone which was easily measured fluorometrically. This reaction, presumably due to the action of α- l -iduronidase, has a maximum hydrolytic activity at pH 3.25. The apparent K M value of α- l -iduronidase in leukocyte whole cell homogenates for this substrate was 179 μmol/l compared to 353, 41 and 116 μmol/l for other α- l -iduronidase substrates phenyl α-L-iduronide, iduronosyl anhydro [l- 3 H]mannitol 6-sulfate and iduronosyl anhydro [l- 3 H] mannitol respectively; the corresponding V max values were 617, 394, 158 and 10 pmol/min/mg protein respectively. Incubation of the 4-methylumbelliferyl α-L-iduronide with whole cell homogenates prepared from cultured skin fibroblasts and leukocytes from a Hurler patient gave 4-methylumbelliferone at a rate more than 20 times less than found for control normal preparations. 4-Methylumbelliferyl α-L-iduronide is a sensitive, convenient and superior substrate to phenyl α-L-iduronide for the assay of α-L-iduronidase activity, but is not a suitable replacement for the radiolabelled substrate iduronosyl anhydro [1−su3H] mannitol 6-sulfate.

Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles

6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.

Diastereoisomeric forms of methyl 4,6-O-benzylidene-2,3-di-O-methyl-α-d-gluco- and α- and β-d-galactopyranoside

Abstract Treatment of methyl 2,3-di- O -methyl-α- d -glucopyranoside with benzylidene bromide, toluene, potassium tert -butoxide, and tert -butanol gave two diastereoisomeric 4,6- O -benzylidene derivatives ( A and B ). The complete conversion A → B was effected with carbon tetrachloride-hydrogen chloride. Only diastereoisomer B was formed on acid-catalysed benzylidenation. Closely parallel results were obtained on benzylidenation of methyl 2,3-di- O -methyl-α- and β- d -galactopyranoside under basic conditions. The possible conformations of the benzylidene derivatives are considered on the basis of n.m.r. spectroscopic data. At equilibrium in the acid-catalysed benzylidenation of methyl α- d -galactopyranoside the only product present is the 4,6- O -benzylidene derivative, m.p. 168–170°, [α] d + 144° in chloroform. In the early, kinetically-controlled, stages of the reaction, small amounts of the diastereoisomeric 3,4- O -benzylidene derivatives were detected.

Reinvestigation of the synthesis of 4-methylcoumarin-7-yl 5-acetamido-3,5-dideoxy-α-d-glycero-d-galacto-2-nonulopyranosidonic acid, a fluorogenic substrate for neuraminidase

A crystalline tetrabutylammonium salt of 7-hydroxy-4-methylcoumarin was prepared and shown to contain two coumarin residues for each ammonium group. Condensation of this salt with the glycosyl chloride of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate in dry acetonitrile at room temperature gave the corresponding alpha-glycoside in higher yield and purity than previously reported methods. Removal of the acetyl and methyl ester blocking-groups gave the free glycoside, which was shown to have the alpha configuration by n.m.r. spectroscopy. In contrast, the reaction of the free coumarin derivative with the chloro sugar in refluxing, dry toluene in the presence of cadmium carbonate as acid acceptor gave none of the above glycoside, but gave the corresponding glycal in good yield.

Further observations on the acid-catalysed benzaldehyde-glycerol reaction

Abstract The proportions of cis - and trans -5-hydroxy-2-phenyl-1,3-dioxan and cis - and trans -4-hydroxymethyl-2-phenyl-1,3-dioxolan in acid-catalysed, equilibrated, O -benzylidene-glycerol mixtures are critically dependent on solvent and temperature. In carbon tetrachloride, elevation of temperature increases the proportion of dioxolan derivatives and, at a given temperature, diminishing concentration increases the proportion of the cis -1,3-acetal at the expense of the trans -isomer. At equilibrium in dimethyl sulphoxide, the trans -1,3-acetal is preponderant. On acid-catalysed benzylidenation of glycerol under homogeneous conditions ( N , N -dimethylformamide), the 1,2-acetals form rapidly in the initial, kinetic phase, and the 1,3-acetals are preponderant at equilibrium. A rationalisation of some of these observations is presented.

Synthesis of l-iduronic acid derivatives by epimerisation of anancomeric d-glucuronic acid analogues

Abstract Derivatives of l -iduronic acid were prepared by epimerisation of d -glucuronic acid derivatives that were constrained to adopt a conformation having C-6 in an axial position, so that the l -iduronic acid derivatives would be thermodynamically more stable. Methyl 3,5- O -benzylidene-1,2- O -isopropylidene-β- l -idofuranuronate was conveniently obtained in 23% yield by crystallisation from the epimerising mixture and was characterised either by reduction to the corresponding alcohol or by catalytic hydrogenation followed by spontaneous cyclisation to give 1,2- O -isopropylidene-β- l -idofuranurono-6,3-lactone. A synthesis of methyl 3,5- O -benzylidene-1,2- O -isopropylidene-α- d -glucofuranuronate in two steps from d -glucofuranurono-6,3-lactone was devised. Other candidates for epimerisation, including other esters, amides, and nitriles, were also examined. In all cases, the yields were reduced by competing decomposition, probably involving β-elimination.

Synthesis of luciferin glycosides as substrates for novel ultrasensitive enzyme assays

Abstract Condensation of 2-cyano-6-hydroxybenzothiazole with acetohalogeno derivatives of d -glucose, d -galactose, and 2-acetamido-2-deoxy- d -glucose gave the corresponding β-glycosides. Attempted basic deacetylation caused methanolysis of the nitrile group. Condensation of the first two acetylated glycosides with d -cysteine, followed by deacetylation, gave the firefly luciferin β-glycosides that were substrates for the corresponding glycohydrolases. The liberated luciferin was determined by fluorescence spectroscopy and, in one instance, by coupled-bioluminescence assay with firefly luciferase. The amount of luciferin released and determined by bioluminescence assay, was only ∼65% of that determined by fluorescence spectroscopy, which suggested that the luciferin was partly racemised. Because of the great sensitivity of bioluminescence detection, these novel substrates provide potentially ultrasensitive assays for glycohydrolases, but their syntheses are more difficult than those of the corresponding fluorogenic substrates.

Synthesis of some aryl 2,3,4,6-tetra-O-acetyl-l-idopyranosides and of 4-methylcoumarin-7-yl α-l-idopyranosiduronic acid

Abstract Several routes for synthesis of 3,5,6-tri-O-acetyl-1,2-O-isopropylidene-β- l -idofuranose have been evaluated. Previously described routes, which involved selective sulphonylation, were not reproducible on a 100-g scale. To overcome this difficulty, a new variation was developed, involving complete tosylation of 1,2-O-isopropylidene-α- d -glucofuranurono-6,3-lactone followed by reduction and acetylation. The idofuranose derivative was converted into the desired 1,2,3,4,6-penta-O-acetyl-α- l -idopyranose via 1,6-anhydro-β- l -idopyranose. Fusion of 1,2,3,4,6-penta-O-acetyl-α- l -idopyranose with 4-nitrophenol, 1- or 2-naphthol, or 4-methylcoumarin-7-ol, using freshly fused zinc chloride as catalyst, gave an anomeric mixture of glycosides, with the α anomer being preponderant. The major 4-methylcoumarin-7-yl glycoside was deacetylated and converted, by catalytic oxidation, into 4-methylcoumarin-7-yl α- l -idopyranosiduronic acid, a fluorogenic substrate for α- l -iduronidase.

Synthesis and some reactions of 1,3:4,6-di-O-benzylidene-d-threo-2,5-hexodiulose

Abstract Oxidation of 1,3:4,6-di- O -benzylidene- d -mannitol in ethyl acetate with ruthenium tetraoxide gave the corresponding diketone as its hydrate, which was dehydrated to the free diketone. These compounds were treated with a variety of reagents containing hydride, oxygen, nitrogen, and carbon nucleophiles. Only in reactions with methylmagnesium iodide and with diazomethane was the symmetrical bis-adduct obtained. In all other cases, the reactivity of the two carbonyl groups was different. Hydride addition occurred with opposite stereoselectivity at each carbonyl group. With oxygen and nitrogen nucleophiles, cyclic adducts were obtained, arising from bridging across the two carbonyl groups by one molecule of nucleophilic reagent. The configurations of the ring fusions were deduced by p.m.r spectroscopy, and the cis,cis -structure was preferred in compounds containing one five- and two six-membered rings. In the reactions with other carbon nucleophiles, complex mixtures were obtained due, at least in the case of the reaction with phenylmagnesium bromide, to elimination reactions.

Asymmetric reduction of ketones by using complexes of lithium tetrahydridoaluminate(III) with 1,4:3,6-dianhydro-D-mannitol and 1,3:4,6-di-O-benzylidene-D-mannitol

The use of chiral diols each containing a two-fold axis of symmetry to achieve asymmetric reductions of some ketones with metal hydrides is described. The symmetry of these complexes simplifies interpretation of the observed enantioselectivity.