Syntheses of methyl 2,6-diacetamido-2,3,6-trideoxy-α-d-ribo-hexopyranoside (methyl di-N-acetyl-α-d-tobrosaminide) and methyl 2-acetamido-2,3,6-trideoxy-β-l-lyxo-hexopyranoside

The title glycosides were synthesised from d-glucose, via the common intermediate methyl 2-acetamido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-α-d-ribo-hexopyranoside.     

Studies on aroyl- and aryl-hydrazide derivatives from d-glycero-d-gulo-heptono-1,4-lactone

A series of aroyl- and aryl-hydrazide derivatives was prepared from d-glycero-d-gulo-heptono-1,4-lactone (1). The reactivity of the NH proton in these hydrazides, in terms of their dissociation constants (pK_a), was determined from their electronic spectra, and correlated to the Hammett σ values of the substituents. Comparable reactivities of the NH protons for the compounds, and the effect of the substituent, were studied by n.m.r. spectroscopy. Decomposition of the aroylhydrazides with copper(II) sulfate or nitrous acid resulted in the regeneration of 1.     

The structure of d-threo-2,5-hexodiulosonic acid and derivatives in solution

The structure of d-threo-2,5-hexodiulosonic acid (1) and various derivatives in solution was determined by ¹³C-n.m.r. spectroscopy to be a hydrated, pyranose form. The structures of the methyl ester of 1 and of its 5-(dimethyl acetal) were confirmed by chemical means and by X-ray structure analysis.     

Selenium derivatives of L-arabinose, D-ribose,and D-xylose

Attempted cyclization of 2,3,4-tri-O-methyl-5-seleno-L-arabinose dimethyl acetal in acidic solution gave the corresponding diselenide. Intramolecular attack by the selenobenzyl group at C-5 of 5-O-p-tolylsulfonyl-L-arabinose dibenzyl diseleno-acetal resulted in the formation of benzyl 1,5-diseleno-L-arabinopyranoside. Similarly, 2,3,5-tri-O-methyl-4-O-p-tolylsulfonyl-D-xylose dibenzyl diselenoacetal gave benzyl 2,3,5-tri-O-methyl-1,4-diseleno-L-arabinofuranoside, and 2,3,4-tri-O-acetyl-5-O-p-tolylsulfonyl-D-xylose (or ribose) dibenzyl diselenoacetal gave benzyl 2,3,4-tri-O-acetyl-1,5-diseleno-D-xylo- (or ribo-)pyranoside. The glycosylic benzylseleno group was removed from the pyranoside with mercuric acetate, but attempted deacetylation of the product led to decomposition and not to the expected 5-seleno-D-xylopyranose.     

Syntheses of 2-acetamido-2-deoxy-4-O-α-D-glucopyranosyl-α-d-glucopyranose (n-acetylmaltosamine) and 2-acetamido-2-deoxy-4-O-β-d-glucopyranosyl-α-d-glucopyranose

Condensation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside with 2,3,4,6-tetra-O-benzyl-1-O-(N-methyl)acetimidoyl-β-D-glucopyranose gave benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside which was catalytically hydrogenolysed to crystalline 2-acetamido-2-deoxy-4-O-α-D-glucopyranosyl-α-D-glucopyranose (N-acetylmaltosamine). In an alternative route, the aforementioned imidate was condensed with 2-acetamido-3-O-acetyl-1,6-anhydro-2-deoxy-β-D-glucopyranose, and the resulting disaccharide was catalytically hydrogenolysed, acetylated, and acetolysed to give 2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-α-D-glucopyranose Deacetylation gave N-acetylmaltosamine. The synthesis of 2-acetamido-2-deoxy-4-O-β-D-glucopyranosyl-α-D-glucopyranose involved condensation of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide in the presence of mercuric bromide, followed by deacetylation and catalytic hydrogenolysis of the condensation product.     

Equilibria of the anomeric and ring forms of ammonium 3-deoxy-d-manno-octulosonate in deuterium oxide

The tautomeric composition of a solution of ammonium 3-deoxy-d-manno-octulosonate (KDO, 1a) in D₂O at 28° was assessed by means of ¹³ C-F.t.-n.m.r. spectroscopy. The results revealed the presence of 6?0 and 11 % of the α and β anomers of the pyranose, and 20 and 9 % of the two furanoses, and suggested, but did not unequivocally prove, that the major furanose form is the α anomer. To facilitate interpretation of the spectral results for 1, ammonium 3,5-dideoxy-d-arabino(or ribo)-octulosonate (3a) was prepared by the reaction of 5-deoxy-d-erythro-pentose with sodium oxalacetate at pH 11. A chromatographically homogeneous, noncrystalline sample of 3 was obtained by lyophilization, and characterized as its (4-nitrophenyl)hydrazone (m.p. 162-163°). The ¹³C-n.m.r. spectrum of a solution of 3a in D₂O revealed it to be substantially all in the α-pyranose form. No signals were obtained for the possible 1,4-lactone of 3. As the 1,5-lactone and furanose forms are impossible for 3, it exhibited no signals analogous to those attributed to furanoid 1. On the basis of these results for 3, the two lactone forms of 1 were excluded from consideration, and the three pairs of ¹³C-n.m.r. signals observed at ≈45, 86, and 104 p.p.m. were assigned to the furanose forms of 1.     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

Synthesis and characterization of propyl O-β-d-galactopyranosyl-(1→4)-O-β-d-galactopyranosyl-(1→4)-α-d-galactopyranoside

Allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl-α-d- galactopyranoside was O-deallylated to give the 1-hydroxy derivative, and this was converted into the corresponding 1-O-(N-phenylcarbamoyl) derivative, treatment of which with dry HCl produced the α-d-galactopyranosyl chloride. This was converted into the corresponding 2,2,2-trifluoroethanesulfonate, which was coupled to allyl 2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside, to give crystalline allyl 4-O-[4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di- O-benzyl-β-d-galactopyranosyl]-2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside (15) in 85% yield, no trace of the α anomer being found. The trisaccharide derivative 15 was de-esterified with 2% KCN in 95% ethanol, and the product O-debenzylated with H₂-Pd, to give the unprotected trisaccharide. Alternative sequences are discussed.     

Structure of the D-fructan isolated from garlic (Allium sativum) bulbs

Extraction of defatted garlic bulbs with hot water yielded a mixture of polysaccharides containing pectic acid, a D-galactan, and a fructan component. The pectic acid was partially removed as calcium pectate, and the galactan-enriched portion was separated by fractional precipitation with alcohol; on concentration and several fractionations, the supernatant liquor furnished the fructan component, which contained fructose (94.4%) and glucose (4.3 %). Methanolysis and hydrolysis of the permethylated fructan gave (a) 1,3,4,6-tetra-O-methyl-D-fructose, (b) 2,3,4,6-tetra-O-methyl-D-glucose, (c) 2,4,6-tri-O-methyl-D-glucose, and (d) 3.4,6-tri-O-methyl-D-fructose in the ratio (a + b): (d) = 1:20.3. On periodate oxidation, the fructan reduced one molar equivalent of the oxidant per hexosyl residue, and liberated one molar equivalent of formic acid per 51 hexosyl residues. On Smith degradation, the major product was glycerol, and ～2 % of the glucose survived. From these results, and from the fact that the fructan is hydrolyzed by β-D-fructofuranosidase, a linear, inulin-type of structure is suggested for it.     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).     

The dependence of the conformation of a (1→3)-β-D-glucan on chain-length in alkaline solution

(1→3)-β-D-Glucans of various degrees of polymerization were prepared by degradation of a gel-forming D-glucan with formic acid. The degraded D-glucans were separated into a water-soluble fraction (soluble D-glucan) and an insoluble fraction (insoluble D-glucan). Both D-glucans were further fractionated. The optical rotation including determination of the o.r.d. curves of the fractions and of the original gel-forming D-glucans was measured at various sodium hydroxide concentrations (0–5M). The results indicate that (1→3)-β-D-glucans of $\text{DP}$n below ca. 25 (the soluble D-glucan) took a disordered form in both neutral and alkaline solutions, whereas the D-glucans of higher $\text{DP}$n (the insoluble and the original D-glucans) took an ordered structure in dilute alkaline solution (0.1M). The proportion of ordered structure in the insoluble D-glucan increases with $\text{DP}$n to attain a maximum value at a $\text{DP}$n of around 200; this may be the lower limit of $\text{DP}$n to permit gel formation in neutral media. The formation of complexes with Congo Red in alkaline solutions by the soluble and the insoluble D-glucans supports the same conclusions.     

Kinetics and mechanism of oxidation of d-erythrose and dL-glyceraldehyde by chromium(VI) and vanadium(V) in perchloric acid medium

The kinetics of oxidation of d-erythrose and dL-glyceraldehyde by chromium (VI) and vanadium(V) in perchloric acid medium have been investigated spectrophotometrically. Each reaction was first-order with respect to [oxidant] and [substrate]. The reactions were catalysed by acid, but their dependence on acidity was complex. Sodium perchlorate accelerated the rate of each reaction. The oxidation rates follow the order glyceraldehyde > erythrose. The activation parameters were calculated and mechanisms consistent with the experimental observations are proposed.     

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Two l-arabino-d-galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l-arabinose, d-galactose, l-fucose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β-d-galactosyl residues, to which side chains consisting of α-(1→6)-linked d-galactosyl residues were attached. The α-l-arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β-d-galactosyl residues of the side chains. Single α-l-fucopyranosyl end groups were linked to O-2 of the l-arabinofuranosyl residues, and the 4-O-methyl-β-d-glucopyranosyluronic acid end groups were linked to d-galactosyl residues. The O-α-l-fucopyranosyl-(1→2)-α-l-arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l-arabino-d-galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O-d-galactosylserine linkage.     

Reaction of some D-glucobioses with 2,2-dimethoxypropane

Laminarabiose, cellobiose, and gentiobiose were acetonated with 2,2-dimethoxy-propane under various conditions. Two isopropylidene acetals in which the reducing D-glucose residue had the furanoid form were obtained from laminarabiose, and two, in which the reducing D-glucose residue formed the acyclic dimethyl acetal, from cellobiose. Gentiobiose gave both types of isopropylidene compound.     

Derivatives of S-α- and -β-d-hexopyranosyl thiophosphates

Di-tert-butyl esters of the tetra-O-acetyl and tetra-O-benzyl derivatives or S-α- and -β-D-glucopyranosyl thiophosphates were prepared by reaction of di-tert-butyl triethylammonium phosphorothioate with tetra-O-acetyl- or tetra-O-benzyl-hexopyranosyl halides.     

An enzymic synthesis of purine d-Arabinonucleosides

A method is described for the synthesis of purine d-arabinonucleosides that uses purine bases and 2,2′-anhydro-(1-β-d-arabinofuranosylcytosine), AraC-an, as the starting materials. AraC-an was chosen as the precursor to the d-arabinosyl donor, because it is more readily available than any of the products that may be sequentially derived from it, namely, 1-β-d-arabinofuranosylcytosine (AraC), 1-β-d-arabinofuranosyluracil (AraU), and α-d-arabinofuranosyl-1-phosphate (Araf 1-P), a d-arabinofuranosyl donor. Four reactions were involved in the overall process; (a) AraC-an was nonenzymically hydrolyzed at alkaline pH to AraC which was then (b) deaminated by cytidine deaminase to AraU, a nucleoside, (c) phosphorylyzed by uridine phosphorylase to Araf 1-P, and (d) this ester caused to react with a purine base to afford a purine d-arabinonucleoside, the reaction being catalyzed by purine nucleoside phosphorylase. All four reactions occurred in situ, the first and second being performed sequentially, whereas the third and fourth were combined in a single step. The three enzyme catalysts were purified from Escherichia coli. The efficiency of the method is exemplified by the synthesis of the d-arabinonucleosides of 2,6-diaminopurine and adenine; the overall yields, based on AraC-an, were 60 and 80%, respectively.     

Reactions of 4,6-disulphonates of methyl d-glucopyranosides and methyl d-galactopyranosides with thio-nucleophiles

The reactions of some 4,6-disulphonates of methyl 2,3-di-O-acyl-(and di-O-methyl)-d-glucopyranosides and -galactopyranosides, with thiocyanate, thioacetate, and thiobenzoate anions, have been studied under a variety of conditions. In the glucoside series, somewhat similar reactivity is shown by isomers differing only in anomeric configuration, and there is no very great difference between the reactivities of a 2,3-dibenzoate and its 2,3-di-O-methyl analogue. In contrast to the situation in the β-d-galactoside series, the presence of O-benzoyl groups in an α-d-galactoside does not have an unfavourable effect on displacement at C-4. Two hexose derivatives containing the novel 4,6-epithio bridge are described.     

A sensitive,specific radioisotope assay for l-glutamine-d-fructose-6-phosphate aminotransferase

A sensitive and specific radioassay for l-glutamine-d-fructose-6-phosphate aminotransferase (EC 5.3.1.19) activity is presented. Picomoles of product are measurable, and the assay can be applied to systems having limited quantities of available protein, particularly in extracts of either cell or organ cultures. The assay is at least 10,000 times more sensitive under K₁ concentrations of fructose 6-phosphate than the modified Elson-Morgan colorimetric assay and 20 times more sensitive under saturating conditions of fructose 6-phosphate. As little as 0.5 μg of cell-extract protein will yield measurable product. In contrast, 280 μg of crudeextract protein from colon is required with the modified Elson-Morgan colorimetric assay.     

α-l-arabinofuranosidases and β-d-galactosidases in germinating-lupin cotyledons

Extraction of germinating-lupin cotyledons, followed by ion-exchange and gel chromatography, gave two α-l-arabinofuranosidases and three β-d-galactopyranosidases. Some fractions were further purified by using Concanavalin A-Sepharose. The changes in the activities of the enzymes during germination have been determined. Some kinetic and physical properties of these enzymes are described, and their role in the modification of cell-wall polysaccharides is discussed.