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.     

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.     

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.     

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.     

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.     

Synthesis of aminocyclitols from l-quinic acid

A variety of 1,3-diamino and 1,4-diaminocyclitols, manoaminocyclitols, and triaminocyclohexanol have been synthesized starting with the chiral ketone intermediate, 2, derived from l-quinic acid. Reduction of 2 with lithium borohydride afforded two epimeric diols (4 and 5), both of which were transformed by straight-forward but distinctly different chemical procedures into potentially useful aglycons for preparing novel tupes of bioactive, aminocyclitol glycoside antibiotics. The disposition of the substituents at C-1, C-3, C-4, and C-5 in 19 and 37 is identical with that present in the 2-deoxystreptamine nucleus in the naturally occurring antibiotics     

The synthesis of D-xylo-hexos-4-ulose and some derivatives

D-xylo-Hexos-4-ulose has been synthesised, characterised chromatographically, and methyl α-D-xylo-hexopyranosid-4-ulose has been shown to be stable in neutral aqueous solution, contrary to a previous report. Glycosyl phosphate derivatives are also reported.     

Synthesis of glycopeptide derivatives containing the 2-acetamido-N-(L-aspart-4-oyl)-2-deoxy-β-D-glucopyranosylamine linkage and having the amino acid sequences 32–34, 33–35,33–37, and 33–38 of bovine ribonuclease

The synthesis is described of the glycotripeptide derivatives 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L--seryl-L-nitroarginyl-L-aspart-4-oyl]-2-deoxy-β-D-glucopyranosylamine, 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-seryl-L-nitroarginyl-L-aspart-1-oyl-(1-p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-nitroarginyl-L-aspart-1-oyl-(L-leucine methyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine, and of the glycopentapeptide and glycohexapeptide derivatives 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-nitroarginyl-L-aspart-1-oyl-(L-leucyl-L-threonyl-threonyl-N^ε-tosyl-L-lysine-(p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glycopyranosylamine and 2-acetamido-3,4,6-tri-O-acetyl-N-[N-(benzyloxycarbonyl)-L-nitroarginyl-L-aspart-1-oyl-(L-leucyl-L-threonyl-N^ε-tosyl-L-lysyl-L-aspartic 1,4-di-p-nitrobenzyl ester)-4-oyl]-2-deoxy-β-D-glucopyranosylamine.     

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).     

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.     

Synthesis of 4,5-dideoxy-4-C-[(R,S)-phenylphosphinyl]-d-ribo- and l-lyxo-furanose and their 1,2,3-triacetates

2,3-O-Isopropylidene-d-ribose diethyl dithioacetal, prepared from d-ribose, was converted in three steps into the corresponding dimethyl acetal, which was monotosylated at O-5, and the ester oxidized at C-4 with pyridinium chlorochromate; addition of methyl phenylphosphinate to the resulting pentos-4-ulose derivative then provided (4R,S)-4,5-anhydro-2,3-O-isopropylidene-4-C-[(R,S)-(methoxy)phenylphosphinyl]-d-erythro-pentose dimethyl acetal. Hydrogenation of this compound in the presence of Raney Ni, followed by reduction with SDMA, hydrolysis, and acetylation, yielded the title compounds (seven kinds), the structures of which were established on the basis of their 400-MHz, ¹H-n.m.r. and mass spectra. A general dependence of the ²J_PH and ³J_PH values on the OPCH and PCCH dihedral angles provided an effective method for the assignment of the configurations and conformations of these 4-deoxy-4-phosphinyl-pentofuranoses.     

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.     

Determination of the position of linkage of 2-acetamido-2deoxy-D-galactose and 2-acetamido-2-deoxy-D-glucose residues in oligosaccharides and glycoproteins. Synthesis of 2-acetamido-2deoxy-D-xylitol and 2-acetamido-2-deoxy-L-threitol

A method has been studied for the determination of the position of the linkage of the 2-acetamido-2-deoxy-D-galactose and 2-acetamido-2-deoxy-D-glucose residues in oligosaccharides and glycoproteins that is based on the borohydride reduction of the reducing terminal residues to the corresponding alditol derivatives periodate oxidation, borohydride reduction, hydrolysis (eventually followed by borohydride reduction), separation of the fragments as per-O-(trimethylsilyl) or per-O-(trifluoroacetyl) derivatives, and identification of the fragments as derivatives of 2-acetamido-2-deoxyglycerol, 2-acetamido-2-deoxy-L-threitol, 2-acetamido-2-deoxy-L-arabinitol, 2-acetamido-2-deoxy-D-xylitol, 2-acetamido-2-deoxy-D-galactitol, and 2-acetamido-2-deoxy-D-glucitol by gas-liquid chromatography-mass spectrometry. New syntheses for the standard compounds 2-acetamido-2-deoxy-L-threitol and 2-acetamido-2-deoxy-D-xylitol are described.     

A kinetic analysis of d-xylose transport in rhodotorula glutinis

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for d-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low K_m carrier was repressed in rapidly growing cells and depressed by starvation of the cells.Several hexoses were observed to inhibit d-xylose transport. In the studies reported here, the inhibitions produced by d-galactose and 2-deoxy-d-glucose were examined in some detail in order to define the interactions of these sugars with the d-xylose carriers. 2-Deoxy-d-glucose competitively inhibited both of the d-xylose carriers. In contrast, only the low-K_m carrier was competitively inhibited by d-galactose.     

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.     

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.     

Determination of the d and l configuration of neutral monosaccharides by high-resolution capillary g.l.c.

Capillary g.l.c. on SE-30 of the trimethylsilylated (-)-2-butyl glycosides of d and l monosaccharides gives multiple peak patterns, which can be used for the assignment of the absolute configurations. (-)-2-Butyl glycosides can be prepared from monosaccharides or their methyl glycosides; consequently, for the analysis of oligo- or poly-saccharides, hydrolysis as well as methanolysis can be applied. Provided that the peaks of the (-)-2-butyl glycosides do not completely overlap, mixtures of monosaccharides can be analysed directly, as illustrated for the constituents of the cell-wall lipopolysaccharide from Salmonella typhimurium LT-2.     

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.