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.     

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.     

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.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

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.     

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.     

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.     

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.     

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 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     

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.     

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.     

l-Myoinositol-1-phosphate synthase from Neurospora crassa: Purification to homogeneity and partial characterization

The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH₄)₂SO₄ and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH₄)₂SO₄ and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg²⁺ and Mn²⁺ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD⁺ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD⁺-independent activity.     

Aromatic and heterocyclic 1-C-substituted derivatives of 1,5-anhydro-d-glucitol

Reaction of anomeric 1-O-acyl and 1-halide derivatives of 2,3,4,6-tetra-O-benzyl-d-glucose with anisole, ferrocene, thiophene, furan, and 1,3,5-tri-methoxybenzene in the presence of a Lewis acid gives the corresponding C-β-d-glucopyranosyl derivatives.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

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.     

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.     

A re-investigation of the kinetics of alkaline degradation of phenyl β-d-glucopyranoside and some other glycosides

Previous investigators of mechanisms of alkaline degradation of glycosides in which the 2-hydroxyl group is trans to the aglycon concluded that the major reaction-pathway occurs via an SnicB substitution by the C-2 oxyanion at C-1. Recent reports also suggested that, for the phenyl d-glucopyranosides, direct Sn2 substitution by hydroxyl ion at C-1 competes isgnificantly with the SnicB pathway. We now conclude that the latter hypothesis was in error, because of failure to allow for variation in activity values for hydroxyl ion and water. For the nitrophenyl glycosides, however, we confirm that Sn2AR reactions compete with the SnicB pathway.     

Synthesis of β-l-fucopyranosyl phosphate andl-fucofuranosyl phosphates by the MacDonald procedure

Fusion or β-l-fucopyranose tetraacetate with phosphoric acid for 1 min at 50° gives a 9:1 anomeric mixture of the α-and β-pyranosyl phosphates. Longer fusion times give the α-anomer exclusively. The l-fucofuranose tetraacetates were synthesized for the first time by acetolysis or methyl-2,3,5-tri-O-acetyl-β-l-fucofuranoside. Fusion or the furanose tetraacetates with phosphoric acid gave a mixture or the fucofuranosyl phosphates in which the β-anomer predominated (β/α = 2.4). Anomeric pairs in the fucofuranose series appear to be distinguishable by the chemical shift of the C-6 methyl protons, as already shown by Sinclair and Sleeter in the pyranose series.