José H. Duarte

Properties of 13C-N.M.R. spectra of O-(1-carboxyethylidene) derivatives of methyl β-d-galactopyranoside: models for determination of pyruvic acid acetal structures in polysaccharides

Abstract 13 C-N.m.r. spectra were recorded of compounds containing O -(1-carboxyethylidene) groups linked to galactopyranose and fucopyranose derivatives. These compounds are useful as aids in determination of the positions and configurations of pyruvic acid acetal substituents in polysaccharides. Chemical shifts of non-protonated acetal carbons depend on whether the acetal ring is 5-membered (δ c 107–109.5) or 6-membered (δ c 100.5–102.4). The C-3 signals of 3,4-(1-carboxyethylidene) acetals are typical, being at ∼δ c 81 and in the case of the barium salt of the methyl β- d -galactopyranoside derivative. The exact value depends on the configuration, whether it is as in 6 (δ c 81.1) or 5 (δ c 80.4). The CH 3 signals of proton-n.m.r. spectra are also diagnostically useful, falling at δ 1.97 and 2.07 respectively. (The foregoing shift-values are pH-dependent). The pyruvylated galactan from the snail, Pomacea lineata , was shown to contain some residues that could be assigned a structure corresponding, in the positions of acetal substitution and acetal configuration, to structure 6 . Compound 6 (barium salt) is of interest as its 13 C-n.m.r. spectrum lacks non-protonated carbonyl and acetal carbon resonances, when obtained by the usual procedures. While this is principally because of long T 1 values, the non-protonated acetal carbon signals are comparatively broad, possibly through slow conformational interchange. In the case of the carbonyl resonance, the lack of sensitivity is because of a low n.O.e. value of 1.4, approximately one half that of other carbon atoms in the molecule.

Structure of a galactan found in the albumen gland of Biomphalaria glabrata

Abstract A neutral galactan, found in the albumen gland of B. glabrata , has been studied by acid fragmentation, by periodate oxidation, and by Smith and Barry degradations. The galactan has a highly branched structure and contains D - (64 %) and L -galactopyranose residues (36%) linked β-(1→3) and β-(1→6), in approximately the same proportion. The release, on mild hydrolysis with acid, of two disaccharides, 3- O -galactopyranosylgalactopyranose and 6- O -galactopyranosylgalactopyranose, is discussed in relation to the occurrence of these sugar sequences in the polysaccharide structure.

Some structural studies on the fucogalactan from egg masses of the snail ampullarius sp

Abstract The heteropolysaccharide isolated from egg masses of the snail Ampullarius sp. is composed of d -galactose and l -fucose residues. The fully methylated polysaccharide gave 2,3,4,6-tetra-(27 mole%), 2,4,6-tri-(5 mole%), 2,3,4-tri-(26 mole%), 3,4,6-tri-(11 mole%), and 2,4-di-O-methyt- d -galactose (29 mole%), and 2,3,4-tri-O-methyl- l -fucose (2 mole%). The results of periodate oxidation and Smith degradation are in satisfactory agreement with the methylation data, and indicate that the fucogalactan is a highly branched polysaccharide containing d -galactose and l -fucose as non-reducing end-groups and branching points at C-3 and C-6 of the d -galactose residues. In addition to a preponderance of (1→6) over (1→3) linkages, the fucogalactan contains part of the internal residues of d -galactose linked (1→2), which differentiates it from the polysaccharides of other molluscs that have been studied previously. No appreciable difference between polysaccharides isolated from freshly collected egg masses and from albumen glands of the Ampullarius sp. was observed.

Synthesis and chromatographic properties of isomeric O-β-d-galactopyranosyl-d-galactoses, and of diastereo-isomers of 3,4-O- and 4,6-O-(1-carboxyethylidene)-d-galactose

Abstract Partial hydrolysis of β- d -galactopyranans and d -galactose-containing polysaccharides having pyruvic acetal groups gives isomeric O -β- d -galactopyranosyl- d -galactoses and O -(1-carboxyethylidene)- d -galactoses, respectively. Their chromatographic properties in various systems were investigated as an aid in identification. The best method was a combination of silica gel t.l.c., which separates the 3,4- and 4,6- O -(1-carboxyethylidene) derivatives, and paper electrophoresis in borate buffer, which resolves the 2- O -, 3- O -, 4- O -, and 6- O -linked galactobioses after removal of the O -(1-carboxyethylidene) derivatives. 2- O - and 4- O -β- d -Galactopyranosyl- d -galactoses were synthesized, as well as the 4 diastereoisomeric 3,4- and 4,6- O -(1-carboxyethylidene)- d -galactoses.

Methylation studies of the polysaccharides resulting from sequential Smith-degradations of the galactan from the snail strophocheilus oblongus☆

When the galactan from the albumen glands of the snail Strophocheilus oblongus was submitted to three Smith-degradation, the degraded polysaccharide, isolated in 6% yield, was much more linear. Methylation analysis showed that the Smith-degraded polysaccharide gave an increased percentage of 2,4,6-tri, decreased percentages of 2,3,4,6-tetra-and 2,4-di, and a large variation in the amount of 2,3,4-tri-O-methyl-D-galactose. The sugars in the polysaccharide which result in the formation of 2,3,4,6-tetra- and 2,3,4-tri-O-methyl-D-galactose are destoryed in subsequent degradation procedures. The above observations suggest that the degradation by periodate oxidation proceeds via non-reducing end-groups and though some internal residues that are exposed as the degradation proceeds. As a result of the overall process, new non-reducing end-groups are formed and new (1 leads to 6)-linked D-galactose residues are then exposed. The isolation of glycosides of low molecular weight supports the suggestion that the molecule, in the main, is sequentially degraded from the external layers.

Occurrence of β-d-galactopyranosyl units esterified at O-6 with 2-aminoethylphosphonic acid in the d-galactan of albumen glands of the snail Megalobulimus paranaguensis☆

Abstract The albumen glands of the snail Megalobulimus paranaguensis were previously found to contain a branched β- d -galactopyrananan consisting mainly of nonreducing end-groups and 3,6-di- O -substituted residues with smaller proportions of 3- O - and 6- O -substituted units. 13 C-n.m.r. spectroscopy has now shown that 2-aminoethylphosphonic acid ( 1 ) is an additional component and its presence was confirmed by cellulose-plate chromatography. Furthermore, the 2-aminoethylphosphonic acid-substituted oligosaccharides, formed on partial hydrolysis of the polysaccharide, were isolated, examined by fast-atom-bombardment mass spectrometry, and found to have molecular weights consistent with monophosphonic esters of 1 . The 13 C-n.m.r. spectrum of this mixture indicated substitution of d -galactopyranosyl units at O-6 with esters of 1 , and this was confirmed by spectral examination of a monosubstituted d -galactose isolated after strong acid hydrolysis of the polymer. The snail d -galactan contained one ester of 1 for every 34 residues, it was free of other components such as protein, lipid, and nucleic acid, and is the first polysaccharide of this type that has been identified.

Homogeneous guluronic and mannuronic acid blocks in the alginate of the brown seaweed Laminaria brasiliensis

The alginate from fronds of the brown seaweed Laminaria brasiliensis contains residues of (1 → 4)-linked β-d-mannopyranosyl (uronic acid) and α-l-gulopyranosil, (uronic acid) in a molar ratio 1.2:1. Strong acid hydrolysis gave rise to oligosaccharide blocks of β-d-ManpA (yield 6%; DP 19–26) and of α-l-GulpA (yield 17%; DP 16–20). These contained 8%; and < 2%; of the other uronic acid respectively, indicating preferential hydrolysis of glycosidic bonds in the region containing mixed uronic acids.

Structure and biodegradation of the polysaccharide components of egg masses isolated from snails of an Ampullarius species

Abstract A polysaccharide preparation, isolated from egg masses deposited by snails of an Ampullarius species, was purified via precipitation with Cetavlon in the presence of sodium borate, and found to contain d -galactose and a smaller proportion of d -glucose, and to have two components with sedimentation coefficients of 10S and 40S. A polysaccharide, isolated from freshly laid egg masses, was highly branched and was shown to contain nonreducing α- d -glucopyranosyl and β- d -galactopyranosyl end-groups, and 3,6-di-O-substituted β- d -galactopyranosyl residues. One or more of the polysaccharide components was a d -glucopyrano- d -galactopyranan with non-reducing α- d -glucopyranosyl end-groups (1→4)-linked to β- d -galactopyranosyl residues. The polysaccharide preparations, obtained from freshly laid egg masses and from those that were left for 10 and 15 days after being laid, were structurally different from each other. With the passage of time, progressive diminution of the 10S component and the proportion of d -glucose in the polysaccharide took place, suggesting that each constituent was consumed preferentially by the snail embryos as an energy source.

Some structural studies on the galactose-containing polysaccharide from bovine placenta

Polysaccharides were extracted from 8-month-old placenta with aqueous HgCl2. The protein-free material was purified by selective precipitation with Cetavlon in the presence of sodium borate at pH 8.5 and was homogeneous on molecular-sieve chromatography, electrophoresis, and on treatment with Concanavalin A. The preparation contained galactose and glucose as principal monosaccharides with 5 per cent of hexosamines. Methylation studies suggested that D-gluco and D-galactopyranosyl units may be constituents of glucan and galactan respectively which form a molecular aggregate that does not dissociate during the fractionation procedures. After treatment of the fraction with beta-amylase, the proportion of glucose in the polysaccharide diminished, indicating the presence of (1-->4)-linked alpha-D-glucopyranosyl residues. Also, when the fraction was treated with a crude protease having glucosidase activity a residual alpha-D-galactopyranan was isolated and found to contain non-reducing end-groups (30.0 per cent), 3-O-(39.5 per cent) and 3,6-di-O-substituted (30.5 per cent) units. The structure of the galactan was not modified according to methylation data, on removal of the glucosyl component. The polysaccharide fraction (pH 8.5 Cetavlon), isolated from bovine placenta, thus contains a glycogen-like material associated with a galactan as molecular aggregate. This galactan has not been previously recognized in bovine placenta and its occurrence in this organ supports the hypothesis that galactose-containing polysaccharides are involved in foetal development.