Synthesis and immunoadjuvant activities of the repeating,disaccharide-dipeptide unit of the bacterial,cell-wall peptidoglycan and of some carbohydrate analogs

The repeating disaccharide-dipeptide units of the bacterial, cell-wall peptidoglycan, one being O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→4)-2-acetamido-2-deoxy-d-glucose, and the other, O-(2-acetamido-2-deoxy-β-d-glucosyl)-(1→4)-N-acetyl-muramoyl-l-alanyl-d-isoglutamine, have been synthesized. Some carbohydrate analogs, such as O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)- (1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, O-β-d-glucosyl-(1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, and O-(6-acetamido-6-deoxy-β-d-glucosyl)-(1→4)-N-acetylmuramoyl-l-alanyl-d-isoglutamine, were also synthesized. Their immunoadjuvant activities were examined in guinea-pigs.     

Conversion of amylose into a (1-4)-2-amino-2-deoxy-alpha-D-glucopyranuronan and its 2-N-sulfo derivative, a heparin analog

By a modification of a previously established reaction-sequence involving successive oxidation with methyl sulfoxide-acetic anhydride, oximation, and reduction with lithium aluminum hydride, 6-O-tritylamylose (1) was converted into a 6-O-tritylated (1→4)-α-D-linked glucan (3) containing 2-amino-2-deoxy-D-glucose residues and some O-(methylthio)methyl groups. Removal of the ether groups from this product gave a 2-aminated amylose (4) of degree of substitution (d.s.) by amine of 0.54 that underwent cleavage by fungal alpha-amylase to give oligosaccharides containing amino sugar residues. N-Trifluoroacetylation of 3 followed by removal of the ether groups, oxidation at C-6 with oxygen-platinum, and removal of the N-substituent, gave a (1 →4)-2-amino-2-deoxy-α-D-glucopyranuronan 7 having d.s. by amine of up to 0.65, and by carboxyl, of 0.46. Sulfation of this product with sulfur trioxide-pyridine and then with chlorosulfonic acid-pyridine gave a (1→4)-2-deoxy-2-sulfoamino-α-D-glucopyranuronan, isolated as its sodium salt 8, which showed appreciable blood-anticoagulant activity.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [¹⁴C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is $\text{Gal}\text{β1 → 3([}{}^{14}\text{C}\text{]}\text{Gal}\text{β1 → 4}\text{GlcNac}\text{β1 → 6)}\text{GalNAcol}$. Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.     

Mass spectrometry of pertrimethylsilyl nueraminic acid derivatives

The mass spectra of the trimethylsilyl (TMS) derivatives of the methyl and trideuteriomethyl esters of N-acetylneuraminic acid, the methyl ester of N-glycolylneuraminic acid, the methyl ester methyl β-glycoside of N-acetylneuraminic acid, the trideuteriomethyl ester trideuteriomethyl β-glycoside of N-acetylneuraminic acid, and the methyl esters of the (2→3)- and (2→6)-linked isomers of N-acetylneuraminic acid—lactose are discussed. The characteristic fragmentation patterns of the sialic acid derivatives can be used for the identification of this type of carbohydrate. The (2→3)- and (2→6)-linked isomers of N-acetylneuraminic acid—lactose can be differentiated.     

Water- and alkali-soluble glucans from oak lichen

A water-soluble glucan, [α]²_D +217° (water), and an alkali-soluble glucan,

+152° (sodium hydroxide), have been isolated from the oak lichen Evernia prunastri (L.) Ach. On the basis of methylation analysis, periodate oxidation, and partial acid hydrolysis, the water-soluble polysaccharide has been shown to be a neutral, slightly branched glucan with a main chain composed of (1→3)- and (1→4)- linked glucopyranose residues in the ratio 1?:1. Branching occurs most probably at position 2 of (1→4)-linked glucopyranose residues. On the basis of optical rotation and i.r. spectral data, and enzymic hydrolysis, the α-D configuration has been assigned to the glycosidic linkages. Likewise, the alkali-soluble polysaccharide was shown to be a neutral, branched glucan with a main chain composed of (1→3)- and (1→4)-linked α-D-glucopyranose residues in the ratio 6:1. Each of the (1→4)-linked units was a branch point involving position 6. The presence of some β-D linkages is not excluded since hydrolysis with β-D-glucosidase occurred to a small extent.     

The preparation of a crystalline guanosine trialcohol and its sodium salt

A polysaccharide has been extracted from Cassia corymbosa seeds with cold, acidulated water, and purified to give a water-soluble product containing d-galactose and d-mannose in 4:7 molar ratio. Acid-catalyzed fragmentation, periodate oxidation, methylation, and enzymic hydrolysis showed that the seed gum has a branched structure consisting of a linear chain of β-d-(1→4)-linked mannopyranosyl units, some of which are substituted at O-6 by two α-d-(1→6) galactopyranosyl units mutually linked glycosidically. Methylation analysis of the galactomannan afforded 2,3,4-tri- and 2,3,4,6-tetra-O-methylgalactose, along with 2,3-di- and 2,3,6-tri-O-methylmannose, in the molar ratios of 2:2:2:5. Both the methylation and the periodate-oxidation studies showed ～36.4% of end groups. The significance of these results, together with the findings of partial hydrolysis with acid, are discussed, in relation to ascertaining the structure of the repeating unit of the polysaccharide.     

Structure of the ceramide octadekahexoside isolated from gastric mucosa

A fucose-containingceramide octadekahexoside exhibiting blood-group (A+H) activity has been isolated from hog gastric mucosa. Based on the results of partial acid hydrolysis, sequential degradation with specific glycosidases, oxidation with periodate and chromium trioxide, and permethylation analysis, we propose that the carbohydrate chain of this fucolipid contains four branches. Two of the branches are terminated by βGall→4βGlcNAc, one by $\text{αFucl→2βGall→}\text{3}\text{4}\text{βGlcNAc}$ and one by $\text{αGalNAcl→3(αFucl→2)βGall→}\text{3}\text{4}\text{βGlcNAc}$.     

Structure of the d-galacto-d-mannan isolated from the seeds of Melilotus indica All

A d-galacto-d-mannan ([α]_D +72.0 and d-galactose-to-d-mannose ratio 1:1.14) was isolated from the seeds of Melilotus indica All., syn. M. parviflora Desf. The ¹H- and ¹³C-n.m.r., and i.r. spectra indicated the presence of α-d-galactopyranosyl and β-d-mannopyranosyl residues. Methylation of the polysaccharide, followed by hydrolysis, afforded, 2,3,4,6-tetra-, 2,3,6-tri-, 2,3-di-, and 3,4-di-O-methyl-d-mannose, and 2,3,4,6-tetra- and 2,3,6-tri-O-methyl-d-galactose in the molar ratios of 1:2:22:6:27:3. Periodate oxidation of the polysaccharide, followed by reduction and hydrolysis, gave erythritol (1 mol) and glycerol (1.24 mol). Partial acid hydrolysis of the polysaccharide afforded O-β-d-mannopyranosyl-(1→2)-d-mannopyranose, O-β-d-mannopyranosyl-(1→4)-d-mannopyranose, O-α-d-galactopyranosyl-(1→6)-d-mannopyranose, O-α-d-galactopyranosyl-(1→4)-d-galactopyranose, and O-α-d-galactopyranosyl-(1→6)-O-β-d-mannopyranosyl-(1→4)-d-mannopyranose. A highly branched structure having a mannan backbone composed of 36% of (1→4)- and 10% of (1→2)-linked β-d-mannopyranosyl units is proposed for the galactomannan.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.     

The structure of an α-D-glucan from Cyttaria harioti Fischer

A homogeneous glucan has been isolated from the fruiting bodies of Cyttaria harioti Fischer. Partial acid hydrolysis produced major amounts of isomaltose, whereas acetolysis gave maltose and maltotriose. Enzymic hydrolysis with amylo-glucosidase and pullulanase indicated a structure based on maltotriose residues connected by (1→6)-α-D linkages. This conclusion was supported by periodate-oxidation data which also showed that 3–7% of the glucose resisted oxidation. Methylation analysis confirmed the presence of (1→6) and (1→4) linkages in the ratio 1:2.4.     

Isolation and analysis of neutral glucans from Ecklonia radiata and Cystophora scalaris

Neutral glucans were isolated from the stipes and fronds of Eklonia radiata and Cystophora scalaris. Partial acid hydrolysis revealed the presence of gentiobiose and laminara-oligosaccharides. Methylation analysis, periodate oxidation, and enzyme studies indicated that the glucans contain β-(1→3) and β-(1→6) linkages. Methylation studies showed that branching in these glucans occurs via a 1,3,6-tri-O- substituted residue with a frequency of one branch point per seven glycosyl residues. In contrast to laminaran from Laminaria digitata, the intrachain (1→3)- and (1→6)- glucopyranoside occur in a molar ratio of 1:1. Enzymic hydrolysis confirmed the absence of long segments of (1→3)-linked residues in the glucans.     

Bis(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)amine obtained by a fusion reaction

A unique, alkali-soluble polysaccharide has been isolated from the cell walls of the basidiomycete Coprinus macrorhizus microsporus. The polysaccharide, which is primarily a glucan, contains a large proportion of α-(1→4)-linked d-glucose residues and a smaller amount of β-(1→3) and (1→6) linkages, as suggested by methylation, partial acid hydrolysis, periodate oxidation, and enzymic studies. Hydrolysis of the methylated polysaccharide gave equimolar amounts of 2,4-di- and 2,3-di-O-methyl-d-glucose; no 2,6-di-O-methyl-d-glucose was identified, indicating the absence of branch points joined through O-1, O-3, and O-4. The isolation and identification of 2-O-α- glucopyranosylerythritol from the periodate-oxidized polysaccharide suggests that segments of the a-(1→4)-linked d-glucose residues are joined by single (1→3)-linkages. An extracellular enzyme-preparation from Sporotrichum dimorphosporum (QM 806) containing both β-(1→3)- and α-(1→4)-d-glucanohydrolase activity released 76% of the reducing groups from the polysaccharide. The polysaccharide also contains minor proportions of xylose, mannose, 2-amino-2-deoxyglucose, and amino acids.     

Immunochemical studies on the combining site of the d-galactopyranose and 2-acetamido-2-deoxy-d-galactopyranose specific lectin isolated from Bauhinia purpurea alba seeds

The combining site of the Bauhinia purpurea alba lectin was studied by quantitative precipitin and precipitin inhibition assays. Of 45 blood group substances, glycoproteins, and polysaccharides tested, 35 precipitated over 75% of the lectin. Precursor blood group substances with I activity (Cyst OG 10% from 20% and Cyst OG 20% from 10%), desialized fetuin, and desialized ovine salivary glycoprotein, in which more than 75% of the carbohydrate side chains have dGalN Ac linked through α1 → to the OH group of Ser or Thr of a protein core, completely precipitated the lectin. The poorly reactive blood group substances after mild acid hydrolysis or Smith degradation, as well as sialic acid-containing glycoproteins after removal of sialic acid, had substantially increased activity so that more than 80% of the lectin was precipitated. Precipitability with various blood group substances and glycoproteins is ascribable to the terminal nonreducing dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 3 or 4dGlcNAc, and dGalβ1 → 3 or 4dGlcNAcβ1 → 3dGal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, dGalNAc and its p-nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding dGal compounds but methyl βDGalNAcp was only about 40% more active than methyl βdGalp. The α-anomers of p-nitrophenyl DGalNAcp and dGalp, were twice as active as the corresponding β-anomers. Methyl αDGalNAcp was four times as active as the β-anomer but the inhibitory power of the methyl α- and β-anomers of dGal were about equal. Among the oligosaccharides tested, dGalβ1 → 3dGalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as dGalβ1 → 3dGalNAc, which was somewhat more active than dGalNAcα1 → 6dGal and dGalNAc, and 2.5 and 5 times as active as dGalNAcα1 → 3dGalβ1 → 3dGlcNAc and dGalNAcαl → 3dGa1, respectively (blood group A specific). These findings suggest that a subterminal dGalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that dGalβ1 → 3dGlcNAc and dGalβ1 → 6dGal were poorer inhibitors although dGalβ1 → 3dGlcNAc was two to three times as active as glycosides of dGal. Oligosaccharides with terminal nonreducing dGal and subterminal α-linked dGal were as active or less active than dGal. dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc (lacto-N-tetraose) and dGalβ1 → 3dGlcNAcβ1 → 3dGal-β1-O-(CH₂)₈COOCH₃ were equally active and 1.5 times as potent as dGalβ1 → 3dGlcNAc whereas dGalβ1 → 3dGlcNAcβ1 → 6dGal was only 40% as potent as dGalβ1 → 3dGlcNAc suggesting that a third sugar may be part of the determinant. Substitution of dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc on the subterminal dGlcNAc by lFucα1 → 4 in lacto-N-fucopentaose II reduced activity fourfold; if the nonreducing dGal is substituted by lFucα1 → 3 as in lacto-N-fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing dGal as well as subterminal dGlcNAc are contributing to binding. The β → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4dGlcNAc is a poorer inhibitor. Although the available data suggest that the combining site of the lectin Bauhinia purpurea alba may be most complementary to the structure dGalβ1 → 3dGalNAcβ1 → 3dGal, several other possibilities remain to be tested when suitable oligosaccharides become available.     

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 carbohydrate analogs (positional,configurational, and optical) of N-acetylmuramoyl-l-alanyl-d-isoglutamine,and their immunoadjuvant activities

2-Acetamido-2-deoxy-4- and -6-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranose, 2-acetamido-2-deoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-allopyranose, -d-gulopyranose, -d-galactopyranose, -d-mannopyranose, and -l-idopyranose, and 3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d- and -l-glucopyranose were synthesized, in order to clarify the structural requirements for the immunoadjuvant activity of the carbohydrate moiety in N-acetylmuramoyl-l-alanyl-d-isoglutamine. Immunoadjuvant activity of the N-acetylmuramoyl-dipeptide analogs was examined in guinea-pigs.     

Structural studies on an antitumor polysaccharide from Microellobosporia grisea

The structure of the antitumor polysaccharide from the actinomycete Microellobosporia grisea has been investigated. By methylation and periodate-oxidation studies, the polysaccharide was shown to consist of (nonreducing)d-mannosyl groups, (1→4)-linkedd-glucosyl residues, and 3,6-branched, (1→4)-linkedd-glucosyl residues in the approximate molar ratios of 2:1:1. Periodate oxidation of the polysaccharide, followed by borohydride reduction and mild hydrolysis with acid yielded glycerol, erythritol, 2-O-β-d-glucopyranosyl-d-erythritol, and 5-O-β-d-glucopyranosyl-2,4-bis(hydroxymethyl)-1,3-dioxane, which were isolated in the molar ratios of 2.0:0.14:0.74:0.35. Partial hydrolysis of the polysaccharide gave α-d-Man p-(1→6)-d-Glcp, β-d-Glcp-(1→4)-d-Glcp, α-d-Man p-(1→3)-d-Glcp, and β-d-Glcp-(1→4)-[α-d-Man p-(1→3)-]-d-Glcp. From these results, it is proposed that the polysaccharide is mainly composed of tetrasaccharide repeating-units having the following structure.     

Chemical structures in a lignin-carbohydrate complex isolated from the bovine rumen

The soluble, lignin-carbohydrate complex (LCC) from the rumen fluid of steers fed a diet of pure spear grass (Heteropogon contortus) has been purified by gel filtration. The purified LCC contained 7.4% of carbohydrate which, on hydrolysis, gave d-glucose, d-xylose, l-arabinose, l-rhamnose, and traces of d-galactose and d-mannose. The structure of the LCC was examined by methylation analysis, using g.l.c.-m.s. for the unequivocal classification of the sugar derivatives. d-Glucose, d-xylose, and l-rhamnose were shown to be glycosidically linked to lignin. Some of the d-glucosyl residues carry other (1→4)-linked d-glucose units, and some of the d-xylosyl residues bear other (1→4)-linked d-xylose units and (1→3)-linked l-arabinofuranosyl groups. The major carbohydrate component is a single d-glucopyranosyl group. The LCC was subjected to various chemical treatments in an investigation of the chemical nature of the bonding between lignin and the carbohydrates. d-Glucose could be enzymically hydrolyzed from the LCC, but only with a very high concentration of β-d-glucosidase. The presence of lignin in rumen LCC has been confirmed by nitrobenzene oxidation, vanillin and syringaldehyde being identified by g.l.c.-m.s. as oxidation products from both the original spear grass and the LCC.     

Action of alpha-1,6-glucan glucohydrolase on oligosaccharides derived from dextran

The action of α-1,6-glucan glucohydrolase on α-(1→6)-D-glucosidic linkages in oligosaccharides that also contain an α-(1→2)-, α-(1→3)-, or α-(1→4)-D-glucosidic linkage has been investigated. The enzyme could hydrolyse α-(1→6)-D-glucosidic linkages from the non-reducing end, including those adjacent to an anomalous linkage. α-(1→6)-D-Glucosidic linkages at branch points were not hydrolysed, and the enzyme could neither hydrolyse nor by-pass the anomalous linkages. These properties of α-1,6-glucan glucohydrolase explain the limited hydrolysis of dextrans by the exo-enzyme. Hydrolysis of the main chain of α-(1→6)-D-glucans will always stop one D-glucose residue away from a branch point. The extent of hydrolysis by α-1,6-glucan glucohydrolase of some oligosaccharide products of the action on dextran of Penicillium funiculosum and P. lilacinum dextranase, respectively, has been compared. Differences in the specificity of the two endo-dextranases were revealed. The Penicillium enzymes may hydrolyse dextran B-512 to produce branched oligosaccharides that retain the same 1-unit and 2-unit side-chains that occur in dextran.     

Multiple Smith-degradations of carcinoembryonic antigen (CEA) and of asialo CEA

Carcinoembryonic antigen (CEA) and asialo CEA were subjected to multiple Smith-degradation (i.e., for each degradation, application in sequence of periodate oxidation, borohydride reduction, and mild hydrolysis with acid; borohydride-t was substituted for unlabelled borohydride). High yields of modified glycoproteins were obtained at each stage. After three complete degradations and a further periodate-borohydride-t treatment, the carbohydrate content of CEA and of asialo CEA had decreased from 45–50% to 11–12% (i.e., 90% removal of carbohydrate). Glycerol was always one of the products obtained after each degradation, but threitol and erythritol were not detected. The second degradation caused a substantial loss of 2-acetamido-2-deoxyglucose, which is consistent with the location of some of this monosaccharide towards the terminal (non-reducing) end of the oligosaccharides. The “core” region of the oligosaccharides is composed of galactose, mannose, and 2-acetamido-2-deoxyglucose. After the fourth oxidation, 2-acetamido-2-deoxyglucose was 50–60% of the total content of residual carbohydrate. After the first degradation, there was a progressive loss in antigenic activity, but this was associated with a small amount of hydrolysis of the protein moiety of CEA.