Akira Misaki

Structure of the dextran of Leuconostoc mesenteroides B-1355

Abstract The structure of dextran B-1355-S (soluble fraction) has been investigated. Acid hydrolysis of the methylated dextran yielded 2,3,4,6-tetra-, 2,3,4-tri-, 2,4,6-tri-, and 2,4-di-O-methyl- d -glucose, in the molar ratios of 1.0:4.6:3.6:1.0, indicating that the dextran has a branched structure containing (1→6)- and (1→3)-α- d -glucosidic linkages with an average repeating-unit of ten sugar residues. The dextran-poly-alcohol, derived by successive periodate oxidation and borohydride reduction, gave, on complete hydrolysis with acid, glycerol and glucose (molar ratio, 1.0:0.83); mild hydrolysis (0. 1 m acid at 25°) gave glycerol and 1-O-α- d -glucosylglycerol (molar ratio, 1:1.45). Exo-G2-dextranase of Arthrobacter globiformis T6 acted on the dextran to release isomaltose and O-α- d -glucopyranosyl-(1→3)-O-α- d -glucopyranosyl-(1→6)- d -glucose in the molar ratio of 5.7:1.0, leaving a degraded dextran (limit dextran). Methylation analysis indicated the limit-dextran to be highly branched, with a repeating unit of six glucose residues, consisting mainly of alternate (1→6)- and (1→3)-α- d -glucosidic linkages. These findings confirm the overall arrangement of alternating (1→6)- and (1→3)-linked α- d -glucose residues, and the absence of consecutive α-(1→3)-linked sugar residues; the latter factor may be related to the water insolubility of α- d -glucans of Streptococcus mutans.

Effects of branch distribution and chemical modifications of antitumor (1→3)-β-D-glucans

Abstract A potent antitumor-active branched (1 → 3)-β- d -glucan (VVG) purified from fruiting body of Volvariella volvacea and some other glucans were chemically modified to study the enhancement of inhibitory activity on the growth of mouse-transplanted tumors. Conversion of the glucosyl groups substituted at O -6 atoms of the (1 → 3)-linked d -glucose residues into the corresponding polyhydroxyl groups gave significant enhancement of the original activities both on allogeneic and syngeneic tumors, whereas deletion of the polyhydroxyl groups by mild acid treatment resulted in a great reduction of the activity. When d -glucose residues of the branches were modified to the 3,6-anhydro d -glucose residues by partial sulfation and then alkali treatment, the resulting modified VVG showed essentially no antitumor activity. In connection of the modifications of the branches, a linear (1 → 3)-β- d -glucan was modified to epoxylated glucan (degree of substitution, 0·14), which lost its original activity. On the contrary, conversion of the epoxy groups to hydrophilic glycerol groups remarkably enhanced the original antitumor activity. These results confirmed the previous findings that, besides the conformation of (1 → 3)-β-glucan backbone, the molecular shape and the distribution pattern of the substituted groups located outside the backbone chains, must also play an important role in exhibiting antitumor action.

Purification of an antitumor-active, branched (1→3)-β-d-glucan from Volvariella volvacea, and elucidation of its fine structure☆

A (1--3)-beta-D-glucan branched by O-6 substitution (FCAP), obtained from the cold-alkali extract of the fruiting body of V. volvacea, exhibited potent growth-inhibitory activity against implanted tumors in mice. It contained protein and appeared to be heterogeneous. Fractionation by DEAE-Toyopearl column chromatography yielded an unbound, protein-free glucan fraction ([alpha]D - 30 degrees in M NaOH, mol. wt. 1.5-2 x 10(6)), which showed the highest antitumor activity. The polysaccharide had a moderately branched structure, consisting of a backbone chain of beta-(1--3)-linked-D-glucose residues, one out of five or six being substituted at O-6 with single glucosyl of beta-(1--6)-linked diglucosyl groups. Digestion of the glucan with exo-(1--3)-beta-D-glucanase yielded glucose and ++gentiobiose (molar ratio, 8:2:1.0), and a highly branched (d.b. 1/3), degraded glucan. Digestion with endo-(1--3)-beta-D-glucanase gave D-glucose, laminarabiose, a trisaccharide beta-D-Glcp-(1--6)-beta-D-Glcp-(1--3)-D-Glc, a tetrasaccharide beta-D-Glcp-(1--3)-beta-D-Glcp-(1--3)-[beta-D-Glcp-(1--6)]-D-Glc, and a highly branched (d.b. 1/2), enzyme-resistant glucan. The results suggest that the Volvariella glucan is structurally heterogeneous with regard to the distribution of branches, having less branched, moderately branched, and highly branched segments.

Synthesis of water-soluble, branched polysaccharides having d-mannopyranose, d-arabinofuranose, or oligo-d-arabinofuranose side-chains and their antitumor activity☆

Branched polysaccharides having D-mannopyranose, D-arabinofuranose, or oligo-D-arabinofuranose side-chains were synthesized by the reaction of 3,4,6-tri-O-acetyl-(1,2-O-ethylorthoacetyl)-beta-D-mannopyranose, 3,5-di-O-benzoyl-(1,2-O-ethylorthobenzoyl)-beta-D-arabinofuranose, or 3-O-benzoyl-(1,2,5-O-orthobenzoyl)-beta-D-arabinofuranose with cellulose acetate or curdlan acetate, followed by desterification. The structure and antitumor activity of the water-soluble portion of the polysaccharides thus obtained were investigated. Polysaccharides synthesized from (1----3)-beta-D-glucan as the main chain with oligo-D-arabinofuranose side-chains exhibited high antitumor activity.

A New Fungal α-D-glucan, elsinan, elaborated by Elsinoe leucospila

Abstract A new α- D -glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri- O -methyl- D -glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di- O -methyl- D -glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α- D -glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2- O -α- D -glucosyl- D -erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O -α- D -glucopyranosyl-(1→3)- O -α- D -glucopyranosyl (1→4)- D -glucopyranose, O -α- D -glucopyranosyl-(1→4)- O -α- D -glucopyranosyl-(1→3)- D -glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α- D -Gl cp -(1→4)-α- D -Gl cp -(1→.The unique structural features of elsinan are discussed in comparison with other glucans.

Degradation of ALPHA-linked D-gluco-oligosaccharides and dextrans by an isomalto-dextranase preparation from Arthrobacterglobiformis T6

Abstract Further studies were made on the action of isomalto-dextranase from Arthrobacter globiformis (T6) using gluco-oligosaccharides and dextrans. This enzyme releases isomaltose from some oligosaccharides by splitting not only the (1→6)-α-linkage but also (1→2)-α-, (1→3)-α- and (1→4)-α-linkages. Its exo-lytic mode of action was confirmed using isomaltohexaitol. The enzyme was also found to recognize both O-α-D-Glcp-(1→2 and 3)-O-α-D-Glcp-(1→6)-D-Glc as isomaltose and to split these trisaccharides from a gluco-tetrasaccharide and dextrans. A possible mechanism of action of this isomalto-dextranase on dextrans is proposed.

Structure of the water-insoluble α-d-glucan of streptococcus salivarius HHT

Water-insoluble, non-adherent α-d-glucans have been obtained from Streptococcus salivarius HHT under two sets of conditions: from a growing culture, or synthesized enzymically by using a glucosyltransferase. In the former case, the glucan ([α]d + 197°) was shown by methylation analysis to have a slightly branched structure containing a relatively high proportion (80 %) of (1→3)-α-d-glucosidic linkages, together with small proportions of (1→6)- and (1→4)-α-d-glucosidic linkages. The enzymically synthesized glucan had a much less-branched structure, containing 88 % of (1→3)-α-d-glucosidic linkages. Both glucans, on Smith degradation (sequential periodate oxidation, borohydride reduction, and mild acid hydrolysis), gave linear, (1→3)-α-d-glucosidic polysaccharides (yields, 82-90%) that constitute the backbone chains. The presence of small proportions of glycerol, erythritol, 1-O-α-d-glucosyl-d-glycerol, and also 2-O-α-d-glucosyl-d-erythritol in the products of Smith degradation suggests that the short side-chains are attached to the backbone chain by (1→4)-, (1→6)-, and (1→3)-α-d-glucosidic linkages

The structure of the glycan moiety of tora-bean (phaseolus vulgaris)lectin

Abstract The structure of the carbohydrate unit of Tora-bean lectin, which contains 7.8% of neutral carbohydrate, was elucidated by isolation of the glycopeptide from the digest by the action of proteolytic enzymes. The purified glycopeptide (mol. wt. 2,700) comprises 9.2 moles of d -mannose, 2.0 moles of 2-acetamido-2-deoxy- d -glucose, 0.6 mole of l -fucose, and 0.7 mole of d -xylose per mole of asparagine. Methylation analysis, Smith degradation and enzymic-degradation studies permitted formulation of a possible structure of the glycan of the glycopeptide, as follows.

Fine structural features of oyster glycogen: mode of multiple branching

Abstract The fine structural features of oyster glycogen, especially its mode of multiple branching, was investigated by repeated enzymic treatment with β-amylase and pullulanase, followed by the precise analysis of the α-1,4-linked glucosyl unit-chains by high performance anion exchange chromatography (HPAEC). The purified glycogen (average mol. wt 8.5 × 10 5 , CL 11) obtained by DMSO-extraction from fresh oysters ( Crassostrea gigas ) collected in February (a time when the oysters are edible) showed a distribution of α-1,4- d -glucosyl unit-chains, with degrees of polymerization (dp) in the range 2–35 (dp 6, dominant), as measured by HPAEC after complete enzymic debranching. The oyster glycogen was subjected to stepwise degradations with β-amylase and pullulanase, and this procedure was repeated until complete hydrolysis was achieved (extent and degradation of 98% after five treatments). The yield of the limit dextrin formed at each trimming step and quantitative analysis of the unit-chain distributions indicated that the oyster glycogen has a highly branched structure (A:B-chain, 0.7:1), involving five or six times interlinkings of the chains (B-chains). Assuming that B1 chain carrying only A-chains, attaches by α-1,6-bonds to another B-chain (B2 chain), which in turn attaches to a B3-chain, and so on, the molar ratios of the unit-chains (A, B1, B2-) of the dextrins during successive enzymic trimming showed that the ratio of A:B1:B2:B3:B4:B5-chain was 34:25:11:5:5:1, confirming the multiple ramified molecule. In connection with the digestion of oyster glycogen in the mammalian digestive tract, the glycogen was hydrolyzed by salivary and pancreatic α-amylase, and several branched maltosaccharides in the digestion product were fractionated, and their structures determined using HPAEC.

Chain Conformation of a Glucurono-xylo-mannan Isolated from Fruit Body of Tremella fuciformis Berk

Abstract A possible chain conformation of the acidic heteropolysaccharide isolated from the fruit body of Tremella fuciformis Berk (Shirokikurage) was proposed by the X-ray diffraction study combined with the computational model building technique. The polysaccharide consists of a linear backbone of 1,3-linked α-D-mannose which is highly substituted with β-D-xylose, β-D-glucuronic acid, and 1,2-linked β-D-xylobiose units at the C2 position of the mannose residue. It was suggested from the X-ray data, together with the knowledge of the chain conformations proposed for other 1,3-linked α-D-glycans, that the backbone conformation has the left-handed, three-fold helical symmetry. Orientations of the side group residues on the mannan backbone were suggested from the theoretical calculations. The proposed conformation of a repeating unit of the helical model consisted of six mannose residues and three side group residues in the 2.42 nm distance along the fiber axis. Two interresidue hydrogen bonds were observed...