Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

A 13C-N.M.R.-Spectral study of a gel-forming,branched (1→3)-β-d-glucan, (lentinan) from lentinus edodes, and its acid-degraded fractions. Structure,and dependence of conformation on the molecular weight

The structure and conformation of lentinan, an anti-tumor, branched (1→3)-β-d-glucan from Lentinus edodes, and its acid-degraded, lower molecular-weight fractions have been investigated by ¹³C-n.m.r. spectroscopy. It is found that their ¹³C-n.m.r. spectra are considerably changed, depending on the molecular weight. The conformational behavior as studied by ¹³C-n.m.r. spectroscopy is consistent with that revealed by a study of the shift in the absorption maximum of Congo Red complexed with lentinan and its acid-degraded fractions. It is found that the water-soluble fraction II (mol. wt. 3,640) gives rise to well-resolved ¹³C-n.m.r. spectra; the ¹³C-signals are assigned to (1→3)-β-d-glucan and branch points at C-6. The branched structure is also confirmed by examination of the ¹³C-n.m.r. spectra of the compounds in dimethyl sulfoxide. For the gel state of the fractions of higher molecular-weight, lentinan (mol. wt. 1,000,000) and fraction IV (mol. wt. 16,200), however, ¹³C-n.m.r. spectra of considerably attenuated signal-amplitude are observed. The fact that the ¹³C-signals of the β-d-(1→3)-linked main chain and side chains are completely suppressed is explained as a result of immobilization caused by their taking an ordered conformation. The ¹³C-resonances observed in the gel state, which are assigned to β-d-(1→6)-linkages, are unequivocally assigned to the side chains (of disordered conformation). Finally, the ordered conformation of both the β-d-(1→3)-linked main chain and side chains is identified as the single-helix conformation, which tends to form multiple helixes as junction zones for gel structure.     

The galactan sulfate from the edible,red alga Porphyra columbina

A galactan sulfate has been isolated from the seaweed Porphyra columbina, and its structure established by a combination of methylation, methanolysis, treatment with alkali followed by methylation, and ¹³C-n.m.r. spectroscopy. The polysaccharide belongs to the porphyran class, and consists of 3-linked β-d-galactosyl residues and 4-linked α-l-galactosyl residues. 3,6-Anhydro-l-galactose and l-galactose 6-sulfate residues total approximately half of the sugar units, the other half being made up of d-galactose and 6-O-methyl-d-galactose residues. Some evidence is presented that suggests that the galactan sulfate does not have a completely alternating structure.     

An arabinoglucuronomannan from suspension-cultured cells of Nicotiana tabacum

From the soluble polysaccharides of suspension-cultured, tobacco cells (Nicotiana tabacum), an arabinoglucuronomannan (AGM) was isolated from the extract with phosphate buffer. Application of partial hydrolysis with acid, carboxyl reduction, methylation analysis, and ¹H- and ¹³C-n.m.r. spectroscopy indicated AGM to consist of the tetrasaccharide repeating-unit

.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype 55

The structure of the capsular polysaccharide from Klebsiella type 55 has been investigated by using the techniques of methylation, Smith periodate oxidation, and partial, acid hydrolysis. The anomeric configurations of the glycosidic linkages were determined by performing ¹H-n.m.r. and ¹³C-n.m.r.spectroscopy on the polysaccharide and derived poly- and oligo-saccharides obtained through degradative procedures. The position of the O-acetyl group was located by devising an improved method for its replacement by a methyl ether group. The structure was shown to consist of the following tetrasaccharide repeating unit.     

Structural investigation of the capsular polysaccharide from Klebsiella serotype K-34 and its characterization by N.M.R. spectroscopy

The repeating unit of the capsular polysaccharide from Klebsiella type K-34 has been established by methylation, partial hydrolysis, and Smith degradation to consist of a hexasaccharide repeating-unit built up of four l-rhamnose, one d-glucose, and one d-galacturonic acid residues. The anomeric configurations of the linkages was determined by proton and ¹³C-n.m.r. spectroscopy at each step of the degradation procedures. Further evidence for the configurations of the glycosidic linkages involved the use of proton T₁ relaxation-times and oxidation by chromium trioxide. The data allowed assignment of the following structure for the repeating unit:     

Structural investigation of the capsular polysaccharide of klebsiella serotype K12

Klebsiella K12 capsular polysaccharide has been investigated by the techniques of methylation, Smith degradation—periodate oxidation, uronic acid degradation, and partial hydrolysis, in conjunction with ¹H-n.m.r. spectroscopy at 100 and 220 MHz, and ¹³C-n.m.r. spectroscopy at 20 MHz. The structure has been found to consist of the hexasaccharide repeating-unit shown, having a d-galactofuranosyl residue at the branch point. In this series, a d-galactofuranosyl residue has previously only been found in the polysaccharide from Klebsiella K41.

     

An improved synthesis of 1,6-anhydro-2,4-dideoxy-β-D-threo-hexopyranose

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-1299 and B-1399, and the native, structurally homogeneous dextrans from L. mesenteroides. B-640, B-1396, B-1422, and B-1424, were examined by ¹³C-n.m.r. spectroscopy at 34 and at 90°, and by g.l.c.-m.s. The ¹³C-n.m.r. data indicate that the dextrans of this series branch exclusively through α-d-(1→2)-linkages, and differ from one another only in degree of linearity. Diagnostic, ¹³C-n.m.r resonances, correlating with 2,6-di-O-substituted α-d-glucosyl residues at branch points, have chemical shifts that are independent of the degree of linearity of the dextran. The intensities of these diagnostic resonances from branching residues, compared to the resonances associated with linear dextran (low degree of branching), are generally proportional to the degree of branching established by methylation-fragmentation analysis. The validity of assignment of the diagnostic, ¹³C-n. m.r. resonances is substantiated by a critical review of methods previously used to provide structural information on dextrans having α-d-(1→2)-linkages, and by evaluation of the corresponding results on the basis of the ultimate standard-methylation structural analysis.     

Analysis of bromine-oxidised dextran by 13C-n.m.r. spectroscopy

Dextran T 10, elaborated by Leuconostoc mesenteroides NRRL B-512, was oxidised with aqueous bromine at pH 7.0. The resulting oxodextran and its methoximated derivative were analysed by ¹³C-n.m.r. spectroscopy. The total amount of keto groups and their positions were established. Assignments of the ¹³C signals were made by referring to spectra of the corresponding methyl glucosiduloses and an oxodextran having most of the carbonyl groups at position 3 of the glycopyranosyl residues. In accordance with the mechanism for bromine oxidation of mono- and di-saccharides, the glucopyranosyl residues of dextran were oxidised mainly at C-2 and C-4. Over-oxidation resulted in a small proportion of acidic, ring-cleavage products.     

Chemical and n.m.r.-spectroscopic investigation of the capsular polysaccharide of klebsiella serotype k41

The structure of the repeating unit of the capsular polysaccharide from Klebsiella type 41 has been investigated by methylation analysis of the original and the carboxyl-reduced polymer, uronic acid degradation, Smith degradation, and graded acid hydrolysis. Proton- and ¹³C-n.m.r. spectroscopy of the original polysaccharide and of the fragments obtained by various methods confirmed some structural features and allowed determination of the anomeric configuration of the glycosidic linkages. This polysaccharide is shown to have the following heptasaccharide repeating-unit:

This is the first polysaccharide antigen K of the Klebsiella series found to have seven sugar residues in its repeating unit, and to contain a galactose residue in its furanose form.     

Structural characterisation of a neutral antitumour β-d-glucan extracted with hot sodium hydroxide from cultured fruit bodies of Grifola frondosa

The structural characterisation of an antitumour β-d-glucan (grifolan-7N), obtained from the hot sodium hydroxide extract of Grifola frandosa, is described. Grifolan-7N, purified by digestion with alpha-amylase, precipitation with ethanol, and chromatography on Con A-Sepharose, gave a single and symmetrical peak on gel filtration with Sepharose CL-4B (0.2m NaOH/8m urea) and had a molecular weight of ～1,200,000. The results of methylation analysis, ¹³C-n.m.r. spectroscopy, Smith degradation, and enzymic digestion indicated grifolan-7N to be a (1→3)-linked β-d-glucan having a single β-d-glucopyranosyl group attached to position 6 of almost every third backbone unit. Grifolan-7N showed potent activity against the solid Sarcoma 180 in mice.     

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus, contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and ¹³C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β-d-galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d-galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.     

13C-N.M.R.-Spectral study of the mode of binding of Mn2+ and Gd3+ to N-acetyl-α-neuraminic acid

Natural-abundance, ¹³C-n.m.r. spectroscopy was used to study the binding of Gd³⁺ and Mn²⁺ to N-acetyl-2-O-methyl-α-neuraminic acid (2) and to methyl N-acetyl-2-O-methyl-α-neuraminate (3). The results showed that Gd³⁺ and Mn²⁺ bind in the region of the glycerol-1-yl side-chain and the 5-acetamido group of compound 3. When the α-NeuAc derivative contains a carboxylate anion, as in compound 2, multiple, metal-ion-binding sites occur, involving the head (the carboxyl end) and the tail (the glycerol-1-yl and 5-acetamido groups) of the molecule.     

Structural features of a rye-bran arabinoxylan with a low degree of branching

Two fractions (AX-1 and AX-II) of an l-arabino-d-xylan with a low degree of branching were isolated from rye bran by different extraction procedures with and without a chlorite-delignification step. The yields and compositions of AX-I and AX-II were similar, but the d.p. of AX-II was ∼100 units higher. AX-I was shown by partial hydrolysis, methylation analysis, and ¹³C-n.m.r. spectroscopy to contain a backbone of (1→4)-linked β-d-xylopyranosyl residues substituted with single α-l-arabinofuranosyl groups at position 3 of every sixth or seventh d-xylosyl residue.     

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.     

Arabinans from the roots of horsebean (Vicia faba)

Young roots from the horsebean (Vicia faba L.) show a very high content of arabinose among their constituent cell-wall carbohydrates. Two water-soluble arabinans have been isolated from the cell-wall material. Their structures have been established by chemical methods and by ¹³C-n.m.r. spectroscopy and showed to consist of an α-(1→5)-linked backbone of l-arabinofuranose residues; arabinopyranose residues are absent. The latter feature make these polysaccharides slightly different from arabinans from other origins that are characterized by their high degree of branching.     

Glycosylation by d-fructofuranose thio-orthoesters

The capsular polysaccharide from Klebsiella type K54, containing both O-formyl and O-acetyl groups, has been investigated by using the techniques of methylation analysis (by gas-liquid chromatography), periodate oxidation-Smith degradation, and both ¹H- and ¹³C-n.m.r. spectroscopy. Degradation of the native polysaccharide with a bacteriophage-induced glucosidase generated a formylated, as well as a formylated and acetylated, tetrasaccharide, whereas similar depolymerization of the deacetylated polysaccharide yielded a single tetrasaccharide; the corresponding, O-acylated octasaccharides were also isolated and characterized. These oligosaccharides, utilized in chemical and spectroscopic studies in order to determine the location of the O-acyl substituents in the repeating sequence, indicated formylation at O-4 of each lateral d-glucosyl group and acetylation at O-2 of alternate l-fucosyl residues. A new structure for the repeating unit in the polysaccharide is proposed.     

Structure of the capsular polysaccharide of Klebsiella K- type 63

Structural investigation of the capsular polysaccharide from Klebsiella K type 63 by methylation analysis, periodate oxidation, and uronic acid degradation showed the repeating unit to consist of →3)-α-D-Galp-(1→3)-α-D-GalpA-(1→3)-α-L-Fucp(1→. This structure is identical to that of Escherichia coli serotype K-42 capsular polysaccharide. The ¹H- and¹³C-n.m.r. spectra of the original and modified polysaccharide are consistent with the foregoing structure.     

Structure of a new galactomannan from the ascocarps of Cordyceps cicadae shing

A water-soluble galactomannan (C-3), [α]_D²⁰ +30°, isolated from the rod-like ascocarps of Cordyceps cicadae, was determined to be homogeneous, and the molecular weight was estimated by gel filtration to be 27,000. The polysaccharide is composed of d-mannose and d-galactose in the molar ratio of 4:3. The results of methylation analysis, Smith degradation, stepwise hydrolysis with acid, and ¹³C-n.m.r. spectroscopy indicated that the polysaccharide is of highly branched structure, and composed of α-d-(1→2)-linked and α-d-(1→6)-linked mannopyranosyl residues in the core; some of these residues are substituted at O-6 and O-2 with terminal β-d-galactofuranosyl and α-d-mannopyranosyl groups, and with short chains of β-d-(1→2)-linked d-galactofuranosyl units.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype k23

Klebsiella K23 capsular polysaccharide has been investigated by the techniques of hydrolysis, methylation, Smith degradation-periodate oxidation, and base-catalysed degradation, either on the original or the carboxyl-reduced polysaccharide. The structure was found to consist of a tetrasaccharide repeating-unit, as shown below. The anomeric configurations of the sugar residues were determined by ₁H-and ¹³C-n.m.r. spectroscopy on the original and degraded polysaccharides.