Toshiko Utamura

Analyses of homogeneous d-gluco-oligosaccharides and -polysaccharides (degree of polymerization up to about 35) by high-performance liquid chromatography and thin-layer chromatography

Abstract Conditions for the separation of (1→2)-, (1→3)-, (1→4)- and (1→6)-linked homogeneous d -gluco-oligosaccharides and -polysaccharides were investigated by high-performance liquid chromatography on a 3-μm chemically modified amine column (ERC-NH-1171) and by thin-layer chromatography (TLC) on three kinds of silica gel plates. Saccharide samples were prepared by partial hydrolyses or partial acetolyses of cyclosophoraose [cyclic (1→2)-β- d -glucan], curdlan [(1→3)-β- d -glucan], amylose [(1→4)-α- d -glucan], cellulose [(1→4)-β- d -glucan], dextran [(1→6)-α- d -glucan], and luteose [(1→6)-β- d -glucan]. Each series of saccharides, other than β- d -(1→3)- and β- d -(1→4)-linked ones, whose soluble higher oligomers could not be obtained, was well resolved from glucose to the polymer having a degree of polymerization (DP) of about 35 on the amine column by using simple isocratic elution with acetonitrile—water. TLC analyses were performed on silica gel 70 and silica gel 60 TLC plates with a concentration zone using n -butanol—ethanol—water as the developing solvent, and on Si 50000 HPTLC plates using n -butanol—pyridine—water. These methods made it possible to separate simultaneously each series of homogeneous saccharides of DP up to 20–30.

Isolation and characterization of branched cyclodextrins

Abstract Three branched cyclodextrins (CDs) were isolated by high-performance liquid chromatography (l.c.) from the mother liquors of a large-scale preparation of the unbranched CDs with Bacillus ohbensis cyclomaltodextrin glucanotransferase. Evidence from chromatographic behavior on three l.c. columns of different separation modes, fragmentation analysis, 13C-n.m.r. spectroscopy, methylation analysis, and fast-atom bombardment-mass spectrometry (f.a.b.—m.s.) indicated that these compounds were 6-O-α- d -glucopyranosylcyclomaltohexaose (1), 6-O-α- d -glucopyranosylcyclomaltohepataose (2), and 6,6‴-di-O-α- d -glucopyranosylcyclomaltoheptaose (3).

Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase

Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG6 less than cG7 less than cG8). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-alpha-maltotriosylmaltotriose (branched G6) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G6 was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and 13C-n.m.r. spectroscopic analyses.

Analysis of heptakis(2,6-di-O-methyl)-β-cyclodextrin by thin-layer, high-performance liquid and gas chromatography and mass spectrometry

Abstract Methods for the analysis and isolation of heptakis(2,6-di-O-methyl)-β-cyclodextrin (CD) by thin-layer and high-performance liquid chromatography (HPLC) were investigated. By using these methods a commercial and two synthetic samples of heptakis(2,6-di-O-methyl)-β-CD were analysed and it was found that they contained two major and at least four minor components, of which the two major and one minor components were isolated by semi-preparative HPLC. Careful fragmentation analyses, which consisted of successive hydrolysis, reduction, acetylation and characterization of the partially methylated d -glucitol peracetates by gas chromatography—mass spectrometry and determination of molecular weights by fast-atom bombardment mass spectrometry indicated that these compounds were heptakis(2,6-di-O-methyl)-β-CD, hexakis(2,6-di-O-methyl)-mono(2,3,6-tri-O-methyl)-β-CD and pentakis(2,6-di-O-methyl)-bis(2,3,6-tri-O-methyl)-β-CD.

Separation of cyclic (1→2)-β-D-glucans (cyclosophoraoses) produced by agrobacterium and rhizobium, and determination of their degree of polymerization by high-performance liquid chromatography

Conditions for the separation of the eight components contained in cyclic (1→2)-β-D-glucans (cyclosophoraoses) from Agrobacterium and Rhizobium were investigated by high-performance liquid chromatography on a chemically modified amine column (μBondapak CH) with mixtures of acetonitrile and water as eluent, and on a reversed-phase column (Dextro-Pak cartridge) with methanol—water (4:96) as eluent. Although retention on the amine column was related to molecular mass, that on the reversed-phase column differed from it, and was probably related to the solubility in water. The degree of polymerization of each cyclosophoraose was determined by high-performance liquid chromatography of its partial hydrolysate. Sophoro-oligomers having degrees of polymerization up to 24 were well resolved on a different amine column (Finepak SIL NH2, 10 μm) within 27 min by using a simple isocratic elution of acetonitrile-water (55:45). On hydrolysis under the appropriate conditions the longest straight chain sophoro-oligomer was present in sufficient amount to be able to recognize it in the partial hydrolysate; the distinguishing last peaks in the chromatograms of the eight cyclosophoraose hydrolysates could be detected clearly as the 17th–24th peaks.

Structural studies on cyclic (1→2)-β-d-glucans (cyclosophoraoses) produced by Agrobacterium and Rhizobium☆

Abstract Eight cyclic (1→2)-β- d -glucans (cyclosophoraoses) of different molecular weights were isolated from culture filtrates of Agrobacterium and Rhizobium . By 6.2-MPa, liquid chromatography of their partial hydrolyzates, it was concluded that they were cyclosophoroheptadecaose, cyclosophoro-octadecaose, cyclosophorononadecaose, cyclosophoroeicosaose, cyclosophoroheneicosaose, cyclosophorodocosaose, cyclosophorotricosaose, and cyclosophorotetracosaose. Cyclic (1→2)-β- d -glucans from 19 strains of Agrobacterium and Rhizobium tested were divided into four classes on the basis of differences in the distribution patterns of the eight cyclosophoraoses.

Further studies on the separation of cyclic (1→2)-β-d-glucans (cyclosophoraoses) produced by rhizobium meliloti ifo 13336, and determination of their degrees of polymerization by high-performance liquid chromatography

Abstract Eight pure cyclic (1→2)-β- d -glucans (cyclosophoraoses) varying in size from 17 to 24 residues were previously isolated from culture filtrates of Agrobacterium and Rhizobium. Thereafter further studies on the separation of cyclosophoraoses by high-performance liquid chromatography (HPLC) with small-particle columns showed an occurrence of many cyclosophoraoses having degrees of polymerization (DPs) of more than 24. One sample prepared from Rhizobium meliloti IFO 13336 contained large amounts of higher cyclosophoraoses of up to at least DP 40, which were separated clearly on a 3-μm chemically modified amine column (ERC-NH-1171). Some pure cyclosophoraoses with higher DPs were isolated by liquid chromatography using reversed-phase columns, and their DPs were determined by HPLC of their partial hydrolysates.

Retention behaviour of cyclodextrins and branched cyclodextrins on reversed-phase columns in high-performance liquid chromatography

Abstract The theoretical plate numbers and the asymmetry factors of eight columns packed with C18-bonded phases were measured to obtain a reliable indication of column performance and for column-to-column comparisons. Among those columns, including silicone-coated silica gel- and porous polymer gel-based new C18 columns, silica-based, endcapped and monomeric phase columns showed the best performance. Cyclodextrins (CDs) and branched CDs were separated satisfactorily on six C18 columns with 3–7% aqueous methanol. Furthermore, multi-branched CDs could be separated from their isomers having the same molecular size. The elution order of CDs and branched CDs on C18 columns with aqueous methanol may or may not coincide with their solubilities in the mobile phases.

Enzymic syntheses of doubly branched cyclomaltoheptaoses through the reverse action of Pseudomonas isoamylase

Abstract Two and three new cyclomaltoheptaose (β-cyclodextrin, cG 7 ) derivatives, respectively, were identified among the products obtained by the action of Pseudomonas isoamylase on maltose and maltotriose, and cG 7 . They were 6 A ,6 D -di- O -α-maltosyl-cG 7 and 6- O -α-(6 2 - O -α-maltosyl)maltosyl-cG 7 , and 6 A ,6 D -di- O -α-maltotriosyl-cG 7 , 6- O -α-(6 3 - O -α-maltotriosyl)maltotriosyl-cG 7 , and 6- O -α-(6 2 - O -α-maltotriosyl)maltotriosyl-cG 7 . In addition, 6 1 - and 6 2 - O -α-maltosylmaltose were identified as mutual condensation products of maltose. Maltose was the smallest substrate to act as both an acceptor and a donor for the action of Pseudomonas isoamylase.

Preparation of cyclosophoraose-A and its complex-forming ability

Cyclosophoraoses (CySs) are unbranched cyclic (1→2)-β-D-glu-cans produced by many strains of Agrobacterium and Rhizobium. Pure CyS-A, the group member having the smallest molecular size (degree of polymerization 17), was efficiently prepared by liquid chromatography using charcoal and ODS columns from the culture fluid of the mutant strain RA-12 from R. phaseoli AHU 1133. The complex-forming ability of CyS-A was estimated from its enhancement of the solubilities of slightly soluble guest molecules in water using methods [I], [II], and [III]. In [I], an aqueous solution of CyS-A was shaken with the guest molecule, while, in [II], it was shaken with an acetone solution of the guest compound. In method [III], freeze-dried CyS-A powder was stirred with an acetone solution of the guest compound. The CyS-A cavity is thought to be able to accommodate three-dimensionally extended guest molecules, e.g., indomethacin. Method [II] was the best for obtaining CyS-A inclusion complexes, while method [III] would be recommended if the guest molecule is labile in the presence of water. Crystalline CyS-A inclusion complexes have not been obtained, but CyS-A complexes are expected to greatly enhance the solubilities of slightly soluble or insoluble guest molecules in water, because CyS-A is much more soluble than β-cyclodextrin. Method [II] or [III] may afford a useful means of obtaining oily drug, e.g., vitamin E and K1, in an amorphous state.