Toshiko Tanimoto

High-performance anion-exchange chromatography of homogeneous D-gluco-oligosaccharides and -polysaccharides (polymerization degree >50) with pulsed amperometric detection

Abstract High-performance anion-exchange chromatography under alkaline conditions with pulsed amperometric detection was applied to the analyses of (1 → 2)-, (1 → 3)-, (1 → 4)- and (1 → 6)-linked homogeneous α- or β- d -gluco-oligosaccharides and -polysaccharides up to a degree of polymerization (DP) of ⩾ 50. Each series of homogeneous d -gluco-oligomers and -polymers showed a linear relationship between log k′ and DP in isocratic elution using 150 mM sodium hydroxide solution containing 100 mM sodium acetate as the eluent. An effective separation of individual members of an homologous series of linear glucans was achieved using gradient elution, accomplished by maintaining the sodium hydroxide concentration at 150 mM and increasing the sodium acetate concentration during the analysis. The detector response per HCOH group in d -gluco-oligomers (DP 2–7) was almost the same.

Isolation and characterization of novel heterogeneous branched cyclomalto-oligosaccharides (cyclodextrins) produced by transgalactosylation with α-galactosidase from coffee bean

Transgalactosylated derivatives of cyclomalto-hexaose (alpha CD), -heptaose (beta CD), and -octaose (gamma CD) were synthesized by alpha-galactosidase from coffee bean using melibiose as a donor and alpha CD, beta CD or gamma CD as an acceptor. Mono- and di-O-alpha-D- galactosylated CDs were isolated and purified by HPLC. Their structures were elucidated by fast-atom bombardment mass spectrometry (FABMS) and 13C NMR spectroscopy. For structural determination of positional isomers of 6(1),6n-di-O-alpha-D-galactosyl-CDs, digestion products with cyclodextrin glucanotransferase were analyzed by HPLC and FABMS.

Determination of cyclic glucans by anion-exchange chromatography with pulsed amperometric detection

Abstract Anion-exchange chromatography with pulsed amperometric detection was applied to the determination of cyclodextrins ( CD s), branched CD s and cyclosophoraoses. These cyclic glucans with degree of polymerization 6–25 were well resolved in each series by using simple isocratic elution with 150 m M sodium hydroxide solution containing 140–200 m M sodium acetate. The separation mode was not only simple anion exchange, but also involved some hydrophobic interactions and, moreover, inclusion interactions also seemed to take part in the retention. The detector response per glucose unit of these cyclic glucans was almost the same, regardless of the molecular weight and linkage form. The limit of determination of the cyclic glucans was 5–10 pmol and the detection limit was 2.5–5 pmol with a signal-to-noise ratio of 3.

Isolation and characterization of heptakis(2,6-di-O-methyl)cyclomaltoheptaose and over-methylated homologues

Abstract From a mixture of methylation products of cyclomaltoheptaose (β-cyclodextrin, βCD) containing heptakis(2,6-di- O -methyl)-βCD ( 1 ) and its over-methylated homologues, two major components [ 1 and hexakis(2,6-di- O -methyl)-(2,3,6-tri- O -methyl)-βCD ( 2 )] and fourteen minor components were isolated by h.p.l.c. The solubilities and inclusion behaviour of 1 and a commercial DM-βCD (a mixture containing 1 and 2 ) were compared. A 3- O -methyl group had little effect on the inclusion behaviour of 2,6-di- O -methyl-βCD. Fourteen minor over-methylated homologues were characterized.

Preparation, isolation, and characterization of novel heterogeneous branched cyclomalto-oligosaccharides having β-d-galactosyl residue(s) on the side chain

Transgalactosylated products of branched cyclodextrins (glucosyl-alpha CD, -beta CD, -gamma CD, and maltosyl-alpha CD, -beta CD, -gamma CD) were synthesized by beta-D-galactosidases from Bacillus circulans and Penicillium multicolor using lactose as a donor substrate and branched CDs as acceptors. Eighteen beta-D-galactosylated branched CDs were isolated and purified by HPLC. Their structures were elucidated by FABMS and 13C NMR spectroscopies, and methylation analysis. The chromatographic behavior of these novel heterogeneous branched CDs on three HPLC columns of different separation modes was compared.

Analyses of isomeric mono-O-methyl-d-glucoses, d-glucobioses and d-glucose monophosphates by high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract To investigate the contribution of each hydroxyl group of d -glucose to retention on pellicular quaternary amine-bonded resins and to the pulsed amperometric detector response, all sets of isomeric mono-O-methyl- d -glucoses and d -glucobioses and three isomeric d -glucose monophosphates were analysed by high-performance anion-exchange chromatography under alkaline conditions with pulsed amperometric detection. The results showed that the reduction of retention time on mono-O-methylation follows the order 2-OH > 3-OH > 6-OH > /4-OH > -O Although the elution order of 1-, 3- and 6-phosphates was the same as obtained for the corresponding methyl esters, in the case of glucobioses the elution position was somewhat affected by the configurational contribution and hydrophobic interaction. The pulsed amperometric detector response was little affected by the acidity of each hydroxyl group of d -glucose. The suppression of the pulsed amperometric detector response by substitution of hydroxyl group seemed to be due to the inhibitory effect of the substituent group on the vicinal hydroxyl group and, as a rule, the degree of suppression caused by 6-O-substitution was the smallest.

Preparation and reactivity of a novel disaccharide, glucosyl 1,5-anhydro-d-fructose (1,5-anhydro-3-O-α-glucopyranosyl-d-fructose)

A novel disaccharide, glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-alpha-glucopyranosyl-D-fructose, GAF) was enzymatically prepared from 1,5-anhydro-D-fructose (1,5-AF) and cyclomaltoheptaose (beta-cyclodextrin). Cyclodextrin glucanotransferase transferred various sizes of maltooligosaccharide to 1,5-AF. Glucoamylase digested the maltooligosyl chain of the products to a glucosyl residue giving a final product, GAF. An NMR analysis of GAF elucidated that the glucose residue was linked to C-3 of the 1,5-AF residue with an ether linkage. Reactivity on the aminocarbonyl reaction of GAF with bovine serum albumin was lower than that of 1,5-AF, but was higher than that of glucose.

Separation of basic drug enantiomers by capillary electrophoresis using methylated glucuronyl glucosyl β-cyclodextrin as a chiral selector

Separations of basic drug enantiomers by capillary electrophoresis have been investigated using methylated glucuronyl glucosyl β-cyclodextrin (Me GUG β-CD) as the chiral selector in the background electrolyte. Methylation was performed by reaction with methyl iodide, barium oxide, and barium hydroxide in dimethylformamide, varying the reaction temperature and time. The resulting Me GUG β-CD derivatives differed in their degree of substitution and were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectra. Among the Me GUG β-CD derivatives prepared, the one with a degree of substitution of 2.8 showed the highest resolution abilities for basic drug enantiomers. Chiral resolution of 16 basic drugs was attained using the Me GUG β-CD derivative (10 mM solution in 40 mM sodium phosphate buffer at pH 3.5). The chiral recognition abilities of Me GUG β-CD were compared with those of GUG β-CD. Me GUG β-CD showed higher resolution for bupivacaine, clorprenaline, isoprenaline, pindolol, salbutamol, and terbutaline than GUG β-CD, while GUG β-CD showed higher resolution for atenolol, chlorpheniramine, dimethindene, homochlorcyclizine, ketamine, piperoxan, promethazine, propranolol, trimetoquinol, and verapamil than Me GUG β-CD. Thus, Me GUG β-CD and GUG β-CD can be complementarily used for the resolution of basic drug enantiomers.

Characterization of five isomers of branched cyclomaltoheptaose (β CD) having degree of polymerization (d.p.) = 9: Reinvestigation of three positional isomers of diglucosyl-β CD

It has been confirmed by methylation analyses and chemical syntheses that three isomers of branched cyclomaltoheptaose (beta CD) isolated from the mother liquors of a large-scale preparation of beta CD with Bacillus ohbensis cyclomaltodextrin glucanotransferase are 6(1),6(4)-di-O-(alpha-D-glucopyranosyl)-cyclomaltoheptaose (1), 6(1),6(3)-di-O-(alpha-D-glucopyranosyl)-cyclomaltoheptaose (2), and 6-O-(alpha-isomaltosyl)-cyclomaltoheptaose (4) instead of 6(1),6(2)-di-O-(alpha-D-glucopyranosyl)-cyclomaltoheptaose (3), which was erroneously characterized in an earlier paper. Compound 3 has been newly isolated from a glucosyl-beta CD mixture prepared by hydrolysis with glucoamylase of a maltosyl-beta CD mixture, synthesized from maltose and beta CD through the reverse action of pullulanase. Chromatographic behavior and spectral data (13C-n.m.r. and f.a.b.-m.s.) of these isomers of branched beta CD (1-4), as well as those of another isomer prepared by the reverse action of hydrolytic enzymes, 6-O-(alpha-maltosyl)-cyclomaltoheptaose (5), were compared.

Preparation of 61,62-, 61,63-, 61,64-, and 61,65-di-O-(α-d-glucopyranosyl) cyclomalto-octaoses

Abstract Four positional isomers of 6 1 ,6 n - di -O-( d - glucopyranosyl)cyclomalto-octaoses (cG 8 s) ( n = 2, 3, 4, and 5) were chemically synthesized by using the authentic compounds, 6 1 ,6 n -di- O -trityl-cG 8 s or 6 1 ,6 n -di- O -( tert -butyldimethylsilyl)-cG 8 s ( n = 2−5) as the key glucosyl intermediates, and the glucosyl donor, 2,3,4,6- tetra -O- benzyl -α- d - glucopyranosyl trichloroacetimidate.