SEPARATION AND CHARACTERIZATION OF 5 POSITIONAL ISOMERS OF TRIMALTOSYL-CYCLOMALTOHEPTAOSE (TRIMALTOSYL-BETA-CYCLODEXTRIN)

Trace amounts of trimaltosyl-cyclomaltoheptaoses (trimaltosyl-beta-cyclodextrins, trimaltosyl-beta CDs) were found in a mixture of maltosyl-cyclomaltoheptaoses (maltosyl-beta-cyclodextrins, maltosyl-beta CDs) prepared from maltose and cyclomaltoheptaose (beta-cyclodextrin, beta CD) through the reverse action of Klebsiella pneumoniae pullulanase. Five positional isomers of trimaltosyl-beta CD were isolated by highperformance liquid chromatography (HPLC) on a reversed-phase column and a graphitized carbon column. For the structural analysis of 6(1), 6(2), 6(3)-, 6(1), 6(2), 6(5)-, and 6(1), 6(3), 6(5)-tri-O-maltosyl-beta CDs, an enzymic method using glucoamylolysis, followed by hydrolysis with Bacillus subtilis saccharifying alpha-amylase, was applied. Although 6(1), 6(2), 6(4)- and 6(1), 6(2), 6(6)-substituted isomers were indistinguishable by this method, these isomers were distinguished clearly by digestion of branched oligosaccharides produced from each isomer by the aforesaid method, with B. stearothermophilus neopullulanase or with glucoamylase. The resulting hydrolysates were analyzed by HPLC on an amino derivatized column and by fast-atom bombardment spectrometry (FABMS). The chromatographic behavior and spectral data (C-13 NMR and FABMS) of five positional isomers of trimaltosyl-beta CD are described.