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The exact positions of the acetyl groups in the sugar moieties of the glycosidic antibiotics, the glykenin (GK) components, were elucidated using MS/MS technique under high energy collision conditions, because the frit-FAB LC/MS technique that was described in a preceding paper and other techniques were insufficient for the purpose. In the negative product ion spectra of (M-H)-, a charge-remote fragmentation based on a terminal carboxylic acid of the aglycone was observed, whereas many cleavage ions were observed in the positive product ion spectra of (M+Li)+. The diagnostic ions were selected from the cleavage ions of two bonds within the glycosidic ring in the product ion spectra of the (M+Li)+ and (M-H)-. The positions of the acetyl groups for each component could be determined by the presence or absence of these diagnostic ions, which enabled us to determine the structure of each of the GK components.

Three new glycosidic antibiotics, glykenin (GK)-III A, B, and C (2a-c), were isolated as a mixture from a strain of Basidiomycetes sp. and identified as glycosides of C26-fatty acids and a diacetylated trisaccharide composed of glucose and two xyloses. The locations of the two acetyl groups were elucidated by secondary ion mass spectrometry (SIMS), double quantum filter (DQF), and relayed chemical shift correlation spectroscopy (COSY) spectral analysis.

The structures of three aglycones of glykenins, produced by Basidiomycetes sp., were determined to be (2S, 16R, 17S, 21R)-2, 16, 17, 21-, (2S, 17R, 18S, 22R)-2, 17, 18, 22-, and (2S, 17S, 18S, 22R)-2, 17, 18, 22-tetrahydroxyhexacosanoic acids (3a-c). The absolute configurations of two of the four hydroxy groups in 3a-c were established by chiral synthesis of the degradation products (6a-c and 7a-c). Chemical transformation of 3a-c to 6, 8-dioxabicyclo[3.2.1]octane derivatives (18-c) revealed the relative and absolute configurations of the acyclic 1, 2-diol moieties in 3a-c.

The glycosidic antibiotics of the glykenin (GK) family produced by Basidiomycetes sp. were separated into nine components (GK-I-VII and DG) by normal-phase chromatography. It was found that these components differ in the number and location of the acetyl groups in the sugar moiety. Each component (GK-I-VII and DG) was further separated into three isomers (A, B and C), which possess different aglycones, by reversed-phase chromatography on an ODS column with methanol-acetonitrile as eluent. The best composition of the eluent was found to be methanol-acetonitrile-1% trifluoroacetic acid (4:3.5:2.5). The profile analysis of GK-III-VII and DG was also carried out using a modified mobile phase. The combination of normal- and reversed-phase chromatography separated all components of the GK mixture except GK-I and II. The relationship between structure and separation behaviour of GK is discussed.