Many types of glycosides are widely distributed as major constituents in medicinal plants. However, due to their hydrophilic properties, they are poorly absorbed from gastro intestinal tract into body fluid, but their aglycones formed by intestinal bacterial hydrolysis are readily absorbed. Therefore, intestinal bacteria play an important role in increasing absorption and efficacy of drugs including glycosides. In nature, there is a special type of glycoside, C-glycosides, such as those of flavone, isoflavone, chromone, xanthone, anthrone, and gallic acid. Since C-1 of the sugar ring is directly connected to the aglycone by C-C bonding, C-glycosides are usually resistant against acidic and enzymatic hydrolysis in contrast with the corresponding O-glycosides. Thus, with the prospect of improving the stability, various C-glycoside analogues mimicking bioactive but labile O-glycosides have been prepared for developing the new drugs.2) In addition, a lot of publications describing in detail about O-glycosidase appeared, but that of C-glycosidase did not.On the other aspects, in the last two decades, C-glucosyl bond-cleaving reactions by human intestinal bacteria were reported. 1,[3][4][5][6][7][8][9][10][11] Despite of the growing importance of C-glycosides in the field of pharmaceuticals, characteristic features of C-glucosyl bond-cleaving enzymes involved in its metabolism were still remain to be unclear.Recently, we isolated a new bacterial species named strain PUE that transforms a C-glucoside puerarin to its aglycone daidzein.1) The aglycone moiety is normally detected easily because of UV active, however, the liberated sugar segment from C-glucoside has not been yet identified. The structural elucidation of the liberated sugar moiety could yield a valuable clue in revealing the reaction mechanism (Fig. 1). In the present paper, we describe the structural elucidation of the sugar moiety after cleavage of a C-glucosyl bond of puerarin by anaerobic incubation with a human intestinal bacterium, strain PUE, and a cell-free extract.
Results
Synthesis of [6؆,6؆-D 2 ]puerarin (8)Since puerarin (1) has only one primary alcohol at C-6 of the sugar moiety, we targeted it for labeling with deuterium. First, 1 was treated with benzaldehyde and zinc chloride to give 2. Other hydroxyl groups of 2 were acetylated with acetic anhydride in pyridine to obtain 3. Benzylidene acetal of 3 was hydrolyzed under an acidic condition to afford 4. The primary hydroxyl of 4 was oxidized to carboxylic acid with a Jones reagent to give 5. The acetyl groups of 5 were removed by alkali to give 6, then, the carboxyl group was esterified to yield 7 by a usual method. We failed the reduction of 7 to the corresponding alcohol with LiAlD 4 due to decomposition, but 8 was prepared from 7 by slow addition of CD 3 OD into the reaction mixture containing NaBD 4 . 12) In this reaction, the addition of CH 3 OH instead of CD 3 OD gave a mixture of 8 and [6Љ-H,6Љ-D]puerarin (ca. 1 : 1 ratio), because hydrogen atoms originated from CH 3 OH were also incorp...
