Calystegins of Physalis alkekengi var. francheti (Solanaceae). Structure determination and their glycosidase inhibitory activities

Five calystegins were extracted from the roots of Physalis alkekengi var. francheti (Solanaceae) with hot water and purified to homogeneity by the combination of a variety of ion-exchange column chromatographies. Their structures have been determined from the 1H- and 13C-NMR spectral data, and two of the compounds were identified as calystegins A3 and B2, which have been isolated from the roots of Calystegia sepium (Convolvulaceae). Two of the remaining three were found to be 1 alpha,3 alpha,4 beta-trihydroxy-nortropane and 1 alpha,2 alpha,3 alpha,4 beta-tetrahydroxy-nor-tropane and given the trivial name calystegins A5 and B3, respectively. The last calystegin was assigned as 1 alpha,2 beta,3 alpha,6 alpha-tetrahydroxy-nor-tropane, which was the same as the relative configuration proposed in the literature for calystegin B1 isolated from C. sepium. However, the 13C-NMR spectral data for the compound from C. sepium differed substantially from our results. From a personal communication with the authors of the original paper on calystegins, it was clarified that the 13C-NMR chemical shifts of calystegin B1 in the original paper had been erroneous. Since their corrected 13C-NMR data of calystegin B1 and its 1H-NMR chemical shifts in the original paper are very close to our present data, we concluded that both compounds from C. sepium and P. alkekengi are identical. Calystegin B2 has been known to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.2 micromolar) and coffee bean alpha-galactosidase (Ki = 0.86 micromolar). In this study calystegin B1 (1 alpha,2 beta,3 alpha,6 alpha-tetrahydroxynor-tropane) proved to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.9 micromolar) and bovine liver beta-galactosidase (Ki = 1.6 micromolar), but not an inhibitor of alpha-galactosidases. Calystegin A3 was found to be a weaker inhibitor compared to calystegin B2 but with the same inhibitory spectrum. Calystegin A5 a 2-deoxy derivative of calystegin B2, showed no activity against any glycosidases tested. Since calystegin B3, a 2-epimer of calystegin B2, also exhibited only a weak inhibitory activity, it was concluded that the equatorially oriented OH group at C2 is the essential feature for recognition and strong binding by the active site of glycosidases. Based on the structure/activity relationships for the five calystegins isolated from P. alkekengi var. francheti and calystegin C1 from Morus alba, we propose that the OH group at C6 of calystegin B1 or C1, in place of the beta-glycoside oxygen, is protonated by an acidic group in the active site of the beta-glycosidase.