Butenolide ring formation in cardenolide biosynthesis: Spontaneous cyclization of pregnane-21-O-malonyl hemiesters

Abstract

Cardiac glycosides are valuable drugs in the medication of patients suffering from cardiac insufficiency [1]. Although features of the biosynthetic pathway of cardenolides have been elucidated with the use of labelled and unlabelled precursors, its biosynthesis still not fully understood [2]. The incubation of a 21-hydroxy-20-oxo-pregnane precursor with malonyl-coenzyme A in the presence of cell-free extracts from cardenolide-producing plants leads to the formation of its 21-O-malonyl hemiester [3]. To investigate the next step in cardenolide biosynthesis, namely the butenolide ring formation, it is necessary to have mg quantities of the respective substrates. We developed a simple, fast and efficient chemical method for the synthesis of 21-hydroxypregnane-21-O-malonyl hemiesters and applied 1D and 2D NMR techniques for their complete 1H and 13C assignments. The incubation of 21-hydroxypregnane-21-O-malonyl hemiesters in the presence of cell-free extracts obtained from cardenolide-producing plants leads to the hydrolysis of the 21-O-ester linkage of the substrate. On the other hand, spontaneous butenolide ring formation of pregnane 21-O-malonyl hemiester was observed in buffer (100 mM KPi buffer, pH 7.5) and in the mixture of buffer + DMSO at 60°C. The reaction was dependent on temperature and substrate concentration. Butenolide ring formation from the 21-hydroxypregnane-21-O-malonyl hemiester with the hydroxyl group at the position C-14β (typical of cardenolide aglycone) was higher than from compounds without this particular group. Although only spontaneous butenolide ring formation has been observed so far, the presence of a butenolide ring cyclase in cardenolide containing-plants can not be ruled out.