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

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.

Acknowledgments: We are grateful to the German Academic Exchange Service (DAAD) for the doctoral fellowship to R.M.P.

References: [1] Wasserstrom, AJ. et al. (2005) Am. J. Physiol. Heart Circ. Physiol. 289: 1781–1793. [2] Kreis, W. et al. (1998) Planta Med. 64: 491–499. [3] Stuhlemmer U. et al. (1996) Tetrahedron Lett. 37: 2221–2224.