Synthesis of a putative substrate for malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase, an enzyme involved in cardenolide formation, and development of an HPLC method for the quantification of its malonylated derivative

The butenolide ring is the main common characteristic of all cardenolides. Its formation is supposed to be initiated by the transfer of a malonyl moiety from malonyl-coenzyme A to the 21-hydroxypregnane [1, 2]. Since all methods (HPLC, TLC, GC) tried so far for the determination of malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase activity had their weak points, a reliable, fast and sensitive method had to be developed. A surrogate substrate was synthesized (Fig.1) containing a side chain resembling the sugar side chain attached to C-3 of putative cardenolide precursors and a chromophor allowing UV/HPLC detection. We synthesized 3β-benzoyloxy-5β-pregnane-14β, 21-dihydroxy-20-one and its 21-O-malonylated derivative, the latter being the expected product of the enzyme reaction. The new substrate was well accepted by the enzyme. An UV/HPLC method has been established for the detection and quantification of 3β-benzoyloxy-5β-pregnane-14β, 21-dihydroxy-20-one and its 21-O-malonylated derivative, 3β-benzoyloxy-5β-pregnane-14β-hydroxy-20-one 21-O-malonyl hemiester, the product of enzyme action. The method was validated.

Acknowledgments: German Academic Exchange Service (DAAD) for the PhD Grant awarded to SPK.

References: [1] Stuhlemmer, U. and Kreis, W. (1996) Tetrahedron Lett., 37: 2221–2224. [2] Kreis, W. et al. (1998) Planta Med. 64: 491–499.

Fig.1: Main compounds involved in the preparation of 3β-benzoyloxy-5β-pregnan-14β-hydroxy-20-on-21-malonylhemiester (4) from digitoxigenin (1). Benzoyldigitoxigenin (2), 3β-benzoyloxy-5β-pregnan-14β, 21-dihydroxy-20-one (3).