Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1215676 | Journal of Chromatography B | 2007 | 7 Pages |
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 an appropriate 21-hydroxypregnane. A new, reliable, fast and sensitive method to determine malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase activity had to be developed since previous attempts employing HPLC, TLC or GC did not prove successful. A surrogate substrate was synthesized containing a side chain resembling the sugar side chain attached to C-3 of putative cardenolide precursors and containing a chromophor allowing UV detection. 3β-benzoyloxy-5β-pregnane-14β,21-dihydroxy-20-one and its 21-O-malonylated derivative were synthesized, the latter being the expected product of the enzyme reaction. The new substrate was well accepted by the enzyme. An HPLC method has been established to detect and quantify 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-malonylhemiester. The method was validated.