title: Synthesis of a putative substrate for malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase and development of an HPLC method for the quantification of the enzyme reaction.
abstract: 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. 3beta-benzoyloxy-5beta-pregnane-14beta,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 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and its 21-O-malonylated derivative, 3beta-benzoyloxy-5beta-pregnane-14beta-hydroxy-20-one 21-O-malonylhemiester. The method was validated.
authors.affiliation: Department of Biology, Friedrich-Alexander University of Erlangen-Nuremberg, Staudtstr. 5, D-91058 Erlangen, Germany.
authors.forename: Serge Philibert
authors.initials: SP
authors.lastname: Kuate
authors.forename: Rodrigo M
authors.initials: RM
authors.lastname: Pádua
authors.forename: Hervé Martial P
authors.initials: HM
authors.lastname: Poumale
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kreis
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D000217:Acyltransferases; C527691:malonyl-coenzyme A-21-hydroxypregnane 21-O-malonyltransferase