Does the malonyl-coenzyme A:21-hydroxypregnane 21-hydroxymalonyltransferase catalyze the first step in the formation of the butenolide ring of cardenolides?

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

“…In an earlier study products of malonyl-coA: 21-hydroxypregnane 21-O-malonyltransferase action were identified by GC-MS and their spectra compared to those of pregnane acetyl esters as the malonylhemiesters were decarboxylated to the corresponding acetyl esters at the high temperatures used in GC analysis [15]. Alternative methods identifying the putative substrates and products, such as HPLC and TLC, were also flawed.…”

“…The peaks at m/z 357 and 339 correspond to the loss of two molecules of water, which confirms the presence of two hydroxy groups in compound 3. The 1 H NMR data (Table 1) (7), 162 (14), 147 (15), 124 (11), 111 (17), 85 (14).…”

“…Gas chromatography has also been used, but due to the decarboxylation of the product 3␤-acetoxy-5␤-pregnane-14␤-hydroxy-20-on-21-malonylhemiester (2) at high temperatures, only the acetylated derivative (7) is identified ( Fig. 1) [15]. The synthesis of a new putative substrate, incorporating a chromophor, and its corresponding reaction product, allowed the development of a simple and reliable HPLC method.…”

Spontaneous butenolide ring formation of pregnane-21-O-malonyl hemiesters under mild reaction conditions is facilitated by the 14β-hydroxy group present in all natural cardenolides

“…Subsequent decarboxylation and dehydration then yields the butenolide ring. 5 Actually, spontaneous butenolide ring formation from pregnane 21-O-malonyl hemiesters was observed after heating or storage at room temperature for an Abbreviations: APCI, Atmosphere pressure chemical ionization; PPB, Potassium phosphate buffer; SCRF, Self-Consistent Reaction Field; DTF, Density functional theory. extended period of time.…”

“…extended period of time. 5 Butenolide ring forming enzymes ('butenolide cyclases') using pregnane 21-O-malonic hemiesters or norcholenoic acids as substrates have not yet been described. However, a malonyltransferase, termed malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) has been purified from D. purpurea.…”