Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Abstract

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation-the Phase II conjugation reaction-of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3'-phosphoadenosine-5'-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.

Sulfated Metabolites of Flavonolignans and 2,3-Dehydroflavonolignans: Preparation and Properties

“…In accordance with our previous study, efficient sulfation was only found for silybin B (up to 48%, Table 1) with the formation of silybin B 20- O -sulfate [18]. When silychristin or silydianin were tested as substrates in this AST IV catalyzed reaction, both consumption of the sulfate donor p -nitrophenyl sulfate ( p -NPS) and formation of p -nitrophenol ( p -NP) were observed (Table 1), which clearly proved the sulfation reactions.…”

“…packed and eluted with 80% methanol. Only one sulfated product was obtained; silybin B 20- O -sulfate (14.5 mg, 0.03 mmol, yield 25%), whose identity was confirmed by comparison with previously published data [18]. …”

“…Silybin (natural mixture of diastereomers A and B), silychristin, and silydianin were sulfated using whole E. coli cells transfected with and overexpressing the gene for AST IV [18]. In accordance with our previous study, efficient sulfation was only found for silybin B (up to 48%, Table 1) with the formation of silybin B 20- O -sulfate [18].…”

“…Recently reported sulfates of silybins A and B and of isosilybins A and B were obtained using bacterial aryl sulfotransferase from Desulfitobacterium hafniense (AST DH), which selectively catalyzed C-20 OH sulfation of the parent compounds [17]. On the other hand, aryl sulfotransferase IV from rat liver (AST IV) heterologously expressed in E. coli , used for the same reactions, exhibited a strict stereoselective sulfating reaction of silybin isomers; silybin B was sulfated yielding silybin B 20- O -sulfate, whereas silybin A proved to be completely resistant to the sulfating reaction [18]. In the present work, we focused on the sulfation of six flavonolignans of silymarin, namely silybin A, silybin B, 2,3-dehydrosilybin, silychristin, 2,3-dehydrosilychristin, and silydianin.…”

“…The enzyme was expressed in E. coli BL21 (DE3) Gold as described [18], with modifications reported in our previous work [19]. Whole E. coli cells producing the enzyme, freshly prepared according to the literature [19], were employed in the biotransformation experiments as a catalyst.…”