Effective In Vivo One-pot Multienzymes Synthesis of trans-Androsterone-glycosides

Meng-Ru Wang (王孟儒), Prof. Tung-Kung Wu

Department of Biological Science and Technology, National Yang Ming Chiao Tung University

Steryl-glycosides are amphiphilic compounds consisting of a hydrophobic steroidal backbone and one or more hydrophilic sugar moieties. They have a variety of biological activities in anti-bacterial, anti-tumor, and immune response regulation, making them to have great potential in carbohydrate-based drugs. Glycosylation can improve the water solubility and stability of sterols and triterpenoids, further enhancing their bioavailability and activity. Glycosyltransferases (GTs) are common glycosylated enzymes that transfer glycosyl donors to a variety of specific acceptors and form glycosidic bonds. For the traditional synthesis of steryl-glycosides, the use multiple enzymes, including sugar kinase, nucleotidyltransferase, and glycosyltransferase in stepwise reactions is a time-consuming approach. The one-pot multi-enzyme system we have constructed can co-express four enzymes, including N-acetylhexosamine 1-kinase (NahK), inorganic pyrophosphatase (PmPpA), Bifidobacterium longum UDP‐Sugar pyrophosphorylase (BLUSP) and glycosyltransferase HP0421/Bs-YjiC in E. coli BL21(DE3). Previously, 7β-hydroxy-epiandrosterone, an androgenic derivative of trans-Androsterone (tAND), showed anti-estrogenic effects on breast cancer, therefore, tAND may have anti-breast cancer effects. In this study, two kinds of monosaccharides (glucose and 2-deoxyglucose) were selected as sugar donors, and tAND was used as a sugar acceptor to synthesize various tAND-glycosides through a one-pot reaction in vivo. The synthesized tAND-glycosides were purified by silica gel column chromatography and HPLC, and their molecular weights and structures were further analyzed by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (NMR). The synthesized compounds will be further tested for their anti-tumor cell activity.