Radix Scutellariae In The Glycosyl Transferase Gene Clone, Prokaryotic Express And Enzyme Catalysis Research

The natural flavonoids are a large group of natural products with a variety of biologicalactivities and pharmacological effects. These compounds are of significant benefits to human’shealth. These flavonoids have been used to treat cardiovascular diseases, and also possessantioxidant and anticancer activities. However, the water-solubility of those compounds can notmeet the needs of drug development. Some of researches have been carried out to solve thisproblem. Among them, glycosylation reactions are selected for enhancing their solubility andstability.Because flavonoids contain more than one phenolic hydroxyl group, so the chemicalmethods of glycosylations often suffer from poor yields and side reaction. As a result, thereactions require one or more deprotection steps to liberate free glucoside. Enzymatic methods ofsynthesis employ uridine-5’-diphosphoglycosyl transferases (UGTs): a superfamily of enzymesresponsible for glycurosylation in vivo. This procedure provides a mild, green and stereospecificsynthesis in a single step. The study focused on the glycosylations of flavonoids catalyzed byrecombinant glycosyltransferase expressed in E. coli. No much work has been reported yet onthis field so far.Four glycosyltransferases genes have been cloned from Scutellaria baicalensis using theteconology of RT-PCR. The genes have not yet been reported. The recombinant proteins of fourgenes were expressed in E. coli and then purified on the chromatography of chitin affinity andnickel affinity. The activities of four recombinant glycosyltransferases were analyzed bysolid-phase catalytic technology, respectively. These four recombinant glycosyltransferases wereevaluated in parallel for their ability on10different flavonoids. Consequently, the fourglycosyltransferases all worked with different flavonoids relative to control. No conspicuousdifferences were observed on the main products and by-products among different enzymes andsubstrates. It was noticed that the number of by-products is related to the number of hydroxylgroup on the A ring and B ring of the favonoids substrates. And regarding the flavonoidscombined with four or more hydroxyl groups, the products are complex and less. Theglycosylations products of5,7–Dihydroxy-4’-methoxyisoflavone and baicalein wereelucidated by LC-ESI-MS spectroscopic analysis and assigned as glycation products. Accordingto the structures and the numbers of products, it could be tentatively concluded that the mainproducts of the ten flavonoids are the7-O-glucosides. The extract structures of products are stillneeded to be elucidated by NMR and MS spectrum.In conclusion, the data in this study demonstrate the isolation from Scutellaria ofglycosyltransferases expressed in E. coli, which are able to catalyze the transfer of a glucosylmoiety from UDP-glucose to the hydroxy group of flavonoids. The elucidation of UGT enzymatic activity provide an efficient, single-step synthesis of glucosides under mild conditions,and it will be important to improve the water-solubility of flavonoids. This enzymatic methods ofglycurosylation synthesis could be used in the development of flavonoids drug.