Efficient Enzymatic Synthesis Of Novel Glycyrrhetinic Acid Glycoside Derivatives Based On UDP Recycling

Glycyrrhetinic acid,which is derived from licorice,is a pentacyclic triterpenoid compound that is widely used as its various pharmacological activities.But its poor water solubility,low bioavailability and a variety of clinical toxic and side effects after long-term use and other defects limit the further application of glycyrrhetinic acid.Therefore,the strategy of using glycyrrhetic acid as a skeleton to synthetize low toxicity,high efficacy and novel glycyrrhetinic acid derivative drugs has drawn much attention.Among them,glycosylation modification plays an important role in natural products modification applications.In view of the fact that glycosylation of glycyrrhetinic acid has many disadvantages through chemical method,such as complex group protection/deprotection,complicated process and low yield.Enzymatic method has drawn much attention because of its good orientation,mild conditions and environmental friendliness.We previously achieved the biosynthesis of 3-O-monoglucosyl glycyrrhetinic acid using the glycosyltransferase BvEc in vitro,but the glycosyltransferase BvEc relies on expensive UDP-glucose as a glycosyl donor and the efficiency of enzyme catalysis is low,furthermore the glycosylation products is relatively monotonous,which limited the further application of synthesis of glycyrrhetinic acid derivatives by glycosyltransferase in vitro.In this study,we systematically analysis and deeply mine the sucrose synthases in Glycyrrhiza uralensis.And a UDP recycle system has been constructed by conjugating Glycyrrhiza uralensis sucrose synthase in order to synthesize glycyrrhetinic acid glycosylated derivatives efficiently and obtained several glycyrrhetinic acid glycosylated derivatives,which provide a new idea for glycosylated modification of glycyrrhetinic acid.The main findings are as follows:(1)Through the systematically analysis and mining of genome information in Glycyrrhiza uralensis,we obtained two sucrose sucrose synthase genes-Gu Su S1 and Gu Su S2.We verified the activity of sucrose synthesizing and sucrose cleavage of these two enzymes by prokaryotic expression in vitro.The system evolutionary relationship of Gu Su S1 and Gu Su S2 was analyzed.The recombinant proteins of Gu Su S1 and Gu Su S2 were purified,then the effects of p H and temperature on catalyzing the sucrose hydrolysis catalyzed by Gu Su S1 and Gu Su S2 were investigated.The optimal p H of the reaction was 5.5 and the optimal temperature was50℃.In addition,the enzymatic characteristics of Gu Su S1 and Gu Su S2 were also characterized.Through the three-dimensional structural homology simulation of Gu Su S1 and docking analysis with substrate sucrose and UDP molecules,13 potential key amino acid sites were identified.Combined with alanine scanning mutagenesis experiment,we found that seven of these sites is very important for Gu Su S1 to catalyze sucrose cleavage process and we explore the reaction mechanism preliminarily.(2)Combine the sucrose synthase Gu Su S1 with glycosyltransferase BvEc to construct UDP recycle and regeneration system,we obtained 3-O-monoglucosyl glycyrrhetinic acid and two other glycyrrhetinic acid glycosylated derivatives from this system.Through optimization of the reaction conditions,the optimal reaction temperature was 40 ℃ and the optimal p H was 6.5.It was found that the overall reaction efficiency was not affected when the ratio of Gu Su S1 and BvEc was adjusted from 1: 1 to 1: 5,and ethanol or DMSO was used as a cosolvent also had no effect on this system.Two kinds of glycyrrhetinic acid glycosylated derivatives were obtained by semi-preparative liquid chromatography.The structures of these two compounds were confirmed by LC-MS and NMR and we call them 30-O-monoglucosyl-3-O-mon oglucosyl glycyrrhetinic acid and 30-O-monoglucosyl-3-O-monoglucuronosyl glycyrrhetinic acid.And the latter is the firstly synthetized through checking on the chemicals database.Through the time-course experiment,it was found that the conversion of glycyrrhetinic acid was completed within 5h with UDP recycle and regeneration system.The content of 30-O-monoglucosyl-3-O-monoglucosyl glycyrrhetinic acid reached 70.84%,while that of 3-O-monoglucosyl glycyrrhetinic acid accounted for 34.33%.Using UDP recycle and regeneration system can not only improve the efficiency of glycyrrhetinic acid glycosylation greatly,but also unlock the potential of glycosyltransferase for catalysis.