One-pot Enzymatic Synthesis Of Cardiac Glycosides With Varied Sugar Chain Lengths And Its Selective Inhibition On The ?2 Isoform Of Na~+,K~+-ATPase

Cardiotonic steroids(CTS),an important class of natural product,have been widely used for the treatment of congestive heart failure for over 200 years due to their strong Na~+,K~+-ATPase inhibitory effect.Poor water solubility and narrow therapeutic window limit their clinical applications.It has been reported that the?2 isoform-selective CTS could induce cardiac contraction with only minimal systemic toxicity,and could be used as safer cardiotonic agents as compared to conventional CTS.Structure-activity analyses showed that the oligosaccharide at C3 of CTS is pivotal for their?2 isoform-selective effects.However,chemical synthesis of oligosaccharides at the C-3 position of CTS remains restricted by poor regio-and stereo-selectivity and tedious protection and deprotection of functional groups.The application of microbial glycosyltransferases(GTs)is faced with poor regiospecific and only mono-or di-O-glycoside(minor)of CTS could be obtained.Therefore,development of an applicable approach for the synthesis of diverse cardiac glycosides with varied sugar chain lengths at C-3 could be of great value.In this thesis,research work was carried out on the C-3 glycosylation and the extension of sugar chain of CTS.(1)In order to achieve the C-3 mono-glycosylation of CTS,the steroid 3-O-glycosyltransferase UGT74AN3 was cloned from Catharanthus roseus for the first time after analysis of the transcriptome database,which explained why the suspension cells of C.roseus could glycosylate bufadienolides.A cyclodextrin glycosyltransferase(CGTase)was successfully cloned and purified from Bacillus licheniformis,and the extension of the C-3 sugar chain of the CTS was achieved by the catalysis of CGTase.(2)We report an efficient one-pot enzymatic system for the synthesis of cardiac glycosides with varied length of sugar chains.This system couples UGT74AN3 and CGTase to achieve one-pot glycosylation of CTS.By optimizing the reaction parameters of this system,the yields of glycosylated product are increased,and the conversion rates are between 75.2%-99.5%.(3)The system has broad applications in the glycosylation of cardiotonic drugs,and exhibits robust substrate tolerance for the different structures of CTS.Altogether twenty cardiac glycosides were obtained by this system,of which 16 cardiac glycosides were new compounds.(4)We selected two representative series of compounds,including bufadienolides(arenobufagin)3/3a/3b/3c/3d/3e and cardenolides(digitoxigenin)7/7a/7b/7c/7d/7e for biological assay.For the bufadienolides,compound 3b showed the highest?2 isoform selectivity(K?1/K?2=3.07)which was approximately 3-fold than that of 3,indicating that two sugar units were optimal.For the cardenolides,compound 7c showed the highest?2 isoform selectivity(K?1/K?2=5.4),which was approximately greater 5.5-fold than that of 7.(5)We have selected the most selective compound 7c for further toxicology studies,and observed lower toxicity on zebrafish embryo compared to 7.The one-pot enzymatic method reported in this thesis provides a powerful tool for the synthesis of safer cardiac glycosides,and also highlights the important role of tolerant biocatalysts in drug development.The selective inhibition of NKA?2 isoform provides a scientific basis for the search for novel cardiotonic agents with high-efficiency and low-toxicity.