Synthesis And Biological Applications Of Stimuli-Responsive Puromycin Prodrugs And Puromycin Derivatives

Puromycin(Puro)is a protein synthesis inhibitor that mimics the function of aminoacyl-t RNA,and has a wide range of applications in cell screening,detection and selective labeling of nascent proteins,and subcellular localization of translating ribosomes.However,the non-selective cytotoxicity of Puro limits its development as a therapeutic agent.Stimuli-responsive prodrugs can selectively release anticancer drugs in tumor cells using endogenous/exogenous stimuli,which not only significantly improves the efficacy of cancer treatment,but also reduces toxicity to normal tissues.Therefore,designing Puro as an endogenous/exogenous stimulus-activated prodrug will promote its development as an anticancer drug.Disulfide and diselenide bonds are commonly used reaction sites for endogenous or exogenous stimuli.Linear disulfide bonds having been applied to various thiol-activated therapeutic prodrugs,while cyclic diselenide bonds have unique selectivity for thioredoxin reductase(Trx R).In addition,diselenide bonds have higher reaction activity than disulfide bonds,and many stimuliresponsive drug delivery systems are designed based on the redox properties of linear diselenide bonds.In this paper,a series of endogenous/exogenous stimuli-activated Puro prodrugs were designed and synthesized by protecting the amino group of Puro through disulfide/diselenide scaffolds,and a series of Puro derivatives containing 1,2,3-triazole were synthesized by click reaction.The mechanism of action and anticancer effect of Puro prodrugs and Puro derivatives were investigated.The details are as follows:Chapter 1: Introduction.Firstly,we summarized the application of Puro and its derivatives as tool molecules in chemical biology.Then,we classified and discussed the application of disulfide/diselenide scaffolds in prodrug design and drug delivery system.Finally,we briefly reviewed the research progress of endogenous stimuliactivated prodrugs based on tumor microenvironment and exogenous stimuli-activated prodrugs based on bioorthogonal cleavage reaction and photocontrolled release reaction.Chapter 2: Trx R is a selenoprotein that is overexpressed in tumor cells and plays an important role in maintaining cellular redox homeostasis.The design of prodrugs specifically activated by Trx R will promote the development of anticancer prodrugs with Trx R as an endogenous stimulus.We designed endogenous stimuli-activated Puro prodrugs S1-Puro and S2-Puro by protecting the amino group of Puro through a cyclic disulfide scaffold and a linear disulfide scaffold.S2-Puro with linear disulfide scaffold can be activated by endogenous small molecule thiols and protein thiols,while S1-Puro with cyclic disulfide scaffold can be specifically activated by endogenous Trx R.S1-Puro has higher cytotoxicity in tumor cells overexpressing Trx R,which provides a new design idea for Puro to become an anticancer drug targeting Trx R.Chapter 3: Prodrugs activated by exogenous stimuli can achieve spatiotemporal controlled release of drugs,reduce drug off-target phenomenon,and improve antitumor effect.We designed exogenous stimuli-activated Puro prodrug Se1-Puro and endogenous stimuli-activated Puro prodrug Se2-Puro by protecting the amino group of Puro through a cyclic diselenide scaffold and a linear diselenide scaffold.Se2-Puro with linear diselenide scaffold can be activated by endogenous small molecule thiols and protein thiols,while Se1-Puro with cyclic diselenide scaffold can be activated by exogenous dithiothreitol(DTT)but not by endogenous biothiols.This unique response mechanism of Se1-Puro provides a new design strategy for the five-membered cyclic diselenide scaffold as an exogenous stimulus-responsive site.Chapter 4: Theranostic prodrugs are a new prodrug design strategy that combines chemotherapy and tissue imaging.They can monitor the release and distribution of drugs by detecting reporter molecules,and have great application potential in the precise treatment of cancer.We covalently linked Puro and fluorophores with different emission wavelengths to both sides of the linear disulfide scaffolds by carbamate bond,and synthesized three glutathione(GSH)-activated theranostic prodrugs AFC-SS-Puro,ANA-SS-Puro and ADM-SS-Puro.All three prodrugs can be activated by GSH in cells and release fluorescence,which can monitor the release and distribution of Puro.Chapter 5: The non-selective cytotoxicity of Puro is the key to limiting its use as a therapeutic drug.We synthesized a series of Puro derivatives containing 1,2,3-triazole by derivatizing 3’-azido-3’-deoxy-N,N-dimethyladenosine(puromycin azide nucleoside)via click reaction,and performed preliminary cytotoxicity tests on these compounds using four different tumor cells.All Puro derivatives have almost no cytotoxicity at low concentrations(5 μM),indicating that the amino acid moiety of Puro is the key to its function of inhibiting protein synthesis.Chapter 6: The work of this paper is summarized,and the design of endogenous/exogenous stimuli-activated prodrugs with Puro as an antitumor drug is prospected.