Chemical Synthesis Of Ubiquitin Reagents For Biochemical Study Of Specific Proteases

Posttranslational modifications of proteins with ubiquitin(Ub)and ubiquitin-like(Ubl) modifiers widely presents in organisms and regulates diverse cellular processes in eukaryotes.Protein ubiquitination is a dynamic,reversible process.Ubiquitin(Ub)is added to the lysine residues of the substrate protein through the cascade of E1 activating enzymes(E1s),E2 conjugating enzymes(E2s),and E3 ligases(E3s),whereas the deubiquitinases(DUBs)reverse this process by removing Ub from the substrate protein.The activity of related enzymes is strictly regulated during the ubiquitination process,while the dysfunction is closely related to the occurrence of many major diseases,such as neurodegenerative diseases and cancer.Thus,DUBs are considered promising drug targets.It is necessary to explore the activity of deubiquitinases(DUBs)and screen small molecule inhibitors for DUBs.An important requirement is to obtain the homogeneous ubiquitin protein samples and fluorogenic ubiquitin reagents.Protein chemical synthesis enables us to build the desired proteins at atomic level and provides the powerful technical platform for preparing the ubiquitinated tool molecules,which plays an irreplaceable role in deciphering the DUBs.In this thesis,we focused on study the kinetics of DUBs and identify inhibitors for some members of these enzymes.Firstly,we took Ubiquitin(Ub)and Small ubiquitin-like modifier-2(SUMO2)as the research objects and we prepared fluorogenic ubiquitin(ubiquitin-like)reagents(Ub-Rho110-G and SUMO-Rho110-G)efficiently and conveniently through aminolysis of Boc-protected thioester counterparts.Then we confirmed that the synthesized fluorescent reagents are highly sensitive and can be effectively recognized and hydrolyzed by related enzymes through the enzymatic hydrolysis experiments.It can provide an effective tool for the activity detection of specific protease.Based-on the above-mentioned ubiquitin fluorescent reagent Ub-Rho110-G,we further tried to apply it to the high-throughput screening of deubiquitinating enzyme USP7 small molecule inhibitors.As a result,we screened three lead molecules that have potential to develope into USP7 small molecule inhibitors(compound 13,compound 14 and compound 40).Thereby it can provide important reference value for the treatment of diseases in related pathways and provide a powerful tool for the development of DUBs inhibitors associated with cancer.Finally,we took the K27-linked chain as the backbone and obtained K27-linked mixed triubiquitin(K27_C/48-linked mixed tri Ub,K27_C/63-linked mixed tri Ub and K27_C/M1-linked mixed tri Ub)efficiently through a combination of enzymatic reaction with the cysteine-aminoethylation assisted chemical ubiquitination(CAACU).We also proved that the synthetic tri Ub had correct secondary structure and defined-linkage for the study of DUBs.The synthetic tri Ub lays a foundation for further understanding of the role played by the K27-linked chain.It can also be used to monitor the activity of distinct DUBs.