Study On The Chemical Synthesis Of Polyubiquitin Chains And Ubiquitin Derivatives

Protein ubiquitination,as a kind of important post-translational modification of the important protein with various effects and diverse results,is involved in almost all life activities of eukaryotes.Such as protein degradation,cell division,growth,cell signal transduction,cell movement and apoptosis.Former people developed a variety of synthetic strategies for obtaining ubiquitinated proteins.Enzymatic methods fail to obtain certain ubiquitinated proteins due to the lack of specific enzyme systems.Protein in vitro chemical synthesis provides an effective way to obtain precise size and different types of ubiquitin chains because of its advantages of being capable of accurately constructing proteins at the atomic scale,but the synthesis steps are complicated and the workload is large.In order to solve the above-mentioned problems,previous researchers proposed a method for constructing non-natural isopeptide ubiquitin chains based on chemical ligation reactions.However,this strategy requires that the ubiquitin fragment be first chemically engineered and the ligation reaction be less efficient.In addition,the vast majority of mimic isopeptide bonds differ greatly from the true structure.Based on this,it is great scientific significance to develop a new strategy for efficiently obtaining uniform ubiquitin chains.In this paper,protein chemical synthesis technology is used to study the chemical synthesis of ubiquitin chains and ubiquitin derivatives.In the first work,we first performed efficient loading between prosthetic and ubiquitin fragments by Thiol-ene coupling between small molecules and protein fragment sulfhydryls,and then through the natural chemical ligation reaction,the ubiquitin fragments loaded with auxiliary and the ubiquitin hydrazides are linked to achieve efficient synthesis of ubiquitin chains,overcoming the previously low coupling efficiency between macromolecules and macromolecules.By this method,we efficiently synthesize K48 and K27-linked diubiquitin chains.In the second work,we developed a new method for the efficient and rapid synthesis of ubiquitin probe Ub-AMC.As an ubiquitin probe,Ub-AMC has been widely used in deubiquitinase enzymatic assays and high-throughput screening for deubiquitinase inhibitors.However,the current methods for the synthesis of Ub-AMC mainly include chemical total synthesis and fragment ligation.Both of these methods have the drawbacks of tedious synthesis steps,heavy workload,and high cost.Based on this,we develop a new method for the efficient synthesis of one-hundred milligrams of Ub-AMC that can be efficiently digested by deubiquitinase.The experiment verified that the synthesized ubiquitin probe can be well applied to explore the dynamic mechanical properties of DUB enzyme and the screening of DUB inhibitors.