Chemical Synthesis Of The Sulfotyrosine Modified Hirudin And Elucidation Of Its Anticoagulant Mechanism

Thrombin is a serine protease that plays a central role in maintaining coagulation homeostasis in the body.Thrombin hydrolyzes soluble fibrinogen to form insoluble fibrin.However,when blood coagulation homeostasis is destroyed due to some physiological or pathological factors,excessive clotting substances will lead to thrombotic diseases.Therefore,thrombin has become an important target for the development of anticoagulant drugs.As various thrombin inhibitors have been developed and put into clinical treatment,various limitations have gradually emerged,such as weak inhibitory activity,narrow drug window,high risk of bleeding,easy accumulation in vivo and other safety problems.Therefore,the existence of natural thrombin inhibitors in the body or saliva of many blood-sucking insects has attracted the attention of researchers.Hirudin is the most potent natural thrombin inhibitor known at present.Its carboxyl terminus(C-terminus)is homologous to fibrinogen and can produce strong interaction with thrombin.More importantly,the presence of a sulfated tyrosine at the C-terminus of natural hirudin enhances its inhibitory activity.However,the use of recombinant expression method to obtain hirudin modified with sulfated tyrosine has some problems,such as heterogeneous target products,difficult separation and sulfate instability.Therefore,most of the current studies are limited to recombinant hirudin without this modification.In addition,the regulatory effect of sulfated tyrosine on the thrombin inhibition mechanism of hirudin has not been fully resolved.In this work,a chemical synthesis method of HIRV1 containing sulfated tyrosine modification was developed.First,we achieved the synthesis of sulfated HIRV1 polypeptide fragments by introducing a sulfated modified tyrosine monomer protected by a neopentyl group,and found that the neopentyl group could be removed automatically in native chemical ligation.Then,we explored the synthesis strategy of HIRV1,and finally synthesized four different HIRV1 proteins using the native chemical ligation and refolding one-pot strategy,and confirmed the correctness of the refolding of the four HIRV1 proteins through enzyme digestion experiments.Then,by inhibiting fibrinogen hydrolytic activity of thrombin and short peptide hydrolytic activity of thrombin,we found that the four synthetic HIRV1 proteins all showed good thrombin inhibitory activity,among which the sulfated tyrosine modified HIRV1 showed the strongest inhibitory activity.Finally,by introducing photocross-linking tags in the synthesis process,we resolved the regulatory mechanism of tyrosine sulfation modification on hirudin-thrombin inhibitory activity.In summary,we have developed an efficient synthesis method for HIRV1 containing tyrosine sulfation modification.In addition,on the basis of this method,the way in which sulfated modification regulates the mechanism of HIRV1 thrombin inhibition was studied,which promoted the study of the chemical synthesis of sulfated protein and laid a foundation for the analysis of the mechanism of sulfated modification regulating the physiological function of protein.