Thiol-ene Click,Oxo-ester Mediated Native Chemical Ligation:A Methodology For The Synthesis Of Cysteine Modified Hyaluronic Acid For In Situ Hydrogel Formation

Hyaluronic acid(HA),or hyaluronan, is a natural non-sulfated anionic linear glycosamino-glycan (GAG),ubiquitous in the human body,especially in the synovia of joints,the corpus vitreum of the eyes and the dermis of the skin, plays many important physiological roles ,suggesting that HA is a biomolecule of considerable importance.However, HA is rapidly turned over in the body by hyaluronidase,with tissue half-lives ranging from minutes to days ,limiting its applications as a biocompatible, biodegradable and non-immunogenic polymer. The process of chemical modification and crosslinking of results in larger,more stable molecules that have been used to achieve longer residence time in vivo and miantianed pharmacological and therapeutic properties,biocompatibilitym,viscoelastic properties similar to those of the naturally occurring substance. We present herein a novel,efficient and mild approach to functionalize the hyaluronic acid via its C-6 hydroxyl groups and obtained a series of cysteine-hyaluronic acid conjugates through a stable ether bond linkage.For the first time, Seven concentration gradient ene-functional hyaluronic acids were prepared by etherification of the C-6 hydroxyl groups with allyl glycidyl ether,and performed by lyophilized, and formed spongy slice product. The hydroxyl groups of HA was modified by allyl glycidyl ether and formed stable ether linkages in the process of vacuum freeze drying. The method is the first applied to the modified Hyaluronic acid by small molecules.The alkene hydrothiolation reaction, also called the thiol-ene coupling reaction(TEC) is high yielding and compatible with oxygen and an aqueous environment. The modified polysaccharides were then through thiol-ene radical chemistry (TEC)reacted with cysteine derivatives to obtain a N-cysteine hyaluronic acid conjugates under photoinitiator and UV light.The oxo-ester mediated native chemical ligation(OMNCL) reaction between an alcohol ester and an N-terminal-Cys yields an S-acyl covalent intermediate that spontaneously undergoes an S- to N-acyl migration to form a new amide bond through a five-member ring intermediate,which overcomes many of the earlier limitations of NCL, including cytotoxicity of thiol leaving groups and slow reaction kinetics, and represents a promising strategy for chemical cross-linking of hydrogels in a biological context. Multivalent four-armed poly(ethylene glycol) precursors containing N-hydroxysuccinimide activated oxo-esters and N-cysteine hyalur onic acid conjugates formed hydrogels cross-linked by oxo-ester mediated native chemical ligation (OMNCL) under mild conditions.A useful feature of this hydrogen strategy is the regeneration of thiol functional groups as a result of the OMNCL reaction, thereby allowing fictionalization of the polymer hydrogen with biomolecules.The kinetics of gel formation and the viscoelastic behavior of hydrogels were further studied by oscillatory rheology, which demonstrated gelation times from minutes to hours. Hydrogels were demonstrated by conjugation of cell adhesion peptide,permitting the attachment and maintains glucose-stimulated insulin release by islet RIN m5f cell. The methodology represents a promising strategy for chemical cross-linking of hydrogels in a biological context and is an attractive candidate for cell , tissue graft function in transplantation , tissue engineering and regenerative medicine applications.The pentapeptide Thymopentin (TP5) is the fragment of thymopoietin II(32-36)with basic sequence H-Arg-Lys-Asp-Val-Tyr-OH. It has the same biological activityas the thymopoietin II, which has the double-direction immunities to our bodies as immune regulator. Now Thymopentin (TP5) has been represented a promising,effective medicine appling on market. Therefore, researches on the solid phase synthesis of TP5 are not only has good market prospect, but also provide new method and idea for the establishing the solid peptide phase synthesis (SPPS)technical platform.So far, in the majority of Fmoc solid phase peptide synthesis literatures and patents, piperidine solution is considered to be an universally acceptable strategy on removing Fmoc protecting group on amino acids. However, piperidine has many drawbacks in industrial and scientific research: piperidine as II classe precursor chemical need to strict control under the public security department; piperidine is a colorless liquid, hygroscopicity, vapors with water and on sensitives on carbon dioxide , result in inconvenience on storage and transportation.Therefore,we need a simple, effective, low cost, and easy to obtain deprotection agent on the solid peptide phase synthesis of thymopentin (TP5)In this paper, we used piperazine and piperidine as the Fmoc deprotection agent,synchronous solid peptide phase synthesis of thymopentin, and each step of intermediate,final thymopentin product and standard product monitored by HPLC and MS. There were not different between piperazine and piperidine method.It is innovative and breakthrough on the selection piperazine as deprotection agent on solid peptide phase synthesis. In contrast with piperidine,the advantages of piperazine are more convenient to transport and store. under the same concentration,the Fmoc depriving efficiency of the piperazine is higher than that of piperidine; The strategy has great stimulative significance in production and research of peptides,and provides a new choice and ideas for researchers.