Synthesis Of The Cell Adhesive Motif Tripeptide RGD Using A Novel Chemical Method And Trypsin-Catalyzed Synthesis Of Bz-RGD-(OEt)₂

Peptides, which play very important roles in the life activity as basicconditioners, such as hormone, tropic hormone, nerve peptides and manyfunctional regulators which are synthesized or released by many organs.They are all essential biology active substances regulating economyfunction and maintaining vital movement. These peptides are intituledbiologically active peptides or biopeptides. It is easy to find biopeptides inhuman's organic systems,which act as the function of regulating thisorganic systems by cell receptor in the target organs. The importantfunctions of biopeptides for the organs have made them become good studyobjects to many research and development personnel. Many biopeptideshave been synthesized and separated, which have widely applicationforeground in medicament field.Peptides containing Arg-Gly-Asp widely exist in many multiorganismsand different tissues of organism. Early in 1984, Pierschbacher andRuoslahti have firstly reported RGD sequence within fibronectin, which is acell recognition site, the RGD sequence was further affirmed cell identifysystematic essence units by Erkki Ruoslash et al in 1987, and they areintermedium of adhesive action for cell and ECM. Henceforth, scientistsstudy RGD sequence and their receptors further, finding that these proteinsparticipate in many physiological process, such as transfer of tumors,thrombosis, inflammation, failing kidneys, et al.. Protein or peptides whichcontain RGD sequence can repress these physiological process, whichmade it possibility that peptide of RGD can cure medical persistent ailment.In recent years, many researchers tried to synthesize RGD andRGD-containing peptides by chemical or enzymatic methods. Chemicalmethods are classical synthesis methods of peptides, which always needshort reaction time and high yield. The principle of the enzymatic methodsis on the bases of reverse of the hydrolyzation of peptide bonds. Ascompared to the chemical method, enzymatic peptide synthesis have manyimportant benefits such as mild conditions of the reaction, highregiospecifity of enzyme allowing the use of minimally protected substrates,few of side reactions, small toxicity et al., therefore enzymatic methodshave a good applicable foreground. However, the method also exist in somelimit in the employment, the organic solvents must be adopted instead ofliquid solvents for the reverse of peptides hydrolyzation, under this instance,we need to consider the problem that hydrophilic amino acids are lowsolubility in organic solvents. RGD tripeptide is composed of three aminoacids in which two are hydrophilic and one is neutral amino acid, whichmake the synthesis of RGD difficult. Consequently, the study of synthesisof RGD in organic solvents has important theory significance and practicalapplication value.In this study, we firstly synthesized free RGD tripeptide by a novelchemical method, not only inheriting the benefits of classical chemicalmethods but also simple and cheapness. In the first place, we synthesizedGD dipeptide using a novel chemical method in two steps includingchloroacetylation of L-aspartic acid and ammonolysis of chloroacetylatedL-aspartic acid with low cost at large scale. Sencondly the synthesis ofRGD was carried out by chemical methods, which include the preparationof NCA-Arg and synthesis of RGD. The methods are benefit to quickreaction rate, shorter reaction time for the organs, high yield with low castat a large scale;reaction amino acid need not to be protected;and the freetripeptide can be adopted to the farther polypeptide synthesis directly.Compared to other method, this method is simple, speediness, cheapnessand high yield with about 45%, which is very fit for industrial exploiture.In the next place, owing to the chemical structure characteristic, wesucceed in synthesizing the tripeptide RGD ester by combining a novelchemical method with enzymetic method. First of all, free dipeptide,Gly-Asp was synthesized by a novel chemical method only using glycine ata large scale with low cast, followed by esterification of Gly-Asp. Thelinkage of the third amino acid Bz-Arg-OEt to Gly-Asp-(OEt)2 wascompleted by enzymatic method in ethanol/ This-HCl buffer system,because ethanol/ This-HCl buffer system is suitable as the reaction mediumfor the synthesis of hydrophilic amino acid-containing peptides because ofbetter solubility of substrates. RGD tripeptide was in the form of two ethylester [Bz-RGD-(OEt)2], which can act as both acyl donor and nucleophilefor carrying out peptide chain extension directly withoutdeprotection/activation. The effects of water content, TEA, molar ratio ofsubstrates and reaction time on the yield of Bz-RGD-(OEt)2 synthesis wereexamined.