Synthesis Of The Precursor Tripeptide Of Thymopentin By Chemo-enzymatic Mathod

Peptides, as a series of elementary substance of life, have many important functions in organisms, some of them, for example hormones, trophic hormones, cell factors, neutropeptide and many other functional regulative factors, are all very important bioactive substance to maintain and regulate life activities. These peptides with regulative functions are called bioactive peptides. They distribute among all systems of human body, such as neural system, immune system, digestive system, etc., and regulate organism's activities through connecting with acceptors on the target cells. There are many researchers pay attention to bioactive peptides due to their important functions. Lots of peptides with definitive biological activity have been separated and synthesized for the usage as medicine or functional food, this help people to improve their life qualities.Thymopoietin is a polypeptide hormone produced by Thymus, consisting of 49 amino acids.The pentapeptide thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, corresponding to amino acids 32-36 of thymopoietin, appears to represent the active site of thymopoietin so that it has all the biological activities of the native Thymopoietin.TP-5 has many functions, it can induce the secretion of many hormones, mediate the cytodifferentiation of T-cell, regulate immune system and etc., Studies show that Thymopentin has clinical efficiency in the treatments of rheumatoid arthritis and atopic dermatitis and genital warts. It also can be used as adjunctive therapy of genital system infection and chemotherapy & radiotherapy. TP-5 have the biological characteristics of being immunonormalizing which, whether the immune disbalance be in the direction of hyper- or hyporesponsiveness, points to its potential utility in human diseases characterized by immune disbalance. Researches have proved that TP-5 can cure lymphadenopathy of drug addicts, inhibit the accumulation of hyperoxide in the cells, and relieve the insulin dependence of diabetic. A large amount of researches have proved that TP-5 is safe in treatments and can be widely used in therapy and adjuvant therapy.Three main approaches of peptide synthesis are available at present,a) chemical synthesis; b)enzymatic synthesis; c)recombinant DNA technology. We synthesized dipeptide Val-Tyr-OH by chemical method. Valine is an amino acid which contains a long aliphatic side chain that makes it easily to be transformed to L-Valine-N-carboxyanhydride (NCA-Val), NCA-Val is a high activated intermediate of Valine. It can be linked with Tyrosine to form our objective dipeptide. During this procedure, the choice of reation system is very important. Yield of dipeptide is very low and many other by-products existed no matter how to control the reaction condition under the traditional aqueous phase system. Separation of the by-products is very difficult. In order to solve these problems, we built a new two-phase reaction that synthesis can be carried out under a mild condition and got a good yield.There are many factors affected the results of reaction, such as solvents, stirring speed, temperature, and pH etc. With the two-phase reaction system and certain substrates, we optimized the reaction condition of the dipeptide synthesis. The best yield(91.36%) of Val-Tyr-OH was gained under the condition of -10℃, NCA-Val concentration of 0.06M, activating at pH10.0, stirring speed >1500rpm, acidificating at pH6.0, reaction time of 2 h.As to the synthesis of tripeptide Z-Asp-Val-Tyr-OH, it is difficult to find an enzyme that has high specificity for Z-Asp-OMe, so we chose Alcalase enzyme as catalyst and studied its ability of catalyzing this tripeptide's synthesis through orthogonal experiments, there is a initiatory result of reaction conditions: Alcalase as catalyst, water content 5% in acetic ether, 25℃,pH10.0 , substrate ratio=1:3, reaction time of 3h, the new peak compares with control is 80.84% in all.The separation and purification of the dipeptides were carried out mainly using the method of solvent extraction. Because Val-Tyr-OH is easily dissolved in both water and ethanol, so the salt in the product could be removed through ethanol extraction. Generally, the purity coefficient of the product is more than 90%, after extraction the product could be used directly for next step reaction. There is no need to take more steps for separation and purification. Val-Tyr-OEt is a slimy liquid that can be dissolved in ethanol and methanol but water. We used Sephadex LH-20 column to make the separation of Cbz-Val-Tyr-OH and got a good yield (92.3%). The best synthesis method of tripeptide has not been established so far, so we didn't study the separation method of it.The dipeptide products could be identified only using the method of MS not by means of other methods because of the simple reaction products without racemation during peptide synthesis. So we identified the target dipeptides only by their molecule weights.In a word, we designed a route of synthesis of TP-5 according to characteristics of itself, the convenience and low cost is also considered into our designing scheme.We have gotten some delectable result and established a good basis for the synthesis of thymopentin pentapeptide.