Study On The Synthesis Of Thymopentin (TP-5) By A Novel Chemo-enzymatic Method

Bioactive peptides, 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.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. Lots of peptides with definitive biological activity have been separated and synthesized. They have been widely used as drugs, health care products and food additive. Moreover, the market of bioactive peptides concerned by more people is continuously growing in the pharmaceutical and food industries.Synthetic pentapeptide thymopentin (TP-5) is a bioactive peptide which corresponds to the amino acids 32³6 of thymopoietin and exhibits the full biological activity of the natural hormone. TP-5 has many functions. It can promote the thymus and peripheral T cell differentiation and development, bidirectional regulate the immune function confirmed by laboratory animal pharmacology and clinical pharmacology studies and increase the content of SOD in the blood. TP-5 has a good effect on the treatment of rheumatoid arthritis, skin diseases, tumor formation and certain infectious diseases, as well as diseases such as immunodeficiency syndrome because of its non-toxic, very short half-life, with a long time biological effects and the security. In addition, it can also be used as anti-aging drug. The results of animal and clinical experiments have proved that TP-5 is a safe and reliable drug and has good application prospects.Now the synthesis methods about TP-5 are almost chemical methods, including solid-phase and solution-phase chemical synthesis, especially solid-phase synthesis method is widely used. As we all known, solid-phase synthesis is a convenient and simple method, and the technology is quite mature, but solid-phase synthesis still has some limitations, such as protection of side chains during the reaction followed by an increasing in the cost of the reaction, excessive chemical reagents which will bring about a series of issues, including environmental pollution, toxicity, etc., causing a lot of restrictions for its application and production. As compared to the chemical method, enzymatic approach has been proved to be a promising alternative in peptide synthesis in regard to its numerous advantages over the chemical ones, such as mild reaction conditions, the needs for expensive protecting groups, organic solvents or hazardous chemicals greatly reduced, and almost no damage to the environment, reducing the post-processing steps of the target product and the cost of reaction. Of course, the method has some shortcomings including an undesirable hydrolysis of the growing peptide chain due to an amidase activity,a narrow substrate specificity,and deactivation due to organic solvents still hamper its use in peptide synthesis with long sequence.Based on the above research background, in this paper, specical attention was given to study the synthesis methods for TP-5. After analyzing the composition of TP-5, we successfully synthesized Z-Asp-Val-Tyr-NH₂, Z-Asp-Val-Tyr-OEt, as the precursors of TP-5, and Z-Arg-Lys-Asp-Val-Tyr-NH₂ by enzymatic approach with a combination of chemical method.In the study about the synthesis of the tripeptide precursors (Z-Asp-Val-Tyr-OEt/NH₂), the first step is the synthesis of the nucleophiles (Val-Tyr-OEt/NH2) for enzymatic synthesis. The second step is the linkage of the third amino acid that is Z-Asp-OH as the acyl donor to Val-Tyr-OEt/NH₂ completed by enzymatic method under thermodynamical control in organic media. Thermoase PC 10F was selected to synthesize the peptide bond. The purpose of the first step is to accumulate enough substrate for the next step. In this paper, N-carboxy anhydride method was used to prepare the free dipeptide (Val-Tyr-OH) in a two-phase reaction system, because that 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) and no racemization, NCA-Val is a high activated intermediate of Valine. It can be linked with Tyrosine to form our objective dipeptide at large scale. And then, we synthesized Val-Tyr-OEt using the traditional ROH-HCl method and Val-Tyr-NH₂ mainly using N-carboxy anhydride method. After receiving a certain amount of crude product, we purified them by gel column chromatography and preparative high performance liquid chromatograp- hy. The purity coefficient of the product is more than 90% by HPLC detection. In the second step, we chose Thermoase PC 10F as catalyst in accordance with the substrate specificity. It is suitable for the synthesis of the target products because that it is an industrial enzyme and very cheap, catalyzes industrial production of aspartame, no esterase activity, amino acid ester selected as nucleophiles, and has a very good thermal stability. In this paper, we only investigated the effects of reaction conditions on the tripeptide amide because of the low yield of the tripeptide ester and taking into account of the next enzymatic reaction (amide is more conducive to the synthesis of target peptide). The synthesis reaction conditions of the tripeptide amide were optimized by examining the effects of several factors including water content,temperature, pH, molar ratio of substrate and reaction time on the yields of the peptide. The optimum conditions for Z-DVY-NH2 were pH 6.5, 40℃,in MES/NaOH (containing 5mM CaCl₂) of n-Butanol system(15/85, V/ V), Thermoase 100mg, for 20h in the maximum yield of 27.02%.As to the synthesis of Z-Arg-Lys-Asp-Val-Tyr-NH2, we chose Trypsin and Alcalase to catalyze reaction under kinetic control in organic media.Trypsin was selected due to its substrate specificity suitable for the synthesis of the peptide bond with Z-Arg-Lys-OMe as the acyl donor. In addition,when the trypsin pre-treated with a buffer had memory function with a pH,the yield of the target peptide was increased markedly. Alcalase was used as the biocatalyst because it was the most stable in organic solvents among the family of proteases and has a suitable specificity for hydrophilic amino acid. We studied their ability of catalyzing TP-5's synthesis through elementary experiments and established two reaction models: The conditions for Z-GKDVY-NH₂ were pH 8.0,30℃,in ethanol/ Tris-HCl buffer system(97/3, V/V) for 9h in the maximum conversion rate of 67.03% with pre-treated trypsin power. The conditions for Z-GKDVY-NH2 were pH 10.0, 35℃, in acetonitrile/Na2CO3-NaHCO3 buffer system(93/7,V/ V) for 3h in the maximum conversion rate of 56.44% with pre-treated Alcalase .