Enzymatic Synthesis Of Containing Hydrophilic Amino Acids ASP Of Small Bioactive Peptide

Bioactive peptides are important substance to maintain and regulate life activies, distributing among all systems of human body, such as human central nervous system, endocrine system, immune system and others. They regulate organism's activities through connecting with acceptors on the target cells. Nowdays, peptide synthesis methods are mainly three methods: chemical methods, enzymatic synthesis and recombinant DNA technology.Nowdays, the main bioactive peptides synthesis method is chemical methods. Enzymatic synthesis is a new method rising in recent years. The principle of Enzymatic synthesis is the use the reverse reaction of proteolytic enzymes to form peptide bond. Enzymatic synthesis method has the following advantages: mild reaction conditions; the high regiospecifity of enzyme allowing the use of minimally protected substrates; the reaction being stereospecificity without racemization.In our study of small bioactive peptides, we found that many functional peptides contain hydrophilic amino acid Asp, such as the world's first industrial bioactive peptides aspartame application of enzymatic synthesis,thymopeptide, corn bioactive small peptides, cell adhesion RGD. In order to solve the solubility problem of hydrophilic amino acids in the hydrophobic organic solvents, So we choose hydrophilic organic solvents as reaction media. But enzyme easy to lose activity in organic solvents and other problems, so hydrophilic small peptides enzymatic sythesis is a difficult problem.In this study, we successfully got small function peptides of containing hydrophilic amino acids ASP through Enzymatic synthesis, and summed up the law of enzymatic synthesis of functional peptides contain hydrophilic amino acid Asp, build the optimized reaction model.We successfully synthesized dipeptide Z-Asp-Val-NH2 of TP-5, precursor Tripeptide Z-Asp-Val-Tyr-OH of TP-5. Firstly, using traditional chemical method to synthesis the nucleophile (Val-NH2, Val-Tyr-OH) at large scale with low cost. Secondly, the linkage of the acyl donor and the nucleophile was completd by using Alcalase under kinetic control condition in organic solvents. The synthesis reaction conditions were optimized by examining the effects of several factors, including organic solvents, water content, pH, temperature reaction time and concentration of the acyl donor and the nucleophile, build the optimized reaction model.The yield reached to 63% and 71.3% respectively. At the same time we also enzymatic synthesized precursor dipeptide Z-Asp-Tyr-NH2 of corn peptide with Alcalse as catalyst,and got the target product, but reaction condition need to be further optimized. Separation and purification by Sepadex G-10 column. Quantitative analysis of the target dipetide product was carried out by HPLC and the products of the target product was carried out by HPLC and the product were confirmed by LC-MS.The optimum conditions for Z-Asp-Val-NH2 synthesis using Alcalase as the catalyst were Z-Asp-OMe (0.05M),Val-NH2 (0.25M), triethylamine (14μL/mL), in acetonitrile /0.1M Na2CO3-NaHCO3 pH 10.0 buffer system (90:10, V/V), 35℃, 5h with the dipeptide yield of 63%.When synthesizing the right side precursor tripeptide Z-Asp-Val-Tyr-OH of TP-5 with Z-Asp-OMe as the acyl donor and Val-Tyr-OH as the nucleophile, alcalse was used on the bases of the synthesis of Z-Asp-Val-NH2. The optimum conditions for Z-Asp-Val-Tyr-OH synthesis were Z-Asp-OMe (50 mmol), Val-Tyr-OH (150 mmol), triethylamine(50μl/ml), in acetonitrile/0.1M Na2CO3- NaHCO3 pH 10.0 buffer system (85:15, V/V), 35℃, 2.5h with the tripeptide yield of 71.3%.In the synthesis of Z-Asp-Tyr-NH2, through a preliminary experiment under kinetic control condition in organic solvents, we synthesized corn peptide precursor dipeptide Z-Asp-Tyr-NH2. From the above we can draw the following conclusions: Alcalase have a very good stability and catalytic activity in hydrophilic solvents contains a small amount of water. In Enzymatic synthesis of containing hydrophilic amino acids ASP of function peptide precursors small peptide course, under the control of danamic, hydrophilic amino acid substrate have a good solution in the hydrophilic organic solvent Contains a small amount of water, in this kind Of system we could get a high yield. This offer a new thought to the research of Enzymatic Synthesis of Bioactive Peptides...