Peptide synthesis by enzymatic method, especially its application for total synthesis of human insulin

以人胰岛素的全合成为目的，采用酶促方法将A、B链片段缩合，通过A、B链偶联制备单链胰岛素，用岛津PSSM-8型多肽合成仪合成8个胰岛素片段。金黄色葡萄球菌V8蛋白水解酶(简称SAP)、胰酶(简称T)和赖氨酰内肽酶(简称L)用于偶联。反应是在高浓度有机共溶剂(40%二甲基甲酰胺)存在下进行的。反应混合物含有2 mM的胺组分和10 mM的羧基组分，pH 6~7，室温放置1~3d。其他实验条件如下所述。采用高效液相色谱法测定偶联产物的产率。结果表明，在以下所有偶联反应中，胺组分的峰都消失了，并出现了新的峰。对各新峰的氨基酸分析表明，新峰为预期的偶联产物。：A；胰岛素A链，Cys6-Cys11(S-S桥)，Cys7-ACm，Cys20-Acm.B；胰岛素B链，Cys7-Acm，Cys19-Acm，乙酰胺甲基1.括号中的数字是各自序列中的氨基酸数字。

For the purpose of total synthesis of human insulin, enzymatic method was applied for the fragment condensation of A or B chain, together with the preparation of single chain insulin by coupling of fragments A and B chains.Eight insulin fragments were synthesized using a peptide synthesizer (Shimadzu model PSSM-8). For coupling, Staphylococcus aureus V8 protease (abbrev.SAP), trypsin (abbrev.T) and lysyl endopeptidase (abbrev.L) were used. The reaction was performed in the persence of high concentration of organic cosolvent (40% dimethylformamide). The reaction mixture contained 2 mM amine component and 10 mM carboxyl component, pH 6-7, which was kept for 1-3 days at room temperature. The other experimental conditions are described below. The coupling yield was determined using high performance liquid chromatography (HPLC). The result indicated that the peak of amine component disappeared and a new peak appeared in HPLC in all the coupling reactions below. Amino acid analysis of the respective new peaks in HPLC indicated that the new peaks were the coupling products as had been expected.Abbrev. : A ; insulin A chain, Cys6-Cys11 (S-S bridge), Cys7-Acm, Cys20-Acm.B ; insulin B chain, Cys7-Acm, Cys19-Acm.Acm, acetoamidemethy1..The numbers in parentheses are the amino acid ones in the respective sequences.