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

为实现人胰岛素的全合成，采用酶法将A链或B链进行片段缩合，并将A链和B链偶联制备单链胰岛素，用岛津PSSM-8型肽合成仪合成了8个胰岛素片段。为了偶联，使用金黄色葡萄球菌V8蛋白酶（焦v.SAP）、胰蛋白酶（焦v.T）和赖氨酰内肽酶（焦v.L）。反应在高浓度有机助溶剂（40%二甲基甲酰胺）存在下进行。反应混合物含有2 mM胺组分和10 mM羧基组分，pH 6-7，将其在室温下保持1-3天。其他实验条件如下所述。使用高效液相色谱法（HPLC）测定偶联产率。结果表明，在以下所有偶联反应中，胺组分的色谱峰消失，并出现一个新的色谱峰。HPLC中各新峰的氨基酸分析表明，新峰是预期的偶联产物。缩写：A ;胰岛素A链，Cys 6-Cys 11（S-S桥），Cys 7-Acm，Cys 20-Acm.B ;胰岛素B链，Cys 7-Acm，Cys 19-Acm.Acm，乙酰胺甲基。括号中的数字是相应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.