Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary
Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary
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DOI：
10.1073/pnas.121178598
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发表时间：
2001-06-05
影响因子：
11.1
通讯作者：
Botti, P
中科院分区：
文献类型：
--
作者：
Low, DW;Hill, MG;Botti, P
我们已经完成了细胞色素b562和轴向配体类似物的全化学合成。Cyt b562。通过硫酯介导的化学连接未受保护的多肽片段。使用了一种新的辅助介导的天然化学连接，使肽连接能够应用于缺乏半胱氨酸的蛋白质序列。一种可裂解的含硫醇的辅助基团。1-苯基-2-硫代乙基。被添加到一个肽段的α-氨基上，以促进酰胺键的连接。氨基连接的1-苯基-2-硫代乙基助剂对固相肽合成后用于裂解和脱保护肽的无水氟化氢是稳定的。在与含有硫代酯的片段进行天然化学连接之后。通过用无水氟化氢处理，将辅助基团从新形成的酰胺键上干净地移除，得到全长未修饰的多肽产品。得到的多肽与血红素重组并折叠形成功能蛋白分子。人工合成的野生型细胞色素b562具有与重组蛋白相同的光谱和电化学性质，而工程化的[Semet(7)]细胞色素b562类似蛋白在光谱和功能上是不同的，还原电位移动了约45 mV。本文报道的1-苯基-2-硫代乙基可拆卸助剂的使用将极大地扩展通过天然化学连接无保护的多肽片段来合成总蛋白的适用性。
We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue. [SeMet(7)]cyt b562. by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group. 1-phenyl-2-mercaptoethyl. was added to the alpha -amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment. the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximate to 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.