Department of Chemistry, University of Science and Technology of China, Hefei 230026, China.
Chem Asian J. 2011 May 2;6(5):1241-51. doi: 10.1002/asia.201000760. Epub 2011 Mar 1.
The method of native chemical ligation between an unprotected peptide α-thioester and an N-terminal cysteine-peptide to give a native peptide in aqueous solution is one of the most effective peptide ligation methods. In this work, a systematic theoretical study was carried out to fully understand the detailed mechanism of ligation. It was found that for the conventional native chemical ligation reaction between a peptide thioalkyl ester and a cysteine in combination with an added aryl thiol as catalyst, both the thiol-thioester exchange step and the transthioesterification step proceed by an anionic concerted S(N)2 displacement mechanism, whereas the intramolecular rearrangement proceeds by an addition-elimination mechanism, and the rate-limiting step is the thiol-thioester exchange step. The theoretical method was then extended to study the detailed mechanism of the auxiliary-mediated peptide ligation between a peptide thiophenyl ester and an N-2-mercaptobenzyl peptide in which both the thiol-thioester exchange step and intramolecular acyl-transfer step proceed by a concerted S(N)2-type displacement mechanism. The energy barrier of the thiol-thioester exchange step depends on the side-chain steric hindrance of the C-terminal amino acid, whereas that of the acyl-transfer step depends on the side-chain steric hindrance of the N-terminal amino acid.
在水溶液中，未保护的肽α-硫酯与 N 端半胱氨酸肽之间的天然化学连接方法是最有效的肽连接方法之一。在这项工作中，进行了系统的理论研究，以充分了解连接的详细机制。结果发现，对于常规的肽硫烷基酯与半胱氨酸的天然化学连接反应，结合外加的芳基硫醇作为催化剂，硫醇-硫酯交换步骤和转硫酯步骤均通过阴离子协同 S(N)2 取代机制进行，而分子内重排通过加成-消除机制进行，限速步骤是硫醇-硫酯交换步骤。然后，该理论方法被扩展到研究肽硫苯基酯与 N-2-巯基苄基肽之间的辅助介导肽连接的详细机制，其中硫醇-硫酯交换步骤和分子内酰基转移步骤均通过协同 S(N)2 型取代机制进行。硫醇-硫酯交换步骤的能垒取决于 C 末端氨基酸的侧链空间位阻，而酰基转移步骤的能垒取决于 N 末端氨基酸的侧链空间位阻。
