Insights into the mechanism and catalysis of the native chemical ligation reaction
Insights into the mechanism and catalysis of the native chemical ligation reaction
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DOI：
10.1021/ja058344i
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发表时间：
2006-05-24
影响因子：
15
通讯作者：
Kent, Stephen B. H.
中科院分区：
文献类型：
--
作者：
Johnson, Erik C. B.;Kent, Stephen B. H.
未受保护的肽片段的天然化学连接涉及肽-α-硫酯和半胱氨酸-肽之间的反应，以产生在连接位点具有天然酰胺键的产物。肽-α-硫代烷基酯由于其易于制备而被普遍使用。这些硫代烷基酯是相当不反应的，因此连接反应是通过用硫醇添加剂原位硫代酯交换催化的。迄今为止使用的最常见的硫醇催化剂是苯硫酚/苄基硫醇的混合物，或烷基硫醇MESNA。尽管使用这些硫醇催化剂，连接反应通常需要24-48小时。为了深入了解天然化学连接的机理，寻找更好的催化剂，我们研究了一些硫醇化合物的使用。发现pK（α）> 6的取代的硫酚最好地联合收割机结合了与硫代烷基酯快速和完全交换的能力，然后在肽-硫代酯与半胱氨酸-肽的硫醇侧链的反应中充当有效的离去基团。一种高效实用的催化剂是（4-羧甲基）苯硫酚（“MPAA”），一种无恶臭的水溶性硫醇。使用MPAA在天然化学连接的模型研究和小蛋白质火鸡卵类粘蛋白第三结构域（OMTKY 3）的合成中给出了一个数量级更快的反应。MPAA应该在天然化学连接和蛋白质的全合成中找到广泛的用途。
Native chemical ligation of unprotected peptide segments involves reaction between a peptide-alpha-thioester and a cysteine-peptide, to yield a product with a native amide bond at the ligation site. Peptide-alpha-thioalkyl esters are commonly used because of their ease of preparation. These thioalkyl esters are rather unreactive so the ligation reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts used to date have been either a mixture of thiophenol/benzyl mercaptan, or the alkanethiol MESNA. Despite the use of these thiol catalysts, ligation reactions typically take 24-48 h. To gain insight into the mechanism of native chemical ligaton and in order to find a better catalyst, we investigated the use of a number of thiol compounds. Substituted thiophenols with pK(a) > 6 were found to best combine the ability to exchange rapidly and completely with thioalkyl esters, and to then act as effective leaving groups in reaction of the peptide- thioester with the thiol side chain of a cysteine-peptide. A highly effective and practical catalyst was (4-carboxylmethyl) thiophenol ('MPAA'), a nonmalodorous, water-soluble thiol. Use of MPAA gave an order of magnitude faster reaction in model studies of native chemical ligation and in the synthesis of a small protein, turkey ovomucoid third domain (OMTKY3). MPAA should find broad use in native chemical ligation and in the total synthesis of proteins.