Traceless ligation of cysteine peptides using selective deselenization.
Traceless ligation of cysteine peptides using selective deselenization.
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DOI：
10.1002/anie.201001900
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发表时间：
2010-09-17
影响因子：
16.6
通讯作者：
Dawson, Philip E.
中科院分区：
文献类型：
--
作者：
Metanis, Norman;Keinan, Ehud;Dawson, Philip E.
具有完全天然序列的蛋白质的合成是蛋白质化学中的持续挑战。天然化学连接（NCL）方法已被证明在半胱氨酸（Cys）残基被适当定位的情况下是普遍适用的，[1，2]然而，许多蛋白质的合成通常需要在多肽序列中的非Cys位点处进行连接。[3-7]以前，我们引入了一种基于Cys的全局脱硫的在Ala位点连接的还原策略[7][8]，该策略已被广泛用于通过NCL合成复杂蛋白质。[9，10]选择性脱硫可以受到雷尼镍和Pd/C/H2 [7]的影响，最近还受到自由基引发剂VA-044与水溶性膦TCEP组合的影响。[11]然而，由于这些条件导致蛋白质中所有巯基的整体脱硫，因此该方法需要天然序列中所有其他Cys残基的保护和脱保护。[6a这些额外的步骤使较大多肽的合成复杂化，并限制天然Cys残基用于连接。[13]硒代半胱氨酸（Sec，U）已被证明可以将NCL方法扩展到Xaa-Sec位点，允许合成硒蛋白。[14-16]此外，所得的硒肽可以在与用于含Cys的肽的条件类似的条件下脱硒，以产生相应的Ala肽序列。[11我们推断硒醇形成自由基的高倾向性[18]可以用于在硫醇存在下选择性还原硒醇（方案1），从而避免对保护/脱保护步骤的需要。这种方法的灵感来自于我们的观察，即含有Sec的谷氧还蛋白3（Grx 3）的合成类似物与水溶性还原剂TCEP的还原不相容，[19]导致产生显著水平的脱硒副产物。相比之下，发现wt-Grx 3对TCEP稳定。事实上，肽和蛋白质中Sec对TCEP还原的敏感性先前已在开发
The synthesis of proteins with a fully native sequence is an ongoing challenge in protein chemistry. Native chemical ligation (NCL) approaches have proven to be generally applicable where cysteine (Cys) residues are appropriately positioned,[1, 2] however, the synthesis of many proteins often require ligation at non-Cys sites in the polypeptide sequence.[3-7] Previously, we introduced a reductive strategy for ligation at Ala sites [7] based on global desulfurization of Cys [8] that has found widespread utility for the synthesis of complex proteins by NCL.[9, 10] Selective desulfurization can be affected by both Rainey Ni and Pd/C/H2 [7] and, more recently, by the radical initiator VA-044 in combination with the water soluble phosphine TCEP.[11] However, since these conditions result in global desulfurization of all thiols in the protein, the method requires protection and deprotection of all other Cys residues in the native sequence.[6a, 12] These additional steps complicate the synthesis of larger polypeptides and limit the use of natural Cys residues for ligation.[13]Selenocysteine (Sec, U) has been shown to expand the NCL method to Xaa-Sec site, allowing the synthesis of selenoproteins.[14-16] Additionally, the resulting selenopeptides can be deselenized under similar conditions to that used for Cys containing peptides to yield the corresponding Ala peptide sequences.[11, 17] We reasoned that the high propensity of selenols to form radicals [18] could be harnessed for selective reduction of selenols in the presence of thiols (Scheme 1), thus avoiding the need for protection/deprotection steps. This approach was inspired by our observation that synthetic analogs of glutaredoxin 3 (Grx3) containing Sec were incompatible with reduction by the water soluble reducing agent TCEP,[19] leading to the generation of significant levels of a deselenized side products. By contrast, the wt-Grx3 was found to be stable to TCEP. Indeed, the sensitivity of Sec in peptides and proteins to reduction by TCEP has been previously noted in the development of
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影响因子：
16.6
作者：
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