Native chemical ligation of thioamide-containing peptides: development and application to the synthesis of labeled α-synuclein for misfolding studies.
Native chemical ligation of thioamide-containing peptides: development and application to the synthesis of labeled α-synuclein for misfolding studies.
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DOI：
10.1021/ja2113245
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发表时间：
2012-06-06
影响因子：
15
通讯作者：
Petersson, E. James
中科院分区：
文献类型：
--
作者：
Batjargal, Solongo;Wang, Yanxin J.;Goldberg, Jacob M.;Wissner, Rebecca F.;Petersson, E. James
肽主链的硫代酰胺修饰用于扰乱二级结构、抑制蛋白水解、作为光开关和光谱标记。到目前为止，它们的掺入仅限于固相合成的单一肽。我们在 C 端硫酯或 N 端 Cys 片段中生成了硫代酰胺，并检查了它们与天然化学连接条件的相容性。大多数序列变体可以与 TCEP 或 DTT 作为还原剂以良好的产率偶联，但在延长 TCEP 孵育时观察到一些副产物。此外，我们发现硫代酰胺与 N 末端 Cys 的噻唑烷保护相容，因此可以使用多重连接来构建更大的蛋白质。由于硫代酰胺的酸不稳定性阻碍了使用 Boc 化学在树脂上合成硫酯，因此我们设计了一种合成硫代酰胺肽的方法，该方法具有原位显示的掩蔽 C 末端硫酯。最后，我们证明了硫代酰胺肽可以与表达的蛋白质片段偶联，通过合成用于蛋白质折叠研究的淀粉样蛋白 α-突触核蛋白的标记版本，生成带有主链硫代酰胺标记的大蛋白质。在原理验证实验中，我们证明硫代酰胺的荧光猝灭可用于跟踪标记的 α-突触核蛋白聚集过程中的构象变化。
Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.
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DOI：
10.1016/0006-291x(84)91449-9
发表时间：
1984-01-01
影响因子：
3.1
作者：
CLAUSEN, K;SPATOLA, AF;LAWESSON, SO
通讯作者：
LAWESSON, SO
影响因子：
--
作者：
BONDI, A
通讯作者：
BONDI, A
影响因子：
15
作者：
Deechongkit, S;Powers, ET;Kelly, JW
通讯作者：
Kelly, JW
影响因子：
3.6
作者：
BURNS, JA;BUTLER, JC;WHITESIDES, GM
通讯作者：
WHITESIDES, GM
影响因子：
2.1
作者：
Flemer, Stevenson, Jr.
通讯作者：
Flemer, Stevenson, Jr.