Post-translational site-selective protein backbone α-deuteration
Post-translational site-selective protein backbone α-deuteration
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DOI：
10.1038/s41589-018-0128-y
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发表时间：
2018-10-01
影响因子：
14.8
通讯作者：
Davis, Benjamin G.
中科院分区：
文献类型：
--
作者：
Galan, Sebastien R. G.;Wickens, James R.;Davis, Benjamin G.
同位素替代技术在小分子领域有着久经考验的应用。然而，在蛋白质中的应用在很大程度上仅限于生物合成策略或可交换(例如，N-H/D)不稳定位点。蛋白质中的生物后合成、C-H-1-和GT；C-H-2/D替换的发展可以使探索机制成为可能，以及其他用途。在这里，我们描述了一种选择性蛋白质α-碳氢代反应的化学方法(从半胱氨酸到脱氢丙氨酸(DHA)再到重氢半胱氨酸)，允许在一个不可交换的主干位置进行整个H-1-和H-2/D交换。这里用它来探索蛋白质生物偶联反应的机理。这一分析表明，结合量子力学计算，在半胱氨酸转化为DHA的过程中，通过蛋白质上的碳离子和意想不到的硫叶立德逐步去质子化，符合Carba-Swern机制。在现有的完整蛋白质结构上的现成应用(没有专门的培养或遗传方法)表明这种C-D标记策略在蛋白质机制、结构、生物技术和医学中可能是一种工具。
Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-H-1 -> C-H-2/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein alpha-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall H-1 -> H-2/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a 'carba-Swern' mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C-D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine.