Selective and Efficient Cysteine Conjugation by Maleimides in the Presence of Phosphine Reductants
Selective and Efficient Cysteine Conjugation by Maleimides in the Presence of Phosphine Reductants
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DOI：
10.1021/acs.bioconjchem.6b00371
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发表时间：
2016-10-01
影响因子：
4.7
通讯作者：
Frey, Andreas
中科院分区：
文献类型：
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作者：
Henkel, Maik;Roeckendorf, Niels;Frey, Andreas
含巯基氨基酸的巯基官能团是标记、配体或药物与蛋白质化合物缀合的主要连接位点。通常，硫醇被提供来自待连接的分子的异生亲电子部分，例如马来酰亚胺官能团。由于巯基倾向于氧化成二硫化物，因此它们必须在缀合前被还原。在生物科学中流行的硫醇还原试剂是取代的膦三（2-羧乙基）膦（TCEP）。然而，膦也是亲核的，因此可能与硫醇竞争马来酰亚胺的贫电子烯烃部分，导致复杂的产物混合物。为了克服这个缺点，我们开发了一种方法，以消除过量的还原剂在反应混合物中的选择性氧化的膦与叠氮苯甲酸偶联前。这导致马来酰亚胺对半胱氨酸的选择性和有效标记。
Sulfhydryl functions of thiol-containing amino acids are prime attachment sites for conjugation of labels, ligands, or drugs to proteinaceous compounds. Usually the thiol is offered a xenobiotic electrophilic moiety from the molecule to be attached such as a maleimido function. As sulfhydryls tend to oxidize into disulfides they must be reduced before conjugation. A popular thiol reduction reagent in biosciences is the substituted phosphine tris(2-carboxyethyl)phosphine (TCEP). Yet, phosphines are nucleophilic, too, and thus potentially compete with thiols for the electron-poor alkene moiety of maleimide resulting in complex product mixtures. To overcome this shortcoming we developed a method to eliminate excess reducing agent in the reaction mixture by selective oxidation of the phosphine with azidobenzoic acid before coupling. This results in a selective and efficient labeling of cysteines by maleimides.