Selective Reagent for Detection of N-ε-Monomethylation of a Peptide Lysine Residue through SNAr Reaction
Selective Reagent for Detection of N-ε-Monomethylation of a Peptide Lysine Residue through SNAr Reaction
复制标题
通过 SNAr 反应检测肽赖氨酸残基 N-ε-单甲基化的选择性试剂
DOI：
10.1002/ejoc.201700488
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发表时间：
2017
影响因子：
2.8
通讯作者：
Kagechika Hiroyuki
中科院分区：
文献类型：
--
作者：
Mori Shuichi;Hirano Tomoya;Takaguchi Asuka;Fujiwara Takashi;Okazaki Yusuke;Kagechika Hiroyuki
组蛋白特定赖氨酸残基的甲基化由组蛋白甲基转移酶 (HMT) 催化，在基因表达的表观遗传控制中发挥关键作用。为了评估 HMT 的活性、开发抑制剂和鉴定底物，已经建立了几种检测赖氨酸残基 N-ε-甲基化的方法。然而，它们大多采用特异性抗体或酶（例如肽酶），其可靠性和重现性通常取决于蛋白质试剂的质量和反应条件。在这里，我们描述了一种通过简单的化学反应检测赖氨酸残基的 N-ε-单甲基化的便捷方法。我们重点研究芳香族亲电子试剂和伯氨基或单甲基化氨基之间的亲核芳香族取代反应（SNAr 反应）。各种亲电子试剂的筛选表明，4-氟-2-硝基苯乙酮（1g）对赖氨酸的N-ε-单甲基化氨基具有较高的选择性。此外，1g与赖氨酸和N-ε-单甲基化赖氨酸、5g和6g的反应产物分别表现出不同的吸收光谱，即6g在350 nm处的吸光度比5g大13倍。我们表明，1g 的这些特性可用于选择性检测 HMT 底物肽中赖氨酸残基的甲基化状态，并用于测定 HMT 活性。
Methylations of specific lysine residues of histone proteins are catalyzed by histone methyltransferases (HMTs) and play key roles in the epigenetic control of gene expression. Several methods to detectN‐ε‐methylation of the lysine residue have been established in order to evaluate the activity of HMTs, to develop inhibitors, and to identify substrates. However, they mostly employ specific antibodies or enzymes such as peptidases, and their reliability and reproducibility often depend on the quality of the protein reagents and the reaction conditions. Here, we describe a convenient method to detectN‐ε‐monomethylation of the lysine residue through a simple chemical reaction. We focused on nucleophilic aromatic substitution reaction (SNAr reaction) between an aromatic electrophile and a primary or monomethylated amino group. Screening of various electrophiles indicated that 4‐fluoro‐2‐nitroacetophenone (1g) has high selectivity for theN‐ε‐monomethylated amino group of lysine. Furthermore, the reaction products of1gwith lysine andN‐ε‐monomethylated lysine,5gand6g, respectively, show different absorption spectra, that is, the absorbance at 350 nm of6gis 13 times larger than that of5g. We show that these characteristic properties of1gcan be utilized for the selective detection of the methylation state of lysine residues in HMT substrate peptides, and for an assay of HMT activity.