Genomics Institute of the Novartis Research Foundation (GNF), Biotherapeutics & Biotechnology, 10675 John Jay Hopkins Drive, San Diego, CA, 92121, USA.
Chembiochem. 2018 Apr 16;19(8):799-804. doi: 10.1002/cbic.201700611. Epub 2018 Mar 15.
Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins.
活化酯广泛用于在赖氨酸侧链和 N 末端标记蛋白质。这些试剂对于标记几乎任何蛋白质都很有用，但对伯胺的强反应性通常排除了选择性修饰。在一个独特的情况下，氟代苯酯被证明优先标记人κ抗体在轻链恒定域中的单个赖氨酸（Lys188）上。邻近残基 His189 和 Asp151 有助于相对于 ≈40 个其他赖氨酸位点加速 Lys188 的标记速率。通过以下任何一种方法都可以将富集的 Lys188 标记从 50-70%提高到 >95%：降低反应温度、应用流动化学或周围蛋白质环境中特定残基的突变。我们的结果表明，具有氟取代芳香离去基团的活化酯，包括氟萘酯，可以作为抗体和其他免疫球蛋白型蛋白质的赖氨酸选择性标记的一般有用试剂。
