Chemo- and Regioselective Lysine Modification on Native Proteins.
Chemo- and Regioselective Lysine Modification on Native Proteins.
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DOI：
10.1021/jacs.7b12874
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发表时间：
2018-03-21
影响因子：
15
通讯作者：
Bernardes GJL
中科院分区：
文献类型：
--
作者：
Matos MJ;Oliveira BL;Martínez-Sáez N;Guerreiro A;Cal PMSD;Bertoldo J;Maneiro M;Perkins E;Howard J;Deery MJ;Chalker JM;Corzana F;Jiménez-Osés G;Bernardes GJL
合成分子与蛋白质的位点选择性化学偶联扩展了它们的功能和治疗能力。目前基于合成和生化技术的蛋白质修饰方法可以实现位点选择性，但这些技术通常需要大量的序列工程或仅限于N端或c端。在这里，我们展示了计算机辅助设计的丙烯酸磺酰酯试剂，用于修饰天然蛋白质序列上的单个赖氨酸残基。所设计的磺酰基丙烯酸酯的这一特征，加上每个赖氨酸周围独特的局部微环境所赋予的固有和微妙的反应性差异，有助于观察到反应的区域选择性。此外，通过计算预测了这种位点选择性，其中具有最低pKa的赖氨酸在微碱性下是动力学上有利的残基。化学选择性也被观察到，即使在存在其他亲核残基（如半胱氨酸）的情况下，试剂也优先与赖氨酸反应。反应速度快，在生物相容性条件下（37℃，pH 8.0）使用一摩尔当量的丙烯酸磺酯试剂进行。该技术通过五种不同蛋白质的定量和不可逆修饰得到证明，包括临床使用的治疗性抗体曲妥珠单抗，而无需事先进行序列工程。重要的是，在修改后，它们原有的二级结构和功能被保留。这种区域选择性赖氨酸修饰方法允许进一步的生物偶联，通过aza-Michael添加到丙烯酸酯亲电试剂上，该亲电试剂是在赖氨酸标记后由甲磺酸自发消除产生的。我们发现，在赖氨酸上特异安装荧光团的蛋白质-抗体偶联物可分别用于凋亡细胞的选择性成像和Her2+细胞的检测。这种简单、可靠的方法不需要基因工程，通常可用于获取多种、定义良好的蛋白质偶联物，用于基础生物学和治疗研究。
Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.
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