Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents.
Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents.
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DOI：
10.1038/ncomms13128
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发表时间：
2016-10-26
影响因子：
16.6
通讯作者：
Bernardes GJ
中科院分区：
文献类型：
--
作者：
Bernardim B;Cal PM;Matos MJ;Oliveira BL;Martínez-Sáez N;Albuquerque IS;Perkins E;Corzana F;Burtoloso AC;Jiménez-Osés G;Bernardes GJ
马来酰亚胺仍然是制备治疗和成像蛋白偶联物的首选试剂，尽管已知产物在体内进行巯基交换反应时存在不稳定性。本文介绍了用于化学选择性半胱氨酸生物偶联的羰基丙烯酸试剂的合理设计。这些试剂在生物相容性条件下以化学计量量进行快速硫醇迈克尔加成。当使用配备PEG或荧光基团的羰基丙烯酸试剂时，该方法可以获得在预定位点精确修饰的蛋白质和抗体偶联物。重要的是，形成的缀合物在血浆中不易降解，并且具有生物学功能，分别通过凋亡细胞和HER2+细胞的选择性成像和检测证明了这一点。羰基丙烯酸试剂的简单制备，化学计量学的使用和精细的半胱氨酸选择性，结合产品的稳定性和半胱氨酸标记蛋白的生物学相关性，使该方法适用于体内应用的化学定义的偶联物的常规制备。目前用于蛋白质-药物偶联物合成的半胱氨酸生物偶联策略通常产生不均匀和不稳定的产物。本文作者利用羰基丙烯酸衍生物选择性修饰半胱氨酸残基，合成了在血浆中高度稳定的具有生物功能的抗体偶联物。
Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications. Current cysteine bioconjugation strategies for protein-drug conjugates synthesis often yield heterogeneous and poorly stable products. Here, the authors use carbonylacrylic derivatives to selectively modify cysteine residues and synthesize biologically functional antibody conjugates highly stable in plasma.