Matos Maria J, Jiménez-Osés Gonzalo, Bernardes Gonçalo J L
Department of Chemistry, University of Cambridge, Cambridge, UK.
Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, Logroño, Spain.
Methods Mol Biol. 2019;2033:25-37. doi: 10.1007/978-1-4939-9654-4_3.
This protocol details a novel bioconjugation strategy that uses a methanesulfonyl acrylate reagent that is directed to the most reactive lysine on human serum albumin, which enables the construction of chemically defined and stable bioconjugates. The reaction proceeds rapidly and a regioselective modification is achieved using a single molar equivalent of the reagent under biocompatible conditions (37 °C, pH 8.0). Importantly, the bioconjugate retains both the secondary structural content and function of the unmodified protein. During the reaction of the amino group of lysine and the sulfonyl acrylate reagent, methanesulfinic acid is released after the conjugate addition, which then generates an electrophilic acrylate moiety on the protein. This acrylate can be further used for site-specific protein labeling using a synthetic molecule bearing a reactive amine under biocompatible conditions (21 °C, pH 8.0).
本方案详细介绍了一种新型生物共轭策略，该策略使用一种甲磺酸丙烯酸酯试剂，该试剂靶向人血清白蛋白上反应性最强的赖氨酸，从而能够构建化学定义明确且稳定的生物共轭物。反应迅速进行，在生物相容条件（37°C，pH 8.0）下使用单摩尔当量的试剂即可实现区域选择性修饰。重要的是，生物共轭物保留了未修饰蛋白质的二级结构含量和功能。在赖氨酸的氨基与磺酸丙烯酸酯试剂反应过程中，共轭加成后会释放甲磺酸，然后在蛋白质上产生亲电丙烯酸酯部分。在生物相容条件（21°C，pH 8.0）下，这种丙烯酸酯可进一步用于使用带有反应性胺的合成分子进行位点特异性蛋白质标记。
