Toward a Traceless Tag for the Thiol-Mediated Uptake of Proteins

Dr. John R. J. Maynard, Dr. Saidbakhrom Saidjalolov, Marie-Claire Velluz, Stefania Vossio, Prof. Charlotte Aumeier, Dr. Dimitri Moreau, Dr. Naomi Sakai, Prof. Stefan Matile
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Abstract

The emergence of thiol-mediated uptake (TMU) as a powerful strategy to penetrate cells calls for the development of practical small-molecule TMU tools for traceless delivery. Toward this goal, esters are explored here as bioreversible linkers between dynamic covalent cascade exchangers accounting for TMU and the protein of interest (POI). The method relies on α-aryl-α-diazoamides that react with carboxylic acids of the POI to form esters that can be enzymatically hydrolyzed inside cells to release the native POI. A two-step protocol is established for bioreversible conjugation of TMU tags to the POI. Despite the small number of tags attached to POIs to prevent isoelectric precipitation, POIs with traceless TMU tags are shown to efficiently enter cells not only in 2D culture but also in 3D spheroids mimicking deep tissue, confirming a key advantage of TMU. Uptake inhibition by various thiol-reactive agents confirms the participation of cell-surface thiols in cell penetration, i. e., the occurrence of TMU.

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巯基介导的蛋白质摄取的无迹标记
巯基介导摄取(TMU)作为一种穿透细胞的强大策略的出现,要求开发实用的小分子TMU工具,用于无痕迹递送。为了实现这一目标,本文探讨了酯类作为动态共价级联交换器(TMU)和感兴趣蛋白(POI)之间的生物可逆连接物。该方法依赖于α -芳基- α -重氮酰胺与POI的羧酸反应形成酯,该酯可以在细胞内酶解释放天然POI。建立了TMU标签与POI生物可逆偶联的两步方案。尽管附着在poi上的标签数量很少,以防止等电沉淀,但具有无痕TMU标签的poi不仅可以在2D培养中有效进入细胞,还可以在模拟深层组织的3D球体中有效进入细胞,这证实了TMU的一个关键优势。各种硫醇活性物质的摄取抑制证实了细胞表面硫醇参与细胞渗透,即TMU的发生。
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