Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Angew Chem Int Ed Engl. 2024 Aug 5;63(32):e202401733. doi: 10.1002/anie.202401733. Epub 2024 Jun 19.
Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.
细胞的存活、生长和分化在很大程度上依赖于对外源营养物质的摄取。然而，定量检测单细胞中小分子营养物质的摄取是很困难的。在这里，我们提出了一种使用逆电子需求 Diels-Alder（IEDDA）化学来研究活单细胞中营养物质摄取的新方法。我们用四嗪对羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）进行了修饰，CFSE 是一种猝灭荧光团，可以与蛋白质共价结合，只有在细胞内酯酶活性后才会在细胞质中被打开。四嗪是连接荧光团的第二个猝灭剂。当与在 IEDDA 反应中带有富电子或应变二烯体的营养素反应时，这个猝灭基团被破坏，从而使探针能够发出荧光。这使我们能够使用流式细胞术和活细胞显微镜监测各种含有二烯体的营养素在原代免疫细胞群体中的摄取。
