Click‐Functionalized Cyanine Fluorogenic Dimers for Improved Detection of GPCRs: Application to Imaging of ApelinR in Living Cells

Abstract Fluorogenic dimers enable background‐free imaging of biological targets under wash‐free conditions owing to a strong fluorescence enhancement in the apolar cell microenvironment. However, it is crucial that the imaging probe interacts solely with the target receptor to avoid nonspecific interactions and ensure detection with a high signal‐to‐noise ratio. Herein, we describe a convenient and rapid approach for the synthesis of various functionalized cyanine dyes by click chemistry allowing the fine‐tuning of the physicochemical and fluorogenic properties of the dimers. A structure‐interaction relationship study was conducted for the fluorogenic dimers in the presence of bovine serum albumin (BSA) and liposomes as models of serum proteins and cell membranes. We identified d─Cy─E which combined the lowest nonspecific interactions with the optimal fluorescence turn‐on properties. By conjugating d─Cy─E to a peptide ligand of the apelin GPCR, we developed Ap─d─Cy─E , the first fluorescent turn‐on probe for the background‐free imaging of this receptor in living cells.