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

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.