Fluorogenic probes are important tools to image proteinsw ith high contrast and no wash protocols. In this work, we rationally designed and synthesized as mall set of four protein fluorogens with red or near-infrared emission. The fluorophores were characterizedinthe presence of albumin as am odel protein environment and exhibited good fluorogenicitya nd brightness (fluorescenceq uantum yield up to 36 %). Oncec onjugatedt oahaloalkanel igand, the probes reactedw ith the protein self-labelingt ag HaloTag with ah igh fluorescencee nhancement (up to 156-fold). The spectroscopic properties of the fluorogens and their reaction with HaloTag were investigated experimentally in vitro and with the helpo fm olecular dynamics. The two most promising probes, one in the red and one in the near-infrared range,w eref inally applied to image the nucleus or actin in live-cell and in wash-free conditions using fluorogenica nd chemogenetic targeting of HaloTag fusion proteins.

Ten borylated bipyridines (BOBIPYs) have been synthesized and selected structural modifications have been made that allow useful structure-optical property relationships to be gathered. These systems have been further investigated using DFT calculations and spectroscopic measurements, showing blue to green fluorescence with quantum yields up to 41%. They allow full mapping of the structure to determine where selected functionalities can be implemented, to tune the optical properties or to incorporate linking groups. The best derivative was thus functionalised with an alkyne linker, which would enable further applications through click chemistry and in this optic, the stability of the fluorophores has been evaluated.

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths...