Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

Fluorescent labelling of specific proteins in complex biological systems remains an important challenge in chemical biology. One promising approach comprises the use of small molecules designed to react specifically with a targeted protein of interest and to increase in fluorescent intensity following this reaction. This kind of fluorogenic reaction generally derives from fluorescence quenching in the unreacted probe that is abrogated over the course of the reaction. Herein, we review the mechanistic principles of three major photophysical quenching mechanisms involving Forster resonance energy transfer (FRET), through-bond energy transfer (TBET), and photoinduced electron transfer (PeT). We then present design principles for novel fluorogenic probes based on an understanding of these quench mechanisms, with emphasis on the emerging utility of density functional theory (DFT) calculations in the design process.