Fluorescently labeled small molecules are an important class of probes for studying biology. Tethering fluorophores to compounds of interest can allow for visualizing reversible and temporal perturbations of biological systems in their endogenous state. From these agents, real-time information can be gathered on a compound's cellular localization as well as other indicators such as the quantitative expression levels of an intended target. In the field of chemical biology, the synthesis of such tagged, small molecules has been fruitful, allowing for the exploration of critical modes of action in disease states. These fluorescent probes have also served an equally pivotal role in the mechanistic exploration of promising clinical candidates and established chemotherapies.
Selection of the organic fluorophore for these small molecules is a central aspect of probe development. While there are many commercially available dyes, only a few can be used for chemical biology, and even from this select pool, many suffer from at least one key drawback. A fluorophore for imaging in chemical biology has a particular ideal target profile and should possess a specific set of attributes. Of primary importance is the brightness of the dye. This is a quantifier that allows for side-by-side comparison of different fluorophores and is defined as the product of the quantum yield and the extinction coefficient. Additionally, the fluorophore should have good aqueous solubility while retaining a favorable partition coefficient (log P value). An appropriate balance of these two factors is important to ensure that the dye can diffuse through membranes and organelles easily while concurrently engaging in minimal, non-specific membrane staining. Tied with these attributes is the necessity of the dye to remain a neutral, uncharged species in aqueous environments. Finally, the fluorophore should demonstrate long-term stability to aqueous and cellular imaging media, minimal consumption of chemical space, and a modular functional group for bioconjugate chemistry. The premise of these stipulations is the need for the fluorophore to maintain chemical stability with ideal optical properties in the cell while contributing minimal drug-like attributes that would interfere with the visualization and mechanism of the probe of interest.