Synthesis of Cyanine Dyes with Nucleophilic Functional Groups and Their Application as Activatable Molecular Probes

Fluorescence imaging is a powerful technique for visualizing and quantifying dynamically changing biological events with high spatial resolution and sensitivity, and it has been widely applied in both fundamental biological research and clinical practice. However, conventional “always-on” fluorescent probes emit signals irrespective of their localization, often resulting in high background fluorescence and low imaging contrast in biological environments. To address these limitations, activatable fluorescent probes, which switch from a non-emissive “off” state to an emissive “on” state only in response to specific biological stimuli, have attracted increasing attention owing to their high signal-to-noise ratios and improved detection sensitivity. A variety of activation mechanisms have been developed, including the removal of quenching groups, changes in molecular planarity, and modulation of π-conjugation length through elimination reactions. Among these strategies, probes activated by recovery of the π-conjugated system provide unique opportunities for rational molecular design. In particular, cyanine dyes incorporating nucleophilic functional groups represent a promising yet relatively underexplored platform for activatable probe development. In this account, we summarize our recent efforts on the design and synthesis of nucleophile-containing cyanine-based activatable fluorescent probes. We focus on enzyme-responsive systems, including probes activated by esterases and aldehyde dehydrogenase, and discuss their photophysical properties, activation mechanisms, and applications in biological imaging.