Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging

Heptamethineindocyanines are invaluable probes for near-infrared(NIR) imaging. Despite broad use, there are only a few synthetic methodsto assemble these molecules, and each has significant limitations.Here, we report the use of pyridinium benzoxazole (PyBox) salts asheptamethine indocyanine precursors. This method is high yielding,simple to implement, and provides access to previously unknown chromophorefunctionality. We applied this method to create molecules to addresstwo outstanding objectives in NIR fluorescence imaging. First, weused an iterative approach to develop molecules for protein-targetedtumor imaging. When compared to common NIR fluorophores, the optimizedprobe increases the tumor specificity of monoclonal antibody (mAb)and nanobody conjugates. Second, we developed cyclizing heptamethineindocyanines with the goal of improving cellular uptake and fluorogenicproperties. By modifying both the electrophilic and nucleophilic components,we demonstrate that the solvent sensitivity of the ring-open/ring-closedequilibrium can be modified over a wide range. We then show that achloroalkane derivative of a compound with tuned cyclization propertiesundergoes particularly efficient no-wash live cell imaging using organelle-targetedHaloTag self-labeling proteins. Overall, the chemistry reported herebroadens the scope of accessible chromophore functionality, and, inturn, enables the discovery of NIR probes with promising propertiesfor advanced imaging applications.