Unsymmetric Heptamethine Cyanines as a General Strategy to Construct Nonaggregating Fluorophores for Biomedical...

Heptamethine cyanine dyes are widely used as near-infrared (NIR) fluorophores in biomedical imaging. However, conventional heptamethine cyanine fluorophores are symmetric in nature, which favors fluorophore self-assembly from π-π stacking in aqueous media. This H-aggregation significantly reduces the fluorescence intensity and hampers imaging performance. Although a few nonaggregating heptamethine cyanines have been reported, no general strategy has been established for synthesizing nonaggregating heptamethine cyanine. Here, we introduce a broadly applicable approach to prevent H-aggregation by breaking the molecular symmetry of heptamethine cyanines through selective substitution along the polymethine backbone. This structural asymmetry disrupts π-π stacking interactions without adversely affecting the desirable photophysical properties of the dyes. This strategy accommodates a wide range of indolenine and polymethine substituents that enable the development of a diverse library of nonaggregating heptamethine cyanines (15 examples in total) including nonaggregating ICG analogs. One of the unsymmetrical heptamethine cyanine dyes, SAT-NIR-746, was further characterized for its aggregation behavior. SAT-NIR-746 remained monomeric in water and across a range of salt concentrations, indicating strong resistance to salt-induced H-aggregation. Furthermore, a carboxylic acid derivative of SAT-NIR-746 was converted to an NHS ester and conjugated to monoclonal antibodies (mAbs) via lysine residues. The resulting IgG-SAT-NIR-746 conjugates showed no signs of H-aggregation, further confirming the dye's stability in both solution and protein-bound states. Finally, we show the advantages of SAT-NIR-746 for quantitative bioimaging in vitro, in vivo, and ex vivo phantom imaging.