UnsymmetricHeptamethine Cyanines as a General Strategyto Construct Nonaggregating Fluorophores for Biomedical Imaging

Abstract 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.