A General Synthetic Approach for Polymethine Modified Trimethine Cyanines Gives Access to Viscosity Sensitive Probes

Polymethine functionalization of trimethine cyanine (Cy3) fluorophores remains challenging due to limited synthetic strategies. Here, we report a versatile synthetic strategy for accessing both symmetric and unsymmetric meso-substituted Cy3 fluorophores using activated carboxyl intermediates. This three-step, high-yielding approach enables polymethine modified Cy3. Through systematic variation of acyl chloride precursors and indolenine derivatives, we generated a structurally diverse library of Cy3 dyes featuring aliphatic, aromatic, and electron-withdrawing meso-substituents. The approach is compatible with a broad range of indolenines. The Cy3 fluorophores have absorption and emission maxima between 550–590 nm and 560–650 nm, respectively. Unsymmetric Cy3 derivatives have larger Stokes shifts (up to 100 nm) as compared to symmetrical Cy3. Additionally, we demonstrate a new approach to synthesize viscosity sensitive probes. We show that meso-substituted benzothiazole-containing analogs exhibit up to 20-fold enhancement in quantum yield in high-viscosity environments, and, as such, could be used as molecular rotors for viscosity sensing. Complementary time-dependent density functional theory (TD-DFT) calculations further support the experimental observations by accurately reproducing the substituent‐dependent trends in absorption, emission, and Stokes shifts across the series. The computed absorption wavelengths and Stokes shifts are consistent with the experimental results. This work establishes a general synthetic scheme for meso-functionalized Cy3, offering new opportunities to fine-tune photophysical properties for imaging, sensing, and bioconjugation applications.