A General Strategy for Photostable and Bioconjugatable Pentamethine Cyanine Fluorophores

Abstract

Pentamethine cyanine (Cy5) fluorophores are widely used in biological imaging but are often limited by rapid photobleaching under prolonged illumination and oxidative conditions. Here, we report a general strategy to enhance the photostability of pentamethine cyanines through electronic modulation of the indolenine scaffold. Specifically, we introduce electron-withdrawing substituents, including sulfonate groups on the benzene ring for aqueous solubility and difluoro- or trifluoroethyl groups at the N-alkyl position, to obtain H1 and H2. Importantly, the incorporation of these electron-withdrawing groups preserves the photophysical properties of the dyes while significantly reducing photooxidative degradation under continuous irradiation and in reactive oxygen and sulfur species-rich environments compared to commercial standards, including Alexa Fluor 647. Additionally, our strategy can be modulated to incorporate bioconjugation handles for biomolecule labeling. We demonstrate that bioconjugatable versions of H1 and H2 can be conjugated to antibodies (mAbs) at a range of labeling densities and retain their enhanced photostability. Overall, this work demonstrates that tuning the electronic properties of indolenine building blocks provides a general and effective approach to designing pentamethine cyanine fluorophores with improved photostability for advanced biological imaging applications.