Azaindole-based asymmetric pentamethine cyanine dye for mitochondrial pH detection and near-infrared ratiometric fluorescence imaging of mitophagy

Mitochondria are crucial organelles responsible for maintaining cell growth, and their homeostasis is closely linked to pH regulation. Physiologically, mitochondria exhibit a weakly alkaline state (pH similar to 8.0). However, when subjected to stress stimuli that cause damage, cells initiate the process of mitophagy, resulting in mitochondrial acidification. Therefore, monitoring changes in mitochondrial pH to comprehend the physiological processes associated with mitophagy is essential. In this study, we developed an asymmetric pentamethine cyanine dye Cy5.5-H-CyN as a probe for continuous monitoring of mitophagy in living cells. By incorporating an azaindole structure into the dye molecule, a ratiometric fluorescence response was achieved that is specifically responsive to pH variations while preserving its ability to target mitochondria and emit near-infrared fluorescence. Through various methods inducing mitophagy, Cy5.5H-CyN was employed to determine mitochondrial pH quantitatively, demonstrating its suitability as an ideal probe for continuous monitoring of mitophagy in living cells. (c) 2025 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.