Synthesis of spectrally tunable cyanine analogs of MitoTracker Deep Red for mitochondrial imaging

Mitochondrial fluorescent probes remain essential tools for studying mitochondrial structure and function in live cells. However, access to structurally diverse and synthetically accessible dyes remains limited. Here, we report a robust preparative strategy for the synthesis of MitoTracker Deep Red and a series of structurally related cyanine-based mitochondrial probes. The approach is based on the direct introduction of chlorobenzyl group combined with a two-step assembly of the cyanine fluorophore, enabling the preparation of six probes spanning the Cy3 - Cy7.5 spectral range, as well as a Cy5-based derivative bearing two chlorobenzyl moieties. The photophysical properties of all synthesized dyes were characterized, and four representative probes were further evaluated in live-cell mitochondrial labeling experiments. Optimal staining concentrations and incubation times were established for mammalian cells. We assessed staining stability during prolonged cultivation, probe sensitivity to mitochondrial membrane potential dissipation, and compatibility with chemical fixation. The results demonstrate that the synthesized dyes provide robust mitochondrial labeling in live cells and exhibit predictable behavior under standard experimental conditions, offering a versatile and accessible platform of cyanine-based mitochondrial probes for fluorescence imaging applications.