π-Spacer modulation unlocking tunable photophysics and organelle-specificity in 2,6 disubstituted BODIPY-cyanostilbene fluorophores

Abstract

Simultaneous visualization of two distinct organelles using a single fluorescent probe offers powerful opportunities for unravelling cellular signalling pathways and inter-organelle interactions. However, this goal remains challenging due to the complex microenvironment of subcellular compartments and the absence of robust design principles. In this study, we designed and synthesized two new donor–acceptor (D–A) BODIPY-cyanostilbene fluorophores, functionalized at the 2,6-positions with tailored π-spacers: phenyl (compound 7) and thiophene (compound 8) units. Strategic π-spacer engineering enabled precise control over both optical behavior and organelle-targeting selectivity. The thiophene analogue (compound 8) exhibited a remarkable Stokes shift (∼112 nm) and far-red/NIR emission (600–800 nm), overcoming two key limitations of conventional BODIPYs, particularly visible-range emission (490–510 nm) that restricts deep-tissue imaging, and narrow Stokes shifts that lead to spectral overlap and self-quenching. In contrast, the phenyl analogue (compound 7) emitted at 570 nm with a moderate 35 nm Stokes shift, primarily due to the extended π-conjugation framework. The photophysical properties were supported by DFT calculations (B3LYP/6-31G(d), Gaussian 09W). Both probes showed excellent photostability, minimal cytotoxicity, high chemical and pH stability, and non-interference from competitive species, making them highly suitable for cellular studies. Interestingly, both probes achieved dual-targeting of lipid droplets (LDs) and lysosomes, enabling real-time monitoring of dynamic changes in these organelles. This work highlights π-spacer engineering as a powerful strategy to unlock far-red/NIR dual-organelle probes, paving the way for advanced organelle-targeting imaging, and early-stage disease diagnostics.