Visualizing the Invisible: Dual Click Imaging of Ruthenium-Based Photoactivated Chemotherapy Agents and Their DNA Synthesis Inhibition in Fixed Cancer Cells

Like many drugs, ruthenium-based photoactivated chemotherapy (PACT) complexes are hard to follow in cells due to their absence of emissive properties. Here, two alkyne-functionalized Ru-based PACT compounds with the formula [Ru(HCC-tpy)(N̂N)(Hmte)](PF₆)₂ were synthesized, where HCC-tpy = 4'-ethynyl-2,2':6',2″-terpyridine, N̂N = 3,3'-biisoquinoline (i-biq, [<b>2</b>](PF₆)₂) or di(isoquinolin-3-yl)amine (i-Hdiqa, [<b>4</b>](PF₆)₂), and Hmte = 2-(methylthio)ethanol. Their challenging synthesis involved a protection-deprotection strategy to avoid the reaction of the free alkyne group with the coordinatively unsaturated ruthenium center. The thermal stability and photosubstitution quantum yield (Φ_[<b>2</b>] = 0.022 and Φ_[<b>4</b>] = 0.080) of the PACT complexes were essentially preserved upon alkyne functionalization. Interestingly, however, cellular uptake was doubled after alkyne functionalization, resulting in increased cytotoxicity against A549 cancer cells for both complexes in the dark and after green light activation (EC_50,light = 5 and 7 μM, respectively). To follow the complexes and see the effect of light activation, post-treatment fluorophore labeling via copper-catalyzed azide-alkyne cycloaddition was realized in fixed cells at 2 different time points, which allowed for imaging the otherwise invisible molecules. The images showed that the Ru complexes accumulated in the cytoplasm only after light irradiation and that they colocalized with the lysosomes and the Golgi apparatus. Moreover, we combined this approach with metabolic labeling of DNA, and showed by dual click imaging that DNA replication was inhibited by complex <b>4</b>. The strategy described herein, pioneered for nonemissive, photosubstitutionally active ruthenium complexes, opens a new avenue for investigating the selective attack of lung cancer cells by PACT.