Dual Organelle Targeting for Intra-Organelle Click: Mitochondria and Endoplasmic Reticulum-Directed Benzothiophene-Fused Cycloalkyne Probes

Organelle targeting is an important area of research for studying organelle function and morphology, organelle signaling, and organelle-targeted drug delivery. Here, we report the development of dual organelle-targeted cycloalkyne reagents that can undergo click reactions with azides directly in the organelle of choice (mitochondria and endoplasmic reticulum (ER))—Mito-ER click systems. The design of the Mito-ER click systems is based on the benzothiophene-fused azacyclononyne BT9N, featuring a mito-targeting (thiophene) and mitochondria-maintaining (thiourea) functional group, along with a sulfonamide moiety responsible for delivery to the ER. We have demonstrated that the addition of a mitochondrial membrane potential (MMP)-dependent mito-directing group to the reagent alters the probe distribution towards mitochondria. Therefore, the combination of MMP-independent groups with the sulfonamide moiety is preferable for dual organelle targeting. The intracellular localization of the developed Mito-ER click systems in both organelles of choice was demonstrated by fluorescence microscopy, using colocalization with ER and mito trackers, and quantified using colocalization coefficients. The intra-organelle clickability of the developed Mito-ER click systems was confirmed by the intracellular click reaction with fluorogenic azide.