Mitochondria-Targeting Moieties Based on N-Tethered Pyridinium Cations

Mitochondria-targeting moieties (MTMs) are molecular fragments designed to deliver covalently tethered functional cargo to mitochondria, providing a modular strategy for chemical biology tools, imaging agents, and mitochondria-targeted therapies. Phosphonium- or nitrogen cation-based MTMs are not inert vectors and exhibit intrinsic bioactivity on mitochondrial and cellular levels to various extents. Here, we systematically evaluated a panel of N⁺-based cations to determine how structural features influence subcellular distribution and inherent bioactivity. Live-cell imaging of fluorescent dye conjugates revealed that 3,5-diphenylpyridinium (DPPy⁺) exhibits cellular uptake and mitochondrial targeting comparable to the benchmark triphenylphosphonium (TPP⁺), whereas conjugates with unsubstituted pyridinium preferentially accumulate in lysosomes. Profiling of inert cargo derivatives showed that DPPy⁺ has lower intrinsic activity on mitochondrial membrane potential and oxidative phosphorylation, as well as on cellular respiration and viability than TPP⁺. The combination of efficient mitochondrial delivery and low intrinsic bioactivity translated to bioactive cargo: a Kv1.3 inhibitor conjugate with DPPy⁺ induced apoptosis in cancer cell lines and demonstrated improved cancer selectivity relative to the TPP⁺ conjugate in pancreatic organoid models. These results position lipophilic pyridinium cations as effective TPP⁺ surrogates with enhanced biocompatibility for mitochondria-targeted therapeutic and diagnostic agents, while revealing the structure-dependent competing lysosomal accumulation of permanent nitrogen cations.