Mitochondria-Targeting Carriers that Do Not Induce Mitochondrial Uncoupling and Depolarization Even at High Concentrations

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Advantages

• Less likely to induce mitochondrial uncoupling ¹⁾
• Less likely to induce depolarization
• Lower toxicity than non-trifluoromethyl-substituted triphenylphosphonium (= TPP⁺)
• Carboxylic acid and NHS ester forms ― easily conjugatable to fluorescent dyes and other compounds

Reference Data 1

Comparison of the functionality of unsubstituted TPP⁺ and CF₃-substituted TPP⁺

HeLa cells were cultured overnight with either the unsubstituted TPP⁺ compound (1) or the CF₃-substituted TPP⁺compound (2) at the indicated concentrations. Mitochondrial membrane potential was observed using JC-1. Compound 2 maintained the membrane potential even at 5000 nM, resulting in red staining. Compound 1 showed almost no detectable red color.

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Reference Data 2

HeLa cells were cultured overnight with the indicated concentrations of unsubstituted TPP⁺ compound (1) and CF₃-substituted TPP⁺ compound (2). Relative cell numbers were measured using an MTT assay. The relative cell count for each condition was calculated as a ratio of the survival rate of cells cultured without additives. Compound 2でmaintained high survival rates at all concentrations, while compound 1 showed a concentration-dependent decrease in survival rate.

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Reference Data 3

Confirmation that CF₃-substituted TPP⁺ compounds accumulate in mitochondria using Coumarin 343 (Product No. C2899), a fluorescent dye conjugated with CF₃-substituted TPP⁺.

Top row: HeLa cells were co-stained with a CF₃-substituted TPP⁺ coumarin 343 conjugate (3) and MitoTracker® Deep Red.
Bottom row: HeLa cells were co-stained with Coumarin 343 (4) and MitoTracker® Deep Red.
Staining was observed at an excitation wavelength of 470 nm and an emission wavelength of 525 nm for Coumarin 343, while MitoTracker® Deep Red was observed at an excitation wavelength of 640 nm and an emission wavelength of 690 nm. Compound 4 showed almost no staining; however, the staining patterns of compound 3 and MitoTracker® Deep Red largely overlapped, suggesting that they target the same cellular regions.

† MitoTracker® is a registered trademark of Molecular Probes, Inc.
‡ The mitochondrial targeting of molecules conjugated with TPP⁺ is known to be influenced by the charge and hydrophobicity of the molecular species.⁴⁾

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We confirmed that, while accumulating in mitochondria, CF₃-substituted TPP⁺ compounds are less likely to induce mitochondrial depolarization and exhibit lower cytotoxicity than unsubstituted TPP⁺ compounds. There are reports of cases where metformin modified with CF₃-substituted TPP⁺ showed reduced in vivo toxicity while maintaining antitumor efficacy,²⁾ and cases where a CF₃-substituted TPP⁺-based phenylbutyric acid (PBA) conjugate displayed greatly enhanced mitochondrial accumulation compared to PBA alone, resulting in strong tumor growth inhibition.²⁾ These compounds are also valuable for studying heart failure, ischemia-reperfusion injury, and Alzheimer's disease, as reactive oxygen species (ROS) arising from mitochondrial dysfunction or abnormalities are implicated in these conditions.

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References

1. 1) A Novel Triphenylphosphonium Carrier to Target Mitochondria without Uncoupling Oxidative Phosphorylation
   • C. A. Kulkarni, R. J. Kerns, et al., J. Med. Chem. 2021, 64, 662.
2. 2) Fluorinated triphenylphosphonium analogs improve cell selectivity and in vivo detection of mito-metformin
   • M. AbuEid, M. B. Dwinell, et al., iScience. 2022, 25, 105670.
3. 3) A Mitochondria-Targeted Phenylbutyric Acid Prodrug Confers Drastically Improved Anticancer Activities
   • D. Huang, Y. He, et al., J. Med. Chem. 2022, 65, 9955.
4. 4) Reaction-based fluorescent sensor for investigating mobile Zn²⁺ in mitochondria of healthy versus cancerous prostate cells
   • W. Chyan, R. J. Radford, et al., Proc. Natl. Acad. Sci. USA. 2014, 111, 143.

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