Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.

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Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

Coumarins are a very large family of compounds containing the unique 2H-chromen-2-one motif, as it is known according to IUPAC nomenclature. Coumarin derivatives are widely found in nature, especially in plants and are constituents of several essential oils. Up to now, thousands of coumarin derivatives have been isolated from nature or produced by chemists. More recently, the coumarin platform has been widely adopted in the design of small-molecule fluorescent chemosensors because of its excellent biocompatibility, strong and stable fluorescence emission, and good structural flexibility. This scaffold has found wide applications in the development of fluorescent chemosensors in the fields of molecular recognition, molecular imaging, bioorganic chemistry, analytical chemistry, materials chemistry, as well as in the biology and medical science communities. This review focuses on the important progress of coumarin-based small-molecule fluorescent chemosensors during the period of 2012-2018. This comprehensive and critical review may facilitate the development of more powerful fluorescent chemosensors for broad and exciting applications in the future.

Coumarin-Based Small-Molecule Fluorescent Chemosensors.

Metal cations and anions are essential for versatile physiological processes. Dysregulation of specific ion levels in living organisms is known to have an adverse effect on normal biological events. Owing to the pathophysiological significance of ions, sensitive and selective methods to detect these species in biological systems are in high demand. Because they can be used in methods for precise and quantitative analysis of ions, organic dye-based ratiometric fluorescent probes have been extensively explored in recent years. In this review, recent advances (2015-2019) made in the development and biological applications of synthetic ratiometric fluorescent probes are described. Particular emphasis is given to organic dye-based ratiometric fluorescent probes that are designed to detect biologically important and relevant ions in cells and living organisms. Also, the fundamental principles associated with the design of ratiometric fluorescent probes and perspectives about how to expand their biological applications are discussed.

Synthetic ratiometric fluorescent probes for detection of ions

Bioactive species, including reactive oxygen species (ROS, including O2˙-, H2O2, HOCl, 1O2, ˙OH, HOBr, etc.), reactive nitrogen species (RNS, including ONOO-, NO, NO2, HNO, etc.), reactive sulfur species (RSS, including GSH, Hcy, Cys, H2S, H2S n , SO2 derivatives, etc.), ATP, HCHO, CO and so on, are a highly important category of molecules in living cells. The dynamic fluctuations of these molecules in subcellular microenvironments determine cellular homeostasis, signal conduction, immunity and metabolism. However, their abnormal expressions can cause disorders which are associated with diverse major diseases. Monitoring bioactive molecules in subcellular structures is therefore critical for bioanalysis and related drug discovery. With the emergence of organelle-targeted fluorescent probes, significant progress has been made in subcellular imaging. Among the developed subcellular localization fluorescent tools, ROS, RNS and RSS (RONSS) probes are highly attractive, owing to their potential for revealing the physiological and pathological functions of these highly reactive, interactive and interconvertible molecules during diverse biological events, which are rather significant for advancing our understanding of different life phenomena and exploring new technologies for life regulation. This review mainly illustrates the design principles, detection mechanisms, current challenges, and potential future directions of organelle-targeted fluorescent probes toward RONSS.

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Fluorescent probes for organelle-targeted bioactive species imaging

Fluorescence imaging is a powerful approach for noninvasive and real-time visualization and tracking of biomolecules and biological processes in living systems. The fluorescent chemosensors with dual/triple interplaying sensing mechanisms tend to provide diverse fluorescence signals or amplify the response signals, which are propitious to simultaneously track multiple analytes or to improve the selectivity and sensitivity of the chemosensors. Thus, the development of dual/triple sensing mechanism-based chemosensors has attracted great interest recently. This review highlights the representative cases of the fluorescent chemosensors with dual/triple interplaying sensing mechanisms published since 2010, and these chemosensors are classified according to the types of the interplaying sensing mechanisms, including ICT-FRET, PET-FRET, PET-ICT-ESIPT, etc.

Fluorescent chemosensors manipulated by dual/triple interplaying sensing mechanisms

A new ratiometric fluorescent probe for rapid, sensitive and selective detection of endogenous hydrogen sulfide in mitochondria.

In this study, a novel ratiometric pH probe RNL based on fluorescence resonance energy transfer (FRET) was well developed. It was fabricated by integrating the naphthalimide moiety as an FRET donor with the rhodamine moiety as an FRET acceptor. Meanwhile, 4-(2-aminoethyl)morpholine, which was a lysosome-locating group, was introduced. The sensing mechanism was the integration of PET and FRET processes and the comprehensive effect led to the simultaneous intensity enhancement of naphthalimide and rhodamine along with the pH value decrease. With a pKa of 4.82, the fluorescence intensity ratio (I529/I580) of the probe changed significantly within the pH range from 4.50 to 5.50. The probe showed excellent selectivity among various metal cations, amino acids and ATP. Moreover, RNL has been successfully applied in HeLa cells, and the results demonstrated that it could be used to detect lysosomal pH changes. The probe could also selectively stain lysosome in HeLa cells. Besides, the probe exhibited low cytotoxicity and satisfactory photostability in living HeLa cells.

A ratiometric lysosomal pH probe based on the naphthalimide–rhodamine system

An effective colorimetric and ratiometric fluorescent probe for bisulfite in aqueous solution

In this study, we developed a coumarin–rhodamine based fluorescence resonance energy transfer (FRET) system RC1 as a ratiometric pH probe. The probe with a pKa of 4.98 was constructed by integrating a coumarin moiety as an FRET donor with a rhodamine moiety as an FRET accepter. Upon addition of H+, the coumarin emission at 477 nm decreased and the rhodamine emission at 582 nm increased simultaneously. The fluorescence intensity ratio (I477/I582) displayed excellent pH-dependent performance and responded linearly to minor pH changes in the range of 4.20–6.00. The probe exhibited brilliant selectivity among different amino acids, metal cations and the ATP. Moreover, it has been successfully applied in fluorescence imaging in HeLa cells and the results indicated that the probe could selectively stain lysosomes with low cytotoxicity and excellent photostability. We also applied RC1 to monitor intracellular pH variations induced by dexamethasone. Therefore, RC1 could act as a practical tool for the detection of pH in weakly acidic conditions and provide essential information in medicinal analysis and real biological systems.

A ratiometric lysosomal pH probe based on the coumarin–rhodamine FRET system

A ratiometric fluorescent probe DPCA based on coumarin and pyrazoline was designed and synthesized for sensing cysteine (Cys) in physiological pH. The probe can detect Cys by fluorescent spectrometry within 40 min with a detection limit of 5.08 μM. The mechanism of DPCA sensing Cys involved conjugate addition/cyclization which was confirmed by HRMS and fluorescence spectra analysis. Moreover, DPCA was able to be applied to complicated conditions with negligible interference from these species and successfully applied in cell imaging.

Tao Zhang

Papers

A new ratiometric fluorescent probe for rapid, sensitive and selective detection of endogenous hydrogen sulfide in mitochondria.

A ratiometric lysosomal pH probe based on the naphthalimide–rhodamine system

An effective colorimetric and ratiometric fluorescent probe for bisulfite in aqueous solution

A ratiometric lysosomal pH probe based on the coumarin–rhodamine FRET system

A ratiometric fluorescent probe for cysteine and its application in living cells