Strategies in Developing Fluorescent Probes for Live Cell Imaging and Quantitation of Hydrogen Sulfide
- 1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, USA
The involvement of hydrogen sulfide (H2S) in a number of cellular signal transduction pathways has triggered great interest in the development of selective and sensitive detection methods for H2S. This mini-review summarizes three current strategies used in the design of hydrogen sulfide-reactive fluorescent probes, including copper complexation, redox reaction, and nucleophilic cyclization. Examples from these three categories of fluorescent probes are also compared and discussed briefly.
Peng H (2013) Strategies in Developing Fluorescent Probes for Live Cell Imaging and Quantitation of Hydrogen Sulfide. JSM Biotechnol Bioeng 1(3): 1018.
• Fluorescent probes
• Hydrogen sulfide
• Cell imaging
Hydrogen sulfide (H2 S), known as a toxic gas with unpleasant rotten egg smell, is the most recently discovered gasotransmitter, joining nitric oxide (NO) and carbon monoxide (CO) . H2 S is found to participate in a number of cell signaling pathways . Its concentration is closely related to cardiovascular diseases , Down syndrome  and inflammation [5,6]. It shows protective effects in multiple systems including cardiovascular (CV)  and central nervous systems (CNS) .
Although the significance of H2 S has been revealed in a variety of systems, the accurate determination of its concentration in biological samples remains unsolved. Real-time imaging of H2 S in live cell is challenging, too. Concentration of hydrogen sulfide in blood and tissues has been reported to be 50-150 µM [9,10], while some studies indicated a much lower (nanomolar) concentration . This discrepancy could be attributed to differences in sampling techniques and detection methods as well as the actual analyte (free H2 S or total sulfide, including HS- and S2-). One of the difficulties in accurate detection is due to its volatility and reactive nature. Current detection methods of hydrogen sulfide include chromatographic, electrochemical and colorimetric/fluorometric methods. Chromatographic methods include gas chromatography (GC)  and high performance liquid chromatography (HPLC) . Electrochemical methods include sulfide ion selective electrode  and polarographic methods . Colorimetric method include the methylene blue method [15,16], which has been used as one of the standard methods for sulfide quantitation in water samples. Fluorescent probes form an attractive new method for hydrogen sulfide detection due to the convenient low-cost measurement and perfect compatibility with live cell imaging. Sulfide reactive fluorescent probes are based on metal-sulfide interaction, redox reaction and nucleophilic cyclization. Mechanisms and examples of these probes will be briefly introduced in this mini-review. For detailed mechanisms and discussion on development of H2 S reactive fluorescent probes, readers are referred to previously published reviews [17-21].
The sulfide anion shows strong intrinsic affinity for transition metals such as copper (Ksp (CuS) = 6×10-36) and mercury (Ksp (HgS) = 2×10-53). The formation of copper sulfide complex has been utilized in the design of fluorescent probes for H2 S. This was first published in 2009 by Chang and co-workers (Compound 1) for the sensing of sulfide in aqueous media . Later a number of other probes are published. These probes are composed of a fluorophore attached to heterocyclic ligands such as di-(2- picolyl)amine (DPA) (Compound 2) [22,23], cyclen (Compound 3)  and others . Chelation of Cu2+ to the ligands quenches the fluorescence. When exposed to trace amount of sulfide, Cu2+ is extracted and the fluorescence is restored. Although these metal containing sulfide reactive probes have disadvantages such as irreversible reaction and heavy metal toxicity, they are especially useful to quantitatively determine real-time H2 S concentration due to fast response and high sensitivity (LOD< 1 µM).
The reduction of azido group by sulfide  was found to be a useful strategy in the development of fluorescent probes for H2 S. In 2011, the Chang  and Wang  groups reported rhodamine (SF1 and SF2) and dansyl-based (DNS-Az) fluorescent probes, respectively. Both probes are consisted of a fluorophore attached to an azido group. The fluorescence was found to be efficiently quenched. When the azido group is reduced by sulfide anion to the corresponding amino group, the fluorescence is recovered to generate highly fluorescent products, which can be used for quantitation of H2 S. DNS-Az was used for determination of endogenous H2 S concentration in blood. Its second-generation probe 2,6-DNS-Az shows much higher fluorescence quantum yield . SF1 and SF2 were used for live cell imaging. Later the Chang group reported a series of bis-azido analogues (SF4- SF7) with improved sensitivity and cell trapping ability . Among them SF7-AM was used for imaging of endogenous H2 S generation in HUVEC cells. Other azido-based H2 S fluorescent probes have also been reported showing various features such as near infrared (NIR)  ratiometric detection (Cy-N3 ) and twophoton spectroscopic properties . In addition, other redoxsensitive moieties such as nitro , hydroxylamine  and selenium  have also proven successful for H2 S sensing. Due to the unique redox-reactivity feature, these fluorescent probes show exclusive selectivity for H2 S over other none-reducing species. Fast reaction rates (e.g. DNS-Az) also allow accurate detection H2 S.
Sulfur as exist in thiols represents one of the strongest nucleophiles in biological molecules. Unlike other biological thiols such as cysteine and glutathione, H2 S could be deprotonated twice, thus exhibits the “dual-nucleophilicity” and can be used for nucleophilic cyclization. This feature distinguishes H2 S from thiols and has been utilized for selective H2 S sensing in a number of probes. The first example was published in 2003 by the MartinezManez group, taking advantage of the pyrylium cycle reaction for sensing of sulfide anion . However, this probe only showed color change and limited sensitivity. In 2011, the Xian  and Qian  group reported two nucleophilic cyclization-based fluorescent probes 8 and 9 (SFP-1), respectively. They were both successfully used in live cell imaging for H2 S. SFP-1 was also used to monitor H2 S production by recombinant CBS. Xian group reported in 2012 the second generation probe 11 and 12 with improved selectivity over thiols due to the reversible Micheal addition site. A cyanine-based probe was also reported by Tang and co-workers showing NIR fluorescent property . The cyclization-based probes are showing good biocompatibility and exclusive selectivity for sulfide. On the other hand, due to the two-step reaction process, their reaction rates are generally low, thus are unable to accurately quantitate H2 S. However, they are still very useful in the live cell imaging.
In summary, due to the biological significance of H2 S, a great deal of research interest has been invested in developing sensitive and accurate detection methods for this small molecule. A number of fluorescent probes have been reported for live cell imaging and blood serum quantitation of H2 S. These probes are designed based on different strategies including CuS formation, redox reactions, and nucleophilic cyclization. The molecular mechanisms involving cellular functions of H2 S remains to be revealed. It is believed that these probes will serve as useful tools in H2 S detection in both research and clinical practice.
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