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Review
Fluorescent probes for hydrogen sulfide (H2S) and sulfane sulfur and their applications to biological studies Q1 Kazuhito Shimamoto, Kenjiro Hanaoka * Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
A R T I C L E
I N F O
Article history: Received 9 September 2014 Revised 18 October 2014 Accepted 10 November 2014 Available online Keywords: Hydrogen sulfide Sulfane sulfur Fluorescent probes
A B S T R A C T
Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, plays key roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. The most commonly used methods or detecting H2S are the methylene blue method and the electrode method, but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (R—S—SH) and polysulfides (R—S—Sn—S—R). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH). Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies. © 2014 Published by Elsevier Inc.
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Contents 1. 2. 3.
4. 5. 6. 7. 8.
Introduction ............................................................................................................................................................................................................................................................. Chemical characteristics of H2S ......................................................................................................................................................................................................................... Development of fluorescent probes for H2S ................................................................................................................................................................................................. 3.1. Fluorescent probes based on reduction of azide group to amine ........................................................................................................................................... 3.2. Fluorescent probes based on reduction of nitro group to amine ............................................................................................................................................ 3.3. Fluorescent probes based on the nucleophilicity of HS– ............................................................................................................................................................ 3.4. Fluorescent probes based on the quenching effect of copper ion (Cu2+) .............................................................................................................................. Physiological functions of sulfane sulfur ....................................................................................................................................................................................................... Development of fluorescent probes for sulfane sulfur ............................................................................................................................................................................. Biological applications of developed fluorescent probes for H2S and sulfane sulfur ..................................................................................................................... Applications for development of chemical tools such as enzymatic inhibitors and caged H2S donors .................................................................................. Conclusions and prospects ................................................................................................................................................................................................................................. Acknowledgments ................................................................................................................................................................................................................................................. References ................................................................................................................................................................................................................................................................
1. Introduction
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* Corresponding author. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan. Fax: +81 3 5841 4855. E-mail address: [email protected] (K. Hanaoka).
62 63 Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, Q2 64 65 plays key roles in many physiological processes, including relax66 ation of vascular smooth muscles [1,2], mediation of neurotrans67 mission [3,4], inhibition of insulin signaling [5], and regulation 68 of inflammation [6,7]. The most commonly used methods for
http://dx.doi.org/10.1016/j.niox.2014.11.008 1089-8603/© 2014 Published by Elsevier Inc.
Please cite this article in press as: Kazuhito Shimamoto, Kenjiro Hanaoka, Fluorescent probes for hydrogen sulfide (H2S) and sulfane sulfur and their applications to biological studies, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.11.008
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detecting H2S are the methylene blue method [8], the electrode method [9] and the monobromobimane method [10,11], but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported [12,13]. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (R—S—SH) and polysulfides (R—S—Sn—S—R). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH) [2,14]. Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration [15]. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies. 2. Chemical characteristics of H2S H2S is highly water-soluble, with pKa1 of 6.8 and pKa2 of over 13 [16]; thus, at pH 7.4, 80% of H2S exists as HS–, which is strongly nucleophilic. In addition, H2S itself shows reducing ability, changing the oxidation state of sulfur from –2 (H2S) to 0 (S); for example, it can reduce azide and nitro groups [17,18]. These properties of H2S have been utilized to design fluorescence off/on switching mechanisms for the development of fluorescent probes. 3. Development of fluorescent probes for H2S 3.1. Fluorescent probes based on reduction of azide group to amine The first fluorescent probes utilizing azide reduction were SF1 and SF2, reported by Chang et al. (Fig. 1a) [19]. Reduction of the azide group of the xanthene moiety to amine causes opening of the intramolecular spirocycle of the rhodamine scaffold; this restores the conjugated system of the xanthene moiety, leading to strong fluorescence (ФFl = 0.50 and 0.60, respectively). SF1 and SF2 showed seven- and ninefold fluorescence increases, respectively, within 1 hr after addition of 100 μM NaHS. Chang et al. subsequently developed improved probes (SF4–7, Fig. 1a), with enhanced sensitivity and cellular retention, using the same strategy [20]. This approach can also be applied to other fluorophores; indeed, the azidesubstituted dansyl fluorophore, DNS-Az (Fig. 1b), showed a faster reaction rate than the SF series [21]. H2S fluorescent probes targeting specific organelles have also been developed. Kim et al. reported two fluorescent probes, SHS-M1 and SHS-M2, which have a triphenylphosphonium group as a mitochondrial targeting moiety (Fig. 1c) [22]. Yang et al. reported SulpHensor, which possesses a morpholine group as a lysosome targeting moiety and showed strong fluorescence in the presence of H2S only under acidic conditions, as found in lysosomes (Fig. 1d) [23]. Thus, various types of H2S fluorescent probes utilizing azide group reduction have been reported [12,13]. 3.2. Fluorescent probes based on reduction of nitro group to amine Like the azide group, the nitro group can be reduced by H2S, and this reaction has also been used as a fluorescence off/on switching mechanism for fluorescent probes targeting H2S. Pluth and Montoya reported a fluorescent probe, HSN1, based on this design concept (Fig. 2 left) [24], and Yu et al. reported colorimetric and ratiometric
probes (Fig. 2 right) [25]. Thus, nitro group reduction is also a useful strategy for the molecular design of H2S fluorescent probes. 3.3. Fluorescent probes based on the nucleophilicity of HS– H2S (HS–) has strong nucleophilicity, and this was utilized by Qian et al. to design fluorescent probes, SFP-1 and SFP-2, that target HS– (Fig. 3a) [26]. Fluorescence switching occurs via HS– addition to aldehyde, followed by Michael addition of the resulting hemithioacetal to unsaturated acrylate ester to form a thioacetal, under physiological conditions. The resulting stable tetrahydrothiophene shows strong fluorescence. Xian et al. reported a fluorescein-based probe, WSP1-5 (Fig. 3b) [27,28], in which the disulfide bond is cleaved by H2S followed by intramolecular nucleophilic attack of the persulfide group on the ester moiety to release the fluorophore, with a large fluorescence increase. Guo et al. reported a reversible fluorescent probe, CouMC (Fig. 3c) [29], based on the merocyanine scaffold; this simple spiropyran structure undergoes a rapid, reversible nucleophilic addition, and the fluorescence reaches a plateau within seconds. 3.4. Fluorescent probes based on the quenching effect of copper ion (Cu2+) It is well known that heavy metal ions such as iron (III) ion (Fe3+) and Cu2+ quench the fluorescence of a nearby fluorophore [30]. Chang et al. used this phenomenon to obtain a H2S fluorescent probe (Fig. 4a) [31]. In this molecular design, a dipicolylamine moiety was used as a chelator for Cu 2+ , which served as the fluorescence quencher, and the fluorescein scaffold was used as the fluorophore. This probe showed a rapid fluorescence increase after addition of 100 μM Na2S, but it also reacted with GSH (glutathione) at millimolar concentration, which is typically present under physiological conditions, showing a rapid and large fluorescence increase. So, this fluorescent probe lacks selectivity for H2S among biothiols and is not suitable for biological applications. On the other hand, our group hypothesized that this low selectivity was related to the stability of the chelator–Cu2+ complex, and we synthesized a H2S fluorescent probe, HSip-1, in which Cu2+ is complexed with an azamacrocyclic ring (Fig. 4b) [32]. We expected that Cu2+ would be released from the azamacrocyclic ring when H2S binds to the Cu2+ center, resulting in a large fluorescence enhancement. Indeed, this fluorescent probe showed a large and fast fluorescence increase after addition of 10 μM Na2S. Further, this probe showed good water-solubility, so that DMSO (dimethylsulfoxide) or detergents are not required as cosolvents. Therefore, this probe is expected to be useful for biological studies in vitro and in vivo. Although these types of probes release CuS which may show toxicity to live cells, HSip-1 showed no cytotoxicity [32]. Some applications will be described later. 4. Physiological functions of sulfane sulfur One mechanism of activity regulation of proteins by H2S is thought to be sulfhydration of cysteine residues (RSH→RSSH) [2,14]. However, sulfhydration of cysteine is an oxidation reaction, and H2S is basically a reductant [17]. So, it is thought that the sulfhydration cannot occur by H2S directly (Fig. 5a). An oxidant is probably needed for the reaction of H2S with a thiol to form a persulfide. On the other hand, sulfane sulfur is a partially-oxidized form of sulfur, i.e., it contains a form of sulfur with six valence electrons, no charge (S0), and it shows the unique ability to reversibly bind to other sulfur atoms, as seen in elemental sulfur (S8), persulfides (R—S—SH), and polysulfides (R—S—Sn—S—R). Thus, as shown in Fig. 5b, it is considered that sulfhydration reactions involving H2S may be mediated by sulfane sulfur.
Please cite this article in press as: Kazuhito Shimamoto, Kenjiro Hanaoka, Fluorescent probes for hydrogen sulfide (H2S) and sulfane sulfur and their applications to biological studies, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.11.008
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Fig. 1. Fluorescent probes for H2S utilizing reduction of the azide group, based on xanthene dye (a) and dansyl dye (b), including probes targeted to mitochondria (c) and lysosomes (d).
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6. Biological applications of developed fluorescent probes for H2S and sulfane sulfur
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Fig. 2. Fluorescent probes for H2S utilizing reduction of a nitro group.
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nucleophilic attack of persulfide on the ester moiety occurs, releasing the fluorophore (Fig. 6c). They further reported a fluorescent probe for hydrogen polysulfide (H2Sn, n > 1), which can be considered as oxidized forms of H2S (Fig. 6b) [34]. H2S and sulfane sulfur are redox partners and therefore are very likely to coexist in biological systems. From the viewpoint of reactivity, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration, and the above-mentioned fluorescent probe utilized this reactivity (Fig. 6d). Thus, nucleophilic aromatic substitution of H2Sn (H2S2) with the probe affords the perfulfide intermediate, in which intramolecular nucleophilic attack of persulfide on the ester moiety takes place, releasing the fluorophore. This probe is specific for hydrogen polysulfide (–S—Sn—S–).
5. Development of fluorescent probes for sulfane sulfur Xian et al. reported fluorescent probes for sulfane sulfur (Fig. 6a) [33]. The probe design strategy is as follows: sulfane sulfur attaches a sulfur atom to the thiol group of the thiobenzene moiety of the probe, affording persulfide (R—SS–). Then, intramolecular
Various biological applications of fluorescent probes for H2S and sulfane sulfur, including fluorescence imaging, have been reported. So far, almost all live-cell fluorescence imaging experiments have employed an artificial H2S-releasing system, i.e., NaHS (Na2S) was added to the medium of cultured cells to artificially increase
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Fig. 3. Fluorescent probes for H2S utilizing nucleophilic reaction of the sulfide.
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Fig. 4. Fluorescent probes based on the fluorescence quenching effect of Cu2+.
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the concentration of H2S inside the cells. However, a few fluorescent probes are able to image endogenous concentrations of H2S in live cells, and reported applications of these probes will be reviewed later. Chang et al. reported azide-based H2S fluorescent probes with high sensitivity and high intracellular retention, and used them to detect endogenous H2S in live cells [20]. Their earliest probes, SF1 and SF2, were insufficiently sensitive to detect endogenous H2S production, and to improve the detection limit, they incorporated two azide groups, and also introduced two acetoxymethyl (AM) ester-protected carboxyl groups to improve membrane permeability and obtain high retention of the probe inside cells after intracellular hydrolysis of the AM moieties. One of the improved probes, SF7-AM, was loaded into human umbilical vein endothelial cells (HUVECs) and then the cells were stimulated with VEGF (vascular endothelial growth factor). The probe successfully visualized VEGF-triggered H2S production in live cells. They also investigated the signal pathway by using both SF7-AM and VEGF2 inhibitor (AAL993). When AAL-993 was added to the cells, the fluorescence intensity of SF7 was decreased, which indicates that VEGF stimulation promotes H2S production inside the cells. They hypothesized that H2S production through this pathway is related to H2O2 production, because H2O2 has been implicated as an early-response second messenger in growth factor signaling [35]. In order to test the idea that VEGF stimulation activates NADPH oxidase (Nox) to produce H2O2, they used an H2O2 scavenger (PEG-catalase), Nox inhibitors (diphenyleneiodonium chloride and gp21ds-tat), and fluorescent probe SF7-AM. They found that generation of H2O2 by Nox enhanced phosphorylation of VGEF2, and this triggered a positive feedback loop for H2S production. These results demonstrated crosstalk between VEGF stimulation and H2S production mediated by H2O2. Furthermore, another fluorescent probe in the SF series, SF5, was used by Wallace et al. to investigate the relationship between inflammation and H2S production in vitro and in vivo [36]. They found that the fluorescence intensity of SF5 was decreased both in vitro and in vivo by inflammatory stimulation, and this result
indicated that the activities of CBS (cystathionine β-synthase) and CSE (cystathionine γ-lyase) were also decreased by inflammatory stimulation. However, other researchers have reported both positive [6,37] and negative [38,39] regulation of H2S production and CSE expression by inflammatory stimulation. Thus, new chemical tools, including fluorescent probes and inhibitors, are needed to fully elucidate the mechanisms of these H2S-related biological events. HSip-1, which we developed, has also been used for biological studies. For example, Stipanuk et al. examined the production of H2S from cysteine in primary hepatocytes with HSip-1 [40]. Hepatocytes were cultured with HSip-1, and the medium was transferred to a 96well plate. The fluorescence of HSip-1 was measured using a plate reader to evaluate the amount of extracellular H2S. This approach makes use of the fact that H2S is cell-membrane-permeable, and H2S released from cells can be trapped by the probe in the external medium. It is considered that the HS−/H2S chemical equilibrium is maintained in the medium, and H2S still exhibits nucleophilicity toward the Cu2+ center of HSip-1 in the extracellular environment [41,42]. The results of these experiments indicated that hepatocytes from cysteine dioxygenase (CDO)-knockout mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. Oxidation of cysteine in mammalian cells occurs via two routes: the highly regulated direct oxidation pathway, in which the first step is catalyzed by CDO, and the desulfhydration–oxidation pathway catalyzed by CSE and CBS, in which the sulfur is released in a reduced oxidation state (such as H2S). Knockout of CDO in hepatocytes increases the level of cysteine, which serves as a substrate of CSE and CBS, resulting in increased levels of H2S and thiosulfate (an intermediate in the oxidation of H2S to sulfate). Thus, HSip-1 was useful for measuring H2S produced by CSE and CBS in live cells. Ichiose et al. used both HSip-1 and SSP4 to measure H2S and sulfane sulfur in SH-SY5Y cells and in the medium [43]. They investigated the protective effects of H2S-releasing N-methyl-D-aspartate receptor antagonists against 1-methyl-4-phenylpyridinium (MMP+, a metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine))-induced cell death. MPTP induces neurodegeneration of dopaminergic neurons in mammalian midbrain, which leads to Parkinson’s disease-like symptoms. Based on their experiments, they suggested that H2S-donor compounds increased intracellular sulfane sulfur levels and are potentially useful as neuroprotective agents against neurodegenerative diseases.
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Fig. 5. S-sulfhydration reaction. (a) Hydrosulfide is ineffective, whereas (b) sulfane sulfur is much more reactive.
Fluorescent probes for H2S are useful not only for biological applications, but also for the development of chemical tools for H2S studies. One example would be high-throughput screening (HTS) to find inhibitors of the three H2S-producing enzymes, CSE, CBS and
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Fig. 6. (a) Fluorescent probes for sulfane sulfur and (b) selective fluorescent probes for hydrogen persulfide. The reaction mechanisms of (c) SSP and (d) DSP with sulfane sulfur and hydrogen persulfide are shown.
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3MST (3-mercaptopyruvate sulfurtransferase). Inhibitor screening assay based on fluorescence detection has great advantages in terms of speed and convenience, and is suitable for HTS of large chemical libraries. Prior to HTS, some inhibitors of CBS and CSE had been reported, but they have disadvantages such as poor selectivity and relatively weak affinity for the targeted enzyme [44–50]. No inhibitor of 3MST had been reported. Barrios et al. performed CBS inhibitor screening of 1900 chemical compounds by using 7-azide-4-methylcoumarin (AzMC) (Fig. 7a), and found several inhibitors [51]. As we presented at the Third International Conference on Hydrogen Sulfide in Biology and Medicine (H2S2014), we also performed HTS for inhibitors of CSE and 3MST in a chemical library containing about 160,000 compounds by using HSip-1 [52]. In the assay, purified 3MST or CSE and the corresponding substrate (3-mercaptopyruvate (3-MP) or cysteine) were used, and HSip-1 reacted with H2S released by the enzymatic reaction. Compounds that suppressed the fluorescence increase of HSip-1 in response to H2S were selected as inhibitors (Fig. 7b,c). After further examination of the selectivity of the hit compounds, we obtained
both 3MST- and CSE-selective inhibitors. We are currently investigating the utility of these inhibitors for biological studies. HSip-1 was also useful during the development of H2S-caged compounds. For example, Nakagawa et al. developed a photocontrollable hydrogen sulfide donor and during this research, they used HSip-1 for the detection of H2S released from the developed H2S-caged compound [53]. 8. Conclusions and prospects Here, we have reviewed selective fluorescent probes for H2S and sulfane sulfur, and their biological applications. From the viewpoint of probe design, properties such as reducing capacity, nucleophilicity and ability to form a complex with Cu2+ have been utilized for fluorescence off/on switching. Fluorescent probes are expected to be useful tools for nondestructive, real-time detection of H2S or sulfane sulfur in biological samples such as live cells or tissues, but although many H2S fluorescent probes have been reported, only a few probes are yet capable of detecting endogenous
Please cite this article in press as: Kazuhito Shimamoto, Kenjiro Hanaoka, Fluorescent probes for hydrogen sulfide (H2S) and sulfane sulfur and their applications to biological studies, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.11.008
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(b) Negative compounds L-cysteine or 3-MP (Substrate)
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(c) Positive compounds (hit compounds) compound
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Fig. 7. (a) Chemical structure of AzMC. (b,c) Screening schemes for 3MST and CSE inhibitors.
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H2S production in biological contexts. Further, although some fluorescent probes for sulfane sulfur have been developed, there is no selective fluorescent probe for persulfides, such as cysteine persulfide (Cys—S—S–), glutathione persulfide (G—S—S–). This is important, because Akaike et al. reported that cysteine persulfide (Cys—S—S–) formation from cystine was catalyzed by CSE [54]. Thus, there is a need for fluorescent probes that can distinguish various biothiol species for further studies on the biological functions of H2S and sulfane sulfur. We are engaged in developing new fluorescent probes for this purpose.
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Q3 Acknowledgments This work was supported in part by MEXT (Grant Nos. 24689003, 24659042 and 26104509 to K.H.). K.H. was also supported by Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Naito Foundation, the Asahi Glass Foundation, and Takeda Science Foundation.
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Review
Fluorescent probes for hydrogen sulfide (H2S) and sulfane sulfur and their applications to biological studies Q1 Kazuhito Shimamoto, Kenjiro Hanaoka * Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
A R T I C L E
I N F O
Article history: Received 9 September 2014 Revised 18 October 2014 Accepted 10 November 2014 Available online Keywords: Hydrogen sulfide Sulfane sulfur Fluorescent probes
A B S T R A C T
Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, plays key roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, and regulation of inflammation. The most commonly used methods or detecting H2S are the methylene blue method and the electrode method, but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (R—S—SH) and polysulfides (R—S—Sn—S—R). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH). Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies. © 2014 Published by Elsevier Inc.
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Contents 1. 2. 3.
4. 5. 6. 7. 8.
Introduction ............................................................................................................................................................................................................................................................. Chemical characteristics of H2S ......................................................................................................................................................................................................................... Development of fluorescent probes for H2S ................................................................................................................................................................................................. 3.1. Fluorescent probes based on reduction of azide group to amine ........................................................................................................................................... 3.2. Fluorescent probes based on reduction of nitro group to amine ............................................................................................................................................ 3.3. Fluorescent probes based on the nucleophilicity of HS– ............................................................................................................................................................ 3.4. Fluorescent probes based on the quenching effect of copper ion (Cu2+) .............................................................................................................................. Physiological functions of sulfane sulfur ....................................................................................................................................................................................................... Development of fluorescent probes for sulfane sulfur ............................................................................................................................................................................. Biological applications of developed fluorescent probes for H2S and sulfane sulfur ..................................................................................................................... Applications for development of chemical tools such as enzymatic inhibitors and caged H2S donors .................................................................................. Conclusions and prospects ................................................................................................................................................................................................................................. Acknowledgments ................................................................................................................................................................................................................................................. References ................................................................................................................................................................................................................................................................
1. Introduction
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* Corresponding author. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan. Fax: +81 3 5841 4855. E-mail address: [email protected] (K. Hanaoka).
62 63 Hydrogen sulfide (H2S), a toxic gas with the smell of rotten eggs, Q2 64 65 plays key roles in many physiological processes, including relax66 ation of vascular smooth muscles [1,2], mediation of neurotrans67 mission [3,4], inhibition of insulin signaling [5], and regulation 68 of inflammation [6,7]. The most commonly used methods for
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detecting H2S are the methylene blue method [8], the electrode method [9] and the monobromobimane method [10,11], but these methods require destructive sampling, e.g., homogenization of biological samples. On the other hand, the fluorescence detection method has been widely used in biological studies to study the physiological roles of H2S, because this technology provides real-time, easy-to-use, nondestructive detection in live cells or tissues. Many selective fluorescent probes for H2S have been reported [12,13]. Sulfane sulfur compounds contain divalent sulfur atoms bonded to other sulfur atom(s), as in persulfides (R—S—SH) and polysulfides (R—S—Sn—S—R). They are currently attracting increasing interest because one of the mechanisms of activity regulation of proteins by H2S is sulfhydration of cysteine residues (RSH→RSSH) [2,14]. Since H2S and sulfane sulfur are redox partners, they are very likely to coexist in biological systems, and from a reactivity point-of-view, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration [15]. Therefore, sulfane sulfur may be involved in mediating at least some of the biological activities of H2S. In this review, we summarize recent work on fluorescent probes selective for H2S and/or sulfane sulfur, and we briefly review their applications to biological studies. 2. Chemical characteristics of H2S H2S is highly water-soluble, with pKa1 of 6.8 and pKa2 of over 13 [16]; thus, at pH 7.4, 80% of H2S exists as HS–, which is strongly nucleophilic. In addition, H2S itself shows reducing ability, changing the oxidation state of sulfur from –2 (H2S) to 0 (S); for example, it can reduce azide and nitro groups [17,18]. These properties of H2S have been utilized to design fluorescence off/on switching mechanisms for the development of fluorescent probes. 3. Development of fluorescent probes for H2S 3.1. Fluorescent probes based on reduction of azide group to amine The first fluorescent probes utilizing azide reduction were SF1 and SF2, reported by Chang et al. (Fig. 1a) [19]. Reduction of the azide group of the xanthene moiety to amine causes opening of the intramolecular spirocycle of the rhodamine scaffold; this restores the conjugated system of the xanthene moiety, leading to strong fluorescence (ФFl = 0.50 and 0.60, respectively). SF1 and SF2 showed seven- and ninefold fluorescence increases, respectively, within 1 hr after addition of 100 μM NaHS. Chang et al. subsequently developed improved probes (SF4–7, Fig. 1a), with enhanced sensitivity and cellular retention, using the same strategy [20]. This approach can also be applied to other fluorophores; indeed, the azidesubstituted dansyl fluorophore, DNS-Az (Fig. 1b), showed a faster reaction rate than the SF series [21]. H2S fluorescent probes targeting specific organelles have also been developed. Kim et al. reported two fluorescent probes, SHS-M1 and SHS-M2, which have a triphenylphosphonium group as a mitochondrial targeting moiety (Fig. 1c) [22]. Yang et al. reported SulpHensor, which possesses a morpholine group as a lysosome targeting moiety and showed strong fluorescence in the presence of H2S only under acidic conditions, as found in lysosomes (Fig. 1d) [23]. Thus, various types of H2S fluorescent probes utilizing azide group reduction have been reported [12,13]. 3.2. Fluorescent probes based on reduction of nitro group to amine Like the azide group, the nitro group can be reduced by H2S, and this reaction has also been used as a fluorescence off/on switching mechanism for fluorescent probes targeting H2S. Pluth and Montoya reported a fluorescent probe, HSN1, based on this design concept (Fig. 2 left) [24], and Yu et al. reported colorimetric and ratiometric
probes (Fig. 2 right) [25]. Thus, nitro group reduction is also a useful strategy for the molecular design of H2S fluorescent probes. 3.3. Fluorescent probes based on the nucleophilicity of HS– H2S (HS–) has strong nucleophilicity, and this was utilized by Qian et al. to design fluorescent probes, SFP-1 and SFP-2, that target HS– (Fig. 3a) [26]. Fluorescence switching occurs via HS– addition to aldehyde, followed by Michael addition of the resulting hemithioacetal to unsaturated acrylate ester to form a thioacetal, under physiological conditions. The resulting stable tetrahydrothiophene shows strong fluorescence. Xian et al. reported a fluorescein-based probe, WSP1-5 (Fig. 3b) [27,28], in which the disulfide bond is cleaved by H2S followed by intramolecular nucleophilic attack of the persulfide group on the ester moiety to release the fluorophore, with a large fluorescence increase. Guo et al. reported a reversible fluorescent probe, CouMC (Fig. 3c) [29], based on the merocyanine scaffold; this simple spiropyran structure undergoes a rapid, reversible nucleophilic addition, and the fluorescence reaches a plateau within seconds. 3.4. Fluorescent probes based on the quenching effect of copper ion (Cu2+) It is well known that heavy metal ions such as iron (III) ion (Fe3+) and Cu2+ quench the fluorescence of a nearby fluorophore [30]. Chang et al. used this phenomenon to obtain a H2S fluorescent probe (Fig. 4a) [31]. In this molecular design, a dipicolylamine moiety was used as a chelator for Cu 2+ , which served as the fluorescence quencher, and the fluorescein scaffold was used as the fluorophore. This probe showed a rapid fluorescence increase after addition of 100 μM Na2S, but it also reacted with GSH (glutathione) at millimolar concentration, which is typically present under physiological conditions, showing a rapid and large fluorescence increase. So, this fluorescent probe lacks selectivity for H2S among biothiols and is not suitable for biological applications. On the other hand, our group hypothesized that this low selectivity was related to the stability of the chelator–Cu2+ complex, and we synthesized a H2S fluorescent probe, HSip-1, in which Cu2+ is complexed with an azamacrocyclic ring (Fig. 4b) [32]. We expected that Cu2+ would be released from the azamacrocyclic ring when H2S binds to the Cu2+ center, resulting in a large fluorescence enhancement. Indeed, this fluorescent probe showed a large and fast fluorescence increase after addition of 10 μM Na2S. Further, this probe showed good water-solubility, so that DMSO (dimethylsulfoxide) or detergents are not required as cosolvents. Therefore, this probe is expected to be useful for biological studies in vitro and in vivo. Although these types of probes release CuS which may show toxicity to live cells, HSip-1 showed no cytotoxicity [32]. Some applications will be described later. 4. Physiological functions of sulfane sulfur One mechanism of activity regulation of proteins by H2S is thought to be sulfhydration of cysteine residues (RSH→RSSH) [2,14]. However, sulfhydration of cysteine is an oxidation reaction, and H2S is basically a reductant [17]. So, it is thought that the sulfhydration cannot occur by H2S directly (Fig. 5a). An oxidant is probably needed for the reaction of H2S with a thiol to form a persulfide. On the other hand, sulfane sulfur is a partially-oxidized form of sulfur, i.e., it contains a form of sulfur with six valence electrons, no charge (S0), and it shows the unique ability to reversibly bind to other sulfur atoms, as seen in elemental sulfur (S8), persulfides (R—S—SH), and polysulfides (R—S—Sn—S—R). Thus, as shown in Fig. 5b, it is considered that sulfhydration reactions involving H2S may be mediated by sulfane sulfur.
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Fig. 1. Fluorescent probes for H2S utilizing reduction of the azide group, based on xanthene dye (a) and dansyl dye (b), including probes targeted to mitochondria (c) and lysosomes (d).
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6. Biological applications of developed fluorescent probes for H2S and sulfane sulfur
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Fig. 2. Fluorescent probes for H2S utilizing reduction of a nitro group.
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nucleophilic attack of persulfide on the ester moiety occurs, releasing the fluorophore (Fig. 6c). They further reported a fluorescent probe for hydrogen polysulfide (H2Sn, n > 1), which can be considered as oxidized forms of H2S (Fig. 6b) [34]. H2S and sulfane sulfur are redox partners and therefore are very likely to coexist in biological systems. From the viewpoint of reactivity, sulfane sulfur seems likely to be much more effective than H2S in S-sulfhydration, and the above-mentioned fluorescent probe utilized this reactivity (Fig. 6d). Thus, nucleophilic aromatic substitution of H2Sn (H2S2) with the probe affords the perfulfide intermediate, in which intramolecular nucleophilic attack of persulfide on the ester moiety takes place, releasing the fluorophore. This probe is specific for hydrogen polysulfide (–S—Sn—S–).
5. Development of fluorescent probes for sulfane sulfur Xian et al. reported fluorescent probes for sulfane sulfur (Fig. 6a) [33]. The probe design strategy is as follows: sulfane sulfur attaches a sulfur atom to the thiol group of the thiobenzene moiety of the probe, affording persulfide (R—SS–). Then, intramolecular
Various biological applications of fluorescent probes for H2S and sulfane sulfur, including fluorescence imaging, have been reported. So far, almost all live-cell fluorescence imaging experiments have employed an artificial H2S-releasing system, i.e., NaHS (Na2S) was added to the medium of cultured cells to artificially increase
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Fig. 3. Fluorescent probes for H2S utilizing nucleophilic reaction of the sulfide.
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Fig. 4. Fluorescent probes based on the fluorescence quenching effect of Cu2+.
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the concentration of H2S inside the cells. However, a few fluorescent probes are able to image endogenous concentrations of H2S in live cells, and reported applications of these probes will be reviewed later. Chang et al. reported azide-based H2S fluorescent probes with high sensitivity and high intracellular retention, and used them to detect endogenous H2S in live cells [20]. Their earliest probes, SF1 and SF2, were insufficiently sensitive to detect endogenous H2S production, and to improve the detection limit, they incorporated two azide groups, and also introduced two acetoxymethyl (AM) ester-protected carboxyl groups to improve membrane permeability and obtain high retention of the probe inside cells after intracellular hydrolysis of the AM moieties. One of the improved probes, SF7-AM, was loaded into human umbilical vein endothelial cells (HUVECs) and then the cells were stimulated with VEGF (vascular endothelial growth factor). The probe successfully visualized VEGF-triggered H2S production in live cells. They also investigated the signal pathway by using both SF7-AM and VEGF2 inhibitor (AAL993). When AAL-993 was added to the cells, the fluorescence intensity of SF7 was decreased, which indicates that VEGF stimulation promotes H2S production inside the cells. They hypothesized that H2S production through this pathway is related to H2O2 production, because H2O2 has been implicated as an early-response second messenger in growth factor signaling [35]. In order to test the idea that VEGF stimulation activates NADPH oxidase (Nox) to produce H2O2, they used an H2O2 scavenger (PEG-catalase), Nox inhibitors (diphenyleneiodonium chloride and gp21ds-tat), and fluorescent probe SF7-AM. They found that generation of H2O2 by Nox enhanced phosphorylation of VGEF2, and this triggered a positive feedback loop for H2S production. These results demonstrated crosstalk between VEGF stimulation and H2S production mediated by H2O2. Furthermore, another fluorescent probe in the SF series, SF5, was used by Wallace et al. to investigate the relationship between inflammation and H2S production in vitro and in vivo [36]. They found that the fluorescence intensity of SF5 was decreased both in vitro and in vivo by inflammatory stimulation, and this result
indicated that the activities of CBS (cystathionine β-synthase) and CSE (cystathionine γ-lyase) were also decreased by inflammatory stimulation. However, other researchers have reported both positive [6,37] and negative [38,39] regulation of H2S production and CSE expression by inflammatory stimulation. Thus, new chemical tools, including fluorescent probes and inhibitors, are needed to fully elucidate the mechanisms of these H2S-related biological events. HSip-1, which we developed, has also been used for biological studies. For example, Stipanuk et al. examined the production of H2S from cysteine in primary hepatocytes with HSip-1 [40]. Hepatocytes were cultured with HSip-1, and the medium was transferred to a 96well plate. The fluorescence of HSip-1 was measured using a plate reader to evaluate the amount of extracellular H2S. This approach makes use of the fact that H2S is cell-membrane-permeable, and H2S released from cells can be trapped by the probe in the external medium. It is considered that the HS−/H2S chemical equilibrium is maintained in the medium, and H2S still exhibits nucleophilicity toward the Cu2+ center of HSip-1 in the extracellular environment [41,42]. The results of these experiments indicated that hepatocytes from cysteine dioxygenase (CDO)-knockout mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. Oxidation of cysteine in mammalian cells occurs via two routes: the highly regulated direct oxidation pathway, in which the first step is catalyzed by CDO, and the desulfhydration–oxidation pathway catalyzed by CSE and CBS, in which the sulfur is released in a reduced oxidation state (such as H2S). Knockout of CDO in hepatocytes increases the level of cysteine, which serves as a substrate of CSE and CBS, resulting in increased levels of H2S and thiosulfate (an intermediate in the oxidation of H2S to sulfate). Thus, HSip-1 was useful for measuring H2S produced by CSE and CBS in live cells. Ichiose et al. used both HSip-1 and SSP4 to measure H2S and sulfane sulfur in SH-SY5Y cells and in the medium [43]. They investigated the protective effects of H2S-releasing N-methyl-D-aspartate receptor antagonists against 1-methyl-4-phenylpyridinium (MMP+, a metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine))-induced cell death. MPTP induces neurodegeneration of dopaminergic neurons in mammalian midbrain, which leads to Parkinson’s disease-like symptoms. Based on their experiments, they suggested that H2S-donor compounds increased intracellular sulfane sulfur levels and are potentially useful as neuroprotective agents against neurodegenerative diseases.
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80 7. Applications for development of chemical tools such as enzymatic inhibitors and caged H2S donors
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Fig. 5. S-sulfhydration reaction. (a) Hydrosulfide is ineffective, whereas (b) sulfane sulfur is much more reactive.
Fluorescent probes for H2S are useful not only for biological applications, but also for the development of chemical tools for H2S studies. One example would be high-throughput screening (HTS) to find inhibitors of the three H2S-producing enzymes, CSE, CBS and
Please cite this article in press as: Kazuhito Shimamoto, Kenjiro Hanaoka, Fluorescent probes for hydrogen sulfide (H2S) and sulfane sulfur and their applications to biological studies, Nitric Oxide (2014), doi: 10.1016/j.niox.2014.11.008
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Fig. 6. (a) Fluorescent probes for sulfane sulfur and (b) selective fluorescent probes for hydrogen persulfide. The reaction mechanisms of (c) SSP and (d) DSP with sulfane sulfur and hydrogen persulfide are shown.
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3MST (3-mercaptopyruvate sulfurtransferase). Inhibitor screening assay based on fluorescence detection has great advantages in terms of speed and convenience, and is suitable for HTS of large chemical libraries. Prior to HTS, some inhibitors of CBS and CSE had been reported, but they have disadvantages such as poor selectivity and relatively weak affinity for the targeted enzyme [44–50]. No inhibitor of 3MST had been reported. Barrios et al. performed CBS inhibitor screening of 1900 chemical compounds by using 7-azide-4-methylcoumarin (AzMC) (Fig. 7a), and found several inhibitors [51]. As we presented at the Third International Conference on Hydrogen Sulfide in Biology and Medicine (H2S2014), we also performed HTS for inhibitors of CSE and 3MST in a chemical library containing about 160,000 compounds by using HSip-1 [52]. In the assay, purified 3MST or CSE and the corresponding substrate (3-mercaptopyruvate (3-MP) or cysteine) were used, and HSip-1 reacted with H2S released by the enzymatic reaction. Compounds that suppressed the fluorescence increase of HSip-1 in response to H2S were selected as inhibitors (Fig. 7b,c). After further examination of the selectivity of the hit compounds, we obtained
both 3MST- and CSE-selective inhibitors. We are currently investigating the utility of these inhibitors for biological studies. HSip-1 was also useful during the development of H2S-caged compounds. For example, Nakagawa et al. developed a photocontrollable hydrogen sulfide donor and during this research, they used HSip-1 for the detection of H2S released from the developed H2S-caged compound [53]. 8. Conclusions and prospects Here, we have reviewed selective fluorescent probes for H2S and sulfane sulfur, and their biological applications. From the viewpoint of probe design, properties such as reducing capacity, nucleophilicity and ability to form a complex with Cu2+ have been utilized for fluorescence off/on switching. Fluorescent probes are expected to be useful tools for nondestructive, real-time detection of H2S or sulfane sulfur in biological samples such as live cells or tissues, but although many H2S fluorescent probes have been reported, only a few probes are yet capable of detecting endogenous
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(b) Negative compounds L-cysteine or 3-MP (Substrate)
HS-
compound 3MST or CSE
HSip-1
Fluorescence increase
(H2S production)
(No inhibition)
(c) Positive compounds (hit compounds) compound
L-cysteine or 3-MP (Substrate)
3MST or CSE
HS-
HSip-1
No fluorescence
(H2S suppression)
(Inhibition)
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Fig. 7. (a) Chemical structure of AzMC. (b,c) Screening schemes for 3MST and CSE inhibitors.
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H2S production in biological contexts. Further, although some fluorescent probes for sulfane sulfur have been developed, there is no selective fluorescent probe for persulfides, such as cysteine persulfide (Cys—S—S–), glutathione persulfide (G—S—S–). This is important, because Akaike et al. reported that cysteine persulfide (Cys—S—S–) formation from cystine was catalyzed by CSE [54]. Thus, there is a need for fluorescent probes that can distinguish various biothiol species for further studies on the biological functions of H2S and sulfane sulfur. We are engaged in developing new fluorescent probes for this purpose.
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Q3 Acknowledgments This work was supported in part by MEXT (Grant Nos. 24689003, 24659042 and 26104509 to K.H.). K.H. was also supported by Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Naito Foundation, the Asahi Glass Foundation, and Takeda Science Foundation.
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