Reversible ratiometric detection of highly reactive hydropersulfides using a FRET-based dual emission fluorescent probe† †Electronic supplementary information (ESI) available: Full experimental procedures, synthesis and characterization of compounds. See DOI: 10.1039/c6sc03856e Click here for additional data file.

A ratiometric fluorescent probe that can visualize endogenously produced hydropersulfides has been developed.


Introduction
Reactive sulfur species (RSS) are a family of sulfur-containing molecules endogenously produced in biological systems. Among RSS, hydropersuldes (R-SSHs) such as hydrogen per-sulde (H 2 S 2 ) and cysteine hydropersulde (CysSSH) are increasingly recognized as an important class of RSS that modulate a variety of physiological events in mammals. For instance, hydropersuldes are found to play a role in S-sulydration of Cys residues of proteins as signalling molecules. 1,2 Previous reports proposed that protein S-sulydration is mediated by the reaction of hydrogen sulde (H 2 S) with oxidised cysteine residues such as S-sulfenic acid (S-OH) and S-nitrosothiol (SNO). [3][4][5][6][7] However, recent reports suggested that hydropersuldes serve as the main reactive species that directly S-sulydrate numerous proteins. [8][9][10][11][12][13] In particular, it has been revealed that S-sulydration regulates the functions of important classes of proteins involved in cell redox homeostasis, [8][9][10] metabolism, 11 and signal transduction. 12 Meanwhile, Akaike suggested that hydropersuldes such as S-sulydrated glutathione (GSSH) serve as potent reducing agents in redox signalling, and may provide a primary and potent antioxidant defence in cells. 1 Hydropersuldes (R-SSHs) are generated by different pathways in biological systems. It has been reported that hydrogen persulde (H 2 S 2 ) can be formed by the oxidation of endogenous H 2 S by reactive oxygen species (ROS). 14 Akaike found that CysSSH is biosynthesized from cystine (CysS-SCys) by two major enzymes: cystathionine b-synthetase (CBS) and cystathionine g-lyase (CSE). 1,15 He also proposed that the enzymatically generated CysSSH is converted to GSH-based hydroper-/hydro-polysuldes (e.g., GSSH, GSSSH, etc.) through persulde interchange reactions. Meanwhile, Banerjee proposed that sulde oxidation pathways in mitochondria are the important source of RSS, such as GSSH. 16 Despite the extensive study of the hydro-persulde formation pathways, regulation of their levels in cells, especially their reducing mechanism, remains largely elusive. Recent reports proposed thioredoxin (Trx) as an important enzyme that reduces CysSSH, although clear evidence for this process has not yet been provided. 17 To understand the varied roles of hydropersuldes in biological systems, it is critical to develop a new analytical tool that allows us to detect the formation and consumption of these RSS species. Fluorescent probes which are available for real-time cell imaging could meet this requirement. [18][19][20][21][22][23] In this regard, Xian et al. recently reported a series of selective uorescent probes for hydroper-/hydropolysuldes (H 2 S n , n > 1). [18][19][20] They ingeniously exploited the high nucleophilic activity of H 2 S n to develop reaction-based turn-on uorescence probes, which were successfully applied to the visualization of intracellular H 2 S n . However, due to the irreversible nature of the reactions, it was intrinsically difficult to monitor reversible concentration dynamics of intracellular H 2 S n using these probes. In this paper, we report the development of a ratiometric uorescent probe for detecting hydropersuldes, based on intramolecular uorescence resonance energy transfer (FRET) (Fig. 1A). 24 The sensing mechanism of this probe involves a reversible nucleophilic attack of a highly reactive hydropersulde species on the pyronine uorophore. This adduct formation disrupts the conjugation structure of the xanthene ring, decreasing the intramolecular FRET efficiency due to a change in the spectral overlap between the coumarin uorescence (FRET donor) and the xanthene absorbance (FRET acceptor), which causes a clear dual-emission signal change. Taking advantage of this reversible sensing property, the probe was successfully applied to detect the concentration dynamics of hydropersuldes in living cells, demonstrating the utility of the probe as a chemical tool in RSS research.

Molecular design of the probe
In the previous study, we reported that the uorescence of the xanthene derivative 1 signicantly decreased upon addition of a large excess of glutathione (GSH, $10 mM) under neutral aqueous conditions. 25 The spectroscopic analyses revealed that xanthene 1, which lacks a C9 aromatic substituent unlike uorescein, was susceptible to nucleophilic attack by thiol species and readily converted to a non-uorescent adduct. Since hydropersuldes (RSSHs) are more nucleophilic than stable thiols such as GSH and H 2 S, 26 we thought that this reactionbased uorescence quenching could be exploited for selective detection of hydropersuldes. As an initial attempt, we synthesized a series of xanthene derivatives bearing the different substituents (Fig. 1B), and evaluated their uorescence responses toward several biological thiol species. The results are summarized in Fig. 1C and S1. † Compound 1 showed a marked decrease in uorescence (F/F 0 ¼ 40%) upon treatment with 50 mM sodium disulde (Na 2 S 2 ), the extent of which is much larger than that induced by addition of the same concentration of Na 2 S (F/F 0 ¼ 93%) and a high concentration (1 mM) of cysteine (F/F 0 ¼ 91%). However, 1 also responded to a biologically relevant concentration of GSH (5 mM) with a high quenching efficiency (F/F 0 ¼ 56%), indicative of the low selectivity of 1 among biologically relevant thiols. The rhodol-type compound 2 exhibited a rather non-selective weak uorescence response to the thiol species. The pyronine-type compound 3,    Time trace plot of the ratio value in A549 cells upon the treatment with Na 2 S 2 (5 mM) (red square, n ¼ 6), Na 2 S 2 (5 mM) and the subsequent addition of NEM (100 mM) at 15 min (blue circle, n ¼ 4), without Na 2 S 2 and NEM (green diamond, n ¼ 6).
possessing two six-membered piperidine rings, is highly susceptible to thiol species with signicant uorescence quenching efficiencies (F/F 0 < 30%), except for L-cysteine. However, pyronines 4 and 5, which possess one and two vemembered pyrrolidine rings, respectively, showed selective uorescence responses (F/F 0 ¼ 6% and 56%, respectively) toward Na 2 S 2 (50 mM). The formation of the H 2 S 2 adduct with 5 was conrmed by a 1 H-NMR experiment (Fig. S2 †). The uorescence response of 4 and 5 toward Na 2 S 2 was further evaluated by titration with different concentrations of Na 2 S 2 . As shown in Fig. 1D and S3, † 4 was more sensitive than 5 and showed a substantial decrease in uorescence with a low concentration of Na 2 S 2 (below 10 mM). The varied uorescence response of these pyronine-type probes, depending on the substituents, would be reasonably explained by the different electron donating abilities of the cyclic amines. That is, the vemembered pyrrolidine can act as a stronger electron donating substituent than the six-membered piperidine, 25 so that the tolerance to nucleophilic attack by Na 2 S 2 is in the order of 5 > 4 > 3. We selected pyronine 4 as a uorescent subunit of the ratiometric probe for hydropersuldes, on account of its selective and sensitive detection of Na 2 S 2 , as shown in Fig. 1. The structure of the newly designed dual-emission probe 6 is shown in Fig. 2A. The probe possesses a coumarin as the FRET donor, which is conjugated to a pyronine unit as the FRET acceptor through a rigid linker. The two carboxylate groups are introduced into the coumarin unit in order to increase the hydrophilicity of the probe, which prevents its leakage from cells during imaging experiments. The synthesis of probe 6 is shown in Scheme 1. The radical bromination of 7 with Nbromosuccinimide (NBS) and the subsequent nucleophilic reaction with N-Boc-piperazine yielded 8. Aer the deprotection, 8 was converted to bis-triate 10, which was sequentially reacted with pyrrolidine and piperidine to give 11 as a mixture of the substitution isomers. The keto-reduction of 11 with borane-SMe 2 and the subsequent oxidation using DDQ yielded the pyronine 13. Aer the removal of the Boc group, 13 was subjected to a conjugation reaction with the N-hydroxysuccinimide ester of coumarin 14 to give 15. Finally, the deprotection of the tert-butyl ester groups of 15 and the following HPLC purication provided 6 as a mixture of the isomers.

Ratiometric uorescence sensing of hydropersuldes
The functional analysis of probe 6 was initially conducted in a neutral aqueous solution. A solution of 6 (5 mM) in 50 mM HEPES buffer (pH 7.4) showed two distinct UV peaks at 400 nm and 562 nm (Fig. 2B), which correspond to the absorbance of coumarin and pyronine, respectively. Upon titration with sodium disulde Na 2 S 2 (0-100 mM), the absorbance at 562 nm decreased gradually to ca. 25% of its original intensity, whereas the absorbance at 400 nm scarcely changed. In the uorescence spectrum, a solution of 6 (5 mM) showed two distinct emissions at 479 nm and 584 nm due to the coumarin and pyronine units, respectively, when excited at 410 nm (Fig. 2C). This dual-emission spectrum changed dramatically in a see-saw manner upon addition of Na 2 S 2 (0-100 mM). The large decrease in emission at 584 nm and the concomitant increase in emission at 479 nm strongly suggest that FRET between the coumarin and xanthene units is cancelled as a result of a decrease in the spectral overlap between the coumarin emission and the pyronine absorption (Fig. S4 †). The FRET efficiency of 6 was calculated to be 60% in the initial state, which decreased to 31% in the presence of 100 mM Na 2 S 2 . Fig. 2D shows the time-lapse detection of the uorescence response of 6 toward Na 2 S 2 . The ratio value R (F 479 nm /F 584 nm ) rapidly increased upon addition of Na 2 S 2 (0-30 mM) and reached a plateau almost within 60 s. The plot of R value against the concentration of Na 2 S 2 (0-30 mM) shows a linear relationship (Fig. 2E), indicative of the highly quantitative nature of the ratiometric detection of hydropersuldes using 6. Probe 6 was able to detect as low as 1.0 mM Na 2 S 2 based on the calculation of the detection limit (3s). This sensitivity is sufficiently high for detection of intracellular hydropersuldes, the concentration of which was reported to be around ten micromolar under basal conditions. 1 The sensing selectivity of 6 for various biologically relevant thiol species was evaluated (Fig. 3A). In contrast to the large increase in R value induced by Na 2 S 2 (lane 3), 6 showed a negligible uorescence response upon addition of the same concentration of Na 2 S (lane 2). This sensing selectivity is reasonably ascribed to the lower pK a value of H 2 S 2 (pK a ¼ 5.0) compared to that of H 2 S (pK a ¼ 6.9), rendering H 2 S 2 highly nucleophilic as a thiolate anion (HS 2 À ) under neutral conditions (pH ¼ 7.4) (Fig. 3B). 26 A moderate R value change was observed upon addition of hydropolysulde Na 2 S 4 (lane 4), though this change was apparently smaller than that induced by Na 2 S 2 (lane 3). The weak uorescence response of 6 for Na 2 S 4 might be ascribed to the rather poor nucleophilic activity of the HS 4 À anion due to its extremely low pK a value (pK a ¼ 3.8). 26 Probe 6 also showed a moderate increase in the R value upon treatment with CysSSH (lane 5) and GSSH (lane 6), which were generated in situ from Cys and GSH, respectively, by the reaction with Na 2 S and NO donor (NOC7). 1 In a similar manner, the mixture of Na 2 S (50 mM) and NaOCl (50 mM) in a basic solution (0.1 M NaOH), which generates Na 2 S 2 in situ, induced a moderate increase of R value (lane 7), while their mixture in a neutral solution (50 mM HEPES, pH 7.4), which mainly produces Na 2 S 4 and Na 2 S 5 , 14 resulted in a small increase in the R value (lane 8). This difference in signal change is consistent with the results obtained by the direct titration with these thiols (lanes 3 and 4). Probe 6 showed a negligible R value change upon addition of biologically relevant concentrations of GSH (5 mM, lane 9), L-cysteine (1 mM, lane 10), and cystine (0.5 mM, lane 11). It is noteworthy that the sensing selectivity of 6 for Na 2 S 2 is apparently higher than that of 5 among these thiol species (Fig. 1C), probably due to steric hindrance and/or the electron conguration effect of the coumarin unit conjugated to the pyronine unit of 6. Therefore, 6 was able to detect Na 2 S 2 with a sufficient sensitivity (detection limit ¼ 4.4 mM) even in the presence of 5 mM of GSH (Fig. S5 †). We also conrmed that 6 scarcely responded to various redox-relevant compounds, including ROS such as NaOCl and H 2 O 2 , reactive nitrogen species (NOS), ascorbic acid, and KCN (Fig. S6 †). All of these data suggest that probe 6 primarily serves as a selective uorescent probe for hydropersuldes. It was conrmed that 6 did not show a signicant ratio change over the physiological pH range (5.0 to 8.5; Fig. S7 †).
The uorescence response of 6 towards a change in the hydropersulde level was examined. As shown in Fig. 3C, the R value of 6 (5 mM) increased stepwise upon the repeated addition of 30 mM Na 2 S 2 , and reached a plateau almost within 60 s. The subsequent addition of N-ethylmaleimide (NEM, 500 mM) to consume Na 2 S 2 induced a large decrease in the R value. These data clearly indicate that 6 can reversibly change its R value depending on the concentration of hydropersulde according to the binding equilibrium shown in Fig. 1A. The reverse uorescence response of 6 was further evaluated upon addition of other reactive species (Fig. 3D). Addition of NaOCl (100 mM) to the mixed solution of 6 (5 mM) and Na 2 S 2 (50 mM) also induced a signicant decrease in the R value (lane 3), as observed with NEM (lane 4). This change is reasonably ascribed to the decrease in Na 2 S 2 level due to the formation of oxidized sulfurs and hydropolysuldes (H 2 S n , n > 2). Conversely, a small change in the R value was induced upon addition of GSH (5 mM, lane 5) and L-Cys (1 mM, lane 6), suggesting that these biologically abundant thiols do not interfere with the hydropersulde sensing of 6.

Ratio imaging of hydropersuldes in living cells
We next applied probe 6 to the ratiometric uorescence imaging of hydropersuldes (R-SSH) in living cells. For cell imaging, 6 was chemically modied with acetoxymethyl (AM) groups to enhance its membrane permeability. 27 It was expected that the AM-modied probe, 6-AM ( Fig. 2A), could be readily hydrolyzed by intracellular esterases to liberate 6, which is unlikely to leak from cells due to its highly polar character. When A549 cells were treated with 6-AM (5 mM) for 20 min, bright uorescence of the probe was observed in the cytosolic region of the cells (Fig. 4A(a-d)). A cell viability assay revealed that 6-AM showed low cytotoxicity to A549 cells at a concentration below 25 mM (Fig. S8 †). Addition of 5 mM Na 2 S 2 to the cell medium induced an obvious change in the uorescence ratio (R ¼ 430-480 nm/ 550-630 nm) (Fig. 4A(e)) of cells, relative to that observed in the control experiment without Na 2 S 2 treatment (Fig. 4A(f)). Titration of the 6-AM pre-stained cells with Na 2 S 2 (0-5 mM) showed a linear dependence between Na 2 S 2 concentration and R value ( Fig. 4B and S9 †), demonstrating the accurate hydropersulde sensing property of 6 in living cells. The time-lapse imaging revealed that the R value increased immediately aer the addition of 5 mM Na 2 S 2 and reached a steady state (R ¼ $1.4) aer 10 min (Fig. 4C). The subsequent treatment of the cells with 100 mM N-ethylmaleimide (NEM) induced a signicant decrease in the R value ( Fig. 4A(g, h) and C) due to the decrease of the hydropersulde level by the nucleophilic reaction with NEM, demonstrating the reversible sensing property of 6 in living cells.
The probe 6-AM was further applied to the ratiometric detection of endogenous hydropersuldes produced by enzymes in living cells. It has been reported that CSE and CBS are the major enzymes responsible for generation of cysteine hydropersulde (CysSSH) from cystine (CysSSCys) in A549 cells. 1 When A549 cells, pre-stained with 6-AM, were treated with cystine (CysSSCys), a gradual increase in the R value was observed ( Fig. 5B and C). This change was effectively suppressed on treatment of the cells with aminooxyacetic acid (AOAA), an inhibitor of CSE and CBS ( Fig. 5B and D, lane 3). 28 Conversely, treatment of the cells with auranon, an inhibitor for thioredoxin reductase (TrxR), induced a statistically signicant increase in the R value (Fig. 5D, lane 4 and S10 †), suggesting an increase in the intracellular hydropersulde level as a result of the inhibition of Trx activation by TrxR (Fig. 5A). This result is consistent with the recent report that Trx catalyses reduction of Cys-SH as a major regulator of its intracellular level. 17 It has been reported that hydropersulde is also produced in living cells by the oxidation of H 2 S, which is generated from L-Cys by CSE and CBS. 20,22 To conrm this point, uorescence imaging of A549 cells treated with L-Cys (200 mM) was performed using 6-AM. As shown in Fig. 5E and F, the R value largely increased in a time-dependent manner in the living cells. Treatment of the cells with AOAA effectively suppressed the increase in R value induced by L-Cys ( Fig. 5E and G, lane 3). Unlike the case of the cysteine experiment (Fig. 5D), inhibition of TrxR by auranon did not cause an increase in the R value (Fig. 5G, lane 4 and S11 †), implying that the direct conversion of L-Cys to CysSSH is not a major pathway of the hydropersulde formation in living cells. All of these data suggest that hydropersuldes are generated from L-Cys through the enzyme-mediated H 2 S formation and subsequent oxidation in living cells. Finally, the addition of high concentration NaOCl (300 mM) induced a signicant decrease in the R value in cells (Fig. 5H and S12 †), indicating that the decrease in hydropersulde level was a result of its oxidative degradation to oxidized sulfurs and hydrogen poly-suldes (H 2 S n , n > 2).

Conclusion
In conclusion, we have developed a ratiometric uorescent probe 6 that can visualize the endogenously produced hydro-persuldes in living cells. To our knowledge, probe 6 is the rst example of a ratiometric uorescent probe that can reversibly detect intracellular hydropersulde levels. Since the research eld of reactive sulfur species (RSS) is still in its infancy, probe 6 would serve as a versatile analytical tool, not only for understanding the chemical nature of hydropersulde in biological systems, but also for elucidating the roles of hydropersulde in cell signalling and redox homeostasis. For this purpose, further functional improvements in probe 6, which include sensing selectivity for a single hydropolysulde species and/or localizability to a certain cell compartment, would be desirable to realize more precise and quantitative analysis of RSS. Research along these lines is currently underway in our laboratory.

Synthesis and characterization of the compounds
The syntheses and characterization of probes 1-6 and 6-AM are described in the ESI. †

Fluorescence measurement
Fluorescence titration was conducted with a solution (3 mL or 0.5 mL) of the probe in a quartz cell. Typically, a freshly prepared aqueous stock solution of Na 2 S 2 was added to a solution of 6 (5 mM) in 50 mM HEPES, 10 mM NaCl, 1 mM MgSO 4 , pH 7.4 with 0.4% Tween at 25 C and the uorescence emission spectra were measured aer 10 min (l ex ¼ 410 nm) with a Perkin Elmer LS55 uorescence spectrometer.
For the uorescence bioimaging, cells were cultured for 2 days in a 35 mm glass-bottomed dish (Iwaki Scitech).

Fluorescence imaging of exogenous H 2 S 2 in A549 cells
Fluorescence imaging was conducted with a confocal laser scanning microscope (LSM 780, Zeiss) equipped with a 63Â objective lens. The following detection channels were chosen for the ratiometric imaging; Ch1 l ex ¼ 405 nm, l em ¼ 430-480 nm, and Ch2 l ex ¼ 405 nm, l em ¼ 550-630 nm. In a glass-based dish, A549 cells in HBS buffer (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO 4 , 2.0 mM CaCl 2 , 11.5 mM glucose, 20 mM HEPES, pH 7.4) were incubated with 6-AM (5 mM) for 20 min at 37 C under a humidied atmosphere of 5% CO 2 in air. Aer removal of excess probe and washing with HBS buffer, the cells were treated with Na 2 S 2 (0-5 mM, nal conc.) and subjected to the uorescence imaging. For the imaging of the endogenously produced hydropersuldes, A549 cells, pre-stained with 6-AM (5 mM), were treated with cystine (200 mM) or L-cysteine (200 mM). For inhibition of CSE and CBS enzymes, the cells were pretreated with AOAA (aminooxyacetic acid, 1 mM, Sigma) in HBS buffer for 1 h before staining with 6-AM. For inhibition of TrxR, the cells were pre-treated with auranon (2 mM, Wako) in HBS buffer for 1 h before staining with 6-AM.