Fluorogenic hydrogen sulfide (H₂S) donors based on sulfenyl thiocarbonates enable H₂S tracking and quantification

Abstract

Hydrogen sulfide (H₂S) is an important cellular signaling molecule that exhibits promising protective effects. Although a number of triggerable H₂S donors have been developed, spatiotemporal feedback from H₂S release in biological systems remains a key challenge in H₂S donor development. Herein we report the synthesis, evaluation, and application of caged sulfenyl thiocarbonates as new fluorescent H₂S donors. These molecules rely on thiol cleavage of sulfenyl thiocarbonates to release carbonyl sulfide (COS), which is quickly converted to H₂S by carbonic anhydrase (CA). This approach is a new strategy in H₂S release and does not release electrophilic byproducts common from COS-based H₂S releasing motifs. Importantly, the release of COS/H₂S is accompanied by the release of a fluorescent reporter, which enables the real-time tracking of H₂S by fluorescence spectroscopy or microscopy. Dependent on the choice of fluorophore, either one or two equivalents of H₂S can be released, thus allowing for the dynamic range of the fluorescent donors to be tuned. We demonstrate that the fluorescence response correlates directly with quantified H₂S release and also demonstrate the live-cell compatibility of these donors. Furthermore, these fluorescent donors exhibit anti-inflammatory effects in RAW 264.7 cells, indicating their potential application as new H₂S-releasing therapeutics. Taken together, sulfenyl thiocarbonates provide a new platform for H₂S donation and readily enable fluorescent tracking of H₂S delivery in complex environments.

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Introduction

Hydrogen sulfide (H₂S) is an important gaseous molecule that plays critical roles in living systems.^1–3 Endogenous H₂S is primarily produced from cysteine (Cys) and homocysteine (Hcy) by four main enzymes, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfur transferase (3-MST), and cysteine aminotransferase (CAT).^4–8 Due to its bioregulatory and protective roles, H₂S is considered as an important cellular signaling molecule, much like nitric oxide (NO) and carbon monoxide (CO).^8–11

Although many H₂S-related biological functions have been discovered in the past two decades, many investigations have been limited due to the lack of controllable and refined H₂S delivery systems (H₂S donors). Inorganic sulfide salts, such as sodium sulfide (Na₂S) and sodium hydrosulfide (NaHS), are widely used in H₂S investigations, but they release H₂S quickly in aqueous media, thus failing to mimic the slow and well-regulated in vivo H₂S generation.¹² By contrast, the H₂S release from GYY4137, a phosphorodithioate-based H₂S donor, is slow in aqueous buffer, and the low H₂S releasing efficiency remains as a major concern when applying it to the living systems.¹³ Building from these donor scaffolds, a series of synthetic H₂S donors have been developed in the last decade.^14–19 These donors are activated by different triggers, such as enzymes,^20,21 cellular thiols,^22–27 pH modulation,^28,29 and photo activation,^30–33 and the released H₂S exhibits promising activities in different physiological and pathological processes.^17,18

To expand the library of H₂S donors, we recently developed the carbonyl sulfide (COS)-based platform for H₂S donation. In this approach, caged-thiocarbamates are activated and the resultant intermediates undergo a cascade reaction to release COS, which is quickly converted to H₂S by the ubiquitous enzyme carbonic anhydrase (CA) (Fig. 1a).³⁴ Expanding on this initial report, we, as well as others, have applied the similar caged-COS systems to include a series of triggerable COS-based H₂S donors which can be activated by different mechanisms, such as reactive oxygen species (ROS),^35–37 esterase,^38,39 cellular nucleophiles,⁴⁰ click chemistry,⁴¹ light,^42–44 and Cys,⁴⁵ to release H₂S under various conditions.^45,46

Fig. 1 (a) General design of previous self-immolative COS/H₂S donors. (b) COS/H₂S release from fluorogenic caged-sulfenyl thiocarbonate donor motifs.

Although H₂S release from these donors has been measured using different methods, the real-time tracking of donor activation and H₂S delivery in living systems remains a key challenge due to the inherent limitations of current H₂S detection methods. For example, the colorimetric methylene blue (MB) assay has been widely used to measure H₂S levels, but requires strongly acidic conditions, which may trigger H₂S release from acid labile sulfide pools. Additionally, MB is an end-point assay which destroys experimental samples, thus making it not feasible for in vivo H₂S detection.⁴⁷ Similarly, H₂S-selective electrodes are most often used in bulk measurements rather than non-homogenized biological samples.⁴⁷ By contrast, H₂S fluorescent probes have attracted much attentions due to their high sensitivities and have been used to sense and visualize H₂S in biological samples.⁴⁸ Albeit promising, H₂S fluorescent probes are prone to react with reactive cellular species, such as Cys or glutathione (GSH), which results in either probe consumption or false positive signals. In addition, all of the above methods consume the H₂S being measured. Recently, we reported a series of γ-ketothiocarbamates (γ-KetoTCM) that release p-nitroaniline as a colorimetric indicator; however, although the colorimetric response enables monitoring of COS/H₂S release in cuvettes, the direct tracking in live-cell environments remains an un-met need.⁴⁹ Taken together, a key advancement compatible with live-cell and tissue experiments would be the development of H₂S donors that deliver H₂S with a concomitant fluorescence response to enable tracking of H₂S delivery by common microscopy techniques.

Aligned with these needs, we report here the development of a new H₂S-releasing strategy based on caged sulfenyl thiocarbonates and apply this donor platform to access fluorescent turn-on H₂S donors. In this approach, cellular thiols (i.e. Cys and GSH) activate the sulfenyl thiocarbonates through thiol-mediated disulfide reduction to release COS, which is quickly converted to H₂S by CA (Fig. 1b). To the best of our knowledge, the sulfenyl thiocarbonate reduction strategy provides a new activation pathway that has not been used to trigger COS/H₂S release from donor platforms. Unlike currently-available donors that function through initial COS release, the sulfenyl thiocarbonate system does not generate reactive electrophile byproducts upon activation, which provides a significant advance in the field. In addition, we leverage this new donor strategy to access systems in which a concomitant fluorescence turn-on occurs upon donor activation, thus allowing for real-time monitoring and quantification of H₂S release using fluorescence spectroscopy.

Results and discussion

To test our hypothesis that caged-sulfenyl thiocarbonates could serve as thiol-triggered fluorescent COS/H₂S donors, four donors (FLD-1–4) were prepared by reacting fluorescein or 3-O-methylfluorescein with ((benzyl)dithio)carbonyl chloride 1 or ((phenol)dithio)carbonyl chloride 2 in the presence of DIPEA (Scheme 1a). A caged thiocarbonate (TCN-1) was synthesized as a triggerless control compound, which is expected to be stable toward thiol activation due to the lack of a disulfide functional group (Scheme 1b).

Scheme 1 Synthesis of fluorescent COS/H₂S donors (a) and thiocarbonate control compound (b).

With these compounds in hand, we first evaluated their spectroscopic properties in PBS buffer (pH 7.4, 10 mM). As expected, FLD-1–3 and TCN-1 are not absorptive in the visible region and are all nonfluorescent because the fluorescein unit is locked in the closed lactone form. By contrast, FLD-4 shows a prominent absorbance band in the visible region (λ_max = 449 nm, ε = 27 300 ± 2500 M⁻¹ cm⁻¹) with measurable fluorescence (λ_em = 514 nm, Φ = 0.11 ± 0.01) due to the free hydroxyl group (Table S1†). Based on the promising spectroscopic properties, large dynamic range, and efficient release of two equivalents of COS/H₂S, we chose to use FLD-1 as the model donor for initial reactivity and selectivity evaluations.

To test the reactivity of FLD-1 towards Cys-induced activation, FLD-1 (10 μM) was incubated with Cys (100 μM) in PBS buffer (pH 7.4, 10 mM) containing physiologically-relevant concentrations of CA (25 μg mL⁻¹), and the fluorescence intensity was measured using a fluorescence spectrometer. As expected, Cys successfully activated FLD-1 and resulted in a 500-fold fluorescence turn on over 2 h, demonstrating the release of the fluorescein upon FLD-1 activation (Fig. 2a). Fluorescein formation was also confirmed by UV-vis spectroscopy under the identical conditions (Fig. S1†). A Cys-dependent fluorescence enhancement was observed when treating FLD-1 (10 μM) with increasing concentrations of Cys (1–20 equiv.), indicating a high sensitivity of FLD-1 towards Cys. No fluorescent signal was observed in the absence of Cys, suggesting that FLD-1 is stable in aqueous buffer, and that it is not hydrolyzed to provide false signals (Fig. 2b). In addition, to confirm the H₂S delivery from FLD-1, we treated FLD-1 (10 μM) with Cys (100 μM) in PBS containing CA (25 μg mL⁻¹) and quantified H₂S release using the MB assay. In this experiment, 15 μM of H₂S was detected (75% releasing efficiency), which is consistent with our hypothesis that 2 equivalents of COS/H₂S would be released upon FLD-1 activation (Fig. S2†). Taken together, these experiments demonstrate that FLD-1 is highly responsive to Cys activation.

Fig. 2 (a) Time-dependent fluorescence spectra of FLD-1 (10 μM) in PBS (pH 7.4, 10 mM) containing Cys (100 μM) and CA (25 μg mL⁻¹). (b) Cys-dependent (0–200 μM) fluorescence turn on of FLD-1 (10 μM) in PBS. λ_ex = 490 nm, λ_em = 500–650 nm, and slit width = 0.3 mm. The experiments were performed in triplicate and results are expressed as mean ± SD (n = 3).

To determine whether H₂S release correlated directly with the observed fluorescence response, we measured the fluorescent response from FLD-3 in the presence of Cys and CA and quantified H₂S release using the MB assay. We chose to use FLD-3 as the model compound for these investigations because it only contains one sulfenyl thiocarbonate moiety, and therefore should simplify the reaction kinetics. In comparison, FLD-1 contains two sulfenyl thiocarbonate groups, and the cleavage of one sulfenyl thiocarbamate would generate FLD-4 as a reaction intermediate, which exhibits moderate fluorescence (see Fig. 4). Incubation of FLD-3 (10 μM) with Cys (100 μM) resulted in a rapid fluorescence response with 96% of the H₂S release measured by MB. At extended time points, we observed a slight decrease in measured H₂S, possibly due to volatilization of H₂S in the headspace of the closed system or adventitious oxidation of released H₂S. Negligible H₂S was detected in the absence of CA, indicating that Cys-triggered H₂S delivery from FLD-3 proceeds through intermediate COS formation (Fig. 3a). Importantly, the strong linear correlation between the measured fluorescence and H₂S measured from the MB method detection (first 25 min, R² = 0.988) demonstrates that fluorescent readouts can serve as reliable optical tools to track COS/H₂S release from FLD donors with temporal resolution (Fig. 3b). Moreover, this linear correlation suggests that choice of other fluorophores with different brightnesses and photophysical properties could be used to access different dynamic ranges of H₂S release, thus enabling this approach to be translated to different types of experimental designs.

Fig. 3 (a) Time-dependent fluorescence turn on (red) and H₂S release (blue) upon FLD-3 (10 μM) activation in PBS (pH 7.4, 10 mM) containing Cys (100 μM) and CA (25 μg mL⁻¹). No H₂S was detected in the absence of CA (black). λ_ex = 454 nm, λ_em = 500–650 nm, and slit width = 0.3 mm. (b) Correlation between fluorescence measurement and MB detection. The experiments were performed in triplicate. The results are expressed as mean ± SD (n = 3).

Having confirmed the efficiency of Cys-triggered fluorescence turn on and COS/H₂S release, we next evaluated the reactivity of other FLD donors towards Cys activation. Treating FLD-1–3 (10 μM) with Cys (100 μM) in PBS buffer (pH 7.4, 10 mM) resulted in a 120–500-fold fluorescence turn on over 2 h. We attribute the faster response of FLD-2 to the more electrophilic phenyl sulfenyl thiocarbonate in comparison to the less electrophilic benzyl sulfenyl thiocarbonate in FLD-1. FLD-4, however, provided minimal fluorescence enhancement due to its strong background fluorescence. No fluorescence response from TCN-1 (10 μM) was observed under the identical conditions, which demonstrates the stability of the thiocarbonate group in the presence of Cys (Fig. 4).

Fig. 4 Fluorescence turn on of FLD-1 (gray), FLD-2 (red), FLD-3 (blue), FLD-4 (black), and TCN-1 (dark yellow) (10 μM) in PBS (pH 7.4, 10 mM) containing Cys (100 μM). λ_ex = 490 nm for FLD-1, 2, 4, and TCN-1, λ_ex = 454 nm for FLD-3, λ_em = 500–650 nm, and slit width = 0.3 mm. The experiments were performed in triplicate. The results are expressed as mean ± SD (n = 3).

To further support our proposed activation mechanism in Fig. 1b, as well as the activation of the sulfenyl thiocarbonate group by other thiols, we incubated FLD-1 (10 μM) in PBS (pH 7.4, 10 mM) with 10 equivalents of benzyl mercaptan (100 μM) for 1 h and analyzed the reaction products by HPLC (Fig. S3†). Consistent with our proposed activation mechanism, we observed FLD-1 consumption and the formation of both benzyl disulfide and fluorescein, which supports the proposed mechanism.

In addition to Cys and BnSH, other cellular thiols and biological nucleophiles were tested to determine whether they resulted in donor activation. In these experiments, FLD-1 (10 μM) was incubated with GSH, homocysteine (Hcy), N-acetyl cysteine (NAC), penicillamine (PEN), or bovine serum albumin (BSA) (100 μM), in PBS buffer (pH 7.4, 10 mM) containing CA (25 μg mL⁻¹) and the fluorescent intensity was measured after 2 h. As expected, FLD-1 is stable in PBS at physiological pH in the absence of thiols (Fig. 5 bar 1). Incubation of FLD-1 with Cys, NAC, GSH, and Hcy, however, led to a significant fluorescence enhancement, indicating successful donor activation and COS/H₂S release (Fig. 5 bars 2–5 and S4†). In addition, these results demonstrate that the sulfenyl thiocarbonate group is responsive to different types of thiols. In comparison, PEN resulted in only minimal fluorescence response and BSA failed to activate the donor presumably due to the bulkiness of these two thiol species, which hindered their reactions with FLD-1 (Fig. 5 bars 6 and 7). Additionally, N-ethylmaleimide (NEM) pretreatment of Cys samples significantly reduced the fluorescence enhancement from FLD-1, confirming the necessity of the thiol-induced reduction for the donor activation (Fig. 5 bar 8).

Fig. 5 Fluorescence turn on of FLD-1 (10 μM) in the presence of cellular RSONs (100 μM): (1) FLD-1 only, (2) Cys, (3) NAC, (4) GSH, (5) Hcy, (6) PEN, (7) BSA, (8) Cys + NEM, (9) H₂O₂, (10) ClO⁻, (11) O₂⁻, (12) TBHP, (13) Ser, (14) Lys, (15) Gly, (16) GSSG, (17) GSNO, and (18) PLE (1 U mL⁻¹). Fluorescence intensity was measured after 2 h incubation. The experiments were performed in triplicate. The results were expressed as mean ± SD (n = 3).

We also tested the response of the FLD donors in the presence of other cellular reactive sulfur, oxygen, and nitrogen species (RSONs). FLD-1 (10 μM) was incubated with RSONs (100 μM), such as hydrogen peroxide (H₂O₂), hyperchlorite (ClO⁻), superoxide (O₂⁻), tert-butyl hydroperoxide (TBHP), serine (Ser), lysine (Lys), glycine (Gly), oxidized glutathione (GSSG), and S-nitrosoglutathione (GSNO), and COS/H₂S release was monitored by tracking the fluorescein formation. As expected, minimal fluorescence was observed in these experiments, confirming the stability of FLD-1 to common RSONs (Fig. 5 bars 9–17). Because the carbonate functional group may be sensitive to esterase-catalyzed hydrolysis, we also tested the esterase stability of the sulfenyl thiocarbonate group by incubating FLD-1 (10 μM) with porcine liver esterase (PLE, 1 U mL⁻¹). Although a slight fluorescence turn on was observed after a 2 h incubation, the observed response was much lower than that from thiol activation (Fig. 5 bar 18). Taken together, these studies demonstrate that FLD-1 is highly responsive and selective to thiol activation and common cellular RSONs do not trigger FLD-1 to release COS/H₂S.

After confirming the thiol-triggered COS/H₂S release from FLD-1 in aqueous buffer, we next investigated the H₂S delivering capacity of FLD-1 in cellular environment. In these experiments, HeLa cells were treated with FLD-1 (50 μM) or TCN-1 (50 μM) and H₂S release was monitored using C7-Az, a H₂S-responsive fluorescent probe.^50,51 Incubation of HeLa cells with C7-Az (50 μM) alone resulted in a negligible fluorescence response, indicating minimal endogenous H₂S (Fig. 6 top row). Treatment with TCN-1 also failed to provide a fluorescence signal, suggesting that the thiocarbonate group was stable and did not decompose to generate false signals in cellular environments (Fig. 6 middle row). By contrast, a strong C7-Az fluorescent signal was observed when incubating HeLa cells with FLD-1, suggesting that H₂S release was successfully triggered by endogenous thiols. In addition, a strong fluorescence signal was also observed from activated FLD-1 in the fluorescein channel, confirming the fluorescence response upon donor activation (Fig. 6 bottom row). Taken together, these results demonstrate that the FLD donors not only function as efficacious H₂S donors in live cells, but also provide a fluorescence signal that enables observing H₂S release.

Fig. 6 H₂S delivery from FLD-1 in HeLa cells. HeLa cells were treated with NucRed nuclear dye and C7-Az (50 μM) in DMEM only (top row) or DMEM containing TCN-1 (50 μM) (middle row) or FLD-1 (50 μM) (bottom row) for 30 min. Cells were then washed with PBS and cell images were taken in PBS using a fluorescent microscope. Bar scale: 50 μm.

To further demonstrate the H₂S-releasing fidelity of the FLD donors we also investigated the anti-inflammatory activities of FLD-1. We pretreated macrophage RAW 264.7 cells with FLD-1 (0–25 μM) for 2 h, followed by a 24 h incubation with lipopolysaccharide (LPS, 0.5 μg mL⁻¹) to trigger the inflammatory response. The inflammation event usually results in the NO generation, which can be monitored by measuring nitrite (NO₂⁻) accumulation. We chose to use concentrations of FLD-1 up to 25 μM because these concentrations did not induce cytotoxicity (Fig. S6†).

As expected, the pretreatment of RAW 264.7 cells with FLD-1 showed a dose-dependent inhibition of NO₂⁻ accumulation, indicating anti-inflammatory activity from FLD-1. Although GYY4137 has shown anti-inflammatory effects at higher concentration and longer incubation time (i.e. 100–1000 μM and 24 h incubation),⁵² such cytoprotection was not observed at the 25 μM concentration used for comparison, highlighting the efficacious H₂S release from FLD-1 in the cellular environment (Fig. 7). To further confirm that the observed effects were due to H₂S rather than other components of donor activation, we treated cells with 25 μM of TCN-1, fluorescein or benzyl mercaptan and measured NO₂⁻ production. None of these compounds attenuated NO₂⁻ generation, confirming that the anti-inflammatory activities of FLD-1 is due to H₂S release (Fig. S7†). Overall, these studies demonstrate that FLD-1 releases COS/H₂S in complex cellular environment and exhibits promising anti-inflammatory protections, indicating potential applications of FLD-1 as H₂S-releasing therapeutics.

Fig. 7 Effects of FLD-1 on LPS-induced NO₂⁻ accumulation. RAW 264.7 cells were pretreated with FLD-1 (5–25 μM) or GYY4137 (25 μM) for 2 h, followed by a 24 h treatment of LPS (0.5 μg mL⁻¹). Results are expressed as mean ± SD (n = 4). ***P < 0.001 vs. the control group; ^##P < 0.01 vs. vehicle-treated group; ^###P < 0.001 vs. vehicle-treated group.

Conclusions

In conclusion, we prepared and evaluated a class of caged sulfenyl thiocarbonates as new controllable and fluorescent COS/H₂S donors. Thiol-triggered COS/H₂S release from these molecules has been detected and visualized in both aqueous buffer and in live cells. Importantly, the concomitant release of a fluorescein reporter after H₂S release enables the real-time monitoring of H₂S release dynamics. In addition, we demonstrate that FLD-1 exhibits a dose-dependent inhibition of the LPS-induced NO formation, which is consistent with anti-inflammatory activities of H₂S. Taken together, caged-sulfenyl thiocarbonates are promising new class of COS/H₂S donors with high potential for accessing H₂S-related protective activities, and the developed fluorescent donors provide new methods for monitoring H₂S release in real-time in complex environments.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Research reported in this publication was supported by the NIH (R01GM113030) and Dreyfus Foundation. NMR, fluorescence microscopy, and MS instrumentation in the UO CAMCOR facility is supported by the NSF (CHE-1427987, CHE-1531189, and CHE-1625529).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: H₂S release curves, cytotoxicity data, NMR spectra. See DOI: 10.1039/c8sc05200j

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