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. Author manuscript; available in PMC: 2020 Apr 28.
Published in final edited form as: J Am Chem Soc. 2019 Aug 14;141(34):13610–13618. doi: 10.1021/jacs.9b06319

Cyclic Sulfenyl Thiocarbamates Release Carbonyl Sulfide (COS) and Hydrogen Sulfide (H2S) Independently in Thiol-Promoted Pathways

Yu Zhao 1, Andrea K Steiger 1, Michael D Pluth 1,*
PMCID: PMC7023849  NIHMSID: NIHMS1555959  PMID: 31373809

Abstract

Hydrogen sulfide (H2S) is an important signaling molecule that provides protective activities in a variety of physiological and pathological processes. Among the different types of H2S donor compounds, thioamides have attracted attention due to prior conjugation to non-steroidal anti-inflammatory drugs (NSAIDs) to access H2S-NSAID hybrids with significantly-reduced toxicity, but the mechanism of H2S release from thioamides remains unclear. Herein, we reported the synthesis and evaluation of a class of thioamide-derived sulfenyl thiocarbamates (SulfenylTCMs) that function as a new class of H2S donors. These compounds are efficiently activated by cellular thiols to release carbonyl sulfide (COS), which is quickly converted to H2S by carbonic anhydrase (CA). In addition, through mechanistic investigations we establish that COS-independent H2S release pathways are also operative. In contrast to the parent thioamide-based donors, the SulfenylTCMs exhibit excellent H2S releasing efficiencies of up to 90% and operate through mechanistically well-defined pathways. In addition, we demonstrate that the sulfenyl thiocarbamate group is readily attached to common NSAIDs, such as naproxen, to generate YZ-597 as an efficient H2S-NSAID hybrid, which we demonstrate releases H2S in cellular environments. Taken together, this new class of H2S donor motifs provide an important platform for new donor development.

Graphical Abstract

graphic file with name nihms-1555959-f0001.jpg

Introduction

Gasotransmitters, such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), are small gaseous signaling molecules that are produced endogenously and transmit chemical signals within the organism, tissues, and cells by acting on specific targets.1–3 H2S, which is the youngest member of the gasotransmitter family, is generated from cysteine (Cys) and homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and cysteine aminotransferase (CAT)/3-mercaptopyruvate sulfur transferase (3-MST), which work either individually or in concert to regulate H2S levels under physiological conditions.4–6 Once generated, H2S plays important roles in a variety of physiological and pathological events.7–10

To deliver H2S in complex environments, numerous H2S releasing agents (H2S donors) have been developed. These donors function as important chemical tools to both mimic H2S biosynthesis and also to investigate H2S chemistry and biology in contextually-rich environments. Although sodium sulfide (Na2S) and sodium hydrosulfide (NaSH) are the most commonly-used sources of exogenous H2S, both of these compounds release H2S instantly and spontaneously in aqueous media, making the controlled release of sulfide unfeasible.11–12 Ideal H2S donors should only release sulfide upon activation and deliver H2S with slower, but controllable kinetics. In response to this need, chemists have developed different types of H2S donors in the last decade, which can be activated by different triggers, such as hydrolysis,13–15 cellular thiols,16–26 light,27–31 pH modulation,32–33 and enzymes34–35 (Figure 1).36–43 Among these donors, aryl thioamides have attracted attention due to their synthetic simplicity and ease of incorporation into common pharmaceutical compounds.24 For example, when compared to regular non-steroidal anti-inflammatory drugs (NSAID), thioamide-coupled NSAIDs, such as ATB-346, have retained promising anti-inflammatory activities while significantly reducing side GI damage, suggesting potential applications of these donors as H2S-related therapeutics.38, 44 Although thioamides provide promising donor motifs, the H2S releasing efficiency of thioamide-based donors remains at relatively low levels (typically 1 – 2%), and the detailed mechanism of H2S remains unclear (Figure 1 and Scheme 2 top).

Figure 1.

Figure 1.

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Selected synthetic H2S donors.

Scheme 2.

Scheme 2.

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(Top) Cys-Activated H2S release from thioamides. (Bottom) Proposed thiol-triggered COS/H2S release from SulfenylTCMs.

To further diversify available H2S donor platforms and also to develop mechanistically well-defined donors, our group has recently reported new strategies to access H2S donors through the intermediate release of carbonyl sulfide (COS). In our initial approach, COS was caged in a self-immolative thiocarbamate system. Upon removal of the protecting group, the cascade decomposition of the thiocarbamate released COS, which is quickly hydrolyzed to H2S by the ubiquitous mammalian enzyme carbonic anhydrase (CA) with an associated rate constant of 2.2 × 104 M−1 s−1 (for bovine CA II) (Scheme 1 top).45–47 Following our initial report, our group, as well as others,48 have developed a series of COS-based H2S donors that can be triggered through different mechanisms, such as cellular reactive oxygen species (ROS),49–51 esterases,46, 52–53 nucleophiles,54 light,55–57 click chemistry,58 and Cys.59 More recently, we have broadened our approach to include different activation strategies and core motifs to develop colorimetric60 and fluorescent61 COS-based H2S donors, such as γ-KetoTCM-1 and FLD, which released COS/H2S with a concomitant change in optical readout (Scheme 1 bottom).

Scheme 1.

Scheme 1.

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Examples of COS-based H2S donors.

Advancing from this prior work, we envisioned that hybrid COS-releasing constructs based on thioamide cores could be used to leverage the increased H2S-releasing efficiency from COS donors with the beneficial properties of thioamides, such as synthetic simplicity and ease of incorporation into common pharmaceutical compounds. Here we report the design, synthesis, evaluation, and mechanistic investigation of thioamide-derived cyclic sulfenyl thiocarbamates (SulfenylTCMs, also known as 1,2,4-dithiazolin-3-ones).62,63 The SulfenylTCMs are stable in aqueous solutions but are activated by cellular thiols to cleave the cyclic disulfide and release COS, which is quickly converted to H2S by CA. The resultant iminodisulfide intermediate then reacts further with cellular thiols to generate a thioamide, which can further release H2S (Scheme 2 bottom).

Results and Discussion

Donor synthesis

To test our hypothesis that cyclic sulfenylTCMs can function as thiol-activated COS/H2S donors, we first prepared sulfenylTCMs by treating corresponding thioamides with chlorocarbonylsulfenyl chloride (Scheme 3). Briefly, chlorocarbonylsulfenyl chloride (2.0 equiv.) was added to anhydrous THF containing the desired thioamide (1.0 equiv.) at 0 °C. The resultant solution was stirred at room temperature until the completion of the reaction as indicated by TLC. The reaction solution was then concentrated and the sulfenylTCM was isolated and purified by flash column chromatography. Five donors with aryl (SulfenylTCM-1 – 4) or alkyl (SulfenylTCM-5) substituents were prepared, and SulfenylTCM-1 was selected as the model donor for COS/H2S releasing and mechanistic evaluations.

Scheme 3.

Scheme 3.

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Synthesis of sulfenyl thiocarbamates.

GSH-Activated COS/H2S release from SulfenylTCM-1

To evaluate thiol-activated H2S delivery from the donor motifs, we used the colorimetric methylene blue (MB) assay to monitor H2S production from SulfenylTCM-1 (25 μM) in the presence of GSH (0 – 1000 μM) in PBS buffer (pH 7.4, 10 mM) containing cellularly-relevant concentrations of CA (25 μg/mL). GSH was used as the model thiol trigger due to its cellular abundance (typically 5 – 10 mM) and high nucleophilicity. The MB assay was chosen to measure H2S production since it has been widely used to detect H2S from a variety of H2S donors. Treating SulfenylTCM-1 in PBS in the absence of GSH failed to provide detectable H2S signal, indicating negligible spontaneous H2S delivery from SulfenylTCM-1. In the presence of GSH, however, SulfenylTCM-1 exhibited a dose-dependent COS/H2S release response (Figure 2). These results demonstrate that SulfenylTCM-1 is activated by GSH in aqueous buffer, and that the resultant COS is quickly converted to H2S by CA.

Figure 2.

Figure 2.

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COS/H2S Release from SulfenylTCM-1 (25 μM) in the presence of 0 μM0020(black), 250 μM (red), 500 μM (blue), and 1000 μM (green) GSH. The experiments were performed in triplicate and results are expressed as mean ± S.D. (n = 3).

Thiol-activated COS/H2S release from SulfenylTCM-1

Because disulfide bonds are readily cleaved by thiol species, we anticipated that SulfenylTCM-1 should be triggered by not only GSH, but also by other cellular thiols, such as Cys, Hcy, N-acetylcysteine (NAC), and penicillamine (PEN). To test this hypothesis, we treated SulfenylTCM-1 (25 μM) with each thiol trigger (500 μM) in PBS buffer (pH 7.4, 10 mM) containing CA (25 μg/mL) and monitored H2S generation using the MB assay. As expected, a time-dependent COS/H2S release was observed in the presence of Cys, Hcy, or NAC, indicating a successful activation of SulfenylTCM-1. In comparison, H2S delivery was significantly reduced (~10% COS/H2S release) in the presence of PEN, presumably due to the increased steric bulk and resultant decrease in nucleophilicity, which prohibited its reaction with SulfenylTCM-1 (Figure 3). Taken together, these studies demonstrated that SulfenylTCM-1 can be activated by a variety of cellular thiol species, such as GSH, Cys, Hcy, NAC and PEN. In addition, COS/H2S delivery from SulfenylTCM-1 can be controlled by using different triggers due to their different reactivities towards SulfenylTCM-1.

Figure 3.

Figure 3.

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Thiol-dependent (500 μM) COS/H2S release from SulfenylTCM-1 (25 μM). The experiments were performed in triplicate and results are expressed as mean ± S.D. (n = 3).

Effects of cellular nucleophiles on COS/H2S release from SulfenylTCM-1

To investigate whether COS/H2S release can be triggered by other species, SulfenylTCM-1 (25 μM) was treated with biologically relevant species (500 μM), including oxidized glutathione (GSSG), lysine (Lys), serine (Ser), glycine (Gly), thiosulfate (S2O32–), sulfite (SO32–), and sulfate (SO42–), in PBS buffer (pH 7.4, 10 mM) containing CA (25 μg/mL) and COS/H2S release was monitored using the MB assay. Compared to GSH-induced donor activation, which led to 70% H2S production during a 4-hour reaction period, none of the above species triggered SulfenylTCM-1 (Figure 4). These studies confirmed that SulfenylTCM-1 is stable towards common biological nucleophiles and reactive sulfur species, but also highly sensitive towards thiol activation to deliver COS/H2S.

Figure 4.

Figure 4.

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COS/H2S Release from SulfenylTCM-1 (25 μM) in the presence of cellular nucleophiles (500 μM). H2S concentration was measured after 4-h incubation. The experiments were performed in triplicate and the results were expressed as mean ± S.D. (n = 3).

GSH-Activated COS/H2S release from Other SulfenylTCMs

Having evaluated COS/H2S production from SulfenylTCM-1, we next investigated the COS/H2S releasing efficiency of other SulfenylTCMs (SulfenylTCM-2 – 5) using GSH as the model trigger. In these experiments, the SulfenylTCM donors (25 μM) were incubated with GSH (500 μM) in PBS buffer (pH 7.4, 10 mM) containing CA (25 μg/mL). As expected, all donors were activated by GSH and COS/H2S release was detected by the MB assay. Importantly, the rate of COS/H2S release from these donor motifs was changed by structural modifications, which demonstrates that release rates can be tuned by use of differently-substituted thioamide precursors. For example, even though SulfenylTCM-1 – 3 showed similar COS/H2S releasing profile, donors with strong electron withdrawing groups (i.e. CF3 in SulfenylTCM-4) or small alkyl group (i.e. CH3 in SulfenylTCM-5) provided significantly enhanced COS/H2S release (Figure 5). During the 4-hour time course of these experiments, we were surprised to observe over 100% H2S release from SulfenylTCM-4 (130%) and SulfenylTCM-5 (126%). Although the extra H2S could potentially be attributed to H2S release from the 4-methoxythiobenzamide and thioacetamide byproducts, the low H2S releasing efficiency of thioamide-based H2S donors suggested to us that an alternative COS-independent H2S releasing pathway was plausible in these SulfenylTCM systems.

Figure 5.

Figure 5.

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COS/H2S release from SulfenylTCM-1 – 5 (25 μM) in the presence of GSH (500 μM) in PBS (pH 7.4, 10 mM) containing CA (25 μg/mL). The experiments were performed in triplicate and the results are expressed as mean ± S.D. (n = 3).

To determine whether SulfenylTCM donors also released H2S directly through a COS-independent pathway, we treated SulfenylTCM-1 – 5 (25 μM) with GSH (500 μM) in PBS (pH 7.4, 10 mM) in the absence of CA. For each donor, direct H2S release was observed using the MB assay, which confirmed the existence of direct H2S releasing pathway(s) (Figure 6). Although certain aryl thioamides have been reported previously as Cys-activated H2S donors, we did not observe H2S release from thioamides (25 μM), such as thiobenzamide, in the presence of GSH (500 μM) using the MB assay under our conditions (Figure S1).24 Taken together, these studies demonstrated that SulfenylTCM donors can release H2S through a COS- and thioamide-independent pathway (vide infra).

Figure 6.

Figure 6.

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COS-Independent H2S release from SulfenylTCM-1 – 5 (25 μM) in the presence of GSH (500 μM) in PBS (pH 7.4, 10 mM). The experiments were performed in triplicate and the results were expressed as mean ± S.D. (n = 3).

Mechanistic investigation on COS/H2S release from SulfenylTCM-1

To further investigate the operative direct H2S release mechanism from SulfenylTCMs, we next conducted NMR experiments to determine which intermediates and products were formed during the course of the reaction in the absence of CA. We first confirmed the stability of SulfenylTCM-1 in DMSO-d6/D2O (9:1) over the course of the standard experiments (Figure S3). Next, we prepared a DMSO-d6/D2O (9:1) solution containing SulfenylTCM-1 (10 mM) and 5 equiv. of benzyl mercaptan (BnSH, 50 mM) and monitored the reaction by NMR spectroscopy. This experiment showed that the reaction was complete with full consumption of SulfenylTCM-1 within 30 min. The two major products of the reaction were thiobenzamide and benzyl disulfide (BnSSBn), which were confirmed by comparison to authentic samples (Figures 7 and S4). In related experiments using substoichiometric amounts of BnSH (0.1 – 0.5 equiv.), we also observed complete SulfenylTCM-1 consumption, which indicated that the thiol triggers could serve as promotors for SulfenylTCM activation (Figures S5 and S6). This observation is consistent with a mechanism in which activation of SulfenylTCM-1 by BnSH generates reactive thiol intermediates, which would further react with the donor motif to release COS/H2S. In addition, we also treated SulfenylTCM-1 (5.0 mM) with GSH (10.0 mM) in DMSO/PBS (pH 7.4, 10 mM) (1:1) for 4 h. Thiobenzamide was then isolated as the final product (yield 98%), which is consistent with our NMR study using BnSH as a model trigger (Figure S7).

Figure 7.

Figure 7.

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13C{1H} NMR spectra of the reaction between SulfenylTCM-1 (red star) and BnSH (green square). Thiobenzamide (purple circle) and BnSSBn (blue triangle) were identified as major products using authentic samples.

Based on the MB measurements and NMR experiments, three main observations drive the mechanistic requirements of donor activation: (1) SulfenylTCM donors can be activated by thiols to release COS, which functions as an H2S precursor in the presence of CA; (2) SulfenylTCM donors can also directly release H2S in the absence of CA; and (3) thiobenzamide and disulfide (RSSR) are the two major products when treating SulfenylTCM-1 with either substoichiometric or excess RSH (0.1 – 5 equiv.), such as BnSH. According to these results, we proposed donor activation and H2S releasing pathways in Scheme 4. Briefly, thiol triggers (RSH) can react with the SulfenylTCM to cleave the disulfide bond, which results in a dethiocarboxylation to release COS, which is quickly converted to H2S by CA (COS-dependent pathway). This step also generates iminodisulfide intermediate 1, which can undergro thiol/disulfide exchange to generate RSSR and thiobenzamide (pathway A). A thiol can also react with iminodisulfide 1 at the electrophilic imine carbon to yield iminothioether 2 and a persulfide (RSSH), which can react further with thiols to generate H2S (pathway B – COS-independent H2S releasing pathway). Adding further complexity to this reactivity, the generated H2S or persulfides could also likely intercept 1 to yield the thiobenzamide product and generate an additional persulfide or polysulfide, respectively. In addition, any persulfide intermediates can also likely react with the donor motif directly to propagate this chain of reactions to yield H2S.

Scheme 4.

Scheme 4.

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Proposed mechanism of thiol-triggered COS/H2S release from SulfenylTCM-1.

In our investigations, we did not observe iminothioether 2 directly in our NMR experiments (Figures 7, S4, S5, S6), but we suspected that 2 would likely react directly with H2S to generate RSH, which would further react with SulfenylTCM-1. This general reaction scheme supports the role of thiols acting as a promotor for SulfenylTCM activation (Figures S5 and S6). To test our hypothesis, we synthesized iminothioether 2 and treated it (10 mM) with NaSH (100 mM) in DMSO-d6/D2O (9:1). NMR experiments showed the full consumption of 2 by H2S within 10 min, and the formation of thiobenzamide and RSH as the final products (Figure S8), which is consistent with proposed reaction pathway B. This reactivity suggests that H2S generated in the system may go on to propagate donor activation. Furthermore, this reactivity is consistent with the recently-reported Cys-triggered H2S donation from iminothioether derivatives.25 Moreover, our studies here provided useful mechanistic insights on internal H2S scavenging by iminothioether donor motifs, which also helps to explain the low H2S releasing efficiency observed in the recently-reported iminothioether donor systems.25

Generation of a COS-hybrid naproxen as new COS/H2S-releasing NSAID

To demonstrate the generality of sulfenyl thiocarbamate incorporation into biologically-active platforms, we envisioned that this donor motif could be readily incorporated into compounds used for anti-inflammatory activity, such as non-steroidal anti-inflammatory drugs (NSAIDs). One of the major limitation of NSAIDs is the potential gastrointestinal (GI) and cardiovascular toxicity.64–65 Recent efforts to improve the therapeutic profile and reduce the GI toxicity of these drugs have included the generation of a series of H2S-releasing hybrid NSAIDs (H2S-NSAIDs), many of which include thioamides (Figure 8a–c).38, 66–67 For example, one of the most successful H2S-NSAIDs that progressed into clinical development is ATB-346, a naproxen derivative coupled with thiobenzamide as an H2S releasing moiety. This hybrid H2S-NSAID exhibited similar anti-inflammatory activities to naproxen but with significantly-reduced GI damage.38, 44, 68 Aligned with this high therapeutic potential, ATB-346 has advanced to two clinical trials in 201469 and 2017.70 One limitation of ATB-346, much like other thioamide-based donors, is the poorly-understood H2S releasing mechanism and the relatively low efficiency of H2S release, which was reported to be only 10 – 20 μM H2S from 1 mM of the drug motif.38 We viewed that developing related H2S-releasing NSAIDs with more efficient and mechanistically-understood donor motifs could provide a useful platform for further leveraging these hybrid H2S donor / drug system.

Figure 8.

Figure 8.

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Representative thioamide-containing H2S-NSAIDs, such as (a) GIC-1001, (b) NBS-1121, and (c) ATB-346, and (d) COS/H2S-NSAID YZ-597.

To investigate whether cyclic sulfenyl thiocarbamates could be used to enhance H2S release from such systems, we prepared YZ-597 from ATB-346 (Figure 8d). Our expectation, based on our work described above, was that this compound would be activated much more efficiently than the parent H2S-NSAID conjugate. Consistent with this hypothesis, when we incubated YZ-597 (25 μM) in the presence of GSH (500 μM) in PBS buffer (pH 7.4, 10 mM) containing CA (25 μg/mL) and 1 mM CTAB to help solubilize the components we observed rapid and efficient H2S release (90%). Although H2S release from ATB-346 has been reported to be favored in the presence of reducing agents, such as Cys and GSH or in the presence of biological materials, we did not observed efficient H2S release from ATB-346 under our experimental conditions (Figure 9).68 These data suggest that YZ-597 may have more accessible H2S releasing pathways than the thioamide-derived ATB-346 motif.

Figure 9.

Figure 9.

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GSH (500 μM)-triggered H2S release from YZ-597 or ATB-346 (25 μM). The experiments were performed in triplicate and results are expressed as mean ± S.D. (n = 3).

Having confirmed H2S release from YZ-597 in the presence of GSH, we next investigated whether this conjugate could release H2S efficiently in live cells. We first incubated HeLa cells with FBS-free DMEM containing the H2S-responsive fluorescent probe SF7-AM (5 μM), and after washing then added either YZ-597 (50 μM) or vehicle.71 As shown in Figure 10, the HeLa cells displayed negligible H2S fluorescent signal in the absence of YZ-597, suggesting minimal endogenous H2S levels. In comparison, cells treated with YZ-597 revealed a strong fluorescent signal, indicating that YZ-597 was activated to release H2S in a cellular environment. We also treated HeLa cells with the donor motif SulfenylTCM-1 under the identical condition and observed similar fluorescence enhancement (Figure S9). Taken together, we view that YZ-597 and related sulfenyl thiocarbonate motifs can provide a useful platform for investigating the action of H2S-hybrid NSAID and related pharmacologically-active compounds.

Figure 10.

Figure 10.

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H2S Delivery from YZ-597 in HeLa cells. HeLa cells were treated with SF7-AM (5 μM) and Hoechst (10 μg/mL) for 5 min. After removal of extracellular SF7-AM and Hoechst, cells were incubated in FBS-free DMEM in the absence (Top row) or presence (Bottom row) of YZ-597 (50 μM) for 30 min. Cells were then washed and imaged in PBS. Scale bar: 50 μm.

Conclusions

Thioamide-derived sulfenyl thiocarbamates function as thiol-activated COS/H2S donors. These compounds can be activated by cellular thiol species, such as GSH, Cys, Hcy, and NAC, to deliver H2S. We demonstrated that the H2S-releasing efficiency of SulfenylTCMs is significantly enhanced in comparison to the parent thioamide compounds. Moreover, COS and H2S release from SulfenylTCM compounds is mechanistically well-defined, which provides the ability to tune release parameters. Leveraging this design platform, we also prepared the COS/H2S-NSAID, YZ-597, which is built off of the ATB-346 platform, and demonstrated the efficient H2S release both in vitro and in live cells from YZ-597. In addition, we anticipate that YZ-597 and related compounds will also serve as a promising chemical tools to provide insights into the relationship between H2S release and efficacy of H2S-hybrid compounds. Further investigations into the activity of YZ-597 and related H2S hybrids are currently ongoing in our laboratory.

Experimental Section

Materials and Methods

Reagents were purchased from Sigma Aldrich, Tokyo Chemical Industry (TCI), Fisher Scientific, and VWR and used directly as received. Carbonic anhydrase (CA) from bovine erythrocytes was purchased from Sigma-Aldrich (C2624). Silica gel (SiliaFlash F60, Silicycle, 230−400 mesh) was used for column chromatography. Deuterated solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, Massachusetts, USA). 1H, 19F, and 13C{1H} NMR spectra were recorded on Bruker 500 MHz NMR instrument at the indicated frequencies. Chemical shifts are reported in ppm relative to residual protic solvent resonances. Mass spectrometric measurements were performed by the University of Illinois, Urbana Champaign MS facility, or on a Xevo Waters ESI LC/MS instrument. Methylene blue absorbance was monitored by an UV−vis spectrometer (Cary 100, Agilent Technologies, Santa Clara, California, USA) in PBS buffer. SF7-AM,71 ATB-346,72 and iminothioether 225 were synthesized by following the literature report. HeLa cells were purchased from ATCC (Manassas, Virginia, USA). Cell imaging experiments were performed on a Leica DMi8 fluorescence microscope, equipped with an Andor Zyla 4.2+ sCMOS detector.

Synthesis

SulfenylTCM-1. Thiobenzamide (135 mg, 1.00 mmol) was dissolved in anhydrous THF (15 mL) followed by the addition of chlorocarbonylsulfenyl chloride (262 mg, 2.00 mmol) at 0 °C. The resultant solution was stirred at 0 °C for 10 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. for 1 h. The reaction solution was then concentrated under vacuum and the crude product was purified by column chromatography. SulfenylTCM-1 was isolated as yellow solid (70%). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.16 (d, J = 10.0 Hz, 2H), 7.80 (d, J = 10.0 Hz, 1H), 7.66 (t, J = 10.0 Hz, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ (ppm): 189.3, 186.1, 135.5, 131.4, 130.3, 128.5. IR (cm−1): 1680, 1655, 1592, 1501, 1479, 1445, 1306, 1235, 1086, 1065, 923, 766, 683, 669. HRMS m/z [M+H]+ calcd for [C8H6NOS2]+ 195.9891; found 195.9891.

SulfenylTCM-2 was prepared from 4-methoxythiobenzmide by following the procedure described above (47 mg, 41% yield). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.14 (d, J = 10.0 Hz, 2H), 7.17 (d, J = 10.0 Hz, 2H), 3.91 (s, 3H). 13C{1H} NMR (125 MHz, DMSO-d6) δ (ppm): 188.2, 185.8, 165.3, 130.9, 123.9, 115.6, 56.4. IR (cm−1): 2963, 2835, 1668, 1597, 1574, 1521, 1484, 1421, 1308, 1245, 1168, 1078, 1023, 925, 828, 666. HRMS m/z [M+H]+ calcd for [C9H8NO2S2]+ 225.9996; found 225.9999.

SulfenylTCM-3 was prepared from 4-chlorothiobenzmide by following the procedure described above (91 mg, 81% yield). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.17 (d, J = 10.0 Hz, 2H), 7.73 (d, J = 10.0 Hz, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ (ppm): 188.1, 186.0, 140.3, 130.3, 130.2. IR (cm−1): 3068, 3033, 1660, 1590, 1504, 1477, 1400, 1306, 1281, 1239, 1178, 1008, 827, 666. HRMS m/z [M+H]+ calcd for [C8H5ClNOS2]+ 229.9505; found 229.9497.

SulfenylTCM-4 was prepared from 4-trifluoromethylthiobenzmide by following the procedure described above (104 mg, 79% yield). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.35 (d, J = 10.0 Hz, 2H), 8.01 (d, J = 10.0 Hz, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ (ppm): 188.0, 186.0, 134.9, 129.4, 127.1, 125.0, 122.9. 19F NMR (470 MHz, DMSO-d6) δ (ppm): −61.8. IR (cm−1): 3047, 1661, 1517, 1493, 1408, 1330, 1305, 1190, 1171, 1118, 1064, 1011, 924, 845, 669. HRMS m/z [M+H]+ calcd for [C9H5F3NOS2]+ 263.9765; found 263.9760.

SulfenylTCM-5 was prepared from thioacetamide by following the procedure described above (35 mg, 40% yield). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 2.79 (s, 3H). 13C{1H} NMR (125 MHz, DMSO-d6) δ (ppm): 192.9, 187.0, 22.9. IR (cm−1): 1662, 1516, 1494, 1165, 1071, 1000, 668, 628, 589. HRMS m/z [M+H]+ calcd for [C3H4NOS2]+ 133.9734; found 133.9735.

YZ-597 was prepared from ATB-346 by following the procedure described above (105 mg, 87% yield). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 8.19 (d, J = 5.0 Hz, 2H), 7.88 (s, 2H), 7.86 (s, 1H), 7.54 (d, J = 5.0 Hz, 1H), 7.34 (s, 2H), 7.32 (s, 1H), 7.19 (d, J = 10.0 Hz, 1H), 4.29 (q, J = 5.0 Hz, 1H), 3.88 (s, 3H), 1.63 (d, J = 10.0 Hz, 3H). 13C{1H} NMR (125 MHz, DMSO-d6) δ (ppm): 188.2, 186.0, 172.8, 157.8, 155.9, 135.3, 134.0, 130.3, 129.7, 129.0, 127.8, 126.7, 126.4, 123.5, 119.4, 106.3, 55.7, 45.0, 18.8. IR (cm−1): 1742, 1677, 1662, 1485, 1412, 1206, 1165, 1135, 1117, 1087, 1028, 893, 853, 844, 814. HRMS m/z [M+H]+ calcd for [C22H18NO4S2]+ 424.0677; found 424.0657.

H2S Release from SulfenylTCMs in PBS

A SulfenylTCM stock solution (50.0 μL, 10.0 mM in DMSO) was added to 20.0 mL of PBS (pH 7.40, 10.0 mM) containing CA (25.0 μg/mL) in a 25-mL scintillation vial. A thiol stock solution (0.100 M in H2O) was then added to generate the desired thiol working concentrations as shown in Figures 2, 3, and 5. For H2S release in the absence of CA, the measurement was set up by following the procedure as described above in PBS with no CA addition (Figure 6). For H2S release from YZ-597, the measurement was set up by following the procedure as described above in PBS containing CA (25.0 μg/mL) and CTAB (1.00 mM) (Figure 9). Next, 0.300 mL aliquots of the reaction mixture were transferred to UV cuvettes containing 0.300 mL of MB cocktail (0.060 mL zinc acetate (1.00% w/v), 0.120 mL FeCl3 (30.0 mM in 1.20 M HCl), and 0.120 mL N,N-dimethyl-p-phenylene diamine (20.0 mM in 7.20 M HCl)) at different time points. The absorbance at 670 nm was then measured after 30 min and was converted to H2S concentration by using the H2S calibration curve.

Selectivity investigations on H2S release

A SulfenylTCM-1 stock solution (50.0 μL, 10.0 mM in DMSO) was added to 20.0 mL of PBS (pH 7.40, 10.0 mM) containing CA (25.0 μg/mL) in a 25-mL scintillation vial. An analyte stock solution (100 μL, 100 mM in H2O) was then added. The reaction was stirred at r.t. for 4 h. Next, 0.300 mL aliquot of the reaction mixture was transferred to UV cuvettes containing 0.300 mL of MB cocktail (0.060 mL zinc acetate (1.00% w/v), 0.120 mL FeCl3 (30.0 mM in 1.20 M HCl), and 0.120 mL N,N-dimethyl-p-phenylene diamine (20.0 mM in 7.20 M HCl)). The absorbance at 670 nm was then measured after 30 min and was converted to H2S concentration by using the H2S calibration curve.

Mechanism investigations of H2S release

SulfenylTCM-1 (0.0200 M) and BnSH (0.100 M) solutions were prepared by adding SulfenylTCM-1 (3.90 mg) and BnSH (12.4 mg) in DMSO-d6/D2O (9:1, 1.00 mL), respectively. Then SulfenylTCM-1 (0.0200 M, 0.500 mL) was added to BnSH (0.100 M, 0.500 mL) to reach the concentrations of 0.0100 M of SulfenylTCM-1 and 0.0500 M of BnSH, respectively. The reaction process was then monitored by using a Bruker 500 MHz NMR instrument.

Cellular imaging of H2S release from YZ-597

HeLa cells were plated in poly-D-lysine coated plates (MatTek) containing 2 mL of DMEM and incubated at 37 °C under 5% CO2 for 24 h. The confluent cells were washed with PBS and then incubated with SF7-AM (5.00 μM) and Hoechst dye (10.0 μg/mL) for 5 min. The cells were then washed with PBS and incubated with in FBS-free DMEM in the absence or presence of YZ-597 (50.0 μM) for 30 min. Prior to imaging, cells were washed with PBS and bathed in 2 mL of PBS. Cell imaging was performed on a Leica DMi8 fluorescent microscope using DIC for bright field and a standard DAPI and GFP filter cubes for fluorescence imaging, respectively. The scale bar represents 50 μm.

Supplementary Material

supporting information

ACKNOWLEDGMENT

Research reported in this publication was supported by the NIH (MDP; R01GM113030) Dreyfus Foundation, and NSF/GRFP (AKS; DGE-1309047). NMR, Fluorescence microscopy, and MS instrumentation in the UO CAMCOR facility is supported by the NSF (CHE-1427987, CHE-1531189, and CHE-1625529).

Footnotes

ASSOCIATED CONTENT

Supporting Information. H2S release data, NMR experiments, cell imaging, and spectra. This material is available free of charge via the Internet at http://pubs.acs.org

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