2H-Thiopyran-2-thione sulfine, a compound for converting H2S to HSOH/H2S2 and increasing intracellular sulfane sulfur levels

Cui, Qi; Shieh, Meg; Pan, Tony W.; Nishimura, Akiyuki; Matsunaga, Tetsuro; Kelly, Shane S.; Xu, Shi; Jung, Minkyung; Ogata, Seiryo; Morita, Masanobu; Yoshitake, Jun; Chen, Xiaoyan; Robinson, Jerome R.; Qian, Wei-Jun; Nishida, Motohiro; Akaike, Takaaki; Xian, Ming

doi:10.1038/s41467-024-46652-7

Download PDF

Article
Open access
Published: 19 March 2024

2H-Thiopyran-2-thione sulfine, a compound for converting H₂S to HSOH/H₂S₂ and increasing intracellular sulfane sulfur levels

Nature Communications volume 15, Article number: 2453 (2024) Cite this article

1387 Accesses
Metrics details

Subjects

Abstract

Reactive sulfane sulfur species such as persulfides (RSSH) and H₂S₂ are important redox regulators and closely linked to H₂S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H₂S to HSOH, and then to H₂S₂ in the presence of excess H₂S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H₂S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H₂S donors, the corresponding donors of H₂S₂ (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting

Article 16 September 2021

Mechanism-based and computational modeling of hydrogen sulfide biogenesis inhibition: interfacial inhibition

Article Open access 04 May 2023

Nucleophilic covalent ligand discovery for the cysteine redoxome

Article 29 May 2023

Introduction

Since the discovery of hydrogen sulfide (H₂S) as a nitric oxide (NO)-like signaling molecule in recent years, research on H₂S and its related reactive sulfur species has exploded^1,2,3. A hot topic in this field is to develop compounds or materials that can precisely control the release of H₂S or its related sulfur species and to explore their applications^4,5,6,7,8. In this regard, hydrogen persulfide (H₂S₂), a highly reactive sulfane sulfur species, has received much attention as it is believed to have distinct regulatory functions in redox biology and is closely linked to H₂S-signaling^9,10,11. Endogenous H₂S₂ is produced indirectly by enzymes like 3-mercaptopyruvate transferase (3-MST) and cysteinyl-tRNA synthetase (CARS), via persulfides as the key intermediates. It can also be produced from H₂S via H₂S-NO cross talk or hemeprotein-catalyzed oxidation. H₂S₂ appears to be more efficient than H₂S in inducing protein persulfidation (forming P-SSH), and this is suggested to be one of the main functions of H₂S₂ in biology^12,13,14,15.

While the research on H₂S₂ is rapidly growing, a major challenge is the use or handling of the H₂S₂ source in studies. H₂S₂ is much more reactive and unstable than H₂S. Therefore, there is a pressing need to develop suitable H₂S₂ donors. Unlike H₂S, whose donors have been extensively studied with a large number of such compounds already reported^4,5,6,7,8, the donors of H₂S₂ are still very limited. Currently, most studies use Na₂S₂ as the equivalent of H₂S₂, but Na₂S₂ is considered to be an uncontrollable and instant H₂S₂ donor. A few synthetic H₂S₂ donors have been reported, and their structures are shown in Fig. 1-A^{16,17,18,19,20}. These donors rely on protected disulfide (-S-S-) structures, which have inherent limitations: 1) disulfides are sensitive to disulfide-exchange reactions with cellular thiols (Cys or GSH) and would change their identity (e.g. not form H₂S₂), and 2) acyl disulfides (as shown in I and BW-HP, Fig. 1-A) are highly reactive toward cellular nucleophiles (-SH, -NH₂, etc.), which can diminish their efficiency. In theory, H₂S₂ can be produced from in situ reactions of H₂S with oxidants such as H₂O₂ and HClO₄²¹. However, this is not a suitable strategy for H₂S₂ delivery as the presence of oxidants can compromise the biological systems under investigation. In addition, the oxidation can hardly be controlled, so over-oxidation could occur to produce the stable and much less reactive adduct, elemental sulfur (S₈). The specificity of oxidation is also a problem as many biomolecules (such as proteins) can be modified by oxidation conditions. Given the aforementioned considerations, exploring novel strategies in the design of H₂S₂ donors has been a top priority in our lab. Herein, we report a unique booster system based on 2H-thiopyran-2-thione sulfine (TTS), which can effectively and specifically convert H₂S to HSOH, and then to H₂S₂ via non-oxidation and non-disulfide conditions. The reaction mechanism has been studied, and its applications in protein and cell models are evaluated. Interestingly, the reaction product 2H-thiopyran-2-thione (TT) can be converted back to TTS by biologically relevant oxidants, highlighting this as a potentially regenerable system. Furthermore, TTS can be applied in conjunction with H₂S-donor systems, essentially turning the H₂S donor system into an HSOH/H₂S₂-donor system.

**Fig. 1: Summary of the idea and chemistry of TTS.**

Results

Design and preparation of the sulfine-based booster TTS

In our search of possible new chemistry for H₂S₂ generation, we identified sulfines (thiocarbonyl oxides) as having great potential. Sulfines are oxidative intermediates of thiocarbonyl compounds^22,23,24,25. Aliphatic sulfines were found to be normally unstable and could decompose to form the corresponding carbonyl and thiocarbonyl compounds with half-lives of 1–4 days (Fig. 1B). When UV light was introduced, atomic sulfur could release²³. Nucleophiles such as amines could react with sulfines to form imines and HSOH. It is worth noting that HSOH is the smallest sulfenic acid, which can be expected to possess H₂S₂-like reactivity (such as inducing persulfidation). However, the chemistry of HSOH has not been well-studied due to its instability. To the best of our knowledge, there is no HSOH donor reported thus far. Based on these analyses, sulfines may serve as unique HSOH (or sulfane sulfur) donors. However, the poor stability of common sulfines would limit their applications. We envisioned that if the sulfine moiety was introduced to an aromatic heterocyclic system, its stability would be significantly enhanced. That could prevent its reaction with less reactive cellular nucleophiles (-OH, -NH₂, etc.) and only allow the most reactive nucleophiles (such as H₂S) to react. More importantly, once HSOH is generated, it could further react with H₂S to form H₂S₂, thereby creating a system for H₂S₂ generation.

Our recent work showed that thiocarbonyl-containing heterocycles such as 2H-thiopyran-2-thiones (TT) could serve as CS₂ donors upon bio-orthogonal cycloaddition with strained alkynes²⁶. Thus, these heterocycles may be appropriate substrates to use for sulfine preparation and allow us to test our hypothesis. As shown in Fig. 1C, we found that the sulfine derivative (TTS) of 2H-thiopyran-2-thiones could be readily prepared from TT upon the treatment of m-CPBA. The structure of TTS was unambiguously determined by single-crystal X-ray diffraction studies of the analog, TTS-Br (Fig. 1C and Supplementary Figs. 1 and 2). Both the heterocyclic core and sulfine fragment were effectively coplanar (∠S(2)–C(1)–S(1)–O(1): 0.1(7)˚), where the S–O bond was oriented towards the endocyclic sulfur. The S–O bond distance in TTS-Br (1.381(10) Å) supported significant double bond character and was shorter than that of aryl sulfines (1.427(4)–1.477(2) Å)^27,28 and close to those found in interstitial SO₂ (~1.380 Å)^29,30,31.

Reactions of TTS

Next, we tested TTS’s reaction with H₂S. In a mixed solvent system with PBS buffer (pH 7.4) and acetonitrile (1:1), the reaction between TTS and H₂S (using Na₂S as the equivalent) produced TT in an almost quantitative yield (94%). The formation of H₂S₂ in this reaction was also demonstrated by trapping H₂S₂ with phenyl 2-fluoro-5-nitrobenzoate³², which gave the desired product 5-nitro-3H-1,2-benzodithiol-3-one 1 in an 80% isolated yield. Since HSOH was expected to be the key intermediate in the reaction, we attempted to trap HSOH by chemicals such as dimedone. However, we were unable to obtain the desired HSOH-trapped product. This might be attributed to the much faster reaction between HSOH and H₂S than the trapping reaction. Nevertheless, the mechanistic analyses (vide infra) still support the formation of HSOH in this process.

It is worth noting that the reaction between TTS and H₂S was a clean and fast process. TTS and TT possess different UV-Vis absorbance spectra at 380 and 339 nm, respectively, so the reaction could be easily monitored by UV-Vis spectrophotometry (Fig. 1D). By measuring time-dependent spectra changes, the second-order rate constant, k₂, was calculated to be 2.7 M⁻¹s⁻¹, indicating that this was a fast reaction comparable to some click reactions³³.

The reaction between TTS and H₂S is unique as it produces not only H₂S₂, but also TT, the precursor of TTS. As such, this molecule might be regenerated in biological systems upon reaction with oxidants. To test this idea, we first treated TT with H₂O₂, the most prevalent oxidant in biology. As shown in Fig. 2A, the reaction was clean and reached completion in ~24 h. The progress of the reaction could also be monitored by UV-Vis spectrophotometry, where the reaction rate constant (k₂) was measured to be 0.1 M⁻¹s⁻¹. We further measured the oxidation of TT by several other oxidants, including t-BuOOH, AcOOH, and OCl^–, and the results were summarized in Fig. 2B. t-BuOOH showed weaker reactivity than H₂O₂ while AcOOH and OCl^– were efficient and caused rapid oxidation. These results demonstrated that the TTS-based booster system could be regenerated in the presence of appropriate oxidants.

**Fig. 2: Mechanistic studies of TTS reactions.**

The TTS/H₂S reaction mechanism was proposed in Fig. 2C, which was also studied by DFT (M06-2X) calculations. The reaction was initiated by the nucleophilic addition of HS^– to the thiocarbonyl carbon of TTS via TS1 to give intermediate INT1. This rate-determining step had an activation Gibbs free energy of 15.2 kcal/mol. Then, the [1,4]-H shift on INT1 occurred to give thermodynamically favored INT2. A sulfur(0) species HOS^- was released next via TS2 (ΔG^‡ = 11.5 kcal/mol), which could react with another molecule of H₂S to give HSS^-, H₂O, and TT.

To provide additional experimental support for the proposed mechanism, we carried out a sulfur isotope labeling experiment (Fig. 2D). TTS was treated with ³⁴S-labeled Na₂S in PBS buffer (pH 7.4). As expected, ³⁴S-labeled product TT-³⁴S was obtained and characterized by MS analysis (Supplementary Fig. 3). It should be noted that TT-³⁴S could be converted into TTS-³⁴S (Supplementary Fig. 4) upon oxidation. Therefore, TTS-³⁴S could be a useful reagent for the in-situ generation of ³⁴S-labeled HSOH/H₂S₂, and we believe that this could have some useful applications in understanding the biological functions of these species. As a proof of concept, we used 2-fluoro-5-nitrobenzoate to trap ³⁴S-labeled H₂S₂. As expected, ³⁴S-labeled product 1-³⁴S was obtained, albeit with the possibility of having two isomers (the HRMS spectrum of 1-³⁴S is shown in Supplementary Fig. 5).

Validation of H₂S₂ formation from TTS by fluorescence studies

To further evaluate the specificity of TTS in converting H₂S to H₂S₂ under our conditions (e.g. w/ excess H₂S), we used SSP4 to monitor the formation of H₂S₂ in this reaction and compared the results with a series of controls. SSP4 is a well-established fluorescent sensor for sulfane sulfurs including H₂S₂³⁴. As shown in Fig. 3I-A, only the co-existence of H₂S and TTS yielded significant fluorescence response. We did not observe any fluorescence in the cases of TTS/TT only, TTS+Cys, TT + H₂S, and TT+Cys. Interference experiments with Cys or GSH were also tested (Fig. 3I-B). The presence of thiols led to decreased but still significant fluorescence. This was expected as thiols would compete with SSP4 to react with H₂S₂, thereby causing decreased signals. Taken together, these results demonstrated that TTS could effectively convert H₂S to H₂S₂. The results also showed that the byproduct TT would not cause unwanted reactions with Cys or H₂S.

**Fig. 3: Fluorescence-based studies of H₂S₂ formation from TTS.**

To demonstrate that the reaction between TTS and H₂S could proceed under cellular environments, we carried out live cell imaging studies. As shown in Fig. 3II, HeLa cells were treated with TTS, TTS + H₂S, or TT for 30 min before the fluorescent sensor SSP4 was applied to the cells to monitor the formation of H₂S₂. As expected, we only observed significant green fluorescence in cells with both TTS and H₂S (Fig. 3II-D). The other two treatments (TTS only or TT only) did not show obvious fluorescence. Na₂S₂ was used as the positive control in this study (Fig. 3II-F). Other control studies including TT + H₂S, H₂S only, and SSP4 only were also performed, and we did not observe obvious fluorescence in these samples (Fig. 3II-G~I). These results demonstrated that TTS could effectively convert H₂S to H₂S₂ in complex biological systems. It should be noted that the actual cellular reactions might be more complex due to the formation of HSOH in this process. However, HSOH should possess H₂S₂-like reactivity and rapidly react with cellular thiols to form persulfides (RSSH), which would yield the same result as that from H₂S₂. All these reactive sulfane sulfur species (H₂S₂, HSOH, RSSH) can be detected by SSP4 and can be considered as H₂S₂ equivalents here. Therefore, in the following biological and biologically-relevant studies, mentions of H₂S₂ should be understood as those of H₂S₂ and/or its equivalents in those systems.

To determine whether TTS could also convert endogenous H₂S to H₂S₂ in biological systems, we decided to induce hypoxia in cells because hypoxic environments are known to elevate H₂S levels^35,36,37. Neonatal rat cardiomyocytes (NRCMs) were used in this study. Hypoxia-induced elevated H₂S in NRCMs was first confirmed by SF7-AM, an H₂S-specific fluorescent probe³⁸ (Supplementary Fig. 8). Next, NRCMs were treated with TTS or TT (10 μM) under hypoxic conditions and then incubated with SSP4 (5 μM) for imaging (Fig. 4). For comparison, the same experiments were also performed with cells under normoxia. As expected, strong fluorescence from SSP4 was observed in the cells treated with TTS with significantly lower fluorescence in TT-treated or untreated cells, or in cells under normoxia. These results demonstrated the applicability of TTS as a booster for endogenous H₂S₂ (or its equivalents) in cellular systems.

**Fig. 4: Representative fluorescence images of normoxic and hypoxic neonatal rat cardiomyocytes.**

Quantitative determination of intracellular sulfane sulfur levels with TTS treatment

We further conducted sulfur metabolome analyzes using beta-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) to quantitatively measure the levels of sulfane sulfurs levels in TTS-treated cells. Mouse embryonic fibroblasts (MEFs) were treated with 100 µM TTS or 50 µM Na₂S₂ for 1 h at 37 °C, followed by washing and application to the sulfur metabolome. The results shown in Fig. 5 revealed that TTS alone was able to preferentially increase the level of certain persulfides such as GSSH, H₂S₂ and H₂S₃ in MEF cells. Na₂S₂ also significantly increased the levels of these persulfides. These findings thus suggest that TTS indeed promoted sulfur catenation, leading to the production of reactive sulfane sulfurs such as persulfides and polysulfides, even in the cellular context.

**Fig. 5: The effect of TTS on the sulfur metabolome in MEFs.**

Preparation and validation of protein-bound TTS

The reaction between TTS and H₂S can be considered a bio-orthogonal reaction, so we expected that TTS could be delivered to certain cellular locations or conjugated with certain biomolecules to achieve specific/localized H₂S to H₂S₂ transformation. To test this idea, we prepared a TTS-conjugated p-nitrophenol carbonate 2 and treated it with a model protein –lysozyme C. As shown in Fig. 6, the TTS moiety was successfully linked to the protein. When the modified protein (Lyso-TTS) was then treated with H₂S, the desired product Lyso-TT was obtained (as demonstrated by MS). The formation of H₂S₂ in this reaction was also confirmed (by the SSP4 assay). These results further demonstrated the stability and applicability of TTS in biological systems. For example, protein-bound TTS can be engineered for controlled H₂S₂ delivery or controlled induction of protein modifications via protein-protein interactions.

**Fig. 6: Demonstration of TTS conjugation with proteins.**

TTS-induced protein persulfidation

One of the primary functions of H₂S₂ is to induce protein S-persulfidation. To achieve this, researchers usually treat proteins with Na₂S₂ directly to obtain the persulfidated proteins. However, it is known that the use of continuous and lower concentrated oxidants is often more effective than that of the bolus addition of the oxidants to cause biological responses^39,40,41. We thus wondered if the TTS + H₂S system would be more efficient at persulfidating proteins than Na₂S₂. GAPDH was used as the protein model, and its persulfidation was evaluated by SSP4 using a protocol we recently established^34,39. In this study, the reduced GAPDH (100 μM) was individually treated with 10 eq. of TTS+Na₂S or Na₂S₂, as well as control reagents (TTS or Na₂S only). The protein samples were then purified using Zeba Spin Desalting Columns (7 K MWCO), diluted to 10 μM, and treated with SSP4 (5 μM). Fluorescence signals were recorded to reflect persulfidation levels. As shown in Fig. 7A, control samples did not give any notable fluorescence. Samples treated with TTS+Na₂S or Na₂S₂ yielded strong fluorescence while statistical analyses revealed that the combination of TTS and Na₂S gave significantly higher signals than that of Na₂S₂. To demonstrate that the observed fluorescence signals were not due to the insufficient removal of Na₂S₂ in the desalting step, we carried out control studies using -SH blocked GAPDH, small molecules only samples, and cysteine-mutated recombinant human GAPDH under the same conditions. These samples (shown in Supplementary Fig. 9) did not give any fluorescence. To provide further evidence of persulfidation in the GAPDH system, TTS+Na₂S-treated GAPDH was alkylated with HPE-IAM to stabilize the protein persulfides. The protein was then digested with trypsin, and the peptides were subjected to LC-MS/MS analysis (Supplementary Fig. 10), which indicated successful protein persulfidation. Together, these results confirm the efficiency of the TTS booster system in achieving protein persulfidation.

**Fig. 7: TTS-mediated protein persulfidation and H₂S donor to H₂S₂ donor conversion.**

Converting H₂S donors to H₂S₂ donors by TTS

So far, a large number of small molecule H₂S donors have been developed while known HSOH or H₂S₂ donors are still very limited. The unique bio-orthogonality of TTS suggested that TTS could be used in conjunction with any H₂S donor to create the corresponding HSOH/H₂S₂ donor system. We believe this will be a useful application of this compound. To prove this idea, we first tested the combination of TTS and JK-1, a hydrolysis-based H₂S donor developed by our lab with demonstrated promising activities in cardiovascular disease models^42,43,44. As shown in Fig. 7B, we envisioned that simply mixing JK-1 and TTS in solution would create HSOH/H₂S₂. To this end, the solution of JK-1 was prepared and then treated with TTS. After a 30 min incubation, the formation of HSOH/H₂S₂ was measured by SSP4. As shown in Fig. 7C, only the mixture of JK-1 and TTS gave strong fluorescence and was comparable to the signal obtained from Na₂S₂.

To further prove that TTS could turn an H₂S donor into an HSOH/H₂S₂ donor, we designed and synthesized a TTS-TAGDD hybrid compound 3 (Fig. 7D). TAGDD is a gem-dithiol based H₂S donor which can be triggered by biothiols such as cysteine to release H₂S⁴⁵. We expected that cysteine could still trigger the release of H₂S from the TAGDD component of 3 while the presence of TTS would allow for the formation of H₂S₂ (or its equivalent). As shown in Fig. 7E when 3 was treated with Cys, we observed steady and continuous H₂S₂ formation (monitored by SSP4 signals). Control experiments (e.g. 3 only or TTS only) did not give any notable fluorescence. Thus, the results shown in Fig. 7 clearly demonstrated that TTS can boost H₂S₂ formation from H₂S donors.

TTS compensated for certain aspects of SQR deficiency

In cells, sulfide:quinone oxidoreductase (SQR) is one of the key enzymes responsible for H₂S metabolism. SQR catalyzes the two-electron oxidation of H₂S to form sulfane sulfurs (S⁰) via persulfide and H₂S₂ intermediates^46,47,48. Since TTS could promote the conversion of H₂S to H₂S₂, we envisioned that TTS might be able to offset some features of SQR deficiency. To test this idea, cell imaging studies employing wild-type (WT) and SQR knockdown (SQR-KD) MEFs⁴⁹ were carried out. Briefly, the WT and SQR-KD MEFs were treated with several concentrations of Na₂S or the mixture of Na₂S and TTS in PBS for 20 min at 37 °C. Then, cellular sulfane sulfur levels were monitored and compared by fluorescence imaging with SSP4. As shown in Fig. 8, TTS was found to significantly increase intracellular sulfane sulfur levels, as evidenced by the elevation of fluorescence from both WT and SQR-KD MEFs treated with TTS (in an exogenously applied Na₂S concentration-dependent manner). Intriguingly, such a Na₂S-enhanced fluorescence was not observed with SQR-KD MEFs (Fig. 8A). The attenuated fluorescence signal by the SQR-KD was, however, almost completely restored by administering TTS to the SQR-KD cells in culture, as demonstrated by Fig. 8B. In addition, we also checked TTS’s impact on mitochondrial function as assessed by the membrane potential formation through a JC-1 fluorescence imaging of the mitochondria. The results showed that TTS efficiently augmented the mitochondrial membrane potential of both WT and SQR-KD MEFs in a concentration-dependent manner (Fig. 8C). Notably, the membrane potential that was significantly impaired by SQR KD was found to be almost completely recovered by the TTS treatment to a level similar to that of the WT MEFs. The activity of TTS in this case might be explained by: 1) TTS could detoxify H₂S by rapidly removing H₂S from cellular media, and/or 2) the persulfide or H₂S₂ generated via the TTS reaction might mediate sulfur respiration, as proposed by several studies reported recently^11,48,50. All these results suggest that TTS may be able to counteract some characteristics of SQR deficiency, and this might have some interesting applications. However, it should be noted that, unlike SQR, the reaction of TTS is not via a catalytic process. The regeneration of TTS from TT by cellular oxidants like H₂O₂ is slow. Structural modifications on TTS may lead to compounds with faster regeneration kinetics, and this will be an interesting future research direction.

**Fig. 8: The effects of TTS on sulfane sulfur generation and mitochondrial membrane potential in MEFs.**

Discussion

While the biological significance of H₂S₂ has been recognized in recent years, the study of H₂S₂ in biological systems is still a challenge due to the inherent instability of H₂S₂ and the lack of suitable donor compounds. In this work, we discovered 2H-thiopyran-2-thione sulfine (TTS), a unique and redox regenerable booster compound that can efficiently convert H₂S to H₂S₂. Mechanistic studies reveal that HSOH is the key intermediate from the reaction between TTS and H₂S. HSOH is also a highly reactive sulfane sulfur species, which is expected to behave like H₂S₂. So far, reliable methods for specific production of HSOH are still lacking. Thus, TTS may also be considered as a unique HSOH donor as well. The presence of biothiols would quickly react with HSOH to form persulfides, leading to the same result as that from H₂S₂. TTS could even be conjugated to proteins to achieve specific delivery for localized HSOH/H₂S₂ formation. We also demonstrated that the combination of TTS and H₂S led to efficient protein persulfidation through the generation of H₂S₂ or its equivalents. Most interestingly, two examples were used to show that TTS could turn established H₂S donors into H₂S₂ donors. In addition, TTS was found to be able to offset some effects of SQR deficiency in cells and promote the conversion of sulfide to sulfane sulfurs. These results demonstrate that TTS is a conceptually new strategy for the design of donor systems for H₂S₂ and/or its equivalents. Thus, we expect TTS to be a useful tool for elucidating the functions of H₂S₂, HSOH, and persulfides.

Methods

Syntheses and characterization of key substrates

TTS: To a stirred solution of methyl 2-thioxo-2H-thiopyran-5-carboxylate TT (186 mg, 1.0 mmol) in CH₂Cl₂ (10 mL), NaHCO₃ (84 mg, 5.0 mmol, 5.0 eq) and m-CPBA (70%, 246 mg, 1.0 mmol, 1.0 eq) were added. After being stirred at room temperature for 30 min, the reaction was quenched by sat. NaHCO₃ solution and extracted by ethyl acetate (EA). Combined organic layers were washed with brine and dried by Na₂SO₄. The solvent was removed under reduced pressure. The crude material was purified by flash column chromatography (HEX/EA = 5/1–3/1) to yield TTS (153 mg, yield = 76 %) as a dark red powder. TTS: R_f: 0.3 (HEX/EA = 2/1), m.p. = 130–131 °C. ¹H NMR (600 MHz, CD₂Cl₂) δ 8.32 (dd, J = 1.2, 1.2 Hz, 1H), 7.06 (dd, J = 10.5, 1.2 Hz, 1H), 6.93 (dd, J = 10.5, 1.2 Hz, 1H), 3.85 (s, 3H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 184.7, 163.3, 139.1, 126.8, 124.8, 120.2, 53.0. HRMS (ESI, m/z): [M + H]⁺ calculated for C₇H₇O₃S₂⁺: 202.9837; found: 202.9836.

TTS-Br: TTS-Br was prepared from S1 (see the SI) via esterification. R_f: 0.33 (HEX/EA = 2/1), m.p. = 163–165 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.00 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 10.5 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H), 7.02 (dd, J = 10.5, 1.3 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 185.0, 160.9, 149.5, 142.2, 132.5, 124.9, 124.5, 124.2, 120.5, 118.6. HRMS (ESI, m/z): [M]⁺ calculated for C₁₂H₇BrO₃S₂⁺: 341.9020; found: 341.9050.

Compound 2: To a stirred solution of S3 (see the SI) (165 mg, 0.39 mmol) in CH₂Cl₂ (5 mL), NaHCO₃ (164 mg, 1.95 mmol, 5.0 eq) and m-CPBA (70%, 123 mg, 0.5 mmol, 1.3 eq) were added. The mixture was stirred at room temperature for 10 min. Then, the reaction was quenched by sat. NaHCO₃ solution and extracted by EA. Combined organic layers were washed by brine and dried by Na₂SO₄. The solvent was removed by rotary evaporator. The crude material was purified by flash column chromatography (HEX/EA = 2/1–1/2) to yield 2 (105 mg, yield = 61 %) as a dark red powder. 2: R_f: 0.5 (EA), m.p. = 81 – 83 °C. ¹H NMR (600 MHz, CD₂Cl₂) δ 8.35 (s, 1H), 8.26 (d, J = 9.2 Hz, 2H), 7.39 (d, J = 9.2 Hz, 2H), 7.04 (d, J = 10.5 Hz, 1H), 6.93 (d, J = 10.5 Hz, 1H), 4.47–4.27 (m, 4H), 4.06–3.51 (m, 4H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 184.6, 162.8, 155.9, 152.9, 145.9, 139.5, 126.7, 125.7, 124.7, 122.2, 120.2, 69.3, 69.0, 68.6, 65.0. HRMS (ESI, m/z): [M + H]⁺ calculated for C₁₇H₁₆NO₉S₂⁺: 442.0266; found: 442.0262.

Compound 3: To a stirred solution of S5 (see the SI) (163 mg, 0.4 mmol) in CH₂Cl₂ (5 mL), NaHCO₃ (168 mg, 2.0 mmol, 2.0 eq) and m-CPBA (70%, 78 mg, 0.4 mmol, 1.0 eq) were added. After being stirred at room temperature for 10 min, the reaction was quenched by sat. NaHCO₃ solution and extracted by EA. Combined organic layers were washed by brine and dried by Na₂SO₄. The solvent was removed by rotary evaporator. The crude material was purified by flash column chromatography (HEX/EA = 3/1–2/1) to yield 3 (119 mg, yield = 70 %) as a dark red powder. 3: R_f: 0.2 (HEX/EA = 1/1), m.p. = 97 – 98 °C. ¹H NMR (600 MHz, CD₂Cl₂) δ 8.53 (dd, J = 1.2, 1.2 Hz, 1H), 7.41 – 7.34 (m, 2H), 7.28 – 7.23 (m, 1H), 7.12 (dd, J = 10.5, 1.2 Hz, 1H), 7.10 – 7.08 (m, 1H), 7.01 (dd, J = 10.5, 1.2 Hz, 1H), 6.20 (s, 1H), 2.32 (s, 6H). ¹³C NMR (151 MHz, CD₂Cl₂) δ 192.8, 184.2, 161.4, 150.8, 142.1, 140.8, 130.2, 126.12, 126.08, 124.5, 121.7, 121.3, 120.5, 47.7, 30.3. HRMS (ESI, m/z): [M + H]⁺ calculated for C₁₇H₁₅O₅S₄⁺: 426.9802; found: 426.9797.

X-ray crystallography

Crystals of TTS-Br were grown from solvent diffusion. ~3 mg of solid TTS-Br was dissolved in ~2 mL of acetone and placed in a 1 dram glass vial. ~2 mL of pentane was layered on top and the vial was parafilmed. Dark brown-red needle-like crystals suitable for X-ray crystallography formed after approximately three weeks.

SSP4 fluorescence assays

SSP4 was dissolved in DMSO to obtain a 200 μM stock solution. CTAB was dissolved in ethanol to yield a 2 mM stock solution. TTS and TT were dissolved in DMSO to make 1 mM stock solutions. Na₂S, Na₂S₂, L-Cys, and GSH were prepared in MilliQ H₂O to obtain stock solutions at 2 mM, 1 mM, 4 mM or 8 mM (for the interference assay), and 40 mM (for the interference assay), respectively. 50 mM PBS buffer was added to individual wells of black polystyrene, flat-bottomed clear 96-well plate, followed by TTS (25 μM) or TT (25 μM), and Na₂S (50 μM), cysteine (100 μM), or Na₂S₂ (25 μM). For the interference assay, TTS or TT (25 μM) were added to wells with or without Na₂S (50 μM) next. Samples were mixed by pipetting up and down and incubated in the dark at room temperature (rt) for 30 min. Next, CTAB (50 μM) was added to all wells followed by SSP4 (5 μΜ), and samples were mixed by pipetting up and down. For the interference assay, Cys (200 μM) or GSH (1 mM) were first mixed with SSP4 (5 μM) and CTAB (50 μM) and then added to the appropriate wells. The final volume was 200 μL in each well. Samples were then incubated in the dark at rt for another 30 min before being measured on a Molecular Devices SpectraMax iD3 Multi-Mode Microplate Reader (emission 525 nm; excitation 485 nm; integration time 400 s, low PMT sensitivity, attenuation 1, read height 1.00 mm).

Procedures for cell imaging

HeLa cells were obtained from ATCC (#CCL-2) and were seeded at ~7000 cells/well in a 96-well clear flat-bottomed BioLite plate (ThermoFisher Scientific, #130188) in DMEM/F12 (1:1) (Gibco, Invitrogen, #11330-032) medium supplemented with 10% fetal bovine serum (FBS) at 37 °C, 5% CO₂ overnight.

Preparation before cell treatment

Na₂S and Na₂S₂ stocks were prepared at 10 mM in sterile 1X PBS. TTS and TT were prepared at 10 mM concentrations in DMSO. They were then diluted to 50 μM concentrations in serum-free Fluorobrite DMEM in individual Eppendorf tubes (0.5% DMSO). Na₂S (final concentration = 100 μM) was added to the Eppendorf tube containing Fluorobrite DMEM and TTS or TT immediately before being added to the cells. Compounds were mixed by pipetting up and down prior to cell addition.

Cell treatment

Culturing media was aspirated from all wells, and cells were washed twice with 1X PBS. Compounds were added to cells (100 μL/well) and incubated for 30 min at 37 °C, 5% CO₂. While cells were incubating, SSP4 treatment was prepared by first adding 5 mM CTAB in ethanol to Fluorobrite followed by 6.125 mM SSP4 in DMSO to final concentrations of 100 μΜ and 5 μΜ, respectively, in a tube. The final volume was 5 mL (0.08% DMSO). Compounds were mixed by pipetting up and down prior to cell addition. After incubation, media was aspirated, and cells were washed twice with 1X PBS. SSP4 treatment was added to all wells (100 μL/well), and cells were incubated for 30 min at 37 °C, 5% CO₂ for 30 min. Media was then aspirated, and cells were washed three times with 1X PBS. Fluorobrite DMEM was added to all wells before imaging on the Keyence All-in-One Fluorescence Microscope (BZ-X810) (excitation: 470/40 nm; emission: 525/50 nm).

Cell imaging under hypoxia and normoxia

Neonatal rat cardiac myocytes (NRCMs) were prepared from the ventricles of 2-day-old SD rats⁵¹. All experiments using NRCMs were approved by the ethics committees at the National Institutes of Natural Sciences (Japan) and performed according to approved protocol code: 23A015. NRCMs were seeded on a matrigel-coated glass bottom dish and cultured in DMEM (low glucose) supplemented with 2% FBS. Two days after plating, NRCMs were incubated under normoxia or hypoxia (1% O₂) for 6 h. TT or TTS (10 µM) was added during the last 30 min of normoxia or hypoxia. Untreated cells served as controls.

For H₂S₂ imaging, NRCMs were incubated with 5 µM SSP4 in HBSS containing 0.04% Pluronic F-127 and 2 μg/mL Hoechst for 30 min³⁷. NRCMs were washed with HBSS three times and imaged by the BZ-X710 (Keyence). The SSP4 fluorescence intensities from the cell images were calculated using Fiji and ImageJ (National Institutes of Health). Data are expressed as mean ± standard error of the mean (SEM). Statistical evaluations were performed on GraphPad Prism with ordinary one-way ANOVA.

For H₂S imaging, cells (without TT/TTS treatment) were incubated with 2.5 µM SF7-AM in HBSS containing 2 μg/mL Hoechst for 30 min. NRCMs were washed with HBSS three times and imaged by the Keyence BZ-X710. The results are shown in the Supplementary Fig. 8.

Preparation of lysozyme-TTS conjugate

To a 600 μL solution of chicken lysozyme (14.3 kDa, 300 μM in 1X PBS buffer, pH 9), 2 (30 μL, 60 mM in DMSO, 10 eq) was added. The mixture was incubated at room temperature for 16 h. Excess 2 was removed by a Zeba 7 K MWCO (ThermoFisher Scientific) spin column using PBS buffer pH 7.4 solution as the eluent. The final concentration of Lyso-TTS was 80 μM as determined by the Nanodrop One Spectrophotometer. Lyso-TTS was characterized by LC-MS (Fd. +0 mod: 14304.82; +1 mod: 14607.11; +2 mod: 14909.24).

The reaction between lysozyme-TTS and H₂S

To a solution of Lyso-TTS (80 μM, 300 μL in PBS buffer pH 7.4), Na₂S (30 μL, 10 mM in PBS pH 7.4 buffer) was added. The mixture was incubated at room temperature for 30 min to give Lyso-TT, which was then purified by a Zeba 7 K MWCO spin column using PBS buffer pH 7.4 as the eluent. The final concentration of the modified lysozyme (Lyso-TT) was 10 μM as determined by the Nanodrop One Spectrophotometer. Lyso-TT was characterized by LC-MS (Found. +0 mod: 14305.20; +1 mod: 14591.33; +2 mod: 14877.66).

Computational method

All calculations were performed with the Gaussian 09 program⁵². Geometry optimizations of all minima and transition states involved were carried out using M06-2X⁵³ functional and SMD⁵⁴ solvation model in H₂O solvent and the basis set was maug-cc-pVTZ⁵⁵. It was labeled as SMD(H₂O)/M06-2X/maug-cc-pVTZ level. Frequency calculations at the same level were performed to validate each structure as either a minimum (the number of imaginary frequencies = 0) or a transition state (the number of imaginary frequencies = 1) and to evaluate its zero-point energy and thermal corrections at 298 K. Standard states are the hypothetical states at 1 mol/L.

Fluorescence measurement of the reaction between JK-1 with TTS

JK-1 (1.1 mg) was added to a 1 dram vial and 4.484 mL of 50 mM PBS (pH 6.0) was then added to obtain a 1 mM stock solution with no headspace in the vial. The solution was vortexed to mix and then incubated in the dark at rt for 20 min. Based on the H₂S-release profile reported⁴² under the same conditions, 400 μM of H₂S (40% of 1 mM) should be released. The pH of the solution was then adjusted to ~7.4 by adding 16 μL 2 N NaOH. The same volume of 50 mM PBS (pH 6.0) buffer was added to an empty 1 dram vial and 16 μL of 2 N NaOH was added to adjust the pH of the buffer similarly for the controls. After incubation, 500 μL of the pH-adjusted PBS was aliquoted to nine 1.5 mL Eppendorf tubes while the same volume of JK-1 solution was aliquoted into six 1.5 mL Eppendorf tubes. TTS (10 mM stock in DMSO, 10 μL) was then added to three of the JK-1-containing tubes and to three of the pH-adjusted PBS only tubes. Na₂S₂ (10 mM stock in MilliQ H₂O, 10 μL) was added to three of the pH-adjusted PBS only tubes. Tubes were closed and vortexed to mix. Incubation occurred for 30 min at rt in the dark. CTAB (100 mM stock in EtOH, 2 μL) was then added to all 15 tubes followed by SSP4 (9.44 mM stock in DMSO, 2.11 μL). Tubes were closed, vortexed to mix, and incubated for 30 min in the dark at rt. A total of 200 μL of each tube was then added to separate wells on a 96-well black flat clear-bottomed plate before being measured on a Molecular Devices SpectraMax iD3 Multi-Mode Microplate Reader (emission 535 nm; excitation 485 nm; integration time 400 s, low PMT sensitivity, attenuation 1, read height 3.00 mm).

Fluorescence measurement of the reaction of TTS-TAGDD hybrid compound 3

TTS and 3 were prepared as 1 mM solutions in DMSO. L-cysteine and Na₂S₂ were prepared at 4 mM and 1 mM in MilliQ H₂O, respectively. SSP4 was dissolved in DMSO to obtain a 200 μM stock solution. CTAB was dissolved in ethanol to yield a 2 mM stock solution. 50 mM PBS buffer (pH 7.4) was added to individual wells of a black polystyrene, flat-bottomed clear 96-well plate, followed by TTS (25 μM) or 3 (25 μM) then cysteine (100 μM) or Na₂S₂ (25 μM). Samples were mixed by pipetting up and down and incubated in the dark at room temperature (rt) for 30 min. Next, CTAB (50 μM) was added to all wells followed by SSP4 (5 μΜ), and samples were mixed by pipetting up and down. Samples were then incubated in the dark at rt for another 30 min before being measured on a Molecular Devices SpectraMax iD3 Multi-Mode Microplate Reader (fluorescence mode; kinetics measurement type; emission 535 nm; excitation 485 nm; integration time 400 s, low PMT sensitivity, attenuation 1, read height 1.00 mm; total run time: 12 h; 25 cycles; interval: 0.5 h). Data was graphed as a time-dependent fluorescence response, with F₀ as the SSP4 only condition. The fluorescence of Na₂S₂ over time was not represented in the line graph presented due to the compound’s known instability. Data obtained from the 12 h time point, including from Na₂S₂ was also represented in a bar graph for greater clarity. Results were expressed as mean ± SEM (n = 3 distinct samples).

Generation of SQR knockdown (KD) cell lines

Generation of SQR-KD cell lines were according to the method we previously reported⁴⁹. Briefly, short hairpin RNA sequences targeting mouse SQR were designed using BLOCK iT^TM RNAi design software (Thermo Fisher Scientific). A pair of oligos for SQR shRNA, comprising sense: 5’-GATCCCCGGAGAGTTGGAGCAGAGAATGTTCAAGAGACATTCTCTGCTCCAACTCTCCTTTTTA-3’ and anti-sense: 5’-AGCTTAAAAAGGAGAGTTGGAGCAGAGAATGTCTCTTGAACATTCTCTGCTCCAACTCTCCGGG-3’ were synthesized, annealed and ligated into the Bgl II and Hind III sites of pSUPER retro puro vector. MEFs were transfected with the pSUPER-shRNA (SQR) plasmid using Lipofectamine 3000. After 24 h, the medium was replaced, and cells were replated onto 10-cm dishes containing puromycin-supplemented medium. Puromycin-resistant clones were selected to establish stable SQR-KD cell lines.

Imaging studies of TTS in the WT and SQR-KD MEFs

WT and SQR KD MEFs were seeded in eight-well glass chamber slides coated with poly-D-lysine (Sigma-Aldrich). The SQR-KD MEFs were produced according to the shRNA-mediated knockdown method reported recently⁴⁹. The WT and SQR KD MEFs were treated with several concentrations of Na₂S or mixture of Na₂S and TTS in PBS for 20 min at 37 °C. The cells were washed once with PBS, followed by incubation with 20 μM SSP4 in PBS (+) containing 200 μM CTAB (cetyltrimethylammonium bromide) for 30 min at 37 °C. After removing the excess probes from the cells, cells were washed twice with PBS, followed by the measurement of fluorescence in images (excitation wavelength of 488 nm) with a confocal laser scanning microscope (Nikon C2 plus, NIS elements version 5.01 software). The fluorescence intensity of image was calculated as fluorescence intensity per cell (arb.units/cell) by using ImageJ software (National Institutes of Health) and dividing the intensity value by the number of cells.

Measurements of mitochondrial membrane potential

To determine the mitochondrial membrane potential (ΔΨm) of MEFs under several experimental conditions, tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining was carried out following a previously reported method with some modifications¹¹. The accumulation of the cell-permeable JC-1 probe (Abcam) within the mitochondria correlates with the membrane potential, which triggers a fluorescence emission shift from green to red. In brief, both WT and SQR-KD MEFs were cultured in 8-well slide chamber coated with poly-D-lysine and were treated with 1 or 10 µM TTS for 30 min at 37 °C. For JC-1 staining, the treated cells were washed with HKRB buffer (20 mM HEPES, 103 mM NaCl, 4.77 mM KCl, 0.5 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM KH₂PO₄, 25 mM NaHCO₃ and 15 mM glucose, pH 7.3), incubated with 20 μM JC-1 for 30 min at 37 °C, rinsed twice with HKRB buffer, and examined using a Nikon EZ-C2 Plus confocal laser microscope. ImageJ software was employed for image processing and quantification of the JC-1 fluorescence signals.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Data generated and analyzed in this study are included in this article and in its Supplementary Information files. The supplementary Cartesian Coordinates and MS files are included in the Supplementary Data 1 file. Source data are provided with this paper. The X-ray crystallographic coordinates for the structure reported in this work have been deposited at the Cambridge Crystallographic Data Center (CCDC). Accession code: CCDC 2259484. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. The protein mass spectrometry data have been deposited to the MassIVE repository with the dataset accession: MSV000094034. The data will also be available through the ProteomeXchange with the accession number: PXD049256. Source data are provided with this paper.

References

Wallace, J. L. & Wang, R. Hydrogen sulfide-based therapeutics: exploiting a unique but ubiquitous gasotransmitter. Nat. Rev. Drug. Discov. 14, 329–345 (2015).
Article CAS PubMed Google Scholar
Wang, R. Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol. Rev. 92, 791–896 (2012).
Article CAS PubMed Google Scholar
Kimura, H. Signaling molecules: hydrogen sulfide and polysulfide. Antioxid. Redox Signal. 22, 362–376 (2015).
Article CAS PubMed PubMed Central Google Scholar
Zheng, Y. et al. Toward hydrogen sulfide based therapeutics: critical drug delivery and developability issues. Med. Res. Rev. 38, 57–100 (2018).
Article CAS PubMed ADS Google Scholar
Powell, C. R., Dillon, K. M. & Matson, J. B. A review of hydrogen sulfide (H₂S) donors: Chemistry and potential therapeutic applications. Biochem. Pharmacol. 149, 110–123 (2018).
Article CAS PubMed Google Scholar
Zhao, Y., Biggs, T. D. & Xian, M. Hydrogen sulfide (H2S) releasing agents: chemistry and biological applications. Chem. Commun. 50, 11788–11805 (2014).
Article CAS Google Scholar
Pluth, M. D. (Editor), Wang, B. (Series Editor). Hydrogen sulfide: chemical biology basics, detection methods, therapeutic applications, and case studies (Wiley Series in Drug Discovery and Development) 1st Edn. (2022).
Bora, P., Chauhan, P., Pardeshi, K. A. & Chakrapani, H. Small molecule generators of biologically reactive sulfur species. RSC Adv. 8, 27359–27374 (2018).
Article CAS PubMed PubMed Central ADS Google Scholar
Kimura, H. Physiological roles of hydrogen sulfide and polysulfides. In Chemistry, Biochemistry and Pharmacology of Hydrogen Sulfide; Moore, P. K., Whiteman, M., Eds.; Springer International Publishing: Cham, 230, 61–81 (2015).
Liu, H. et al. Inorganic hydrogen polysuflides: chemistry, chemical biology, and detection. Br. J. Pharmacol. 176, 616–627 (2019).
Article CAS PubMed Google Scholar
Akaike, T. et al. Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics. Nat. Commun. 8, 1177 (2017).
Article PubMed PubMed Central ADS Google Scholar
Greiner, R. et al. Polysulfides link H₂S to protein thiol oxidation. Antioxid. Redox Signal. 19, 1749–1765 (2013).
Article CAS PubMed PubMed Central Google Scholar
Filipovic, M. R. et al. Chemical biology of H₂S signaling through persulfidation. Chem. Rev. 118, 1253–1337 (2018).
Article CAS PubMed Google Scholar
Yang, C. T. et al. S-Persulfidation: chemistry, chemical biology, and significance in health and disease. Antioxid. Redox Signal. 33, 1092–1114 (2020).
Article CAS PubMed PubMed Central Google Scholar
Ida, T. et al. Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc. Natl Acad. Sci. USA 111, 7606–7611 (2014).
Article CAS PubMed PubMed Central ADS Google Scholar
Xu, S. et al. Strategies for the design of donors and precursors of reactive sulfur species. Chem. Eur. J. 25, 4005–4016 (2019).
Article CAS PubMed Google Scholar
Xu, S. et al. Diacyl disulfides as the precursors for hydrogen persulfides (H₂S₂). Bioorg. Med. Chem. Lett. 30, 126903 (2020).
Article CAS PubMed Google Scholar
Kang, J. et al. O→S relay deprotection, a general approach for the design of controllable donors of reactive sulfur species. Angew. Chem. Int. Ed. 57, 5893–5897 (2018).
Article CAS Google Scholar
Roy, B. et al. A single component photo-responsive template for the controlled release of NO and H₂S₂. J. Am. Chem. Soc. 145, 277–287 (2023).
Article CAS PubMed Google Scholar
Yu, B. et al. Toward direct protein S-persulfidation: a prodrug approach that directly delivers hydrogen persulfide. J. Am. Chem. Soc. 140, 30–33 (2018).
Article CAS PubMed Google Scholar
Li, Q. & Lancaster, J. R. Jr. Chemical foundation of hydrogen sulfide biology. Nitric Oxide 35, 21–34 (2013).
Article PubMed Google Scholar
Cerreta, F., Leriverend, C. & Metzner, P. Carbon versus sulfur addition of nucleophilics to sulfines: the case of amines. Tetrahedron Lett. 34, 6741–6742 (1993).
Article CAS Google Scholar
Chevrie, D. & Metzner, P. Oxidation of dithiocarbamates and synthesis of a stable sulfine. Tetrahedron Lett. 39, 8983–8986 (1998).
Article CAS Google Scholar
Akihiko Ishii, A. et al. Synthesis and photophysical properties of conjugated thioketone, thioketone S-oxide (Sulfine), and related compounds incorporated in a dibenzobarrelene skeleton. J. Sulfur Chem. 41, 238–257 (2020).
Article Google Scholar
Tamagaki, S. Hotta, K. & Kozuka, S. Sulfines in the oxidation of 1,2-dithiole-3-thiones. Chem. Lett. 9, 619–620 (1980).
Cui, Q. et al. Controllable cycloadditions between 2H-(thio)pyran-2-(thi)ones and strained alkynes: a click-and-release strategy for COS/H₂S generation. Org. Lett. 24, 7334–7338 (2022).
Article CAS PubMed Google Scholar
Kappert, W., Sander, W. & Landgrafe, C. Structure, conformation, and thermochemistry of dimethyl methanethial S-oxide (dimesityl sulfine). Liebigs Ann. 2519–2524 (1997).
Hasserodt, J., Pritzkow, H. & Sundermeyer, W. Uber (Pentafluorpheny1)thiocarbonyl-Verbindungen sowie die Synthese und Struktur von (Pentafluorpheny1)phenylsulfin. Chem. Ber. 126, 1701–1706 (1993).
Article CAS Google Scholar
Deacon, G. B. et al. Stabilising small clusters: synthesis and characterisation of thermolabile [Gd₄F₇(15-crown-5)₄][AsF₆]₅·6SO₂. Dalton Trans. 2237–2238 (2005).
Akkus, O. N., Decken, A., Knapp, C. & Passmore, J. Complexes formed by the reactions of fluorinated and non-fluorinated organonitriles with [Zn(SO₂)₂][AsF₆]₂: A structural study. J. Chem. Cryst. 36, 321–329 (2006).
Article CAS Google Scholar
Muller, M. & Ruchner, M. R. Understanding the localization of berylliosis: interaction of Be²⁺ with carbohydrates and related biomimetic ligands. Chem. Eur. J. 25, 16257–16269 (2019).
Article PubMed Google Scholar
Liu, C. et al. Rational design and bioimaging applications of highly selective fluorescent probes for hydrogen polysulfides. J. Am. Chem. Soc. 136, 7257–7260 (2014).
Article CAS PubMed PubMed Central Google Scholar
Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).
Article CAS Google Scholar
Shieh, M. et al. Shining a light on SSP4: a comprehensive analysis and biological applications for the detection of sulfane sulfurs. Redox Biol. 56, 102433 (2022).
Article CAS PubMed PubMed Central Google Scholar
Peng, Y.-J. et al. H₂S mediates O₂ sensing in the carotid body. Proc. Natl Acad. Sci. USA 107, 10719–10724 (2010).
Article CAS PubMed PubMed Central ADS Google Scholar
Olson, K. R. et al. Extended hypoxia-mediated H2S production provides for long-term oxygen sensing. Acta Physiol. 228, e13368 (2020).
Article CAS Google Scholar
Tang, X. et al. Echinochrome prevents sulfide catabolism-associated chronic heart failure after myocardial infarction in mice. Mar. Drugs 21, 52 (2023).
Article CAS PubMed PubMed Central Google Scholar
Lin, V. S., Lippert, A. R. & Chang, C. J. Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production. Proc. Natl Acad. Sci. USA 110, 7131–7135 (2013).
Article CAS PubMed PubMed Central ADS Google Scholar
Ni, X. et al. A sweet H₂S/H₂O₂ dual release system and specific protein S-persulfidation mediated by thioglucose/glucose oxidase. J. Am. Chem. Soc. 143, 13325–13332 (2021).
Article CAS PubMed Google Scholar
Stancill, J. S. et al. Pancreatic β-cells detoxify H₂O₂ through the peroxiredoxin/thioredoxin antioxidant system. J. Biol. Chem. 294, 4843–4853 (2019).
Article CAS PubMed PubMed Central Google Scholar
Marinho, H. S. et al. H₂O₂ delivery to cells: steady-state versus bolus addition. Meth. Enzymol. 526, 159–173 (2013).
Article CAS Google Scholar
Kang, J. et al. pH-Controlled hydrogen sulfide release for myocardial ischemia reperfusion injury. J. Am. Chem. Soc. 138, 6336–6339 (2016).
Article CAS PubMed Google Scholar
Li, Z. et al. Mitochondrial H₂S regulates BCAA catabolism in heart failure. Circulation Res. 131, 222–235 (2022).
Article CAS PubMed Google Scholar
Li, Z. et al. Hydrogen sulfide attenuates renin angiotensin and aldosterone pathological signaling and improves exercise tolerance in heart failure. J. Am. Coll. Cardiol. Basic Transl. Sci. 3, 796–809 (2018).
Google Scholar
Zhao, Y. et al. Thio-activated gem-dithiols: a new class of controllable hydrogen sulfide donors. Org. Lett. 16, 4536–4539 (2014).
Article CAS PubMed PubMed Central Google Scholar
Libiad, M., Yadav, P. K., Vitvitsky, V., Martinov, M. & Banerjee, R. Organization of the human mitochondrial hydrogen sulfide oxidation pathway. J. Biol. Chem. 289, 30901–30910 (2014).
Article CAS PubMed PubMed Central Google Scholar
Jackson, M. R., Melideo, S. L. & Jorns, M. S. Human sulfide:quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite. Biochemistry 51, 6804–6815 (2012).
Article CAS PubMed Google Scholar
Alam, M. M. et al. Contribution of NRF2 to sulfur metabolism and mitochondrial activity. Redox Biol. 60, 102624 (2023).
Article CAS PubMed PubMed Central Google Scholar
Matsunaga, T. et al. Supersulphides provide airway protection in viral and chronic lung diseases. Nat. Commun. 14, 4476 (2023).
Article CAS PubMed PubMed Central ADS Google Scholar
Marutani, E. et al. Sulfide catabolism ameliorates hypoxic brain injury. Nat. Commun. 12, 3108 (2021).
Article CAS PubMed PubMed Central ADS Google Scholar
Nishimura, A. et al. Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload. Sci. Signal. 12, eaaw1920 (2019).
Article PubMed Google Scholar
Frisch, M. J. et al. Gaussian 09, Revision D.01 (Gaussian, Inc., 2013).
Zhao, Y. & Truhlar, D. G. The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theor. Chem. Acc. 120, 215 (2008).
Article CAS Google Scholar
Marenich, A. V., Cramer, C. J. & Truhlar, D. G. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B, 113, 6378 (2009).
Article CAS PubMed Google Scholar
Papajak, E., Leverentz, H. R., Zheng, J. & Truhlar, D. G. Efficient diffuse basis sets: cc-pVxZ+ and maug-cc-pVxZ. J. Chem. Theory Comput. 5, 3330 (2009).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This work was supported by the NIH (R01GM125968/R35GM149170 to M.X. and R01DK122160 to W.J.Q.), Grants-in-Aid for [Transformative Research Areas (A), International Leading Research, Scientific Research (S), (B) and (C) and Challenging Research (Exploratory)] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, to T.A. (21H05263, 22K19397, and 23K20040), T.M. (22K06893), J.M. (23K14341), S.O. (23K14333), M.M. (23K06145), and M.N. (21H05269, 22H02772, and 22K19395); Japan Science and Technology Agency (JST), CREST Grant Number JPMJCR2024 to M.N., T.A. and A.N., Japan; a grant from the Japan Agency for Medical Research and Development (AMED), Grant Number JP21zf0127001 to T.A. M.S. is supported by a NIH F31 Predoctoral Fellowship (F31HL170516). The NSF is gratefully acknowledged for its support of the acquisition of a single crystal X-ray diffractometer (CHE-2117549). DFT calculations were conducted using resources and services at the Center for Computation and Visualization (CCV) at Brown University. Part of the mass spectrometry experiments described herein were performed in the Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, a national scientific user facility sponsored by the Department of Energy under Contract DE-AC05-76RL0 1830.

Author information

These authors contributed equally: Qi Cui, Meg Shieh.

Authors and Affiliations

Department of Chemistry, Brown University, Providence, RI, 02912, USA
Qi Cui, Meg Shieh, Tony W. Pan, Shi Xu, Xiaoyan Chen, Jerome R. Robinson & Ming Xian
Division of Cardiocirculatory Signaling, National Institute for Physiological Sciences (NIPS) and Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, 444-8787, Japan
Akiyuki Nishimura & Motohiro Nishida
Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan
Tetsuro Matsunaga, Minkyung Jung, Seiryo Ogata, Masanobu Morita, Jun Yoshitake & Takaaki Akaike
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA
Shane S. Kelly & Wei-Jun Qian
Department of Physiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan
Motohiro Nishida

Authors

Qi Cui
View author publications
You can also search for this author in PubMed Google Scholar
Meg Shieh
View author publications
You can also search for this author in PubMed Google Scholar
Tony W. Pan
View author publications
You can also search for this author in PubMed Google Scholar
Akiyuki Nishimura
View author publications
You can also search for this author in PubMed Google Scholar
Tetsuro Matsunaga
View author publications
You can also search for this author in PubMed Google Scholar
Shane S. Kelly
View author publications
You can also search for this author in PubMed Google Scholar
Shi Xu
View author publications
You can also search for this author in PubMed Google Scholar
Minkyung Jung
View author publications
You can also search for this author in PubMed Google Scholar
Seiryo Ogata
View author publications
You can also search for this author in PubMed Google Scholar
Masanobu Morita
View author publications
You can also search for this author in PubMed Google Scholar
Jun Yoshitake
View author publications
You can also search for this author in PubMed Google Scholar
Xiaoyan Chen
View author publications
You can also search for this author in PubMed Google Scholar
Jerome R. Robinson
View author publications
You can also search for this author in PubMed Google Scholar
Wei-Jun Qian
View author publications
You can also search for this author in PubMed Google Scholar
Motohiro Nishida
View author publications
You can also search for this author in PubMed Google Scholar
Takaaki Akaike
View author publications
You can also search for this author in PubMed Google Scholar
Ming Xian
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Q.C. and M.X. conceived the idea of the chemistry of TTS. Q.C., M.S., M.N., T.A. and M.X. designed the studies. Q.C. and T.W.P. carried out syntheses and mechanistic analyzes. Q.C. did DFT calculations. M.S. performed SSP4-based fluorescence assays on small molecules, proteins, HeLa cells, kinetic studies, and prepared the TTS-Br crystals. A.N. and M.N. performed NRCM-based studies. T.M. and T.A. performed MEF-based and mitochondrial membrane potential studies. S.S.K. and W.J.Q. performed LC-MS/MS studies. S.X. did kinetic studies. M.J. and M. M. produced recombinant GAPDH and SQR-KD MEFs. S.O. and J.Y. performed sulfur metabolome MS analyzes. X.C. performed protein MS studies. J.R.R. performed X-ray crystallography analysis. All authors analyzed and interpreted data. Q.C., M.S., M.N., T.A. and M.X. prepared the figures and wrote and edited the paper. All authors reviewed the paper.

Corresponding authors

Correspondence to Takaaki Akaike or Ming Xian.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Reporting Summary

Peer Review File

Description of Additional Supplementary files

Supplementary Data 1

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Cui, Q., Shieh, M., Pan, T.W. et al. 2H-Thiopyran-2-thione sulfine, a compound for converting H₂S to HSOH/H₂S₂ and increasing intracellular sulfane sulfur levels. Nat Commun 15, 2453 (2024). https://doi.org/10.1038/s41467-024-46652-7

Download citation

Received: 27 July 2023
Accepted: 26 February 2024
Published: 19 March 2024
DOI: https://doi.org/10.1038/s41467-024-46652-7

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.