Diacyl Disulfides as the Precursors for Hydrogen Persulfide (H₂S₂)

Abstract

While hydrogen polysulfides (H₂S_n, n≥2) are believed to play regulatory roles in biology, their fundamental chemistry and reactivity are still poorly understood. Compounds that can produce H₂S_n are useful tools. In this work we found that H₂S₂ could be effectively produced from diacyl disulfide precursors, triggered by certain nucleophiles, in both aqueous solutions and organic solvents. This method was used to explore redox reactions of H₂S₂, such as scavenging 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and reduction of tetrazines.

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Reactive sulfur species (RSS) are a group of sulfur-containing molecules that play regulatory roles in biological systems. Among all RSS, hydrogen sulfide (H₂S) is most well studied.^1–5 In the past decade, H₂S has been identified as an important gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO). Very recently hydrogen polysulfides (H₂S_n, n≥2) have received increased attention as H₂S_n are also believed to have regulatory functions and are linked to H₂S signaling. Endogenous H₂S_n are proposed to be produced enzymatically by 3-mercaptopyruvate transferase (3-MST)⁶ and cysteinyl-tRNA synthetases (CARS).⁷ H₂S_n can also be generated from H₂S via the H₂S-NO cross talk and haem protein-catalyzed oxidation.^8–11 Recent studies suggested some biological activities that are originally attributed to H₂S may be actually mediated by H₂S_n. For example, H₂S_n were found to be hundreds of times more potent at activating transient receptor potential A1 channels than H₂S.¹² H₂S_n are also very effective in inducing S-sulfhydration in Keap1, the key regulating protein in Nrf2 signaling.¹³ It is likely that H₂S and H₂S_n work collectively to maintain sulfur redox balance.

To further understand the function and biological roles of H₂S_n, compounds that deliver H₂S_n are needed. In current studies, researchers often use commercially available inorganic salts (such as Na₂S₂) as H₂S_n equivalents. These salts release H₂S_n uncontrollably in solutions. Additionally, the preparation of these salts involves treating sodium with stoichiometric amounts S₈ under high temperature. Impurities such as Na₂S and S₈ seems inevitable,¹⁴ making it hard to attribute the observed bioactivities solely to H₂S_n. As such, methods or compounds that can controllably produce H₂S_n are highly desirable. However, this area remains underdeveloped and only a few such examples have been reported. For example, the O to S relay deprotection based donors developed by our lab could release H₂S₂ under acid or F^- activation (Scheme 1a).¹⁵ Wang et al. reported BW-HP donors that were triggered by esterase or phosphatase to produce H₂S₂ (Scheme 1b).¹⁶ It should be noted that BW-HP donors are based on the acyl disulfide template. Acyl disulfides are highly reactive species, they can form persulfides and release H₂S upon thiol activation.¹⁷ It is also known that acyl disulfides are highly reactive toward amines to form amides (Scheme 1c).¹⁸ It is expectable that H₂S₂ is released as a byproduct in the process while this has not been characterized. We envisioned the reaction between diacyl disulfides and certain nucleophiles like amines could produce H₂S₂ in a clean and controlled way. This would allow chemistry investigation of H₂S₂ under certain conditions. In this work, we studied the generation of H₂S₂ in this system and evaluated the reactivity of H₂S₂ in organic solutions.

Scheme 1.

Possible ways for H₂S₂ generation

In our studies three diacyl disulfides (2a-c, Scheme 2) were prepared by oxidizing the corresponding thioacids with iodine. With these compounds in hand we first tested their stability. 2a-c were fairly stable when stored as neat materials at 4 °C. No obvious decomposition was observed after 3 weeks. In organic solvents such as DCM and THF, they were found to be stable for days at room temperature. However, when dissolved in buffers, slow but noticeable decomposition was observed. This could be due to hydrolysis of diacyl disulfides in aqueous solutions. One possible pathway is simultaneous hydrolysis of both acyl groups on diacyl disulfides, forming H₂S₂ as the product (Figure 1–a). It is also possible that one acyl group gets hydrolyzed first to form an acylated persulfide intermediate (Figure 1–b). This intermediate further degrades to form other sulfur species. To determine these two possibilities, we incubated diacyl disulfides in PBS buffer and used three specific fluorescent probes to identify the sulfur species formed in hydrolysis. As shown in Figure 1–c, although diacyl disulfides showed some degree of fluorescence turn-on with DSP-3¹⁹ (a H₂S₂ probe) and WSP-5²⁰ (a H₂S probe), the most prominent fluorescence enhancement came from SSP-4²¹ (a sulfane sulfur probe). These results suggested sulfane sulfur was the major end product generated from hydrolysis of diacy disulfides. The formation of sulfane sulfur could be attributed to the either the decomposition of the acylated persulfide intermediate or H₂S₂. Interestingly, NMR study (Figure S1) showed both acetic acid and thioacetic acid were formed from 2a. Based on these observations, we feel both pathway a) and b) were possible. The decomposition of the acyl persulfide intermediate should provide thioacetic acid, S₈ (a sulfane sulfur), and H₂S while the detailed mechanism is still unclear. Disproportionation or radical reaction could be involved. It is also noted that the more sterically hindered substrate 2c did not undergo hydrolysis as all those fluorescent probes did not give significant signals.

Scheme 2.

Synthesis and structures of diacyl disulfides

Figure 1.

a) and b) Possible hydrolysis pathways of diacyl disulfides. c) Decomposition of diacyl disulfides monitored by fluorescent probes. 2a-c (50 μM) were incubated with WSP-5, SSP-4, DSP-3 (10 μM) in PBS (pH 7.4, 50 mM, with CTAB 25 μM).

Next we turned to test if diacyl disulfides could release H₂S₂ under amine activation in organic solutions. 2a was treated with different amines (3a-e) in THF (Figure 2). H₂S₂ generation was monitored by an H₂S_n specific fluorescent probe DSP-3. As expected, time-dependent fluorescence increases were observed when 2a was treated with 3a-d, indicating H₂S₂ was indeed produced. In theory, diacyl disulfides should release H₂S₂ only when both acyl groups were removed simultaneously. We expected that diamines would facilitate the reaction and make H₂S₂ release faster. However, little difference in the rates of H₂S₂ formation from 3a-c was observed (Figure 2–b). Presumably the reactions of all unhindered primary amines were equally fast. We did observe slower H₂S₂ formation with a secondary amine 3d and almost no H₂S₂ formation with aniline 3e. These suggest varied nucleophilicity of amines could tune H₂S₂ release profiles from acyl disulfides. On the other hand, when 3a was treated with different diacyl disulfides (2a-c) substitution effects were obvious (Figure 2–c) as more sterically hindered substrates gave slower H₂S₂ release. These results suggest the rates of H₂S₂ production from diacyl disulfides can be controlled. Additionally, 2a and 3a can generate H₂S₂ in a variety of organic solvents with different rates (Figure S2). Generally, polar aprotic solvents could promote H₂S₂ production. For consistency, we use THF as the solvent for following reactions of 2a and 3a to generate H₂S₂.

Figure 2.

a) Structures of amines used in reactions with acyl disulfides. b) Time-dependent H₂S₂ release from 2a (5 mM) when treated with amines 3a, 3b (5 mM), 3c - 3e (10 mM) in THF and monitored by DSP-3. c) Time-dependent H₂S₂ release from 2a - 2c when treated with 3a and monitored by DSP-3.

To further confirm H₂S₂ release from diacyl disulfides, we used a model compound 4 to trap H₂S₂. 4 should react with H₂S₂ via an aromatic nucleophilic substitution followed by an intramolecular cyclization to form 5 (Scheme 3).¹⁹ In this experiment 2a and 3a were mixed in THF in the presence of 4 for 1 h. As expected, the desired product 5 was isolated in 25% yield. The low yield however, may suggest the formation of other RSS in addition to H₂S₂, such as persulfide RSSH, or the instability of H₂S₂. Compound 4 could also react with the RSSH intermediate generated from 2a and 3a to form the corresponding disulfide adduct. Monobormobimane (MBB), which has been used in capturing and charactering biologically generated H₂S₂,²² was used in our studies. The corresponding disulfide adduct 6 was obtained in 19% yield. It should be noted in this experiment, the tri- and mono-sulfide adducts of MBB were also identified by mass spectroscopy. Additionally, elemental sulfur was detected in both reactions. While the formation of 5 and 6 clearly indicates the generation of H₂S₂, the formation of other byproducts also suggests H₂S₂ may disproportionate into other RSS and its decomposition seems to be unavoidable.

Scheme 3.

Capturing H₂S₂ generated from 2a and 3a.

Having demonstrated diacyl disulfides’ capability of generating H₂S₂ in organic solvents, we wondered if they could release H₂S₂ when interacting with cells. Cell-imaging experiments were then conducted. PC3 cells were first loaded with DSP-3 and then treated with 2a. As shown in Figure 3–b, a significant fluorescence enhancement was observed. However, when cells were pre-treated with a thiol-blocking reagent N-ethylmaleimide (NEM), the fluorescence enhancement was attenuated (Figure 3–c). Interestingly, when NEM-treated cells were subsequently treated with lysine and 2a, fluorescent enhancement resumed (Figure 3–d). Similar results were obtained when 2a was applied to HeLa cells (Figure S4). These results suggest under physiological conditions, 2a was mainly activated by cellular nucleophiles such as cysteine and GSH. This process should outweigh its hydrolysis as the hydrolysis is much slower. Of note, it is known that NEM can react with amines^{23, 24} and we found lysine could reactivate 2a in NEM-treated cells. These results suggested that cellular amines could also activate 2a. Previous study showed diacyl disulfides were able to produce H₂S when reacting with thiols.¹⁷ Therefore, it is likely that 2a produced both H₂S and H₂S₂ in cells. Overall, these results indicate diacyl disulfides are activate species vulnerable to cellular nucleophiles and can release more than one RSS under physiological conditions. Caution should be taken if diacyl disulfides are used to study the bioactivity of a single RSS.

Figure 3.

Fluorescence imaging of 2a in PC3 cells. Cells were incubated with DSP3 (10 μM) for 20 min, then washed and subjected to different treatments. a) control (No 2a added); b) 100 μM 2a for 30 min; c) 500 μM NEM for 20 min, then 100 μM 2a for 30 min. d) 500 μM NEM 20 min; 200 μM Lys 30 min; 100 μM 2a for 30 min. e) Mean fluorescence intensities of images a)-d).

We then utilized diacyl disulfides to study the properties of H₂S₂. Although H₂S₂ is of higher oxidation state than H₂S, it is also reported to be a stronger reductant.^25,26 We used DPPH assay to verify the reducing ability of H₂S₂. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) is a stable radical which has a strong purple color. In the presence of species that can donate one electron, DPPH can be reduced and decolored. This process can be monitored by UV spectrometry.²⁷ In our studies, we compared in-situ generated H₂S₂ with H₂S and cysteine (Figure 4). It should be noted that we tried to directly use Na₂S and Na₂S₂ in this study. However, no DPPH reduction was noted with these inorganic salts, presumably due to low solubility of these salts in ethanol. While 2a and 3a showed no DPPH reduction alone, a pre-mixed solution of 2a and 3a reduced 40% DPPH in 7 min. On the other hand, about 20% DPPH was reduced by cysteine. We also used compounds 7 and 8, which could release H₂S and H₂S₂ respectively in the presence of fluoride.¹⁵ H₂S₂ generated from 8 reduced about 30% of DPPH, While H₂S generated from 7 also reacted with DPPH quickly, it only scavenged about 15% of DPPH, presumably due to one less reactive sulfur atom in the molecule. In these experiments 20 μM analyte was first prepared and then added to an ethanol solution containing 100 μM DPPH. It is known that persulfides are more reactive toward radicals compared to H₂S and thiols.²⁶ Based on the similarity of H₂S₂ and persulfides, they should have similar redox reactivity. Therefore, it is not surprising that H₂S₂ generated from 2a and 3a showed better DPPH reducing ability than H₂S and cysteine. It should be noted that H₂S₂ may decompose to form H₂S and sulfane sulfur, which may also reduce DPPH. However, the observed DPPH reduction should mainly come from H₂S₂ due to short reaction time.

Figure 4.

a) Reduction of DPPH. b) Generation of H₂S and H₂S₂ by 7 and 8. c) DPPH scavenging of various RSS. 100 μM DPPH was mixed with 20 μM RSS, UV absorption at 517 nm was measured.

Tetrazines are often used to perform ‘click’ reactions with trans-cyclooctenes.^28,29 A recent report found that H₂S could reduce tetrazines to dihydrotetrazines.³⁰ Based on the results of DPPH experiments, we speculated H₂S_n could reduce tetrazine, likely more effectively than H2S. To test this hypothesis, tetrazine 9, which has a characteristic purple color and UV absorption at 500~550 nm, was used as the model substrate. Upon reduction by H₂S or H₂S_n, 9 would be converted to 10 with the disappearance of UV absorption. This allowed us to monitor the progress of the reduction. We first used Na₂S and Na₂S₂ as the equivalents of H₂S and H₂S₂. To ensure the solubility of both 9 and Na₂S/Na₂S₂, these experiments were performed in a mixed solvent (20% H₂O, 80% THF). As shown in Figure 5–b, Na₂S₂ (25 mM) was able to reduce 90% of 9 (5 mM) in about 10 min. Although Na₂S also showed ability to reduce 9, the process was much slower (~30% after 50 min). Next, we tested if in-situ generated H₂S₂ could reduce 9. To this end, 2a and 3a were incubated for 30 min and treated with 9. Since 2a, 3a and 9 were all soluble in organic solvents, these experiments were done in THF. As shown in Figure 5–c/d, a dose-dependent decrease of UV absorption at 548 nm was detected, indicating the reduction of 9, whereas 2a and 3a alone could not reduce 9. Interestingly, in-situ generated H₂S by 7 and TBAF could not reduce 9 under this condition. These results suggest that H₂S₂ is much more potent than H₂S in reducing tetrazines. This finding may be used to develop tetrazine-based H₂S_n sensors.

Figure 5.

a) Reduction of tetrazine. b) Time-dependent reduction of 9 (5 mM) by Na₂S or Na₂S₂ (25 mM) in THF w/ 20% water. c) Dose-dependent reduction of 9 (1 mM) by H₂S₂ generated from 2a and 3a in THF. d) Reduction of 9 (1 mM) by various species (4 mM) in THF.

In summary, we demonstrated here that diacyl disulfides could be used as precursors to produce H₂S₂ in both aqueous buffers and organic solvents. H₂S₂ generated by this method could potently reduce DPPH and tetrazines. These results will aid future understanding of H₂S_n.