Reactions of Isolated Persulfides Provide Insights into the Interplay between H₂S and Persulfide Reactivity

Abstract

Hydrogen sulfide is ubiquitous in biological systems and exerts function over a wide range of important physiological processes. Complementing free H₂S, the reductant-labile sulfur pool plays significant roles in the translocation and action of sulfide, however the chemistry of reductant-labile sources of sulfide have not been studied systematically. Using a combination of NMR and UV-Vis spectroscopy, we investigated the spectroscopic properties and reactivity of three isolated organic persulfides and report a simple model for persulfide reactivity, including their roles as nucleophiles, electrophiles, and sulfide donors.

graphical abstract

1. Introduction

Hydrogen sulfide (H₂S) continues to generate significant interest as an important biomolecule due to its role as a ubiquitous signaling molecule in diverse biological systems and processes.[1, 2] Produced by both enzymatic[3] and non-enzymatic processes, H₂S plays important roles in maintaining immune, cardiovascular, neuronal, as well as other functions.[1, 4] Although sulfide signaling mechanisms remain an active area of investigation, they also face significant challenges due to the inherent complexity of the chemical processes occurring during the course of such mechanisms. Cellular concentrations of free H₂S are now understood to be in the low to mid-nanomolar range, [5] which is significantly lower than the initial reports of physiologically-unreasonable micromolar levels of free sulfide.[6, 7] This significant revision of endogenous sulfide levels is due in part to better tools for measuring and differentiating sulfide pools, and also reflects a broadened appreciation of the importance of reductant-labile forms of sulfide.[8] Contrasting acid-labile sulfide pools, which often consist of metal sulfides such as iron sulfur clusters,[9-11] the reductant-labile sulfane-sulfur pools are comprised primarily of oxidized thiols such as hydrodisulfides/persulfides (RSSH), hydropolysulfides (RSS_nSH), and both organic and inorganic polysulfides (RS(S)_nSR).[2, 12-15]

The reductant-labile sulfane sulfur pool is hypothesized to play significant roles in the observed biological action associated with H₂S and also provides access to redox-neutral pathways for transsulfuration and transpersulfidation processes.[16, 17] In addition to providing a significant source of redox-labile sulfide, proteins bearing persulfide modified cysteine residues show altered activity, such as increased nucleophilicity, by comparison to the parent thiols.[18-23] Persulfides may also play important roles in the observed antioxidant properties of H₂S, based on the higher stability of the persulfide radical (RSS•) compared with the thiyl radical (RS•).[24]

Despite the widespread importance of persulfides in biological processes, understanding the different reactivity of persulfides, thiols, and sulfide remains a significant challenge, in part due to the inherent complexity of the sulfane sulfur pool and in part due the difficulty of disentangling the innate chemistry of its constituents, because the chemistries of persulfides, thiols, and sulfide are largely intertwined. Further complicating our understanding of persulfide chemistry, many of the previous studies on persulfides have relied on persulfides generated in situ rather than purified compounds.[25-28] Although isolated persulfides have appeared sporadically in the literature and have been the focus of previous studies,[29] little work has been done to systematically characterize persulfide reactivity under controlled and comparable conditions. The difficulty in differentiating persulfide reactivity from other sulfhydryl-containing biomolecules has been reflected by the significant challenges in designing selective methods for labeling persulfides.[30-32] A more complete understanding of the inherent chemical properties and reactivity of persulfides would allow for the development of more reliable persulfide detection methodologies and would also provide valuable insights into the roles of persulfides in biological signaling and regulation. To address this need, we have synthesized three organic persulfides (Figure 1) and characterized their physical properties and reactivity with a variety of reactive small molecules under controlled conditions.

Figure 1.

Chemical structure of the prepared persulfides: trityl persulfide (TrtSSH), adamantyl persulfide (AdSSH), and benzyl persulfide (BnSSH).

2. Materials and Methods

2.1. Materials

Diacetylsulfide (2), tritylacetyldisulfide (4c), and tritylpersulfide (TrtSSH) were prepared as described previously.[33] Both benzylpersulfide (BnSSH)[34] and adamantylpersulfide (AdSSH)[35] have been reported previously, but we report modified synthetic procedures and complete characterization data below. Thioacetic acid, acetyl chloride, sulfuryl chloride and the required thiols were purchased from commercial suppliers and used as received. Triphenylphosphine, [NBu₄⁺][CN⁻], [NBu₄⁺][Cl⁻], [NBu₄⁺][I⁻], and [NBu₄⁺][OAc⁻] were handled in an inert atmosphere glove box and used at received. [NBu₄⁺][SH^±] was prepared by reaction of NaSH with [NBu₄⁺][Cl⁻] in MeCN in the absence of both water and air. Concentration of the solution under vacuum, followed by trituration of the resulting solid with Et₂O produces [NBu₄⁺][HS⁻] as a white solid. Dithiopropane was degassed using three freeze-pump-thaw cycles and stored under nitrogen. TEA, was distilled under vacuum prior to use and stored under nitrogen. Trityl thiolate was generated by deprotonation of trityl thiol with sodium hydride. NO_(g) was prepared by the slow addition of H₂SO₄ to a concentrated aqueous solution of NaNO₂. The evolved gas was purified by passing through a column of NaOH pellets and low-boiling impurities were condensed by passage through a glass wool filled coil at −78 °C, and collected in a storage bulb. H₂S_(g) was purchased from Sigma Aldrich and transferred through a custom-built stainless steel transfer line into a glass storage bulb prior to use. Note: Hydrogen sulfide and its salts are highly toxic and should be handled carefully to avoid exposure. Deuterated solvents were purchased from Cambridge Isotope Laboratories, degassed using three freeze-pump-thaw cycles and stored under nitrogen over 4Å molecular sieves. Diethyl ether and toluene were degassed by sparging with argon followed by passage through a Pure Process Technologies solvent purification system. All air-free manipulations were performed using standard Schlenk techniques or by use of an Innovative Technology glove box.

2.2 Spectroscopic methods

NMR spectra were acquired on a Brüker Avance-III-HD 600 spectrometer with a Prodigy multinuclear broadband CryoProbe or a Varian INVOA-500 spectrometer at 25.0 °C. Chemical shifts are reported in parts per million (δ) and are referenced to residual protic solvent resonances. The following abbreviations are used in describing NMR couplings: (s) singlet, (d) doublet, (b) broad, and (m) multiplet. Persulfide reactivity was assessed by NMR using anhydrous and anaerobic CD₂Cl₂ at room temperature with 18 mM persulfide and reactant (Table 2). UV-vis spectroscopic measurements were performed on a Cary 60 spectrophotometer equipped with a Quantum Northwest cuvette temperature controller under anaerobic conditions in 1.0 cm path length septum-sealed cuvettes obtained from Starna Scientific. IR spectra were measured in the solid phase on a Thermo Scientific Nicolet 6700 RT-IR using an ATR attachment. High resolution mass spectrometry (HRMS) measurements were performed by the Biomolecular Mass Spectrometry Core of the Environmental Health Sciences Core Center at Oregon State University.

Table 2.

Reactivity of persulfides in the presence of different reductants, nucleophiles, gases, and bases. Experiments were performed anhydrously and anaerobically in CD₂Cl₂ at room temperature with 18 mM persulfide and reactant. All reaction products were confirmed by ¹H or ³¹P{¹H} NMR spectroscopy. S₈ formation was confirmed and quantified by titration with PPh₃ at the end of the reaction. Abbreviations: nr: no reaction, DTP: 1,3-dithiopropane. Reactions taking > 48 hours to occur are considered exhibit no reactivity.

Reactant	TrtSSH		BnSSH		AdSSH
	Products	time	Products	time	Products	time
PPh3	TrtSH, Ph3PS	< 2 min	BnSH, BnSSSBn, H2S, Ph3PS	16 hr	AdSH, Ph3PS	> 24 hr
DTP	Nr	–	nr	–	nr	–
H2S(g)	Nr	–	nr	–	nr	–
NO(g)	Nr	–	nr	–	nr	–
[Na+][TrtS−]	TrtSH, S8	< 2 min	Polysulfides	< 2 min	AdSH, S8	< 2 min
[NBu4+][HS−]	TrtSH, S8, H2S	> 24 hr	H2S, polysulfides	< 2 min	nr	–
[NBu4+][CN−]	TrtSH, S8	< 2 min	H2S, BnSH, polysulfides	< 2 min	AdSH, S8	< 2 min
[NBu4+][AcO−]	TrtSH, S8	< 2 min	Polysulfides	< 2 min	AdSH, S8	< 2 min
[NBu4+][Cl−]	TrtSH, S8	1 hr	polysulfides	< 2 min	nr	–
[NBu4+][I−]	nr	–	polysulfides	> 24 hr	nr	–
NEt3	TrtSH, S8	< 2 min	polysulfides	< 2 min	AdSH, S8	30 min

2.3 Synthesis of acetyl sulfenylchloride

Acetyl sulfenylchloride (3) was prepared using a procedure modified from the literature [33]. In rigorously dried glassware using anhydrous reagents, sulfuryl chloride (3.73 mL, 46.2 mmol) was added dropwise to diacetylsulfide (5.3 g, 45 mmol) at −30 °C in a dry ice/methanol bath under nitrogen. The reaction mixture was warmed from −30 °C to −5 °C over the course of 1 h, during which time the color of the solution changed from pale yellow to orange. The generated SO₂ was removed at −15 °C under vacuum at 20 torr. Vacuum distillation at 100 mtorr of the remaining mixture at 0 °C into a receiving flask at −40 °C produced 3.10 g (28.0 mmol, 62.3%) of a bright yellow liquid, with spectroscopic properties matching previous reports. This compound was used immediately.

2.4 Synthesis of Acetyldisulfides

General Procedure: The desired thiol (1.82 mmol) was dissolved in 1.0 mL of dry Et₂O and was added dropwise at room temperature to acetylsulfenyl chloride (2, 221 mg, 2.00 mmol) dissolved in 1.0 mL of dry Et₂O under nitrogen. For best results, use freshly distilled 2. The reaction mixture was allowed to stir for 1.5 h. The remaining volatiles were removed under vacuum to give the corresponding acetyl disulfide as a solid. These compounds are all generally stable on the bench top and appear to be stable indefinitely in the freezer.

Benzylacetyldisulfide (4a). The general procedure described above was modified in the following ways: the volatiles were removed at 0 °C instead of at rt. Benzylacetyldisulfide was isolated as a pale white solid in 86.5% yield (300 mg). ¹H NMR (600 MHz, CDCl₃) δ: 7.31 (m, 5H), 3.93 (s, 2H), 2.34 (s, 3H). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ: 195.2, 136.2, 129.6, 128.7, 127.9, 43.0, 28.8.

Adamantylacetyldisulfide (4b). The general procedure yields 287 mg (57.5%) of a loose white solid. ¹H NMR (600 MHz, CDCl₃) δ: 2.47 (s, 3H), 2.07 (bs, 3H), 1.83 (d, 6H, J = 2.4 Hz), 1.66 (q, 6H, J = 12 Hz). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ: 195.9, 50.5, 42.2, 36.0, 29.9, 28.9.

2.5 Synthesis of BnSSH and AdSSH

General Procedure: Methanolic HCl (5 N) was prepared by adding acetyl chloride to dry degassed methanol in an ice bath. The acetyldisulfide (100 mg,) was dissolved in 1.0 mL of methanol and cooled in an ice bath under nitrogen. Methanolic HCl (5 N, 342 μL) was added to the solution dropwise, and the resultant reaction mixture was allowed to stir overnight. It is important that no metals be in the reaction vessel at this time. The disulfide does not immediately dissolve fully, and takes up to 3 hr to fully dissolve. After stirring for 24 h, the solvents were removed under vacuum to yield the desired persulfide.

Benzylpersulfide (BnSSH). The general procedure yields 74.0 mg (94%) of a viscous liquid. This compound has a half-life of approximately 2 weeks when stored at −20 °C in the absence of both water and oxygen. ¹H NMR (600 MHz, CD₂Cl₂) δ: 7.32 (m, 5H), 3.91 (s, 2H), 2.96 (bs, 1H, SH). ¹³C{¹H} NMR (150 MHz, CD₂Cl₂) δ: 137.5, 129.8, 129.0, 128.0, 43.4. IR (cm⁻¹): 3061, 3028, 2914, 2505, 1493, 1452, 1070, 763, 697, 665. HRMS-ESI (m/z): [M-H] calcd for [C₇H₈S₂] 156.0068; found 156.0060.

Adamantylpersulfide (AdSSH). The general procedure yields 74.4 mg (90%) of a viscous oil that quickly solidifies on cooling. Adamantyl persulfide appears stable indefinitely when stored at −20 °C, and solution samples do not significantly degrade over the course of a few days. ¹H NMR (600 MHz, CD₂Cl₂) δ: 2.70 (s, 1H, SH), 2.13 (bs, 3H), 1.88 (d, 6H, J = 2.5 Hz), 1.73 (q, 6H, J = 12 Hz). ¹³C{¹H} NMR (150 MHz, CD₂Cl₂) δ: 46.7, 41.5, 36.0, 30.0. IR (cm⁻¹): 2904, 2849, 2674, 2655, 2491, 1439, 1341, 1311, 1297, 1254, 1102, 1041, 977, 867, 686. HRMS-ESI (m/z): [M-H] calcd for [C₁₀H₁₆S₂] 200.0694; found 200.0695.

3. Results and Discussion

We recently reported the isolation, structural characterization, and preliminary reactivity studies of trityl persulfide (TrtSSH).[33] We note that these investigations, as well as those presented herein, were performed in organic solution under anaerobic conditions to enable isolation of specific protonation states of both the persulfide and other reactants as well as to prevent unwanted oxidation chemistry. By contrast to proposed persulfide reactivity with thiols in water,[36] we did not observe H₂S release upon treatment of TrtSSH with thiols. We surmised that this difference could be due to steric constraints of the trityl group, differences in proton transfer in organic versus aqueous solution, or changes in persulfide electronics due to the stability of the trityl cation. For example, the trityl group is partially electron donating due to the high stability of the trityl cation, thus reducing the electrophilicity of the α-sulfur of the persulfide by polarizing C-S bond, which should reduce its reactivity toward thiol nucleophiles. To further investigate these reactivity differences, we have prepared and isolated two additional organic persulfides, benzyl persulfide (BnSSH) and adamantyl persulfide (AdSSH), both of which are known in the literature and have significantly different physicochemical properties than TrtSSH.

AdSSH is similar to TrtSSH in that both are tertiary persulfides. Unlike TrtSSH, however, AdSSH has an entirely aliphatic backbone that is conformationally locked, which significantly reduces the electron donating ability by contrast to TrtSSH. Additionally, AdSSH is significantly less sterically hindered than TrtSSH. To compare the reactivity differences between tertiary and primary persulfides, we also prepared benzyl persulfide (BnSSH), which provides a less sterically hindered persulfide than AdSSH or TrtSSH and also provides an accessible electrophilic α-carbon. If the observed reactivity of these three dissimilar persulfide models provide identical reactivity, then the reactivity differences between biological persulfides and the synthetic persulfides are to be likely attributed to solvents and/or proton transfer chemistry. The described persulfides were synthesized using the general method (Scheme 1). The thiol of the desired persulfide is treated with acetylsulfenyl chloride (3) to yield the corresponding acetyldisuflide (4). Acidic methanolysis of the acetyldisulfide yields the desired persulfide.

Scheme 1.

General synthetic route to access organic persulfides.

With isolated persulfides in hand, we measured and compared their spectroscopic properties and compared these parameters to those of the parent thiols (Table 1). The sulfhydryl ¹H NMR resonance on each persulfide was measured in CD₂Cl₂ and was observed to be 3.02 ppm for BnSSH, 2.70 ppm for AdSSH, and 2.75 ppm for TrtSSH. For both BnSSH and AdSSH, the sulfhydryl proton shifts approximately 1.15 ppm downfield by comparison to the parent thiol, whereas for a 0.35 ppm upfield shift is observed for TrtSSH. Adding to the spectroscopic characterization, we also measured the S-H stretch of each compound using IR spectroscopy. All three of the persulfides show S-H stretches between 2490 and 2508 cm⁻¹, which in each case is about ~60 cm⁻¹ lower in energy than the parent thiol. The combination of IR and ¹H NMR spectroscopic data provide general spectroscopic characteristics of persulfides when compared to their parent thiols, which should facilitate future characterization and identification of small molecule persulfides.

Table 1.

Spectroscopic properties for the ¹H NMR resonance (δ) and IR stretching frequency (υ) of the S–H bond for AdSSH, BnSSH, and TrtSSH compared with their parent thiols.

	δ (ppm)		υSH (cm−1)
R	RSSH	RSH	RSSH	RSH
Trt	2.75	3.10	2508	2574
Bn	3.02	1.86	2504	2560
Ad	2.70	1.57	2491	2567

Having characterized the spectroscopic properties of TrtSSH, AdSSH, and BnSSH, we next systematically investigated the chemical reactivity of the isolated persulfides with a variety of reductants, nucleophiles, bases, and gases (Table 2). Treatment of the persulfides, which contain sulfur atoms in the −1 oxidation state, with one equivalent of triphenylphosphine (PPh₃), a nucleophilic reductant, generates the parent thiol and triphenylphosphine sulfide (Ph₃PS). This reaction constitutes an overall 2-electron reduction and produces two sulfur atoms in the −2 oxidation state. Although TrtSSH and AdSSH react to cleanly afford Ph₃PS and the parent thiols as the only product, BnSSH also generates benzyltrisulfide (BnSSSBn, δ = 4.07 ppm) and H₂S (δ = 0.9 ppm, Figure S7). The ratio of BnSSSBn to BnSH is 3:4 by ¹H NMR spectroscopy and the ³¹P{¹H} NMR spectrum of the reaction mixture shows unreacted PPh₃ in solution, suggesting that the trisulfide-generating reaction pathway does not consume PPh₃. One explanation for this difference in reactivity is that BnSSH may be sufficiently acidic (vide infra) such that deprotonation of the persulfide by PPh₃ becomes competitive with the rate of PPh₃-mediated reduction chemistry. Another interesting observation is that BnSSSBn is the only observed polysulfide formed during the reaction, and that higher order polysulfides, such as tetra- and pentasulfides, are not observed. This selectivity is consistent with extraction of sulfur from trisulfides by PPh₃ in methylene chloride being slow[37] by comparison to either higher order polysulfides or persulfides.

Exposure of any of the isolated persulfides to other reducing agents, such as H₂S_(g), NO_(g), or dithiopropane (DTP) failed to produce any measurable reaction over a 48 hr period. The observed persulfide stability in the presence of DTP and H₂S_(g) suggests that either these sulfhydryl-containing reductants are either not sufficiently reducing to release sulfide, or that proton transfer steps, which are not favorable in non polar solvents but should be facilitated by solvation in water, are important mechanistic components of the reduction process. To probe whether proton inventory was important to the reduction of persulfides by thiols, we treated AdSSH, TrtSSH, and BnSSH with [Na⁺][TrtS⁻]. We observed that instead of acting as a reducing agent and producing H₂S, treatment of the persulfides with [Na⁺][TrtS⁻] produced TrtSH (δ = 3.1 ppm, Figures S11, S22), indicating that the thiolate was deprotonating the persulfides and generating a persulfide anion.

To further explore the observed persulfide reactivity, we treated each persulfide with a variety of both anionic and neutral bases. For the series CN⁻, AcO⁻, Cl⁻, and I⁻, we observed that persulfides react with the anions at a rate increasing with anion basicity, which is consistent with a mechanism dependent on deprotonation. We observed a slower reaction rate for NEt₃ than with the charged bases, which we attribute to solvation effects. Because the experiments were conducted in CD₂Cl₂, charged species are solvated much more poorly than neutral species, and this property makes NEt₃ a weaker base by comparison to an anion such as acetate. The reaction of HS⁻ with BnSSH was fast, however no reaction was observed with either AdSSH or TrtSSH. If HS⁻ is acting as a base, it is unclear why the reactivity of HS⁻ with AdSSH and TrtSSH is so much lower than the other tested bases.

Upon treatment with each of the bases, BnSSH reacted the fastest, followed by TrtSSH, and then AdSSH. We theorize that the increase in reaction rate corresponds to a greater generation of the persulfide anion during the reaction, suggesting that BnSSH is most acidic, followed by TrtSSH, and then AdSSH. Additionally the products observed for the reaction of persulfide with base changed depending on the amount of steric hindrance surrounding the persulfide. AdSSH and TrtSSH are both tertiary persulfides, and in each case their reaction with base produced only the parent thiol and elemental sulfur. By contrast, for the primary persulfide BnSSH, we observed a variety of benzyl polysulfide products (n=2-6) in addition to the parent thiol (Table 3). When stronger bases were used, we observed H₂S generation from BnSSH during the reaction.

Table 3.

Distribution of products observed in the reactions of benzyl persulfide as measured by integration of the peaks present in the product spectra. The polysulfides were identified using their reported chemical shifts.[38]

Reactant	BnSH	BnSSBn	BnS3Bn	BnS5Bn	BnS6Bn
PPh3	0.54	0.07	0.39	-	-
[Na+][TrtS−]	0.41	-	0.30	0.22	0.07
[NBu4+][HS−]	-	-	0.58	0.33	0.09
[NBu4+][CN−]	0.28	0.15	0.37	0.14	0.06
[NBu4+][AcO−]	0.26	0.08	0.48	0.18	-
[NBu4+][Cl−]	-	-	0.61	0.28	0.11
[NBu4+][I−]	-	-	0.5	0.35	0.15
NEt3	0.28	0.03	0.39	0.21	0.09

To verify that the persulfide anion was present over the course of the reaction, we monitored the reaction of the persulfides with base by UV-Vis spectroscopy (Figure 2). Previous work in aqueous buffers above pH 8.0 has shown a peak attributed to the cysteine persulfide anion appears between 335 – 350 nm with a molar absorptivity of ~400 M⁻¹cm⁻¹, although analytical standards for such compounds remain unavailable.[18, 39] Upon treatment of BnSSH with AcO⁻, we initially only observed a minimal change in the large shoulder sweeping down from the UV range. After a short incubation period, however, a peak centered near 340 nm appeared, which we attribute to the persulifde anion, which reaches a maximum intensity of 0.065 AU after 30 min. The solution was monitored for a total of 4 hr, after which the intensity of the peak centered near 340 nm returned to background levels, consistent with complete reaction of the persulfide anion. Further supporting the assignment of the 340 nm absorbance band as a persulfide anion, addition of anhydrous HCl in Et₂O to the reaction mixture immediately abolishes the 340 nm absorbance, which is consistent with protonation of the persulfide anion to generate the parent persulfide. We also observed that the UV-vis reactions proceeded at a significantly slower rate than the NMR experiments, which is consistent with our proposed bimolecular persulfide decomposition reaction because the UV-Vis experiments were conducted at a significantly lower concentration.

Figure 2.

UV-Vis spectrum of 100 μM BnSSH reacting with 10 mM [NBu₄⁺][AcO⁻] in dichloromethane. Immediately after addition of base (black), the intensity of the peak centered around 340 nm increases for 30 min (blue), before returning to background levels (red). Inset: Intensity of persulfide anion (340 nm) with respect to time.

On the basis of the observed reactivity of the three persulfides investigated here, we propose a general mechanism for the observed base-mediated decomposition (Figure 3). For the reaction between two persulfides under basic conditions (5), if R is sufficiently sterically hindered or is a tertiary center, such as for AdSSH and TrtSSH, only the terminal sulfur atom of the persulfide is accessible for nucleophilic attack by other persulfides. This reaction results in formation of one equivalent of hydropolysulfide anion (6) and one equivalent of thiol. The resultant hydropolysulfide anions (6) can then react with additional persulfides to further elongate the hydropolysulfide chain (7). Once the sulfur chain in the hydropolysulfide anion is of sufficient length, an intramolecular nucleophilic attack results in extrusion of elemental sulfur (such as S₈) and generates the parent thiol. By contrast, when the R-group is not sterically hindered or is primary center, such as for BnSSH, the α-sulfur atom becomes accessible for nucleophilic attack. Attack on the α-sulfur by a persulfide anion or hydropolysulfide anion results in formation of a symmetrical polysulfide (8) and HS⁻, thus terminating the chain.

Figure 3.

Proposed reaction mechanism for the reaction of persulfides (RSSH) with reductants and bases.

Conclusions

In summary we have prepared three organic persulfides, TrtSSH, AdSSH, and BnSSH, and investigated their reactivity in aprotic organic solvents using NMR and UV-vis spectroscopic to determine how differences in persulfide structure relate to differences in observed reactivity. Although our experiments were performed in organic solution under anaerobic conditions, and thus may not directly represent the exact reactivity of organic persulfides in aerobic aqueous environments, we believe that the reaction profiles of these isolated persulfides contributes positively to unravelling the complex reactivity associated with biological persulfides. For example we observed that persulfide stability inversely correlates with persulfide acidity, with acidic persulfides decomposing more rapidly than less-acidic persulfides. This observation may help explain why although cysteine persulfides in enzyme active sites act as highly-reactive nucleophiles, less-acidic glutathione persulfide (GSSH) is present and stable in micromolar concentrations in the cytoplasm. Furthermore, differences in solvation and hydrogen bonding environments available to GSSH compared to the cysteine persulfides in enzyme pockets may also contribute to these differences in reactivity. In our model studies, we also observed that the steric bulk near the persulfide significantly impacts the reactivity, and this observation may have consequences in determining the relative electrophilicity and nucleophilicity of the sulfur atoms of the persulfide. This observation may also suggest that specific proteins or protein pockets may be sufficiently shielded to prevent the formation of longer-chain polysulfides, thus imparting selectivity on polysulfide chain length and associated changes in reactivity based on which hydropolysulfides can be generated. Additionally, we observed that persulfides can only release H₂S following reduction by another species, including another persulfide, which supports the general hypothesis that persulfides may play important roles for sulfide storage and transport, whereas free H₂S/HS⁻ is prone to reaction with other reactive biological species. Taken together, these results provide new insights into persulfide reactivity gleaned from simple model compounds investigated under identical conditions.

Highlights.

• -Persulfides are biologically important molecules
• -Persulfide reactivity remains underinvestigated
• -Adamantyl and benzyl persulfides are prepared and fully characterized
• -Persulfide reactivity is studied in non-polar anaerobic solvents
• -A model for the reactivity of the studied persulfides is proposed