Kinetic Insights into Hydrogen Sulfide (H₂S) Delivery from Caged-Carbonyl Sulfide (COS) Isomeric Donor Platforms

Abstract

Hydrogen sulfide (H₂S) is a biologically-important small gaseous molecule that exhibits promising protective effects against a variety of physiological and pathological processes. To investigate the expanding roles of H₂S in biology, researchers often use H₂S donors to mimic enzymatic H₂S synthesis or to provide increased H₂S levels under specific circumstances. Aligned with the need for new broad and easily-modifiable platforms for H₂S donation, we report here the preparation and H₂S release kinetics from a series of isomeric caged-carbonyl sulfide (COS) compounds, including thiocarbamates, thiocarbonates, and dithiocarbonates, all of which release COS that is quickly converted to H₂S by the ubiquitous enzyme carbonic anhydrase. Each donor is designed to release COS/H₂S after the activation of a trigger by activation by hydrogen peroxide (H₂O₂). In addition to providing a broad palette of new, H₂O₂-responsive donor motifs, we also demonstrate the H₂O₂ dose-dependent COS/H₂S release from each donor core, establish that release profiles can be modified by structural modifications, and compare COS/H₂S release rates and efficiencies from isomeric core structures. Supporting our experimental investigations, we also provide computational insights into the potential energy surfaces for COS/H₂S release from each platform. In addition, we also report initial investigations into dithiocarbamate cores, which release H₂S directly upon H₂O₂-mediated activation. As a whole, the insights on COS/H₂S release gained from these investigations provide a foundation for the expansion of the emerging area of responsive COS/H₂S donors systems.

Graphical Abstract

INTRODUCTION

Hydrogen sulfide (H₂S) is an important biological gaseous molecule that exhibits promising protective functions in mammals against various disease states. Due to its role in different physiological processes, H₂S is now recognized as the third member of the gasotransmitter family, along with nitric oxide (NO) and carbon monoxide (CO).^1–7 Endogenous H₂S is generated enzymatically from cysteine and homocysteine by several enzymes, such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), cysteine aminotransferase (CAT), and 3-mercaptopyruvate sulfur transferase (3-MST), which can work either individually or in concert.^8–12 Once produced, H₂S causes relaxation of smooth muscle cells and can induce vasodilation by activating K_ATP channels.^13–14 Endogenous H₂S formation, as well as exogenous H₂S administration, has been demonstrated to rescue cells, tissues, and organs from a variety of severe damages, thus raising the therapeutic potential of H₂S-releasing materials.^15–16 Although H₂S is toxic at higher concentrations, lower levels often exert beneficial effects. For example, H₂S exhibits anti-inflammatory activities in animal models¹³ and cardioprotective effects against oxidative stress by scavenging cellular reactive oxygen species (ROS).^17–20

Due to the significant contributions of H₂S in different (patho)physiological processes, researchers often use H₂S releasing agents (H₂S donors) to modulate H₂S levels and enable new investigations.^21–28 Common commercially-available H₂S sources, such as sodium sulfide (Na₂S) and sodium hydrogen sulfide (NaHS), generate H₂S immediately upon addition to water and have been widely used in H₂S studies. Although convenient, the instantaneous and uncontrollable H₂S release from these salts does not mimic controlled enzymatic H₂S synthesis and can often lead to contradictory results (i.e. anti-inflammation and pro-inflammation effects) depending on experimental conditions, handling, or commercial sources.^29–30 Complementing these inorganic salts, garlic-derived polysulfide compounds, such as diallyl trisulfide (DATS, Figure 1), release H₂S in the presence of reduced glutathione (GSH), and the resultant H₂S from such compounds has been demonstrated to exhibit protective activities in animal models.³¹ Because such compounds generate persulfides en-route to H₂S release, it remains unclear whether the observed protective effects are due to H₂S alone or whether other sulfur-containing intermediates or products contribute to the observed activities.

Figure 1.

Selected small molecule H₂S donors and motifs.

In addition to naturally-occurring H₂S donors, researchers have also developed different classes of synthetic H₂S releasing molecules with different properties and release mechanisms.^21–23,25 For example, GYY4137, synthesized from Lawesson’s reagent, has been used widely as a hydrolysis-based H₂S donor in systems ranging from simple cell culture experiments to animal models.^32–37 Although GYY4137 exerts H₂S-related outcomes in a wide array of systems, its H₂S release is inefficient and unreacted GYY4137, or its H₂S-depleted analog, may cause other side effects. Furthering the potential biomedical applications of synthetic donors, H₂S-releasing ADT-OH has been coupled to a series of nonsteroidal anti-inflammation drugs (NSAIDs). Although the resultant H₂S-hybrid NSAIDs exhibit greatly reduced NSAID-induced GI damage while maintaining NSAID activity in GI system, the detailed mechanism of H₂S release from ADT-OH derived systems remains uncertain.^38–40 Building on the need for mechanistically-understood systems, different controllable H₂S donors have been designed and evaluated.^21–23,25 Such donors are typically activated by specific triggers, such as cellular thiols,^41–47 light,^48–49 esterase,⁵⁰ nitroreductase,⁵¹ or pH,^52–53 to release H₂S (Figure 1). In addition to their small-molecule counterparts, H₂S-releasing biomaterials, including H₂S releasing polymers, peptides, and microfibers, are also emerging as an important class of donor materials with modifiable macromolecular properties.^54–59

Building from the need for new donor motifs that function through well-defined release mechanisms, we recently reported the first examples of H₂S donors that function through the initial release of carbonyl sulfide (COS).⁶⁰ Although COS has only recently emerged as a potential biologically-important molecule, it is the most prevalent sulfur-containing gas in the Earth’s atmosphere and has a long history in geological, atmospheric, and agricultural chemistry.^61–62 In the global sulfur cycle, COS is generated from both abiotic sources, including volcanos, hot springs, and oceans, as well as biotic sources, including plants, soils, and biomass burning,⁶³ and is eventually oxidized to sulfur dioxide (SO₂) and sulfate (SO₄²⁻) in the atmosphere.^61,64 In mammalian systems, however, COS functions as a competent substrate for the ubiquitous enzyme carbonic anhydrase (CA), which results in rapid conversion to H₂S (k_cat/K_M for bovine carbonic anhydrase II: 2.2 × 10⁴ M⁻¹s⁻¹).^65–68 This property enables COS to serve as a H₂S precursor and provides new avenues for H₂S donor development based on COS-releasing motifs.⁶⁸

Expanding from our initial report of using caged thiocarbamates as COS/H₂S donor motifs, we recently reported a class of COS/H₂S-releasing donors that were activated by reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), peroxynitrite (ONOO⁻), and superoxide (O₂⁻), with H₂O₂ as the most potent trigger.⁶⁹ H₂S can scavenge H₂O₂ directly, albeit with modest second-order rate constants (0.73 M⁻¹s⁻¹ for HS⁻/H₂O₂ reaction at 37 °C, pH 7.4), and it is also well-established to react with other cellular oxidants.⁷⁰ More recently, Kimura and co-workers demonstrated that NaSH significantly rescued Neuro2a cells from H₂O₂-induced oxidative stress, suggesting promising anti-oxidative effects of H₂S.⁷¹ In our initial H₂O₂-triggered donor systems, COS was caged as an O-alkyl thiocarbamate, which upon activation of an H₂O₂-activated trigger, underwent a self-immolative decay to release COS/H₂S in an H₂O₂ dose-dependent manner. Cellular investigations demonstrated that these donors, but not carbamate control compounds that release CO₂/H₂O, rescued cells from H₂O₂-induced oxidative stress, thus demonstrating that COS-releasing donors can access activities associated with H₂S-releasing motifs. In addition to H₂O₂ activation, our group as well as other researchers have expanded the COS-releasing landscape to include donors activated by nucleophilic attack,⁷² light,⁷³ click chemistry,⁵³ and cellular esterases.^74–75 Because such donors function by the intermediate release of COS en-route to H₂S generation, it is possible that such donors could also reveal activities associated with COS directly, although the CA-mediated conversion of COS to H₂S is fast. Despite the rapid expansion of this research area, the structural motifs comprising the caged COS core structures have remained somewhat limited. To expand our understanding of COS-based H₂S donor systems to enable future expansion of this emerging area, we report here the preparation and COS/H₂S-releasing capacities of six different isomeric cores, including O- and S-alkyl thiocarbamates and thiocarbonates, as well as dithiocarbonate derivatives (Figure 2). We complement our H₂S releasing investigations with DFT investigations on the energetics on the self-immolative and COS-extrusion potential energy surfaces for each isomeric derivative. For the best releasing core structure, we demonstrate how electronic substitution can be used to tune the rate of COS/H₂S release and highlight design requirements for efficient donor function. In addition, we also include an initial expansion of our platform to include dithiocarbamate motifs that are designed to release H₂S and/or CS₂, which has been recently reported to be protective in biosystems^76–77 and may be an emerging small molecule of biological interest.

Figure 2.

H₂S release from H₂O₂-activated isomeric caged COS/H₂S donors

RESULTS AND DISCUSSION

The general design of the H₂O₂-activated donor motifs requires H₂O₂-mediated boronate cleavage to generate a phenolic intermediate, which undergoes a subsequent self-immolative decomposition to release COS (Figure 2). In our initial report, we focused exclusively on the O-alkyl thiocarbamate core structure, although many other isomeric derivatives are accessible and should still release COS. To investigate the H₂S releasing profile of such isomeric caged-COS molecules, we prepared isomeric O-alkyl and S-alkyl peroxy-sensitive thiocarbamates (OA-PeroxyTCM and SA-PeroxyTCM), thiocarbonates (OA-PeroxyTCN and SA-PeroxyTCN), and the S-alkyl dithiocarbonate (SA-PeroxyDTCN) with an arylboronate trigger. Upon activation, each of these donor platforms releases COS, which is quickly hydrolyzed to H₂S by CA. In addition to these COS-releasing molecules, we also prepared peroxy-labile dithiocarbamate (PeroxyDTCM) compounds, which could potentially release CS₂ or H₂S directly by different pathways vide infra).

Donor Synthesis

One benefit of the caged COS donor platforms investigated is the simplicity and modularity of their preparation. The thiocarbamate-based OA-PeroxyTCMs were synthesized by treating the corresponding 4-(hydroxymethyl)phenylboronic acid pinacol ester with the desired aryl isothiocyanate in the presence of sodium hydride (Scheme 1a). Similarly, SA-PeroxyTCM-1 and the caged-CS₂ PeroxyDTCMs were synthesized by treating 4-(thiomethyl)phenylboronic acid pinacol ester with the desired aryl isothiocyanate reagents (Scheme 1b). The thiocarbonates OA-PeroxyTCN-1 and SA-PeroxyTCN-1, were prepared by treating the desired pinacol ester benzyl alcohol or thiol starting materials with the corresponding aryl chloroformate or chlorothionoformate reagents (Scheme 1c). Similarly, SAPeroxyDTCN-1 was prepared by treating 4-(thiomethyl)phenylboronic acid pinacol ester with the aryl chloroformate in the presence of pyridine (Scheme 1c).

Scheme 1.

Synthesis of the isomeric thiocarbamate, thiocarbonate, and dithiocarbamate donors.

H₂S Release from Caged Donors

Electronic Effects of H₂S Release from O-alkyl Thiocarbamates

To evaluate the substituent effects on H₂S release from OA-PeroxyTCMs, we chose to focus on the OA-PeroxyTCM family because of the well-defined release behavior, our previous investigations with O-alkyl thiocarbamate derivatives, and their high release efficiencies (vide infra). To measure rate data, we used an H₂S-selective electrode to monitor H₂S release from OA-PeroxyTCM-1 through 6 (50 μM) in the presence of excess H₂O₂ in PBS buffer (pH 7.4, 10 mM) containing cellularly-relevant concentrations of CA (25 μg/mL). We used excess H₂O₂ in this study to ensure the boronate cleavage was under pseudo-first order condition. The pseudo-first order rate constant (k_obs) was obtained by plotting the H₂S releasing response versus the time of measurement, and the second order rate constant (k₂) was obtained by plotting the k_obs versus H₂O₂ concentrations. For each donor in the series, we observed a dose-dependent increase in the rate of COS/H₂S released with increased H₂O₂, but also a lower total peaking amount of H₂S, which is consistent with an increase in H₂S scavenging by H₂O₂ at higher concentrations (Figure S1). The OA-PeroxyTCM donors exhibited different k₂ values for COS/H₂S release ranging from 0.22 to 1.16 M⁻¹s⁻¹ (Table 1, Figure S3).

Table 1.

Comparison of H₂S releasing efficiencies from differently substituted O-alkyl thiocarbamates.^a

Donor	Strucutre	k2(M−1s−1)	rel. rate	H2S (%)a
OA-PeroxyTCM-1		1.16 ± 0.05	1.27	60
OA-PeroxyTCM-2		0.59 ± 0.03	0.65	90
OA-PeroxyTCM-3		0.91 ± 0.01	1.00	80
OA-PeroxyTCM-4		0.42 ± 0.02	0.46	76
OA-PeroxyTCM-5		0.22 ± 0.02	0.24	64
OA-PeroxyTCM-6		0.37 ± 0.02	0.41	46

^aH₂S releasing efficiency was evaluated in the presence of 500 μM of H₂O₂. The H₂S releasing yield (%) was calculated by using a NaSH calibration curve.

To better compare the effects of electronic substitution on COS/H₂S release from the OA-PeroxyTCM donors, we normalized each rate to the parent OA-PeroxyTCM-3 (R = H) donor (Table 1). Our initial expectation was that inclusion of electron withdrawing groups (EWGs) would facilitate COS release, therefore accelerating H₂S generation, whereas electron donating groups (EDGs) would lead to slower H₂S release (Scheme 2a). Although OA-PeroxyTCM-2 (k_rel. = 0.65) and OA-PeroxyTCM-3 (k_rel. = 1.27) followed this trend, we observed slower H₂S release from OA-PeroxyTCM-4 to 6 with k_rel. of 0.46, 0.24, and 0.41, respectively (Table 1). We attribute this slower H₂S release to the acidification of the thiocarbamate NH proton by EWGs, thus potentially leading to isothiocyanate formation in aqueous buffer (Scheme 2b). Williams and co-workers have reported the similar degradation of thiocarbamates to isothiocyanates in aqueous solution, which supports our hypothesis.⁷⁸ Although aryl isothiocyanates are known H₂S donors, the H₂S release from these compounds less efficient than from the OA-PeroxyTCM core and thus would be expected to reduce the overall rate of H₂S formation.^79–80 H₂S Releasing efficiency was calculated by using a NaSH calibration curve. The low efficiency of H₂S release EWG-containing donors, especially for OA-PeroxyTCM-6, highlighting a limitation of including highly electron withdrawing moieties on thiocarbamate-based donor motifs.

Scheme 2.

Competing H₂S release pathways for O-alkyl thiocarbamates with electron withdrawing groups. (a) H₂O₂-mediated COS/H₂S release and (b) base-mediated isothiocyanate formation followed by hydrolysis to generate H₂S.

H₂S Release from S-Alkyl Thiocarbamates

Having investigated the impacts of electronic substitution on the O-alkyl thiocarbamate scaffolds, we next compared the O- versus S-alkyl isomeric differences for thiocarbamate donors by evaluating the H₂S releasing from SA-PeroxyTCM-1. We note that Chakrapani and co-workers recently reported esterase-cleavable S-alkyl thiocarbonate and thiocarbamate COS donor motifs, but their activities were not compared with O-alkyl donor analogues.⁷⁵ Our expectation was that the S-alkyl isomer should have a greater ground state stability due to enhanced resonance stabilization in the amide moiety of the thiocarbamate by comparison to the thioamide moiety in the O-alkyl derivative. Although SA-PeroxyTCM-1 is more thermodynamically stable than the corresponding O-alkyl isomer, it is less stable in solution, likely due to the better leaving group ability of the benzyl thiol versus benzyl alcohol. For example, incubation of model compounds lacking the boronate trigger revealed complete recovery of the O-alkyl isomer from buffer after 24 hours, whereas only 40% of the S-alkyl isomer was recovered. In addition, over 80% of the S-alkyl isomer decomposed after 24-h incubation with ROS, such as H₂O₂, ONOO⁻, and O₂⁻, suggesting that the S-alkyl thiocarbamate functional group is reactive toward ROS, which may lead to relatively lower COS/H₂S releasing efficiency (Figure S6). As in the case of O-alkyl OA-PeroxyTCM-1, we observed dose-dependent H₂O₂-mediated COS/H₂S release from SA-PeroxyTCM-1 (Figures 4 and S2a). The rate of COS/H₂S release, however, was significantly slower (k_rel = 0.09) with respect to OA-PeroxyTCM-1 (k_rel = 1.00) with 42% of the expected H₂S being captured (Table 2, Figure S3), indicating that H₂S release from caged-thiocarbamate compounds can be tuned by isomeric changes to the caged COS core.

Figure 4.

Representative H₂S Release from caged-COS donors in the presence of H₂O₂.

Table 2.

H₂S Release from caged-COS molecules^a

Donor	Strucutre	k2(M−1s−1)	rel. rate	H2S (%)a
OA-PeroxyTCM-1		1.16 ± 0.05	1.0	60
SA-PeroxyTCM-1		0.10 ± 0.01	0.09	42
OA-PeroxyTCN-1		0.60 ± 0.04	0.52	36
SA-PeroxyTCN-1		0.16 ± 0.01	0.14	36
SA-PeroxyDTCN-1		0.36 ± 0.02	0.31	46

^aH₂S releasing efficiency was evaluated in the presence of 500 μM of H₂O₂. The H₂S releasing yield (%) was calculated by using a NaSH calibration curve.

H₂S Release from O- and S-Alkyl Thiocarbonates

In addition to thiocarbamates, thiocarbonate derivatives also provide access to phenolic payloads and also eliminate the potentially-acidic thiocarbamate proton. We envisioned that thiocarbonate compounds, including S- and O-alkyl isomers, with an arylboronate trigger would be new H₂O₂-responsive COS/H₂S donors. Therefore, in addition to caging COS as thiocarbamates, thiocarbonates were also investigated as alternative COS/H₂S donor motifs. We expected that COS/H₂S release from these donors would still be triggered by the H₂O₂-mediated cleavage of the aryl boronate, and that the COS/H₂S releasing kinetics would be different from that of thiocarbamate-based donors based on the inherent electronic differences between the thiocarbamate and thiocarbonate cores. To investigate our hypothesis, we measured the H₂S release from the O-alkyl and S-alkyl isomers OA-PeroxyTCN-1 and SA-PeroxyTCN-1. As expected, both isomers exhibited an H₂O₂-dependent H₂S release in the presence of CA, with the O-alkyl isomer exhibiting faster H₂S-releasing kinetics (Figures 4, S2b, S2c, and S3) and also exhibiting excellent stability in buffer in the absence of H₂O₂ over a 24 hour period. Moreover, the O-alkyl thiocarbonate functional group exhibits better stability in the presence of ROS (i.e. H₂O₂, O₂⁻, and ONOO⁻) compared to other isomeric counterparts, such as O-alkyl and S-alkyl thiocarbamates, making it a promising candidate for future COS/H₂S donor design (Figure S6). In addition, we also prepared SA-PeroxyDTCN-1, in which the phenol payload was replaced with the corresponding thiophenol to determine whether dithiocarbonate cores were still functional as COS/H₂S releasing moieties. We expected that the thiophenol would be a better leaving group than the parent phenol, thus leading to enhanced COS/H₂S release rates. As expected, SA-PeroxyDTCN-1 exhibited faster COS/H₂S release kinetics than the parent SA-PeroxyTCN-1 derivative (k_rel = 0.30 vs 0.14), and also established that dithiocarbonate cores are functional COS/H₂S releasing donor motifs (Figures 4, S2d, S3 and Table 2).

H₂S/CS₂ Release from Dithiocarbamates

Building from the positive impacts that we envision COS donors will have on the biological roles of COS biology, we also envisioned that our general donor design approach could facilitate access to CS₂-releasing motifs. Recent studies suggest that CS₂ may have has specific protective activities in biological systems, and may be an emerging biologically-relevant sulfur-containing small molecule.⁷⁶ Aligned with this idea, we prepared PeroxyDTCM-1 and 2, both of which cage CS₂ into dithiocarbamate compounds. Much like H₂O₂-labile PeroxyTCMs and PeroxyTCNs, the PeroxyDTCMs should be activated by H₂O₂ to generate anionic dithiocarbamate intermediates, which could release CS₂ instead of COS. (Scheme 3). It is also possible, however, that such dithiocarbamates could spontaneously extrude H₂S en route to isothiocyanates generation by a pathway in which proton transfer from the nitrogen to the sulfur in the anionic dithiocarbamate intermediate results in the formation of aryl isothiocyanate and extrusion of H₂S (Scheme 3).

Scheme 3.

Possible competing pathways for (a) H₂S and (b) CS₂ releasing pathways from caged dithiocarbamates.

To determine which of these pathways was operable, we treated PeroxyDTCM-1 with H₂O₂ and observed only a small amount of H₂S release using the methylene blue assay (Figure 5). This result suggested that direct H₂S extrusion may be possible, but was likely inefficient for PeroxyDTCM-1. To further investigate this platform, we also prepared PeroxyDTCM-2, which contains a nitro group in the para position, which should significantly acidify the NH proton, therefore facilitating isothiocyanate formation and H₂S extrusion. As expected, we observed an enhanced H₂S release from PeroxyDTCM-2 after addition of H₂O₂ (Figure 5). To rule out the possibility that released CS₂ was reacting with H₂O₂ to produce H₂S, we incubated authentic CS₂ sample directly with H₂O₂ under the identical condition but failed to observe H₂S release (Figure S4). Our attempts to measure CS₂ released from the PeroxyDTCM donors using different CS₂-responsive colorimetric methods^81–83 as well as by GC have remained unsuccessful, suggesting that CS₂ release is not facile from these compounds. This lack of CS₂ release could be due, in part, to the greater stability of the dithiocarbamate anion formed after the initial self-immolative decay by comparison to the corresponding anionic thiocarbamate anions. Taken together, these dithiocarbamate platforms may provide a future scaffold for efficacious H₂S or CS₂ release after fine tuning of the intermediate stabilities.

Figure 5.

H₂S release from PeroxyDTCMs in the presence of H₂O₂.

Computational Investigations

To provide further insights into the energetics of COS/H₂S release from the different isomeric donor motifs described above, we conducted conformational searches for the ground state, intermediate, and transition state geometries using Gaussian 09 at the B3LYP/6–311++G(d,p) level of theory applying the IEF-PCM water solvation model. Each potential energy surface (PES) begins from the phenol product generated after boronate cleavage by H₂O₂ to provide equivalent starting structures for each energy surface. Graphical summaries of the potential energy surfaces for each donor motif are shown in Figure 6. Attempt to locate the transition state for quinone methide extrusion from the neutral phenol for any of the structures were unsuccessful, which is consistent with prior reports that the self-immolative decomposition proceeds from the anionic phenolate.^84–85

Figure 6.

Calculated potential energy surfaces for thiocarbamate, thiocarbonate, and dithiocarbonate donors at the B3LYP/6-311++G(d,p) level of theory with PCM solvation (H₂O). (a) O-Alkyl and S-alkyl thiocarbamates OA-PeroxyTCM and SA-PeroxyTCM. (b) O-Alkyl and S-alkyl thiocarbonates OA-PeroxyTCN and SA-PeroxyTCN (c) Dithiocarbonate SA-PeroxyDTCN-1. (d) Hypothetical S-alkyl dithiocarbonate SA-PeroxyDTCN-2, which should release CS₂ upon cleavage. All energies are reported in kcal/mol and referenced to the lowest energy phenolate generated after initial donor deprotonation.

For OA-PeroxyTCM, initial proton transfer from the phenol generates the anionic phenolate intermediate and H₃O⁺, which then proceeds through TS1 to generate the monothiocarbamate intermediate and the quinone methide with a barrier of 5.3 kcal/mol. This local barrier is lower than that for SA-PeroxyTCM (8.2 kcal/mol), which is consistent with the different inherent stabilities of the thiocarbamate isomers. During our searches for COS extrusion pathways from the monothiocarbamate anionic intermediate, we were surprised to observe a significant barrier of 42.3 and 28.4 kcal/mol for the O-alkyl and S-alkyl derivatives, respectively. These barriers are in contrast with our lack of observable intermediates during the course of these reaction. Because prior work by Ghadiri and co-workers has highlighted the importance of different additives and/or Lewis acids in COS extrusion from thiocarbamates en route to peptide bond formation,⁸⁶ we also investigated whether lower energy assisted pathways might be present. To simplify such investigations, we focused on water-assisted COS extrusion, in which proton transfer from H₂O occurs concomitantly with COS extrusion (TS2′), and also on general acid catalyzed COS extrusion, in which proton transfer from H₂PO₄⁻ accompanies COS extrusion (TS2″). Both assisted pathways eliminate the direct formation of the high-energy deprotonated amine intermediate that results from direct COS extrusion, which would be unlikely in an aqueous environment. Notably, we were able to locate transition states corresponding to concomitant proton transfer and COS release for both assisted reactions, which are 8.5 and 15.6 kcal/mole lower in energy than the un-assistant pathway for the H₂O and H₂PO₄⁻ assisted routes, respectively. The ease of locating these assisted COS extrusion pathways, as well as their lower energy barriers, suggesting the viability of other solvent or other additive assisted routes of COS release. For both the O-alkyl and S-alkyl isomers, COS extrusion from the monothiocarbamate anion intermediate is exotheric. To complete the final PES for each isomer, we assumed that the produced quinone methide would be scavenged by H₂O to form the p-hydroxy benzyl alcohol, which is consistent with previous experimental observations in similar systems. In total, the overall reaction is only exothermic for the O-alkyl thiocarbamate but endothermic, and thus entropically-driven, for the S-alkyl thiocarbamate donor.

We carried out similar calculations for isomeric O-alkyl and S-alkyl thiocarbonate donor derivatives and observed similar ground state energy differences between the two isomers (Figure 6b). The S-alkyl isomer, SA-PeroxyTCN, was 15.7 kcal/mol lower in energy than the O-alkyl isomer, OA-PeroxyTCN, which we attribute to better orbital overlap between the ester moiety with the aromatic ring. Much like the thiocarbamate counterparts, the O-alkyl and S-alkyl isomers both proceed through self-immolative transition states TS1 (2.6 kcal/mol) and TS1′ (7.2 kcal/mol) respectively, en route to the common thiocarbonate anion intermediate. Direct COS extrusion from the monothiocarbonate anion intermediate proceeds through a significantly lower transition state TS2 (17.2 and 1.5 kcal/mol for the S-alkyl and O-alkyl isomers, respectively) than for the thiocarbamate analogues, which we attribute to the greater leaving group ability of the phenolate by comparison to the analogous deprotonated amine product for the corresponding thiocarbamate donors. Attempts to locate transition states for H₂O or H₂PO₄⁻ assisted COS extrusion were unsuccessful, likely due to the lower basicity of the developing phenolate by contrast to the adeprotonated amine. Subsequent COS extrusion, proton transfer to generate the neutral phenol, and p-hydroxy benzyl alcohol formation complete the overall PES. Similar to the overall thermodynamics of the PES for the thiocarbamate isomers, only the O-alkyl thiocarbonate donor is exothermic, with the more thermodynamically stable S-alkyl thiocarbonate being endothermic, and entropically controlled. One additional insight afforded from the thiocarbonate energy surfaces is that the ground state destabilization of the O-alkyl thiocarbonate generates a system in which the initial self-immolative step to release the quinone methide is rate-limiting, rather than COS extrusion from the anionic thiocarbonate intermediate.

In contrast to the two thiocarbonate isomers, the two dithiocarbonate isomers proceed through different intermediates that should result in COS and CS₂ extrusion from the OA-PeroxyDTCN-1 and SA-PeroxyDTCN-2 isomers, respectively. OA-PeroxyDTCN-1, proceeds through a modest 5.6 kcal/mol activation barrier (TS1) to generate the anionic dithiocarbonate intermediate, which proceeds through TS2 to generate thiophenol and extrude COS en route to an overall endothermic reaction (Figure 6c). Although we were unable to prepare the SA-PeroxyDTCN-2, our DFT investigations show that such donors could potentially function as CS₂-releasing molecules. SA-PeroxyDTCN-2 proceeds through a modest activation barrier of 6.0 kcal/mol en route to the dithiocarbonate intermediate, which requires a significantly higher (17.9 kcal/mol) activation barrier to result in CS₂ release (Figure 6d). Attempts to locate transition states for H₂O or H₂PO₄⁻ assisted pathways for CS₂ extrusion pathways were unsuccessful. After CS₂ extrusion, proton transfer to generate the phenol product, and generation of the p-hydroxy benzyl alcohol, the overall reaction is endothermic by 3.8 kcal/mol. As a final point of comparison, the OA-PeroxyDTCN-1 is isomeric to SA-PeroxyDTCN-2, allowing for the energetics of the two reactions to be compared directly. Interestingly, referencing to the more thermodynamically-stable SA-PeroxyDTCN-1 isomer reveals that the anionic dithiocarbamate intermediate formed from SA-PeroxyDTCN-1 is 15.3 kcal/mol higher than the dithiocarbamate intermediate formed in the SA-PeroxyDTCN-2 PES. Additionally, TS2 is 19.9 kcal/mol higher in energy for SA-PeroxyDTCN-2 than for SA-PeroxyDTCN-1, suggesting that CS₂ release is less kinetically favorable than COS release from the corresponding isomers (see Supporting Information for a direct energy comparison).

To further interrogate the observed H₂S release from the dithiocarbamate PeroxyDTCM, we employed similar computational methods. The initial self-immolative decay from the phenolate intermediate proceeds with a modest activation barrier of 5.7 kcal/mol en route to formation of the dithiocarbamate anion intermediate (Figure 7). Direct CS₂ extrusion from this intermediate, however, is enthalpically unfavorable with a high-energy TS2 of 41.9 kcal/mol. In parallel to our investigations on the COS-releasing thiocarbamate donors, we also located transition states for H₂O and H₂PO₄⁻ assisted CS₂ release pathways, which maintain activation enthalpies of 34.0 and 27.8 kcal/mol, respectively. The final product of all of these routes, which include CS₂, PhNH₂, and p-hydroxy benzyl alcohol, is endothermic by 6.2 kcal/mol. We were also able to locate a PES for direct HS⁻ release, which proceeds through initial proton transfer from the NH to RS⁻ in the anionic dithiocarbamate intermediate, followed by direct HS⁻ extrusion and isothiocyanate formation with an activation barrier (TS3) of 25.4 kcal/mol. The resultant isothiocyanate should also release COS through hydrolysis, leading to a slightly exothermic product formation of H₂S, COS, PhNH₂, and p-hydroxy benzyl alcohol. Notably, this transition state for direct HS⁻ extrusion is lower in energy than any of the assisted CS₂ extrusion pathways, suggesting that sulfide extrusion, which in solution is almost certainly assisted through hydrogen bonding, is a lower energy pathway than CS₂ release. We also investigated whether a similar direct HS⁻ extrusion pathway was energetically accessible from the thiocarbamate-based donors, but found that the transition state for HS⁻ to be 44.9 kcal/mol, which is even higher than the unassisted COS-release pathway. As a whole, these computational insights support the observed direct H₂S release from the caged dithiocarbamate donor motifs, but also suggest that such platforms may be modifiable to access CS₂ releasing systems as well.

Figure 7.

Calculated potential energy surfaces for CS₂ and H₂S extrusion from the dithiocarbamate donor PeroxyDTCM at the B3LYP/6-311++G(d,p) level of theory with PCM solvation (H₂O). All energies are reported in kcal/mol and referenced to the phenolate generated after initial donor deprotonation.

CONCLUSIONS

By preparing and directly comparing thiocarbamate, thiocarbonate, and dithiocarbonate donor platforms, we demonstrate access to a wide array of structural motifs that function as controllable COS-based H₂S donors. In addition to providing access to new H₂O₂-responsive H₂S donors, the combined experimental and computational investigations provide a cohesive platform for understanding how donor core modifications impact efficiency, stability, and rates. Finally, our initial investigations into dithiocarbamate motifs suggest that these platforms provide access to new classes of triggerable H₂S releasing motifs, which could also possibly be tuned to release CS₂. In conclusion, the insights on COS/H₂S release gained from these investigations provide a foundation for the expansion of the emerging area of responsive COS/H₂S donors systems.

EXPERIMENTAL SECTION

Materials and Methods

Reagents were purchased from Sigma-Aldrich, Tokyo Chemical Industry (TCI), Fisher Scientific, and VWR and used directly as received. Carbonic anhydrase (CA) from bovine erythrocytes was purchased from Sigma-Aldrich (C2624). Silica gel (SiliaFlash F60, Silicycle, 230–400 mesh) was used for column chromatography. Deuterated solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, Massachusetts, USA). ¹H, ¹⁹F and ¹³C{¹H} NMR spectra were recorded on Varian INOVA 500 MHz, Bruker 500 MHz, or Bruker 600 MHz NMR instruments at the indicated frequencies. Chemical shifts are reported in ppm relative to residual protic solvent resonances. H₂S Detection was monitored by using an H₂S electrode (ISO-H₂S-2, World Precision Instruments, Inc. Sarasota, Florida, USA) or UV-Vis spectrometer (Cary 60, Agilent Technologies, Santa Clara, California, USA) by following methylene blue assay in PBS buffer. Stability test was performed on HPLC (1260 Infinity II, Agilent Technologies, Santa Clara, California, USA). OA-PeroxyTCM-1, OA-PeroxyTCM-2, OA-PeroxyTCM-3, and OA-TCM-1 were synthesized by following our previous study.⁶⁹

Synthesis

General procedure for the synthesis of O-alkylthiocarbamate compounds

4-(Hydroxymethyl)phenylboronic acid pinacol ester (1.0 equiv.) was combined with substituted phenyl isothiocyanate (1.0 equiv.) in anhydrous THF (15 mL) at 0 °C, followed by the addition of NaH (60% in paraffin liquid, 1.2 equiv.). The resultant mixture was stirred at 0 °C for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. until the completion of the reaction indicated by TLC. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (3 ×15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography. We note that thiocarbamates show two sets of NMR resonances at room temperature due to slow rotation around the thiocarbamate functional group.

OA-PeroxyTCM-4 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester and 4-bromophenyl isothiocyanate using the general synthetic procedure described above (125 mg, 37% yield). ¹H NMR (600 MHz, DMSO-d₆) δ (ppm): 11.35 (s, 1H), 7.70 (s, 3 H), 7.51 (m, 4H), 7.29 (br, 1H), 5.59 (br, 2H), 1.30 (s, 12H). ¹³C{¹H} NMR (150 MHz, DMSO-d₆) δ (ppm): 187.8, 187.4, 139.6, 139.2, 138.4, 137.3, 135.0, 132.1, 131.8, 128.7, 127.8, 125.1, 124.3, 117.5, 84.2, 72.6, 70.6, 40.5, 25.1. IR (cm⁻¹): 3208, 3097, 3031, 2975, 1615, 1590, 1532, 1487, 1390, 1360, 1325, 1273, 1168, 1141, 1091, 1007. HRMS m/z [M - H]⁻ calcd. for [C₂₀H₂₂BBrNO₃S]⁻ 446.0597; found 446.0599.

OA-PeroxyTCM-5 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester and 4-trifluoromethylphenyl isothiocyanate using the general synthetic procedure described above (100 mg, 46% yield). ¹H NMR (500 MHz, Acetone-d₆) δ (ppm): 10.45 (s, 1H), 7.95 (br, 2H), 7.80 (d, J = 10.0 Hz, 2H), 7.72 (d, J = 10.0 Hz, 2H), 7.51 (d, J = 10.0 Hz, 2H), 5.66 (s, 2H), 1.36 (s, 12H). ¹³C{¹H} NMR (125 MHz, Acetone-d₆) δ (ppm): 188.0, 138.8, 134.8, 127.4, 127.1, 125.9, 125.3, 123.5, 121.9, 83.7, 59.7, 24.3. ¹⁹F NMR (470 MHz, Acetone-d₆) δ (ppm): −62.6. IR (cm⁻¹): 3208, 3097, 3031, 2975, 1615, 1590, 1532, 1487, 1390, 1360, 1325, 1273, 1184, 1168, 1141, 1091, 1017, 816. HRMS m/z [M - H]⁻ calcd. for [C₂₁H₂₂BF₃NO₃S]⁻ 436.1366; found 436.1369.

OA-PeroxyTCM-6 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester and 4-nitrophenyl isothiocyanate using the general synthetic procedure described above (82 mg, 24% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.50 (s, 1H), 8.18 (d, J = 10.0 Hz, 2H), 7.85 (d, J = 10.0 Hz, 2H), 7.56 (br, 2H), 7.42 (d, J = 10.0 Hz, 2H), 5.62 (s, 2H), 1.35 (s, 12H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 187.7, 144.1, 142.6, 137.3, 135.2, 127.7, 125.0, 120.6, 118.7, 84.0, 74.0, 24.9. IR (cm⁻¹): 3265, 2973, 2926, 1609, 1598, 1553, 1508, 1493, 1396, 1359, 1332, 1319, 1268, 1169, 1144, 1068, 1015, 857, 842, 656. HRMS m/z [M - H]⁻ calcd. for [C₂₀H₂₂BN₂O₅S]⁻ 413.1342; found 413.1345.

Synthesis of S-alkylthiocarbamate compounds

The corresponding benzyl thiol species (1.0 equiv.) was combined with 4-fluorophenyl isocyanate (1.0 equiv.) in anhydrous THF (15 mL) at 0 °C, followed by the addition of triethylamine (1.2 equiv.). The resultant solution was stirred at 0 °C for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. until the completion of the reaction indicated by TLC. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (3 ×15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography.

SA-PeroxyTCM-1 was prepared from 4-(thiomethyl)phenylboronic acid pinacol ester (2) using the general synthetic procedure described above (87 mg, 62% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.76 (d, J = 10.0 Hz, 2H), 7.36 (m, 4H), 7.21 (s, 1H), 6.99 (t, J = 5.0 Hz, 2H), 4.21 (s, 2H), 1.34 (s, 12H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 165.5, 160.6, 158.6, 141.0, 135.2, 133.5, 128.3, 121.9, 115.9, 115.7, 83.9, 34.6, 29.7, 24.9. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −117.8. IR (cm⁻¹): 3293, 2982, 2920, 1691, 1611, 1530, 1508, 1398, 1382, 1303, 1270, 1136, 1084, 854, 833. HRMS m/z [M - H]⁻ calcd. for [C₂₀H₂₂BFNO₃S]⁻ 386.1397; found 386.1398.

SA-TCM-1 was prepared from benzyl thiol using the general synthetic procedure described above (120 mg, 49% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.39 (m, 4H), 7.34 (t, J = 10.0 Hz, 2H). 7.28 (m, 1H), 7.07 (br, 1H), 7.04 (t, J = 10.0 Hz, 2H), 4.25 (s, 2H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 165.6, 160.6, 158.6, 137.8, 133.5, 128.9, 128.7, 127.4, 121.9, 115.9, 34.5. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −117.6. IR (cm⁻¹): 3254, 3143, 3060, 2924, 1644, 1613, 1538, 1504, 1453, 1407, 1301, 1211, 1153, 1096, 698. HRMS m/z [M + H]⁺ calcd. for [C₁₄H₁₃FNOS]⁺ 262.0702; found 262.0700.

Synthesis of SA-PeroxyTCN-1

4-(Thiomethyl)-phenylboronic acid pinacol ester (160 mg, 0.64 mmol, 1.0 equiv.) was combined with 4-fluorophenyl chloroformate (113 mg, 0.64 mmol, 1.0 equiv.) in anhydrous DCM (15 mL) at 0 °C, followed by the addition of NEt₃ (65 mg, 0.64 mmol 1.0 equiv.). The resultant solution was stirred at 0 °C for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. for 16 h. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (3 ×15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography (90 mg, 36% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.82 (d, J = 5.0 Hz, 2H), 7.41 (d, J = 5.0 Hz, 2H), 7.13 (m, 2H), 7.08 (t, J = 5.0 Hz, 2H), 4.20 (s, 2H), 1.37 (s, 12H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 169.9, 161.4, 159.4, 147.0, 139.7, 135.2, 128.3, 122.8, 116.3, 116.1, 83.9, 35.8, 24.9. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −116.2. IR (cm⁻¹): 2979, 2922, 1724, 1610, 1498, 1403, 1355, 1322, 1175, 1143, 1105, 1010. HRMS m/z [M + H]⁺ calcd. for [C₂₀H₂₃BFO₄S]⁺ 389.1394; found 389.1387.

4-Fluorophenyl thiochloroformate (3)

4-Fluorophenyl mercaptan (384 mg, 3.0 mmol, 1.0 equiv.) was dissolved in DCM (20 mL), followed by the addition of triphosgene (356 mg, 1.2 mmol, 0.4 equiv.) at 0 °C. To this solution was then added pyridine (237 mg, 3.0 mmol, 1.0 equiv.) dropwise. The resultant solution was stirred at r.t. for 3 h. The reaction was quenched by adding HCl (10 mL, 1 M) and the aqueous solution was extracted with DCM (3 × 15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography (3, 404 mg, 71%). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.55 (t, J = 5.0 Hz, 2H), 7.18 (t, J = 5.0 Hz, 2H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 165.3, 163.2, 136.4, 122.6, 117.0. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −107.9. IR (cm⁻¹): 1759, 1589, 1489, 1400, 1231, 1158, 1093, 1013, 800. HRMS m/z M⁺ calcd. for [C₇H₄ClFOS]^+• 189.9655; found 189.9652.

Synthesis of SA-PeroxyDTCN-1

4-(Thiomethyl)phenylboronic acid pinacol ester (2) (154 mg, 0.60 mmol, 1.0 equiv.) was combined with 4-fluorophenyl thiochloroformate (3, 114 mg, 0.60 mmol, 1.0 equiv.) in anhydrous DCM (15 mL) at 0 °C, followed by the addition of NEt₃ (61 mg, 0.60 mmol 1.0 equiv.). The resultant solution was stirred at 0 °C for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. for 16 h. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (3 ×15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography (150 mg, 62% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.79 (d, J = 5.0 Hz, 2H), 7.53 (m, 2H), 7.32 (d, J = 5.0 Hz, 2H), 7.14 (t, J = 10.0 Hz, 2H), 4.22 (s, 2H), 1.37 (s, 12H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 188.9, 165.0, 163.0, 139.7, 137.7, 135.2, 128.3, 122.3, 116.8, 116.6, 83.9, 35.3, 24.9. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −109.5. IR (cm⁻¹): 2980, 2920, 1730, 1644, 1610, 1587, 1488, 1396, 1354, 1320, 1270, 1225, 1143, 1087, 962. HRMS m/z [M + H]⁺ calcd. for [C₂₀H₂₃BFO₃S₂]⁺ 405.1166; found 405.1165.

Synthesis of O-alkylthiocarbonate

The benzyl alcohol species (1.0 equiv.) was combined with 4-fluorophenyl chlorothionoformate (1.0 equiv.) in anhydrous DCM (15 mL) at 0 °C, followed by the addition of pyridine (1.0 equiv.). The resultant solution was stirred at 0 °C for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. until the completion of the reaction indicated by TLC. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (3 × 15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography.

OA-PeroxyTCN-1 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester using the general synthetic procedure described above (106 mg, 55% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.89 (d, J = 10.0 Hz, 2H), 7.48 (d, J = 5.0 Hz, 2H), 7.12 (d, J = 5.0 Hz, 4H), 5.60 (s, 2H), 1.39 (s, 12H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 195.0, 161.7, 159.7, 149.4, 149.3, 137.0, 135.1, 127.5, 123.5, 123.4, 116.4, 116.2, 84.0, 75.5, 24.9. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −115.6. IR (cm⁻¹): 2978, 2921, 1615, 1500, 1400, 1359, 1292, 1183, 1140, 1008. HRMS m/z [M + Na]⁺ calcd. for [C₂₀H₂₂BFNaO₄S]⁺ 411.1214; found 411.1216.

OA-TCN-1 was prepared from benzyl alcohol using the general synthetic procedure described above (60 mg, 85% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.47 (m, 4H), 7.15 (m, 4H), 5.60 (s, 2H). ¹³C{¹H} NMR (125 MHz, CDCl₃) δ (ppm): 195.0, 161.7, 159.7, 149.4, 149.3, 134.1, 128.9, 128.8, 128.6, 123.5, 116.4, 116.2, 75.8. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −115.6. IR (cm⁻¹): 3034, 2926, 1500, 1454, 1379, 1274, 1174, 1144, 1088, 1009, 836. HRMS m/z M^+• calcd. for C₁₄H₁₁FO₂S^+• 262.0464; found 262.0472.

Synthesis of dithiocarbamate compounds

The corresponding benzyl thiol species (1.0 equiv.) was combined with aryl isothiocyanate (1.0 equiv.) in anhydrous THF (15 mL) at 0 °C, followed by the addition of triethylamine (1.2 equiv.). The resultant solution was stirred at 0 °C for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. until the completion of the reaction indicated by TLC. The reaction was quenched by adding brine (30 mL), after which the resultant aqueous solution was extracted with ethyl acetate (3 × 15 mL). The organic layers were combined, dried over MgSO₄, and evaporated under vacuum. The crude product was purified by column chromatography.

PeroxyDTCM-1 was prepared from 4-fluorophenyl isothiocyanate using the general synthetic procedure described above (173 mg, 70% yield). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.87 (s, 1H), 7.77 (d, J = 10.0 Hz, 2H), 7.38 (m, 4H), 7.09 (t, J = 5.0 Hz, 2H), 4.58 (s, 2H), 1.35 (s, 12H). ¹³C{¹H} NMR (125 MHz, DMSO-d₆) δ (ppm): 196.2,140.9, 136.5, 135.1, 129.0, 127.8, 126.3, 115.8, 84.1, 39.0, 25.1. ¹⁹F NMR (470 MHz, CDCl₃) δ (ppm): −112.0. IR (cm⁻¹): 3307, 2975, 2922, 1605, 1506, 1451, 1352, 1319, 1268, 1204, 1142, 1086, 994. HRMS m/z [M - H]⁻ calcd. for [C₂₀H₂₂BFNO₂S₂]⁻ 402.1169; found 402.1166.

PeroxyDTCM-2 was prepared from 4-nitrophenyl isothiocyanate using the general synthetic procedure described above (88 mg, 41% yield). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 12.17 (s, 1H), 8.27 (d, J = 10.0 Hz, 2H), 8.08 (d, J = 10.0 Hz, 2H), 7.64 (d, J = 10.0 Hz, 2H), 7.43 (d, J = 10.0 Hz, 2H), 4.61 (s, 2H), 1.29 (s, 12H). ¹³C{¹H} NMR (125 MHz, DMSO-d₆) δ (ppm): 197.2, 145.8, 144.3, 140.5, 135.1, 129.1, 125.0, 123.4, 84.1, 39.1, 25.1. IR (cm⁻¹): 2982, 1610, 1594, 1514, 1355, 1328, 1312, 1270, 1193, 1087, 804 HRMS m/z [M - H]⁻ calcd. for [C₂₀H₂₂BN₂O₄S₂]⁻ 429.1114; found 429.1116.

H₂S Release Measurements

H₂S Release measurement from PeroxyTCMs and PeroxyTCNs

A donor stock solution (50 μL, 20 mM in DMSO) was added to 20 mL of PBS (pH 7.4, 10 mM) containing CA (25 μg/mL) in a scintillation vial. After stirring at room temperature for 5 min, the H₂O₂ stock solution (1.0 M in H₂O) was added to reach the desired final H₂O₂ concentration, and the H₂S release was monitored using an H₂S microsensor. The k₂ value of each donor was obtained by plotting k_obs vs H₂O₂ concentrations. Relative rates were obtained by dividing k₂ of each donor by that of PeroxyTCM-3.

H₂S Release measurement from PeroxyDTCMs

A donor stock solution (200 μL, 10 mM in DMSO) was added to 20 mL of PBS (pH 7.4, 10 mM)/DMSO (9:1) in a scintillation vial. After stirring at room temperature for 5 min, the H₂O₂ stock solution (20 μL, 1.0 M in H₂O) was added, and the H₂S release was monitored using methylene blue (MB) protocol. Briefly, 0.5 mL of reaction aliquots were taken to 1-cm UV cuvettes containing 0.5 mL of MB cocktail (0.1 mL zinc acetate (1% w/v), 0.2 mL FeCl₃ (30 mM in 1.2 M HCl), and 0.2 mL N,N-dimethyl-p-phenylene diamine (20 mM in 7.2 M HCl)) periodically. The absorbance at 670 nm was then measured after 30 min and was converted to H₂S concentration by using the H₂S calibration curve.

Stability Investigations

An OA-TCM-1, SA-TCM-1, or OA-TCN-1 stock solution (5 μL, 10 mM in DMSO) was added to 500 μL of PBS (pH 7.4, 10 mM)/CH₃CN (1:1) in a 1.5 mL GC vial, followed by the addition of the ROS stock solution (5 μL, 30 mM in H₂O). The reaction was analyzed by HPLC, using the following conditions: HPLC gradient: 50% – 0% solvent A (5% MeOH in H₂O) in solvent B (MeCN) over 4 min then 100% solvent B over 4 min and 0% – 50% solvent A in solvent B over 4 min. Flow rate: 1 mL/min, 2 μL injection. Column: Agilent Poroshell 120 EC-C18, 4.6 × 100 mm, 2.7 μm (particle size)

Computational Methods

Calculations were performed using the Gaussian 09 software package.⁸⁷ Conformational searches were conducted through the geometry optimizations of all reasonable input dihedral angles using the 6–311++G(d,p) basis set at the B3LYP level of theory applying the IEFPCM solvation model for water. Frequency calculations were performed on each located stationary point to ensure that it was a local minimum or saddlepoint. Relative energies were calculated using the zero-point-energy-corrected electronic energies obtained in the frequency calculations. A superfine integration grid in Gaussian was used for all computations. Graphical representations of optimized structures were prepared using VESTA.⁸⁸

Figure 3.

Representative H₂S release from OA-PeroxyTCM-1 to 6 in the presence of H₂O₂.