Development and Application of Carbonyl Sulfide-Based Donors for H2S Delivery

Abstract

In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H₂S) has been recently recognized as an important biological signaling molecule with implications in a wide variety of processes, including vasodilation, cytoprotection, and neuromodulation. In parallel to the growing number of reports highlighting the biological impact of H₂S, interest in developing H₂S donors as both research tools and potential therapeutics has led to the growth of different H₂S-releasing strategies. Many H₂S investigations in model systems use direct inhalation of H₂S gas or aqueous solutions of NaSH or Na₂S; however, such systems do not mimic endogenous H₂S production. This stark contrast drives the need to develop better sources of caged H₂S. To address these limitations, different small organosulfur donor compounds have been prepared that release H₂S in the presence of specific activators or triggers. Such compounds, however, often lack suitable control compounds, which limits the use of these compounds in probing the effects of H₂S directly. To address these needs, our group has pioneered the development of carbonyl sulfide (COS) releasing compounds as a new class of H₂S donor motifs. Inspired by a commonly used carbamate prodrug scaffold, our approach utilizes self-immolative thiocarbamates to access controlled release of COS, which is rapidly converted to H₂S by the ubiquitous enzyme carbonic anhydrase (CA). In addition, this design enables access to key control compounds that release CO₂/H₂O rather than COS/H₂S, which enables delineation of the effects of COS/H₂S from the organic donor byproducts. In this Account, we highlight a library of first-generation COS/H₂S donors based on self-immolative thiocarbamates developed in our lab and also highlight challenges related to H₂S donor development. We showcase the release of COS in the presence of specific triggers and activators, including biological thiols and bio-orthogonal reactants for targeted applications. We also demonstrate the design and development of a series of H₂O₂/reactive oxygen species (ROS)-triggered donors and show that such compounds can be activated by endogenous levels of ROS production. Utilizing approaches in bio-orthogonal activation, we establish that donors functionalized with an o-nitrobenzyl photocage can enable access to light-activated donors. Similar to endogenous production by cysteine catabolism, we also prepared a cysteine-selective COS donor activated by a Strongin ligation mechanism. In efforts to help delineate potential differences in the chemical biology of COS and H₂S, we also report a simple esterase-activated donor, which demonstrated fast COS-releasing kinetics and inhibition of mitochondrial respiration in BEAS-2B cells. Additional investigations revealed that COS release rates and cytotoxicity correlated directly within this series of compounds with different ester motifs. In more recent and applied applications of this H₂S donation strategy, we also highlight the development of donors that generate either a colorimetric or fluorescent optical response upon COS release. Overall, the work described in this Account outlines the development and initial application of a new class of H₂S donors, which we anticipate will help to advance our understanding of the rapidly emerging chemical biology of H₂S and COS.

Figure 1.

Representative physiological processes involving H₂S and associated mechanisms of action.

Figure 2.

(a) Design and mechanism of self-immolative carbamates and (b) self-immolative thiocarbamates. (c) Conversion of COS to H₂S by CA in the presence of acetazolamide at varying concentrations measured by a sulfide-selective electrode.

Figure 3.

(a) Mechanism of self-immolation and subsequent conversion of COS to H₂S by CA. (b) Development of analyte-replacement fluorescent probes. (c) Current examples self-immolation-based COS/H₂S donors reported to date by our lab.

Figure 4.

(a) Representative reaction scheme and mechanism for H₂O₂-triggered COS/H₂S release. (b) Cytotoxicity of H₂O₂ in RAW 264.7 cells, Cytotoxicity of Bpin-triggered thiocarbamate donor, Bpin-triggered carbamate control, and triggerless thiocarbamate control at various concentrations in RAW 264.7 cells when co-incubated with 100 μM H₂O₂. (c) Imaging ROS-scavenging upon stimulation with PMA in RAW 264.7 cells.

Figure 5.

Mechanism of self-immolation from photocleavable thiocarbamate COS/H₂S donors.

Figure 6.

Mechanism of COS-release from OA-CysTCM-1 in the presence of cysteine with subsequent hydrolysis of COS to H₂S by carbonic anhydrase.

Figure 7.

Altered cytotoxicity via steric modulation for esterase-triggered COS/H₂S donors. (a) Mechanism of self-immolation of esterase-triggered COS donors. (b) Library of different ester size thiocarbamates prepared. (c) Inhibition of mitochondrial bioenergetics with the tert-Butyl triggered COS donor in BEAS2B cells. (d) The relationship between COS release rate and observed cytotoxicity at 100 μM in HeLa cells for a library of different esterase-triggered COS donors of varying ester size.

Figure 8.

Mechanism of ‘click-and-release’ biorthogonal COS donor, and measured H₂S release in the presence of mammalian blood and plasma, without exogenous CA.

Figure 9.

(a) Mechanism of COS/H₂S release from γ-KetoTCM1 (b) Conditions for measuring H₂S release from γ-KetoTCM1 (c) Measurement of PNA formation over time by UV/Vis spectroscopy (d) Correlation between measured [H₂S] and PNA formation by the methylene blue assay and UV/Vis spectroscopy.

Figure 10.

(a) Mechanism of thiol-mediated, COS/H₂S release from sulfenyl thiocarbonates. (b) Structure of FLD-1 and release of H₂S from FLD-1 (10 μM) in the presence of cysteine (100 μM, 10 equiv.) in the presence of carbonic anhydrase in 10 mM PBS (pH 7.4) monitored by fluorescence spectropscopy (λ_ex = 490 nm, λ_em = 500–650 nm). (c) Imaging of cellular H₂S release from FLD-1 (50 μM) in HeLa cells.