Nanohoop Rotaxane Design to Enhance the Selectivity of Reaction-Based Probes: A Proof-of-Principle Study.

Mechanical interlocking of a nanohoop fluorophore and a reactive thread couples the benefits of a reaction-based probe with a sterically congested active site for enhanced selectivity. Advantageously, the thread design uses dual function stoppers that act as both a quencher and a trigger for sensing. In progress toward expanding this approach to biologically relevant analytes, this system is used to demonstrate steric differentiation and provide a selective turn-on fluorescent response with size selectivity for HS- rather than larger thiolates.

N-Methylation of Self-Immolative Thiocarbamates Provides Insights into the Mechanism of Carbonyl Sulfide Release.

Hydrogen sulfide (H2S) is an important biomolecule, and self-immolative thiocarbamates have shown great promise as triggerable H2S donors with suitable analogous control compounds; however, thiocarbamates with electron-deficient payloads are less efficient H2S donors. We report here the synthesis and study of a series of N-methylated esterase-triggered thiocarbamates that block the postulated unproductive deprotonation-based pathway for these compounds. The relative reaction profiles for H2S release across a series of electron-rich and electron-poor N-Me aniline payloads are examined experimentally and computationally. We show that thiocarbamate N-methylation does block some side reactivity and increases the H2S release profiles for electron-poor donors. Additionally, we show that isothiocyanate release is not a competitive pathway, and rather that the reduced efficiency of electron-poor donors is likely due to other side reactions.

Direct Comparison of Triggering Motifs on Chemiluminescent Probes for Hydrogen Sulfide Detection in Water.

Hydrogen sulfide (H2S) is an important biomolecule and significant efforts have focused on developing chemical tools to aid different biological investigations. Of such tools, there are relatively few chemiluminescent or bioluminescent methods for H2S detection. Here we report two dioxetane-based chemiluminescent probes for H2S detection. With these probes, we directly compare the probe response to H2S-mediated azide reduction and nucleophilic displacement of 2,4-dinitrophenyl motifs and demonstrate that the SNAr cleavage of the DNP group results in a larger response and greater stability in water.

Activatable Small-Molecule Hydrogen Sulfide Donors.

Significance: Hydrogen sulfide (H2S) is an important biological signaling molecule involved in many physiological processes. These diverse roles have led researchers to develop contemporary methods to deliver H2S under physiologically relevant conditions and in response to various stimuli. Recent Advances: Different small-molecule donors have been developed that release H2S under various conditions. Key examples include donors activated in response to hydrolysis, to endogenous species, such as thiols, reactive oxygen species, and enzymes, and to external stimuli, such as photoactivation and bio-orthogonal chemistry. In addition, an alternative approach to release H2S has utilized the catalyzed hydrolysis of carbonyl sulfide (COS) by carbonic anhydrase to generate libraries of activatable COS-based H2S donors. Critical Issues: Small-molecule H2S donors provide important research and pharmacological tools to perturb H2S levels. Key needs, both in the development and in the use of such donors, include access to new donors that respond to specific stimuli as well as donors with well-defined control compounds that allow for clear delineation of the impact of H2S delivery from other donor byproducts. Future Directions: The abundance of reported small-molecule H2S donors provides biologists and physiologists with a chemical toolbox to ask key biological questions and to develop H2S-related therapeutic interventions. Further investigation into different releasing efficiencies in biological contexts and a clear understanding of biological responses to donors that release H2S gradually (e.g., hours to days) versus donors that generate H2S quickly (e.g., seconds to minutes) is needed.

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

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.

Esterase-Triggered Self-Immolative Thiocarbamates Provide Insights into COS Cytotoxicity.

Hydrogen sulfide (H2S) is an important gasotransmitter and biomolecule, and many synthetic small-molecule H2S donors have been developed for H2S-related research. One important class of triggerable H2S donors is self-immolative thiocarbamates, which function by releasing carbonyl sulfide (COS), which is rapidly converted to H2S by the ubiquitous enzyme carbonic anhydrase (CA). Prior studies of esterase-triggered thiocarbamate donors reported significant inhibition of mitochondrial bioenergetics and toxicity when compared to direct sulfide donors, suggesting that COS may function differently than H2S. Here, we report a suite of modular esterase-triggered self-immolative COS donors and include the synthesis, H2S release profiles, and cytotoxicity of the developed donors. We demonstrate that the rate of ester hydrolysis correlates directly with the observed cytotoxicity in cell culture, which further supports the hypothesis that COS functions as more than a simple H2S shuttle in certain biological systems.