Hydrogen Sulfide Deactivates Common Nitrobenzofurazan-Based Fluorescent Thiol Labeling Reagents

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H₂S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H₂S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H₂S. Although H₂S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H₂S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H₂S addition. Furthermore, the addition of H₂S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H₂S. Taken together, these studies highlight the differential reactivity of thiols and H₂S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H₂S due to the potential for both false-positive and eroded responses

Understanding the Effects of Preorganization, Rigidity, and Steric Interactions in Synthetic Barbiturate Receptors

Synthetic barbiturate receptors have been utilized for many applications due to their high binding affinities for complementary guests. Although interest in this class of receptors spans from supramolecular to materials chemistry, the effects of receptor steric bulk and preorganization on guest binding affinity has not been studied systematically. To investigate the roles that steric bulk and preorganization play in guest binding, we prepared a series of 12 deconstructed Hamilton receptors with varying degrees of steric bulk and preorganization. Both diethylbarbital and 3-methyl-7-propyl­xanthine were investigated as guests for the synthetic receptors. The stoichiometry of guest binding was investigated using Job plots for each host–guest pair, and ¹H NMR titrations were performed to measure the guest binding affinities. To complement the solution-state studies, DFT calculations at the B3LYP/6-31+G­(d,p) level of theory employing the IEF-PCM CHCl₃ solvation model were also performed. Calculated guest binding energies correlated well with the experimental findings and provided additional insight into the factors influencing guest binding. Taken together, the results presented highlight the interplay between preorganization and steric interactions in establishing favorable interactions for self-assembled hydrogen-bonded systems

Chemiluminescent Detection of Enzymatically Produced Hydrogen Sulfide: Substrate Hydrogen Bonding Influences Selectivity for H₂S over Biological Thiols

Hydrogen sulfide (H₂S) is now recognized as an important biological regulator and signaling agent that is active in many physiological processes and diseases. Understanding the important roles of this emerging signaling molecule has remained challenging, in part due to the limited methods available for detecting endogenous H₂S. Here we report two reaction-based ChemiLuminescent Sulfide Sensors, CLSS-1 and CLSS-2, with strong luminescence responses toward H₂S (128- and 48-fold, respectively) and H₂S detection limits (0.7 ± 0.3, 4.6 ± 2.0 μM, respectively) compatible with biological H₂S levels. CLSS-2 is highly selective for H₂S over other reactive sulfur, nitrogen, and oxygen species (RSONS) including GSH, Cys, Hcy, S₂O₃^2–, NO₂^–, HNO, ONOO^–, and NO. Despite its similar chemical structure, CLSS-1 displays lower selectivity toward amino acid-derived thiols than CLSS-2. The origin of this differential selectivity was investigated using both computational DFT studies and NMR experiments. Our results suggest a model in which amino acid binding to the hydrazide moiety of the luminol-derived probes provides differential access to the reactive azide in CLSS-1 and CLSS-2, thus eroding the selectivity of CLSS-1 for H₂S over Cys and GSH. On the basis of its high selectivity for H₂S, we used CLSS-2 to detect enzymatically produced H₂S from isolated cystathionine γ-lyase (CSE) enzymes (<i>p</i> < 0.001) and also from C6 cells expressing CSE (<i>p</i> < 0.001). CLSS-2 can readily differentiate between H₂S production in active CSE and CSE inhibited with β-cyanoalanine (BCA) in both isolated CSE enzymes (<i>p</i> < 0.005) and in C6 cells (<i>p</i> < 0.005). In addition to providing a highly sensitive and selective reaction-based tool for chemiluminescent H₂S detection and quantification, the insights into substrate–probe interactions controlling the selectivity for H₂S over biologically relevant thiols may guide the design of other selective H₂S detection scaffolds

Mechanistic Insights into the H₂S‑Mediated Reduction of Aryl Azides Commonly Used in H₂S Detection

Hydrogen sulfide (H₂S) is an important biological mediator and has been at the center of a rapidly expanding field focused on understanding the biogenesis and action of H₂S as well as other sulfur-related species. Concomitant with this expansion has been the development of new chemical tools for H₂S research. The use of H₂S-selective fluorescent probes that function by H₂S-mediated reduction of fluorogenic aryl azides has emerged as one of the most common methods for H₂S detection. Despite this prevalence, the mechanism of this important reaction remains under-scrutinized. Here we present a combined experimental and computational investigation of this mechanism. We establish that HS^–, rather than diprotic H₂S, is the active species required for aryl azide reduction. The hydrosulfide anion functions as a one-electron reductant, resulting in the formation of polysulfide anions, such as HS₂^–, which were confirmed and trapped as organic polysulfides by benzyl chloride. The overall reaction is first-order in both azide and HS^– under the investigated experimental conditions with Δ<i>S</i>^⧧ = −14(2) eu and Δ<i>H</i>^⧧ = 13.8(5) kcal/mol in buffered aqueous solution. By using NBu₄SH as the sulfide source, we were able to observe a reaction intermediate (λ_max = 473 nm), which we attribute to formation of an anionic azidothiol intermediate. Our mechanistic investigations support that this intermediate is attacked by HS^– in the rate-limiting step of the reduction reaction. Complementing our experimental mechanistic investigations, we also performed DFT calculations at the B3LYP/6-31G­(d,p), B3LYP/6-311++G­(d,p), M06/TZVP, and M06/def2-TZVPD levels of theory applying the IEF-PCM water and MeCN solvation models, all of which support the experimentally determined reaction mechanism and provide cohesive mechanistic insights into H₂S-mediated aryl azide reduction

Organelle-Targeted H₂S Probes Enable Visualization of the Subcellular Distribution of H₂S Donors

Hydrogen sulfide (H₂S) is an essential biological signaling molecule in diverse biological regulatory pathways. To provide new chemical tools for H₂S imaging, we report here a fluorescent H₂S detection platform (<b>HSN2-BG</b>) that is compatible with subcellular localization SNAP-tag fusion protein methodologies and use appropriate fusion protein constructs to demonstrate mitochondrial and lysosomal localization. We also demonstrate the efficacy of this detection platform to image endogenous H₂S in Chinese hamster ovary (CHO) cells and use the developed constructs to report on the subcellular H₂S distributions provided by common H₂S donor molecules AP39, ADT–OH, GYY4137, and diallyltrisulfide (DATS). The developed constructs provide a platform poised to provide new insights into the subcellular distribution of common H₂S donors and a useful tool for investigating H₂S biochemistry

Kinetic Insights into Hydrogen Sulfide Delivery from Caged-Carbonyl Sulfide Isomeric Donor Platforms

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 donor systems

The Intersection of NO and H₂S: Persulfides Generate NO from Nitrite through Polysulfide Formation

Hydrogen sulfide (H₂S) and nitric oxide (NO) are important biosignaling molecules, and their biochemistries are increasingly recognized to be intertwined. Persulfides are an oxidized product of biological H₂S and have emerged as important species involved in the biological action of reactive sulfur species. Using isolated persulfides, we employed a combination of experimental and computational methods to investigate the contribution of persulfides to H₂S/NO crosstalk. Our studies demonstrate that isolated persulfides react with nitrite to produce NO via polysulfide and perthionitrite intermediates. These results highlight the importance of persulfides, polysulfides, and perthionitrite as intertwined reactive nitrogen and sulfur species

Design, Synthesis, and Characterization of Hybrid Metal–Ligand Hydrogen-Bonded (MLHB) Supramolecular Architectures

Despite the prevalence of supramolecular architectures derived from metal–ligand or hydrogen-bonding interactions, few studies have focused on the simultaneous use of these two strategies to form discrete assemblies. Here we report the use of a supramolecular tecton containing both metal-binding and self-complementary hydrogen-bonding interactions that upon treatment with metal precursors assembles into discrete hybrid metal–ligand hydrogen-bonded assemblies with closed topology. ¹H NMR DOSY experiments established the stability of the structures in solution, and the measured hydrodynamic radii match those determined crystallographically, suggesting that the closed topology is maintained both in solution and in the solid state. Taken together, these results demonstrate the validity of using both hydrogen-bonding and metal–ligand interactions to form stable supramolecular architectures

Understanding Hydrogen Sulfide Storage: Probing Conditions for Sulfide Release from Hydrodisulfides

Hydrogen sulfide (H₂S) is an important biological signaling agent that exerts action on numerous (patho)­physiological processes. Once generated, H₂S can be oxidized to generate reductant-labile sulfane sulfur pools, which include hydrodisulfides/persulfides. Despite the importance of hydrodisulfides in H₂S storage and signaling, little is known about the physical properties or chemical reactivity of these compounds. We report here the synthesis, isolation, and characterization (NMR, IR, Raman, HRMS, X-ray) of a small-molecule hydrodisulfide and highlight its reactivity with reductants, nucleophiles, electrophiles, acids, and bases. Our experimental results establish that hydrodisulfides release H₂S upon reduction and that deprotonation results in disproportionation to the parent thiol and S⁰, thus providing a mechanism for transsulfuration in the sulfane sulfur pool

Self-Immolative Thiocarbamates Provide Access to Triggered H₂S Donors and Analyte Replacement Fluorescent Probes

Hydrogen sulfide (H₂S) is an important biological signaling molecule, and chemical tools for H₂S delivery and detection have emerged as important investigative methods. Key challenges in these fields include developing donors that are triggered to release H₂S in response to stimuli and developing probes that do not irreversibly consume H₂S. Here we report a new strategy for H₂S donation based on self-immolation of benzyl thiocarbamates to release carbonyl sulfide, which is rapidly converted to H₂S by carbonic anhydrase. We leverage this chemistry to develop easily modifiable donors that can be triggered to release H₂S. We also demonstrate that this approach can be coupled with common H₂S-sensing motifs to generate scaffolds which, upon reaction with H₂S, generate a fluorescence response and also release caged H₂S, thus addressing challenges of analyte homeostasis in reaction-based probes