An Expanded Palette of Fluorescent COS/H2S-Releasing Donors for H2S Delivery, Detection, and In Vivo Application.

Hydrogen sulfide (H2S) is an important reactive sulfur species that is involved in many biological functions, and H2S imbalances have been indicated as a potential biomarker for various diseases. Different H2S donors have been developed to deliver H2S directly to biological systems, but few reports include donors with optical responses that allow for tracking of H2S release. Moreover, donor systems that use the same chemistry to deliver H2S across a palette of fluorescent responses remain lacking. Here we report five thiol-activated fluorescence turn-on COS/H2S donors that utilize blue, yellow, orange, red, and near infrared-emitting dyes functionalized with an H2S-releasing sulfenyl thiocarbonate scaffold. Upon treatment with thiols, each donor provides a fluorescence turn-on response (3-310-fold) and high H2S release efficiencies (>60 %). Using combined electrode and fluorescence experiments, we directly correlate the measured H2S release with the fluorescence response. All donors are biocompatible and release H2S in live cell environments. In addition, we demonstrate that the NIR donor allows for imaging H2S release in live rats via subcutaneous injection of the donor loaded into an alginate gel, which to the best of our knowledge is the first in vivo tracking of H2S release from a fluorogenic donor in non-transparent organisms.

Activity-Based Fluorescent Probes for Hydrogen Sulfide and Related Reactive Sulfur Species.

Hydrogen sulfide (H2S) is not only a well-established toxic gas but also an important small molecule bioregulator in all kingdoms of life. In contemporary biology, H2S is often classified as a "gasotransmitter," meaning that it is an endogenously produced membrane permeable gas that carries out essential cellular processes. Fluorescent probes for H2S and related reactive sulfur species (RSS) detection provide an important cornerstone for investigating the multifaceted roles of these important small molecules in complex biological systems. A now common approach to develop such tools is to develop "activity-based probes" that couple a specific H2S-mediated chemical reaction to a fluorescent output. This Review covers the different types of such probes and also highlights the chemical mechanisms by which each probe type is activated by specific RSS. Common examples include reduction of oxidized nitrogen motifs, disulfide exchange, electrophilic reactions, metal precipitation, and metal coordination. In addition, we also outline complementary activity-based probes for imaging reductant-labile and sulfane sulfur species, including persulfides and polysulfides. For probes highlighted in this Review, we focus on small molecule systems with demonstrated compatibility in cellular systems or related applications. Building from breadth of reported activity-based strategies and application, we also highlight key unmet challenges and future opportunities for advancing activity-based probes for H2S and related RSS.

Reversible Hydrosulfide (HS-) Binding Using Exclusively C-H Hydrogen-Bonding Interactions in Imidazolium Hosts.

H2S is a physiologically important signaling molecule with complex roles in biology and exists primarily as HS- at physiological pH. Despite this anionic character, few investigations have focused on the molecular recognition and reversible binding of this important biological anion. Using a series of imidazole and imidazolium host molecules, we investigate the role of preorganization and charge on HS- binding. Using a macrocyclic bis-imidazolium receptor, we demonstrate the unexpected 2:1 host-guest binding of HS-, which was characterized both in solution and by X-ray crystallography. To the best of our knowledge, this is the first example of this binding stoichiometry for HS- binding. Moreover, the short C-H···S distances of 2.53, 2.54, 2.76, and 2.79 Å are well within the sum of the van der Waals radii of the interacting atoms, which is consistent with strong C-H···S interactions.

Advances and Opportunities in H2S Measurement in Chemical Biology.

Hydrogen sulfide (H2S) is an important biological mediator across all kingdoms of life and plays intertwined roles in various disciplines, ranging from geochemical cycles to industrial processes. A common need across these broad disciplines is the ability to detect and measure H2S in complex sample environments. This Perspective focuses on key advances and opportunities for H2S detection and quantification that are relevant to chemical biology. Specifically, we focus on methods for H2S detection and quantification most commonly used in biological samples, including activity-based H2S probes, the methylene blue assay, the monobromobimane assay, and H2S-sensitive electrode measurements. Our goal is to help simplify what at first may seem to be an overwhelming array of detection and measurement choices, to articulate the strengths and limitations of individual techniques, and to highlight key unmet needs and opportunities in the field.

2-λ5-Phosphaquinolin-2-ones as Non-cytotoxic, Targetable, and pH-Stable Fluorophores.

Several phosphaquinolinone derivatives have been synthesized and characterized to explore their usefulness in the realm of cell imaging. Solution-state photophysical properties in both aqueous and organic solutions were collected for these derivatives. Additionally, CCK-8 cell viability assays and fluorescent imaging in HeLa cells incubated with the new heterocyclic derivatives show evidence of favorable cell permeability, cell viability, and moderate intracellular localization when appended with the well-known morpholine targeting motif.

Benchmarking the placement of hydrosulfide in the Hofmeister series using a bambus[6]uril-based ChemFET sensor.

Hydrosulfide (HS-) is the conjugate base of gasotransmitter hydrogen sulfide (H2S) and is a physiologically-relevant small molecule of great interest in the anion sensing community. However, selective sensing and molecular recognition of HS- in water remains difficult because, in addition to the diffuse charge and high solvation energy of anions, HS- is highly nucleophilic and readily oxidizes into other reactive sulfur species. Moreover, the direct placement of HS- in the Hofmeister series remains unclear. Supramolecular host-guest interactions provide a promising platform on which to recognize and bind hydrosulfide, and characterizing the placement of HS- in the Hofmeister series would facilitate the future design of selective receptors for this challenging anion. Few examples of supramolecular HS- binding have been reported, but the Sindelar group reported HS- binding in water using bambus[6]uril macrocycles in 2018. We used this HS- binding platform as a starting point to develop a chemically-sensitive field effect transistor (ChemFET) to facilitate assigning HS- to a specific place in the Hofmeister series. Specifically, we prepared dodeca-n-butyl bambus[6]uril and incorporated it into a ChemFET as the HS- receptor motif. The resultant device provided an amperometric response to HS-, and we used this device to measure the response of other anions, including SO42-, F-, Cl-, Br-, NO3-, ClO4-, and I-. Using this response data, we were able to experimentally determine that HS- lies between Cl- and Br- in the Hofmeister series, which matches recent theoretical computational work that predicted a similar placement. Taken together, these results highlight the potential of using molecular recognition coupled with ChemFET architectures to develop new approaches for direct and reversible HS- detection and measurement in water and further advance our understanding of different recognition approaches for this challenging anion.

Understanding Reactive Sulfur Species through P/S Synergy.

We investigated the differential oxidative and nucleophilic chemistry of reactive sulfur and oxygen anions (SSNO-, SNO-, NO2-, S42-, and HS-) using the simple reducing electrophile PPh2Cl. In the case of SSNO- reacting with PPh2Cl, a complex mixture of mono and diphosphorus products is formed exclusively in the P(V) oxidation state. We found that the phosphine stoichiometry dictates selectivity for oxidation to P=S/P=O products or transformation to P2 species. Interestingly, only chalcogen atoms are incorporated into the phosphorus products and, instead, nitrogen is released in the form of NO gas. Finally, we demonstrate that more reducing anions (S42- and HS-) also react with PPh2Cl with P=S bond formation as a key reaction driving force.

Direct hydrogen selenide (H2Se) release from activatable selenocarbamates.

Hydrogen selenide (H2Se) is a possible bioregulator, potential gasotransmitter, and important precursor in biological organoselenium compound synthesis. Early tools for H2Se research have benefitted from available mechanistic understanding of analogous small molecules developed for detecting or delivering H2S. A now common approach for H2S delivery is the use of small molecule thiocarbamates that can be engineered to release COS, which is quickly converted to H2S by carbonic anhydrase. To expand our understanding of the chemical underpinnings that enable H2Se delivery, we investigated whether selenocarbamates undergo similar chemistry to release carbonyl selenide (COSe). Using both light- and hydrolysis-activated systems, we demonstrate that unlike their lighter thiocarbamate congeners, selenocarbamates release H2Se directly with concomitant isocyanate formation rather than by the intermediate release of COSe. This reaction mechanism for direct H2Se release is further supported by computational investigations that identify a ΔΔG‡ ∼ 25 kcal mol-1 between the H2Se and COSe release pathways in the absence of protic solvent. This work highlights fundamentally new approaches for H2Se release from small molecules and advances the understanding of reactivity differences between reactive sulfur and selenium species.

Taming the Dichalcogenides: Isolation, Characterization, and Reactivity of Elusive Perselenide, Persulfide, Thioselenide, and Selenosulfide Anions.

Reactive sulfur species (RSS) and reactive selenium species (RSeS) play integral roles in hydrogen sulfide (H2S) and hydrogen selenide (H2Se) biological signaling pathways, and dichalcogenide anions are proposed transient intermediates that facilitate a variety of biochemical transformations. Herein we report the selective synthesis, isolation, spectroscopic and structural characterization, and fundamental reactivity of persulfide (RSS-), perselenide (RSeSe-), thioselenide (RSSe-), and selenosulfide (RSeS-) anions. The isolated chalcogenides do not rely on steric protection for stability and have steric profiles analogous to cysteine (Cys). Simple reduction of S8 or Se by potassium benzyl thiolate (KSBn) or selenolate (KSeBn) in the presence of 18-crown-6 afforded [K(18-crown-6)][BnSS] (1), [K(18-crown-6)][BnSeSe] (2), [K(18-crown-6][BnSSe] (3), and [K(18-crown-6][BnSeS] (4). The chemical structure of each dichalcogenide was confirmed by X-ray crystallography and solution-state 1H, 13C, and 77Se NMR spectroscopy. To advance our understanding of the reactivity of these species, we demonstrated that reduction of 1-4 by PPh3 readily generates E═PPh3 (E: S, Se), and reduction of 1, 3, and 4 by DTT readily produces HE-/H2E. Furthermore, 1-4 react with CN- to produce ECN-, which is consistent with the detoxifying effects of dichalcogenide intermediates in the Rhodanese enzyme. Taken together, this work provides new insights into the inherent structural and reactivity characteristics of dichalcogenides relevant to biology and advances our understanding of the fundamental properties of these reactive anions.

Solubilization of elemental sulfur by surfactants promotes reduction to H2S by thiols.

Elemental sulfur (S8) may contribute to sulfane sulfur (S0) storage in biological systems. We demonstrate that surfactants can solubilize S8 in water and promote S8 reduction to H2S by thiols. Moreover, anionic and cationic surfactants interact differently with intermediate S0 carriers, highlighting how specific hydrophobic microenvironments impact reactive sulfur species.

Thiol-Activated 1,2,4-Thiadiazolidin-3,5-diones Release Hydrogen Sulfide through a Carbonyl-Sulfide-Dependent Pathway.

Recent efforts have expanded the development of small molecule donors that release the important biological signaling molecule hydrogen sulfide (H2S). Previous work on 1,2,4-thiadiazolidin-3,5-diones (TDZNs) reported that these compounds release H2S directly, albeit inefficiently. However, TDZNs showed promising efficacy in H2S-mediated relaxation in ex vivo aortic ring relaxation models. Here, we show that TDZNs release carbonyl sulfide (COS) efficiently, which can be converted to H2S by the enzyme carbonic anhydrase (CA) rather than releasing H2S directly as previously reported.

Synthesis and reactivity of a tris(carbene) zinc chloride complex.

The [PhB(tBuIm)3]1- ligand has gained increased attention since it was first reported in 2006 due to its ability to stabilize highly reactive first row transition metal complexes. In this work, we investigate the coordination chemistry of this ligand with redox-inert zinc to understand how a zinc metal center behaves in such a strong coordinating environment. The PhB(tBuIm)3ZnCl (1) complex can be formed via deprotonation of [PhB(tBuIm)3][OTf]2 followed by the addition of ZnCl2. Salt metathesis reaction with nucleophilic n-BuLi yields the highly carbon-rich zinc coordination complex PhB(tBuIm)3ZnBu (2) with three carbene atom donors and one carbanion donor. In contrast, reaction of complex 1 with a less nucleophilic polysulfide reagent, [K.18-C-6]2[S4], leads to the formation of a tetrahedral zinc tetrasulfido complex via protonation of one carbene donor to form PhB(tBuIm)2(tBuImH)Zn(κ2-S4) (3).

Subcellular Delivery of Hydrogen Sulfide Using Small Molecule Donors Impacts Organelle Stress.

Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with important roles in regulating organelle function and stress. Because of its high reactivity, targeted delivery of H2S using small molecule H2S donors has garnered significant interest to minimize off-target effects. Although mitochondrially targeted H2S donors, such as AP39, have been reported previously and exhibit significantly higher potency than nontargeted donors, the expansion of targeted H2S delivery to other subcellular organelles remains largely absent. To fill this key unmet need, we report a library of organelle-targeted H2S donors that localize H2S delivery to specific subcellular organelles, including the Golgi apparatus, lysosome, endoplasmic reticulum, and mitochondria. We measured H2S production in vitro from each donor, confirmed the localization of H2S delivery using organelle-specific H2S responsive fluorescent probes, and demonstrated enhanced potency of these targeted H2S donors in providing protection against organelle-specific stress. We anticipate this class of targeted H2S donors will enable future studies of subcellular roles of H2S and the pathways by which H2S alleviates subcellular organelle stress.

Reactive sulfur and selenium species in the regulation of bone homeostasis.

Reactive oxygen species (ROS) are important modulators of physiological signaling and play important roles in bone tissue regulation. Both reactive sulfur species (RSS) and reactive selenium species (RSeS) are involved in ROS signaling, and recent work suggests RSS and RSeS involvement in the regulation of bone homeostasis. For example, RSS can promote osteogenic differentiation and decrease osteoclast activity and differentiation, and the antioxidant activity of RSeS play crucial roles in balancing bone remodeling. Here, we outline current research progress on the application of RSS and RSeS in bone disease and regeneration. Focusing on these investigations, we highlight different methods, tools, and sources of RSS and RSeS, and we also highlight future opportunities for delivery of RSS and RSeS in biological environments relating to bone.

Supramolecular Activation of S8 by Cucurbiturils in Water and Mechanism of Reduction to H2S by Thiols: Insights into Biological Sulfane Sulfur Trafficking.

Reactive sulfur species (RSS) play critical roles in diverse chemical environments. Molecules containing sulfane sulfur (S0) have emerged as key species involved in cellular redox buffering as well as RSS generation, translocation, and action. Using cucurbit[7]uril (CB[7]) as a model hydrophobic host, we demonstrate here that S8 can be encapsulated to form a 1:1 host guest complex, which was confirmed by solution state experiments, mass spectrometry, and X-ray crystallography. The solid state structure of CB[7]/S8 shows that the encapsulated S8 is available to nucleophiles through the carbonyl portals of the host. Treatment of CB[7]/S8 with thiols results in efficient reduction of S8 to H2S in water at physiological pH. We establish that encapsulated S8 is attacked by a thiol within the CB[7] host and that the resultant soluble hydropolysulfide is ejected into solution, where it reacts further with thiols to generate soluble sulfane sulfur carriers and ultimately H2S. The formation of these intermediate is supported by observed kinetic saturation behavior, competitive inhibition experiments, and alkylative trapping experiments. We also demonstrate that CB[7]/S8 can be used to increase sulfane sulfur levels in live cells using fluorescence microscopy. More broadly, this work suggests a general activation mechanism of S8 by hydrophobic motifs, which may be applicable to proteins, membranes, or other bimolecular compartments that could transiently bind and solubilize S8 to promote reaction with thiols to solubilize and shuttle S8 back into the redox labile sulfane sulfur pool. Such a mechanism would provide an attractive manifold in which to understand the RSS translocation and trafficking.

A Cell Trappable Methyl Rhodol-Based Fluorescent Probe for Hydrogen Sulfide Detection.

Hydrogen sulfide is a biologically important molecule and developing chemical tools that enable further investigations into the functions of H2 S is essential. Fluorescent turn-on H2 S probes have been developed for use in cellulo and in vivo, but the membrane permeability of these probes can lead to probe leakage and signal attenuation over time. Here we report a cell trappable fluorescent probe for H2 S, CT-MeRhoAz, which is based on a methylrhodolazide scaffold derivatized with an acetoxymethyl ester group. Prior to ester cleavage, the CT-MeRhoAz probe generates a 2500-fold turn-on response to H2 S, which is enhanced to a 3000-fold response for the carboxylic acid form of the probe. Additionally, the probe is highly selective for H2 S over other biologically relevant sulfur, oxygen, and nitrogen-based analytes. Live cell imaging experiments confirmed the biocompatibility of CT-MeRhoAz and also that it is cell trappable, unlike the parent MeRhoAz scaffold.

Thionitrite (SNO- ) and Perthionitrite (SSNO- ) are Simple Synthons for Nitrosylated Iron Sulfur Clusters.

S/N crosstalk species derived from the interconnected reactivity of H2 S and NO facilitate the transport of reactive sulfur and nitrogen species in signaling, transport, and regulatory processes. We report here that simple Fe2+ ions, such as those that are bioavailable in the labile iron pool (LIP), react with thionitrite (SNO- ) and perthionitrite (SSNO- ) to yield the dinitrosyl iron complex [Fe(NO)2 (S5 )]- . In the reaction of FeCl2 with SNO- we were able to isolate the unstable intermediate hydrosulfido mononitrosyl iron complex [FeCl2 (NO)(SH)]- , which was characterized by X-ray crystallography. We also show that [Fe(NO)2 (S5 )]- is a simple synthon for nitrosylated Fe-S clusters via its reduction with PPh3 to yield Roussin's Red Salt ([Fe2 S2 (NO)4 ]2- ), which highlights the role of S/N crosstalk species in the assembly of fundamental Fe-S motifs.

Efficient inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by sulfuration with solubilized elemental sulfur.

Hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO) have garnered increasing scientific interest in recent decades due to their classifications as members of the gasotransmitter family of signaling molecules. Due to the versatility of sulfur redox chemistry in biological systems, H2S specifically is being studied for its ability to modulate cellular redox environments, particularly through the downstream production of oxidized sulfur species. A major mechanism of this regulation is through a posttranslational modification known as persulfidation, where oxidized sulfur atoms are appended to free cysteine in proteins. Currently, it is difficult to discern the activity of H2S itself versus these oxidized sulfur species, particularly sulfane sulfur (S0). We have previously developed a method of solvating S8, a source of pure S0, to more accurately study persulfidation and sulfuration in general. Here, we apply this pure S0 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has previously been shown to be inhibited by S0-containing polysulfides via persulfidation. Using solvated S0, we demonstrate that native, reduced GAPDH can be completely inhibited by sulfuration with S0. Further, oxidized GAPDH activity cannot be rescued using S0, demonstrating that it is the oxidation of reduced GAPDH by S0 that curtails its activity. We also compare inhibition of GAPDH by pure S0 to different polysulfides and demonstrate the modulating effects that pendant alkyl groups have on GAPDH inhibition. These results highlight the promise of this novel, simplified system for the study of S0.

C-H⋯S hydrogen bonding interactions.

The short C-H⋯S contacts found in available structural data for both small molecules and larger biomolecular systems suggest that such contacts are an often overlooked yet important stabilizing interaction. Moreover, many of these short C-H⋯S contacts meet the definition of a hydrogen bonding interaction. Using available structural data from the Cambridge Structural Database (CSD), as well as selected examples from the literature in which important C-H⋯S contacts may have been overlooked, we highlight the generality of C-H⋯S hydrogen bonding as an important stabilizing interaction. To uncover and establish the generality of these interactions, we compare C-H⋯S contacts with other traditional hydrogen bond donors and acceptors as well as investigate how coordination number and metal bonding affect the preferred geometry of interactions in the solid state. This work establishes that the C-H⋯S bond meets the definition of a hydrogen bond and serves as a guide to identify C-H⋯S hydrogen bonds in diverse systems.

Esterase-Activated Perthiocarbonate Persulfide Donors Provide Insights into Persulfide Persistence and Stability.

Persulfides (RSSH) are important reactive sulfur species (RSS) that are intertwined with the biological functions of hydrogen sulfide (H2S). The direct study of persulfides is difficult, however, due to their both nucleophilic and electrophilic character, which leads to the generation of an equilibrium of different RSS. To investigate the effects of persulfides directly, especially in biological systems, persulfide donors are needed to generate persulfides in situ. Here, we report the synthesis of esterase-activated perthiocarbonate persulfide donors and investigate the effects of structural modifications on persulfide release. Although steric bulk of the ester did not significantly alter persulfide release kinetics, increased steric bulk of the thiol increased the persulfide release rate. In addition, we found that the steric bulk and identity of the thiol significantly impact persulfide persistence. Further mechanistic investigations into different competing reaction pathways from perthiocarbonates revealed that multiple RSS can be delivered (i.e., H2S, COS, or RSSH) depending on the persulfide donor structure and activator identity.