Hydrogen Sulfide Donors Activated by Reactive Oxygen Species

Hydrogen sulfide (H₂S) exhibits promising protective effects in many (patho)physiological processes, as evidenced by recent reports using synthetic H₂S donors in different biological models. Herein, we report the design and evaluation of compounds denoted PeroxyTCM, which are the first class of reactive oxygen species (ROS)‐triggered H₂S donors. These donors are engineered to release carbonyl sulfide (COS) upon activation, which is quickly hydrolyzed to H₂S by the ubiquitous enzyme carbonic anhydrase (CA). The donors are stable in aqueous solution and do not release H₂S until triggered by ROS, such as hydrogen peroxide (H₂O₂), superoxide (O₂^?), and peroxynitrite (ONOO^?). We demonstrate ROS‐triggered H₂S donation in live cells and also demonstrate that PeroxyTCM‐1 provides protection against H₂O₂‐induced oxidative damage, suggesting potential future applications of PeroxyTCM and similar scaffolds in H₂S‐related therapies.     

A Novel Fluorescent Probe for Hydrogen Peroxide and Its Application in Bio-Imaging

Hydrogen peroxide (H₂O₂) plays an important role in the human body and monitoring its level is meaningful due to the relationship between its level and diseases. A fluorescent sensor (CMB) based on coumarin was designed and its ability for detecting hydrogen peroxide by fluorescence signals was also studied. The CMB showed an approximate 25-fold fluorescence enhancement after adding H₂O₂ due to the interaction between the CMB and H₂O₂ and had the potential for detecting physiological H₂O₂. It also showed good biocompatibility and permeability, allowing it to penetrate cell membranes and zebrafish tissues, thus it can perform fluorescence imaging of H₂O₂ in living cells and zebrafish. This probe is a promising tool for monitoring the level of H₂O₂ in related physiological and pathological research.     

A Persulfide Donor Responsive to Reactive Oxygen Species: Insights into Reactivity and Therapeutic Potential

Persulfides (RSSH) have been hypothesized as critical components in sulfur‐mediated redox cycles and as potential signaling compounds, similar to hydrogen sulfide (H₂S). Hindering the study of persulfides is a lack of persulfide‐donor compounds with selective triggers that release discrete persulfide species. Reported here is the synthesis and characterization of a ROS‐responsive (ROS=reactive oxygen species), self‐immolative persulfide donor. The donor, termed BDP‐NAC, showed selectivity towards H₂O₂ over other potential oxidative or nucleophilic triggers, resulting in the sustained release of the persulfide of N‐acetyl cysteine (NAC) over the course of 2 h, as measured by LCMS. Exposure of H9C2 cardiomyocytes to H₂O₂ revealed that BDP‐NAC mitigated the effects of a highly oxidative environment in a dose‐dependent manner over relevant controls and to a greater degree than common H₂S donors sodium sulfide (Na₂S) and GYY4137. BDP‐NAC also rescued cells more effectively than a non‐persulfide‐releasing control compound in concert with common H₂S donors and thiols.     

Antioxidant Activity and Anti-Apoptotic Effect of the Small Molecule Procyanidin B1 in Early Mouse Embryonic Development Produced by Somatic Cell Nuclear Transfer

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H₂O₂) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H₂O) and oxygen (O₂) in order to reduce the harm caused by H₂O₂. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H₂O₂ accumulates excessively, it produces radical-(HO-) through Fe^2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H₂O₂ by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.     

Tandem Payne/Dakin Reaction: A New Strategy for Hydrogen Peroxide Detection and Molecular Imaging

Hydrogen peroxide (H₂O₂) has been recognized as one of the most significant ROS (reactive oxygen species) in human health and disease. Because of the intrinsic attributes of H₂O₂—such as its low reactivity under physiological pH—it is exceedingly challenging to develop small‐molecule fluorescent probes with high selectivity and sensitivity for visualization of H₂O₂ in an intricate biological milieu. To address this gap, a rationally designed tandem Payne/Dakin reaction is reported that is specific to molecular recognition of H₂O₂. New H₂O₂ probes based on this unique chemical strategy can be easily synthesized by a general coupling reaction, and the practical applicability of those probes has been confirmed by the visualization of endogenously produced H₂O₂ in living cells. In particular, starvation‐induced H₂O₂ production in mouse macrophages has been detected by the novel probe in both confocal imaging and flow cytometry. This tandem Payne/Dakin reaction provides a basis for developing more sophisticated molecular tools to interrogate H₂O₂ functions in biological phenomena.     

Orthogonal Activation of RNA‐Cleaving DNAzymes in Live Cells by Reactive Oxygen Species

RNA‐cleaving DNAzymes are useful tools for intracellular metal‐ion sensing and gene regulation. Incorporating stimuli‐responsive modifications into these DNAzymes enables their activities to be spatiotemporally and chemically controlled for more precise applications. Despite the successful development of many caged DNAzymes for light‐induced activation, DNAzymes that can be intracellularly activated by chemical inputs of biological importance, such as reactive oxygen species (ROS), are still scarce. ROS like hydrogen peroxide (H₂O₂) and hypochlorite (HClO) are critical mediators of oxidative stress‐related cell signaling and dysregulation including activation of immune system as well as progression of diseases and aging. Herein, we report ROS‐activable DNAzymes by introducing phenylboronate and phosphorothioate modifications to the Zn²⁺‐dependent 8–17 DNAzyme. These ROS‐activable DNAzymes were orthogonally activated by H₂O₂ and HClO inside live human and mouse cells.     

Electroactivity of Superoxide Anion in Aqueous Phosphate Buffers Analyzed with Platinized Microelectrodes

The reactivity of platinized ultramicroelectrodes (Pt-black UMEs) towards superoxide anion O₂^.−, an unstable Reactive Oxygen Species (ROS), and its relatives, H₂O₂ and O₂, was studied. Voltammetric studies in PBS demonstrate that Pt-black UMEs provide: i) a well-resolved reversible redox signature for O₂^.− detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H₂O₂ at pH 7.4. The oxygen reduction reaction (ORR) at Pt-black surfaces solely yields H₂O₂ (2 electrons/2 H⁺) at physiological pH. Consequently, Pt-black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico-chemical investigations.     

O→S Relay Deprotection: A General Approach to Controllable Donors of Reactive Sulfur Species

Reactive sulfur species (RSS) are biologically important molecules. Among them, H₂S, hydrogen polysulfides (H₂S_n, n>1), persulfides (RSSH), and HSNO are believed to play regulatory roles in sulfur‐related redox biology. However, these molecules are unstable and difficult to handle. Having access to their reliable and controllable precursors (or donors) is the prerequisite for the study of these sulfur species. Reported in this work is the preparation and evaluation of a series of O‐silyl‐mercaptan‐based sulfur‐containing molecules which undergo pH‐ or F^?‐mediated desilylation to release the corresponding H₂S, H₂S_n, RSSH, and HSNO in a controlled fashion. This O→S relay deprotection serves as a general strategy for the design of pH‐ or F^?‐triggered RSS donors. Moreover, we have demonstrated that the O‐silyl groups in the donors could be changed into other protecting groups like esters. This work should allow the development of RSS donors with other activation mechanisms (such as esterase‐activated donors).     

A 3,7‐Dihydroxyphenoxazine‐based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide

A novel N‐borylbenzyloxycarbonyl‐3,7‐dihydroxyphenoxazine fluorescent probe (NBCD) for detecting H₂O₂ in living cells is described. The probe could achieve high selectivity for detecting H₂O₂ over other biological reactive oxygen species (ROS). In addition, upon addition of H₂O₂, NBCD exhibited color change from colorless to pink, which makes it a “naked‐eye” probe for H₂O₂ detection. NBCD could not only be used to detect enzymatically generated H₂O₂ but also to detect H₂O₂ in living systems by using fluorescence spectroscopy, with a detection limit of 2 μm . Importantly, NBCD enabled the visualization of epidermal growth factor (EGF)‐stimulated H₂O₂ generation inside the cells.     

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

The C‐terminal octapeptide of cholecystokinin (CCK8) includes some easily oxidizable amino acids. The oxidation of CCK8 by reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^?) was investigated using reversed‐phase high performance liquid chromatography (RP‐HPLC) and subsequent electrospray ionization mass spectrometry. The mechanism of oxidation of CCK8 in the H₂O₂ system differed from that of CCK8 in the Fenton system, in which OH^? are produced. In the H₂O₂ system, ²⁸Met and ³¹Met were oxidized to methionine sulfoxide, and no further oxidation or degradation/hydrolysis occurred. On the other hand, in the Fenton system, ²⁸Met and ³¹Met residues were oxidized to methionine sulfone via the formation of methionine sulfoxide. In addition, the oxidized product was observed at the Trp residue but not at the Tyr residue, and small peptide fragments from CCK8 were observed in the Fenton system. From these results, it was concluded that ²⁸Met and ³¹Met residues of CCK8 are susceptible to oxidation by ROS. Copyright © 2009 John Wiley & Sons, Ltd.     

Design of a novel mitochondria targetable turn-on fluorescence probe for hydrogen peroxide and its two-photon bioimaging applications

Considering that hydrogen peroxide (H₂O₂) plays significant roles in oxidative stress, the cellular signal transduction and essential biological process regulation, the detection and imaging of H₂O₂ in living systems undertakes critical responsibility. Herein, we have developed a novel two-photon fluorescence turn on probe, named as Pyp-B for mitochondria H₂O₂ detection in living systems. Selectivity studies show that probe Pyp-B exhibit highly sensitive response toward H₂O₂ than other reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as biologically relevant species. The fluorescence colocalization studies demonstrate that the probe can localize in the mitochondria solely. Furthermore, as a bio-compatibility molecule, the highly selective and sensitive of fluorescence probe Pyp-B have been confirmed by its cell imaging application of H₂O₂ in living A549 cells and zebrafishes under the physiological conditions.     

A Detailed Insight into the Effects of Morphologies of Cerium Oxide on Fenton-like Reactions for Different Applications

As an exceptional Fenton-like reagent, cerium oxide (CeO₂) finds applications in biomedical science and organic pollutants treatment. The Fenton-like reaction catalyzed by CeO₂ typically encompasses two distinct processes: one resembling the classical Fenton reaction, wherein cerium (Ce³⁺) triggers the decomposition of hydrogen peroxide (H₂O₂) to yield reactive oxygen species (ROS), and the other involves the complexation of H₂O₂ on the Ce³⁺ surface, leading to the formation of peroxides. However, the influence of diverse CeO₂ morphologies on these two reaction pathways has not been comprehensively explored. In this study, CeO₂ exhibiting three typical morphologies, rods, cubes, and spheres, were prepared. The generation of ROS and peroxides was evaluated using the 3,3,5,5-tetramethylbenzidine (TMB) oxidation reaction and the reduction current of H₂O₂, respectively. Moreover, the impacts of pH variations and CeO₂/H₂O₂ concentrations on the production and conversion of these two reaction products were investigated. To corroborate the distinctions between the resultant products and their applicability, apoptosis assays and acid orange 7 (AO7) degradation analyses were performed. Notably, CeO₂ rods exhibited the highest proportion of Ce³⁺, predominantly engaging in complexation with H₂O₂ to foster peroxide formation, thereby facilitating the robust degradation of AO7. However, the generated peroxides appeared to occupy Ce³⁺ sites, thereby impeding the H₂O₂ decomposition process. Conversely, Ce³⁺ species on the surface of CeO₂ cubes were primarily involved in H₂O₂ decomposition, leading to heightened ROS production, and thus showcasing substantial potential for damaging A549 tumor cells. It is worth noting that the ability of these Ce³⁺ species to form peroxides through complexation with H₂O₂ was comparatively reduced. In summation, this study sheds light on the intricate interplay between distinct CeO₂ morphologies and their divergent impacts on Fenton-like reactions. These findings expand our comprehension of the influences on its reactivity of CeO₂ morphologies and open new insights for applications in diverse domains, from organic dye degradation to tumor therapy.     

Upconverting Carbon Nanodots from Ethylenediaminetetraacetic Acid (EDTA) as Near-Infrared Activated Phototheranostic Agents

This work describes the synthesis of nitrogen-doped carbon nanodots (CNDs) synthesized from ethylenediaminetetraacetic acid (EDTA) as a precursor and their application as luminescent agents with a dual-mode theranostic role as near-infrared (NIR) triggered imaging and photodynamic therapy agents. Interestingly, these fluorescent CNDs are more rapidly and selectively internalized by tumor cells and exhibit very limited cytotoxicity until remotely activated with a NIR illumination source. These CNDs are excellent candidates for phototheranostic purposes, for example, simultaneous imaging and therapy can be carried out on cancer cells by using their luminescent properties and the in situ generation of reactive oxidative species (ROS) upon excitation in the NIR range. In the presence of CNDs, NIR remote activation induces the in vitro killing of U251MG cells. Through the use of flow imaging cytometry, we have been able to successfully map and quantify the different types of cell deaths induced by the presence of intracellular superoxide anions ( ^. O₂⁻) and hydrogen peroxide (H₂O₂) ROS generated in situ upon NIR irradiation.     

Use of Dithiasuccinoyl-Caged Amines Enables COS/H2S Release Lacking Electrophilic Byproducts

The enzymatic conversion of carbonyl sulfide (COS) to hydrogen sulfide (H₂S) by carbonic anhydrase has been used to develop self-immolating thiocarbamates as COS-based H₂S donors to further elucidate the impact of reactive sulfur species in biology. The high modularity of this approach has provided a library of COS-based H₂S donors that can be activated by specific stimuli. A common limitation, however, is that many such donors result in the formation of an electrophilic quinone methide byproduct during donor activation. As a mild alternative, we demonstrate here that dithiasuccinoyl groups can function as COS/H₂S donor motifs, and that these groups release two equivalents of COS/H₂S and uncage an amine payload under physiologically relevant conditions. Additionally, we demonstrate that COS/H₂S release from this donor motif can be altered by electronic modulation and alkyl substitution. These insights are further supported by DFT investigations, which reveal that aryl and alkyl thiocarbamates release COS with significantly different activation energies.     

A Highly Water- and Air-Stable Iron-Containing MRI Contrast Agent Sensor for H2O2

A highly water- and air-stable Fe(II) complex with the quinol-containing macrocyclic ligand H₄qp4 reacts with H₂O₂ to yield Fe(III) complexes with less highly chelating forms of the ligand that have either one or two para-quinones. The reaction increases the T₁-weighted relaxivity over four-fold, enabling the complex to detect H₂O₂ using clinical MRI technology. The iron-containing sensor differs from its recently characterized manganese analog, which also detects H₂O₂, in that it is the oxidation of the metal center, rather than the ligand, that primarily enhances the relaxivity.     

Electrochemical Formation of Fe(IV)=O Derived from H2O2 on a Hematite Electrode as an Active Catalytic Site for Selective Hydrocarbon Oxidation Reactions

The high-valence iron species (Fe(IV)=O) in the cytochrome P450 enzyme superfamily is generated via the activation of O₂, and serves as the active center of selective hydrocarbon oxidation reactions. Furthermore, P450 can employ an alternate route to produce Fe(IV)=O, even from H₂O₂ without O₂ activation. Meanwhile, Fe(IV)=O has recently been revealed to be the reactive intermediate during H₂O oxidation to O₂ on hematite electrodes. Herein, we demonstrated the generation of Fe(IV)=O on hematite electrodes during the electrochemical oxidative decomposition of H₂O₂ using in situ UV-visible absorption spectra. The generation of Fe(IV)=O on hematite electrodes from H₂O₂ exhibited 100 mV lower overpotential than that from H₂O. This is because H₂O₂ serves not only as the oxygen source of Fe(IV)=O, but also as the additional oxidant. Finally, we confirmed that the Fe(IV)=O generated on hematite electrodes can serve as the catalytic site for styrene epoxidation reactions.     

A Neutrophil‐Inspired Supramolecular Nanogel for Magnetocaloric–Enzymatic Tandem Therapy

Neutrophils can responsively release reactive oxygen species (ROS) to actively combat infections by exogenous stimulus and cascade enzyme catalyzed bio‐oxidation. A supramolecular nanogel is now used as an artificial neutrophil by enzymatic interfacial self‐assembly of peptides (Fmoc‐Tyr(H₂PO₃)‐OH) with magnetic nanoparticles (MNPs) and electrostatic loading of chloroperoxidase (CPO). The MNPs within the nanogel can elevate H₂O₂ levels in cancer cells under programmed alternating magnetic field (AMF) similar to the neutrophil activator, and the loaded CPO within protective peptides nanolayer converts the H₂O₂ into singlet oxygen (¹O₂) in a sustained manner for neutrophil‐inspired tumor therapy. As a proof of concept study, both the H₂O₂ and ¹O₂ in cancer cells increase stepwise under a programmed alternating magnetic field. An active enzyme dynamic therapy by magnetically stimulated oxygen stress and sustained enzyme bio‐oxidation is thus shown with studies on both cells and animals.     

Reactive oxygen species, cell growth, and taxol production of Taxus cuspidata cells immobilized on polyurethane foam

Dynamic changes in reactive oxygen species (ROS) of Taxus cuspidata cells immobilized on polyurethane foam were investigated and the relation between ROS content and taxol production was discussed. Immobilization shortened the lag period of cell growth and moderately increased H₂O₂ and O₂ ^−• contents inside the microenvironment within the first 15 d. After 20 d, excessive production of H₂O₂ and O₂ ^−• was observed accompanied by marked increases in membrane lipid peroxidation and cell membrane permeability. The taxol content of immobilized cells was fourfold that of suspended cells at d 35. The addition of exogenous H₂O₂ barely affected malondialdehyde content and cell membrane permeability but led to an obvious accumulation of taxol. It is inferred that the intracellular and extracellular H₂O₂ inside the microenvironment might be one factor promoting taxol biosynthesis under the immobilization stress.     

A Multi-responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super-resolution Imaging

Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H₂O₂), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H₂O₂ level leads to disassociation of nucleic acid–protein complexes.     

On the Influence of Oxygen on the Degradation of Fe‐N‐C Catalysts

Fe‐N‐C catalysts containing atomic FeN_x sites are promising candidates as precious‐metal‐free catalysts for oxygen reduction reaction (ORR) in proton exchange membrane fuel cells. The durability of Fe‐N‐C catalysts in fuel cells has been extensively studied using accelerated stress tests (AST). Herein we reveal stronger degradation of the Fe‐N‐C structure and four‐times higher ORR activity loss when performing load cycling AST in O₂‐ vs. Ar‐saturated pH 1 electrolyte. Raman spectroscopy results show carbon corrosion after AST in O₂, even when cycling at low potentials, while no corrosion occurred after any load cycling AST in Ar. The load‐cycling AST in O₂ leads to loss of a significant fraction of FeN_x sites, as shown by energy dispersive X‐ray spectroscopy analyses, and to the formation of Fe oxides. The results support that the unexpected carbon corrosion occurring at such low potential in the presence of O₂ is due to reactive oxygen species produced between H₂O₂ and Fe sites via Fenton reactions.