Multi‐Fluorinated Azido Coumarins for Rapid and Selective Detection of Biological H2S in Living Cells

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. In order to visualize the endogenous in situ production of H₂S in living cells in real time, here we developed multi‐fluorinated azido coumarins as fluorescent probes for the rapid and selective detection of biological H₂S. Kinetic studies indicated that an increase in fluorine substitution leads to an increased rate of H₂S‐mediated reduction reaction, which is also supported by our theoretical calculations. To our delight, tetra‐fluorinated coumarin 1 could react with H₂S fast (t_1/2≈1 min) and selectively, which could be further used for continuous enzymatic assays and for visualization of intracellular H₂S. Bioimaging results obtained with 1 revealed that d ‐Cys could induce a higher level of endogenous H₂S production than l ‐Cys in a time‐dependent manner in living cell.     

FRET‐Based Mitochondria‐Targetable Dual‐Excitation Ratiometric Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio‐H₂S 1/2 are the first FRET‐based mitochondrial‐targetable dual‐excitation ratiometric fluorescent probes for H₂S on the basis of H₂S‐promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H₂S; this is a 7.5‐fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria‐targetable dual‐excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

A Dual‐Response Fluorescent Probe Reveals the H2O2‐Induced H2S Biogenesis through a Cystathionine β‐Synthase Pathway

The two signaling molecules H₂S and H₂O₂ play key roles in maintaining intracellular redox homeostasis. The biological relationship between H₂O₂ and H₂S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single‐ and dual‐response fluorescent probes for detecting both H₂O₂ and H₂S in living cells. The dual‐response probe was shown to be capable of mono‐ and dual‐detection of H₂O₂ and H₂S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H₂O₂ could induce H₂S biogenesis in living cells. By using gene‐knockdown techniques with bioimaging, the H₂S biogenesis was found to be majorly cystathionine β‐synthase (CBS)‐dependent. Our finding shows the first direct evidence on the biological communication between H₂O₂ (ROS) and H₂S (RSS) in vivo.     

Dual‐Reactable Fluorescent Probes for Highly Selective and Sensitive Detection of Biological H2S

Hydrogen sulfide (H₂S) is an important endogenous signaling molecule with a variety of biological functions. Development of fluorescent probes for highly selective and sensitive detection of H₂S is necessary. We show here that dual‐reactable fluorescent H₂S probes could react with higher selectivity than single‐reactable probes. One of the dual‐reactable probes gives more than 4000‐fold turn‐on response when reacting with H₂S, the largest response among fluorescent H₂S probes reported thus far. In addition, the probe could be used for high‐throughput enzymatic assays and for the detection of Cys‐induced H₂S in cells and in zebrafish. These dual‐reactable probes hold potential for highly selective and sensitive detection of H₂S in biological systems.     

Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three‐Channel Imaging Correlation

Enzyme activity in live cells is dynamically regulated by small‐molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long‐standing challenge. Herein, we report a three‐channel imaging correlation by a single dual‐reactive fluorescent probe to measure the dependence of phosphatase activity on the H₂S level in cells. The two sites of the probe reactive to H₂S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three‐channel imaging correlation shows that cells have an ideal level of H₂S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H₂S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H₂S in cellular signaling and in various human diseases.     

Highly Selective and Sensitive Near‐Infrared‐Fluorescent Probes for the Detection of Cellular Hydrogen Sulfide and the Imaging of H2S in Mice

Herein, we report the development of two fluorescent probes for the highly selective and sensitive detection of H₂S. The probes take advantage of a Cu^II? cyclen complex, which acts as a reaction center for H₂S and as a quencher of BODIPY (boron‐dipyrromethene)‐based fluorophores with emissions at 765 and 680 nm, respectively. These non‐fluorescent probes could only be turned on by the addition of H₂S, and not by other potentially interfering biomolecules, including reactive oxygen species, cysteine, and glutathione. In a chemical system, both probes detected H₂S with a detection limit of 80 nM . The probes were successfully used for the endogenous detection of H₂S in HEK 293 cells, for measuring the H₂S‐release activity of dietary organosulfides in MCF‐7 cells, and for the in vivo imaging of H₂S in mice.     

A Single Fluorescent Probe to Visualize Hydrogen Sulfide and Hydrogen Polysulfides with Different Fluorescence Signals

Hydrogen sulfide (H₂S) and hydrogen polysulfides (H₂S_n, n>1) are endogenous regulators of many physiological processes. In order to better understand the symbiotic relationship and cellular cross‐talk between H₂S and H₂S_n, it is highly desirable to develop single fluorescent probes which enable dual‐channel discrimination between H₂S and H₂S_n. Herein, we report the rational design, synthesis, and evaluation of the first dual‐detection fluorescent probe DDP‐1 that can visualize H₂S and H₂S_n with different fluorescence signals. The probe showed high selectivity and sensitivity to H₂S and H₂S_n in aqueous media and in cells.     

Dual Mechanism of an Intramolecular Charge Transfer (ICT)–FRET‐Based Fluorescent Probe for the Selective Detection of Hydrogen Peroxide

A dual‐mechanism intramolecular charge transfer (ICT)–FRET fluorescent probe for the selective detection of H₂O₂ in living cells has been designed and synthesized. This probe used a coumarin–naphthalimide hybrid as the FRET platform and a boronate moiety as the recognition group. Upon the addition of H₂O₂, the probe exhibited a redshifted (73 nm) fluorescence emission, and the ratio of fluorescence intensities at λ =558 and 485 nm (F ₅₅₈/F ₄₈₅) shifted notably (up to 100‐fold). Moreover, there was a good linearity (R ²=0.9911) between the ratio and concentration of H₂O₂ in the range of 0 to 60 μm , with a limit of detection of 0.28 μm (signal to noise ratio (S/N)=3). This probe could also detect enzymatically generated H₂O₂. Importantly, it could be used to visualize endogenous H₂O₂ produced by stimulation from epidermal growth factor.     

Synthesis and fluorescent properties of 2‐(1H‐benzimidazol‐2‐yl)‐phenol derivatives

A high yield one pot synthesis of 2‐(2‐hydroxyaryl)‐1H‐benzirrndazole derivatives by 2‐hydroxy aromatic aldehydes with aromatic 1,2‐diamines in the presence of manganese(III) acetate at room temperature was developed. Nine fluorescencers 2‐(2‐hydroxyaryl)‐1H‐benzirrndazoles with substituent(s) X (X = H, CH₃, CH₃O, Cl) and two fluorescencers 2‐(2‐hydroxyaryl)‐1H‐naphth[2,3‐d]imidazoles with substituent of H or Cl were prepared in 38–87% yield and the ultraviolet absorption and fluorescent spectra of the eleven compounds synthesized were measured in methanol. The fluorescent characteristics of the 2‐(2‐hydroxyaryl)benzimidazole derivatives prepared were investigated on the basis of excited‐state intramolecular proton transfer mechanism, Stokes' shift, quantum yield, and the relationship between fluorescent intensity and the substituents were derived.     

Enzyme‐Triggered Fluorescence Turn‐on: A Probe for Specifically Imaging Ovarian‐Cancer‐Related γ‐Glutamyltranspeptidase

A fluorescent turn‐on probe for specifically targeting γ ‐glutamyltranspeptidase (GGT ) was designed and synthesized by integrating boron‐dipyrromethene (BODIPY ) as a chromophore and glutathione (GSH ) as the GGT substrate. GGT ‐catalyzed the cleavage of the γ ‐glutamyl bond and generated the aromatic hydrocarbon transfer between the sulfur and the nitrogen atom in BODIPY , leading to distinct optical changes. Such specific responsiveness provides an easily distinguishable fluorescence signal to visualize the GGT activity in living cells and differentiate GGT ‐positive cancer cells from GGT ‐negative cells.     

Fluorogenic Probe Using a Mislow–Evans Rearrangement for Real‐Time Imaging of Hydrogen Peroxide

Hydrogen peroxide (H₂O₂) mediates the biology of wound healing, apoptosis, inflammation, etc. H₂O₂ has been fluorometrically imaged with protein‐ or small‐molecule‐based probes. However, only protein‐based probes have afforded temporal insights within seconds. Small‐molecule‐based electrophilic probes for H₂O₂ require many minutes for a sufficient response in biological systems. Here, we report a fluorogenic probe that selectively undergoes a [2,3]‐sigmatropic rearrangement (seleno‐Mislow‐Evans rearrangement) with H₂O₂, followed by acetal hydrolysis, to produce a green fluorescent molecule in seconds. Unlike other electrophilic probes, the current probe acts as a nucleophile. The fast kinetics enabled real‐time imaging of H₂O₂ produced in endothelial cells in 8 seconds (much earlier than previously shown) and H₂O₂ in a zebrafish wound healing model. This work may provide a platform for endogenous H₂O₂ detection in real time with chemical probes.     

Active‐Site‐Matched Fluorescent Probes for Rapid and Direct Detection of Vicinal‐Sulfydryl‐Containing Peptides/Proteins in Living Cells

Vicinal‐sulfydryl‐containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (–CGPC–) of human thioredoxin, two active‐site‐matched fluorescent probes, o‐Dm‐Ac and m‐Dm‐Ac, were developed for real‐time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W‐6), WCGGPCK (W‐7), and WCGGGPCK (W‐8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o‐Dm‐Ac displays a higher binding sensitivity with the above‐mentioned peptides and proteins, especially with W‐7 and rTrx. Furthermore, o‐Dm‐Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe‐design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ.     

Design of Ratiometric Fluorescent Probes Based on Arene–Metal‐Ion Interactions and Their Application to CdII and Hydrogen Sulfide Imaging in Living Cells

Non‐coordinative interactions between a metal ion and the aromatic ring of a fluorophore can act as a versatile sensing mechanism for the detection of metal ions with a large emission change of fluorophores. We report the design of fluorescent probes based on arene–metal‐ion interactions and their biological applications. This study found that various probes having different fluorophores and metal binding units displayed significant emission redshift upon complexation with metal ions, such as Ag^I, Cd^II, Hg^II, and Pb^II. X‐ray crystallography of the complexes confirmed that the metal ions were held in close proximity to the fluorophore to form an arene–metal‐ion interaction. Electronic structure calculations based on TDDFT offered a theoretical basis for the sensing mechanism, thus showing that metal ions electrostatically modulate the energy levels of the molecular orbitals of the fluorophore. A fluorescent probe was successfully applied to the ratiometric detection of the uptake of Cd^II ions and hydrogen sulfide (H₂S) in living cells. These results highlight the utility of interactions between arene groups and metal ions in biological analyses.     

BODIPY-based Fluorescent Probe for Fast Detection of Hydrogen Sulfide and Lysosome-targeting Applications in Living Cells

Hydrogen sulfide (H₂S) is recognized as an endogenous gaseous signaling agent in many biological activities. Lysosomes are the main metabolic site and play a pivotal role in cells. Herein, we designed and synthesized two new fluorescent probes BDP-DNBS and BDP-DNP with a BODIPY core to distinguish H₂S. The sensing mechanism is based on the inhibition-recovery of the photo-induced electron transfer (PET) process. Through comparing the responsive behaviors of the two probes toward H₂S, BDP-DNBS showed a fast response time (60 s), low limit of detection (LOD, 51 nM), high sensitivity and selectivity. Moreover, the reaction mechanism was demonstrated by mass spectrometry and fluorescence off-on mechanism was proved by density functional theory (DFT). Significantly, confocal fluorescence imaging indicated that BDP-DNBS was successfully used to visualize H₂S in lysosomes in living HeLa cells.     

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.     

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.     

2,3‐Dihydro‐3‐methyl‐2‐nitrimino‐1,3‐thiazole

The title compound {alternatively, 3‐methyl‐2‐[oxido(oxo)hydrazono]‐2,3‐dihydro‐1,3‐thiazole}, C₄H₅N₃O₂S, was obtained by methyl­ation of N‐(2‐thia­zolyl)­nitr­amine. The molecule lies on a mirror plane and the thia­zole ring is planar, regular in shape and aromatic. The S atom participates in the aromatic sextet via an electron pair on the 3p_z orbital. In the crystal, the mol­ecules are arranged in parallel layers, bound to each other by weak C—H?O and C—H?N hydrogen bonds and by S?O dipolar interactions, with an interlayer separation of 3.23 Å.     

The low‐melting compounds 1,4‐diethyl‐, 1,2‐diethyl‐ and ethylbenzene

Crystals of 1,4‐diethyl‐ and 1,2‐diethylbenzene, both C₁₀H₁₄, and ethylbenzene, C₈H₉, have been grown in situ. The molecules of 1,4‐diethyl‐ and 1,2‐diethylbenzene are located about a centre of inversion and across a twofold axis, respectively. In both molecules, the terminal methyl groups are located on opposite sides of the plane of the aromatic ring. In the crystal structures of all three compounds, molecules are linked together by (Ar)C—H...π and CH₂...π contacts. The methyl H atoms do not form close contacts with any of the aromatic π systems.     

Quinolinium‐Based Fluorescent Probes for the Detection of Thiophenols in Environmental Samples and Living Cells

A new type of fluorescent probes for thiophenols, 6HQM‐DNP and 7HQM‐DNP, containing 6‐ or 7‐hydroxy quinonlinium as fluorophore and 2,4‐dinitrophenoxy (DNP) as nucleophilic recognition unit were constructed. As ethers, these non‐fluorescent probe molecules can release the corresponding fluorescent quinolinium (6HQM and 7HQM) through aromatic nucleophilic substitution (S_NAr) by thiolate anions from thiophenols. The sensing reaction is highly sensitive (detection limit of 8 nM for 7HQM‐DNP) and highly selective to thiophenols over aliphatic thiols and other nucleophiles under neutral conditions (pH 7.3). The probes respond rapidly to thiophenols, with second‐order rate constants k=45 M ^?1 s^?1 for 7HQM‐DNP and 24 M ^?1 s^?1 for 6HQM‐DNP. Furthermore, the selective detection of thiophenols in living cells by 7HQM‐DNP was demonstrated by confocal fluorescence imaging. In addition, these quinolinium salts show excellent chemical and thermal stability. In conclusion, this type of probes may find use in the detection of thiophenols in environmental samples and biosystems.     

Synthesis of 4‐Aryl‐8‐fluoro‐3a,4,5,9b‐tetrahydro‐3H‐cyclopenta[c]quinolines and Their Ozonides

4‐Aryl‐8‐fluoro‐3a,4,5,9b‐tetrahydro‐3H‐cyclopenta[c]quinolines are synthesized by acid‐catalyzed (CF₃CO₂H) three‐component cyclocondensation of 4‐fluoroaniline with aromatic aldehydes and cyclopentadiene. Stable ozonides with (1R*,4S*,5aR*,6S*,11bS*)‐configurations are obtained by ozonolysis of corresponding trifluoroacetyl derivatives.