A fluorescent probe for monitoring Cys fluctuations in the oxidative stress environment simulated by Cu2+ or H2O2.

Redox balance is the core of holding the good physiological state of the body. Cysteine (Cys) is one of the important biomolecules, which plays an indispensable role in maintaining the body's redox homeostasis. The redox of organisms is mainly the result of the dynamic balance between reactive oxygen species (ROS) and biological reducing agents (such as Cys). Fluorescent probes have the advantages of simple operation, good specificity and high sensitivity, and have become a common tool for bio-sensing in complex systems. In this article, we designed a probe NF-O-SBD that can specifically detect Cys. The chlorine atom of NF-O-SBD was easily substituted by sulfhydryl as a reaction site. After the formation of sulfur substitution products, intramolecular rearrangement occurred and fluorescent signal was emitted in the yellow channel at 550 nm. It can be seen from the spectroscopy experiment that the content of Hcy in organisms (15 µM) basically did not cause significant fluorescence changes, Therefore, based on the practical application in biology, we further used NF-O-SBD to visualize endogenous and exogenous Cys in HepG-2 cells and zebrafish. Simultaneously, we used Cu2+ or H2O2 induction to simulate the oxidative stress environment of cells and zebrafish, under which the concentration variation of Cys was monitored.

Rapid Reaction, Slow Dissociation Aggregation, and Synergetic Multicolor Emission for Imaging the Restriction and Regulation of Biosynthesis of Cys and GSH.

Biosynthesis is a necessary process to maintain life. In recent years, research has fully shown that three kinds of biothiols (Cys, Hcy, GSH) mainly play the role in oxidative stress and maintaining cell homeostasis in cells, and that abnormal concentrations will lead to the occurrence of cardiovascular diseases, cancers, etc. Various fluorescent probes have shown unprecedented advantages in detecting their concentrations and studying their biological functions. As a matter of fact, these three kinds of biothiols are generated in the process of biosynthesis in vivo. It is of great significance to understand their biosynthetic pathways and elucidate their synthetic relationships. In this work, to α,ß-unsaturated ketones conjugated ethylenediamine coumarin and pyrandione was introduced boron fluoride and, through its strong electron deficiency effect, afforded a molecule having near-infrared emission and regulated the rigidity of molecules. At the same time, the conjugated double bond is used to respond to molecular rigidity. The rapid response of the probe to biothiols and the slow dissociation aggregation of the probe itself through the response environment could monitor the absence of biothiols in cells. In addition, based on the difference in sensitivity of response of Cys and GSH to the probe, this work studied the interaction between biosynthetic pathways of Cys and GSH in cells through enzyme inhibition for the first time. The relationship of restriction and regulation of biosynthesis in vivo was revealed.

A coumarin-based fluorescence sensor for rapid discrimination of cysteine/homocysteine and glutathione under dual excitation wavelengths.

Biological thiols (Cys, Hcy and GSH) are crucial biomolecules in living cells and play indispensable roles in maintaining the redox homeostasis of organisms. But due to their similar molecular structure, the development of effective tools for distinguishing two or three of them remains a great difficulty. Herein, we constructed a sensitive sensor (CB) by connecting the bifunctional fluorescent reagent with coumarin derivatives for simultaneous recognition of these three thiols through different pathways. Free CB had no fluorescence; however, with gradual addition of thiols, the chlorine unit was replaced by sulfhydryl. Furthermore, the intramolecular rearrangement occurred between the amino and sulfhydryl groups of Cys/Hcy and yellow fluorescence was observed at 570 nm. However, GSH with a large structure could not undergo intramolecular rearrangement, and green fluorescence was excited at 505 nm. In this way, Cys/Hcy and GSH can be detected distinctively. Under dual excitation wavelengths, CB exhibited high selectivity and fast response to the three thiols. Furthermore, CB was successfully applied to imaging endogenous and exogenous thiols in living cells and zebrafish, providing us with a reliable tool for thiols recognition.

Sulphide activity-dependent multicolor emission dye and its applications in in vivo imaging.

Reactive sulfur species (RSS) play pivotal roles in various pathological and physiological processes. There exists an intricate relevance in generation and metabolism among these substances. Although they are nucleophilic, there are still some differences in their reactivity. There are many methods to detect them by using reactive fluorescent probes, but the systematic study of their reactivity is still lacking. In our study, we designed a multiple reaction site fluorescent probe based on benzene conjugated benzopyrylium and NBD. The study revealed that besides both biothiols and hydrogen sulfide, sulfur dioxide (SO2) can cleave the ether bond. There are two reaction forms for GSH with low reactivity: cutting the ether bond and adding the conjugated double bond of benzopyrylium. Nevertheless, Cys/Hcy with higher activity can further rearrange with NBD after cutting the ether bond. In addition, SO2 can not only cleave the ether bond, but also continue to add the conjugated double bond of benzopyrylium. The above processes lead to multicolor emission of the probe, thus realizing the characteristic analysis of different sulfides. Thus the probe can be used for the detection of sulfide in mitochondria, and further for the imaging of sulfide in cells and zebrafish. This effective analysis method will provide a broad application prospect for practical applications.

A Pyrene-Based Fluorescent Probe for Specific Detection of Cysteine and its Application in Living Cell.

Cysteine (Cys) is an essential amino acid in organism, which is transformed from methionine in vivo and participates in protein synthesis and cell redox process. Therefore, the detection of Cys is of great significance. In this work, a novel fluorescent probe, (E)-3-(2-chloroquinolin-3-yl)-1-(pyren-3-yl) prop-2-en-1-one (PAQ) was designed and synthesized to specifically detect Cys. The response mechanism of the reaction between PAQ and Cys was due to the addition reaction of Cys to α,ß-unsaturated ketone of PAQ. Interestingly, the addition of Cys induced significant fluorescence intensity enhancement at 462 nm. PAQ exhibited favorable sensing properties towards Cys such as the low limit of detection (0.27 µM) and fast response speed (2 min). In addition, PAQ displayed high selectivity and anti-interference ability toward Cys among various analytes. Notably, PAQ has been successfully used to image exogenous and endogenous Cys in HeLa cells.

High-Specific Fluorescence Probe for SO32- Detection and Bioimaging.

It is well known that sulfite (SO32-) plays an indispensable role in various physiological processes. Abnormal levels of SO32- can trigger a wide variety of diseases involving respiratory, nervous and cardiovascular systems. Hence, it is necessary to find an efficient approach for detection of SO32-. In this study, a pyrene derivative, (E)-4-(3-oxo-3-(pyren-1-yl)prop-1-en-1-yl)phenyl acrylate (PPA), was designed and synthesized for monitoring SO32-. The probe possessed simple synthetic steps, excellent anti-interference ability and specific response to SO32- in the presence of other substances. The reaction between PPA and SO32- was ascribed to Michael addition and the detection mechanism was confirmed by HRMS spectra analysis and FTIR analysis. Additionally, PPA responded linearly to detect SO32- within the rang of 0-100 µM. The limit of detection was calculated as low as 0.17 µM in accordance with the recommendation of IUPAC (CDL =3sb/m). Notably, PPA was further applied in biological imaging in HepG2 cells, which provided a possibility to monitor SO32- in vivo.

A New Strategy: Distinguishable Multi-substance Detection, Multiple Pathway Tracing Based on a New Site Constructed by the Reaction Process and Its Tumor Targeting.

In recent years, it has become a trend to employ organic molecular fluorescent probes with multireaction sites for the distinguishable detection and biological imaging of similar substances. However, the introduction of multireaction sites brought great challenges to organic synthesis, and at the same time, often destroyed the conjugated structure of the molecules, leading to an unsatisfactory fluorescence emission wavelength not conducive to practical application. As the eternal theme of life, metabolism goes on all the time. Metabolism is a series of ordered chemical reactions that occurs in the organism to maintain life. Chemical reactions in metabolism can be summarized as metabolic pathways. Simultaneous monitoring of different metabolic pathways of the same substance poses a lofty challenge to the probe. Here, we developed a new strategy: to construct new sites through the preliminary reactions between probes and some targets, which can be used to further distinguish among targets or detect their metabolites, so as to realize the simultaneous visualization tracer of multiple metabolic pathways. By intravenous injection, it revealed that the probe containing benzopyrylium ion can target tumors efficiently, and thiols are highly expressed in tumors compared to other tissues (heart, lung, kidney, liver, etc.). The consumption of thiols by the probe could not prevent tumor growth, suggesting that the tumor cure was not correlated with thiol concentration. The construction of new sites in the reaction process is a novel idea in the pursuit of multiple reaction sites, which will provide more effective tools for solving practical problems.

Heat Stroke in Cell Tissues Related to Sulfur Dioxide Level Is Precisely Monitored by Light-Controlled Fluorescent Probes.

Heat stroke (HS) can cause serious organism damage or even death. Early understanding of the mechanism of heat cytotoxicity can prevent or treat heat stroke related diseases. In this work, probe Ly-NT-SP was synthesized, characterized, and used for sulfur dioxide (SO2) detection in lysosomes. PBS solutions of probe Ly-NT-SP at pH 5.0 present a marked broad emission band in the green zone (535 nm). After UV irradiation, the spiropyran group in Ly-NT-SP isomerizes to the merocyanine form (Ly-NT-MR), which presented a weak red-shifted emission at 630 nm. In addition, photocontrolled isomerization of Ly-NT-SP to Ly-NT-MR generated a C═C-C═N+ fragment able to react, through a Michael addition, with SO2 to yield a highly emissive adduct with a marked fluorescence in the green channel (535 nm). In vitro studies showed a remarkable selectivity of photoactivated Ly-NT-MR to SO2 with a limit of detection as low as 4.7 µM. MTT viability assays demonstrated that the Ly-NT-SP is nontoxic to HeLa cells and can be used to detect SO2 in lysosomes. Taking advantage of this, the sensor is successfully applied to image increasing SO2 values in lysosomes during heat shock for the first time. Moreover, we also confirmed that the increased SO2 can protect the small intestine against damage induced by heat shock through regulating oxidative stress in cells and mice.

Thiol "Click" Chromene Ring Opening and Subsequent Cascade Nucleophilic Cyclization NIR Fluorescence Imaging Reveal High Levels of Thiol in Drug-Resistant Cells.

As the structural unit of natural products, chromene derivatives show a wide range of biological activity and pharmacological activity due to their unique photophysical and chemical properties. Ten years ago, our research group discovered the "thiol-chromene" click reaction, which achieved the selective detection of thiols through the change of the optical spectrum. Afterward, we attempted to develop various chromene-based fluorescent probes for imaging including near-infrared (NIR) probe, ratiometric probe, and multifunctional probe. However, how to integrate the fluorophore and reaction sites into the chromene-based skeleton remains challenging. In this work, we connected the chromene motif with the NIR fluorophore methylene blue utilizing a carbamate spacer to provide a new fluorescent probe (CM-NIR), which is triggered by thiols to open the pyran ring followed by attacking the carbamate by phenolate to releases the methylene blue. This novel cascade mechanism avoids the formation of para-quinone methides, which proved to be toxic to normal cells. CM-NIR also showed the specific imaging of thiols in living cells and mice. More importantly, the thiols level in drug-resistant cancer cells was found to be significantly higher than that in the corresponding cancer cell, which indicated that the thiols level may have an important role in cancer cells developing drug resistance.

A practical pH-compatible fluorescent sensor for hydrazine in soil, water and living cells.

The excellent water solubility of hydrazine (N2H4) allows it to easily invade the human body through the skin and respiratory tract, thereby damaging human organs and the central nervous system. To realize the monitoring of N2H4 effectively, first, coumarin was used to construct an inner alicyclic ring as the reaction site, extending the conjugation and strengthening the rigidity of the probe Co-Hy to improve its luminescence performance and enhance its ability to resist acids and alkalis. Second, we introduced a carboxyl group at the ortho position of the inner alicyclic ring to improve the water solubility of Co-Hy, and its strong electron pulling effect increased the activity of the reaction site. Spectroscopy experiments showed that Co-Hy featured excellent water solubility, high pH resistance (pH 4-11), excellent selectivity, fast analysis speed (within 5 minutes), and a low detection limit toward N2H4 (69 nM, 2.2 ppb). In addition, test-strip, spray, and cell-imaging experiments confirmed the outstanding application potential of Co-Hy for convenient N2H4 analysis in a variety of environments.

Formal enantioselective total synthesis of bisdehydroneostemoninine.

A formal enantioselective total synthesis of bisdehydroneostemoninine employing L-glutamic acid as the chiral pool is described. The key features of the synthesis include regioselective and enantioselective opening the chiral epoxide with dimethylsulfonium methylide and tandem Friedel-Crafts cyclization followed by lactonization to form the 5-7-5 tricyclic core of the target stemona alkaloids. The synthetic route provides opportunities to explore the biological behavior of enantiopure bisdehydroneostemoninine. [Formula: see text].

Colorimetric and NIR Fluorescence Probe with Multiple Binding Sites for Distinguishing Detection of Cys/Hcy and GSH in Vivo.

Although some progress has been made in distinguishing the detection of biothiols, NIR biothiol fluorescent probes for simultaneously distinguishing detection of Cys, Hcy, and GSH in vivo have not been reported. The design of these probes involves the introduction of NIR fluorophores and multiple binding sites; thus, the integrated design of probes remains a challenge. Although Cys, Hcy, and GSH have common functional groups, a sulfydryl group and an amino group, due to their differences in spatial structure, they may react with multiple binding site probes to produce different reaction products in different bonding mechanisms, resulting in different colors and fluorescent signal changes of the system. Therefore, multiple binding site fluorescent probes can realize their discrimination detection. For an NIR fluorescent probe, it is easier to realize in vivo imaging to promote the research of biothiols in clinical diagnosis. In our work, not only were multiple binding sites constructed in the compound but also NIR fluorophores were introduced. This enables the probe to not only efficiently distinguish detection of Cys/Hcy and GSH but also achieve fluorescence imaging in vivo. We believe this result is a milestone in the discrimination detection of biothiols.

A dimethoxypillar[5]arene/azastilbene host-guest recognition motif and its applications in the fabrication of polypseudorotaxanes.

Pillar[n]arenes, known as the fifth generation of host macrocycles since 2008, have become a popular topic over the past ten years. Until now, the studies of pillar[n]arenes were mainly focused on pillar[5]arenes owing to their easy synthesis and high yields. In particular, 1,4-dimethoxypillar[5]arene (DMP5), which shows a simple structure, efficient synthesis and high yield, has played important roles in the construction of various advanced supramolecular architectures. However, DMP5 has only displayed host-guest binding properties towards some guests. Therefore, the investigation of the host-guest chemistry of DMP5 should be able to greatly promote the development of pillararene chemistry. Herein, a photosensitive azastilbene derivative was chosen as a neutral guest to study the host-guest binding and stimuli-responsive behavior with DMP5. In addition, the binding behavior of DMP5 towards a series of analogous neutral guest molecules was investigated to study the driving forces of the host-guest interaction between DMP5 and the azastilbene guest. Moreover, the [2]pseudorotaxane based on DMP5 and the azastilbene guest was used to construct a polypseudorotaxane via metal coordination.

A Fluorescent Probe Based on Pyrene Ring for Detecting Cys and its Application in Biology.

The identification of thiols has become a research hotspot due to its role in biological systems. In this work, we simply constructed a turn-on fluorescent probe named 3-(5-bromopyridin-3-yl)-1-(pyren-1-yl) prop-2-en-1-one, that a combination of pyrene ring and substituted pyridine via the connection of α, ß-unsaturated ketone. Cys can destroy the space effect by Michael addition reaction, which makes the fluorescence intensity changes. Furthermore, the probe featured excellent selectivity and high sensitivity (the detection limit was 0.52 µM) by addition of Cys. Moreover, this probe suggested a potential for imaging in vivo owing to the successful imaging of the probe in HepG2 cells, zebrafish, and Arabidopsis thaliana.

A Simple Fluorescent Probe for Sensing pH and its Application in E. coli Cells.

We designed and synthesized a simple fluorescent probe, (E)-2-(2-(3,4,5-Trimethoxybenzylidene)hydrazinyl) benzothiazole (probe), which could be applied to the detection of strongly acidic and alkaline pH in DMSO/water (1/4, v/v) system. It could be used to quantitatively detect strong acid in the range of 2.60-3.53 with a pKa of 2.78. Meanwhile, it also showed an excellent linear relationship between the fluorescence intensity and alkaline pH values over the range of 9.98-10.95 with a pKa of 9.32. The probe exhibited excellent properties to pH with high selectivity and sensitivity. The mechanism studies showed that the H+ binding with the N atom of benzothiazole moiety and hydrazine moiety in acid solution while the deprotonation of N atom in hydrazine group in basic environment. Importantly, the probe was successfully applied for imaging the strongly acidic and alkaline in E.coil cells.

Dual-Site Fluorescent Probe for Visualizing the Metabolism of Cys in Living Cells.

Fluorescent probes, as noninvasive tools for visualizing the metabolism of biomolecules, hold great potential to explore their physiological and pathological processes. For cysteine (Cys), however, none of the reported fluorescent probes could image the metabolic processes in living cells. To achieve this goal, we developed a coumarin derivative based on rational design of the dual recognition sites for Cys and its metabolite, SO2. The probe displayed distinct two channels with turn-on fluorescent emission toward Cys and SO2, which were successfully applied for imaging both A549 cells and zebrafish. Further, with reversible fluorescent responses toward Cys, the probe could image the enzymatic conversion of Cys to SO2 in living A549 cells in a ratiometric manner. The present work reports the first probe to image the endogenous generated SO2 without incubation of the SO2 donors.

Curcumin-Based "Enhanced SNAr" Promoted Ultrafast Fluorescent Probe for Thiophenols Detection in Aqueous Solution and in Living Cells.

We report herein a highly selective and sensitive turn-on fluorescent probe (compound 1) with a fast response time (less than 2 min) for thiophenol detection based on an "enhanced SNAr" reaction between thiophenols and a sulfonyl-ester moiety covalently attach to curcumin. Reaction of 1 in Hepes-MeOH (1:1, v/v, pH 7.4) in the presence of 4-methylthiophenol (MTP) resulted in a remarkable enhancement of the fluorescence. A linear response in the presence of MTP of the relative fluorescent intensity (F - F0) of 1 at 536 nm in the 0-40 µM MTP concentration range was found. A limit of detection (LOD) for the detection of MTP of 26 nM, based on the definition by IUPAC (CDL = 3 Sb/m), was calculated. Probe 1 was applied to monitor and imaging exogenous MTP in live cells and to the detection of MTP in real water samples.

A Dual Colorimetric/Fluorescence System for Determining pH Based on the Nucleophilic Addition Reaction of an o-Hydroxymerocyanine Dye.

Owing to their ability to monitor pH in a precise and rapid manner, optical probes have widely been developed for biological and nonbiological applications. The strategies thus far employed to determine pH rely on two types of processes including reversible protonation of amine nitrogen atoms and deprotonation of phenols. We have developed a novel dual, colorimetric/fluorescence system for determining the pH of a solution. This system utilizes an o-hydroxymerocyanine dye that undergoes a nucleophilic addition reaction that subsequently causes reversible structural changes interconverting a merocyanine to a spirocyanine and a spirocyanine to a spiropyran. It was demonstrated that the dye can be employed to measure the pH of solutions in the 2.5-5.75 and 9.6-11.8 ranges with color changes from yellow to dark blue and then to lavender. Moreover, the fluorescence response associated with the spirocyanine-spiropyran transformation of the dye occurring in alkaline solutions provides a precise method.

Potential of the Endophytic Fungus Phialocephala fortinii Rac56 Found in Rhodiola Plants to Produce Salidroside and p-Tyrosol.

2-(4-Hydroxyphenyl)ehyl-ß-D-glucopyranoside (salidroside) and 4-(2-hydroxyethyl)phenol (p-tyrosol) are famous food and medicine additives originally derived from alpine Rhodiola plants. Salidroside or p-tyrosol production by the endophytic fungus Rac56 (Phialocephala fortinii) was confirmed by UPLC/Q-TOF-MS and ¹H-NMR. The fermentation conditions were optimized by orthogonal design using data processing system software. The broth fermentation results showed that salidroside and p-tyrosol extraction yields from Rac56 were stable and reached 1.729 ± 0.06 mg and 1.990 ± 0.05 mg per mL of culture medium, respectively. The optimal conditions for salidroside and p-tyrosol production in fermentation culture of Rac56 were determined to be 25 °C, pH values of 7 and 5, Czapek-Dox culture medium volumes of 150 mL and 50 mL in 250 mL flasks, rotation speeds of 100× g and 200× g, and fermentation durations of 7 and 15 days, respectively. Under these optimal conditions, stable yields of 2.339 ± 0.1093 mg and 2.002 ± 0.0009 mg per mL of culture medium of salidroside and p-tyrosol, respectively, were obtained, indicating that the P. fortinii Rac56 strain is a promising source of these compounds.

The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin.

Centrin is a member of the EF-hand superfamily that is phosphorylated during mitosis and is associated with alterations of contractile fibers. To obtain insight into the structural basis for the functional effects of phosphorylation, we found that the serine residue at position 166 of Euplotes octocarinatus centrin (EoCen) can be phosphorylated by protein kinase A (PKA) in the absence or presence of metal ions using (31)P-NMR spectroscopy. Cations of Ca(2+) and Tb(3+) bound to EoCen resulted in an important structural transition from a closed to an open state. EoCen in both the closed and the open state can be phosphorylated by PKA. After phosphorylation, secondary and tertiary structural changes of EoCen, mainly on its C-terminal domain (C-EoCen), were noted through circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and 2-p-toluidinylnaphthalene-6-sulfonate fluorescence. After the protein was phosphorylated, the α-helix content and the extent of the exposed hydrophobic surface on EoCen were decreased. Phosphorylated EoCen has higher affinity for the peptide melittin than nonphosphorylated EoCen. In addition, binding of melittin with phosphorylated C-EoCen was enthalpy-driven.