Real-time imaging of intracellular cysteine level fluctuations during Cu2+ or H2O2 induced redox imbalance using a turn-on fluorescence sensor.

Redox balance is a necessary guarantee to maintain the normal physiological activities of organisms. Cysteine (Cys), a critical biological thiol, has the effect of maintaining redox balance in the body. The concentration of intracellular Cys is abnormal under redox imbalance, thereby resulting in multiple diseases. Additionally, studies have revealed that Cu2+ can stimulate the body to produce excess reactive oxygen species (ROS, similar to H2O2), and the generated ROS will consume reducing substances (such as Cys) in the body, leading to redox imbalance. Thus, finding a simple and effective method to monitor Cys under redox imbalance is pressing. Here, a turn on probe (DDNO) was proposed by connecting SBD-Cl to a red dye (HDM). The probe can specifically recognize Cys with rapid response (180 s) and low detection limit (0.61 µM) through substitution-rearrangement reaction between sulfhydryl and chlorine atom. Bioimaging experiments indicated that the probe has good biocompatibility and cell membrane permeability, which can be applied to monitor the fluctuation of Cys levels in live cells and zebrafish under the redox imbalance induced by Cu2+ or H2O2.

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.

A sequentially activated bioluminescent probe for observation of cellular H2O2 production induced by cysteine.

We report herein a caged luciferin probe Cy-Hy as a sequentially activated probe to selectively and sensitively sense L-Cys and H2O2. The probe displayed fluorescence and bioluminescence responses toward the two analytes. Utilizing the present probe, cellular excess L-Cys-induced H2O2 up-regulation was observed for the first time in living MDA-MB-231 cells.

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 reversible coumarin-based sensor for intracellular monitoring cysteine level changes during Cu2+-induced redox imbalance.

Biological thiols are crucial small molecule amino acids widely existing in cells, which play indispensable roles in maintaining redox homeostasis of living systems. Owing to their abnormal levels have close relation with many diseases, thus, developing more convenient, rapid and practical in-vivo detection tools is imminent. Herein, a reversible coumarin-based probe (HNA) was successfully constructed through a simple two-step synthesis. HNA can detect Cys/Hcy with high response speed and desirable selectivity based on Michael addition recognition mechanism. Free HNA has an orange emission at 580 nm, but after addition of Cys/Hcy, the conjugated structure of probe HNA was destroyed by the attack of sulfhydryl, resulting in a new green emission at 507 nm. Further, HNA has been applied to monitor Cys/Hcy in HeLa cells and zebrafish. Notably, HNA has also been successfully applied for real-time tracing Cys levels changes in living cells and zebrafish during the imbalance in redox status caused by copper (II). This provides a new strategy for studying the process of oxidative stress in cells.

A near-infrared fluorescent probe targeting mitochondria for sulfite detection and its application in food and biology.

Sulfur dioxide (SO2) is the main air pollutant in the environment, causing great harm to human health. Abnormal SO2 levels are usually associated with some respiratory diseases, cardiovascular diseases, and neurological disorders (even brain cancer). Therefore, monitoring SO2 levels is helpful to better understand its special physiological and pathological role. Although many fluorescent probes for SO2 have been reported, many of them were not ideal for in vivo imaging due to the short emission wavelength. In this work, a near-infrared fluorescent probe NIR-BN with emission wavelength of 680 nm was constructed by conjugating the benzopyrylium moiety and 6-hydroxy-2-naphthaldehyde. NIR-BN had high selectivity and rapidity for SO2 detection. In addition, the detection limit of NIR-BN was relatively low, which can be used for the determination of sulfite in different sugar samples with high accuracy. Of course, due to the excellent spectral and structural properties of NIR-BN, we have applied NIR-BN to the detection of SO2 in biological systems.

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.

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.

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.

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 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.

Independent bi-reversible reactions and regulable FRET efficiency achieving real-time visualization of Cys metabolizing into SO2.

In this work, we synthesized an independent bi-reversible reaction sensor BPC for simultaneously detecting cysteine (Cys) and sulfur dioxide (SO2), showing multi-fluorescence signal modes due to the regulable FRET efficiency, and finally achieving real-time process visualization of Cys metabolizing into SO2 in subcellular organelles and tumors.

Mitochondria-targeted reversible ratiometric fluorescent probe for monitoring SO2/HCHO in living cells.

Sulfur dioxide (SO2) maintains a certain steady state balance in the body, high concentration SO2 will be harmful to human health. Seeking a suitable detection method to monitor sulfur dioxide in real time becomes an urgent requirement owing to the transient nature of sulfur dioxide in organisms. Here, a novel NIR ratiometric fluorescent probe for detection of SO2 was developed based on a conjugation of coumarin and indol salt with excellent water solubility. The probe Mito-CI displayed highly sensitive (69 nM), fast response time (30 s), large Stokes shift (174 nm) and the NIR fluorescence emission wavelength (655 nm). In the reversibility process of the SO3--probe Mito-CI system induced by HCHO in vitro was also detected. Besides, cell imaging showed that Mito-CI possesses mitochondria-targeted ability. Particularly, Mito-CI was proved to reversibly detect SO2/HCHO in living cells.

Fast detecting hypochlorous acid based on electron-withdrawing group promoted oxidation and its biological applications in cells and root tips of plants.

Hypochlorous acid, a type of reactive oxygen species, has been shown to play an important role in organisms. Nowadays, there are many kinds of fluorescence detecting mechanisms to detect hypochlorous acid in vivo. Due to the high selectivity, the mechanism of using the strong oxidation of hypochlorous acid to break carbon­carbon double bonds has been favored by many scientists. However, the reported probes of breaking carbon­carbon double bonds still had drawback such as slow response. Based on this, we introduced electron-withdrawing group malonitrile to accelerate the oxidation of hypochlorous acid, resulting in reaction time less than 150 s. Meanwhile, the probe exhibited excellent selectivity, optical stability, high sensitivity and the detection limit as low as 0.19 µM. More importantly, we also successfully proved the potential application of the probe for the detection of intracellular ClO- living cells and Arabidopsis root tip by fluorescence imaging.

NEM assisted real-time fluorescence detection of Cys in cytoplasm and mice imaging by a Coumarin probe containing carboxyl group.

Alterations of the homeostasis balance of cysteine (Cys) are associated with a variety of diseases and cellular functions, and therefore, Cys dynamic real-time living cell intracellular imaging and quantification are important for understanding the pathophysiological processes. Thus, Cys probe that can permeate high efficiently is the first one to be affected. In fact, it is difficult for organic molecular probes to infiltrate cells because of the unique structure of the cell membrane. In this work, we found that probe containing-carboxyl just stagnated in cytomembrane due to carboxyl of probe and amino group of membrane protein forming peptide chains, nevertheless, the addition of NEM, improved membrane permeability by NEM reacting with sulfhydryl of membrane protein, which made probe permeate high efficiently and sequentially real-time detect the Cys in cytoplasm. It is the first time noted that NEM can regulate Cys probe containing-carboxyl for high efficient detection in cytoplasm. Additionally, probe was successfully applied to image Cys in mouse.

A new strategy for the fluorescence discrimination of Cys/Hcy and GSH/H2S simultaneously colorimetric detection for H2S.

The development of fluorescent probes enabling distinguishable detection Cys, Hcy, GSH and H2S is still a considerable challenge owing to their similar functional group with comparable reactivity. In this work, a novel fluorescent probe FHC-O-NBD has been synthesized, and a practicable strategy for the fluorescence discrimination of Cys/Hcy and GSH/H2S, especially the colorimetric detection for H2S have been presented. FHC-O-NBD reacted with Cys/Hcy to produce two fluorescent emissions at 486 nm and 550 nm, while for GSH/H2S, only one fluorescent signal at 486 nm appeared. And, only upon addition of H2S, the color of the system changed from colorless to pink. So it can serve as a colorimetric probe for H2S by "naked eye". Furthermore, FHC-O-NBD can selectively distinguish Cys/Hcy and GSH/H2S in living cells, meaning it has great potential in biological applications.

A novel strategy: A consecutive reaction was used to distinguish in the presence of statins between normal cells and cancer cells.

Statins, as the most commonly drugs could reduce the concentration of low-density lipoprotein cholesterol, have been proved to elevate the H2S generation in cells. Besides, the abnormal levels of biothiols might lead to cancer. Therefore, it is worth considering how to combine the characteristics of the two diseases to realize the detection of cancer cells. Based on this view, we developed a multiresponse fluorescent probe for the detection of hydrogen sulfide (H2S) and biothiols successively based on theoretical calculation. It is interesting that the fluorescence intensity of the probe reacting H2S and biothiols successively was significantly higher than that of probe reacting either of them. Based on this view, we further explored the biological application of the probe and found that the probe had obvious signal response to cancer cells than the normal cells in the presence of fluvastatin. This interesting finding might provide a new insight into cancer cell recognition.

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.

Four- and five-coordinate aluminum complexes supported by N,O-bidentate ß-pyrazylenolate ligands: synthesis, structure and application in ROP of ε-caprolactone and lactide.

In this paper we report a series of Al(iii) complexes supported by N,O-bidentate ß-pyrazyl functionalized enolate ligands HL1-HL5 (L = (6-Me-2,5-C4H2N2)-CH[double bond, length as m-dash]C(R)-O-), (R = tBu, Ph, p-tolyl, p-OMePh, o-tolyl) and their exploitation for the ring-opening polymerization of ε-caprolactone (ε-CL) and rac-lactide (rac-LA). The structures of the form LAlMe2 (1a-5a) or L2AlMe (1b-4b) were isolated depending on the ligand or stoichiometry of complexation. The investigation of these complexes toward the ROP of both ε-CL and rac-LA under the same conditions showed that dimethyl-aluminum complexes LAlMe2 (1a-5a) gave a higher activity than monomethyl-aluminum complexes L2AlMe (1b-4b). Meanwhile, monomethyl-aluminum complexes 1b-4b promoted the ring-opening polymerization with a better control over molecular weights and polydispersities than 1a-5a. Moreover, all of the LAlMe2 and L2AlMe produced PLA with strong isotactic bias (Pm up to 0.77) and narrow distributions. Generally, a ß-pyrazyl enolate system exhibited significantly higher catalytic activity for the ROP of ε-CL and rac-LA than the analogous ß-quinolyl enolate systems reported recently. The results are discussed in terms of electronic and steric properties affected by the substituents on the ligands.