dicyanomethylene-4H-pyran Research Articles

Discovery of a novel photosensitizer based on the enediyne antibiotic N1999A2 and its application as a glutathione-activatable theranostic agent

Abstract The 2-naphthol derivative 2, which corresponds to the aromatic moiety of the enediyne antibiotic N1999A2, was found to degrade protein under irradiation with long-wavelength UV light in the absence of any additives. Structure–activity relationship studies of 2 indicated that 3, in which the primary hydroxyl group at the C5 position of 2 is modified with a t-butyldiphenylsilyl group, has strong protein photodegradation ability. Furthermore, the theranostic molecule 5 was designed and synthesized. Compound 5 comprises a disulfide moiety linked to the hydroxyl group at the C2 position of 3 and to the fluorescent molecule dicyanomethylene-4H-pyran (DCM) chromophore derivative 6. The disulfide moiety is cleaved in the presence of glutathione (GSH), 5 showed significantly reduced photolytic activity and fluorescence compared to 3 and 6, but produced 3 and 6 when reacted with GSH. 5 showed selective fluorescence and photocytotoxicity against cancer cells that highly express GSH.

Amphiphilic Molecules Exhibiting Zwitterionic Excited-State Intramolecular Proton Transfer and Near-Infrared Emission for the Detection of Amyloid β Aggregates in Alzheimer's Disease.

Chromophores with zwitterionic excited-state intramolecular proton transfer (ESIPT) have been shown to have larger Stock shifts and red-shifted emission wavelengths compared to the conventional π-delocalized ESIPT molecules. However, there is still a dearth of design strategies to expand the current library of zwitterionic ESIPT compounds. Herein, a novel zwitterionic excited-state intramolecular proton transfer system is reported, enabled by addition of 1,4,7-triazacyclononane (TACN) fragments on a dicyanomethylene-4H-pyran (DCM) scaffold. The solvent-dependent steady-state photophysical studies, pKa measurements, and computational analysis strongly support that the ESIPT process is more efficient with two TACN groups attached to the DCM scaffold and not affected by polar protic solvents. Impressively, compound DCM-OH-2-DT exhibits a near-infrared (NIR) emission at 740 nm along with an uncommonly large Stokes shift. Moreover, DCM-OH-2-DT shows high affinity towards soluble amyloid β (Aβ) oligomers in vitro and in 5xFAD mouse brain sections, and we have successfully applied DCM-OH-2-DT for the in vivo imaging of Aβ aggregates and demonstrated its potential use as an early diagnostic agent for AD. Overall, this study can provide a general molecular design strategy for developing new zwitterionic ESIPT compounds with NIR emission in vivo imaging applications.

Desirable photo-physicochemical properties of dicyanomethylene-4H-pyran probe enables long-term fluorescent tracking of lysosomal dynamics in live cells

Most currently used small molecular lysosomal dyes usually fail in tracing the lysosomal long-term dynamics because of their inappropriate photophysical and photochemical properties, such as pH dependency and weak photostability. Therefore, it is of great interest to develop other lysosomal probes with desired photo-physicochemical properties for visualizing and tracking lysosomal dynamics. Here, the popular dicyanomethylene-4H-pyran (DCM) derivative DCM-OH was found to image lysosomes selectively with bright far-red fluorescence. Importantly, DCM-OH imaging was demonstrated to be pH-insensitive, highly photostable and low (photo-) cytotoxic, and it also exhibited high resistance to various other lysosomal microenvironmental changes (polarity and viscosity). All these properties enabled its qualification for long-term trace of lysosomal dynamics in live cells. At last, the applications of DCM-OH in photo-tracking lysosomal dynamic events, including fusion-fission, kiss-and-run process and mitophagy, were successfully observed. Therefore, the study demonstrates DCM-OH molecule is highly qualified for fluorescent visualization and real-time trace of lysosomes by overcoming the limitations of most commercial Lysosomal dyes thanks to its desirable photo-physicochemical properties.

A theranostic abscisic acid-based molecular glue.

Molecular glues, capable of selectively controlling the interactions between specific pairs or groups of proteins and the associated downstream effects, have become a promising strategy for manipulating cellular functions and developing novel therapies for human diseases. Theranostics with both diagnostic and therapeutic capabilities acting at disease sites has become a powerful tool to achieve both functions simultaneously with high precision. To selectively activate molecular glues at the desired site and monitor the activation signals at the same time, here we report an unprecedented theranostic modular molecular glue platform integrating signal sensing/reporting and chemically induced proximity (CIP) strategies. We have demonstrated for the first time the integration of imaging and activation capacity with a molecular glue on the same platform to create a theranostic molecular glue. A theranostic molecular glue ABA-Fe(ii)-F1 was rationally designed by conjugating a NIR fluorophore dicyanomethylene-4H-pyran (DCM) with a CIP inducer abscisic acid (ABA) using a unique carbamoyl oxime linker. We have also engineered a new version of ABA-CIP with an enhanced ligand-responding sensitivity. We have validated that the theranostic molecular glue can sense Fe2+ and produce turn-on NIR fluorescence for monitoring as well as releasing the active inducer ligand to control cellular functions including gene expression and protein translocation. This novel molecular glue strategy paves the way to building a new class of molecular glues with theranostic capacity for research and biomedical applications.

Metabolically Specific In Situ Fluorescent Visualization of Bacterial Infection on Wound Tissues.

The ability to effectively detect bacterial infection in human tissues is important for the timely treatment of the infection. However, traditional techniques fail to visualize bacterial species adhered to host cells in situ in a target-specific manner. Dihydropteroate synthase (DHPS) exclusively exists in bacterial species and metabolically converts p-aminobenzoic acid (PABA) to folic acid (FA). By targeting this bacterium-specific metabolism, we have developed a fluorescent imaging probe, PABA-DCM, based on the conjugation of PABA with a long-wavelength fluorophore, dicyanomethylene 4H-pyran (DCM). We confirmed that the probe can be used in the synthetic pathway of a broad spectrum of Gram-positive and negative bacteria, resulting in a significantly extended retention time in bacterial over mammalian cells. We validated that DHPS catalytically introduces a dihydropteridine group to the amino end of the PABA motif of PABA-DCM, and the resulting adduct leads to an increase in the FA levels of bacteria. We also constructed a hydrogel dressing containing PABA-DCM and graphene oxide (GO), termed PABA-DCM@GO, that achieves target-specific fluorescence visualization of bacterial infection on the wounded tissues of mice. Our research paves the way for the development of fluorescent imaging agents that target species-conserved metabolic pathways of microorganisms for the in situ monitoring of infections in human tissues.

Fluorescence Enhancement of Dicyanomethylene-4H-Pyran Derivatives in Solid State for Visualization of Latent Fingerprints.

The efficient development of latent fingerprint (LFP) is attractively important for criminal investigation. The low-cost and high-contrast developer is still a challenge. In this study, we designed and synthesized dicyanomethylene-4H-pyran (DCM) derivatives PZ-DCM and Boc-PZ-DCM by introducing of large steric hindrance group Boc, the solid-state fluorescence of DCM derivatives was greatly enhanced. The low-cost fluorescent LFP developers were prepared by blending with different proportion of montmorillonite (MMT). As a result, clear and high contrast fingerprint patterns were obtained with dusting method by the developer with 3% content of Boc-PZ-DCM. Furthermore, we employed the developer with 3% content of Boc-PZ-DCM to develop the sweat latent fingerprints on different substrates by powder dusting, and collected clear fingerprint patterns, indicating that the developer is universal. In a word, the Boc-PZ-DCM/MMT powder is a promising candidate for LFP developer.

Progress of Dicyanomethylene-4H-Pyran Derivatives in Biological Sensing Based on ICT Effect

As one of the typical fluorescent cores, dicyanomethylene-4H-pyran (DCM) derivatives exhibit excellent photophysical and photochemical properties, such as large Stokes shift, excellent light stability, and tunable near-infrared (NIR) emission. The luminescence mechanism of DCM probes mainly depends on the intramolecular charge transfer (ICT). Hence, by regulating the ICT process, the probes can specifically act on the target molecule. Accordingly, a series of NIR DCM probes have been constructed to detect the ions, reactive oxygen species (ROS), and biological macromolecules in cells. However, there is no relevant review to summarize it at present. This minireview mainly summarizes the NIR DCM probes based on ICT effect and their applications in biosensors and biological imaging in recent years. This will be beneficial to innovatively construct new DCM probes and actively promote their application in the future.

A new near-infrared fluorescence sensor based on dicyanomethylene-4H-pyran for the detection of Al3+, Cr3+, Fe3+ and Cu2+

A novel near-infrared (NIR) fluorescence sensor DCM-PSI based on dicyanomethylene-4H-pyran(DCM) was designed and synthesized to selectively detect Al3+, Cr3+, Fe3+ and Cu2+. It had a large Stokes shift (λabs = 450 nm, λex = 490 nm, λem = 690 nm), low detection limits and large binding constants. With the addition of Al3+, Cr3+, Fe3+ and Cu2+, the fluorescence quenched and the emission peaks blue shifted to different extent. Experimental analysis and quantum chemical calculations showed that the luminescence of DCM-PSI experienced an excited state intramolecular proton transfer (ESIPT) and partly intramolecular charge transfer (ICT) process, while the complexation of Al3+, Cr3+, Fe3+ and Cu2+ caused ligand-to-metal electron transfer, and altered the “push–pull” effect in DCM-PSI. Furthermore, the Al3+ and Fe3+ in drinking water was also measured with DCM-PSI to test the practical application of the sensor.

A double donor-π-acceptor type hydrogen sulfide fluorescent probe with nanomolar level sensitivity and second level response time for evaluating metformin-induced hepatotoxicity

A dicyanomethylene-4H-pyran (DCM)-based fluorescent probe capable of hydrogen sulfide (H2S) sensing with limit of detection down to 3.89 nM, response time less than 1 s, two-photon excitation fluorescence (TPEF) feature has been developed. This probe was constructed by tethering an electron-withdrawing nitrobenzooxadiazole (NBD) moiety to the skeleton periphery of an amine-π-DCM system via ether bond. The H2S-mediated cleavage of NBD ether bond restored the electron-donating ability of amine and simultaneously generated an electron-donating phenol moiety, which enabled significantly strengthened π-delocalization over the D-π-A molecule skeleton of the reaction product and therefore turn-on fluorescence with tremendous contrast for H2S sensing with unprecedented sensitivity. The charge distribution calculation results of the probe molecule indicated the low electron density of the target carbon atom vulnerable to nucleophilic attack, which was expected to facilitate H2S-mediated nucleophilic substitution reaction and therefore contribute to the unprecedented response rate of the probe to H2S. The superior performance of such probe in terms of recognition specificity, biocompatibility and TPEF for bioimaging with satisfactory depth in mapping H2S level in physiological milieu was verified. The superior advantages of such probe regarding detection sensitivity and response rate as well as the preliminary in vitro experiment results regarding mapping the H2S level in liver organ unequivocally proclaimed its potential for reliable detection of highly elusive H2S species, a potential footprint for evaluating metformin-induced hepatotoxicity, and diagnosis of H2S-associated liver injury in practical applications.

Highly sensitive light-up near-infrared fluorescent probe for detection and imaging of β-glucuronidase in human serum, living cells and tumor-bearing mice

β-Glucuronidase (GUS) plays a key role in tumor initiation, metastasis, and progression, and thus, has been proposed as a promising cancer biomarker. In this study, we designed an enzyme-activatable near-infrared (NIR) fluorescent probe (DCM-βGlcA) for the rapid and accurate detection of GUS activity in vitro, in vivo and ex vivo. The DCM-βGlcA was prepared by linking a glucuronic acid residue to dicyanomethylene-4H-pyran (DCM). This probe exhibited significant light-up NIR fluorescent signals at 680 nm after reacting with GUS and the Stokes shift could reach 150 nm. The DCM-βGlcA showed a high sensitivity toward GUS and an excellent linear relationship at concentrations ranging between 0 and 4 U L−1 (R2 = 0.9974) with the limit of detection as low as 0.19 U L−1. We used the DCM-βGlcA to identify GUS serum levels in both cancer patients and healthy individuals with a similar accuracy as that of an enzyme-linked immunosorbent assay (ELISA) while being easier and faster to perform. Moreover, the DCM-βGlcA was used for tracking endogenous GUS in living cells, thereby discriminating GUS-overexpressed liver cancer from normal cells. Additionally, the DCM-βGlcA was able to detect and image endogenous GUS in liver cancer tissue and tumor-bearing mouse models. These findings demonstrate the potential of the DCM-βGlcA as a promising tool for detecting and monitoring GUS activity in preclinical applications.

Indication of Dynamic Peroxynitrite Fluctuations in the Rat Epilepsy Model with a Near-Infrared Two-Photon Fluorescent Probe.

Epilepsy is a chronic neurodegenerative disease that has seriously threatened human health. Accumulating evidence reveals that the pathological progression of epilepsy is closely related to peroxynitrite (ONOO-). Unfortunately, understanding the physiological roles of ONOO- in epilepsy is still challenging due to the lack of powerful imaging probes for the determination of the level of fluctuations of ONOO- in the epileptic brain. Herein, a near-infrared (NIR) two-photon (TP) fluorescent probe [dicyanomethylene-4H-pyran (DCM)-ONOO] is presented to trace ONOO- in living cells and in kainate (KA)-induced rat epilepsy models with satisfactory sensitivity and selectivity. The probe is composed of a NIR TP DCM fluorophore and a recognition moiety diphenylphosphinamide. The phosphoramide bond of the probe is interrupted after reacting with ONOO- for 10 min, and then, the released amino groups emit strong fluorescence due to the restoration of the intramolecular charge transfer process. The probe can effectively detect the changes of endogenous ONOO- with excellent temporal and spatial resolution in living cells and in rat epileptic brain. The imaging results demonstrate that the increasing level of ONOO- is closely associated with epilepsy and severe neuronal damage in the brain under KA stimulation. In addition, the low-dose resveratrol can effectively inhibit ONOO- overexpression and further relieve neuronal damage. With the assistance of TP fluorescence imaging in the epileptic brain tissue, we hypothesize that the abnormal levels of ONOO- may serve as a potential indicator for the diagnosis of epilepsy. The TP fluorescence imaging based on DCM-ONOO provides a great potential approach for understanding the epilepsy pathology and diagnosis.

Highly Photostable Fluorescent Tracker with pH-Insensitivity for Long-Term Imaging of Lysosomal Dynamics in Live Cells

Visualizing and tracking lysosomal dynamic changes is crucially important in the fields of physiology and pathology. Most currently used pH-dependent small-molecule lysotrackers and sensors usually fail to visualize and track the changes due to (1) their leakage from lysosomes when the lysosomal pH increases and (2) their low photostability. Therefore, it is of significant interest to develop lysosomal probes for visualizing and tracking lysosomal dynamics independent of pH fluctuations and with high photostability. Herein, we found that the popular dicyanomethylene-4H-pyran (DCM) derivative DCM-NH2 can selectively target and label lysosomes with bright red fluorescence regardless of pH changes. The fluorescence enhancement in lysosomes has probably resulted from their microenvironment of polarity and viscosity. Compared with the commonly used commercial lysosomal molecular probes (LysoTracker Deep Red (LTDR) and LysoTracker Red DND-99), DCM-NH2 was demonstrated to exhibit a much stronger tolerance in lysosomes against various treatments and microenvironmental changes, and lysosomal membrane permeability could not cause DCM-NH2 to lose imaging of their targets as well. Moreover, DCM-NH2 exhibited a superior anti-photobleaching ability and low (photo-) cytotoxicity, which, along with pH-insensitivity, ensured its capability of long-term visualizing and tracking lysosomal dynamics. Lysosomal dynamic events such as the kiss-and-run process, fusion-fission, and mitophagy were successfully recorded with DCM-NH2. Our study thus confirms that DCM-NH2 is highly competitive for lysosomal imaging by overcoming the limitations of the commercial LysoTrackers and highlights the unexplored application of DCM-NH2 in bioimaging.

A novel DCM-based NIR fluorescent probe for detecting ozone and its bioimaging in live cells

Nowadays, ozone has been widely applied in industry and medical therapies. However, excessive exposure to ozone can lead to lung dysfunction and many respiratory symptoms. As a member of reactive oxygen species (ROS), ozone was also involved in various physiology and pathology process. Given the fact of this, the effective detection of ozone in the atmosphere and biological system is of vital significance. Herein, we reported a novel dicyanomethylene-4H-pyran (DCM)-based fluorescent probe DCM-O3 with butenyl being the recognition moiety for monitoring ozone. The probe displayed high selectivity towards ozone, and its response towards ozone could be completed within 5 min under the optimal condition. Besides, a good linear correlation was obtained between the ozone concentrations (0–50 μM) and the corresponding fluorescent intensity at 560 nm, and the limit of detection (LOD) was calculated to be 6.2 × 10−7 M. Moreover, the probe DCM-O3 showed low cytotoxicity and was successfully applied to detect ozone in live cells. Given all the merits, the probe DCM-O3 could function as a robust tool for researchers to investigate ozone-related diseases in the complex biological environment.

Supramolecular Assembly of TPE-Based Glycoclusters with Dicyanomethylene-4H-pyran (DM) Fluorescent Probes Improve Their Properties for Peroxynitrite Sensing and Cell Imaging.

Two red-emitting dicyanomethylene-4H-pyran (DM) based fluorescent probes were designed and used for peroxynitrite (ONOO- ) detection. Nevertheless, the aggregation-caused quenching effect diminished the fluorescence and restricted their further applications. To overcome this problem, tetraphenylethylene (TPE) based glycoclusters were used to self-assemble with these DM probes to obtain supramolecular water-soluble glyco-dots. This self-assembly strategy enhanced the fluorescence intensity, leading to an enhanced selectivity and activity of the resulting glyco-dot comparing to DM probes alone in PBS buffer. The glyco-dots also exhibited better results during fluorescence sensing of intracellular ONOO- than the probes alone, thereby offering scope for the development of other similar supramolecular glyco-systems for chemical biological studies.

A dicyanomethylene-4H-pyran-based fluorescence probe with high selectivity and sensitivity for detecting copper (II) and its bioimaging in living cells and tissue.

Copper (Cu) plays a significant role in the process of oxygenic photosynthesis in living systems. The detection of copper ion (Cu2+) is valuable and meaningful for further investigating the functions of Cu2+ under physiological and pathological conditions. In this paper, a novel fluorescence probe DCM-Cu based on the near-infrared (NIR) fluorophore dicyanomethylene-4H-pyran (DCM) was designed for Cu2+ detection. The probe DCM-Cu possessed characteristic of "turn-on" fluorescent signal in the presence of Cu2+ through the enhanced ICT process. It exhibited satisfactory sensitivity and selectivity toward Cu2+. A good linear correlation was observed between the concentrations of Cu2+ and the fluorescence intensities at 700nm. The detection limit (LOD) of DCM-Cu toward Cu2+ was calculated to be 2.54×10-8M. Importantly, DCM-Cu was successfully applied in the detection of Cu2+ in living MCF-7 cells and tumor tissue with low cytotoxicity. Therefore, this probe would have the potential to monitor cellular Cu2+ in the living system and be applied to the diagnosis of related diseases.

Enhanced cellular uptake efficiency of DCM probes or SN38 conjugating with phenylboronic acids

High-level of sialic acid (SA) expression on the surface of cancer cells is observed extremely common. Phenylboronic acids (PBAs) have a high affinity with SA. The cellular uptake efficiency could be enhanced by the strategy of introducing PBA fragments to the compounds. In this work, we synthesized five new probes with the Dicyanomethylene-4H-pyran (DCM) fluorophore, three of them conjugated with different phenylboronic acid fragments. By cellular uptake experiments, DLCB and DLAB showed enhanced cellular uptake abilities compared with DLN and DLO. These two effective phenylboronic acid fragments were then conjugated with SN-38 and the conjugates showed enhanced cellular uptake abilities by 3-fold or 7-fold compared with irinotecan. In summary, the strategy of introducing 4-carboxyphenylboronic acid and 3-amino-benzoxaborole groups shows great potential in drug delivery system. Moreover, the released linkers between boric acid and drugs deserve further studies.

A highly selective and ultrafast near-infrared fluorescent turn-on and colorimetric probe for hypochlorite in living cells

Hypochlorous acid (HOCl)/hypochlorite (OCl−), important reactive oxygen species, play essential roles in many physiological and pathological progresses. Accordingly, we developed a novel dicyanomethylene-4H-pyran (DCM)-based probe DCM-OCl for colorimetric and near-infrared fluorescent turn-on detection of OCl−. The probe exhibited excellent selectivity and sensitivity for OCl− over other bio-related analytes with a detection limit of 80 nM. The excellent selectivity of DCM-OCl for OCl− was ascribed to specific oxidative cleavage of the dimethylthiocarbamate (DMTC) recognition unit by OCl−. Moreover, DCM-OCl exhibited an ultrafast turn-on response (<3 s) to OCl−, potentially allowing real-time detection of OCl−. Furthermore, DCM-OCl was successfully used to image endogenous/exogenous OCl− in living cells.

Near-infrared fluorescent probe for imaging nitroxyl in living cells and zebrafish model

Nitroxyl (HNO) is one of the important derivatives of nitric oxide (NO). It was intertwined with various biological and pharmacological events as a well-defined active molecule. Developing fluorescent probes for high specific and in situ trapping of HNO in living samples is still challenging. In this project, we constructed a near-infrared (NIR) metal-free fluorescent probe (DCM-P) for monitoring HNO in vitro and in vivo. The novel probe, DCM-P, contains a dicyanomethylene-4H-pyran (DCM) fluorophore as the reporter and a triarylphosphine as the recognition moiety. Upon exposure to HNO, the probe emits fluorescence at the wavelength of 688 nm, which belongs to near-infrared region and endows great beneficial for imaging in vivo. Moreover, DCM-P shows high chemoselective detection and fast response to HNO in the presence of various biological relevant reductants, and is successfully employed to trap nitroxyl in different living cells and zebrafish models. These results suggest that DCM-P is a suitable near-infrared metal-free fluorescent probe for motoring nitroxyl in living samples.

FRET-Based Upconversion Nanoprobe Sensitized by Nd3+ for the Ratiometric Detection of Hydrogen Peroxide in Vivo.

The exorbitant level of hydrogen peroxide is closely related to many human diseases. The development of novel probes for H2O2 detection will be beneficial to disease diagnosis. In this study, a novel Nd3+-sensitized upconversion nanoprobe based on Förster resonance energy transfer was first developed for sensing H2O2. This nanosystem was made of core-shell upconversion nanoparticles (emission at 540 and 660 nm), dicyanomethylene-4 H-pyran (DCM)-H2O2, and poly acrylic acid (PAA)-octylamine. Obviously, upconversion nanoparticles (UCNPs) doped with Nd3+ acted as an energy donor, and DCM-H2O2, transferring to DCM-OH with the reaction of H2O2, acted as an energy acceptor. The ratiometric upconversion luminescence (540 nm/660 nm) signal could be utilized to visualize the H2O2 level, and the LOD of the nanoprobe for H2O2 was quantified to be 0.168 μM. Meanwhile, owing to the dope of Nd3+, the nanoprobe would not induce the overheating effect in biological samples and could possess deeper tissue penetration depth, compared with the UCNPs excited by 980 nm light during bioimaging. The nanoprobe could also play an important role in detecting the exogenous and endogenous H2O2 in living cells with ratiometric UCL (upconversion luminescence) imaging. Furthermore, our nanoprobe could function in detecting the H2O2 in a tumor-bearing mouse model. Therefore, this novel nanoprobe along with the ratiometric method for responding and bioimaging H2O2 could serve as a new model that promotes the emergence of novel probes for H2O2 detection.

Triphenyl phosphate end-capped dicyanomethylene-4H-pyran as a near infrared fluorescent sensor for lysozyme in urine sample

A dicyano-methylene-4H-pyran (DCM)-based fluorescent probe DCPOP was designed for detecting lysozyme in urine sample. DCPOP showed obvious NIR emission (> 650 nm) avoiding the background fluorescence of urine (450 ˜ 600 nm). Compared to its mimic (DCPO), DCPOP has a big end-capped triphenyl phosphate (TPP) moiety, which induced good stability in several aqueous with different pH value. Furthermore, the end-capped TPP moiety showed weak interaction with lysozyme, which could modulate the emission signals of DCPOP. As a result, DCPOP could detect the lysozyme in both PBS buffer and real urine samples.