Nanoscale Covalent Organic Frameworks with Donor-Acceptor Structures as Highly Efficient Light-Responsive Oxidase-like Mimics for Colorimetric Detection of Glutathione.

Although organic artificial enzymes have been reported as biomimetic oxidation catalysts and are widely used for colorimetric biosensors, developing organic artificial enzymes with high enzymatic activity is still a challenge. Two-dimensional (2D) covalent organic frameworks (COFs) have shown superior potential in biocatalysts because of their periodic π-π arrays, tunable pore size and structure, large surface area, and thermal stability. The interconnection of electron acceptor and donor building blocks in the 2D conjugated COF skeleton can lead to narrower band gaps and efficient charge separation and transportation and thus is helpful to improve catalytic activity. Herein, a donor-acceptor 2D COF was synthesized using tetrakis(4-aminophenyl)pyrene (Py) as an electron donor and thieno[3,2-b]thiophene-2,5-dicarbaldehyde (TT) as an electron acceptor. Under visible light irradiation, the donor-acceptor 2D COF exhibited superior enzymatic catalytic activity, which could catalyze the oxidation of chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) by the formation of superoxide radicals and holes. Based on the above property, the photoactivated donor-acceptor 2D COF with enzyme-like catalytic properties was designed as a robust colorimetric probe for cheap, highly sensitive, and rapid colorimetric detection of glutathione (GSH); the corresponding linear range of GSH was 0.4-60 µM, and the limit of detection was 0.225 µM. This study not only presents the construction of COF-based light-activated nanozymes for environmentally friendly colorimetric detection of GSH but also provides a smart strategy for improving nanozyme activity.

A solvent-assisted ESIPT fluorescent dye for F-/Ag+ sensing and high-resolution imaging of the cilia in live cells.

A solvent-assisted ESIPT fluorescent dye was synthesized and used as a probe (2-PPN) for the detection of F-/Ag+ and high-resolution imaging of the cilia in live cells. The developed ESIPT fluorophore exhibited strong tautomeric fluorescence in protic solvents and normal emission in aprotic solvents, which is a significant departure from that of conventional intramolecular ESIPT compounds. The H-binding interaction of F- and the chelation of Ag+ with the ESIPT module of 2-PPN resulted in significant tautomeric emission quenching. From this basis, the 2-PPN-based assays for the detection of F- and Ag+ were established. The detection limit for F- and Ag+ sensing is 2.4 nM and 1.5 nM, respectively. The selective experimental results showed that no tautomeric fluorescence change of 2-PPN could be observed in the presence of the other inorganic ions in the same medium, revealing high selectivity of 2-PPN to F- and Ag+. Furthermore, MTT assay experiments proved that the probe 2-PPN exhibited low cytotoxicity and good cell membrane permeability. The probe was also further successfully utilized to image the cilia in vitro MCF7 cells, displaying its high-resolution imaging performance.Graphical abstract.

Self-Assembled Pyrene Stacks and Peptide Monolayers Tune the Electronic Properties of Functionalized Electrolyte-Gated Graphene Field-Effect Transistors.

Aromatic molecules such as pyrenes are a unique class of building units for graphene functionalization, forming highly ordered π-π stacks while peptides provide more complex, biocompatible linkers. Understanding the adsorption and stacking behavior of these molecules and their influence on material properties is an essential step in enabling highly repeatable 2D material-based applications, such as biosensors, gas sensors, and solar cells. In this work, we characterize pyrene and peptide self-assembly on graphene substrates using fluorescence microscopy, atomic force microscopy and electrolyte-gated field-effect measurements supported by quantum mechanical calculations. We find distinct binding and assembly modes for pyrenes versus peptides with corresponding distinct electronic signatures in their characteristic charge neutrality point and field-effect slope responses. Our data demonstrates that pyrene- and peptide-based self-assembly platforms can be highly beneficial for precisely customizing graphene electronic properties for desired device technologies such as transport-based biosensing graphene field-effect transistors.

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.

Peptide Vectors Carry Pyrene to Cell Organelles Allowing Real-Time Quantification of Free Radicals in Mitochondria by Time-Resolved Fluorescence Microscopy.

Real-time quantification of reactive nitrogen and oxygen species (ROS) in cells is of paramount importance as they are essential for cellular functions. Their excessive formation contributes to the dysfunction of cells and organisms, ultimately leading to cell death. As ROS are mostly produced in the mitochondria, we have synthesized a fluorescent probe able to reach this organelle to detect and quantify, in real time, the variation of ROS by time-resolved microfluorimetry. The new probes are based on the long fluorescence lifetime of pyrene butyric acid (PBA). Two PBA isomers, attached at their 1- or 2-positions to a peptide vector to target mitochondria, were compared and were shown to allow the measurement of free radical species and oxygen, but not non-radical species such as H2 O2 .

Novel Pyrene Excimer and Fluorogenic Probe for the Detection of Alkylating Agents.

A pyrene-containing salicylic acid derivative (4) was found to be low in fluorescence, but its derivative pyrene-containing methyl salicylate (3) was found to be highly fluorescent in aqueous solution. This derivative has been tested in solution and found to be superior in the fluorogenic assay of pharmaceutical compounds, detection of chemical warfare agents, a preliminary toxicology test, mutagenicity of medicinal compounds, and other chemical analyses, including trimethylsilyl diazomethane; alkyl bromides and iodides; a sulfur mustard mimic 2-chloroethyl ethyl sulfide; and anticancer drugs, busulfan and pipobroman. The salicylic acid derivative (4) was applied as a fluorogenic probe for the detection of alkylating agents by esterification and generating fluorescence at 475 nm in solutions at low concentrations.

Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells.

Lipid droplets are dynamic subcellular organelles that participate in a range of physiological processes including metabolism, regulation and lipid storage. Their role in disease, such as cancer, where they are involved in metabolism and in chemoresistance, has emerged over recent years. Thus, the value of lipid droplets as diagnostic markers is increasingly apparent where number and size of droplets can be a useful prognostic. Although diverse in size, LDs are typically too small to be easily enumerated by conventional microscopy. The advent of super-resolution microscopy methods offers the prospect of detailed insights but there are currently no commercial STED probes suited to this task and STED, where this method has been used to study LDs it has relied on fixed samples. Here, we report a pyrene-based ceramide conjugate PyLa-C17Cer, that stains lipid droplets with exceptionally high precision in living cells and shows excellent performance in stimulated emission depletion microscopy. The parent compound PyLa comprises a pyrene carboxyl core appended with 3,4-dimethylaminophenyl. The resulting luminophore exhibits high fluorescent quantum yield, mega-Stokes shift and low cytotoxicity. From DFT calculations the Stokes shifted fluorescent state arises from a dimethylaminophenyl to pyrene charge-transfer transition. While the parent compound is cell permeable, it is relatively promiscuous, emitting from both protein and membranous structures within the living mammalian cell. However, on conjugation of C17 ceramide to the free carboxylic acid, the resulting PyLa-C17Cer, remains passively permeable to the cell membrane but targets lipid droplets within the cell through a temperature dependent mechanism, with high selectivity. Targeting was confirmed through colocalisation with the commercial lipid probe Nile Red. PyLa-C17Cer offers outstanding contrast of LDs both in fluorescence intensity and lifetime imaging due to its large Stokes shift and very weak emission from aqueous media. Moreover, because the compound is exceptionally photochemically stable with no detectable triplet emission under low temperature conditions, it can be used as an effective probe for fluorescence correlation spectroscopy (FCS). These versatile fluorophores are powerful multimodal probes for combined STED/FCS/lifetime studies of lipid droplets and domains in live cells.

Bispyrene-Based Self-Assembled Nanomaterials: In Vivo Self-Assembly, Transformation, and Biomedical Effects.

Self-assembled nanomaterials show potential high efficiency as theranostic agents for high-performance imaging and therapy. However, superstructures and properties of preassembled nanomaterials are somewhat compromised under complicated physiological conditions. Given the advantages of the dynamic nature and adaptive behavior of self-assembly systems, we propose an "in vivo self-assembly" strategy for in situ construction of nanomaterials in living objects. For the proof-of-concept study of in vivo self-assembly, we developed a bispyrene (BP) molecule as a multifunctional building block. BP molecules show nonfluorescence in the monomeric state. Quantum-chemical calculations indicate that BP forms twisted intramolecular charge transfer states, which are separated into two orthogonal units, preventing the fluorescence emission. Interestingly, the typical excimeric emission of BP is observed with the formation of J-type aggregates, as confirmed by single-crystal X-ray diffraction. Packing of the BP molecules generates parallel pyrene units that interact with adjacent ones in a slipped face-to-face fashion through intermolecular π-π interactions. BP and/or its amphiphilic derivatives are capable of self-aggregating into nanoparticles (NPs) in aqueous solution because of the hydrophobic and π-π interactions of BP. Upon specific biological stimuli, BP NPs can be transformed into variable self-assembled superstructures. Importantly, the self-assembled BP NPs exhibit turn-on fluorescence signals that can be used to monitor the self-assembly/disassembly process in vitro and in vivo. On the basis of the photophysical properties of BP and its aggregates, we synthesized a series of designed BP derivatives as building blocks for in situ construction of functional nanomaterials for bioimaging and/or therapeutics. We observed several new biomedical effects, e.g., (i) the assembly/aggregation-induced retention (AIR) effect, which shows improved accumulation and retention of bioactive nanomaterials in the regions of interests; (ii) the transformation-induced surface adhesion (TISA) effect, which means the BP NPs transform into nanofibers (NFs) on cell surfaces upon binding with specific receptors, which leads to less uptake of BP NPs by cells via traditional endocytosis pathway; and (iii) transformation of the BP NPs into NFs in the tumor microenvironment, showing high accumulation and long-term retention, revealing the transformation-enhanced accumulation and retention (TEAR) effect. In this Account, we summarize the fluorescence property and emission mechanism of BP building blocks upon aggregation in the biological environment. Moreover, BP-derived compounds used for in vivo self-assembly and transformation are introduced involving modulation strategies. Subsequently, unexpected biomedical effects and applications for theranostics of BP based nanomaterials are discussed. We finally conclude with an outlook toward future developments of BP-based self-assembled nanomaterials.

A simple pyrene-based fluorescent probe for highly selective detection of formaldehyde and its application in live-cell imaging.

A novel and simple pyrene-based fluorescent probe (B1) was synthesized and used for the detection of formaldehyde (FA). When B1 was added to FA in CH3CH2OH/HEPES (10 mM, pH = 7.4, 1:99, v/v) solution, the solution system strongly enhanced the fluorescence. Over various small molecular species, probe B1 showed high sensitivity and selectivity for detecting FA, with a low limit of detection (0.107 µM). Furthermore, probe B1 was successfully applied for visualizing FA in human embryonic kidney (HEK293T) cells, demonstrating its value of application in living biomedical samples.

PyA-cluster system for the detection and imaging of miRNAs in living cells through double-three-way junction formation.

Herein, we describe an extended version of a fluorescence probe for detecting miRNAs through the novel application of a PyA-cluster system. By testing various (CG)n sequences in the middle of the oligonucleotide strand of the probe, we obtained an optimal sequence that formed a double-three-way-junction structure, with two PyA units positioned close together, in the presence of the target miRNA. This system readily detected the locations of target miRNAs in living cells and allowed visualization of structural changes through variations in the color of the fluorescence.

Supramolecular nanofiber of pyrene-lactose conjugates and its two-photon fluorescence imaging.

A lactose modified pyrene derivative (Py-Lac) was synthesized, with which novel twisted supramolecular nanofibers in diameter about 20 nm were constructed by self-assembly. The nanofibers showed solid-state fluorescence between 400 nm and 650 nm with the maximum emission at 495 nm. Furthermore, its recognition reaction with PNA lectin was investigated by fluorescence spectra and turbidity assays. It is interesting found that the supramolecular assembly as multivalent glycocluster exhibited unique and selectively binding interactions with PNA lectin with the binding constant of 5.74 × 106 M-1. Moreover, compound Py-Lac showed two-photon fluorescence imaging with Hep G2 cells.

Exploring Orthogonal Hydrogen Bonding towards Designing Organic-Salt-Based Supramolecular Gelators: Synthesis, Structures, and Anticancer Properties.

A series of primary ammonium monocarboxylate (PAM) salts derived from ß-alanine derivatives of pyrene and naphthalene acetic acid, along with the parent acids, were explored to probe the plausible role of orthogonal hydrogen bonding resulting from amide⋅⋅⋅amide and PAM synthons on gelation. Single-crystal X-ray diffraction (SXRD) studies were performed on two parent acids and five PAM salts in the series. The data revealed that orthogonal hydrogen bonding played an important role in gelation. Structure-property correlation based on SXRD and powder X-ray diffraction data also supported the working hypothesis upon which these gelators were designed. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell migration assay on a highly aggressive human breast cancer cell line, MDA-MB-231, revealed that one of the PAM salts in the series, namely, PAA.B2, displayed anticancer properties, and internalization of the gelator salt in the same cell line was confirmed by cell imaging.

Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Anti-proliferative and nuclease activities.

A series of tripodal ligands based on the 2-tert-butyl-4-R-6-phenol was synthesized, where R=aldehyde (HL1), R=putrescine-pyrene (HL2) and R=putrescine (HL3). A dinucleating ligand wherein a putrescine group connects two tripodal moieties was also prepared (H2L4). The corresponding copper complexes (1, 2, 3, and 4, respectively) were prepared and characterized. We determined the phenol's pKas in the range 2.47-3.93. The DNA binding constants were determined at 6×106, 5.5×105 and 2.7×106 for 2, 3 and 4, respectively. The complexes display a metal-centered reduction wave at Epc,red=-0.45 to -0.5V vs. saturated calomel electrode, as well as a ligand-centered oxidation wave above 0.57V at pH7. In the presence of ascorbate they promote an efficient cleavage of DNA, with for example a concentration required to cleave 50% of supercoiled DNA of 1.7µM for 2. The nuclease activity is affected by the nature of the R group: putrescine-pyrene≈bis-ligating>putrescine>aldehyde. The species responsible for strand scission is the hydroxyl radical. The cytotoxicity of the complexes was evaluated on bladder cancer cell lines sensitive or resistant to cis-platin. The IC50 of complexes 2 and 4 span over a short range (1.3-2µM) for the two cell lines. They are lower than those of the other complexes (3.1-9.7µM) and cis-platin. The most active compounds block the cell cycle at the G0/1 phase and promote apoptosis.

Aggregate Formation of Oligonucleotides that Assist Molecular Imaging for Tracking of the Oxygen Status in Tumor Tissue.

The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.

Comparative binding to DR4 and DR5 receptors of TRAIL and BNNTs/PAHE/mPEG-DSPE/TRAIL nanoparticles.

TRAIL is a member of the tumor necrosis factor family of cytokines, which induces apoptosis of cancer cells, thanks to its binding to its cognate receptors DR5 and DR4. We have recently demonstrated that nanovectorization of TRAIL with single-walled carbon nanotubes enhanced TRAIL affinity to DR5. In this paper, 1-pyrenebutyric acid N-hydroxysuccinimide ester functionalized boron nitride nanotubes (BNNTs) were used to anchor the TRAIL protein. The resulting BNNT/1-pyrenebutyric acid N-hydroxysuccinimide ester nanotubes were mixed with methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-conjugates so as to allow a good dispersion of these nanoparticle TRAIL (NPT) in aqueous solution. The difference of binding between NPT and soluble TRAIL to DR4 and DR5 receptors was then studied by the use of affinity chromatography. DR4 and DR5 receptors were thus immobilized on a chromatographic support, and the binding of the 2 ligands TRAIL and NPT to DR4 and DR5 was studied in the temperature range 30°C to 50°C. Negative enthalpy (ΔH) values indicated that van der Waals interactions and hydrogen bonding are engaged favorably at the ligand-receptor interface. It was shown that their rank-ordered affinities were strongly different in the sequence TRAILDR4  < NPTDR4  < TRAILDR5  < NPTDR5 , and the highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on BNNTs. Taken together, our results demonstrated that nanovectorization of TRAIL with BNNTs enhanced its binding to both DR4 and DR5 receptors at 37°C. Our novel nanovector could potentially be used for delivering TRAIL to cells for cancer treatment.

Photochemically-assisted synthesis and photophysical properties of difluoroboronated ß-diketones with fused four-benzene-ring chromophores, chrysene and pyrene.

We investigated the photophysical properties of difluoroboronated ß-diketones (BF2DK) with chrysene and pyrene skeletons (ChB and PyB, respectively) in solution and in the solid state. Acetylchrysenes, as the key precursors to ChBs, were photochemically prepared from the corresponding (acetylphenyl)naphthylethenes by means of a modified photocyclization method. The absorption and emission spectra of the BF2DKs were obtained in chloroform and acetonitrile, and the quantum yields and lifetimes of the fluorescence were determined. Excimeric fluorescence from PyB was absent even in highly concentrated solution. Based on the Lippert-Mataga analysis of the absorption and fluorescence features, the photophysical properties of the ChBs were discussed in comparison with those of PyB. The fluorescence states of the studied BF2DKs are shown to be of a charge-transfer character. The fluorescence quantum yields decrease with increasing the solvent polarity due to the enhanced internal conversion process. The fluorescence quantum yields in the solid state of the studied BF2DKs were determined, and it was found that PyB is fluorescent, whereas the fluorescence quantum yields of the ChBs depend on the substituted position of the chrysene moiety.

Spirolactone and spirothiolactone rhodamine-pyrene probes for detection of Hg²âº with different sensing properties and its application in living cells.

Two new rhodamine B-based fluorescent probes PyRbS and PyRbO containing pyrene moiety were designed and synthesized. Both of the probes showed colorimetric and fluorometric sensing abilities for Hg(2+) with high selectivity over other metal ions. The binding analysis using Job's plot suggested 1:1 stoichiometry for the complexes formed for Hg(2+). Compared with PyRbO, the PyRbS showed higher selectivity and sensitivity due to the thiophilic property of Hg(2+) ion. The PyRbS exhibited the linear fluorescence quenching to Hg(2+) in the range of 0.3 to 4.8 µM (λ(ex)=365 nm) and 0.3 to 5.4 µM (λ(ex)=515 nm), and the detection limit was 0.72 µM. Moreover, ratiometric changes of PyRbS with Hg(2+) in absorption spectrum were observed, which could not be obtained in the combination of PyRbO with Hg(2+). In addition, the methyl thiazolyl tetrazolium (MTT) assay demonstrated that RbPyS had low cytotoxicity and was successfully used to monitor intracellular Hg(2+) levels in living cells.

Enhancement of TFO Triplex Formation by Conjugation with Pyrene via Click Chemistry.

This paper reports the preparation of 14-mer triplex-forming oligonucleotides (TFOs) containing a 2-O-methyl-1-ß-phenyl-α-propargyl-ribose unit, which was conjugated with azide-modified molecules via a click reaction. Modification of these TFOs with pyrene assisted triplex formation, improving the stability of the triplex DNA and the anti-proliferative effects against A549 cells.

Synthesis and evaluation of a novel Rhodamine B pyrene [2]rotaxane as an intracellular delivery agent for doxorubicin.

A novel Rhodamine B-derivatized host [2]rotaxane, containing a dibenzyl-24-crown-8 (DB24C8) ring as the wheel and a pyrene as another fluorophore blocking group, was designed, synthesized and structurally characterized. A comparison of the (1)H NMR spectra of RhBPy [2]rotaxane with those of and DB24C8, nuclear Overhauser effect spectroscopy (NOESY), mass spectrometry and fluorescence spectroscopy confirmed the interlocked nature of RhBPy [2]rotaxane. The temperature dependence of the rotaxane studied by (1)H NMR spectroscopy further demonstrated that RhBPy [2]rotaxane can be applied as a molecular switch. RhBPy [2]rotaxane has also been demonstrated to be an efficient transport agent for delivering the cancer drug doxorubicin (DOX) into tumor cells. Indeed, DOX delivered by RhBPy [2]rotaxane could effectively inhibit tumor cell growth.

Design, synthesis and biological evaluation of novel pyrenyl derivatives as anticancer agents.

Polycyclic aromatic hydrocarbons are widespread in nature with a toxicity range from non-toxic to extremely toxic. A series of pyrenyl derivatives has been synthesized following a four-step strategy where the pyrene nucleus is attached with a basic heterocyclic moiety through a carbon linker. Virtual screening of the physicochemical properties and druggability has been carried out. The cytotoxicity of the compounds (1-8) have been evaluated in vitro against a small panel of human cancer cell lines which includes two liver cancer (HepG2 and Hepa 1-6), two colon cancer (HT-29 and Caco-2) and one each for cervical (HeLa) and breast (MCF-7) cancer cell lines. The IC50 data indicate that compound 6 and 8 are the most effective cytotoxic agents in the present set of pyrenyl derivatives, suggesting that having a 4-carbon linker is more effective than a 5-carbon linker and the presence of amide carbonyl groups in the linker severely reduces the efficacy of the compound. The compounds showed selectivity toward cancer cells at lower doses (<5 µM) when compared with the normal hepatocytes. The mechanism of action supports the cell death through apoptosis in a caspase-independent manner without cleavage of poly (ADP-ribose) polymerase (PARP), even though the compounds cause plasma membrane morphological changes. The compounds, whether highly cytotoxic or mildly cytotoxic, localize to the membrane of cells. The compounds with either a piperidine ring (6) or an N-methyl piperazine (8) in the side chain were both capable of circumventing the drug resistance in SKOV3-MDR1-M6/6 ovarian cancer cells overexpressing P-glycoprotein. Qualitative structure-activity relationship has also been studied.