Recognition Moiety Research Articles

Europium(III) Macrocyclic Chelates Appended with Tyrosine-based Chromophores and Di-(2-picolyl)amine-based Receptors: Turn-On Luminescent Chemosensors Selective to Zinc(II) Ions.

Zinc ions play an important role in many biological processes in the human body. To selectively detect Zn2+ , two EuDO3A-based complexes (DO3A=1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid) appended with tyrosine as a chromophore and di-(2-picolyl)amine (DPA) as the Zn2+ recognition moiety were developed as suitable luminescent sensors. Their luminescence intensity is affected by the photoinduced electron transfer mechanism. Upon addition of Zn2+ , both probes display an up to sevenfold enhancement in Eu3+ emission. Competition experiments demonstrated their specificity toward Zn2+ over other metal ions, while also revealing the nonspecificity of the derivatives lacking the DPA-moiety, thus confirming the essential role of the DPA for the recognition of Zn2+ . The induced emission changes of Eu3+ allow for precise quantitative analysis of Zn2+ , establishing these lanthanide-based complexes as viable chemosensors for biological applications.

Selectively Light-up Detection of Phosgene with an Aggregation-Induced Emission-Based Fluorescent Sensor.

Phosgene, a widely used but highly toxic substance, may pose a serious risk to public safety and health because of the potential abuse and possible accidental leakage. Consequently, it is of great significance to develop a rapid, reliable, and sensitive detection method for this noxious agent. In this work, an aggregation-induced emission-based sensor, 3,6-bis(1,2,2-triphenylvinyl)benzene-1,2-diamine (DATPE), has been rationally designed for detecting phosgene by conjugation of o-phenylenediamine (OPD) core as the reactive recognition moiety decorated with two peripheral triphenylethylene (TPE) units. A light-up fluorescence response is achieved by the fast cyclization reaction of OPD part and phosgene along with the formation of 2-imidazolidinone ring, thus inhibiting the intramolecular charge transfer quenching process in the sensor. Moreover, an easy-to-use test paper with DATPE is fabricated for onsite visual detection of phosgene in the gas phase even at a concentration of as low as 0.1 ppm.

Fluorescent Single-Walled Carbon Nanotubes for Protein Detection.

Nanosensors have a central role in recent approaches to molecular recognition in applications like imaging, drug delivery systems, and phototherapy. Fluorescent nanoparticles are particularly attractive for such tasks owing to their emission signal that can serve as optical reporter for location or environmental properties. Single-walled carbon nanotubes (SWCNTs) fluoresce in the near-infrared part of the spectrum, where biological samples are relatively transparent, and they do not photobleach or blink. These unique optical properties and their biocompatibility make SWCNTs attractive for a variety of biomedical applications. Here, we review recent advancements in protein recognition using SWCNTs functionalized with either natural recognition moieties or synthetic heteropolymers. We emphasize the benefits of the versatile applicability of the SWCNT sensors in different systems ranging from single-molecule level to in-vivo sensing in whole animal models. Finally, we discuss challenges, opportunities, and future perspectives.

Synthesis and Self-Assembly of Multistimulus-Responsive Azobenzene-Containing Diblock Copolymer through RAFT Polymerization.

This paper gathered studies on multistimulus-responsive sensing and self-assembly behavior of a novel amphiphilic diblock copolymer through a two-step reverse addition-fragmentation transfer (RAFT) polymerization technique. N-Isopropylacrylamide (NIPAM) macromolecular chain transfer agent and diblock copolymer (poly(NIPAM-b-Azo)) were discovered to have moderate thermal decomposition temperatures of 351.8 and 370.8 °C, respectively, indicating that their thermal stability was enhanced because of the azobenzene segments incorporated into the block copolymer. The diblock copolymer was determined to exhibit a lower critical solution temperature of 34.4 °C. Poly(NIPAM-b-Azo) demonstrated a higher photoisomerization rate constant (kt = 0.1295 s−1) than the Azo monomer did (kt = 0.088 s−1). When ultraviolet (UV) irradiation was applied, the intensity of fluorescence gradually increased, suggesting that UV irradiation enhanced the fluorescence of self-assembled cis-isomers of azobenzene. Morphological aggregates before and after UV irradiation are shown in scanning electron microscopy (SEM) and dynamic light scattering (DLS) analyses of the diblock copolymer. We employed photoluminescence titrations to reveal that the diblock copolymer was highly sensitive toward Ru3+ and Ba2+, as was indicated by the crown ether acting as a recognition moiety between azobenzene units. Micellar aggregates were formed in the polymer aqueous solution through dissolution; their mean diameters were approximately 205.8 and 364.6 nm at temperatures of 25.0 and 40.0 °C, respectively. Our findings contribute to research on photoresponsive and chemosensory polymer material developments.

A sensitive fluorescent probe for imaging biothiol in zebrafish

Due to their highly significant biological activities, in vivo monitoring biothiols, including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), draws great attention. Among them, GSH is the representative nonprotein thiol inside the cell with the high abundance, protecting cells from oxidative stress and toxins. In this work, we developed a fluorescent probe NI for sensitive mapping endogenous biothiol distribution in zebrafish. We used 1,8-naphthalimide scaffold as easy modifiable fluorophore and 2-thiophenecarbonxyl as recognition moiety. After the cleavage of ester bond by GSH addition, NI exhibited strong emission with large Stokes shift (110 nm) and its LOD is 0.120 μM. We also studied the difference of kinetic response of GSH over Cys and Hcy. Finally, the described probe was successfully applied in sensing endogenous biothiol in zebrafish, showing its potential utility in vivo imaging.

Recent Progress in the Development of Fluorescent Probes for Thiophenol.

Thiophenol (PhSH) belongs to a class of highly reactive and toxic aromatic thiols with widespread applications in the chemical industry for preparing pesticides, polymers, and pharmaceuticals. In this review, we comprehensively summarize recent progress in the development of fluorescent probes for detecting and imaging PhSH. These probes are classified according to recognition moieties and are detailed on the basis of their structures and sensing performances. In addition, prospects for future research are also discussed.

Synthesis and Evaluation of New Cyclodextrin Derivatives as Amyloid‐β Aggregation Inhibitors

AbstractThe presence of extracellular amyloid‐beta (Aβ) plaques is one of the pathological hallmarks of Alzheimer's disease. Appropriately functionalized compounds can modulate amyloidogenesis. Here, we report a new class of cyclodextrin (CyD) derivatives rationally designed to improve the Aβ recognition and to modulate its aggregation. In particular, a dimethylamino aromatic moiety was conjugated to βCyD or γCyD to explore how different structural aspects of these derivatives could influence the recognition and the aggregation of Aβ. The conjugation of the Aβ recognition moiety to CyDs resulted in a significant suppression of the self‐assembly propensity of Aβ when the dimethylamino aromatic moiety was attached to the upper rim of βCyD. The activity of the derivatives toward Aβ confirms that the conjugation of CyDs with Aβ recognition moieties may be considered a promising approach for designing new molecules that interfere with Aβ aggregation.

Experimental and theoretical study of the role of CH/π interactions in the aminolysis reaction of acetyl galactoside

Molecular recognition of saccharides is a growing field, which has many implications in cancer therapy, drug discovery, and cellular communication among others. The participation of CH/π interactions in this event is well known. Nevertheless, the intrinsic role of CH/π for modulating chemical reactions is still far from being applicable. In this experimental and computational work we have evaluated the participation of CH/π interactions in the aminolysis reaction of acetyl galactoside promoted with different 6-substituted 2(1H)-pyridones. Two features have been incorporated to the promoter molecular structure, on one end the promoting pyridone group and on the other end the recognition moiety, joined together by an alkyne spacer. The small increment in the observed pseudo-first-order rate constant values (kobs) was related to the stability of the transition state provided by noncovalent interactions, including CH/π interactions. A longer alkyne spacer was necessary to improve the molecular recognition of the galactoside substrate. The trend of the calculated activation energy values (ΔERTS) was in good accordance with the experimental rate constant values.

Michael Addition/S,N-Intramolecular Rearrangement Sequence Enables Selective Fluorescence Detection of Cysteine and Homocysteine.

Acrylate has been widely used as the recognition unit for Cys fluorescent probes. Despite this widespread use, a potential drawback of this probe type is that the ester linkage between the fluorophore and acryloyl recognition unit is liable to be hydrolyzed by abundant esterase in the cytosol, thus affording a high background signal. To solve this problem, we herein put forward a new strategy to construct a selective fluorescent probe for cysteine (Cys)/homocysteine (Hcy) with propynamide as the recognition moiety. The free probe CPA displays weakly fluorescent emission in aqueous media because of the donor-excited photoinduced electron transfer (d-PET) process within the molecule. The Michael addition of Cys (or Hcy) thiols to the conjugated alkyne of CPA gives the expected β-sulfido-α,β-unsaturated amides (1a/1b), which subsequently undergo an intramolecular S,N rearrangement, yielding β-amino-α,β-unsaturated amides (2a/2b) as the final products. The above cascade reaction results in the blockage of d-PET within CPA, thus affording a dramatic fluorescence enhancement at 495 nm. The involvement of the sulfhydryl and the adjacent amino groups in the sensing process renders CPA high selectivity for Cys/Hcy over glutathione as well as other amino acids. The probe has been successfully applied to image Cys in different cell lines. Further, CPA shows two-photon fluorescence properties, and its ability to monitor Cys in deep tissues has been demonstrated by using two-photon microscopy.

Recognition Moieties of Small Molecular Fluorescent Probes for Bioimaging of Enzymes.

Enzymes are a class of important substances for life, and their abnormal levels are associated with many diseases. Thus, great progress has been made in the past decade in detecting and imaging enzymes in living biosystems, and in this respect fluorescent probes combined with confocal microscopy have attracted much attention because of their high sensitivity and unrivaled spatiotemporal resolution. Fluorescent probes are usually composed of three moieties: a signal or fluorophore moiety, a recognition or labeling moiety, and an appropriate linker to connect the two aforementioned moieties. At present, however, research and reviews on enzymatic probes mostly focus on fluorophores and/or linkers, whereas those on the recognition moiety are relatively few. Moreover, current enzymatic probes with some recognition moieties have drawbacks such as poor selectivity, high background fluorescence, or/and low sensitivity and are unsatisfactory for practical applications. Thus, developing new recognition moieties with higher specificity or/and sensitivity to the enzyme of interest is very desirable but still challenging. In this Account, we introduce the recognition moieties of fluorescent probes for several enzymes, including tyrosinase, monoamine oxidase A (MAO-A), nitroreductase (NTR), and aminopeptidases. Highlights are given on how new specific recognition moieties of tyrosinase and MAO-A were designed to eliminate the interference by reactive oxygen species (ROS) and MAO-B, respectively. Here we present four recent examples in which designed fluorescent probes are employed to image enzymes in living biosystems. The first example shows that 3-hydroxyphenyl can serve as a new and more specific recognition moiety than the traditional 4-hydroxyphenyl group for tyrosinase, enabling the development of a highly selective fluorescent probe for imaging of tyrosinase without interference by ROS. The second presents a general design strategy for fluorescent probes specific for an enzyme, which involves combining the characteristic structure of an inhibitor of the target enzyme along with its traditional reactive group as a new recognition moiety, and successfully demonstrates it by selective detection of MAO-A in the presence of its isomeric MAO-B. The third mainly illustrates that 5-nitrothiophen-2-yl alcohol with a stronger electron-donating S atom is a better fluorescence quenching and recognition moiety than 5-nitrofuran-2-yl alcohol for NTR, leading to the development of a highly sensitive method for NTR assay. Lastly, on the basis of known observations, we show that besides the specific interaction with the target, another function of some recognition moieties may be responsible for tuning the fluorescence signal, which is exemplified by the linking of several aminopeptidases' recognition moieties to the free hydroxyl or amino group of different fluorophores. It is our wish that this Account will promote the appearance of more specific recognition moieties and fluorescent probes with excellent properties and that new biofunctions of the enzymes will be uncovered.

Red-Emitting Fluorescence Sensors for Metal Cations: The Role of Counteranions and Sensing of SCN- in Biological Materials.

The spatiotemporal sensing of specific cationic and anionic species is crucial for understanding the processes occurring in living systems. Herein, we developed new fluorescence sensors derived from tetrapyrazinoporphyrazines (TPyzPzs) with a recognition moiety that consists of an aza-crown and supporting substituents. Their sensitivity and selectivity were compared by fluorescence titration experiments with the properties of known TPyzPzs (with either one aza-crown moiety or two of these moieties in a tweezer arrangement). Method of standard addition was employed for analyte quantification in saliva. For K+ recognition, the new derivatives had comparable or larger association constants with larger fluorescence enhancement factors compared to that with one aza-crown. Their fluorescence quantum yields in the ON state were 18× higher than that of TPyzPzs with a tweezer arrangement. Importantly, the sensitivity toward cations was strongly dependent on counteranions and increased as follows: NO3- < Br- < CF3SO3- < ClO4- ≪ SCN-. This trend resembles the chaotropic ability expressed by the Hofmeister series. The high selectivity toward KSCN was explained by synergic association of both K+ and SCN- with TPyzPz sensors. The sensing of SCN- was further exploited in a proof of concept study to quantify SCN- levels in the saliva of a smoker and to demonstrate the sensing ability of TPyzPzs under in vitro conditions.

Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

Dual Mode Chip Enantioselective Express Discrimination of Chiral Amines via Wettability-Based Mobile Application and Portable Surface-Enhanced Raman Spectroscopy Measurements.

A dual-mode functional chip for chiral sensing based on mobile phone wettability measurements and portable surface-enhanced Raman spectroscopy (SERS) is reported. The plasmon-active regular gold grating surface was covalently grafted with chiral recognition moieties, l- or d-enantiomers of tartaric acid, making stereoselective discrimination of chiral amines possible. Chiral sensing of amines includes two modes of analysis, performed subsequently on the one chip surface with portable instruments (mobile phone equipped with a camera and developed application (app) Dropangle and a portable Raman spectrometer). First, the wettability changes, caused by enantioselective entrapping of chiral amines, are monitored and analyzed via our mobile phone app, allowing detection of the optical configuration and concentration of enantiomers with 1 order of magnitude accuracy. Second, SERS measurement on the same chip provides information about the chemical structure of entrapped amines and allows calculation of the enantiomeric excess with great accuracy. The applicability of the developed chip is demonstrated on a variety of chiral amines, including tyrosine, cysteine, dopamine (DOPA), and dextromethorphan in analytical solutions and in commercially available DOPA-containing drug. Moreover, we demonstrate that the chips could be regenerated and used repeatedly for at least five cycles.

A highly sensitive two-photon fluorescent probe for glutathione with near-infrared emission at 719 nm and intracellular glutathione imaging.

A near-infrared turn-on two-photon fluorescent probe ST-BODIPY for glutathione-specific detection was designed and synthesized by attaching triphenylamine to BODIPY skeleton through the Knoevenagel condensation to prolong the maximum emission wavelength to the NIR region. And 2,4-dinitrobenzenesulfonyl group (DNBS), as the fluorescence quencher and thiol recognition moiety, was modified in 8 position of BODIPY. In the presence of GSH, the probe afforded an "off-on" signal response with a significant NIR fluorescence enhancement centered at 719 nm accompanying by quantum yield increased to 0.44, which was ascribed to the glutathione-induced SNAr (aromatic substitution) reaction. Surprisingly, we found that the probe could discriminate GSH from other biothiols including Cys and Hcy upon the addition of intracellular concentrations of them. Time-dependence also demonstrated that the probe could distinguish GSH from Cys and Hcy under physiological environment. The limit of detection (LOD) for GSH was calculated as 25.46 nM from the titration experiments, which is lower than most previously reported GSH-selective probes. Under the Ti:sapphire pulsed laser's 800 nm irradiation, ST-BODIPY toward GSH generated an "off-on" signal response with a significant enhancement of fluorescence emission at 719 nm after treatment with GSH. Besides, the 2PA cross section value (σ2) was calculated to be 410 GM, suggesting that it could not only function well as an excellent two-photon fluorescent probe for the detection of intracellular GSH, but also be applied for two-photon imaging with high sensitivity in living cells. Moreover, ST-BODIPY probe has been successfully employed for monitoring exogenous and endogenous GSH in MCF-7 cells with satisfying results, perhaps it was feasible for detecting abnormal contents of GSH in a biological system and accomplishing the goal of maintaining normal human activities.

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.

An acetate-based NIR fluorescent probe for selectively imaging of hydrogen peroxide in living cells and in vivo

Hydrogen peroxide (H2O2), as an important biomolecule, is involved in various physiology and pathologic processes such as cellular signal transduction, host defense, and pathogen invasion. Thus, convenient and efficient detection of H2O2 in living organisms is of paramount significance and has drawn tremendous interest from researchers worldwide. In the present study, a novel H2O2-recognition moiety, the acetyl group, was proposed. Based on this unique recognition moiety, a new near-infrared (NIR) fluorescence probe was designed and synthesized for detecting H2O2. With numerous advantages including high selectivity and sensitivity, good compatibility and low cytotoxicity, this probe not only has the great competence to monitor endogenous H2O2 in cells, tissues, and in vivo but also has the huge potentiality to be employed in the diagnosis of H2O2-related cancers.

A new turn-on fluorescent probe with ultra-large fluorescence enhancement for detection of hydrogen polysulfides based on dual quenching strategy.

Based on dual quenching strategy (ESIPT inhibited quenching and PET quenching), we have developed a new turn-on fluorescent probe 1. Combining 3-(benzo[d]thiazol-2-yl)-10-butyl-10H-phenothiazin-2-ol (dye 2) as the fluorophore and 2-fluoro-5-nitro-benzoic as the recognition moiety, probe 1 had feature of notable large Stokes shift, highly sensitivity and selective for monitoring H2Sn with remarkable fluorescence enhancement (328-fold) response at 534 nm. Probe 1 exhibited excellent performance in the quantitative detection of H2Sn with a 137 nm Stokes shift and a low detection limit of 26 nM in solution. Finally, probe 1 was successfully utilized to image H2Sn in living A549 cells and zebrafish.

A FRET-based ratiometric two-photon fluorescent probe for superoxide anion detection and imaging in living cells and tissues.

The superoxide anion (O2˙-) plays a crucial role in several physiological processes and many human diseases. Developing new methods for O2˙- detection in biological systems is very important. A FRET-based two-photon (TP) fluorescent probe with a ratiometric signal, TFR-O, was developed. A naphthalene derivative based TP fluorescent group was selected as the energy donor group, and a rhodol fluorescent group was chosen as the energy acceptor; the trifluoromethanesulfonate group was chosen as the recognition moiety. After reacting with O2˙-, the recognition moiety was removed and the fluorophore was released, leading to a fluorescence intensity decrease at the wavelength of 425 nm and a significant enhancement of the fluorescence intensity at 550 nm. The fluorescence intensity ratio between 550 and 425 nm (I550/I425) varied from 0.15 to 6.72, with the O2˙- concentration increasing from 0 to 50 μM. The detection limit of the TFR-O was 83 nM. Moreover, TFR-O was applied for detecting and imaging O2˙- in cells and liver tissues.

Monitoring mitochondrial pH with a hemicyanine-based ratiometric fluorescent probe.

Mitochondria as essential organelles play critical roles in cellular metabolism. Mitochondrial pH is a vital parameter that directly affects the unique function of mitochondria. Herein, we present a new ratiometric fluorescent probe M-pH for monitoring the pH within the mitochondria. M-pH consists of a stable and large π-electron conjugated merocyanine system. The lipophilic cationic benzyl group will facilitate the accumulation of M-pH in mitochondria. The phenol unit is the recognition moiety, achieving the ratiometric sensing of pH changes. The experimental results indicate that M-pH displays ratiometric fluorescence response to different pH values. Meanwhile, M-pH shows negligible response to common species, and has high stability and low cytotoxicity. In biological experiments, M-pH can solely accumulate in mitochondria and visualize the pH changes during mitophagy and cell apoptosis. We thus believe that M-pH has great potential as a practical tool for real-time monitoring of pH changes of mitochondria, contributing to revealing the pathogenesis of mitochondrial pH associated diseases.

A novel fluorescent off-on probe for the sensitive and selective detection of fluoride ions.

A highly sensitive and selective fluorescent probe for fluoride ions has been developed by incorporating the dimethylphosphinothionyl group as a recognition moiety into the fluorophore of coumarin. The detection mechanism is based on the fluoride ion-triggered cleavage of the dimethylphosphinothionyl group, followed by the release of coumarin, which leads to a large fluorescence enhancement at 455 nm (λex = 385 nm). Under the optimized conditions, the fluorescence enhancement of the probe is directly proportional to the concentration of fluoride ions in the range of 0–30 μM with a detection limit of 0.29 μM, which is much lower than the maximum content of fluoride ions guided by WHO. Notably, satisfying results have been obtained by utilizing the probe to determine fluoride ions in real-water samples and commercially available toothpaste samples. The proposed probe is rather simple and may be useful in the detection of fluoride ions in more real samples.