Design and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in immune cells

Cannabinoid CB2 receptor has emerged as a very promising target over the last decades. We have synthesized and evaluated a new fluorescent probe designated NMP6 based on 6-methoxyisatin scaffold, which exhibited selectivity and K(i) value at hCB2 of 387 nM. We have demonstrated its ability to be an effective probe for visualization of CB2 receptor binding using confocal microscopy and a flow cytometry probe for the analysis of CB2 protein expression. Furthermore, NMP6 was easily obtained in two chemical steps from commercially available building blocks.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

A highly sensitive,cell-membrane-permeable fluorescent probe for glutathione

Glutathione (GSH) is a primary intracellular antioxidant. Here, we developed a novel, highly sensitive fluorescent probe for GSH, designated DNs-HMRG, whose fluorescence is regulated by two distinct switching mechanisms, intramolecular spirocyclization and photo-induced electron transfer (PeT). DNs-HMRG showed good cell permeability, and a rapid increase in fluorescence intensity was observed when it was applied to living cells. Further, taking advantage of the fact that the intracellular GSH level in tumor tissue is higher than that in normal tissue, we employed this probe for rapid (within a few tens of seconds) in vivo detection of tiny tumor nodules (less than 1 mm in diameter) in tumor-bearing mice. This probe is expected be a powerful tool in various biological applications, especially studies on redox status.     

Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, (1)H NMR, and (13)C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials.     

A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells

An approach of high sensitivity and selectivity for hydrogen peroxide (H₂O₂) detection is highly demanded due to its important roles in regulating diverse biological process. In this work, we introduced an easily synthesized fluorescent “turn off” probe, BNBD. It is designed based on the core structure of 4-chloro-7-nitrobenzofurazan as a fluorophore and incorporated with a specific H₂O₂-reactive group, aryl boronate, for sensitive and selective detection of H₂O₂. We demonstrated its selectivity by incubating the probe with other types of ROS, and measured the limit of detection of BNBD as 1.8 nM. BNBD is also conducive to H₂O₂ detection at physiological conditions. We thus applied it to detect both exogenous and endogenous changes of H₂O₂ in living cells by confocal microscopy, supporting its future applications to selectively monitor H₂O₂ levels and identify H₂O₂-related physiological or pathological responses from live cells or tissues in the near future.     

A novel colorimetric fluorescent probe for detecting hydrazine in living cells and zebrafish

Hydrazine (NH₂NH₂) is a highly toxic organic substance that poses a threat to human health. Monitoring hydrazine with high sensitivity and selectivity is very important. Here, a simple colorimetric fluorescent probe for hydrazine detection, which is a seminaphthorhodafluor derivative containing thiophene-2-carboxylic acid ester reaction site, was rationally constructed. The probe itself exhibits weak fluorescence. The fluorescence is significantly enhanced when hydrazine is added. The probe exhibited a broad linear range (0–1 mM) with satisfactory selectivity and sensitivity (limit of detection 36.4 μM), which turned out to be an excellent fluorescent probe for monitoring hydrazine. Additionally, the probe was used to track hydrazine in living cells and zebrafish with great success, and the detection performance was satisfying. These results proved that this type of fluorescent probe with the thiophene-2-carboxylic acid ester structure can detect hydrazine with higher selectivity and sensitivity.     

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe

Background

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking.

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1 . LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.     

Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors

Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K_d) and the dissociation off-rates (k_off) of the enzyme–inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ_ex = 325 nm, λ_em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme’s tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K_d and k_off values.     

Development of a fluorescent cardiomyocyte specific binding probe

Cardiomyocytes are the major component of the heart. Their dysfunction or damage could lead to serious cardiovascular diseases, which have claimed numerous lives around the world. A molecule able to recognize cardiomyocytes would have significant value in diagnosis and treatment. Recently a novel peptide termed myocyte targeting peptide (MTP), with three residues of a non-natural amino acid biphenylalanine (Bip), showed good affinity to cardiomyocytes. Its selectivity towards cardiac tissues was concluded to be due to the ability of Bip to bind cardiac troponin I. With the aim of optimizing the affinity and the specificity towards cardiac myocytes and to better understand structure–activity relationship, a library of MTP derivatives was designed. Exploiting a fluorescent tag, the selectivity of the MTP analogs to myocardium over skeletal and stomach muscle tissues was assayed by fluorescence imaging. Among the tested sequences, the peptide probe Bip2, H-Lys(FITC)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Ser-Gly-Ser-Bip-Bip-NH₂, displayed the best selectivity for cardiomyocytes.     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Nitroxidative chemistry interferes with fluorescent probe chemistry: Implications for nitric oxide detection using 2,3-diaminonaphthalene

Simultaneous production of nitric oxide (NO) and superoxide generates peroxynitrite and causes nitroxidative stress. The fluorometric method for NO detection is based on the formation of a fluorescent product from the reaction of a nonfluorescent probe molecule with NO-derived nitrosating species. Here, we present an example of how nitroxidative chemistry could interact with fluorescent probe chemistry. 2,3-Naphthotriazole (NAT) is the NO-derived fluorescent product of 2,3-diaminonaphthalene (DAN), a commonly used NO-detecting molecule. We show that NO/superoxide cogeneration, and particularly peroxynitrite, mediates the chemical decomposition of NAT. Moreover, the extent of NAT decomposition depends on the relative fluxes of NO and superoxide; the maximum effect being reached at almost equivalent generation rates for both radicals. The rate constant for the reaction of NAT with peroxynitrite was determined to be 2.2 × 10³ M⁻¹ s⁻¹. Further, various peroxynitrite scavengers were shown to effectively inhibit NO/superoxide- and peroxynitrite-mediated decomposition of NAT. Taken together, the present study suggests that the interference of a fluorometric NO assay can be originated from the interaction between the final fluorescent product and the formed reactive nitrogen and oxygen species.     

Development of a fluorescent probe for detection of citrulline based on photo-induced electron transfer

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine residues to form citrulline residues. Aberrant citrullination of histones by one of the PAD isozymes, PAD4, is associated with various diseases, including rheumatoid arthritis, so high-throughput screening systems are needed to identify PAD4 inhibitors as chemical tools to investigate the role of PAD4, and as candidate therapeutic agents. Here, we utilized the addition-cyclization reaction between phenylglyoxal and citrulline under acidic conditions to design turn-on fluorescent probes for citrulline based on the donor-excited photoinduced electron transfer (d-PeT) mechanism. Among several derivatives of phenylglyoxal bearing a fluorescent moiety, we found that FGME enabled detection of citrulline without a neutralization process, and we used it to establish a simple methodology for turn-on fluorescence detection of citrulline.     

Quinolizinium as a new fluorescent lysosomotropic probe

We have synthesized a collection of quinolizinium fluorescent dyes for the purpose of cell imaging. Preliminary biological studies in human U2OS osteosarcoma cancer cells have shown that different functional groups appended to the cationic quinolizinium scaffold efficiently modulate photophysical properties but also cellular distribution. While quinolizinium probes are known nuclear staining reagents, we have identified a particular quinolizinium derivative salt that targets the lysosomal compartment. This finding raises the question of predictability of specific organelle targeting from structural features of small molecules.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Synthesis,characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.     

Rhodamine-based “turn-on” fluorescent probe with high selectivity for Fe imaging in living cells

A rhodamine-based “turn-on” fluorescent probe 1 was synthesized with high yield. The recognizing behavior displays high selectivity of 1 toward Fe²⁺ with a 2:1 complex, and 1 exhibits a stable response for Fe²⁺ over a concentration range from 2 μM to 24 μM. Most importantly, probe is hardly interfered by other transition metal ions. Their fluorescent enhancement is observed in the presence of Fe²⁺ because of the ring-open interactions of spirocyclic. All measurements are made in PBS buffer environments simulating biological conditions to make them suitable candidates for fluorescent labeling of biological systems. Confocal laser scanning microscopy experiments have proven that probe can be used to monitor Fe²⁺ in living cells.     

A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258, our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT assays.