Synthesis and biological applications of two novel fluorescent proteins-labeling probes

Two novel chlorinated fluoresceins 2′,4′,5′,7′-tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein succinimidyl ester (1G) and 2′,4′,5′,7′-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein succinimidyl ester (2G) were synthesized as fluorescent probes for labeling proteins. Structures of target compounds and intermediates were determined via IR, MS, ¹H NMR and element analysis. The investigation in immunofluorescence histochemistry showed them had strong fluorescence, high photostability and good biocompatibility.     

The fluorescent markers based on oxazolopyridine unit for imaging organelles

Bioactive oxazolopyridine unit was used in the synthesis of fluorescent markers for specific organelles in this paper. The compounds 1a-c are linked with double bond between oxazolopyridine ring and photogenic precursors (3a-c). Compound 1a showed higher fluorescence yield (0.86 in THF), compounds 1b-c showed larger stokes shifts in DMSO. In lipid vesicles environment, they also showed good optical properties. In addition, the three compounds are biomarkers with lower cytotoxicity. Among them, compound 1a based on oxazolopyridine and coumarin unit is a dual targetable fluorescent marker for mitochondria and lipid droplets; while the other two compounds 1b-c are only biomarkers for lipid droplets.     

Development of 6-arylcoumarins as nonsteroidal progesterone antagonists. Structure–activity relationships and fluorescence properties

Progesterone is involved in multiple physiological processes, including female reproduction, via binding to the progesterone receptor (PR). We have developed 6-arylcoumarins such as 5 and 6 as non-steroidal PR antagonists with receptor-binding-dependent fluorescence. In this study, we investigated the structure–activity relationships and fluorescence properties of coumarin derivatives bearing a heterocyclic aromatic moiety. Among these derivatives, 7c (IC₅₀: 34 nM) and 10b (IC₅₀: 24 nM) showed more potent PR-antagonistic activity than lead compounds 5 (IC₅₀: 500 nM) and 6 (IC₅₀: 65 nM) in alkaline phosphatase (AP) assay. Compound 9b showed solvent-dependent fluorescence intensity, exhibiting strong fluorescence in the presence of PR LBD only in buffer solution. On the other hand, 10b showed a solvent-dependent shift of the fluorescence maximum wavelength in the presence of PR LBD. These results indicate that 6-arylcoumarin will be a useful scaffold for PR antagonists and fluorescent probes targeting PR.     

Direct incorporation and extension of a fluorescent nucleotide through rolling circle DNA amplification for the detection of microRNA 24-3P

We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene, which have different sizes, and screened their incorporation and extension capability during the rolling circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incorporate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP, and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA 24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P. This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P and also exhibited highly sensitive and selective detection properties.     

Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis,biological activity and cellular uptake kinetics

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.     

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and 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.     

Fluorescent sensors based on quinoline‐containing styrylcyanine: determination of ferric ions,hydrogen peroxide,and glucose,pH‐sensitive properties and bioimaging

A novel styrylcyanine‐based fluorescent probe 1 was designed and synthesized via facile methods. Ferric ions quenched the fluorescence of probe 1, whereas the addition of ferrous ions led to only small changes in the fluorescence signal. When hydrogen peroxide was introduced into the solution containing probe 1 and Fe²⁺, Fe²⁺ was oxidized to Fe³⁺, resulting in the quenching of the fluorescence. The probe 1/Fe²⁺ solution fluorescence could also be quenched by H₂O₂ released from glucose oxidation by glucose oxidase (GOD), which means that probe 1/Fe²⁺ platform could be used to detect glucose. Probe 1 is fluorescent in basic and neutral media but almost non‐fluorescent in strong acidic environments. Such behaviour enables it to work as a fluorescent pH sensor in both the solution and solid states and as a chemosensor for detecting volatile organic compounds with high acidity and basicity. Subsequently, the fluorescence microscopic images of probe 1 in live cells and in zebrafish were achieved successfully, suggesting that the probe has good cell membrane permeability and a potential application for imaging in living cells and living organisms. Copyright © 2014 John Wiley & Sons, Ltd.     

Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization

A cyclic decapeptide (1,

), which acts on the extracellular region of the EGF receptor, preventing it from dimerizing, has been developed. Peptide 2, which was labeled with fluorescein at the N-terminus of peptide 1, was synthesized based on structure–activity relationship studies. Peptide 2 essentially retained the inhibitory activity of peptide 1 against the receptor autophosphorylation. Confocal microscopy studies revealed that in carcinoma cells, the fluorescence of peptide 2 was localized inside some vesicles. Treatment of intact cells by peptide 1 in combination with peptide 2 decreased the fluorescence intensity significantly compared to treatment with only peptide 2. These results indicate that peptide 2 competes with peptide 1 for binding to the cellular surface. Six derivatives of peptide 2, in which constituent amino acids, with the exception of two cysteines and proline were randomized, were synthesized and used to treat the cells. Peptides 6 and 9 showed the highest fluorescence intensity in cells. From the results of the EGF receptor autophosphorylation assay, these two derivatives were proven to have higher inhibitory activity than peptide 2, which would therefore be a useful delivery peptide and fluorescent probe to find new inhibitors against the EGF receptor. Peptides 6 and 9 are promising leads for EGF receptor inhibitors.     

Ligand-based design of GLUT inhibitors as potential antitumor agents

Glucose transporters (GLUTs) regulate glucose uptake and are often overexpressed in several human tumors. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against a NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and antiproliferative activities. From these, we identified that compounds 2, 5, 6 and 13 inhibited the cell viability in a dose-dependent manner and that the IC₅₀s of 2 and 6 are<10 µM concentration in the HCT116 colon cancer cell line. Lead compound 13 (NSC295720) was a GLUT1 inhibitor. Docking studies show that GLUT1 residues Phe291, Phe379, Glu380, Trp388, and Trp412 were important for inhibitor binding.     

Isolation and antioxidant activity evaluation of two new phthalate derivatives from seahorse, Hippocampus Kuda Bleeler

Seahorse (Hippocampus Kuda Bleeler) has been used as traditional medicine for thousands of years, in Eastern Asia. In this study of the methanol extract of fresh Hippocampus Kuda, the new compounds 2-ethyldecyl 2-ethylundecyl phthalate (1), 2, 12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecylphthalate (2), along with a known Bis(2-ethylheptyl) phthalate (3) were isolated. They were tested for their antioxidant activities, including lipid peroxidation inhibitory activity, DPPH radical scavenging, hydroxyl radical scavenging, superoxide anion radical scavenging, alkyl radical scavenging, and cellular radicals; these can be detected using a fluorescence probe, 2??,7??-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW264.7 cell. Compound (2) exhibited the highest antioxidant activity and inhibitory intracellular ROS than another compounds (1, 3). Furthermore, MTT assay showed no cytotoxicity on mouse macrophages cell (RAW264.7) and human fetal lung fibroblast cell line (MRC-5). This antioxidant property depends on concentration and increasing with increased amount of the compound.     

Studies on interactions of carbazole derivatives with DNA,cell image,and cytotoxicity

DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV–Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2. Circular dichroism spectra showed that both compounds had no influence on the conformation of G-quadruplexes. Fluorescence titrations indicated that 2 had the potential to be a G-quadruplex selective fluorescent probe, while 1 could be used as a fluorescent probe for duplex DNA. Confocal fluorescence images indicated that both compounds could enter the living HepG2 cells, and 1 mainly located in nucleus whereas 2 mainly distributed in cytoplasm. DNase and RNase digest tests indicated that both compounds could enter into the nucleus and interact with duplex DNA, especially, 2 could interact with RNA and/or G-quadruplex DNA. They also exhibited an obvious antiproliferative activity to HepG2 by using MTT assays, with IC₅₀ values of 2.7 and 9.5?μM for 1 and 2, respectively.     

Naphthalene-based environmentally sensitive fluorescent 8-substituted 2′-deoxyadenosines: Application to DNA detection

We synthesized various C8-naphthylethynylated 2′-deoxyadenosine derivatives and investigated their photophysical properties. Among them, cyano- and N,N-dimethylamino-substituted 8-naphthylethynylated 2′-deoxyadenosine derivatives (^cnA and ^dnA) showed strong fluorescence with high quantum yields and a remarkable solvatofuorochromicity. In particular, fluorescence of N,N-dimethylamino-substituted ^2,6dnA was not quenched by neighboring guanines (Gs) when incorporated in DNA duplexes, in contrast to ^cnA. We developed a new fluorescent probe containing ^2,6dnA that can be used for the detection of target DNA via a bulge formation regardless of the neighboring sequences.     

Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405 nm, the hoeFL–DNA complex displayed two fluorescence bands around 460 nm and 520 nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F₅₂₀/F₄₆₀) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.     

A novel nitro-substituted benzothiadiazole as fluorescent probe for tumor cells under hypoxic condition

Most of solid tumor cells are hypoxic and hard to trace and measure. A new compound, 4,7-bis(4-dodecylthiophen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO₂), was synthesized for labeling the hypoxic cells specially in this paper. BTTD-NO₂ showed no cytotoxicity to MG63 cells by MTT method. When MG63 cells were cultured with BTTD-NO₂ under hypoxic condition for 24 h, strong red fluorescence distribution in cytoplasm was observed. Flow cytometry results showed that 65% of MG63 cells were labeled with strong red fluorescence in hypoxic condition while only 2.4% in oxic condition. Furthermore, Real time RT-PCR proved that BTTD-NO₂ could stimulate high gene expression of the nitroreductase in the cells which could improve the conversion rate of BTTD-NO₂ to BTTD-NH₂ in turn. It proved that the fluorescence of BTTD-NO₂ was quenched by its two nitro groups, however, strong red fluorescence could emit in the cytoplasm after the reduction of its nitro groups to amino groups in the tumor cells under hypoxic condition. These results suggested that BTTD-NO₂ had the potential as a superior fluorescent probe for tumor detection.     

A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs

A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration-dependent self-quenching (dequenching) of fluorescence of 5-N-(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self-quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA-injected eggs; 3) Ca²⁺ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and dequenching of AF18 fluorescence by using a simple one-excitation and two-emission wavelength system.     

Synthesis and evaluation of a novel ‘off-on’ chemical sensor based on rhodamine B and the 2,5-pyrrolidinedione moiety for selective discrimination of glutathione and its bioimaging in living cells

A new “turn-on” fluorescent probe, RDMBM, based on the rhodamine B dye and the 2,5-pyrrolidinedione moiety was synthesized and characterized. Its sensing behavior toward various amino acids was evaluated via UV–vis and fluorescence spectroscopic techniques. The observed spectral changes showed that RDMBM displays high selectivity and sensitivity toward GSH in MeOH/H₂O (1:2, v/v, pH 7.40, Tris-HCl buffer, 1?mM) solution and that it undergoes 1:1 covalent binding with GSH. More importantly, the hydrogenation and ring-opening of the nitrogen atom in the spirane structure of rhodamine B derivatives were tightly bound to the induction effects of different groups. Furthermore, fluorescence imaging applications demonstrated that RDMBM can be successfully used for the detection of GSH in human breast cancer cells MCF-7.     

Carbazole-based fluorescent probes for G-quadruplex DNA targeting with superior selectivity and low cytotoxicity

G-quadruplex DNA plays a very important role in clinical diagnosis and fluorescence analysis has attracted extensive attention. A class of carbazole-based fluorescent probes for the detection of G-quadruplex DNA was established in this work. In this system, the installation of an oligo(ethylene glycol) chain on the scaffold will improve the water-solubility and biocompatibility. The presence of styrene-like different side groups could tune the selectivity toward G-quadruplex DNA binding. Results revealed that the substitution pattern and position gave a great influence on the ability for the discrimination of the G-quadruplex from other DNA structures. Especially, probe E1 bound to G-quadruplex DNA with superior selectivity, which exhibiting almost no fluorescence response in the presence of non-G-quadruplex DNA structures. Comprehensive analyses revealed that E1 could bind both ends of the G-quadruplex, resulting in a significant increase of fluorescence emission intensity. Cellular uptake assay suggested that E1 could pass through membrane and enter living cells with low cytotoxicity.     

A selective and sensitive turn-on chemosensor for detection of Fe3+ in aqueous solution and its cell imaging in dorsal root ganglia neurons and MKN-45 cells

A new turn-on fluorescent chemosensor (RBTM) for Fe³⁺ was designed based on Rhodamine B and a thiocarbonylimidazole moiety. The spectroscopic probe used for characterization of the synthesized system showed 300-fold fluorescence enhancement for the detection of Fe³⁺ with a 1:1 stoichiometry in EtOH/H₂O solution (2:1, v/v, HEPES buffer, 1 mM, pH 7.30). Upon addition of Fe³⁺ in aqueous ethanol, the probe displayed a significant fluorescence enhancement and a distinct color change (colorless to pink) that can be detected by the naked eye. The binding constant between the probe and Fe³⁺ was determined to be 1.16 × 10⁴ M⁻¹ and the corresponding detection limit was calculated to be 0.256 µM. In addition, the energy gaps between the HOMO and LUMO in RBTM and RBTM-Fe³⁺ were calculated using DFT calculations to be 92.93 kcal/mol and 37.49 kcal/mol, respectively. The results indicate that binding of Fe³⁺ to RBTM lowered the HOMO–LUMO energy gap of the complex and stabilized the system. Fluorescence imaging experiments demonstrated that RBTM can be used as a fluorescent probe to detect Fe³⁺ in MKN-45 cells and dorsal root ganglia, thus revealing that RBTM could be used for biological applications.