Design, synthesis and cell imaging of a simple peptide-based probe for the selective detection of RNA.

Here we present a novel peptide-based fluorescent "turn-on" molecule P1 for detecting RNA, in a double or single strand, AU-rich or CG-rich. Both computational and experimental studies indicate that the detection efficiency depends on the binding affinity of P1 and conformational changes. P1 could be applied for cell imaging without any additional transfection vectors. Selective detection of RNA in cells was determined by RNase digestion. Successful application of P1 for RNA imaging in cell mitosis reveals that it may have broad applications in research, biotechnology and medical science.

A simple and effective dansyl acid based "turn-on" fluorescent probe for detecting labile ferrous iron in physiological saline and live cells.

Labile ferrous iron (Fe2+) plays important biochemical functions in many physiologically essential processes. It is very important to find an effective method to detect Fe2+. Herein, a simple and effective Fe2+ fluorescent probe (FeP1) has been constructed via a unique strategy of Fe2+-induced reducing reaction. As expected, FeP1 exhibited a 'turn-on' fluorescence response toward Fe2+ over various small analytes, with high selectivity and excellent sensitivity (DL = 18 nM) for the detection of Fe2+ in Tris-DMSO (4:1, pH = 7.4, v/v) solution. Moreover, the probe can act in different real samples, such as physiological saline and living cells.

Optical Supramolecular Sensing of Creatinine.

Calix[4]pyrrole phosphonate-cavitands were used as receptors for the design of supramolecular sensors for creatinine and its lipophilic derivative hexylcreatinine. The sensing principle is based on indicator displacement assays of an inherently fluorescent guest dye or a black-hole quencher from the receptor's cavity by means of competition with the creatinine analytes. The systems were thermodynamically and kinetically characterized regarding their 1:1 binding properties by means of nuclear magnetic resonance spectroscopy (1H and 31P NMR), isothermal titration calorimetry, and optical spectroscopies (UV/vis absorption and fluorescence). For the use of the black-hole indicator dye, the calix[4]pyrrole was modified with a dansyl chromophore as a signaling unit that engages in Förster resonance energy transfer with the indicator dye. The 1:1 binding constants of the indicator dyes are in the range of 107 M-1, while hexylcreatinine showed values around (2-4) × 105 M-1. The competitive displacement of the indicators by hexylcreatinine produced supramolecular fluorescence turn-on sensors that work at micromolar analyte concentrations that are compatible with those observed for healthy as well as sick patients. The limit of detection for one of the systems reached submicromolar ranges (110 nM).

A sensitive pH fluorescent probe based on triethylenetetramine bearing double dansyl groups in aqueous solutions and its application in cells.

pH fluorescent probes possess many advantages, including intracellular detection, rapid response time and nondestructive testing. In this paper, a highly selective and sensitive fluorescent pH probe based on triethylenetetramine bearing double dansyl groups (1) was synthesized. This probe offers fluorescent measurement of pH value in the range of 5.81-7.21 in aqueous solution, with an 8.64-fold enhancement of fluorescent emission intensity over the unmodified probe. Probe 1 shows a fluorescent color change from a pale yellow to bright green when the pH is increased from 5.81 to 7.21. In addition, probe 1 shows good potential as a fluorescent visualizing sensor for pH values in living GS cells of epinepheluscoioides. The mechanism of the fluorescent response of probe 1 to solution pHs was further clarified by NMR, fluorescent spectra, and UV-vis absorption spectra. The results indicate that the fluorescent emission will shift with an increase in solution pHs, due to increasing deprotonation of the nitrogen atom on the sulfonamides. Deprotonation of the sulfonamide group will inhibits the intramolecular charge transfer process between the imino group and the naphthalene ring, resulting in the recognition phenomenon of blue shift and enhancement of fluorescent emission intensity.

Two-Photon Fluorescence Probe for Selective Monitoring of Superoxide in Live Cells and Tissues.

The abnormal location or generation of superoxide radical anion (O2•-) are implicated in many diseases, including cancers; thus, development of an efficient method to detect O2•- is of great importance. Inspired by the fluorophore-governed selective manner to O2•- and peroxynitrite (ONOO-) of previously reported phosphinate-based fluorescence probes, in this contribution, a phosphinothioate-containing probe, TPP, was designed. The probe exhibited easy accessibility through a one-step sequence and good photostability and biocompatibility. Interestingly, TPP showed high specificity and sensitivity to O2•- over other reactive oxygen species/nitrogen species including ONOO-. Furthermore, with the assistance of two-photon microscopy, TPP was successfully applied for imaging endogenous O2•- in live cells and tissues.

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.

A novel peptide-based fluorescent chemosensor for Cd(II) ions and its applications in bioimaging.

Nowadays, it is of great significance to develop a novel fluorescent chemosensor for Cd(II) ions detection with cost-effective, rapid, facile and applicable to environment and biological milieus. Herein, we report a new peptide-based fluorescent chemosensor DSC (Dan-Ser-Cys-NH2) based on dipeptide (Ser-Cys-NH2) conjugated with dansyl group, which was synthesized using solid phase peptide synthesis (SPPS) technology. As designed, DSC exhibited fluorescent "turn-on" response for Cd2+ in 100% aqueous solution over a wide range of pH values based on photoinduced electron transfer (PET). The stoichiometry binding of DSC and Cd2+ was determined to be 2:1 by Job's plot and ESI-MS analysis. Furthermore, DSC showed highly sensitive for Cd2+ and a low detection limit of 13.8 nM. What's more, DSC has cell permeability and low cytotoxicity, and fluorescence imaging experiments demonstrated that DSC was capable of monitoring Cd2+ in living HK2 cells by confocal microscopy.

A sequential and reversibility fluorescent pentapeptide probe for Cu(II) ions and hydrogen sulfide detections and its application in two different living cells imaging.

In this study, we report a sequential and reversibility fluorescent probe (DP5) based on pentapeptide conjugated with dansyl groups using the solid phase peptide synthesis (SPPS) technology. DP5 showed immediate "turn off" response toward Cu2+ ions at an excitation wavelength of 330 nm with detection limits of 23.5 nM. The 2:1 binding ratio between DP5 and Cu2+ were confirmed using Job's plot method and fluorescence titration study, and DP5-Cu complex was observed with an association constant of 6.76 × 108 M-2. As designed, DP5-Cu complex as a promising analytical probe exhibited highly selective for H2S detection in aqueous solutions. The detection limit for H2S was obtained to be 17.2 nM, and lower than EPA and WHO guidelines. In addition, the reversibility and cyclicity were imparted to the DP5 during the detection of Cu2+ and H2S, and cycle effect is very good. Furthermore, DP5 displayed better biocompatibility and low biotoxicity, and sequential fluorescence "on-off-on" responses of DP5 to Cu2+ and H2S were successfully applied in two different living cells.

Labeling of Nanofiber-Forming Peptides by Site-Directed Bioconjugation: Effect of Spacer Length on Self-Assembly.

BACKGROUND: The conjugation of small organic molecules to self-assembling peptides is a versatile tool to decorate nanostructures with original functionalities. Labeling with chromophores or fluorophores, for example, creates optically active fibers with potential interest in photonic devices. AIM AND OBJECTIVE: In this work, we present a rapid and effective labeling procedure for a self-assembling peptide able to form nanofibers. Rapid periodate oxidation of the N-terminal serine residue of the peptide and subsequent conjugation with dansyl moiety generated fluorophore-decorated peptides. RESULTS: Three dansyl-conjugated self-assembling peptides with variable spacer-length were synthesized and characterized and the role of the size of the linker between fluorophore and peptide in self-assembling was investigated. Our results show that a short linker can alter the self-assembly in nanofibers of the peptide. CONCLUSIONS: Herein we report on an alternative strategy for creating functionalized nanofibrils, able to expand the toolkit of chemoselective bioconjugation strategies to be used in site-specific decoration of self-assembling peptides.

Discovery of a Turn-On Fluorescent Probe for Myeloid Cell Leukemia-1 Protein.

The environment-sensitive probe 2 with turn-on switch for Mcl-1 protein was developed herein. After careful evaluation, this small molecule fluorescent probe revealed a selective binding affinity with Mcl-1 protein with a Ki value of 2.6 µM and can be well applied to imaging Mcl-1 protein or detecting the cellular distribution of Mcl-1 protein inhibitors. Compared with other imaging approaches, such as the immunofluorescence and fluorescent protein-based techniques, this fluorescent method is rapid, convenient, and affordable, especially since a washing procedure is no longer needed. This environment-sensitive "off-on" design strategy may present a case in point for developing small-molecule fluorescent probe of Bcl-2 family proteins.

A self-assembled amphiphilic imidazolium-based ATP probe.

A novel amphiphilic imidazolium-based probe containing a dansyl fluorophore and a long cetyl chain has been developed for ATP recognition. The probe forms self-assembled micelle-like aggregates at low concentration in its aqueous solution and can selectively recognize ATP among other bioactive anions with a significant enhancement in fluorescence emission.

Synthesis of a dansyl-labeled inhibitor of 17ß-hydroxysteroid dehydrogenase type 3 for optical imaging.

The steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a therapeutic target in the management of androgen-sensitive diseases such as prostate cancer and benign prostate hyperplasia. In this Letter, we designed and synthesized the first fluorescent inhibitor of this enzyme by combining a fluorogenic dansyl moiety to the chemical structure of a known inhibitor of 17ß-HSD3. The synthesized compound 3 is a potent fluorogenic compound (λex=348 nm and λ em=498 nm). It crosses the cell membrane, keeps its fluorescent properties and is distributed inside the LNCaP cells overexpressing 17ß-HSD3, where it inhibits the transformation of 4-androstene-3,17-dione into the androgen testosterone (IC50=262 nM).

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes.

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity.

A peptide-based fluorescent chemosensor for multianalyte detection.

A novel multifunctional peptide fluorescent chemosensor (DP-3) with a lysine backbone and double sides conjugated with histidine and dansyl groups has been designed and synthesized by solid phase synthesis. This chemosensor is a promising analytical tool for detecting Zn(2+), Cu(2+), and S(2-) based on different mechanisms in 100% aqueous solutions, and intracellular biosensing has been successfully actualized. The peptide beacon structure of DP-3 makes it more stable and capable of achieving multianalyte detection, especially for sulfide ions. Until now, there have been few examples of using a peptide fluorescent chemosensor to detect anions with a continuous method. As designed, DP-3 exhibits excellent cell permeation and low biotoxicity and displays high selectivity and sensitivity, with Zn(2+) and Cu(2+) detection limits of 82 nM and 78 nM, respectively. This study raises the new possibility of a highly selective peptide fluorescent chemosensor for multifunctional detection, including cation and anions, by different mechanisms in environmental and biological systems. We expect that this work will inspire the development of a multifunctional beacon peptide-based fluorescent chemosensor library using modifiable lateral and terminal groups for a variety of practical applications in physiological and pathological events.

Dansyl-labeled anionic amphiphile with a hexadecanoic carbon chain: synthesis and detection for shape transitions in organized molecular assemblies.

The probing properties of a new fluorophore-labeled anionic surfactant, sodium 16-(N-dansyl)aminocetylate (16-DAN-ACA) were investigated systematically in molecular assemblies, especially in the transitions between micelles and vesicles. 16-DAN-ACA can efficiently differentiate the two different aggregate types in mixed cationic and anionic surfactant systems. The fluorescence anisotropy of 16-DAN-ACA was found to be sensitive for directly detecting the micellar growth in micelles containing oppositely charged surfactants; both cationic cetyltrimethylammonium bromide (CTAB) systems and anionic sodium dodecyl sulfate (SDS) systems were studied. The results indicated that the 16-DAN-ACA is a good fluorescent probe for differentiating the different aggregates, and even more can be used to detect the micellar growth.

Dansyl-8-aminoquinoline as a sensitive pH fluorescent probe with dual-responsive ranges in aqueous solutions.

A sensitive pH fluorescent probe based on dansyl group, dansyl-8-aminoquinoline (DAQ), has been synthesized. The probe showed dual-responsive ranges to pH changes, one range from 2.00 to 7.95 and another one from 7.95 to 10.87 in aqueous solution, as it showed pKa values of 5.73 and 8.56 under acid and basic conditions, respectively. Furthermore, the pH response mechanism of the probe was explored successfully by using NMR spectra. The results indicated that the responses of DAQ to pH changes should attribute to the protonation of the nitrogen atom in the dimethylamino group and deprotonation of sulfonamide group.

Development of a fluorescent probe for the study of the sponge-derived simplexide immunological properties.

Simplexide is a glycolipid of marine origin, endowed with immunological properties, composed of a long chain secondary alcohol glycosylated by an α-d-glucosyl-(1→4)-ß-D-galactosyl disaccharide residue. Herein we describe the preparation of a fluorescent derivative of simplexide, labeled at position 6 of the distal glucose with a dansyl group, as a probe for future studies on the mechanism by which simplexide affects the immune system. Fluorescent simplexide was prepared from a 6″-amino functionalized compound, which in turn was obtained through a highly efficient glycosylation between the preformed activated disaccharide and the long chain secondary alcohol 18-pentatriacontanol.

Synthesis and properties of molecular probes for the rescue site on mutant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is a genetic disease caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In vitro experiments have demonstrated that 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (VRT-532, 1) is able to partially restore the function of mutant CFTR proteins. To help elucidate the nature of the interactions between 1 and mutant CFTR, molecular probes based on the structure of 1 have been prepared. These include a photoreactive aryl azide derivative 11 and a fluorescent dansyl sulfonamide 15. Additionally, a method for hydrogen isotope exchange on 1 has been developed, which could be used for the incorporation of radioactive tritium. Using iodide efflux assays, the probe molecules have been demonstrated to modulate the activity of mutant CFTR in the same manner as 1. These probe molecules enable a number of biochemical experiments aimed at understanding how 1 rescues the function of mutant CFTR. This understanding can in turn aid in the design and development of more efficacious compounds which may serve as therapeutic agents in the treatment of cystic fibrosis.

An improved synthesis of dansylated α-galactosylceramide and its use as a fluorescent probe for the monitoring of glycolipid uptake by cells.

A highly efficient synthesis of the biologically important fluorescent probe dansyl α-GalCer is presented. Key in our strategy is the incorporation of the fluorescent dansyl group at an early stage in the synthesis to facilitate in the monitoring and purification of intermediates via TLC and flash column chromatography, respectively, and the use of a high yielding α-selective glycosylation reaction between the phytosphingosine lipid and a galactosyl iodide donor. The ability of dansyl α-GalCer to activate iNKT cells and to serve as a fluorescent marker for the uptake of glycolipid by dendritic cells is also presented.

Fluorescent substrates for polyamine catabolic enzymes and transport.

The most widely used methods for measuring polyamine enzyme activities are radioisotope methods that measure the radioactivity of compounds produced from radiolabeled substrate by the enzyme reaction. Several fluorescent polyamines have been developed for the measurement of the polyamine transport system (PTS) or transglutaminase. Although fluorophores in the fluorescent polyamines may affect the affinity of the polyamine moiety to the enzyme protein, the assays that use fluorescent substrate are sensitive and simple for common laboratory usage.In this chapter, the uses of dansyl polyamines with a simple high-performance liquid chromatography system for the measurement of the PTS and polyamine catabolic enzymes including spermidine/spermine N¹-acetyltransferase and N¹-acetylpolyamine oxidase are described.