Synthetic macrocyclic ligands. VI. Lithium ion-selectivefluorescent emission with crowned benzo- and naphtho-thiazolylphenols

Synthesis of fluorescent crowned benzo- and naphtho-thiazolylphenols are described and lithium ion-selective fluorescent emission is observed under the restricted conditions.     

Quantum dots for single bio-molecule imaging.

The emerging nanomaterial, quantum dots or QDs, offers numerous potential applications in the biological area. As cell labeling probes, QDs become now an alternative of existing organic fluorescent dyes and fluorescent proteins. In this short review, we cover typical and successful applications of QDs as fluorescent probes in cell labeling and genomic diagnosis. As a future important application, biomolecular detection at a single molecule level utilizing QDs is also discussed.     

Studies on Fluorescamine. Part II - Thin-Layer Chromatographic Mobilities of Fluorescamine Positive Drugs

Abstract

Fluorescamine reacts with primary amines, it does not react with secondary or tertiary amines, to yield bright acquamarine fluorescent products. This reaction has been developed into a spot test useful for differentiation of amphetamine from methamphetamine; previous spot tests did not have this degree of specificity. The fluorescent products of several primary amines have been demonstrated to have sufficient stability to permit thin-layer chromatography comparisons with authentic materials. The fluorescent derivative of amphetamine has been demonstrated to be capable of differentiation from other fluorescent products formed from fluorescamine based on its unique migration in three different TLC systems.     

Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells.

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3 beta-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37 degrees C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Fluorescent iminodiacetamide derivatives as potential ionophores for optical zinc ion-selective sensors.

Fluorescent sensor molecules were synthesized by conjugation of iminodiacetamide derivatives with fluorescent moieties of different structures and their UV-visible and fluorescent properties were characterized in acetonitrile solvent. The fluorescent measurements revealed that the N-(2-naphthyl) and N-phenyl derivatives exhibit a distinct zinc ion-selectivity over alkali and alkaline earth metal ions, while N-(anthrylmethyl) and N-(3-methoxyphenyl) derivatives do not possess any ion-selectivities. In contrast to the fluorescent measurements, all ligands show Zn(2+) selectivity over Ca(2+) and Mg(2+) ions in plasticized PVC membranes using potentiometric signal transduction. This observation found for N-(anthrylmethyl) and N-(3-methoxyphenyl) derivatives can be ascribed to the more hindered interaction between the signalling group of the ionophore and the central metal ion in PVC membranes than in acetonitrile solution upon complexation. From the fluorescent measurements it can also be concluded that the ligands with metal ions form complexes mainly with 2:1 stoichiometry (L(2)M). On complex formation a considerable decrease in the fluorescent intensity was observed for all ligands except the N-(anthrylmethyl) derivative, where a 25 - 30 fold fluorescence enhancement was found, which is explained by the photoinduced electron transfer (PET) mechanism. All ionophores exhibited serious hydrogen ion interference, therefore complexation-induced spectral changes were measured in aprotic acetonitrile solution.     

Pyrrolopyridazine derivatives as blue organic luminophores: synthesis and properties. Part 2

Two environmentally friendly methods, one in liquid and other in solid phase, for preparation of highly fluorescent pyrrolopyridazine (PP) derivatives under microwave (MW) irradiation is presented. The first synthesis in solid phase of fluorescent PP derivatives using activated alkynes under MW and conventional heating is also reported. Under MW irradiation the yields are higher, the amount of used solvent in liquid phase is at least five-fold less while solid phase does not use solvents, so these reactions may be considered as environmentally friendly. Eight new blue fluorescent 2-aryl-pyrrolopyridazine derivatives were synthesised. A certain influence of the PP substituents concerning absorption and fluorescent properties was observed. Introduction of a π-conjugated system in the second position of PP moiety increase the fluorescent properties. In the absorption spectra were observed a gradual decrease of wavelengths simultaneously with an increasing of molar extinction coefficient, due to increased number of ester groups from pyrrolic ring.     

8-Hydroxyquinoline derivative as a fluorescent chemosensor for zinc ion.

8-Hydroxyquinoline derivative 1 as a fluorescent chemosensor for Zn2+ was synthesized. Because Cd2+ is often found with Zn2+ in the environment and can form fluorescent complexes with chelating fluorophores, a potentially important property of chemosensors for Zn2+ is their selectivity for Zn2+ over Cd2+. The Zn2+ or Cd2+ complexes of 1 gave an emission band from the 1:1 complex, but the fluorescence intensity for Cd2+ was a half of that for Zn2+. Ligand 1 is suited for use as a fluorescent chemosensor for Zn2+.     

Spectrofluorometric determination of kynurenic acid with horseradish peroxidase in the presence of hydrogen peroxide.

The catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide has been investigated for the fluorescent derivatization of kynurenic acid under conditions with no exposure to light. Non-fluorescent kynurenic acid was converted into a fluorescent compound (Ex: 367 nm, Em: 470 nm) with HRP in the presence of hydrogen peroxide, and the optimum conditions of this fluorescent derivatization were investigated. Moreover, this fluorescent derivatization was developed for a spectrofluorometric determination of trace amounts of kynurenic acid by measuring the fluorescence intensity of the fluorescent compound. The calibration curve obtained was linear from 1.0 to 10.0 nmol of kynurenic acid in a 1.0 mL sample solution. The relative standard deviation at 5.0 nmol of kynurenic acid was 5.71% (n=5). By adjusting the bandwidths for both the excitation and emission to 15 nm, the calibration curve was also linear in the range between 0.1 to 1.0 nmol of kynurenic acid in a 1.0 mL sample solution. This method was applied to the fluorometric determination of trace amounts of kynurenic acid in the control sera.     

Reactant concentrations from fluorescence correlation spectroscopy with tailored fluorescent probes. An example of local calibration-free pH measurement

The present account is concerned with the measurement of local reactant concentrations by observing specific fluorescent probes in fluorescence correlation spectroscopy (FCS). The Theoretical Analysis section revisits the photophysical, thermodynamic, and kinetic information that is contained in the corresponding FCS correlation curves. In particular, we examine the conditions under which FCS is revealed as a superior tool to measure concentrations of reactive species. Careful molecular engineering of the specific fluorescent probes that simultaneously integrates photophysical, thermodynamic, and kinetic constraints will be required to benefit most from FCS. We illustrate the FCS titration approach with a series of fluorescent probes that we tailored to measure pH at around 4-6 by FCS after two-photon excitation. We show that an optimal design allows one to access pH without any preliminary calibrations such as the determination of the protonation constant or the photophysical properties of the fluorescent probe.     

Changes in surface charge density of lecithin liposomes by lipid peroxidation. A fluorescence study with 8-anilino-1-naphthalenesulfonate.

Treatment of lecithin liposomes with 100 microM ascorbic acid and 10 microM ferrous ion resulted in the formation of fluorescent products exhibiting an emission maximum at 430 nm and a decrease in the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) bound to the liposomes without change in the emission maximum. The degree of ascorbic acid/Fe(2+)-induced decrease in the ANS fluorescence was dependent on the extent of fluorescent product formation. The results of kinetic studies on ANS-binding to the liposomes showed that treatment of the liposomes with ascorbic acid/Fe2+ causes an increase of the apparent dissociation constant (Kd) of ANS-liposome complex. This indicates that lipid peroxidation of the liposomes by treatment with ascorbic acid/Fe2+ decreases the binding affinity of ANS to the liposomes. In addition, it was also found that there is a good correlation between degrees of the Kd value and the formation of fluorescent products. The fluorescence properties, i.e. emission maximum and response of the fluorescence intensity for borohydride reduction, of the products formed by lipid peroxidation of the liposomes were similar to those derived from modification of the liposomes with monofunctional aldehydes such as acetaldehyde and heptaldehyde. From these results, it is suggested that the decrease of ANS-binding affinity to the liposomes by treatment with ascorbic acid/Fe2+ may be due to changes in the surface charge density of the liposomes relating to the formation of fluorescent products.     

Quantitation of chlordiazepoxide and its metabolites in biological fluids by thin-layer chromatography.

Chlordiazepoxide and its 4 major metabolites were assayed after separation by thin-layer chromatography following extraction from biological fluids. The compounds become intensely fluorescent in the presence of red, fuming nitric acid. The resulting compounds are quantitated with a spectrodensitometer with a fluorescent attachment. The sensitivity varies between 0.05 and 0.1 microgram. The coefficient of variation is 1.4% for assays in urine and 6.4% in serum.     

Interaction of cyanine dyes with nucleic acids. XXV. Influence of affinity-modifying groups in the structure of benzothiazol-4-[2,6-dimethylpyridinium] dyes on the spectral properties of the dyes in the presence of nucleic acids.

Novel monomethine pyridinium cyanine dyes of similar structure and containing 'affinity-modifying' groups of different chemical nature were studied by spectral-luminescent methods as possible fluorescent probes for the nucleic acids detection. It was shown that the nature of the functional groups in the dye linker influences the fluorescent properties of the dye-nucleic acids complexes. Incorporation of a hydroxyl group into the linker structure leads to a significant increase in the fluorescence intensity of the dye--double-stranded DNA complexes relative to the parent dye Cyan 40.     

Phospholipid-linked coumarin: a fluorescent probe for sensing hydroxyl radicals in lipid membranes.

A fluorescent probe, DPPEC (1,2-dipalmitoylglycerophosphorylethanolamine labeled with coumarin) was developed for detecting hydroxyl radical (*OH) in lipid membranes. The coumarin moiety contributes to the fluorescent detection of *OH and the phospholipids moiety gives a driving force to localize the probe in lipid membranes. DPPEC in liposomal membranes rapidly reacted with *OH and increased the fluorescence intensity, depending on the concentration of *OH. The increase in the fluorescence intensity induced by *OH was effectively suppressed by the addition of DMSO. The probe exhibited a higher fluorescence response to *OH over other reactive oxygen species, such as hydrogen peroxide, nitric oxide, peroxynitrite, alkylperoxyl radical, and hypochlorite. DPPEC would be useful as a new type of fluorescent probe that can localize in lipid membranes and detect *OH efficiently.     

Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles.

New silica-based europium fluorescent nanoparticles having surface amino groups were prepared by a covalent binding-copolymerization technique. In the nanoparticles, the fluorescent Eu3+ chelate molecules were covalently bound to silicon atoms to protect the nanoparticles from dye leaking in bio-applications. The amino groups on the surface of nanoparticles made the surface modification and bioconjugation of nanoparticles easier. The nanoparticles were characterized and developed as a new type of fluorescence probe for a highly sensitive time-resolved fluoroimmunoassay (TR-FIA) of human hepatitis B surface antigen (HBsAg).     

Simultaneous imaging of multiple fluorescent probes in bio-cells.

In order to obtain the full spectrum from 400 to 800 nm of each pixel of a microscopic image, a unique spectro-imaging system was developed using an image slicer. The image slicer is composed of 100 photo fibers which are arranged in a matrix of 10 x 10 at the entrance and 100 x 1 at the exit. A line of this 100 signals is passed through a glism and projected onto a CCD. This system was applied to the fluorescent imaging of bio-cells. One of the demonstrative examples was simultaneous measurements of the Ca2+ concentration and the pH using of respective fluorescent probes. An electric signal was applied to BY-2 protoplasts and the fluorescent spectrum from 500 nm to 800 nm was measured every 5 s. The spectrum of the BY-2 protoplasts changed in response to the electric signal and the Ca2+ concentration, and the pH changes could be monitored. The wavelength resolution was satisfactory, but the space resolution was still rough in comparison with the usual microscopic systems. Notwithstanding these conditions, we could obtain discrete data from more than several tens of sites in a single-cell or a chain of several cells.     

Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis.

BACKGROUND: The caspase-mediated proteolysis of many cellular proteins is a critical event during programmed cell death or apoptosis. It is important to determine which caspases are activated in mammalian cells, and where and when activation occurs, upon receipt of specific death stimuli. Such information would be useful in the design of strategies to regulate the activation of caspases during apoptosis. RESULTS: We developed two novel fluorescent substrates that were specifically cleaved by caspase-1 or caspase-3. For in vitro studies, four-amino-acid recognition sequences, YVAD for caspase-1 and DEVD for caspase-3, were introduced between blue fluorescent protein (BFP) and green fluorescent protein (GFP), expressed in bacteria and purified. For in vivo studies, YVAD and DEVD were introduced between cyan fluorescent protein and yellow fluorescent protein, and expression was monitored in live mammalian cells. The proximity between fluorophores was determined using fluorescence resonance energy transfer. Purified substrates were cleaved following exposure to purified caspase-1 and caspase-3. In Cos-7 cells, caspase-1 and caspase-3 substrates were cleaved upon induction of apoptosis with staurosporine, a protein-kinase inhibitor, whereas caspase-3 but not caspase-1 substrate was cleaved upon treatment of cells with the DNA-damaging agent mitomycin c. CONCLUSIONS: These substrates allow the spatial activation of specific members of the caspase family to be deciphered during the initiation and execution phase of programmed cell death, and allow activation of specific caspases to be monitored both in vivo and in vitro. This technology is also likely to be useful for high-throughput screening of reagents that modulate caspase activity.     

Operation of the proton wire in green fluorescent protein. A quantum dynamics simulation

A nuclear quantum dynamical simulation of the proton shuttle operating in the green fluorescent protein has been carried out on a high-quality, high-dimensionality potential energy surface describing the photoactive pipi* excited state, and including motion of both the three protons and of the donor and acceptor atoms of the hydrogen bonds in a closed proton wire. The results of the simulations show that proton transfer along the wire is essentially concerted, synchronous, and very fast, with a substantial amount of the green fluorescent species forming within several tens of femtoseconds. In this regard, analysis of the population of the fluorescent species indicates that at least two dynamical regimes are present for its formation. Within the first hundreds of femtoseconds, dynamics is very fast and impulsive. Later on, a slower pace of formation appears. It is discussed that the two largest decay times for the protonated chromophore reported experimentally (Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8362-8367) might correspond to some irreversible process occurring after formation of the fluorescent species, rather than to cleavage of the chromophore's phenolic O-H bond.     

Spectrofluorimetric study of the degradation of alpha-amino beta-lactam antibiotics catalysed by metal ions in methanol.

The alpha-aminopenicillins ampicillin and amoxicillin and a cephalosporin, cephalothin, give rise to a fluorescent product when their methanolic solutions are incubated for prolonged time periods. The process also occurs in the presence of the metal ions Cd2+, Co2+ and Zn2+. The effects of the different ions on the emission and excitation wavelengths and the appearance rate of the fluorophore were studied. The appearance of the fluorescent product was zero order for ampicillin and amoxicillin in metal ion-free solution and solutions with Cd2+ and Zn2+, whereas in the presence of Co2+ ion it was first order under the experimental conditions used; for cephalothin it was first order in all cases. Apparent fluorescent compound formation rates were calculated in the zero-order reactions and rate constants in the first-order reactions. The activation energy of the formation reaction of the fluorescent products of amoxicillin and ampicillin was calculated from a study of the reactions at four temperatures; all the values recorded were between 34 and 118 kJ mol-1. As a possible mechanism for the formation of these products, cyclization of the penamaldic derivative of the antibiotic, which is formed in the first stage of the methanolytic reaction, is proposed.     

Rosafluin,ein neues Diapocarotindiol aus Rosenblüten. 8. Mitteilung über Rosenfarbstoffe

Rosafluine, a Novel Diapocarotenediol from Rose Petals A strongly fluorescent trace compound from rose petals was identified as the as yet unknown 10,10′-diapocarotene-10,10′-diol ( = 4,9-dimethyldodeca-2,4,6,8,10-pentaene-1,12-diol; 2 ).     

Simultaneous light emitting diode-induced fluorescence and contactless conductivity detection for capillary electrophoresis.

A combined detection system of simultaneous contactless conductometric and fluorescent detection for capillary electrophoresis (CE) has been designed and evaluated. The two processes share a common detection cell. A blue light-emitting diode (LED) was used as the excitation source and an optical fiber was used to collect the emitting fluorescence for fluorescent detection (FD). Inorganic ions, fluorescein isothiocyanate (FITC)-labeled amino acids and small molecule peptides were separated and detected by the combined detector, and the detection limits (LODs) of sub-microM level were achieved.