Thermal stability of solid dispersions of naphthalene derivatives with β-cyclodextrin and β-cyclodextrin polymers

The thermal stabilities of some naphthalene derivatives (1-naphthyl acetate, 2-acetylnaphthalene, 1-naphthol) in β-cyclodextrin (β-CD) inclusion complexes and in β-CD-containing polymeric systems (Polyβ-CD) have been studied using thermal and thermogravimetric analyses and infrared spectroscopy. In β-CD systems, the stability of the 1-naphthyl acetate complex is lower than that of the 2-acetylnaphthalene complex, and both are more stable than the corresponding physical mixtures. For Polyβ-CD systems, the solid dispersions result much more stable than the corresponding β-CD ones, both at room temperature and at 60 °C. In the case of Polyβ-CD, besides the inclusion within CD cavities, the interaction of the guest with the crosslinking network confers an additional stability against volatilization. In contrast, an analogous crosslinked polymer prepared using sucrose instead of β-CD does not retain noticeable amounts of the naphthalene derivatives.     

Sensing of Fe(III) ion via turn-on fluorescence by fluorescence probes derived from 1-naphthylamine

Fluorescence probes NA1 and NA2 derived from 1-naphthylamine (NA) as fluorophore have been synthesized and characterized by different spectroscopic studies. Identification behaviour of these probes towards various metal ions has been investigated. Both the fluorescent probes are selective as well as sensitive towards Fe(III) ion. Novel fluorescence probe NA2 afforded turn-on fluorescence behaviour for Fe(III) ion over other metal ions such as Ca(II), Mg(II), Mn(II), Fe(II), Co(II), Fe(III), Ni(II), Cu(II), Zn(II) and Hg(II).     

Novel probes showing specific fluorescence enhancement on binding to a hexahistidine tag

The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel-nitrilotriacetic acid (Ni(2+)-NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal-NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal-NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni(2+) and Co(2+) were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni(2+) and Co(2+) complexes of NTACs in pH 7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)(6)-Tyr-NH(2); Tyr was included to allow convenient concentration measurement) in pH 7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co(2+) complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide.     

Study of the chromatographic behavior and inclusion constants of new fluorescence agents by use of cyclodextrin additives under conditions of steady- and dynamic-state equilibrium and with fluorescence detection

Summary We report four new derivatization agents, acridone-N-acetic acid (ARC), carbazole-9-ylacetic acid (CRA), carbazole-9-ylpropionic acid (CRP), and 2-methyl-2-carbazole-9-ylacetic acid (MCRA), with strong fluorescence emission which has low dependence on solvent polarity. The emission maxima for ARC, CRA, CRP, and MCRA were 430 nm (λ_ex 404 nm), 368 nm (λ_ex 335 nm), 356 nm (λ_ex 340 nm) and 360 nm (λ_ex 330 nm), respectively. The effects of mobile-phase composition, pH, and temperature on the liquid chromatographic retention behavior of the four fluorescence agents were investigated. An experimental model was established for calculating the inclusion constants of cyclodextrin (CD) complexes in the dynamic state, using β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) as examples, and different mobilephase compositions. On the basis of the model, the inclusion constants of the solutes in pure water (K _fw) were determined by extrapolation. The thermodynamic parameters (ΔH ^o and ΔS ^o) and dissociation constantsK _am for the solutes in this chromatographic system were obtained by means of capacity factor (k) values using a corresponding model formulation.     

Aggregation numbers of SDS micelles formed on EHEC. A steady state fluorescence quenching study

The present investigation proves that in the interaction between an uncharged polymer and a negatively charged amphiphilic ion (surfactant) clusters are actually formed and it provides data for the cluster concentration and the cluster size and their variation with composition. The polymer bound cluster size increases after a certain critical surfactant concentration and passes through a maximum. This maximum cluster size decreases with decreasing polymer concentration and attains a limiting value at infinite dilution. For the highest polymer concentration the cluster size is close to the size of normal surfactant micelles. The cluster concentration was determined by a fluorescence quenching technique and the amount surfactant adsorbed to the polymer by dialysis equilibrium measurements. Combining these independent sets of data permits the cluster aggregation number to be unambiguously determined. Solubilization experiments indicate the possibility to regulate the amount solubilized by varying the polymer concentration. The molecular properties of the system are sensitively monitored by the variation in two vibronic peaks in the pyrene fluorescence emission spectrum which defines a hydrophobic index. Very good agreement is found between all three experimental methods. Finally, the model suggested is analyzed in terms of coil size and cluster-cluster distance. Depending upon the degree of adsorption saturation and the density of polymer segments in solution the interaction may switch from being intramolecular to becoming intermolecular.     

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C_BZC/C_Lys < 9) and a combined quenching process at higher concentration of BZC (C_BZC/C_Lys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn²⁺ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.     

Computational study on the molecular inclusion of andrographolide by cyclodextrin

Due to the poor water solubility of andrographolide (andro), an inclusion technique has been developed to modify its physical and chemical properties so as to improve its bioavailability. In contrast with the immense experimental studies on the inclusion complexes of andro:cyclodextrin, no computational study has so far been carried out on this system. In this work, preliminary docking experiments with AutoDock were performed. Density Functional Theory (DFT) and Austin Model 1 (AM1) calculations upon the docking instances were applied to investigate the two possible modes of molecular inclusions between andro and x-cyclodextrin (xCD, where x is alpha, beta or gamma). Atoms-in-Molecules (AIM) analysis based on the B3LYP/cc-pVDZ wavefunction was applied to verify the existence of the intermolecular hydrogen bonds. It was found that the most stable complex among the six possible inclusion complexes was the one formed between andro and betaCD with andro's decalin ring moiety wrapped by CD at a ratio of 1:1. The hydrogen bonds between andro and CD were responsible for the stability of the inclusion complexes. The calculated data were found to be consistent with the experimental results. Thus, the results of this study can aid new drug design processes.     

Accessibility of the gel phase in macroporous network polymers: A comparison of the fluorescence probe and inverse steric exclusion chromatography techniques

Two independent methods of analysis were employed to evaluate accessibility of the gel phase in macroporous network polymers. Fluorescence probes, covalented incorporated into the gel during polymerization, can provide a qualitative assessment of accessible surface area from an analysis of the solvatochromic shift of fluorescence emission. These findings are corroborated by inverse steric exclusion chromatography (ISEC). The data permits quantification of the polymers surface character in the solvated state. The results call attention to the inadequacy of dry surface area technique (e.g., BET analysis) in providing information of this type.     

Preliminary study on dual fluorescence behavior of porphyrin

It is well known that porphyrin derivatives play a key role in the primary process of photo-synthesis[1], in which porphyrins directly absorb the sunlight or indirectly acquire excitation en-ergy from light-harvesting antenna system to reach their excited state, and then donate electrons to quinone acceptors to yield a series of charge-separated species. In general, only first singlet ex-cited state of porphyrins is involved in energy transfer process[2]. However, highly excited state (S2 stat…     

Quantitative determination of oxidized carbon nanotube probes in yeast by capillary electrophoresis with laser-induced fluorescence detection

Short oxidized multi-walled carbon nanotubes were functionalized with fluorescein isothiocyanate to form carbon nanotube probes (CNTP). The distribution of CNTP in yeast was quantitatively determined by capillary electrophoresis coupled with laser-induced fluorescence detection. The detection sensitivity for CNTP was greatly improved comparing with UV absorbance and Raman detection. The time- and temperature-dependent influx patterns of CNTP into yeast were obtained. The apparent permeability coefficient for influx of CNTP into yeast was calculated, which suggested that CNTP might permeate into yeast through endocytosis. This study implies that CNTP could be a fine drug transporter and might be wildly used in multidrug resistance research and microorganism detection.     

Light scattering and fluorescence photobleaching recovery study of poly(amidoamine) cascade polymers in aqueous solution

Diffusion coefficients of polyamidoamine cascade polymers (PAMAMs) were measured in aqueous solutions by dynamic light scattering and, after labeling with fluorescein isothiocyanate, by fluorescence photobleaching recovery. The dynamic light scattering results depended weakly on pH at a high salt concentration, but varied strongly with the concentration of added salt in the low-salt limit. The fluorescence photobleaching recovery values were almost independent of salt concentration. The difference between the two techniques is that thermodynamic nonideality strongly affects light scattering at the concentrations that are experimentally accessible. The hydrodynamic sizes from fluorescence photobleaching recovery were somewhat smaller than those from dynamic light scattering in the high-salt limit, despite attachment of the dye. Nevertheless, fluorescently tagged PAMAMs should make suitable markers and diffusion probes. © 1996 John Wiley & Sons, Inc.     

Evidence for covalent binding of epicocconone with proteins from synchronous fluorescence spectra and fluorescence lifetimes

Synchronous fluorescence and time-resolved fluorescence spectroscopic studies that reveal the interaction of epicocconone with human serum albumin is significantly different from its interaction with surfactant assemblies. This observation, along with steady-state fluorescence data, indicates ground-state interaction between the fluorophore epicocconone and the protein. Similarity in fluorescence properties with the adduct of the fluorophore with n-butylamine indicates that bonding occurs at the N-terminus of the protein.     

Effect of the reaction temperature on the transglycosylation reactions catalyzed by the cyclodextrin glucanotransferase from Bacillus macerans for the synthesis of large-ring cyclodextrins

The synthesis of cyclodextrins with from 6 to more than 50 glucose units by cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus macerans was investigated. Analysis of the synthesized cyclic α-1,4-glucan products showed that a higher yield of large-ring cyclodextrins were obtained with a reaction temperature of 60 °C compared to 40 °C. The yield of large-ring cyclodextrins obtained at 60 °C represented about 50% of the total glucans employed in the reaction. Analysis of the cyclodextrin-forming cyclization reaction and of the coupling reaction of the CGTase resulting in the degradation of mainly the larger cyclic α-1,4-glucans indicated higher rates of the cyclization reaction at 60 °C compared to 40 °C while the opposite was found for the coupling reaction.     

A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.     

Curing of linear and crosslinked epoxy systems: A fluorescence study with dansyl derivatives

The curing of diglycidyl ether of bisphenol A (DGEBA) with N,N′-dimethylethylenediamine (N,N′-DMEDA) or ethylenediamine (EDA) was monitored by fluorescence spectroscopy and Fourier transform infrared (in the near-infrared region). 5-Dimethylamino-naphthalene-1-sulfonamide (DNS) derivatives were used as probes (fluorophores added to the reaction mixture) and labels (fluorophores attached by covalent bonds to diglycidyl reactants). The term containing the ratio of the reaction rate constants for the addition of the secondary and primary amine hydrogens to the epoxide was included in the reduced reaction rate term for the autocatalyzed and catalyzed epoxide curing reactions. The changes in the integrated fluorescence intensities of the labels during the epoxy group conversion indicated, in some cases, the most important changes in the chemical transformations of the reaction mixture: the epoxy group conversion, during which a rapid increase in the tertiary amino group concentration was first observed; the gel point (for EDA); and the entry of the system into the glassy state (for N,N′-DMEDA and EDA). The fluorescence probes monitored neither the gel point nor the threshold of the glassy state. For the DGEBA–N,N′-DMEDA system, a wavy dependence of the integrated fluorescence intensities of the DNS labels on the epoxy group conversion might reflect the molar concentrations of polymer homologues (referred to the initial number of moles in the system) in the reaction mixtures of the diepoxide and secondary diamine. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 64–78, 2004     

The art of fluorescence imaging with chemical sensors

Fluorescence imaging techniques involving chemical sensors are essential tools in many fields of science and technology because they enable the visualization of parameters which exhibit no intrinsic color or fluorescence, for example, oxygen, pH value, CO(2), H(2)O(2), Ca(2+), or temperature, to name just a few. This Review aims to highlight the state of the art of fluorescence sensing and imaging, starting from a comprehensive overview of the basic functional principles of fluorescent probes (or indicators) and the design of sensor materials. The focus is directed towards the progress made in the development of multiple sensors and methods for their signal read out. Imaging methods involving optical sensors are applied in quite diverse scientific areas, such as medical research, aerodynamics, and marine research.     

Alternative approach to the design of thermosensitive polymers: The addition of hydrophobic groups to the ends of hydrophilic polyether

We synthesized thermosensitive polymers by adding hydrophobic groups to the ends of a hydrophilic polyether, poly(ethylene oxide‐co‐propylene oxide). The cloud points of the polymers were controlled by the molecular weight of the polyether and the kind of hydrophobic end group. The polymers showed relatively sharp phase transitions, as observed by turbidimetry. The order of the cloud points of the polymers with various end groups did not follow that of the critical micelle concentrations of the polymers determined with fluorescence measurements with pyrene as a probe. The cloud points of the polymers linearly decreased with increasing concentrations of salts (Na₂SO₄ and Na₃PO₄). The slopes of the linear lines were almost constant, regardless of the kind of hydrophobic group and the molecular weight of the polyether. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1007–1013, 2005     

Comparative effect of cyclodextrin nanocavities versus organic solvents on the fluorescence of carbamate and indole compounds

The effect of the addition of different amounts of organic solvents (S) on the fluorescence of aromatic compounds (C) and their inclusion complexes with β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPCD) has been examined using steady-state measurements. Carbamate pesticides with different aromatic moiety, such as carbofuran (CF), promecarb (PC), carbaryl (CY) and bendiocarb (BC) were used, as well as indole derivatives with different polarity in their lateral chains, such as melatonin (M, neutral), 5-methoxytryptamine (MT, cation) and auxin (IA, anion). Their complexes in water show a fluorescence signal higher than that obtained for the free substrates in solvent:water mixtures (30%, v/v n-propanol or acetonitrile, and 50%, v/v methanol). The isofluorescent point (IF), the %IF and the F_85% are defined in order to evaluate the use of CD nanocavities as a non-polluting alternative for the analysis of the compounds analyzed.Apparent formation constants (K_AP, M⁻¹) for the complexes of C:HPCD at different solvent percentages were determined for CF and PC with methanol (MeOH), n-propanol (ProOH) and acetonitrile (ACN), and for indole compounds with ACN. A decrease in the K_AP values for the CF:HPCD (120–30) and PC:HPCD (2000–400) complexes occurs in accordance with the solvent affinities for CDs (MeOH < ACN < ProOH). Nevertheless, in the indolic series, the polar characteristics of MT, IA and M determine their behaviour in the presence of ACN. For the neutral substrate M, K_AP decreases with the increasing percentage of ACN (100–10). In contrast, for IA and MT (ionic substrates) K_AP increases (10–100).These results may be accounted for by two different mechanisms: the competition between C and S for the cavity of the receptor or the formation of ternary complexes C:S:CD with additional stabilization.     

水溶性CdTe量子点的合成及其用于荧光猝灭法测定肌红蛋白

以3-巯基丙酸为稳定剂,合成了具有特殊光学性质的水溶性CdTe量子点,其最大发射波长位于544 nm.利用荧光光谱、紫外可见光谱及圆二色光谱法系统的研究了CdTe量子点与肌红蛋白(Mb)二者结合前后体系光谱的变化,从而证实了CdTe量子点与Mb之间静电结合反应的特征.在pH 7.0的PBS缓冲液中,用CdTe量子点作为荧光探针研究了肌红蛋白与量子点的相互作用,并基于肌红蛋白对CdTe量子点有显著的荧光猝灭作用,建立了肌红蛋白的快速检测方法.在最佳实验条件下,该体系荧光强度的猝灭程度(△F)与肌红蛋白质量浓度呈良好的线性关系,线性范围为0.3～24 μg/mL,检出限为0.13 μg/mL.该方法已对合成样品中肌红蛋白进行检测,并用于人体尿样中肌红蛋白的测定.     

Quantitative structure-property relationship study on the determination of binding constant by fluorescence quenching

Models to predict binding constant (logK) to bovine serum albumin (BSA) should be very useful in the pharmaceutical industry to help speed up the design of new compounds, especially as far as pharmacokinetics is concerned. We present here an extensive list of logK binding constants for thirty-five compounds to BSA determined by florescence quenching from the literature. These data have allowed us the derivation of a quantitative structure-property relationship (QSPR) model to predict binding constants to BSA of compounds on the basis of their structure. A stepwise multiple linear regression (MLR) was performed to build the model. The statistical parameter provided by the MLR model (R = 0.9200, RMS = 0.3305) indicated satisfactory stability and predictive ability for the model. Using florescence quenching spectroscopy, we also experimentally determined the binding constants to BSA for two bioactive components in traditional Chinese medicines. Using the proposed model it was possible to predict the binding constants for each, which were in good agreement with the experimental results. This QSPR approach can contribute to a better understanding of structural factors of the compounds responsible for drug-protein interactions, and be useful in predicting the binding constants of other compounds.