Cross-linked polyvinylpyrrolidones with increased affinity and specificity for methyl orange and its homologs

Polyvinylpyrrolidones of various degrees of cross-linkage have been prepared by radical polymerization of N-vinylpyrrolidone with methylenebisacrylamide to regulate the fraction of cross-linkage. The insoluble polymers obtained were examined for their ability to bind methyl orange and its homologs, methyl orange, ethyl orange, propyl orange, and butyl orange at 5, 15, 25, and 35°C, respectively, in an aqueous solution. The first binding constants and the thermodynamic parameters that accompanied the binding were calculated. For any particular dye the extent of binding, the absolute magnitude of ΔF°, and the value of ΔS° increased as the degree of cross-linkage increased, starting with water-soluble polyvinylpyrrolidone (zero cross-linkage) and proceeding to the polymer with high cross-linking density. This behavior can be accounted for in terms of more extensive hydrophobic domains in the cross-linked polymeric matrix that enhances hydrophobic interactions in the binding process. Moreover, the cross-linked macromolecule polymerized in the presence of methyl orange and then stripped of the bound methyl orange shows substantially stronger binding for this small molecule than the polymer cross-linked in the absence of methyl orange. In contrast, the cross-linked polymer prepared similarly in the presence of the larger molecule, butyl orange, exhibits decreased affinity toward the smaller consolute, methyl orange, than either of the other polymers described. It seems, therefore, that the polymeric matrix provides favorable binding sites or pockets that can accommodate a specific small molecule. The preparative procedure, which uses a small-molecule template, molds into the polymer some structural specificity in the binding of small molecules.     

Binding methyl orange and hydrophobic fluorescent probes by poly(2-dimethylaminoethyl methacrylate) and copolymers of 2-dimethylaminoethyl methacrylate and N-vinyl-2-pyrrolidone: pH dependence of the binding and fluorescence intensity

The pH dependence of the interaction of poly(2-dimethylaminoethyl methacrylate) and copolymers of 2-dimethylaminoethyl methacrylate and N-vinyl-2-pyrrolidone with methyl orange, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), and 1,6-diphenyl-1,3,5-hexatriene (DHT) was studied by equilibrium dialysis and fluorescence measurements at pH's 7–10. The first binding constant accompanying the binding of methyl orange and TNS by the polymers, in particular the homopolymer, shows a maximum around pH 8 and maximal fluorescence intensity of TNS is obtained around pH 8.5 in the presence of the polymers. To elucidate these observations the pH-induced conformational changes of the homopolymer were examined by potentiometric titration and viscosity measurements and the thermodynamic parameters that accompany the binding were calculated. The polymer was found to change from an extended coil at lower pH to a compact coil at higher pH. The electrostatic attraction between the sulfonate group of the small molecule and the protonated nitrogen atoms on the polymer is increased at lower pH and the hydrophobic interaction between the hydrophobic moieties of the polymer and the small molecule is enhanced at higher pH. The results obtained for the dye binding and fluorescence intensity were discussed in terms of the electrostatic and hydrophobic interactions.     

Thermodynamics of binding methyl orange and its homologs by polyvinylpyrrolidone: The effect of hexamethylenetetramine

Hexamethylenetetramine (HMT) has been examined for its effect on the binding of methyl orange homologs, methyl orange, ethyl orange, propyl orange, and butyl orange by polyvinylpyrrolidone (PVP). In the presence of HMT the entropy changes associated with the binding tend to become more positive and the absolute magnitude in the enthalpy changes becomes smaller compared with those in the absence of HMT. These tendencies are accounted for in terms of the water-structure-promoting effect of HMT, hence the enhancement of hydrophobic interactions in the binding. PVP undergoes changes in conformation on the addition of HMT and its conformation becomes more compact. This also increases the contribution of the hydrophobic interactions to the macromolecule-small molecule interaction. Some other effects exerted by the added HMT on the binding system are also described.     

Peculiar temperature dependence on the binding of methyl orange and its homologs by 2-hydroxyethyl methacrylate-N-vinyl-2-pyrrolidone copolymers

2-Hydroxyethyl methacrylate (HEMA)-N-vinyl-2-pyrrolidone (VPy) copolymers of various compositions have been prepared. The copolymers obtained were examined for their ability to bind a homologous series of methyl orange derivatives, methyl orange, ethyl orange, propyl orange, and butyl orange, at 5, 15, 25, and 35°C, respectively, in an aqueous solution. The first binding constants and the thermodynamic parameters that accompanied the binding were evaluated. The binding ability of the copolymer for the small cosolute was enhanced with an increase of the HEMA content in the copolymer. Moreover, a bell-shaped curve appeared in the binding of butyl orange by the copolymers having higher HEMA residues when the first binding constant was plotted as a function of temperature, whereas no such phenomenon was detected for the copolymers with less HEMA content or for the less hydrophobic dye, methyl orange, ethyl orange, or propyl orange. This peculiar temperature dependence of the first binding constant shows that the enthalpy of the binding varies from a positive (unfavorable) value below ca. 15°C to a negative (favorable) one above this temperature. This behavior can be accounted for in terms of more hydrophobic effects involved in the binding process.     

Thermodynamics of binding of methyl orange and its homologs by poly(vinylpyrrolidone): The effect of inorganic electrolytes

The extent of binding of methyl orange, ethyl orange, propyl orange, and butyl orange by poly(vinylpyrrolidone) has been measured in aqueous solutions of inorganic electrolytes such as NaCl, LiCl, NaSCN, and NaClO₄ by an equilibrium dialysis method. The effect of the salts on the first binding constants and the thermodynamic functions which are accompanied by the dye—polymer association process was investigated relative to the corresponding values in the absence of such salts. It was found that in aqueous solutions of NaCl and LiCl the enthalpy change accompanying the binding is small and the largest contribution to the free energy of binding is from the positive entropy gain. For NaSCN and NaClO₄, the values of ΔF° and ΔH° were both large and negative and the value of ΔS° was small and negative. Thus, the favorable free energy for the complex formation was due entirely to the negative enthalpy term. These characteristics of the thermodynamic quantities are discussed in terms of changes in structural properties of water in the vicinity of the binding entities and conformational changes of the polymer to which the dye is bound due to the added foreign electrolytes.     

Binding of methyl orange and its homologs by polycations containing apolar pendant groups

The binding of methyl orange, ethyl orange, and propyl orange by polycations involving various apolar pendant groups such as methyl, ethyl, benzyl, or dodecylbenzyl groups has been examined quantitatively by an equilibrium dialysis method at 5, 15, 25, and 35°C. The first binding constants and the thermodynamic parameters in the course of the binding have been calculated. The favorable free energy of the binding is accompanied by an entropy gain and an exothermic enthalpy change. The shorter the alkyl chain of the dyes or the polymers, the more negative is the enthalpy change and hence the smaller is the entropy change. Furthermore, an increase in binding affinity can be created in the polycation upon introduction of hydrophobic groups. In particular, the binding ability of the polycation containing a dodecylbenzyl group for methyl orange is almost 300-fold that of bovine serum albumin. Therefore it is clear that hydrophobic interactions, as well as electrostatic ones, are involved in the binding.     

Spectral studies of N-nonyl acridine orange in anionic, cationic and neutral surfactants

The spectroscopic and photophysical properties of N-nonyl acridine orange - a metachromatic dye useful as a mitochondrial probe in living cells - are reported in water and microheterogeneous media: anionic sodium dodecylsulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB) and neutral octylophenylpolyoxyethylene ether (TX-100). The spectral changes of N-nonyl acridine orange were observed in the presence of varying amount of SDS, CTAB and TX-100 and indicated formation of a dye-surfactant complex. The spectral changes were also regarded to be caused by the incorporation of dye molecules to micelles. It was proved by calculated values K(b) and f in the following order: K(bTX-100)>K(bCTAB)>K(bSDS) and f(TX-100)>f(CTAB)>f(SDS). NAO binds to the micelle regardless the micellar charge. There are two types of interactions between NAO and micelles: hydrophobic and electrostatic. The hydrophobic interactions play a dominant role in binding of the dye to neutral TX-100. The unexpected fact of the binding NAO to cationic CTAB can be explained by a dominant role of hydrophobic interactions over electrostatic repulsion. Therefore, the affinity of NAO to CTAB is smaller than TX-100. Electrostatic interactions play an important role in binding of NAO to anionic micelles SDS. We observed a prolonged fluorescence lifetime after formation of the dye-surfactant complex tau(SDS)>tau(TX-100)>tau(CTAB)>tau(water), the dye being protected against water in this environment. TX-100 is found to stabilize the excited state of NAO which is more polar than the ground state. Spectroscopic and photophysical properties of NAO will be helpful for a better understanding of the nature of binding and distribution inside mammalian cells.     

Interaction of crosslinked polyethylenimine with a homologous series of methyl orange derivatives in aqueous solution

Polyethylenimine (PEI) was crosslinked with dichloroethane, glyoxal, or glutaraldehyde and polymers of various degrees of crosslinkage were made. The insoluble polymers obtained were examined for their ability to bind methyl orange and its homologs, methyl, ethyl, propyl, and butyl orange at 5, 15, 25, and 35°C, respectively, in an aqueous solution. PEI crosslinked with glutaraldehyde showed markedly increased binding affinity toward these cosolutes compared with the polymers crosslinked with dichloroethane or glyoxal. The extent of the binding increased with an increase in the degree of crosslinkage. These results suggest that the enhancement of the binding by the crosslinking is due mainly to a dual effect, introduction of hydrophobic moieties and proximity of neighboring polymer chains. The first binding constants and the thermodynamic parameters that accompanied the binding were calculated. The thermodynamic data show that the binding process is athermal and is stabilized entirely by the entropy term. Water-soluble PEI exhibited stronger cooperative interactions than the crosslinked polymer because the mobilities of the chains of the former are greater than those of the latter.     

Peculiar temperature dependence of the binding of methyl orange and its homologs by 2-diethylaminoethyl methacrylate-N-vinyl-2-pyrrolidone copolymers

2-Diethylaminoethyl methacrylate (DEAEMA)–N-vinyl-2-pyrrolidone (VPy) copolymers of various compositions have been synthesized. The resultant copolymers were examined for their ability to bind methyl orange and its homologs, in particular butyl orange, at 5, 15, 25, and 35°C in aqueous solutions. The amount of binding of butyl orange is much higher with the copolymers than with polyvinylpyrrolidone or with 2-hydroxyethyl methacrylate–N-vinyl-2-pyrrolidone copolymers. Introduction of only 3% of the hydrophobic DEAEMA residue increases markedly the binding affinity toward the cosolute. Maximal binding is obtained at 15°C in the temperature range measured. This peculiar temperature dependence of the extent of binding is explicable on the basis of hydrophobic effects involved in this binding. The peculiar temperature dependence disappeared in aqueous solution of NaSCN which acts as a water-structure breaker: the extent of binding changes regularly with temperature. This is interpretable only in terms of reduction of hydrophobic contribution to the binding. With propyl orange, which is a less hydrophobic cosolute than butyl orange, the peculiarity of the binding was not detected.     

Interplay of supramolecular organization, metallophilic interactions, phase changes, and luminescence in four polymorphs of IrI(CO)2(OC(CH3)CHC(CH3)N(p-tol))

Four polymorphs of IrI(CO)2(OC(CH3)CHC(CH3)N(p-tol)) have been characterized by single crystal X-ray crystallography. While all contain the same molecular unit with no significant structural variations within the molecules, all show different degrees of metallophilic interactions between the planar molecules. Three of these (the amber, the pale yellow, and the orange forms) are stable at room temperature, while the fourth, the L. T. orange form, is only obtained by cooling the orange polymorph. At 77 K, the amber, pale yellow, and L. T. orange polymorphs show intense luminescence. The variations in the luminescence among the polymorphs are considered in the context of the structural differences between them and the nature of the metallophilic interactions between the iridium centers. These results demonstrate how subtle variations in molecular organization can affect the physical properties of planar d8 transition metal compounds, which are an important class of lumiphores.     

Binding of methyl orange and its homologs by powdered nylon 612: Peculiar temperature dependence on the binding

The ability of powdered Nylon 612 to bind methyl orange, ethyl orange, propyl orange, and butyl orange was investigated at 5, 15, 25 and 35°C in an aqueous solution. The amount of binding of the dye is much higher with this polyamide than with powdered Nylon 66 reported previously,¹ although the former polymer has fewer amide end groups. The Van't Hoff plots of the first binding constant for the binding of butyl orange and propyl orange by powdered Nylon 612 exhibit a bell-shaped curve, whereas the plots for methyl orange and ethyl orange do not. Maximal binding occurs at approximately 15°C for propyl orange and at about 25°C for butyl orange. This is the first instance where the peculiar temperature dependence of the binding constant has been found in the binding of propyl orange, whose hydrophobicity is less than that of butyl orange. These tendencies can be accounted for in terms of increased hydrophobic of butyl orange. These tendencies can be accounted for in terms of increased hydrophobic domains in powdered Nylon 612 and enhanced hydrophobic contributions in the binding process.     

Binding of methyl orange and its homologs by polyion complexes consisting of a piperidinium cationic polymer and various polyanions in aqueous solution

A study was made of the formation of polyion complexes between a piperidinium cationic polymer and polyanions and of the binding of azo-dye anions (methyl, ethyl, propyl, and butyl orange) by these complexes. Sodium poly(acrylate), poly(styrenesulfonate), dextran sulfate, and carboxy-methylcellulose were used as polyanions. The resultant polyion complexes (insoluble in aqueous solutions) were compared for their ability to bind the small organic molecules in aqueous solutions, for example, of urea and an inorganic electrolyte (KCI), and exhibited a strong binding affinity toward these small anions. Polyion complexes that consisted of sodium poly(acrylate), dextran sulfate, and carboxymethylcellulose as polyanions cooperated in the binding, whereas the polyion complex of sodium poly(styrenesulfonate) did not. It was suggested that small organic anions interact with the polyion complexes primarily through electrostatic and hydrophobic forces.     

人血白蛋白结合位点II的分子结合模式研究

人血白蛋白是血浆中含量最高的蛋白,是一种重要的载体蛋白,能与多种内源性和外源性物质结合。人血白蛋白主要有两个药物结合位点,位点I和位点II,其中位点II的柔性较大,对药物分子的亲和性较高。本文采用分子模拟方法,基于12种结合于位点II的小分子-人血白蛋白晶体结构,分析了相互作用能,发现12种分子的结合以疏水作用为主,静电作用为辅。进一步采用丙氨酸扫描和结合能评价,分析结合部位的关键氨基酸残基,探究结合模式的规律性,发现从位点入口到空腔内部存在静电、疏水和杂合的三层相互作用分布,共同形成了稳定的分子结合。最后采用分子对接和分子动力学模拟,预测了L-色氨酸的结合模式。研究结果有助于深入了解人血白蛋白药物位点II的小分子结合模式,为基于位点II的药物和分离配基的优化设计提供指导。     

Confocal fluorescence imaging of photosensitized DNA denaturation in cell nuclei

The double-stranded helical structure of DNA is maintained in part by hydrogen bonds between strands and by stacking interactions between adjacent purine and pyrimidine bases in one strand. The transition (denaturation) from a double-stranded (ds) to a single-stranded (ss) form can be induced in isolated DNA or fixed cells by exposure to elevated temperatures, alkali or acids, aprotic or nonpolar solvents or some drugs. We report here that DNA denaturation can occur in situ in cell nuclei as a result of interaction between light and an intercalated dye, acridine orange or ethidium bromide. This DNA photodenaturation was probed using metachromatic properties of acridine orange and imaged by fluorescence confocal microscopy. Furthermore, an empirical kinetic model was developed to separate changes of acridine orange luminescence intensities caused by photobleaching from those that were a result of DNA denaturation. We investigated the influence of oxygen on these phenomena and propose a mechanism by which photodenaturation may occur.     

噻唑橙类菁染料与生物大分子结合的光谱特性的研究

研究了两个噻唑橙类菁染料TO-1和TO-2与核酸、蛋白质结合的光谱特性.发现染料喹啉环中氮上取代基对染料标记生物大分子的荧光特性有着很大的影响.羧基的引入,使染料TO-2与核酸结合后的荧光性能下降,但同时也提高了它与蛋白质结合的光物理性能.研究表明:在喹啉环氮上引入适当的亲水性取代基,可以使噻唑橙类菁染料的应用范围拓展到蛋白质标记领域.并初步研究了蛋白质浓度的变化对染料TO-2的荧光光谱的影响以及TO-2在不同pH值的蛋白质溶液中的荧光性质.     

SO2-binding properties of cationic η6,η1-NCN-pincer arene ruthenium platinum complexes: spectroscopic and theoretical studies

The SO(2)-binding properties of a series of η(6),η(1)-NCN-pincer ruthenium platinum complexes (NCN = 2,6-bis[(dimethylamino)methyl]phenyl anion) have been studied by both UV-visible spectroscopy and theoretical calculations. When an electron-withdrawing [Ru(C(5)R(5))](+) fragment (R = H or Me) is η(6)-coordinated to the phenyl ring of the NCN-pincer platinum fragment (cf. [2](+) and [3](+), see Scheme 1), the characteristic orange coloration (pointing to η(1)- SO(2) binding to Pt) of a solution of the parent NCN-pincer platinum complex 1 in dichloromethane upon SO(2)-bubbling is not observed. However, when the ruthenium center is η(6)-coordinated to a phenyl substituent linked in para-position to the carbon-to-platinum bond, i.e. complex [4](+), the SO(2)-binding property of the NCN-platinum center seems to be retained, as bubbling SO(2) into a solution of the latter complex produces the characteristic orange color. We performed theoretical calculations at the MP2 level of approximation and TD-DFT studies, which enabled us to interpret the absence of color change in the case of [2](+) as an absence of coordination of SO(2) to platinum. We analyze this absence or weaker SO(2)-coordination in dichloromethane to be a consequence of the relative electron-poorness of the platinum center in the respective η(6)-ruthenium coordinated NCN-pincer platinum complexes, that leads to a lower binding energy and an elongated calculated Pt-S bond distance. We also discuss the effects of electrostatic interactions in these cationic systems, which also seems to play a destabilizing role for complex [2(SO(2))](+).     

Study of molecular interactions between lipids and proteins using dynamic surface tension measurements: a review

Molecular interactions between small molecules and proteins, such as binding of lipids to proteins, are of fundamental importance in various biological processes. A recently-developed method based on dynamic surface tension measurement is efficient and versatile in detecting such molecular interactions: Axisymmetric Drop Shape Analysis (ADSA) provides a tool for measuring the surface tension (γ) response to surface area changes. Through the analysis of the γ response pattern, surface competitive adsorption between small organic molecules and protein molecules can be detected. Surface squeeze-out of small molecules by proteins can also be observed. Molecular binding of lipids to proteins manifests itself in a modification of the γ response which is not compatible with a simple superposition of the two individual patterns. The specific binding can be studied in terms of dose effects and specificity.     

Environmental effects on the enhancement in natural and damaged DNA nucleobase acidity because of discrete hydrogen-bonding interactions

The present study uses density functional theory to carefully consider the effects of the environment on the enhancement in (natural and damaged) DNA nucleobase acidities because of multiple hydrogen-bonding interactions. Although interactions with one small molecule can increase the acidity of the nucleobases by up to 60 kJ mol-1 in the gas phase, the maximum increase in enzymatic-like environments is expected to be approximately 40 kJ mol-1, which reduces to approximately 30 kJ mol-1 in water. Furthermore, the calculated (simultaneous) effects of two, three, or four molecules are increasingly less than the sum of the individual (additive) effects with an increase in the number and acidity of the small molecules bound or the dielectric constant of the solvent. Regardless of these trends, our calculations reveal that additional hydrogen-bonding interactions will have a significant effect on nucleobase acidity in a variety of environments, where the exact magnitude of the effect depends on the properties of the small molecule bound, the nucleobase binding site, and the solvent. The maximum increase in nucleobase acidity because of interactions with up to four small molecules is approximately 80 kJ mol-1 in enzymatic-like environments (or 65 kJ mol-1 in water). These results suggest that hydrogen-bonding interactions likely play an important role in many biological processes by changing the physical and chemical properties of the nucleobases.     

Temperature dependence on the binding of butyl orange by bovine serum albumin: The effect of urea

The effect of urea on the extent of the binding of butyl orange by bovine serum albumin has been examined by an equilibrium dialysis method. The first binding constants and the thermodynamic parameters for the formation of the first dye anion–protein complex have been calculated. Addition of urea to the binding system causes a marked decrease in the absolute magnitude of the free energy change. The enthalpy change during binding becomes more exothermic, and the entropy change tends to decrease with increasing concentration of urea. These results can be interpreted in terms of the concept that urea reduces the structure of the aqueous environment and hence lowers the tendency of apolar groups of the dye and the albumin to participate in the formation of hydrophobic interactions.     

Interaction of poly(vinylpyrrolidone) with methyl orange and its homologs in aqueous solution over the temperature range 60–90°c

The binding of methyl orange, ethyl orange, propyl orange, and butyl orange by poly(vinylpyrrolidone) has been examined by a technique of equilibrium dialysis over a high temperature range (60–90°C). The first binding constants and the thermodynamic parameters in the course of the binding were evaluated. The results obtained at these temperatures were compared to those at lower ones (5–35°C) described previously in order to estimate the contribution of hydrophobic bonds to the binding. It was found that at the 60–90°C range complex formation between the dye and the macromolecule is associated with an exothermic enthalpy change and a positive entropy change. The enthalpy and entropy changes of the binding are of the order of ?4.5 kcal/mole and 6 eu, respectively, for each dye measured. Thus the binding is mainly enthalpy-controlled. Furthermore the effect of the alkyl chain length of the dye on both the ΔH° and ΔS° values is not pronounced. Also temperature dependences of the ΔH° and ΔS° terms were not observed. All these observations in the higher temperature range can be explained as a result of the disruption of water structure in the binding environment and hence a decrease in hydrophobic bond formation between the dye and the polymer.