Vis Titration Research Articles

Flavonoids in plant nuclei: detection by laser microdissection and pressure catapulting (LMPC), in vivo staining, and uv–visible spectroscopic titration

Previous studies in our laboratory have indicated that the nuclei of a number of trees are associated with flavonoids, especially flavan‐3‐ols. In the present study, three techniques were applied to verify that flavonoids are naturally incorporated into nuclei. These were histochemistry, UV–visible (UV‐VIS) titration and laser microdissection. Nuclei from intact seed wings of Tsuga canadensis were isolated from their cells using laser microdissection and pressure catapulting (LMPC). Thereafter, the excised nuclei were stained with p‐dimethylamino‐cinnamaldehyde (DMACA), which resulted in a blue coloration due to the presence of flavanols. Thus, there is no doubt that the nuclei were, prior to staining, associated with flavanols. The nuclei of the coniferous species Abies lasiocarpa, Cedrus deodara, Cedrus libani, Juniperus communis, Picea abies, Picea orientalis and Pseudotsuga menziessii(Douglas fir) showed a yellow fluorescence typical for flavonols from the beginning of bud break over the entire growing season. However, after the bud‐breaking period, the nuclei of all species, except for Cedrus deodara, showed additionally a blue reaction for flavanols. Rather late, in midsummer, blue‐stained flavanols in nuclei were found in Picea orientalis. Generally, zeatin intensified the flavanol association with the nuclei. The main components of nucleosomes are DNA and the histone proteins. The nature of their association with the flavonols quercetin and rutin was investigated by UV‐VIS spectroscopic titration. The data were evaluated by means of the Mauser (A and AD) diagrams. The results indicate that DNA shows largely no spectroscopically detectable association equilibria under the experimental conditions chosen. However, association (aggregation) equilibria can be observed with rutin or quercetin and histone sulphate in Tris buffer (pH 8.0, 7.4 and 7.0). In phosphate buffer, rutin shows spectroscopically no or only weak association with histone sulphate, in contrast to its behaviour towards quercetin.

Complexes of carboxymethylcellulose in water. 1: Cu 2+, VO 2+ and Mo 6+

The importance of finding alternative materials to remediation of natural and waste waters is undoubted nowadays. The search for inexpensive and renewable substances that could grab metal ions and safely return the used waters to be used again is a matter of great importance. The properties of carboxymethylcellulose (CMC) renewable, inexpensive and water soluble biopolymer, in the absence and in the presence of some metal ions that have varying industrial applications, are reported in this work. Potentiometric and ultraviolet–visible titrations were performed in aqueous solutions of CMC in the absence and in the presence of Cu 2+, VO 2+ and Mo 6+ obtaining the values of the logarithms of the binding constants (stepwise formation constants) considering the complexation towards the monomeric sugar repeating units of CMC (HL) as follows, 10.63 ± 0.08, 12.08 ± 0.02 and 15.89 ± 0.03 for ML, when M was Cu 2+, VO 2+ and Mo 6+, respectively. For ML 2, the calculated values were 8.54 ± 0.08 and 9.1 ± 0.1 for Cu 2+ and VO 2+, respectively. Other binding constants were detected and their values calculated. The quantitatively obtained infrared spectra of the solids precipitated from aqueous systems revealed decreasing intensity in the –OH and –COOH bands as the pH values of the solution in which the solids were obtained were raised. These results matched the species distribution diagrams obtained in the aqueous studied systems. Also the solids were inspected for their thermal behavior and the results showed a more thermally stable complex species than the CMC alone. Scanning Electron Microscopy showed stable, compact and homogeneous films. CMC will be employed as a remediation agent in different aqueous solutions containing metal ions and the results will be released soon.

Synthesis and DNA binding of μ-[2,9-bis(2-imidazo[4,5-f][1,10]phenanthroline)-1,10-phenanthroline]bis[1,10-phenanthrolinecopper(II)

A binuclear complex [(phen)Cu(μ-bipp)Cu(phen)](ClO 4) 4, where phen = 1,10-phenanthroline and bipp = 2,9-bis(2-imidazo[4,5-f][1,10]phenanthroline)-1,10-phenanthroline, has been synthesized and its interaction with calf-thymus DNA in the buffer containing 5 mM Tris and 50 mM NaCl has been studied by means of electronic absorption titration, luminescence titration and viscometric measurements. The absorbance of the complex in the range of 320–400 nm, which is mainly based on bipp showed no obvious change upon addition of DNA, while the peak at 270 nm, which is determined by both phen and bipp decreased by up to 18%. The emission band of the complex around 360 nm decreased remarkably in presence of DNA. The emission quenching of this complex by [Fe(CN) 6] 4− was depressed greatly when bound to DNA. The relative viscosity of DNA was increased by this complex more significantly than a bipp directed intercalating reagent. These results suggest that this complex binds to calf thymus DNA by intercalation of the two phenanthrolinecopper terminals. The apparent intrinsic binding constant of the complexes with DNA was 1.6 × 10 4 M −1 as determined by UV–visible titration.

Modular Approach toward Supramolecular Functional Assemblies: Characterization of Donor−Spacer−Acceptor Ternary Complexes

It is shown that the noncovalent donor-spacer-acceptor (DSA) motif is useful in constructing an electron-transfer assembly. As a representative example, the equilibrium and structure of one of the DSA assemblies, consisting of Zn-tetraphenylporphyrin, a spacer unit bearing pyridine and amidinium moieties, and 3,4-dinitrobenzoic acid, were studied in detail by the extensive use of UV-vis titration, fluorescence spectroscopy, and 1H NMR, with the help of a three-component equilibrium model. Complex formation and fluorescence quenching in 20 different DSA complexes constructed from a library of five donors, two spacers, and two acceptors were investigated. It has been experimentally shown that supramolecular modular approach is useful for a systematic and quick search for a functionally optimized assembly.

New Azothiacalix[4]arenes Containing Biheterocyclic Subunits: Extraction and Complexation Properties

New thiacalix[4]arenes 2a, 3a, 4 and 5 functionalized with biheterocyclic and azophenyl groups at the lower rim and the upper rim of the macrocycle, respectively, were synthesized and their extraction properties towards Cu2+, Ag+, Ni2+ and Pb2+ studied. The complexation properties of the bithiazolyl receptors 2a, 4 and the bipyridyl receptors 3a, 5 were investigated by fluorescence and UV–visible titrations, respectively. The stoichiometries of the complexes were determined. A stoichiometry of 1:1 was found for the Cu-2a, Ag-3a complexes and 2:1 for the Cu2-4, Ag2-5 complexes as a function of the number of grafted bithiazolyl groups. The association constants for the 1:1 complexes were calculated using the Benesi–Hildebrand plot and by linear regression analysis.

Light-harvesting macroring accommodating a tetrapodal ligand based on complementary and cooperative coordinations.

A porphyrin macroring, mimicking the light-harvesting complex of photosynthetic purple bacteria, was synthesized by self-assembling trisporphyrinatoZn(II) complexes with imidazolyl substituents at both molecular terminals. Very strong complementary coordination of imidazolyl to Zn(II) afforded exclusively the cyclic trimer of trisporphyrin under dilute conditions at 27 degrees C in CHCl3/MeOH = 9/1 (v/v). 1H NMR spectra of the macroring indicate the existence of two topological isomers, one symmetric and one asymmetric. Use of three noncoordinated porphyrinatoZn(II) sites allows a tetrapodal ligand to be incorporated into the cavity of the macroring. A Job plot and the clear bending behavior on UV-vis titration indicated the formation a 1:1 complex; the heterogeneity of two topological isomers was not observed, and both isomers similarly accommodated the tetrapodal ligand. The association constant obtained by curve fitting analysis was 8 x 108 M-1 in toluene. This large association constant reflects the cooperative nature of three coordination sites.

Equilibrium and kinetic studies of ligand BMXD complexation with copper(II) and glycylglycine

Equilibrium studies of BMXD complexation with copper(II) and glycylglycine were performed by potentiometric and UV–visible titration. The equilibrium constants determined by potentiometry for the ternary system are: [LCu 2P 3+]/[LCu 2 4+][P −]=8.11(3), [LCu 2HP 4+]/[LCu 2P 3+][H +]=5.39(2), [LCu 2H −1P 2+][H +]/[LCu 2P 3+]=−6.33(2), [LCu 2(OH)H −1P +][H +]/[LCu 2H −1P 2+]=−8.20(1) and [LCu 2(OH) 2HH −1P][H +]/[LCu 2(OH)H −1P −1]=−10.33(3), which are in good agreement with the values determined by UV–visible titration. The species formed were confirmed by electrospray mass spectrometry. The association constants, determined by kinetic studies, for the association of the second molecule of glycylglycine with the LCu 2P complex, for two ionic media, are 1.29 (KCl) and 1.27 (KNO 3). These are in good agreement with the values determined by thermodynamic measurements 1.41 (KCl) and 1.26 (KNO 3).

Amino acid recognition of pyridine bis(oxazoline)–copper(II) complex in aqueous solvent

Enantioselective recognition of amino acids has been studied with C 2-symmetric chiral pyridine bis(oxazoline)–copper(II) complexes at physiological pH condition. UV–visible titration revealed strong binding of submillimolar dissociation constant between pyridine bis(oxazoline)–copper(II) complex and amino acids in aqueous solution. Moderate selectivity of up to 2:1 between d- and l-amino acids was achieved. The enantiomers were baseline resolved by capillary electrophoresis, using the bis( l-lysine)–copper(II) complex as a chiral selector.

Interaction of oligopyrrole macrocycles with aromatic acids: spectroscopical, quantum chemical and chromatographic aspects

The study of the behavior of oligopyrrole macrocyclic compounds in solution and covalently bond on silica surface were carried out. For this purpose ultraviolet–visible titrations and chromatography measurements were employed. Interactions of porphyrin and sapphyrin with nitrobenzoic, aminobenzensulfonic, hydroxybenzoic and terephthalic acids were studied. The spectroscopic results were compared with quantum chemical calculations at semiempirical level and with the data obtained by HPLC measurements. Chromatographic data showed that interaction of the analytes with the metalled porphyrin and the sapphyrin core led to significant increase of retention factors of the aromatic acids and selectivity improvement of the separation.

NON-COVALENT INTERACTIONS IN HOST-GUEST COMPLEXES WITH FLUORINATED PHENYL COMPOUNDS

Complexation constants with the macrocyclic azoniacyclophane CP44 and phenyl guest compounds with at least four fluorine atoms or alternatively protons at the ring were obtained by NMR shift titrations in water. The fluorinated compounds show free energies of complexation which are smaller by ΔΔG=3·4–7·7 kJ mol−1 in comparison with the protonated compounds. The NMR shifts induced upon 100% complexation (CIS values) were obtained simultaneously from non-linear least-squares fitting and indicate intra-cavity inclusion in all cases. The CIS values agree roughly with screening constants calculated from aromatic ring current and linear electric field effects, the latter resulting from the permanent charges at the host compound. Molecular mechanics calculations (CHARMm) indicate that intracavity inclusion is possible with all compounds with negligible strain induced (<1 kJ mol−1) in the macrocycle upon complexation. In contrast, α-cyclodextrin can accommodate fluorinated phenyl compounds only at the rim of the cavity without larger strain. Preliminary data with α-cyclodextrin, obtained by competitive UV–visible titration with methyl orange, indicate again a smaller association free energy (ΔΔG=1·-7 kJ mol−1) for pentafluorphenol compared with normal phenol as guest. © 1997 John Wiley & Sons, Ltd.

Interactions between metal cations and the ionophore lasalocid. Part 6.—Potentiometric and electron spin resonance study of the complexation of Gd3+ in methanol by lasalocid and simpler carboxylic acids

Complexes formed in methanol between the ionophore lasalocid and the Gd3+ ion have been studied using pHmetric, e.s.r. and u.v.–visible titration against tetrabutylammonium methoxide solution. The same has been done for salicylic and benzoic acids. In all cases the formation of the successive complexes AGd2+, A2Gd+ and A3Gd between the acid AH and the Gd3+ion has been observed and the corresponding formation constants were determined. More or less strong variation of the height, width and intensity of the e.s.r. line has been observed with the formation of the complexes; individual e.s.r. parameters could be had for most of the complexes. Comparison of data obtained for lasalocid and salicylic or benzoic acid suggests that in lasalocid anion–gadolinium cation complexes in methanol, the carboxylate group is the main and possibly the sole binding site of the gadolinium ion. Other complexes are formed in more basic media in methanol; these may involve the salicylate dianion, as shown for salicylic acid–gadolinium complexes, or, more probably, result from successive substitution of a methoxide ion for a lasalocid anion in A3Gd.

Stoichiometry of electron uptake and oxidation-reduction midpoint potentials of NADH:nitrate reductase.

Microcoulometric titrations of NADH:nitrate reductase at 25 degrees C in Mops buffer, pH 7.0, showed that the native enzyme, containing functional FAD, haem and Mo, required addition of five electrons for complete reduction. Reduction of the native enzyme occurred in three waves corresponding to addition of reducing equivalents to the centres in the order: Mo, haem, FAD. Oxidation-reduction midpoint potentials (E'0) for the various redox couples were calculated to be as follows: MoVI/MoV, +16 mV; MoV/MoIV, -27 mV; haemoxidized/haemreduced, -172 mV; FAD/FADH2, -283 mV. The values for the haem and flavin are in excellent agreement with those obtained by visible titrations, namely -164 mV and -288 mV respectively. In contrast, the results for the Mo centre are 28-50 mV more positive than the values previously determined by e.p.r. analysis of frozen enzyme samples poised at defined potentials at 25 degrees C and suggest different pH-dependencies or entropies of reduction for the Mo couples.

Biomolecular dynamics and electron spin resonance spectra of copper complexes of antitumour agents in solution

For the purpose of developing new antitumour agents which are more efficacious and have less generalized toxicity than existing ones, the free-radical generation and metal complexation of well known anticancer agents have been studied. Copper(II) ion complexes are readily formed with several members of a class of hydroxyurea derivatives which are known to be effective ribonucleotide reductase inhibitors. E.s.r. measurements and u.v.–visible titration illustrate weak binding for 3,4-dihydroxybenzohydroxamic acid and tight binding in complex formation for gallohydroxamic acid and 2,3,4-trihydroxybenzohydroxamic acid. These data were used in a preliminary investigation of cytotoxicity, and the results are consistent with single phase cell cycle killing.

Ferrometric determination of vanadium in VN1−x using visible and potentiometric equivalence point detection

Techniques for the decomposition of VN1−x and determination of the vanadium content by ferrometric titration of V5+ are presented. For the method of visible titration the sodium salt of diphenylamine-4-sulphonic acid was used as an internal indicator. The influence of ortho-phosphoric acid and urea on the titration was investigated by potentiometric measurements. The results of the titration are compared with the oxidation method for determining the metal content in transition metal nitrides.

Base hydrolysis of amino-acid esters and amides in the co-ordination sphere of cobalt(III). Part 3. Hydrolysis of methyl and ethyl 4-amino-butanoate

The base hydrolysis of cis-[CoCl(en)2{NH2(CH2)3CO2Me}]2+(en = ethylenediamine) has been studied by pH-stat and stopped-flow spectrophotometry at 25 °C and l= 0.1 mol dm–3. Two consecutive reactions are observed in the pH-state measurements. Following loss of Cl–(kOHCl 15.2 dm3 mol–1 s–1) a slower base hydrolysis of the ester function occurs (kOH 0.72 dm3 mol–1 s–1). In the pH-stat measurements 2 mol of base are consumed per mol of complex and the final product of the reaction is the penta-amine hydroxo-complex which has been identified by visible spectra and potentiometric titration. In the stopped-flow measurements a single kinetic process is observed spectrophotometrically corresponding to chloride hydrolysis (kOHCl 15.6 dm3 mol–1 s–1). For the analogous cis-[CoBr(en)2{NH2(CH2)3CO2Me}]2+, kOHBr= 125 ± 3 dm3 mol–1 s–1 and kOHBr: kOHCl= 8 : 1. Complete base hydrolysis of the complex cis-[CoCl(en)2{NH2(CH2)3CO2}]+ containing the N-co-ordinated amino-acid requires 1 mol of base (kOHCl 10.4 dm3 mol–1 s–1 determined by pH-stat; kOHCl 9.83 dm3 mol–1 s–1 determined spectro-photometrically at l= 0.1 mol dm–1 and 25 °C). For the unprotonated ester NH2(CH2)3CO2Me, lactamisation to give 2-pyrrolidinone competes with base hydrolysis, but kOH for ester hydrolysis is ca. 0.1 dm3 mol–1 s–1 at 25 °C. Thus a small enhancement (ca. 7.2 times) occurs for hydrolysis of the N-co-ordinated ester.

Base hydrolysis of amino-acid esters and amides in the co-ordination sphere of cobalt(III). Part II. Hydrolysis of 2-aminoethyl acetate

Base hydrolysis of the complex cis-[Co(en)2X(NH2CH2CH2OCOMe)]2+(en = ethylenediamine; X = Cl or Br) has been studied by pH-stat and spectrophotometry at 25 °C and I= 0·1M. Two consecutive reactions occur in basic media. Following loss of halide ion (kOHCl= 1·38 × 103, kOHBr= 8·7 × 103|mol–1 min–1), slower base hydrolysis of the ester function occurs (kOHester= 1·44 × 102|mol–1 min–1). Initial base hydrolysis of halide ion is consistent with the observations that (a) rate constants for the slow hydrolysis steps in the bromo- and chloro-complexes are identical, within experimental error, and (b) a single rate process is observed spectrophotometrically with kOH= 1·42 × 102|mol–1 min–1. In the pH-stat measurements 2 mol of base are consumed per mol of complex and the final product of the reaction is the penta-aminohydroxo-complex which has been identified by visible spectra and potentiometric titration. The pKap value for ionisation (i) is 6·1 at 25 °C and I= 0·1M. [Co(en)2OH2(NH2CH2CH2OH)]3+⇌[Co(en)2OH(NH2CH2CH2OH)]2++ H+(i) Hydrolysis of the complex cis-[Co(en)2Cl(NH2CH2CH2OCOMe)]2+ in ca. 6M-hydrochloric acid gives the corresponding penta-aminechloro-complex of 2-aminoethanol. Base hydrolysis of the penta-aminechloro-complex. containing N-co-ordinated 2-aminoethanol requires 1 mol of base for complete hydrolysis (kOH= 1·23 × 103|mol–1 min–1 by pH-stat and spectrophotometry at 25 °C and I= 0·1M). It is estimated that base hydrolysis of 2-aminoethyl acetate in the dipositive cobalt(III) complex is some 18 times faster than that for the free ligand and some 2 times faster than that for the protonated ligand. Mercury(II)-catalysed aquations of the complex cis-[Co(en)2Cl(NH2CH2CH2OCOMe)]2+ and the corresponding 2-aminoethanol derivative have also been studied. The reactions follow a rate law, rate =k[HgII][Complex] in 0·57M-HClO4. There is evidence for neighbouring-group participation (NGP–5OH) in aquation of the 2-aminoethanol derivative.

Base hydrolysis of amino-acid esters and amides in the co-ordination sphere of cobalt(III). Part I. Hydrolysis of methyl 6-aminohexanoate

The base hydrolysis of cis-{Co(en)2Cl[NH2(CH2)5CO2Me]}2+ has been studied by pH-stat and stopped-flow spectrophotometric techniques at 25 °C and I= 0·1M. Two consecutive reactions occur in base. Following loss of Cl–(kOH= 12·8 l mol–1 s–1 by pH-stat, kOH= 13·6 ± 1 l mol–1 s–1 by stopped flow), a slower base hydrolysis of the ester function occurs (KOH= 0·22 l mol–1 s–1). In the pH-stat measurements 2 moles of base are consumed per mole of complex and the final product of the reaction is the hydroxypenta-ammine which has been identified by visible spectra and potentiometric titration. Base hydrolysis of the complex cis-{Co(en)2Cl[NH2(CH2)5CO2]}+ containing the N-co-ordinated amino-acid requires 1 mole of base for complete hydrolysis (kOH= 9·5 l mol–1 s–1 for loss of chloride by pH-stat, kOH= 9·2 l mol–1 s–1 determined spectrophotometrically in borax buffers at l= 0·1 M and 25 °C). For base hydrolysis of the unprotonated form of the free ester, NH2(CH2)5CO2Me + OH–→ NH2(CH2)5CO2–+ MeOH in the pH range 11·0–11·5 the value of kOH= 0·148 l mol–1 s–1. Thus a slight rate enhancement (ca. 1·5 times) occurs for hydrolysis of the N-co-ordinated ester. Such an effect is understandable owing to the length of the alkyl chain separating the cobalt(III) centre from the ester function. The present results establish a further hydrolytic pathway for N-co-ordinated amino-esters in halogenopenta-ammine complexes of cobalt(III), quite different from the reactions of glycine esters where intramolecular pathways occur.