Macrocyclic systems containing 2,6,9-trioxabicyclo[3.3.1]-nona-3,7-dienes as chiral spacer groups: Synthesis, stereochemical features and preliminary complexation properties

Novel 2:2-macrocycles bearing bridged concave 2,6,9-trioxabicyclo[3.3.1]nona-3,7-dienes as chiral spacer units were obtained by cyclocondensation reaction of the chiral bisacid chloride and the corresponding diols, while use of methylene diamines instead of diols afforded 1:1 macrocycles only. Applying the same, but now template-assisted, experimental procedure to the reaction of the bisacid chloride with triethylene glycol brought about a significant increase in yield as well as a suitable simplification of the work-up during preparation and separation of the corresponding 1:1 as well as 2:2 macrocycles, when compared to results reported previously. HPLC separation on chiral columns revealed the presence of diastereoisomers [RR(S,S)- and RS-(meso)-forms] for all 2:2 macrocycles, which was further evidenced by the CD spectrum of one of those species as an example. Preliminary ESI-MS experiments indicated strong complexation abilities of the sulphur-containing ligand towards Ag(I), Cu(II) and Au(III) ions.

Effects of iron complexation and indiscriminate uptake on shoot iron of barley

The free-ion model (FIM) describes iron ( Fe) uptake by barley [ Hordeum vulgare ( L.) 'Grammett'] as being controlled by the activity of the buffered, free, uncomplexed Fe3+ in solution. Chelators' effect on Fe uptake by barley was evaluated and the rate of exchange of Fe between chelators was examined. Barley was grown for two weeks in a low-Fe nutrient solution and transferred to solutions varying in Fe and chelators for 6 h assays. Shoot Fe-59 was higher in barley grown in citrate (7743 and 1928 Fe-59 Bq g(-1)) than in NTA(3220 and 1113 Fe-59 Bq g(-1); P = 0.045) despite similar free-Fe3+ activities. A comparison of Fe uptake by barley from solutions with pFe(3+) activities of 17.1 and 24.6 showed < 5% was from indiscriminate apoplastic-flow uptake (3250 Fe-59 Bq g(-1) vs. 160 Fe-59 Bq g(-1)). Using nutrient solutions from the barley studies but without plants, Fe exchange between chelators and a simulant for the barley phytosiderophore occurred within hours ( for NTA and citrate), or days ( EDTA and HEDTA). Results were similar between the barley and Fe-exchange experiments for the two nutrient-solution treatments where the same Fe3+ activities but different total-Fe concentrations were used: the higher total-Fe treatment resulted in six-fold higher shoot Fe-59, while in the Fe-exchange study that treatment had six-fold more Fe bound to the phytosiderophore simulant after 2 d. Results indicated deviations from the FIM were not explained by indiscriminate-flow uptake, and that sluggish Fe-exchange reactions between chelate and phytosiderophoresimulant, not FIM guidelines, may be more important in explaining Fe uptake from synthetic chelates by Fe-deficient barley.

The effect of complexation on characteristics and drug release from PLGA microspheres loaded by cyclosporine-cyclodextrin complex

The purpose of this study was to evaluate the effect of cyclosporine (CyA)-cyclodextrin (CD) complex incorporated within PLGA inicrospheres on microsphere characteristics, with particular emphasis on drug release kinetics. For this purpose, microspheres encapsulated with CyA and those loaded by CyA-CD complex were prepared by solvent evaporation and multiple emulsification solvent evaporation methods, respectively. Morphology, size, encapsulation efficiency and drug release pattern from microspheres were evaluated. Also, physicochemical properties of drug inside microspheres were characterized by differential scanning calorimetry (DSC) and infrared spectroscopy (IR) studies. Scanning electron microscopy (SEM) studies showed that microspheres encapsulated with CyA had islands on the microsphere surface but the islands were not seen on the surface of microspheres loaded by complex. Size range varied from 1 to 25 mu m for CyA encapsulated microspheres and 1 to 50 mu m for complex loaded microspheres. The release of CyA was biphasic with an initial more rapid release phase followed by a slower phase but drug release was twice as fast for complex loaded microspheres. IR studies did not indicate any chemical interaction between the components of microspheres and DSC thermograms revealed that CyA was present either in its amorphous state in microspheres or the presence of CyA as an inclusion complex within microspheres loaded by complex. In conclusion, using CyA as an inclusion complex with CD within microspheres can affect microsphere characteristics and drug release and it is possible to modify microsphere properties like drug release by incorporating CDs as complexing agents.

Studies of the interaction of Potassium(I), Calcium(II), Magnesium(II), and Copper(II) with Cyclosporin A

Metal ion binding properties of the immunosuppressant drug cyclosporin A have been investigated. Complexation studies in acetonitrile solution using H-1 NMR and CD spectroscopy yielded 1:1 metal-peptide binding constants (log(10)K) for potassium(l), < 1, magnesium(II), 4.8 +/- 0.2. and calcium(II), 5.0 +/- 1.0. The interaction of copper(II) with cyclosporin A in methanol was investigated with UV/visible and electron paramagnetic resonance (EPR) spectroscopy. No complexation of copper(II) was observed in neutral solution. In the presence of base, monomeric copper(II) complexes were detected. These results support the possibility that cyclosporin A has ionophoric properties for biologically important essential metal ions. (C) 2003 Elsevier Inc. All rights reserved.

Structural studies of copper(II)-amine terminated dendrimer complexes by EXAFS

The three-dimensional branched nature of dendritic macromolecules provides many potential sites per molecule for the complexation of metal ions. Therefore, dendrimers may act as hosts for metals with coordination potentially occurring at the periphery, the interior, or both. To understand further the complexation of dendrimers with metal ions EXAFS experiments were carried out. In this work, the interaction of amine-terminated polyamido(amine), PAMAM, dendrimer with copper(II) ions determined by EXAFS is reported. It was found that a model consisting of the copper(II) ion forming five- and six-membered rings by chelating with the primary amine, amide, and tertiary amine nitrogen donors of the PAMAM dendrimer could describe the experimental EXAFS data well. Corroborative evidence for binding to amide nitrogen donors comes from the broadening of NMR resonances of a copper(Il)-PAMAM mixture revealing the presence of paramagnetic copper(II) ions at these sites. The significance of the results presented in this paper is that copper(II) ions form complexes within the dendrimer structure and not just at the periphery. The current study may have implications for the use of PAMAM dendrimers as effective ligands in sensing systems.

Iron catalysed assembly of an asymmetric mixed-ligand triple helicate

The 2-pyridinecarbaldehyde isonicotinoyl hydrazone (HPCIH) family of ligands are typically tridentate N,N,O chelators that exhibit very high in vitro activity in mobilizing intracellular Fe and are promising candidates for the treatment of Fe overload diseases. Complexation of ferrous perchlorate with HPCIH in MeCN solution gives the expected six-coordinate complex Fe-II(PCIH)(2). However, complexation of Fe-II with 2-pyridinecarbaldehyde picolinoyl hydrazone (HPCPH, an isomer of HPCIH) under the same conditions leads to spontaneous assembly of an unprecedented asymmetric, mixed-ligand dinuclear triple helical complex Fe-2(II)(PCPH)(2)(PPH), where PPH2- is the dianion of bis(picolinoyl) hydrazine. The X-ray crystal structure of this complex shows that each ligand binds simultaneously to both metal centres in a bidentate fashion. The dinuclear complex exhibits two well separated and totally reversible Fe-III/II redox couples as shown by cyclic voltammetry in MeCN solution.

Self-assembly of a charge-neutral molecular square

Complexation of cadmium(II) by the ditopic (bis-tridentate) thiocarbazone ligand 1,5-bis(6-methyl-2-pyridylmethylene) thiocarbonohydrazide, H2L1, results in the self-assembly of a charge-neutral 2 x 2 molecular grid, [Cd-4(L-1)(4)], comprising four metals and four ligands in an interlocked cyclic array. The solid-state structure of this tetramer has been established by X-ray crystallography and in solution by H-1 NMR spectroscopy. The presence of lower molecular weight oligomers was identified by both NMR and ESI-MS.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(H) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1muM of complexed Hg(II), and for inhibition of motility it was 0.05 muM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 muM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned. (C) 2004 Elsevier B.V. All rights reserved.

Tuning the photophysical behavior of luminescent cyclam derivatives by cation binding and excited state redox potential

The emission from two photoactive 14-membered macrocyclic ligands, 6-((naphthalen-1-ylmethyl)-amino)trans-6,13-dimethyl- 13-amino- 1,4,8,11 -tetraaza-cyclotetradecane (L-1) and 6-((anthracen-9-ylmethyl)-amino)trans-6,13 -dimethyl - 13 -amino- 1,4,8, 1 1-tetraaza-cyclotetradecane (L-2) is strongly quenched by a photoinduced electron transfer (PET) mechanism involving amine lone pairs as electron donors. Time-correlated single photon counting (TCSPC), multiplex transient grating (TG), and fluorescence upconversion (FU) measurements were performed to characterize this quenching mechanism. Upon complexation with the redox inactive metal ion, Zn(II), the emission of the ligands is dramatically altered, with a significant increase in the fluorescence quantum yields due to coordination-induced deactivation of the macrocyclic amine lone pair electron donors. For [ZnL2](2+), the substituted exocyclic amine nitrogen, which is not coordinated to the metal ion, does not quench the fluorescence due to an inductive effect of the proximal divalent metal ion that raises the ionization potential. However, for [ZnL1](2+), the naphthalene chromophore is a sufficiently strong excited-state oxidant for PET quenching to occur.

An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid

To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (K-I and k(inact)) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (K-I and k(inact)) for isoniazid were 48.6 mu M and 0.042 min(-1) and 79.3 mu M and 0.039 min(-1). Clorgyline was a selective inactivator of CYP1A2 (6.8 mu M and 0.15 min(-1)). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors.

Enzyme Electrochemistry - Biocatalysis on an electrode

Oxidoreductase enzymes catalyze single- or multi-electron reduction/oxidation reactions of small molecule inorganic or organic substrates, and they are integral to a wide variety of biological processes including respiration, energy production, biosynthesis, metabolism, and detoxification. All redox enzymes require a natural redox partner such as an electron-transfer protein ( e. g. cytochrome, ferredoxin, flavoprotein) or a small molecule cosubstrate ( e. g. NAD(P)H, dioxygen) to sustain catalysis, in effect to balance the substrate/product redox half-reaction. In principle, the natural electron-transfer partner may be replaced by an electrochemical working electrode. One of the great strengths of this approach is that the rate of catalysis ( equivalent to the observed electrochemical current) may be probed as a function of applied potential through linear sweep and cyclic voltammetry, and insight to the overall catalytic mechanism may be gained by a systematic electrochemical study coupled with theoretical analysis. In this review, the various approaches to enzyme electrochemistry will be discussed, including direct and indirect ( mediated) experiments, and a brief coverage of the theory relevant to these techniques will be presented. The importance of immobilizing enzymes on the electrode surface will be presented and the variety of ways that this may be done will be reviewed. The importance of chemical modification of the electrode surface in ensuring an environment conducive to a stable and active enzyme capable of functioning natively will be illustrated. Fundamental research into electrochemically driven enzyme catalysis has led to some remarkable practical applications. The glucose oxidase enzyme electrode is a spectacularly successful application of enzyme electrochemistry. Biosensors based on this technology are used worldwide by sufferers of diabetes to provide rapid and accurate analysis of blood glucose concentrations. Other applications of enzyme electrochemistry are in the sensing of macromolecular complexation events such as antigen - antibody binding and DNA hybridization. The review will include a selection of enzymes that have been successfully investigated by electrochemistry and, where appropriate, discuss their development towards practical biotechnological applications.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Three classes of starch granule swelling: Influence of surface proteins and lipids

The role of non-carbohydrate surface components of granular starch in determining gelatinisation behaviour has been tested by treatment of native starches with a range of extractants. Resulting washed starches were analysed for (bio)chemical, calorimetric and theological properties. Sodium dodecyl sulphate (SDS) was the most efficient extractant tested, and resulted in major changes to the subsequent theological properties of wheat and maize starches but not other starches. Three classes of starch granule swelling behaviour are identified: (i) rapid swelling (e.g. waxy maize, potato), (ii) slow swelling that can be converted to rapid swelling by extraction of surface proteins and lipids (e.g. wheat, maize), and (iii) limited swelling not affected by protein/lipid extraction (e.g. high amylose maize/potato). Comparison of a range of extractants suggests that all of protein, lipid and amylose are involved in restriction of swelling for wheat or maize starches. Treatment of starches with SDS leads to a residue at comparable (low) levels of SDS for all starches. C-13 NMR analysis shows that this SDS is present as a glucan inclusion complex, even for waxy maize starch. We infer that under the conditions used, glucan inclusion complexation of SDS is equally likely with amylopectin as with amylose. (c) 2006 Elsevier Ltd. All rights reserved.