Bis(3-hydroxy-4-pyridinone)-EDTA derivative as a potential therapeutic Al-chelating agent. Synthesis, solution studies and biological assays

The 3-hydroxy-4-pyridinones are chelating agents of current interest due to their high affinity for hard metal ions and potential clinical applications as metal-decorporation agents. A new bis-(3-hydroxy-4-pyridinone) derivative of EDTA have been developed, and herein we describe the results of solution studies to determine the protonation constants and the partition coefficient. Biodistribution studies, performed with 67Ga-overload mice, showed rapid clearance of the radiotracer from the body, thus indicating that the new ligand should be a quite effective agent for the in vivo aluminium removal.     

Pictet-Spengler condensation of the antiparkinsonian drug L-DOPA with D-glyceraldehyde. Opposite kinetic effects of Fe3+ and Cu2+ ions and possible implications for the origin of therapeutic side effects

In 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C. L-DOPA, a widely used antiparkinsonian drug, reacted smoothly with D-glyceraldehyde to afford diastereoisomeric (1R, 1'S,3S)-3-carboxy-1-(1',2'-dihydroxyethyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline (1) and (1S,1'5S,3S)-3-carboxy-1-(1',2'-dihydroxyethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (2) in an approx. 3:2 ratio. The prevalent formation of 1 over 2 reflects stereoselective cyclisation of a transient Schiff base in accord with the Felkin-Anh model. Fe3+ ions, present at relatively high levels in parkinsonian brains, markedly accelerated formation of 1 and 2, whereas Cu2+ decreased the reaction rate, due apparently to different sites of chelate formation between L-DOPA and the metal ions. Both metal ions markedly decreased the stereoselectivity of the reaction. Product 1 exhibited chelating properties toward metal ions comparable or stronger than those of L-DOPA. These results throw new light on the effects of transition metal ions on the Pictet-Spengler reaction and suggest a possible role of tetrahydroisoquinoline products from L-DOPA and carbohydrate metabolites in the severe side effects of the drug.     

Theoretical insights into the metal chelating and antimicrobial properties of the chalcone based Schiff bases

The emergence of multi-drug resistant pathogens in infectious disease conditions accentuates the need for the design of new classes of antimicrobial agents that could defeat the multidrug resistance problems. As a new class of molecules, the Heterocyclic Schiff base is of considerable interest, owing to their preparative accessibility, structural flexibilities, versatile metal chelating properties, and inherent biological activities. In the present study, CAM-B3LYP/LANL2DZ and M062X/DEF2-TZVP level of density functional method is used to explore the complexation of chalcone based Schiff base derivatives by Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ metal ions. The HL(1-3)-Co²⁺, HL(1-3)-Ni²⁺ and HL(1-3)-Zn²⁺ complexes formed the distorted tetrahedral geometry. Whereas, the HL(1-3)-Cu²⁺ complexes prefers distorted square-planar geometry. The BSSE corrected interaction energies of the studied complexes reveals that Cu²⁺ ion forms the most stable complexes with all three chalcone based Schiff bases. Of the three Schiff bases studied, the HL2 Schiff base acts as a potent chelating agent and forms the active metal complexes than the HL1 and HL3 Schiff bases. Further, the strength of the interaction follows the order as Cu^2+?>?Ni^2+?>?Co^2+?>?Zn²⁺. The QTAIM analysis reveals that the interaction between the metal ions and coordinating ligand atoms are electrostatic dominant. The metal interaction increases the π-delocalisation of electrons over the entire chelate. Hence, the antimicrobial activity of the metal complexes is more effective than the free Schiff bases. Moreover, the HL(1-3)-Cu²⁺ complexes shows higher antimicrobial activities than the other complexes studied.     

Preparation of new chiral borane-protected P,N-ferrocenyl ligands via a methoxy directed ortho-lithiation

We have shown that the readily prepared (ferrocenyl)benzylic methyl ethers of type 1 can be ortho-metalated with tert-BuLi with high diastereoselectivity. This reaction has been used to prepare new borane-protected P,N-substituted ferrocenes of type 2 in high diastereomeric and enantiomeric purity. The chiral heterocyclic aminophosphines 2 may be of interest as chelating ligands for asymmetric metal catalysis.     

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.

A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.     

N-Carboxyalkyl derivatives of 3-hydroxy-4-pyridinones: synthesis,complexation with Fe(III), Al(III) and Ga(III) and in vivo evaluation

A set of three N-carboxyalkyl 3-hydroxy-4-pyridinones has been studied as bidentate M(III) chelators (M=Fe, Al, Ga), with potential for oral administration. After preparation of the ligands, their protonation constants (log K(i)) and the stability constants of their metal complexes have been determined. The distribution coefficients of these compounds, between 1-octanol and Tris buffer pH 7.4, were measured. The effect of these compounds on the biodistribution of 67Ga-citrate loaded rats was investigated and compared with that of the administered 67Ga-complexes. Results indicated that, among these chelating agents, the N-carboxyethyl derivative has the highest affinity towards this set of metal ions, irrespective of the metal, and that it could even compete with transferrin, the main Fe-plasma protein. The binding affinity and the hydrophilic character decrease with the increase in the size of the alkylic chain. The biological assays indicate that the complex formation in vivo is characterized by a high kinetics and thermodynamic stability, suggesting a competition with the transferrin. All the ligands were found to enhance the excretion of the gallium. Noteworthy is the observed Ga bone fixation, mostly with the ethyl derivative, thus suggesting the potential use of the complex as a bone seeking agent.     

Synthesis and characterization of metal binding pseudotripeptides.

Metal complexes with peptide or pseudopeptide type ligands can serve as good model compounds for a deeper understanding of enzymatic catalysis, but ligands with a high selectivity for different transition metal cations are hard to find due to the rather flexible nature of peptides. Since such ligands would be the sine qua non condition for the synthesis of heterodinuclear peptide metal complexes with catalytic activity, the search for small, affine and selective metal chelating sequences is of interest. Using four different amino acids (His, Lys, Asp, Glu) a set of 16 pseudotripeptides of the common structure Bz-AS1-Sar-AS2-NH2 has been synthesized, purified and characterized by mass spectrometry and 1H-NMR. Their ability to form metal complexes has been investigated leading to short motifs capable of selectively binding only one or two transition metal cations with high affinity. As expected, the complexation of transition metal cations by pseudotripeptides is strongly dependent not only on the amino acid composition, but also on the sequence with regard to the stability of the resulting complexes, as well as the selectivity of the ligands towards Cu2+, Co2+, Ni2+, Zn2+ and Mn2+.     

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.     

Inhibition of glutathione-insulin transhydrogenase by metal ions and activation by histidine and other chelating agents

The catalytic activity of purified glutathione-insulin transhydrogenase (thiol:protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2) from bovine pancreas is markedly stimulated by histidine and other chelating agents. The activation produced was highest with EDTA, followed by EGTA, 8-hydroxyquinoline and 1,10-phenanthroline. Of the many amino acids tested, histidine was the only one that activated the enzyme; the structurally related compounds, 3-methylhistidine and imidazole also stimulated the enzyme, but 1-methylhistidine and histamine were without effect. The activation of EDTA was negated by metal ions, most effectively by Se2+, Hg2+, Cu2+ and Zn2+, and less effectively by Ca2+ and Ni2+. Likewise, activation by histidine was negated by Zn2+ but not by Ca2+ or Mg2+. Thus, activation of glutathione-insulin transhydrogenase is apparently achieved in part by the chelation of inhibitory metal ion(s). These findings are consistent with a regulatory scheme for glutathione-insulin transhydrogenase in which (a) the enzyme is inhibited by selenium and heavy metal ions normally present in tissues and (b) this inhibition can be relieved by the addition of histidine or chelating agents.     

Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.     

Chelation ability of spironaphthoxazine with metal ions in silica gel

Spironaphthoxazine (SNO) and three metal ions, Mg(2+), Zn(2+), and Al(3+), were dispersed in silica gels by the sol-gel method. The chelation ability of SNO with the metal ions in silica gels was investigated by measuring the fluorescence spectra and was compared to that of 8-hydroxyquinoline (8-HQ) in ethanol and silica gels. A merocyanine-type isomer photoderived from SNO as well as 8-HQ easily formed complexes of the metal ions in the order of Al(3+), Zn(2+), and Mg(2+) because the coordination ability of the metal ions to such ligands depended on their electron affinity. The changes in the fluorescence spectra of the silica gel samples during light irradiation were also investigated. The relative band intensity due to the intermediate species between the original SNO and the merocyanine species decreased and that of the complex increased with the UV irradiation time. The reverse process was observed during visible irradiation. The UV irradiation effects on the chelation of SNO and its photochromic property also depended on the electron affinity of the metal ions.     

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)₃ and KEMPBu(3,4-HP)₃) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)₃ ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with ⁶⁷Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.     

Ratiometric and selective fluorescent chemodosimeter for Cu(II) by Cu(II)‐induced oxidation

A new rhodamine derivative, rhodamine B 4‐N,N‐dimethylaminobenzaldehyde hydrazone (1), was designed for ratiometric sensing of Cu(II) selectively. A red‐shift from 515 to 585 nm was observed in the fluorescence spectrum when Cu(II) was added to 1 in acetonitrile. Other metal ions of interest showed no ratiometric response. The interaction between Cu(II) and 1 was found to be the Cu(II)‐induced oxidation of 1. Copyright © 2008 John Wiley & Sons, Ltd.     

Phenyl substituted 3-hydroxypyridin-2(1H)-ones: Inhibitors of influenza A endonuclease

Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is recognized as an attractive target for the development of new agents for the treatment of influenza infection. Our earlier study employing small molecule fragment screening using a high-resolution crystal form of pandemic 2009 H1N1 influenza A endonuclease domain (PA_N) resulted in the identification of 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating ligand at the active site of the enzyme. In the present study, several phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity as measured by a high-throughput fluorescence assay. Two of the more potent compounds in this series, 16 and 18, had IC₅₀ values of 11 and 23 nM in the enzymatic assay, respectively. Crystal structures revealed that these compounds had distinct binding modes that chelate the two active site metal ions (M1 and M2) using only two chelating groups. The SAR and the binding mode of these 3-hydroxypyridin-2-ones provide a basis for developing a new class of anti-influenza drugs.     

The effect of chemicals on the activity of deglycosylated Aureobasidiumβ-fructofuranosidases

The effects of chemicals such as metal ions and typical organic enzyme inhibitors on the activity of deglycosylated β-fructofuranosidases ( P -1 and P -2) from Aureobasidium were observed and compared with those of native enzymes. P -1 and P -2 enzymes became sensitive to metal ions, such as Fe²⁺, Co²⁺, Ni²⁺ and Al³⁺, after deglycosylation. The enzymes were also inhibited by a sulphydryl reagent (monoiodoacetic acid), a peptide-hydrolysing reagent (hydroxylamine) and chelating reagents (sodium citrate, EDTA and sodium azide) after deglycosylation. The importance of deglycosylation for the determination of the true characteristics of the enzymes against chemicals is discussed.     

Combined chelation of bi-functional bis-hydroxypiridinone and mono-hydroxypiridinone: Synthesis, solution and in vivo evaluation

3-Hydroxy-4-pyridinones (3,4-HP) are well known iron-chelators with applications in medicinal chemistry, mainly associated with their high affinity towards trivalent hard metal ions (e.g. M³⁺, M = Fe, Al, Ga) and use as decorporating agents in situations of metal accumulation. The polydenticity and the extra-functionality of 3,4-HP derivatives have been explored, aimed at improving the chelating efficacy and the selectivity of the interaction with specific biological receptors. However, the ideal conjugation of both features in one molecular unity usually leads to high molecular weight compounds which can have crossing-membrane limitations.Herein, a different approach is used combining a arylpiperazine-containing bis-hydroxypyridone (H₂L¹) with a biomimetic mono-hydroxypyridinone, ornithine-derivative (HL²), to assess the potential coadjuvating effect that could result from the administration of both compounds for the decorporation of hard metal ions. This work reports the results of solution and in vivo studies on their chelating efficacy either as a simple binary or a ternary system (H₂L¹:HL²:M³⁺), using potentiometric and spectrophotometric methods. The solution complexation studies with Fe(III) indicate that the solubility of the complexes is considerably increased in the ternary system, an important feature for the metal complex excretion, upon the metal sequestration. The results of the in vivo studies with ⁶⁷Ga-injected mice show differences on the biodistribution profiles of the radiotracer, upon the administration of each chelating agent, that are mainly ascribed to the differences of their extra-functional groups and lipo/hydrophilic character. However, administration of both chelating agents leads to a more steady metal mobilization, which may be attributed to an improved access to different cellular compartments.     

NMR spectroscopy of Group 13 metal ions: biologically relevant aspects

In spite of the fact that Group 13 metal ions (Al(3+), Ga(3+), In(3+) and Tl(+/3+)) show no main biological role, they are NMR-active nuclides which can be used in magnetic resonance spectroscopy of biologically relevant systems. The fact that these metal ions are quadrupolar (with the exception of thallium) means that they are particularly sensitive to ligand type and coordination geometry. The line width of the NMR signals of their complexes shows a strong dependence on the symmetry of coordination, which constitutes an effective tool in the elucidation of structures. Here we report published NMR studies of this family of elements, applied to systems of biological importance. Special emphasis is given to binding studies of these cations to biological molecules, such as proteins, and to chelating agents of radiopharmaceutical interest. The possibility of in vivo NMR studies is also stressed, with extension to (27)Al-based MRI (magnetic resonance imaging) experiments.     

Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates

Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination as well as the success of metal ion removal on attempted purification. We compared the use of Empore chelating disks and Chelex 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore chelating disks were found to give much higher recoveries of the probes under the conditions used. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.     

Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion. II. Applications

The relative importance of the individual effects of precipitation and chelation of metal ions in anaerobic digestion is assessed. Experimentally determined soluble metal ion levels are compared with predicted levels obtained by using a previously described methodology.(1) It is found that soluble metal complexes may increase the level of soluble metals in the presence of CO(3) (2-) and S(2-) by a factor of up to 10(4). The formation of a soluble complex may increase or decrease the availability of the metal ion in question for microbial uptake. Two case studies are presented, one using a defined medium and one a complex medium. It is possible, in the case of the defined medium, to accurately predict the free metal ion concentration using the methodology previously developed.(1) While the identification of the presence of natural chelating compounds in a complex medium is not routinely possible, the significant discrepancy between the measured level of the soluble metal ion Fe(2+) and the calculated level in the case studies presented indicates that natural chelating compounds may play a vital role in providing available metal ions to the microorganisms of an anaerobic digester.     

Interaction of RNase A with VO3- and VO2+ ions. Metal ion binding mode and protein secondary structure

Some of vanadyl complexes have shown potential to inhibit RNase activity by acting as transition state analogue, while at the same time not inhibiting DNase. To gain an insight into the interaction of protein with vanadate (VO3-) and vanadyl (VO2+) ions, the present study was designed to examine the binding of ribonuclase A (RNase A) with NaVO3 and VOSO4 in aqueous solution at physiological pH with metal ion concentrations of 0.001 mM to 1 mM, and protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second derivative resolution enhancement were used to determine the cation binding mode, association constant and the protein secondary structure in the presence of vanadate and vanadyl ions in aqueous solution. Spectroscopic results show that an indirect metal ion interaction occurs with the polypeptide C = O, C-N (via H2O) with overall binding constants of K(VO3-) = 3.93x10(2) M(-1) and K(VO2+) = 4.20x10(3) M(-1). At high metal ion concentrations, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 23-24%; beta-sheet (pleated and anti) 50% (free enzyme) to 64-66% and turn 21% (free enzyme) to 10-12% in the metal-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of high metal ion concentration.