DNA cleavage by a Chromium(III) complex

A new Cr(III) complex with the empirical formula [Cr(Schiff base) (H(2)O)(2)]ClO(4), where the Schiff base is 2, 3-bis?[(2-hydroxy-4-diethylamino) (phenyl) (methylene)]amino?2-butenedinitrile has been synthesized and characterized spectroscopically. Binding of this complex to DNA has been studied using UV-visible spectroscopy. The complex has been found to bind to the major groove of DNA with a binding constant, K = (1.3 +/- 0.2) x 10(3) M(-1). The induced CD spectrum of the complex in the presence of DNA is also indicative of major groove binding. Gel electrophoresis of plasmid DNA in the presence of the complex shows that the complex brings about nicking of the DNA.     

NADPH-cytochrome-P-450 reductase promoted hydroxyl radical production by the iron(III)-ochratoxin A complex

The Fe3+ complex of ochratoxin A has been shown to produce hydroxyl radicals in the presence of NADPH and NADPH-cytochrome-P-450 reductase. ESR spin-trapping experiments carried out in the presence of the hydroxyl radical scavenger ethanol and the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide) produced ESR spectra characteristic of the hydroxyl radial-derived carbon-centered DMPO-alkoxyl radical adduct. Thus hydroxyl radicals produced by the Fe3(+)-ochratoxin A complex in the presence of an enzymatic reductase may be be partly responsible for ochratoxin A toxicity.     

Oxidative DNA cleavage induced by an iron(III) flavonoid complex: synthesis, crystal structure and characterization of chlorobis(flavonolato)(methanol) iron(III) complex

A flavonol iron(III) complex, [Fe(flavonolato)(2)Cl(MeOH)], has been prepared. The compound has been characterized by X-ray crystallography, spectroscopy, magnetism and electronic paramagnetic resonance (EPR) at X- and Q-band. The geometrical environment around the metal is best described as rhombic distorted octahedral. This distortion has also been inferred from the magnetic measurements and from the EPR spectra at different temperatures, E/D(rhombicity parameter) approximately 0.06. The DNA cleavage activity of the iron(III) complex with and without ascorbate/hydrogen peroxide is reported. Mechanisms of the oxidative cleavage have been proposed when DNA strand scission is performed both with and without ascorbate/hydrogen peroxide activation.     

Inhibition of lipid peroxidation promoted by iron(III) and ascorbate.

Peroxidation of rat liver microsomes and of phospholipid isolated from them was studied using iron(III) and ascorbate initiation. One-half equivalent of citrate per iron equivalent maintained solubility of the metal ion at neutral pH. Several metal chelators, including additional citrate, blocked peroxidation, but catalase did not. These characteristics are consistent with those reported by others (D. M. Miller and S. D. Aust (1989) Arch. Biochem. Biophys. 271, 113-119). Several antioxidants, principally tocopherol analogues and nitroxides, and, as well, a nonenzymatic component of "thymol-free" catalase, potently blocked lipid peroxidation, or, equivalently, dioxygen depletion from suspensions of peroxidizing microsomes. Chromanols were the most active antioxidants. No thiol studied had significant antioxidant activity in the test system.     

Self-reduction of the iron(III)-doxorubicin complex

The Fe³⁺-doxorubicin complex undergoes reactions that suggest that the complex self-reduces to a ferrous oxidized-doxorubicin free radical species. The Fe³⁺-doxorubicin system is observed to reduce ferricytochrome c, consume O₂ and react with 2,2′-bipyridine. Bipyridine acts as a “ferrous ion scavenger” as it reacts with the ferrous ion produced by Fe³⁺-doxorubicin self-reduction. In the absence of O₂, a ferrous doxorubicin complex accumulates. In the presence of oxygen, Fe²⁺ recycles back to Fe³⁺. The rates of these reactions were measured and the Fe³⁺-doxorubicin self-reduction was determined to be the rate-determining step. The Fe³⁺-doxorubicin induced inactivation of cytochrome c oxidase and NADH cytochrome c reductase on beef heart submitochondrial particles occurs at a rate similar to Fe³⁺-doxorubicin self-reduction. Thus the rate at which damage to these mitochondrial enzymes occurs may be controlled by a nonezymatic Fe³⁺-doxorubicin self-reduction.     

Selective cleavage of N-terminal amino acid in a peptide by polymeric cobalt (III) triene complex

Cellulose was functionalized to incorporate triethylenetetramine group. This was in turn converted into the polymeric analogue of cobalt(III)triene complex. The polymeric complex reacts with peptides resulting in the cleavage of amino end amino acid, thus suggesting the applicability of the polymeric reagent as a solid phase reagent for N-terminal determination.     

Phosphate ester cleavage in ribose-5-phosphate induced by OH radicals in deoxygenated aqueous solution. The effect of Fe(II) and Fe(III) ions.

The reaction of OH radicals and H atoms with ribose-5-phosphate (10(-2) M) in deoxygenated aqueous solution at room temperature (dose-rate 2-1 X 10(17) eV/ml-min, dose 5 X 10(18)-15 X 10(18) eV/ml) leads to the following dephosphorylation products (G-values): ribo-pentodialdose 1 (0-2), 2-hydroxy-4-oxoglutaraldehyde 2 (0-06), 5-deoxy-erythro-pentos-4-ulose 3 (0-1) and 3-oxoglutaraldehyde 4 (0-06). In addition, some minor phosphate free products (total G=0-09) are formed. G(inorganic phosphate) =1-3 and G(H2O2)=0-3. On the addition of 10(-3) M (Fe(III) ions, G (1) and G (3) increase to 0-6 and 0-4 respectively. In the presence of 10(-3) M Fe(II), G(1) and G(3) change to 0-4 and 0-8, respectively. The other dephosphorylation products are suppressed by the iron ions. G(1) also increases on the addition of increasing amounts of H2O2. Each product can be assigned a precursor radical formed by hydrogen abstraction from C-5, C-4 or C-3 of the ribose-5-phosphate molecule. Products 1 and 2 are formed by oxydative dephosphorylation of an alpha-phospho radical with preceeding H2O elimination for product 2. Elimination of H3PO4 from a beta-phospho radical leads to product 3; product 4 is formed by elimination of two molecules of H2O from its precursor radical and hydrolytic cleavage of an enol phosphate bond. Deuterium-labelling experiments and the effects of the iron ions and of H2O2 support the mechanisms proposed. The importance of the dephosphorylation mechanisms for the formation of strand breaks in DNA is discussed with special reference to the effects of the radiosensitizers.     

Facile cleavage of RNA via phosphodiester linkage fission by Co(III) complex.

Adenylyl (3'-5')adenosine (ApA) is effectively cleaved to two adenosine molecules by [Co(trien)(H2O)2]3+ complex (trien: triethylenetetramine). The complex (0.20 M) accelerates the cleavage by 10(5) fold, decreasing half-life of ApA from 4000 years to 9.3 days. The reaction involves general base catalysis by the hydroxide ion bound to the Co(III) ion for the formation of adenosine 2',3'-cyclic phosphate (A greater than p), followed by the prompt cleavage of the intermediate to adenosine.     

Phosphodiester cleavage by yttrium(III) peroxide complexes

Potentiometric titrations of hydrogen peroxide in the presence of Y(III) revealed formation of dinuclear Y₂(O₂)₂ ²⁺ and Y₂(O₂)₂(OH)₂ complexes. Kinetics of the cleavage of bis(4-nitrophenyl) phosphate (BNPP) in the presence of Y(III) and H₂O₂ was studied at 25 °C in pH range 6-8 and at variable metal and H₂O₂ concentrations. Comparison of the pH-dependence of the reaction rate with the species distribution diagram shows that Y₂(O₂)₂(OH)₂ is the reactive species. The reaction kinetics is second-order in Y(III) at low metal concentration, but is of a ‘saturation’ type at high metal concentrations. A reaction mechanism, which agrees with such kinetics, involves intermediate reversible dimerization of Y₂(O₂)₂(OH)₂ to a tetranuclear complex capable to bind BNPP anion and to cleave it intramolecularly.     

Efficient enhancement of DNA cleavage activity by introducing guanidinium groups into diiron(III) complex

Inspired by the structures of natural nucleases, guanidinium groups were introduced into binuclear iron(III) systems. Compared with the corresponding analogue without guanidinium groups, the new diiron(III) system led to considerable rate enhancement on DNA cleavage. The cooperativity between metal ions and guanidine groups was evidenced by the fact that no significant cleavage was observed after incubating pBR322 plasmid DNA with non-metalated ligands or free Fe3+ ion. DNA binding experiments indicated that introduction of positively charged guanidinium groups can obtain more than one order of magnitude enhancement in the affinity of complex with DNA.     

Hydrolysis of 2,4-dinitrophenylphosphate promoted by hydroxoaquatetraaminecobalt(III) ions

The hydrolysis of 2,4-dinitrophenylphosphate (DNPP) to orthophosphate and 2,4-dinitrophenolate (DNP) is accelerated in the presence of excess tn₂Co(H₂O)_2³⁺ or trpnCo(H₂O)_2³⁺ at rates which maximize at pHs close to those at which the hydroxoaquatetraaminecobalt(III) complex concentrations peak (tn₂, pH 6.4; trpn, pH 6.0; tn = trimethylenediamine; trpn = 3,3′,3″-triaminotripropylamine). For dilute DNPP solutions (10⁻⁴ M) the hydrolysis rates (25°C, 0.50 M NaClO₄) increase with increasing Co/DNPP ratio in ways that are qualitatively as well as quantitatively different for the two systems (trpn: steady increase moving toward rate saturation, higher rates; tn₂: ‘S’-shaped curve with very low rates at low ratios, lower rates compared to trpn for comparable ratios). For the trpn system the results are interpreted on the basis of pre-equilibrium formation of the 1:1 monodentate-DNPP cobalt complex by substitution of the labile water on cobalt, and rate-determining attack by the cis-coordinated hydroxide on the phosphorus center to affect hydrolysis. For the tn₂ system the main path to hydrolysis is through a 2:1 cobalt to DNPP complex in which attack by a cis-coordinated hydroxide is again involved. The more complex rate behavior and the slower hydrolysis rates observed for tn₂ system result from the formation of cis and trans isomers in which trans arrangements of coordinated DNPP and hydroxide leave the latter unavailable to participate in intramolecular hydrolysis. Computer fitting of the observed rate data provides values of equilibrium and rate constants for the two systems. Detailed mechanistic schemes are proposed. For the trpn system at pH 6.0 and a 25:1 cobalt to DNPP ratio (5 × 10⁻⁵ M DNPP) the observed acceleration over hydrolysis in the absence of the cobalt complex is 3 × 10³; the calculated specific rate constant for hydrolysis in the reactive 1:1 complex (k 0.2 s⁻¹) represents an acceleration over the unpromoted rate of 3 × 10⁴.     

Initial iron oxidation in horse spleen apoferritin. Characterization of a mixed-valence iron(II)-iron(III) complex.

In ferritin, iron is stored by oxidative deposition of the ferrous ion to form a hydrous ferric oxide mineral core. Two intermediates, formed during the initial stages of iron accumulation in apoferritin, have been observed previously in our laboratory and have been identified as a mononuclear Fe3(+)-protein complex and a mixed-valence Fe2(+)-Fe3(+)-protein complex. The physical characteristics of the mixed-valence Fe2(+)-Fe3+ complex and its relationship to the mononuclear Fe3+ complex in horse spleen apoferritin samples to which 0-240 iron atoms were added was examined by EPR spectroscopy. The results indicate that the mononuclear complex is not a precursor to the formation of the mixed-valence complex. Competitive binding studies with Cd2+, Zn2+, Tb3+, and UO2+(2) suggest that the mixed-valence complex is formed on the interior of the protein in the vicinity of the 2-fold axis of the subunit dimer. The mixed-valence complex could be generated by the partial oxidation of Fe2+ in apoferritin containing 120 Fe2+ or by the addition of up to 120 Fe2+ to ferritin already containing 18 Fe3+/protein molecule. The fact that the complex is generated during early Fe2+ oxidation suggests that it may be a key intermediate during the initial oxidative deposition of iron in the protein. The unusual EPR powder lineshape at 9.3 GHz of the mixed-valence complex was simulated with a rhombic g-tensor (gx = 1.95, gy = 1.88, gz = 1.77) and large linewidths and g-strain parameters. The presence of significant g-strain in the complex probably accounts for the failure to observe an EPR signal at 35 GHz and likely reflect considerable flexibility in the structure of the metal site. The temperature dependence of the EPR intensity in the range 8-38 K was modeled successfully by an effective spin Hamiltonian including exchange coupling (-2JS1.S2) and zero-field terms, from which an antiferromagnetic coupling of J = -4.0 +/- 0.5 cm-1 was obtained. This low value for J may reflect the presence of a mu-oxo bridge(s) in the dimer.     

Bisintercalator-containing dinuclear iron(III) complex: An efficient artificial nuclease

Two acridine groups were successfully introduced into di-iron(III) complex. DNA cleavage experiments indicated that complex conjugating bisacridine groups can enhance 300-fold for the cleavage efficiency compared with complex lacking of acridine conjugation. Further ligation assay of DNA segments provided the evidence for hydrolytic mechanism of DNA cleavage.     

DNA cleavage promoted by 2,9-dimethyl-4,7-diazadecane-2,9-dithiol (DDD) derivatives

Three piperidine derivatives of 2,9-dimethyl-4,7-diazadecane-2,9-dithiol (DDD), NEPDDD, NEMPDDD, and NEMMPDDD, were synthesized and used as catalysts in DNA cleavage. Under physiological conditions, a series of experiments have been done. The effects of DNA cleavage with three ligands were studied under different concentrations, cleavage time, and pH values. The results strongly suggested that the plasmid DNA (pUC 19) can be cleaved efficiently by these ligands. For the cleavage reaction catalyzed by NEMPDDD, Form I DNA could convert to Form II completely, and the DNA-cleavage mechanism involved an oxidative pathway.     

Effect of substituents on complex stability aimed at designing new iron(III) and aluminum(III) chelators

The solution equilibria of iron(III) and aluminum(III) with two classes of hard ligands (catechol, salicylic acid and their nitro-derivatives) have been reliably studied by potentiometric, spectrophotometric and NMR spectroscopy. The effect of the nitro substituent on the binding properties of catechol and salicylic acid has been examined thoroughly. The inductive and resonance properties of the substituent that, as expected, lower the basicity of the phenolic and carboxylic groups, lead to a general decrease in both protonation and complex formation constants. This decrease causes an increase in pM of between 0.2 and 1.1 pM units for the nitro-substituted salicylates and of about 4 units for 4-nitrocatechol, with a significantly higher chelating efficacy. The influence of the substituent on catechol and salicylic acid is discussed in detail on the basis of conditional constants at pH 7.4.     

Complexation of iron(III) by catecholate-type polyphenols

We report here a complete physico-chemical study of the chelation of iron(III) by catechin (L¹), an abundant polyphenol in green tea. Using a fruitful combination of electrospray mass spectrometry, absorption spectrophotometry and potentiometry, we have characterized three ferric complexes of catechin (L¹Fe, and (L¹)₃Fe) as well as a ternary complex L¹FeNTA when an exogenous ligand (nitrilotriacetic acid) is added to the medium. Thanks to this study, we discuss the influence of an exogenous tetradentate ligand in the ferric recognition processes by catecholate-type polyphenols.     

Phosphate group effects upon the equilibrium of iron(III) meso-tetrakis (4-N-methylpyridiniumyl) porphyrin in aqueous solution

Iron(III) meso-tetrakis (4-N-methylpyridiniumyl) porphyrin (FeTMPyP) undergoes a complex equilibrium in aqueous solution as a function of pH. Use of phosphate buffers, a common practice in biomedical applications of porphyrins, suggests the complexation of phosphate anion at the sixth coordination position to the iron, which contributes to the complexity of the equilibrium in the pH range from 1 to 4. In the absence of phosphate the equilibrium is simplified in a similar way as in the presence of high salt concentrations. Combined use of optical absorption, (1)H NMR and infrared spectroscopies, together with the literature data, suggest the formation of hexacoordinated monoaqueous-phosphate FeTMPyP complex in a limited acidic pH range. Discussion of the behavior of cationic FeTMPyP as compared to anionic iron(III) meso-tetrakis (4-sulfonatophenyl) porphyrin (FeTPPS(4)) is presented in regard to equilibrium of different species to explain the observed complex equilibrium.     

Hydrolytic cleavage of DNA by quercetin zinc(II) complex

Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.