[Crystallochemistry of copper (II) and zinc (II) chelates by nonsteroidal antiinflammatory drugs]. / Cristallochimie des complexes à visée thérapeutique du cuivre (II) et du zinc (II) par des anti-inflammatoires non-stéroïdiens.

Copper(II) and zinc(II) chelates by some non-steroidal antiinflammatory drugs NSAIDs (niflumic acid, indomethacin) and 3,5-diisopropylsalicylic acid (DIPS) were characterized by single X-ray diffraction methods. Copper(II) complexes by these two types of chelates are binuclear compounds, with Cu(2)(DIPS)(4)L(2) or Cu(2)(AINS)(4)L(2) formula (L=axial non-NSAID ligand such as diethylether, dimethylsulfoxide DMSO). In zinc(II) complex by DIPS, the metal ion is tetrahedrally coordinated and the corresponding compound is mononuclear with Zn(DIPS)(2)(DMSO)(2) formula. These copper(II) and zinc(II) complexes were found to be more active than their parent drugs from the antiinflammatory and anticonvulsant properties. It was pointed out that the Cu(2)(DIPS)(4)L(2) complexes (L=diethylether, N,N-dimethylformamide) exhibited no rotorod toxicity when examined for anticonvulsant activity using the seizure produced by maximal electroshock, following oral administration to rats.

Low-temperature crystal structures of tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodico pper(II) and tetrakis-mu-3,5-diisopropylsalicylatobis-diethyletheratodicopp er(II) and their role in modulating polymorphonuclear leukocyte activity in overcoming seizures.

Two binuclear copper(II) complexes of 3,5-diisopropylsalicylic acid were characterized by single crystal X-ray diffraction methods and examined for anti-inflammatory activity using activated polymorphonuclear leukocytes and for anticonvulsant activities using electroshock and metrazol models of seizures. These complexes were crystallized from dimethylformamide (DMF) or diethylether. Tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodicop per(II) [Cu(II)2(3,5-DIPS)4(DMF)2] I is in space group P 1; a = 10.393 (2), b = 11.258 (2), c = 12.734 (2) A, alpha = 96.64 (2), beta = 92.95 (2), gamma = 94.90 (2) degrees; V = 1471.7 (4) A3; Z = 1. Tetrakis-mu-3,5-diisopropylsalicylatobis-etheratodicopper(II ) [Cu(II)2(3,5-DIPS)4(ether)2] II is in space group P 1; a = 10.409 (3), b = 11.901 (4), c = 12.687 (6) A, alpha = 91.12 (5), beta = 90.84 (5), gamma = 100.90 (4) degrees; V = 1542 (1) A3; Z = 1. The structure of I was determined at 140 K from 4361 unique reflections (I > 2sigma(1)) and refined on F2 to R1 = 0.04 and wR2 = 0.09. The structure of II was determined at 180 K from 4605 unique reflections (I > 2sigma(I)) and refined on F2 to R1 = 0.05 and wR2 = 0.13. Each compound is a crystallographically centrosymmetric binuclear complex with Cu atoms bridged by four 3,5-diisopropylsalicylate ligands related by a symmetry center [Cu-Cu(i): 2.6139 (9) A in I and 2.613 (1) in II]. The four nearest O atoms around each Cu atom form a nearly rectangular planar arrangement with the square pyramidal coordination completed by the dimethylformamide (or diethylether) oxygen atom occupying an apical position, at a distance of 2.129 (2) A in I and 2.230 (3) A in II. Each Cu atom is displaced towards the DMF (or diethylether) ligand, by 0.189 A in I and 0.184 A in II, from the plane of the four O atoms. The crystal structures of I and II are essentially similar to each other, except for the DMF or diethylether accommodation. Many disorder phenomena were found in the crystal structure of I. Copper(II)2(3,5-DIPS)4(DMF)2 inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism in vitro. This effect was concentration related and significant for concentrations higher than 10 microg or 0.68 nmol/ml. Copper(II)2(3,5-DIPS)4(DMF)2 was more active than the parent ligand, 3,5-DIPS, as has been demonstrated with copper complexes of other non-steroidal anti-inflammatory drugs. The DMF and diethylether ternary complexes of Cu(II)2(3,5-DIPS)4 were found to have anticonvulsant activity in the maximal electroshock model of grand mal epilepsy in doses ranging from 26 to 258 micromol/kg of body mass following intraperitoneal, subcutaneous, or oral treatment. The DMF ternary complex was also found to be effective in the subcutaneous injection of metrazol model of petit mal epilepsy. We conclude that both ternary copper complexes are lipophilic and bioavailable, capable of facilitating the inflammatory response to brain injury and causing the subsidence of this response in bringing about remission of these disease states.

Copper(II) complexes of a nonsteroidal anti-inflammatory drug niflumic acid. Synthesis, crystal structure of tetrakis-mu-(2-[3-(trifluoromethyl) phenyl]aminonicotinato)bis(dimethylsulfoxide)-dicopper(II) complex at 190 K. Anti-inflammatory properties.

The synthesis and characterization of three complexes with a potent nonsteroidal anti-inflammatory drug niflumic acid {2-[3-(trifluoromethyl)phenyl]aminonicotinic acid} with formula [Cu(niflumato)2L] (L = H2O, DMSO = dimethylsulfoxide, DMF = N,N-dimethylformamide) were investigated. The crystal and molecular structure of the {Cu(niflumato)2(DMSO)}2 was reported. Crystallographic data are as follows: monoclinic system, space group P2(1)/n, Z = 2, a = 11.1318(8), b = 17.513(2), c = 15.336(1) A, beta = 103.316(8) degrees, V = 2909.4(4) A3. The structure was refined to R = 0.030 and wR = 0.037 for 3702 reflections with I > sigma (I). It consists of centrosymmetric binuclear units with the Cu-Cui (symmetry code i: 1-x, -y, 1-z) distance between two centrosymmetrically related ions of 2.6272(5) A. Each Cu(II) ion in [Cu2(DMSO)2(mu-niflumato)4] is coordinated to an apical dimethylsulfoxide O atom on the one hand and to the equatorial carbonyl and carboxylic O atoms of two crystallographically independent niflumate moieties and their centrosymmetric counterparts on the other hand. In spite of the low-temperature (190 K) crystal measurements, one L-CF3 grouping exhibits some disorder. The biological activities of these complexes were compared to that of niflumic acid. Niflumic acid and its various copper complexes significantly inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism, as assessed by chemiluminescence and O2- generation measurement. This effect was dose-dependent. All copper complexes exerted a similar inhibiting effect which was always significantly higher than that exerted by the parent drug.

Interaction of Cu(II) 3,5-diisopropylsalicylate with human serum albumin--an evaluation of spectroscopic data.

The copper(II) complex of 3,5-diisopropylsalicylate is a lipophilic water-insoluble binuclear complex, Cu(II)2(3,5-DIPS)4, that has attracted interest because of a wide range of pharmacological activities. This study was undertaken to examine bonding interactions between the complex and human serum albumin (HSA) to help elucidate the mode of transport of the complex in vivo. Electron paramagnetic resonance, numerical magnetic resonance and UV-visible absorption spectroscopic studies were performed using 200 microM aqueous solutions (pH 7.5) of HSA to which had been added up to three molar equivalents of CuCl2, CuSO4, or Cu(II)2(3,5-DIPS)4. Both EPR and UV-visible spectra demonstrated the presence of more than one copper bonding site on HSA, and proton NMR spectra showed that the 3,5-DIPS ligand is also bonded to HSA. These results indicate that there is no observable direct coordination of the ligand to copper in the presence of HSA, and that the majority of the copper and 3,5-DIPS bond to HSA at separate sites. Addition of solid Cu(II)2(3,5-DIPS)4 to HSA at pH 7.5 similarly resulted in spectra suggest that there are no ternary Cu(II)(3,5-DIPS), Cu(II)(3,5-DIPS)2, or Cu(II)2(3,5-DIPS)4 complexes formed with HSA. It is concluded that any ternary complexes formed in the presence of HSA are below the spectroscopic detection limits and represent less than 5% of the total copper.

Mechanism-based inactivation of porcine kidney diamine oxidase by 1,4-diamino-2-butene.

Cis- and trans-1,4-diamino-2-butene are substrates and potent inactivators of porcine kidney diamine oxidase. Evidence from absorption and NMR spectra indicates that both are oxidized to pyrrole. Both substrates are irreversible mechanism-based inactivators of the enzyme, although the trans isomer is more potent and results in complete inactivation in a reaction which follows pseudo-first-order kinetics with an apparent Ki of 0.34 mM and a second-order inactivation constant of 500 M-1 s-1. Under the same conditions, 46% of the activity remains when the enzyme is reacted with cis-1,4-diamino-2-butene. Trans-4-amino-2-butenal, the product of oxidation of the trans diamine, has been synthesized and shown to undergo cyclization to pyrrole in a concentration-dependent manner, approaching second-order at low concentrations. Trans-4-amino-2-butenal is itself a potent irreversible inhibitor with IC50 of 2.5 microM. We propose that the irreversible inactivation by both cis- and trans-1,4-diamino-2-butene involves attack by a protein-based nucleophilic residue on the unsaturated aminoenal products of the enzymatic reactions, resulting in a covalent adduct. Cyclization of the cis-aminoenal to pyrrole is much more rapid than in the trans case, thus it is less available for inhibitory reaction with the protein.

Cyanide inhibition of porcine kidney diamine oxidase and bovine plasma amine oxidase: evidence for multiple interaction sites.

The interactions of cyanide and phenylhydrazine with porcine kidney diamine oxidase (PKDAO) and bovine plasma amine oxidase (BPAO) (EC 1.4.3.6) have been investigated. Cyanide displays mixed noncompetitive inhibition against amine substrates and also against O2. EPR spectroscopy shows that cyanide binds to an equatorial site on Cu(II) and can be displaced by chloride, which is not an inhibitor, without recovery of activity, indicating that Cu(II)-bound cyanide is not inhibitory. 14CN-. studies have shown that one cyanide in PKDAO and two in BPAO are covalently and irreversibly bound per enzyme dimer at an unknown site, even under conditions where cyanide is not bound to Cu(II). These cyanides have no effect on activity or on binding of phenylhydrazine to the enzymes. Cyanide also binds reversibly to the organic cofactor in both enzymes, presumably as a cyanohydrin, leading to the observed mixed noncompetitive inhibition against substrate. In both enzymes, two phenylhydrazines react per enzyme dimer. The kinetics of phenylhydrazine titration are affected by cyanide, which indicates that phenylhydrazine and cyanide react at the same carbonyl group in the enzymes. The results suggest that inhibition of amine oxidases by cyanide is through a carbonyl reagent and a Cu(I) ligand rather than through a Cu(II) ligand.

Hydroxyl radical production in the reactions of copper-containing amine oxidases with substrates.

Solutions of porcine kidney diamine oxidase, PKDAO, and bovine plasma amine oxidase, BPAO, were saturated with the spin-trapping agent alpha-phenyl-N-t-butylnitrone, PBN, and incubated with cadaverine or benzylamine substrate, respectively, under aerobic conditions. EPR spectra due to trapped hydroxyl radicals were obtained for both enzymes with no evidence of superoxide formation. Under anaerobic conditions, hydroxyl radicals were formed only when H2O2 was present as well as substrate. Catalase prevented hydroxyl radical formation by PKDAO but not BPAO. The results indicate that hydroxyl radical is produced in the reaction of the product H2O2 with the reduced enzymes and therefore may be important in turnover-related enzyme degradation, but is not a true reaction intermediate.

Spectral energy effects in ESR bone dosimetry: photons and electrons.

The spectral energy-dependence of the radiation-induced ESR signal has been studied in ovine cortical bone. Crushed bone samples were irradiated using photon beams with effective energies in the range from 0.06 to 6 MeV, and electron beams with mean energies in the range from 2 to 10 MeV. The photon and electron data were normalized to a dose to bone of 50 Gy and the results are reported as response relative to the ESR signal for photon irradiation at 1.25 MeV (60Co). The photon irradiation results show that the ESR response is greatest at low energies with a relative value of 1.2 at 0.06 MeV. The relative response decreases, as the energy increases, to approximately 0.85 in the region of 2 to 3 MeV. These variations in the relative ESR responses are significantly less than the ESR energy-dependent responses reported in the literature for human tooth enamel and synthetic hydroxyapatite. An explanation for this difference is offered. For electron beam irradiations, the ESR signal is fairly constant with energy, and approximately equal to that at a photon energy of 1.25 MeV. Implications of these results are discussed.

Hydrated electron formation on laser excitation of P-pyridoxyl amino acids and proteins.

Evidence from a characteristic transient spectrum, its lifetime and quenching by N2O demonstrates that XeCl excimer laser excitation of the reduced form of pyridoxal 5'-phosphate bound to lysine residues will produce the hydrated electron. The phenoxyl-type radical coproduct was also observed. The results identify a useful label of protein structures for site-specific electron generation which can occur outside the region of light absorption by aromatic residues of protein. The results also represent the first observation of hydrated electron formation from flash excitation of a vitamin B6 cofactor.

EPR studies of spin-labeled bovine plasma amine oxidase: the nature of the substrate-binding site.

The carbonyl cofactor of bovine plasma amine oxidase (EC 1.4.3.6), recently shown to be 6-hydroxydopa (also known as topa), has been spin labeled to the extent of one label per enzyme dimer molecule, using 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) and 4-hydrazino-TEMPO followed by reduction with borohydride. By studying the EPR spectra of the labeled enzyme, it has been deduced that there is no magnetic interaction between the copper and the spin label, and that the spin label is at least 1.3 nm distant from the copper(II) ion in the resting enzyme. The bound label is strongly immobilized, is in a sterically constricted environment, and is not accessible to small anions. Removal of the copper does not alter the EPR spectrum of the label. The results are similar to results for porcine plasma amine oxidase, and show that the copper is not close to, and does not directly interact with, the topa-bound substrate.

Structural and catalytic properties of copper in lysyl oxidase.

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.

Vicinal diamines as pyrroloquinoline quinone-directed irreversible inhibitors of lysyl oxidase.

The observation that aliphatic diamines become poor substrates as the carbon chain length decreases and that ethylenediamine, the shortest diamine, is an irreversible inhibitor of lysyl oxidase led to the investigation of the mechanism of inhibition by ethylenediamine. The cis but not the trans isomer of 1,2-diaminocyclohexane was also a potent irreversible inhibitor of lysyl oxidase, consistent with the interaction of both amino groups of vicinal diamines with an enzyme moiety. Both cis-1,2-diaminocyclohexane and ethylenediamine but not trans-1,2-diaminocyclohexane markedly perturbed the spectrum of free pyrroloquinoline quinone (PQQ), a covalently linked form of which is the carbonyl cofactor of lysyl oxidase. cis-1,2-Diaminocyclohexane also induced similar changes in the spectrum of lysyl oxidase. The perturbations of the spectra of PQQ or of lysyl oxidase by cis-1,2-diaminocyclohexane or ethylenediamine as well as the development of irreversible inhibition of the enzyme by cis-1,2-diaminocyclohexane or ethylenediamine were all markedly reduced under anaerobic conditions. Moreover, approximately 1 mol of H2O2 was released per mol of PQQ or lysyl oxidase upon aerobic incubation with cis-1,2-diaminocyclohexane, while approximately 2 mol of 3H+ were released from cis-[1,2-3H] 1,2-diaminocyclohexane per mol of PQQ or lysyl oxidase under corresponding conditions. A proposal for the mechanism of inhibition of lysyl oxidase by vicinal diamines is presented which involves limited oxidation of the diamine linked to PQQ at the active site so that the PQQ-diamine complex is finally stabilized by a conjugated 6-membered ring.

Fluorescence lifetime and polarization anisotropy studies of membrane surfaces with pyridoxal 5'-phosphate.

The fluorescence lifetime and depolarization of the pyridoxal 5'-phosphate label demonstrated different environments at the structure-solvent interface for micelles, liposomes, proteins and membranes. A short lifetime and rotational correlation time for the micelles and liposomes proved that the label was strongly associated with the water solvent and rotated freely about the covalent bond. The proteins provided a more buried or hydrophobic site as shown by an increase in the lifetimes. Rotational correlation times of 4-6 ns for sarcolemma and erythrocyte membranes suggested restricted rotation for the pyridoxal 5'-phosphate label. Lower values of the rotational correlation time for rod outer segment and myelin sheath proved that the protein epsilon-amino groups are at the solvent interface which allows for more rotation.

The binding of copper(II) and zinc(II) to oxidized glutathione.

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.

Transition-metal binding site of bleomycin A2. A carbon-13 nuclear magnetic resonance study of the zinc(II) and copper(II) derivatives.

The 13C NMR spectra at 25.2 MHz of the Zn(II) and Cu(II) complexes of the antitumor antibiotic bleomycin A2 are discussed. Complexation of the drug to Zn(II) causes 38 of the 52 resonance lines of bleomycin A2 to shift to new positions. All but ten of these shifted lines have been assigned in the Zn(II) bleomycin complex. Although the specific donor sites of the drug cannot be identified from the 13C NMR data, the analysis clearly shows that the pyrimidine-imidazole portion of the molecule is affected by chelation. This finding is in agreement with the previously reported metal-binding site of the antibiotic. The analysis also shows that carbon atoms which have large through-bond distances from the binding site can experience substantial chemical-shift changes upon metal binding. Complexation of the drug to Cu(II) eliminates 23 resonances from the spectrum of the molecule. All of these resonances emanate from carbon atoms which are located in the pyrimidine-imidazole portion of the drug.

A spectroscopic investigation of the metal binding site of bleomycin A2. The Cu(II) and Zn(II) derivatives.

Using a combination of ultraviolet-visible absorption, 1H NMR and ESR techniques we have established that N(1) of the imidazole and N(1) of the pyrimidine residues of bleomycin A2 bind to Cu(II) and Zn(II). The observations coupled with the earlier results that the alpha-amino group of the alpha-amino carboxamide function and the carbamoyl moiety are also Cu(II)-ligating groups makes it possible to reconstruct the detailed geometry and stereochemistry of the metal binding site of bleomycin A2.

An EPR study of the lineshape of copper in cytochrome c oxidase.

The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220 degreesK, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx = 1.99, gy = 2.03, gz = 2.185, / Ax(Cu) / = 0.0020 cm-1, / Ay(Cu) / = 0.0025 cm-1, / Az(Cu) / = 0.0030 cm-1. The principal values of the g tensor and the small value of /Az(Cu) / are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distrotion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values deltaHx = 45G, deltaHy = 65 G, deltaHz = 85 G. A weak dipolar interaction with a low-spin ferric species stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time has been observed near g = 14 and g = 3 at 5 degrees K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.