Transition metal complexes of amino acids and derivatives containing disulphide bridges

The interaction of cobalt(II), nickel(II), copper(II) and zinc(II) with D-penicillamine disulphide, oxidized glutathione and L-cysteinylglycine disulphide were studied by pH-metric, spectrophotometric and EPR methods. D-Penicillamine disulphide forms binuclear complexes with all the metal ions studied. The formation of 1:1 complexes is characteristic of oxidized glutathione. L-Cysteinylglycine disulphide behaves like dipeptides, but the presence of two separate peptide moieties also results in the formation of various binuclear complexes. Metal ion-disulphide binding was not observed in any case.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala2 and Ala3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala2 and Ala3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala2 hydrolysis by EDTA-treated cells was inhibited by the Ca2+/H+ ionophore, tetronasin. Ala3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B(1)4 had activities which ran at the same R(f); strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R(f) for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M(r) of the dipeptidases from strains M384 and B(1)4 were 115,000 and 114,500 respectively, and 112,500 and 121,500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Interaction between cytochrome C and di- and tripeptides.

By observing changes in the absorbance spectrum between 340 and 650 nm, we found that tyrosyltyrosylphenylalanine (TTP) interacts with cytochrome C (CC). TTP caused the characteristic changes of CC reduction, namely, an increased optical density at 524 and 550 nm and a hyperchromic shift at 416 nm. The apparent dissociation constant (Kd) was 2.9 x 10(-3) M. Most of the reducible CC at 20 uM concentration was reduced by 10 mM TTP. TTP was more potent than all other peptides tested, including the previously reported tyrosylphenylalanine. That the carboxyl terminal phenyl group was essential for reduction was shown by comparing variously substituted di- and tripeptides. Reduction by TTP increased with increasing pH and buffer concentration at constant pH. A combination of superoxide dismutase and catalase failed to inhibit the reduction. We found no effect of TTP on O2 consumption of isolated intact mitochondria. Our data demonstrate that small peptides can serve as probes of the topography and electron density of CC and that the TTP-CC interaction may provide insight as an analog of in-vivo processes.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Transition metal complexes bearing atropisomeric saturated NHC ligands

From achiral imidazolinium salts, chiral transition metal complexes containing an N-heterocyclic carbene (NHC) ligand were prepared (metal = palladium, copper, silver, gold, rhodium). Axial chirality in these complexes results from the formation of the metal-carbene bond leading to the restriction of rotation of dissymmetric N-aryl substituents about the C–N bond. When these complexes exhibited a sufficient configurational stability, a resolution by chiral high-performance liquid chromatography (HPLC) on preparative scale enabled isolation of enantiomers with excellent enantiopurities (>99% ee) and good yields. A study of the enantiomerization barriers revealed the effect of the backbone nature as well as the type of transition metal on its values. Nevertheless, the evaluation of palladium-based complexes in asymmetric intramolecular α-arylation of amides demonstrated that the ability to induce an enantioselectivity cannot be correlated to the configurational stability of the precatalysts.     

Transition metal complexes with pyrazole derivatives as ligands

Degradation reactions of scorpionates were observed in the presence of transition metal salts MX₂ to give complexes of transition metal and pyrazole derivatives. Otherwise, pyrazolato complexes of transition metals and weakly coordinating anions such as nitrates have been synthesized from transition metal nitrates and 3-phenyl- and 4-phenyldiazo-pyrazole. A number of complexes with pyrazole derivatives as ligands, [Zn(3-tBupzH)₂Cl₂], [Fe₂(3-Phpz)₆Cl₄], [Cu(pzH)₄Br₂], [Ni(py)₂(pzH)₂Cl₂], [Li(THF)₄][Ti₂(μ-pz)₃Cl₄(NMe₂)₂], [Zn₂(μ-3-Phpz)₂(3-PhpzH)₂][(NO₃)₂], [M(3-PhpzH)₄(NO₃)₂] (M = Co, Ni, Cu, Zn, Cd), [Zn(3-PhpzH)₂(NO₃)₂], [Zn(4-PhNNpzH)₂(NO₃)₂](H₂O), and [Cd(4-PhNNpzH)₂(NO₃)₂(H₂O)₂], have been crystallized and characterized by single-crystal X-ray diffraction.     

Transition metal complexes IX. Nickel complexes with a chiral azaphosphole ligand

The reaction of (−)-(R)-myrtenal and (+)-(R)-phenylethylamine gave a Schiff base 1 which was reacted with MePBr₂ in the presence of a base to give under dehydrohalogenation of an intermediate McCormack product a salt 2. Treatment of 2 with sodium led to the formation of the azaphosphole 4. η³-C₃H₅NiCl and 4 gave a 1:1 adduct 5 and nickel(0) gave a 1:4 complex 6. Compounds 4–6 were characterized by NMR spectroscopy as well as by single crystal X-ray structure determination.     

Thermolysin and alpha-chymotrypsin mediated synthesis of tripeptides containing proline

The tripeptides Z-Pro-Leu-Gly-OEt, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OBzl were synthesized by thermolysin and alpha-chymotrypsin catalysis. The optimum conditions for the couplings between Z-Pro-OH and H-Leu-OEt or H-Leu-Gly-OEt catalysed by thermolysin were determined by a systematic study involving analysis of pH effect, ammonium sulfate concentration, reaction time, enzyme concentration, and relative proportion of the carboxyl and amine components. The best yield obtained for Z-Pro-Leu-OEt was 77% and for Z-Pro-Leu-Gly-OEt, 100%. Z-Pro-Leu-OEt was coupled to H-Gly-OEt, H-Gly-NH2 and H-Gly-OBzl. The best conditions to obtain Z-Pro-Leu-Gly-OEt and Z-Pro-Leu-Gly-NH2 were determined by the study of some factors that affect the reaction yield, such as organic solvent presence, substrate ratio and aqueous and organic solvent ratio. The yield obtained under optimum synthesis conditions was 55% for Z-Pro-Leu-Gly-OEt and 61% for Z-Pro-Leu-Gly-NH2. Z-Pro-Leu-Gly-OBzl was synthesized with 42% yield.     

Transition metal complexes as mediator-titrants in protein redox potentiometry

A selection of nine macrocyclic Fe^III/II and Co^III/II transition metal complexes has been chosen to serve as a universal set of mediator-titrants in redox potentiometry of protein samples. The potential range spanned by these mediators is approximately from +300 to −700 mV vs the normal hydrogen electrode, which covers the range of most protein redox potentials accessible in aqueous solution. The complexes employed exhibit stability in both their oxidized and their reduced forms as well as pH-independent redox potentials within the range 6 < pH < 9. The mediators were also chosen on the basis of their very weak visible absorption maxima in both oxidation states, which will enable (for the first time) optical redox potentiometric titrations of proteins with relatively low extinction coefficients. This has previously been impractical with organic mediators, such as indoles, viologens and quinones, whose optical spectra interfere strongly with those of the protein.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Transition metal complexes of diclofenac with potentially interesting anti-inflammatory activity

An overview is given of the results of metal ion-diclofenac interactions. Several complexes have been synthesized at the University of Ioannina. Binuclear complexes, [Cu(L)2(H2O)]2 x 2H2O and [CuL2(S)]2 where S is H2O, EtOH, DMSO, (CH3)2CO and DMF, and mononuclear complexes, [MnL2(H2O)], [FeL2(H2O)2], [CoL2(H2O)2] x 0.5H2O, [CoL2(H2O)], [NiL2(H2O)2] x 2H2O, [NiL2] and [PdL2] x 2H2O, have been characterized by spectroscopy, X-ray crystallography and electrochemical studies. The catalytic activity of these complexes was correlated to the reduction potential. Some of the complexes of diclofenac exhibit very promising anti-inflammatory activity and act as antioxidant compounds, a property that is absent from diclofenac.     

Transition metal ion complexes of 2-thio-6-picoline N-oxide

Complexes of the anion 2-thio-6-picoline N-oxide (6MOS) have been isolated with the following stoichiometry: M(6MOS)₃ (M = Cr, Fe, and Co) and M(6MOS)₂ (M = Co, Ni, Cu and Zn). The spectral properties of these complexes are compared with those of 2-thiopyridine N-oxide in order to determine the stereochemical effect of the 6-methyl substituent. The nature of the Ni(I) species formed on exposure to high energy radiation, and the nature of the heterocyclic amine adducts to both the Ni(II) and Cu(II) complexes are also reported.     

Heat capacity changes and partial molal heat capacities of some di- and tripeptides in water

Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔC at 30°C. Limiting partial molal heat capacities ΔC have been determined by combining ΔC with C_p2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group ? CONH? was semiquantitatively estimated.     

Lysine side-chain dynamics derived from 13C-multiplet NMR relaxation studies on di- and tripeptides

Summary ¹³C NMR relaxation data have been used to determine dipolar auto- and cross-correlation times for the di- and tripeptides GK, KG and GKG, primarily to analyze lysine side-chain motional dynamics. In general, correlation times are largest for backbone positions and decrease on going through the lysine side chain, consistent with the idea of increased mobility at C and C methylenes. Correlation times, however, vary with the peptide ionization state. In the zwitterionic state of GK, for example, both auto-and cross-correlation times are at their maximum values, indicating reduced internal motions probably resulting from intramolecular electrostatic interactions. Modifying the charge state increases motional fluctuations. Activation energies determined from the temperature dependence of CH rotational autocorrelation times at neutral pH are approximately equal for glycine and lysine C and lysine C and C positions (4.1±0.2 to 4.5±0.2 kcal/mol) and tend to decrease slightly for lysine C and C (3.8±0.2 to 4.3±0.2 kcal/mol). The sign of lysine side-chain cross-correlations could not be explained by using any available rotational model, including one parameterized for multiple internally restricted rotations and anisotropic overall tumbling. Molecular and stochastic dynamics calculations were performed to obtain insight into correlated internal rotations and coupled overall tumbling and internal motions. Relatively strong correlations were found for i,i+1 backbone and lysine side-chain internal bond rotations. Stochastic dynamics calculations were more successful at explaining experimentally observed correlation times. In the fully charged state, a preferred conformation was detected with an all-trans lysine side chain.Abbreviations rf radio frequency - GK dipeptide glycine-lysine - KG dipeptide lysine-glycine - GKG tripeptide glycine-lysine-glycine     

Transition metal half-sandwich complexes as redox mediators to glucose oxidase

Chromium and manganese half-sandwich complexes are evaluated as mediators to glucose oxidase (GOx) since they are of similar size to ferrocene derivatives (sandwich complexes) and contain a single pi-ligand for interaction with the enzyme co-factor. A series of seven amino derivatives of [(eta-C(6)H(6))Cr(CO)(3)] were investigated of which only [[eta-C(6)Me(4)(NH(2))(2)]Cr(CO)(3)] (7), with the lowest oxidation potential of +40 mV (versus SCE), was found to display reversible electrochemistry. Small catalytic currents were recorded in the presence of GOx and glucose when complex (7) was incorporated in a screen-printed carbon electrode. Manganese cyclopentadienyl (Cp) half-sandwich complexes were found to be more effective GOx mediators and comparable in efficacy to ferrocene derivatives. A mediator rate constant k(M) of 2.1 x 10(5)M(-1)s(-1) was determined for the water-soluble complex [(eta-MeC(5)H(4))Mn(NO)(CN)(2)]Na (11) compared to a range of 3 x 10(4) to 8 x 10(6)M(-1)s(-1) previously determined for ferrocenes under the same experimental conditions. beta-Cyclodextrin (beta-cd) was found to be helpful in solubilising hydrophobic complexes such as [(eta-MeC(5)H(4))Mn(NO)(S(2)CNMe(2))] (15) and the neutral oxidised form of [MeCpMn(NO)[(SCCN)(2)]]NEt(4) (14), either directly as an inclusion adduct or in situ during cyclic voltammetry. Screen-printed amperometric electrodes, containing a mediator and GOx immobilised in an organic conducting carbon layer, were useful in assessing the mediation ability of complex (15) where aqueous insolubility precluded any kinetic studies with GOx in solution. This work was briefly extended to other oxidoreductase enzymes apart from GOx. Thus, rotating ring-disk voltammetry demonstrated that the beta-cd complex of compound (15) is also a useful mediator to Horseradish peroxidase (HRP) since it displays an identical catalytic current to the ferrocene ethanolamine derivative (1) used in the MediSense ExacTech and Precision QID blood glucose biosensor electrodes.     

Complementary role of surface hydrolysis and intact transport in the intestinal assimilation of di- and tripeptides

The small intestine is known to possess mechanisms for intact transport and membrane hydrolysis of oligopeptides. To determine the relative role of these processes in peptide assimilation the fate of two model peptides known to be high-affinity substrates for the brush border aminooligopeptidase were studied in rat small intestine in vivo. Both 20 mM Gly-L-Pro, a potent inhibitor of peptide transport, and specific inhibitors of the aminopeptidase, 10 mM L-Ala-β-naphthylamide or the phthalimido derivative of 0.1 mM L-leucine bromomethyl ketone, reduced assimilation of L-Leu-Gly-Gly and L-Leu-L-Leu. Further inhibition was found when both transport and peptidase inhibitors were included in the intestinal perfusate suggesting that the model di- and tripeptides utilize both intact transport and surface hydrolysis for their assimilation. Although comparative kinetic parameters of intact transport (K_m = 22 mM; V = 1.9 · 10^?3μmol · s^?1 · cm^?2) and surface hydrolysis (K_m = 8.7; V = 1.1 · 10^?3) for l-Leu-l-Leu differed markedly, the relationship of peptide concentration to assimilation rate was nearly identical for intact transport and surface hydrolysis in the physiological range of 1–10 mM substrate. Both intact peptide transport and surface hydrolysis appear to be efficient and complementary processes that promote efficient assimilation of dipeptides and tripeptides. The relative importance of each assimilation process appears to depend upon the amino acid composition of the peptide nutrient.     

Bioorganometallics: First examples of cyclometalated iridium(III) complexes containing di- and tripeptide ester ligands

The synthesis of bis-cyclometalated [Ir(ptpy)₂(gly-gly-OEt)] (2, ptpy = 2-(p-tolyl)pyridinato; gly-gly-OEt = glycylglycine ethyl ester) and [Ir(ptpy)₂(gly-gly-gly-OEt)] (3, gly-gly-gly-OEt = glycylglycylglycine ethyl ester) from the reaction of [{Ir(μ-Cl)(ptpy)₂}₂] (1) with the corresponding peptide ester hydrochlorides in the presence of NaOMe is described. The molecular structure of 2 was confirmed by a single-crystal X-ray diffraction study. The compound crystallized from dichloromethane/iso-hexane in the space group P2₁/a. In the crystal packing the molecules of 2 exhibit N–H?O hydrogen bonds to the neighbor molecules to form dimeric units. The absorption and emission spectra of 2 and 3 were recorded and exhibit these compounds as strong green-emitting complexes.     

Synthesis and evaluation of fluorobenzoylated di- and tripeptides as inhibitors of cyclooxygenase-2 (COX-2)

A series of fluorobenzoylated di- and tripeptides as potential leads for the development of molecular probes for imaging of COX-2 expression was prepared according to standard Fmoc-based solid-phase peptide synthesis. All peptides were assessed for their COX-2 inhibitory potency and selectivity profile in a fluorescence-based COX binding assay. Within the series of 15 peptides tested, cysteine-containing peptides numbered 7, 8, 11 and 12, respectively, were the most potent COX-2 inhibitors possessing IC(50) values ranging from 5 to 85 μM. Fluorobenzoylated tripeptides 7 and 8 displayed some COX-2 selectivity (COX-2 selectivity index 2.1 and 1.6), whereas fluorobenzoylated dipeptides 11 and 12 were shown not to be COX-2 selective. Fluorbenzoylated tripeptide FB-Phe-Cys-Ser-OH was further used in molecular modeling docking studies to determine the binding mode within the active site of the COX-2 enzyme.