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Teicoplanin, a member of the "last chance" antibiotic family has a similar structure and the same mechanism of action as parent drug vancomycin, which is proved to be an effective binder of Cu(II) ions. However, the potentiometric and spectroscopic studies (UV-visible, CD, NMR) have shown that the modification of the N-terminal structure of the peptide backbone in teicoplanin affects considerably the binding ability towards Cu(II) ions. While vancomycin forms almost instantly the stable 3N complex species involving the N-terminal and two amide nitrogen donors, in case of teicoplanin only two nitrogen donors derived from the N-terminal amino group and adjacent peptide bond are coordinated to Cu(II) ion within the whole pH range studied. The major factor influencing the binding mode is most likely the structure of the N-terminus of the peptide unit in the antibiotic ligand.

The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).

Three model dipeptides containing 3-(2,3-di(pyridin-2-yl)quinoxalin-6-yl)alanine, 3-(dipyrido[3,2-a:2,3-c]phenazin-11-yl)alanine, and 3-(2,3-diphenylquinoxalin-6-yl)alanine were studied with respect to their ability to bind selected transition metal ions, such as Cu(II), Fe(II), Ni(II), Co(II), Mn(II), and Cr(III). It was found that only Cu(II) and Fe(II) ions could form stable complex species with the studied compounds. The ability to form the complexes correlated well with DNA damage experiments. Only the ferrous and cupric complexes are capable of generating both single- and double-strand scissions. However, double-strand breakages appear to be dominating lesions in the presence of hydrogen peroxide, especially for copper(II) containing systems. The quantity of breakage products in the presence of N-(3-(dipyrido[3,2-a:2,3-c]phenazine-11-yl)alanyl)glycine complexes was the highest as compared to the complexes of the remaining compounds. Moreover, this ligand was the only one that cleaved DNA in the absence of either Cu(II) or Fe(II) ions.

The interaction between metal ions and nucleotides is well characterized, as is their importance for metabolic processes, e.g. in the synthesis of nucleic acids. Hence, it is surprising to find that no detailed comparison is available of the metal ion-binding properties between nucleoside 5'-phosphates and 2'-deoxynucleoside 5'-phosphates. Therefore, we have measured here by potentiometric pH titrations the stabilities of several metal ion complexes formed with 2'-deoxyadenosine 5'-monophosphate (dAMP2-), 2'-deoxyadenosine 5'-diphosphate (dADP3-) and 2'-deoxyadenosine 5'-triphosphate (dATP4-). These results are compared with previous data measured under the same conditions and available in the literature for the adenosine 5'-phosphates, AMP(2-), ADP(3-) and ATP(4-), as well as guanosine 5'-monophosphate (GMP(2-)) and 2'-deoxyguanosine 5'-monophosphate (dGMP(2-)). Hence, in total four nucleotide pairs, GMP(2-)/dGMP(2-), AMP(2-)/dAMP(2-), ADP(3-)/dADP(3-) and ATP(4-)/dATP(4-) (= NP/dNP), could be compared for the four metal ions Mg2+, Ni2+, Cu2+ and Zn2+ (= M2+). The comparisons show that complex stability and extent of macrochelate formation between the phosphate-coordinated metal ion and N7 of the purine residue is very similar (or even identical) for the AMP(2-)/dAMP(2-) and ADP(3-)/dADP(3-) pairs. In the case of the complexes formed with ATP(4-)/dATP(4-) the 2'-deoxy complexes are somewhat more stable and show also a slightly enhanced tendency for macrochelate formation. This is different for guanine nucleotides: the stabilities of the M(dGMP) complexes are clearly higher, as are the formation degrees of their macrochelates, than is the case with the M(GMP) complexes. This enhanced complex stability and greater tendency to form macrochelates can be attributed to the enhanced basicity (DeltapKaca. 0.2) of N7 in the 2'-deoxy compound. These results allow general conclusions regarding nucleic acids to be made.

The structure of vancomycin coordinated to Cu(2+) ions is presented and structural aspects upon metal coordination are discussed. The asymmetric part of unit cell comprises two independent molecules of vancomycin-Cu(2+) complex, one of them is partially disordered. The binding site involves one imino nitrogen atom, two amide nitrogen atoms delivered by peptide bonds, and carboxyl oxygen from the peptide moiety. The identical set of donor atoms is not reflected in identical coordination geometry around individual metal ions. The studied complex presents two distinct types of conformation. Additionally, leucinyl side chain in one conformer is disordered leading to another type of conformation. The complex molecules form heterodimer with antiparallel hydrogen bonding.

The extent to which the replacement of a ribose unit by a 2'-deoxyribose unit influences the acid-base properties of nucleotides has not hitherto been determined in detail. In this study, by potentiometric pH titrations in aqueous solution, we have measured the acidity constants of the 5'-di- and 5'-triphosphates of 2'-deoxyguanosine [i.e., of H(2)(dGDP)(-) and H(2)(dGTP)(2-)] as well as of the 5'-mono-, 5'-di-, and 5'-triphosphates of 2'-deoxyadenosine [i.e., of H(2)(dAMP)(+/-), H(2)(dADP)(-), and H(2)(dATP)(2-)]. These 12 acidity constants (of the 56 that are listed) are compared with those of the corresponding ribose derivatives (published data) measured under the same experimental conditions. The results show that all protonation sites in the 2'-deoxynucleotides are more basic than those in their ribose counterparts. The influence of the 2'-OH group is dependent on the number of 5'-phosphate groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is most significantly enhanced (by about 0.2 pK units), while the effect on the phosphate groups and the N1H or N1H(+) sites is less pronounced but clearly present. In addition, (1)H NMR chemical shift change studies in dependence on pD in D(2)O have been carried out for the dAMP, dADP, and dATP systems, which confirmed the results from the potentiometric pH titrations and showed the nucleotides to be in their anti conformations. Overall, our results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact basis for the calculation, determination, and understanding of perturbed pK(a) values in DNAzymes and ribozymes, as needed for the delineation of acid-base mechanisms in catalysis.

Protonation equilibria of puromycin (PM) and puromycin aminonucleoside (PAN) and their coordination by copper(II) ion were studied in solution by potentiometry, electronic absorption spectroscopy (UV-Vis), circular dichroism (CD), electron paramagnetic resonance (EPR) and mass spectrometry. For puromycin four mononuclear complexes were found, with stoichiometries Cu(PM)2+, CuH(-1)(PM)+, CuH(-2)(PM) and CuH(-3)(PM)(-). In each of them the Cu(II) ion was bound in the peptidic-like manner, the differences of stoichiometries are a consequence of subsequent deprotonations of the sugar C2'-OH group and the coordinated water molecule. The coordination mode for puromycin aminonucleoside was aminosugar-like. Two dimeric complexes, Cu2H(-1)(PAN)2(2+) and Cu2H(-2)(PAN)2+, and one monomeric CuH(-2)(PAN)2 were found. The N6,N6-dimethyladenine moiety of PAN was not involved in the coordination process due to steric hindrance.

Sinefungin (SFG) is an antifungal and antiparasitic nucleoside antibiotic composed by ornithine and adenosine moieties both having the potential to bind copper(II). NMR studies performed at physiological pH have shown that the alpha-amino and the carboxylate groups in the ornithine unit are the preferred donor sites for Cu(II) binding. On the contrary, at acidic pH, Cu(II) complexation starts from adenosine nitrogen being the alpha-amino group still protonated and not available for metal binding. The proton paramagnetic relaxation enhancements measured at neutral pH allowed to obtain the 3D structure of the 1:2 Cu(II)-SFG complex. Molecular dynamics calculations were revealing for the existence of secondary Cu(II) interaction with the purine nitrogens of the adenosine moiety.

Interactions between sinefungin and copper(II) ions were investigated. Stoichiometry and stability constants of the metal-free system and two mononuclear complexes present in solution were determined on the basis of potentiometric data analysis. The results were compared to the Cu(II)-ornithine system due to structural similarities between both molecules. Combined spectroscopic and theoretical studies allowed for determination of coordination pattern for the Cu(II)-sinefungin complexes. At acidic pH, copper is bound in "glycine-like" coordination mode, identical with that of ornithine. This involves alpha-amino group and the carboxyl oxygen. At higher pH, a "bis-complex" is formed by two sinefungin molecules. The second ligand binds in equatorial position displacing two water molecules, what results in the stable {2N,2O} coordination. Both axial positions are supposed to be occupied by N1 nitrogen donors of adenine moiety, what is confirmed by DFT calculations. They interact indirectly with copper(II) through water molecules as the result of dominant syn conformation of purine.

The effect of selected 10 antibiotics and their complexes with Cu(2+) ions on the catalytic activity of the trans-acting antigenomic delta ribozyme was investigated. Sisomicin, vancomycin, and actinomycin D displayed weak inhibitory properties. However, much stronger effects were detected with complexes of these antibiotics with Cu(2+) ions. The strongest inhibition was observed with actinomycin D-Cu(2+) complex, for which the calculated K(i) value was reduced ca. 35-fold upon metal ion complexation. We postulate that the antibiotic-Cu(2+) complexes are guided to the ribozyme metal ion binding site(s) presumably displacing the catalytically important metal ion(s). Moreover, we assume that, once positioned in appropriate distances to RNA phosphate groups and bases, the coordinated Cu(2+) ions become positively charged factors that enhance the affinity of the antibiotics to the ribozyme. These observations indicate that coordination of metal ions to antibiotics substantially changes their properties which might also have a biological relevance inside the cell.

The oxidation-promoting reactivity of the Cu(II)-sinefungin complex in the presence of hydrogen peroxide was studied at pH 7.4, using N,N-dimethyl-p-nitrosoaniline (NDMA), as well as plasmid DNA as target molecules. Mixture of the complex with H(2)O(2) was found to be an efficient oxidant, bleaching NDMA solution, and generating single- and double-strand breaks in DNA. The oxidative DNA damage was investigated also in the presence of varying amounts of glutathione, histidine, Gly-Gly-His peptide, H2A histone, and ascorbic acid, showing diverse influence of those substances on the cleavage extension.

Vancomycin, a "last chance" antibiotic, is a glycopeptide consisting of an oligopeptide unit being potentially the effective binder of Cu2+ ions. The potentiometric and spectroscopic studies (UV-Vis, CD, EPR, NMR) have shown that, indeed, the peptide unit binds cupric ions very effectively forming almost instantly the 3N complex involving the N-terminal nitrogen donors in the metal ion coordination. The comparison of the binding ability of vancomycin with other peptide chelators clearly shows the efficiency of this antibiotic in metal ion coordination. It is very likely that Cu2+ ions may play a crucial role in the pharmacology of vancomycin, particularly when administered in high doses.

Potentiometric measurements of ATP-His system proved an existence of five adducts in the solution with stoichiometries ranging from H(5)(ATP)(His) to H(ATP)(His)(4-). Their formation is a consequence of electrostatic interactions only. In the ternary Ni(II)-ATP-His system, two complex species NiH(ATP)(His)(2-) and Ni(ATP)(His)(3-), were found. In the former, stacking interaction between the aromatic moiety of ATP and the imidazole ring of l-histidine is crucial to the adduct stability. All studied systems are able to generate single strand lesions of plasmid DNA in the presence of hydrogen peroxide. However, only binary systems produce linear form of DNA, which is a consequence of the accumulation of the single-stranded breaks.

Redox properties of Cu(II) complexes of the terminally blocked hexapeptide -TESHHK- and a series of its alanine substituted analogs: -TASHHK-, -TEAHHK-, -TESAHK-, -TESHAK-, were investigated in their reactions with hydrogen peroxide in solution and by cyclic voltammetry in a broad range of pH. The formation of reactive oxygen species was followed with the use of spectrophotometric indicators, NDMA and NBT. The results indicate that the ability of these complexes to generate hydroxyl-like radicals correlates with the formation of active Cu(III) complexes resulting from the oxidation of Cu(II) by H2O2, which interact with further H2O2 molecules specifically.

This communication reports the characteristics of the mechanism of highly specific tRNA(Phe) cleavage, which occurs in the anticodon loop in the presence of aminoglycoside antibiotic-neomycin B. The data prove that the cleavage requires previous depurination of the polynucleotide chain at position 37, which is occupied by a hypermodified guanine base-wybutine. The results suggest that the phenomenon, previously considered as selective with respect to the presence of tRNA hypermodification, may concern far more RNA molecules, namely the ones carrying abasic sites.

The formation of binary Ni(II) complexes with 2'-deoxyguanosine 5'-triphosphate (dGTP, L) as well as ternary complexes thereof with L-histidine (His, A) was studied with the use of potentiometry and electronic absorption spectroscopy. In the binary and ternary systems, the complexes with stoichiometries NiH2L-, NiHL2-, NiL3- and NiH2LA2-, NiHLA3-, NiLA4- respectively, were detected. The ternary complexes are very stable at pH 7.4 and thus may constitute biologically relevant Ni(II) carriers in the cell. In the presence of hydrogen peroxide, the binary and ternary systems both generate hydroxyl radical-like species and undergo dGTP degradation with the formation of the 8-oxo-dGTP intermediate. The latter, along with dGTP complexation and degradation, may lead to mutagenesis and carcinogenesis due to base-mispairing properties of 8-oxoguanine and the disturbance in the physiological balance among the four canonical triphosphodeoxynucleotide substrates for DNA synthesis.

Actinomycin D (AD) is a potent anticancer drug widely applied in therapy, which however exhibits very high toxicity in humans. As the character of donors present in the AD molecule seems to be very favorable for Cu(II) ions, we undertook the coordination study on the Cu(II)-AD system. Potentiometric experiments proved a formation of very stable complexes and with the use of spectroscopic methods the identification of the binding sites was made. The values of potential energy minima, provided by theoretical modeling, confirmed the feasibility of formation of the complexes in water solution. We also demonstrated a significant effect of Cu(II) ions on AD interactions with DNA. The strand-nicking activity was observed. This process could be correlated with the speciation of complex forms. We also found out that in the presence of H2O2, low levels of Cu(II)-AD complexes induce the formation of considerable amounts of linearised plasmid. In consequence, the hypothesis is proposed that the physiologically available cupric ions may participate in the drug-induced toxic effects.

The formation of binary and ternary complexes of Ni(II) with two biologically relevant molecules, 2'-deoxyguanosine 5'-monophosphate (dGMP) and l-histidine (histidine or His) was characterized by potentiometry and UV-visible spectroscopy. For dGMP, the mononuclear complexes with stoichiometries NiH(2)L(+), NiHL and NiL(-) were found. In the mixed system the ternary complexes NiH(2)LA, NiHLA(-) and NiLA(2-) were detected. In binary systems, the Ni(II) ion coordinates to dGMP through the N-7 atom of its purine ring and indirectly through a water molecule bonded to the phosphate group, while in ternary complexes Ni(II) is bonded to all three histidine donors and directly to the phosphate group of dGMP. Both binary and ternary complexes are susceptible to oxidation by H(2)O(2), with the increased formation of 8-oxo-dGMP in the ternary system. The toxicological relevance of these findings stems from possible disturbance by the major biological Ni(II)-His complex of the nucleotide pools homeostasis through the formation of ternary species and oxidation promotion, as well as from 8-oxo-dGMP capacity to inhibit enzymatic elimination of promutagenic oxidized nucleotides from such pools.

The oxidative DNA damage by the cupric complexes of sisomicin was investigated in the presence of varying amounts of histidine, ATP, Mg(II) ions or phosphates. We found that by very low concentrations, the amino acid is able to inhibit the cleavage totally. This occurs both by its competition with antibiotic for copper(II) binding, what was proved by spectroscopic measurements, as well as by ROS scavenging by the imidazole ring. ATP and magnesium also exert an influence on the yield of the DNA destruction by decreasing the amount of the single strand breaks, however only their significant excess is able to break this process. The influence of ATP on the plasmid damage has in this case a similar chemical mechanism to that one observed for histidine. Mg(II) ions, however, interact with DNA and thus prevent the complex binding. Only phosphate anions, in the range of their physiological concentrations, exert no influence on the cleavage process.

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.