S-Ribosylhomocysteine Analogues Modified at the Ribosyl C-4 Position.

4-C-Alkyl/aryl-S-ribosylhomocysteine (SRH) analogues were prepared by coupling of homocysteine with 4-substituted ribofuranose derivatives. The diastereoselective incorporation of the methyl substituent into the 4 position of the ribose ring was accomplished by addition of methylmagnesium bromide to the protected ribitol-4-ulose yielding the 4-C-methylribitol in 85% yield as single 4R diastereomer. The 4-C hexyl, octyl, vinyl, and aryl ribitols were prepared analogously. Chelation controlled addition of a carbanion to ketones from the (Si-face) was responsible for the observed stereochemical outcome. Oxidation of the primary alcohol of the 4-C ribitols with the catalytic amount of tetrapropylammonium perruthenate in the presence of N-methylmorpholine N-oxide produced 4-C-alkylribono-1,4-lactones in high yields. Mesylation of the latter compounds at the 5-hydroxyl position and treatment with a protected homocysteine thiolate afforded protected 4-C-alkyl/aryl-SRH analogues as the lactones. Reduction with lithium triethylborohydride and successive global deprotections with TFA afforded 4-C-alkyl/aryl SRH analogues. These analogues might impede the S-ribosylhomocysteinase(LuxS)-catalyzed reaction by preventing ß-elimination of a homocysteine molecule, and thus depleting the production of quorum sensing signaling molecule AI-2.

Activity of fluconazole and its Cu(II) complex towards Candida species.

Candida species, although they are present as commensal organisms in the digestive tract of healthy individuals, can produce a broad spectrum of serious illnesses in compromised hosts. Fluconazole, a water-soluble triazole with bioavailability greater than 90 %, has been extensively used to treat a wide range of Candida infections. However, a growing resistance of microorganisms in the treatment leads to the discovery of new drugs or modifications of existing ones. The aim of the present study was to investigate whether coordination of Cu(II) ions to fluconazole affects its antifungal activity. The in vitro susceptibility tests and antifungal studies were performed with two Candida spp.: Candidaglabrata and Candida albicans. Overall, 34 strains of the former and 16 strains of the latter were treated with fluconazole, its Cu(II) complex and free Cu(II) ions. The obtained MIC values in 16 cases of the C. glabrata and in 5 cases of the C. albicans were lower for the complex in comparison to the drug. This implies that the complex is more effective against particular strains than the parent drug. The most significant improvement in the complex drug efficacy was observed for fluconazole-resistant species.

Interaction of methotrexate, an anticancer agent, with copper(II) ions: coordination pattern, DNA-cleaving properties and cytotoxic studies.

The acid-base properties and the Cu(II) binding processes of methotrexate (MTX) were characterized by selected spectroscopic techniques and potentiometric measurements. The pH titration data showed that MTX behaves as a triprotic ligand. The deprotonation constants were determined for α-COOH and γ-COOH groups and (N1)H+ from the pteridine ring. Taking all the obtained results into consideration, a coordination pattern was proposed. The DNA-cleaving activity and reactive oxygen species (ROS) generation were investigated for both MTX and the Cu(II)-MTX system. The complex displayed a promising nuclease activity toward plasmid DNA in the presence of hydrogen peroxide. Interestingly, the induction of ROS, such as hydroxyl radicals, superoxide anions or singlet oxygen, was excluded and a different mechanism of DNA degradation was proposed. As MTX is now commonly used in anticancer therapy i.e. against lung cancer, basic cell-based studies were carried out to establish if its Cu(II) complex exhibits higher cytotoxic properties than the ligand alone. Activities of both compounds were also tested against colon carcinoma. Moreover, the determined values of IC50 were confronted with the cytotoxic activity of cisplatin.

Acid-base characterization, coordination properties towards copper(II) ions and DNA interaction studies of ribavirin, an antiviral drug.

We have studied processes of copper(II) ion binding by ribavirin, an antiviral agent used in treating hepatitis C, which is accompanied usually by an increased copper level in the serum and liver tissue. Protonation equilibria and Cu(II) binding were investigated using the UV-visible, EPR and NMR spectroscopic techniques as well as the DFT (density functional theory) calculations. The spectroscopic data suggest that the first complex is formed in the water solution at pH as low as 0.5. In this compound Cu(II) ion is bound to one of the nitrogen atoms from the triazole ring. Above pH6.0, the metal ion is surrounded by two nitrogen and two oxygen atoms from two ligand molecules. The DFT calculations allowed to determine the exact structure of this complex. We found that in the lowest energy isomer two molecules of the ligand coordinate via O and N4 atoms in trans positions. The hypothetical oxidative properties of the investigated system were also examined. It proved not to generate plasmid DNA scission products. However, the calf thymus (CT)-DNA binding studies showed that it reacts with ribavirin and its cupric complex. Moreover, the interaction with the complex is much more efficient.

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids.

BACKGROUND: Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA. METHODS: In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5' ends with (32)P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined. RESULTS AND CONCLUSIONS: Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg(2+), Mn(2+) or Zn(2+) are absolutely necessary. GENERAL SIGNIFICANCE: The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.

Revised coordination model and stability constants of Cu(II) complexes of tris buffer.

2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krezel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50µM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Mapping the interactions of selected antibiotics and their Cu2+ complexes with the antigenomic δ ribozyme.

The interactions of selected antibiotics with the trans-acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all-RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb(2+) -induced cleavage, nuclease digestion, and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu(2+) ions were bound to the same ribozyme regions, but more effectively, showing lower Kd values. On the other hand, the Cu(2+) complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double-stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all-RNA and deoxycytidine-modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.

Selective control of Cu(II) complex stability in histidine peptides by ß-alanine.

The cooperativity of formation of 5-membered and 6-membered chelate rings is the driving force for specificity and selectivity in Cu(II) peptidic complexes. α-Amino acids enable the formation of 5-membered rings, while a 6-membered ring is provided by the coordination of the His side chain imidazole. Introduction of ß-alanine is another way of creating a 6-membered ring in the Cu(II) complex. The potentiometric and spectroscopic (UV-vis and CD) study of Cu(II) complexation by a series of four peptides, AAH-am, ABH-am, BAH-am, and BBH-am (where B stands for ß-alanine, and -am for C-terminal amide) revealed a very strong effect of the sizes of individual rings, with the order of complex stability AAH-am (5,5,6)>BAH-am (6,5,6)>ABH-am (5,6,6)≫BBH-am (6,6,6). The stabilities of ABH-am and BAH-am complexes are intermediate between those of strong His-3 peptides but these complexes are still able to saturate the coordination sphere of the Cu(II) ion at neutral pH. This fact opens up new possibilities in engineering specific peptide-based chelates.

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics.

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.

Di-µ-iodido-bis-[(dimethyl 2,2'-biquinoline-4,4'-dicarboxyl-ate-κ(2)N,N')copper(I)].

In the centrosymmetric dinuclear title complex, [Cu(2)I(2)(C(22)H(16)N(2)O(4))(2)], the Cu(I) atom is coordinated in a distorted tetra-hedral geometry by an N,N'-bidentate dimethyl 2,2'-biquinoline-4,4'-dicarboxyl-ate ligand and two symmetry-related I atoms, which act as bridges to a symmetry-related Cu(I) atom. The distance between the Cu(I) atoms within the dinuclear unit is 2.6723 (11) Å.

The Cu(II)-fluconazole complex revisited. Part I: Structural characteristics of the system.

Protonation equilibria and Cu(II) binding processes by an antifungal agent fluconazole, α-(2,4-difluorophenyl)-α-(1H-1,2,4-triazol-1-yl-methyl)-1H-1,2,4-triazole-1-ethanol, were studied using the UV-Vis, EPR and NMR spectroscopic techniques. The protonation constant of fluconazole was determined from NMR titration and attributed to N4' nitrogen atoms using the DFT methods. The spectroscopic data suggest that at pH as low as 0.4 the first complex is formed, in which one or two Cu(II) ions are bound to one of the nitrogen atoms (N4') from triazole rings. Above pH 1.5 each Cu(II) ion is surrounded by two nitrogen atoms (also N4') from two different ligand molecules, forming primary monomeric complexes and above pH=5, both dimeric or oligomeric species occur, which is well registered by the EPR technique. The mixture of Cu(NO(3))(2) with fluconazole in a 1:1 molar ratio in a water (pH=4.5)/ethanol solution gave crystals of [Cu(2)(H(2)O){(C(6)H(3)-2,4-F(2))(CH(2)N(3)C(2)H(2))(2)C-OH}{(C(6)H(3)-2,4-F(2))(CH(2)N(3)C(2)H(2))(2)C-O}(NO(3))](NO(3))(2)·9(H(2)O). This complex is the first example of a cupric 3D polymeric structure with a fluconazole ligand coordinated via both N2' and N4' atoms from the same triazole rings. At higher pH values, we obtained a binuclear complex [Cu(2)(L)(2)(H(2)O)(2)(NO(3))(2)], in which the copper(II) atoms were bridged by the oxygen atoms of the deprotonated OH group of fluconazole. The hypothetical oxidative properties of this system were also examined, however it failed to generate either reactive oxygen species or DNA scission products.

Coordination pattern, solution structure and DNA damage studies of the copper(II) complex with the unusual aminoglycoside antibiotic hygromycin B.

The aminoglycosidic antibiotic hygromycin B presents a peculiar chemical structure, characterized by two sugar rings joined via a spiro connection. The Cu(ii) complex of hygromycin B in water solution was characterized by (1)H-NMR, UV-Vis, EPR and CD spectroscopy, combined with potentiometric measurements. The spin-lattice relaxation enhancements were interpreted by the Solomon-Bloembergen-Morgan theory, allowing us to calculate copper-proton distances that were used to build a model of the complex by molecular mechanics and dynamics calculations. The fidelity of the proposed molecular model was checked by ROESY maps. Moreover DNA damage by the Cu(ii)-hygromycin B system was also investigated, showing single and double strand scissions exerted by the complex at concentrations in the range 1-5 mM. Addition of either hydrogen peroxide or ascorbic acid to each sample resulted in the shift of the cleavage potency towards lower concentrations of the complex.