Speciation of the complexes of zinc(II) with a dodecapeptide (Ac-SCPGDQGSDCSI-

Several metal-based carbon monoxide-releasing molecules (CORMs) are active CO donors with established antibacterial activity. Among them, CORM conjugates with azole antibiotics of type [Mn(CO) 3 (2,2′-bipyridyl)(azole)] + display important synergies against several microbes. We carried out a structure−activity relationship study based upon the lead structure of [Mn(CO) 3 (Bpy)(Ctz)] + by producing clotrimazole (Ctz) conjugates with varying metal and ligands. We concluded that the nature of the bidentate ligand strongly influences the bactericidal activity, with the substitution of bipyridyl by small bicyclic ligands leading to highly active clotrimazole conjugates. On the contrary, the metal did not influence the activity. We found that conjugate [Re(CO) 3 (Bpy)(Ctz)] + is more than the sum of its parts: while precursor [Re(CO) 3 (Bpy)Br] has no antibacterial activity and clotrimazole shows only moderate minimal inhibitory concentrations, the potency of [Re(CO) 3 (Bpy)(Ctz)] + is one order of magnitude higher than that of clotrimazole, and the spectrum of bacterial target species includes Gram-positive and Gram-negative bacteria. The addition of [Re(CO) 3 (Bpy)(Ctz)] + to Staphylococcus aureus causes a general impact on the membrane topology, has inhibitory effects on peptidoglycan biosynthesis, and affects energy functions. The mechanism of action of this kind of CORM conjugates involves a sequence of events initiated by membrane insertion, followed by membrane disorganization, inhibition of peptidoglycan synthesis, CO release, and break down of the membrane potential. These results suggest that conjugation of CORMs to known antibiotics may produce useful structures with synergistic effects that increase the conjugate's activity relative to that of the antibiotic alone.

The essential Cu(i) and the toxic Hg(ii) ions possess similar coordination properties, and therefore, similar cysteine rich proteins participate in the control of their intracellular concentration. In this work we present the metal binding properties of linear and cyclic model peptides incorporating the three-cysteine motifs, CxCxxC or CxCxC, found in metallothioneins. Cu(i) binding to the series of peptides at physiological pH revealed to be rather complicated, with the formation of mixtures of polymetallic species. In contrast, the Hg(ii) complexes display well-defined structures with spectroscopic features characteristic for a HgS2 and HgS3 coordination mode at pH = 2.0 and 7.4, respectively. Stability data reflect a ca. 20 orders of magnitude larger affinity of the peptides for Hg(ii) (log βpH7.4HgP ≈ 41) than for Cu(i) (log βpH7.4CuP ≈ 18). The different behaviour with the two metal ions demonstrates that the use of Hg(ii) as a probe for Cu(i), coordinated by thiolate ligands in water, may not always be fully appropriate.

Copper(I) binding features of oligopeptides, mimicking the metal binding loops (MBL) of the bacterial copper efflux regulator protein CueR, were investigated with an aim of exploring potential candidates for Cu(I)-sequestration. Two of the studied ligands comprise the MBL of E. coli CueR (Ac-ACPGDDSADCPI-NH 2 ; EC) and V. cholerae CueR (Ac-SCPGDQGSDCPI-NH 2 ; VC) while the third peptide is a His-containing variant of VC (Ac-SCHGDQGSDCSI-NH 2 ; HS). UV-titrations of the ligands by Cu(I) at pH = 7.4 and monitoring the characteristic S Cys − → Cu(I) charge transfer bands, together with CD-experiments, indicated the exclusive formation of monomeric Cu(I)-complexes (CuL) up to a 1:1 Cu(I):L ratio and the presence of oligometallic/ cluster type complexes at Cu(I)-excess. Such a change in speciation and the domination of the latter species was also supported by comparing the ESI-MS spectra obtained at 0.9:1 and 2:1 Cu(I):peptide ratios. pH-dependence of the UV spectra of the Cu(I):peptide 1:1 complexes reflected the formation of the two thiolate-Cu(I) bonds well below neutral pH, as two successive deprotonation processes could be fitted by apparent pK a values falling in the pH range of 1.8-3.0 and 4.3-5.5. Cu(I)-binding affinity of the ligands was determined by competition experiments applying the bicinchoninate ion for the displacement of the Cu(I)-ligated peptides. The obtained conditional stability constants (= 15.3, 15.8 and 16.3 for Cu(EC), Cu(VC) and Cu(HS), respectively) indicate a strong affinity of Cu(I) to each ligand and suggest a slight destabilizing effect of charge repulsion between sidechain groups in EC while a possible Cu(I)-coordination of the histidine residue in HS.