Being able to easily determine the Cu(II) affinity for biomolecules of moderate affinity is important. Such biomolecules include amyloidogenic peptides, such as the well-known amyloid-β peptide involved in Alzheimer’s disease. Here, we report the synthesis of a new water-soluble ratiometric Cu(II) dye with a moderate affinity (10⁹ M^–1 at pH 7.1) and the characterizations of the Cu(II) corresponding complex by X-ray crystallography, EPR, and XAS spectroscopic methods. UV–vis competition was performed on the Aβ peptide as well as on a wide series of modified peptides, leading to an affinity value of 1.6 × 10⁹ M^–1 at pH 7.1 for the Aβ peptide and to a coordination model for the Cu(II) site within the Aβ peptide that agrees with the one mostly accepted currently.

The thermodynamic stability of the metallacrown complexes formed by picolinehydroxamic acid (Picha) with Cu(II), Ni(II) and Zn(II) in aqueous solution has been determined by potentiometry, and the speciation models were validated by ESI-MS and UV-visible spectrophotometry. Cu(II) and Zn(II) form 12-MC-4 species as the unique metallacrowns present in the solution. While for Cu(II) the 12-MC-4 is slightly less stable than that obtained with alaninehydroxamic acid (Alaha), the opposite was found for Zn(II). Moreover, with Cu(II) unprecedented 15-MC-5 and 18-MC-6 species were identified under ESI-MS conditions. Picha with Ni(II) forms, in contrast, a 15-MC-5 complex as a unique metallacrown species. Structural studies of the framework of the 12-MC-4 complexes by ab initio methods were also carried out. The results of our investigations allowed us to rationalize not only the different behaviour of Picha in the formation of metallacrowns with the three metal ions, but also the reasons which underpin the strategies for stabilization of these species reported in the literature using ancillary ligands such as pyridine.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes’ first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d⁷ electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H₂O/OH⁻)]_N(TRIL2WL23H)₃ⁿ⁺ that behaves similarly to native Co(II) substituted human‐CAs. Like the Zn(II) analogue, the cobalt‐derivative at slightly basic pH is incapable of hydrolyzing p‐nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH⁻)]_N(TRIL2WL23H)₃⁺ peptide complex. X‐ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5‐coordinate species by pH 11. UV‐vis spectroscopy can monitor this transition, giving a pK_a for the conversion of 10.3. We assign this conversion to the formation of a 5‐coordinate Co(II)(N_imid)₃(OH)(H₂O) species. The pH dependent kinetic analysis indicates the maximal rate (k_cat), and thus the catalytic efficiency (k_cat/K_m), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

ROS (i.e., reactive oxygen species) scavenging is a key function of various Mn-based enzymes, including superoxide dismutases (SODs) and catalases, which are actively linked to oxidative stress-related diseases. In this study, we synthesized and characterized two novel Mn(III)-based synzymes (i.e., synthetic enzymes), designated C1 ([MnL1Cl(H₂O)]Cl·3H₂O) and C2 ([MnL2Cl₂]·2H₂O), which differ in the presence of a bridging aliphatic or aromatic group in the chelator. Using a range of analytical techniques, we found that the aromatic C2 bridge significantly influences the Mn(III) center’s cis-β configuration, unlike C1, which adopts a trans configuration. We then thoroughly evaluated the oxidation-reduction properties of C1 and C2, including their redox potentials (by cyclic voltammetry) and capacity to consume various ROS species (using DPPH, hydroxyl radical, hydrogen peroxide, and superoxide UV–visible spectrophotometric assays). The specific kinetics of the H₂O₂ dismutation process, as measured by a Clark-type electrode and time-resolved ESI-MS, revealed that both synzymes possess catalytic activity. Toxicological experiments using the Galleria mellonella larval model demonstrated the compounds’ innocuous nature towards higher eukaryotic organisms, while cytotoxicity assays confirmed their selective efficacy against lung cancer cells. Additional cytological assays, such as the thiobarbituric acid reactive substances assay and caspase-3 activity and p53 expression analysis, reported that C1 and C2 induce cytotoxicity against cancer cells via apoptosis rather than necrosis and behave very differently towards redox substances and ROS-regulating enzymes in vivo. These findings suggest that the structural differences between C1 and C2 lead to distinct redox properties and biological activities, highlighting the potential of these novel Mn(III)-based synzymes as therapeutic agents for the treatment of oxidative stress-related diseases, particularly lung cancer. Further studies are warranted to elucidate the underlying mechanisms of action and explore their clinical applications.