Design and redox properties characterization of metal/tetrabranched peptide adducts as possible superoxide dismutase mimics

The alteration in the human physiological redox balance can lead to the increase of reactive oxygen species (ROS), that results in oxidative stress condition, which can cause molecular damage and tissue injury. The protection from ROS naturally occurs by processes mediated by superoxide dismutases (SODs) and catalases (CATs). To reduce oxidative stress damage exogenous SOD and CAT have been used as therapeutic agents [1], although with limited success [2]. Their use is limited by some restrictions, such as low thermal and chemical stability, low adaptation to extreme environments, and expensive preparation and purification processes [3]. To overcome these limitations, investigations have been directed to the design of low molecular-weight antioxidant catalyst. Enzyme mimics (EMs) usually replicate the binding and catalytic activities of natural enzymes, employing: i) metal complexes with comparable catalytic properties, ii) oligopeptides that reproduce the active site structure of naturals enzymes [4]. In this work both strategies were integrated to produce a EMs based on a synthetic tetrabranched structure composed by a scaffold (PWT2 – based on cyclam (1,4,8,11-tetraazacyclotetradecane)) bound to four oligopeptides (Scheme 1) able to bind metal cations [5,6]. The synthesis of the tetrabranched PWT2-peptides has been achieved by binding four identical peptides on the PWT2 scaffold. The final tetrabranched systems is obtained in solution by reacting the maleimide functionalized scaffold PWT2 with the chosen peptides in a classic thiol-Michael reaction at room temperature and in the presence of a weak base, ensuring environmental and economical sustainable synthesis conditions. The redox behaviour of single peptides and/or tetrabranched systems, and their complexes with Cu, Mn, Ni and V(IV)/V(V), have been characterized by means of cyclic voltammetry with the aim to evaluate what chemical system can be considered good candidates to become EMs. In order to simulate the redox behaviour of SOD, in fact, the chemical species redox signals must be between the values -0.37 V and 0.71 V vs Ag/AgCl, with an optimal value at 0.16 V vs Ag/AgCl [7]. The CV measurements conditions were optimized. The main efforts were focused in increasing the sensitivity of the technique and reducing the needed sample volumes. Solid and screen printed electrodes were tested: Glassy Carbon Electrode, (GCE), Gold Electrode, (AuE), Gold Screen Printed Electrode (AuSPE), Carbon Screen Printed Electrode (CSPE), and Multi Wall Carbon Nano Tubes – Carbon Screen Printed Electrode (MWCNT-CSPE). Voltammetric signals were collected on aqueous solutions with: (i) peptide (1.0 mmol L-1), (ii) metal cation 1.0 mmol L-1 and peptide 1.0 mmol L-1; (iii) metal cation and tetrabranched systems 1.0 mmol L-1. Tetrabranched systems were tested at different metal-to-ligand ratios. The solutions were prepared in NaCl 0.2 mol L-1 at pH 7.3. MOPS (3-(N-morpholino)propanesulfonic acid) was used as pH buffer. Since with the screen-printed electrodes it was possible to work with low amount of solution, they resulted to be particularly suitable to achieve the analytical goal. On the basis of the voltammetric response obtained with CSPE a priority level was assigned to each metal-peptides or tetrabranched system. The voltammetric parameters taken into account to assign the priority are the reversibility of the observed electrochemical process and the potential value related to this process. On the basis of the experimental results the most promising systems are those involving P1 and P3 peptides with copper and manganese, and the tetrabranched system composed with P3 peptide with copper. Financial support of the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.1 – NextGenerationEU (grant PRIN PNRR 2022-P2022EMY52; CUP F53D23008960001; project title: “EnzyMime – Biocompatible and sustainable enzyme mimics based on metal complexes with peptide derivatives: synthesis, characterization and potential biological applications”) is gratefully acknowledged.