Exploring metal ion sequestering abilities of carnosine and carnosine derivatives in aqueous solutions for analytical applications

L-carnosine is drawn attention as a bioactive dipeptide and has been extensively studied during the last years for its promising benefits for human health and employed in several research fields, such as medicine, cosmetics, nutraceuticals and food additives. Albeit there is no clear evidence in the literature on the specific physiological roles and mechanisms of action of carnosine, its bioactivity seems to be strictly dependent on its own metal ion coordination ability. The antioxidant effect of carnosine could also be attributed to its high hydrophilicity and enhanced by its chelating properties, as it interacts with most bivalent and transition metal cations, such as Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Ru2+ and Cd2+. Advantageously, carnosine is also a heavy metal chelator. Therefore, this thesis focuses on this ambiguous dipeptide highlighting and exploiting its chelating abilities toward metal cations, with the express purpose of using this property for analytical applications. However, it is well known that knowledge of the total metal concentration does not sufficiently explain the effect or impact of an element in a multicomponent system and, on the other hand, a speciation study performed on a ligand, as carnosine, aims to define the different forms under which it is present in natural systems. The starting point was an in-depth speciation study on carnosine in NaCl aqueous solutions. The study was carried out under different conditions of ionic strength (0.15 ≤ I/mol L-1 ≤ 1) and temperature (288.15 ≤ T/K ≤ 310.15) by potentiometry, UV-Vis spectrophotometry and 1H NMR spectroscopy. Experimental data and literature ones were combined to obtain a stanch speciation model and trustworthy values of protonation equilibria. In addition, structural information and fragmentation pathways of carnosine were explored by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS) and tandem Mass Spectrometry (MS/MS) techniques. Quantum-mechanical calculations were also performed to appreciate the protonation capabilities of functional groups (Chapter 3). Once the acid-base properties of the dipeptide were defined, thermodynamic interactions parameters with bivalent metal cations (M2+) were investigated, together with their dependence on ionic strength and temperature. For this purpose, a potentiometric study was performed on Ca2+, Mg2+, Mn2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+-carnosine systems. Determination of the thermodynamic interaction parameters (logβ, ΔG, ΔH, TΔS) regarding the M2+-carnosine complexes was very useful for predictive purposes and allowed to simulate the distribution of the species in different real systems. Furthermore, sequestering ability of carnosine toward the metal cations under study was assessed in conditions simulating real systems, such as physiological (I = 0.15 mol L-1 and pH = 7.4) and seawater (I = 0.7 mol L-1 and pH = 8.1) solutions (Chapter 4). During pandemic COVID-19, literature research on dipeptide electroactivity intrigued me. However, the literature data related to the voltammetric detection of carnosine are not well detailed and currently there is no clear evidence on its electrochemical mechanisms. Therefore, several tests on carnosine by voltammetry were performed during my research stay at the Department of Chemical Engineering of the Universitat Rovira i Virgili (Tarragona, Spain). This investigation was mainly performed using bare Screen-Printed Carbon Electrodes (SPCEs) and modifying them with suitable materials in order to improve electrochemical properties of carnosine. Although this study did not provide reproducible data neither on bare SPCEs nor on modified ones, it inputs the idea of synthetizing more electroactive carnosine derivatives and studying the sensing capability toward metal cations. Functionalization of carnosine was readily achievable as it has multiple recognition groups, such as the imidazole ring, amino and carboxylic groups, which pose itself as an ideal candidate for modification with more versatile molecular units. Synthetic approaches include: 1) Conjugation of carnosine (CAR) with ferrocene (Fc). This strategy is well exploited in the literature as it leads to redox-active materials with interesting electron transfer properties (Chapter 5). 2) Conjugation of carnosine (CAR) with Pyrene (Py). The derivative, PyCAR, has been designed as a possible fluorescent metal sensor (Chapter 5). Analytical approach on FcCAR and PyCAR derivatives included: i) Determination of the acid-base behavior by potentiometry and UV-Vis spectrophotometry in NaCl aqueous solutions at I = 0.15 mol L-1 and T = 298.15 K. ii) Determination of the complexing ability toward some bivalent metal cations. FcCAR has been therefore investigated as a candidate for electrochemically recognizing metal cations and, thus, as a potential metal probe. Among the cations, FcCAR showed a greater affinity toward Hg2+ and Hg2+-FcCAR system was used as model to study how to improve electrochemical performance. Therefore, MultiWalled Carbon NanoTubes Modified with Cyclodextrins (MWCNT-CD) dispersions were cast on Screen-Printed Carbon Electrodes (SPCEs). In this way, an amplification of the peak current signals was achieved (Chapter 5). Study on PyCAR using is still ongoing. 3) Grafting of carnosine on a commercial polyacrylamide/azlactone copolymer (AZ) and study of the resulting carnosine-based resin, AZCAR, in the Pb2+ removal procedures under conditions simulating natural fluids. In this case, preliminary speciation study on the Pb2+-CAR system was helpful in establishing the best conditions of pH, ionic strength and temperature for adsorption experiments. The adsorption capacity of AZCAR toward Pb2+ was studied under conditions simulating the ionic strength and pH of various natural waters, and the best results were obtained at pH > 7.0 and 0.001 ≤ I/ mol L-1 ≤ 0.7 (Chapter 6). Additional studies on further analytical applications have already been undertaken in this thesis but some aspects remain to be evaluated.