Aqueous Solution Equilibria and Spectral Features of Copper Complexes with Tripeptides Containing Glycine or Sarcosine and Leucine or Phenylalanine

Copper(II) complexes of glycyl-L-leucyl-L-histidine (GLH), sarcosyl-L-leucyl-L-histidine (Sar-LH), glycyl-L-phenylalanyl-L-histidine (GFH) and sarcosyl-L-phenylalanyl-L-histidine (Sar-FH) have potential anti-inflammatory activity, which can help to alleviate the symptoms associated with rheumatoid arthritis (RA). From pH 2–11, the MLH, ML, MLH-1 and MLH-2 species formed. The combination of species for each ligand was different, except at the physiological pH, where CuLH-2 predominated for all ligands. The prevalence of this species was supported by EPR, ultraviolet-visible spectrophotometry, and mass spectrometry, which suggested a square planar CuN4 coordination. All ligands have the same basicity for the amine and imidazole-N, but the methyl group of sarcosine decreased the stability of MLH and MLH-2 by 0.1–0.34 and 0.46–0.48 log units, respectively. Phenylalanine increased the stability of MLH and MLH-2 by 0.05–0.29 and 1.19–1.21 log units, respectively. For all ligands, 1H NMR identified two coordination modes for MLH, where copper(II) coordinates via the amine-N and neighboring carbonyl-O, as well as via the imidazole-N and carboxyl-O. EPR spectroscopy identified the MLH, ML and MLH-2 species for Cu-Sar-LH and suggested a CuN2O2 chromophore for ML. DFT calculations with water as a solvent confirmed the proposed coordination modes of each species at the B3LYP level combined with 6-31++G**

Applications of CW and pulsed EPR spectroscopy for the characterization of copper(II) complex stereochemistry and of Beta-peptide secondary structure

Electron Paramagnetic Resonance (EPR) is a spectroscopic technique, based on the magnetic resonance principles, that allows the characterization of systems containing unpaired electrons to achieve information on their chemical environment with high resolution. Considering the peculiar class of samples that can be investigated, nowadays, EPR spectroscopy finds applications in several areas of science. Thanks to the modern Site-Directed Spin Labelling (SDSL) approaches, it is possible to introduce spin labels into defined positions of a natural diamagnetic system making it detectable by EPR. This method has further extended the applications towards samples which are naturally EPR silent. The first part of the thesis offers an overview of the main theoretical key concepts required to understand the set-up and the outcomes of an EPR experiment. In the second part of the thesis CW-EPR spectroscopy is employing to characterize the geometries adopted in aqueous solution by some copper(II) complexes with important biological ligands. The study of biochemical processes, in fact, cannot be performed neglecting the inorganic biometals dissolved in biological fluids. These metal ions are involved in the cell biochemistry coordinated by several biomolecules forming metal complexes which are the real players with specific biological activities. The functions of these systems are strictly related with the arrangement of the ligands around the metal centre and with the overall geometry of the complex. The experimental results presented in this part of the thesis enable to develop a more detailed picture of these copper(II) species in solution in order to better clarify their structure-function relationships for further biochemical considerations about their role. Additionally, voltammetric measurements are performed on the same systems to support the spectroscopic data. In the third part of the thesis, the results of a project developed in the Electron Spin Resonance research group at the Max Planck Institute for Biophysical Chemistry (Göttingen Germany), under the supervision of Professor Marina Bennati, is presented. It is well-known that the structural characterization of membrane proteins in their natural environment is a challenging task. EPR spectroscopy in combination with SDSL approaches is emerging as a powerful biophysical tool to reveal biomolecular structural information at atomic resolution. In particular, Pulsed Electron Double Resonance (PELDOR) spectroscopy, called also Double Electron Electron Resonance (DEER), is a pulsed EPR method which enables to detect distances between two paramagnetic centres in a biological system in order to characterize its structure. Measuring the dipolar coupling between two unpaired electrons, PELDOR allows to probe their intramolecular distances with high resolution and reliability. In this project, CW-EPR and PELDOR/DEER spectroscopy are employed for the structural characterization of a transmembrane peptide in solution and in a lipid environment. The samples are prepared introducing two semi-rigid TOPP nitroxide spin labels into the peptide s backbone in order to make it detectable by EPR. The experimental results of this part of the thesis demonstrate the great potential of EPR spectroscopy in structural biology to characterize biomolecular structures and encourage the employment of TOPP spin label as useful tool for the EPR investigation of peptides foldamers in solution and in lipid bilayer