Aguinaldo Souza1 Izabelle Amorin Ferreira Boza1 Valdecir Ximenes1 Maurício Yoguim1 Nelson Morgon2 Ignez Caracelli3

1, Chemistry, Sao Paulo State University - UNESP, Bauru, São Paulo, Brazil
2, Chemistry, Campinas State University, Campinas, São Paulo, Brazil
3, Physics, Federal University at São Carlos, São Carlos, São Paulo, Brazil

Human serum albumin (HSA) plays an important role in the transport of substances with pharmacological properties due to their high plasma concentration and specificity, placing it as the fundamental protein responsible for the pharmacokinetic implication of the drugs. The dansylglycine amino acid derivative (DG) is a fluorescent marker specific for the II site on the HSA. In addition, when binding to the protein, DG acquires chirality, a feature that can also be used to characterize the binding site of new compounds in albumin. This work aimed to elucidate the induction of chirality in the DG by its bonding to the HSA. Experimental Electronic Circular Dichroism (ECD) spectra of DG (100 μM) in the absence or presence of HSA (30 μM) in 50 mM sodium phosphate buffer pH 7.0 were obtained in a Jasco J-815 spectropololarimeter at 25 o C. The spectra were obtained with a resolution of 1 nm and a scanning speed of 50 nm/min. The theoretical ECDs were simulated using the Density Functional Theory (DFT) approach with the hybrid functional B3LYP and CAM-B3LYP in the base set 6-311 ++ G (2d, p) and the implicit Solvation Model based on Density (SMD) for the solvents ethanol, methanol, acetonitrile, water and tetrahydrofuran; the calculations were performed with the Gaussian09 program. The DG-HSA complex formation in buffer resulted in the appearance of a positive ECD spectrum centered at 346 nm. Considering that the ICD signal should arise from a chiral conformation of the DG acquired by binding in the protein, calculations were initially performed to obtain the stable conformations focusing on the inversion of configuration centered on the nitrogen atom: alpha-amino group. However, both configurations: R and S showed similar and positive ECD signals. Calculations of the potential energy surface (PES) of the DG were performed, focusing on the dihedral angles formed by the bonding of the -N(CH3)2 group of the naphthalene ring. The analysis of the various conformations obtained and their respective theoretical ECDs revealed that the 150 ° dihedral (ECD positive centered at 320 nm) presented excellent similarity with the experimental spectrum. On the other hand, the 80° dihedral showed a signal of the ECD spectrum opposite to that observed experimentally. In addition, we observed that the nitrogen atom of the -N(CH3)2 group presented the greatest contribution to the HOMO-LUMO transition that gives rise to the n-pi * electronic transition involved in the generation of the ECD signal. The molecular docking analysis, using the GOLD 5.5 program, of the complexation between DG and HSA revealed a conformation with a dihedral similar (150 °) to that obtained theoretically (DFT) and whose ECD is in agreement with the experimental result. In conclusion, through the study of the possible conformations for dansylglycine and the calculation of its theoretical ECD, it was possible to identify the origin of the ECD signal obtained by the complexation between DG and HSA.