In this study, we aimed to elucidate the molecular interactions in deep eutectic solvents (DESs) and acquire additional experimental evidence to increase our understanding of the molecular mechanisms responsible for the preparation of DES. We investigated three N-methylthiourea (NMTU)-based DESs with choline chloride (ChCl), allyltrimethylammonium chloride (ATMAC), and benzyltriethylammonium chloride (BTEAC) as hydrogen bond acceptors (HBA). To accomplish this, we used both experimental techniques including isopiestic, differential scanning calorimetry (DSC), and FTIR measurements as well as molecular dynamics (MD) simulations. The thermal behaviors of the prepared DESs were investigated by DSC method. In order to study the interaction and hydrogen bonding between the DESs constituents, FT-IR analysis and isopiestic measurements were carried out. Constant solvent activity lines of ternary HBA + HBD + solvent mixtures were determined by the isopiestic technique. The large positive deviation of the isosolvent activity lines from the semi-ideal behavior indicates a strong interaction between HBA and HBD, which is much stronger than HBA-HBA and HBD-HBD interactions. Classical MD simulations were performed at 298.15 K to analyze the nanoscopic properties and structure of the DESs. To investigate the interaction between the components and visualize the three-dimensional structure of the DESs, radial distribution functions (RDFs), coordination numbers (CNs), and combined (CDFs), and spatial (SDFs) distribution functions were determined by MD simulations. The obtained theoretical results confirmed the importance of hydrogen bonds in the preparation of DESs and also showed that these systems exhibit different structural arrangements. MD simulations were also used to determine the density of the DESs and the results showed good agreement with the experimental density.
