Solvation effects of ionic liquid/water mixtures on biomolecules

Ionic liquids (ILs) belong to a broad class of ionic compounds that, differently from conventional salts, are usually liquid at T < 100°C. They are characterized by vanishing vapour pressure, good thermal stability, high ion density and ionic conductivity. Thanks to the large variety of available ions, the physico-chemical properties of ILs can be modulated by careful selection of both cation and anion with specic characteristics for tailored applications. A more convenient strategy for an ecient tuning of the performances of ILs consists in mixing ILs with other ionic or molecular liquids, such as e.g. water. It is observed that addition of water to ILs allows to improve some of their properties and performances, especially for applications in biological eld. For instance, recent studies reported on the capability of IL/water solutions to enhance the structural stability of proteins, enzymes and deoxyribonucleic acid (DNA) also at high temperatures. This PhD thesis aims to show the usefulness of synchrotron-based UV Resonance Raman (SR-UVRR) spectroscopy for investigating i) the structural dynamics of IL/water solutions and ii) the solvation efects of these IL-based solvents on bio-molecules, such as peptides and DNA. UVRR exhibits several advantages with respect to conventional spontaneous Raman spectroscopy, as the signicant increment of the detection limit that allows to study the samples in very high diluted conditions and the selective enhancement of the Raman cross section of vibrations associated to specic molecular groups of the same system. Thanks to the unique tunability of the synchrotron emission, the UVRR spectra of aqueous solutions of imidazolium-based ILs have been excited at diferent wavelengths nely matching with the resonance transitions occurring in the system. These spectra showed to have good sensitivity to the modications induced on the local structure of solutions of ILs by i) the change of the anion and ii) the substitution on the imidazolium ring of progressively longer alkyl-chains. Additionally, some UVRR signals are specically informative on the effect induced by addition of water on the strength of hydrogen bonds (H-bonds) in IL-water solutions. The molecular view provided by SR-UVRR experiments has been further complemented by the structural parameters extracted by Small Angle Neutron Scattering (SANS) measurements performed on the same IL/water mixtures. The investigation of the structure-dynamic relationship in IL/water solutions is the preliminary step for the deep comprehension of the effects of these mixtures on the solvation dynamics of molecules of biological interest, such as peptides and DNA. This is an issue of special interest by considering that the solute-solvent interactions are strongly related to the biological activity of bio-macromolecules. Some results will be presented in this thesis, concerning the case of two different type of bio-systems: i) small peptides dissolved in IL/water solutions: the UVRR spectra of peptides contain several spectroscopic markers of the structural rearrangement induced by the hydration shell on peptides, such as the Amide bands that are usually not well detectable in spontaneous Raman spectra. The analysis of these spectral features can provide insights on the peculiar effect induced on the hydration dynamics of peptides by different ILs; ii) DNA dissolved in IL/water solutions: a suitable choice of the exciting radiation allows to collect UVRR spectra of DNA where the vibrational signals associated to the different nitrogenous bases are selectively enhanced. This gives the unique opportunity to disentangle specic bands in the Raman spectra of DNA that appear usually very complex. Such approach can be conveniently used to obtain insights on the molecular mechanism responsible of the different thermal stability exhibited by DNA structure in the presence of different IL/water solutions.

onic liquids (ILs) belong to a broad class of ionic compounds that, differently from conventional salts, are usually liquid at T < 100°C. They are characterized by vanishing vapour pressure, good thermal stability, high ion density and ionic conductivity. Thanks to the large variety of available ions, the physico-chemical properties of ILs can be modulated by careful selection of both cation and anion with specic characteristics for tailored applications. A more convenient strategy for an ecient tuning of the performances of ILs consists in mixing ILs with other ionic or molecular liquids, such as e.g. water. It is observed that addition of water to ILs allows to improve some of their properties and performances, especially for applications in biological eld. For instance, recent studies reported on the capability of IL/water solutions to enhance the structural stability of proteins, enzymes and deoxyribonucleic acid (DNA) also at high temperatures. This PhD thesis aims to show the usefulness of synchrotron-based UV Resonance Raman (SR-UVRR) spectroscopy for investigating i) the structural dynamics of IL/water solutions and ii) the solvation effects of these IL-based solvents on bio-molecules, such as peptides and DNA. UVRR exhibits several advantages with respect to conventional spontaneous Raman spectroscopy, as the signicant increment of the detection limit that allows to study the samples in very high diluted conditions and the selective enhancement of the Raman cross section of vibrations associated to specic molecular groups of the same system. Thanks to the unique tunability of the synchrotron emission, the UVRR spectra of aqueous solutions of imidazolium-based ILs have been excited at different wavelengths nely matching with the resonance transitions occurring in the system. These spectra showed to have good sensitivity to the modications induced on the local structure of solutions of ILs by i) the change of the anion and ii) the substitution on the imidazolium ring of progressively longer alkyl-chains. Additionally, some UVRR signals are specically informative on the effect induced by addition of water on the strength of hydrogen bonds (H-bonds) in IL-water solutions. The molecular view provided by SR-UVRR experiments has been further complemented by the structural parameters extracted by Small Angle Neutron Scattering (SANS) measurements performed on the same IL/water mixtures. The investigation of the structure-dynamic relationship in IL/water solutions is the preliminary step for the deep comprehension of the effects of these mixtures on the solvation dynamics of molecules of biological interest, such as peptides and DNA. This is an issue of special interest by considering that the solute-solvent interactions are strongly related to the biological activity of bio-macromolecules. Some results will be presented in this thesis, concerning the case of two dierent type of bio-systems: i) small peptides dissolved in IL/water solutions: the UVRR spectra of peptides contain several spectroscopic markers of the structural rearrangement induced by the hydration shell on peptides, such as the Amide bands that are usually not well detectable in spontaneous Raman spectra. The analysis of these spectral features can provide insights on the peculiar effect induced on the hydration dynamics of peptides by dfferent ILs; ii) DNA dissolved in IL/water solutions: a suitable choice of the exciting radiation allows to collect UVRR spectra of DNA where the vibrational signals associated to the different nitrogenous bases are selectively enhanced. This gives the unique opportunity to disentangle specic bands in the Raman spectra of DNA that appear usually very complex. Such approach can be conveniently used to obtain insights on the molecular mechanism responsible of the dierent thermal stability exhibited by DNA structure in the presence of different IL/water solutions.