We explore the influence of the two osmolytes ectoine and hydroxyectoine on the structure of pure
water and aqueous NaCl solutions using non-resonant X-ray Raman scattering spectroscopy at the
oxygen K-edge. Both ectoine and hydroxyectoine are naturally occurring organic osmolytes synthesized
by halophilic organisms that live in high-salt and other extreme environments. We find that X-ray
spectroscopic data at the oxygen K-edge are consistent with a scenario where both osmolytes affect
the hydrogen bonding network of water on a local scale to in effect increase tetrahedral order. This
supports the proposed stabilizing mechanism of the osmolytes for proteins: preferential exclusion of the
osmolytes from the proteins’ surface and preferential hydration of the macromolecules instead of complex alterations to the structure of water on a bulk scale. The effect of NaCl on water, a disruption
of hydrogen bonds and tetrahedral order, acts in opposition to the localized water-binding effects of ectoine and hydroxyectoine. For ternary mixtures of osmolyte in the presence of NaCl, the effects seen
in the spectra are found to be additive such that the mixed solutes generate a level of oppositional frustration in the water network.