Phytosphingosine and Sphingosine Ceramide Headgroup Hydrogen Bonding: Structural Insights through Thermotropic Hydrogen/Deuterium Exchange

Abstract

IR spectroscopic studies are reported for the phytosphingosine class of ceramides and are compared with two analogous sphingosine ceramides. The phytosphingosine class of molecules, not previously widely investigated by physical techniques, constitutes ∼30% of the total ceramide content of the stratum corneum, the barrier to permeability in skin. The current measurements utilize temperature-controlled horizontal attenuated total reflectance spectroscopy of hydrated films to study H → D exchange in the polar regions of the molecules as well as chain conformational order and packing properties. Analysis of the methylene stretching and scissoring vibrations reveals that the chains of the two phytosphingosine derivatives (ceramides 3 and 7) are much more poorly packed at room temperature than their sphingosine counterparts (ceramides 2 and 5 respectively), despite having order → disorder transitions some 15−20 degrees higher. This unanticipated relative stability of the phytosphingosines is traced to enhanced headgroup H-bonding interactions manifest by lower Amide I and higher Amide II frequencies. Water penetration into the polar regions is monitored by the temperature dependence of the Amide II and O−H/N−H stretching intensities as a function of H→D exchange. Neither ceramide 2 nor 3 exchanges N−H or O−H protons until relatively high temperatures (>65 °C). However, addition of an α-hydroxy group on the fatty acid chain in ceramides 5 or 7 results in exchange events observed at temperatures much closer to physiological. These measurements reveal that the relative contributions of chain packing and H-bonding under physiological conditions differ markedly for the phytosphingosines compared to the sphingosines. The former are characterized by hexagonal chain packing with relatively strong H-bonding; the latter by orthorhombic chain packing and weaker H-bonding. The implications of these molecular structure data for lipid organization in the stratum corneum are briefly discussed.