Self-association as a solubility limiting factor of riboflavin in aqueous media

Single crystal X-ray diffraction analysis of needle shaped riboflavin (RF) crystals revealed π-stacking of RF's isoalloxazine units (distance: 3.643-3.313 Å) with syn-orientated ribityl chains. In line with this, classical molecular dynamics (MD) (400 ns) using an isobaric-isothermal (NPT) ensemble of eight RF in a water box (〈V〉 ∼ 508.62 nm³, 〈p〉 = 1.11 bar) revealed anti-aligned aggregation of RF in water (COM-distance: 4 Å). Comparing umbrella sampling for the separation of two RF molecules to the separation of two lumichrome molecules, the similar mean potential force for the separation of RF and lumichrome (22.8 kJ mol^-1; 24.4 kJ mol^-1) proved dispersive interactions as the origin of RF's aggregation. Though stacking of RF is the major water-solubility limiting factor, the conformation of RF's ribityl chain may alter the solubility in water. Both, MD (in water) and COSMO-RS (in water continuum) predicted that conformations of RF with an extended ribityl chain are thermodynamically preferred over any conformations with internal hydrogen bonds between hydroxyl groups and nitrogen/oxygen atoms of the pyrimidine moiety of the flavin ring. Interestingly, COSMO-RS predicted the solubility of the extended conformation to be significantly lower than the latter leading to the very low average solubility of RF. Nuclear Overhauser effect measurements (NOESY) of the structurally related sodium riboflavin 5'-monophosphate (RF-PO₄) in deuterium oxide confirmed π-stacking of the isoalloxazine rings. In conformity with the 350 times higher water-solubility of RF-PO₄, NOESY also indicated a contorted conformation of the ribityl phosphate chain, whereas, for RF, indications for a contorted chain were not observed.