Two inclusion modes were realized in the complexation between phenobarbital (PHB) and 2-hydroxypropyl-β-cyclodextrin (HPCyD) in aqueous solution using 13C-NMR spectroscopic and isothermal titration microcalorimetric studies. The geometries of the complexes were estimated by molecular dynamics calculations. Results of 13C-NMR spectrometry showed that the chemical shifts of barbituric acid and phenyl rings were significantly shifted upfield due to penetrating both rings into HPCyD cavity. And the affinity constants of the carbons of the phenyl ring were higher than those of the barbituric acid ring. The signals of ethyl side-chain contrarily moved downfield by the repulsions with the carbons situated around the rim of HPCyD. The calorimetric data indicates that two different types of PHB-HPCyD complexes at 1 : 1 stoichiometry are formed in unionized PHB, whereas, single type of inclusion at ionized PHB. The first type of inclusion complexes was formed with high affinity by an entropy-driven reaction associated with nearly constant values of -ΔG1 (26.9±1.0kJ/mol), -ΔH1 (3.73±0.86kJ/mol), and large positive ΔS1 (77.5±1.5J/mol/K) in all pH. It seems that the phenyl ring gets included within the HPCyD cavity and that the hydrophobic interaction dominates the stabilization of the complex. However, the second type of inclusion complexes with lower affinity was characterized by large values of -ΔH2 and small values of -ΔS2 at pH lower than 7.0, reflecting the van der Waals'or electrostatic interactions. The large inflections around pKa 7.4 were observed in all thermodynamic parameters. Especially, the values of -ΔG2 were lessened at pH > 8.0 due to decreases in the stability constants to the order of 102M-1. The barbituric acid ring seems to penetrate the cavity at lower pH than pKa. In both cases, the ethyl side-chain rests outside of the cavity.
