15N NMR Study of Intramolecular Hydrogen Bonding in Imidazole-4-Propanoic Acid and Its Relevance to the α-Lytic Protease Hydrogen Bonding Network

The presence of an intramolecular hydrogen bond in imidazole-4-propanoic acid in acetonitrile has been verified using ¹⁵N NMR and ¹H NMR. Hydrogen bonding (zwitterion) and non-hydrogen bonding (cation) cases were studied in acetonitrile-d₃ using a ¹⁵N-labeled 2-mercaptoimidazole-4-propanoic acid compound. Studies showed a downfield shift difference for N3 of 9 ppm, likely the result of an intramolecular hydrogen bond between H3 and the carboxylate anion. A CD₃CN/H₂O mixed solvent study, where the fraction gauche was measured as water was added to a solution of imidazole-4-propanoic acid in CD₃CN in 5 μL increments, revealed the amount of water necessary to break the intramolecular hydrogen bond in imidazole-4-propanoic acid. When a ratio of 12 moles of water to one mole of imidazole-4-propanoic acid is reached, the fraction of gauche conformers reverts to statistical, indicating that the hydrogen bond is broken.  The enzyme α-lytic protease utilizes an extensive hydrogen-bonding network at its active site that contains an aspartic acid – histidine hydrogen bond and the compounds studied here are excellent model compounds for this interaction. The results reported here imply that only a moderate concentration of water is required to disrupt the aspartic acid – histidine hydrogen bond. Because water is disruptive to the hydrogen bond, it is possible that the concentration of water near the active site is relatively small compared to elsewhere in the protein envelope.