11B NMR experiments are reported for seven boronic acid inhibitors, including a very potent slow-binding peptide-based one, in the presence of excess chymotrypsin. Under these conditions, for several inhibitors slow-exchange is evident between chymotrypsin-bound and free inhibitor from the observation of separate signals only on addition of the enzyme. This new condition enables assignment of the bound boron to tetrahedral symmetry based solely on its chemical shift. Surprisingly, one of the inhibitors shows two enzyme-bound tetrahedral boron signals, only one of which is removed by acylation of the active center Serl95 with phenylmethanesulfonyl fluoride. To demonstrate the requirement for Ser for binding of the inhibitors, anhydroSerl95 chymotrypsin was synthesized and found not to show any bound boron signals under any conditions tried so far. In addition to the tetrahedral site in the slow-exchange regime, there was in a few cases also demonstrated the existence of a boron resonance in fast-exchange between the free and enzyme-bound forms, in accord with a previous report from this laboratory (Adebodun, F.; Jordan, F. J. Am. Chem. Soc. 1988,110, 309–310). These new observations are applicable to inhibitors with any binding affinity, so long that the boron is in slow-exchange between the free and bound forms. The study is therefore complementary to the one reported earlier, in which the boron nucleus was found in rapid exchange between the free and bound forms, necessitating extraction of the bound chemical shift and relaxation properties (to enable calculation of the quadrupolar constant and thence the hybridization) from the weighted average values.
ASJC Scopus subject areas
- Colloid and Surface Chemistry