The interaction of (R)-1-acetamido-2-(4-fluorophenyl)ethane-1-boronic acid with alpha-chymotrypsin at pH 4 was studied by a variety of 19F-NMR experiments. It was demonstrated that this compound forms a complex with a 1:1 stoichiometry, probably because the boronic acid acts as a 'transition state' inhibitor of the enzyme. Analysis of fluorine T1 relaxation behavior and 19F[1H] NOE data shows that the rate constant for dissociation of the complex is 1.3 s-1 and that the motion of the 4-fluoroaromatic ring within the complex can be characterized by an overall rotational correlation time of 13 ns and a correlation time for rotation about its local C2 axis of 110 ns. Enzyme-induced fluorine chemical shifts, fluorine relaxation times, line width data and 2D 19F[1H] NOE results suggest that the structure of the complex in the vicinity of the fluoroaromatic ring is similar to that found in a closely similar acylated enzyme. However, the dynamics of 4-fluoroaromatic ring motions are different in the two systems, with the ring being slightly more mobile in the boronic acid complex than in the acylenzyme.

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