Screening of boronic acids for strong inhibition of the hydrolytic activity of a-chymotrypsin and for sugar sensing associated with a large fluorescence change

The inhibitory effect of arylboronic acids which act as a transition state analogue for certain peptidases is efficiently intensified by added saccharides: this finding enables us to control the enzyme activity by a combination of arylboronic acids and polyols including sugars. In particular, the combined system of 3-biphenylboronic acid and D-glucose strongly inhibited the hydrolysis reaction mediated by achymotrypsin, the inhibitory effect ( Ki = 1.1 X mol dm'3) being stronger than that of a specific inhibitor, chymostatin (Ki = 4.8 XlU7 mol d~n-~) . Hence, saccharides act as a 'co-inhibitor' in the boronic acid inhibition system. To search for such fluorescent boronic acids that are useful for sensitive detection of sugars in water, 8 aromatic boronic acids have been screened. Among them biphenyl-3-boronic acid and naphthalene-2-boronic acid could fully satisfy three prerequisites for sugar sensing: strong fluorescence intensity, large pH-dependent change in I-, and shift of the pHZ, , profile to lower pH region in the presence of sugars. INTRODUCTION Trigonal boron compounds contain a vacant 2p orbital which easily reacts as a Lewis acid with nucleophiles such as hydroxide, alkoxide, or imidazole to give a tetrahedral boron adduct. This adduct formation also occurs in the active site of certain hydrolytic enzymes such as subtilisin and a-chymotrypsin and the active site serine (or histidine) is usually the fourth ligand of the tetrahedral structure (1-4). Apparently, phenylboronic acid acts as a novel 'transition state analogue' for these enzymes and the K, values for phenylboronic acid are estimated to be 2.3 X l04-8.OX mol dm-3 for a-chymotrypsin (1 and 2). mol dm-3 for subtilisin (1 and 2) and 1.9 X Meanwhile, it is known that boronic acids form cyclic esters with saccharides and the reaction occurs reversibly and rapidly at ambient temperature. It was recently demonstrated by a few groups that boronic acids serve as a useful interface to selectively recognize saccharides in water (5-14). For example, saccharides in water can be spectrophotometrically detected by boronic-acid-appended porphyrins (7) or boronic-acid-appended fluorophores (8 and 12). Saccharide detection with boronic-acid-appended porphyrins is based on the idea that absorption and fluorescence spectra of porphyrins change sensitively in response to a shift in the aggregation-deaggregation equilibrium and the complexation of saccharides with the boronic acid moieties changes this equilibrium to deaggregation because of the enhanced hydrophilicity of the complexed porphyrin. Here, it occurred to us that phenylboronic acid working as an inhibitor in the enzyme active site should be withdrawn upon complexation with saccharides because of the enhanced hydrophilicity of the saccharide-complexed inhibitor. As a result the enzyme activity should be regenerated. To test this intriguing hypothesis we investigated the influence of added saccharides on the inhibition ability of phenylboronic acid in a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine-