N-Benzylgalactonoamidines as potent β-galactosidase inhibitors

Illuminating the binding interactions of galactonoamidines during the inhibition of β-galactosidase (E. coli)

“…The kinetic parameters were deduced by application of the Michaelis-Menten model as described for related studies. 6,9 The catalytic rate constant ( k cat ) of the hydrolysis of 18 by β-galactosidase ( E. coli ) was determined to be 91 ± 2 s −1 and the substrate affinity (K M ) was found to be 0.040 ± 0.004 mM confirming previous results of others under comparable conditions ( k cat 90 ± 1 s −1 ; K M = 0.040 ± 0.002 mM, 30 mM TES buffer, 145 mM NaCl, pH 7.0, 25 °C). 29,30 The apparent rate constant ( k ’ cat ) and the apparent Michaelis-Menten constant (K’ M ) were then determined in presence of at least three different inhibitor concentrations for each amidine.…”

“…29,30 The apparent rate constant ( k ’ cat ) and the apparent Michaelis-Menten constant (K’ M ) were then determined in presence of at least three different inhibitor concentrations for each amidine. 6,9 The corresponding inhibition constants (K i ) were calculated from the recorded data, and the obtained values were averaged (Table 1). …”

“…7,6 All glyconoamidines were shown to inhibit the selected enzyme competitively; parent amidine 1 shows inhibition ability in the picomolar and derivatized amidines 1a–c in the low nanomolar concentration range rendering 1 one of the most potent glycosidase inhibitor known today. Removal of the hydroxyl group at C-2, as accomplished in 1a , or derivatizations at the hydroxyl groups of C-2 and C-4, as accomplished in 1b and 1c , respectively, diminish the hydrogen bonding and inhibition ability of the glyconoamidines.…”

“…6 We elaborated a gram-scale synthesis toward the galactoamidine precursor compounds, 7 and evaluated the contributions of the respective aglycon to the overall compound inhibition ability in structureactivity relationship studies with a small library of 22 amidines. 8 Hydrophobic interactions of the aglycon were identified as driving force for the inhibition ability of the most competitive inhibitors during the enzymatic hydrolysis of model substrates (K i = 8–12 nM).…”

Evaluating N-benzylgalactonoamidines as putative transition state analogs for β-galactoside hydrolysis

“…6 Not all of these compounds may fulfill the experimental requirements for a TSA, but they may resemble fortuitous binders in the active site of β-galactosidase ( A. oryzae ) instead. Prior to the preparation of catalytic microgels from such aryl-D-galactonoamidines, it is thus imperative to experimentally select a compound with true transition state-like character for the hydrolysis of glycosidic bonds.…”