Funct. Mater. 2023; 30 (4): 494-505.
4′-Benzyloxyflavonol glucoside as fluorescent indicator for β-glucosidase activity
1Institute of Chemistry and School of Chemistry, V. N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61022, Ukraine.
2State Scientific Institution "Institute for Single Crystals′′, National Academy of Sciences of Ukraine, 60 Nauky Ave., Kharkiv 61072, Ukraine.
Fluorescent flavonols and their glucosylated derivatives are promising tools for studying protein structure and enzyme activity screening. The fluorescence properties of such probes are tunable by electron-withdrawing substituents in 2-aryl ring, while a role of bulky hydrophobic groups has not been examined in-depth yet. Here, we examine the application of benzyloxy-substituted flavonol β-D-glucoside as a fluorescent indicator for the activity screening of the β-glucosidase enzyme in an aqueous solution. The rate constant of the enzymatic cleavage of an O-glycosidic bond in benzyloxy-substituted and un-substituted flavonol glucosides was compared using fluorescence kinetic measurements. We found that introducing a hydrophobic bulky benzyloxy group in the 4′-position of flavonol glucoside resulted in a 2.3-fold decrease in the hydrolysis rate constant. The molecular docking calculations allowed us to reveal critical molecular interactions in a flavonol-enzyme complex and identify favorable binding modes and the binding affinity of the flavonols towards the β-glucosidase protein. We found that upon adding a bulky benzyloxy group, the flavonol glucoside′s binding affinity towards the enzyme was increased from -9.9 to -10.8 kcal/mole. However, the stronger binding of the substituted glucoside would require a higher activation energy barrier to form an appropriate substrate-receptor transition state for the hydrolysis reaction, seen as some decrease in the rate constant. Finally, these findings promise that flavonol glucosides can be utilized as easy-to-use fluorescent indicators for rapid activity screening of other enzymes from a glucosidase family.
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