Xanthene-based dyes such as rhodamines and fluorescein are widely used for labeling and sensing biomolecules. They have a high extinction coefficient and fluorescence quantum yield in aqueous solution, and their excitation and emission wavelengths are in the visible range. Fluorescein esters are nonfluorescence, but they could be cleaved by intracellular enzymes to yield fluorescent products. In order to search for a fluorogenic substrate for lipase, we evaluated the fluorgenic behavior of fluorescein esters to the assay of lipase. In this study, nine different aromatic and aliphatic esters of fluorescein were synthesized using P2O5/SiO2 reagent and rate of enzyme hydrolytic and non-enzyme hydrolytic of ester were evaluated using fluorimetry. Our results, were shown, that among seven of aromatic esters of fluorescien, derivation of P- chloro benzoic acid, P- methyl benzoic acid and benzoic acid have higher hydrolytic rate than aliphatic derivation such as acetate and alpha- chloro-acetate. Therefore, aromatic derivation of fluorescein is better substrates for the activity of lipase with the higher rate of hydrolysis.

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