A series of fluorescein aralkyl esters were synthesized through esterification of fluorescein with carboxylic acids or acyl chlorides and applied to the fluorometric assay of lipase. The rate of spontaneous hydrolysis and enzymatic hydrolysis was measured by monitoring of fluorescence intensities of the solution and correlated with the enzyme activity. In addition, the chain length effect of aralkyl fluorescein esters was studied. Fluorescein di(5-phenylpentanoate) was found to be a better substrate than their homologs for the fluorometric assay of lipase with the higher rate of hydrolysis and better Km value. As little as 15 ng mL-1 of lipase can be detected with fluorescein di(5-phenylpentanoate). Substitute’s effects on the enzymatic hydrolysis of phenyl acetic acid and replacement of phenyl with other polycyclic aromatics such as biphenyl or naphthyl showed a decrease in enzymatic hydrolysis rate.

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