New Monofunctionalized Fluorescein Derivatives for the Efficient High‐Throughput Screening of Lipases and Esterases in Aqueous Media

Monoalkylation or acylation of fluorescein ( 1 ) with various acyloxymethyl or acyl halides afforded, respectively, a series of ether‐ ( 2 ) and ester‐functionalized ( 3 ) fluorogenic probes. The highly reactive and water‐soluble substrates release fluorescein ( 1 ) upon reaction with lipases and esterases within seconds or minutes, both under fully aqueous conditions or in the presence of DMSO (20%) as a co‐solvent. The most‐reactive substrates in the two series were the octanoic acid derivatives 2f (= 2‐{6‐[(octanoyloxy)methoxy]‐3‐oxo‐3H‐xanthen‐9‐yl}benzoic acid) and 3a (= 2‐[6‐(octanoyloxy)‐3‐oxo‐3H‐xanthen‐9‐yl]benzoic acid). Esterases were found to generally react faster under aqueous conditions, while lipases were more reactive in the presence of DMSO as a co‐solvent.