A sensitive and selective high-throughput screening fluorescence assay for lipases and esterases

Long-chain fatty acid esters of 7-(3,4-dihydroxybutyloxy)-2H-1-benzopyran-2-one (6) such as octanoate 2a are shown to be exceptionally sensitive and selective fluorogenic substrates for lipases and esterases. Umbelliferone (8) is released upon hydrolysis of the ester function in 2a in the presence of bovine serum albumin and sodium periodate. These substrates are at least by one order of magnitude more sensitive to lipases than the commercial fluorogenic substrate 4-methylumbelliferyl heptanoate. Furthermore, they are stable to a broad range of pH-induced- and thermal-hydrolysis conditions and do not react with non-catalytic proteins such as bovine serum albumin (BSA).