Fluorogenic substrates for lipases, esterases, and acylases using a TIM-mechanism for signal release

3-Acyloxyl-2-oxopropyl ethers of umbelliferone were investigated as new fluorogenic substrates for lipases and esterases. The aliphatic primary alcohol-leaving group released the fluorescent product umbelliferone by an enolization/beta-elimination reaction similar to the triose phosphate isomerase (TIM) reaction. A similarly designed phenylacetamide provided a fluorescent probe for penicillin G acylase, whereby the enolization/beta-elimination sequence from the intermediate aminoketone was very fast and spontaneous even under acidic conditions. The corresponding epoxyketone was not fluorogenic with epoxide hydrolases (EH). These substrates represent periodate-free Clips-otrade mark substrates.