Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates

•Synthesis of three racemic chromogenic triglycerides is described.•The method of chemoselective couplings to triglyceride scaffolds is generally applicable.•The triglycerides are used as substrates for lipase activity assays by means of FRET.•Kinetic parameters are determined using Thermomyces lanuginosus lipase.

Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460 s−1 M−1, 59 s−1 M−1 and 346 s−1 M−1, respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204 s−1 M−1. Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide