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.

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