title: Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.
abstract: 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 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant.
authors.affiliation: Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark.
authors.forename: Rokhsana J
authors.initials: RJ
authors.lastname: Andersen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, 2880 Bagsværd, Denmark. Electronic address: jebk@novozymes.com.
authors.forename: Jesper
authors.initials: J
authors.lastname: Brask
mesh_terms: D060326:Chemistry Techniques, Synthetic; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D013379:Substrate Specificity; D014280:Triglycerides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D014280:Triglycerides; D008049:Lipase
keywords: EnzChek; FRET; Fluorogenic substrate; Lipase assay; Triglyceride synthesis