Design of a new fluorogenic molecular probe of lipases that will be specific for monoacylglycerol lipase

Homeostatic imbalances in enzyme activities have been linked to an array of human pathological conditions. Evidence exists for the role of monoacylglycerol lipase (MAGL) in the development and progression of malignant forms of cancers, including breast, prostate, melanoma and ovarian, as well as tuberculosis, Alzheimer’s, and other forms of neurological disorders. There is great need to develop improved sensing methods for the determination of activity of lipases, including MAGL, to probe mechanisms of disease development and progression. This research successfully developed two rhodamine B-based FRET (Fluorescence Resonance Energy Transfer) molecular probes (JA4 and JA7) targeted at lipases. The probes were achieved by a convergent synthesis in which a novel piperazine-linked rhodamine fluorophore (as donor) and an aminoanthraquinone ‘black-hole’ acceptor (as quencher) were coupled via a key ester bond destined to be the site of cleavage (activation) by lipase hydrolytic action. All new target compounds and intermediates were characterized by high resolution electrospray (+) mass spectrometry. The structures of the target compounds JA1 (fluorophore), JA4 (probe 1) and JA7 (probe 2) were confirmed by NMR spectroscopy. Novel substrate FRET probes, JA4 and JA7 were demonstrated to be smart probes in which rhodamine fluorescence was completely quenched until activated by lipase(s) as demonstrated by the efficient restoration of rhodamine fluorescence emission upon incubation with probes JA4 and JA7 by UV fluorescence spectroscopy experiments. The identities of the PPL-induced cleavage products of JA4, i.e., JA1 and JA3, were determined by HPLC using UV/Visible detection; this was possible because standards of JA1 and JA3 were already available by synthesis (and their structures determined, originally by mass spectrometry and latterly by NMR spectroscopy). Efficient restoration of intense rhodamine fluorescence upon incubation of the probes with porcine pancreatic lipase (PPL) enzyme, as a model for human lipases, served as a proof of concept that the probes could be activated by lipase action. The probes have potential to determine over-expressed lipase activity in human tissues ex vivo, without any interference from molecules of the biological matrix, to aid diagnosis, and monitor stage and progression of disease