A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione

Abstract

Butyrylcholinesterase (BChE) is a biomarker in human blood. Aberrant BChE activity has been associated with human diseases. Here we developed a fluorescence resonance energy transfer (FRET) chemical probe to specifically quantify BChE activity in serum, while simultaneously discriminating against glutathione (GSH). The FRET chemical probe 11 was synthesized from a key trifunctional bicyclononyne exo-6 and derivatives of 5-(2-aminoethylamino)-1-naphthalenesulfonic acid (EDANS) and 4-[4-(dimethylamino)phenylazo]benzoic acid (DABCYL). EDANS fluorescence visualization and kinetic analysis of 11 in the presence of diverse compounds confirmed the outstanding reactivity and specificity of 11 with thiols. The thiol-dependent fluorescence turn-on property of 11 was attributed to a general base-catalyzed SN2 nucleophilic substitution mechanism and independent of metal ions. Moreover, all thiols, except GSH, reacted swiftly with 11. Kinetic studies of 11 in the presence of covalently modified GSH derivatives corroborated that the steric hindrance of 11 imposing on GSH was the likely cause of the distinguished reactivity. Since GSH commonly interferes in assays measuring BChE activity in blood samples, the 11-based fluorescent assay was employed to directly quantify BChE activity without GSH interference, and delivered a linear range of 4.3–182.2 U L−1 for BChE activity with detection limit of 4.3 U L−1, and accurately quantified serum BChE activity in the presence of 10 μM GSH. Finally, the 11-based assay was exploited to determine Ki of 5 nM for tacrine inhibition on BChE catalysis. We are harnessing the modulated characteristics of 6 to synthesize advanced chemical probes able to more sensitively screen for BChE inhibitors and quantify BChE activity in serum.