Two Fluorescent Probes for Recognition of Acetylcholinesterase: Design, Synthesis, and Comparative Evaluation

In this study, two "on-off" probes (BF₂-cur-Ben and BF₂-cur-But) recognizing acetylcholinesterase (AChE) were designed and synthesized. The obtained probes can achieve recognition of AChE with good selectivity and pH-independence with a linear range of 0.5~7 U/mL and 0.5~25 U/mL respectively. BF₂-cur-Ben has a lower limit of detection (LOD) (0.031 U/mL), higher enzyme affinity (K_m = 16 ± 1.6 μM), and higher inhibitor sensitivity. A responsive mechanism of the probes for AChE was proposed based on HPLC and mass spectra (MS) experiments, as well as calculations. In molecular simulation, BF₂-cur-Ben forms more hydrogen bonds (seven, while BF₂-cur-But has only four) and thus has a more stable enzyme affinity, which is mirrored by the results of the comparison of K_m values. These two probes could enable recognition of intracellular AChE and probe BF₂-cur-Ben has superior cell membrane penetration due to its higher log p value. These probes can monitor the overexpression of AChE during apoptosis of lung cancer cells. The ability of BF₂-cur-Ben to monitor AChE in vivo was confirmed by a zebrafish experiment.