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Comparative Study
. 2024 Apr 25;29(9):1961.
doi: 10.3390/molecules29091961.

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

Xia Lin  1   2   3 , Qingyuan Yi  1 , Binyang Qing  4 , Weisen Lan  1 , Fangcheng Jiang  5 , Zefeng Lai  5 , Jijun Huang  6 , Qing Liu  6 , Jimin Jiang  6 , Mian Wang  4 , Lianjia Zou  2 , Xinbi Huang  2 , Jianyi Wang  1   3
Affiliations
Comparative Study

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

Xia Lin et al. Molecules. .

Abstract

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 (Km = 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 Km 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.

Keywords: acetylcholinesterase; apoptosis; comparative evaluation; lung carcinoma cell; probe; visualized monitoring.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Design principle of the aimed probes.
Figure 2
Figure 2
pH adaptation (A,B), fluorescence titration (C,D) and standard curve (E,F) of BF2-cur-Ben and BF2-cur-But to AChE, respectively. Fluorescence response of BF2-cur-Ben (G) and BF2-cur-But (H) with different interferences. 1. blank, 2. K+, 3. Na+, 4. Ca2+, 5. Fe3+, 6. Fe2+, 7. Mg2+, 8. Zn2+, 9. Cu2+, 10. Ba2+, 11. NH4+, 12. CO32−, 13. Br−, 14. C2O42−, 15. CH3COO−, 16. ClO−, 17. HS−, 18. Lys, 19. Pro, 20. Met, 21. Gln, 22. Thr, 23. Ala, 24. Phe, 25. Ile, 26. Gly, 27. Leu, 28. Glu, 29. Arg, 30. His, 31. Tyr, 32. Vc, 33. GSH, 34. Cys, 35. CEs, 36. BChE, 37. α-chymotrypsin, 38. Lysozyme, 39. Phospholipase, 40. Glucose Oxidase, 41. Elastase, 42. Trypsin, 43. BSA, 44. AChE. Michaelis-Menten curve (I,J) and Lineweaver–Burk curve (K,L) of BF2-cur-Ben and BF2-cur-But catalysed by AChE. Effect of different concentrations of inhibitor neostigmine on fluorescence intensity (M,N). Error bars are ± SD (n = 3).
Figure 3
Figure 3
Recognition mechanism of the two probes with AChE. HPLC monitoring of the reaction process of BF2-cur-Ben (A) and BF2-cur-But (B). Mass spectra of BF2-cur-Ben (C) and BF2-cur-But (D) to AChE. (E) The possible responsive mechanism.
Figure 4
Figure 4
Binding model of (A) BF2-cur-Ben and (B) BF2-cur-But to AChE.
Figure 5
Figure 5
DFT and TD-DFT calculations of frontier molecular orbitals of (A) BF2-cur-Ben, (B) BF2-cur-But and (C) BF2-cur.
Figure 6
Figure 6
A549 and HeLa cells after incubation with different concentration of the two probes, respectively. (A) Confocal fluorescence images. (B,C) Quantification of the relative fluorescence intensities of the microscope images. Error bars are ± SD (n = 3), scale bar = 25 μm.
Figure 7
Figure 7
BF2-cur-Ben monitors accelerant/inhibitor-induced regulation of AChE in A549 cells. (A) Confocal fluorescence images. (B) Quantification of the relative fluorescence intensities of microscope images. Error bars are ± SD (n = 3), scale bar = 25 μm.
Figure 8
Figure 8
In vivo fluorescence imaging of BF2-cur-Ben for detection of AChE in zebrafish. (A) Confocal fluorescence images. (B) Quantification of relative fluorescence intensity of the microscopic images. Error bars are ± SD (n = 3).
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