Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates

Abstract

Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (k(i)) ranging from 0.3 x 10(5)M(-1)min(-1) to 10.4 x 10(5)M(-1)min(-1). When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE's ( congruent with 1 x 10(7)M(-1)min(-1)) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling.

Figure 1

Proposed design for chromophore-linked OPs using click reaction.

Figure 2

UV-Vis of rMAChE inhibited by dabsyl-FP 10b.

Figure 3

Visualizations of AChE (turquoise) inhibited by dabsyl-FP (magenta) showing a phosphonylated enzyme bound at Ser-203. The chromophore is positioned near P-site residues (e.g., Trp-286). Only residues that are 4Å from dabsyl-FP and polar hydrogens shown. Views A and B are rotated 90 degrees about the vertical axis.

Scheme 1

Synthesis of a propyl- and butyl-tethered, chromophore-linked fluorophosphonates: (i) TEA, CH₂Cl₂ (ii) NaN₃, CH₃CN/H₂O (iii) NaOH, 50–60 °C; H⁺ (iv) C₃N₃F₃, CH₂Cl₂ (v) CuSO₄, sodium ascorbate, t-BuOH/H₂O. All compounds were fully characterized.