Interaction between glyphosate pesticide and amphiphilic peptides for colorimetric analysis

Abstract

The large-scale use of glyphosate pesticides in food production has attracted attention due to environmental damage and toxicity risks. Several regulatory authorities have established safe limits or concentrations of these pesticides in water and various food products consumed daily. The irreversible inhibition of acetylcholinesterase (AChE) activity is one of the strategies used for pesticide detection. Herein, we found that lipopeptide sequences can act as biomimetic microenvironments of AChE, showing higher catalytic activities than natural enzymes in an aqueous solution, based on IC₅₀ values. These biomolecules contain in the hydrophilic part the amino acids l-proline (P), l-arginine (R), l-tryptophan (W), and l-glycine (G), covalently linked to a hydrophobic part formed by one or two long aliphatic chains. The obtained materials are referred to as compounds 1 and 2, respectively. According to fluorescence assays, 2 is more hydrophobic than 1. The circular dichroism (CD) data present a significant difference in the molar ellipticity values, likely related to distinct conformations assumed by the proline residue in the lipopeptide supramolecular structure in solution. The morphological aspect was further characterized using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM), which showed that compounds 1 and 2 self-assembly into cylindrical and planar core-shell structures, respectively. The mimetic AchE behaviour of lipopeptides was confirmed by Ellman's hydrolysis reaction, where the proline residue in the peptides act as a nucleophilic scavenger of organophosphate pesticides. Moreover, the isothermal titration calorimetry (ITC) experiments revealed that host-guest interactions in both systems were dominated by enthalpically-driven thermodynamics. UV-vis kinetic experiments were performed to assess the inhibition of the lipopeptide catalytic activity and the IC₅₀ values were obtained, and we found that the detection limit correlated with the increase in hydrophobicity of the lipopeptides, implying the micellization process is more favorable.

Fig. 1. Molecular structures of the lipopeptides PRWG–C₁₈H₃₇ (compound 1), PRWG–(C₁₈H₃₇)₂ (compound 2).

Fig. 2. Fluorescence intensity results using pyrene as probe (378 nm) for compounds 1 and 2 (A) as a function of the concentration. The intersection point of the two straight lines determines the cac values. (B) CD spectra for 0.033 wt% 1 and 2 in water solutions at the pH 7.

Fig. 3. (A) SAXS data spectra of 1 and 2 at 0.2 wt%. The green line represents the power law slope at low q values, corresponding to cylinder (q⁻¹) and planar (q⁻²) assemblies. The red line is the fitting using a cylinder core–shell or bilayer as form factor. Cryo-TEM images were obtained for (B) compound 1 and (C) compound 2 at pH 7.0.

Fig. 4. Reactant complex (left), transition state (center) and product (right) for the N-methyl-l-prolinamide and acetylthiocholine reaction. Geometries were obtained using the B3LYP/def2-TZVP/SMD/D3BJ methodology as detailed in the text. Distances (shown in figure) for the nascent C–N and S–H bonds are; reactant: C–N 3.90 Å, S–H 2.88 Å; transition state C–N 1.52 Å, S–H 2.35 Å; product C–N 1.35 Å, S–H 1.35 Å.

Fig. 5. (A) ITC data for molecules 1 and 2 in the presence of PNG and the correspondent SAXS data (B) and (C), respectively.

Fig. 6. Absorbance intensity as a function of the time at different [P/L] ratios for compounds 1 (A) and 2 (B), respectively. (C) Inhibition ratio (ΔI) as a function of [P/L] for compounds 1 (A) and 2, the red and black lines are fitted using a sigmoidal function to obtain the IC₅₀ values. (D) The ln(ΔI) as a function of the PNG molar concentration for compounds 1 (A) and 2, the red and black lines are linear fits to determine the limit of detection (LOD).