Turn-off fluorescence probe for the selective determination of pendimethalin using a mechanistic docking model of novel oxacalix[4]arene

Abstract

A novel bidansylated oxacalix[4]arene (BDO) fluoroionophore for the selective determination of pendimethalin (PM) was carried out in the linear range of detection between 0.4 μM and 20 μM. Furthermore, computational studies were performed to assess the binding and stability of the complex. For the predicted model, PM faces laterally and interacts via weak intermolecular forces with BDO with a E value of −176.00 kJ mol⁻¹.

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Pesticides are continuously employed to increase the production of crops by controlling different types of plagues/pests. However, their overuse leads to high perseverance in the environment and the residues being adequately present in soil, river sediments, natural water and edible items.¹ A plethora of research has proven that their persuasive accumulation in the environment leads to a detrimental impact on the biosphere.^2,3 Therefore, owing to such perilous non-target effects, a quick, on-time and satisfactory detection of pesticides is an imperative need.

Several techniques, such as gas chromatography,⁴ thin-layer chromatography,⁵ solid-phase micro extraction-HPLC,⁶ high-performance liquid chromatography (HPLC)⁷ and immuno-affinity chromatography (coupled column liquid chromatography/mass spectrophotometry),⁸ have been used to detect the presence of pesticides, but these methods are tedious and time-consuming. Intriguingly, the fluorescence method for detecting pesticides is considered to be the key state-of-the-art technique that gives rapid results in a highly selective and sensitive manner.^9–12

Pendimethalin (PM) (N-(1-ethylpropyl)-2,6-dinitro-3-4-xylidine) is a dinitroaniline group-containing herbicide (Fig. 1) that is widely used to regulate the growth of susceptible species by inhibiting cell division and elongation by blocking mitotic division, followed by the accumulation of abnormal microtubular structures.¹³ Ominously, PM possesses a low volatility and has low solubility in water,¹⁴ therefore the chances of its accumulation in soil and water body deposits due to non-leaching features are higher.² A large number of investigations and pesticide glossaries have shown that PM is highly toxic to soil micro biota and terrestrial, aquatic and plant life. PM restrains the plant cell division process that is responsible for chromosome separation and cell wall formation during cellular division.¹⁵ Therefore, a simple, rapid and effective method is needed to detect the presence of PM. Different electroanalytical,² supercritical fluid extraction¹⁶ and gas chromatography¹⁷ methods have been used for the detection of PM. In the present text, we have developed a spectrofluorimetry method for the selective and sensitive detection of PM.

Fig. 1 Chemical structure of PM (N-(1-ethylpropyl)-2,6-dinitro-3-4-xylidine).

Calixarenes are characterized by a central annulus surrounded by a wide upper rim and a lower rim with a high level of preorganization and conformational preferences. Calix[n]arenes are third-generation supramolecules with inherent hollow and hydrophobic cavities to hold a variety of guest analytes, such as cations, anions and neutral molecules.¹⁸ Different mechanisms, such as photoinduced electron transfer (PET), Forster (fluorescence) resonance energy transfer (FRET), photoinduced charge transfer (PCT) and intramolecular charge transfer (ICT) are proposed for mediating chemosensors functioning.¹⁹ Calixarenes with dansyl groups have been used for sensing various guest analytes^20–22 and pesticides, such as dalapon, glyphosate, methiocarb, benomyl and carbendazim, which can be detected spectrofluorometrically.^23–26 In particular, oxacalix[4]arenes (OC), which are heteracalixarenes in which the methylene bridge is replaced by oxygen, hold additional recognition features. Oxacalix[4]arenes can easily bind with anions, cations and neutral guests due to the capability of H-bonding and the possession of a constitutional hollow cavity to form inclusion complexes.²⁷ Dansyl chloride (DC) is a commonly used fluorogenic unit that can be easily attached to an organic moiety. DC possesses excellent fluorescence with an observable Stoke's shift and a relatively long emission wavelength between 400 and 600 nm.²⁸ DC is therefore used as a synthetic receptor for selectively binding various guest molecules/ions and for the formation of host–guest complexes.²⁹ Thus, a new fluoroionophore, bidansylated oxacalix[4]arene(BDO), comprising a calix system with dansyl chloride was designed, which showed excellent selectivity for PM.

The combination of experimental and molecular modelling studies has been recognized as a powerful tool for the study of the complexation geometry of the inclusion system.^30–33 Recently, our group reported docking studies of biological activities by novel biologically-pertinent oxacalix[4]arene derivatives.³⁴ However, as far as the fluorescence detection of pesticides is concerned, no one has reported molecular docking for OC.

In the present work, we demonstrate for the first time, a novel and facile synthesis of an oxacalixarene-based fluoroionophore, namely 2,8,14,20-tetraoxapentacyclo[19.3.1.1^3,7.1^9,13.1^15,19]octacosa-1(25),3,5,7(28),9,11,13(27),15,17,19(26),21,23-dodecaene-25,27-(di-dansyl),4,6,16,18-tetranitro, receptor, BDO from OC³⁵ (Scheme 1).

Scheme 1 Synthetic route for the preparation of BDO.

This fluoroionophore was applied to the selective neutral molecular recognition of PM in the presence of other pesticides. The sensitivity of our fluorophore was determined within the linear range of detection of 0.4 μM to 20 μM.

The absorption spectra of BDO were recorded in methanol in the presence of various pesticides, namely ametryn, simetryn, sulfosulfuron, propanil, pretilachlor, tebuconazole, terbutryn, metalaxyl, clodinafop propargyl, simazine, thiophanate methyl, PM, tricyclazole and atrazine. The absorption band of BDO (251 nm) was shifted to 244 nm during the addition of PM (4 × 10⁻⁵ M) in BDO (4 × 10⁻⁵ M) (Fig. 2).

Fig. 2 Absorption spectra of BDO (4 × 10⁻⁵ mol L⁻¹) upon the addition of different pesticides in methanol (4 × 10⁻⁵ mol L⁻¹).

Compared to other pesticides, only PM showed substantial changes in the absorption spectra. This blue-shift indicates an interaction of PM with the ligand BDO. The emission study of BDO was conducted with different pesticides, which showed emission at 666.42 nm under excitation at 251 nm (Fig. 3).

Fig. 3 Fluorescence spectra of BDO (4 × 10⁻⁵ mol L⁻¹) upon the addition of different pesticides in methanol (4 × 10⁻⁵ mol L⁻¹).

Strong quenching of the emission intensity was observed only in the presence of PM. Thus, based on the absorption studies, the observed blue-shift for PM was attributed to the increase of the electronic density on the naphthalene ring due to the complexation process between BDO and PM, which shifted the amine-to-naphthalene charge transfer state in the dansyl chromophore to higher energy.³⁶ Based on the emission studies, it is proposed that an intramolecular charge transfer occurs when the DC (donor) moiety is attached to the OC (acceptor) scaffold. Thus, it is predicted that the analyte (PM) is in close interaction with the donor or the acceptor moiety, which changes the photophysical properties and efficiency of the fluorophore. Therefore, quenching occurs.^37–39 Among the different pesticides, only PM underwent a considerable change in emission intensity; therefore, its emission titration was considered to assess its binding constant with BDO using the procedure described in ref. 40 and 41. The fluorescence spectra of BDO showing the changes in emission intensities upon the addition of an increasing concentration of the lone PM are shown in Fig. 4. The fluorescence intensity increased with the increasing PM concentration. The plot of log[(F₀ − F)/(F − F₁)] versus log[P] is shown as an inset in Fig. 4.

Fig. 4 Fluorescence spectra change of BDO (4 × 10⁻⁵ mol L⁻¹) upon the addition of PM in methanol. Inset: linear regression fit (double-logarithmic plot) of the titration data as a function of the concentration of the pesticide.

The binding constant of the BDO–PM complex was calculated to be 1.26 × 10⁵ M⁻¹. The fluorescence quantum yield was also determined based on the reported literature.^42,43 The quantum yield of DC was nearly 0.7, as described in the literature, and the quantum yield of BDO was 0.79. Thus, the number of emitted photons decreased as the concentration of PM increased, i.e. quenching occurred. A strong fluorescence quenching for PM (89.40%) was found compared to the other pesticides, as shown in Fig. 5.

Fig. 5 Graphical representation of the percentage quenching of BDO with various pesticides.

Stern–Volmer plots were used to determine the quenching mechanism.^44,45 In the case of linearity in the I₀/I plot, the fluorescence quenching mechanism can be regarded as purely static or purely dynamic, the latter mechanism being due to the formation of a ground-state, non-fluorescent complex. If linearity is not observed in the I₀/I plot, the fluorescence quenching mechanism can be regarded as a simultaneous static and dynamic linear fluorescence quenching. In our case, a linear plot for PM with BDO was obtained, which indicates that the fluorescence quenching mechanism was either purely static or purely dynamic (Fig. S3†). Job's method of continuous variation (Fig. S4†) was used for the determination of the stoichiometric ratio of the complex of PM and BDO. The analysis confirmed the 1 : 1 stoichiometry ratio of PM and BDO. To further verify the 1 : 1 complex formation between BDO (the host) and PM (the guest), electrospray ionization mass spectrometry (ESI-MS) analysis was recorded in methanol, where the spectrum of BDO showed a molecular ion peak at m/z 1046.3, and in the presence of PM showed a peak at 1328.2 [BDO + PM + H]⁺ (Fig. 6).

Fig. 6 ESI mass spectrum showing the peak pattern of the 1 : 1 complex formed between BDO and PM.

Prior to understanding the exact molecular dynamics of the BDO–PM complex, ¹H NMR spectroscopic titration studies were performed to analyze the interaction between BDO (the host) and PM (the guest). As shown in Fig. 7, when 7 mM concentration of BDO was added to the same concentration of PM, the proton signals of PM shifted downfield. The resonances of the protons of PM corresponding to aromatic H_a at δ = 8.11 ppm and methyl H_c proton at δ = 0.79 ppm underwent a downfield shift of Δδ = 0.02 ppm, while the NH H_b proton at 7.10 ppm underwent downfield shifts of Δδ = 0.03 ppm. Moreover, ¹H NMR titration showed that with increasing concentrations of the host^46,47 (BDO), the guest (PM) aromatic H_a proton, –NH H_b proton and methyl H_c proton underwent downfield shifts (Fig. S5†). These chemical shift changes of the PM proton resonances indicate that a well-integrated complex was formed between BDO and PM.

Fig. 7 ¹H NMR spectra (DMSO-d₆, 500 MHz, 298 K): (a) 7 mM BDO; (b) 7 mM BDO + 7 mM PM; (c) 7 mM PM, which show that BDO can bind PM, selectively.

Molecular docking studies were used to study the mechanism behind the binding of PM with BDO. By translating the ligand flexibly and comprehending the significant interactions, the resulting pose was captured and imported for the molecular dynamics studies. The docked pose, comprising the best score according to the Hex energy scheme E_total = −176.00 kJ mol⁻¹, was selected from the enormous delineated orientations. For comparison purposes, docking simazine was reported to have an E_total = −181.91 kJ mol⁻¹. The energetic difference between the two complexes was not very large and this could, in principle, be due to shape selectivity of the group present in the complex. The occurrence of the lowest energy within the complex was mainly due to the stabilization by the maximum non-bonding and shape complementarity features. Congruently, the characterizations and their accord with the binding constant were inconsistent with previous reports.^47–49 However, visual observation clearly showed that the BDO ligand interacts laterally with PM, which also suggests that the cavity size was not sufficient to form an intercavity complex with the guest (PM). PM was reported to exhibit π–π stacking and various hydrophobic interactions, including π-alkyl, π-lone pair and π-donor with BDO, as depicted in Fig. 8. Contrary to simazine, we were able to observe that the presence of nitro and isopentyl groups facilitates the interactions in PM (i.e. a better shape fit).

Fig. 8 Docked complex of BDO with (a) PM and (b) simazine.

To gauge the structural integrity and stability of the complex, we performed MD simulations (10 ns) of the docked inclusion complex in a water solvent system. The simulations were executed with a constant shape and volume at room temperature to study the structural features and behaviour of the host–guest inclusion system. Subsequently, a resultant solvated low-energy inclusion complex was created from a built-in energy minimization technique which was analysed during the simulation trajectory by computing the root mean square deviation (RMSD), intramolecular H-bond and radius of gyration (r_Gyr) as a function of time.

A molecule is expected to possess different conformations if its initial conformation is varied from its final by RMSD > 4 Å.⁵⁰ The inclusion complex in our case displayed negligible RMSD fluctuations (a maximum of 1.2 Å was observed) throughout the trajectory, which supports the compatibility of the developed inclusion structure. The time-dependent fluctuations of the RMSD based on the backbone atoms during the complete 10 ns simulation period is graphically depicted in (Fig. 9).

Fig. 9 RMSD, radius of gyration and intramolecular hydrogen bonds of the complex over a time period of 10 ns.

Noticeably, the only significant change in the RMSD was captured during 7 to 8 ns, which did not affect the geometry of the inclusion complex, even in different conformations. The low RMSD across the simulated molecules over time indicated that the modelled conformation was stable and energetically favourable. Furthermore, the radius of gyration (r_Gyr) was used to study the ‘extendedness’ of a ligand in the inclusion complex, and is equivalent to the principle moment of inertia. Moderate compaction (2.96 to 3.25 Å) in the inclusion complex throughout the trajectory indicated the constancy and firmness of the mass distribution of the complex around the rotational axis. In addition, we recognized a small change in the gyration radius of 2.5 nm to 2.8 nm (Fig. 9). This observation indicated that compaction was a slow process in the case of complexation and could be attributed to the fluctuations in the inter-dihedral angles. Only a single intramolecular H-bond in the complex was developed during the simulation time. The decrease in H-bonds was captured at approximately 7.5 ns, when the RMSD fluctuation was the highest (0.1 to 1.1 Å), and was coupled with a decrease in the compactness (r_Gyr = the lowest at 2.96). During this instant, no intra H-bonding was present in the complex.

In conclusion, we reported for the first time a novel bidansylated oxacalix[4]arene (BDO) fluoroionophore as a selective “turn-off” probe for PM, with a linear range of detection of 0.4 μM to 20 μM. Also the detection limit of the synthesized receptor was found to be 3.81 nM for PM. The geometry and stability of the inclusion complex was studied using molecular docking and dynamic simulations, which showed that a low-energy complex is feasible, with an optimum of one intra H-bond and numerous hydrophobic contacts. Furthermore, the ESI-MS and ¹H NMR titration results displayed a successful interaction between BDO and PM. This spectrofluorimetric method thus provides us with great interest for developing a routine analysis of PM.

Acknowledgements

The authors thank the financial assistance provided by DRDO (New Delhi) and University Grant Commission-Basic Scientific Research (UGC-BSR) New Delhi. Mohd. Athar and Anita Kongor would like to thank DST, New Delhi for providing INSPIRE-JRF fellowship and P. C. J would like to thank UGC for start-up grants. The authors also acknowledge Gujarat Forensic Science University-Gandhinagar (GFSU), Central Salt and Marine Chemicals Research Institute Bhavnagar (CSMCRI), Central University of Gujarat-Gandhinagar (CUG), National Facility for Drug Centre Discovery-Rajkot (NFDD), Oxygen Healthcare-Ahmedabad (O₂h) for providing instrumental facilities, and UGC-Info net & INFLIBNET for e-journals.

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Footnote

† Electronic supplementary information (ESI) available: Materials and methods, synthesis procedure, competitive titration, Job's plot, quantum yield, computational details of BDO ligand with PM, NMR titration, details of docking and MD is given in ESI. See DOI: 10.1039/c6ra05707a

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