Benzene Sulfonyl Linked Tetrasubstituted Thiacalix[4]arene for Selective and Sensitive Fluorometric Sensing of Sulfosulfuron along with Theoretical Studies

Herein, we designed two fluorescent tetrasubstituted benzene sulfonyl appended Thiacalix[4]arene receptors named L1 and L2, which sensitively and selectively detect Sulfosulfuron among other herbicides and pesticides. The detection limit (LOD) was found to be 0.21 ppm and 0.35 ppm, and the enhancement constant (Ks) was determined to be 7.07 X 104 M−1 and 5.55 X 104 M−1 for L1 and L2, respectively. Using the non-linear regression method, the association constant was obtained as 2.1 X 104 M−1 and 2.23 X 104 M−1 whereas, the binding ratio was found to be 1:1 for both L1 and L2, respectively. Additionally, the interference studies show the selective nature of receptors for Sulfosulfuron among its sulfonylurea family. To further confirm the interaction mechanism, 1H-NMR spectroscopy, and a computational investigation were carried out, which validates the 1:1 binding ratio. The receptors were found to be recyclable in nature with simple acid–base treatment. This new approach of using supramolecules as fluorescent probes for sensitive and selective detection of herbicides is rare in the literature.


Introduction
Food is an important source of energy for living organisms, and its upsurging demand has led to the use of pesticides that have ramped up globally as they protect crop-damaging species, hence increasing the yield.But such heavy uses of pesticides result in anthropogenic activities such as discarding wastewater into water bodies leading to contamination, and further scarcity of potable drinking water.Conversely, the extravagant use of these pesticides results in large-scale environmental deterioration affecting the soil, plants, water, edible product, and human beings.The permissible level of pesticides in drinking water was set at 0.1 µg/L by European Union (EU) standards of drinking water.However, various reports claim the concentration level of pesticides in water systems exceeded the permissible limit set by the EU [1][2][3][4].
Wheat is a widely sought crop across the globe owing to its source of nutrients in food; its production is of prime importance.The most widely used herbicide for wheat production is Sulfosulfuron as it retards the growth of brome, phalaris minor, quack grass, grip grass, broadleaf weeds, wild oats, apera, spica-venti and is found safe on wheat for its application in pre-and post-emergence to weeds [5,6].Other than wheat it has also demonstrated efficient selective controlling of weeds in a variety of field crops such as cotton, rapeseed, corn, alfalfa, vegetables like potato and tomato, and orchards including olive groves [7].Owing to the elevated demand for Sulfosulfuron, its global consumption is estimated to be above USD 91.27 million in 2020-2025.Ultimately, all these pesticides and their residue get accumulated in the environment, causing terrestrial and aquatic pollution and entering the food chain, affecting the ecosystem as a whole.The reports show that Sulfosulfuron possesses acute toxic effects on aquatic organisms [8,9].Hence, the rapid, reliable, precise, and effortless detection of Sulfosulfuron is a need of the moment.
Thiacalix [4]arene are modified class of Calix[n]arene molecules replacing methyl bridges with sulphur atom providing additional co-ordination site, ease in modification and even flexibility to the system resulting in more pre-organization and selectivity towards guest motifs [29][30][31].Despite this, the literature on Thiacalix [4]arene based pesticides sensors is scarce and only a few reports on sensing pesticides using Thiacalix [4]arene scaffold are available [32,33].To the best of our knowledge, no fluorescent sensor has been reported in the literature that selectively senses Sulfosulfuron using fluorescence spectroscopy.Thus, in an endeavor to develop a sensitive and selective sensor for Sulfosulfuron, herein, we report two novel Benzene sulfonyl appended Thiacalix [4]arene based fluorescence "Turn-on" sensors named L 1 and L 2 which offers sensitive and selective sensing of Sulfosulfuron amongst the other common pesticides in this study.

Experimental Section
Receptors L 1 and L 2 were synthesized using Thiacalix [4] arene.In a 100 ml RBF (round bottom flask), a heterogenous mixture of Thiacalix [4]arene, potassium carbonate, and potassium iodide was taken in acetonitrile (MeCN) solvent and refluxed for the next 1 h.After the solution cools down, to this 2,4-dimethyl benzene sulfonyl chloride (for L 1 ) and 4-isopropyl benzene sulphonyl chloride (for L 2 ) were added slowly and the reaction was refluxed for 24 h to offer L 1 and L 2 respectively.Chemical structure of the receptors is shown in Fig. 1.The synthetic procedure mentioned briefly in Materials and Methods section (SI Page No. S4-S6).

Photophysical Studies
The selective sensing ability of both receptors L 1 and L 2 were evaluated using both UV-visible and fluorescence spectroscopic studies in the presence of various pesticides in aqueous system.For spectroscopic competitive studies pesticides such as Ametryn, Atrazine, Clodinafop, Chlorsulfuron, Metalaxyl, Pretilachlor, Propanil, Propargyl, Pendimethalin, Simazine, Simetryn, Sulfosulfuron, Tebuconazole, Terbutryn, and Tricyclazole were considered.The stock solution of 0.05 mM of both the receptors was prepared in MeCN:Water system (9:1).The UV-Visible spectra show absorbance maxima at 230 nm and 283 nm for receptor L 1 and 273 nm for receptor L 2 (Fig. 2 and SI Fig. S13).Significant changes in the absorbance spectra (Bathochromic shift) for receptor L 1 and L 2 were observed only in the presence of Sulfosulfuron, whereas no remarkable changes were observed with other pesticides.Similarly, in the fluorescence spectra, strong emission maxima were observed at 373 nm and 393 nm for receptor L 1 and at 410 nm and 431 nm for receptor L 2 , upon excitation at 300 nm and 290 nm for L 1 and L 2 respectively.For both L 1 and L 2 , a "Turn-ON" fluorescence in the spectra was observed in the presence of Sulfosulfuron.However, no changes were observed for the rest of the pesticides (Fig. 2 and SI Fig. S13).Both UV-visible and fluorescence titration experiments were carried out to further confirm the selectivity of L 1 and L 2 with an incremental concentration of Sulfosulfuron (0.1 mM-1.0 mM).A total 15-fold time rise in the emission intensity was observed for Sulfosulfuron (0.5 mM) in comparison with various pesticides in the presence of receptor L 1 and around a 14-fold rise in the emission intensity for L 2 .(Fig. 2E, F) Detection limit (LOD) was calculated using the 3σ-method [34] with incremental addition (0.1 mM-0.4 mM) of Sulfosulfuron (SI Fig. S14) and tabulated in Table 1.The quantum yield for L 1 and L 2 were found out to be 0.061 and 0.113 respectively using tryptophan (quantum yield = 0.14) as standard Fig. 1 Chemical structures of receptors L 1 and L 2 [35] for the emission spectra of L 1 and L 2 whereas, for L 1 -SS and L 2 -SS interaction, the quantum yield was determined to be 0.093 and 0.133 respectively.Similarly, the enhancement constant (Ks) [36] was determined for both the receptors (Table 1).A linear fitting suggests that enhancement is due to a static mechanism (SI Fig. S14 and Fig. 3).

Theoretical Studies
To gain detailed insight into the binding process and validate the experimental results, further computational work has been performed using Becke's three-parameter hybrid exchange (B3LYP) in the Gaussian 16, Revision B.01b [37][38][39][40].The 6-311G (d,p) and 6-31G (d,p) basis sets were utilized for all the elements.The geometric structures of the complex in the ground state (singlet) were fully optimized at the B3LYP levels.The effect of solvent (Acetonitrile: Water) was systematically monitored for all the steps via a conductor-like Polarizable Continuum Model (PCM).Docking images have been visualized using Biovia discovery studio viewer [41,42].
The optimized structures (SI Figs.S15 and 16) have been unitized for the docking study.The optimization of geometry for receptors in solvent and gas phases is presented in the Table 2.The binding energy of L 1 -SS (Fig. 4) and L 2 -SS (Fig. 5) are -215.44kcal/Joule and -220.85 kcal/Joule, respectively.The Binding behavior has been confirmed through a docking study which suggests that strong binding between ligand and analyte is the result of π-π stacking and non-covalent π-sulfur interactions.Docking results reveal the change in the bond length of ligands upon introduction of the analyte, this suggests the disruption in the bond energy of ligands.

Binding Constant
A nonlinear regression approach was used to understand the Host (L 1 and L 2 ) -Guest (pesticide) interaction for more reliable association parameters [43].An open-access program i.e.; supramolecular.orgwas used to obtain association constant and ratio using absorption titration data [].From the binding model, the fitting prefers 1:1 stoichiometry and the association constant for L 1 and L 2 were calculated as 2.1 X 10 4 M −1 and 2.23 X 10 4 M −1   respectively.The Association constant values are summarized in Table 3 and their direct links are available in Supporting Information (Table S1).

MALDI-TOF Studies
To further confirm the 1:1 binding of Sulfosulfuron and both the receptors, the MALDI-TOF spectrometric technique was used [34].In Acetonitrile: Water

Probable Interaction Mechanism
The emission peaks of L 1 and L 2 can be attributed to intraligand Π-Π* transition from sulfonyl lone pair to phenyl group.In the presence of Sulfosulfuron, the triplet peak (in fluorescence spectra) merges into a single peak (Fig. 8A).To examine this behavior, 1 H-NMR studies* were evaluated for receptor L 2 as shown in Fig. 7B [11].
A shift of (Δ) 0.17 ppm (downfield) phenylic calixarene protons (denoted as A) was observed showing the interaction of guest is maybe outside the ring system.Moreover, a significant change of (Δ) 0.11 ppm (downfield) was observed for the sulfonyl phenyl group (denoted as B and C) of receptors and indenyl group (denoted as D and E) of Sulfosulfuron.This shows a significant Π-Π stacking  between the rings as proposed in Fig. 7A.No notable changes in the aliphatic region were observed in the spectra. 1 H-NMR studies were carried out in the MeCN-D 3 system and the ratio of Host and Guest was taken 1:1 for better peak resolution.

Interference Studies
In the interference studies, along with Sulfosulfuron, five other relevant pesticides were considered (Ametryn, Chlorsulfuron, Metalaxyl, Pendimethalin, and Terbutryn).No notable change in the fluorescence spectra was observed (Fig. 8) and the peak at 378 nm and intensity in fluorescence similar to [L 1 + SS] was obtained even similar behavior was confirmed using absorption spectra.To confirm more selectivity, Chlorsulfuron (CS) which belongs to same herbicide family with Sulfosulfuron was taken, but no change in the fluorescence spectra with [L 1 + CS] was observed (Supporting Information Fig. S18).

Recyclability Studies
The reversible nature of receptors with Sulfosulfuron was analyzed with acid-base treatment.In the following process, the quantitative fluorescence studies were carried out with Sulfosulfuron using both receptors L 1 and L 2 in MeCN: Water system (9:1 v/v) (Fig. 9 and SI Fig. 19).In the emission spectra, the band at 393 nm undergoes blue shift to 373 nm and the intensity in the fluorescence spectra rises upon the addition of Sulfosulfuron due to interaction with it.0.5N HCl was added to the above solution resulting in a decrease in the intensity of fluorescence spectra and the band shifts from 373 to 390 nm.To this, a freshly prepared 0.5N NaOH solution was added which results in the shift in the band again to its original position.This observation led us to the conclusion of the reversible nature of the binding process of ligands towards Sulfosulfuron which can be employed using simple acid-base treatment [45, 46.The probable pictorial representation for the rise and fall in the emission intensity due to acid-base treatment is displayed in the Fig. 10.

Conclusion
In conclusion, we designed two fluorescent probes for the selective detection of Sulfosulfuron among other relevant pesticides by aqueous phase studies.UV-visible, Fluorescence, 1 H-NMR spectroscopy, and Mass spectrometric techniques conclude the selective detection of Sulfosulfuron in 1:1 stoichiometric complex formation, which was further confirmed through geometry optimization and docking study of receptors and Sulfosulfuron using the same solvent system.The binding constant values for L 1 and L 2 were 2.1 X 10 4 M −1 and 2.23 X 10 4 M −1 respectively whereas the LOD values were determined to be 0.21 ppm and 0.35 ppm respectively.The enhancement constant (Ks) value was found out to be 7.07 X 10 4 M −1 and 5.55 X 10 4 M −1 for L 1 and L 2 respectively.The receptors were found to be completely recyclable in nature using simple acid-base treatment.This new approach of sensing toxic pesticides at micro-level concentration develops interest for further modification in the field of supramolecular sensors.

Fig. 2 A
Fig. 2 A Competitive Absorption spectra of L 1 along with Pesticides.B UV-Visible titration of L 1 with an incremental concentration of Sulfosulfuron.C Competitive Emission spectra of L 1 with various Pesticides.D Change in Fluorescence intensity of L 1 (Host) with an incremental concentration of Sulfosulfuron (Guest).E

Fig. 4 Fig. 5
Fig. 4 Docking Image of L 1 with Sulfosulfuran a Side view; b Top View represent π-π staked interaction in pink color and π-sulfur interaction in yellow color

Table 1
Representation of Enhancement constant (K S ) and Detection limit (LOD) for L 1 and L 2 obtained using Fluorescence titration spectra

Table 2
Geometry optimization table of receptors L 1 and L 2

Table 3
Representation of Binding constant (K a ) value obtained using free online software (Supramolecular.org)accessed on November 2022