In Silico Studies and Design of Scrupulous Novel Sensor for Nitro Aromatics Compounds and Metal Ions Detection

A Novel calix[4]pyrrole system bearing carboxylic acid functionality [ABuCP] has been synthesized and its interaction towards various nitroaromatics compounds [NACs] were investigated. ABuCP showed significant color change with 1,3-dinitro benzene (1,3-DNB) in comparison to the solution of other nitroaromatic compounds such as 2,3-dinitro toluene (2,3-DNT), 2,4-dinitro toluene (2,4-DNT), 2,6-dinitro toluene (2,6-DNT), 4-NBB (4-nitrobenzyl bromide) and 4-nitro toluene (4-NT). The ABuCP-1,3-DNB complex produces a red shift in absorption spectra based on charge transfer mediated recognition. Additionally, the density functional theory calculation confirmed the possible mechanism for the binding of 1,3-DNB as a guest is well supported by the calculation of other parameters such as hardness, stabilization energy, softness, electrophilicity index and chemical potential. The TDDFT calculation facilitates the understanding of the proper binding mechanism in reference to experimental results. Additionally we have also developed its derivative which acts as a new fluorescent sensor which can selectively recognize Sr(II) ions. In this view its aminoanthraquinone derivative of calix[4]pyrrole i.e. ABuCPTAA is synthesized which also results in generation of high fluorescence capability sensor.


Introduction
Nitroaromatics (NACs: hazardous electrophilic species) in the conspicuous matter in this century because of its harmfulness to human health and environment [1]. The production, Storage abuse of this nitro aromatic compounds mainly their uses in terrorist and war activity pose great to public security of the whole world [2][3][4][5]. As one kind of important pollutions, NACs such as trinitrophenol, nitrophenol, dinitrobenzene and trinitrotoluene also cause health problems in both animals and human beings, including anaemia, abnormal liver function, cataract development and skin irritation [6,7]. Even after degradations, the by-products of the common nitro aromatic compounds are still potent carcinogenic and due to this they are referred as priority pollutants [8,9]. Some di-nitroaromatics, especially m-dinitrobenzene (m-DNB) finds application in industry as an intermediate in the chemical synthesis of some rubber chemical, pesticides, dyes, and nitro aromatic compounds [10] and they have been detected also in groundwater near industrial waste disposal sites [2]. Compared with other nitroaromatic compounds, m-DNB could cause methemoglobinemia in animals and humans [11]. It could also produce testicular toxicity [12], and brainstem damage [10,11]. Abundant papers have been published on the identification and quantification of nitro aromatic compounds. At present, numerous kinds of modern instruments such as GCMS, LCMS, Ion mobility spectroscopy, fluorescence, X-ray dispersion, Raman spectroscopy and chemiluminescence [13][14][15][16] are being engaged for the sensitive and selective sensing of NAC, but usually are torment from the disadvantages like high operational cost and portability units during in-field use, and this has limited production of small, low-power units suitable for use in the field which greatly limits their extensive application. But for the same if a molecular sensor is designed and synthesized such that it will be sensitive to our m-DNB that produces a visible optical change in the presence of nitro aromatic compounds even at very low concentration under rather elementary conditions. Such systems, which rely on changes in absorption, can be employed in the absence of instrumentation via simple "naked eye" detection. They can also be incorporated as faster, cheaper, and safer analysis.
In this view, calixarene based chemosensors have been reported to exhibit unique response towards any analyte of curiosity based on the optimized structural as well as geometrical factors [17][18][19]. Modification of calixarene structures can be made to detect nitro aromatic compounds in its ingredient solutions [20][21][22]. Nowadays, pyrrole-based macrocycles are on the forefront for the development of such selective receptors. Calix [4]pyrrole and its substituents derivatives holds a great perspective in the fields of sensors [23][24][25][26], and their distinctive behaviors attributed due to its structural flexibility [27,28]. It is well-known that non-covalent interaction such as hydrophobic effects, pi-pi stacking, van der Waals forces, Hydrogen bonding, metal coordination, cation-pi interactions, are responsible for the function of synthetic or natural supramolecular in biological processes, such as immune response, protein enzyme inhibitors, signal transduction or normal function of cellular/ organelle structure [29][30][31]. One of the approaches which would fit our detection of nitro aromatic compounds involves charge transfer (CT) donor-acceptor complexes [32][33][34]. For the same, our present study is to signify the neutral molecular recognition ability of calix [4]pyrrole tetracarboxylic acid (ABuCP) ligands. Over the last decade, our group has synthesized several calix based chemosensors [5,[35][36][37] but this application can be either an extension to our effort to build up a sensor for NACs. Our goal was to use optical and more specifically colorimetric means of signalling to selectively detect nitro aromatic compounds with low concentration limits and ABuCP serves as an excellent colorimetric sensor. Moreover, the In-silico insights could provide a theoretical as well as preparative chemistry towards host-guest interaction of ABuCP and 1,3-Dinitrobenzene (1,3 DNB).
Additionally, several advanced analytical methods are available today for the identification and quantification of ions and molecules of environmental importance. The most prominent techniques involving metal ion and anion recognition include atomic absorption spectroscopy (AAS), ICP-OES, ICP-MS, Ion chromatographic imagery (IC), X-ray fluorescence spectroscopy (ICP-MS), induced plasma mass spectroscopy (ICP-MS) (XRF). In addition to these approaches, some electroanalytical techniques like as potentiometry amperometry, some electroanalytical techniques like as potentiometry, amperometry and cyclic voltammetry are often employed for the identification of different guest analytes, These processes include costly tools, skilled personal and arduous preparatory techniques for the sample to get extremely accurate findings. The chromogenic sensors [38][39][40][41][42] are thus the main prerequisite for one-site analytics detection. Active field of study is the detection of fluorescence of chemical and biological analytes. The aim of such initiatives is to reduce the costly usage and disposal of radioactive tracers. Fast and cost-effective diagnostic techniques for a variety of clinic, bioprocess and environmental applications are also required. In general, Fluorometry is regarded preferable to spectrophotometry since it is particularly more sensitive. Techniques of fluorescence can measure correctly up to one million times lower concentration than measures of absorbance. Furthermore, via time-resolved measurements, analyte distinguishing ability is higher.
So, our view is to develop a new fluorescent sensor which can selectively recognize cations. In an attempt to execute our vision aminoanthraquinone derivatives [25] of calix [4] pyrrole i.e. ABuCPTAA is synthesized which will result in generation of high fluorescence capability sensors.

Chemical and Materials
All metals salts, nitroaromaic compounds (NACs), and ingredients, such as 1-aminoanthraquinone, 4-acetylbutyric acid and pyrrole were obtained from Sigma-Aldrich, while additional solvents and reagents were obtained from commercial sources. Merck provided TLC plates with fluorescence active (F-254). Magnetic stirrer (REMI-5MLH) and micro-pipette (VAR VOL 100-1000 µl, Kasablanka-Mumbai) were used. All the glassware was very well calibrated before use.

General Method for Spectroscopic Detection of Nitro Aromatic Compounds and Cations
Absorption spectra of ABuCP stock solution (2 × 10 -5 M) and those of various nitroarmatic compounds (2 × 10 -5 M) with 100 equivalents were prepared in MeOH solvent and were recorded and compared. Absorption spectra of resulting solution mixture of same stock solution of ligand were used and nitro aromatic compounds solutions of pet concentration (1.0-100 equiv.) were prepared by dilution of stock solution.
Emission spectra were recorded using ABuCPTAA stock solution (2 × 10 -5 M) and different desired concentration (1.0 -100 equivalents) of corresponding cations (2 × 10 -5 M) such as Mg (II), Co (II), Ni (II), Cd (II), Pb (II), Sr (II), Cu (II), Bi (III), Cr (III) and Al (III) were taken by fitting dilution with same stock solution. Spectra were recorded and compared. The binding constant of the complex was also established using the method described in the literature.
Here the interaction of Sr (II) with ligand ABuCPTAA is studied.

Density Functional Theory Calculation
The Density Functional theory calculation have been performed using Hartree-Fock (HF), Becke's three-parameter hybrid exchange (B3LYP) and Coulomb-attenuating method-Becke's three-parameter hybrid exchange (CAM-B3LYP) [43] in the Gaussian 09 (G09 B.01) software package [44,45]. The geometric structures of the complex have been fully optimized at HF, B3LYP and CAM-B3LYP levels as well as 6-31 g(d,p), 6-31 g + + (d,p) in the ground state (singlet). The ABCPs were demonstrated using Avogadro version 1.2.0 software [46]. The conductor-like polarizable Continuum Model (CPCM) has been systematically monitored for all the steps for the effect of solvent (methanol). The Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), electron density (ED), electrostatic potential (ESP), and Molecular electrostatic potential (MEP) was performed at Cam-B3LYP method with 6-31G(d,p) basis sets.

Host-Guest Interaction
The molecular level interaction of selective nitro aromatic compounds was employed in the glide module of the Maestro suite of Schrodinger software [47,48]. Glide extra precision (XP) was applied for the molecular docking which includes the false positive rates that are removed by more thorough screening and enhanced scoring, resulting in much greater enrichment. The receptor grid was generated using the grid generation wizard in Glide to build a three-dimensional (3D) grid with a maximum scale of 20 × 20x20 and 0.5 spacing. Ligand preparation has been performed using the Ligprep Module [49]. After that the binding affinity and interactions were examined.

Conventional Method
Parent 4-acetyl substituted calix [4]pyrrole skeleton (Scheme 1) was synthesized using general method of acid catalyzed condensation reaction of pyrrole and aliphatic acid. Distilled pyrrole (1.0 ml, 0.0153 mmol) was placed in a 250 ml round bottom flask equipped with magnetic stirrer and filled with acetonitrile (100 ml). Boron trifluoride etherate (2.11 ml) was then added to the reaction flask and stirred for 15 min. 4-acetyl butyric acid (2.00 ml, 0.0153 mmol) was dissolved in acetonitrile (100 ml) and added stepwise to the reaction flask. Reaction mixture was left to stir for 8 h and was monitored through TLC. After completion of reaction, the reaction mixture was poured slowly into another 500 ml round bottom flask equipped with a magnetic stirrer and distilled water (200 ml) containing triethyl amine to obtain a light brown colored residue. It was filtered, collected, and dissolved in diethyl ether (400 ml) to get rid of the black tar. The solvent was evaporated to obtain light brown-black residue, which was then recrystallized from methanol to obtain pure product (58%).

Microwave Irradiation
A mixture of pyrrole (0.125 ml, 0.0018 mol) and boron trifluoride etherate (0.26 ml) in acetonitrile (10 ml) were exposed to microwave irradiation for 10 min. A solution of 4-acetylbutyric acid (0.3058 gm, 0.0018 mol) in acetonitrile (10 ml) was then added to the reaction mixture and was subjected to microwave irradiation for 10 min with a slight pause after every 2 min. After completion of the reaction, a light brown-black reaction mixture was poured into cold water (50 ml) to obtain brown black colored precipitates. The residue was filtered off, collected, dried and dissolved in diethyl ether (25 ml × 2). The solution was again filtered gravitationally to get rid of the black tar. The solvent was evaporated to obtain light brown colored residue which was then recrystallized from a methanol mixture to get pure product for analysis. Step I: Synthesis of 2 -ch lor o-N -(9 ,10 -di oxo -9,

Absorption Spectra
Absorption spectroscopy has been utilized for the investigation of the overall binding property of 1,3-DNB with ligand ABuCP. Bar diagram (Fig. 1) describes no absorption spectrum change when other nitro aromatic compounds were added. The UV-visible absorption spectrum of the ABuCP a peak was observed at a wavelength of 210 nm. Absorption band shift from 210 to 234 nm with red shift of 24 nm (Fig. 2) was observed upon addition of 1,3-DNB. Also, variation of absorption intensity as well as wavelength upon the addition of gradually increasing concentration of 1,3-DNB was studied (Fig. 3). A charge transfer between ABuCP and 1,3-DNB occurs which is responsible for inducing shifting of the absorption band towards higher wavelength. Moreover, regarding the chemical potential it was found that, in the direction of pyrrole ring more + ve potential Is found and that the more negative potential is in the direction of carboxylic group of butyric acid as well as 1,3-DNB compared to that ABuCP and 1,3-DNB, thus there are maximum possibilities of ABuCP to attract 1,3-DNB via hydrogen bonding as well as electrostatic interaction which is described later in in-silico studies. Interestingly, ABuCP-1,3-DNB complex forms a novel supramolecular recognition host system that can discriminate 1,3-DNB from other nitro aromatic compounds. It can be postulated that the synergistic effects of Also, investigation of ABuCPTAA as a fluorescent probe was very well studied by absorption (Fig. 4) and emission spectra of ABuCPTAA with various metal ions: Mg (II), Co (II), Ni (II), Cd (II), Pb (II), Sr (II), Cu (II), Bi (III), Cr (III) and Al (III) as their nitrate salts were recorded in MeOH as solvent. Sr (II) ions were the only cation to show interaction with ABuCPTAA among other tested cations. Also variation of absorption intensity as well as wavelength upon the addition of gradually increasing concentration of Sr (II) ions was studied (Fig. 5). Emission spectra (Fig. 6) showed a remarkable quenching of 80%, which indicates charge transfer mechanism. Carbonyl group on aminoanthraquinone acts as an excellent donor and experiences ICT [25,42,51]. Electron rich oxygen Scheme 2 Synthesis scheme of ABuCPTAA provides electron to electron deficient ring. This ICT mechanism [52] continues until Sr (II) is not added, the time when Sr (II) is added ICT decreases as the formation of a bond between carbonyl oxygen of anthraquinone group that was initially directly attached with anthraquinone ring. The charge transfer from the oxygen of the anthraquinone group to the electron deficient ring stops when the charge density on the oxygen atom decreases, resulting in a drop in emission intensity leading to quenching [25,50]. Also, Emission spectra were increased in respect to decrease in concentration Sr (II) ions Fig. 7.

Stoichiometry of Complex
Job's method [53] of continuous variation was employed for the determination of stoichiometry ratios of the metal ligand complex.
ΔA values were plotted against mole fraction of Sr (II) ions a/a + b (Fig. 8). The obtained value of 0.50 for ΔA at a/a + b indicates a 1:1 ratio single formation of complex. The precision of this result implies the formation of a single complex. To ensure its reliability the measurement was done at different wavelengths. This gave the same values for a/a + b ratio.

Binding Constant and Quantum Yield of ABuCPTAA
With a previously published procedure and given equation [23,54,55], the binding constant of fluoroionophore was established.
A good linear fit of R = 0.98 and 0.97 is obtained using the above equation for ABuCPTAA. Fluorescence quantum yields (ɸ F ) [23] is obtained using following method.
Here F and F std indicates the areas under the fluorescence emission curves of Sr (II) ions complexes with ABuCPTAA  Approximately reported quantum yield of amino-anthraquinone is 0.075 [25] and that of ABuCPTAA is obtained as 0.71 which is showing the occurrence of quenching phenomenon due to increased addition of guests resulting in decreasing the number of emitted photons (Table 1).
Stability of ABCP has been investigated by change in their UV Visible spectrum intensity at different pH (4.0-11.0). UV-Visible band of ABuCP displays slight change at pH other than 7.0 -8.0 and is liable to being some-what turbid. However, if it is exploited to sonicating process for 10-15 min, they retain their ingenuity with insignificant compromise in their UV spectrum. Likewise their absorption intensity of ligand ABCP is slightly quenched at pH other than 7.0. Hence, pH 7.0 was preferred to carry out all experiments on ABuCP and it can be concluded that ligand shows maximum stability (Fig. 10) with wavelength and absorption intensity at pH 7.0 also the same was showing good results i.e. our moiety shows operational stability at: pH 7.0, at room temperature and in the region of 90 days (Figs. 11 and 12).

In-silico Optimization
The Ligand ABuCP as well as other Nitroaromatic nitro aromatic compounds (NACs) was optimized in gas as well as Solvent to get an insight of binding posture of the complex. Table 2 represents the Optimization value Gas phase and Solvent phase.

Docking Interaction
The molecular docking interaction has been performed using Schrodinger glide docking [47]. In the docking study, as an input structural the geometry optimized host structure (ABuCP) (Fig. 13) and guest (All NACs) has been utilized. The highest glide docking energy play a crucial role for the establishment of the tunable and finest docking of ABuCP with all NACs (Fig. 14)which advocate the stable complex formation between the ABuCP and 1,3-DNB having two different poses. (Figs. 15 and 16). The energy of post 1 is -13.771 kcal/mol and Pose 2 is -13.389 kcal/mol which is higher than others. In the docking, mainly two types of interaction play crucial roles for the complex formation and docking energy. One is noncovalent interaction (classical hydrogen bonding, and Aromatic H bonding) and π-Interaction (π-cation) interaction. The other information about the glide docking properties has been depicted in Table 3. The resultant docking bond distance and interaction type in ABuCP and 1,3-DNB (Figs. 15 and 16) has been representing in Tables 4 and 5.

In-Silico Complexation Behavior
The optimization of the complex (having hydrogen bonding interaction) has proceeded for two different types of docking  poses, but at the end we are receiving the same optimization step (Fig. 17) through which we had carried out our further studies. The optimization energy has been utilized for complexation behavior of ABuCP_1,3-DNB in both the gas well as solvent phase at higher level of basis sets depicted in (Table 6) The binding energy (E b ) ( Table 7) has been calculated using below equation: where, E r = complex (ABuCP_1,3-DNB) optimization energy (Hartree); E L = ABuCP optimization energy (Hartree); E A = Analyte (NACs) optimization energy (Hartree).

Other Molecular Properties
Further study of the Frontier molecular orbital (FMOs) energy such as Higher occupied molecular orbital (HOMO) and Lowest Unoccupied molecular orbital (LUMO) energy and other molecular properties to understand the nature of complexation among ABuCP and 1,3-DNB was carried out. The investigation of other molecular properties involves Chemical reactivity [like Hardness and Softness] and electronic properties Exploration of the global electronic descript such as hardness (η), chemical potential (μ), and Softness (S) through the Frontier molecular orbital (FMOs) difference i.e. The energy difference between HOMO and LUMO is of utmost importance for detailed understanding of chemical selectivity and reactivity in the concept of DFT [58][59][60][61]. The molecule with a larger energy gap lacks the possibility to proceed the electron in excited state which indicates the relative higher stability of the molecule [62].
The band gap energy (E g ) is a critical factor for the stability [63], softness (S) [60], hardness (η) [58,64] and chemical potential (μ) has been calculated using below equations:     Maximum hardness principle (MHP) suggests that the system in their ground or valence state would make all the possible efforts to arrange themselves to be as hard as possible [65].
Electrophilicity is yet another global index which is a crucial term for energy reduction due to the maximum current of the electron between donor and acceptor [63].
The stabilization energy ( ΔE ) calculated as follow: Table 8 represents the energy of HOMO and LUMO of ABuCP, 1,3-DNB, and ABuCP_1,3-DNB complexes. As per the results, the following point directs important chemistry through the global electronic descriptor (a) The direction of electron transfer has been determined through the Electron chemical potential (μ). Also, the chemical potential value of the 1,3-DNB (-6.092 a.u) is more electronegative compared to the ABuCP (-2.7285 a.u) Which directs the electron transfer from the occupied of the ABuCP_1,3-DNB complex is responsible for the stability of it. (b) The lower the electrophilicity value acts as donor. The electrophilicity value of 1,3-DNB is higher compared to the ABuCP, denotes the greater electrophilicity ABuCP due to electrophilicity changes in ABuCP_1,3-DNB utilized as an acceptor during the encapsulation process. (c) The global harness (η) of the ABuCP reduced after the complexation with 1,3-DNB. Therefore, the theoretical results predict that the charge transfer interaction has a mandate role for the stabilization of the complexation process. Figure 18 represents the HOMO and LUMO diagram of ABuCP, 1,3-DNB and ABuCP_1,3-DNB complex in solvent phase which indicates the affluence of electron transfer from HOMO of ABuCP to LUMO of 1,3-DNB through solvent media. The TDDFT section delivers more outcomes on the electron transfer process.
The electrostatic potential (ESP) (Figs. 19,20 and 21) was higher slightly at hydroxy oxygen at carboxylic acid and Nitrogen in ABuCP. The Electron density (ED) (Figs. 19,20 and 21) has been uniformly distributed to ABuCP, 1,3-DNB as well as ABuCP_1,3-DNB complex. Figure 21 represents that in the direction of the pyrrole ring more + ve potential is found and that the more negative potential is in the direction of the carboxylic group of butyric acid as well as 1,3-DNB compared to ABuCP and 1,3-DNB.
For the recognition and interaction of hydrogen bonding in complexation has been identified through Molecular electrostatic potential (MEP). In addition to these, MEP plays a vital role in predicting binding sites and relative relativities towards electrophilic and nucleophilic attack [66][67][68].
As per the Figs. 22, 23 and 24 the lone pair of the oxygen attached with Nitrogen in the -NO 2 group in 1,3-DNB forms a hydrogen bond with the hydrogen atom attached with pyrrole N-atom.

Time Dependent Density Functional Theory Calculation
To further evaluate the electron transfer and the mechanism, the Time dependent density functional theory (TD-DFT) was carried out in solvent phase (Methanol) for the first six excited states at the levels CAM-B3LYP with the 6-31G(d,p) basis set. In addition to this, TD-DFT was used to nitro aromatic compounds the UV-Vis absorption spectra of the ABuCP as well as ABuCP_1,3-DNB complex. Table 9 represents the excitation energy (E), oscillator strength (f), wavelength (λ), key transition and the % contribution of the transition with their total energy which was compared with the experimental excitation energy. The frontier molecular orbitals (FMO's) and maximum wavelength absorption of ABuCP has been depicted in Fig. 25 and Table 9. The UV-Vis spectra were obtained due to the electron transition of HOMO (HOMO-1 and HOMO-3) to LUMO (LUMO-4, LUMO-6, and LUMO-7). Among all transitions, the % contribution of more than 15% of HOMOLUMO + 7, HOMO-1LUMO + 4, HOMOLUMO + 6 were responsible for the UV-Vis spectra of ABuCP. The FMO's and maximum wavelength absorption of ABuCP_1,3-DNB complex was  depicted in Fig. 26 and Table 9. Also transitions as HOMO-12LUMO + 1, HOMO-10LUMO + 1 contribute more than 15% and they also contribute a key role for the development of the UV-Vis spectra of the ABuCP_1,3-DNB complex.
Practically, 24 nm red shift appeared in ABuCP_1,3-DNB complex in methanol solvent while the TDDFT study has a resemblance of about 30 nm red shift for the ABuCP_1,3-DNB complex in methanol as a solvent.

Conclusion
A unique and novel 1,3-DNB sensor has been designed and developed using ABuCP as host ligand. Additionally, ABuCP acts as a rapid, highly sensitive, and selective receptor towards 1,3-DNB. Molecular modelling studies favours the complexation behaviour of the synthesized host and guest. In addition, the Host-guest interaction analysis through in-silico method favors the complexation with 1,3-DNB via H-bonding and ππ interaction. Other molecular properties support the charge transfer from 1,3-DNB to ABuCP. The TDDFT support the charge transfer from HOMOLUMO-7, HOMO-1LUMO + 4, HOMO-1LUMO + 6, HOMO-12LUMO + 1, HOMO-10LUMO + 1 transition with 24 nm red shift. Such sensing devices can be prepared from ABuCP to detect 1,3-DNB in a very low concentration range also. Also, its novel derivative; calix [4]pyrrole carrying anthraquinone i.e. ABuCPTAA has been successfully synthesized and characterized. Its complexation behavior with various metal ions is studied by absorption and emission spectroscopy. It exhibited high selectivity for Sr (II) ion with a good quenching. The binding studies were further supported by stoichiometric studies in which 1:1 binding was found. Also binding constant was found around 1.81 ± 0.10 with limit of detection up to 10 µM. Further quantum yield was found for the same. In addition to Sr (II) ions the turn-off mechanism arises, the reason behind it is prohibition of charge transfer from oxygen of anthraquinone group to electron deficient ring.
Authors' Contributions ALD Synthesized molecules and purified. NPP characterised and interpreted the data. JHP Wrote data in manuscript (Editor) form using structure software's. KMM analysed the application in silico using software. KDB major contributor in writing the manuscript and analysis the structural data.
Funding This research received no external funding.
Availability of Data and Material/ Data Availability No repository.