Development of tBu-phenyl Acetamide Appended Thiacalix[4]arene as “Turn-ON” Fluorescent Probe for Selective Recognition of Hg(II) Ions

Herein, a novel N-(4-(tert-butyl)-phenyl)-2-chloroacetamide functionalized thiacalix[4]arene architecture, viz TCAN2PA has been synthesized and the sensing behaviour towards metal ions were explored. The probe, TCAN2PA displayed “turn-on” fluorescence response towards Hg(II) ions in acetonitrile over a series of competing common metal ions. A bathochromic shift in absorption band along with a significant “Turn-On” fluorescence behaviour of TCAN2PA was observed upon interaction with Hg(II) ions. The lower rim modification of thiacalixarene with N-(4-(tert-butyl)-phenyl)-2-chloroacetamide actively contributes toward the fluorescence property due to the presence of strong electron-donating aryl amido substituent. Fluorescence titration experiments were conducted to find out the limit of detection and to understand binding stoichiometry as well. The electron transfer interactions between the electron rich TCAN2PA host with Hg(II) ions have been postulated which is also supported by computational modelling insights.


Introduction
In recent years, there has been a curiosity in the invention of fluorescent sensors for detection of various heavy metal ions concerning its toxicity and negative environmental impact [1,2]. Among different hazardous species and specifically metal ions, mercury is a highly toxic heavy metal [3]. Mercury is broadly used for manufacturing of various materials, agricultural chemicals due to which pollution of air and water originates. Mercury containing compounds, particularly methyl mercury, accumulates in human brain and dislocates the blood-brain wall disturbing the metabolism of the nervous system. Inorganic mercury, during the anaerobic conditions can be converted into methyl mercury [4]. Mercury causes various diseases like kidney failure, lack of coordination, sensory impairment, allergic reactions, negative reproductive effects, acrodynia, and Mina Mata disease [5,6]. Mercury can be determined by various methods i.e. spectrophotometry [7], neutron activation analysis [8], anodic stripping voltammetry [9], X-ray fluorescence spectrometry [10], electrothermal atomic absorption spectrometry [11], atomic fluorescence spectrometry [12], atomic absorption spectrometry (AAS) [16], potentiometric ionselective electrodes [17], inductively coupled plasma mass spectrometry (ICP-MS), capillary electrophoresis and highperformance liquid chromatography (HPLC) and inductively plasma-atomic emission spectroscopy (ICP-AES) [22]. Fluorescence based measurements are mostly used due to its high sensitivity and simplicity, which converts molecular recognition into actual fluorescence signals. Cation binding by fluorescent sensors occurs through the mechanisms like photo-induced charge transfer (PCT), photo-induced electron transfer (PET) [13][14][15], formation of monomer/excimer and recently most of fluorescence resonance energy transfer (FRET) [18][19][20][21].
The third generation "calixarene" is an interesting research area in supramolecular chemistry which functions as a receptor for cations, anions and neutral analytes. In recent decades, various developments are seen, for molecular recognition of cations species [23]. Thiacalix [4]arene containing four sulfur atoms is easily available and therefore imparts new features compared to conventional calixarenes [24][25][26][27]. The thiacalix [4]arene assembly has an inherent hydrophobic cavity that allows recognition of various cationic, anionic, and neutral guests [28][29][30][31][32][33]. The selectivity to bind any particular guest or metal ion by thiacalix [4]arene can be controlled by functionalizing with several binding units as amides, acids, imines, esters, amines and thioethers. The incorporation of soft binding sites such as -N and -S into the thiacalix [4]arene scaffold shifts the binding ability from hard to soft metal ions. Therefore, the thiacalix [4] arene scaffold exhibits many interesting binding features as chemosensor for metal ions [34].
In this article, a novel thiacalixarene derivative, i.e., thiacalix [4]arene N-(4-(tert-butyl) phenyl)-2-chloroacetamide (TCAN2PA) has been synthesized and characterized. N-(4-(tert-butyl)phenyl)-2-chloroacetamide moiety has been functionalized to the lower rim of the parent thiacalix [4] arene and photochemical measurements including absorption and emission techniques were introduced to investigate its selectivity and sensitivity toward Hg(II) ions. Spectrophotometric and spectrofluorimetric results confirmed the binding of Hg(II) ions with TCAN2PA. The stoichiometry, binding constant and quantum yield of the TCAN2PA-Hg(II) complex have also been calculated. Additionally, molecular docking calculation helps to find the bind interaction between TCAN2PA and Hg (II) ions.

Materials and Instrumentations
All metal salts, chemicals, reagents and solvents of analytical grade were purchased from commercial sources and used without further purification. 4-tert-Butyl aniline and chloroacetyl chloride were obtained Sigma Aldrich. Fluorescence active TLC plates (F-2009) were obtained from the Merck. Fluorescence spectra were recorded on a Jasco FP-8300 spectrofluorometer (Tokyo, Japan) with EHCS-813. Absorption spectra were recorded using a Jasco V-750 UV-Vis recording spectrophotometer (Tokyo, Japan) with EHCS-750 in the range of 200-800 nm. [1] H NMR, [13] C NMR, and [13] C DEPT spectra were obtained on a Bruker AV-(III) -400 MHz spectrometer using a BBFO probe. Mass spectra were recorded on a Micromass Q-TOF micro mass spectrometer with a capillary voltage of 3000 V and a source temperature of 120 °C .

Synthesis of TCAN2PA
To a solution of thiacalix [4]arene (0.5 g, 0.00069 mol), K 2 CO 3 (0.19 g, 0.0014) in dry acetone, N-(4-(tert-butyl) phenyl)-2-chloroacetamide (0.31 g, 0.0014 mol) was added. The reaction mixture was then refluxed for 8 h. The progress of the reaction was monitored through TLC. After completion, the reaction mixture was cooled to room temperature, and the solvent was evaporated to dryness. The pure compound (Scheme 1) was obtained by silica gel column chromatography by using (hexane: ethyl acetate) as eluents (9:1) to get the solid product with 70% yield.

Spectrophotometric and Spectrofluorimetric Measurements
Stock solutions of TCAN2PA (2 × 10 -4 M) and a series of common metal ions (2 × 10 -3 M) including Fe(III), Cu(II), Cd(II), Zn(II), Cr(III), Ca(II), Co(II), Mg(II), Ag(I), Pb(II), Sr(II), Hg (II), Ba(II), K(I), Na(I), Bi(III) were prepared in acetonitrile. The stock solution of TCAN2PA was diluted to 5 µM for spectrophotometric study. The change in absorption spectra with the aforementioned cations (10 µM) were Scheme 1 Synthetic scheme for the preparation of TCAN2PA recorded in the range of 200 -500 nm. Moreover, the ligand was excited at 295 nm and the variation in emission responses were also investigated within the range of 350 -550 nm. The emissiontitration experiments were carried out by successive addition of mercury into the solution of TCAN2PA A Job's plot study was performed based on the absorption studies.

In Silico Insight
Molecular docking is an outstanding tool for computational modeling. Molecular docking can also be used to estimate the stability of host-guest complex that provides the structural descriptions of the host-guest complex to gain insights into the binding conformations, mechanistic information and the elucidation of ligand-induced conformational changes [35,36]. Designing of ligands with specific electrostatic and stereo chemical attributes can be developed by molecular docking to procure high receptor binding affinity. It imparts three-dimensional structures for scrupulous inspection of the topology at the binding site for the presence of clefts, cavities and sub-pockets. With molecular docking careful inspection of the electrostatic properties such as charge distribution can also be done [37,38].
Initially the chemical structures of TCAN2PA and Hg(II) were developed by using the geometry optimization technique. The developed structures of TCAN2PA and Hg(II) were then set as starting host and guest, respectively using the Schrodinger software (Maestro 12.0). 3D knowledgebased shapes from the superimposed pair of the host and guest are utilized to calculate the energy scores for the host-guest complex. The best docked score of the complex was retrieved by Hex 8.0 software. Identification of the complex possessing the lowest free energy is done. The equation used to calculate the binding energy is as under: The insights for the possible host-guest interactions are procured by the molecular docking, particularly the noncovalent interactions as they are not explained by the spectrofluorimetric results. Accelry's Discovery Studio visualizer version 16.1 is used to observe the non-covalent interactions that play a major role within the various host-guest interactions. It reveals that Π-lone pair, Π-alkyl, Π-Π stacking and Π-Π T-shape stacking, metal-donor interactions are the main interactions for the host-guest complex formation.

Molecular Dynamics (MD) Simulation
MD simulation was carried out using the Desmond v3.6 (Academic version) [39] package taking the complex with lowest free energy as an input. It is performed to know the ΔE total (kcalmol) = ΔE complex − Δ receptor − ΔE ligand thermodynamic stability of the complex. As per classical mechanics, MD implements Newton's equations of motion, to acquire the details of speed and position of each atom in the system under investigation. Thus, examination of the trajectory and temporal evolution of host-guest complex can be examined [40]. In the first step, a particular configuration is attributed to the atoms with the purpose of replicating the conditions like temperature and pressure of the real system. The position and velocity of each of the atoms at some points later are attainable from the computation of the forces that act on each particle. For the integration of the molecular trajectories for a given time interval, these calculations are repeatedly performed [41]. The simulation cell was created using the device builder module TIP3P (transferable intermolecular potential with three points) water model and the cubic periodic box containing the Simple Point Charge (SPC) (10 × 10 Å) with the Integrated Liquid Simulations Potential (OPLS) all-atom force field 2005. MD simulation for this case was carried out for a duration of 20 ns with a relation time if 1 ps at a constant temperature of 300 K, along with constant volume and shape (NVT) with a Nose-Hoover thermostat 9, constant volume, Smooth Particle Mesh Ewald (PME) method. After that root mean square deviation (RMSD), root means square fluctuation (RMSF), Hydrogen bond, a radius of gyration (Rg), the histogram for torsional bonds were analyzed for the identification of structural changes with the dynamic role of the selected host-guest complex [42,43].

Selectivity Towards Metal Ions
In order to obtain the selectivity of the probe, absorption as well as emission studies were performed. The receptor TCAN2PA exhibits an absorption band at 295 nm. The characteristic absorption band of TCAN2PA at 295 nm indicates a strong n → π transitions in both the phenolic groups of thiacalix [4]arene. Upon interaction with a series of common metal ions, including [Fe(III), Cu(II), Cd(II), Zn(II), Cr(III), Ca(II), Co(II), Mg(II), Ag(I), Pb(II), Sr(II), Hg(II), Ba(II), K(I), Na(I), Bi(III)] a significant red shift of 39 nm was observed only in the case of Hg(II), as shown in (Fig. 1a). The electronic perturbation induced by the coordination with -NH and -OH groups of TCAN2PA accelerates the sensing of Hg(II) ions [44,45]. This certain change in absorption band indicates strong interaction of mercury ions with the receptor TCAN2PA, whereas no change in the absorption band of TCAN2PA were observed upon interaction with other metal ions. On similar lines, upon excitation at 325 nm, the receptor TCAN2PA exhibits an emission maximum at 399 nm (Fig. 1b). A sharp enhancement in emission intensity of TCAN2PA was observed only with the addition of Hg (II) ions, whereas no change in emission maxima was observed with other metal ions. This enhancement in fluorescence intensity may be attributed to the transfer of electrons between electronic rich -NH and -OH group of the receptor to Hg(II) ions.

Sensitivity Towards Hg (II) Ions
Further to gain in depth insight about the sensitivity of TCAN2PA towards Hg(II) ions, fluorescence titration experiments were carried out (Fig. 2a). A significant enhancement in fluorescence intensity was observed upon gradual addition of mercury ions to the solution of TCAN2PA. The Stern-Volmer plot further shows that the probe TCAN2PA can detect mercury ions over a wide range of concentrations (0-10.0 µM) and the value of R 2 was determined to be 0.9970 (Fig. 2a, insert). The jobs plot analysis further reveals that the receptor TCAN2PA interacts with Hg(II) ions in 1:1 stoichiometry (Fig. S14). A plot of log [(F 0 -F)/ (F-F 1 )] versus log [M] help us to find out the binding constant (K S ), where F 0 and F 1 denotes the relative fluorescence intensities of the complex without addition of guest metal ion and with maximum concentration of metal ion [M], respectively. The binding constant for TCAN2PA:Hg(II) system was determined to be 1.8344 × 10 [6] M −1 , which indicates a strong binding interactions between host molecule and guest analyte [46][47][48][49][50]. It is noteworthy to mention that no significant interference of the competitive metal ions was observed during the sensing process (Fig. 2b). The LOD was determined to be 52.9 nM based on the emission titration plot along with the equation DL = 3σ/slope (where, σ is standard deviation of blank reading). A comparison between some recently reported probes toward the recognition of Hg(II), their LOD values and detection methods have been incorporated as Table 1.

In Silico Metal Ion Sensing
Initially, the geometry of the host molecule was optimized and then the Hg(II) ion was incorporated during the docking study. The pose with the lowest energy and most favourable geometry have been captured using Accelry's Discovery Studio visualizer. The geometry optimized structures of the receptor have been displayed as (Fig. 3). Among all the cations, the lowest docking energy were obtained for the TCAN2PA-Hg(II) system, suggesting stable complex formation between them. This lowest energy complex formation only with Hg(II) ions further support the experimental observation and signifies the selectivity of the probe. Host-guest interactions reveal that the complex formation between TCAN2PA and Hg(II) ions is due to one type of interaction namely, metal-donor in two pair with the distance of 2.22 and 2.43 Å (Fig. 4).   respectively. Further, the statistical analysis has revealed the fluctuation and contribution of its higher stability except total energy which possessed exponential growth Fig. S15.
To assess the structural integrity, favourable conformational space and stability of the host-guest inclusion complex MD simulations were performed in a water solvent system. During this event, various parameters were identified which contained intramolecular hydrogen bonds, coulomb, van-der Waals interactions and total energy at 20 ns time interval (Fig. 5).

Sensing Mechanism
The receptor TCAN2PA exhibits weak fluorescence at 399 nm. Upon interaction with Hg(II) ions, an enhancement in fluorescence intensity was observed. This may be due to the inhibition of photo induced electron transfer (PET) from receptor to mercury ions. The molecular docking studies further indicates that the electron rich -OH and -NH sites of the receptor involve in complexation with Hg(II) ions (Fig. 6). This study indicates that upon complexation with mercury ions, the receptor became more rigid, which may lead toward the enhancement in emission maxima.

Conclusion
In summary, a novel thiacalix [4]arene N-(4-(tert-butyl) phenyl)-2-chloroacetamide (TCAN2PA) has been designed and synthesized toward the selective recognition of Hg(II) ions. A red shift in absorption maxima followed by the significant enhancement in emission intensity indicates its exceptional selectivity toward Hg(II) ion over a series of common metal ions. The emission titration experiments reveal that this fluorescent "Turn-ON" sensing probe strongly bind Hg(II) ions, as the binding constant was found to be 1.8344 × 10 6 M −1 . The binding stoichiometry for TCAN2PA-Hg(II) system was determined to be 1:1. Additionally, this thiacalix based probe can determines Hg(II) concentrations up to (0-10 µM) The molecular docking studies further confirms that the -OH groups associate to the lower rim of thiaxalix and the "N" sites of the fluorophore strongly involve in binding interaction of mercury ions. Theoretical studies further predict a lowest energy complex for Hg(II) ions which supports the enhancement in the emission intensity by Hg(II) signifying a stable complex of TCAN2PA with Hg(II) ions. Therefore, this probe can act as an efficient probe towards the sensing of Hg(II) ions.