Remarkable sensing behavior of pyrazole-based chemosensor towards Cu(II) ion detection: Synthesis, characterization and theoretical investigations

Graphical Abstract


Introduction
Cost effective colorimetric chemical sensors with simple design have attracted a tremendous interest in monitoring many environmental and biologically relevant species such as metal ions over the past few years [1][2][3][4].When compared with electrochemical and fluorescent chemo sensors, the colorimetric methods are more promising for the detection of metal ions especially in field works because of naked eye detection.The Cu 2+ ion is one of the most important divalent metal ions in the human body [5].The pivotal role of Cu 2+ ion in most of the living organisms includes its key role in metabolism and immune system [6].On the other hand, the presence of excess of Cu 2+ ion concentration in the organisms may cause lethal issues [7,8].Several methods such as atomic absorption spectroscopy [9], inductively coupled plasma mass spectrometry (ICP-MS) [10], inductively coupled plasma atomic emission spectrometry (ICP-AES) [11] are available for the determination of metal ions with some major disadvantages like sample destruction, high cost, necessary pretreatment procedures etc.Furthermore, there are many reports about the synthesis and use of chemosensors as metal ion probes with low to moderate detection ability [12][13][14][15].Therefore, the design and synthesis of low cost, non-natural sensors for Cu 2+ ion detection is important and being continuous interest for researchers as it is a challenging task in terms of obtaining selective and specific sensor.
Considerable attention has been extended over a decade to the heterocycles containing more than one hetero-atom, namely, imidazole, pyrazole, pyrazine, pyrimidine etc. with potential donor sites to form various metal coordination complexes.In these type of interaction, the π-electrons on the heteroatom actively participate in the formation of stable metal-ligand (M-L) bonds during metallation [16].Since, pyrazole and its derivatives act as stronger π-donor and weaker π-acceptor than the six membered heterocyclic analogues they behave as hard donors during complexation with metal ion.The multidimensional applications of pyrazole derivatives and their coordination compounds have witnessed in diverse disciplines of sciences [17][18][19][20].The electronic behavior of pyrazole based ligands and their metal complexes are of prime interest because of typical behavior of pyrazole backbone in stabilizing the electronic and optical properties of metal complexes [21][22][23].DFT calculations have been widely accepted as indispensable computational tools for prediction of reactive properties of new molecules, complexes and nanomaterials [24][25][26][27][28][29][30][31].
In the present study, we have synthesized and characterized HL and its Cu(II) complexes (1 and 2).Further, the HL is used as colorimetric sensor for the detection of Cu 2+ ion in the presence of various competing ions.We also report the sensitivity of HL in Cu 2+ ion detection, and is found to be 1.6 μM which is much lower than the recommended value (31.5 μM) of the World Health Organization for drinking water.We also performed the calculations of quantum-molecular descriptors that describe local reactivity included molecular electrostatic potential (MEP) and local average ionization energies (ALIE) obtained by mapping of their values to the electron density surface.In order to better understand fundamental properties of HL ligand, additionally Fukui functions and bond dissociation energies for hydrogen abstraction (H-BDE) have been investigated as well.

Material and methods
3-Amino-5-hydroxypyrazole and m-anisaldehyde were purchased from Sigma Aldrich and were used as received.The solvents were purchased from Merck and used without further purification.
The completion of reaction was monitored by thin layer chromatography (TLC) performed on precoated silica-gel plates (Merck, India) and spots were visualized by UV irradiation.FT-IR spectral measurements were recorded on Perkin-Elmer spectrometer version 10.03.09 (KBr pellet technique). 1 H and 13 C NMR were obtained using a 500 MHz Bruker Avance DPX spectrometer with TMS as internal standard.HR-ESI-MS analysis was performed on a Thermo Scientific Exactive mass spectrometer by electrospray ionization technique.The electronic absorption spectra were recorded using UV1800 spectrophotometer (Shimadzu).Steady-state fluorescence experiments were performed with a SPEX Fluorolog F112X spectrofluorimeter by using optically dilute solutions.

Computational Details
DFT calculations on HL ligand and its corresponding Cu(II) complexes have been performed with Jaguar [32][33][34] program.Prior to DFT calculations all possible conformers of HL were generated with MacroModel [35] program using OPLS3 [36][37][38][39] force field.Obtained conformers were then geometrically optimized within the framework of DFT with B3LYP [40] exchange-correlation functional.The lowest energy conformation was checked for its ground state by vibrational analysis which yielded only positive frequencies.Vibrational analysis was also used for 1 and 2 complexes in order to confirm their true ground states, which also yielded only positive frequencies.For all DFT calculations a LACVP(d,p) basis set was employed.Maestro [41] program was used for preparation of input files and visualization of results.Maestro, MacroModel and Jaguar programs were used as incorporated in Schrödinger Materials Science Suite 2017-4 [42].

Chemical synthesis 2.3.1. Synthesis of (E)-3-((3-methoxybenzylidene)amino)-1H-pyrazol-5-ol (HL)
The title compound HL was synthesized as per Scheme 1.To a solution of 3-amino-1H-pyrazol-5-ol (100 mg, 1 mmol) in 20 ml of ethanol, a solution of m-anisaldehyde (137.8 mg, 117 μl, 1 mmol) in ethanol (15 ml) was added with continuous stirring and the reaction mixture was refluxed for 10 h in the presence of catalytic amount of acetic acid.The solid product formed was washed with ethanol and recrystallized with hot ethanol to obtain desired product in pure form.

Results and discussion
The ligand (HL) was obtained according to the synthetic route showed in Scheme 1.The spectroscopic data provided information in deducing molecular structure of compounds.The proposed structures of 1 and 2 are showed in Fig. 1.

NMR studies
One of the surprising observations is that the ligand existed in dimeric state which is evident from  A clear evidence for dimer formation could be seen by analyzing the NMR spectra of HL (Fig. 2).The aromatic peaks of the compound are shown as inset which consists of four pairs of peaks with similar integration and similar splitting nature.Surprisingly, all these peaks had an integration of one proton, suggesting that each pair of these peaks belong to same kind of protons from two different parts of dimer.The picture was more clear when we recorded the HRMS of the HL, which even in dilute solution showed two prominent mass peaks which corresponds to monomeric and dimeric species (Fig. 3).

IR studies
The IR spectra for both liand and its copper complexes are shown in Fig. 4. The IR band CH=N which appeared at 1592 cm -1 for the free ligand (HL) showed a considerable shift towards higher wave numbers (1609 cm -1 in 1 and 1693 cm -1 in 2) which suggests that the azomethine group is involved in the coordination to copper ion in both the complexes.Further, the peak around 3200 cm -1 due to -OH and -NH of ligand became further broad possibly due to the involvement of -NH group for the coordination of copper centre.

Photophysical investigations
In order to evaluate the photophysical properties of ligand and metal complexes, the electronic absorption and emission spectra were recorded using their DMSO solution.As shown in Fig. 5 the HL showed characteristic absorption around 250 nm to 290 nm with emission extending up to 400 nm.Further, the absorption spectrum of 1 showed a broad peak extending upto 450 nm while the complex 2 showed a small new band near 420 nm.The emission profile of the two complexes were similar to parent ligand but the presence of metal has essentially quenched the ligands emission.

Figure 5:
The absorption (i) and emission (ii) spectra of HL and its complexes, 1 and 2 recorded in DMSO.

Local reactivity properties of investigated structures
MEP surfaces are frequently used descriptors for description of local reactivity properties.This descriptor is visualized by mapping of its values to the electron density surface and is regarded as a fundamental quantity describing reactivity of molecules.MEP, V(r), is defined according to the next equation: neglecting polarization and nuclear rearrangement effects due to the presence of a unit test charge at distance r.In equation ( 1 [45,46], according to the following equation: 2) represents the electronic density of the i-th molecular orbital at the point r  , i  represents the orbital energy, while   r   is the total electronic density function.
The identification of the molecule sites prone to electrophilic attacks is certainly the most efficient if both MEP and ALIE surfaces are employed, which has been provided in Fig. 6 for HL ligand and Fig. 7 for its complexes (1 and 2).MEP surfaces of investigated complexes indicate that 1 might have somewhat higher sensitivity towards electrophilic attacks.Namely, the lowest MEP value in case of the 1 is lower for 5 kcal/mol in comparison with 2. However, 2 has much lower minimal ALIE value, more than attacks.Concerning locations of the minimal MEP values for 1 complex, they are located in the near vicinity of Cu atom and nitrogen atoms of pyrazole rings.These locations are also recognized by ALIE surface, however this descriptor also recognizes certain locations above benzene rings also to be sensitive to electrophilic attacks.Concerning locations of minimal values of MEP and ALIE in case of the 2, they are practically identical and located in the near vicinity of Cu atom, except the fact that MEP also indicates electrophilic sensitivity of oxygen atom connecting methyl group.
Additionally, local reactive properties of HL ligand were investigated employing the Fukui functions as well.In Jaguar program for DFT calculations, Fukui functions are calculated in finite difference approximation according to the following equations: where N denotes the number of electrons in the reference state of the molecule and  represents the fraction of electron, which is set to be 0.01 [47].Fukui functions of HL ligand are presented in Fig. 8. Taking into account the importance of oxidative reactions, in this work we have also used DFT calculations to locate molecular sites of HL ligand possible sensitive towards the autoxidation mechanism.Autoxidation mechanism is of great industrial importance and the ability to predict sensitivity of molecular structure towards it is very significant.Autoxidation is correlated with bond dissociation energies for hydrogen abstraction (H-BDE), a parameter which can be used for prediction of sensitivity of molecule towards this mechanism.Molecular sites where H-BDE takes values between 70 kcal/mol to 85 kcal/mol are considered to be highly sensitive towards autoxidation [48,49], while H-BDE values between 85 kcal/mol to 90 kcal/mol might indicate certain sensitivity towards autoxidation, but other influences might prevent the autoxidation [49].
It should also be mentioned that H-BDE values lower than 70 kcal/mol are not indicating sensitivity towards autoxidation mechanism [24,27,48].H-BDE values for HL ligand have been summarized in Fig. 9. H-BDE values provided in Fig. 9 indicate that HL ligand is highly sensitive towards autoxidation mechanism due to the fact that BDE for hydrogen abstraction of hydrogen atom H27 is 76 kcal/mol.Other H-BDE values are much higher than the desired upper border level of 90 kcal/mol.Second lowest H-BDE value has been calculated for hydrogen atom of pyrazole ring, but this value is almost 7 kcal/mol higher than the upper border level.

Cation sensing studies of HL
The cation sensing of the chemosensor HL was studied by UV-Visible spectroscopy in their water/DMSO (9:1, V/V) solutions.The HL shows two characteristic absorption bands located near 275 and 282 nm in its water/DMSO (9:1, V/V) solution.The change in the absorption spectrum of HL (10 μM) upon addition of aqueous Cu 2+ solution is showed in Fig. 10.We observed a linear behavior (Fig. 10b) in this mole ratio plot until the ratio of ligand to metal concentration is 1:2, after which absorption showed a sharp dip with further increase in the concentration of metal ions (Figure 11b).The mass spectrometry suggested that the stoichiometry of complex is 2:1 (ligand:metal) but the linear behavior of the reaction until the [ Cu 2+ ] × 4 times excess, suggests that more metal may be required in order to break the dimeric structure of HL to be available for complex formation.The selectivity of HL towards Cu 2+ ions (red trace) and no such distinguishable changes could be observed during the reaction of HL with other tested metal ions (Fig. 12).The phenomenon is more evident from the picture taken from the solutions of HL and target metal ions.We could see immediate brown colorization of the solution only in case of Cu 2+ ions.We also by UV-Visible studies, the detection limit of HL for Cu 2+ was found to be 1.6 μM, which is much lower than the recommended value (31.5 μM) of the World Health Organization (WHO) in drinking water.

Conclusions
To sum up, a Schiff base ligand, 3-((3-methoxybenzylidene)amino)-1H-pyrazol-5-ol, HL and its Cu(II) complexes, 1 and 2 were synthesized and characterized.This study has presented a colorimetric sensor for Cu 2+ ion detection.The electronic absorption studies shown that the detection limit of novel ligand, HL for Cu 2+ ion was found to be 1.6 μM, which is much lower

its 1 H
and ESI-MS spectra.[The repeated purification of the ligand resulted in similar NMR and Mass spectra suggesting that the observed peaks are not due to impurities].The formation of stable 6 membered ring structures because of hydrogen bonding interaction between pyrazole moieties may be the reason for dimer formation and the proposed structure of dimer is shown along with NMR spectra.

Figure 2 :
Figure 2: The proton NMR spectrum of compound HL.

Figure 3 :
Figure 3: The ESI-MS spectrum of compound HL showing monomeric and dimeric species.

Figure 4 :
Figure 4: The IR spectra of the HL and Cu(II) complexes, 1 and 2.

Figure 10 :
Figure 10: (a) The change in absorption spectrum of HL (10 μM) upon addition of aqueous Cu 2+

Figure 11 :
Figure 11: (a) the change in absorption spectrum of HL (10 μM) upon addition of aqueous Cu 2+

Figure 12 :
Figure 12: The UV-Visible spectra of HL in the presence of two equivalents of Cu 2+ (red trace) than the recommended value (31.5 μM) of the World Health Organization in drinking water, thus, can find significant application in the field work as a promising probe.According to MEP and ALIE surfaces HL has several important reactive molecular sites sensitive to electrophilic attacks.
metal ionsThe lowest ALIE values were ~188 kcal/mol and this descriptor predicted more reactive sites.Fukui f+ function of HL indicates that electron density increases in the near vicinity of C9-H17 bond, while Fukui f-function indicates that electron density decreases in the near vicinity of C1-C2 bond.H-BDE descriptor indicates very high sensitivity of HL to autoxidation mechanism.While both MEP and ALIE descriptors have low minimal values, indicating sensitivity towards electrophilic attacks, complex 2 has minimal ALIE values lower for more than 15 kcal/mol comparing to complex 1, indicating much higher reactivity.