DFT calculation , biological activity , anion sensing studies and crystal structure of ( E )-4-chloro-2-[ ( pyridin-2-ylimino )-methyl ] phenol

(E)-4-Chloro-2-[(pyridin-2-ylimino)methyl]phenol was synthesized in the reaction of 2-aminopyridine with 5-chlorosalicylaldehyde. The structure of compound was investigated by FTIR, UV–Vis, 1H-NMR, 13C-NMR and X-ray data. In addition, characterization of the compound was realized using theoretical quantum mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV–Vis, which were in good agreement with the structure predicted by the theoretical calculations using the density functional theory (DFT). Moreover, the antimicrobial activity of the compound was investigated against some bacteria and yeast cultures by the broth microdilution test. UV–Vis spectroscopy studies of the interactions between the Schiff base and calf thymus DNA (CT-DNA) showed that the compound interacts with CT-DNA via electrostatic binding. The colorimetric response of the compound receptors was investigated before and after the addition of an equivalent amount of each anion to evaluate anion recognition properties.


INTRODUCTION
2][3][4][5] Applications of them as chemical nuc-leases is the focus of current research.][12] The tautomeric balance and intramolecular hydrogen bond in Schiff bases occur through proton transfer, leading the compound to exhibit interesting physical and biological properties that have attracted the attention of researchers.Studies on the physical and chemical properties of Schiff bases in solid state and in solution continue.Molecules creating intramolecular bonds 13 are used in highly specific systems because of their high thermodynamic stability. 14chiff bases are important to synthesize sensors for recognizing and sensing anions, and to enlarge their areas of use.][17] It could be specifically stated that the keto-amine and enol-imine tautomer balances contribute to the bonding and selection of anions.Undoubtedly, colorimetric anion sensors are of more importance, because such materials are useful as they provide visual information more readily.Various studies have been performed in order to develop these materials. 18,19n the present work, the Schiff base (E)-4-chloro-2-[(pyridin-2-ylimino)methyl]phenol (Fig. S-1, of the Supplementary material to this paper) was prepared and studied by experimental (UV-Vis, FTIR, NMR and X-ray diffraction) and computational (density functional theory (DFT)) methods.The molecular structure, HOMO-LUMO analysis, molecular electrostatic potential (MEP) and nonlinear optical (NLO) effects of the compound were investigated using DFT calculations.Additionally, the Schiff base was tested for its potential sensor ability to sense anions selectively and for its interaction with DNA.

Materials and methods
The 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance-500 spectrometer operating at 400 and 101.6 MHz, respectively.The infrared absorption spectra were obtained in KBr discs using a Perkin Elmer BX II spectrometer and are reported in cm -1 units.The UV-Vis spectra were measured using a Shimadzu 1800 series spectrometer.Elementary analyses were performed on a Vario EL III CHNS elemental analyzer.The melting points were measured with an Electro Thermal IA 9100 apparatus using a capillary tube.2-Aminopyridine, 5-chlorosalicylaldehyde, DMSO, EtOH, CHCl Analytical and spectral data are given in the Supplementary material.
The product was obtained by evaporation of the EtOH.It was crystallized from CHCl 3 :n-hexane (3:2 volume ratio) as orange crystals.

X-Ray crystallography
A suitable single crystal of the title compound was mounted on a goniometer and the data were collected at 293 K on a Bruker Kappa APEXII CCD diffractometer using graphite monochromated MoK α radiation (λ = 0.71073 Å).The cell parameters of the compound were determined using SAINT software. 20Absorption correction (μ = 0.34 mm -1 ) was obtained by the multi-scan method via SADAPS V2012/1 software. 21The compound is solved by direct methods using SHELXS-97 22 and refined with SHELXL-97. 22The molecular figures were prepared with the help of Mercury and ORTEP-3 program packages. 23,24Details of the data collection conditions and the parameters of the refinement process are given in Table I. 0.65, -0.60

Computational procedures
All theoretical computations were realized using Gauss-View5 25 molecular visualization and the Gaussian 09W program package. 26For the calculation of the molecule geometry, the atomic coordinates obtained from the X-ray geometry were used.The geometry optimization of the title molecule was performed using the DFT method with the Becke three parameters hybrid exchange-correlation functional (B3LYP) 27 at the 6-311++G(d,p) basis set. 28To investigate the reactive sites and to identify sites of intra and intermolecular interactions of the compound, the molecular electrostatic potential surface was evaluated using the B3LYP/6--311++G(d,p) method.The HOMO-1, HOMO, LUMO and LUMO+1 energy values and their shapes were calculated and simulated using the same basis set.The inocula were adjusted to a turbidity of 0.5 McFarland standard and diluted to a final concentration from 4×10 5 to 8×10 5 CFU mL -1 .The compound was dissolved in DMSO (dimethyl sulfoxide) and serially diluted in cation-adjusted Mueller-Hinton broth for the bacteria or RPMI medium for the yeasts in 96 well plates to a final volume of 100 µL in each well.Then, an equal volume of inocula was added to each well.The concentration of the compound, including control antibiotics ampicillin and antifungal fluconazole ranged between 0.5-256 µg mL -1 .The plates were incubated at 37 °C and the results were evaluated after 16-20 h for the bacteria and 24±2 h for the fungi and the MIC values were recorded.

DNA-binding experiments
The UV-Vis spectra titrations were performed in Tris-HCl/NaCl buffer at room temperature to investigate the binding affinity between CT-DNA and the Schiff base.The UV-Vis absorbance at 260 and 280 nm of the CT-DNA solution in Tris buffer give a ratio of 1.8-1.9,indicating that the DNA was sufficiently free of protein. 30Tris-HCl/NaCl buffer (3 mL) and the solutions of Schiff base of buffered CT-DNA solution were added to each cuvette in order to eliminate the absorbance of DNA itself.Before the absorption spectra were recorded, the Schiff base-DNA solutions were incubated at room temperature for 5 min.

Description of the crystal structure
The title compound crystallizes in the monoclinic space group P2 1 /n with Z = 4 in the unit cell.The asymmetric unit in the crystal structure contains only one molecule.It is known that tautomeric forms of Schiff base compounds have two types of intramolecular hydrogen bonds, which belong to O-H … N in phenol-imine form and N-H … O in the keto-amine form.The single-crystal X-ray study showed that the title compound adopts the phenol-imine tautomeric form.The C7-N1 and C2-O1 bond lengths have a significant influence in determining the tautomeric form.The C7-N1 bond distance (1.278(3) Å) is consistent with a C=N double bond and the C2-O1 bond (1.353(3) Å) is consistent with a C-O single bond.2][33] The dihedral angle between the C1-C6 and pyridine rings (C8-N2) is 4.38(8)°.The molecular structure is stabilized by an intra-molecular O1-H1 … N1 hydrogen bond (Fig. 1).In the crystal structure, molecules are linked to each other by an intermolecular C-H … O hydrogen bond.Atom C7 in the reference molecule at (x, y, z) acts as a hydrogen--bond donor, via H7, to atom O1 in the molecule at (x-1/2, -y+3/2, z-1/2), so forming a C(5) chain running parallel to the [001] direction (Fig. S-6 of the Supplementary material).Details of the hydrogen bonds are summarized in Table II.Optimized structure Geometric optimization of the investigated compound was performed using the DFT/B3LYP method with the 6-311++G(d,p) basis set (Fig. S-7 of the Supplementary material).Some of the bond lengths, bond angles and torsion angles of the optimized structure are listed in Table III and compared with the experimental data of the compound.As can be seen from Table III, most of the molecular geometric parameters are slightly different from the experimental ones.The biggest differences between experimental and calculated bond lengths and angles are 0.058 Å for the C10-C11 bond and 5.3° for the C10-C9-C8 angle, respectively.According to crystallographic studies, the dihedral angle between the C1-C6 and pyridine rings is 4.38(8)°, while this angle was calcul-ated as 41.75° for optimized structure.In order to compare the theoretical results with the experimental values, the root mean square error (RMSE) was used.The calculated RMSE for bond lengths and bond angles are 0.026 Å and 2.34°, respectively.These differences could result from the environments in which experimental and theoretical data were obtained.Namely, the theoretical computations were performed in gaseous phase for isolated molecules, whereas the experimental analyses were recorded in the solid phase of the title molecule.The inter--molecular hydrogen bonding interactions in the solid phase (the C7-H7 … O1 hydrogen bonds depicted in Fig. S-6) were ignored in the theoretical calculations.Due to the O1-H1 … N1 intra-molecular hydrogen bonding interaction, the C2-C1-C7-N1 torsional angle was recorded and calculated as -2.3 and 0.6°, respectively.However, the N2-C8-N1-C7 torsional angles between other groups and pyridine ring (do not exposure any interaction within the theoretical calculations) were measured and computed as 178.0 and 140.4°, respectively.

HOMO-LUMO analyses and tautomeric stability
The highest occupied molecular orbital (HOMO) represents the outermost orbital filled by electrons and behaves as an electron donor, while the lowest unoccupied molecular orbital (LUMO), considered as the first empty innermost orbital unfilled by electron, behaves as an electron acceptor.In this study, the HOMO-1, HOMO, LUMO and LUMO+1 energies and their shapes in the compound are shown in Fig. 2. As seen from Fig. 2, HOMO-1, HOMO and LUMO electrons are localized over almost the whole molecule, whereas LUMO+1 ones are placed on other groups, excepting the 5-chloro-2-hydroxyphenyl ring.The concept of chemical hardness is quite useful in explaining chemical stability.Molecules having a large HOMO-LUMO energy gap will be more stable and less reactive than soft molecules having a small HOMO-LUMO energy gap. 34,35o investigate the tautomeric stability, optimization calculations at B3LYP/6--311++G(d,p) level were performed for O-H•••N in the phenol-imine (OH) and for N-H•••O in the keto-amine (NH) forms of the compound.In addition, the total energy, I (ionization potential), A (electron affinity), χ (electronegativity), ζ (softness), ψ (electrophilicity index) and η (chemical hardness) 36 were calculated at the same level and the results are given in Table IV.The total energy of the OH form is lower than that of the NH form, while the chemical hardness of the OH form is greater than that of the NH form, which indicates that the OH form of the compound is more stable than its NH form in the gas phase. 15,37vailable on line at www.shd.org.rs/JSCS/The molecular electrostatic potential V(r) is created in the space around a molecule by its nuclei and electrons.It is defined by Eq. (1): in which Z A is the charge of nucleus A, located at R A , ) (r′ ρ is the electronic density function of the molecule, and r′ is the dummy integration variable. 38P is related to the electronic density and is a very useful descriptor in determining sites for electrophilic and nucleophilic reactions as well as hydrogen bonding interactions. 39,40The MEP at the B3LYP/6-311++G(d,p) optimized geometry was calculated.
The red color parts represent the negative electrostatic potential regions or electrophilic reactivity, while blue ones represent the positive ones or nucleophilic reactivity. 41As could be seen in Fig. 3, a negative region of the compound was observed around the pyridine rings N2 atom.The negative V(r) value is -0.048 a.u.for the N2 atom.A maximum positive region localized on the C7-H7 bond, indicates a possible site for nucleophilic attack.These values give information about the region from where the compound could have intermolecular interactions.Thus, Fig. 3 confirms the existence of the intermolecular C-H … O hydrogen bond interactions.

Non-linear optical effects
NLO materials have potential applications in optical technology and industrial applications. 42The NLO values for the compound were calculated at the B3LYP/6-311++G(d,p) level using the Gaussian 09W program package.The equations for calculating the magnitude of total static dipole moment μ total , the mean polarizability <α>, the anisotropy of the polarizability ∆α, and the mean first hyperpolarizability (β) using the x, y, z components are defined as: 43,44 3 It is well known that higher values of the linear polarizability and the first hyperpolarizability are important key factors for molecules with effective NLO properties.The polarizabilities and first hyperpolarizability are reported in terms of atomic units (a.u.) and the calculated values were converted using 1 a.u = = 0.1482×10 -24 electrostatic unit (esu) for α and 1 a.u = 8.6393×10 -33 esu for β.
The calculated mean polarizability <α>, the anisotropy of the polarizability (∆α) and the first hyperpolarizability (β) for the studied compound are 28.5354266×10 -2 , 55.741745×10 -24 and 77.5621354×10 -31 esu, respectively (Table V).Urea is one of the essential molecules used for the determination of the NLO properties of molecular systems.Therefore, it is usually used as a reference molecule in NLO studies.The calculated values of urea with the same basis set are 4.9066694×10 -24 esu for <α> and 7.8781964×10 -31 esu for β.According to these results, the mean polarizability and first hyperpolarizability of the studied compound are approximately 5.81 and 9.845 times greater than those of urea, which implies that the title compound is a good candidate material for NLO applications.FTIR, 1 H-NMR, 13 C-NMR and UV spectroscopy The vibration bands with the wave numbers 3433 cm -1 (broad strong, O-H); 3107-3046 cm -1 (weak, Ar-H); 1560 cm -1 (strong, C=C); 1478 cm -1 (strong, C-N) and 1356 cm -1 (strong, Ar-O) were observed for the compound (Fig. S-2).The C=N bond was observed at 1616 cm -1 for the compound.The stretching frequency observed at 2852-2750 cm -1 in the spectrum of the compound showed the presence of O-H … N intramolecular hydrogen bonds. 45,46The C=N bond, which is partially accountable for the existence of the phenol-imine form, can also be inferred from the IR spectrum of the compound.A compound with strong band at 1274 cm -1 possessed high percentages of the phenol-imine tautomer due to stabilization of the phenolic C-O bond. 47he 1 H-NMR data for the compound show that the tautomeric equilibrium favors the phenol-imine form in DMSO (Fig. S-3).The OH proton was observed as a singlet at 12.49 ppm for the compound.The azomethine proton was observed as a singlet at 8.94 ppm for the compound.The phenyl protons of the compound resonated as multiplet between 8.60-6.96ppm.
According to the proton de-coupled 13 C-NMR spectra, the compound has 12 signals in deutero-DMSO (Fig. S-4).The azomethine carbon (ArCH=N-) is observed at δ = 164.15ppm for the Schiff base.In the investigated compound, the aromatic-C chemical shifts (δ / ppm) are 164.15 The UV-Vis spectra of the compound were studied in DMSO (Fig. S-5).Schiff bases exhibited absorptions in the range greater than 400 nm in polar and nonpolar solvents.6][47] The title compound showed no absorption above 400 nm in DMSO, indicating that the compound exists in the phenol-imine form in DMSO.In conclusion, UV-Vis, 1 H-NMR and 13 C-NMR results showed that the compound existed in the phenol-imine form in DMSO.

Minimum inhibitory concentration (MIC)
The MIC was evaluated by the broth micro dilution test.The MIC values were read as the lowest concentration of the drug in the series that prevents the development of visible growth of the test organism.The data reported in Table VI are the average from three experiments.
The antimicrobial activity spectrum of the Schiff base varied in the concentration range 32-128 µg mL -1 .It could be observed from Table VI that the Schiff base exhibited a high antifungal effect on C. albicans and C. tropicalis, while the compound had a low effect on the bacteria.However, the compound had stronger antibacterial effect against S. aureus, E. faecalis, B. cereus NRRL B-3711, E. coli ATCC 25922, E. coli ATCC 35218, P. aeruginosa and P. hauseri compared to B. subtilis.Furthermore, this compound showed similar activity against the tested microorganisms, despite the fact that the cell wall in Gram-positive bacteria have a single layer, whereas the Gram-negative cell wall is a multi-layered structure, and the yeast cell wall is quite complex.The antifungal activity of the compound was found to be dose dependent with MIC of 32 µg mL -1 .

DNA-binding
The potential binding ability of the compound to CT-DNA was characterized by UV spectroscopy.The absorption spectra of the ligand in the absence and presence of CT-DNA at different concentrations are given in Fig. 4. Absorption spectroscopy is one of the most used methods for investigating the effects of any material on DNA.If it has an intercalation effect against DNA, generally a hypochromic effect is observed.However, if the interaction of a material with DNA is electrostatic or partially intercalative, a hyperchromic effect is observed. 4,5,15oreover, a red shift of the absorption maximum indicates that the difference between HOMO and LUMO energy levels decreases, and that the complex interacts with DNA. 4 The absorption spectra of the Schiff base in the absence and the presence of CT-DNA are shown in Fig. 4. In the UV region, two intense bands absorbed at 269 and 386 nm for the ligand.In the presence of CT-DNA, an increase in the peak intensities was observed in the absorption spectra of Schiff base.In addition to the increase in intensity, a small red shift (bathochromism; 1-7 nm) was also observed in the spectra for Schiff base.The absorption intensity of the band at 269 nm of the ligand increased (hyperchromism) evidently after the addition of CT-DNA (0, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 µL), which indicated the interactions between DNA and the Schiff base (Fig. 4).The extent of red shift and hyperchromism are commonly found to correlate with the electrostatic binding strength.Consequently, the observation of hyperchromic effect in the absorption spectra implies that the ligand established an electrostatic bond with DNA.In the electrostatic binding, DNA breaks phenol protons from the more acidic Schiff base.As a result, the negative charge distribution is changed in the DNA chain, thereby, leading to disruption of the DNA molecules.

Colorimetric anion-sensing
In order to investigate whether the Schiff base could be used for visual confirmation of anions in a sample, real color photographs of the corresponding DMSO solutions of the compound with F -, Br -, I -, CN -, SCN -, ClO 4 -, HSO 4 -, ________________________________________________________________________________________________________________________ (CC) 2018 SCS.
Available on line at www.shd.org.rs/JSCS/CHARACTERIZATION AND APPLICATION OF (E)-4-CHLORO-2-[(PYRIDIN-2-YLIMINO)METHYL]PHENOL 719 N 3 -, AcO -, H 2 PO 4 -and OH -as tetrabutylammonium salts were taken and are shown in Fig. 5.After addition of the anions to the Schiff base in DMSO, the color of the solution changed from colorless to yellow and orange with F -, AcO -, OH -and CN -with fast response time (< 1 s), indicating that receptors in the title compound could serve as a "naked-eye" indicator for F -, AcO -, OH -and CN -.
The more acidic phenol proton would deprotonate upon exposure to more basic OH -, F -and AcO -and therefore, intramolecular proton transfer occurs into the keto-amine form.In contrast, CN -has much weaker hydrogen bonding ability in comparison with OH -, F -and AcO -with a stronger nucleophilicity toward the imine group, which results in the addition reaction of CN -to the carbon atom of an electron deficient imine group and, subsequently, fast proton transfer of the phenol hydrogen to the neighboring nitrogen anion through an intramolecular hydrogen bond. 15The formation of the keto-amine form of the compound leads to higher wavelength absorptions.

Fig. 2 .
Fig. 2. The molecular orbital surfaces and their energy values of the title compound.

Fig. 4 .
Fig. 4. Absorption spectra of the compound in the absence and presence of increasing amounts of CT-DNA at room temperature in Tris-HCl/NaCl buffer (pH 7.2).
720 Y I L D I R I M et al.

TABLE I .
Crystallographic data and structure refinement for the title compound Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 35218, Bacillus cereus NRRL B-3711, Proteus hauseri ATCC 13315, Candida albicans ATCC 60193 and Candida tropicalis ATCC 13803 were used as the test microorganisms.The MIC tests were performed by the broth microdilution method in triplicate as outlined in the CLSI guidelines.

TABLE III .
Some selected molecular structure parameters

TABLE IV .
The calculated quantum molecular descriptors of the title compound

TABLE VI .
MIC value (µg mL -1 ) of the compound