Spectroscopic Evaluation of Chalcone Derivatives and their Zinc Metal Complexes: A Combined Experimental and Computational Approach on the Binding of the Complexes with the Serum Albumin

Three chalcone derivatives (L 1 , L 2 , L 3 ) were synthesized using Claisen-Schmidt condensation reaction. Their molecular structures and spectroscopic properties (IR, UV-vis, 1 HNMR), were calculated at B3LYP level. Electrostatic interactions and HOMO-LUMO properties were calculated using TD-DFT method. Molecular docking was used to compare the HSA (human serum albumin) interactions with the ligands and their Zn complexes (C 1 , C 2 , C 3 ) which were synthesized by interaction between the ligands and the Zn (II) ion in a 2:1 M ratio. Elemental analysis, FT-IR, and UV–Vis spectroscopy studies investigated the structure of the synthesized complexes. UV–Vis, molecular docking and molecular dynamics were used to study the interactions of the Zn complexes with the BSA (bovine serum albumin). The biological activity of the Zn-Chalcone complexes was generally higher than the chalcones when evaluated spectroscopically and theoretically.

HSA is responsible to maintaining stable osmotic pressure and to carrying endogenous or exogenous compounds. Additionally, most lipophilic drugs are substrates of HSA and are transferred to target tissues via the bloodstream. The chemical structure of HSA includes three homologous α-helical domains (I, II and III), each of which possesses two subdomains (A and B) [14]. Various binding and denaturation studies have shown to be a rapid and effective tool for the characterization of albumin binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein caused by interaction between different ligands [15][16][17][18].
The last years more and more scientists are using computational chemistry and theoretical tools to evaluate their molecular structures [19][20][21]. Moreover, some researchers are using computational tools to evaluate the biological activity of the molecules of interest [22][23][24][25][26]. This is because of the quick results that the computational tool is giving to the researcher, and the added scienti c value to the ndings. All that, at minimum cost and resources. As the technological improvements are running fast, more and more theoretical tools are going to save time to the researcher and give a different perspective, using the predictive character of the computerized models.
Herein, we propose the synthesis of some chalcone analogues, and their complexation with Zn (II) metal ions. In total, 6 molecules were prepared and evaluated spectroscopically, both in situ and theoretically. More speci cally, we used TD-DFT studies [26][27][28][29][30][31], to evaluate our chalcone derivatives in terms of structure and activity, molecular docking [32][33][34][35], to evaluate their interaction with human serum albumin (HSA) and spectroscopy to evaluate their binding interactions with bovine serum albumin (BSA). The biological activity of the chalcone derivatives, was compared with the biological activity of their counter Zn complexes.

Instruments
All the chalcone analogues and their zinc complexes were characterized by 1 HNMR, recorded on Bruker 300 MHz spectrometer using DMSO-d6 as solvent and TMS as an internal standard. The chemical shifts were expressed in δ ppm. The absorption spectrum of all the reaction mixtures was then taken in a range of 200-400 nm in a JASCO (Tokyo, Japan) UV-visible spectrophotometer using a 1cm path length quartz cuvette.

Synthesis of Zinc (II) Complexes
Zn (II) complexes with ligands L 1 -L 3 An ethanolic solution (30 ml) of Zn (II) chloride (0.01 mol) was added to a re uxing solution of appropriate chalcone analogue L 1 -L 3 (0.02 mol) in ethanol (30 ml DFT methods, the hybrid functional B3LYP, as a good compromise between computational cost, coverage, and accuracy of results [36]. It has become a standard method used to study organic chemistry in the gas phase. UV-vis spectrums at the same level calculated by time-dependent density functional theory (TD-DFT) method. ORCA input les were created by AVOGADRO version 1.2.0 software. HOMO energy (E HOMO ) and LUMO energy (E LUMO ) were taken from the output le. Chalcone analogues and their zinc complexes were docked against human serum albumin. Molecular docking studies were carried out by using iGEMDOCK 2.1 software [11]. The HSA coded crystal structure was selected from the Protein Data Bank (www.rcsb.org). The population size was = 200, generations = 70, number of solutions = 3.

Effect of the ligand and the complex on the absorption spectrum of BSA
The effect of the ligands and their corresponding complexes, on the absorption spectrum of BSA, was studied using UV-visible spectrophotometry. Brie y, BSA (5 μM) were incubated in the absence and presence of 2-9 μΜ of L 1 , L 2 , L 3 , C 1 , C 2 and C 3 for 30 min at RT.

Synthesis of Chalcone Derivatives and their Zinc Complexes
Chalcone derivatives were synthesized based on Claisen-Schmidt condensation reaction. Acetophenones analogues reacted in (1:1) ratio with benzaldehydes analogues in methanolic solutions resulting the chalcone derivatives (L 1 , L 2 and L 3 ). The synthetic routes and conditions are depicted in Fig.   1. Reactions took place at room temperature resulting colourful precipitates after 20 h mixing. Any precipitate formed was removed by ltration before acidi cation. The formation of carbanion or enolate ions is considered to be the rst stage of the condensation reaction. Aromatic ketones having α hydrogen if treated with alkaline solutions (in this case NaOH), the hydroxide ion from the base will attack hydrogen α from the ketone so that a carbanion is formed which can be stabilized by resonance and release the H 2 O molecule. Followed by the second stage which is a nucleophilic addition reaction. Here, the enolate or carbanion ion formed at stage one acts as a nucleophile that attacks the carbonyl group of benzaldehyde. An alkoxide ion is formed which has an excess of electron charge in the O atom. Next, is the formation of an aldol. Aldol is a compound formed from aldehydes and ketones where aldol takes protons from solvent molecules, H 2 O. Alkoxide ions take hydrogen protons from H 2 O molecules to form βhydroxyketone (aldol). Then the hydroxide ion from H 2 O binds to the sodium ion and returns to form a NaOH base catalyst. Finally, the dehydration reaction of the aldol compound takes place in Claisen-Schmidt reaction. Dehydration reaction is a reaction of the release of water molecules. Carbonyl βhydroxy like aldol is easily dehydrated, because the double bonds in the compound conjugate with the carbonyl group. The disappearance of the chemical shift at 2.5 ppm attributed to -CH 3 hydrogens of acetophenone, it is a good evidence of the resulted chalcone derivatives. Moreover, the increase of the melting points from 47 °C to 57 °C is another indicator of the successful synthesis.
Zn (II) complexes (C 1 , C 2 and C 3 ) resulted after mixing ethanolic chalcone derivative solution (L 1 , L 2 and L 3 ) with ZnCl 2 at (1:2) ratio, using re ux for 6 h. Coloured complexes obtained after ltration. The three complexes are soluble to DMSO and DMF. Elemental analyser indicated that the complexes have 1:2 metal to ligands stoichiometry of the types [ZnL 2 (Cl) 2 ], whereas L are the chalcone derivatives resulted after the condensation of the correct acetophenone analogue with the correct benzaldehyde analogue. In addition, low molar conductance values indicate that the complexes are not electrolytes.

Molecular Geometry
The ground state optimization structures of the synthesized chalcone molecules were obtained in the aqueous phase at B3LYP def2-TZVP Grid5 level and are given in Fig. 2.
The geometrical parameters have been procured from optimized molecular structure and summarized in Table 1, giving the main theoretically calculated bond lengths and bond angles of the molecules.

Spectroscopy
The spectroscopic UV-vis spectrum of the three ligand molecules, and their electronic transitions can be computerized and analyzed by time-depended density functional theory or TD-DFT. Additional information in the molecular structure prediction can be taken by electronic spectroscopy. In Fig. 3 we can see the calculated UV-vis spectrum of the molecules taken in the aqueous phase using B3LYP def2-TZVP Grid5 level algorithm.
As seen in Fig. 3, for L 1 and L 3 are observed similar spectrums giving two bands for πàπ* and nà π* transitions respectively. Most absorption spectroscopy of organic compounds is based on transitions of n or π electrons to the π* excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 -700 nm). These transitions need an unsaturated group in the molecule to provide the π electrons. For L 2 molecule only one transition is observed the πàπ*. The similarity of the bands shapes and close wavelengths show that the structures of the studied molecules are quite similar as they are belonging to the same family of the chalcones. 1 HNMR spectrum calculated data also suggest the similarity of the structures.
For structural characterization 1 HNMR spectra of chalcone molecules were calculated and peaks were attributed to the correct chemical groups. Chemical shifts were given according to the TMS reference and calculated from δ=Σ TMS -Σ relation , whereas Σ TMS corresponds to the shielding of proton to the TMS and Σ, the shielding of proton in the sample. The 1 HNMR spectrum calculated at B3LYP DEF2-TZVPP DEF2 level in aqueous phase and are given in Fig. 4.
Chemical shifts of 1 HNMR, are given in Table 2. As can be seen in Fig. 4, the spectra are similar for molecules L 1 , L 3 same as the UV-vis spectra. The only difference is the shift of the rst chemical shift from 3.83 to 2.34 ppm due to the presence of the extra methyl group on L 3 . The presence of the 3.06 ppm chemical shift that it is characteristic for the L 2 and corresponds to the -CH 3 group of the molecule, dominates the anomeric region. Because they are attached to carbon atoms with low s-character hybridization (sp 3 ) the found in a low ppm chemical shift. The aromatic region of the spectra of the three chalcone derivatives are quite similar. As can be seen from Table 2. the -OH group is present at three molecules as well, at the same chemical shift 5.35 ppm. The hydrogens of the phenol group of the chalcones can be found at 8.02 and 7.66 ppm for L 1 , 8.33 and 7.42 ppm for L 2 , and 8.62 and 7.66 ppm for L 3 .
An additional way to verify the structures of the chalcone derivatives, is the vibrational spectrum. It has been calculated by BP86 DEF2-SVP FREQ algorithm. The high intensity peaks with the harmonic vibration frequencies are given in Fig. 5.
The characteristic high frequency peak at 3500 cm -1 , of s(O-H) is common for the three analogues, while the peak at 1200 cm -1 for L 1 , indicating the s(N-H) vibration. The methoxy group of L 3 , is responsible for the t(H-O-C-C) vibration at 800cm -1 which is absent for L 1 and L 2 . The 1700 cm-1 peak is responsible for the s(N-C) of the molecules, while the 1500 cm -1 peak is responsible for s(N-O) that is why it is absent from L 2 .

Molecular Orbital Studies
The molecular orbital studies revealed the energy gap between the highest molecular orbital (HOMO) and the lowest molecular orbital (LUMO). The value of the energy difference between HOMO and LUMO as well as the highest occupied molecular orbital (EHOMO) and lowest unoccupied molecular orbital (ELUMO) energies plays a very important role in stability and reactivity of molecules. The EHOMO energies of molecules show the molecule's ability to give electrons. On the other hand, ELUMO characterizes the ability of the compound to accept electrons. Thus, the one is nucleophile and the other electrophile. Molecules with small energy gaps are considering to have a higher chemical reactivity and softer structures, while molecules having larger energy gaps are considering to be more stable and chemically harder. The computed values of the three chalcone derivatives can be found in Fig. 6.
It seems that L 3 , has the highest reactivity (ΔΕ= 0.38 eV) followed by L 1 (ΔΕ= 2.24 eV) and L 2 (ΔΕ= 2.34 eV). In Table 3. we can see all the calculated energy features of the three molecules including, enthalpy, entropy and Gibbs energy. Any process in which the number of particles in the system increases consequently results in an increase in disorder. This is why we can observe an increase in the entropy of the molecules. The individual charge on each atom on the molecule, it is another factor used to characterised molecular structures and it is presented by the Mulliken population study. The Mulliken atomic charges have been calculated by DFT method and presented in Table 4. The O20 atom has the highest negative charge for L 1 , while for L 2 the highest negative charge belongs to C2 atom. For L 3 the highest negative charge is that of C2 atom as well. On the other hand, N18 and H33 atoms are having the highest electropositive charge for L 1 , and C7 and H33 atoms the highest electropositive charge for L 2 . Finally, for L 3 , C3, N19 and H31 are the most electropositive atoms. The negative charges are due to the electron withdrawing groups that the atoms are attached with and the positive charges are because of the negative charges of the adjacent groups.

Molecular Docking
The examination of the biological activity of the three ligand molecules (L 1 , L 2 , L 3 ) and their corresponding complexes (C 1 , C 2 , C 3 ), calculated theoretically by molecular docking studies. Using this technique, we can predict the best drug candidate in terms of protein inhibition, on a speci c targeted protein. By molecular docking, we can predict binding energies, types of interactions and the amino acid pro le residue of the protein that interacts with the drug molecule. In this study, we investigated the binding a nity of our studied molecules with HSA (human serum albumin). HSA, is the main transport protein in human organisms, were drugs bind and transported throughout the blood transportation. The interaction types between the chalcone molecules and their zinc complexes are given in Table 5. The energy function can be dissected into the following terms: (1) where E bind is the empirical binding energy used during the molecular docking; E pharma is the energy of binding-site pharmacophores; and E ligpre is a penalty value if the ligand unsatis ed the ligand preferences. E pharma and E ligpre were used to improve the number of true positives by discriminating active compounds from hundreds of thousands of non-active compounds. It can be seen that C 2 , exhibit the highest binding affinity on the transport protein, followed by C 3 and L 3 . The highest binding affinity exhibited by C 2 is because of the CH 3 -N-CH 3 group which seems to interact better with the protein with van der Waals forces. The lowest binding affinity exhibited by L 2 , which means that in general, the complexed zinc (II) molecules are more active molecules with only exception the C 1 . In Table 6. the amino acid residue of the protein that interact with the studied molecules can be seen. Table 6. Interactions formed between the studied molecules with the amino acids of the transport protein.

BSA Binding
The structural changes of the protein and the complexation with the studied molecules has been done using UV-vis absorption measurements. The compounds interacted on the site I of the protein. We performed binding studies on the BSA protein because it has a similar shape with the HSA. The UV-vis absorption spectrums in Fig. 8 shows the effect of L 1 , L 2 , L 3 , C 1 , C 2 and C 3 molecules on the BSA spectrum. Strong absorption peaks at 250 nm and 350 nm can be seen for L 1 , L 2 , L 3 which are increased in intensities as the concentration of the molecules increases. The red shift observed at 250-255 nm indicates the complex formation of the protein with the ligands. When the L 1 , L 2 and L 3 complexed with Zn (II), we observe in the spectrum of the protein only one strong absorption peak at 210-220 nm depending on the complex molecule. Again, the red shift of 1-5 nm corresponds to the ligation of the molecules in the protein structure. The Zn (II) ions are responsible for the loosening and unfolding of the protein backbone with an increase of the hydrophobicity of its environment, more drastically than the of L 1 , L 2 , L 3 molecules which Zn is absent. Thus, the drastic change in the spectrum was happened by the complexed C 1 , C 2 , C 3 molecules. Additionally, the results indicated that the interaction of the Zn (II) complexed chalcones with BSA molecule has caused some conformational changes in the microenvironment around chromophore of BSA, that is why we cannot observe any peak at the 300 nm.

Conclusions
In this work, the synthesis of three chalcone derivatives and their corresponding Zn (II) molecules was presented and their structures evaluated spectroscopically and theoretically. Their spectroscopic and theoretical evaluation indicated that the Zn-chalcone molecules exhibited higher binding activity than their corresponding chalcone ligands. The binding activity was predicted with molecular docking studies and con rmed by spectroscopic BSA interactions of L 1 , L 2 , L 3 , C 1 , C 2 and C 3 . From the highest to lowest activity the molecules are C 2 >C 3 >L 3 >C 1 >L 1 >L 2 . Chalcones are biological active molecules and their interactions with Zn metal ion increases their binding activity on transport proteins. Additionally, chalcones could be used as biological chelators to reduce the toxic zinc concentrations in biological systems.

Declaration of competing interest
The author declares that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
[35] Nazia Siddiqui, Saleem Javed, Quantum computational, spectroscopic investigations on ampyra (4aminopyridine) by df t/td-df t with different solvents and molecular docking studies, Journal of Molecular  Figure 4