Synthesis and spectroscopic interpretations of Co(II), Ni(II) and Cu(II) decxycholate complexes with molecular docking of COVId-19 protease

Co(II), Ni(II) and Cu(II) decxycholate complexes are interesting due to their biologically active and deliberate interest in the research due to their coordination properties. The microanalytical ‘elemental analysis’, molar conductivity, (infrared and Raman) spectroscopy, thermal analyses (TGA/DSC), UV-vis spectra, and ESR for copper(II) decxycholate complex investigations were performed in the structural assignments of Co(II), Ni(II) and Cu(II) decxycholate complexes. Reaction of the sodium deoxycholate ligand (C 24 H 39 O4Na) with three transition metal ions form the complexes of formulae, [M(C 24 H 39 O 4 ) 2 (H 2 O) 2 ] . xH 2 O where M = Co(II), Ni(II) and Cu(II) where x = 2 for Cu(II) and x = 4 in case of M = Co(II) or Ni(II) metal ions. The FTIR spectra of the complexes show that decxycholate molecule is present as bidentate ligand. Molecular docking utilizing to additionally examine the interaction of COVID-19 (6LU7) with different complexes of deoxycholic acid with Co(II), Ni(II) and Cu(II). Furthermore, in the case of Co(II) deoxycholate complex, the probe is surrounded by amino residues Met235, Pro241, Glu240, Pro108, Gln110, Phe294, and Ile152. The probe molecule of Ni(II) deoxycholate complex is sited close to amino acids Tyr126, Tyr239, Leu287, Leu272, and Lys137. For, Cu(II) deoxycholate complex, the residues of amino acids comprise of Pro132, Pro108, Gln110, Gly109, Ile200, Asn203, Val202, His246, Pro293 and Tyr154. The binding energy was determined from the docking reads for Co(II)–6LU7, Ni(II)–6LU7 and Cu(II)–6LU7 deoxycholate compounds were found to be − 446.99, − 500.52, − 398.13 kcal mol − 1 individually. of deoxycholic acid. A clear understanding of the structure and spectroscopic properties of metal complexes usable for their biological applications is the aim of the present work. The present work deals with the synthesis and characterization of Co, Ni, and Cu(II) complexes of deoxycholic acid. Also, we aimed to study the interaction of COVID-19 protease (6LU7) by molecular docking studies, with all three complexes of deoxycholic acid. Molecular docking was performed to determine the binding sites and binding energy. From this study, we can obtain the difference in the inhibitory effect of COVID-19 protease for the biological process.


INTRODUCTION
Deoxycholic acid, commonly known as choline acid, and bile acid. Deoxycholic acid is a secondary bile acid, which is byproducts of intestinal bacteria. The two primary bile acids secreted by the liver are cholic acid and chenodeoxycholic acid. Deoxycholic acid is soluble in alcohol and acetic acid. When it is pure, it comes in the form of a white to yellowish-white crystalline powder 1 . Deoxycholic acid has been used since its discovery in various fi elds of human medicine. Deoxycholic acid is used in the human body to emulsify fats for absorption in the intestine. It is licensed in some countries as an emulsifi er in the food industry 2 . It is us ed outside the body to prevent and dissolve gallstones. Deoxycholic acid is used in research as a mild detergent to isolate membrane-bound proteins 3 . Sodium deoxycholate is often used as a biological cleanser to leach cells and dissolve cellular and membrane components 4 . Sodium deoxycholate mixed with phosphatidylcholine, is used in mesotherapy injection to produce lipolysis, and has been used as an alternative to surgical excision in treating lipomas. Deoxycholates and bile acid derivatives, in general, are actively being studied as structures for inclusion in nanotechnology. They also found applications in microprinting as photoresist ingredients 5 . Some publications indicate the effect of deoxycholic acid as an immunostimulant 6, 7 of the innate immune system, activating the main actors, macrophages. According to these publications, enough deoxycholic acid in the human body corresponds to a good immune reaction of the nonspecifi c immune system.
The carboxylic group is capable to interaction with different metal ions which can have a synergistic or antagonistic effect on the anti-microbial activity. It was found that anti-infl ammatory and anti-bacterial activity of metal complexes was higher than that of the parent carboxylic acids 8 . The carboxylate group as a substituent serves as a site to a complex targeting group that can aid in the delivery of the compound to bacteria cells in the body 9 . The design and synthesis of transition metal complexes with carboxylic bonds have received much attention in coordination chemistry because this type of complex has potential applications in molecular magnets, catalysis, and supramolecular chemistry and biological systems 10, 11 . The carboxylate anion can adopt a wide range of bonding modes (monodentate symmetric and asymmetric chelating and bidentate and monodentate bridging) 12 . The literature survey of the past few years reveals the fact that a signifi cant development in the fi eld of biological activity of metal chelates plays a vital role in the case and treatment of cancer 13-16 . Metal complexes of carboxylic acids have been studied extensively 17, 18 , but there are not any works on complexes of deoxycholic acid. A clear understanding of the structure and spectroscopic properties of metal complexes usable for their biological applications is the aim of the present work. The present work deals with the synthesis and characterization of Co, Ni, and Cu(II) complexes of deoxycholic acid. Also, we aimed to study the interaction of COVID-19 protease (6LU7) by molecular docking studies, with all three complexes of deoxycholic acid. Molecular docking was performed to determine the binding sites and binding energy. From this study, we can obtain the difference in the inhibitory effect of COVID-19 protease for the biological process.

Instrumentals
The carbon, hydrogen and nitrogen elements have been performed using Perkin Elmer CHN 2400. The determination of the percentages of metal ions was estimated based on the thermal gravimetric analysis technique. Melting points were carried out using MPS10−120 Melting point apparatus. FTIR spectra of the synthesized complexes were performed on Bruker FTIR Spectrophotometer. At room temperature with freshly prepared solutions, molar conductance of 10 -3 M solutions in dimethyl sulfoxide (DMSO) solvent were measured using a Jenway 4010 conductivity meter. The electronic spectra were scanned in situ dimethyl sulfoxide within 200-800 nm range by UV2 Unicam UV/Vis Spectrophotometer. The effective magnetic moment (μ eff ) of the complexes was measured using Gouy's method by the help of a magnetic susceptibility balance from Johnson Metthey and Sherwood model. The ESR spectrum of the diamagnetic copper(II) complex was scanned by a JES-FE2XG EPR-Spectrometer. Thermogravimetric analysis (TGA) experiments were measured using TGA/DSC-50H Shimadzu analyzer. All experiments were performed using a single loose top-loading platinum sample pan under nitrogen atmosphere at a fl ow rate of 30 mL/min and a 10 o C/ min heating rate for the temperature range 25-800 o C.

Molecular docking
Molecular docking has been used to understand the best binding site of the synthesized metal complexes with the COVID-19 protease (6LU7) using the HEX 8.0 program 19 . The structure of the COVID-19 protease was downloaded from the online protein data bank (http:// www.rcsb.org) in the pdb format 19 . The 3D structure of Co (II), Ni (II) and Cu (II) complexes was designed utilizing the Chem3D software. Avogadro version 1.2 was utilized for the optimization of the structure. The docked conformations were visualized utilizing the Discovery Studio 19 or Chimera of the complex.

Elemental analysis and conductance data
Analytical data of the Co, Ni, and Cu(II) deoxycholate complexes are summarized above in Table 1, and the results obtained are reliable with those calculated for the proposed formulae. All these complexes were stable at atmospheric conditions, non-hygroscopic, and soluble in organic solvents such as dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF). The molar conductivity of the deoxycholate complexes in dimethyl sulfoxide (10 −3 M) at 25 o C is proportionate with their non-electrolytic nature (15-20 Ω −1 cm 2 mol −1 ) 19 . However, the analytical, spectroscopic, and magnetic data do enable us to predict the possible structure of the synthesized complexes.
The infrared band assignments of sodium deoxycholate salt and its Co(II), Ni(II), and Cu(II) deoxycholate complexes are tabulated in Table 2. To identify the coordination modes of the carboxylato group, the  2 ] . 4H 2 O, showed that this complex has an octahedral geometry. There are two electronic bands at 23529 cm -1 and 28571 cm -1 attributed to 4 T 1g → 4 A 2g and 4 T 1g → 4 T 1g (P) transitions, respectively. The magnetic moment of the cobalt(II) complex was found at 3.84 B.M. at room temperature which supported the octahedral geometry (Fig. 1). The electronic spectra of the synthesized nickel(II) exhibited two electronic absorption bands at 14970 and 29239 cm -1 correspond to the 3 A 2g (F)→ 3 T 1g (F) (ν2) and 3 A 2g (F)→ 3 T 1g (P)(ν2) transitions respectively. These transitions revealed that the nickel(II) complex has an octahedral geometry 22 . The effective magnetic moment value (μ eff ) of the Ni(II) complex was 3.20 BM. This value indicated that there were two unpaired electrons so that the complex was paramagnetic. The values of μ eff 3.20 BM in the nickel complex indicated an octahedral complex 23 (Fig. 2). The trend g || (2.1294) > g ┴ (1.5971) observed in copper(II) complex indicate that the Cu(II) is in a distorted octahedral coordination environment, and the unpaired electron most likely resides predominantly in the d x2-y2 orbital of the Cu(II) ion and is the characteristic feature for the axial symmetry. The deviation of "g" values from the free-electron value (2.0023) is by angular momentum contribution in the complexes. The average "g" value for overall distortion is calculated using the equation: g av (1.7745) = 1/3(2g ┴ + g || ) 24 . Molecular orbital coeffi cients in-plane σ-bonding (α 2 ), in-plane π-bonding (β 2 ), and out-ofplane π-bonding (γ 2 ) are the covalency parameters for the metal to ligand bond which were evaluated using the following expressions: α 2 = (A || /0.036) + (g || -2.0027) + 3/7 (g ┴ -2.0023) + 0.04) β 2 = (g || -2.0023)E / -8λα 2 γ 2 = (g || -2.0023)E / -2λα 2 where λ = −829 cm −1 for the free copper ion and E is the electronic transition energy. The observed values of α 2 and β 2 indicate that the complex has covalent bonding character. The smaller the β 2 , the larger the covalency of the bonding. Furthermore, it has appeared that the covalency of the out-of-plane is greater than of the inplane π-bonding. difference between the asymmetric and symmetric carboxylato vibrations (Δυ= υ asym -υ sym ) was calculated. The carboxylate group acts as a monodentate manner, when the difference between the ν as COO-ν s COO is larger than ionic compounds 19, 20 . When the Δν is considerably smaller than the ionic compound, the carboxylate group coordinated towards metal ions as a bidentate fashion. In the spectra of the deoxycholate sodium salt and complexes, characteristic infrared bands arising from the frequencies of the carboxylate anion appeared. The stretching vibrations of the symmetric carboxylate anion ν sym (COO) are present at the wavenumbers 1566 cm −1 , 1552 cm −1 , 1542 cm −1 , and 1596 cm −1 in deoxycholate sodium salt, 20 cobalt complex, nickel complex, and copper complex respectively, and the stretching asymmetric ν as (COO) at the wavenumbers 1302 cm −1 , 1332 cm −1 , 1376 cm −1 , and 1377 cm −1 IR in deoxycholate sodium salt, cobalt complex, nickel complex, and copper complex respectively (Table 3). Based on the position of the ν as COO and ν s COO bands in the IR spectra of the synthesized complexes compared to the position of these bands in the sodium salt, it was found that the differences between the frequencies were determined as 264, 220, 166 and 219 for deoxycholate sodium salt, cobalt complex, nickel complex, and copper complex respectively. These results for Δ values indicated that the carboxylato group participates in a bidentate manner for the complexes (Fig. 1).
New vibration bands have been noticed in spectra of prepared complexes at wave number ranges 615 cm −1 assigned to υ(M-O) vibrations 20 .

UV-Vis spectra, ESR and magnetic susceptibility
The electronic refl ectance spectra of Co(II), Ni(II), and Cu(II) deoxycholate complexes were recorded in solid-state. The octahedral cobalt(II) complexes have a pink or reddish brown 21 but most tetrahedral Co(II) complexes have an intense blue or green color, herein the electronic spectrum of the red color complex, [Co-

Thermal analysis
The  Table 4  It is evident that different complexes of sodium deoxycholate with Co(II), Ni(II) and Cu(II) may have a unique impact on COVID-19 protease. However, it is hard to decide the differences through the experiments. Henceforth, to determine the difference, a computational study could be utilized 25, 26 . So, the molecular docking was performed    Table 6. The value of binding energy was calculated from the docking studies for Co(II)-6LU7, Ni(II)-6LU7 and Cu(II)-6LU7 deoxycholate complexes were − 446.99, − 500.52, − 398.13 kcal mol −1 respectively. The binding affi nity of Co(II)-COVID-19 protease was found to be − 446.99 kcal mol −1 indicating the slightly higher binding energy than Cu(II)-6LU7 and slightly lower than Ni(II)-6LU7. The estimated inhibition to identify the interaction of 6LU7 with Co(II), Ni (II) and Cu(II) deoxycholate complexes. The most possible docking pose between 6LU7 and different complexes of sodium deoxycholate and related data are shown in Table  5, 6 and Fig. 4-6. The binding sites of prepared complexes show that interactions are considerably different in each case as represented from Fig. 4-6. In the case of Co(II) deoxycholate complex, the probe molecule is bounded   Constant, Ki is 48.39 mM (millimolar) which is found in the case of Cu(II) complex 27 . So, this complex has a higher ability to inhibit the biological process of target COVID-19 protease (6LU7).

CONCLUSION
The synthesis of the complexes between sodium deoxycholate and essential metal ions (Co(II), Ni(II) and Cu(II)) were studied to investigate the complexation behavior of these systems as it could mimic many biological interactions. The complexes appear to be superior in biological properties dependent on the molecular docking that utilizing to additionally examine the interaction of COVID-19 (6LU7). The complete elucidation molecular structures of the synthesized deoxycholate complexes were confi rmed by detailed microanalytical 'elemental analysis', molar conductivity, (infrared and Raman) spectroscopy, thermal analyses (TGA/DSC), UV-vis spectra, and ESR techniques. The spectroscopic analysis of the complexes shows that decxycholate molecule acts as a bidentate ligand with octahedral geometry.