COMPLEXATION

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INTRODUCTION
Adrenaline (Adr, Scheme 1) is neurotransmitter hormone (known as epinephrine) [1]. It makes increasing in the rate of heart, vessels of blood constrict, and passages of air dilate in the nervous sympathetic system that contributes to the fight-or-flight response. Based on chemical information, adrenaline is a monoamine catecholamine, formed adrenal glands from phenylalanine and tyrosine amino acids [2][3][4][5]. The information regarding the complexing behavior of catecholamines give an indication for adrenaline solid compounds referring to their antimicrobial properties, with ions of heavy of divalent meta-, such as Mg 2+ , Ca 2+ , Sr 2+ and Ba 2+ . A thermoanalytical study of different adrenaline coordination compounds with some divalent transition metal ions such as Co 2+ , Ni 2+ and Cu 2+ has been reported. The combinatorial interactions of levodopa, dopamine, adrenaline, noradrenaline and tyramine with Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ and Pb 2+ were studied by pH titration method at 20 °C. While levodopa has been found to act as a simple dimeric α-amino acid, dopamine, adrenaline, and norepinephrine coordinate with metal ions via catecholic OH groups. Composition and equilibrium constants of the complexes of Mg 2+ , Co 2+ , Ni 2+ , Cu 2+ , Cd 2+ , and Cd 2+ with two catechol double bonds: dopamine and adrenaline, in aqueous solution, using protomometric and spectrophotometric techniques. The degree of coordination of the metal ion is no more than two, with the stability order Cu 2+ > Pb 2+ > Ni 2+ , Co 2+ , Cd 2+ > Mg 2+ [3,4].
The goal of this work is to study the chelation behavior and antimicrobial efficiency between Adr hormone as a vital biomolecule and bivalent metal ions Mg 2+ , Ca 2+ , Sr 2+ and Ba 2+ . The reason of the selection of alkaline earth metal ions, because Mg 2+ and Ca 2+ metal ions are some important and essential elements which already important to the human body. Also, the work aimed to compare and study the biological and spectroscopic properties of the Adr solid complexes produced from solutions of CH3OH after precipitation via molar ratios of some divalent chloride/adrenaline (M/L). The interpretation of the solid complexes was carried out using elemental analysis, conductance measurements, mid infrared, Raman laser, UV-Vis., X-ray powder diffraction, scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDX).

Materials
From Aldrich Company MgCl2·6H2O, CaCl2·6H2O, SrCl2·2H2O, BaCl2·6H2O, and CH3OH were obtained and from Fluka chemical company, adrenaline was obtained. In this paper high grade of purity chemicals were used.

Preparation of adrenaline complexity
The precipitated complexes were formed in CH3OH solutions, with the (M II )/Adr ratios 1:1 or 1:2.

Measurements
The C and H analyses were carried out at Cairo University, Egypt, in microanalysis unit, using a CHN 2400 instrument (Perkin Elmer, USA). For the soluble adrenaline complexity using conductivity meter Jenway 4010 1.0×10 -2 g/5 cm 3 the freshly prepared conductivities of solutions of dimethyl formamide (DMF) were detected. FT-IR spectra were recorded as potassium bromide discs on Bruker Spectrophotometer within range (4000-400 cm -1 ), while Raman laser spectra of samples were recorded on Bruker FT-Raman with laser 50 mW. UV spectra of adrenaline complexes in DMSO were measured on UV2 Unicam UV/Vis Spectrophotometer using a 1.0 cm path length quartz cell. The scanning electron microscopy (SEM) were recorded on Joel JSM-6390 equipment with an accelerating voltage of 20 KV images and energy dispersive X-ray detection (EDX) were measured. For adrenaline free ligand and its Ca 2+ complexity on X 'Pert PRO PANanalytical X-ray powder diffraction, target copper with secondary monochromate the X-ray diffraction patterns was recorded.

Antibacterial and antifungal activities
The antimicrobial activity of the target compounds was measured using a modified Kirby-Bauer disc diffusion method [6]. Briefly, for fresh media in 10 mL, 100 μL of the best bacteria/fungi were grown till arriving 105 cells/mL count for fungi and approximately108 cells/mL for bacteria [7]. Onto agar plates 100 μL of suspension microbial corresponding to the broth was spread that were maintained. It should be selected isolated colonies of each organism that might be playing a pathogenic role from primary agar plates and then make a test by disc diffusion method for susceptibility [8][9][10][11][12][13][14][15][16][17]. Plates inoculated with filamentous fungi as Aspergillus flavus at 25 o C for 48 hours; Gram (+) bacteria, Bacillus subtilis, Streptococcus pneumonia and Staphylococcus aureus; Escherichia coli and Pseudomonas aeruginosa Gram (-) bacteria, were incubated at 35-37 o C for 1-2 days and Candida albicans as yeast incubated at 30 o C for 1-2 days, then measuring the inhabitation zones diameters in millimeters. Standard discs of tetracycline (antibacterial agent), amphotericin B (antifungal agent) served as positive controls for antimicrobial activity, but filter disc impregnated with 10 μL of solvent (distilled water, chloroform, DMSO) were used as a negative control.

RESULTS AND DISCUSSION
The measured molar conductivity, C and H analysis of adrenaline complexes with Mg 2+ , Ca 2+ , Ba 2+ , and Sr 2+ are given in Table 1 In DMF and dimethylsulfoxide, the complexes were soluble, have high melting points and air stable. Based on conductivity measurements in DMF using solutions of 10 -2 g/5 mL, the complexes have variation data with an electrolytic nature (Table 1).

Conductance measurements
In DMF, the values of molar conductance for the adrenaline complexes of (10 -2 g/5 mL solution) were 245, 228, 220 and 321 Ω -1 cm 2 mol -1 for [Ca(Adr)2(H2O)2](H2O)4Cl2, [Sr(Adr)2(H2O)2]Cl2, [Ba(Adr)2(H2O)6]Cl2 and [Mg2(Adr)2(H2O)7]Cl4 complexes, respectively, confirming that complexes have an be electrolytic nature ( Table 1). The mode of chelation of adrenaline complexes can be deduced from conductivity measurements within the lower limits of their solubility. The degree of ionization of the complexes can be tested, and the liberation of the ions of the complexes in solution (for external coordination anions), the more molar conductivity value can be used and vice versa. From the conductivity measurements, [Mg2(Adr)2(H2O)7]Cl4 complex is more electrolyte than [Ca(Adr)2(H2O)2](H2O)4Cl2, [Sr(Adr)2(H2O)2]Cl2 and [Ba(Adr)2(H2O)6]Cl2 complexes. For all adrenaline complexes the values of molar conductance, referring that all Cl anions are outside the sphere of chelation. This result was detected from the data of analysis of Clwhich precipitated on addition of AgNO3 solution, this experimental test is in a good match with CH data. All adrenaline complexes will have an electrolytic character which refer to presence of Cl -. The stoichiometry of the complexes based on these data was established, which are consisted with the suggested general formulas.

Infrared and Raman spectra
The Cs is a point group represented free ligand molecular structure of adrenaline [18]. The (CH2NHCH3) secondary N-methyl amino group of adrenalines, act as point mass. There are fortyeight active IR and Raman spectra represented the modes of vibration for adrenaline that can be divided as vib = 33A' + 15A'' of Cs point group which are shown in Figures 1 and 2. The interpretation of the adrenaline spectrum is difficult, due to it has low symmetry and high degree of complexity where 29 modes of vibrations only determined that divided into vibrations of 19A' and 10 A''. Based on value and relative intensities for observed bands in similar complexity, for adrenaline, the interpretation of the vibrational frequencies has been discussed [19] (Table 2)      By comparing between free adrenaline ligand and its Mg 2+ , Ca 2+ , Sr 2+ and Ba 2+ complexes using IR and Raman laser spectra, we conclude that, there are a shift to lower wavenumbers for bands due to ν(O-H), ν(C-O) and δ(C-O-H) as decreasing the intensities, confirming that the coordination of heavy metal ions towards adrenaline carried out through two OH catechol moiety of phenolic groups. For adrenaline complexity, there are a sharp broadening with a distorted in the vibrational stretching bands in Raman spectra for Mg 2+ , Ca 2+ , Sr 2+ , and Ba 2+ (Figure 2), which can be explained based on the overlap of fluorescence which, even when very weak, can overwhelm the inherently weak Raman scattering signal where Raman analysis of fluorescent materials and compounds is a challenging task experimentally [20,21]. Based on the above discussion we can conclude that adrenaline act as a bidentate ligand and can form a stable chelating structure through coordination of the phenolic oxygen's groups.

UV spectra
There are two essential UV bands observed for free ligand hormone adrenaline (Adr) appeared at 299 and 376 nm (Figure 3a) due to π→π*, n→π* transitions, r, for aromatic ring, Ar-OH, OH, -NH secondary amine and CH3, with wavenumbers more or less shifting in the adrenaline complexes spectra (Figure 3b   Abs.

X-ray
For free adrenaline ligand and [Ca(Adr)2(H2O)2](H2O)4Cl2 ,the X-ray powder diffraction patterns are in Figures 4a and 4b, respectively. Based on these patterns, we can conclude that there are semi crystalline systems that are well. From XRD patterns by applying FWHM of the peak's characteristic through Deby-Scherrer equation, the size of the crystallite of complexity could be calculated. Where, size of particle of the crystal gain is symbol D, the value 0.94 for Cu grid is a constant K, 1.5406 Ǻ is the X-ray wavelength λ, Bragg diffraction angle is θ and the peak integral width is β. The size particle was calculated based on to the value highest intensity of comparing to other peaks. These information data gave an amorphous structure (Figure 4b).

SEM and EDX studies
The simple tool of scanning electron microscopy explains aspects of microscopic of physical behavior of adrenaline complexes ( Figure 5). This technique is not considered a basic method to elucidate formation of complex, but it can be taken as a guide for the presence of a single component in the prepared complexity. The SEM for [Sr(Adr)2(H2O)2]Cl2 and [Ba(Adr)2(H2O)6]Cl2 complexes gave a homogenous feature products with irregular structures. The elemental analysis data collected by EDX analysis for the prepared complexes between adrenaline and metal ions are listed in Table 3

Biological evaluation
For adrenaline complexity, the antibacterial and anti-fungal activities against (Bacillus subtilis, Streptococcus pneumonia and Staphylococcus aureus) gram-positive and (Escherichia coli and Pseudomonas aeruginosa) gram-negative and two fungus strains of (Aspergillus flavus and Penicillium). From the diffusion tests agar disc, result of adrenaline complexes for antimicrobial activities are presented in Table 4, and illustrated in Figure 6a