Physicochemical and biological activity studies on complexes of some transition elements with mixed ligands of glycine and urea

The synthesis and study of mixed ligand transition metal complexes have been of growing interest . New materials with useful properties such as electrical conductivity photoluminescence, magnetic exchange, nonlinear optical property and antimicrobial activity can be provided by using mixed ligand transition metal complexes. Urea (CO(NH2)2) plays an important role in many biological processes such as decomposition of proteins and amino acids catabolism. In 1828, Wöhler discovered urea, when organic materials were prepared from inorganic substances. All living things contain building blocks of amino acids, which were first discovered as constituents of natural products and then observed to be the major components of proteins. All life forms on earth consist of the simplest proteinaceous amino acid, called glycine or amino acetic acid. Glycine is a neutral, aliphatic, optically inactive nonessential amino acid and it is the only protein amino acid that does not have optical isomers. Most of the metal ions form mono, bis and tris complexes with glycine that acts as a bidentate ligand forming stable 5membered chelating rings via the N atom of the amino group and O atom of carboxylate group. The mixed ligand complexes of urea and glycine acid with Co(II), Ni(II) and Cu(II) ions were synthesized, characterized and thermally studied for the first time in this work.


Introduction
The synthesis and study of mixed ligand transition metal complexes have been of growing interest 1, 2 . New materials with useful properties such as electrical conductivity photoluminescence, magnetic exchange, nonlinear optical property and antimicrobial activity can be provided by using mixed ligand transition metal complexes 3-5 .
Urea (CO(NH2)2) plays an important role in many biological processes such as decomposition of proteins and amino acids catabolism. In 1828, Wöhler discovered urea, when organic materials were prepared from inorganic substances.
All living things contain building blocks of amino acids 6 , which were first discovered as constituents of natural products and then observed to be the major components of proteins.
All life forms on earth consist of the simplest proteinaceous amino acid, called glycine or amino acetic acid 7 . Glycine is a neutral, aliphatic, optically inactive nonessential amino acid 8 and it is the only protein amino acid that does not have optical isomers 9 . Most of the metal ions form mono, bis and tris complexes with glycine that acts as a bidentate ligand forming stable 5-membered chelating rings via the N atom of the amino group and O atom of carboxylate group 10 .
The mixed ligand complexes of urea and glycine acid with Co(II), Ni(II) and Cu(II) ions were synthesized, characterized and thermally studied for the first time in this work.

Materials
ABSTRACT: The reaction of urea (ur) and glycine (gly) with the metal ions Co(ΙΙ), Ni(ΙΙ) and Cu(ΙΙ) in ethanolic solution of 1M:1L1:1L2 molar ratio (where M= Co(II), Ni(II) and Cu(II), and L1 = urea L2 = glycine) led to the preparation of complexes of the general formula [M(ur)(gly)(H2O)2]Cl. Elemental microanalysis (CHN), molar conductivity measurements, IR, 1 HNMR, Mass and UV-VIS spectroscopic, and magnetic susceptibility measurements were used for the characterization of the compounds. Thermal analyses were used for the complexes degradation characterization. The complexes have an octahedral geometry and are of electrolytic nature in DMSO solvent with the absence of inner-sphere coordination of the chloride anion. An inhibition zone was observed for Ni-urea-glycine complex against Escherichia coli when the biological activity was considered. All Chemical reagents used were purchased from BDH and used as provided.

Synthesis of the complexes
Generally, the solid complexes were prepared by the same methodology previously described 11 . Briefly, an ethanolic solution of hydrated metal chloride (0.01 mol) was dropwise added in an ethanolic solution of the first ligand (urea 0.01 mol) with stirring. The mixture was refluxed for 12 h with constant stirring. A hot solution of 0.01 mol glycine in 1:1 ethanol / water mixture ratio was dropwise added to the urea / metal mixture and drops of 1 mol L -1 NaOH solution were used to adjust pH 7.0 -7.5 to deprotonate NH3 + of the glycine to NH2. The mixture was refluxed for 2 h until resulting in the formation of a colored precipitate. The resulting product was filtered off and then washed with distilled water to remove NaCl. The product was further washed with absolute ethanol/dimethylformamide (DMF) and left to dry. Acceptable yield percentage was obtained (52-66%).

Instrumentation
Glass capillary tubes were used to measure the melting points of the metal complexes in degrees celsius on a Stuart Scientific electrothermal melting point apparatus. Silica Gel GF254 plates (mn-kieselgel G., 0.2 mm thickness) was used for TLC. Vario ELFab instrument was used for elemental analysis (carbon, hydrogen and nitrogen) of complexes. Chloride was volumetrically or gravimetrically determined by silver nitrate. The amount of water was determined gravimetrically using weight loss method and also from thermal analysis. Perkin-Elmer 2380 flame atomic absorption spectrophotometer was used to measure the metal content. Jenway conductivity meter model 4510 was used to measure the molar conductance of 10 -3 mol L -1 solutions of the metal complexes in dimethylsulfoxide (DMSO) solvent. IR spectra of the metal complexes were measured by using FT/IR-140 (Jasco, Japan). A Varian FT-300 MHz spectrometer in d6-DMSO solvent was used for obtaining proton 1 HNMR spectra, using TMS as internal standard. Mass spectra were recorded on a JEOL JMS600 spectrometer. The electronic spectra of the complexes were measured in the range 400-800 nm using an UV-VIS spectrophotometer Specord 200, Analytilk Jena (Germany). The mass susceptibility (g) of the solid complexes was measured at room temperature using Gouy's method on a magnetic susceptibility balance from Johnson Metthey and Sherwood model. Differential Thermal Analysis (DTA) and Thermogravimetric Analysis (TGA) were performed using the Shimadzu DTA-50 and Shimadzu TGA-50H thermal analyzers. The experiments were carried out in the temprature range from 25 to 800 o C under nitrogen atmosphere in a platinum pan, heating rate of 10 o C / min and flow rate of 30 mL min -1 . The antibacterial activity against four species of bacteria (Staphylococcus aureus, Bacillus spp., Escherichia coli and Pseudomonas aeruginosa) was tested by agar diffusion method. 1000 µg mL -1 concentration for each of these compounds were individually prepared in DMSO, then the filter paper disc (whatman No.1.5 mm diameter) was saturated with the solution of these compounds. The discs were placed on the surface of Millar Hinton agar dishes seeded with the strains of bacteria. The inhibition zones (mm) were measured after 24 h at 37 o C. DMSO and gentamicin (120 μg mL -1 ) were used as control and reference, respectively.

Results and discussion
Complexes of Co(II), Ni(II) and Cu(II) with urea (ur) and glycine (gly) ligands have been prepared and characterized. Analytical data, physical properties, molar conductivity, and composition of the synthesized complexes are given in Tables 1 and 2. The molar conductivity values (135-149 S cm 2 mol -1 ) reflect the electrolytic properties of these complexes. The single spot appearance in the TLC proves the purity of these complexes.

IR spectra of urea -glycine complexes
The coordination sites of urea and glycine ligands in their complexes were investigated. The infrared spectra show that urea acts as a neutral bidentate ligand through C=O and NH2 groups while glycine behaves as a bidentate anion ligand through COOand NH2 groups. IR spectra of ureaglycine complexes are represented in Figures 1, 2 and 3. Assignments of the characteristic bands are summarized in Table 3. As it was postulated, the metal complexes were quite different when compared with the free ligands.     Table 3. Main IR bands (cm -1 ) of the urea-glycine complexes The infrared spectral data of the complexes are as follows: (1) All the complexes spectra show a broad band at 3422-3432 cm -1 that corresponds to the stretching mode of water existing in the complexes as identified by thermal and elemental analysis. The coordinated water is identified by the appearance of ρr (rocking) and ρw (wagging) at 925 cm -1 and 511 cm -1 , respectively 12 .
(2) The amino groups of urea show lower-shift of 123-103 cm -1 and of 120-90 cm -1 for symmetrical and asymmetrical stretching υ(NH2) frequencies, respectively. This strongly suggests that the nitrogen atom of amino group must be involved in complexation, and the appearance of a new band in the range of 406-460 cm -1 , assigned to υ(M-N) vibration, confirms this proposition 13, 14 .
(4) The characteristic bands in complexes spectra occur in the ranges 3185-3160 cm -1 and 3376-3290 cm -1 for symmetrical and asymmetrical υ(NH2) group of glycine, respectively, which appears at lower wave number than the free υ(NH2). Hence, coordination through nitrogen of the amino group is involved 15 .

1 HNMR spectra of urea-glycine complexes
Complexes were investigated by using 1 HNMR spectra in d6-DMSO and TMS (tetramethyl silane) as standard and data are in Table 4 19,20 . The methylene group of glycine (-CH2-) in Co(ΙΙ), Ni(ΙΙ) and Cu(ΙΙ) complexes absorbs near 3.2, 3.2 and 3.1 ppm, respectively. NH2 group shows signals at 2.9, 2.5 and 2.6 ppm, respectively 21,22 . In urea, one amine and the carbonyl groups are coordinated to the central metal ion without displacement of NH2 proton,
The mass spectrum of [Ni(ur)(gly)(H2O)2]Cl complex exhibited a peak at m/z 244.90, indicating the loss of H2 and NH3, then this molecular ion loses H2O and ½Cl2 leaving an ion at m/z 192.83, which further loses one more H2NCH2COOaffording an ion at m/z 118.87. The complex [Cu(ur)(gly)(H2O)2]Cl loses [CO, ½Cl2] and H2O to give ions at m/z 205.68 and 251.19, respectively.

Electronic and magnetic spectral analysis
The magnetic moments of the Co(ΙΙ), Ni(ΙΙ) and Cu(ΙΙ) complexes as well as their electronic spectra data have provided good evidence for the structures of these complexes as shown in Table 5.
[Co(ur)(gly)(H2O)2]Cl hexa-coordination is suggested. This is based on the spectrum ( Figure 5) recorded in DMSO solution which shows bands at 17985 cm -1 and 14482 cm -1 , due to transition of 4 T1g → 4 T1g(P) (υ3) and 4 T1g→ 4 A2g (υ2), respectively 23 . The third band of the spectrum, assigned to υ1, could not be observed due to the limited range of the used instrument (200-1100 nm). [Co(ur)(gly)(H2O)2]Cl has a magnetic moments of 4.76 B.M; this value is due to a highspin octahedral geometry around the Co(II) ion as reported previously 24 . Moreover, the violet colour of octahedral Co(II) complexes is in good agreement with those previously reported 25      The magnetic moment data as well as the electronic spectrum data of the nickel complex are given in Table  5. The complex [Ni(ur)(gly)(H2O)2]Cl has a magnetic moment value of 3.2 B.M consistent with an octahedral geometry around the Ni(ΙΙ) ion with a 3 A2g ground term, which lies in the range reported in the literature 27 . In addition, the complex has three bands in the UV-VIS recorded in DMSO solution ( Figure 6): 21459 cm -1 may be due to the 3 A2g→ 3 T1g (υ3); 14970 cm -1 due to 3 A2g→ 3 T1g (υ2); 13477 cm -1 in the transition range of an octahedral structure around the Ni(ΙΙ) ion (υ1) (Figure 5) 28 . The green colour is also an additional evidence for the octahedral structure 26 . The band at 23419 cm -1 may be attributed to the charge transfer transition of [Ni(ur)(gly)(H2O)2]Cl complex 23 .
[Cu(ur)(gly)(H2O)2]Cl (structure in Figure 5) has an electronic spectrum (Figure 7) that shows a strong band at 12987 cm -1 due to 2 Eg → 2 T2g transition, suggesting a distorted octahedral geometry 26 . The broadness in the band may be due to Jahn-Teller effect 29 and the proposed geometry is also supported by the blue colour of this complex 27 . The magnetic moment value of this complex 1.43 B.M agrees with the d 9 system containing one unpaired electron 26

Thermal analysis of Cu-urea-glycine complex
The thermal and kinetic parameters for each step in the decomposition sequences of the Cucomplex were determined by using the integral Coast-Redfern equation. The Coats-Redfern method is linearized for a correctly-chosen order of reaction (n) and the activation energy (Ea) is obtained from the slope of the log [y] versus T -1 plot from Equation: where: α = fraction of mass loss, T = temperature (K), Z = pre-exponential factor, R = molar gas constant, q = heating rate and n = reaction order; estimated by Horovitz-Metzger method. The thermodynamic parameters of the thermal degradation step: enthalpy (ΔH * ), entropy (ΔS * ), and Gibbs energy (ΔG * ) of activation are calculated using the following standard equations: max ln kT where z, k, and h are the pre-exponential factor, Boltzmann and Planck constant, respectively.

Antibacterial assay of synthesized complexes
Urea showed activity against the Bacillus spp. and Escherichia coli with inhibitory zones of 12 mm and 10 mm, respectively and glycine against the Bacillus with inhibitory zone of 9 mm. But no inhibition zone was observed for all the complexes against the four studied strains (Bacillus spp., Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) excepting the complex [Ni(ur)(gly)(H2O)2]Cl which was active against Escherichia coli with inhibitory zone 5 mm. This is probably because urea denatures protein when dissolved, and for the presence of amino and carbonyl groups. However, after complexes formation there would be no activity, due to the coordination of the amino and carbonyl groups 31 .

Conclusions
The formulae and the stoichiometry of the complexes of urea and glycine with Co(II), Ni(II) and Cu(II) metal ions are suggested based on the analytical data and TGA results. Neutral bidentate behavior of the urea coordination through the amine nitrogen and carbonyl oxygen is identified by IR spectra. Glycine behaved as an anionic bidentate ligand through the carboxylate group and the neutral amino group. The electrolytic nature of the complexes was confirmed by the molar conductance values. All the complexes have an octahedral geometry, as revealed the spectral and magnetic results. The thermal decomposition studies of [Cu(ur)(gly)(H2O)2]Cl allowed to access the kinetic parameters for the successive steps of its decomposition. antibacterial activities against the four strains of bacteria, except the Ni-complex, which is active against Escherichia coli, probably due to protein denaturation.