Metal complexes of Proline-Azo Dyes, Synthesis, Characterization, Dying Performance and Antibacterial Activity Studies

Introduction

The expansions of new structure of azo compounds have been a subject of regard and much novel structure of these compounds¹. Azo compounds with the hetrocyclic diazo component from colored complexes with universality metal ions² and are an important for industry and biological system³. In addition metal chelates have been studies and attracted much attention due to their interesting electronic and geometrical features in connection with their application various fields⁴. This type of azo compounds which are characterized with (л-acidic) azo imine (-N=N-C-N) which giving results with good rates and great stability as well as characterized with ease of purification and deep colors which have displacements in the wavelength when they are consistent with transition metal ions^{5, 6}. The chelate complexes that have five-membered or six-membered chelate rings are the most stable complexes⁷. In the fact that some drugs including discouraging the growth of germs^{8, 9}. In addition, the important uses of these compounds are analytical reagents^10-12for solvent extraction to determine some metal ions. The azo imidazole compounds have important role in spectral determination field to identify the amount of elements especially with transition metal ions because of its sensitivity and selectivity ¹³. In our research we were able to prepare new ligand (BMP) and its complexes with [Cu(II), Ag(I) and Au(III)] taking into account a spectral study in order to obtain optimum condition (concentration and pH) and metal to ligand ratio for preparation the complexes.

Experimental

Materials and Instruments   

All elements and solvent are of best purity and used as found from the productions.  Microelemental analysis (C.H.N) was gained on a (Eure EA 3000 Elemental analyzer). UV-Vis Spectra were performed in ethanol on a (Shimadz UV-160A) ultra violet-visible spectrophotometer. FTIR-spectra were recorded on a (Shimadz FTIR-8400s Fourier Transform Infrared) spectrophotometer (200-4000) cm^-1 using CsI discs. The ¹HNMR spectra were gained on a (¹HNMR Spectrometer 4000 MHz, Avance III 400 Bruker, Germany”.  The molar Conductivity for the complexes was determined in (10^-3 M) DMSO at room temperature using (HANNA instruments / Conductivity Tester). pH measurement was  performed using (HANNA instruments pH Tester / Pocket pH Tester). Melting points have been gained via using (Stuart Melting Point Apparatus). The percentage of metal in complexes was done by using a “GBC 933 _Plus “Flam Atomic Absorption Spectrophotometer. Thermal Analysis measurements TG, DTG and DSC were performed by using (Mettler) Thermogravimetric Analyzer.

Synthesis of 4-[(2-Benzimidazolyl)azo] Proline (BMP)

This ligand was synthesized according to the method reported in the literature¹⁴ with some modifications as shown in scheme (1)

Scheme 1: preparation of BMP ligand Click here to View Scheme

 

Preparation of solutions

Ammonia acetate as a buffer solution was prepared with a range (5-9) by dissolving (0.01 M , 0.77 gm ) in one litter of deionized water. pH was detected by adding acetic acid or ammonia solution. As for standard solutions for selected metal salts and ligand were prepared by dissolving appropriate weight to the extent (1×10^-5 M- 3×10^-5M).

Synthesis of Complexes

Three complexes were prepared via adding regularly with stirring hot ethanolic solution of (2 mmole) ligand to stoichiometry mole ratio of (1:2) (M: L) for [(Cu(II) and (Ag(I)] and (1:1) for [Au(III)] which were dissolved in the prepared buffer solution at optimum pH. Then the mixture was heated to (60ᵒC) with stirring for (3hour), after that left to cool at room temperature. The colored precipitates were filtered, washed and desiccated in vacuum desiccator. The suggested stoichiometry structure of the prepared complexes is shown in scheme (2).

Scheme 2: The suggested structure for the prepared complexes.   Click here to View Scheme

 

Results and Discussions

The reaction of the prepared ligand (BMP) with selected transition metal ions [Cu(II), Ag(I) and Au(III)] at optimum pH and concentration, which mains to formation of the complexes with following formula [Cu(BMP)₂Cl₂], [Ag(BMP)₂]NO₃ and [Au(BMP)Cl₂]Cl. The ligand (BMP) doings as N, N’-chelator, where N of (azo) and N’ of (benzimidazole). The structure of the complexes is supported via the analytical and spectral studies results. All complexes were stable in air and moisture as well as soluble in most organic solvents such as ethanol, DMSO, DMF, acetone …etc. By studying the molar conductivity of the prepared complexes which were measured at (10^-3 M) in DMSO found that Cu(II) complex was  non electrolyte while Ag(I) and Au(III) complexes are (1:1) electrolyte, supporting the ionic behavior for Ag(I) and Au(III) complexes,   (Table (1)).

Table 1: physiochemical properties, elemental analysis, mole ratio, molar conductance for the ligand (BMP) and selected metal ions.

Complexes (M.wt) (gm/mol)	pH (M:L)	Color λ (nm)	%Found %(Calculated)					ᴧ (S.mol-1.cm2)
			C	H	N	M	Cl
BMP (C12H13N5O2) (260.13)	ـــــــ	Yellow-green	56.87 (56.907)	5.38 (5.471)	25.47 (25.535)	ــــــ	ــــــــ	ـــــ
[Cu(C24H26N10O4)Cl2] (654.8)	7 (1:2)	Blue-violet (610)	44.79 (44.02)	4.43 (3.97)	20.77 (21.70)	9.77 (9.70)	10.91 (10.84)	10
[Ag(C24H26N10O4)]NO3 (690.13)	8.5 (1:2)	Red (497)	40.66 (41.73)	4.19 (3.76)	22.71 (22.31)	15.60 (15.63)	ـــــــ	34
[Au(C12H13N5O2)Cl2]Cl (563.6)	8 (1:1)	Red (496)	25.43 (25.55)	2.19 (2.129)	11.97 (12.42)	34.90 (34.94)	18.99 (18.89)	35

The UV-Vis spectra of the prepared ligand (BMP) and its complexes under examination as was shown in Figures (1 and 2) were shown mainly two peaks observed in ethanol (10^-4) within the range (250-1100) nm. The first peak at (339) nm for ligand (BMP)  was owing to the π→π* transition of the aromatic rings. The second peak (λ_max) at (449) nm ligand (BMP) was related to the π→π* this is assigned to the transition of (GT) intramolecular charge-transfer taken place through the azo moiety (-N=N-)¹⁵.

It has been saw red shift in the visible region when a competitive study between the spectra of mixing aqueous solutions for [Cu(II), Ag(I) and Au(III)] with ethanolic solutions of ligand (BMP) which ranged between (250-1100)nm. The change in the appearance color of free ligand (BMP) solution and high shift in the (λ_max) gives a good sign for coordination and formation of complexes.

Figure 1: UV-Vis spectrum of  the ligand (BMP).   Click here to View Figure

Figure 2: UV-Vis spectra of  the ligand BMP and Au-BMP complexes solution. Click here to View Figure

 

On the other hand the UV-Vis spectra were studied for mixed solutions of [Cu(II), Ag(I) and Au(III)] and ligand (BMP) within the range of concentration from (10^-3M-10^-6M), only 10^-5 obey Lambert Beer low, while the pH sequence was from (5-9). Figure (3) was appeared best suitable straight lines, with correlation factor(r>0.989) when the absorbance plotted against molar concentration in the rang from (1×10^-5M- 3×10^-5M).

Figure 3: Linear relationship between molar concentration and absorbance for metal ion-BMP complexes solution at  optimum pH & λmax.   Click here to View Figure

 

The pH effect was also examined at the range (5-9). Figure (4) was shown the absorbance-pH curves which are completed at (λ_max) and various concentrations for each metal ion solution under study. A high band on the pH curves it is considered as a mark of the complex formation and acceptance as optimum pH for all preparation complexes. But the descent part of the curves may be referring to the dissociation of complex at this point. Therefore, we conclude that all prepared chelate complexes with selected metal ions are formed in neutral or basic medium^{16, 17}:

Figure 4: Effect of pH on absorbance at λmax for metal ions-BMP complexes solution.  Click here to View Figure

 

Spectrophotometry is one of the most useful tools for elucidation of the composition of complexes in solution. The power of the technique lies in the fact that quantitative absorption measurements can be performed without fear of disturbing the equilibria under consideration. Two of the most common techniques employed for identify the composition of the complexes in solution without isolation are the mole ratio procedure and continuous variations method or Job method¹⁸ (Figures (5 and 6)):

Figure 5: Job Method plot of metal ion-BMP ligand complexes solution at optimum pH  and λmax.   Click here to View Figure

Figure 6: Mole ratio plot for metal ion-BMP complex solution at optimum pH and λmax.   Click here to View Figure

 

The two methods support same result about mole ratio of the complexes. It will be taking into account all the result we have obtained to install optimal condition to prepare the complexes of [Cu(II), Ag(I) and Au(III)] with the ligand (BMP).

Determination of Stability Constant and Gibbs free energy of Prepared Complexes

The stability constant (K) for Cu(II) and Ag(I) complexes in a mole ratio (M:L)(1:2) was computed based on the equation¹⁹:

As for the stability constant for Au(III) complex in a mole ratio (M:L) (1:1) was computed based on the equation¹⁹:

Where:

α= degree of dissociation.

Am= the absorption of solution containing the same volume of metal and excess of ligand.

As = the absorption of solution containing a stoichiometric volume of ligand and metal ion.

c = the concentration of the complex solution in mole/ L.

From Table (3) we calculated that Cu (II) and Ag (I) complexes more stable than Au (III) complex. The thermodynamic parameters of Gibbs free energy (DG) were also studied. The DG data have been calculated from the equation below²⁰:

ΔG = -R T ln k

Where R = gas constant = 8.3 J.mol^-1.K, T = absolute temperature (Kelvin).

Table (3) was shown that the formations of all prepared complexes are spontaneous.

Table 2: stability constant (K) and Gibbs free energy (∆G) for the prepared complexes

Electronic Spectra and Magnetic Measurement for Solid Complexes

The UV-Vis absorption spectra of the coordination compounds provide a convenient method for determining the magnitude of the effect of ligand filed on the d-orbitals of the metal ions. The UV-Vis spectrum of Cu-Complex with ligand (BMP) was shown in Figure (7) showed three absorption bands. The first band looked at (610) nm, (16393) cm^-1. This is assigned to the transition (²B₁g →²A₁g). The second band was observed at (427)  nm, (23419) cm^-1 which is assigned to the transition (²B₁g →²B₂g). The third band at (291) nm (34362) cm^-1 and was assigned to C.T transition. These three transitions characterized distorted octahedral (d⁹) geometry D₄h²¹. With paramagnetic properties (Table (4)).

Figure 7: UV-Vis spectrum of the Cu-BMP complexClick here to View Figure

As for the Ag-complexes (d¹⁰), one absorption band at (497)nm (20120)cm^-1 which is related to (л→л*) (MLCT) transition (Figure(8)). These complexes are to be tetrahedral²². The magnetic properties are diamagnetic for Ag-complexes (Table (4)).

Figure 8: UV-Vis spectrum of the Ag-BMP complex   Click here to View Figure

Table 3: The UV-Vis spectra for the prepared complexes at (10^-4M)

Compounds	λ nm	Wave Number cm-1	ϵo× 104 L.mol-1.cm-1	Assignment	Magnetic properties
[Cu(BMP)2Cl2]	610 427 291	16393 23419 34362	0.124 0.028 0.731	2B1g→2A1g 2B1g→2B2g MLCT	Paramagnetic
[Ag(BMP)2]NO3	497	20120	0.414	л→л* (MLCT)	Diamagnetic
[Au(BMP)Cl2]Cl	980  861  653 496	10204  11614  15313 20161	0.096 0.018 0.040 1.305	1A1g→2A2g  1A1g→1Eg  1A1g→1A2u 1A1g→1B1g	Diamagnetic

 

Recently the UV-Vis spectrum for Au-complexes d⁸ low spin square planer structure, was appeared (d-d) transition. The [Au(BMP)Cl₂]Cl have four absorption bands at (980, 861, 653 and 496)nm (10204, 11614, 15313 and 20161)cm^-1 which are due to transition of  ¹A₁g→²A₂g, ¹A₁g→¹Eg, ¹A₁g→¹A₂u and ¹A₁g→¹B₁g respectively as was shown in (Figure(9))²³. With diamagnetic properties (Table (4)).

Figure 9: UV-Vis spectrum of the Au-BMP complex.   Click here to View Figure

 

IR Spectra of Prepared Ligands and Complexes

For identification and detect the coordination site that may be involved in complexation. The FTIR spectra of all prepared complexes using CsI were compared with the prepared ligand (BMP) (Figures (10-13)). The spectra of the prepared complexes presented the bands specific to the ligand with some differences in the shape and positions of the bands mention to the formation and coordination of complex, Table ( 5) induced the main infrared spectral bands of the free ligand and its complexes. The FTIR spectrum of the ligand (BMP) has been appeared a band at (3440 cm^-1 that refer to ν(O-H) of carboxylic group in proline moiety²⁴. Another band was appeared at (3328 cm^-1) refer to ʋ(N-H) group of proline moiety, these two bands stay unchanged in position in the spectra of prepared complexes mention that there were no coordination through N-H and COOH group for proline. A doublet bands was prepared at (1620 and 1591) cm^-1, these bands have been refer to ʋ(C=N) in imidazole moiety which were showed changes in shape and shifted to low wave number in the spectra of all complexes due to coordination through N3 for imidazole moiety. The ligand (BMP) also showed triplet bands at (1450, 1448 and 1429) cm^-1 which refer to (N=N) azo moiety²⁵. These bands were a specific feature of azo composites. The strength of these band reduced in the complexes spectra, in spite of were shifted to lower frequency by (43) cm^-1 for ligand (BMP) which is due to coordination through azo moiety.  The band of     (-C-N=N-C-) was appeared in the spectrum of the  ligand and on complexation was showed a negative shift of order (74-23)cm^-1 for ligand (BMP) with changing in intensity and shape, this another indicating the engagement of azo moiety in the coordination with the metal ion. In addition there are new set of bands do not exist in the spectrum of free  prepared ligand but display in the spectra of the complexes such as ʋ(M-N)_azo, ʋ(M-N)_im, ʋ(M-Cl) in Cu and Au complexes and (M-NO₃)²⁶. These mention to the coordination places of the prepared ligand (BMP) with metals ion.

Table 4: Main band of FTIR for the ligand (BMP) and its Complexes Click here to View Table

Figure 10: FTIR spectrum of BMP ligand.  Click here to View Figure

Figure 11: FTIR spectrum of Cu-BMP Complex.  Click here to View Figure

Figure 12: FTIR spectrum of Ag-BMP Complex. Click here to View Figure

Figure 13: FTIR spectrum of Au-BMP Complex. Click here to View Figure

 

¹H NMR Spectra of Prepared Ligands and Complexes

¹H NMR spectra for the prepared ligand (BMP) and its complexes with )Cu(II), Ag(I) and Au(III)( in DMSO are shown in (Figures(14-17)) and the peak assignments are explained in Table (6). A singlet peak was appeared at δ(15.3) ppm in the spectrum for ligand (BMP) which was due to one proton of free carboxyl moiety in proline²⁷. Another singlet signal peak noted at δ(5.5) ppm in these spectrum for the ligand (BMP) owing to a proton for (NH) imidazole. These signal peaks don’t affect in coordination but stay almost without any chemical shift. The multiple signals noted in the region δ (8.4-7.2) ppm for ligand (BMP), these were denoted to aromatic proton (4H) for benzene ring in benzimidazole moiety. So little shift happen when complex formation. Furthermore a singlet signal detected in the region δ (2.6) ppm for ligand (BMP) which were attributed to (-NH) proton of proline ring²⁷, there are no shift were looked in the position in the spectra of the complexes. Also another signal peaks were looked in the spectra of the prepared ligand (BMP) and its complexes which were described in Table (6):

Table 5 : ¹HNMR spectra of BMP and its Complexes

Compound	-COOH	Ar-H	-NH(imd.)	-NH(Pro.)	Aliph-H(Pro.)
BMP	15.3	(8.4-7.2)	5.5	2.5	(1.1-1.0)
[Cu(BMP)2Cl2]	15.4	(7.5-6.8)	5.5	2.6	(1.2-1.0)
[Ag(BMP)2]NO3	15.4	(8.1-7.0)	5.5	2.6	(1.2-1.0)
[Au(BMP)Cl2]Cl	15.4	(7.7-7.0)	5.5	2.6	(1.15-1.0)

 

Figure 14: 1HNMR Spectrum for the BMP Ligand Click here to View Figure

Figure 15: 1HNMR Spectrum for the Cu-BMP Complex.   Click here to View Figure

Figure 16: 1HNMR Spectrum for the Ag-BMP Complex.   Click here to View Figure

Figure 17: 1HNMR Spectrum for the Au-BMP Complex. Click here to View Figure

 

Thermal Analysis of the Ligand (BMP)

Figure (18) have been appeared the thermo gravimetric analysis (TG and DTG) curves for the prepared ligand (BMP) at room temperature up to 900C⁰ under helium as inert gas and differential scanning calorimetric (DSC) curve (Figure (19)). The ligand (BMP) with the formula (C₁₂H₁₃N₅O₂), it showed three decomposition steps at the temperature range (60-800) C⁰. The first step of (50-310) C⁰ with mass loss of benzimidazole and azo moiety (60.14%) (Calculated 60.76%) indicated by the DTG peak at (273) C⁰ with an endothermic peak in DSC curve at (131.4) C^o.The second temperature of range (310-450) C⁰ the decomposition step assigned to mass loss (COOH) group (18.82%) (Calculated 17.30%) and the DTG peak at temperature (370) C^o and an endothermic peak at (271.73) C⁰ in DSC curve. And finally the third step was represented by mass loss for (C₂H₃^•) (11.00%) (Calculated 11.15%) fragment at temperature range (450- 700) C⁰. The DTG peak was appeared at (555) C⁰ while the DSC curve was shaw exothermic peak at (527.22 C^o). The residue represented the fragment (CH₃N^•) (10.04 %) (Calculated 10.36%).

C₁₂H₁₃N₅O₂ →C₈H₆N₄^•+COOH^•+CH₃N^•+C₂H₃^•.

Figure 18: TG and DTG for BMP Ligand   Click here to View Figure

Figure 19: DSC for BMP Ligand. Click here to View Figure

Figure 20: Antibacterial Activity of prepared Ligand (BMP) and its Complexes.   Click here to View Figure

 

Table 6:  The Diameters (mm) of deactivation of two bacteria of the prepared  ligand (BMP) and its complexes

No.	Compound	G+( Staph)	G-( E-Coli)
2	BMP	9	ــــ
4	[Cu(BMP)2Cl2]	10	ــــ
6	[Ag(BMP)2]NO3	15	10
8	[Au(BMP)Cl2]Cl	11	15
9	Ciprofloxacin	18.15	18.5
10	Control (C) (DMSO)	0	0

 

Antibacterial Activity of Prepared Ligands and its Complexes

Two types of bacteria were used in this experiment, Gram Negative Bacteria, Escherichia Coli (E-Coli) and Gram Positive Bacteria, Staphylococcus aureus (Staph) and Ciprofloxacin used as reference. The ligand (BMP) and its complexes have been appeared low antibacterial activity against Staphylococcus and no antibacterial activity against E-coli exception of Ag-complex had low activity while Au-complex had high activity when comparable with the activity for Ciprofloxacin. Generally the (ZI) mm compounds were in the following order;

Figure 21: Dying of prepared Ligand (BMP) and Au-BMP complex. Click here to View Figure

 

Ciprofloxacin > [Au(BMP)Cl₂]Cl >  [Ag(BMP)₂]NO₃ >>[Cu(BMP)₂Cl₂] > BMP>(DMSO)

The increased inhibition activity of the metal complexes can be explained on the basis of Tweedy’s chelation theory ²⁸, Further; it increases the delocalization of π- electrons over the whole chelate ring ^{29, 30}.

Dying performance

The dying method of the Cu-BMP and Au-BMP complex were studies and applied on acrylic fabric. These set dyes were given colors purple and brown. Figure (21) show a clear color.

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