Synthesis , Characterization and in vitro Antibacterial Studies of Novel Transition Metal ( II ) Complexes of 2 , 5-Diamino-2-( difluoromethyl ) pentanoic Acid Hydrochloride Hydrate

The synthesis and characterization of novel transition Metal (Cu(II), Co(II), Ni(II) and Zn (II)) complexes of 2,5diamino-2-(difluoromethyl) pentanoic acid hydrochloride hydrate (DPH) have been described. The ligand and metal complexes were characterized by Melting point, Conductivity measurement, Elemental analysis, Fourier Transform infrared (FTIR) spectroscopy, Electronic spectroscopy, Magnetic susceptibility measurement and Electrospray Ionization Mass Spectrometry (ESI-MS). The FTIR spectral data suggests that the ligand behaves as a bidentate ligand coordinates to the metal ions through an oxygen atom of the carboxylate and a nitrogen atom of amino group. The terminal amino group of the ligand is protonated to form NH3+ while the carboxylic moiety is deprotonated forming zwitterionic complexes. Electronic spectral and magnetic studies data suggest that the complexes of Cu(II), Co(II) and Ni(II) have octahedral geometry around metal ions while tetrahedral geometry was proposed Zn (II) ion. In vitro anti-bacterial activities of the ligand and metal complexes were carried out using agar diffusion method against two gram-positive bacteria (Staphylococcus aureus, Bacillus substilis) and two gram-negative bacteria (Escherichia coli, Klebsiella pneumonia). The results obtained revealed that the metal complexes showed enhanced antibacterial activities against the four micro-organisms with lowest minimum inhibitory concentration (MIC) when compared to parent compound.


Introduction
Metal carboxylates have attracted much attention owing to their great biological and industrial applications [1][2][3][4][5].Organic ligands containing carboxyl groups have been utilized in the preparation of many useful materials such as metal organic frameworks (MOFs) as a result of affinity of carboxylate anions to the metal ions.It is a well known fact that carboxylate or dicarboxylate ligands exhibit a versatile coordination behavior by displaying distinct bonding modes towards metal ions [6][7][8][9][10][11][12].A literature survey has revealed the synthesis and characterization of complexes with ligand in which carboxylate anion and nitrogen atom formed coordination bonds with metal ions, some of these complexes have been found to possess good antimicrobial activities [13][14][15][16][17][18][19][20][21].Transition metals are the best known cations usually used in the preparation of metal-organic complex due to their electrical, magnetic, catalytic and luminescence properties [22][23][24][25].
The choice of copper, cobalt, nickel, zinc in this study is as a result of their importance in biological and physiological activities that facilitate a variety of chemical reaction needed for life [20,21,[26][27][28][29][30].
Antimicrobial resistance is fast becoming a global concern with swift increase in multidrugresistance bacteria and fungi [32][33][34].This has necessitated continuous search for new antimicrobial compounds with particular focus on coordination compounds of biological importance [35][36][37].The increased lipophilic character of the coordinated metal complexes with the resultant enhanced ability to permeate the cell membrane of the microbes has been proposed as reason for their improved activity over their parent ligands [38][39].Chelation, which is responsible for the reduction in the polarity of the metal ions by partial sharing of its positive charge with donor group of ligands also support this concept [40][41].Encouraging antimicrobial activities of some chelates with carboxylates groups have been reported [42][43][44][45].To the best of our knowledge, it is interesting to state that extensive studies of 2,5diamino-2-(difluoromethyl) pentanoic acid hydrochloride hydrate metal complexes have not been reported to date.

Results and Discussion
The preparation of DPH-metal complexes can be represented by the general formula thus: The complexes are formed by 2:1 molar condensation of ligand to transition metals and the stoichiometric reactions involved in the complex formation resemble earlier work relating to chlorpromazine hydrochloride [46].The melting point of the metal complexes differed from the starting material (ligand), this indicates the likelihood of the formation of coordination compounds.Also, high purity of the complexes formed can also be predicted from sharp melting points obtained.The colours of the metal complexes are distinctly different from that of the ligand, thus, it can then be inferred that the colours displayed by the metal complexes are regulated by the metal ions, a possible indication to the formation of coordination compounds.All the metal complexes are insoluble in common organic solvents but soluble in DMSO and water.
The results showed the presence of water molecules in all the metal complexes either as coordinated ligand, water of crystallization outside the coordination sphere or both.The presence of uncoordinated water molecules outside the coordination sphere was confirmed using cobalt chloride paper.The droplets of colorless liquid stemmed out by gently heating of metal complexes (3-7) turned cobalt chloride paper from blue to pink confirming the presence of water molecules outside the coordination sphere while complexes 1 and 2 with no uncoordinated water molecules had no effect on cobalt chloride paper.This test further buttressed the molecular formulation proposed for each of the metal complexes.
Two amino groups are present in the ligand (DPH), the terminal one is protonated to form NH3 + while the second one is involved in coordination with metal ions through nitrogen atom.The carboxylic moiety is deprotonated leading to the formation of zwitterionic structure of metal complexes.This assertion is in agreement with our earlier work where crystal structure involving the same ligand was obtained [47].

Fourier Transform Infra-red Spectra (FTIR)
The characteristic FTIR bands of the metal complexes differed from the free ligand (DPH) either by the shift or disappearance of some characteristics frequencies and appearance of some new frequencies.This provided significant indications regarding the coordination and bonding sites of the ligand.Relevant characteristic bands of all the metal complexes together with that of the ligand are listed in Table 1.The principal bands attributed to asymmetric (υas) and symmetric (υs) stretching frequencies of (OCO) groups are reported in Table 2 The sharp absorption bands in the region3352-3311 cm -1 observed in the spectra of metal complexes are assigned to the N-H stretching frequency due to the protonation of terminal amino group of the DPH to form NH3 + .This resembles similar compound of DPH earlier reported where x-ray crystal structure is obtained [47].This may be due to the strong hydrogen bonding between water molecules and amino group [48].
Primary amino groups usually exhibit two N-H stretching frequencies hence the bands in the region 3254 and 3173 cm -1 could be attributed to asymmetric and symmetric stretching frequencies of NH2 in the spectrum of the ligand.These symmetric and asymmetric N-H stretching frequencies of the DPH were shifted bathochromically due to coordination of N-donor ligand to metal ions [20], indicating involvement of NH2 in the chelation [49][50].
The FTIR Spectrum of the ligand showed a medium intensity band at 3048 cm -1 assigned to stretching vibration frequency of (-OH) of carboxylic group.On complexation with metal (II) ions, the corresponding shift bands in the spectra of the metal complexes are 3018-3012 cm -1 (Cu); 3037 cm -1 (Ni); 3073 -3054 cm -1 (Co); and 3065cm -1 (Zn) indicating possible coordination through the carboxylate oxygen atoms by deprotonation [7,[51][52].The involvement of hydroxyl of carboxylate group through oxygen atom in the coordination is further supported by asymmetric and symmetric stretching frequencies of (OCO) groups.The stretching frequencies observed at 1647 cm -1 and 1499 cm -1 in the spectrum of the ligand were attributed to asymmetric (υasy (OCO)) and symmetric υsym(OCO) stretching frequencies respectively.The spectra of the metal complexes show bands, which could be assigned to the asymmetric (υasy) and the symmetric (υsym) stretching vibration of the carboxylate group.These data permit us to deduce that carboxylate group is involved in coordination to metal ions.The separation ∆υ = ∆asy (OCO) -∆sym (OCO) characterizes the nature of the metal-carboxylate bond formed.When ∆υLigand> ∆υcomplexes, the (OCO) group is bidentately coordinated while when the differences of Ligand (∆υL) is less than that of metal complexes (∆υcomplexes), the (OCO) group exhibits monodentate mode of coordination [49,53,54].The differences of asymmetric (∆υasy) and symmetric (∆υsym) stretching vibration of (OCO) group of all themetal complexes were found to be greater than that of the ligand as shown in Table 3.This confirms that carboxylate group is monodentately coordinated to the metal centers through the hydroxyl oxygen atom via deprotonation [49,55].
The FTIR spectra of the ligand and complexes displayed broad absorption band attributed to the presence of coordinated or lattice water molecules.The stretching frequency observed at 3393 cm -1 in the spectrum of ligand due to water molecule vibration undergone shift in the spectra of metal complexes.The corresponding shift in the spectra of metal complexes as a result of coordination are 3457 -3420 cm -1 (Cu); 3441 cm - 1 (Ni); 3453 -3404 cm -1 (Co); 3412 cm -1 (Zn) [7,51].
In the lower frequency region, new bands with medium to weak intensities which provide direct evidence for the complexation (Metal -Ligand bond) were observed in the spectra of the complexes.In the present investigation, the bands in the region698 -651 cm -1 wereassigned to υ (M -N) while the bands in 583 -512 cm -1 region were attributed to υ (M -O) and υ (M -Cl) stretching vibrations [7, 56 57].

Properties of the Complexes
The electronic spectrum of the aqueous solution of free ligand showed absorption bands in the UV-region at 228nm (43, 860 cm-1), 240nm (41, 667 cm -1 ) and 307nm (32, 573 cm -1 ).These bands are assigned to intra-ligand transition due to n-π* transition of the non-bonding electrons present on the oxygen of (C=O) group and nitrogen of the amine groups.The slight bathochromic shift and disappearance of the bands in the spectra of metal complexes relative to the ligand is attributable to coordination.
The Copper (II) complexes of DPH exhibited broad asymmetric bands in the region 14, 286 -15,773 cm -1 (700 -634nm) assignable to 2 Eg 2 T2g  transition.A broad band is expected for d-d transition of the copper (II) complexes in an octahedral environment.The broadness of the band could be attributed to the overlapping of several bands as a result of strong Jahn-Teller distortion expected in a d 9 ion [56,57].The magnetic susceptibility measurements revealed that the copper (II) complexes have an effective magnetic moment of 1.78 -1.84B.M. which indicate that they are magnetically diluted and are in excess of the spin-only value of 1.73 B.M as a result of orbital contribution and spin-orbit coupling.Figure 4 showed plots of magnetic susceptibility (XmT) versus temperature (T) for complex 2.
The absorption spectrum of Ni (II) complex showed three bands corresponding to the electronic transitions for d 8 ion in an octahedral environment [58] similar to those earlier reported [49,56].The effective magnetic moment of 3.26 B.M. was obtained for the Ni (II) complex and thus strengthen the octahedral stereochemistry formulated for the complex with two unpaired electrons [57,58].Figure 5 showed magnetic susceptibility (XmT) versus temperature (T) plots for complex 4. The electronic configuration of Zn(II)ion is d 10 hence its absorption spectra show no bands due to d-d transition, but the absorption bands in its spectrum suffered red shift with hyper or hypo chromic effect.This is a natural occurrence as there is no possibility of transition due to nonavailability of empty d-orbital [59].In view of the well known tendency of Zn(II) to form tetrahedral complexes, by considering the spectrum data and elemental analysis results, tetrahedral geometry is proposed for Zn (II) complex [60].

Electrospray Ionization Mass Spectrometry (ESI-MS) Results
The major fragment ions, peak assignment (theoretical and found) mass per charge ratio (m/z) values and relative abundance of metal complexes 1, 2 and 6 were shown in Tables 4 -6.The m/z values observed in each case compete favourably well with the theoretical values; this is equally an evidence which further support the stoichiometric formulation (1:2 metal-ligand chelates).The peak noticed at m/z = 183.10 in all the spectra of the metal complexes is due to [DPH + H] + which signifies the presence of DPH in the coordination sphere hence suggests a proof for coordination.

Antibacterial Screening Results
The in-vitro antibacterial activity of the ligand (DPH) and seven (7) metal complexes were evaluated using agar well diffusion technique [61,62] against Gram-positive bacteria, Staphylococcus aureus and Bacillus substilis, Gram-negative bacteria, Escherichia coli, and Klebsiella pneumonia and the result is presented in Table 7 and Fig 6 .The Minimum Inhibitory Concentration (MIC) of the ligand (DPH) and metal complexes 2,4 and 6 is as well contained in Table 8 and Fig 7 .The solvent (distilled water) showed no zone of inhibition confirming noninvolvement of solvent on the activity of the metal complexes and the DPH.Generally, the results of the antibacterial screening show appreciable activity of the metal complexes when challenged with the test pathogens.The zone of inhibitions of metal complexes are much larger when compared to the free ligand [57,62] indicating that metal complexes are able to decrease the population of bacterial species than the ligand hence they are more effective as antibacterial agents than the ligand.The synthesized copper complexes (complexes 2 and 3) were found to compete favorably well with antibiotic used particularly at higher concentration.MIC values for complexes 2, 4, and 6 are lower than that of the free ligand, indicating that complexation enhances antibacterial activity of complexes.This process of the antibacterial activity of the metal complexes can be justified using the overtone's concept and the Tweedy chelation theory.In relation to overtone's concept of cell permeability, the lipid membrane that surrounds the cell supports the passage of only lipid soluble materials; as a result, liposolubility is an essential factor that regulates antimicrobial activity [63].On chelation theory, the polarity of the metal ion is reduced to a large extent due to overlap of the ligand orbital and partially sharing positive charge of the metal ion with donor groups.It equally increases the delocalization of π-electrons over the whole chelates ring and enhances the lipophilicity of the metal complexes.Consequently, this increases the lipophilic character of the metal complexes thereby favouring its permeation through the lipid layers of bacterial membrane and facilitates the blocking of metal binding sites of the enzymes of the pathogens [5,13,15,57].The variation in the effectiveness of different compound against different organisms depends either on the impermeability of the cell of the microbes or differences in the ribosomes of microbial cells [57].
It is equally established from the results that the antibacterial potency appears to be concentration dependent which generally agrees with the finding of other researchers who asserted that antimicrobial potency is usually concentration dependent.It was also observed that the most resistant pathogens to the metal complexes were Staphylococcus aureus, Bacillus substilis while the most susceptible were Klebsiella pneumoniae and Escherichia coli.

Materials
The ligand,

Physical Measurements
The elemental (CHN) analyses were performed on Thermo Flask 112 CHNSO elemental analyzer from Micro analytical Laboratory at Medac Limited, Surrey, United Kingdom.The FTIR spectra were collected on FTIR -8501 Shimadzu spectrophotometer over 4000-400cm -1 using KBr pellets.Melting points were determined using MPA100 OptiMelt Automated Melting Point system.Solution electronic absorption spectra of the ligand and complexes were ran in the range of 180-400 nm and 180-1100 nm respectively on Jenway 6405uv/vis.The conductivity of the ligand and their complexes were determined in distilled water using EC214 conductivity meter Hanna instrument with cell constant of 1.013.The electrospray ionization mass spectra were recorded on Micromass Autoseptic Premier/Agilent HP6890GC at Medac Limited, UK.The magnetic susceptibility measurements (XmT) for the transition metal complexes between 5 and 400 K were measured with a superconducting quantum interference device (SQUID) magnetometer (Quantum Design MPMS -5S) available at Chemistry department, Kumamoto University, Japan.

Synthesis of [Cu(DPH)2(Cl)(H2O)].HCl (1):
The complex was synthesized by adding (DPH) (0.473g, 2mmol) dissolved in distilled (15 mL) to stirring methanolic solution (10 mL) of copper(II)chloride dihydrate (0.1705g, 1mmol).The resulting mixture was refluxed for 2 hours.The blue coloured solution was filtered hot and the filtrate kept at room temperature for slow evaporation.The skye-blue powdered formed after three days was washed thrice with methanol, recrystallized in methanol: water

Synthesis of [Co(DPH)2Cl2].2H2O. HCl (5)
This cobalt complex was prepared by dissolving the (0.4734 g, 2 mmol) DPH in 10 mL of distilled water followed by slow addition of (0.2379 g, 1 mmol) cobalt (II) chloride hexahydrate, CoCl2•6H2O in 10 mL methanol.The solution which turned to brown colour and later to wine colour was heated under reflux for 10 h until stable purple solution was obtained.The resulting purple coloured solution formed was cooled and left to evaporate slowly at room temperature.The purple precipitate formed was separated out by filtration, washed with methanol and dried over silica gel.M.wt: 566.

5 COMPLEX 6
Solution electronic spectra of Co (II) complexes of DPH in the visible region displayed three bands typical of octahedral geometry around cobalt (II) ions.The magnetic moments of 4.34 -4.25 B.M. established for cobalt (II) complexes were in agreement with high-spin octahedral (with three unpaired electrons) Co (II) complexes.Thus, corroborates the proposed DPH COMPLEX stereochemistry for Co(II) complexes [57, 59].

Figure 6 .Table 8 .
Figure 6.Inhibitory level of the ligand and metal complexes on bacteria at concentration of 300 ppm.

Table 1 .
. Major FTIR (cm -1 ) data of the ligand and the metal complexes.

Table 3 .
Electronic spectra, magnetic moment and molar conductance data of ligand and metal complexes.

Table 7 .
Zone of inhibition (mm) of ligand and metal complexes on bacteria.
2H2O) were obtained from British Drug House Chemical Limited Co. Poole England.Isolates of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Bacillus substilis were obtained from University of Ilorin Teaching Hospital through Microbiology Department, University of Ilorin, Nigeria.