Equilibrium, Kinetics, DNA Binding and Antimicrobial studies of Trans Amine Cobalt (III) Ethylene Diamine Complex
Padmaja Naishadham1, Satyanarayana S.1*
Department of Chemistry, Osmania University, Hyderabad., Andhra Pradesh, INDIA, PIN-500 007
*Corresponding Author E-mail: ssnsirasani@gmail.com
ABSTRACT:
Trans- [ICH2Co(en)2NH3] Complex was synthesized and characterized by chemical analysis, 1H, 13C NMR and Mass spectra. Pseudo first order reaction kinetics and binding studies of trans- [ICH2Co(en)2NH3] complex with imidazole, substituted imidazoles, histidine, histamine, glycine and ethyl glycine ester has been investigated using spectrophotometric technique. Equilibrium constants were determined as a function of pH at 25°c. Binding and kinetic studies were correlated based on basicity. steric hindrance. From the equilibrium data, it was found that the entering nucleophile participates in the transition state, thereby SN1 mechanism was proposed. The effect of the incoming ligands on the complex was studied by molecular mechanics. The binding property of this complex with DNA was observed using UV/Visible, Fluorescence studies. Antimicrobial property of this complex was also studied using E. coli bacteria.
KEYWORDS: Bioactive compounds, DNA-binding, VitaminB12 models
Coenzyme B12 has long fascinated chemistry and its unique property arises from its different catalytic activity. How the Co –C bond is activated towards homolysis or heterolysis is an enduring subject of research.1,2 Studies on model compounds have continued to complement those on the more complex cobalamin and B12 – based proteins. Steric factors are known to be important in weakening the Co – C bond length and does indeed respond to steric rather than electronic effects in the model compounds.
To understand the mechanistic aspect of coenzyme B12, which actively carries many enzymatic reactions, model complexes of the coenzyme are widely studied. Much of the study with model complexes reveal that the active part of the coenzyme is the Co-C bond cleavage, which initiates the enzymatic reaction. Pentammine (methyl) cobalt (III) complex is the simplest model depicting coenzyme B123, where four amine groups are bound to cobalt in equatorial position and one amine and a methyl group in axial positions trans to each other. Later studies on cobalamins and cobaloximes have been extensively carried out. The carbanion co-coordinated to cobalt (III) is influenced by other ligands in the co-ordination with the cobalt in the cis or trans position.
The cis and trans effect on the Co–C bond was reported with naturally occurring methylcobalamin4 and various synthetic complexes such as tetrapyrroles, imines, oxime, mixed imine-oximes or amines.5–9 The data for the trans-[Co(DH)2(CH3)L] series, the 13C chemical shift and 59Co - 13C coupling constant10 and bond distance are influenced by the trans ligand.11 We have previously studied the trans influence in cobaloxime with varying ligands using spectrophotometric methods and molecular mechanics.12–15 Now, we are reporting in this paper the trans and cis influence of ligands on trans-[ICH2Co(en)2NH3] based on the kinetic and binding studies with different ligands and DNA and antimicrobial activity.
MATERIALS AND METHODS:
t-butylCarbazate, acetone, bromoethane, imidazole, 1-methyl imidazole,2-methyl imidazole, glycine, ethylglycine ester, histidine , histamine and CT DNA were purchased from Sigma-Aldrich Chemicals. Tetrabutyl ammonium hydrogen sulfate (TBAHS), potassium hydroxide, Tris –HCl, magnesium sulfate, conc. HCl, cobalt nitrate, ammonia solution, and methanol were obtained from Merck.
Synthesis of complex:
Trans-[ICH2Co(en)2NH3] has been prepared according to procedure available in the literature3.
To the solution of t-butylcarbazate (10g) in acetone (75ml), MgSO4(2g) and five drops of acetic acid are added. The mixture was heated to reflux for 1hr then cooled, filtered and concentrated in vacuum to give t-butylisopropylidene (white solid).
To the white solid (516mg) in toluene (10ml), solid KOH(218mg) and tetrabutylammonium hydrogen sulfate (100mg) were added and the mixture was stirred continuously and heated to 50°C followed by di Iodo methane(3.6mmol) added slowly. The temperature was increased to 80°C and maintained for 3hrs. The mixture was cooled and washed with water until the aqueous extract had a neutral pH. The organic layer was dried (MgSO4) and concentrated in vacuuo to give light coloured oil.
The oil obtained was converted into hydrochloride salt by heating and refluxing along with 2N HCl (two acid equivalents) in THF for 3hrs. The mixture was cooled and concentrated in vacuo. The residues were brought to dryness by addition and removal of toluene. TLC was used to monitor the formation of the product. Then it was neutralized with ethanol and stored at ambient temperature for further use.
According to Kofod3, the pentamine alkyl cobalt (III)complexes were prepared as follows:
5.82g of cobalt nitrate hexahydrate was dissolved in 3ml of water. At 0°C 35ml of 25% ammonia solution was added, followed by (19.1mmol) of Iodo methyl hydrazine. The temperature of the reaction was slowly increased to 20°C and the mixture was stirred at this temperature for 4hrs while exposing to atmospheric di oxygen. After the mixture was cooled to 0°C, 60ml of ice-cold acetone was added. The orange crystals of pentaammine Iodo methyl cobalt(III) nitrate were filtered off and recrystallized by dissolving it in 6 M aqueous ammonia and precipitated with ice-cold methanol.
A 2.0g amount of pentaammine Iodo methyl cobalt (III) nitrate was dissolved in 6M aqueous ammonia(50 ml) and added to a saturated solution of sodium bromide(50 ml). The pink crystals of pentammine Iodo methyl cobalt(III)bromide were filtered off and washed with concentrated aqueous ammonia followed by washing with acetone
Stage3: Preparation of trans- amine(ethylene diamine ) Iodo methyl cobalt(III)bromide [ICH2Co(en)2OH2] Br2
0.5g of pentammine propyl or Iodo methyl cobalt (III)bromide was added to a solution of 240ml ethylene diamine in 3ml of water. The mixture was stirred at 200C for 16 hrs. At the end of this period Conc. Ammonia (0.5ml) was added and the solution was stirred for another 30 minutes. After filtration, the bromide salt was precipitated by addition of 20ml acetone. The orange brown crystals were filtered off, washed well with acetone and air dried at 200C, and yield was 0.56gm.
This alkyl amine ethylene di amine Cobalt complex was photolabile, particularly in solution. This was soluble in water. Complete work with this complex was performed in diffuse light and solutions were covered with aluminium foil.
Recrystallization followed the same procedure. [ found C , 12.164; H, 4.036; N, 14.132. Calc. for C5 N5 H21 I Co: C, 12.085; H, 4.26; N, 14.066 % ] LC-MS: m/z= 497(APCI-NEG1) in Fig.1, IR: 1055 (CH2(en)), 1580 (NH2 (en)), 491 (Co–N (en), 691 (Co–N) (L) and 3130 (NH- stretching) are given in Fig -2, 1H –NMR 2.102 (-CH2), 4.61(en) are given in Fig -3, and 13C-NMR 44.43(en)is given in Fig-4, UV-Vis peaks at; 22,000, 30,000, 42,000 cm-1 is given in Fig.5, Dependence of formation constants ( logKapp) on the pH for the axial ligation of trans- [ICH2 Co(en)2 NH3] by different ligands (L) is given in fig.6, Dependence of the Rate Constants (kobs) for the axial Ligation of trans- [ICH2 Co(en)2NH3] on the concentration of different ligands (L) is given in fig 7, DTA-TG-MS in Fig 8, Molecular Modeling Structures in Figs 9 - 13, DNA absorption study using U.V spectroscopy in Fig 14, fluorescence study in Fig 15 and 16, Anti Microbial Activity in Fig 17.
Fig1 : LC-MS Spectrum of [ICH2 Co(en)2 NH3]
Fig 2: IR Spectrum of [ICH2 Co(en)2 NH3
Fig 3: 1H NMR Spectrum of [ICH2 Co(en)2 NH3]
Fig 4: 13C NMR Spectrum of [ICH2 Co(en)2 NH3]
Fig 5: U.V. Visible scan of [ICH2 Co (en)2 NH3]
Fig 6: Dependence of formation constants ( logKapp) on the pH for the axial ligation of trans-[ICH2 Co(en)2 NH3] by different ligands (L) at 250C in aqueous solution, ionic strength 1.0 M KCl.
Fig 7: Dependence of the Rate Constants kobs for the Axial Ligation of trans- [ ICH2 Co(en)2 NH3] on the Concentration of different ligands (L) at 250C in aqueous solution, ionic strength 1.0 M KCl
Fig 8: Thermogram of [ICH2 Co(en)2 NH3]
Physical measurements:
The pH of the solution was measured by a Digisun pH meter DI-707. For the calibration of the pH meter, standard buffers of pH 4.0, 7.0, 9.2 were used. Elemental analyses (C, H and N) were performed on Flash EA 1112 series Thermofinnigon, Molecular weight determinations were performed on LC-MS- 2010A Shimadzu with Column- C-18, Detector-UV (254) with MS probe of ESI, 1H and 13C NMR Spectra were recorded on a Bruker ARX-300 NMR Spectrometer with D2O as Solvent at COSIST, University of Hyderabad, Hyderabad. Thermal analysis was carried out using TG8110 thermal analyzer to record simultaneous TG,DTA,MS curves in the temperature range of 20-8000c using platinum crucibles at School of Chemistry, University of Hyderabad, Hyderabad.. Infrared spectra were recorded on a Perkin –Elmer 1600 series-FTIR Spectrometer at Department of Chemistry, Osmania University, Hyderabad, using KBR pellets. UV-VIS spectra were recorded on a Elico BL 198 Model spectrophotometer with temperature control. Models for molecules are drawn with the help of Hyperchem software and Bond lengths, Bond angles and energies are calculated using software. Kinetics and binding studies were performed on a Elico SL 171 model single beam spectrophotometer
Spectroscopic Characterization:
Complex was characterized by IR: 1055.2 (C=C), 1580 (C=N), 491.1 (Co–N) (en),. UV-Vis peaks at; 245,306,437; 1H –NMR 2.102(-CH2), 4.61(en) and 13C-NMR peaks at 44.4(en), LC-MS: (APCI-NEG1) studies with Column - C-18, Detector-UV(254) and MS probe of ESI shows that the calculated m/z value was matching with recorded m/z value (m/z 496). The characterization of the structure and energetic of molecular complexes are essential for understanding many biological functions.
Thermal analysis:
The thermal analysis curves (TG, DTA, MS) for [ICH2 Co(en)2 NH3] complex showed a two step decomposition pattern. In the first step the onset temp ( Ton) at 221.8oC and end set temp ( Ten) at 260.60C has been attributed to the mass change of – 26.25 %. In the second step the onset temp (Ton) at 706.60C and end set temp ( Ten) at 776.50C has been attributed to the mass change of -42.65%. In the DTA curve, there are three peaks. The first endothermic peak appears at 245.50C, the second endothermic peak appears at 4600C and the third endothermic peak appears at 6800C. At the end of decomposition cobalt oxide is present.
Binding and Kinetic Studies:
The binding and kinetic studies were carried out using an Elico single beam spectrophotometer (SL-171 model). Spectra were recorded on a Elico BL198 Model. Concentrations were fixed at 486nm. The sample compartment temperature was maintained at temp25° ± 0.1°.
Determination of Equilibrium Constant:
The apparent binding constants (Kapp) were determined for the axial ligation of trans- [ICH2 Co(en)2 NH3] complex with different ligands. By taking fixed concentration of complex and varying the ligand concentration the absorbance was recorded. Solutions containing, an appropriate buffer (0.2M) to maintain pH, KCl to maintain ionic strength (1.0M) and varying concentration of ligand are taken in a 3 ml cuvette and allowed to equilibrate in a thermostat cell holder at 25± 0.1° for 15 min prior to addition of trans -[ICH2 Co (en)2 NH3].
Absorbance’s were recorded and the apparent equilibrium constants were calculated from the plot of ∆A/[L]f vs. ∆A. Thus, for each ligand Kapp was calculated using Eq. 1.
∆A = ∆Amax [L]f / (1/Kapp + [L]f) ------------ (1)
The least square fit of the above equation after rearrangement is given by Eq. 2:
∆A = ∆Amax – {1 / Kapp (∆A / [L]f)} ------------ (2)
Where
∆A = difference in absorbance between solutions containing only complex with and without ligand.
∆Amax = maximum difference in absorbance recorded at high ligand concentration.
[L]f = the unbound ligand concentration and is calculated from Eq. 3:
[L]f = [L]T – (CT ∆A / ∆Amax) -------------- (3)
[L]T = the total volume of ligand added
CT = the total concentration of trans-[ ICH2 Co(en)2 NH3]
The pH independent equilibrium constants are then calculated from Eq. 4:
Keq = Kapp / aL (4)
Where aL (fraction of ligand as free base) was calculated from Eq. 5:
aL = Ka / (Ka + [H+]) ----------------- (5)
Kinetic Studies:
We have investigated the ligand substitution reactions of trans-[ICH2 Co(en)2 NH3] with imidazole, substituted imidazoles, glycine, ethyl glycine ester, histidine and histamine at 25°c. The reaction rates were determined by maintaining pseudo-first order conditions by taking 10-fold excess of ligand concentration with respect to the complex concentration. The kinetics were studied by varying the concentration of the ligand at 486nm and using appropriate buffer at pH below the pKa of the ligand. The absorbance was monitored at λmax 486 nm.
The first order rate constants (kobs) are obtained by least square fits of the data to Eq. 6 below.
lnAt – ln A∞ = kobst ------------ (6)
Where At is the absorbance at time ‘t’ and A∞ is the final absorbance.
RESULTS AND DISCUSSION:
The ligand substitution reaction of trans-[ICH2Co(en)2NH3] with imidazole, substituted imidazoles, glycine and ethyl glycine ester is shown in equation 2. The Formulae show the ligands used along with their structures. Spectra show the variation of absorption spectra of the complex with different concentration of the ligand. Depending on the pKa values of the ligands, the binding studies are made in the pH range above and below the pKa values. The Kapp values were determined as a function of pH by spectrophotometry. The dependence of Kapp for ligation of trans -[ICH2 Co(en)2 NH3] with imidazoles upon the pH and the data is given in Table 1. Up to pKa of the ligand, the log Kapp increases with pH but above pKa the log Kapp is independent of pH. It is observed that the Kapp value below the pKa value is very low due to the protonation of the ligand and as the pH increases, ligand gets deprotonated and binds strongly to Co (III) and Kapp increases. The Keq values for the binding of Imidazoles to [ ICH2 Co(en)2 NH3] follow the order :
1-MeIMD > IMD > 2-MeIMD
Where as the binding of amino acids follow the order of
Histidine > Histamine > Glycine > Glycine ethyl ester
The stability order can be explained by considering the HSAB principle, basicity of ligands and their ability of π-bonding and σ-donation. Considering imidazole series, for 1-MeIMD and IMD the formation constants are high for higher pKa values, i.e. they follow the basicity order. 2-Me-Imidazole is more basic than Imidazole, but the Keq is smaller. This can be attributed to the steric hindrance due to the presence of methyl group at C2 position. Though histamine is slightly more basic than histidine, histidine forms a more stable complex as it is a better π- acceptor than histamine. Among glycine and glycine ester, both are σ -donors but glycine has more binding constant as it is more basic than ethyl glycine ester. The amino acids baring more basic than Imidazoles, form less stable Complexes. The stability order of Imidazoles is attributed to the ability of imidazoles to bind with Co(III) through dπ -pπ back bonding. Glycine and ethyl glycine ester being only σ donors, cannot accept electrons in similar way.
The rate of the reaction increases drastically near the pKa of the ligand. The slope of the plot of kobs vs. concentration of the ligands gives second order rate constant, kon’ at given pH. The data for the plot is given in Table 2.
The slopes of the least square fit of the Eq. 7 gives the second order rate constant.
kobs = kon’[L]T + koff ----------------------- (7)
[L]T =Total ligand concentration
The pH independent second order rate constants, kon are obtained by dividing K Ion with degree of dissociation α Eq. 8.
kon = kon’ / aL ------------------------------------ (8)
The plot of kobs vs. pH given in Fig. 18 clearly indicates that as the pH increases the kobs increases as the deprotonated form of the ligand is readily available at higher pH. The second order rate constants increase as the nucleophilicity of the ligand increased. This is in accordance with the order of Keq values. The kinetics of substitution of the axial base in alkylcobaloximes and related cobalt complexes has been studied under a variety of conditions.16 The studies on cobalt complexes and adenosylcobaloxime provide evidences for the mechanism of substitution to be dissociative17 (Id or D). In view of the evidence presented above, for the existence of pentacoordinate alkylcobaloximes and the ligation kinetic studies of others, both on alkyl cobalt complexes and on cobaloxime complexes 18,19with other equatorial ligand system,20 an SN1 mechanism may be suggested.
Molecular Mechanistic Studies:
The structural investigation of coordination and organometallic chemistry has been advanced using molecular mechanics.21-24 Using MM2 parameterization, the optimized structure was deduced using Hyper chem. 7.5 software, which shows ball and stick representation of complex. The optimized structure of complex with imidazole, 1-Me- imidazole, 2-Me- imidazole, Glycine, ethyl glycine ester, Histidine, Histamine have been given in Fig 9-13. The bond lengths and bond angles are calculated and given in Tables 3 and 4.
Table 1:
|
pH |
1-Me-imd |
Imd |
2-Me-imd |
gly |
Gly-OEt |
Hist |
Histmn |
|
5.0 |
|
|
– |
– |
– |
1.75 |
1.581 |
|
5.5 |
|
|
– |
– |
– |
|
2.05 |
|
6.0 |
1.67 |
1.75 |
0.994 |
|
0.678 |
2.6 |
2.45 |
|
6.5 |
2.134 |
2.201 |
1.48 |
|
1.162 |
2.825 |
2.76 |
|
7.0 |
2.53 |
2.57 |
1.953 |
0.014 |
1.602 |
2.94 |
2.91 |
|
7.5 |
2.81 |
2.82 |
2.36 |
0.514 |
1.95 |
2.988 |
2.97 |
|
8.0 |
2.95 |
2.94 |
2.66 |
1.01 |
2.166 |
3.0 |
2.99 |
|
8.5 |
3.011 |
2.99 |
2.82 |
1.5 |
2.26 |
3.0 |
3.0 |
|
9.0 |
3.031 |
3.0 |
2.88 |
1.95 |
2.3 |
3.0 |
|
|
10.0 |
3.04 |
3.013 |
2.91 |
2.57 |
2.315 |
3.5 |
|
|
11.0 |
3.04 |
3.14 |
2.91 |
2.73 |
2.32 |
|
|
|
Keq |
1098 |
1032.89 |
823 |
573.44 |
207.45 |
1026.23 |
1018.55 |
Formation constants (Kapp) for the axial ligation of the trans amino (ethylene diamine) Iodo methyl cobalt(III) complex by different ligands at 250 C for different pH Values
Table 2:
|
|
M/L |
1-Me-Imd |
Imd |
2-Me-Imd |
Gly |
Gly-Oet |
Hist |
Histmn |
|
kobs (S-1) |
1:50 |
3×10-5 |
2×10-5 |
1×10-5 |
1×10-5(1:10) |
2×10-5(1:10) |
2×10-5(1:10) |
2×10-5(1:10) |
|
|
1:100 |
3×10-5 |
3×10-5 |
5×10-5 |
2×10-5(1:20) |
2×10-5(1:20) |
2×10-5(1:20) |
2×10-5(1:20) |
|
|
1:150 |
3×10-5 |
3×10-5 |
6×10-5 |
5×10-5(1:30) |
5×10-5(1:30) |
5×10-5(1:30) |
5×10-5(1:30) |
|
|
1:200 |
6×10-5 |
7×10-5 |
9×10-5 |
6×10-5(1:50) |
8×10-5(1:50) |
8×10-5(1:50) |
8×10-5(1:50) |
|
kon’ |
|
4.1x10-4 |
3.8×10-4 |
2.62x10-4 |
1.4 x10-4 |
9.9x10-5 |
3.67x10-4 |
3.59x10-4 |
|
pH |
|
7.5 |
6.5 |
6.5 |
11.0 |
7.5 |
7.5 |
7.5 |
|
α |
|
0.586 |
0.154 |
0.037 |
0.948 |
0.43 |
0.43 |
0.43 |
|
kon (dm3 mol-1sec-1) |
|
6.9x10-4 |
2.5x10-3 |
7.1x10-3 |
1.5×10-4 |
2.3×10-4 |
8.5×10-4 |
8.3×10-4 |
Dependence of the rate constants( kobs) for the axial ligation of trans amino (ethylene diamine)Iodo methyl cobalt( III ) on the concentration of the ligand at 250 C
Table- 3:
|
Complex |
|
Imidazole |
1-Me- Imidazole |
2-Me- Imidazole |
Glycine |
Ethyl glycine ester |
Histidine |
Histamine |
|
[ICH2Co (en)2NH3] |
Co1- N2 |
2.2428 |
2.227
|
2.3148
|
1.8955 |
1.8828 |
1.8927 |
1.88312 |
|
|
Co1- N3 |
2.0557 |
2.029 |
2.2422 |
1.8988 |
1.8934 |
1.8885 |
1.8859 |
|
|
Co1- N4 |
2.3273 |
2.339 |
2.0602 |
1.8947 |
1.90076 |
1.90152 |
1.90097 |
|
|
Co1- N5 |
1.86243 |
1.911 |
1.8612 |
1.9108 |
1.90562 |
1.89951 |
1.9012 |
|
|
Co1- C10 |
2.0931 |
2.066 |
2.1176 |
1.96656 |
1.95722 |
1.96332 |
1.9642 |
|
|
Co1- N11 |
2.3371 |
2.066 |
2.336 |
1.85237 |
1.8737 |
1.8877 |
1.9040 |
|
|
C6- N2 |
1.5244 |
1.4736 |
1.525 |
1.4793 |
1.4721 |
1.4743 |
1.46779 |
|
|
C7- N3 |
1.54345 |
1.628 |
1.5434 |
1.4712 |
1.4712 |
1.4737 |
1.46781 |
|
|
C8- N4 |
1.587 |
1.628 |
1.49923 |
1.4696 |
1.46627 |
1.4679 |
1.4691 |
|
|
C9 – N5 |
1.587 |
1.503 |
1.6256 |
1.47342 |
1.4696 |
1.4726 |
1.46381 |
|
|
C6- C7 |
1.587 |
1.504 |
1.5015 |
1.5275 |
1.4838 |
1.5322 |
1.50542 |
|
|
C8- C9 |
1.5579 |
1.517 |
1.5565 |
1.5354 |
1.5025 |
1.5317 |
1. 499 |
Bond lengths of [ ICH2 Co (en)2NH3]
Table- 4:
|
Complex |
|
Imidazole |
1-Me- Imidazole |
2-Me-Imidazole |
Glycine |
Ethyl glycine ester |
Histidine |
Histamine |
|
[CH2ICo(en)2NH3] |
2-1-3 |
73.464 |
77.133 |
72.5589 |
84.457 |
73.4027 |
82.306 |
84.1484 |
|
|
4-1-5 |
37.102 |
36.33911 |
37.6823 |
86.1876 |
66.6631 |
87.1662 |
84.579 |
|
|
8-4-1 |
122.303 |
119.269 |
126.239 |
110.194 |
110.853 |
112.074 |
109.889 |
|
|
1-5-9 |
152.534 |
148.846 |
159.788 |
111.441 |
111.487 |
112.467 |
112.199 |
|
|
6-2-1 |
123.552 |
2.1604 |
123.24 |
113.607 |
112.014 |
112.074 |
108.539 |
|
|
7-3-1 |
124.898 |
117.959 |
129.043 |
112.527 |
112.014 |
117.049 |
110.329 |
|
|
4-8-9 |
107.379 |
107.856 |
102.06 |
105.208 |
102.085 |
104.343 |
101.698 |
|
|
5-9-8 |
65.7502 |
65.3301 |
65.8354 |
108.34 |
104.78 |
107.31 |
109.795 |
|
|
2-6-7 |
103.507 |
103.452 |
106.319 |
110.764 |
101.061 |
110.604 |
111.908 |
|
|
3-7-6 |
114.579 |
115.352 |
113.964 |
108.715 |
105.458 |
109.08 |
106. 615 |
|
|
10-1-11 |
156.764 |
159.784 |
154.788 |
133.624 |
105.057 |
131.409 |
115.908 |
Bond angles of [ICH2 Co (en)2NH3]
Fig. 9 : Trans amino Iodo methyl ethylene diamine cobalt complex
Fig. 10 :Iodomethyl (Imd) ethylene diamine cobalt complex
Fig. 11 Iodomethyl (2-Meimd) ethylene diamine cobalt complex
Fig. 12: Iodomethyl (Etglyest) ethylene diamine cobalt complex
Fig. 13 :Iodomethyl (Histmn) ethylene diamine cobalt complex
Fig – 14 U.V.Visible scan of DNA binding of [ICH2 Co(en)2NH3]
Fig – 15 Fluorescente emisión spectra of complex [ICH2Co(en)2NH3] 2+ in aqueous buffer Tris 5mM, NaCl 50mM, pH 7.0) in the presence of CT DNA, [Co] = 20μM, [DNA] / [Co] 0,5,10,15,20 (The arrow shows the intensity changes upon increasing concentration)
Inset: Plots of relative integrated emission intensity vs [DNA] / [Co].
Fig – 16 Emission quenching of [ICH2Co(en)2NH3] with increasing [Fe(CN)6]4-In the presence and absence of DNA.
Fig 17 – Anti microbial activity of [ICH2 Co(en)2 NH3]
DNA Binding. Absorption Spectral Studies:
The application of electronic absorption spectroscopy in DNA-binding studies is one of the most useful techniques.25-26 Metal complex binding with DNA through groove mode usually results in hypochromism and bathochromism, due to the groove mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The extent of the hypochromism commonly parallels the groove binding strength. The absorption spectra of the complex in the absence and presence of calf thymus DNA are illustrated in Fig. 14 . In the UV region, the intense absorption bands observed in Co(III) complexes are attributed to intraligand dΠ -pΠ transition of the coordinated groups. Addition of increasing amounts of CT DNA results in hypochromism and moderate bathochromic shift in the UV spectra of the complex [ICH2Co(en)2NH3]2+. These spectral data may suggest a mode of binding that involves a stacking interaction between the complex and the base pairs of DNA. In order to quantitatively compare the binding strength of the two complexes, the intrinsic binding constants K of the complexes with CT DNA were determined according to the following equation 27,28 through a plot of [DNA] / ( εb- εf ) vs. [DNA] (Eq. 9 ).
[DNA] / (εa – εf) = [DNA] / (εb – εf) + 1/(K (εb – εf)) (9)
Where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient εa , εf and εb correspond to Aobs / [Co], the extinction coefficient for the cobalt complex in the free and fully bound form, respectively. Intrinsic binding constants K were obtained about 1.9 x 104 M from the decay of the absorbance. The binding constants indicate that the complex binds more strongly.
Fluorescence Studies:
The complexes can emit luminescence in Tris buffer (pH 7.0) at emission maxima at 558 nm. Binding of complex to DNA was found to increase the fluorescence intensity. The emission spectra of complex in the absence and presence of CT DNA are shown in Fig 15. The plots of the relative intensity versus the ratio of [DNA]/[Co] are also inserted in Fig 16. Upon addition of CT DNA, the emission intensity increases steadily.
This observation is further supported by the emission quenching experiments using [Fe(CN)6]4- as quencher. The ion [Fe(CN)6]4- has been shown to be able to distinguish differentially bound cobalt (III) species and positively charged free complex ions. The complex binding to DNA can be protected from the quencher, because highly negatively charged [Fe(CN)6]4- would be repelled by the negative DNA phosphate backbone, hindering quenching of the emission of the bound complex. The method essentially consists of titrating a given amount of DNA-metal complexes with increasing the concentration of [Fe(CN)6]4- and measuring the change in fluorescence intensity. The ferro-cyanide quenching curves for complex in the presence and absence of CT DNA are shown. Obviously it insert into DNA much deeper. The absorption and fluorescence spectroscopy studies determine the binding of complexes with DNA.
Anti microbial activity:
The antimicrobial screening effects of these complexes were tested against Escherichia coli bacteria by the well diffusion method29, using agar nutrient as the medium. In a typical procedure the agar medium was inoculated with microorganisms. The well was filled with the test solution using a micropipette and the plate was incubated, 24 h for bacteria at 35°C. During this period, the test solution diffused to surrounding medium and the growth of the inoculated microorganisms was affected. Wherever the inhibition zone developed, the concentration was noted. The increase in the delocalization of π- electrons over the whole chelate ring enhances the penetration of the complexes blocking of the metal binding sites in the enzymes of microorganisms.30
It was observed that the complex inhibit the growth31 of E.coli as given in figure 17. The antimicrobial activity has been found to be concentration and substitute dependent as the zone of inhibition increases with increase in concentration of the complex (5nm dia. For 20 μm/L).
CONCLUSIONS:
The ligand substitution reactions were studied by different ligands on trans-amine bis(ethylenediamine)Iodo methyl cobalt(III) followed the basicity order in the series of ligands: 1-MeIMD > IMD > 2-MeIMD > glycine > glycine ethyl ester. The binding and kinetic constants varied with the incoming nucleophile suggesting that the nucleophile is taking part in the transition state. Thus, Id mechanism is suggested. The DNA binding studies suggests that the complex binds with CTDNA.
We gratefully acknowledge the UGC, New Delhi for financial support in the form of major research project.
REFERENCES:
1. Brown K.L. Dalton Trans, 2006, 1123
2. Randiccio L. Comments Inorg. Chem, 1999, 21, 327.
3. P. Kofod, Inorg.Chem. 1995, 34, 2768.
4. ‘Vitamin B12 and B12-Proteins’, Eds: B. Kräutler, D. Arigoni, B. T Golding, Wiley-VCH, Weinheim, Germany, 1998.
5. D. Dolphin, A. W. Johnson, Chem. Commun. (London) 1965, 494.
6. K. Farmery, D. H. Busch, Inorg Chem, 1972, 11, 2901.
7. G. N. Schrauzer, Angew. Chem., Int. Ed. 1976, 15, 417; B. D. Gupta, V. Vijaikanth, V. Singh, Organometallics, 2004, 23, 2069; V. Vijaikanth, B. D. Gupta, D. Mandal, S. Shekhar, Organometallics 2005, 24, 4454.
8. G. Costa, G. Mestroni, E. de Savorgnami, Inorg. Chim. Acta 1969, 3, 323.
9. a) T. S. Roche, J. F. Endicott, J. Am. Chem. Soc. 1972, 94, 8622.;
b) P. Kofod, P. Harris, S. Larsen, Inorg.Chem. 1997, 36, 2258.
10. F. Asaro, L. Liguori, G. Pellizer, Phys. Chem. Chem. Phys. 1999, 1, 4981.
11. L. Randaccio, N. Bresciani-Pahor, E. Zangrando, L. G. Marzilli, Chem. Soc. Rev. 1989, 18, 225.
12. D. R. Sudharshan Reddy, B.. Krishna, P. Pallavi, N. Navaneetha, S. Satyanarayana, Indian. J. of Chem. 2005, 44A, 678.
13. N. Navaneetha, S. Satyanarayana, Indian J. of Chem. 2005, 44A, 1191.
14. J. V. Madhuri, V. Malathi, S. Satyanarayana, J. of Chem. Sci. 2004, 116, 143.
15. J.V. Madhuri, V. Malathi, S. Satyanarayana, J. of Chem. Sci. 2005, 117, 305.
16. K. G. Meyer, Synlett 2004, 2355.
17. C. K. Poon, Coord. Chem. Rev. 1973, 10, 1.
18. A. W. Herlinger, T. L. Brown, J. Am. Chem. Soc. 1972, 94, 388.
19. P. Daublain, J. H. Horner, A. Kuznetsov, M. Newcomb, J.
20. J. Zsako, Z. Finta, C. S. Varhelyl, J. Inorg. Nucl. Chem. 1972, 34, 2887.
21. N. Fey, J. Chem. Technol. Biotechnol. 1999, 74, 852.
22. I. B. Bersukker, M. K. Leong, J. E. Boggs, R. S. Pearl Man, Bull. Soc. Chil. Quim 1997, 42, 405. R. Cini, G. Giorgi, F. Laschi, C. Rossi, L. G. Marzilli, J. Biomol. Struct. Dyn. 1990, 7, 859.
23. N. L. Allinger, J. Am. Chem. Soc. 1977, 99, 8127.
24. J. M. Kelly, A. B. Tossi, D. J. McConnell, C. OhUigin, Nucleic Acids Res. 1985, 13, 6017.
25. S. A. Tysoe, R. J.Morgan, A. D. Baker, T. C. Strekas, J.Phys.Chem. 1993, 97, 1707.
26. A. Wolfe, G. H. Shimer , T. Meehan, Biochemistry, 1987, 26, 6392.
27. Ponuganti Pallavi, Penumaka Nagababu, and Sirasani Satyanarayana, Helvicta Chimica Acta, 2007, 90, 627-639.
28. J.R. lakowicz, priciples of Fluorescence Spectroscopy, Plenum Press, 5523.
29. D.S. Sigman, 1986, Acc.Chem. Res, 19, 180- 186.
30. S.Sitlani, E. C.Long, A.M.Pyle, J.K. Barton, 1992, J.Am.Chem.Soc. 114, 2303- 2312.
Received on 29.01.2013 Modified on 07.02.2013
Accepted on 10.02.2013 © AJRC All right reserved
Asian J. Research Chem. 6(3): March 2013; Page 212-220