syntheses: synthesis of [c h on cl cusn]cl -...
TRANSCRIPT
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Syntheses:
Synthesis of [C16H22ON5Cl4CuSn]Cl
To a methanolic solution of Phenylglycine chloride hydrochloride (10 mM, 2.06 g) 10
mM (1.70 g) of CuCl2.2H2O in MeOH was added dropwise. The fluorescent green
solution obtained was left to stir for 1h. An equimolar amount of dichlorodimethyl-
bis(4-pyrazole N2) tin (IV), dissolved in 5 mL MeOH was added to the stirring
solution and refluxed for 3h. The completion of the reaction was monitored by thin
layer chromatography. The solution was cooled to room temperature and left for two
days, a green colored crystalline product obtained was filtered, washed with CHCl3
and dried in vacuo.
Yield: 68.0 %. mp; 242 ±3 ºC Anal. Calcd. for [C16H22ON5Cl4CuSn]Cl: C, 29.12; H,
3.36; N, 10.61, Found: C, 29.15; H, 3.35; N, 10.52; Selected IR data (KBr, ν cm-1)
3317 (NH2); 1747 (C=O); 1388 (C-H); 1236 (C-N); 1043 (C-C); 765 (Ar); 496 (Cu-
N). Molar Conductance: M (1·10-3 M, H2O): 140.00 -1cm2 mol-1 (1:1 electrolyte).
UV-vis (1·10-3 M, H2O max / nm) 258 nm, 340 nm, 590 nm. ESI-MS (m/z+) 624
[C16H22ON5Cl4CuSn]+.
Synthesis of [C16H22ON5Cl4NiSn]Cl
The complex [C16H22ON5Cl4NiSn]Cl was synthesized using NiCl2.6H2O (2.37 g, 10
mM) by a similar method as described for [C16H22ON5Cl4CuSn]Cl. The yellow
colored product obtained was filtered, washed with CHCl3 and dried in vacuo.
Yield: 58%; mp; 242 ±5 ºC. Anal. Calcd. for [C16H22ON5Cl4NiSn]Cl: C, 29.41; H,
3.40; N, 10.72. Found: C, 29.36; H, 3.41; N, 10.63. Selected IR data (KBr, ν cm-1)
3342 (NH2); 1720 (C=O); 1388 (C-H); 1296 (C-N); 1043 (C-C);778 (Ar); 502 (Ni-
N); 1H NMR (400 MHz, DMSO-d6, 25 ºC, δ) 2.68 (-CH3); 3.83 (-CH); 6.35, 6.43 ( H
phenyl, pyrazole ring); 7.80, 8.05 (pyrazole NH, NH2); 13C NMR (100 MHz DMSO-
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d6, 25 ºC, δ) 50.82-77.11 (CH3, CH2); 125.72-129.63(Ar); 165.96 (C=O); 119Sn NMR
(400 MHz DMSO- d6, 25 ºC, δ) -218.83; Molar Conductance, M (1·10-3 M, H2O):
145.00 -1cm2 mol-1 (1:1 electrolyte) UV-vis (1·10-3 M, H2O, max /nm) 260 nm, 344
nm 578 nm. ESI-MS (m/z+) 619 [C16H22ON5Cl4NiSn]+.
Results and discussion
The complexes [C16H22ON5Cl5CuSn] and [C16H22ON5Cl5NiSn] were synthesized by
reacting dichlorodimethyl-bis(4-pyrazole N2) tin(IV) [188], with the corresponding
1:1 metal complex of phenylglycinechloride hydrochloride as shown in scheme 3.
Scheme 3. Proposed structure of complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl.
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Figure 35. Three dimensional ball and Stick model of complex [C16H22ON5Cl4CuSn]Cl; Colour scheme: C grey, N blue, Cl green, O red, Cu(II), Dark green purple Sn (IV). For clarity H atoms are omitted. Complex [C16H22ON5Cl4NiSn]Cl was synthesized only for the NMR structure
elucidation. The mass spectrometric and elemental analyses were consistent with
proposed molecular formulae of the complexes. Molar conductance measurements
suggest that complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl behave
as 1:1 electrolytes. The complexes are soluble in polar organic solvents, DMSO,
MeOH and H2O. AFM, TEM and XRD studies proved that the nano-sized
heterobimetallic Cu-Sn complex has the ability to form DNA condensates. To validate
specific DNA binding of the designed molecule various spectroscopic studies were
done and binding parameters were calculated.
Infrared Spectroscopy
The IR spectra of the complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl
revealed a characteristic broad band at 3317-3342 cm-1 due to the stretching vibrations
of the -NH2 and NH groups of the phenylglycine chloride and pyrazole ligands. The
frequency shift of the (-NH2) towards lower region in comparison to that observed
for free phenylglycine chloride ligand (3400 cm-1) confirms its coordination to the
metal ion [189]. Similarly, the participation of C=O group in complex formation was
ascertained by the shift of the band at 1720 and 1747 cm-1 which appears at 1765 cm-1
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in the ligand. Other ligand skeletal bands observed in the range 765-778 cm-1, 1155-
1162 cm-1, 1043 cm-1, were ascribed to the out of plane –CH bending of aromatic
rings , -CH3, and –CH2 groups, respectively. Bands appearing near 2900 cm-1 were
attributed to stretching vibrations of the -CH3 group [190a,b].
Nuclear magnetic Resonance spectroscopy
To further elucidate the structure of the heterobimetallic complexes, the diamagnetic
complex [C16H22ON5Cl5NiSn] was characterized by NMR spectroscopy. The 1H
NMR and 13C NMR spectra show aliphatic and aromatic signals with chemical shifts
in accordance with the proposed structure as shown in figure 36 and 37 respectively.
The 1H NMR spectrum of complex [C16H22ON5Cl4NiSn]Cl contained multiplets at
6.35 ppm and 7.80- 8.05 ppm consistent with the presence of the pyrazole and the
aromatic ring of amino acid derivative, respectively [191]. A sharp doublet at 3.87
ppm indicated the proton attached to the chiral carbon next to the benzene ring and
the singlet at 2.68 ppm was associated with the methyl protons [192].
The 13C NMR spectrum reveals distinct signals in the region 50.82, 54.02, 77.11 ppm
representing the -CH2 and -CH3 carbons [193]. The aromatic ring carbon signals were
observed at 125.72 ppm and 129.63 ppm [194]. The peak at 165.96 ppm corresponded
to the carbonyl carbon.
Figure 36. 1H NMR of complex [C16H22ON5Cl4NiSn]Cl in D2O at 25°C.
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Figure 37. 13C NMR of complex [C16H22ON5Cl4NiSn]Cl in D2O at 25°C.
119Sn NMR spectrum recorded a sharp peak at -218.83 ppm in agreement with the
hexa-coordinated environment of tin atom as shown in figure 38[195].
Figure 38. 119Sn NMR of complex [ C16H22ON5Cl4NiSn]Cl in D2O at 25°C
Electron paramagnetic resonance spectroscopy
The X-band electron paramagnetic resonance spectrum of complex
[C16H22ON5Cl4CuSn]Cl was recorded at a frequency of 9.1 GHz under the magnetic
field strength 3000±1000 gauss with tetracyanoethylene (TCNE) as field marker (g =
2.0027) at LNT. The spectrum of complex [C16H22ON5Cl5CuSn] as shown in figure
39 depicted a very broad axial symmetrical line shape with g|| = 2.401, g = 2.33 and
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gav = 2.35 computed from the formula gav2 = (g||
2+2g2) ∕3. These parameters are in
accordance with axially symmetrical square pyramidal Cu (II) systems [196]. The
trend g|| > g > 2 revealed that the unpaired electron is present in the dx2
-y2
orbital
[197]. For a covalent complex, g|| < 2.3 and for an ionic environment, g|| = 2.3 or more.
In the present complex g|| > 2.3 indicates an appreciable metal-ligand ionic character
[198].
Figure 39. X-band polycrystalline powder EPR spectrum of complex [C16H22ON5Cl4CuSn]Cl at room temperature. Electronic absorption spectra
The electronic spectra of the complexes [C16H22ON5Cl4CuSn]Cl and
[C16H22ON5Cl4NiSn]Cl were recorded in MeOH at room temperature. In the UV
region, both complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl exhibit
intense absorption band at 258-260 nm and a shoulder at 340-344 nm attributed to π–
π* [199] or charge transfer transitions of the aromatic chromophore. In the visible
region, complex [C16H22ON5Cl4CuSn]Cl display a broad band at 590 nm assigned to
2B1g→2A1g ligand field transition [200]. Similarly, complex [C16H22ON5Cl4NiSn]Cl
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also displays a low energy metal centered d-d absorption band at 578 nm. These
values are consistent with square pyramidal geometry around Cu (II)/Ni (II) metal
ions [201].
XRD Measurements
Although single crystal of the complex was not obtained, however, the crystalline
nature of the complex was authenticated by XRD measurements. Figure 40 shows the
XRD pattern of the complex [C16H22ON5Cl4CuSn]Cl with peaks at 2θ scattering
angles of 27.22, 33.32, 36.33, 38.42, 44.11, 62.54 assigned to (001) (111), (201),
(200), (212) and (220) crystal planes respectively, characteristic of well ordered
tetragonal arrangement of tin atoms [202]. The lattice parameters are a = 5.79 and c =
0.5322 which are in good agreement with known lattice parameters a = 5.8316 and c
= 0.5455 indicating that the nano particles have the same crystal structure as that of
the bulk [203].
Figure 40. The XRD pattern nanoparticles of complex [C16H22ON5Cl4CuSn]Cl showing peaks at different scattered angles.
Intense reflections derived from (200), (212) planes reveal the existence of copper
atoms in a cubic geometry in good agreement with the known lattice parameters
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[204]. The average grain size of the powder sample was calculated to be 20-30 nm
according to half width of (200) diffraction peaks and the size of the DNA condensed
particles was found in the range of 60-80 nm according to half width of (200) using
Debye Sherrer formula [205].
DNA binding studies
DNA is the primary intracellular target of antitumor drugs. Interaction of the
complexes with DNA can induce DNA damage, which leads to blockage of cell
division and eventually cell death.
Absorption spectral studies
The interaction of the metal complex to DNA is often characterized through
absorption spectral titration followed by the changes in the absorbance and shift in the
wavelength. The absorption spectral traces of the Cu-Sn heterobimetallic complex
[C16H22ON5Cl4CuSn]Cl in the absence and presence of CT DNA are shown in figure
41. Upon the addition of CT DNA to complex [C16H22ON5Cl5CuSn] significant
hyperchromism with a red shift of 3 nm was observed at the intraligand bands.
Hyperchromism and hypochromism are the spectral changes typical of a metal
complex association with the DNA helix [206]. Hypochromism results from
contraction of DNA helix as well as change in its conformation while hyperchromism
results from the damage of DNA double helix structure [207]. The observed
hyperchromism results due to the larger positive charge of cations binding to DNA
presumably by a strong electrostatic attraction to the phosphate group of the DNA
backbone and thereby causing a contraction and overall damage to the secondary
structure of DNA [208]. Complex [C16H22ON5Cl4CuSn]Cl shows better prospects as
an antitumor agent due the presence of two different metal centers having different
specificity at the molecular level.
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The hard Lewis acid Sn (IV) atom binds electrostatically to the phosphate backbone
of DNA helix, while the Cu (II) centre may preferentially coordinate with the N7
position of
Figure 41. Absorption spectral traces of complex [C16H22ON5Cl4CuSn]Cl in Tris-HCl buffer upon addition of CT-DNA. Inset: plot of [DNA]/εa-εf vs [DNA] for the titration of CT-DNA with complex [C16H22ON5Cl4CuSn]Cl:complex [C16H22ON5Cl4CuSn]Cl= 0.40 x 10-4 M, [DNA]= 0.14 x 10-4 M - 0.84 x10-4 M; (▲) experimental data points; full lines, linear fitting of the data. guanine either due to the replacement of the labile chloride ligand or by direct
coordination. Base binding of the complex can perturb the hydrogen bonding between
the base pairs causing destabilization of DNA. To assess the binding ability of the
complex [C16H22ON5Cl4CuSn]Cl with CT DNA, the intrinsic binding constant was
determined using equation I [172,173]. The binding constant Kb value was found to
be 8.42 x104 M-1. The significant binding constant Kb value reflects the average of the
dual binding mode of complex [C16H22ON5Cl5CuSn] to DNA.
[DNA] / | a-f | = [DNA] / | b-f | + 1 / Kb | b-f | (I)
Fluorescence studies
The interaction of complex [C16H22ON5Cl4CuSn]Cl with CT DNA was also
investigated by using emission spectroscopy. Complex [C16H22ON5Cl4CuSn]Cl emits
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strong luminescence with maximum at 530 nm when excited at 260 nm in 0.01 mM
Tris–HCl/50 mM NaCl buffer at ambient temperature. Upon the addition of CT DNA
the intensity of the emission band at 530 nm enhances gradually, which indicates that
the complex could interact with DNA (figure 42). The increase in the emission
intensity usually depends on the degree of exposure of the complex molecule into
DNA double helix. The enhancement of the emission intensity is mainly due to the
change in the environment of the metal complex and is related to the extent to which
the complex is inserted into the hydrophobic environment inside the DNA helix
[209,210]. DNA being hydrophobic molecule restricts the mobility of the solvent
water molecules, at the binding site thereby preventing the quenching effect
particularly in an intercalative binding mode [211]. It is quite obvious that the
Figure 42. Emission spectra of the complex [C16H22ON5Cl5CuSn] in Tris-HCl buffer upon addition of CT DNA. Arrow shows intensity change upon increasing concentration of DNA. Inset: plot of r/Cf vs r: [complex (C16H22ON5Cl5CuSn)] = 0.06 x10-4 M, [DNA] = 0-0.33x10-4M.; (▲) experimental data points; full lines, linear fitting of the data.
complex [C16H22ON5Cl4CuSn]Cl is shielded by the DNA duplex efficiently from the
bulk solution and because of the reduced collision between the solvent and the
complex molecule, a decrease in the vibrational modes of relaxation results and hence
quenching does not occur [212]. The observed increasing emission intensity implies
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binding of the complex to the hydrophobic pocket of the DNA along the major or
minor grooves by electrostatic interactions with the phosphate groups [213].
From a plot of r/cf versus r the binding constant for complex [C16H22ON5Cl4CuSn]Cl
was calculated to be 3.42 x 104 M-1 which is in well agreement with the binding
constant values as determined by UV-vis titrations.
Cyclic Voltammetry
Cyclic voltammetry is a useful technique for studying the interaction of metal
complexes with CT DNA and understanding the nature of DNA binding. The cyclic
voltammogram of complex [C16H22ON5Cl4CuSn]Cl was recorded in MeOH at the
scan rate of 0.2 Vs-1over the potential range of 2.0-1.2V. The cyclic voltammetric
response of complex [C16H22ON5Cl4CuSn]Cl in the absence and in presence of CT
DNA is depicted in figure 43. The CV of complex [C16H22ON5Cl4CuSn]Cl exhibit a
quasireversible redox wave corresponding to CuII/CuI with Epc= -0.514 V and Epa= -
0.287 V.
Figure 43. Cyclic voltammogram (scan rate 0.2 Vs-1, MeOH, 25 °C) of (a) complex [C16H22ON5Cl4CuSn]Cl (b) complex [C16H22ON5Cl4CuSn]Cl in presence of CT DNA.
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For this couple, the difference between cathodic and anodic peak potential ΔEp and
the ratio of anodic and cathodic peak current are –0.227V and 0.98, respectively. The
formal electrode potential E1/2 taken as the average of Epa and Epc was -0.401 V in the
absence of DNA. Addition of CT DNA results in the significant shift in E1/2 and
reduction in peak currents (E1/2=-0.413 and ΔEp=-0.164 V respectively). The ratio of
Ipa/Ipc decreases to 0.84. The shift in formal potential and decreases in the current ratio
suggest strong binding of complex [C16H22ON5Cl4CuSn]Cl to CT DNA [214].
DNA Cleavage activity
The ability of the heterobimetallic complex [C16H22ON5Cl4CuSn]Cl to induce DNA
cleavage was assessed on pBR322 supercoiled plasmid DNA. The cleavage activity
was determined with 0.05-0.25 mM of the complex in 0.01mM Tris HCl/50 mM NaCl
buffer at pH 7.2 in a total volume of 30 µL containing pBR322 plasmid DNA, after an
incubation of 1h at 37 ºC. During electrophoresis when scission occurs on one strand
(nicking), the supercoil relaxes to generate Form II. When both strands are cleaved a
linear Form III is generated which migrates between Form I and Form II. In absence
of any exogenous reductant, the pBR322 plasmid DNA remains primarily supercoiled
(form I); however upon increasing the concentration of the metal complex the DNA
was converted to nicked DNA (Form II) although less efficiently without any
concurrent formation of linearized DNA (Form III), suggesting a single strand
breakage [215]. Complex [C16H22ON5Cl4CuSn]Cl unwinds the DNA duplex by
interacting with supercoiled form of pBR322DNA to form a DNA-complex adduct
and hence reduces the number of supercoils. The decrease in supercoils upon binding
of unwinding agents causes a decrease in the rate of migration through agarose gel
revealing the interaction and cutting effect of complex with DNA at a particular
concentration (figure 44a). The nuclease efficiency of the complex was also
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investigated in presence of ascorbate as an activator. The complex was found to
mediate the rapid degradation of the supercoiled (Form I) plasmid DNA to produce
nicked form (form II) in a concentration dependent manner. With the increase in the
concentration of the complex, the band intensity of form II increases while the band
intensity of Form I decreases indicating the DNA cutting efficiency of the complex
increases in the presence of reducing agent. No cleavage was observed beyond
background for ascorbate alone as shown in figure 44b. To better evaluate the
cleavage mechanism, experiment with the scavenging agent 5% DMSO was carried to
identify the intermediate hydroxyl radical (ROS species) that might form during
cleavage reaction.
Figure 44a. Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA(300 ng) by complex [C16H22ON5Cl4CuSn]Cl; Lane 1: DNA; Lane 2: 0.05 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 3: 0.1 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 4: 0.15 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 5: 0.2 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 6: 0.25 mM [C16H22ON5Cl4CuSn]Cl + DNA.
Figure 44b. Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300ng) by [C16H22ON5Cl4CuSn]Cl in presence of radical scavenger (5%DMSO) and ascorbic acid H2A (0.1-0.25 mmol) Lane 1: DNA; Lane 2: 0.05mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO; Lane 3: 0.1mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO + 0.1 mM H2A; Lane 4: 0.15mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO +0.15 mM H2A; Lane 5: 0.2 mM + DMSO + 0.2 mM H2A [C16H22ON5Cl4CuSn]Cl + DNA; Lane 6: 0.25mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO + 0.25 mM H2A.
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The result showed a little effect on the DNA cleavage ruling out the possibility of
DNA cleavage by hydroxyl radicals [216]. Based on these observation, it is proposed
that the Cu (II) complex is initially reduced to Cu (I) species by ascorbic acid, which,
then binds to DNA to form the Cu (I) complex-DNA adduct.
DNA Condensation
DNA condensation is an integral step involved in many DNA transactions such as
recombination, replication, transcription and repair [217]. To define the
pharmacological action of the drug candidate controllable drug-DNA condensation is
imperative to allow protected packaging from enzymatic degradation and
transportation across the biological barriers to a specific target cell. Complex
[C16H22ON5Cl4CuSn]Cl has the potential to induce DNA condensation due to strong
charge compensation of the anionic phosphate segments by the cationic
heterobimetallic core. In this study, DNA condensation to the nano particulate
structure by complex [C16H22ON5Cl5CuSn] under neutral conditions in aqueous
medium (Tris-HCl buffer/50mM NaCl pH 7.2) was achieved by evaporating an
equimolar mixture of complex [C16H22ON5Cl4CuSn]Cl and CT DNA. The increase in
the size of the complex nano particles after CT DNA condensation was determined by
XRD measurements. To analyze the formation of the drug-DNA condensates,
visualization techniques - transmission electron microscopy (TEM) and atomic force
microscopy (AFM) were employed.
TEM and AFM imaging of Complex-DNA condensates
To recognize the complex-DNA condensate morphology, TEM image of uncondensed
complex nanoparticles is given for comparison in figure 45a. The shape of the
particles is irregular but close to spherical. The particles have wide size distribution
and the average
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Figure 45a: TEM image of the nano particles of complex [C16H22ON5Cl4CuSn]Cl.
Figure 45 (b) TEM image indicating the condensation of CT DNA on nano particles of complex [C16H22ON5Cl4CuSn]Cl (c) TEM image indicating the coagulation of nano particles around CT DNA for 12h. particle diameter is in the range of 20-30 nm. The particle size increases almost thrice
when CT DNA was condensed on it (figure 45b and 45c). This clearly indicates that
the complex nanoparticles have a good ability to condense the free CT DNA (existing
as loose strands) to compact solid particles. To obtain the morphological and
structural information about the free complex [C16H22ON5Cl4CuSn]Cl and the
condensates, tapping mode AFM (atomic force microscopy) experiments were
performed using commercially etched silicon tips as AFM probes with typical
resonance frequency of 300 Hz (RTESP Veeco Innova II nanoscope). Two and three
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dimensional AFM images of the complex [C16H22ON5Cl5CuSn] and the condensate
materials are shown in figure 46 (a and b) and 47 (a and b). Figure 48a shows the
morphology of complex [C16H22ON5Cl4CuSn]Cl condensed with supercoiled pBR322
DNA at a particular time (6h) and figure 48b shows the three dimensional
morphology of the condensate material at the same time. Figure 49a shows the change
in the morphology of complex [C16H22ON5Cl4CuSn]Cl condensed with supercoiled
pBR322 DNA at a particular time (after 12h) and figure 49b shows three dimensional
morphology of the condensate material at the same time. The width of the condensate
material increases with respect to time however there is no change in the vertical
height. The condensation of CT DNA and supercoiled pBR322 DNA on the surface of
these nano particles was studied. The surfaces of these nanoparticles were uneven
structures, and after the DNA condensation the grooves were observed on the plane
surface with little pyramidal shapes of the condensate materials. It is clear from the
figure that the width of the structure in figure 49 (a and b) is about three times the
width of the structures in figure 48 (a and b). It clearly shows that with the
advancement of time, the complex [C16H22ON5Cl4CuSn]Cl has the tendency to
condense on the supercoiled pBR322DNA. Thus, the results are indicative of the good
DNA condensation ability of the heterobimetallic Cu-Sn complex.
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Figure 46. (a) Two dimensional AFM image of the nano particles of complex [C16H22ON5Cl4CuSn]Cl (b) Three dimensional AFM image of the nano particles of complex [C16H22ON5Cl4CuSn]Cl
Figure 47. (a)Two dimensional AFM image of the CT DNA condensed nano particles;50 µM nano complex [C16H22ON5Cl4CuSn]Cl + CT DNA in (1:1) ratio. (b) Three dimensional AFM image of the CT DNA condensed nano particles ; 50 µM nano complex [C16H22ON5Cl4CuSn]Cl + CT DNA in (1:1) ratio.
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Figure 48. (a) Two dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl5CuSn]+ 30 µM nano complex [C16H22ON5Cl4CuSn]Cl pBR322 DNA in (1:1) ratio. (b) Three dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl4CuSn]Cl 30 µM nano complex [C16H22ON5Cl4CuSn]Cl + pBR322 DNA + 30 µM Plasmid DNA for 6h. Figure 49. (a) Two dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl4CuSn]Cl 30 µM nano complex [C16H22ON5Cl4CuSn]Cl pBR322 DNA in (1:1) ratio after 12h. (b) Three dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl4CuSn]Cl 30 µM nano complex [C16H22ON5Cl4CuSn]Cl + pBR322 DNA + 30 µM Plasmid DNA for 12h.
(a) (b)
(a) (b)
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Conclusion
The nanoparticulate heterobimetallic complex has been designed and synthesized with
an aim to develop new efficacious chemotherapeutic agent. By using a combination of
spectroscopic techniques, AFM and TEM imaging techniques, we have validated the
DNA binding and condensation properties of nano complex. DNA binding
experiments reveal that complex [C16H22ON5Cl4CuSn]Cl is an avid DNA binding
agent due to the presence of two metal Cu (II) and Sn (IV) ions which provide a dual
mode of binding at the molecular target site and exhibit novelty due to preferential
selectivity towards DNA. The relatively small size, subtle lipophilicity of the ligand
framework, water solubility and balanced electrostatic interactions play an important
role to induce DNA condensation. The nanoparticulate size of the complex
[C16H22ON5Cl4CuSn]Cl makes it most desirable target –specific chemotherapeutic
drug for cancer inhibition and it warrants evaluation in cell based protocols or in gene
therapy.
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Syntheses:
Synthesis of [C8H14N2Cl2Sn]
The dichlorodimethylbis (imidazole) tin (IV) [C8H14N2Cl2Sn] was synthesized
according to the method reported in the literature [218].
Synthesis of [C16H22ON5Cl4CoSn]Cl
To a methanolic solution of Phenylglycine chloride hydrochloride (5 mM 1.03 g), (5
mM 1.19 g) of CoCl2.6H2O in MeOH was added drop wise. The violet blue colour
solution obtained was left to stir for 3h. An equimolar amount of [C8H14N2Cl2Sn]
dissolved in 5 mL MeOH was added to the stirring solution and refluxed for 8h. The
completion of the reaction was monitored by thin layer chromatography (TLC). The
solution was cooled to room temperature and left for two days, when a violet blue
coloured crystalline product was isolated, washed with CHCl3 and dried in vacuo.
Yield: 62%. mp; 254±3ºC, Anal. Calcd. for [C16H22ON5Cl4CoSn]Cl: C, 29.33; H,
3.38; N, 10.69, Found: C, 29.36; H, 3.41; N, 10.64; Selected IR data (KBr, ν cm-1)
3357 (NH2); 1713 (C=O); 1358 (C-H); 1225 (C-N); 1023 (C-C); 763 (Ar); 463 (Co-
N). Molar Conductance: M (1·10-3 M, DMSO): 89.00 -1cm2 mol-1 (1:1 electrolyte).
UV-vis (1X10-3 M, DMSO max, nm) 257 nm, 296 nm , 325 nm, 656 nm. ESI-MS
(m/z+) 655 [C16H22ON5Cl4CoSn]Cl
Synthesis of [C16H22ON5Cl4ZnSn]Cl The complex [C16H22ON5Cl4ZnSn]Cl was synthesized using anhydrous ZnCl2 (1.36g,
10 mM) by a similar method as described for [C16H22ON5Cl4CoSn]Cl. The white
coloured product obtained was filtered, washed with CHCl3 and dried in vacuo. Yield:
72%; mp; 259±2 ºC. Anal. Calcd. for [C16H22ON5Cl4ZnSn]Cl C, 29.04; H, 3.35; N,
10.58. Found: C, 29.11; H, 3.39; N, 10.61. Selected IR data (KBr, ν cm-1) 3392
(NH2); 1707 (C=O); 1381 (C-H); 1256 (C-N); 1013 (C-C);761 (Ar); 496 (Zn-N); 1H
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NMR (400 MHz, DMSO-d6, 25 ºC, δ) 2.58 (-CH3); 3.63 (-CH); 6.45, 7.93 (phenyl,
imidazole ring); 8.05, 8.21 (imidazole NH, NH2); 13C NMR (100 MHz DMSO- d6, 25
ºC, δ) 45.82-71.13 (CH3, CH2); 122.12-139.33(Ar); 159.23 (C=O); 119Sn NMR (400
MHz DMSO- d6, 25 ºC, δ) -560.23; Molar Conductance, M (1X10-3 M, DMSO):
114.00 -1cm2 mol-1 (1:1 electrolyte) UV-vis (1·10-3 M, DMSO, max /nm) 258 nm,
325 nm. ESI-MS (m/z+) 661 [C16H22ON5Cl4ZnSn]Cl
Results and discussion
The complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl were synthesized
according to the procedure described in the literature [219], by the reaction of
dichlorodimethyl bis(imidazole ) tin (IV) with the corresponding 1:1 metal complex
of phenylglycine chloride hydrochloride as shown in scheme 4. Scheme 4:
Step 1
Step 2
Scheme 4. Schematic representation of the complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl
N
NH
N
NH
SnH3C
ClClCH3
N
NH
CH2Cl2+Stirring
SnClCl
H3C CH3
(1)
2
N
HN
Co
H2NC C Cl
O
H
N
NH
SnH3CClCl
CH3H2NC C Cl
O
H CoCl2.6H2O+MeOH
+ 1 Reflux for 5h
Cl .Cl
77
Complex [C16H22ON5Cl4ZnSn]Cl was synthesized only for the NMR structure
elucidation, and the structure was determined by employing various spectroscopic
techniques such as elemental analysis, molar conductivity, UV-vis., IR, ESI-MS and
multinuclear NMR spectroscopy. The mass spectrometric and elemental analyses
were consistent with proposed molecular formulae of the complexes. Molar
conductance measurements suggest that complexes [C16H22ON5Cl4CoSn]Cl and
[C16H22ON5Cl4ZnSn]Cl behave as 1:1 electrolytes. The complexes are soluble in
MeOH, DMSO and DMF. The nano-sized dimension of the heterobimetallic complex
[C16H22ON5Cl4CoSn]Cl is supported by AFM, TEM and XRD measurements. To
validate specific DNA binding of the designed molecule various spectroscopic studies
were employed and binding parameters were calculated.
Infrared spectroscopy
The IR spectrum of the free phenylglycine chloride hydrochloride exhibits
characteristic bands of the amine (-NH2) at 3400 cm-1. However, on complexation
υ(N-H) shifts towards lower wave number, Complexes [C16H22ON5Cl4CoSn]Cl and
[C16H22ON5Cl4ZnSn]Cl exhibit the broad band at 3341 cm-1 –3342 cm-1 due to the
stretching vibrations of the -NH2 and NH groups of the phenylglycine chloride and
imidazole ligands. The frequency shift of the (-NH2) towards lower region in
comparison to that observed for free phenylglycine chloride ligand (3400 cm-1)
confirms its coordination to the metal ion [189]. The involvement of C=O in metal
complexes is evident from the shift in (C=O) stretch which appears at 1713 and
1707 cm-1 in comparison to free uncomplexed ligand band at 1765 cm-1. Other ligand
skeletal bands observed in the range 689–848cm-1, 1129-1179 cm-1, 1074 cm-1, were
ascribed to the out of plane –CH bending of aromatic rings, -CH3, and –CH2 groups,
respectively. Bands appearing near 2900 cm-1 were attributed to stretching vibrations
78
of the -CH3 group [190b,220]. The IR spectra of [C16H22ON5Cl4CoSn]Cl and
[C16H22ON5Cl4ZnSn]Cl reveal (M-N) and (M-O) stretching vibrations in the range
430-450 and 535-580 cm-1, respectively.
Nuclear magnetic Resonance spectroscopy
1H, 13C, 119Sn NMR was used for the characterization of the diamagnetic complexes
[C16H22ON5Cl4ZnSn]Cl. The 1H NMR and 13C NMR, and 119Sn NMR spectra show
aliphatic and aromatic signals with chemical shifts in accordance with the proposed
structure. The 1H NMR spectrum of complex [C16H22ON5Cl4ZnSn]Cl exhibits
multiplets at 7.36–7.62 ppm and 7.40- 8.75 ppm consistent with the presence of the
imidazole and the aromatic ring of amino acid, respectively [212]. A sharp doublet at
3.12 ppm indicated the proton attached to the chiral carbon next to the benzene ring
and the proton attached to the methyl group of tin atom gives a singlet at 1.01–1.23
ppm as shown in figure 50[221, 222].
Figure 50. 1H NMR of the complex [C16H22ON5Cl4ZnSn]Cl.
The 13C NMR spectrum reveals distinct signals in the region 50.82, 54.02, 77.11 ppm
representing the -CH and -CH3 carbons [223]. The aromatic ring carbon signals were
79
observed at 129.59-137.23 ppm [224]. The peak at 163.34 ppm corresponded to the
carbonyl carbon. 119Sn NMR spectrum recorded a sharp peak at -560.23 ppm in
agreement with the hexa-coordinated environment of tin atom as shown in figure
51[225].
Figure 51. 119Sn NMR of the complex [C16H22ON5Cl4ZnSn]Cl.
Mass spectral analysis
The complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl have been
unambiguously characterized through mass spectral analysis. The ESI mass spectrum
of complex [C16H22ON5Cl5CoSn], exhibits the molecular ion peak m/z at 655 which
was assigned to [C16H22ON5Cl4CoSn]Cl. The complex [C16H22ON5Cl4CoSn]Cl,
showed the prominent peaks m/z at 403 with a relative abundance of 60% which was
assigned to [C14H16ON5Cl2Co-3H+]. The fragmentation peaks obtained at m/z 266,
205 which was obtained by the successive expulsion of imidazole and cobalt metal
ion, respectively. The relatively 60% abundant peak m/z at 161 corresponding to
isotopic peak of free phenylglycine chloride was observed. Similar pattern was
obtained for the complex [C16H22ON5Cl4ZnSn]Cl which exhibits the molecular ion
peak m/z at 661.
80
XRD Measurements
XRD measurement of the powered sample of complex [C16H22ON5Cl4CoSn]Cl was
employed to determine the crystalline nature of the complex [C16H22ON5Cl4CoSn]Cl.
The XRD pattern obtained for the metal complex [C16H22ON5Cl4CoSn]Cl shows well
defined crystalline peaks which evidenced for the inherent crystalline nature of the
metal complex. Figure 52 shows the XRD pattern of the complex
[C16H22ON5Cl4CoSn]Cl with peaks at 2θ scattering angles of 23.23, 24.84, 34.23,
42.86, 48.29, 63.86 and 78.46 assigned to (100) (100), (002), (102), (111) and (202)
and (203) crystal planes respectively, characteristic of well ordered tetragonal
arrangement of tin atoms [203]. The
lattice parameters are a = 4.230 and c = 5.187, which are in good agreement with
known lattice parameters a = 4.109 and c = 5.180 indicating that the nano particles
have the same crystal structure as that of the bulk [204]. The average size of the
Figure 52. The XRD pattern nanoparticles of complex [C16H22ON5Cl4CoSn]Cl showing peaks at different scattered angles. powder sample of complex [C16H22ON5Cl4CoSn]Cl was calculated to be 15-20 nm
according to half width of (002) diffraction peaks and the size of the DNA condensed
particles was found in the range of 60-80 nm according to half width of (002) using
81
Debye Sherrer formula [203].A summary of the refined XRD consisting of unit cell
parameters are given in table 2 .
Table 2. Summary of the XRD data and the refinement parameters for compound [C16H22ON5Cl4CoSn]Cl.
Electronic absorption spectra
The electronic spectra of the complexes [C16H22ON5Cl4CoSn]Cl and
[C16H22ON5Cl4ZnSn]Cl were recorded in DMSO at room temperature. In the UV
region both complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl exhibit
intense absorption band at 256-258 nm and a shoulder at 325-329 nm attributed to
S.No
Compound
2
1. Formula
C16H22ON5Cl5CoSn
2. Molecular weight(gmol-1/)
655
3. Temperature(K)
527
4. Crystal System
Primitive
5. Space Group
P-6
6. Method Micro crystalline
7. Cell Parameters
a = 4.230 b = 4.230 c = 5.187 α = 90 β = 90 γ = 120
8. Z 1
9. Radiation
Cu-Kα
10. 2θ min-max 23.23-78.46
82
π–π* [199, 226] or charge transfer transitions of the aromatic chromophore. In the
visible region complex [C16H22ON5Cl4CoSn]Cl display a broad band at 656 nm,
which is consistent with square pyramidal geometry around Co (II) metal ions [227].
DNA binding studies
Absorption titration studies
The interaction of the metal complex to DNA is often characterized through
absorption spectral titration followed by the changes in the absorbance and shift in the
wavelength. The absorption spectral traces of the Co-Sn heterobimetallic complex
[C16H22ON5Cl4CoSn]Cl in the absence and presence of CT DNA are shown in figure
53. With increasing concentration of CT DNA (0-0.4 X 10-4 M), the absorption bands
of [C16H22ON5Cl4CoSn]Cl were affected, exhibiting hyperchromism (24%) and red
shift, respectively in absorption intensities. “Hyperchromic effect” arises mainly due
to the presence of positively charged transition metal ion and Sn (IV) which bind to
DNA via non-covalent interaction [228]. A strong hyperchromic effect with a
significant red shift in -* transition was observed for [C16H22ON5Cl4CoSn]Cl
suggesting that, this complex possess a higher propensity for DNA binding These
changes are typical of complexes bound to DNA through noncovalent interaction [208].
Since numerous naturally and man- made compounds contained Cobalt at two common
oxidation states Co(II) and Co(III). There is growing interest in investigating cobalt and
other transition metal complexes for their interaction with DNA [229]. This may be partly
influenced by the results of extensive investigation into two areas of research, viz. (i) the
binding specificity of small organic molecules for their possible modulation and
inhibition of DNA replication, transcription and recombination, and (ii) anticancer,
antiviral and antibacterial drugs [230]. The DNA specific interactions of complex
[C16H22ON5Cl4CoSn]Cl shows better prospects as an antitumor agent due the
presence of two different metal centers having different specificity at the molecular
83
level. The hard Lewis acid Sn (IV) atom binds electrostatically to the phosphate
backbone of DNA helix, while the Co+2 ion may preferentially coordinate with the N3
position of guanine either due to the replacement of the labile chloride ligand or by
direct coordination. Base binding of the complex can perturb the hydrogen bonding
between the base pairs causing destabilization of DNA.
Figure 53. Absorption spectral traces of complex [C16H22ON5Cl4CoSn]Cl in Tris-HCl buffer upon addition of CT-DNA. Inset: plot of [DNA]/εa-εf vs [DNA] for the titration of CT-DNA with complex [C16H22ON5Cl4CoSn]Cl. [Complex] = 0.067 X 10-4 M, [DNA]= 0.067 X10-4- 0.466 x10-4 M; (▲) experimental data points; full lines, linear fitting of the data.
To assess the binding ability of the complex [C16H22ON5Cl4CoSn]Cl with CT DNA,
the intrinsic binding constant was determined using equation I [170,171]. The binding
constant Kb value was found to be 5.14 x104 M-1. The significant binding constant Kb
value reflects the average of the dual binding mode of complex
[C16H22ON5Cl4CoSn]Cl to DNA.
[DNA] / |a-f| = [DNA] / |b-f| + 1/ Kb |b-f | (1)
To investigate the specific recognition of complex [C16H22ON5Cl4CoSn]Cl with the
nucleobases/ phosphate sugar backbone of DNA helix, we have carried out binding
studies of [C16H22ON5Cl4CoSn]Cl with 5'-GMP by UV-vis absorption titrations. On
84
addition of increasing amount of 5'-GMP, [C16H22ON5Cl4CoSn]Cl there was a sharp
increase in absorption intensity ‘hyperchromism’ and small blue shift of 2 nm (Figure
54).
Hyperchromic effect is the result of damage caused to the secondary structure of
DNA
Figure 54. Variation of UV-vis absorption for complex [C16H22ON5Cl4CoSn]Cl with increase in the concentration of 5′ GMP (0.067 X10-4 -0.466 X10-4 M) in buffer (5mM Tris–HCl/50 mM NaCl, pH= 7.2) at room temperature. Inset: plot of [5′ GMP]/(εa- εf) vs [DNA] for the titration of 5′ GMP.(■), experimental data points; full linear, linear fit of the data.[complex]= 0.33X10-6. through phosphate backbone interaction while small blue shift is attributed to the
coordinative interaction to N7 of 5'-GMP. This is an inherent characteristic feature of
transition metals.
Fluorescence spectral studies
The interaction of complex [C16H22ON5Cl4CoSn]Cl with CT DNA was also
investigated by using emission spectroscopy. Complex [C16H22ON5Cl4CoSn]Cl emits
luminescence with maximum at 344 nm when excited at 263 nm in 0.01 mM Tris–
HCl/50 mM NaCl buffer at ambient temperature. Upon the addition of CT DNA the
intensity of the emission band at 344 nm enhances gradually, which indicates that the
complex could interact with DNA (Figure 55). As DNA is a highly organized
macromolecular complex and its double helix provides a unique coat: core
85
environment comprising hydrophilic backbone of ribose phosphate around the
hydrophobic core of stacked bases [210, 211].
Figure 55. Emission spectra of the complex[C16H22ON5Cl4CoSn]Cl in Tris-HCl buffer upon addition of CT DNA. Arrow shows intensity change upon increasing concentration of DNA. [Complex] = 0.06 x10-4 M, [DNA] = 0―0.466 x 10-4 M. The enhancement of the emission intensity is mainly due to the change in the
environment of the metal complex and is related to the extent to which the complex is
inserted into the hydrophobic environment inside the DNA helix [231]. Since it is
found that the complexes with increased hydrophobicities exhibit greater increase in
emission intensities upon binding to polyectrolytes like DNA, It is therefore
concluded that the emission intensity increases due to the interaction between the
complex [C16H22ON5Cl4CoSn]Cl and DNA which is due the hydrophobicity of both
the complex [C16H22ON5Cl4CoSn]Cl and the DNA. An additional ionic strength
experiment showed the electrostatic mode of binding.
From a plot of r/cf versus r the binding constant for complex [C16H22ON5Cl4CoSn]Cl
was calculated to be 2.62 x 104 M-1 which is in well agreement with the binding
constant as determined by UV-vis titrations.
86
To evaluate the interacting strength of complex [C16H22ON5Cl4CoSn]Cl emission
quenching experiments using [Fe(CN)6]4- as quencher were also performed. In the
absence of DNA, emission intensity of the complex [C16H22ON5Cl4CoSn]Cl were
Figure 56 . Emission quenching curve of complex [C16H22ON5Cl4CoSn]Cl in absence and presence of DNA
efficiently quenched by [Fe(CN)6]4-. The plots of the complexes
[C16H22ON5Cl4CoSn]Cl gave the value of Ksv = 4.23 X 104 M-1 .In presence of DNA,
the slope was remarkably decreased to 1.92 X 104 for the complex
[C16H22ON5Cl4CoSn]Cl as shown in figure 56 . The greater decrease of the Ksv value
for the complex [C16H22ON5Cl4CoSn]Cl indicates the higher DNA binding propensity
of [C16H22ON5Cl4CoSn]Cl. These results are consistent with the electronic absorption
titration.
Effect of ionic strength
To determine the efficient and distinguishable binding modes between DNA and
small molecules, ionic strength is an important parameter. The addition of cation
weakens the surface binding interactions due to a competition for phosphate anion,
viz electrostatic binding and hydrogen bonding between the CT DNA and the
interacting molecules [232, 233]. The effect of ionic strength on complex –DNA
binding of [C16H22ON5Cl4CoSn]Cl was studied and the results so obtained revealed
87
the strong dependence of fluorescence intensity on ionic strength. Moderate
fluorescence quenching was observed in complex-DNA system for both the
complexes [C16H22ON5Cl4CoSn]Cl. The results indicate that the interaction between
complexes and DNA is predominantly electrostatic viz. DNA phosphate backbone.
Cyclic Voltammetry
The application of cyclic voltammetry to the study of metal complex-DNA interaction
provides a useful complement to the previously used methods of investigations, such
as UV-visible spectroscopy. Equilibrium constant (Kb) for the interaction of the metal
complexes with DNA can be obtained from the shifts in peak potentials, the number
of base pair sites involved in binding via intercalative, electrostatic or hydrophobic
interactions and from the dependence of the currents passed during oxidation or
reduction of the bound species on the amount of the added DNA [234]. The cyclic
voltammogram of complex [C16H22ON5Cl4CoSn]Cl was recorded in DMSO at the
scan rate of 0.3 Vs-1over the potential range of 2.0 — -0.8V. The cyclic voltammetric
response of complex [C16H22ON5Cl5CoSn] in absence and in presence of CT DNA is
depicted in figure 57.
Figure 57. Cyclic voltammogram (scan rate 0.3 Vs-1, DMSO, 25°C) of (a) complex [C16H22ON5Cl4CoSn]Cl (b) complex [C16H22ON5Cl4CoSn]Cl in presence of CT DNA.
a b
88
The cyclic voltammogram of [C16H22ON5Cl4CoSn]Cl in the absence of DNA reveal a
non- Nerstian but fairly reversible/quasireversible one electron redox process
involving Co3+/Co2+ couple as observed from peak potential separation of 0.149V
(0.59V for a one electron transfer process). On addition of CT DNA to the complex
[C16H22ON5Cl4CoSn]Cl, there was a significant shift in formal electrode potential E1/2
0.119mV for the complex. In addition to changes in formal potential, voltammetric
current Ipa / Ipc decreases from 0.89 to 0.71 and separation of peak potential ΔEp also
decreases from -0.313 to -0.334.
Furthermore, the significant shift in the electrode potentials and peak current ratios on
addition of CT DNA can be explained in terms of the diffusion of an equilibrium
mixture of free and DNA bound metal complexes to the electrode surface [208] thus
implying a strong binding of [C16H22ON5Cl4CoSn]Cl with CT DNA
DNA Cleavage activity
Upon gel electrophoresis of the reaction mixture, a concentration dependent DNA
cleavage was observed. A significant conversion of supercoiled pBR322 DNA to
Form II and Form III, was observed with increase in concentration of complex
[C16H22ON5Cl4CoSn]Cl (Figure 58). The most impressive cleavage feature observed
for complex [C16H22ON5Cl4CoSn]Cl is that, Form III DNA appears before the
disappearance of Form I DNA (lanes, 3, 4, 5 and 6). This phenomenon indicates that
the complex [C16H22ON5Cl4CoSn]Cl is capable of performing direct double-strand
scission [235], while many cobalt complexes are only able to cleave single strand
successively. The complex [C16H22ON5Cl4CoSn]Cl shows discernible DNA cleavage
with the increase in concentration, more intensified nicked (Form II) while linear
(Form III) was observed in traces. Minor groove binding agent (DAPI) [236] and
major groove binding agent (Methyl Green) [237] were used to probe the potential
89
interacting sites of complex [C16H22ON5Cl5CoSn] with pBR322 DNA. Figure 59
demonstrates that Co (II) complex [C16H22ON5Cl5CoSn] inhibits the methyl green
(Lane 9) as well as DAPI (Lane 10) of the DNA digestion, In these circumstances,
complex [C16H22ON5Cl5CoSn] may bind to both major as well as minor groove .
Figure 58 . Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300 ng) by complex [C16H22ON5Cl4CoSn]Cl ; Lane 1: DNA; Lane 2: [C16H22ON5Cl4CoSn]Cl 5 µM +DNA; Lane 3: [C16H22ON5Cl4CoSn]Cl 10µM + DNA; Lane 4: [C16H22ON5Cl4CoSn]Cl 15µM + DNA; Lane 5: [C16H22ON5Cl4CoSn]Cl 20µM + DNA; Lane 6: [C16H22ON5Cl4CoSn]Cl 25 µM + DNA; Lane 7: [C16H22ON5Cl4CoSn]Cl 30 µM + DNA; Lane 8: [C16H22ON5Cl4CoSn]Cl 35 µM + DNA
DNA cleavage in presence of activators
The nuclease efficiency of metal complexes is mainly dependent on activators [238].
Thus, further activity of metal complex [C16H22ON5Cl4CoSn]Cl has been done with
different activators viz; H2O2, ascorbate (Asc), 3-mercaptopropionic acid (MPA) and
glutathione (GSH). As shown in figure 59, the cleavage activity of Co (II) complexes
[C16H22ON5Cl4CoSn]Cl was significantly enhanced in presence of these activators.
[239]. However, in the presence of H2O2, the DNA bands of [C16H22ON5Cl4CoSn]Cl
completely diminished. The activating efficacy of [C16H22ON5Cl5CoSn] follows the
order H2O2>MPA>Asc≈GSH.
Form II Form III Form I
1 2 3 4 5 6 7 8 9
90
Figure 59. Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300 ng) by complex [C16H22ON5Cl4CoSn]Cl in presence of different activators and groove binders: Lane1: DNA; Lane 2: [C16H22ON5Cl4CoSn]Cl 35µM + DNA + EtOH; Lane 3: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+ 5% DMSO; Lane 4: [C16H22ON5Cl4CoSn]Cl35 µM + DNA+(0.4mM) GSH; Lane 5: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+(0.4mM) MPA; Lane 6: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+(0.4mM) H2O2; Lane 7: [C16H22ON5Cl4CoSn]Cl 35µM + DNA +(0.4mM) NaN3; Lane 8: [C16H22ON5Cl4CoSn]Cl 35µM + DNA +(15unit) SOD; Lane 9: [C16H22ON5Cl4CoSn]Cl 35µM + DNA +(4 µM) DAPI ; Lane 10: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+(1 µl of 0.01mg/ml) methyl green; Lane 11: [C16H22ON5Cl5CoSn] 35µM + DNA +(0.4mM) Ascorbate
DNA cleavage in presence of reactive oxygen species
Reactive oxygen species generated during the interaction between metal complexes
and dioxygen or redox reagents are believed to be a major cause of DNA damage
To probe the potential mechanism of DNA cleavage mediated by complex, some
standard radical scavengers were used prior to the addition of metal complexes to
DNA solution. On adding tert-butyl alcohol (hydroxyl radical scavenger) [240], to
[C16H22ON5Cl4CoSn]Cl, DNA cleavage is inhibited suggesting the possibility of
hydroxyl radical as one of the reactive species (figure 59, lane 2). Thus, free radicals
participate in the oxidation of the deoxyribose moiety, followed by hydrolytic
cleavage of the sugar phosphate back bone in the absence of the scavengers. On the
other hand, addition of NaN3 as 1O2 scavenger (figure 59, lane 7) [96], also reduced
the same extent of cleavage as in case of tert-butyl alcohol; reveal that 1O2 may also
be activated oxygen intermediate responsible for the cleavage. Addition of SOD, as a
superoxide anion radical (O2•‾) scavenger [104], to the reaction mixture shows no
significant quenching of the cleavage revealing that superoxide anion is not the active
Form II Form III Form I
1 2 3 4 5 6 7 8 9 10 11
91
species. These results suggest that in the DNA strand scission reaction caused by the
complex various diffusible oxygen intermediate species (hydroxyl radical or singlet
oxygen) are probably involved. Since the complex is able to cleave DNA in the
absence of any reducing agent, it may be assume that DNA might be cleaved by a
discernible hydrolytic path.
DNA Condensation
DNA condensation is an essential process to transport a therapeutic gene to its target
[241]. It includes the collapse of extended DNA chain into compact, orderly particles
containing one or more molecule. Many biologically relevant divalent metal ions are
capable of DNA condensation and play a critical role in controlling the compact state
of genomic nucleic acid. Polyamines are also required in DNA catenation
(interlocking) by topoisomerases, presumably for local condensing neighbouring
DNA segments [242]. Many small molecules (such as drugs, ligand etc) are thus able
to recognize and bind to single or double stranded DNA with high affinity and
selectivity which could induce DNA structural alterations. Several metal based drugs
affect a number of physiological and biochemical regulatory functions in humans and
have been reported to be able to bind with DNA and other nucleotides.
Complex [C16H22ON5Cl4CoSn]Cl has facilitates the DNA condensation due to
neutralization of negative charge of the DNA base pairs by the positive charge of the
cationic complex. In this study, DNA condensation to the nano particulate structure
by complex [C16H22ON5Cl4CoSn]Cl under neutral condition in aqueous medium
(Tris-HCl buffer/50mM NaCl pH 7.2) was achieved by evaporating an equimolar
mixture of complex [C16H22ON5Cl4CoSn]Cl and CT DNA which have the tendency to
bind and compact DNA more efficiently at a very low concentration. The increase in
the size of the complex nano particles after CT DNA condensation was determined by
92
employing various visualization techniques viz. Transmission electron microscopy
(TEM) and atomic force microscopy (AFM).
TEM and AFM imaging of Complex-DNA condensates
Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are
useful technique for biological and chemical researches. These techniques have been
used here to analyze the morphology of the complex and complex-DNA condensates.
The TEM images of the complex nanoparticles are given in figure 60.
Figure 60. (a-b) TEM image of the nano particles of complex [C16H22ON5Cl4CoSn]Cl. The shape of the particles is irregular and the particles are of variable diameter
ranging from 15-22 nm. The particles size increases nearly 3-4 times when CT DNA
condensed to it indicating that the complex nanopatrticles can condense free CT DNA
(existing as loose strands) to compact solid DNA drug condensate as shown in figure
61 (a-d). Several numbers of drugs are unable to be effective due to the lack of the
information of their structural and morphological details.
b a
93
Figure 61(a-d) TEM image indicating the condensation of CT DNA on nano particles of complex [C16H22ON5Cl5CoSn] at different time intervals To obtain the morphological and structural information about the free
[C16H22ON5Cl4CoSn]Cl and the condensates, tapping mode AFM (atomic force
microscopy) experiments were performed using commercially etched silicon tips as
AFM probes with typical resonance frequency of 300 Hz (RTESP Veeco). Two and
three dimensional AFM images of the complex [C16H22ON5Cl4CoSn]Cl and the
condensate materials are shown in figure 62. The change in the morphological
structure in complex [C16H22ON5Cl4CoSn]Cl and its condensates with CT DNA
clearly validating that the complex is facilitating the DNA condensation, as the width
and the vertical height of the peaks increases. As the complex nanoparticles are of
different diameters and irregular in size and after condensation they appear to be
regular, very close to spherical as shown in figure 63, these results are indicative of
good DNA condensing ability of the complex.
94
Figure 62 (a-d). Two and Three dimensional AFM image of the nano particles of complex [C16H22ON5Cl4CoSn]Cl at different time interval with variable scales
a b
c d
95
Figure 63(a-d). Two and Three dimensional AFM image of the nano particles of complex –DNA condensates at different time interval with variable scales.
a b
c d
96
Molecular Docking
In order to understand further the binding modes obtained from spectroscopic studies,
rigid molecular docking experiment were performed by placing a small molecule into
the binding site of the target macromolecule mainly through non–covalent
interactions. Literature reveals that the forces maintaining the stability of DNA–
intercalator complex include van der Waals, hydrogen bonding, hydrophobic charge
transfer and electrostatic interactions [243]. A critical issue in docking operations
includes the prediction of correct binding pose and the accurate estimation of the
corresponding binding affinity. Despite the enormous size of the conformational
space for a given moiety, current docking methodologies have been successfully
employed by our group in reproducing putative binding modes. The metal complex
was restrained, by applying five dihedral and twenty-one distance restraints, to
disallow distortion. The initial docked structures were subjected to energy
minimization of -229.6 KJ / mol and examined for hydrogen bond interactions
between the metal complexes and the DNA.
The molecular modeling results so obtained indicate the change of total potential
energies of the process of complex [C16H22ON5Cl4CoSn]Cl docking between DNA
base pairs from major and minor groove as shown in figure 64. The total potential
energies of complex–DNA-binding system after intercalation are much less than those
of the system before intercalation, which may be associate with electrostatic
interaction and space matching. Furthermore, total potential energy of complex–
DNA-binding system formed by complex intercalating into C5G6 region from minor
groove is less than those of other systems, illustrating that the minor groove binding
of the complex in C5G6 region is the most preferential binding modeling of the
interactions between the complex and DNA base pairs [244].
97
Figure 64. The binding mode between the complex [C16H22ON5Cl4CoSn]Cl and B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) in minor groove.
Minor Groove
Complex 2
Major Groove
98
Conclusion
The in vitro DNA binding studies of novel heterobimetallic complex
[C16H22ON5Cl4CoSn]Cl reveal an electrostatic mode of binding as well as major /
minor groove binding. The complex [C16H22ON5Cl4CoSn]Cl exhibited an outstanding
ability to affect DNA double strand scission in oxidative as well as free hydroxyl
radical manner. Our spectroscopic results demonstrate that complex
[C16H22ON5Cl4CoSn]Cl is an avid DNA binder which is well corroborated with the
literature reports. The validation of DNA binding studies and DNA condensation was
carried out by using visualization techniques. Results from these visualization
techniques validate the size of the complex which leads it to be most specific target
drug for cancer inhibition.