Synthesis and structural characterization of dioxomolybdenum and dioxotungsten hydroxamato complexes and their function in the protection of radiation induced DNA damage

Article abstract of DOI:10.1039/c3dt52434e

The synthesis and structural characterization of two novel dioxomolybdenum(vi) (1) and dioxotungsten(vi) (2) complexes with 2-phenylacetylhydroxamic acid (PAHH) [M(O)₂(PAH)₂] [M = Mo, W] have been accomplished. The dioxomolybdenum(vi

Full text of DOI:10.1039/c3dt52434e

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Synthesis and structural characterization of
dioxomolybdenum and dioxotungsten
hydroxamato complexes and their function in the
protection of radiation induced DNA damage†
Cite this: Dalton Trans., 2014, 43,
2835
Shiv Shankar Paul,a Md. Selim,a Abhijit Sahab and Kalyan K. Mukherjea*a
The synthesis and structural characterization of two novel dioxomolybdenum(VI) (1) and dioxotungsten(VI) (2)
complexes with 2-phenylacetylhydroxamic acid (PAHH) [M(O)2(PAH)2] [M = Mo, W] have been accom-
plished. The dioxomolybdenum(VI) and dioxotungsten(VI) moiety is coordinated by the hydroxamate
group (–CONHO−) of the 2-phenylacetylhydroxamate (PAH) ligand in a bi-dentate fashion. In both the
complexes the PAHH ligand is coordinated through oxygen atoms forming a five membered chelate. The
hydrogen atom of N–H of the hydroxamate group is engaged in intermolecular H-bonding with
the carbonyl oxygen of another coordinated hydroxamate ligand, thereby forming an extended 1D chain.
The ligand as well as both the complexes exhibit the ability to protect from radiation induced damage
both in CTDNA as well as in pUC19 plasmid DNA. As the damage to DNA is caused by the radicals
generated during radiolysis, its scavenging imparts protection from the damage to DNA. To understand
the mechanism of protection, binding affinities of the ligand and the complex with DNA were determined
using absorption and emission spectral studies and viscosity measurements, whereby the results indicate
that both the complexes and the hydroxamate ligand interact with calf thymus DNA in the minor groove.
The intrinsic binding constants, obtained from UV–vis studies, are 7.2 × 103 M−1, 5.2 × 104 M−1 and
1.2 × 104 M−1 for the ligand and complexes 1 and 2 respectively. The Stern–Volmer quenching constants
obtained from a luminescence study for both the complexes are 5.6 × 104 M−1 and 1.6 × 104 M−1 respec-
tively. The dioxomolybdenum(VI) complex is found to be a more potent radioprotector compared to
the dioxotungsten(VI) complex and the ligand. Radical scavenging chemical studies suggest that the
complexes have a greater ability to scavenge both the hydroxyl as well as the superoxide radicals
compared to the ligand. The free radical scavenging ability of the ligand and the complexes was further
established by EPR spectroscopy using a stable free radical, the DPPH, as a probe. The experimental
results of DNA binding are further supported by molecular docking studies.
Received 4th September 2013,
Accepted 8th November 2013
DOI: 10.1039/c3dt52434e
www.rsc.org/dalton
1. Introduction
aDepartment of Chemistry, Jadavpur University, Calcutta, Kolkata-700032, India.
E-mail: k_mukherjea@yahoo.com; Fax: +(91) (33)24146223;
Tel: Cell phone: +(91) 9831129321
Radiotherapy is commonly employed as a part of the manage-
ment of a wide variety of malignancies and is frequently used
to achieve local or regional control of it either alone or in com-
bination with other modalities such as chemotherapy or
surgery. It is estimated that half of all cancer patients will
receive radiotherapy by way of their treatment for cancer.1
Irradiation of non-cancerous “normal” tissues during the
course of therapeutic radiation can result in a range of side
bUGC-DAE-CSR, Kolkata Centre, III/LB-8, Bidhannagar, Kolkata 700098, India
†Electronic supplementary information (ESI) available: The ORTEP view and
crystal packing structure for complex 2 [Fig. S1(a) and (b)], the absorption
spectra of the ligand and the complexes 1 and 2 in the absence and presence of
increasing amounts of DNA [Fig. S2(a)–(c)], emission spectra of complex 2 in the
presence of increasing amounts of DNA [Fig. S3], viscometric studies of the
ligand, Mo-complex and W-complex [Fig. S4], fluorescence spectra of the
EB-DNA with different doses of radiation at a dose rate of 69.3 Gy min−1 for
5–60 min [Fig. S5], gel electrophoresis study of pUC19 DNA in the presence of effects including self-limited acute toxicities, mild chronic
symptoms, or severe organ dysfunction.2 Efforts to reduce the
toxicities associated with therapeutic radiation have centred
the ligand, complexes 1 and 2 [Fig. S6], protection of the plasmid pUC19 DNA at
20 Gy with different doses of the ligand, complex 1 and complex 2 on gamma-
radiation induced strand breaks [Fig. S7] and DPPH scavenging activity of com-
both on technological improvements in radiation delivery as
plexes 1 and 2. CCDC 795762 and 878566 for complexes 1 and 2 respectively.
well as improvements in chemical modifiers to control radi-
ation induced injury.3 An alternative mechanism to reduce
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3dt52434e
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damage to normal tissues is the use of radiation modifiers/ complex can interact with the DNA in the groove. The DNA
protectors, present prior to or shortly after radiation exposure binding of the complex has been studied with CT DNA
to alter the response of normal tissues to irradiation.4 This whereas the protection of DNA damage from radiation is
approach has also been viewed as an attractive counter- demonstrated with both CT DNA and plasmid DNA. The radio-
measure for possible nuclear/radiological terrorism. So far, a protecting ability of the complex and the ligand has been
number of compounds have been reported to have radiation established by fluorescence emission spectroscopy as well as
protective properties, only amifostine is currently in clinical gel electrophoresis measurements in appropriate cases. The
use.5 From the above facts it becomes apparent that develop- results of the above studies suggest that the synthetic mole-
ment of radioprotector molecules is highly warranted to take cules used are capable of providing protection from radiation
care of the radiation induced damage to normal tissues induced DNA damage very efficiently.
occurring during radiotherapy. Although the preparation of
metal based radioprotectors6 is of prime importance as they
can prevent normal cells from death during radiotherapy of
cancer, the prevalence of metal based radioprotectors is rare.
The metal complexes can interact covalently and non-
covalently with double helical DNA that include three types of
2. Results & discussion
2.1. Synthetic aspects and spectroscopic characterization of
the complexes
binding modes, viz. intercalative, covalent binding and exter- The 2-phenylacetylhydroxamate (O, O donor) ligand was syn-
nal electrostatic binding, and among the different types of thesized by a literature method.12 The molybdenum and tung-
molecules, the metallointercalators have received particular sten-dioxo complexes were synthesized by the reaction of
attention.7
aqueous MO3·H2O [M = Mo, W] solution with 30% H2O2 result-
Amongst the metals, molybdenum, the 4d transition ing in a non-isolable peroxo species; this on reaction with the
element, has been known to have wide biological functions for methanolic solution of PAHH ligand afforded the dioxo
life on earth. The physiologically active oxidation states of molyb- [M(O)2(PAH)2] [M = Mo, W] as a solid residue. The molybdenum
denum are +4, +5 and +6 while they are commonly co-ordi- complex is designated as complex 1 while the corresponding
nated to the proteins.8,9 The essential role of molybdenum in tungsten complex is designated as complex 2. The complexes
biology has been known for decades and molybdoenzymes are are soluble in methanol, acetonitrile, and highly polar solvents
ubiquitous, but it is only recently that the biological role of like DMF and DMSO and behave as non-electrolytes. Com-
tungsten has been established in prokaryotes.9 Different types plexes 1 and 2 are also diamagnetic at 298 K.
of tungstoenzymes have been purified: formate dehydrogen-
The IR spectra of the dioxo compounds are known to
ase, formyl methanofuran dehydrogenase, acetylene hydratase, show two strong bands13 centred around 907–914 and
and a class of phylogenetically related oxidoreductases that 934–949 cm−1 attributable to the asymmetric and symmetric
catalyze the reversible oxidation of aldehydes.9
[M = O {M = Mo, W}] stretches, respectively, in the cis-dioxo
On the other hand, the chemistry and biochemistry of moieties. In the present case the ν(MovO) and ν(WvO)
hydroxamic acid and its derivatives have attracted considerable vibrations in the complexes appear at 905, 960 cm−1 and 915,
attention, due to their pharmacological, toxicological and 967 cm−1, respectively, which are in agreement with earlier
pathological properties. Hydroxamic acid derivatives generally observations.13 In free PAHH (N-phenylacetyl hydroxamic
have low toxicities with a wide spectrum of activities in all acid), the ν(CvO) vibration occurs at 1634 cm−1, while after
types of biological systems: they act as growth factors, food coordination with metals [Mo(VI) and W(VI)], the ν(CvO) of co-
additives, tumor inhibitors, antimicrobial agents, antitubercu- ordinated PAH− appears at 1597 cm−1 and 1605 cm−1 for com-
lor, antileukemic agents, key pharmacophores10 in many plexes 1 and 2 respectively. The shifting of the ν(CvO)
important chemotherapeutic agents, pigments and cell-divi- absorption band to a lower energy region indicates the
sion factors. Several of them have advanced into human clini- decrease in CvO bond order due to drainage of electron
cal trials as pharmaceutical drugs for the treatment of several density from the carbonyl oxygen to the metal [Mo & W]
diseases.11 They have also been employed as insecticides, anti- centre.
microbials, and plant growth regulators11 and are also used
The electronic spectrum of the free ligand shows a broad
industrially as antioxidants, inhibitors of corrosion, for the peak at 249 nm which corresponds to intraligand transition.
extraction of toxic elements, and as a means of flotation of There appear bands at 207 nm and 210 nm in complex 1 and
minerals. The exploration of the chemistry of the hydroxamic complex 2 respectively which are assigned as the intraligand
acid derivatives relevant to applications in the biomedical π→π* transition of the aromatic ring and another broad band
sciences has been focussed on.11 So, in the present study, the for both the complexes from 240 to 305 nm is due to n→π* of
synthesis and characterization of the potential metal complex the CvO functional group of the coordinated ligand. From the
with the biologically relevant ligand, the hydroxamate, have NMR study it was confirmed that complexes 1 & 2 as well as
been undertaken for its possible application as a radioprotec- the ligand are stable in solution phase. In both complexes 1
tor. The ligand was chosen taking into account the availability and 2 the hydroxamate –OH is deprotonated, which is
of oxygen and nitrogen atoms that can establish hydrogen characterized by the disappearance of the –OH proton signal
bonds with the DNA, and finally, the possibility that the metal at δ 4.8; this suggests that the ligand behaves as a mono
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anionic O, O donor centre. So, Mo and W metal centres are in
the +VI oxidation state which is further supported by the dia-
magnetic nature of the complexes.
Table 2 Selected bond lengths (Å) and bond angles (°) of the complex
Bond lengths (Å)
[Mo(O)2(PAH)2] (1)
[W(O)2(PAH)2] (2)
2.2. Crystal structure
Mo–O(1)
Mo–O(2)
Mo–O(3)
O(3)–C(1)
O(2)–N(1)
N(1)–C(1)
N(1)–H(1)
1.689(14)
1.990(12)
2.245(12)
1.267(18)
1.366(19)
1.298(2)
0.860
W–O(1)
W–O(2)
W–O(3)
O(3)–C(1)
O(2)–N(1)
N(1)–C(1)
N(1)–H(1)
1.705(3)
1.982(2)
2.242(2)
1.270(3)
1.368(3)
1.298(4)
0.860
The molecular structures of both complexes 1 and 2 have
been determined by single crystal X-ray diffraction analysis,
and the data are presented in Table 1 whereas the selected
bond lengths and bond angles are listed in Table 2. Com-
plexes 1 and 2 crystallize in the monoclinic C2/c space group,
and a representative ORTEP view of asymmetric units of
complex 1 is shown in Fig. 1(a). The complexes exhibit dis-
torted octahedral geometry whereas the coordinating sites are
occupied by two dioxo ligands in cis arrangement and the
remaining four sites are coordinated by two oxygen atoms of
each of the two participating (–CONHO−) ligand moieties. An
imposed C2 axis bisects the O(1)–M–O(1A) [M = Mo, W] angle,
application of which generates the full complex. The MvO
[Mo and W] bond lengths for (M–O1) and (M–O1A) were
1.689(14) Å for complex 1 and 1.705(3) Å for complex 2, which
are in the typical range for group 6 metal oxo bonds.14 The
M–O2 (carbonyl oxygen) bond lengths in complex 1 are
2.245(12) Å and 2.242(2) Å in complex 2, while the M–O3 (N–O)
bond lengths are 1.990(11) Å in complex 1 and 1.983(19) Å in
complex 2 which are comparable to other reported hydrox-
amato complexes available in the literature.15,16 The O, O
coordination of chelated hydroxamate ligand fragments (O2,
O3, C1 and N1), excluding the phenyl group, is essentially
Bond angles (°)
O(1)–Mo–O(2)
O(1)–Mo–O(3)
O(1)–Mo–O(1a)
O(1)–Mo–O(2a)
O(2)–Mo–O(3)
O(2)–Mo–O(2a)
O(2)–Mo–O(3a)
O(3)–Mo–O(3a)
Mo–O(3)–C(1)
O(1)–Mo–O(3a)
Mo–O(2)–N(1)
C1–N1–H1
105.78(7)
89.59(6)
105.53(8)
90.68(7)
73.45(4)
152.87(5)
85.51(4)
78.55(5)
112.63(10)
160.73(6)
117.18(9)
121.00
O(1)–W–O(2)
O(1)–W–O(3)
O(1)–W–O(1a)
O(1)–W–O(2a)
O(2)–W–O(3)
O(2)–W–O(2a)
O(2)–W–O(3a)
O(3)–W–O(3a)
W–O(3)–C(1)
O(1)–W–O(3a)
W–O(2)–N(1)
C1–N1–H1
90.90(11)
160.72(10)
105.52(12)
105.46(11)
73.56(8)
153.02(9)
85.41(8)
77.78(8)
112.73(18)
160.72(10)
117.11(17)
121.00
121.00
123.3(3)
O2–N1–H1A
O3–C1–C2
O3–C1–N1
N1–C1–C2
121.00
O2–N1–H1
O3–C1–C2
O3–C1–N1
N1–C1–C2
123.56(15)
117.49(15)
118.94(14)
117.0(3)
119.7(3)
planar and is approximately orthogonal to another hydroxa-
mato group of the ligand having the same coordination. The
dihedral angle between the two planes is 92.86(1) in 1 and
92.79(1) in 2. The bond distances between the metal and the
O, O donor hydroxamato (–CONHO−) moiety of the ligand are
in agreement with the single bond reported elsewhere.17 The
crystal packing arrangement in the structure exhibits weak
intermolecular N–H⋯O hydrogen bond interaction between
N–H of the hydroxamate group and the carbonyl oxygen of
another coordinated hydroxamate ligand, which leads to the
formation of a one dimensional chain (a representative packing
diagram is shown in Fig. 1(b)). The relevant hydrogen bonding
parameters are shown in Table 3. The ORTEP view and packing
pattern of complex 2 are shown in ESI Fig. S1(a) and (b).†
Table 1 Crystal data and structure refinement parameters of the
complex
Parameters
Complex 1
16H16N2O6Mo
428.25
293(2)
0.71073
Monoclinic
C2/c
Complex 2
Empirical formula
Mr
T/K
λ/Å
Crystal system
Space group
Unit cell dimensions
a/Å
b/Å
c/Å
α/°
β/°
C
C16H16N2O6W
516.15
293(2)
0.71073
Monoclinic
C2/c
17.4688(4)
10.5106(4)
9.4600(2)
90.00
103.987(2)
90.00
1685.43(8)
4, 1.688
864
17.4365(16)
10.5304(16)
9.4876(10)
90
104.276(2)
90
1688.3(3)
4, 2.031
992
2.3. DNA binding studies
The present study is directed towards the development of syn-
thetic molecules capable of providing protection from radiation
induced DNA damage. This protection can be imparted primarily
through DNA binding and scavenging of radiolysed radicals. So,
γ/°
V/Å3
Z, Dc/g cm−3
F(000)
Crystal size/mm
θ Range for data collection (°) 2.28, 27.64
Reflection
Independent reflections (Rint
Completeness to θ = θmax (%)
Refinement method
0.05 × 0.07 × 0.15 0.04 × 0.08 × 0.15 establishment of the ability of protection of the double stranded
2.28, 25.0
5789
DNA damage from a given molecule demands a thorough study on
the interaction of DNA with the potential protector molecule.
Hence, the binding of DNA with the synthetic molecules has
been studied, the results of which are being presented herein.
2.3.1. Electronic absorption spectral studies. Electronic
absorption spectroscopy is one of the simplest techniques
usually utilized to examine the binding of metal complexes
with DNA. The electronic spectra of both complexes 1 & 2
and the ligand in the presence and absence of DNA were
12 587
)
1953(0.019)
99.1
1489(0.026)
100
Full-matrix-least-squares on F2
Data/restraints/parameters
1953/0/114
1.109
1489/0/114
1.074
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R1 = 0.0203
wR2 = 0.0535
R1 = 0.0218
wR2 = 0.0544
−0.375, 0.232
R1 = 0.0174
wR2 = 0.0446
R1 = 0.0182
wR2 = 0.0450
−0.412, 0.491
R indices (all data)
Largest diff. peak, hole/Å−3
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Fig. 1 (a) ORTEP representation of the X-ray crystal structure of complex 1, with all non-hydrogen atoms shown as 50% thermal ellipsoids. (b)
Crystal packing of complex 1.
Table 3 Relevant hydrogen bonds in the complex
D–H⋯A
d(D–H)
d(H⋯A)
d(D⋯A)
∠(DHA)
[Mo(O)2(PAH)2] (1)
N(1)–H(1)⋯O(3)(i)
0.86
0.86
2.07
2.09
2.853(17)
2.864(3)
151
150
[W(O)2(PAH)2] (2)
N(1)–H(1) ⋯O(3)(ii)
(i) x, 1 − y, −1/2 + z; (ii) x, –y, −1/2 + z.
monitored. Upon the addition of incremental amounts of
DNA, increases in the absorptivity and red shift (2 nm) of the
ligand and both complexes 1 & 2 were observed. Hoechst
33258 family groove binders also exhibit red shifts of absorp-
tion bands when they bind to the grooves of a DNA helix. A
plot of [DNA]/(εa − εf) versus [DNA] gives the binding constants
which were calculated to be (5.2 0.2) × 104 M−1 for DNA- Fig. 2 Emission spectra of complex 1 in the presence of increasing
complex 1, (1.2 0.2) × 104 M−1 for DNA-complex 2 and (7.2
amounts of DNA (a) 0.0 μM, (b) 10 μM, (c) 20 μM, (d) 40 μM, (e) 60 μM,
(f) 80 μM, (g) 100 μM, (h) 110 μM, (i) 120 μM, ( j) 130 μM, (k) 150 μM,
(l) 180 μM and (m) 200 μM. (Inset: Stern–Volmer plot for MoP.)
0.2) × 103 M−1 for DNA-ligand, respectively, which indicates
that both complexes 1 & 2 and the ligand approach DNA
through the groove.18 The corresponding results of absorbance
be 5.6 × 104 M−1 and 1.6 × 104 M−1 for complexes 1 and 2
spectral studies are provided as ESI [Fig. S2(a)–(c)†].
2.3.2. Emission titrations. The PAHH ligand does not
show any emission, but when it is coordinated to the molyb-
denum or tungsten, it shows emission. [Mo(O)2(PAH)2],
complex 1, on excitation at 250 nm exhibits emission at
307.5 nm, whereas [W(O)2(PAH)2], complex 2, on excitation at
270 nm exhibits emission at 290 nm, so these are intrinsic
fluorophores.
The emission intensities of complexes 1 and 2 decrease
gradually on progressive addition of CT-DNA [Fig. 2 & ESI
Fig. S3†], which suggests binding of DNA with both complexes
1 and 2. The fluorescence intensity becomes constant at a high
concentration of DNA, which indicates that the binding has
reached saturation. The fluorescence quenching constants
(KSV) evaluated using the Stern–Volmer equation were found to
respectively at 25 °C [inset in Fig. 2 & ESI Fig. S3†]. The linear
Stern–Volmer plots with the intercept of 1.0 for both com-
plexes 1 and 2 indicate that only one type of quenching
process occurs, i.e., static quenching in both the cases.
The results were further analysed using eqn (3) to get the
required parameter P, which was determined either by
measurement of the fluorescence emission of a given quantity
of complex 1 or 2 in the presence and absence of a large excess
of DNA or by the addition of DNA to a fixed amount of
complex 1 or 2 until no further change in fluorescence
emission was observed. A value of P = 0.05 was obtained
regardless of the protocol and was used for further calcu-
lations. These experimental data are fitted to the neighbour
exclusion model19 (vide eqn (3)). The best fit to the
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experimental data provides an exclusion parameter, n = 3.5
2.4.2. Estimation of the protection from DNA damage by
and 2 base pairs for both complexes 1 and 2, while the intrin- the fluorometric technique. The amount of retention of
sic binding constants were found to be Ki = 0.8 × 105 M−1 and double stranded DNA after irradiation was calculated from
4.8 × 104 M−1 for complexes 1 and 2 respectively. The uncer- dose–effect curves (ESI Fig. S5†). D50 (50% DNA remains
tainty in these parameters is roughly 15%. The number of unchanged) was calculated from the dose–response curve and
binding sites is obtained from the x-intercept of the Scatchard the time required for it was also determined. After irradiating
plot. This phenomenon can be accounted for with the neigh- the DNA for 35 min, i.e., at D50, the fluorescence intensity of
bour exclusion model,19 which holds that the binding of a the EB bound DNA decreases sharply with respect to the
small molecule to one site influences the binding of sub- control DNA, indicating radiation induced damage. For pro-
sequent molecules, and the subsequent molecules are tecting the DNA from radiation induced damage, DNA was pre-
excluded from binding at nearby sites by either physical block- treated with different concentrations of either the ligand or
age or steric alterations in DNA.
the complex 1 or 2 in appropriate cases and then radiation was
2.3.3. Viscometric studies. Hydrodynamic measurement, given, thereafter the emission intensity of EB was measured. It
mainly viscosity, is a sensitive technique to determine the DNA is observed that the fluorescence intensity of EB-DNA solution
binding mode. The relative viscosity of CT-DNA solution is is increased with an increase in the concentration of either the
known to increase on intercalative binding of substrates, ligand or the complexes 1 & 2, the results of which are shown
because the insertion of intercalators causes the base pairs of in Fig. 3(a)–(c). The concentration of the ligand and the com-
the DNA to get apart and thus causes a lengthening of the plexes used is over the range of 0.4 µM to 20 µM, and it is
DNA helix. On the other hand, agents bound to DNA through observed that with the increase in concentration there occurs a
a groove do not alter the relative viscosity of DNA, and the gradual increase in the fluorescence intensity of EB bound to
partial or non-classical intercalation of ligands may bend or irradiated DNA. This indicates that the ligand as well as both
kink the DNA helix, thereby decreasing its effective length and the complexes impart protection to the CTDNA from the
subsequently viscosity.20 The values of relative specific viscos- damage induced by radiation.
ities of DNA in the absence and presence of complexes 1 & 2
A plot of (I − Ia)/(I0 − Ia) versus [radioprotector]/[CT-DNA]
and the ligand are plotted against [complex]/[DNA]. It is observed has been made. This is shown as the inset in Fig. 3(a)–(c) where
that the addition of either the complex 1 or 2 or the ligand to it is found that with the increase in the concentration of either
the CTDNA solution shows neither any significant increase nor the ligand or both complexes 1 and 2, the damage caused by
any significant decrease in the viscosity of the CTDNA, thereby radiation to DNA is inhibited. Precisely, the percentage of pro-
clearly demonstrating the non-intercalative binding of CTDNA by tection imparted either by the ligand or by the Mo and W-com-
the present complexes 1 & 2 as well as the ligand; on the other plexes was evaluated to be 72, 87 and 80%, respectively. The
hand, it hints at groove binding21 [ESI Fig. S4†].
protection from radiation induced CT DNA damage is very
much pronounced in the case of the Mo-complex compared to
the ligand and W-complex. The ligand imparts 72% protection
2.4. Radiation induced DNA damage and protection from it
2.4.1. Fluorimetric estimation of radiation induced from the damage; the Mo-complex is capable of imparting 87%
damage in CT DNA. The fluorimetric assessment of radiation protection whereas its counterpart, the W-complex, imparts
induced DNA damage was carried out using ethidium bromide 80% protection to the double stranded DNA. It may be men-
(EB) as a probe, which has very feeble emission intensity in tioned here that the binding constant of the complexes is also
the aqueous medium. However, the emission intensity of EB is higher than the ligand. Ionizing radiation on interaction with
much enhanced in the presence of DNA due to its strong inter- water in cells can produce reactive free radicals, such as
calation between the adjacent DNA base pairs. If radiation hydroxyl radicals, hydrogen radicals, ROS, hydrogen peroxide
induces damage to the DNA double helix, the bound EB will and toxic substances, all of which can damage critical macro-
be finding its way to come out of the base pairs in the aqueous molecules. The elimination of the free radical species from the
environment again, or if EB is added immediately after cell environment can inhibit the side effects induced by
irradiation of DNA, because of the damage in the double helix, irradiation. Scavenging of the radiolysed radicals arising out of
relatively more EB will remain in aqueous solution, which will the irradiation of water molecules can confer protection to the
be reflected in a decrease in fluorescence intensity. This rela- cellular materials from radiation. In the present case, as the
tive decrease in fluorescence intensity of EB bound to DNA ligand and both complexes 1 and 2 are groove binders, they are
proves that the damage in the DNA double helix is a function capable of protecting the double stranded DNA from radiation
of dose of radiation. In the present case, irradiation of DNA induced damage. But the abilities of protection of the ligand
with γ-rays causes a decrease in the fluorescence intensity of and complexes 1 and 2 are different. Even at a low concen-
EB. The greater the dose of the radiation, the greater is the tration, the Mo-complex (8 µM) is able to impart about 80% pro-
decrease in fluorescence intensity of EB. These dose–effect tection from radiation induced damage to DNA compared to
curves for gamma radiation induced DNA strand breaks are 72% for the W-complex at the same concentration. Although
purely linear, and the estimation indicates a 68% DNA damage with the increase in concentration the ligand and both
by the radiation at the present experimental set-up (ESI complexes 1 and 2 show enhanced protection, in the case of
Fig. S5†).
the Mo-complex it is more pronounced; precisely, at 15 µM
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Fig. 3 Fluorescence emission spectra of the EB-DNA in presence of increasing amounts of (i) ligand, (ii) MoP, (iii) WoP after 35 min of gamma-
irradiation [EB] = 30.0 μM for (a), [EB] = 30.0 μM + [DNA] = 20.0 μM (unirradiated) for (b); (c–l) [Ligand or Complex]/[DNA]: 0–1 (irradiated). (Inset:
Plot of (I − Ia)/(I0 − Ia) versus [ligand or Complex]/[DNA].)
concentration, the ligand, the W-complex and the Mo-complex SC form plasmid DNA got converted into open circular form at
impart 72%, 80% and 87% protection respectively. So, even at doses of 20 & 25 Gy, respectively, as is evident from the figures
a low concentration, complex 1 responds well to the protection (ESI Fig. S7† & Fig. 4, lane 2). The treatment of radiation
of damaged DNA from radiation, compared to the ligand and induced damaged pUC19 DNA with different concentrations of
complex 2. The potentiality of metal complexes to protect the either complexes 1 and 2 or the ligand shows significant pro-
damaged DNA is greater than the ligand, due to chelation of tection from the damage. The damage caused by radiation is
the ligand to the metal centre. Therefore, complexes 1 and 2 mainly due to the formation of radicals. Complexes 1, 2 and
have a greater ability to protect than the ligand.
2.4.3. The role of the ligand and complexes 1 and 2 in the
protection from gamma-radiation induced strand breaks in
plasmid pUC19 DNA. The gel electrophoresis studies with
pUC19 plasmid DNA show that neither the ligand nor the
complex alone does induce any nicking in the plasmid pUC19
DNA (ESI Fig. S6†) at different concentrations of 1.0 and
2.0 mM. Exposure of the plasmid DNA to gamma-radiation at
different doses leads to strand breaks resulting in relaxation of
plasmid DNA from a supercoiled (SC) form to a nicked
coil (NC) form.22 The plasmid DNA is subjected to doses of
radiation of 20 Gy and 25 Gy, which cause damage to the
supercoiled plasmid DNA. When pUC19 DNA is subjected to
Fig. 4 Protection of plasmid pUC19 DNA at 25 Gy with different doses
of ligand, complex 1 and complex 2 on gamma-radiation induced strand
gamma-radiation induced strand breaks, 50% and 60% of the
breaks.
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Table 4 Extent of DNA SC pUC19 protection by the ligand and the complex
Lane no.
1
Reaction condition
Form I (% SC)
96
Form II (% NC)
4
DNA CONTROL (No radiation)
20 Gy
25 Gy
Radiation dose
Form I (% SC)
Form II (% NC)
Form I (% SC)
Form II (% NC)
2
3
4
5
6
7
8
DNA irradiated
DNA + 1 mM (0.015 µg) ligand
DNA + 1 mM (0.0428 µg) MOP complex
DNA + 1 mM (0.0516 µg) WOP complex
DNA + 2 mM (0.030 µg) Ligand
DNA + 2 mM (0.0956 µg) MOP complex
DNA + 2 mM (0.1032 µg) WOP complex
50
62
73
68
80
95
87
50
38
27
32
20
5
40
70
78
72
75
85
76
60
30
22
28
25
15
24
13
the ligand might be able to eliminate the radicals that cause and vitamin C. The 50% inhibitory concentration (IC50) values
damage to the supercoiled DNA. The results of this experiment of mannitol and vitamin C are about 1 × 10−3 and 0.9 × 10−3
M
are presented in Table 4. The ligand and complexes 1 and 2 respectively. According to the scavenging experiments, the IC50
could afford protection from radiation induced damage to the values of the ligand and the complexes 1 & 2 are found to be
plasmid DNA; at a concentration of 2.0 mM of either the 1 × 10−4 M, 6.5 × 10−5 M, and 1.2 × 10−5 M, respectively, which
ligand or complex 1 or 2, the ligand imparts 80% and 75% pro- implies that the complexes 1 and 2 exhibit better scavenging
tection at doses of 20 and 25 Gy respectively, whereas complex activity than mannitol and vitamin C.24 Moreover, the ligand
1 imparts 95% and 85% protection, and for complex 2 the pro- and both the complexes 1 and 2 are also capable of scavenging
tection is 87% and 76% at doses of 20 and 25 Gy, respectively. the radiolysed radicals.
Thus, it is evident that the ligand as well as the complexes
2.5.2. Superoxide radicals (O2−•) scavenging activity. The
could offer significant protection to DNA against gamma-radi- ability to reduce NBT by superoxide radicals generated from
ation induced damage in vitro by reducing the formation of dissolved oxygen by PMS-NADH coupling25 can be measured
strand breaks.
by a decrease in the absorbance of the reaction mixture at
560 nm. The absorbance at 560 nm is affected when the
control solution is treated with different concentrations of the
ligand as well as complexes 1 and 2. Complexes 1, 2 and the
ligand show a gradual decrease in the absorbance at 560 nm,
2.5. Mechanism of protection from radiation induced DNA
damage
Radiation induced DNA damage is mainly caused by the split- indicating their ability to scavenge superoxide radicals from
ting of water to generate reactive oxygen species (ROS) during the reaction mixture. The ability of the ligand and the com-
radiolysis.23 ROS are mainly free radicals. Scavenging of the plexes 1, 2 to scavenge superoxide radicals from the solution
free radicals which are generated during radiolysis is of prime was found to be higher than that of the standard. The inhibi-
importance as these cause damage to DNA. So, for protection tory concentration (IC50) value for the standard quercetin is
from DNA damage from radiation, it is important to remove about 7 × 10−3 M, while the IC50 values for the corresponding
these free radicals, i.e. ROS (OH• hydroxyl radicals and super- ligand and the complexes were found to be 3.6 × 10−4 M, 4.2 ×
oxide anion radicals), from the system.
10−5 M and 0.9 × 10−5 M, respectively.
As the ligand as well as the complexes exhibit protection
from radiation induced DNA damage, it is considered worth-
while to study other potential aspects, such as radical scaven-
ging activity by the ligand or complexes 1 and 2 to understand
the mechanism of action. The radical scavenging activities of
the ligand and complexes 1 & 2 along with the standards have
been examined. The standards used were mannitol and
vitamin C for hydroxyl radicals (OH•), and quercetin for super-
oxide anion radicals (O2−•).
2.5.1. Hydroxyl radical scavenging activity. The hydroxyl
radicals were generated through a Fenton-type reaction, in
which the degradation of safranin was measured in a Fe(II)–
EDTA–H2O2 reaction mixture. The ability of the complexes 1, 2
and the ligand to scavenge hydroxyl radicals was compared
with those of the well-known natural antioxidants23 mannitol
2.5.3. Assessment of the scavenging of DPPH
2.5.3.1. Assessment of the scavenging of DPPH by EPR spectro-
scopy. The elimination of radicals from the solution by
scavenging will protect other molecules of the solution from
radiation induced damage, if any entity capable of scavenging
free radicals is used during radiolysis. The molecules which we
have used to protect from DNA damage have been shown to sca-
venge free radicals from the system. So, to establish the radical
scavenging activity of our molecules in a more sophisticated
way, EPR spectroscopy was used. The model of scavenging the
stable radical, DPPH, is a widely used method26 to evaluate
antioxidant capacities of natural and synthetic products. The
EPR signal of DPPH was used to monitor the free radical
scavenging activity of our complex and the ligand. The EPR
spectra of the methanolic solutions of DPPH were taken. This
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Fig. 5 (a) EPR spectra of DPPH (60 mM) with different concentrations of ligand in methanol solution (a–f: 0–60 mM). (b) EPR spectra of DPPH
(60 mM) with different concentrations of complex 1 in methanol solution (a–h: 0–60 mM). (c) EPR spectra of DPPH (60 mM) with different concen-
trations of complex 2 in methanol solution (a–h: 0–60 mM).
DPPH was treated with incremental amounts of either the
ligand [Fig. 5(a)] or the complexes 1 and 2 [Fig. 5(b) and (c)],
and EPR was taken under identical conditions. Although it is
found that both the complexes and the ligand can induce
scavenging of free radicals of DPPH, the effect is more pro-
nounced in the case of complex 1 than complex 2 and the
ligand. This has also been exhibited in the actual DNA damage
protection experimental results, where complex 1 has been
found to impart greater protection from damage than complex
2 and the ligand. So, this experiment unequivocally supports
our observation of the ability of protection from radiation
induced DNA damage both by complexes 1, 2 and the
ligand.
2.5.3.2. Assessment of the scavenging of DPPH by electronic
absorbance spectroscopy. The DPPH free radical scavenging
property of the ligand and complexes 1 and 2 was investigated by
electronic absorbance spectroscopy using DPPH as a stable
free radical.27 The radical scavenging was monitored at ence of MoP (complex 1) (0–0.06 μM) in MeOH.
Fig. 6 Changes in the UV-vis spectrum of DPPH (0.1 μM) in the pres-
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517 nm at different concentrations of the ligand and
complexes 1 & 2. The ability of the ligand and complexes 1, 2
[Fig. 6 and ESI Fig. S8†] to scavenge DPPH free radicals from
the solution was found to be higher than that of the standard.
The inhibitory concentration (IC50) value for the standard
vitamin C is about 0.9 × 10−3 M, while the IC50 values for the
corresponding ligand and the complex were found to be 1.6 ×
10−4 M, 1.6 × 10−5 M and 2.8 × 10−5 M respectively. This study
also supports the scavenging of free radical DPPH from the
solution by the ligand and both the complexes 1 & 2. This is
done probably by the adduct formation.
Thus, from the scavenging experiments, it can be con-
cluded that both the complexes 1 and 2 show greater scaven-
ging activity compared to the ligand; this is due to the
chelation of ligands with the central metal atom.28 The lower
IC50 values observed in radical scavenging assays did demon-
strate that complexes 1, 2 and the ligand have strong potential-
ity to be applied as scavengers to eliminate the radicals
generated by radiolysis.
2.6. Molecular docking
Fig. 8 Docked pose of Wop showing interaction with base pairs.
2.6.1. Molecular docking investigation on the interaction
of DNA with the ligand and complexes 1 & 2. The mole-
cular docking technique is an attractive scaffold to understand
the drug–DNA interactions for rational drug design and
discovery, as well as to establish the mechanism of action of
the reactants by placing a small molecule into the binding site
of the target specific region of the DNA, mainly in a non-
covalent fashion. However, a covalent bond may also be
formed between the reactants. Different structural properties
lead to different binding modes; in fact one of the most
important factors governing the binding mode is the mole-
cular shape. The literature29 reveals that the forces
Fig. 9 Docked pose of ligand showing interaction with base pairs.
maintaining the stability of the DNA–intercalator complex
include van der Waals forces, hydrogen bonding, hydrophobic
charge transfer and electrostatic complementarity. In our
experiment, the ligand and the complexes 1 & 2 were succes-
sively docked with the DNA duplex of the sequence d
(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) in order to
predict the chosen binding site along with preferred orien-
tation of the ligand inside the DNA minor groove.30 The ener-
getically most favourable conformation of the docked pose
(Fig. 7–9) revealed that complexes 1 & 2 and the ligand bind to
the minor groove of DNA, thereby slightly adjusting the DNA
structure in such a way that a part of the planar phenyl ring
makes favourable stacking interactions with DNA base pairs
Fig. 7 Docked pose of Mop showing interaction with base pairs.
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and leads to van der Waals interactions with the DNA func- (G.R.) were products of Merck (India) and were used directly.
tional groups that define the stability of the groove. Moreover, DPPH (2,2-diphenyl-1-picrylhydrazyl), phenazine methosulfate
two hydrogen bonding interactions with complexes 1 & 2 and (PMS), reduced nicotinamide adenine dinucleotide (NADH),
three for the ligand in the minor groove have been predicted. nitroblue tetrazolium (NBT), quercetin, EDTA, mannitol and
The resulting relative binding energies of the docked struc- safranin were obtained from SRL, India. All other reagents
tures for complexes 1, 2 and the ligand were found to be used were of G.R. grade and were obtained from Merck (India).
−243.63, −212.42 and −202.02 kJ mol−1, respectively. This Analytical grade solvents used for physico-chemical studies
indicates that a more potent binding between the DNA and were further purified by a literature method before use, wher-
complex 1 takes place than complex 2 and the ligand. This cor- ever necessary. CT DNA and supercoiled plasmid pUC19 DNA
relates well with the experimental DNA binding studies. Thus, were obtained from Sigma Chemical Company, USA, and
the spectroscopic experimental results are harmonized with Genei Bangalore, India, respectively.
the molecular docking study as well.
4.2. Preparation of the ligand and the molybdenum and
tungsten complexes
3. Conclusion
4.2.1. Synthesis of the ligand, N-(phenyl acetyl) hydrox-
The hydroxamato complexes of dioxomolybdenum and dioxo- amic acid (PAHH). The ligand was prepared using a literature
tungsten have been designed, synthesised and structurally method;12 methyl phenylacetate was obtained by refluxing the
characterised using single crystal X-ray diffraction. The DNA mixture of phenylacetic acid, 13.6 g (0.1 mol), in 25 ml of dry
binding studies using absorbance spectroscopy show that the methanol followed by addition of 1 ml of conc. H2SO4. Then to
intrinsic binding constants for complexes 1, 2 and the ligand the above mixture, solid NH2OH·HCl, 14 g (0.2 mol), was
are found to be 5.2 × 104, 1.2 × 104 and 7.2 × 103 M−1 respect- added followed by the addition of a 25% methanolic KOH
ively. Furthermore, the Stern–Volmer quenching constant, KSV
,
(0.4 mol) solution with constant stirring. The reaction mixture
for complexes 1 and 2 is found to be 5.2 × 104 M−1 and 1.6 × was neutralized with 1 N HCl solution, filtered off and the
104 M−1 respectively. Both the complexes and the ligand residue was washed with methanol. After the evaporation of
exhibit effective protecting ability against radiation induced this methanolic solution a white solid crystalline phenylacetyl
DNA damage. From the fluorometric assessment, 87% of the hydroxamic acid was obtained. IR (KBr disc, cm−1): 1634(s) (ν
damaged DNA revives when the concentration of complex 1 is CvO), 1546(m) (ν C–N). 1H NMR (CD3OD, 300 MHz) δ in ppm:
about 15 µM, whereas complex 2 and the ligand impart 80% 4.8 (s, 1H, –OH), 3.44 (t, 2H, –CH2–Ar), 7.226–7.312 (m, 5H,
and 72% protection respectively to damaged DNA at the same H-benzene ring), 5.46 (s, 1H, NH).
concentration. At a relatively higher concentration of the
4.2.2. Synthesis of [Mo(O)2(PAH)2] complex (1). MoO3·2H2O
complex, about 95% of supercoiled plasmid (pUC19) DNA is (0.45 g, 2.5 mmol) was dissolved in 5 ml of H2O and
protected. To establish the mechanism of protection by both then 0.5 ml (6.5 mmol) of 30% H2O2 was added to it with con-
complexes 1 and 2 as well as the ligand, radical scavenging stant stirring at room temperature, which produced a pale
experiments were performed which show that the Mo-complex yellow solution. Addition of a 20 ml methanolic solution of
(1) has a greater ability to scavenge free radicals compared to 0.742 g (5 mmol) phenyl acetyl hydroxamic acid (PAHH) to the
the W-complex (2) and the ligand. The EPR study on DPPH above mixture under stirring conditions precipitated [Mo(O)2-
radical scavenging unequivocally supports the mechanism of (PAH)2] as a yellow solid. The solid was filtered off, washed
protection by both complexes 1 and 2 and the ligand. The thoroughly with water, methanol and diethyl ether, and then
molecular docking study supports the binding of complexes 1 dried under vacuum. Single crystals (shiny yellow) suitable for
and 2 and the ligand to DNA where the relative binding energy X-ray diffraction were obtained by slow evaporation of the
of the lowest docked structure is found to be −243.63 kJ mol−1
,
methanolic solution. Yield was 80%. Anal. calcd for
−212.42 kJ mol−1 and −202 kJ mol−1 for complexes 1 and 2 C16H16N2O6Mo: C, 44.87; H, 3.7; N, 6.54; Mo, 22.40; Found. C,
and the ligand respectively. Therefore, the present study 44.94; H, 3.82; N, 6.59; Mo, 22.44; MS (EI): m/z 428.25; IR (KBr
realises the development of a metal based radioprotector disc, cm−1): 1597 (s) (ν CvO), 1498 (s) (ν C–N), 960 (s) & 905
and paves the way for the next phase trial with these (m) [ν MovO]. UV-vis; λmax nm: 308(sh), 242(sh), 207. 1H NMR
molecules.
(CD3OD, 300 MHz) δ in ppm: 3.54 (s, 4H, –CH2–Ar),
7.236–7.345 (m, 10H, H-benzene ring), 5.24 (s, 2H, NH).
4.2.3 Synthesis of [W(O)2(PAH)2] complex (2). The
[W(O)2(PAH)2] complex was synthesized by the same proce-
dure as described for the synthesis of complex 1, only
WO3·2H2O, 0.65 g (2.5 mmol) was used instead of molybdic
4. Experimental
4.1. Materials and physical methods
Molybdic acid (MoO3·2H2O), tungstic acid and phenyl acetic acid. Complex 2 as a cream coloured solid was separated out,
acid were obtained from S.D. Fine Chem. (India). Hydroxyla- filtered off, washed thoroughly with water, methanol and
mine hydrochloride was of extra pure variety and was obtained diethyl ether, and then dried under vacuum. Yield was 78%.
from Merck (India). Potassium hydroxide pellets and methanol The cream coloured single crystals suitable for X-ray
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diffractometric studies were obtained by slow evaporation of intrinsic binding constant Kb was obtained from the ratio of
the methanol solution. Anal. calcd for C16H16N2O6W: C, 53.69; the slope to the intercept.
H, 4.12; N, 9.4; W, 11.4; Found. C, 53.37; H, 4.22; N, 9.07; W,
4.4.2. Luminescence titrations. The excitation of the
10.97; MS (EI): m/z 516.15; IR (KBr disc, cm−1): 1605 (s) (ν methanolic solution of complex 1 at 250 nm gives emission
CvO), 1508 (s) (ν C–N), 967 & 915 (s) [ν WvO], 906 (m). UV- maxima at 307.5 nm while the excitation of complex 2 at
1
vis; λmax nm; 312, 245(sh), 212. H NMR (CD3OD, 300 MHz) δ 270 nm shows emission with λmax 290 nm. For fluorescence
in ppm: 3.54 (s, 4H, –CH2–Ar), 7.236–7.345 (m, 10H, H- titration experiments, the concentrations of complexes 1 and 2
benzene ring), 5.24 (s, 2H, NH).
were so adjusted that the final concentration of the test
sample was 0.70 µM. This was titrated with increasing
concentrations of DNA over the range 0–200 µM. The titration
procedure was similar to that outlined above for spectrophoto-
metric titrations. For all measurements, the samples were
excited at 250 nm (complex 1) and at 270 nm (complex 2) and
a similar slit width (10/10) was used.
4.3. X-Ray crystal structure determination
X-ray diffraction data for the crystals of complexes 1 and 2
were collected at 296 K on a Bruker AXS SMART APEX II dif-
fractometer equipped with a CCD detector31 with a fine focus of
a 1.75 kW sealed tube using Mo Kα radiation (λ = 0.71073 Å).
Crystallographic data and details of structure determination
are summarized in Table 1. The data were processed using
SAINT, and absorption corrections were made using SADABS.32
The structure was solved by direct methods and refined by full-
matrix least-squares on the basis of F2 using the WINGX soft-
ware, using the SHELX suites.33 The non-hydrogen atoms were
refined anisotropically, while the hydrogen atoms were placed
geometrically (0.97 Å for –CH2 group, 0.86 Å for N–H and 0.93 Å
for aromatic C–H distance for different parent atoms) and
refined using a riding model with isotropic displacements fixed
(with a value equal to 1.2Ueq of its parent atoms). Perspective
views of the molecules were obtained using ORTEP.34
The fluorescence quenching experimental data were further
analysed using the Stern–Volmer equation:36
I0=I ¼ 1 þ KSV ½DNAꢀ
ð2Þ
where I0 and I are the fluorescence intensities in the absence
and presence of DNA, respectively; and KSV is the Stern–Volmer
quenching constant, which is a measure of the efficiency of
quenching by DNA. The fluorescence titration data were also
used to determine the binding constant of complexes with
DNA. The concentration of the free drug was calculated accord-
ing to eqn (3):19
CF ¼ CT ðI=I0 ꢁ PÞ=ð1 ꢁ PÞ
ð3Þ
where CT is the concentration of the drug added, CF is the con-
centration of the free drug, and P is the ratio of the observed
quantum yield of the fluorescence of the totally bound drug to
that of the free drug. The value of P is obtained from a plot of
I/I0 versus 1/[DNA] such that it is the limiting fluorescence
yield given by the y intercept. The amount of bound drug (CB)
at any concentration is equal to CT–CF.
A plot of r/CF versus r, where r is equal to CB/[DNA], was
done by the modified Scatchard equation to understand the
detailed binding pattern with binding sites:16,19
4.4. DNA binding studies
4.4.1. UV-vis spectral study. The solutions of CT DNA in
Tris-HCl/NaCl (50 mM Tris-HCl and 50 mM NaCl, pH 7.2)
buffer medium gave a ratio of A260/A280 of ca. 1.8–1.85, indicat-
ing that the DNA was sufficiently free from protein contami-
nation. The DNA concentration per nucleotide was determined
by absorption spectroscopy using the molar absorption coeffi-
cient at 260 nm. Stock solutions were stored at 4 °C and used
within 4 days.
UV-vis spectra were recorded on a Shimadzu UV-1700 spec-
trophotometer. The electronic spectra of the complexes and
the ligand were monitored in the presence and absence of
DNA. In this absorption titration experiment, a fixed concen-
tration of either complex 1 or 2 or the ligand was titrated with
increasing amounts of DNA over a range of 10–150 μM in
appropriate cases. To eliminate the absorbance of DNA, equal
amounts of DNA were added to the reference solution as
well. The intrinsic binding constant was determined using
eqn (1):35
nꢁ1
2r=Cf ¼ Kbð1 ꢁ nrÞ½ð1 ꢁ nrÞ=f1 ꢁ ðn ꢁ 1Þrgꢀ
ð4Þ
where Kb represents the intrinsic binding constant of the
complex with DNA and n is the size of binding sites in base
pairs.
4.4.3. Viscometric study. The viscosity of sonicated
DNA16,17,37 (average molecular weight of ∼200 base pairs was
measured using a Labsonic 2000 sonicator) was measured by a
fabricated micro viscometer, maintained at 28 ( 0.5) °C in a thermo-
static water bath. The viscosities of the CTDNA, CTDNA-
½DNAꢀ=ðεa ꢁ εf Þ ¼ ½DNAꢀ=ðεb ꢁ εf Þ þ 1=½Kbðεb ꢁ εf Þꢀ ð1Þ
ligand, CTDNA-complex
1 and CTDNA-complex 2 were
Here, [DNA] is the concentration of DNA in base pairs, the measured. Data were presented as (η/ηo)1/3 versus the ratio
apparent absorption coefficients εa, εf and εb correspond to of the concentration of either the ligand or complex 1 or 2
Aobsd/[complex], the extinction coefficient for the free complex, to that of the CT DNA, where ηo is the viscosity of CT DNA
and the extinction coefficient for the complex in the fully solution alone and η is the viscosity of CT DNA solution in the
bound form, respectively. Plots of [DNA]/(εa − εf) versus [DNA] presence of either the complexes or the ligand. Viscosity
gave a slope 1/(εb − εf) with Y-intercept 1/[Kb (εb − εf)]. The values were calculated from the observed flow time of CT DNA
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by the relation η = t − t0, where t and t0 are the values of flow solution. Bands were visualized by UV light and photographed
times for the solution and the buffer respectively.
using the UVP BIO-DOC-IT Gel Documentation System. The
extent of supercoiled (SC) pUC19 DNA damage induced by
radiation and protection induced by the ligand and complexes
was determined by analysing the intensities of the bands
using UVP – BIO-DOC-IT LS Software.
4.5. DNA damage and protection
4.5.1. Gamma irradiation. Gamma radiation was passed
through DNA solution with the help of a GC-900 Gama
Chamber, 2 Killo Courie. The CTDNA and DNA-complex
samples were incubated at 37 °C for 30 min before irradiation;
4.6. Mechanism of protection from radiation induced DNA
damage by the ligand and the complexes
the incubated samples were then irradiated in
a
60Co
4.6.1. Hydroxyl radical scavenging experiment. Hydroxyl
radicals were generated in aqueous media through the Fenton-
type reaction. Aliquots of 1.0 ml of 0.10 mmol aqueous safra-
nin, 1 ml of 1.0 mmol aqueous EDTA-Fe(II), 1 ml of 3%
aqueous H2O2, and different concentrations of either the
ligand or complexes 1 and 2 were added to constitute the reac-
tion mixture (3 ml). Samples without either the ligand or com-
plexes 1 and 2 were used as the control in appropriate cases.
The reaction mixtures were incubated at 37 °C for 30 min and
the absorbances were then measured at 520 nm. All the tests
were run in triplicate and results are expressed as the mean
standard deviation (SD).38 The scavenging effect for OH• was
calculated from the following expression:
γ-chamber at a dose rate of 69.3 Gy min−1 for a total dose of
3.068 kGy. For plasmid DNA samples the irradiation doses of
20 & 25 Gy were used.
4.5.2. Exposure to gamma-radiation and assessment of
DNA damage
4.5.2.1. Estimation of radiation induced damage in CTDNA by
fluorescence spectroscopy. Emission intensity measurements
were carried out using a Perkin Elmer LS-55 spectroflouri-
meter. The radiation induced CTDNA damage was assessed by
fluorescence spectroscopy using ethidium bromide (EB)
bound CTDNA solution in Tris-HCl/NaCl buffer (pH 7.2). In
this binding experiment, CTDNA solution was irradiated by a
60Co-γ source as described earlier. Thereafter, this irradiated
DNA was allowed to bind with EB, and the emission spectra
were recorded at 591 nm after excitation at 500 nm (with exci-
tation and emission slits 10 nm). This was compared with the
fluorescence of the non-irradiated DNA-EB system under the
identical experimental set-up.
The dose–response relation is obtained from the plot of (I −
Ia)/(I0 − Ia) versus dose, where Ia is the fluorescence intensity of
EB, I0 is the fluorescence intensity of the unirradiated EB-DNA
control and I is the fluorescence intensity of the irradiated
EB-DNA sample.
4.5.2.2. Estimation of the protection from radiation induced
DNA damage by the complexes and the ligand by fluorescence
spectroscopy. CT DNA was pretreated with either the complex 1
or 2 or the ligand and was incubated for 30 min at 37 °C and
then irradiated by a 60Co-γ source at a dose rate of 69.3 Gy
min−1 for 1 hour. These irradiated DNA solutions were allowed
to bind with EB, and then the emission intensity at 591 nm
was monitored. The solutions were excited at 500 nm (with
excitation and emission slits 10 nm).
Scavenging ratio ð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0Þꢀ ꢂ 100%
ð5Þ
where Ai = absorbance in the presence of the test compound;
A0 = absorbance of the blank in the absence of the test com-
pound; Ac = absorbance in the absence of the test compound,
EDTA-Fe(II) and H2O2.
4.6.2. Superoxide radical scavenging experiment. The
superoxide radical scavenging assay was based on the ability of
the complex and the ligand to inhibit purple formazan for-
mation by scavenging the superoxide radicals generated in a
non-enzymatic phenazine methosulfate–nicotinamide adenine
dinucleotide (PMS/NADH)–nitroblue tetrazolium (NBT)
system.39 Each 3 ml reaction mixture contained 20 mM phos-
phate buffer (pH 7.4), 20 mM of NADH, 0.45 mM PMS,
0.15 mM of NBT and various concentrations of either the
ligand, complex 1 or complex 2. The above mixtures were incu-
bated for 5 min at 37 °C, and then absorbance was measured
at 560 nm against an appropriate blank to determine the quan-
tity of formazan generated.
4.5.2.3. Protection from radiation induced plasmid pUC19
DNA damage by different concentrations of complexes and the
ligand against different doses of gamma-radiation. The DNA
damage protective ability of complexes 1 and 2 or the ligand
was monitored by the agarose gel electrophoresis technique
All the tests were run in triplicate and results are expressed
as mean standard deviation (SD). The scavenging effect for
superoxide anion radicals was calculated using eqn (5).
4.6.3. DPPH radical scavenging activity
4.6.3.1. DPPH radical scavenging activity by EPR spectro-
wherein the supercoiled pUC19 DNA (0.5 µg per reaction) solu- scopy. Electron paramagnetic resonance (EPR) measure-
tions preincubated for 30 min with different concentrations of ments were performed at room temperature (298 K) on a Jeol
either complex 1, 2 or the ligand [(0–2 mM) diluted with the JES-FA 200 ESR spectrometer equipped with a Jeol microwave
Tris-HCl buffer to a total volume of 15 µl] were exposed to bridge. The spectroscopic parameters were: frequency 9.44 GHz,
different doses, e.g. 20 and 25 Gy, of gamma radiation. After field sweep 100 mT, microwave power 0.998 mW, and
irradiation, they were mixed with loading buffer containing modulation amplitude 3000 mT and EPR were measured in a
25% bromophenol blue, 30% glycerol (3 µl) and finally loaded Jeol Quartz pyrex EPR tube, no. 193 5D. The stability of freshly
on 0.9% agarose gel. Electrophoresis was carried out at 80 V prepared methanol solution of DPPH was monitored40 for
for 3 h in TAE buffer (Tris, acetic acid, and 1 mM EDTA, pH 7.2). 30 min, and no significant loss of signal was detected.
The gel was stained using a 1.0 µg ml−1 ethidium bromide Stock solutions of complexes 1 and 2 or the ligand and
2846 | Dalton Trans., 2014, 43, 2835–2848
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DPPH were prepared in methanol. In 60 mM DPPH solution,
different concentrations (0–60 mM) of either complexes 1 & 2
or the ligand were added and mixed thoroughly. The EPR
signal was recorded 2 minutes after mixing either the com-
plexes or the ligand in appropriate cases with the DPPH solu-
tion under identical instrumental conditions.
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Acknowledgements
The authors are thankful to UGC-DAE-CSR-KC for financial
support in the form of a major research project to KKM [UGC-
DAE-CSR-KC/CRS/09/RC-03/]. The single crystal X-ray diffract-
ometer, EPR, and NMR spectrometers used in this work have
been funded by the Department of Science and Technology
(DST-FIST, Department of Chemistry, J.U.), New Delhi, India,
and the Circular Dichroism spectrometer has been funded by
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