In vitro binding studies of organotin(IV) complexes of 1,2-bis(1H- benzimidazol-2-yl)ethane-1,2-diol with CT-DNA and nucleotides (5′-GMP and 5′-TMP): Effect of the ancillary ligand on the binding propensity

Article abstract of DOI:10.1016/j.jorganchem.2011.08.007

The chiral benzimidazole ligand, 1,2-Bis(1H-benzimidazol-2-yl)ethane-1,2- diol, L, exhibiting coordination mode with an oxygen atom of alcohol group directed towards the metal ion and another -OH group with different molecular axis directed away from the metal center was utilized as a building block for organotin complexes [C₁₈H₁₉N₄O ₂SnCl], [C₂₈H₂₃N₄O₂SnCl] and [C₅₂H₄₂N₄O₂Sn₂] (1-3). Complexes 1 and 3 exhibit a pentacoordinate geometry while the complex 2 reveals hexacoordinated environment around the Sn(IV) metal ions as evidenced by ¹¹⁹Sn NMR studies. The DNA binding ability of benzimidazole ligand and their organotin(IV) complexes 1-3 were examined by employing different biophysical methods. The absorption titration of the complexes with CT-DNA reveal significant hyperchromic effect together with strong bathochromic shift of 4-5 nm which infer substantial binding of the complexes with CT-DNA. The intrinsic binding constant K_b values of the complexes 1-3 were found to be 2.16 ± 0.04 × 10⁴, 3.47 ± 0.04 × 10⁴ and 4.60 ± 0.04 × 10³ M^-1, respectively, suggesting pronounced binding of complex 2 with DNA double helix. The mechanism of binding of the complexes was further ascertained by the interaction studies of these complexes with nucleotides (5′-GMP and 5′-TMP) using absorption spectroscopy suggesting a clear preference for 5′-GMP binding which was further authenticated by NMR (¹H and ³¹P NMR) studies.

Full text of DOI:10.1016/j.jorganchem.2011.08.007

Journal of Organometallic Chemistry 696 (2011) 3836e3845
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
In vitro binding studies of organotin(IV) complexes of 1,2-bis(1H-benzimidazol-
2-yl)ethane-1,2-diol with CT-DNA and nucleotides (50-GMP and 50-TMP): Effect of
the ancillary ligand on the binding propensity
*
Farukh Arjmand , Fatima Sayeed, Shazia Parveen
Department of Chemistry, Aligarh Muslim University, Aligarh, UP 202002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The chiral benzimidazole ligand, 1,2-Bis(1H-benzimidazol-2-yl)ethane-1,2-diol, L, exhibiting coordina-
tion mode with an oxygen atom of alcohol group directed towards the metal ion and another eOH group
with different molecular axis directed away from the metal center was utilized as a building block for
organotin complexes [C18H19N4O2SnCl], [C28H23N4O2SnCl] and [C52H42N4O2Sn2] (1e3). Complexes 1 and
3 exhibit a pentacoordinate geometry while the complex 2 reveals hexacoordinated environment around
the Sn(IV) metal ions as evidenced by 119Sn NMR studies. The DNA binding ability of benzimidazole
ligand and their organotin(IV) complexes 1e3 were examined by employing different biophysical
methods. The absorption titration of the complexes with CT-DNA reveal significant hyperchromic effect
together with strong bathochromic shift of 4e5 nm which infer substantial binding of the complexes
with CT-DNA. The intrinsic binding constant Kb values of the complexes 1e3 were found to be
Received 11 May 2011
Received in revised form
9 August 2011
Accepted 14 August 2011
Keywords:
Organotin(IV) complexes
DNA binding
50-GMP & 50-TMP
Circular dichroism
2.16 ꢀ 0.04 ꢁ 104, 3.47 ꢀ 0.04 ꢁ 104 and 4.60 ꢀ 0.04 ꢁ 103
M
ꢂ1, respectively, suggesting pronounced
binding of complex 2 with DNA double helix. The mechanism of binding of the complexes was further
ascertained by the interaction studies of these complexes with nucleotides (50-GMP and 50-TMP) using
absorption spectroscopy suggesting a clear preference for 50-GMP binding which was further authenti-
cated by NMR (1H and 31P NMR) studies.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
tested in vitro and in vivo, against murine leukemia cell lines [4].
Recent reports in literature reveal synthesis of many organotin
Spectacular success of cisplatin, cis-diamminedichloroplati-
num(II) [1], an archetypical inorganic drug for treating solid
cancers led to the discovery of many other second generation drugs
such as oxaliplatin, carboplatin, picoplatin, etc., however owing to
the limitations of platinum drugs, such as severe side effects,
intrinsic and extrinsic resistance; and also since cancers are derived
from numerous tissues with new multiple etiologies, therefore,
these compounds cannot be potent (cytotoxic) against different the
phenotypes of tumors [2], thus a resurging need for the developing
new non-platinum metal compounds was realized. In the forefront
on therapeutic regime are organotin compounds which have since
1972 demonstrated their cytotoxic activity (one compound tri-
phenyltin acetate was shown to retard tumor growth in mice while
its chloride derivative did not show any potency) [3]. Since then,
a large number of organotin derivatives have been prepared and
metal complexes and their DNA binding studies [5e8]. Most of the
compounds tested earlier exhibited interesting activity in specific
cancer models, but often lacked activity against broad spectrum of
experimental tumors. Nevertheless, there is possibility for variation
of organic moieties and donor ligands linked to metal which can
result in several organotin with high antitumor activity [9]. The
ligand framework plays a significant role in metal-based pharma-
ceuticals via alteration in the biological properties by modifying
reactivity or substitution inertness [10]. The effects of varying
substituents of organotin(IV) metal ions both aliphatic and
aromatic were studied as it is well known that the biological effects
of organotins depends on both the nature and number of organic
groups bound to Sn(IV) cation.
Besides this, introduction of an element of chirality is an
attractive prospect as it could enhance the pharmacological uptake
of the drug entity by adopting specific conformation and stereo-
selective binding with molecular target DNA [11]. We have been
interested in a chiral tridentate ligand 1,2-Bis(1H-benzimidazol-2-
yl)ethane-1,2-diol, as it is a versatile tridentate facially coordinating
* Corresponding author. Tel.: þ91 5712703893.
E-mail address: farukh_arjmand@yahoo.co.in (F. Arjmand).
0022-328X/$ e see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2011.08.007

F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
3837
ligand and substituted benzimidazoles have shown diverse and
complexes corresponding to
n
(SneC),
n
(SneN),
n
(SneO) and
broad spectrum of activities, antihistamine [12], antiulcerative [13],
anti-inflammatory, analgesic [14], antioxidant [15], antibacterial
[16], antitumor [17], antiproliferative [18], etc. These benzimid-
azoles selectively inhibit endothelial cell growth and suppress
angiogenesis in vitro and in vivo [19]. Since DNA has been identified
as the primary molecular target of metal-based anticancer drugs,
interaction of metal complexes with DNA ascertains the extent and
mode of DNA binding and the potential of these complexes to act as
chemotherapeutic agents. There are various modes of DNA inter-
actions broadly, covalent, non-covalent, intercalation, etc. [20]. DNA
targeted metal-based drugs which involve covalent binding to
nucleobase moieties have shown low degree of selectivity. On the
other hand, non-covalent DNA binding metal complexes are known
to strongly influence DNA binding properties and among them
organotin is the prominent class of compounds exhibiting non-
covalent binding mode [21,22]. To evaluate the effect of ancillary
ligand as well as organic substituents on the binding properties of
organotins, we have incorporated three organotins viz. dimethyltin
dichloride, diphenyltin dichloride and triphenyltin chloride in the
benzimidazole ligand scaffold and studied their DNA propensity by
various biophysical spectroscopic methods (viz. UV titrations,
fluorescence titrations and circular dichroism) and viscosity
measurements. Furthermore, specific mode of binding exhibited by
the complexes was ascertained by nucleotide recognition of these
complexes with 50-GMP and 50-TMP using electronic absorption
and NMR techniques.
n(SneCl) appeared at 557e588, 435e446, 517e546 and
279e256 cmꢂ1, respectively [25e27].
2.2. Nuclear magnetic resonance spectroscopy
The conclusions drawn from the 1H NMR spectrum of free ligand
were extrapolated to the complexes 1e3 owing to the data simi-
larity. In complex 1, the characteristic peak of the methyl groups
attached to the tin is observed at 0.7e1.9 ppm [28]. While in
complexes 2 and 3, the presence of SnePh protons at 7.8 and
7.9 ppm [29] confirms the formation of the complexes. The 1H NMR
spectra of all the complexes revealed a resonance for the CH proton
attached to the hydroxyl group at 5.9e5.8 ppm which was shifted
downfield compared to the free ligand at 5.4 ppm [30] showing the
involvement of adjacent hydroxyl group in complexation with tin
metal ion. The most striking difference in the 1H NMR of complex 3
with the other complexes 1 and 2 was the disappearance of the
hydroxyl protons of ligand at 6.4 ppm concomitant with the
formation of complex in 1:2 stoichiometric ratio [30]. The aromatic
protons of the ligand appear as a multiplet in the range of
7.1e7.5 ppm. In addition, due to rapid exchange between the two
nitrogen atoms of imidazole ring, the NH protons of the ligand were
not observed in the NMR spectra. This observation was in accor-
dance with the earlier reports [31,32].
The 13C NMR spectra of the ligand exhibit signals corresponding
to C]N, CeO at 155 and 68e70 ppm, respectively [30] which were
shifted in the downfield in the complexes. In addition, the complex
1 displays the methyl carbons attached to the tin metal ion
(SneCH3) at 10e14 ppm; the other complexes 2 and 3 exhibited
only one set of NMR signals for both the phenyl groups (SnePh) and
for the ligands from 127 to 136 ppm, which provide evidence for
the magnetic equivalence of both the phenyl groups of the ligands
and the complexes on the NMR time scale [33,34].
In order to provide further evidence to establish the structure of
the complexes in solution [35], 119Sn NMR spectra was recorded
(Fig. 1). It is well known that 119Sn chemical shifts are very sensitive
to change in the coordination number of tin and to the nature of
groups directly attached to the tin atom [36]. The chemical shift
values for the complexes reported here are within the expected
range for the complexes 1 and 2 (ꢂ186 and ꢂ222 ppm, respec-
tively) with the coordination number of five while complex 3
2. Results and discussion
2.1. Infrared spectroscopy
In the IR spectra of the ligands the eOH and eNH frequencies
are present as a broad envelope in the region from 3450 to
3199 cmꢂ1 due to intermolecular hydrogen bonding NH and OH
groups [21,23] which display minor changes on complexation.
However, a medium or relatively weak band at 1623 cmꢂ1 with
a small shoulder at 1536 cmꢂ1 corresponding to
n(C]N) in the IR
spectra of the ligands [24] were considerably shifted by
2e3 cmꢂ1 and become larger and sharper indicating the
involvement of the nitrogen atom in complexation with Sn(IV)
metal ion in all the complexes 1e3. Other frequencies in
Fig. 1. 119Sn NMR spectra of complexes (a) 1 (b) 2 and (c) 3.

3838
F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
exhibits six coordinate geometry displaying a chemical shift value
at ꢂ586 ppm [37].
thereby, causing contraction and conformational changes in the
CT-DNA via electrostatic interaction with the phosphate backbone
of DNA double helix. The spectral features reveal that the complex
2 has much higher affinity towards CT-DNA among the other
complexes which could be attributed to the presence of labile
groups of varying steric demand and hydrophobicity thereby,
facilitating multifaceted binding modes. The hydrophobic phenyl
groups leads to more intimate binding and possibly promoting
partial insertion of the complex 2 into the DNA helix. In addition,
hydrolysis of labile chloride atoms in the complex 2 [41] and hard
Lewis acidic property of Sn(IV) together contribute to the vicinal
electrostatic binding of complex with the polyanionic phosphate
backbone of CT-DNA [42]. Furthermore, the presence of eOH
group in the ligand being out of the planar phenyl rings, as vali-
dated by the crystal structure of the ligand [30] forms hydrogen
bonds with DNA. Although complete intercalation between a set
of adjacent base pairs is sterically prohibited, but some type of
partial intercalation can be envisioned for complex 2. Similarly,
complex 1 also exhibit an electrostatic binding mode pertaining to
presence of Sn(IV) central metal ion which substantiate prefer-
ential binding to the DNA phosphate backbone; the discerning
binding exhibited by complex 1 compared to complex 2 is due to
the absence of phenyl group in the complex. The complex 3 has
steric constraints caused by the bulky phenyl groups of the tri-
phenyltin(IV) moiety which accounts for its lower binding
propensity with DNA. The Kb value calculated revealed that the
comparative binding strength of the complexes with CT-DNA were
in the order 2 > 1 > 3 (Table 1).
2.3. Electronic absorption spectroscopy
The absorption spectra of the complexes 1e3 were recorded in
methanol at room temperature and revealed bands at 207e210, 245,
and 275 nm attributed to intraligand (IL) transitions of the ligands.
3. DNA binding studies
3.1. Absorption spectral studies
The absorption spectra of complexes 1 and 2 in presence and in
the absence of CT-DNA are depicted in Fig. 2 (and for complex 3,
Supplementary Information fig. S1). In absence of CT-DNA, all the
complexes exhibit similar absorption spectra with the peaks
centered at 245 and 275 nm corresponding to ne
p* and at
207e210 nm for * IL transitions, respectively, owing to the
pep
presence of benzimidazole ligand scaffold. In presence of incre-
mental amounts of CT-DNA, a significant hyperchromic effect with
a
strong bathochromic shift was observed for all studied
complexes. Complex 2 displayed a significant bathochromic shift
of 4e5 nm supports a higher degree of binding for this complex
towards CT-DNA [38]. Thus, from these spectral changes, we
deduce that the complexes bind to the CT-DNA by a strong elec-
trostatic interaction mode as a result of a high affinity of cationic
Sn(IV) to the polyanionic phosphate backbone and lack of base
specificity in DNA binding. Literature reveals [39,40] strong Lewis
acid Sn(IV) ions neutralize the negative charge of the CT-DNA
3.2. Interaction studies with 50-GMP and 50-TMP
To avoid the macrostructural effects of larger nucleic acid which
might obscure the fundamental biomolecular selectivity of these
complexes, absorption titration with small nucleic acid fragments
such as mononucleotides (50-GMP and 50-TMP) were performed
under physiological conditions. Gellert et al. [43] demonstrated X-
ray structural studies showing predominant binding of thirty
binary and ternary mononucleotide-transition metal complexes by
first two modes through exocyclic nitrogen atom of purine rings
(N1 and N7 atom) and N3 of the pyrimidine and oxygen atom of the
phosphate group present in the nucleotides. Later, extensive work
on the interaction of organotin(IV) complexes DNA and nucleotides
was also carried out by Yang et al. to elucidate the probable mode of
binding of the Sn(IV) complexes [39,40]. The gradual addition of 50-
GMP/50-TMP (0e0.50 ꢁ 10ꢂ4 M) to the metal complexes 1e3 leads
to significant “hyperchromic effect” and strong perturbations in
absorption peak of complexes as depicted in Figs. 3 and 4 (and for
complex 3, Supplementary Information fig. S2 and S3). As evident
from above absorption titration, both the electrostatic binding as
well as steric effects play an eminent role in the determining the
binding potential of these analogous complexes with DNA, so the
binding of the complexes 1 and 2 towards nucleotides is also gov-
erned by these interactions. The hyperchromic effect in the
absorption spectra of the complexes indicates an electrostatic
outside binding of the complexes with nucleotides owing to the
Table 1
The binding constant (Kb) values of complexes 1, 2 and 3 with the CT-DNA (mean
standard deviation of ꢀ0.04).
Complex
Kb (Mꢂ1
)
% Hyperchromism
Red shift (nm)
1
2
3
2.16 ꢁ 104
3.47 ꢁ 104
4.60 ꢁ 103
32
45
20
06
05
05
Fig. 2. Absorption spectra of complexes (a) 1 and (b) 2 in the presence of increasing
amount of CT-DNA. Inset: Plots of [DNA]/(εaꢂεf) vs [DNA] for the titration of CT-DNA
with complexes. [Complex] ¼ 1.3 ꢁ 10ꢂ4 M, [CT-DNA] ¼ 0e1.2 ꢁ 10ꢂ4 M.

F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
3839
Fig. 3. Absorption spectral traces of complexes (a) 1 and (b) 2 upon addition of 50-GMP.
Inset: Plots of [50-GMP]/εaꢂεf vs [50-GMP] for the titration of 50-GMP with complexes.
[Complex] ¼ 1.3 ꢁ 10ꢂ4 M, [50-GMP] ¼ 0e0.33 ꢁ 10ꢂ4 M.
Fig. 4. Absorption spectral traces of complexes (a) 1 and (b) 2 upon addition of 50-TMP.
Inset: Plots of [50-TMP]/jεaꢂεfj vs [50-TMP] for the titration of 50-TMP with complexes.
[Complex] ¼ 1.3 ꢁ 10ꢂ4 M, [50-TMP] ¼ 0e0.50 ꢁ 10ꢂ4 M.
presence of Sn(IV) moiety which exerts predominate phosphate
binding of the complexes with nucleotide. Moreover, as observed
from the binding constant data (Table 2), the interaction of
complexes 1 and 2 with 50-GMP produces more pronounced
changes in the absorption spectra as compared to 50-TMP showing
a clear preference of binding of the complexes with 50-GMP.
To further understand the selectivity of the complex 2 owing to
its stronger binding with the CT-DNA and to the strong perturba-
tions induced in the absorption spectra when treated with nucle-
otide 50-GMP, we have carried out 1H and 31P NMR interaction of
affirmation of phosphate selectivity of complex with biomolecules
(CT-DNA and 50-GMP).
3.3. Fluorescence studies
In the absence of DNA, complex 1e3 emit weak luminescence in
TriseHCl buffer at ambient temperature, with a fluorescence
maximum centered at 710 nm when excited with a wavelength of
354 nm. Upon increasing DNA from 0 to 0.50 ꢁ 10ꢂ4 M, the
enhancement in the emission intensity of the complexes 1 and 2
was observed as depicted in Fig. 6 (and for complex 3, Supple-
mentary Information fig. S4). This observed enhancement in the
emission intensity results from shielding effect of these complexes
1
the complex with 50-GMP. The H NMR spectrum of the 50-GMP
after addition of the complex 2 shows no significant chemical shift
differences (Fig. 5). The absence of such shift of H8 signal suggests
non-involvement of the nucleobase in complex binding thus,
precluding the base specific interactions with the complex. Slight
shift of free ribose protons of 50-GMP after interaction with complex
2 from 5.8 to 3.8 ppm to 5.7e3.9 ppm indicate the binding of the
0
0
Sn(IV) complex through O2 and O3 atoms of the sugar moiety
owing to its high affinity for the backbone binding. Thus, the 1H
NMR studies indicates an electrostatic binding mode of complex 2
with 50-GMP. Additional evidence for the selective and specific
binding of Sn(IV) complex with the phosphate group of 50-GMP was
Table 2
The comparative binding constant (Kb) values of complexes 1, 2 and 3 with the 50-
GMP and 50-TMP (mean standard deviation of ꢀ0.07).
Complex
Kb(Mꢂ1
)
Monitored
at (nm)
% Hyperchromism
50-GMP
50-TMP
provided by 31P NMR. There was significant upfield shift of the 31
P
3.03 ꢁ 104
4.80 ꢁ 104
0.28 ꢁ 104
0.40 ꢁ 104
1.51 ꢁ 104
0.16 ꢁ 104
246
246
246
36
48
23
signal of free 50-GMP from 3.73 to 2.10 ppm which corresponds to
a strong binding exhibited by the complex 2 to the phosphate group
of the mononucleotide [44]. These observations provide a definitive
1
2
3

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F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
1
Fig. 5a. H NMR spectra of (a) 50-GMP alone and (b) the reaction of complex 2
(2.5 mmol) with 50-GMP (5 mmol) at 25 ꢃC.
as a consequence of their unspecific binding to the low affinity
outer sphere of DNA polyanionic phosphate backbone. However,
another possibility envisioned for such an increase is the effective
shielding of these outer sphere complexes to the metal complexes
intercalated inside the DNA pockets [45]. Generally there is inter-
play between electrostatic and hydrophobic interactions; even in
systems in which intercalation is evident. The degree to which
hydrophobic interactions predominate over electrostatic is likely to
be dictated by structural, geometric, and charge considerations for
the binding molecule. Since, the complex 2 displays structural
variability as compared to 1 and 3, the partial intercalative hydro-
phobic interaction along with electrostatic binding predominates
for the complex. The propensity to CT-DNA of the complexes
follows the order 2 > 1 > 3. The values of the binding constant, K
gave similar trend of the DNA binding propensity as observed in
case of absorption spectral studies (Table 3).
Steady-state emission quenching experiments were performed
by using anionic quencher, [Fe(CN)6]4ꢂ, which has been found to be
able to distinguish between the binding modes of the complex with
DNA [46,47]. Upon addition increasing CT-DNA, the quenching of
the fluorescence intensity was observed for the complexes 1e3. It is
speculated that intercalated chromophores are less accessible to
quenching by quencher due to electrostatic repulsion between the
highly negatively charged DNA and anionic quencher [48] whereas
compounds which are bound at the DNA surface (groove binding or
electrostatic binding) are more accessible and therefore, the
emission from these molecules can be quenched more efficiently.
The fluorescence-quenching plots of DNA-bound quencher by
complexes 1 and 2 are shown in Fig. 7 and for complex 3 in
supplementary information fig. S5, which illustrate that quenching
phenomena of DNA-bound [Fe(CN)6]4ꢂ by complexes 1e3 are in
good agreement with the linear SterneVolmer equation. However,
in presence of DNA the slope of the plot of was remarkably
decreased (Table 4). These results corroborate well with the
31
Fig. 5b. P NMR spectra of (a) 50-GMP and (b) the reaction of complex 2 (2.5 mmol)
with 50-GMP (5 mmol) at 25 ꢃC.
observations of absorption titrations indicating that the complex 2
bind with higher affinity towards DNA as compared to other
complexes 1 and 3 predominantly via, electrostatic interactions.
3.3.1. Effect of ionic strength
In order to validate the probable binding mode between the
molecule and DNA, fluorescence titrations were carried out under
the conditions of increasing ionic strength (0e0.50 ꢁ 10ꢂ4 M)
through added amounts of NaCl. The fluorescence intensity of
complexes 1e3 was appreciably quenched with increasing ionic
strength (Fig. 8 and supplementary information fig. S6) [49,50].
This change in CT-DNA is in favor of the electrostatic binding of
complexes 1 and 2 with CT-DNA. As evident from, the effect of salt
concentration on complex 2-DNA system was more pronounced in
case of complex 2 than the other complexes revealing a greater
potency for DNA binding.
3.3.2. Effect of phosphate group
The fluorescence intensity of complexes 1e3 increases with
increase in the K2HPO4 concentration (Fig. 9 and supplementary
information fig. S7). This observation provides supportive evidence
for electrostatic interaction of the complexes binding preferentially
to the phosphate groupof DNA double helix. The spectra depicts that
the interaction of complex 2 is relatively strong than complexes 1
which is consistent with the absorption spectral studies.

F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
3841
a
2.0
1.5
1.0
0.5
0.02 0.04 0.06 0.08 0.10 0.12 0.14
[Fe(CN)6]4-x10-4
M
b
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.02 0.04 0.06 0.08 0.10 0.12 0.14
[Fe(CN)6]4-x10-4 M
Fig. 7. Emission quenching curves of (a) 1 and (b) 2 in the absence of CT-DNA (-) and
in the presence of CT-DNA (C). [Complex] ¼ 1.3 ꢁ 10ꢂ4 M, [DNA] ¼ 1.0 ꢁ 10ꢂ4 M.
towards the DNA such that effective screening of the negative
charge on the N7 base site as well as phosphate oxygen, simulta-
neously along the phosphate backbone is observed which support
a trans conformational change of the DNA double helical confor-
mation [52]. It has been speculated that the electrostatic compo-
nents of interactions are effective in bringing about such trans
conformational changes. Further transformation of the DNA
structure proceeds by removal of labile groups attached in the
complexes which removes the water from the base sites and
grooves of DNA helix resulting in effective binding of the complex
to DNA. The CD spectrum of DNA shows a significant decrease in
intensity of positive band upon addition of the complex 2 which
reveal a right handed conformational change of the DNA double
helix possibly due to its partial intercalative binding of the complex.
In contrast, the complexes 1 and 3 (Fig. 10) exhibit less pronounced
CD spectral changes in the positive ellipticity band. However, on
addition of the complexes, no appreciable change in the negative
helicity band of CT-DNA was observed for all the complexes. The
Fig. 6. Emission spectra of complexes (a) 1 and (b) 2 in TriseHCl buffer (pH ¼ 7.2) in
the absence and presence of CT-DNA. [Complex]
¼
1.3
ꢁ
10ꢂ4 M,
[DNA] ¼ 0e0.50 ꢁ 10ꢂ4 M. Arrows indicate the change in emission intensity upon
increasing the DNA concentration.
3.4. Circular dichroism
The circular dichroism pattern observed for CT-DNA provide
further and definitive confirmation of the probable mode of CT-
DNA binding of complexes 1e3 which is depicted by the pertur-
bation induced in the DNA morphology upon the binding of
complexes to CT-DNA. The two bands of CT-DNA are a net result of
excitation coupling interaction of bases which depend on the
skewed orientation on the CT-DNA backbone [51]. On incubation of
the complexes with CT-DNA, moderate changes in both the positive
and negative bands of CT-DNA were observed. The multifaceted
binding mode observed for complex 2 promotes its higher binding
Table 3
Table 4
Emission properties of complexes 1, 2 and 3 bound to CT-DNA (mean average
deviation of ꢀ0.02).
Emission quenching of CT-DNA bound [Fe(CN)6]4ꢂ by complexes 1, 2 and 3 (mean
average deviation of ꢀ0.02).
Complex
Emission
(nm)
Excitation
(nm)
Monitored at
(nm)
K(Mꢂ1
)
Complex
Emission
(nm)
Excitation
(nm)
Monitored at
(nm)
Ksv1
Ksv2
1
2
3
442
446
442
354
354
354
710
710
710
2.0 ꢁ 104
4.2 ꢁ 104
1.4 ꢁ 104
1
2
3
442
446
442
354
354
354
710
710
710
2.57 ꢁ 104
1.0 ꢁ 104
3.14 ꢁ 104
1.28 ꢁ 104
5.7 ꢁ 104
1.42 ꢁ 104

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F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
Fig. 9. Effect of increasing concentration of K2HPO4 on the fluorescence intensity of
Fig. 8. Effect of different concentration of NaCl on the fluorescence spectra of
complexes (a) 1 and (b) 2 where [Complex] ¼ 1.3 ꢁ 10ꢂ4 M with [DNA] ¼ 1.0 ꢁ 10ꢂ4 M.
the complexes (a)
1
and (b) 2. [Complex]
¼
1.0
ꢁ
10ꢂ4
M with [CT-
DNA] ¼ 1.20 ꢁ 10ꢂ4 M. Arrow indicate the gradual decrease of emission intensity as
Arrow indicate the gradual decrease of emission intensity as
concentration.
a function of NaCl
a function of K2HPO4 concentration.
observation is in accordance with the above spectroscopic studies
revealing avid binding of complex 2 as compared to complexes 1
and 3.
which would produce bends or kinks in the DNA strand and hence,
diminish its effective length along with its viscosity. Secondly,
when the r ratios were increased from 0.20 to 0.40, rapidly
increasing viscosity was observed which indicates the partial
intercalative binding mode of the complexes and supports an
electrostatic binding mode together with some partial inter-
calative interactions for the complexes 1 and 2 [53]. However, the
higher reduction at r ¼ 0.00e0.20 and the higher enhancement at
r ¼ 0.20e0.40 in the DNA viscosity for complex 2 compared to 1
recommends that former complex reveals a greater DNA binding
potential than the latter. However, in contrast to this observation,
there is a gradual decrease in the viscosity of DNA on addition of
complex 3 supporting only an electrostatic association of the
complex towards CT-DNA. These results obtained from viscosity
studies are consistent with those obtained from above spectro-
scopic studies.
3.5. Viscosity measurements
The relative viscosity of CT-DNA (1.20 ꢁ 10ꢂ4 M) in presence of
varying amounts of complexes 1e3 in the [complex]/[DNA] (r)
ratio of 0.00e0.40 with an interval of 0.1 are shown in Fig. 11.
Complex 2 interacts with the CT-DNA more strongly and deeply
than other complexes 1 and 3, leading to the greater decrease in
viscosity of the DNA with an increasing value of r ¼ 0.00e0.20
followed by a substantial increase in the viscosity of the CT-DNA
from r ¼ 0.20e0.40. Firstly, the initial decrease in the relative
viscosity of CT-DNA indicate non-intercalative interaction
presumably electrostatic interactions of the complexes with DNA

F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
3843
Fig. 10c. CD spectra of (a) CT-DNA alone (b) CT-DNA in presence of metal complex 3 in
TriseHCl buffer at 25 ꢃC. [Complex] ¼ 1.0 ꢁ 10ꢂ4 M, [DNA] ¼ 1.20 ꢁ 10ꢂ4 M.
Fig. 10a. CD spectra of (a) CT-DNA alone (b) CT-DNA in presence of metal complex 1 in
TriseHCl buffer at 25 ꢃC. [Complex] ¼ 1.0 ꢁ 10ꢂ4 M, [DNA] ¼ 1.20 ꢁ 10ꢂ4 M.
in agreement with viscometric studies which supports two prob-
able mode of associations of the complexes 1 and 2 with DNA viz;
electrostatic in addition to some partial intercalative interactions,
while the complex 3 binds only via an electrostatic interaction
with DNA.
4. Conclusions
In this paper, we have attempted to unravel the binding
behavior of the complexes 1e3 with CT-DNA by employing various
spectroscopic and biophysical methods. The results suggest
a multifaceted mode of binding of the complexes 1 and 2 with CT-
DNA i.e. electrostatic binding mode in addition to partial inter-
calative interactions owing to the presence of planar architectures
within the molecule as evidenced by absorption titrations and
fluorescence studies. The complex 2 binds to CT-DNA more
strongly than complexes 1 and 3. Further studies of the complexes
1e3 with mononucleotides 50-GMP and 50-TMP by employing
UVevisible absorption titrations as well as 1H and 31P NMR lends
a strong affirmation of electrostatic interaction of complexes with
biomolecules. The CD spectra reveal higher perturbations in DNA
5. Experimental
5.1. Materials and method
All reagents were of the best commercial grade and were used
without further purification. 1,2-diaminobenzene (Sigma),
L(þ)-
tartaric acid (Merck), dimethyltin(IV)dichloride (Sigma), trime-
thyltin(IV)chloride (Sigma), Tris(hydroxymethyl)aminomethane or
Tris buffer (Sigma) were used as received. Disodium salt of Calf
thymus DNA (CT-DNA), guanosine 50-monophosphate disodium
salt (50-GMP) and thymidine 50-monophosphate disodium salt (50-
TMP) were purchased from Sigma Chemical Co and Fluka, respec-
tively and were stored at 4 ꢃC.
helical conformation for complex
2 as compared to other
complexes 1 and 3 suggestive of its higher binding affinity with
DNA leading to strong conformational changes in the double
helical structure of CT-DNA. The above DNA binding results were
Fig. 10b. CD spectra of (a) CT-DNA alone (b) CT-ꢂD4NA in presence of metal complex 2 in
TriseHCl buffer at 25 ꢃC. [Complex] ¼ 1.0 ꢁ 10 M, [DNA] ¼ 1.20 ꢁ 10ꢂ4 M.
Fig. 11. Effect of increasing amount of complexes (a) 1 (b) 2 and (c) 3 on the relative
viscosity of CT-DNA. [DNA] ¼ 1.20 ꢁ 10ꢂ4 M, [Complex] ¼ 0e1.0 ꢁ 10ꢂ4 M.

3844
F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
5.2. Physical measurements
5.4.1. Synthesis of ligand, [C16H14N4O2] (L)
The ligand was prepared by reaction of 1,2-diaminobenzene
(4.6 g, 40 mmol) and
The elemental analyses were determined using Carlo Erba
Analyzer Model 1108. Molar conductance was measured at room
temperature on a Digsun Electronic conductivity Bridge. Infrared
(IR) spectra were recorded on an Interspec 2020 FTIR spectrometer.
Electronic spectra were recorded on UV-1700 PharmaSpec UVevis
spectrophotometer (Shimadzu). The 1H and 13C NMR spectra were
obtained on a Bruker DRX-400 spectrometer. Optical rotations of
chiral complexes were determined on a Polarimeter Rudolf Autopol
III at 25 ꢃC using the sodium D line in DMSO. ESI-MS spectra were
recorded on Micromass Quattro II triple quadrupole mass
spectrometer.
L
(þ)-tartaric acid (3.0 g, 20 mmol), accord-
ing to the procedure reported earlier [30]. Yield, 65%. m.p.
270 ꢀ 2 ꢃC, Anal. Calc. for [C16H14N4O2] (%): C 65.30; H 4.79; N
19.04: found % C 65.32; H 4.81; N 19.01. ½a D25
¼ þ110, ESI-MS (m/z):
ꢄ
295 [C16H14N4O2].
Selected IR data on KBr (n y(OH); 1623 y(C]N);
/cmꢂ1): 3322
1227 y(CeO); 740 y(Ar). UVevis (MeOH) [lmax/nm]: 206, 247, 278.
1H NMR (DMSO-d6, ppm): 5.49e5.67 (eCH); 6.41(eOH); 7.10e7.52
(Aromatic-H). 13C NMR (DMSO-d6, ppm): 155 (C]N); 115e121
(AreC); 71 (CeO).
5.4.2. Synthesis of [C18H19N4O2SnCl] (1)
To a stirring solution of dimethyltin(IV)dichloride (1.07 g,
5 mmol) in methanol (20 ml) was added the ligand [C16H14N4O2]
(1.47 g, 5 mmol). The reaction mixture was refluxed at 80 ꢃC with
constant stirring on the rota mantle for 4 h and then allowed to
stand at room temperature overnight (25 ꢃC). Slow evaporation of
the resulting mixture afforded white amorphous complex. The
above complex was washed with hexane and dried in vacuo. Yield,
56%. m.p. 210 ꢀ 2 ꢃC. Anal. Calc. for [C18H19N4O2SnCl] (%): C 45.27; H
5.3. DNA binding experiments
DNA binding experiments which include absorption spectral
traces, luminescence and circular dichoric experiments conformed
to the standard methods [54e57] and practices previously adopted
by our laboratory [22,58]. While measuring the absorption spectra
an equal amount of DNA was added to both the compound solution
and the reference solution to eliminate the absorbance of the CT-
DNA itself, and the CD contribution by the CT-DNA and Tris buffer
was subtracted through base line correction.
4.01; N 11.73: found % C 45.30; H 4.1; N 11.76. ½a D25
ꢄ
¼ þ193, ESI-MS
(m/z): 478 [C18H19N4O2Sn]. Molar Conductance, L (1 ꢁ 10ꢂ3 M,
M
MeOH): 59 Uꢂ1 cm2 molꢂ1 (non-electrolyte). Selected IR data (
n
/
cmꢂ1): 3220
(Ar); 557
y
(OH); 3042
y
(NH); 1625
(SneN); 435
y
(C]N); 1222
y
(CeO); 759
(SneCl).
5.4. Syntheses
y
y
(SneC); 470
y
y
(SneO); 256 y
UVevis (MeOH) [lmax/nm]: 210, 245, 275. 1H NMR (DMSO-d6,
ppm): 5.9 (eCH); 6.4 (eOH); 7.1e7.8 (Aromatic-H). 13C NMR
(DMSO-d6, ppm): 154 (C]N); 114e128 (AreC); 70 (CeO); 10e12
(eCH3). 119Sn NMR (DMSO-d6, ppm): ꢂ189.
The synthesis of the ligand was a straight forward Phillips
condensation reaction and the new organotin(IV) complexes 1e3
were prepared by the coordination of ligands to the central metal
ion via nitrogen and oxygen donor atom. Nitrogen atoms partici-
pate through the coordinate linkages, while oxygen atom is
involved in coordination under release of HCl in 1:1 stoichiometric
ratio (Scheme 1). These complexes were stable at room tempera-
ture and soluble in various organic solvents such as methanol,
ethanol and DMSO. Molar conductance values of complexes in
methanol were recorded as 59, 34 and 37 Uꢂ1 cm2 molꢂ1 for the
complexes 1, 2 and 3, respectively, suggesting their non-electrolytic
nature. The geometry of tin was ascertained by 119Sn NMR spectra
which revealed a pentacoordinate geometry for complexes 1 and 3,
while hexacoordinate environment for complex 2.
5.4.3. Synthesis of [C28H23N4O2SnCl] (2)
The complex 2 was prepared from diphenyltin(IV)dichloride
(1.71 g, 5 mmol) and ligand [C16H14N4O2] (1.47 g, 5 mmol) according
to the procedure described above for complex 1. Yield, 64%. m.p.
215 ꢀ 2 ꢃC. Anal. Calc. for [C28H23N4O2SnCl] (%): C 55.89; H 3.85; N
9.31: found % C 55.85; H 3.86; N 9.34. ½a D25
ꢄ
¼ þ120, ESI-MS (m/z):
604 [C28H23N4O2SnClþ2H]. Molar Conductance, L (1 ꢁ 10ꢂ3 M,
M
MeOH): 34 Uꢂ1 cm2 molꢂ1 (non-electrolyte). Selected IR data (
n
/
cmꢂ1): 3208
y
(OH); 3046
y
(NH); 1625 y(C]N); 1226 y(CeO); 747
H
N
OH
H
N
N
N
OH
L
Triphenyltin(IV)chloride
reflux, 2h
Dimethyltin(IV) dichloride
reflux, 2h
reflux, 2h
Diphenyltin(IV)dichloride
Sn
H
N
OH
H
N
H
N
H
N
OH
H
N
O
N
N
N
N
N
N
O
O
N
O
H
Sn
Sn
Sn
H3C
CH3
Cl
Cl
1
2
3
Scheme 1. Schematic representation of the formation of complexes 1, 2 and 3.

F. Arjmand et al. / Journal of Organometallic Chemistry 696 (2011) 3836e3845
3845
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y
(Ar); 588 y(SneC); 472 y(SneN); 435 y(SneO); 263 y(SneCl).
UVevis (MeOH) [lmax/nm]: 207, 244, 274. 1H NMR (DMSO-d6,
ppm): 5.5e5.4 (eCH); 6.6 (eOH); 7.1e7.9 (Aromatic-H). 13C NMR
(DMSO-d6, ppm): 154 (C]N); 114e136 (AreC); 69 (CeO). 119Sn
NMR (DMSO-d6, ppm): ꢂ586.
5.4.4. Synthesis of [C52H42N4O2Sn2] (3)
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The complex 3 was prepared from triphenyltin(IV)chloride
(1.92 g, 5 mmol) and ligand [C16H14N4O2] (1.47 g, 5 mmol)
according to the procedure described above. Yield, 69%.
m.p. > 300 ꢃC (decompose). Anal. Calc. for [C52H42N4O2Sn2] (%): C
62.94; H 4.27; N 5.65: found % C 62.86; H 4.22; N 5.66. ½a D25
¼ þ90,
ꢄ
ESI-MS (m/z): 1009 [C52H42N4O2Sn2 þ 0.5CH3OH]. Molar Conduc-
tance, LM (1 ꢁ10ꢂ3 M, MeOH): 37
U
ꢂ1 cm2 molꢂ1 (non-electrolyte).
(C]N); 3062 (NH); 1222 (CeO);
(SneN); 446 (SneO). UVevis (MeOH)
Selected IR data (
n
/cmꢂ1): 1621
y
y
y
735
y
(Ar); 561
y
(SneC); 452
y
y
[
lmax/nm]: 207, 245, 275. 1H NMR (DMSO-d6, ppm): 5.5e5.8 (eCH);
7.1e7.9 (Aromatic-H). 13C NMR (DMSO-d6, ppm): 155 (C]N);
114e136 (AreC); 70 (CeO). 119Sn NMR (DMSO-d6, ppm): ꢂ222.
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The authors are highly indebted to the Regional Sophisticated
Instrumentation Center (RSIC), Central Drug Research Institute,
Lucknow, India for providing CHN analysis data, ESI-Mass and
polarimetry, Sophisticated Analytical Instrumentation Facility
(SAIF), Panjab University, Chandigarh for providing the NMR spectra.
Thanks are also due to Dr. Rizwan H. Khan, Interdisciplinary
Biotechnology Unit, AMU, Aligarh for providing the CD facility.
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