Electrochemical and spectroscopic investigations of carboxylic acid ligand and its triorganotin complexes for their binding with ds.DNA: In vitro biological studies

Article abstract of DOI:10.1016/j.jphotobiol.2013.05.005

A carboxylic acid ligand, (Z)-4-(4-acetylphenylamino)-4-oxobut-2-enoic acid (APA-1), and its triphenyl-(APA-2) and tributyl-tin(IV) (APA-3) compounds have been synthesized and investigated for their binding with ds.DNA using UV-visible spectroscopy, fluor

Full text of DOI:10.1016/j.jphotobiol.2013.05.005

Accepted Manuscript
Electrochemical and spectroscopic investigations of carboxylic acid ligand and
its triorganotin complexes for their binding with ds.DNA: In vitro biological
studies
Nasima Arshad, Shahid Iqbal Farooqi, Moazzam H. Bhatti, Samreen Saleem,
Bushra Mirza
PII:
S1011-1344(13)00105-X
DOI:
http://dx.doi.org/10.1016/j.jphotobiol.2013.05.005
JPB 9496
Reference:
To appear in:
Journal of Photochemistry and Photobiology B: Bi‐
ology
Received Date:
Revised Date:
Accepted Date:
20 February 2013
8 May 2013
10 May 2013
Please cite this article as: N. Arshad, S.I. Farooqi, M.H. Bhatti, S. Saleem, B. Mirza, Electrochemical and
spectroscopic investigations of carboxylic acid ligand and its triorganotin complexes for their binding with ds.DNA:
In vitro biological studies, Journal of Photochemistry and Photobiology B: Biology (2013), doi: http://dx.doi.org/
1
0.1016/j.jphotobiol.2013.05.005
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Electrochemical and spectroscopic investigations of carboxylic acid ligand and
its triorganotin complexes for their binding with ds.DNA: In vitro biological
studies
a,*
a
a
b
b
Nasima Arshad , Shahid Iqbal Farooqi , Moazzam H. Bhatti , Samreen Saleem Bushra Mirza
,
a
Department of Chemistry, Allama Iqbal Open University, Islamabad, Pakistan
b
Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan
a,*
Corresponding author. Tel.: +92 51 9057756; fax: +92 51 9250081.
E-mail address: nasimaa2006@yahoo.com (Nasima Arshad)
A B S T R A C T
A carboxylic acid ligand, (Z)-4-(4-acetylphenylamino)-4-oxobut-2-enoic acid (APA-1), and its
triphenyl- (APA-2) and tributyl-tin(IV) (APA-3) compounds have been synthesized and
investigated for their binding with ds.DNA using UV-visible spectroscopy, fluorescence
spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of
pH and temperature. The experimental results from all techniques i.e. binding constant (Kb),
binding site size (n) and free energy change (ΔG) were in good agreement and inferred
spontaneous compound–DNA complexes formation via intercalation. Among all the compounds
APA-3 showed comparatively greater binding at pH 4.7 as evident from its greater K values
b
4
−1
4
−1
4
−1
{
APA-3: K : 5.63×10 M (UV); 7.94×10 M (fluorescence); 9.91×10 M (CV)}.
b
Electrochemical processes of compounds before and after the addition of DNA were found
diffusion controlled. Among all compounds, APA-3 exhibited best antitumor activity.
Key words: Triorganotin(IV) carboxylates, UV-visible / fluorescence spectroscopy, cyclic
voltammerty, intercalation, binding constant / site size, bioactivities
1

1
. Introduction
A vital role of DNA in life processes is to direct all the cell’s activities including genetic
information related to cellular function, DNA replication and gene expression. However, it can
easily be damaged under some conditions [1]. DNA damage is the fundamental cause of
mutations leading to cancer. Its control is one of the most important and big issue to be resolved.
Numerous researches have been carried out to discover most effective anticancer agents and to
utilize them as drug candidates. Recent trends to control or inhibit nucleic acid damage mainly
emphasis on the interaction of DNA with other molecules; especially certain classes of drugs and
metal complexes. Chemotherapeutic anti-cancerous treatment involves the inhibition of the rapid
cell division by targeting DNA through covalent or non-covalent interactional modes [2]. Among
both interactional modes, reversible types non-covalent binding is preferred over covalent adduct
formation due to less chance of toxic side effects of the drug candidate [3]. Normally, three types
of non-covalent binding modes are considered in DNA binding studies: (i) electrostatic
interaction with the phosphate ions of DNA backbone, (ii) groove binding and (iii) intercalation
of the compound into the stacked base pairs of DNA. Spectroscopic and electrochemical
methods have proven to be more authentic and frequently employed methods to anticipate the
mode of interaction of small molecules as well as metal complexes via non covalent binding
[
4−7].
Among metal complexes, organotins have gained much attention due to their high
therapeutic and biomedical applications and have extensively been studied for their interaction
with DNA in order to develop new and effective candidates as anticancer drug [8−11]. Organotin
compounds containing a carboxylic group are considered to have potential biocidal and cytotoxic
activities [12, 13]. However organotin carboxylates are certainly very promising for cancer
2

chemotherapy [14, 15]. They have extensively been studied and screened in vitro and vivo for
antitumor activity [12, 16−18]. As a result divinyl, di-n-butyl, tri-n-butyl, and triphenyltin
carboxylates are found to be more active than cis-platin in vitro against different human cell lines
[
19]. Generally, triorganotin(IV) carboxylates have shown much higher biological activities
when compare to their mono- and di-organotin(IV) analogues due to high binding ability to
proteins [20].
Hence due to tremendous importance of organotin carboxylates, present research has
been focused to investigate binding interactions of (Z)-4-(4-acetylphenylamino)-4-oxobut-2-
enoic acid (APA-1) and its triphenyl- and tributyl-tin(IV) compounds {(triphenylstannyl (Z)-4-
(4-acetylphenylamino)-4-oxobut-2-enoate (APA-2), tri-n-butylstannyl (Z)-4-(4-acetylphenyl-
amino)-4-oxobut-2-enoate (APA-3)} with ds.DNA using UV-visible spectrometry, fluorescence
spectroscopy, cyclic voltammetry, and viscosity measurements. These compounds were also
investigated for their antioxidant and antitumor potential by using different bioassays.
2
. Experimental
2
.1. Materials
All reagents were procured from Aldrich / Fluka and used without further purification.
All the solvents were dried before use by the literature methods. The ligand (APA-1) and its two
complexes APA-2 and APA-3 were prepared as given in scheme 1. ds.DNA was extracted in the
laboratory from chicken blood by the Falcon method [21] and its concentration was determined
−
1
−1
spectrophotometrically at 260 nm using molar extinction coefficient, ε260 = 6600 cm M [22].
Purity of DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280
nm and solution gave a ratio of A /A > 1.8, indicating that DNA was sufficiently pure and
2
60 280
3

free from protein [23]. The stock solutions of APA-1, APA-2, and APA-3 were prepared by
dissolving them in buffer solution of pH 4.7 (acetate buffer; CH COOH + CH COONa) and pH
3
3
7
.4 (phosphate buffer; Na HPO4 + NaH PO ). Autoclaved water was used to prepare all the
2 2 4
solutions.
2
.2. Instrumentation
The melting points were determined in capillary tubes using a MPD Mitamura Riken
Kogyo (Japan) Electro thermal melting point apparatus and were uncorrected. FTIR spectra

1
(
KBr) were recorded on Nicolet iS 10 FTIR spectrophotometer in the range 4000 − 400 cm .
1
13
The H and C NMR spectra were recorded on a Bruker Avance 400 spectrometer operating at
00 MHz. The electronic absorption spectra were recorded on Shimadzu 1800 spectrophotometer
TCC-240A, Japan) equipped with temperature control device using 1.0 cm matched quartz cells.
4
(
Fluorescent emission spectra were recorded on F-7000 FL spectrophotometer 2133-007. Cyclic
voltammetric experiments were performed using AUTOLAB PGSTAT–302 with GPES version
4
.9 (Eco Chemie, Utrecht, Netherlands).
Electrochemical measurements were carried out in a dried conventional three electrode
cell using a glassy carbon (GCE; d = 3 mm) working electrode, a saturated calomel (SCE; 3.5 M
KCl) reference electrode and a Pt sheet counter electrode. Prior to each experiment the GCE was
polished with alumina powder and rinsed thoroughly with doubly distilled water for 30 seconds.
2
2
.3. Preparation of compounds
.3.1. Synthesis of Ligand acid (APA-1)
The ligand acid was obtained from the reaction of maleic anhydride with p-
aminoacetophonene in ethyl acetate as shown in Scheme 1. A solution of maleic anhydride (1.0
g, 10 mmol) in 50 ml ethyl acetate was added to a solution of p-aminoacetophonene (1.35 g, 10
4

mmol) in 50 ml ethyl acetate in a 250 ml conical flask. The mixture was stirred for three hours at
room temperature. After stirring the precipitates of ligand acid were filtered and recrystallized
with ethanol.
Scheme 1
2
.3.2. Synthesis of of APA-2 and APA-3
The organotin derivatives APA-2 and APA-3 were obtained from the reaction of Ph SnOH /
3
(
Bu Sn) O with ligand acid in 1:1 and 1:2 molar ratio, respectively. All the newly synthesized
3 2
compounds are air stable, crystalline solids and soluble in common organic solvents. The general
chemical reaction is given as Scheme 2.
For the preparation of APA-2, equimolar amounts of ligand acid (2.33 g, 10 mmol) and
triphenyltin hydroxide (3.17 g 10.mmol), while for the preparation of APA-3 molar amounts
(2:1) of ligand acid (2.33 g, 10 mmol) and bis(tributyltin)oxide (3.17 g 10.mmol) respectively
were suspended in 100 ml of dry ethanol / acetone (8:2) solvent mixture and refluxed for 8
hours,. After cooling to room temperature, reaction mixture was filtered and solvents were
evaporated in rotary evaporator. The solid obtained was recrystallized from chloroform with few
drops of n-hexane.
Scheme 2
2
.3.3. Characterization data
-1
Physical data APA-1: Yield 78 %, m.p.: 210-212 °C. FTIR data (KBr, cm ):  (OH) 3300-
2
900,  (NHCO) 1709,  (CH CO) 1674, 
(COO) 1631, sym (COO) 1362,  = 269
3
asym
1
3
1
1
H NMR data (DMSO, ppm J( H, H) in Hz): 12.9 (b, 1H, CONH), 10.6 (s, 1H, COOH), 7.92
(d, 2H, (8.0), aromatic protons), 7.73 (d, 2H, (8.0), aromatic protons), 6.47 (d, 1H, (12), ethylene
proton), 6.31 (d, 1H, (12), ethylene proton), 2.52 (s, 3H, CH CO)
3
5

1
3
C NMR data (DMSO, ppm): 166.9 (C1), 130.3 (C2), 131.5 (C3), 196.5 (C4), 143 (C5), 129.5
(C6), 118.6 (C7), 132.0 (C8), 163.6 (C9), 26.4 (C10)
-1
Physical data APA-2: Yield 67 %, m.p.: 180-182 °C. FTIR data (KBr, cm ):  (CH CO) 1675,
3

(NHCO) 1620, asym (COO) 1590, sym (COO) 1372,  = 218,  (Sn-O) 452
1
3
1
1
H NMR data (CDCl , ppm J( H, H) in Hz): 11.5 (b, 1H, CONH), 7.88 (d, 2H, (8.4), aromatic
3
protons), 7.55 (d, 2H, (8.4), aromatic protons), 7.74-7.70 & 7.50-7.45 (m, 15H, SnPh), 6.36 (d,
1
H, (13.4), ethylene proton), 6.28 (d, 1H, (13.4), ethylene proton), 2.57 (s, 3H, CH CO)
3
1
3
n
119
13
C NMR data (CDCl , ppm, J[ Sn, C] in Hz): 171.9 (C1), 129.2 (C2), 129.6 (C3), 197.0
3
(C4), 142.5 (C5), 127.5 (C6), 119.2 (C7), 132.9 (C8), 162.3 (C9), 26.4 (C10), 138.2 [661] (C),
1
36.8 [46.8] (C), 130.7 [66.4] (C), 132.9 (C)
-
1
Physical data APA-3: Yield 62 %, m.p.: 78-80 °C. FTIR data (KBr, cm ):  (CH CO) 1678, 
3
(NHCO) 1612, asym (COO) 1592, sym (COO) 1368,  = 224,  (Sn-C) 582,  (Sn-O) 450
1
3
1
1
H NMR data (CDCl , ppm J( H, H) in Hz): 12.3 (b, 1H, CONH), 7.90 (d, 2H, (8.4), aromatic
3
protons), 7.71 (d, 2H, (8.2), aromatic protons), 6.28 (d, 1H, (13.6), ethylene proton), 6.23 (d, 1H,
(
13.2), ethylene proton), 2.55 (s, 3H, CH CO), 1.64-1.59 & 1.35-1.29 (m, 18H, SnCH CH CH ),
3 2 2 2
0
.88 (t, 9H, SnCH CH CH CH )
2 2 2 3
1
3
n
117/119
13
C NMR data (CDCl , ppm, J[
Sn, C] in Hz): 171.5 (C1), 129.0 (C2), 129.6 (C3), 197.0
3
(C4), 143.0 (C5), 132.8 (C6), 119.2 (C7), 137.6 (C8), 162.6 (C9), 26.4 (C10), 17.1 [333/349]
(C), 27.7 [21.2] (C), 27.0 [65.4] (C), 13.6 (C)
2
2
.4. Procedure for analysis
.4.1. UV-visible and fluorescence spectroscopic titrations
6

Concentration of DNA determined spectrophotometrically (UV-spectroscopy) at 260 nm
−
4
was found 1.8 × 10 M. Spectroscopic titrations were carried out at stomach pH (4.7) and blood
7.4) under body temperature (37 °C). The absorbance measurements by UV-visible
spectrophotometer and the fluorescence emission spectra by fluorescence spectrophotometer
(
−
5
were recorded by keeping the concentration of APA-1, APA-2 and APA-3 constant (2.8 × 10
M) in the sample cell, while varrying the concentration of ds.DNA from 10 µM to 60 µM in the
sample cell. In order to achive the equilibrium between the compound and DNA, solutions were
allowed to stay for at least 5 minutes before each measurements were made. After placing the
sample solutions within the cell cavity and before runnig the spectra, wait for a few seconds till
required temperature was attained and shown on temperature controlled device.
2
.4.2. Voltammetric assay
First a blank CV was run with the buffer solutions (4.7 and 7.4) at 37 °C, which showed
no electroactivity in the potential range of our interest (−2 V to +2 V). Cyclic voltammograms of
−
5
APA-1, APA-2 and APA-3 (2.8 × 10 M) were recorded from −2.0 V to +1.0V vs. SCE before
and after the addition of different volumes (μl) of the stock DNA solution corresponding to the
final concentration of DNA ranging from 10 μM – 60 μM within the cell. Scan rate of 100 mV/s
was used throughout the experiments. All measurements were made at 37 °C after purging the
solution in the cell with argon gas (99.999%) for at least 10 to 15 min for flushing out oxygen
before every electrochemical assay.
2
.4.3. Viscosity measurements
Initially viscosity of DNA solution (η ) was determined at stomach (4.7) and blood (7.4)
o
pH under physiological temperature (37 °C). Then specific viscosity contribution (η), due to
DNA (10 µM) in the presence of increasing concentration of investigated compound was
7

1
/3
determined. The values of the relative specific viscosities for the compounds i.e. (η / ηo) were
then plotted against the ratio; [compound] / [DNA].
2
.4.4. DPPH free radical scavenging assay
DPPH free radical scavenging activity of test compounds were carried out by already
reported methods [24, 25]. Compounds were examined at four concentrations 7.4, 22.2, 66.6 and
−
1
2
00 µg ml as final concentrations from stock. Reaction mixture was prepared by adding 0.1 ml
of each test compound solution in DMSO, 2 ml of 0.1 mM DPPH in ethanol solution and 0.9 ml
of 50 mM Tris-HCl in capped vials. DMSO was used as negative and ascorbic acid was used as
positive control, respectively. Reaction mixture was incubated in dark for 30 minutes at room
temperature. After incubation, change in DPPH color was observed by spectrophotometric
absorbance at 517 nm. Mixture of all solvents utilized in the assay was used as blank for the
spectrophotometer. Percent scavenging of DPPH free radical for each concentration of each
compound was calculated.
2
.4.5. Potato disc antitumor assay
Potato disc antitumor assay with some modifications was performed to detect the tumor
inhibition activity of compounds under investigation [5, 24, 25]. In this assay 48 hour old single
colony culture of Agrobacterium tumefaciens (At-10) strain was used as tumor inducing agent on
potato discs. Each test sample was evaluated for antitumor activity at four concentrations i.e.,
−
1
7
.4, 22.2, 66.6, and 200 µg ml with DMSO as negative controls and vincristine as standard
drug. Under complete aseptic conditions, potato discs (0.5 cm thickness) were made by using
sterilized instruments from surface sterilized (HgCl 0.1%) healthy potatoes tubers. Fifteen
2
potato discs were transferred on each petriplate containing 1.5% agar–agar in distilled water.
After treatment with test compounds and At-10 strain on each disc, these petriplates were then
8

incubated at 28°C for 21 days. Number of tumors was counted after staining with the Lugol’s
solution (10% KI and 5% I ) with the help of dissecting microscope. Percentage tumor inhibition
2
was calculated. Each experiment was carried out in triplicate and IC values for each compound
5
0
were calculated.
3
. Results and discussion
3
.1. DNA binding study by UV-spectroscopy
UV-visible spectrophotometry is the most significant technique to study the binding
mode of small molecules as well as metal complexes [26]. The comparison of UV-visible spectra
of compounds in the absence and presence of DNA provides an efficient tool for monitoring and
recognition of their interaction and yield information about compound−DNA binding mechanism
and complex formation [26]. Three compounds APA-1, APA-2and APA-3 have been analyzed
spectrophotometrically to get an insight into their interactions with ds-DNA at physiological
conditions of pH and temperature.
At first, UV-spectrum of APA-1, APA-2, APA-3 all having same concentration (2.8 ×
−
5
1
0 M) were recorded separately in the complete UV-visible range (200 – 800 nm), Fig. S1 (in
supplementary information). All solutions were prepared in ethanol-water mixture (7:3). A single
peak appears at λmax of 307.2 nm, 304.4 nm and 314.8 nm respectively for APA-1, APA-2 and
APA-3 compounds respectively. The molar extinction coefficient (ε) values were evaluated
−1
−1
−1 −1
−1 −1
1
1175 cm M , 16292 cm M and 10300 cm M respectively for APA-1, APA-2 and APA-3
*
and predicted that π-π transitions are operative for all compounds in the λ range 240 – 390
max
nm, Figs. S2 and S3 (concentration profile; in supplementary information). Absorption spectrum
of pure chicken blood ds-DNA recorded in double deionized distilled water showed a broad band
9

(200 – 350 nm) in the UV-region with a maximum placed at 260 nm. The observed maximum is
the consequence of the chromophoric groups in purine and pyrimidine moieties. The absorbance
ratio (A260 /A ) was 1.83, which ensured the purity of DNA [23].
280
Addition of varying concentration of DNA (10 µM − 60 µM) on fixed (optimized)
−
5
concentration (2.8 × 10 M) of APA-1, APA-2 and APA-3 caused progressive blue shift of
magnitude 3.0 nm, 4.2 nm and 8 nm respectively at pH 4.7 and 1.2 nm, 3.0 nm and 4.9 nm
respectively at pH 7.4 in the electronic absorption spectra along with the decrease in peak
absorbance intensity (hypochromic effect) at both pH, Fig. 1. Such changes in the spectral
responses are indicative of binding of investigated compounds with DNA [8]. Hypochromism in
absoption spectra after the addition of DNA is suggestive of intercalation of these compounds
into the DNA base pairs and this may probably be due to the interaction of the electronic states
of the intercalating chromophore of the compounds and those of the stacked base pairs of ds-
DNA via overlapping [27, 28]. On the other hand, the coupling π-orbital is partially filled by
electrons, thus decreasing transition probabilities and concomitantly resulting in hypochromism
[
27, 28].
Furthermore, upon titration with DNA the spectral changes at both the pH are characterized by
one isosbestic point, Fig. 1, so presence of species other than the free and the intercalated
complexes could be ruled out [18]. Isosbestic point indicates that there is equilibrium between
bound DNA and the free form of the compounds and further suggested only one mode of binding
i.e., intercalation of compounds with DNA [29].
Fig. 1
3
.2. DNA binding study by fluorescence-spectroscopy
1
0

Fluorescence-spectroscopy is one of the important technique to probe compound-DNA
binding [5, 30]. The intrinsic fluorescence emission spectra of APA-1, APA-2 and APA-3 were
recorded separately and all the compounds were found luminescent, Fig. S4 (in supplementary
information). Hence binding studies were carried out by direct addition of different
concentrations of DNA.
The intrinsic fluorescence emission spectra of all the compounds were recorded
separately before and after the addition of varying concentrations of DNA and the effect on the
fluorescence emission spectra of compounds; APA-1, APA-2 and APA-3 is given in Fig. 2.
Upon addition of DNA (10 µM – 60 µM), the growth of emission intensities of APA-1, APA-2
and APA-3 is recorded larger than those in the absence of DNA as 2.07, 2.55 and 4.67 times
respectively at pH 4.7 and 1.68, 1.85 and 1.98 times respectively at pH 7.4. Increase in the
emission intensity after the addition of DNA indicated that all compounds interacted with DNA
through intercalative mode of interaction [30, 31]. In the case of hydrophobic intercalation,
compound being packed into the base stack of the DNA–helix, will favor deactivation or de-
excitation via fluorescence emission instead of radiation less deactivation which is prominent in
the free rotating molecules [26]. Hence a significant increase in the fluorescence emission
intensity of compound will be observed. This phenomenon directly infers that the interaction of
drug to DNA has resultantly increased the quantum efficiency of interacting compound [26]. The
enhancement in the fluorescence intensities may further be attributed to the fact that all the
compounds are protected from the water molecules by the hydrophobic microenvironment of
nitrogenous bases inside the DNA-helix on one hand and their mobility is restricted at the
binding sites on the other hand. Resultantly, this masking of the compound fluorophore along
with snaring at binding sites, upon interaction between the stacked bases within the helix or
1
1

surface binding at the reactive nucleophilic sites on the heterocyclic nitrogenous base of DNA
molecule reduce the vibrational mode of relaxation after excitation and then the compound
binding to DNA leads to marked increase in fluorescence emission intensity [32].
Fig. 2
3
.3. DNA binding study by cyclic voltammetry
Cyclic voltammetry is one of the important and most popular electrochemical techniques
for DNA binding studies with a fact that compound bound to DNA is redox active [4, 33].
Electrochemical behavior of three compounds APA-1, APA-2 and APA-3 were investigated
using cyclic voltammetric technique in ethanol-water mixture (7:3) at scan rate of 100 mV/s and
at glassy carbon electrode surface. All compounds showed irreversible reduction processes and a
single peak was observed at reduction potential (E ) of −0.897V, −0.926V, −0.874V,
Pc
respectively at pH 4.7 and −0.963V, −0.977V, −0.95V, respectively at pH 7.4. Peak broadening
was observed for all the compounds at both pH, which may be attributed to one step two
electrons reduction process [33].
Cyclic Voltammograms were recorded after the addition of 10 μM to 60 μM DNA on
−
5
optimized concentration (2.8 × 10 M) of APA-1, APA-2 and APA-3 at both pH values.
Voltammetric responses have shown a decrease in peak height along with a shift of reduction
peak towards more negative potential, Fig. 3. This behavior is suggestive of interaction of
investigated compounds with DNA via intercalation [4, 33]. The voltammetic parameters
obtained for the three compounds without and in the presence of DNA are given in Table 1.
Fig. 3 and Table 1
Randles-Sevick equation is used to determine the diffusion coefficient values of APA-1,
APA-2 and APA-3 without and in the presence of DNA [34, 35].
1
2

5
1/2
*
o
1/2 1/2
I = 2.99 x 10 n(αn ) AC D
o

(1)
p
α
where, I is peak current in amperes (A), n is charge transfer number, n is number of electrons
p
α
transferred up to and including the rate determining step, α is transfer coefficient, A is surface
2
*
o
−3
area of the electrode (cm ), C is bulk concentration of the electro active species (mol cm ), υ is
−
1
2
−1
potential scan rate in Vs and D is diffusion coefficient (cm s ).
o
1/2
Ip values were plotted νs. υ for all compounds without and in the presence of DNA
which showed linear dependency of peak currents on the square root of scan rates, Fig. 4 (only
shown for all the compounds at pH 4.7, and at pH 7.4 provided as Fig. S5 in supplementary
information). The linearity of plots verified that main mass transport of these electro active
species and their DNA bound complexes is diffusion controlled [36]. A negative shift in the peak
potential along with increase in peak current by increasing the scan rate may further be
attributed to irreversible nature of redox process [37] occurring in compounds with and without
the addition of DNA, Figs. S6a & S6b (in supplementary information). Transfer coefficient (α)
was calculated using equation; E −E = 47.7 mV/αn. For all the compounds its value was
p
p/2
found lesser than 0.5 which suggests that redox reaction of compounds is irreversible process
and two electrons are involved in the one step reduction process [38].
The diffusion coefficients of compounds APA-1, APA-2 and APA-3 before and after the
addition of DNA were calculated and given in Table 2. The lower diffusion coefficients (Do)
values of DNA bound organotin carboxylates are responsible for the decay of peak current in
cyclic voltammograms, Fig. 4.
Fig. 4 and Table 2
1
3

3
.4. Evaluation of binding constants of compounds to DNA by UV-spectroscopy
Binding constant “K ” of compound–DNA complex can be determined from the variation
b
in absorbance in UV-visible spectra before and after the addition of DNA. Binding constants for
the three compounds were evaluated spectrophotometrically under physiological conditions of
pH (4.7 and 7.4) and temperature (37 °C) by applying Benesi-Hildebrand equation [4, 5, 39].
Ao
A A 
G
G
1
DNA


(2)
the
G H G  K


o
H G
G
b
Where A and A are the absorbance’s of compound and complex respectively, E and E
H-G
o
G
molar extinction coefficients of compound and complex respectively. From the plot of A /(A-
o
A ) to 1/[DNA], the ratio of the intercept to the slope gave the values of binding constant, K ,
o
b
Fig. 5.
The Binding constant values were calculated and given in Table 3. The order of
4
−1
magnitude of binding constant K at both the pH values (10 M ) for all the investigated
b
compounds with DNA revealed their stronger binding via intercalation and is in agreement with
4
−1
that reported for typical intercalator lumazine ( K = 1.74 × 10 M ) and anthracycline
4
5
−1
molecules (K ≈ 10 − 10 M ) [39, 40]. Binding constants at stomach pH (4.7) for all the
compounds were evaluated greater, while at pH 7.4 compound APA-3 showed comparatively
greater binding constants than that of typical intercalator lumazine, Table 3. Further, the spectral
changes in absorption spectra during compound–DNA complex formation in present work
inferred intercalation as possible mode of interaction. In addition, the greater binding constants
of investigated compounds at both the pH values may be attributed to their structural planarity
due to phenyl groups in the investigated compounds.
1
4

Binding constants values are used to assess of stability of compound–DNA complexes. In
present studies all the compounds showed greater values of binding constants and revealed the
formation of their stable complexes with the DNA at both the pH. Among the two pH (stomach;
4
.7 and blood; 7.4), however, all compounds bound to DNA more strongly at stomach pH as
evident from comparatively greater K values at this pH (4.7), Table 3. Conversely, APA-3
b
showed relatively greater K value at both pH than that of APA-1 and APA-2. The strong affinity
b
of APA-3 for DNA as indicated by its greater binding constant value as compared to APA-2
could be attributed to additional hydrophobic interactions of the bulky butyl moiety with the
nucleotide bases of DNA [33]. Furthermore, the greater K value of APA-2 as compared to APA-
b
1
is ascribed to the presence of extended aromatic system which may bind strongly with DNA
[
33]. The order of binding constants at pH 4.7 and pH 7.4 are given below.
KAPA-3 > KAPA-2 > K APA-1
(At both stomach 4.7 and blood 7.4 pH)
and
KAPA-3 (stomach; 4.7 pH) > KAPA-3 (blood; 7.4 pH)
Using the data of binding constant, K , Gibbs free energy changes (ΔG) of compounds–
b
DNA complexes were calculated from Eq. 3. Gibbs free energy change indicates the
spontaneity/non- spontaneity of compound–DNA binding.
−1
ΔG = −RTlnK (kJ mol )
(3)
b
Free energies of all the compounds at both pH were evaluated as negative values showing
the spontaneity of compounds–DNA interaction, Table 3 [4, 5, 41]. However, results indicated
that APA-3 bind to the DNA more spontaneously as compared to other compounds at both pH
values. The order of ΔG values of the compounds–DNA complexes at both pH is evaluated same
as for binding constant values i.e.
1
5

ΔGAPA-3 > ΔGAPA-2 > ΔG APA-1
(At both stomach 4.7 and blood 7.4 pH)
and
ΔGAPA-3 (stomach; 4.7 pH) > ΔGAPA-3 (blood; 7.4 pH)
Fig. 5 and Table 3
3
.5. Evaluation of binding constants and binding sites of compounds to DNA by fluorescence-
spectroscopy
Since fluorescence emission intensity is varied after addition of DNA, the binding
constant “K ” of pro-drug–DNA complex can be determined from the variation in fluorescence
b
emission intensity spectra. Binding constant “K ” and binding stoichiometry have been evaluated
b
spectrophotometrically by using following equation (8) [30].
F  Fo
F
log
 log K  nlog

DNA

(4)
b
where, F and F are the fluorescence intensities of the fluorophore in the absence and in the
o
presence of different concentrations of DNA, respectively. K and n are the binding constant and
b
binding site size (binding stoichiometry) respectively which were determined by plotting log [(F-
Fo)/F] vs. log [DNA], Fig. 6 (only shown for APA-3–DNA at both pH and at 37 °C, others
provided as Fig. S7 in supplementary information).
Binding constant, binding site size values of all the compounds were calculated and given
in Table 4. The data obtained for K from fluorescence spectroscopy was in good agreement with
b
that obtained from UV-results and further verified the formation of most stable APA-3–DNA
complex at stomach pH and good binding of all the investigated compounds with DNA at both
pH values. The binding site size (n) was evaluated slightly greater than 1 (n>1) for all the
compound–DNA complexes, Table 4, hence along with intercalation mode of binding the
1
6

possibility of groove binding and electrostatic binding could not be ignored [4, 28]. This suggest
that ligand acid and its organotin complexes might have binding with DNA double helix through
hydrogen bonding and planar phenyl groups present in investigated compounds [42].
From the binding constant data, the standard Gibbs free energy changes for all the compounds
were calculated, Table 4. The negative values of ∆G through fluorescence results further lend
supported the UV- results of free energy changes and indicated the spontaneity of all the
compounds–DNA binding. The orders of K and ΔG values of the compounds–DNA complexes
b
at both pH were found similar as obtained by UV- spectroscopy.
Fig. 6 and Table 4
3
.6. Evaluation of binding constants and binding sites of compounds to DNA by cyclic
voltammetry
Based upon the decrease in peak current of compounds by the addition of different
concentration of DNA, the binding constants, K , for the three compound–DNA complexes were
b
calculated according to the following equation [43].
1
K [DNA]
2
2
2
2
I 
(I  I )  I [DNA]
(5)
p
p
p
p
o
o
b
Where K is the binding constant, I and I are the peak currents with and without DNA. A plot
b
p
po
2
2
2
of I vs. (I – I )/ [DNA] gave a straight line with a slope equal to the reciprocal of binding
p
po
p
constant, K , Fig. 7 (only shown for APA-3–DNA at both pH and at 37 °C others provided as
b
Fig. S8 in supplementary information).
The Binding constants of all the compounds were calculated. Comparatively stronger binding of
all the compounds with DNA at stomach pH, and among all the compounds the greater K value
b
for APA-3–DNA binding at stomach pH is evident from the data presented in Table 5. K values
b
1
7

obtained through voltammetic parameters were also found in good agreement with that obtained
from UV- and fluorescence results hence confirming the compatibility of binding constant
results from all the three complementary techniques. Gibbs free energy changes evaluated
through K data using CV parameters were found as negative values as obtained through UV-
b
and fluorescence results and further inveterate the involvement of spontaneous process in
compound–DNA binding, Table 5. The order in the values of binding constant as well as free
energy change was found identical as obtained by both spectroscopic techniques at both pH.
Binding site sizes (n) were evaluated through cyclic voltammetry by using following
equation [44].
C / C = K [free base pairs] / s
(6)
b
f
Where, s is the binding site size in terms of base pairs (bp). Measuring the concentration of DNA
in terms of compound concentration, the concentration of the base pairs can be expressed as
[
DNA] / 2. So Eq. (5) can be written as:
C / C = K [DNA] / 2s
(7)
C and C denote the concentration of the free and DNA bound species respectively. The C / C
f
b
f
f
b
b
ratio was determined by the equation given below [45].
C / C = I- I / IDNA
(8)
b
f
DNA
where I and IDNA represent the peak currents of the compound in the absence and in the presence
of DNA, respectively. Putting the value of K as calculated according to Eq. (5), the binding site
b
size was obtained from the plot of I- IDNA / IDNA vs. [DNA], Fig. 8 (only shown for APA-3–DNA
at both pH and at 37 °C, others provided as Fig. S9 in supplementary information).
1
8

The binding site size (n) was evaluated slightly greater than 1 (n>1) for all the compound–DNA
complexes, Table 5, the reason being same as discussed in previous section. The values of n
obtained at both pH are consistent with that obtained through fluorescence spectroscopy.
Fig. 7, 8 and Table 5
3
.7. Verification of DNA-binding studies of compounds by viscosity measurements
The viscosity measurement is one of the most reliable methods to verify different modes
of DNA binding [46]. Generally for intercalation mode the viscosity of DNA solution increases
upon addition of various concentrations of the compound. This increase in relative viscosity is
due to the lengthening of DNA helix as the size of base pair pockets is increased to
accommodate the compound as observed for a classical intercalator, ethidium bromide. In the
present study, an increase in the relative viscosity of DNA at both stomach (4.7) and blood (7.4)
pH was observed when various concentrations of the investigated compound (APA-1, APA-2
and APA-3) were added. This behavior is attributed to intercalative binding mode of the
compounds with DNA and could be ascribed as enhancement in the length of DNA helix [47,
1
/3
4
8]. The values of the relative specific viscosities for the compounds i.e. (η / ηo) were plotted
against [compound] / [DNA], Fig. 9, where η and η are the specific viscosity contributions of
o
DNA in the absence and in the presence of the investigated compounds respectively. Increasing
tendency in the viscosity curve upon addition of various concentrations of compound to DNA
provided stronger evidence for the intercalation and further verified intercalative mode of
binding as investigated though spectroscopic and electrochemical studies in the present work.
Fig. 9
3
.8. Biological studies of compounds
1
9

3
.8.1. DPPH free radical scavenging assay
To estimate the antioxidant potential of test compounds, DPPH free radical scavenging
assay was used. The degree of discoloration of DPPH indicates the scavenging potential of the
antioxidant compound. DPPH assay measures loss of DPPH color after reacting with the test
compounds spectrophotometrically at 517 nm. The absorbance of DPPH at 517 nm decreases as
color changes from purple to yellow due to scavenging of the free radical by the antioxidant. In
this assay none of the synthesized compounds showed significant DPPH free radical scavenging
−
1
activity at the concentrations tested. All the compounds have IC value >200 µg ml .
5
0
3
.8.2. Potato disc antitumor assay
The inhibition of A. tumefaciens-induced tumors (or Crown Gall) in potato disc tissue is
an assay based on antimitotic activity, and can detect a broad range of known and novel
antitumor effects [49]. This assay is based on the hypothesis that antitumor agents might inhibit
the initiation and growth of tumors in both plant and animal systems, because certain
tumorogenic mechanisms are similar in plants and animals [50]. The results obtained from potato
disc antitumor assay and from other most commonly used antitumor screening assays had shown
good correlation as tumor induction mechanisms are reported to be similar in both plants and
animals [51]. All the compounds (APA-1, APA-2, and APA-3) were investigated against
Agrobacterium tumefaciens (At 10) for their antitumor potentials. Assay was carried out at
−
1
different concentrations (7.4, 22.2, 66.6 and 200 µg ml ) of synthesized compounds to examine
whether an increase in concentration of compound influenced the tumor formation ability of
bacterium and to calculate IC50 values. Vincristine (positive control) showed 100% tumor
inhibition at all the concentrations tested whereas DMSO (negative control) has no interference
with the activity of bacterium to induce tumors. Furthermore, the inhibition was observed in dose
2
0

−1
dependent manner with highest inhibition at 200 µg ml concentration. Antitumor activity can
also be determined by 50% inhibitory concentration (calculated by inhibition curves of four
concentrations tested for each compound). Lower the IC50 value more effective is the tested
compound. An IC calculation favors APA-3 to be more active as compared to other compounds
5
0
−
1
and exhibited best tumor inhibitory activity with lowest IC value of 7.37 µg ml , Table 6. The
5
0
effectiveness order of compounds is given as: APA-3 > APA-2 > APA-1.
Table 6
4
. Conclusions
A carboxylic acid ligand (Z)-4-(4-acetylphenylamino)-4-oxobut-2-enoic acid (APA-1)
and its compounds; triphenylstannyl (Z)-4-(4-acetylphenylamino)-4-oxobut-2-enoate (APA-2),
and tri- n-butylstannyl (Z)-4-(4-acetylphenylamino)-4-oxobut-2-enoate (APA-3) were
synthesized and characterized. All compounds were further investigated for their binding with
ds.DNA through complimentary techniques; UV-visible spectroscopy, fluorescence
spectroscopy, cyclic voltammerty and further verified for binding mode by viscosity
measurements. The results obtained from these techniques under physiological conditions of pH
°
{
stomach (4.7) and blood (7.4)} and temperature (37 C) revealed predominant intercalation of
the compounds within DNA. The negative values of standard Gibbs energy changes (∆G)
indicated the spontaneity of the binding of compounds with DNA. Interactional parameter, Kb,
calculated from both spectroscopic techniques and cyclic voltammerty were in good agreement
and indicated stronger binding and formation of compound–DNA complexes. The values of
binding site size (n) calculated from fluorescence and voltammertric data further demonstrated
the dominancy of intercalative mode for all the compound–DNA complexes. K , n and ∆G
b
2
1

values for all the compounds–DNA complexes inferred comparatively greater binding and
spontaneity of APA-3 complex with DNA at both pH. Compounds were also probed for their
antioxidant and antitumor activities through biological assays. All the compounds were found
inactive for their antioxidant activity, while APA-3 showed greater antitumor activity as
compared to other compounds. Results obtained for antitumor potential and DNA binding
studies (K values) are correlated with each other and further justify the significance of present
b
studies. Authors are assured that such studies and further investigations in this direction would
not only help to predict the mechanism of action of investigated compounds with DNA as
intracellular target but may lead to explore new and effective candidates as anticancer drug.
Associated Content
Supporting information
Figures S1 to S9 as supplementary information for DNA binding studies.
Author information
Corresponding Author
*
E-mail: nasimaa2006@yahoo.com
Acknowledgements
This work is supported by Department of Chemistry, Faculty of Science, Allama Iqbal Open
University Islamabad, Pakistan.
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