DNA/BSA binding studies of peripherally tetra substituted neutral azophenoxy zinc phthalocyanine

Article abstract of DOI:10.1016/j.poly.2019.114208

A tetra substituted neutral zinc phthalocyanine (ZnPc-4) bearing 4-phenylazophenoxy group at the periphery had been prepared from zinc acetate dihydrate Zn(CH₃COO)₂·2H₂O and 4-(phenylazophenoxy)phthalonitrile. The interaction of neutral metallophthalocyanine, ZnPc-4 with Calf Thymus (CT) DNA was investigated using absorption titrations, competitive Fluorescent Intercalator Displacement (FID) assay, minor groove binding assay and viscosity measurement. The binding studies revealed a probable intercalative mode of interaction of ZnPc-4 with DNA with an appreciable binding constant. The bovine serum albumin (BSA) binding activity of ZnPc-4 was evaluated by fluorescence titration.

Full text of DOI:10.1016/j.poly.2019.114208

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DNA/BSA binding studies of peripherally tetra substituted neutral azophenoxy
zinc phthalocyanine
G.S. Amitha, Suni Vasudevan
PII:
S0277-5387(19)30653-9
DOI:
https://doi.org/10.1016/j.poly.2019.114208
POLY 114208
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25 October 2019
26 October 2019
Please cite this article as: G.S. Amitha, S. Vasudevan, DNA/BSA binding studies of peripherally tetra substituted
neutral azophenoxy zinc phthalocyanine, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.114208
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Article
DNA/BSA binding studies of peripherally tetra substituted neutral
azophenoxy zinc phthalocyanine
1
1*
G. S. Amitha and Suni Vasudevan
1Dept. of Chemistry, National Institute of Technology Calicut, Calicut 673601, Kerala, India
Corresponding authors, Email: suniv@nitc.ac.in
*
Abstract
A tetra substituted neutral zinc phthalocyanine (ZnPc-4) bearing 4-phenylazophenoxy group at
the periphery had been prepared from zinc acetate dihydrate Zn(CH COO) .2H O and 4-
phenylazophenoxy)phthalonitrile. The interaction of neutral metallophthalocyanine, ZnPc-4 with Calf
3
2
2
(
Thymus (CT) DNA was investigated using absorption titrations, competitive Fluorescent Intercalator
Displacement (FID) assay, minor groove binding assay and viscosity measurement. The binding studies
revealed a probable intercalative mode of interaction of ZnPc-4 with DNA with an appreciable binding
constant. The bovine serum albumin (BSA) binding activity of ZnPc-4 was evaluated by fluorescence
titration.
Keywords: ZnPc-4, CT-DNA, Fluorescent Intercalator Displacement (FID) assay, binding constant, BSA
1
. Introduction
Phthalocyanines (Pcs), which are conventionally used as dyes and pigments[1], are a class of
macrocyclics of great interest with wide range of applications. Studies on the interactions of transition
metal complexes with nucleic acids have gained considerable momentum in the recent past with an aim to
develop new DNA molecular probes and novel chemotherapeutic agents[2,3]. Metallophthalocyanines
(
MPcs) are well known for their use as medicinal agents and as DNA interacting probes, on account of
their large π delocalized planar surface area, which can be easily modified according to the biological
target[4–6]. However, the broad applicability of unsubstituted phthalocyanines can be severely hampered
by their insolubility in common organic solvents and in aqueous media[7,8].This problem can be solved
by the judicious introduction of electron-donating substituents (alkyl, alkoxy, alkylthio chains, or bulky
phenyl, aryl groups), electron- withdrawing substituents (chloro, bromo, or nitro moieties), polar
ionizable groups (anionic sulfonyl, carboxyl or cationic amino or quaternized ammonium groups) and
neutral biomolecules like glucose, lactose, galactose etc. at the peripheral and non-peripheral positions[9–
16]. A majority of the reported DNA targeting Pcs were observed to be cationic, while very few of them
were observed to be anionic in nature. Charged Pcs were found to be aqueous soluble, which was

essential for biological applications. Cationic Pcs have an inclination towards the anionic phosphodiester
backbone of DNA, thus improving the cell permeability and the cleaving effect of cationic
phthalocyanines[17–19]. This prompted us to study the behavior of neutral metallophthalocyanine in a
similar environment, and to the best of our knowledge, very few reports are available on the DNA
interaction studies of neutral MPcs to date. Arslantas et al. have focused on the DNA binding interaction
of various neutral MPcs peripherally substituted with functional groups such as 4-(4-hydroxyphenyl)-1-
phenylethyl) phenoxy group, 3,4,5-trimethoxybenzyloxy substituents, dimethyl- 5-(phenoxy)-
isophthalate, 5-tert-butyl-2-hydroxyphenoxy groups etc [20–23]. In another report, Durantini et al., had
studied the photodynamic application of zinc phthalocyanine peripherally substituted with methoxy and
trifluoromethylbenzyloxy substituents[24].
The photo switching property of azobenzene based compounds often limit their biological
applications, since the strong scattering tendency of UV light reduces the effective penetration of cells
and tissues [25]. Moreover, UV light can trigger unwanted cellular responses like apoptosis, which can
further complicate the studies[26,27]. However, a few reports had described the use of azobenzene in
conjugation with pharmacologically active compounds of high therapeutic index[28,29] and Zhang et al.
in another report had evaluated the photo dependent antibacterial activity of honeycomb patterned
dodecyloxy-azo-zinc phthalocyanine film (daZnPc), towards Escherichia coli (E. coli)[30]. BSA and
human serum albumin (HSA) are blood plasma proteins, which play critical role in the transport of drugs
to specific targets in the blood, and their distribution and metabolism in the body[31,32]. So the BSA-
drug interaction has vital importance in the formulation and the production of novel drug.
In this perspective, we hereby report the DNA/BSA binding studies of zinc phthalocyanine (ZnPc-4)
peripherally substituted with 4-phenylazophenol moiety by oxygen linkage. The mode of interaction of
ZnPc-4 with CT-DNA had been investigated using electronic absorption titrations, fluorescent intercalator
displacement assays, minor groove binding assays and viscosity change studies. The BSA binding affinity
of ZnPc-4 was evaluated by fluorescence spectroscopy.
2
. Experimental
Materials & Instrumentation
CT-DNA and bio-reagent grade lyophilized powder of BSA were purchased from Sigma-Aldrich.
The commercially purchased chemicals were used without further purification. UV-visible absorption
spectra were recorded on a Schimadzu UV-Visible 2600 Spectrophotometer with quartz cuvette and
fluorescence spectra were obtained from Cary-Eclipse Agilent fluorescence spectrophotometer. The
viscosity measurements were taken using a 15 ml Ostwald Viscometer.
3
. Results and discussion

3
.1. Preparation of zinc phthalocyanine, ZnPc-4
The molecular structure of the compound under study, 2,9,16,23-Tetra-(4-phenylazophenoxy)
zinc(II)phthalocyanine (ZnPc-4) is represented in Figure 1.
Figure 1. Molecular structure of 2,9,16,23-Tetra-(4-phenylazophenoxy) zinc(II)phthalocyanine, ZnPc-4
This tetra substituted zinc phthalocyanine was prepared by cyclotetramerization of 4-(4-
o
(
phenylazophenoxy) phthalonitrile by refluxing with Zn(CH
3
COO)
2
.2H
2
O in n-pentanol/DBU at 170 C
1
under N
2
atmosphere. The compound was fully characterized with electronic, FT-IR, HNMR and mass
spectroscopic techniques and the results were concordant with the expected structure. The synthesis and
characterization details had been reported elsewhere[33] and additional data are given in Supplementary
Information (SI).
3
.2. Interaction of ZnPc-4 with duplex DNA
3.2.1. Determination of binding of ZnPc-4 to CT-DNA using absorption titrations
The UV-visible absorption titration of ZnPc-4 with CT-DNA was conducted at room temperature
in a 50mM Tris-HCl buffer containing 50mM NaCl (pH 7.4). DNA was dissolved in the buffer solution
by ultra-sonication. UV absorbance of DNA stock solution in buffer at 260 and 280 nm was measured and
the ratio A260/A280 was found to be 1.87, ensuring the DNA sample was sufficiently free of protein. The
concentrations of DNA per nucleotide phosphate was calculated from the absorbance at 260 nm using the
-1
-1
o
average extinction coefficient value of ε260 = 6600 M cm . DNA was stored at 4 C overnight and was
used within 4 days[34]. The stock solution of ZnPc-4 was prepared in DMF and then diluted to the
desired concentration using Tris-HCl buffer of pH 7.4. The absorption titrations were carried out by

adding increasing amounts of DNA, i.e., 1µM aliquots, to a fixed concentration (10µM) of ZnPc-4
contained in a quartz cell, with phosphate/drug (p/d) ratio ranging from 0.1 to nearly 1.0. Absorption
spectra were recorded after each addition.
Binding constant was calculated by plotting [DNA] in the x-axis and [DNA]/ε
a
f
-ε in the y-axis,
where ε was extinction coefficient of bound complexes and ε was the absorption coefficient of free
a
f
complexes. From the absorption titration data, the binding constant was determined using Wolfe-Schimer
equation (1) [35]:
[
퐃퐍퐀]
[퐃퐍퐀]
훆퐛−훆퐟
ퟏ
=
+
(1)
훆퐚−훆퐟
퐊 (훆 −훆 )
퐛 퐛 퐟
where [DNA] was the concentration of DNA in base pairs and the apparent absorption coefficients, ε
and ε corresponded to Aobsd/[M], the extinction coefficient of the free compounds and the extinction
coefficient of the compound when fully bound to DNA respectively. A linear fitting of the plot of
DNA]/(ε − ε ) versus [DNA] was done and the binding constant K was given by the ratio of slope to the
intercept.
a
, ε ,
f
b
[
a
f
b
With increased addition of CT-DNA, the absorbance of ZnPc-4 got decreased, reaching saturation
beyond the DNA concentration of 10 µM. Interaction of ZnPc-4 with CT DNA was observed by the
hypochromism of three major bands located at λmax of 350, 640 and 686 nm associated with slight red
shift for each band. As shown in Figure 2 , ZnPc-4 exhibited hypochromism of about 26%, 20.8%,
18.73% and bathochromic shift of about 2.5 nm, 1.5 nm, 2 nm for the bands situated at 350, 640, 686 nm
respectively. Three isosbestic points were also seen at 720, 578 and 442 nm on the addition of increasing
concentrations of CT DNA. Hypochromism associated with a slight bathochromic shift usually resulted
from the intercalative mode of binding, involving a stacking interaction between an aromatic
chromophore and the base pairs of DNA[12]. The highly planar central moiety of ZnPc-4 with the
conjugated ring systems, could possibly incorporate an intercalation mode of binding with CT DNA. The
5
-1
binding constant, K value was calculated as 1.58 x10 M .
b

a
Figure 2. Absorption spectra of ZnPc-4 in DMF/Tris-HCl buffer upon addition of CT DNA Inset: plots of [DNA]/ε -
ε
f
verses [DNA] for the titration of DNA with ZnPc-4.
3.2.2. Fluorescent Intercalator Displacement (FID) Assay
FID assay is a non-destructive technique which relies on the change in fluorescence observed on
the displacement of DNA-bound ethidium bromide (EB) by another DNA binding compound. EB is a
classical DNA intercalator, as it binds to the biomolecule by occupying the intercalative sites between
DNA bases and the fluorescence yield of EB is significantly enhanced on DNA binding. The ability of the
compound under study to displace EB from the intercalative sites on DNA can be monitored by a
decrease in EB–DNA fluorescence where the amount of unbound or free EB would be increased[36].
Although the present compound, ZnPc-4 was inherently fluorescent with λem situated at 694 nm (given in
SI S9), its fluorescence got quenched due to aggregation in aqueous/buffer medium[37]. Hence EB
displacement assay had been performed to find out the mode of interaction of compound with DNA. To
study the competitive binding of EB with ZnPc-4, 10µM CT DNA was pretreated with EB for 10 minutes
at room temperature in Tris-HCl buffer of pH 7.4 and excited at 480 nm and the emission spectra were
recorded in the absence, and at various amounts of ZnPc-4 in DMF, up to the ratio of [DNA]/[ZnPc-4]
was reached to unity.

Figure 3. Competitive Intercalator displacement assays: Fluorescence titration of the EB–CT DNA complex with
ZnPc-4. The EB–CT DNA complex was excited at 480 nm and emission spectra were recorded from 500–800 nm.
-
6
o
Inset: Stern-Volmer plots of F /F verses [ZnPc-4]x10 M for the titration of CT DNA with ZnPc-4.
Figure 3 shows the emission spectra of EB-DNA adducts (λex=480 nm; λem=598 nm) in the
presence of increasing amounts of ZnPc-4 in DMF. The emission intensity of EB-DNA adduct was
gradually decreased on the addition of ZnPc-4 and the extend of quenching was quantitatively estimated
by using the Stern–Volmer (S-V)equation (2) [38],
푭풐
(2)
=
ퟏ + 퐊 [퐐]
푺푽
푭
where F
o
and F were the fluorescence intensities in the absence and presence of the quencher ZnPc-4, KSV
is the S-V quenching constant and [Q] the quencher concentration. The quenching constant KSV was
5
-1
obtained as 1.282 x 10 M from the slope of the S-V plot F /F versus [Q] given in the inset of Figure 3.
o
A linear S-V plot as obtained in the inset of Figure 3 suggested only one type of binding or
quenching process, either static or dynamic quenching, which can be differentiated using equation (3).
푲푺푽 = 푲 푿 흉
(3)
풒
풐
The approximate quenching constant (K
q
) can be calculated from the equation (3), where τ is the
o
lifetime of CT DNA-EB adduct was reported as 19.2 ns in the absence of the complex[39]. The calculated
12
-1 -1
12
-1 -1
value of K
q
(6.67x10 M S ) was in the order of 10 M S , which was 100 times larger than the
maximum diffusion collision quenching constant (2 x1010 M S ) of a variety of quenchers with
-1
-1

biomacromolecules[40]. The result unambiguously showed the existence of a static quenching mechanism
rather than dynamic collision. This pointed out that the gradual formation of non-fluorescent DNA-Pc
adducts by replacing EB from the binding site could be the reason for the static quenching, rather than the
collision deactivation process.
푲푬푩 [푬푩]
푲풂풑풑
=
(4)
[
푸]
K
app can be defined as the binding constant at which 50% reduction in fluorescence intensity of
DNA-EB adduct occurs with the addition of quencher. The apparent binding constant[41] Kapp was
6
-1
7
-1
calculated as 3.64 x10 M using the equation (4), where KEB was the binding constant of EB (1x10 M ),
while [EB] and [Q] were the concentrations of EB and quencher ZnPc-4 respectively at 50% reduction in
fluorescence intensity.
3.2.3. Minor groove binder displacement assay
Minor groove binder displacement assay with another classical groove binder Hoechst 33258 was
performed, in order to investigate the possibility of groove binding mode of interaction of ZnPc-4 with
CT DNA. Hoechst 33258 dye (HD) is chemically 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-
bi-1H-benzimidazole trihydrochloride hydrate and is known to be a potential minor groove binder, which
shows great affinity towards A-T (adenine-thymine) rich sequence of DNA double helix. Like EB
displacement assay, a similar fluorescence competition experiment was carried out using HD, as shown in
Figure 4. Initially the HD-DNA binding occurred in the minor groove of DNA double helix. The
fluorescence quantum yield of HD increased significantly on interacting with DNA. The displacement of
bound HD from its binding site on CT DNA was implicated from a decrease in its fluorescence intensity
on addition of the compound.
5
-1
The apparent binding constant Kapp was calculated as 1.27 x10 M using the equation (4), where
5
-1
K
HD was the binding constant of HD (9.6x10 M )[42], while [HD] and [Q] were concentrations of HD
and quencher ZnPc-4 respectively at 50% reduction in fluorescence intensity.

Figure 4. Competitive Minor groove binder displacement assays: Fluorescence titration of the HD-CT DNA
complex with ZnPc-4. HD-CT DNA complex was excited at 355 nm and emission spectra were recorded from 370–
-
6
6
o
50 nm. Inset: Stern-Volmer plots of F /F verses [ZnPc-4]x10 M for the titration of CT DNA with ZnPc-4.
Table 1. Comparison of KSV and Kapp value of ZnPc-4 with Fluorophore-DNA bound adduct to predict the mode of
interaction of DNA binding for [phosphate or DNA]/[drug or compound]([p]/[d]) ratio ranges from 0-1 with 0.1
increment
-1
SI No. Type binding assay
[p]/[d] ratio Linear
correlation
fit KSV (M )
Apparent binding
constant (Kapp
)
-
1
coefficient
Adj.R2
(M )
1
2
EB displacement assay
HD displacement assay
0-1
(0.1 0.991
1.28x105
3.64 x106
1.27 x105
increment)
0-1
(0.1 0.997
0.663x105
increment)
From the Table 1 given above, the quantitatively determined parameters clearly suggested that the
compound ZnPc-4 was presumably interacting with DNA via an intercalative mode of interaction.
3.2.4. Viscosity measurements
Hydrodynamic measurements are considered as the unambiguous and the most reliable test to
elucidate the strength and mode of DNA-drug interactions, unless the crystallographic data of DNA
bound drug conjugate are available. When a molecule interacts with DNA by intercalative interactions, it
can cause a significant increase in the viscosity of a DNA solution, since the insertion of intercalator
causes separation of base pairs, and hence, an increase in the overall DNA molecular length. On the other

hand, non-classical or partial intercalators, such as the [Ru(phen)
DNA helix, decreasing the viscosity of DNA[43,44].
3
]2+ cation usually bend or kink the
Figure 5. Effect of increasing amount of ZnPc-4 on the relative viscosity of CT DNA in 50 mM Tris-HCl buffer.
Viscosity measurements were carried out at room temperature using an Oswald viscometer of 15 ml
capacity. The DNA was sonicated for 10 min and bubbled with nitrogen, for 3 minutes for homogenous
mixing. The DNA concentration was kept constant and the concentration of ZnPc-4 was increased from 0
to 10 µM in different samples. The flow time was measured three times for each sample, viscosity and
(
η/η
presented as (η/η
the complex, η is the viscosity of DNA alone and [X] is the concentrations of either EB or ZnPc-4.
Viscosity values were calculated from the observed flow time of DNA-complex solutions corrected for
o
)1/3 were calculated for each and every trial and finally an average (η/η
o
)1/3 was calculated. Data were
)1/3 versus the ratio of [X]/[DNA], where η is the viscosity of DNA in the presence of
o
o
o
o
the flow time (t ) of buffer alone, i.e., η = t-t . In the same way, the viscometric titration of EB was also
performed with CT DNA and cross plotted with the viscometric response curve of DNA-ZnPc-4. As
shown from Figure 5, the viscosity of DNA solution increased steadily with the addition of ZnPc-4
aliquots, mostly due to the hydrophobic core insertion of the phthalocyanine molecules between the DNA
base pairs, resulting in the lengthening of DNA helix[45].
3
.3. BSA binding experiment of ZnPc-4
BSA-drug interaction has a critical role in pharmacokinetic and pharmacodynamic phases of drug
metabolism. When a compound is administered as a drug, it would immediately encounter blood plasma
proteins BSA and HSA in the blood. BSA displays a significant contribution to the physiological
functions and effective delivery of drug to specific site, hence the investigation on the interaction of
drugs/compound with BSA is of great interest. The binding of ZnPc-4 to BSA was studied by

fluorescence spectroscopy in 50mM Tris HCl buffer at room temperature. The fluorescence of BSA at
48 nm was mainly attributable to the amino acid tryptophan in the macromolecule[32].
3
Figure 6. Fluorescence spectral changes of BSA on addition of varying concentrations of ZnPc-4 in buffer. [BSA] =
-
6
-6
2
7.28×10 M, [ZnPc-4] varies from 0 to 20×10 M. Excitation wavelength = 280 nm.
Figure 6 shows the fluorescence emission spectra of BSA in the presence of different
concentrations of ZnPc-4. The emission intensity of BSA was found to be diminished with the addition of
ZnPc-4, and this observation suggested that the compound ZnPc-4 was interacting with BSA at the
proximity of tryptophan residues and the extend of interaction can be quantified by determining the S-V
quenching constants (KSVBSA) from equation (4).
As discussed in section 3.2.2., the linear S-V plot revealed that either static or dynamic quenching process
was taking place. Likewise the slope KSVBSA of S-V plot (inset of Figure 6) was calculated as 1.99x10 M
1.
The bimolecular quenching constant K of the BSA-complex adduct was estimated using the equation
3); where τBSA, the approximate lifetime of fluorophore BSA in the absence of quencher was usually
5
-
q
(
1
3
-1 -1
taken as 10 ns[46,47]. The K value was estimated as 1.99x10 M s , which was quite higher than the
q
10
-1 -1
proposed value of the order of 10 M s for diffusion controlled dynamic quenching, and hence it can be
assumed that the BSA-ZnPc-4 interaction obeyed static model quenching process. The decrease in the
intrinsic fluorescence intensity of tryptophan with increasing concentration of ZnPc-4 indicated that the
compound readily bound to BSA by the approaching the tryptophan enriched subdomains of BSA. This
also suggested that the primary binding sites of these molecules were very close to tryptophan residues,
since the occurrence of quenching requires molecular contact as per the static quenching model[48].

4
. Conclusions
In summary, DNA/BSA binding activity of the compound peripherally tetra-(4-
phenylazophenoxy) substituted zinc phthalocyanine ZnPc-4 was evaluated by different instrumental
techniques. The DNA binding studies of ZnPc-4 towards CT DNA was carried out in 50mM Tris-HCl-
NaCl buffer of pH 7.4 by UV-visible absorption titrations, competitive fluorescence quenching titration
and viscosity measurements. The competitive fluorescent displacement assay was carried out with the
classical intercalator Ethidium bromide and minor grove binder Hoechst dye and binding mode was
predicted by comparing their S-V quenching constant KSV and apparent binding constant (Kapp). The value
5
-1
of KSV 1.28 x 10 M and Kapp was higher for EB displacement assay than HD displacement assay. The
5
-1
intrinsic binding constant K from absorption titration was 1.58 x 10 M and the trend of relative
b
viscosity of DNA solution was increasing with increase in [ZnPc-4]/[DNA] ratio, and all these
observations clearly pointed out that ZnPc-4 was interacting with DNA via intercalative mode. The BSA
1
3
-1 -
interaction experiment revealed that the bimolecular quenching constant K
q
was of the order of 10 M s
1,
so the binding interaction of ZnPc-4 with BSA was assumed to be following static fluorescence
quenching mechanism. The strong binding interaction of ZnPc-4 with BSA suggested that it can be easily
transported in the blood, hence the synthesized molecule ZnPc-4 can be considered as a potential
candidate for further studies and optimizations as a drug.
Conflict of interest:
The authors declare no conflict of interest.
Acknowledgements
AGS is thankful to NCB-GATE for research fellowship and SV would like to thank SERB – Dept. of
Science and Technology (Grant Sanction No. SERB/F/6178/2014-15) for financial support.
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Figures and Tables
DNA/BSA binding studies of peripherally tetra substituted neutral
azophenoxy zinc phthalocyanine
1
1,*
Amitha G. S. and Suni Vasudevan
1Dept. of Chemistry, National Institute of Technology Calicut, Calicut 673601, Kerala, India
Corresponding authors, Email: suniv@nitc.ac.in
*
Figure 7. Molecular structure of 2,9,16,23-Tetra-(4-phenylazophenoxy) zinc(II)phthalocyanine, ZnPc-4

Figure 8. Absorption spectra of ZnPc-4 in DMF/Tris-HCl buffer upon addition of CT DNA Inset: plots of [DNA]/ε
verses [DNA] for the titration of DNA with ZnPc-4.
a
-
ε
f
Figure 9. Competitive Intercalator displacement assays: Fluorescence titration of the EB–CT DNA complex with
ZnPc-4. The EB–CT DNA complex was excited at 480 nm and emission spectra were recorded from 500–800 nm.
-
6
o
Inset: Stern-Volmer plots of F /F verses [ZnPc-4]x10 M for the titration of CT DNA with ZnPc-4.

Figure 10. Competitive Minor groove binder displacement assays: Fluorescence titration of the HD-CT DNA
complex with ZnPc-4. HD-CT DNA complex was excited at 355 nm and emission spectra were recorded from 370–
-
6
6
o
50 nm. Inset: Stern-Volmer plots of F /F verses [ZnPc-4]x10 M for the titration of CT DNA with ZnPc-4.
Table 2. Comparison of KSV and Kapp value of ZnPc-4 with Fluorophore-DNA bound adduct to predict the mode of
interaction of DNA binding for [phosphate or DNA]/[drug or compound]([p]/[d]) ratio ranges from 0-1 with 0.1
increment
-1
SI No. Type binding assay
[p]/[d] ratio Linear
correlation
fit KSV (M )
Apparent binding
constant (Kapp
)
-
1
coefficient
Adj.R2
(M )
1
2
EB displacement assay
HD displacement assay
0-1
(0.1 0.991
1.28x105
3.64 x106
1.27 x105
increment)
0-1
(0.1 0.997
0.663x105
increment)

Figure 11. Effect of increasing amount of ZnPc-4 on the relative viscosity of CT DNA in 50 mM Tris-HCl buffer.
Figure 12. Fluorescence spectral changes of BSA on addition of varying concentrations of ZnPc-4 in buffer. [BSA]
-
6
-6
=
27.28×10 M, [ZnPc-4] varies from 0 to 20×10 M. Excitation wavelength = 280 nm.



Synthesized peripherally 4-phenylazophenoxy substituted zinc phthalocyanine (ZnPc-4)
DNA binding affinity of ZnPc-4 was studied by UV-visible, fluorescence titrations and
viscosity measurements


ZnPc-4 show intercalative mode of interaction with DNA by an appreciable binding
5
-1
constant of the order of 10 M
Static interaction of ZnPc-4 with BSA was investigated by fluorescence titration

Graphical abstract
DNA/BSA binding studies of peripherally tetra substituted neutral
azophenoxy zinc phthalocyanine
1
1,*
G. S. Amitha and Suni Vasudevan
1Dept. of Chemistry, National Institute of Technology Calicut, Calicut 673601, Kerala, India
Corresponding authors, Email: suniv@nitc.ac.in
*
The DNA binding activity of the newly synthesized peripherally tetra-(4-phenylazophenoxy)
substituted zinc phthalocyanine ZnPc-4 was evaluated by UV-visible absorption titrations,
fluorescence quenching titration and viscosity measurements studies. The inferences from all the
techniques invariably pointed out that ZnPc-4 intercalating into the base pair sequences of DNA
by its large delocalized π surface. ZnPc-4 was also showing strong static interaction with BSA
proven that compound is an effective DNA interacting drug structural candidate with
biocompatibility.