Multispectroscopic studies on the interaction of a copper(ii) complex of ibuprofen drug with calf thymus DNA

Article abstract of DOI:10.1080/15257770.2016.1223305

The interaction of copper(II)–ibuprofenato complex with calf thymus DNA (ct-DNA) has been explored following, UV-visible spectrophotometry, fluorescence measurement, dynamic viscosity measurements, and circular dichroism spectroscopy. In spectrophotometric studies of ct-DNA it was found that [Cu(ibp)₂]₂ can form a complex with double-helical DNA. The association constant of [Cu(ibp)₂]₂ with DNA from UV-Vis study was found to be 6.19 × 10⁴ L mol^?1. The values of K_f from fluorescence measurement clearly underscore the high affinity of [Cu(ibp)₂]₂ to DNA. The experimental results showed that the conformational changes in DNA helix induced by [Cu(ibp)₂]₂ are the reason for the fluorescence quenching of the DNA-Hoechst system. In addition, the fluorescence emission spectra of intercalated methylene blue (MB) with increasing concentrations of [Cu(ibp)₂]₂ represented a significant increase of MB intensity as to release MB from MB-DNA system. The results of circular dichroism (CD) suggested that copper(II)–ibuprofenato complex can change the conformation of DNA. In addition, the results of viscosity measurements suggest that copper(II)–ibuprofenato complex may bind with non-classical intercalative mode. From spectroscopic and hydrodynamic studies, it has been found that [Cu(ibp)₂]₂ interacts with DNA by partial intercalation mode which contains intercalation and groove properties.

Full text of DOI:10.1080/15257770.2016.1223305

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Multispectroscopic Studies on the Interaction of
a Copper(II) Complex of Ibuprofen Drug with Calf
Thymus DNA
Nahid Shahabadi & Farshad Shiri
To cite this article: Nahid Shahabadi & Farshad Shiri (2016): Multispectroscopic Studies on the
Interaction of a Copper(II) Complex of Ibuprofen Drug with Calf Thymus DNA, Nucleosides,
Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2016.1223305
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1223305
Published online: 11 Nov 2016.
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Date: 12 November 2016, At: 23:28

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Multispectroscopic Studies on the Interaction of a Copper(II)
Complex of Ibuprofen Drug with Calf Thymus DNA
Nahid Shahabadia,b and Farshad Shiria
aInorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran; bMedical
Biology Research Center (MBRC) Kermanshah University of Medical Sciences, Kermanshah, Iran
ABSTRACT
ARTICLE HISTORY
Received  March 
Accepted  August 
The interaction of copper(II)–ibuprofenato complex with calf
thymus DNA (ct-DNA) has been explored following, UV-visible
spectrophotometry, fluorescence measurement, dynamic viscos-
ity measurements, and circular dichroism spectroscopy. In spec-
trophotometric studies of ct-DNA it was found that [Cu(ibp)2]2 can
form a complex with double-helical DNA. The association con-
stant of [Cu(ibp)2]2 with DNA from UV-Vis study was found to
be 6.19 × 104 L mol−1. The values of Kf from fluorescence mea-
surement clearly underscore the high affinity of [Cu(ibp)2]2 to
DNA. The experimental results showed that the conformational
changes in DNA helix induced by [Cu(ibp)2]2 are the reason for the
fluorescence quenching of the DNA-Hoechst system. In addition,
the fluorescence emission spectra of intercalated methylene blue
(MB) with increasing concentrations of [Cu(ibp)2]2 represented a
significant increase of MB intensity as to release MB from MB-
DNA system. The results of circular dichroism (CD) suggested that
copper(II)–ibuprofenato complex can change the conformation
of DNA. In addition, the results of viscosity measurements sug-
gest that copper(II)–ibuprofenato complex may bind with non-
classical intercalative mode. From spectroscopic and hydrody-
namic studies, it has been found that [Cu(ibp)2]2 interacts with
DNA by partial intercalation mode which contains intercalation
and groove properties.
KEYWORDS
Copper(II)-ibuprofen
complex; DNA interaction;
partial intercalation; Spectral
probes
GRAPHICAL ABSTRACT
CONTACT Nahid Shahabadi
n.shahabadi@razi.ac.ir
Inorganic Chemistry Department, Faculty of Chemistry,
Razi University, Kermanshah, Iran, and Medical Biology Research Center (MBRC), Kermanshah University of Medical Sci-
ences, Kermanshah, Iran
©  Taylor & Francis Group, LLC

2
N. SHAHABADI AND F. SHIRI
Introduction
Development of cancer drugs is slow and costly. One approach to accelerate the
availability of new drugs is to reposition approved drugs for other indications as anti-
cancer agents.[1] A class of drugs that are produced and consumed in large quantities
and can be used for this purpose is nonsteroid anti-inflammatory drugs or NSAIDs.
Out of several NSAIDs, ibuprofen that derived from propionic acid is widely used
as analgesic and antipyretic.[2] Ibuprofen is often used for the treatment of rheuma-
toid arthritis and osteoarthritis, mild to moderate pain, dysmenorrhea, fever and
also shows anticancer activity.[3,4] The binding mode between ibuprofen and DNA
helix is intercalation that is most effective mode of the drugs targeted to DNA and is
related to the antitumor activity of this compound.[5] In addition, the investigation
of the complexes of metal ions such as Cu, Zn, and Ni which are vital for biological
systems and can be present at the foods, is important because it can serve the clari-
fication of clinical results.[6] Among these ions, copper is an essential trace element
for all living organisms and is a universally important cofactor for many hundreds of
metalloenzymes.[7] Copper(II) complexes have found possible medical uses in the
treatment of many diseases including cancer[8,9] as well as have demonstrated a wide
range of pharmacological activities such as antiviral[10] and anti-inflammatory.[11]
Copper(II) complexes of NSAIDs are often more active and desirable drugs than
the parent ligands themselves.[12] Copper ibuprofenate is a chemical complex con-
sisting of copper(II) and the chelate form of the anti-inflammatory drug ibuprofen
which showed better anti-ulcer protective effect than the parent drug.[13] Therefore,
investigations on copper complexes and drugs such as ibuprofen, which can be used
in the goals that discussed above, are becoming more prominent in the research
area of bioinorganic chemistry. On the other side, the DNA binding properties
of substitution-inert transition-metal complexes have lately captured an extensive
interest due to their possible applications in cancer therapy and molecular biology.
Metallointercalators and transition metal complexes which bind DNA primarily as
classical or non-classical intercalation, are considered as the most effective class of
molecules in these applications.[14,15] In the present study, the interaction between
copper ibuprofenate complex ([Cu(ibp)2]2, [ibp = Ibuprofen]) ( Figure 1) and ct-
DNA was investigated with the use of Hoechst 33258 and methylene blue (MB) dyes
as spectral probes by the application of UV–vis and fluorescence spectroscopic tech-
niques, CD spectroscopy, viscosity measurements, and docking simulation.
Material and methods
Reagents
The ibuprofen drug was supplied by Sigma Co, Cu(CH3COO)2.H2O and NaOH
were obtained from Merck. [Cu(ibp)2]2 were prepared according to the pub-
lished method.[13,16] The ct-DNA, Tris–HCl and Hoechst 33258 were purchased
from Sigma Co and were used without further purification. Methylene blue

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Figure . The chemical structure of [Cu(ibp)] complex (optimized by Spartan  program).
(MB) was provided from Merck. Tris–HCl buffer solution was prepared from
tris-(hydroxymethyl)-amino-methane–hydrogen chloride and pH was regulated to
7.4. The stock solution (1 × 10−3 mol.L−1) of [Cu(ibp)2]2 was prepared by dissolv-
ing its crystals in 80% (v/v) ethanol/Tris–HCl buffer. Calf thymus DNA dissolved in
Tris–HCl buffer as stock solution and was stored at 4°C, the purity of ct-DNA was
checked by monitoring the ratio of A260/A280> 1.80, and the concentration of ct-
DNA was determined by the absorption of ct-DNA at 260 nm (ε260 = 6600 L mol−1
cm−1).[17]
Synthesis of [Cu(ibp)2]2complex
The [Cu(ibp)2]2 complex was prepared according to the following procedure:
2.0 mmol (0.412 g) of ibuprofen reacted with 2.0 mmol (0.08 g) of NaOH in 20 mL
of distilled water, to give the sodium salt of the ibuprofen. Then 1.0 mmol (0.199 g),
Cu(CH3COO)2.H2O was added by stirring. The turquoise precipitate formed was
collected by filtration, washed several times with distilled water and dried at room
temperature. The formation of this complex was confirmed by elemental analysis,
UV-visible and also FT-IR spectroscopy according to the published methods[13,16]
as follows: The UV-vis characteristic bands of ibuprofen alone and along with the
[Cu(ibp)2]2 complex from 200 to 800 nm (Figure 2) are listed in Table 1. Free ibupro-
∗
fen shows bands at 228 and 262 nm assigned to π - π transitions. Clear hyper-
chromic due to metal- ligand charge transfer for the complex is attributed to com-
plexation of ibuprofen. The IR spectra of [Cu(ibp)2]2 complex and free ibuprofen
were measured as KBr discs and the assignments are given in Table 2. The ν(C=O)
and ν(OH) of carboxylic acid appeared at 1721 and 3000–3350 cm−1 for the free lig-
and, but disappeared in the spectrum of the complex. IR spectrum of the complex

4
N. SHAHABADI AND F. SHIRI
Figure . UV- Vis spectra of ibuprofen (ligand) and [Cu(ibp)] complex.
Table . Characteristic bands in UV-vis absorption spectra of ibuprofen and [Cu(ibp)] complex.
Ibuprofen
[Cu(ibp)] complex
Assignment


—


∼ –
π- π∗ transitions
Metal- Ligand charge
transfer
—
∼ 
d-d transition
Table . Principal FT-IR absorption bands for ibuprofen and [Cu(ibp)] complex.
Ibuprofen
[Cu(ibp)]
Assignment
– w
–m-w
—
νOH(HO); νOH(COOH)
νasym. CH,CH sym.
CH,CH
– m-w
vs
—
 m-w
 m-w
 m
—
. vs
 w
 w
 m
νC=O(COOH)
νasym. (COO)
νC-C ring str.
νC-C ring str.
νsym. (COO)
–. m-w
–w
asym. CH,CH,CH
sym. CH,CH,CH
i.p. C-H (ring)
i.p. C-H (ring)
o.p. C-H (ring)
o.p. C-H (ring)
o.p. C-H (ring)
i.p. CH (ring)
νCu-O
. w
 w
 m
 w
. m
 w
—
w
vw
—
 vw
 vw
—
. w
s = strong, v = very, m = medium, w = weak, str = stretching, asym = asymmetric, sym = symmetric, i.p. = in planer,
o.p. = out of planer

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
shows two very strong bands at 1587 cm−1 and 1407 cm−1 which were assigned
to ν(COO) asymmetric and symmetric stretches, respectively, with an average δν
of ∼180 cm−1, indicating bidentate coordination of carboxylate. Coordination via
oxygen of carboxylate is confirmed by ν(Cu–O) band at 553.6 cm−1. Thus, ibupro-
fen is coordinated through oxygen of carboxylates. Elemental analysis also confirms
the synthesis of the complex as, C52H68Cu2O8Calc.: C; 70.98 and H; 7.73. Found:
C, 71.87 and H, 7.43.
Apparatus
The UV−visible absorption measurements were obtained with an Agilent 8453
spectrophotometer, using a 1.0 cm cell. Fluorescence measurements were carried
out with a JASCO spectrophotometer (FP 6200). Viscosity measurements were per-
formed in a viscometer (SCHOT AVS 450), which was kept in a constant temper-
ature bath at 29 0.2°C. CD measurements were recorded on a JASCO (J-810)
spectrophotometer. The elemental analysis was performed using a Heraeus CHN
elemental analyzer. The pH values were measured with a pH meter (Jenway Model:
3020) supplied with a glass-combined electrode.
UV–visible absorption spectral measurements
The UV–vis absorption spectra of ct-DNA in the absence and presence of
[Cu(ibp)2]2 complex and the absorption spectra of corresponding concentrations
of [Cu(ibp)2]2 solutions were measured in the range of 200–350 nm and pH 7.4 in
Tris–HCl buffer solution at room temperature.
Fluorescence emission spectral measurements
The fluorescence intensity was measured with the excitation and emission wave-
lengths at 265 and 292 nm, respectively at 283, 298, and 315 K. The spectra band
widths of excitation and emission slits were both kept at 5.0 nm. To eliminate the
probability of reabsorption and inner-filter effect arising from UV−vis absorption
of ct-DNA, the fluorescence data of [Cu(ibp)2]2 complex were corrected for absorp-
tion of excitation and emitted light according to Eq. 1[18,19]
+ A
)
F = Fm10(A
,
(1)
1
2
2
c
where Fc and Fm are the corrected and measured fluorescence, respectively, and
A1 and A2 are the absorbance of the ct-DNA solutions at 265 and 292 nm,
respectively.
Competitive experiments
The Uv-vis and fluorescence spectra of the competitive reaction between
[Cu(ibp)2]2 complex and the probes (MB and Hoechst) with ct-DNA were
performed in Tris–HCl buffer solution (pH 7.4) at room temperature. This part of

6
N. SHAHABADI AND F. SHIRI
experiment was carried out by titration of constant amounts of the probes and ct-
DNA with increasing amounts of [Cu(ibp)2]2 complex solution and analysis of the
changes in absorbance and fluorescence intensities.
Circular dichroism measurements
The CD spectra of DNA in the presence of [Cu(ibp)2]2 complex were conducted
in the wavelength range of 200–350 nm by keeping the concentration of ct-DNA
constant at 4.81 × 10−5 mol L−1 while varying the [Cu(ibp)2]2 concentration from
0 to 5.76 × 10−5 molL−1.
Viscosity measurements
Viscometric measurements were carried out using a viscometer, which was kept at
(29 0.2)°C by a constant temperature bath. The solution of (DNA + [Cu(ibp)2]2)
was prepared in Tris–HCl buffer and the flow times of ct-DNA alone and its mix-
tures with different ratios of [Cu(ibp)2]2 to ct-DNA through the capillary were then
measured in seven replicates using a digital stopwatch with an accuracy of 0.02 s.
Docking simulation
Docking simulation was performed using MGL tools 1.5.4 with AUTOGRID 4 and
AUTODOCK version 4.2. The DNA sequence obtained from the Protein Data Bank
(PDB NDB: DD0018) and the complex was optimized by SPARTAN 10 program
and both the complex and DNA were prepared for using in AUTODOCK tools.
The DNA was enclosed in a box with number of grid points in x × y × z directions,
40 × 40 × 80.
Results and discussion
UV–Vis absorption spectroscopy
The application of electronic absorption spectroscopy is one of the most use-
ful techniques for studying both the stability of DNA and the interactions with
molecules.[20]
The binding of metal complexes to DNA helix has been characterized through
absorption spectral titrations by following the changes in absorbance and shift in
wavelength.[21] The spectral change process reflects the corresponding changes in
the helix structure and the steric configuration of DNA after a compound binds to
DNA.
The UV spectra of DNA at about 260 nm in the absence and presence
of [Cu(ibp)2]2 complex is shown in Figure 3. The absorption band exhibits
hyporchromism without any clear shifts in the band position, suggesting that there
exists an interaction between the [Cu(ibp)2]2 complex and ct-DNA, which indi-
cates formation of an adduct between DNA and [Cu(ibp)2]2 and change in DNA

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Figure . Changes of UV spectra of ct-DNA in the presence of different concentrations of [Cu(ibp)]
complex at pH . and room temperature. UV-Vis spectra of ibuprofen (ligand) and [Cu(ibp)] com-
plex at different concentrations as depicted in the insets of figures. (A) By titration; (B) Over  h at
ambient temperature.
conformation. The observed large hypochromism during the interaction of the
[Cu(ibp)2]2 complex with DNA strongly indicates that the distance between the
[Cu(ibp)2]2 complex and the DNA bases is small and suggests a strong interac-
tion between the electronic states of the chromophore and DNA bases (Figure 3B).
This hypochromism usually results in complex binding with DNA, through inter-
calation or groove binding mode. In intercalation binding mode, the π∗ orbital
of the intercalated molecule can couple with the π orbital of the DNA base
pairs, the coupling π - π∗orbital is partially filled by electrons, thus, decreas-
ing the transition probabilities and concomitantly resulting in hypochromism. In
the case of the groove binding mode between DNA and drug molecules, the
hypochromic effect can be associated with the overlapping of the electronic states
of the chromophore of the complex with the nitrogenous bases in the grooves of
DNA.[22]
A large part of our existing knowledge of noncovalent binding is based on the
measurement of binding constant. Binding constant afford the scientist information
on the mechanism of the chemical process involved. In general, the basic process

8
N. SHAHABADI AND F. SHIRI
can be distilled down to the association, or binding of one or more ligands to a host
molecule. A scheme for interaction between substrate and drug may be represented
by:
D + nd = dnD,
(2)
where n signifies the maximum number of binding sites on substrate (D or DNA),
d, denotes the drugs that are free to bind to each site and Dnd is DNA-drug complex.
Hence the binding constant (Kb) is:
[Dnd]
D d
[ ] [ ]
Kb =
.
(2a)
n
The binding constant and the number of [Cu(ibp)2]2 complex bound to one DNA
molecule has been developed before (n = 1).[23] It is obvious that all the binding sites
are independent and the Beer’s law is followed by all species. A wavelength is con-
sidered at which the molar absorptivity of the DNA (ε ) and the molar absorptivity
D
of the DNA-drug (εd-D) are not same. At whole concentration of the DNA (D0), and
the light path length (b), the solution absorbance is introduced as in the following
equation (in the absence of drug):
A = ε b D .
[ ]0
(2b)
0
D
As though the absorbance of a solution containing the same concentration of
whole DNAs, by role playing the total concentration of drug ([d]0) is obtained as
follows:
A = ε b D + ε b d + ε b d D ,
[ ] [ ]
(2c)
[ ]
D
d
d−D
n
where [D] is the concentration of the uncomplexed DNA, [d] the concentration of
the free drug, and [dnD] is the concentration of the formed complex. By applying
equation (2c) with the mass balance on [D] and [d], equation (2d) can be intro-
duced:
A = ε b D + ε b d + ꢀε b d D ,
[ ]0 [ ]0
(2d)
[ ]
D
L
d−D
n
And δεd−D = εd−D − ε - nε (ε , molar absorptivity of the drug). By evaluating
D
d
d
absorbance of the solution against a blank containing whole concentration of drug
[d]0, the calculated absorbance is given as:
A = ε b D + ꢀε b d D .
[ ]0
(2e)
[ ]
D
d−D
n
By combining Eq. (2a) and Eq. (2b) with Eq. (2e), whereas δA = A−A0 and A0 is
the absorbance of DNA at 260 nm in the absence of [Cu(ibp)2]2:
n
ꢀA = Kbꢀε b D d .
(2f)
[ ] [ ]
d−D
From this mass balance equation, [D]0 = [D] + [Dnd],

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Table . The Kb, n, and other parameters in Eq. (h) by curve fitting on this equation.
1
A
1
d−D
1
d−D
model
=
+
n
ꢀε
[D]
K ꢀε
[D] [d]
0
0
b
R-square
.
Kbꢀεd−D[D]0
−.E + 
−.
.
ꢀεd−D[D]0
N
Kb
.E +  (L.mol−
)
we get [D] = [D]0/(1 +Kb[d]n):
n
ꢀA
=
Kbꢀε
D
d
d−D[ ]0[ ]
.
(2g)
n
b
K d + 1
b[ ]
The binding isotherm (Eq. 2g) can be introduced which shows the hyperbolic
dependence on not-bonded drug concentration. The Kb and n can be estimated
from the following equation that is 1/(Eq. 2g):
b
ꢀA
1
1
=
+
.
(2h)
n
Kbꢀε
D
d
ꢀε
D
d−D[ ]0[ ]
d−D[ ]0
This equation only yields the binding stoichiometry when there is only one bind-
ing site or in the case of infinite cooperativity; i.e., the occupation of one bind-
ing site favors ligand association. In the otherwise, n is just a phenomenologi-
cal parameter (the Hill coefficient), that is a qualitative measure of the degree of
cooperativity and it is experimentally less than the actual number of binding sites
present.[19,24]
The Kb and n for the [Cu(ibp)2]2–DNA complex were obtained from the opti-
cal absorption at 260 nm at the various [Cu(ibp)2]2 complex concentrations, by the
plotting of 1/ (A–A0) versus C[Cu(ibp)2]2 and curve fitting by use of MATLAB software
(Table 3 and Figure 4).
Figure . Representative binding curve for effect of various [Cu(ibp)] concentrations on ct-DNA
absorption at  nm.

10
N. SHAHABADI AND F. SHIRI
Fluorescence studies
Determination of binding stoichiometry by the job’s plot
To realize the correctness of the above discussion and that really n is near to one, the
continuous variation method was used (or Job plot), that is one potential method
to determine binding stoichiometry.[24] In this method, the total molar concentra-
tion [DNA+ Cu(II) complex] is held constant, but their mole fractions are varied.
A measurable parameter, that is proportional to formed complex concentration, is
plotted against the mole fraction of one of these two components. A special point
is determined from the plot. The binding stoichiometry is calculated from the mole
fraction of DNA and Cu(II) complex at that specific point.[25]
In this case, fluorescence intensity of the complex was selected as the parameter
that is proportional to the complex concentration. The relation between the Cu(II)
complex concentration and the fluorescence intensity can be described by:
Cu
Cu
F
F0
[
[
]
t
=
]
0
,
(3)
where [Cu]0 is the total [Cu(ibp)2]2 concentration and [Cu]t the concentration
of uncomplexed[Cu(ibp)2]2. Defining the concentration of the formed complex as
[D−Cu] = [C]0−[C]t and it can be shown using Eq. 4:
F0 − F∗
D − Cu =
Cu .
(4)
[
]
[
]
0
F0
The plot of [D-Cu] versus mole fraction of [Cu(ibp)2]2 (The Job plot) is shown
in Figure 5. The obtained results, presented in Figure 5, show that the maximum of
the Job’s curve occurs at mole fraction of [Cu(ibp)2]2= 0.5, indicating the existence
of a complex with a 1:1 stoichiometry and n = 1.
Fluorescence quenching studies
To elucidate the mode of binding, fluorescence quenching of DNA bound
[Cu(ibp)2]2 complex was studied in relation to the fluorescence quenching of the
Figure . Job’s plot for DNA–Cu(II) complex. [D–Cu] versus mole fraction of Cu(II) complex at total
concentration [DNA + Cu(II) complex] = . × − mol.L−.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Figure . Emission spectra of the copper(II)–ibuprofenato complex in Tris-HCl buffer at °C in the
absence and presence of ct-DNA at different concentrations. C Cu(II) complex = . × − mol.L−, CDNA
= . to . × − mol.L−. The inset of figure shows [Cu(ibp)] fluorescence intensity versus
CDNA/ C Cu(II) complex
.
drug in pH 7.4 Tris–HCl buffer solution. Figure 6 shows the characteristic changes
in fluorescence emission spectra during the titration of [Cu(ibp)2]2 complex with
ct-DNA. Upon subsequent addition of ct-DNA there is a gradual quenching of the
fluorescence intensity without any significant change in emission maxima which is
direct evidence of the interaction between [Cu(ibp)2]2 complex and ct-DNA.[26]
The fluorescence quenching effect might result from a variety of processes such
as excited state reactions, ground state complex formations, and collisional pro-
cesses. Quenching can occur by different mechanisms which are usually classified
as dynamic and static quenching. The static quenching was due to the formation of
ground state complex between fluorophores and quencher and the dynamic quench-
ing was due to the process in which the fluorophore and the quencher come into
contact during the transient existence of the exited state. Because, the collisional
quenching or dynamic quenching resulted from the collisions between fluorophores
and quencher, it could be mathematically expressed by the Stern–Volmer equation
(eq. 3)[27]
:
F0
F
= 1 + K τ Q = 1 + K Q ,
0 [ ] sv [ ]
(5)
q
where F0 = the initial fluorescence intensity of copper(II)–ibuprofenato complex,
F= the fluorescence intensity of [Cu(ibp)2]2 complex after the addition of the
quencher (DNA), Kq is the apparent bimolecular quenching rate constant, Ksv the
Stern–Volmer quenching constant which is considered to be a measure of efficiency
of fluorescence quenching by DNA, τ is the average lifetime of [Cu(ibp)2]2 com-
0
plex without the quencher[28] and [Q] is the concentration of the quencher, the Ksv
can be obtained by the slope of the plot of F0/F versus [Q] (Figure 7).

12
N. SHAHABADI AND F. SHIRI
Figure . Plots of F/F versus [Q].
Dynamic and static quenching can be distinguished by their different dependence
on temperature. Dynamic quenching depends on diffusion. Since higher tempera-
tures result in larger diffusion coefficients, in the dynamic quenching process, the
elevated temperature is conducive to effective collision between molecules. So the
quenching rates accelerate, Kq or Ksv increases. In contrast, an increase in tempera-
ture is likely to result in a decrease in the stability of complexes, and thus, lower the
values of the static quenching constants.[29]
Also, the limiting diffusion rate constant of biomolecule is known to be around
2.0 × 1010 M−1S−1, thus if the value of Kq is higher than the limiting diffusion rate
constant, the quenching process is static rather than dynamic.[30]
In this work, the Stern–Volmer plot is linear, indicating that only one type of
quenching process occurs, either static or dynamic quenching.[31] The results in
Table 4 indicate that Ksv has been increased with increasing temperature (dynamic
quenching) and Kq is higher than the limiting diffusion rate constant (static quench-
ing). Thus, UV–vis absorption spectrum was used to give some more evidences for
the actual quenching process.
Dynamic quenching only affects the excited state; no changes are expected in the
ground state. Alternatively, a static process only involves complex formation in the
ground state.[32] As shown in Figure 3 there is some changes in the UV–vis spectra of
the DNA in the presence of various concentrations of the complex, so an adduct of
DNA and copper complex forms in static quenching, whereas dynamic quenching
has no such changes and UV spectra of DNA would have no detectable changes if the
quenching was a dynamic mechanism.[33] Therefore, according to the UV spectra,
Table . The quenching constants of copper(II)–ibuprofenato complex by ct-DNA at different
temperatures.
T (K)
KSV (×  L.mol−
)
Kq (×  L.mol−
)
R
283
298
310
.
.
.
.
.
.
.
.
.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
Table . Binding parameters of ibuprofen–Cu(II) complex with DNA at different temperatures.
T(K)
Fc(a.u.)
Kd (× − mol.L−
)
Kf(×  L.mol−
)
R
283
298
310
.
.
.
.
.
.
.
.
.
.
.
.
the fluorescence quenching of the complex in this case seems to be primarily caused
by complex formation between the copper(II)–ibuprofenato complex and ct-DNA
and confirming that the quenching process is static rather than dynamic.
Determination of the binding constant
The binding constant (Kf) for the complex formation between [Cu(ibp)2]2 complex
and ct-DNA were measured according to the published method,[24] using the Eq. 6
that is most generally valid equation to analyze fluorescence changes upon forma-
tion of a 1:1 complex.
ꢀ
ꢁ
ꢂ
Cu + D + K −
[ ]t
Cu + D + K 2 − 4∗ Cu ∗ D
[
]
t
[
]
t
[ ]t
[
]t [ ]t
d
d
F0 − F
F0 − F
=
(6)
(7)
2∗ Cu
[
]
c
t
1
Kf =
,
Kd
where F is the measured fluorescence, F0 the fluorescence in the absence of the DNA,
Fc the fluorescence of the fully complexed [Cu(ibp)2]2, Kd the dissociation constant,
[D]t the concentration of added ct-DNA and [Cu]t the concentration of [Cu(ibp)2]2,
In the following, the values of Kd, Kf, and Fc were obtained for different temperatures
by fitting the experimental data with Eq. 6. The obtained results are presented in
Table 5.
The binding constants calculated for the [Cu(ibp)2]2-DNA are comparable with
those of the K values estimated by Stern–Volmer (discussed above) and suggest high
affinity of [Cu(ibp)2]2-DNA binding by increasing in temperature.[34–36]
Thermodynamic parameters of DNA binding
The interaction forces between drug and biomolecule may involve hydrophobic
forces, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc.
According to the data obtained from enthalpy changes (ꢀH°) and entropy changes
(ꢀS°), the model of interaction between a drug and biomolecule can be con-
cluded[37]
:
(a) ꢀH°> 0 and ꢀS°> 0, hydrophobic forces; (b) ꢀH°< 0 and ꢀS°< 0, van
der Waals interaction and hydrogen bonds; (c) ꢀH°< 0 and ꢀS°> 0, electrostatic
interactions.[38]
If the temperature does not vary significantly, the enthalpy change can be
regarded as a constant. By utilizing the Vant–Hoff relation,[39] which describes the
dependence of equilibrium constant on temperature, thermodynamic parameters of

14
N. SHAHABADI AND F. SHIRI
Figure . Plot of ꢀG (ꢀG = −RTLnKf) versus T.
equilibrium have been determined:
ꢀH◦
RT
ꢀS◦
R
LnKf = −
+
(8)
(9)
ꢀG◦ = ꢀH◦ − TꢀS◦ = −RTLnKf ,
where Kf is the binding constant at the corresponding temperature, ꢀH° is the molar
enthalpy change, ꢀS° is the molar entropy change, R = 8.31 J.mol−1K the universal
gas constant, and T is the Kelvin temperature. A linear regression of ꢀG° versus T
is shown in Figure 8, which determines enthalpy and entropy change of the reaction
from the slope and intercept of the linear plot. The results show that ꢀH°>0 and
ꢀS°> 0 (Table 6), therefore, hydrophobic forces are probably the main forces in the
binding of the complex to ct-DNA.
Competitive binding studies with methylene blue and hoechst 33258
Methylene blue as classical intercalator[40,41] and Hoechst 33258 as groove binder[42]
probes were used, respectively to clarify the nature of the interaction between
[Cu(ibp)2]2 and DNA.
Initially, with the addition of [Cu(ibp)2]2 to a solution of DNA-Hoechst complex,
some Hoechst molecules were released into the solution after displacing with the
[Cu(ibp)2]2, so result was fluorescence quenching (Figure 9), this assert the view
that [Cu(ibp)2]2 could interact as a groove interaction.
Table . Thermodynamic parameters for the binding of copper(II)–ibuprofenato complex to ct-DNA.
T (K)
ꢀG° (×  J.mol−
)
ꢀH° (×  J.mol−
)
ꢀS° (J.mol−K−
)
283
298
310
−.
−.
−.
.
.
.
.
.
.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Figure . Fluorescence spectra of the competition between [Cu(ibp)] and Hoechst . CDNA
=
. × − mol.L−, C Hoechst = . × − mol.L− , C Cu(II) complex = ., ., ., ., ., .,
. and . × − mol.L−.
But it was later shown that the dominant binding mode was not only groove
interaction. The significant change in the fluorescence intensity of the DNA-
methylene blue complex with adding [Cu(ibp)2]2 indicated that MB molecules were
released from the DNA helix after addition of the [Cu(ibp)2]2. So, binding mode of
[Cu(ibp)2]2 and DNA is an intercalative mode, too (Figure 10).
To find out that the changes in fluorescence intensity is only resulted of releasing
probe molecules into the solution after displacing with the [Cu(ibp)2]2 complex and
are not reason of: (1) the binding between [Cu(ibp)2]2 complex and probe molecules
and (2) formation aspecial complex with fluorescence emission and increase in flu-
orescence intensity of the system. This theory was examined by using Uv-vis and
fluorescence spectroscopy. Since there are not any changes in the fluorescence inten-
sities of MB and Hoechst upon adding [Cu(ibp)2]2 when DNA is absence, the first
reason is not true. For testing the second reason UV-vis spectroscopy was used. If
Figure . Fluorescence spectra of the competition between [Cu(ibp)] and MB. CDNA = . × −
mol.L−, C MB = . × − mol.L−, C Cu(II) complex = ., ., ., ., ., ., ., ., .,
., ., ., ., and . × − mol.L−.

16
N. SHAHABADI AND F. SHIRI
Figure . Absorption spectra of MB in the presence of different DNA concentrations.
the increase in fluorescence intensity of MB-DNA system is due to the formation of
acomplex with fluorescence emission, absorption spectra of the MB-DNA should
not be changed upon adding [Cu(ibp)2]2 complex.
The absorption spectra of the MB dye upon addition of DNA are shown in
Figure 11. It is apparent from this figure that the absorption peak of the MB at around
650 nm showed gradual decrease with the increasing concentration of DNA, and a
new band at around 700 nm developed. This was attributed to the formation of the
new DNA-MB complex. An isobestic point at 683 nm confirmed DNA-MB complex
formation.
As shown in Figure 12 upon addition of [Cu(ibp)2]2 complex the spectra exhib-
ited the reverse process. The observed changes in the absorption spectra with
increasing amounts of [Cu(ibp)2]2 to the DNA-MB complex solution suggest that
the addition of complex induces conformational changes on DNA, which lead to the
free form of MB.
Figure . Effect of the [Cu(ibp)] complex on the absorption spectra of MB-DNA.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
So, with the addition of copper(II)–ibuprofenato complex into the solutions of
DNA-MB complex and DNA-Hoechst complex, the MB and Hoechst molecules
were released into the solutions after displacing with the [Cu(ibp)2]2 and results are
changes in the fluorescence intensities. This asserts that [Cu(ibp)2]2 could interacts
as both groove and intercalation interaction.
As to the unexpected and adverse binding mode, it may be concluded that an
unusual DNA binding mode is existed and could be explained by a special type of
binding mode, like partial intercalation or a special type of binding with a partially
intercalation portion and the same in the groove portion[43,44]; an unusual DNA
binding mode with significantly slower association and dissociation rates compared
with classical intercalation. In this interaction the situation is obviously different
than the penetration by ordinary intercalators in the bound state, a part of the
molecule is partially intercalated between the base pairs, and the bulky substituents
are located one in each groove. To reach this final state the propeller-like bulky
end of this molecule must insert through the DNA base pairs, which transiently
requires the opening of base pairs, i.e., local DNA melting, to an unknown extent.
[Cu(N-N)(L)(EtOH)] (NO3)2. 2H2O,[45] and bisantrene[46] fall in this category of
interaction.
Viscosity measurements
In addition to spectroscopic data, viscosity experiment was carried out which is
regarded as less ambiguous and the most critical tests of DNA binding model in solu-
tion.[47] Generally, a classical intercalation binding causes an increase in the viscos-
ity of DNA solution because it demands a large enough space of adjacent base pairs
to accommodate the ligand and to lengthen the double helix. However, a partial or
nonclassical intercalation of the ligand may bend or kink the DNA helix, resulting in
a decrease of its effective length and concomitantly, its viscosity.[48] In contrast, there
is little or no increase of DNA viscosity if electrostatic or groove binding occurs in
the binding process.[49]
The relative viscosity of DNA in the absence and presence of [Cu(ibp)2]2 complex
were calculated from the Eq. 7:
t − t0
η =
,
(10)
t
where t0 and t were the observed flow time in the absence and presence of
[Cu(ibp)2]2 complex. Data has been presented as (η/η ) versus 1/R (R= [copper
0
complex]/[DNA]), where η is the viscosity of DNA in the presence of the complex
and η is the viscosity of DNA alone (Figure 13).
0
As shown in Figure 13 the viscosity of DNA remarkably decreased upon the addi-
tion of [Cu(ibp)2]2 concentration. Such behavior is consistent with bending or kink-
ing of the helix upon binding copper(II)–ibuprofenato complex to DNA. The results
suggest that [Cu(ibp)2]2 complex may bind with a nonclassical intercalative mode
like partial intercalation.

18
N. SHAHABADI AND F. SHIRI
Figure . The effect of increasing amounts of [Cu(ibp)] on the viscosity of ct-DNA; CDNA=  × −
mol.L−, C Cu(II) complex = ., ., ., ., ., and . × − mol.L−.
Circular dichroic (CD) spectral studies
It is well known that the circular dichroism (CD) is very sensitive to the environment
of the metal complexes, so to establish in more detail whether binding of the com-
plex brings about any significant conformational change of the DNA double helix,
CD spectra of ct-DNA were recorded. The changes in CD signals of DNA observed
on interaction with molecules may be assigned to the corresponding changes in
DNA structure, as a positive band at 275 nm from base stacking and a negative
band at 246 nm from helicity of DNA are quite sensitive to the mode of DNA
interactions with molecules.[50] The effect of increasing amounts of copper(II)–
ibuprofenato complex on the conformation of the structure of DNA was studied
(Figure 14).
As shown in Figure 14, in the presence of the [Cu(ibp)2]2 complex, both the pos-
itive and the negative bands decreased. The observed decreases in the positive and
negative bands are consistent with a conformational change in DNA. The observed
reduction in the positive DNA dichroic signal is likely due to a transition from the
extended nucleic acid double helix to the more denatured structure.[51,52] These
Figure . CD spectra of DNA in the presence of increasing amounts of [Cu(ibp)] complex.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
Figure . Moleculardockedmodelsofenergy–minimizedstructureof[Cu(ibp)] complexwithDNA.
changes correlate with helix unwinding and can be associated with a kind of inter-
calative action by the [Cu(ibp)2]2 complex.[53] Also it should be noted that, remark-
ably reductions in molar ellipticity in the negative band (245 nm) are related to
destabilization and helix unwinding.[54,55]
In assumed binding mode, a part of the complex is partially intercalated between
the base pairs, and other substituents are located in the groove position, so both
signals of base stacking and helicity at 275 nm and 246 nm should be changed.
Therefore, these changes in CD signals are in agreement with the proposed mode of
interaction.
Docking simulation results
Molecular docking techniques are well-documented computational tools to under-
stand the Drug–DNA interactions for structure-based drug design and discovery,
as well as mechanistic examination by replacing a small molecule into the bind-
ing site of the target region of the DNA highly in a non-covalent mode.[56,57] Here,
molecular docking studies of the complex were carried out with DNA duplex to
search the proper binding site. The energetically most favorable conformation of
the docked pose is picked up from the 50 runs. As a result of these interactions,
it was clear that the complex was located in both groove and intercalation sites of
double-helix DNA (Figure 15). This again asserts that [Cu(ibp)2]2 could interacts as
both groove and intercalation modes that is in agreement with the proposed mode of
interaction.

20
N. SHAHABADI AND F. SHIRI
Table . Binding constants (Kb) of some of the copper complexes which studied previously.
Complex
Kb / L.mol−
. × 
. × 
. × 
Ref.
[]
[]
[]
[CuL]Cl L = ,-bis(- hydroxyethyl)-,,,,,
hexaazacyclotetradecane
[CuL]Cl L = ,-bis(-propionitrile) - ,,,,,
hexaazacyclotetradecane
[Cu(L)(HO)](ClO) L =
N-(furan--ylmethyl)--pyridinecarboxamide
[Cu(N-N)(L)(EtOH)] (NO). HO
. × 
[]
[]
[Cu(L)(HO)](NO) L =
. × 
N-(thiophen--ylmethyl)--pyridinecarboxamide
Outlook
Conclusion
One goal in the study of molecules that target the reproduction of tumor cells is
the investigation on complexes which have a high affinity to DNA, as well as slow
dissociation from DNA. Based on the results mentioned above it was found that
copper(II)–ibuprofenato complex molecules could bind with DNA by both groove
and intercalation modes, something likes a partial intercalation interaction with
the following properties: (I)Time consumption and temperature dependence of the
interaction (there are more changes in the UV–visible absorption spectra during
the experiment and there is an increase in the binding constant with the tem-
perature increasing). (II) Temperature stable interaction (free energy changes are
decreased with increasing temperature). (III) A part of the complex is partially inter-
calated between the base pairs, and other substituents are located in groove positions
(Whole of the MB (classical intercalator) and Hoechst (groove binder) molecules
were released into solution after displacing with the [Cu(ibp)2]2 complex which
indicates blocking of both locations by copper ibuprofenate complex). As well as
the results of CD spectroscopy and Docking simulation prove exists of both groove
and intercalation modes. In addition of high desire to interaction with DNA by this
complex, because of high equilibrium constant (6.19 × 104 M−1)in contrast to other
copper complexes, (Some of these complexes are shown in Table 7[45,58,59]),it also has
a new binding mode with DNA which has the properties of both the intercalation
and groove binding modes. Therefore this complex can occupy both the active site
of DNA namely groove and intercalation sites and can deeply affect DNA. These
results again can reveal that this complex is more effective than others copper com-
plexes with just groove[60–62] or intercalation[59,63] mode of bindings and can be a
new direction in the research of DNA interaction, cancer therapy and molecular
biology.
Acknowledgments
This work was supported by Razi University of Kermanshah-Iran.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
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