DNA binding, DNA cleavage and HSA interaction of several metal complexes containing N-(2-hydroxyethyl)-N′-benzoylthiourea and 1,10-phenanthroline ligands

Article abstract of DOI:10.1007/s00775-016-1388-1

Abstract: Four novel ternary metal complexes of the type [M(Phen)(L₁)₂)] [phen?=?1,10-phenanthroline, L₁?=?N-(2-hydroxyethyl)-N′-benzoylthiourea, M?=?Ni(II)(1), Co(II) (2), Cu(II) (3), Pd(II) (4)] were synthesized. The org

Full text of DOI:10.1007/s00775-016-1388-1

J Biol Inorg Chem
DOI 10.1007/s00775-016-1388-1
ORIGINAL PAPER
DNA binding, DNA cleavage and HSA interaction of several metal
complexes containing N‑(2‑hydroxyethyl)‑N′‑benzoylthiourea
and 1,10‑phenanthroline ligands
1
1
3
2
1
1
Bo Peng · Zhuantao Gao · Xibo Li · Tingting Li · Guorong Chen · Min Zhou ·
3
Ji Zhang
Received: 2 March 2016 / Accepted: 20 August 2016
©
SBIC 2016
Abstract Four novel ternary metal complexes of the
type [M(Phen)(L ) )] [phen = 1,10-phenanthroline,
of the complexes to human serum albumin (HSA) was
also investigated. Agarose gel electrophoresis study
revealed that the metal complexes could cleave super-
coiled pBR322 DNA to a nicked form in the absence of
external agents. In vitro anti bacterial studies show that
copper complex has weak antibacterial activities. Cop-
per complex exhibits a better biological activity among
all complexes. This study provides a new perspective
and evaluation on the role and importance of the effect
factors on the medicinal properties of benzoylthiourea
compounds.
1
2
L = N-(2-hydroxyethyl)-N′-benzoylthiourea, M = Ni(II)
1
(
1), Co(II) (2), Cu(II) (3), Pd(II) (4)] were synthesized.
The organic ligands and their corresponding organome-
tallic complexes have been characterized using UV–vis
absorption spectroscopy, element analysis, infrared radia-
tion spectroscopy and fluorescence spectra. DNA binding
and cleavage studies of these complexes were conducted
in detail. In vitro DNA-binding properties were studied
by electronic absorption spectra and fluorescence spectra
methods. The results indicate that all of the ternary metal
complexes can efficiently bind to DNA via intercalation
mode. The DNA-binding constants for all ternary com-
Graphical abstract Synchronous fluorescence spectra of
HSA (10 μM) as a function of concentration of the com-
plexes 1–4.
6
−1
pounds are around 4 × 10 M . The binding propensity
*
Bo Peng
pengbo.nwnu@hotmail.com
1
Key Laboratory of Bioelectrochemistry and Environmental
Analysis of Gansu Province, Key Laboratory
of Eco-Environment-Related Polymer Materials, Ministry
of Education, College of Chemistry and Chemical
Engineering, Northwest Normal University, Lanzhou 730070,
China
2
3
Food Test Center of Jiuquan, Jiuquan 735000, China
College of Life Science, Northwest Normal University,
Lanzhou 730070, China
1
3

J Biol Inorg Chem
Keywords Thiourea · Phenanthroline · Complex · DNA ·
Antibacterial activities
derivatives for Cu [14, 15], Co [15], Ni(II) [16, 17], Pd [18,
19], Pt [ [20–22] and Ru [23] attracted a wide attention to
the researcher for their diverse range of antibacterial, anti-
fungal, and anticancer activities. As an universal chelating
ligand scaffold, 1,10-phenanthroline and its derivatives
were widely reported having the ability to act on antifungal
drugs [24, 25] even cytotoxic drugs [22, 26]. Metal com-
plexes with phenanthroline ligand have strong ability of
binding and cleaving DNA [27–29].
Abbreviations
HSA
DNA
Human serum albumin
Deoxyribose nucleic acid
CT-DNA Calf-thymus DNA
DMSO
BSA
Dimethyl sulphoxide
Bovine serum albumin
Tris-HCl Tris(hydroxymethyl)aminomethane
EB Ethidium bromide
Nucleic acid is one of the main compositions of life.
DNA regulates many biochemical processes occurring in
the cellular system. Therefore, understanding and inves-
tigation of the interaction and mechanism of metal com-
plexes binding with nucleic acid (DNA/tRNA) could be a
more robust finding out of the origin of life on a molecular
level and are particularly important for developing of new
chemotherapy drugs in biological science. It is expected to
design different ligands and metal complexes which could
fit well in DNA binding, cleavage, as well as interactions
with the certain protein which is the main cellular target for
anticancer drugs. The interaction of drug with human serum
albumin (HSA) and bovine serum albumin (BSA) will pro-
vide important information on the absorption, distribution,
Introduction
Thiourea and substituted thioureas [1] are extensively
studied because they are a rich source of materials for the
development of agrochemical and pharmaceutical prod-
ucts. Many of their important applications are plant-growth
regulators [2, 3], herbicides [4, 5], insecticides [6], antifun-
gal [7], antibacterial [3, 8], anti-HIV [9], anti tuberculosis
and pharmacodynamics [10–12], protein inhibitors [13],
etc. Recently, complexes containing substituted thiourea
1
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J Biol Inorg Chem
activity and toxicity of a specific drug. In our previous
works, we prepared a series of N-benzoyl-N′-arylthiourea
derivatives and their binary metal (Cu, Pt, Pd) complexes.
The antibacterial properties of the ligands and complexes
against Gram-positive and Gram-negative bacteria were
investigated [30, 31]. The incorporation of transition metal
center into such structures can not only be used to impart
cationic charge, but also to confer new properties on these
molecules [22]. Some acylthiourea binary metal complexes
frequently were synthesized. They usually exhibit cyto-
toxicity against tumour cells, and are more cytotoxic than
the free ligands [13, 14, 20, 22, 23]. However, the practical
result for ternary metal complexes involving benzoylthi-
ourea derivatives has rarely been reported.
form (Form II) was major components was purchased from
Dingguo Changsheng Biotechnology Company of Beijing
(China). Agarose (molecular biology grade) and ethidium
bromide were obtained from Zhongqin chemical company
of Shanghai (China). 10 mM Tris–HCl/10 mM NaCl buffer
solution being used for DNA and HSA binding or pBR322
DNA cleavage (1 μg/μL, 4631 bp) was prepared using
ultrapure water. Tris–acetate–EDTA (TAE) buffer was used
for DNA cleavage study. All of the absorption spectra were
performed using a UV–Vis Spectrophotometer (T6, Beijing
Puxi Instrument Co., Beijing, China) with 1.00 cm quartz
cuvettes. The fluorescence spectra of the compounds were
recorded in solution on a Shanghai F-280 fluorescence spec-
trophotometer (F-280, Shanghai Analytical Instrument Fac-
tory, Shanghai, China). Vibration spectra were performed on
FT–IR–double–beam model IR spectrophotometer (Tianjin
Guangdong Science and Technology Development Limited
Company, Tianjin, China) with samples prepared as KBr
pellets.
To further investigate the effect on the medicinal and
biological properties of the ternary metal (Co, Ni, Cu,
Pd) complexes with benzoylthiourea series, we synthe-
sized four kinds of [M(Phen)(L ) ] complexes using N-(2-
1
2
hydroxyethyl)-N′-benzoylthiourea and 1,10-phenanthro-
line as the ligands. Four different metals were introduced
to contrast and compare the difference of their biological
activity. In addition, the interaction of these complexes
with HSA, cleavage activity with pBR322 plasmid DNA by
agarose gel electrophoresis method. DNA-binding profile
and antibacterial activity were studied in vitro. The results
demonstrated that all of the ternary complexes could inter-
calate with Calf-Thymus DNA (CT-DNA) and bind to HSA
in different degree. The ternary complex of copper exhib-
its a better biological activity out of four metal compounds
synthesized. The aim of this study is to provide a new per-
spective and evaluation on role and importance of the effect
factors on the medicinal properties of benzoylthiourea and
transition metal. The incorporation of thioureas derivatives
or phen as ancillary ligands in mixed compounds is a prom-
ising strategy to design new anticancer metal compounds
with intercalating properties.
Synthesis of N‑(2‑hydroxyethyl)‑N′‑benzoylthiourea
The benzoylthiourea ligands were prepared according to
the reported methods [30–32]. Benzoyl isothiocyanate
was obtained by the reaction of benzoyl chloride (1.410 g,
10 mmol) and ammonium thiocyanate (1.140 g, 15 mmol)
in CH Cl (20 mL) solution using polyethylene glycol-400
2
2
(0.180 g) as solid–liquid phase transfer catalyst. The ben-
zoyl isothiocyanate was then treated with ethanolamine
(0.610 g, 10 mmol) to give the objective compound. The
final solid was separated from the reaction mixtures by fil-
tration, washed with CH Cl , and followed by recrystallisa-
2
2
tion from ethanol then dried at 40 °C.
N-(2-hydroxyethyl)-N′-benzoylthiourea (L ): White nee-
1
dle crystal, m.p., 124–126 °C, and UV–Vis (acetonitrile
in solution): λmax (nm), 242, 280. Fluorescence spectros-
copy (DMSO in solution): λ (nm), 303, λem (nm), 337.
ex
−1
FT-IR (KBr): ν (cm ), 1670 (C=O), 1270 (C=S), 3332,
Materials and methods
3232(N–H).
Chemicals and instrumentation
The ligand of 1,10‑phenanthroline (phen)
All reagents used were of analytical grade unless otherwise
For white needle crystal, UV–Vis (acetonitrile in solution):
stated. Co(OO-CCH ) ·4H O and Ni(OO-CCH ) ·4H O
λ
(nm), 230, 264. Fluorescence spectroscopy (DMSO
3
2
2
3 2
2
max
were obtained from chemical industry company of Tianjin
in solution): λ (nm), 302, λem (nm), 337. FT-IR (KBr): ν
ex
(cm ), 1504 (ring), 1090 (C–H), 736 (C–H).
−
1
Kaixin. Cu(OO-CCH ) ·H O was purchased from chemical
3
2
2
industry company of Shanghai Jianxin. PdCl and Phen were
2
purchased from Damao chemical company of Tianjin. Calf-
Thymus DNA (an average size of 100 to 2000 base pairs,
Synthesis of the complexes
2
mg/ml, Shipping Condition: Dry ice, A260/A280 > 1.9,) was
Ternary complex of Ni, Co, and Cu (complex 1–3)
purchased from Solarbio Life Sciences. HSA was obtained
from Rongsheng Pharmacy Company of Chengdu (China).
Super-coiled pBR322 DNA in which open circular relaxed
1 mmol M(Ac)2 and 1 mmol phen were dissolved in
20 mL 50 % ethanol solution forming colored solution,
1
3

J Biol Inorg Chem
the solution was added to benzoylthiourea (2 mmol) 95 %
ethanol solution. The reaction mixture was heated at 40 °C
with constant stirring. Then, the resulting precipitate was
separated by filtration washing several times with water
and ethanol to afford pure and stable compounds.
to incubate for 10 min before each of measurements was
carried out. Absorption spectral traces and fluorescence
methods were used to determine the binding modes of the
complexes to Calf-Thymus DNA.
For fluorescence-quenching experiments, DNA was pre-
treated with ethidium bromide (EB) for 30 min. The M(II)
complex samples were added to this mixture, and then, the
emission intensity was measured. The emission intensity
was observed between 550 and 750 nm when the samples
were excited at 522 nm.
[
Ni(phen)(L ) ] (1): Purple powder, Anal. calcd for
1 2
C H N S O Ni: C, 55.90; H, 4.70; N, 12.23; S, 9.33.
3
2
32
6
2
4
Found: C, 55.49; H, 3.12; N, 12.66; S, 9.01 (%). UV–Vis
acetonitrile in solution): λmax (nm), 228, 270. Fluores-
cence spectroscopy (DMSO in solution): λ (nm), 376,
(
ex
−
1
λ
(nm), 410. FT-IR (KBr): ν (cm ), 1653 (C=O), 1221
em
(
C=S), 3426 (N–H).
Co(phen)(L ) ] (2): Red Powder, Anal. calcd for
DNA cleavage experiments
[
1
2
C H N S O Co: C, 55.51; H, 4.70; N, 12.22; S, 9.33.
For the gel electrophoresis study, super-coiled pBR322
DNA (1.0 μg/μL) was treated with the M(II) complex in
Tris–HCl buffer, pH 7.42. The solution was incubated for
1 h in a dark chamber at 37 °C. The samples were elec-
trophoresed 0.5 h at 80 V on a 0.9 % agarose gel in TAE
buffer. The gel was stained with 0.5 μg EB before the elec-
trophoresis. Bands were visualized by UV light and pho-
tographed. The extent of cleavage was measured using the
UVITEC Gel Documentation System [34].
3
2
32
6
2
4
Found: C, 56.67; H, 2.92; N, 13.21; S, 9.01 (%). UV–Vis
acetonitrile in solution): λmax (nm), 226,268. Fluorescence
spectroscopy (DMSO in solution): λex (nm), 374, λem (nm),
(
4
3
08. FT-IR (KBr): ν (cm-1), 1627 (C=O), 1222 (C=S),
428(N–H).
[
Cu(phen)(L ) ] (3): Snow blue powder, Anal. calcd for
1 2
C H N S O Cu: C, 55.88; H, 4.67; N, 12.14; S, 9.26.
3
2
32
6
2
4
Found: C, 55.13; H, 3.55; N, 11.46; S, 9.06 (%). UV–Vis
acetonitrile in solution): λmax (nm), 236, 264. Fluores-
cence spectroscopy (DMSO in solution): λ (nm), 376,
(
HSA‑binding experiments
ex
−
1
λ
(nm), 409. FT-IR (KBr): ν (cm ), 1596 (C=O), 1266
em
(
C=S), 3436 (N–H).
On additional of metal complex solution to 10 μM HSA
solution, the solution was determined from absorption
spectra at 278 nm using Tris–HCl buffer solution as ref-
erence. The HSA-binding study was also carried out with
excitation wavelength at 293 nm and emission wavelength
at 330 nm using a fluorescence spectrophotometer. The
excitation and emission slit widths, sensitivity, and scan
rates were constantly maintained for all the experiments.
Synchronous fluorescence spectra were recorded using
the same concentration of HSA and the complexes as men-
tioned above with two different ∆λ (difference between the
excitation and emission wavelengths of HSA) values, such
as 15 and 60 nm.
Ternary complex of Pd (complex 4)
The Pd(II) complex was prepared according to the reported
procedures [33]. The complex [Pd(phen)Cl ] was obtained
2
by the reaction of phen (1 mmol) with K [PdCl ] (1 mmol)
2
4
in methanol solution under reflux about 1 h. The precipi-
tated crystalline powders were recovered by filtration and
dried under vacuum for 2 h. The resulting complex was
suspended again in a water/methanol solution; the respec-
tive benzoylthiourea (1 mmol, L ) was then added to the
1
suspension under reflux. After 1 h, clear yellow to orange
solution was obtained. The solution was filtrated and the fil-
trates were kept for 3–5 days at room temperature for crys-
tallization. As a result, yellow–red crystals were obtained.
Antifungal assay
[
Pd(phen)(L ) ] (4): Orange solid, Anal. calcd for
The antibacterial test was done in vitro against one Gram-
negative bacteria standard strains (Escherichia coli) and
one Gram-positive standard bacteria strains (Staphylococ‑
cus aureus) using the agar well diffusion method [35, 36].
1
2
C H N S O Pd: C, 52.27; H, 4.40; N, 11.43; S, 8.72.
3
2
32
6
2
4
Found: C, 52.11; H, 3.63; N, 11.06; S, 8.46 (%). UV–Vis
methanol in solution): λ (nm), 236, 276. FT-IR (KBr):
(
max
−1
−3
ν (cm ), 1578 (C=O), 1260 (C=S), 3400 (N–H). Fluores-
Each M(II) complex and benzoylthiourea (1 × 10 M) in
cence spectroscopy (methanol in solution): λ = 277 nm,
dimethyl sulphoxide(DMSO)or methanol solution was pre-
pared for testing against spore germination of each fungus.
Filter paper discs (5 mm) were prepared using Whatman
filter paper No.1 (sterilized in an autoclave) which was sat-
urated with 10 μL of the compounds solution. The fungal
culture plates were incubated at 37 ± 0.2 °C for 24 h to
make sure that no contamination would occur in the dish.
ex
λ
= 577 nm.
em
DNA‑binding activity
The DNA-binding experiments were performed at room
temperature. M(II) complex-DNA solutions were allowed
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J Biol Inorg Chem
Fig. 1 a–d UV–Vis spectra of the complexes 1–4 in Tris–HCl buffer
g = 0.98, h = 1.14, and i = 1.30 μM. Arrow shows the absorbance
changes with increasing DNA concentration. Inset plots of [DNA]/
(ε − ε vs. [DNA] for the titration of complexes 1–4 with ct-DNA
−5
upon addition of Calf-Thymus DNA (a–i), [complex] = 2.0 × 10
M; a = 0.00, b = 0.17, c = 0.33, d = 0.50, e = 0.66, f = 0.82,
a
f)
Activity was determined by measuring the diameter of
complete inhibition zone (in mm).
Table 1 DNA-binding constant (Kb), quenching constant (Kq), and
apparent binding constant (Kapp) values
−1
−1
Kapp(M )
Complex
K (M−1)
K (M
)
b
q
6
6
6
6
5
5
5
5
5
5
5
5
1
2
3
4
4.21 × 10
4.07 × 10
3.99 × 10
3.85 × 10
3.32 × 10
2.75 × 10
2.84 × 10
3.68 × 10
1.94 × 10
1.77 × 10
1.60 × 10
2.28 × 10
Results and discussion
Characterization of ligands and metal complexes
IR spectroscopy
In the infrared spectra, the bands of the ring-stretching fre-
hydrogen bonding in benzoylthiourea. The bands observed
−1
−1
quencies [ν (C=C) and ν (C=N)] at 1504 and 1422 cm
of free phenanthroline were shifted to higher frequencies
at 1222 and 1670 cm in L were assigned to C=S and
1
C=O stretching vibrations, respectively. The C=S and
C=O were shifted to lower frequencies about 4–49 and
−1
upon complexation. The C–H band at 852 and 736 cm
−
17–92 cm in the complexes. The carbonyl O atom in L
1
1
in the spectra of phenanthroline was also shifted upon
metal complexation. The shifts can be explained by the
fact that each of the two nitrogen atoms of phenanthro-
line ligand offers a pair of electrons to the central metal
atom forming a coordinate covalent bond [37, 38]. The
lose coordination capability due to intramolecular hydro-
gen bonding [39], so L ligand behaves like a monodentate
1
thiourea ligand [32].
−
1
bands at 3332 and 3232 cm could be assigned to the
N–H stretches of the benzoylthiourea, while the bands
were also shifted to higher frequencies and turned into a
broad peak in the metal complexes due to intramolecular
UV–Vis absorption spectroscopy
The free phen ligand exhibits absorption bands at 230
and 264 nm, whilst two absorption bands in the range
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J Biol Inorg Chem
Fig. 2 a–d Fluorescence emission spectra of the complex 1–4 in
and j = 1.44 μM. Arrow shows the emission intensity changes with
increasing DNA concentration. Inset Stern–Volmer plot of the fluo-
rescence titration data of the complexes 1–4
DMSO or methanol solution upon addition of Calf-Thymus DNA
−
5
(
a–h), [complex] = 4.0 × 10 M; a = 0.00, b = 0.17, c = 0.33,
d = 0.50, e = 0.66, f = 0.82, g = 0.98, h = 1.14 μM, i = 1.30 μM,
2
35–290 nm are observed for the free benzoylthiourea
is higher than the second one located at 428 nm; whilst
for complex 4, this band has higher intensity compared
with the second emission band at 577 nm. Moreover, the
emission band is a clear red shift for complexes and the
fluorescence intensity has an apparent change compared to
the ligands. The results show that the electron of excited
state goes back to ground state resulting in the change of
fluorescence intensity upon complexation for ligands and
metal ions.
ligand (L ). The UV–vis spectra of the complexes (1–4)
1
in methanol or acetonitrile present one to two absorption
bands located in the UV range, which can be assigned to
intraligand n–π* and π–π* transitions (LC) perturbed by
complexation to the M(II) metal. The moderately intense
absorptions around 270 nm for all prepared complexes 1–4
can be attributed to spin-allowed metal-to-ligand charge
transfer MLCT(d –π*) transition in analogy to semblable
M
metal complexes containing phen ligand [40].
DNA‑binding studies
Fluorescence spectroscopy
Electronic absorption study
All the complexes show strong luminescence properties
both in DMSO and methanol solution at room tempera-
ture. The absorption spectra are closely matched with the
excitation spectra. All metal complexes show two emission
bands in the 390–500 nm range except for the emission
band of Pd(II) complex in the 500–650 nm range. The first
one has the same position around 409 nm for complexes
Titration with UV spectrophotometry is well suited for inves-
tigating the binding mode of DNA with metal complexes.
The extent of the hypochromism or hyperchromism in the
metal-to-ligand charge transfer (MLCT) band is in accord-
ance with the strength of intercalative interaction [41].
Upon addition of increasing concentration of DNA to
the fixed concentration of complex (1-4), a concomitant
increase in the absorption intensity (hyperchromism) was
1
–3, but in respect to the position of the complex 4 is
around 539 nm. For complexes 1–3, the first band intensity
1
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J Biol Inorg Chem
Fig. 3 a–d Fluorescence-quenching curves of EB bound to DNA in the presence of complexes 1–4. [DNA] = 25 μM, [EB] = 1.25 μM and
[
complex] = 0, 0.4, 0.8, 1.1, 1.5, 1.8, 2.1 μM (a–g)
Fig. 5 Gel electrophoresis diagram showing the cleavage of super-
coiled pBR322 DNA (1 μg/μL in base pair) by complexes 1–4
(
3
100 μM) in DMSO or methanol/Tris–HCl buffer at pH 7.42 and
7 °C with an incubation time 1 h in the absence of any reducing/oxi-
dative agents. Lane 1 DNA, lane 2 DNA + Ni, lane 3, DNA + Co,
lane 4 DNA + Cu, lane 5 DNA + Pd
Fig. 4 Stern–Volmer plots of fluorescence titrations of the complexes
with ct-DNA
at low concentration level is of intercalation mode which is
most likely through the mode involving a stacking interac-
tion between the aromatic chromophore and the base pairs
of DNA [43].
The DNA-binding affinities for the interaction of the com-
plex with DNA are compared quantitatively by obtaining the
observed, as shown in Fig. 1. In the presence of increas-
ing concentrations of CT-DNA, a hyperchromism effect is
apparent. Hypochromism possibly results from the inter-
action between the electronic states of the intercalating
chromophore and that of the DNA base [42]. The associa-
tion reaction between the ternary complexes and CT-DNA
intrinsic binding constant K using the following the follow-
b
ing equation [44]:
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J Biol Inorg Chem
Fig. 6 a–d Electronic absorption titration curves of HSA (10 μM)
in the absence and presence of the M(II) complex in Tris–HCl buffer
ing the concentration of the complex (a–j), a = 0, b = 0.1, c = 0.3,
d = 0.5, e = 0.7, f = 1.0, g = 1.4, h = 1.9, i = 2.3, and j = 2.7 μM.
(
pH 7.20). The arrow shows the absorbance changes with increas-
Inset plot of 1/(A − A ) vs. 1/[M(II) complex
0
fluorescence emission intensity increased remarkably
with slight change in the shape and position of emission
bands (Fig. 2). The result indicates that complexes 1–3
can strongly interact with DNA and be protected effi-
ciently from the hydrophobic environment inside the DNA
helix. Since DNA being hydrophobic macromolecule binds
to complex, the emission spectrum of complexes is not
quenched. The accessibility of solvent molecules to the
duplex is reduced and the complex mobility is limited at the
binding site, which leads to the decrease in the vibrational
modes of relaxation [45]. However, the decrease of fluo-
rescence intensity for complex 4 is most likely attributed to
energy or electron transfer from the guanine base of DNA to
the MLCT of complex [46].
[
DNA]/(εa − ε ) = [DNA]/(ε − ε ) + 1/K (ε − ε )
f b f b b f
(1)
where [DNA] is the concentration of DNA in base pairs,
ε , ε and ε correspond to the apparent absorption coef-
a
f,
b
ficient Aobs/[complex], the extinction coefficient for the free
metal complex and the extinction coefficient for the metal
complex in the fully bound form, respectively. In plots of
[
DNA]/(ε − ε ) versus [DNA], K is afforded by the ratio of
a
f
b
slope to the intercept. The K values were listed at Table 1.
b
All K values for the complexes are higher than complexes
b
4
−1
like [(Ru(phen) (bpy)](PF ) (K = 0.36 × 10 M ), tri-
2
6 2
b
cationic Co(III) complexes with asymmetric ligand, [Co
3
+
4
−1
(
phen) (pdta)] (K = 2.8 × 10 M ), and [Co(bpy) (CNOI
2
b
2
3+
4
−1
P)] (K = 5 × 10 M ) [43]. Obviously, the rigid structure
b
of ternary complexes would lead to much stronger interaction
with DNA. While the result suggests that different metals do
not influence the intercalating ability of these complexes.
EB displacement assay
To further investigate the binding mode of complexes 1–4
with DNA, competitive-binding experiment using ethidium
bromide (EB) as a probe was implemented. The present
M(II) complexes can quench the EB emission by either
displacing EB of DNA-EB system or involving in partial
Fluorescence spectral studies
On addition of increasing concentration of CT-DNA
(0–1.44 μM) to fixed amount of complexes 1–3,
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J Biol Inorg Chem
Fig. 7 a–d Intrinsic tryptophan fluorescence spectra of HSA
indicate the effect of increasing concentration of complexes on the
(
10 μM) in the presence of complex 1–4 (a–g/h), a = 0, b = 0.1,
tryptophan fluorescence emission of HSA. The inset shows the linear
c = 0.2, d = 0.3, e = 0.4, f = 0.5, g = 0.6, and h = 0.7 μM. Arrows
fit of F /F vs. [complex]
0
intercalation of planar ring at higher concentrations [47].
The fluorescence intensity decreases with the increment
amount of complexes (1–4), as shown in Fig. 3.
The results suggest that complexes might bind to CT-
DNA through intercalated interaction to a large extent,
which is the most possible interaction mode to CT-DNA.
The Stern–Volmer quenching constant K for each complex
q
was calculated using the equation given as F /F = 1 + K
0
q
[
Q]. Here, F and F are the fluorescence intensity in the
0
absence and presence of complexes, respectively, Kq is
a linear Stern–Volmer quenching constant, and [Q] is the
total concentration of a complex to that of DNA. The value
of K is given by the ratio of slope to intercept in a plot of
q
F0/F versus [Q] (Fig. 4).
Fig. 8 Stern–Volmer plots of the fluorescence titrations of the com-
plexes with HSA
The quenching constant values are listed in Table 1.
To quantify the displacement, the concentration of the
complex at which EB fluorescence decreases by 50 %
(
[
assumed to be 50 % displacement of EB) is calculated
48]. From a plot of the observed intensities against com-
of the complex used to obtain 50 % reduction in fluores-
cence intensity of EB. The higher K values reveal that the
q
plex concentration, the values of apparent DNA-bind-
ternary complexes have stronger ability of displacing EB
from DNA-EB system. The capability of the complexes
to quench the fluorescence in EB displacement assay is in
accordance with the UV–Vis titration spectroscopic stud-
ies, which indicate their intercalation in DNA.
ing constant (Kapp) were calculated using the equation
5
−1
KEB[EB] = Kapp[complex], where KEB (4.94 × 10 M
)
is the DNA-binding constant of EB, [EB] is the concentra-
tion of EB (1.25 μM), and [complex] is the concentration
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J Biol Inorg Chem
HSA‑binding studies
Most of the total blood plasma protein is constituted of
serum albumin. The protein plays a vital role in the drug
transport (metal complexes) and drug metabolism. The
binding affinity of complex towards HSA was also investi-
gated to understand the carrier role in body system.
Electronic absorption spectroscopy
Upon addition of complexes 1–4 to HSA of constant con-
centration (10 μM), a gradual increase in absorption inten-
sity and hyperchromism of the intraligand band at 278 nm
was observed, as shown in Fig. 6.
Fig. 9 Scatchard plots of the fluorescence titrations of the complexes
The results indicate that the complexes 1–4 can inter-
act with HSA by non-covalent interaction possibly bind to
HSA by the modes of hydrophobic interaction and elec-
trostatic binding. Electrostatic attraction can be facilitated
by hydrogen-bond formation, which results in the slight
change in the absorption profile deriving from the effect of
the polar solvent (water) and perturbations in the microen-
vironment of the polypeptide backbone of the HSA [50].
with HSA
Table 2 Protein-binding constant (K ), quenching constant (KSV),
and number of binding sites (n) values
b
Complex
K (M−1)
KSV (M
−1
)
n
−1 −1
kq(S M )
b
6
6
6
6
6
6
6
6
14
14
14
14
1
2
3
4
2.16 × 10
1.49 × 10
2.39 × 10
3.52 × 10
1.93 × 10
1.67 × 10
2.37 × 10
3.23 × 10
1.38
1.15
1.22
1.33
1.93 × 10
1.67 × 10
2.37 × 10
3.23 × 10
The plots of 1/(A − A ) vs. 1/[M(II) complex] are linear
0
lines (Fig. 6), and the binding constant (K ) can be obtained
b
from the ratio of the intercept to slope. The binding con-
4
5
5
5
stant (K ) of 5.1 × 10 , 4.4 × 10 , 5.7 × 10 , and 1.3 × 10
b
−1
M
for Ni(II), Co(II), Cu(II) and Pd(II) complex with
HSA were calculated, respectively. The values of K show
b
DNA cleavage studies
that HSA can be considered as a good carrier for transfer of
these complexes in vivo.
To assess the DNA cleavage ability of the complexes,
super-coiled plasmid pBR322 DNA was cultured with
complexes 1–4 in Tris–HCl buffer solution at pH 7.42 for
Fluorescence‑quenching studies with HSA
1
h in the absence of any external agents. In the absence
Binding to the protein may lead to the loss or enhancement
of the biological properties of the original compounds,
or provide paths for their transportation. Fluorescence-
quenching analyses are usually employed to analyze the
interaction of chemical compounds with HSA. A solu-
tion of HSA (10 μM) was titrated with various concentra-
tion of the complexes (0.1–0.6/0.7 μM). The fluorescence
spectrum of tryptophan was recorded in the range from
290 to 500 nm upon excitation at 293 nm. The effects of
complexes (1–4) on the fluorescence spectrum of HSA are
shown in Fig. 7.
Upon the addition of the M(II) complexes to the solution
of HSA, fluorescence intensity at 330 nm decreases with
the initial intensity of HSA, accompanied by a hypsoch-
romatic shift of 6, 9, 4, and 5 nm for complexes 1, 2, 3,
and 4. The observed hypochromicity with blue shift reveals
that the complexes could interact hydrophobically with the
HSA protein and the efficient interaction between metal
complex and HSA results in the change in its secondary
structure (affecting the tryptophan residues of HSA).
of the complex, the DNA remained in its super-coiled
form (Form I). Upon addition of complex when subjected
to agarose gel electrophoresis, an open circular relaxed
form (Form II) was formed and the linear form (Form III)
was found upon further cleavage. When electrophoresis
was performed on the reaction mixture, the compact Form
I migrated relatively faster, while the nicked Form II
migrated slowly. The linearized form (Form III) migrated
between Forms I and II [49]. From Fig. 5, all the present
complexes converted open circular relaxed form (Form II)
into linearized form (Form III) to almost the same extent.
It was found that all the metal complexes could effec-
tively cleave the super-coiled DNA. The nuclease activity
of the Cu(II) complex shows the highest ability of cleav-
ing DNA and with the order Cu > Ni > Pd > Co. It can
be seen that ternary complexes of copper show efficient
chemical nuclease activity and are good DNA intercala-
tive binders [14] and have the potential for applications in
antibacterial agents.
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J Biol Inorg Chem
Fig. 10 a–d Synchronous fluorescence spectra of HSA (10 μM) as a function of concentration of the complexes 1–4 [(a–g/h), a = 0, b = 0.1,
c = 0.2, d = 0.3, e = 0.4, f = 0.5, g = 0.6, and h = 0.7 μM] with ∆λ = 15 nm
The Stern–Volmer and Scatchard graph is used to study
the interaction of a quencher with serum albumins. Accord-
ing to the Stern–Volmer quenching the following equation
of log [(F − F)/F] versus log [Q] (Fig. 9), the number
0
of binding sites (n) and the binding constant (K ) values
b
were obtained. The calculated K , K , and n values were
SV
b
[
51]:
given in Table 2. The value of n was around 1.2 for all of
the complexes. Notably, Pd(II) complex exhibited a better
interaction effect with HSA than other complexes.
F /F = 1 + k t [Q] = 1 + KSV[Q]
0 q 0
(2)
where F is the initial tryptophan fluorescence intensity of
0
HSA, F is the tryptophan fluorescence intensity of HSA
Characteristics of synchronous fluorescence spectra
after the addition of the quencher, k is the quenching rate
q
constants of HSA, KSV is the dynamic quenching constant,
τo is the average lifetime of HSA without the quencher, [Q]
is the concentration of the quencher, KSV = k τ , and tak-
Synchronous fluorescence spectroscopy offers informa-
tion on the molecular microenvironment, particularly
in the vicinity of the fluorophore functional groups. The
presence of tyrosine, tryptophan, and phenylalanine resi-
dues might have an influence on the fluorescence of HSA.
The difference between excitation and emission wave-
lengths (∆λ) reflects the nature of chromosphere [53].
The large (~60 nm) and the small (~15 nm) ∆λ values
are characteristic of tryptophan and tyrosine residues,
respectively.
q
o
ing as fluorescence lifetime (τ ) of tryptophan in HSA at
o
−8
around 10 s, and the dynamic quenching constant (KSV,
−
1
M
) can be calculated using the plot of F /F versus [Q]
0
(
Fig. 8).
If it is assumed that the binding of complexes with HSA
occurs at equilibrium, the equilibrium binding constant can
be analyzed according to the Scatchard equation [52]:
Synchronous fluorescence spectra of HSA were recorded
at both 15 and 60 nm ∆λ with the addition of the M(II)
complexes (1–4) in various concentration to understand the
structural change occurred in HSA (Figs. 10, 11).
log[(F − F)/F] = log K + n log[Q]
0
b
(3)
where K is the binding constant of the compound with
b
HSA and n is the number of binding sites. From the plot
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J Biol Inorg Chem
Fig. 11 a–d Synchronous fluorescence spectra of HSA (10 μM) as a function of concentration of the complexes 1–4 [(a–g/h), a = 0, b = 0.1,
c = 0.2, d = 0.3, e = 0.4, f = 0.5, g = 0.6, and h = 0.7 μM] with ∆λ = 60 nm
Table 3 Antibacterial activities of the benzoylthiourea ligand and
ternary metal complexes
Antibacterial activities
Sample Inhibition zone (mm)
All synthesized compounds were screened for antibacterial
activity against bacterial strains by the inhibition zone method
with agar diffusion. The biological activity for the complexes
was defined by measuring the diameter of the inhibition area
surrounding each bore in millimeters. The experiments were
repeated three parallel times. The values of inhibition zones
of the all compounds were reported as means of at least three
determinations and were represented in Table 3.
Gram-negative (Escherichia Gram-positive (Staphylo‑
coli)
coccus aureus)
1
2
3
4
6.50
8.00
11.2
7.50
5.80
7.00
8.70
10.0
8.70
5.20
L1
The antibacterial activities of the complexes and ligand
were determined against Gram-positive and Gram-negative
pathogenic organisms. Methicillin [R (≤9 mm), I (10–
13 mm), S (≥14 mm)] and trospectomycin (I = 10–16 mm,
S = 15–20 mm) were used as the standard control. The
results show that only copper complexes have nearly
moderate activity against the tested bacterial strains. All
complexes have slightly higher activity than their ligands
against the same microorganism. The antibacterial activi-
ties of copper complex shows more activity against
the tested bacteria than other metals and with the order
Cu > Pd > Co > Ni. Weak antibacterial activity of Pd, Co,
and Ni complexes may attribute to the poor solubility in
solvent. It should be emphasize that the antibacterial study
is very preliminary, since it was based on only two bacteria.
With increasing the concentration of the complexes, the
intensity of emission corresponding to tyrosine (at 293 nm)
was found to decrease in the magnitude of 58.7, 63.5, 53.2,
and 41.5 % for complexes 1–4 accompanied with slightly
red shift in emission wavelength. The tryptophan fluores-
cence emission presented apparent decrease in the intensity
(
at 288 nm) of about 38.8, 40.7, 43.4, and 31.6 % for com-
plexes 1, 2, 3, and 4. The position of the emission band had
a slight shift. The results indicate that the complexes could
affect the microenvironments of both tyrosine and trypto-
phan during the binding process, while the effect was more
towards tyrosine than tryptophan [54].
1
3

J Biol Inorg Chem
1
3. Zeng Y-Q, Cao R-Y, Yang J-L, Li X-Z, Li S, Zhong W (2016)
Eur J Med Chem 119:83–95
Conclusion
1
4. Chetana PR, Srinatha BS, Somashekar MN, Policegoudra RS
N, N′-disubstituted thiourea derivatives and 1,10-phen-
anthroline complexes still receive much attention due to
synthetic convenience and excellent biological activities.
O-, S-, and N-donor atoms and thione, thiol (–N–CS–N–)
groups in the ligands stretch their coordination ability
and popularity towards a variety of metal ions [14]. Four
novel ternary metal complexes of N-benzoylthiourea and
(
2016) J Mol Struct 1106:352–365
15. Singh A, Bharty MK, Bharati P, Bharti A, Singh S, Singh NK
2015) Polyhedron 85:918–925
(
1
1
1
1
6. Selvakumaran N, Pratheepkumar A, Ng SW, Tiekink ERT, Kar-
vembu R (2013) Inorg Chim Acta 404:82–87
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Din, et al (2015) J Biol Inorg Chem 20(3):541–554
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,10-phenanthroline derivatives, [Ni(phen)(L ) ], [Co(phen)
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1 2 1 2 1 2
20. Plutín AM, Alvarez A, Mocelo R, Ramos R, Castellano EE, da
Silva MM et al (2016) Inorg Chem Commun 63:74–80
2
synthesized and characterized by their conventional physi-
cal and chemical analyses. UV–Vis and fluorescence spec-
tral studies indicate that all these complexes exhibit efficient
and strong binding abilities with DNA and HSA. The exper-
iments of fluorescence quenching further reveal the strong
interaction of the complexes with HSA protein. Meanwhile,
all ternary complexes can cleavage DNA efficiently. Among
the complexes examined, copper (II) complex exhibits
the strongest DNA binding and cleavage ability. Although
this study is preliminary, they provide the theoretical basis
and experimental data for the study of the potential drug
with nucleic acid as the target based on disubstituted thio-
ureas. We hope to inspire the role and importance of N, N′-
disubstituted thiourea compounds and further work on their
complexes for biomedical applications.
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Acknowledgments The authors would like to thank the National
Natural Science Foundation of China (21174114, 21167015), the
Innovative Team of Ministry of Education of China (IRT15R56), and
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