DNA/protein binding, cytotoxicity and catecholase activity studies of a piperazinyl moiety ligand based nickel(II) complex

Article abstract of DOI:10.1016/j.poly.2016.05.003

The Ni(II) complex, {[Ni(HL)(SCN)₂(H₂O)]·2(DMF)} (1) [HL = 6-methoxy-2-{[2-(1-piperazinyl)ethylimino]methyl}phenol], was synthesized and characterized by X-ray crystal structure analysis and spectroscopic methods. The crystal structure of complex 1 reveals a distorted octahedral coordination geometry around the nickel centre which forms a supramolecular assembly through hydrogen bonds. The interaction of complex 1 with the calf thymus DNA (CT-DNA) was investigated using electronic absorption and fluorescence spectroscopic methods. The results show that complex 1 has binding affinity to CT-DNA in the order of 6.06 × 10⁵ M^-1. The interactions of complex 1 with bovine serum albumin (BSA) and human serum albumin (HSA) were also studied using electronic absorption and fluorescence spectroscopic techniques and the analysis show that interaction of complex 1 with BSA/HSA occur mainly with ground state association process. The number of binding sites and binding constant were calculated using double logarithm regression equation. Anticancer activity of 1 in human breast (MCF7) cancer cell lines reveals dose dependent suppression of cell viability with IC₅₀ value 64 ± 3.7 μM. Catecholase activity of 1 has been investigated in methanol medium using 3,5-di-tert-butylcatechol (3,5-DTBC) as model substrate and the result shows that 1 is active for catalyzing aerobic oxidation of 3,5-DTBC to 3,5-di-tert-butylbenzoquinone (3,5-DTBQ).

Full text of DOI:10.1016/j.poly.2016.05.003

Accepted Manuscript
DNA / protein binding, cytotoxicity and catecholase activity studies of a piper-
azinyl moiety ligand based nickel(II) complex
Soumen Mistri, Horst Puschmann, Subal Chandra Manna
PII:
S0277-5387(16)30143-7
DOI:
http://dx.doi.org/10.1016/j.poly.2016.05.003
POLY 11974
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
29 February 2016
1 May 2016
Please cite this article as: S. Mistri, H. Puschmann, S.C. Manna, DNA / protein binding, cytotoxicity and catecholase
activity studies of a piperazinyl moiety ligand based nickel(II) complex, Polyhedron (2016), doi: http://dx.doi.org/
10.1016/j.poly.2016.05.003
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DNA / protein binding, cytotoxicity and catecholase activity studies of a
piperazinyl moiety ligand based nickel(II) complex
Soumen Mistri,a Horst Puschmann,b Subal Chandra Manna*,a
a
Department of Chemistry & Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India, E-mail: scmanna@mail.vidyasagar.ac.in, Fax: (91) (03222)
275-329.
b Department of Chemistry, University of Durham, South Road, Durham DH1 3LE, U.K.
ABSTRACT
The Ni(II) complex, {[Ni(HL)(SCN)2(H2O)]·2(DMF)} (1) [HL=6-methoxy-2-{[2-(1-
piperazinyl)ethylimino]methyl}phenol], was synthesized and characterized by X-ray crystal
structure analysis and spectroscopic methods. The crystal structure of complex 1 reveals a
distorted octahedral coordination geometry around the nickel centre which forms a
supramolecular assembly through hydrogen bonds. The interaction of complex 1 with the calf
thymus DNA (CT-DNA) was investigated using electronic absorption and fluorescence
spectroscopic methods. The results show that complex 1 has binding affinity to CT-DNA in
the order of 6.06 ×105 M-1. The interactions of complex 1 with bovine serum albumin (BSA)
and human serum albumin (HSA) were also studied using electronic absorption and
fluorescence spectroscopic techniques and the analysis show that interaction of complex 1
with BSA/HSA occur mainly with ground state association process. The number of binding
sites and binding constant were calculated using double logarithm regression equation.
Anticancer activity of 1 in human breast (MCF7) cancer cell lines reveals dose dependent
suppression of cell viability with IC50 value 64 3.7 µM. Catecholase activity of 1 has been
investigated in methanol medium using 3,5-di-tert-butylcatechol (3,5-DTBC) as model
substrate and the result shows that 1 is active for catalyzing aerobic oxidation of 3,5-DTBC to
3,5-di-tert-butylbenzoquinone (3,5-DTBQ).
1

___________________________________________________________________________
Keywords: Ni(II) complex; crystal structure; DNA binding studies; serum albumin binding
studies; catecholase activity.
__________________________________________________________________________
1. Introduction
Transition metal coordination compounds are attracting considerable interest due to
their potential applications in the field of catalysis [1a-1d], magnetism [1e-1g], fluorescence
sensing [1h,1i], medicinal chemistry [1j-1l], etc. Some Schiff-base transition metal
coordination compounds play an important role in biology due to their antimicrobial [2],
antifungal [3], antibacterial [4], antitumor [5], antiviral [6], antipyretic [3a] and antidiabetic
activities [7]. There are reports in the literature that many transition metal complexes may
potentially be used as therapeutic agents for the treatment of several human diseases including
diabetes, neurological disorders, anti-inflammatory, carcinomas and lymphomas. In addition
to this therapeutic application, these compounds also function as potential free radical
scavengers and hence behave like anti-oxidant agents [2, 8]. Complexes of vanadium and zinc
(antidiabetic agents), platinum and ruthenium (anti-cancer agent), iron, copper and manganese
(anti-oxidant and superoxide dismutase mimetic agents) are widely considered in the
development of new metallopharmaceutics [9]. Limited numbers of mono / dinuclear Ni(II)
complexes have also been reported in the literature and are showing important biological
activities: they can strongly bind to and cleave DNA, show dominant anticancer activities and
can control apoptosis [10].
Study of the interactions of DNA with transition metal coordination compounds under
physiological conditions is important for the design of new metallo-pharmaceuticals. These
compounds have the potential to promote oxidative damage to the 2-deoxyribose moiety and /
or to nucleo base in presence of oxidizing or reducing agent, redox active metal centre or light
[11]. On the other hand, serum albumin is the major soluble protein in the circulatory system
2

and functions as a transporter and disposer of many endogenous and exogenous compounds
like fatty acids, amino acids, drugs, and pharmaceuticals, etc. [12]. Analysis of the kinetics of
interactions of DNA / serum albumin and transition metal complex is important to determine
the affinities of complex to DNA / protein, and this information will help to develop efficient
metallo-pharmaceuticals.
In the present contribution we report the kinetics of the interaction of
{[Ni(HL)(SCN)2(H2O)]·2(DMF)}
(1)
[HL
=
6-methoxy-2-{[2-(1-
piperazinyl)ethylimino]methyl}phenol] with calf thymus DNA (CT-DNA) and serum
albumin. As human serum albumin (HSA) is structurally homologues to bovine serum
albumin (BSA), we have taken both the serum albumin for the purpose of analysis. We have
also studied anti-cancer and catecholase activities of complex 1.
2. Experimental section
2.1. Materials and methods
High purity 2-ethylaminopiperazine and 3,5-di-tert-butylcatecholwas purchased from
the Aldrich Chemical Co. Inc. and were used. All other chemicals were of AR grade. Solvents
used for spectroscopic studies were purified and dried by standard procedures before use [13].
Elemental analyses (carbon, hydrogen and nitrogen) were performed using a Perkin-
Elmer 240C elemental analyzer. IR spectra were recorded as KBr pellets on a Bruker Vector
22FT IR spectrophotometer operating from 400 to 4000 cm–1. ESI-MS spectra were recorded
on a JEOL MS 700 mass spectrometer in fast atom bombardment (FAB) mode. NMR spectra
of ligand recorded on Bruker 400 MHz instrument. Electronic absorption spectra were
obtained with Shimadzu UV-1601 UV-vis spectrophotometer at room temperature. Quartz
cuvettes with a 1 cm path length and a 3 cm3 volume were used for all measurements.
Emission spectra were recorded on a Hitachi F-7000 spectrofluorimeter. Room temperature
3

(300 K) spectra were obtained in methanol solution using a quartz cell of 1 cm path length.
The slit width was 2.5 nm for both excitation and emission.
The fluorescence quantum yield was determined using phenol as a reference and water
medium for phenol. The solvent used for complex is water [refractive index (η), 1]. Emission
spectra were recorded by exciting the complex and the reference phenol at the same
wavelength, maintaining nearly equal absorbance (~0.1). The area of the emission spectrum
was integrated using the software available in the instrument and the quantum yield was
calculated [14] according to the following equation:
2
η
η
A I
s
s
r
Φ =Φ
s
r
2
A I
r
s
r
Where Φs and Φr are the fluorescence quantum yield of the sample and reference,
respectively. As and Ar are the respective optical densities at the wavelength of excitation, Is
and Ir correspond to the areas under the fluorescence curve; and ηs and ηr are the refractive
index values for the sample and reference, respectively.
2.2. Synthesis of 6-methoxy-2-{[2-(1-piperazinyl)ethylimino]methyl}phenol (HL)
A methanolic solution (20 mL) of mixture of 2-hydroxy-3-methoxybenzaldehyde
(0.152 g, 1 mmol) and 2-ethylaminopiperazine (1.292 g, 1 mmol) was refluxed for 3 h. The
resulting yellow color solution cooled to room temperature and solid yellow compound was
obtained after evaporation of solvent under reduced pressure. The compound obtained was re-
dissolved in DCM/MeCN (1:1) and filtered. The solution was left for slow evaporation at
room temperature. After one week, yellow crystals of HL were obtained. C14H21N3O2: HR-
MS: [M + H]+, m/z, 262.1 (100%) calcd: m/z, 263.0. Anal.found: C, 3.90; H, 7.95; N, 15.95;
1
calc.: C, 63.87; H, 7.98; N, 15.96. H NMR (CDCl3, 400 MHz, δ, ppm): 8.29 s (1H), 7.12-
6.74 m (3H), 4.95 s (1H), 3.81 s (3H), 3.75-3.68 m (2H), 2.70-2.67 m (6H), 2.66-2.51 m
4

(5H).13C NMR (CDCl3, 400 MHz, δ, ppm): 165.57 s, 148.53 s, 148.27 s, 124.86 s, 122.86 s,
120.80 s, 119.55 s, 58.50 s, 56.31 s, 56.26 s, 55.80 s, 54.60 s.
2.3. Synthesis of complex
The complex has been synthesized by adopting the procedures schematically given in Scheme
I.
Scheme I. Synthesis of 1
{[Ni(HL)(SCN)2(H2O)]·2(DMF)} (1)
A methanolic solution (5 mL) of NiCl2·6H2O (1.0 mmol, 0.237 g) was added drop
wise to a methanolic solution (10 mL) of mixture of HL (1 mmol, 0.263 g) and Et3N (1
mmol) with constant stirring at room temperature (25 °C). To the resulting mixture an
aqueous solution (1 ml) of KSCN (2mmol, 0.194 g) was added dropwise and resulting green
reaction mixture was stirred for 4h at 25 °C and filtered. The filtrate was left for slow
evaporation at room temperature. After a few days, green compound was separated and the
compound was redissolved in DMF and filtered. Green crystals suitable for X-ray analysis
were obtained after two weeks. Yield 75%. Anal.calc. for C22H37N7NiO5S2 (602.41): C,
43.82; H, 6.14; N, 16.26. Found: C, 43.80; H, 6.15; N, 16.14 %. The infrared spectrum
exhibited the following absorptions (cm-1): 3200-3700 (br,vs), 2975 (s), 2943 (vw), 2092 (s),
1652 (w), 1561 (vs), 1467 (s), 1419 (vs), 1376 (s), 1299 (vs), 1251 (vw), 1124 (vw), 1079
(vs), 1008 (s), 885 (vs), 817 (vs), 652 (s), 509 (s).
5

2.4. Crystallographic data collection and refinement
X-ray diffraction data of complex 1 were collected at 120 K on an Oxford Diffraction
Gemini Ultra diffractometer. Cell refinement, indexing and scaling of the data sets were was
performed with the Agilent CrysAlisPro package, Version 1.171.35.10 [15]. The non-standard
space group Ia was chosen, since it affordas a smaller beta angle than than the standard-
setting space group Cc. The structure was solved by using ShelXS [16] using direct methods
and refined using ShelXL-2014/7 [17] by the full matrix least-squares method based on F2
with all observed reflections. The terminal S atom of one of the two NCS ligands was
modelled as disordered in equal occupancy. The deposited CIF file (CCDC 1021243) contains
the full hkl and res files, thereby fully describing the model. All structural work, including the
generation of packing diagrams and all geometric calculations were performed using Olex2
[18]. Crystal data and details of refinements are given in Table 1.
2.5. DNA binding experiments
2.5.1. Electronic absorption spectral study
Electronic absorption spectral titration was carried out at a fixed concentration of nickel(II)
compound (3 ml, 0.1991M) in water and varying the concentration of CT-DNA from 0 to
4.50 µM. Intrinsic binding constant (Kib) of the complex with CT-DNA was determined using
the equation [19]
[DNA]
[DNA]
1
=
+ꢃ
(εꢀ − εꢁ) (εꢂ − εꢁ) Kꢄꢂ(εꢂ − εꢁ)
where, [DNA] is the concentration of CT-DNA, εa is the extinction co-efficient of the
complex at a given CT-DNA concentration, εf and εb are the extinction co-efficient of the
complex, in free solution and when fully bound to CT-DNA, respectively. The plot of
[DNA]/(εa-εf) vs [DNA] give a straight line with 1/(εb-εf) and 1/[Kib(εb-εf)] as slope and
6

intercept, respectively. From the ratio of the slope to the intercept the value of Kib was
calculated.
2.5.2. Competitive binding fluorescence measurement
The competitive binding nature of EB and nickel(II) compound with CT-DNA was
investigated adopting fluorometric method using aqueous solution of EB bound CT-DNA in
HEPES buffer (pH 7.2) at room temperature. In presence of DNA, ethidium bromide (EB)
exhibits fluorescence enhancement due to its intercalative binding to DNA. Competitive
binding of nickel(II) compound with CT-DNA results fluorescence quenching due to
displacement of EB from CT-DNA. The fluorescence intensities at 598 nm (λex, 500 nm) of
EB bounded CT-DNA with increasing concentration of nickel(II) compound was recorded.
The quenching constant (Stern-Volmer constant, Ksv) was calculated using Stern-Volmer
equation [20]
I0/I = 1 + Ksv[complex]
where I0 and I are the emission intensity in absence and in presence of nickel(II) compound,
Ksv is the Stern-Volmer constant, and [complex] is the concentration of nickel(II) compound.
2.6. Protein binding studies
Stock solutions human serum albumin (HSA) and bovine serum albumin (BSA) were
prepared in 55 mM HEPES buffer (pH 7.2). Aqueous solution of the nickel(II) compound was
prepared by dissolving the compound in water: HEPES buffer (1:99). The interactions of
compound with serum albumins were studied by recording the tryptophan fluorescence of
HSA / BSA. To the solutions of serum albumin (3.109 µM, HAS; 8.214 µM, BSA) in 55 mM
HEPES buffer (pH 7.2) at room temperature, nickel(II) compound was added, and the
7

quenching of emission intensities at 344 nm (λex, 280 nm) were recorded after gradual
addition of 0.1991 M aqueous solution compound.
2.7. Cell Culture
Breast cancer cell line MCF7 was cultured in DMEM medium as monolayers supplemented
with 10 % FBS, 100U/ml penicillin and 100 µg/ml streptomycin at 37 °C in a humid
condition with 5% CO2 atmosphere.
2.8. In vitro Cytotoxicity Assay
The cytotoxicity of the compounds was determined by the MTT [3-(4, 5 dimethylthiazol-2-
yl)-2-5 diphenyltetrazolium bromide] assay. Cells were seeded at 5 x 103 cells per well in 96-
well plates. Next day, cells were treated with different concentrations (0.01 – 100 µM) of the
compounds for 48 hour. Then, MTT solution was added to each well and further incubated for
3.5 h at 37°C in a humid, 5% CO2 atmosphere. Finally, medium containing MTT solution was
replaced by MTT solvent (4 mM HCl and 0.1% Triton X-100) and incubated for 15 minute at
room temperature. The absorbance was read in Thermo Pierce Elisa plate reader at 570 nm.
The percentage of growth inhibition was calculated with respect to vehicle (DMSO) treated
cells. All the experiments were repeated for three times.
2.9. Immunofluorescence study
For immunofluorescence study cells were seeded on poly-lysine coated cover slip one day
prior the treatment. Next day cells were either treated with DMSO or complex for 48 hours.
Then cells were washed three times with cold phosphate buffer saline (PBS) followed by
fixation with 4% paraformaldehyde at room temperature for 30 minutes. Fixed cells were
washed with cold PBS three times, permeabilized with 0.1% Triton X-100 for 10 minutes.
Permeabilized cells were blocked with 5% BSA followed by stained with mouse α-tubulin
8

primary antibody and anti-mouse rodamine conjugated secondary antibody. DNA was stained
with DAPI. Fluorescence image was captured using Olympus IX71 microscope.
3. Results and discussion
3.1. Synthetic aspects
The multisite coordinating ligand, HL, was prepared by a onepot synthesis employing
condensation of the corresponding amine and aldehyde in methanol under reflux condition,
and characterized by NMR (Fig. 1S), ESI mass (Fig. 2S), FT-IR (Fig. 3S), electronic
absorption and emission spectra. Using HL, complex 1 was synthesized at room temperature.
3.2. Molecular structure of {[Ni(HL)(SCN)2(H2O)]·2(DMF)}
Crystal structure determination reveals complex 1 consists of discrete molecule,
[Ni(HL)(SCN)2(H2O)], and two DMF molecules that are form a hydrogen bond with a metal-
bound water molecule. In 1, HL acts as a meridionally bound tridentate N,N,O donor ligand
and the distorted octahedral coordination spere is completed with two N-donor thiocyanate
nitrogen and one oxygen from water molecule, resulting in a NiN4O2 chromophore. The
structure of the metal center with the atom numbering scheme is shown in Figs. 1 and 4S,
while selected geometrical parameters are given in Table 2. The nickel(II) center meridionally
coordinated by the tridentate HL, and completes distorted octahedral coordination sphere with
two N-donor thiocyanate nitrogen and one oxygen from water molecule, resulting in a
NiN4O2 chromophore. The N/O donor [N18, N21, O10] HL and the aqua oxygen O1W define
the equatorial plane around each pseudo octahedral nickel ion [Ni1–N18: 2.021(3) A, Ni1–
N21: 2.255(3) A, Ni1–O10: 2.024(2) A, Ni1–O1W: 2.121(2) Å]
The trans axial positions are occupied by the nitrogen atoms from the two different
thiocyanate [Ni1–N1: 2.059(3), Ni1–N2: 2.063(3) Å] with a N1–Ni1–N2 bond angle of
9

174.13(11)°. Slight distortion of each Ni(II) is also reflected on the cisoid angles, which range
from 81.78(10) to 97.61(10)°. The equatorial mean plane is slightly distorted (RMSD 0.024)
with the metal insignificantly displaced by 0.0368(14) Å towards N2. The packing of
structure 1 reveals that each complex forms two hydrogen bonds to the solvate DMF
molecules via the metal-bound water molecule [O40–O1: 2.704(3), O1W–O50: 2.796(3) Å].
One of these DMF molecules forms another hydrogen bond through the same oxygen atom to
the protonated N atom in a neighboring complex [O50–N24: 2.869(4) Å], which in turn forms
another hydrogen bond (through the other H atom) to the phenolic (and M-bound) O atom of
the original complex [N24–O10: 2.653(4) Å] (Fig. 2, Table 1S). Fig. 3a and 3b shows the top
and side view of the layer respectively.
3.3. ESI mass spectral analysis
The ESI mass spectrometric data for complex 1 was collected in methanol medium and the
result is shown in Fig. 5S. Spectrum of complex 1 shows a peak at m/z = 456.12, which
corresponds to [Ni(HL)(SCN)2(H2O)].+ (calc. m/z = 455.69), indicating that the octahedral
coordination environment of Ni(II) in methanol solution is same as in the solid state structure.
3.4. IR spectral results
IR spectra of ligand HL and complex 1 are shown in Figs. 3S and 6S, and the most
important absorption bands are summarized in the experimental section and in Table 2S. IR
spectra show that ν(O-H) and ν(N-H) stretching vibrations appear in the region 3000-3650
cm-1. Stretching vibrations in the region 2975 – 2992 cm-1 are for Csp2−H bond and bands at
2943 – 2965 cm-1 are for Csp3−H bond. Aromatic ν (C=C) and aliphatic ν(C=N) stretching
vibrations are appears in the region 1419 - 1548 cm-1 (for HL) and 1419- 1561 cm-1 (for 1).
νs(C-O)phenolic stretching vibration appears at 1254cm-1 (for HL) and 1251cm-1 (for 1),
10

whereas ν(C-N)aliphatic appears at 1082cm-1 (for HL) and 1079cm-1 (for 1). For complex 1,
stretching vibrations at 2092 [for ν(CN)], 817 [for ν(CS)] and 509 cm-1 [for δ(NCS)]
corroborate the presence of N-coordinated thiocyanate ligand [21]. The IR spectrum of 1
shows additional bands corresponding to ρr(H2O) at 767 cm-1 and ρw(H2O) at 652 cm-1,
indicating the presence of coordinated water molecule.
3.5. Electronic absorption spectra and fluorescence spectra of complex 1
Electronic absorption spectrum (Fig. 7S) of 10-5 M aqueous solution of complex 1
exhibits bands at 213 nm (0.244 x 105 liter mole-1 cm-1), 241 nm (0.382 x 105 liter mole-1 cm-
1), 269 nm (0.068 x 105 liter mole-1 cm-1), 318 nm (0.059 x 105 liter mole-1 cm-1) and 392 nm
(0.035 x 105 liter mole-1 cm-1). Upon excitation (λex) at 269 nm, 1 exhibits fluorescence (λem)
at 313 nm, 328 nm and 360 nm, in aqueous solution at room temperature. Detailed spectral
data of HL and complex 1 are summarized in Table 3.
3.6. DNA binding studies
3.6.1. Electronic absorption spectral titration
Hypochromism with or without red / blue shift in the electronic spectra of a compound due to
gradual increasing concentration of DNA indicate the intercalation between the compound
and DNA [22]. On the other hand hyperchromism is observed in the absorption spectra of a
compound with increasing concentration of DNA evidences the non-intercalative interaction
between the compound and DNA [23]. In the present study the interaction of Ni(II)
compound with CT-DNA was investigated using UV-vis absorption spectral studies. The
electronic absorption spectra of complex 1 exhibited bands at 213 nm, 241 nm, 269 nm, 318
and 392 nm. Fig. 4 shows the change in the absorption spectra of aqueous solution of complex
1 (3 ml, 0.1991 M) on gradual addition of 10 µl of aqueous solution (28.181 µM) of CT-
11

DNA. The absorbance of 275 nm band gradually increases with blue shifting up to 11 nm and
finally the band appear at 264 nm. On the other hand the absorbance corresponding to peak at
363 nm gradually decreases. Above change in the electronic absorption spectra indicates that
complex 1 bind to the CT-DNA helix via intercalation. The isobestic points at 226 nm, 245
nm and 346 nm in the titration curve (Fig. 4) indicate the presence of more than two species
in the medium [24].
ꢈ
Plot of [DNA]/(ꢅꢆ −ꢃꢅꢇ) vs [DNA] (inset of Fig. 4) is a straight line with
and
(ꢉ ꢋꢃꢉ )
ꢊ
ꢌ
ꢈ
as slope and intercept, respectively. The value of intrinsic binding constant (Kib)
ꢌ
ꢍ
(ꢉ ꢋꢃꢉ )
ꢊ
ꢎꢊ
was calculated from the ratio of slope to the intercept and calculated value of Kib was 6.06 ×
105 L mol-1.
3.6.2. Ethidium Bromide (EtBr) displacement studies
Ethidium bromide (EtBr = 3,8-diamino-5-ethyl-6-phenyl phenanthridinium bromide) shows
fluorescence with an orange colour, when it exposed to ultra violet radiation. The intensity of
EtBr fluorescence increases around 20 fold in presence of CT-DNA due to strong
intercalation of the planar ethidium bromide phenanthridium ring between adjacent base pair
of the double helix [11a, 25]. CT-DNA bounded EtBr shows emission at 598 nm on excitation
at 500 nm. Addition of a compound, which is capable to interact with CT-DNA, to the
solution of a mixture of EtBr-CT-DNA results in the quenching of EtBr-CT-DNA
fluorescence intensity. The quenching of fluorescence occurs due to decrease in the number of
binding sites on the CT-DNA available for EtBr. The fluorescence quenching observed in
presence of compound may be used to study intercalation between CT-DNA and this
compound. Fluorescence titration spectra (Fig. 8S) of EtBr-CT-DNA, upon gradual addition
of 10 µL 0.1991 M solution of complex 1, indicates the reduction of fluorescence intensity
keeping emission wavelength fixed. This observation suggests that complex 1 displaced EtBr
12

molecule from the DNA binding sites [25,26]. From the Stern-Volmer equation [20] (I0/I = 1
+ Ksv[quencher], where I0 and I are the fluorescence intensity in the absence and in the
presence of the quencher, respectively), a linear relationship (Fig. 9S) was obtained from the
titration of EtBr-CT-DNA using complex 1 as quencher. The calculated value of binding
constant Ksv is 44.1753 L mol-1.
3.7. Protein binding studies
Fig. 5 shows the change of electronic absorption spectra of Bovine Serum Albumin (BSA) (3
ml, 8.214 µM aqueous solution) and Human Serum Albumin (HSA) (3 ml, 3.109 µM aqueous
solution) in presence of different concentration of complex at pH 7.2 (using HEPES buffer).
Electronic absorption spectra of both serum albumins show increase in the absorbance at 280
nm with bathochromic shift (8 nm for BSA; 7 nm for HSA). In addition to this spectral
change a new band appears with gradual bathochromic shift for both serum albumins and
finally appears at 336 nm and 365 nm for BSA and HSA, respectively. Bathochromic shift at
280 nm confirms the ground state association of complex 1 with serum albumins. The
apparent association constants (Kapp) were calculated adopting the following equation
1
1
1
=ꢃ
+ꢃ
(ꢏꢔ − ꢏꢓ) ꢕꢆꢖꢖ ꢏꢔ − ꢏꢓ [ꢗꢘꢙꢚꢛꢜꢝ]
(
)
(ꢏꢐꢑꢒ − ꢏꢓ)
where Aobs is the observed absorbance at 280 nm of the solution containing different
concentrations of the complex, A0 is the absorbance of serum albumin only and Ac is the
absorbance of serum albumin with complex 1. The calculated values of apparent association
constants are 2.29 × 104 L mol-1 and 2.43 × 103 L mol-1 for 1-BSA and 1-HSA complex,
respectively.
To examine the interaction of serum albumin (SA) with complex 1, the fluorescence
spectroscopic studies were carried out. In aqueous solution (pH 7.2, HEPES buffer) complex
1 exhibits fluorescence (λem) at 313 nm, 328 nm and 360 nm at room temperature upon
13

excitation (λex) at 280 nm. On the other hand both BSA and HSA exhibit luminescence at 330
nm, on excitation at 280 nm (in aqueous solution, pH 7.2, HEPES buffer) at room
temperature. The change of the fluorescence spectra of BSA [2 ml 8.214 µM (ε at 280 nm
43824M-1cm-1) aqueous solution] and HSA [2 ml 3.109 µM (ε at 280 nm 35700M-1cm-1)
aqueous solution] on the addition of increasing concentration of complex 1 are shown in Fig.
6. Upon gradual addition of 10 µL 0.1991 M aqueous solution of complex 1 to the solution of
serum albumins, significant decrease in fluorescence intensity (up to 64.46 and 43.92 % for
BSA and HSA, respectively) observed at 330 nm. The quenching of fluorescence intensity of
serum albumin upon addition of complex mainly attributed due to association of serum
albumin to complex, serum albumin denaturation or conformational change of the serum
albumin [27]. From the Stern-Volmer equation [20] a linear relationship were obtained (R =
0.9984 for BSA curve; R = 0.9924 for HSA curve) for the titration of serum albumin using
complex 1 as quencher (Fig. 10S). The calculated values of Stern-Volmer quenching
constants (Ksv) are 21.72 × 105 L mol-1 (for BSA) and 21.54 × 105 L mol-1 (for HSA). The ksv
values indicate that complex 1 has good serum albumin fluorescence quenching ability.
Fluorescence quenching may occur either by dynamic or static mode. In the dynamic
quenching process fluorophore and the quencher come into contact during transient existence
of the excited state. Whereas in case of static quenching the ground state complex formation
occurs between fluorophore and quencher. UV-vis absorption spectral study is the important
tool to determine the nature of quenching. From the UV-vis spectral study it is found that the
absorption spectra of BSA/HSA influenced by the addition of complex 1. This phenomenon
evidence the existence of static interaction between BSA/HSA and complex 1.
When a small molecule bind independently in a static mode to a macromolecule in its
set of equivalent sites, the equilibrium between free and bound molecules is expressed by the
Scatchard equation [28]
14

(Fꢓ − F)
F
log
= logkꢂ + n log[complex]
where kb is the binding constant of complex with serum albumin, n is the number of binding
sites per serum albumin molecule. The plot of log(ꢞ ꢋꢞ) vs log[complex] gives a straight line
ꢟ
ꢞ
(inset of Fig. 6 ) with n and logkb as slope and the intercept, respectively. Calculated values of
kb and n are given in Table 4. The values of n for BSA and HSA are 1.12 and 1.25,
respectively, which indicates the existence of static binding sites in BSA/HSA for complex 1.
3.8. Cytotoxicity Assay
Cytotoxic effect of the complex 1 was tested using standard MTT assay. Cells were treated
with the complex 1 for 48 hours. Results demonstrated that the complex 1 has moderate toxic
effect on growth of MCF7 cells (Fig. 7). Complex 1 exhibits 50% growth inhibition at 64
3.7 µM concentration. Further we have studied the cellular morphology of the cells in the
absence and presence of this compound by immunefluorescence study. Our result
demonstrated that cellular organization of microtubules and DNA are unaffected upon
treatment of this compound suggesting that the compound has moderate cytotoxic effect at the
cellular level (Fig. 11S).
3.9. Catechol oxidase study of complex 1
The catecholase activity of complex 1 has been investigated using 3,5-di-tert-butyl catechol
(3,5-DTBC) as model substrate in oxygen saturated methanol medium. At room temperature
1×10-4 M methanolic solution of complex 1 was added to the 1×10-2 M methanolic solution of
3,5-DTBC. The UV-vis spectrum of the mixture was recorded at a five minute interval for up
to 1.5 hour. Absorbance of a new band at 400 nm corresponding to 3, 5-di-tert-butylquinone
(3, 5-DTBQ) [29] was gradually increased after addition of complex 1 to the solution of 3,5-
15

DTBC (Fig. 8). log A∞ /(A∞-At) versus time (t) plot (where A∞ and At are the absorbance at
infinite time and ‘t’ time, respectively) gives a straight line (Fig. 9) passing through origin
with 4.78 × 10-5 min-1 as slope. The rate constant for complex-substrate mixture was
determined from the slope of this curve and its calculated value was 4.78 × 10-5 min-1. Fig. 10,
shows the change of rate of reaction with concentration of substrate. From this figure it is
clear that at low concentration of 3, 5-DTBC a first order dependence of the rate on the
substrate concentration was observed, whereas a saturation kinetics was observed at higher
concentration of 3, 5-DTBC. We have applied Michaelis-Menten model [30] for this system
and the values of Michaelis binding constant (KM) 0.372 litre/mol, 1/V = KM/Vmax[S] + 1/Vmax
where [S] is the concentration of substrate.
The kinetic parameters for the oxidation of 3,5-DTBC by 1 are listed in Table 5. The turnover
number (Kcat), calculated by dividing Vmax with concentration of complex [30]. The calculated
value of turn over number is 7.51 h-1, which is relatively small to those reported values for
nickel(II) complexes [30-32].
ESI-MS spectra (Fig. 12S) of a solution containing 1 : 100 mixture of complex 1 and
3,5 DTBC, exhibits a strong peak at m/z = 621.02 and one small peak at m/z = 618.89. On
the other hand when a mixture of starch-potassium iodide solution treated with a mixture
containing complex 1 and 3,5-DTBC, results blue coloration. But no blue coloration was
observed when starch-potassium iodide solution treated with complex 1 or 3,5-DTBC only.
This observation indicates that hydrogen peroxide was produced during catalytic oxidation of
3,5 DTBC to 3,5 DTBQ. Probable mechanistic path for the formation of H2O2 as by-product
during Ni(II) compound catalyzed oxidation of 3,5-DTBC to 3,5-DTBQ was suggested by
Mukhopadhyay et. al. [32]. Based on the ESI-MS spectral study and considering the
formation of H2O2 as by-product during the course of reaction, we have proposed a possible
mechanism which is shown in Scheme II. Here the catalytic process takes place through
16

radical pathway and the first electron transfer takes place to imine bond of coordinated Schiff-
base, and atmospheric oxygen molecules are reduced to H2O2. The ESI-MS spectral peak at
m/z = 621.02 corresponds to the intermediate 1a (calc. m/z 620.73), whereas the peak at m/z =
621.02 corresponds to intermediates 1b (calc. m/z 619.73) and 1c (calc. m/z 619.73).
Scheme II. Possible mechanism for catalytic oxidation of 3,5-DTBC to 3,5-DTBQ.
4. Conclusion
A nickel(II) complex containing 6-methoxy-2-{[2-(1-piperazinyl)ethylimino]methyl}phenol
and thiocyanate ligands was synthesized and characterized by spectroscopic and single
crystal X-ray diffraction studies with a view to investigate its biological activity. The nickel
complex is mononuclear and extends to a 1D supramolecular chain with hydrogen bonding
interactions. The CT-DNA and protein binding of the nickel(II) complex was investigated
using electronic absorption and fluorescence spectroscopic techniques. The compound binds
effectively to CT-DNA in the order of 105 M-1 through intercalation. Fluorescence
spectroscopic study shows that interaction of complex with serum albumins occur through
ground state association process. Cytotoxicity study reveals that the complex shows dose
17

dependent suppression of human breast (MCF7) cancer cell lines. Nickel(II) complex also
exhibits catecholase activity with turnover number 7.51 h-1 and probable mechanistic path of
catalytic activity has been explained with ESI mass spectroscopy.
Appendix A. Supplementary data
CCDC 1021243 contains the supplementary crystallographic data for complex 1. This data
can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from
the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. Figures and Tables showing IR,
NMR, electronic absorption and fluorescence spectral data are provided as supporting
information.
Acknowledgements
The authors gratefully acknowledge the financial assistance given by the CSIR,
Government of India, to Dr. Subal Chandra Manna [Project No. 01 (2743)/13/EMR-II].
Authors are also thankful to Dr. Manas Kumar Santra (National Centre for Cell Science,
Pune, India) for Cytotoxicity study.
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Table 1. Crystal data and details of structure refinement of complex 1.
Complex
1
Empirical formula
C22H37N7NiO5S2
Formula mass, g mol–1
602.41
Monoclinic
Ia
Crystal system
Space group
a, Å
b, Å
c, Å
11.6906(5)
17.9082(6)
14.4741(6)
90
α, deg
β, deg
109.915(5)
90
2849.0(2)
4
γ, deg
V, Å3
Z
D(calcd), g cm–3
1.404
µ(Mo-Kα), mm–1
F(000)
0.872
1272
Theta range, deg
No. of collected data
No. of unique data
Rint
2.993, 27.999
18613
6709
0.042
Observed reflections [I> 2σ(I)] 6205
Goodness of fit (F2)
1.045
Parameters refined
R1, wR2 (I >2σ(I)) [a]
Residuals, e Å–3
356
0.0332, 0.0672
-0.36, 0.47
[a]R1(Fo) = ΣFo–Fc / ΣFo, wR2(Fo2) = [Σw (Fo2 – Fc 2) 2/ Σw (Fo2) 2 ]
½
23

Table 2. Experimental bond distances (Å) and angles (°) for complex 1.
Bond Distances
Ni1-O10
Ni1-O1W
2.024(2)
2.121(2)
2.059(3)
Ni1-N2
Ni1-N18
Ni1-N21
2.063(3)
2.021(3)
2.255(3)
Ni1-N1
Bond angles
N1-Ni1-N2
N1-Ni1-N18
N1-Ni1-N21
N1-Ni1-O10
N1-Ni1-O1W
N2-Ni1-N18
N2-Ni1-N21
N2-Ni1-O10
174.13(12) N2-Ni1-O1W
85.58(10)
81.78(10)
90.10(10)
179.10(11)
171.26(10)
97.61(10)
90.46(9)
92.13(11)
94.12(11)
89.41(10)
88.58(10)
93.72(11)
87.34(11)
89.96(10)
N18-Ni1-N21
N18-Ni1-O10
N18-Ni1-O1W
N21-Ni1-O10
N21-Ni1-O1W
O10-Ni1-O1W
Table 3. Photophysical parameters of 1.
Compound λex, nm (εcal, M-1cm-1)
λem, nm ∆νa, nm φ
44, 59, 0.125
213 (0.244 x 105), 241 (0.382 x 105), 269 313,
1
(0.068 x 105), 318 (0.059 x 105), 392 328, 360 91
(0.035 x 105)
Bold number indicates the excitation wavelengths. aStoke shift.
Table 4. Kinetic parameters of the interaction BSA /HSA with complex 1.
Complex-Albumin T (K) kapp (Lmol-1)
n
1.25
ksv (Lmol-1)
kb (Lmol-1)
1-HSA
1-BSA
300
300
2.4315 × 103
21.540 × 105 15.992 × 105
21.720 × 105 12.184 × 105
22.9494 × 103 1.12
Table 5. Kinetic parameters for the oxidation of 3,5-DTBC catalyzed by complex 1.
Vmax (M min-1) Std. error
Km [M] Std. error
2.175 x 10-6 0.372 1.02 x 10-4 7.51
Kcat(h-1) Kcat/ Km (M-1h-1)
28.572
1.252 x 10-5
Caption of the Figures
Fig. 1. Ortep view (30%) of complex 1.
Fig. 2. 1D supramolecular chain of complex 1.
Fig. 3. (a) Top view of 1D layer of 1; (b) Side view of 1D layer of 1.
Fig. 4. Electronic absorption spectra of complex 1 (3 ml, 0.1991 M) in HEPES buffer upon
gradual addition of 10 µL 28.181 µM aqueous solution of CT-DNA.
24

Fig. 5. UV-vis absorption spectra of BSA (3 ml 8.214 µM aqueous solution) (figure A) and
HSA (3 ml 3.109 µM aqueous solution) (figure B) upon gradual addition of 20 µL 0.1991 M
aqueous solution of complex 1 at 300K.
Fig. 6. Fluorescence spectra of BSA (2 ml 8.214 µM) (figure A) and HSA (2 ml 3.109 µM)
(figure B) upon gradual addition of 10 µL 0.1991 M aqueous solution of complex 1 at room
temperature (λex = 280 nm and λem = 330 nm).
Fig. 7. Dose dependent suppression of cell viability of human breast cancer cell line MCF7 by
complex 1.
Fig. 8. Increase in 3,5-DTBQ band at 400 nm after addition of 10-4 M methanolic solution of
complex 1 to 100 fold methanolic solution of 3,5-DTBC. The spectra were recorded at an
interval of 5 min.
Fig. 9. Change in absorption maxima at 400 nm with time after addition of 10-4 M methanolic
solution of complex 1 to 100 fold methanolic solution of 3,5-DTBC.
Fig. 10. Rate vs. substrate (left) concentration and Lineweaver-Burk (right) plots for complex
1.
Fig. 1
25

Fig. 2
Fig. 3a
Fig. 3b
26

Fig. 4
Fig. 5
27

Fig. 6
Fig. 7
28

Fig. 8
Fig. 9
29