Binuclear and tetranuclear Zn(ii) complexes with thiosemicarbazones: Synthesis, X-ray crystal structures, ATP-sensing, DNA-binding, phosphatase activity and theoretical calculations

Article abstract of DOI:10.1039/c9ra10549b

Two Zinc(ii) complexes [Zn₄(L¹)₄]·2H₂O (1) and [Zn₂(L²)₂]·2H₂O (2) of pyruvaldehydethiosemicarbazone ligands are reported. The complexes were characterized by elemental analysis, IR, NMR, UV-vis spectroscopy and by single-crystal X-ray crystallography. X-ray crystal structure determinations of the complexes show that though Zn : ligand stoichiometry is 1 : 1 in both the complexes, the molecular unit is tetranuclear for 1 and binuclear for 2. Both the complexes show selective sensing of ATP at pH 7.4 (0.01 M HEPES) in CH₃CN-H₂O (9 : 1) medium in the presence of other anions like AcO^-, NO₃^-, F^-, Cl^-, H₂PO₄^-, HPO₄^2- and P₂O₇^2-. The UV-titration experiments of complexes 1 and 2 with ATP results in binding constants of 2.0(±0.07) × 10⁴ M^-1 and 7.1(±0.05) × 10³ M^-1 respectively. The calculated detection limits of 6.7 μM and 1.7 μM for 1 and 2 respectively suggest that the complexes are sensitive detectors of ATP. High selectivity of the complexes is confirmed by the addition of ATP in presence of an excess of other anions. DFT studies confirm that the ATP complexes are more favorable than those with the other inorganic phosphate anions, in agreement with the experimental results. Phosphatase like activity of both complexes is investigated spectrophotometrically using 4-nitrophenylphosphate (NPP) as a substrate, indicating the complexes possess significant phosphate ester hydrolytic efficiency. The kinetics for the hydrolysis of the substrate NPP was studied by the initial rate method at 25 °C. Michaelis-Menten derived kinetic parameters indicate that rate of hydrolysis of the P-O bond by complex 1 is much greater than that of complex 2, the k_cat values being 212(±5) and 38(±2) h^-1 respectively. The DNA binding studies of the complexes were investigated using electronic absorption spectroscopy and fluorescence quenching. The absorption spectral titrations of the complexes with DNA indicate that the CT-DNA binding affinity (K_b) of complex 1 (2.10(±0.07) × 10⁶ M^-1) is slightly greater than that of 2 (1.11(±0.04) × 10⁶ M^-1). From fluorescence spectra the apparent binding constant (K_app) values were calculated and they are found to be 5.41(±0.01) × 10⁵ M^-1 for 1 and 3.93(±0.02) × 10⁵ M^-1 for 2. The molecular dynamics simulation demonstrates that the Zn(ii) complex 1 is a good intercalator of DNA.

Full text of DOI:10.1039/c9ra10549b

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PAPER
Binuclear and tetranuclear Zn(II) complexes with
thiosemicarbazones: synthesis, X-ray crystal
structures, ATP-sensing, DNA-binding,
Cite this: RSC Adv., 2020, 10, 12735
phosphatase activity and theoretical calculations†
a
a
b
Antonio Frontera, *b
Piyali Adak, Bipinbihari Ghosh, Antonio Bauza,
´
c
a
Steven R. Herron and Shyamal Kumar Chattopadhyay
*
1
2
Two Zinc(II) complexes [Zn
4
(L )
4
]$2H
2
O (1) and [Zn
2
(L )
2 2
]$2H O (2) of pyruvaldehydethiosemicarbazone
ligands are reported. The complexes were characterized by elemental analysis, IR, NMR, UV-vis
spectroscopy and by single-crystal X-ray crystallography. X-ray crystal structure determinations of the
complexes show that though Zn : ligand stoichiometry is 1 : 1 in both the complexes, the molecular unit
is tetranuclear for 1 and binuclear for 2. Both the complexes show selective sensing of ATP at pH 7.4
ꢀ
3ꢀ
ꢀ
ꢀ
3
(0.01 M HEPES) in CH CN–H
2
O (9 : 1) medium in the presence of other anions like AcO , NO , F , Cl ,
H
2
PO , HPO
4ꢀ
42ꢀ
and P
2
O . The UV-titration experiments of complexes 1 and 2 with ATP results in
72ꢀ
4
ꢀ1
3
ꢀ1
M respectively. The calculated
binding constants of 2.0(ꢁ0.07) ꢂ 10
M
and 7.1(ꢁ0.05) ꢂ 10
detection limits of 6.7 mM and 1.7 mM for 1 and 2 respectively suggest that the complexes are sensitive
detectors of ATP. High selectivity of the complexes is confirmed by the addition of ATP in presence of
an excess of other anions. DFT studies confirm that the ATP complexes are more favorable than those
with the other inorganic phosphate anions, in agreement with the experimental results. Phosphatase like
activity of both complexes is investigated spectrophotometrically using 4-nitrophenylphosphate (NPP) as
a substrate, indicating the complexes possess significant phosphate ester hydrolytic efficiency. The
ꢃ
kinetics for the hydrolysis of the substrate NPP was studied by the initial rate method at 25 C.
Michaelis–Menten derived kinetic parameters indicate that rate of hydrolysis of the P–O bond by
ꢀ
1
complex 1 is much greater than that of complex 2, the kcat values being 212(ꢁ5) and 38(ꢁ2) h
respectively. The DNA binding studies of the complexes were investigated using electronic absorption
spectroscopy and fluorescence quenching. The absorption spectral titrations of the complexes with DNA
6
ꢀ1
indicate that the CT-DNA binding affinity (K
b
) of complex 1 (2.10(ꢁ0.07) ꢂ 10
M ) is slightly greater
Received 15th December 2019
6
ꢀ1
than that of 2 (1.11(ꢁ0.04) ꢂ 10
M
). From fluorescence spectra the apparent binding constant (Kapp)
Accepted 13th March 2020
DOI: 10.1039/c9ra10549b
rsc.li/rsc-advances
5
ꢀ1
5
ꢀ1
values were calculated and they are found to be 5.41(ꢁ0.01) ꢂ 10
M
for 1 and 3.93(ꢁ0.02) ꢂ 10 M
for 2. The molecular dynamics simulation demonstrates that the Zn(II) complex 1 is a good intercalator of
DNA.
transition metal complexes mimic the coordination environ-
ment of many biological systems. As a class of nitrogen–sulfur
1
. Introduction
The coordination chemistry of nitrogen–sulphur donor ligands donor ligands, the synthesis of compounds incorporating thi-
1,2
towards transition metal ions has attracted much attention osemicarbazones has attracted widespread attention of chem-
and the interest arises in part from the fact that S-ligated ists as well as biologists, because of the interesting chemical,
biological, structural and electronic properties of their metal
3–9
complexes. Again, Zn(II) ions play important roles in medic-
inal, chemical and biological events, and it is the second most
abundant transition metal next to the iron ion in the body
a
Department of Chemistry, Indian Institute of Engineering Science and Technology,
Shibpur, Howarh-711 103, India. E-mail: shch20@hotmail.com
b
Department of Chemistry, University of the Balearic Islands, Carretera de
(
human beings contain an average of 2–3 g of zinc), being vital
Valldemossa km 7.5, 07122 Palma de Mallorca, IllesBalears, Spain
10
11
c
in many cellular processes, including gene expression,
Department of Chemistry, Utah Valley University, 800W University Pkwy, Orem UT
12
13
14
8
4058, USA
apoptosis, enzyme regulation and neurotransmission. It
†
Electronic supplementary information (ESI) available. CCDC 1911646 and has been suggested that the anti-Alzheimer medicine clioqui-
1
911647. For ESI and crystallographic data in CIF or other electronic format see
nol, a 8-hydroxyquinoline derivative, functions by way of
DOI: 10.1039/c9ra10549b
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chelating Zn(II) along with Cu(II), the two metal ions which are NMR spectra were recorded on a Bruker AVANCE DPX 300 MHz
critically associated with protein aggregation and degeneration spectrometer using, Si(CH3)4 as internal standard. ESI-MS
15
processes in the brain. Zinc complexes with N,S-donor sets are spectra of the samples were recorded on JEOL JMS 600
16
potential mimics of various zinc-containing metalloproteins.
instrument.
The biological activity and the DNA-binding ability of zinc
complexes have been the subject of a large number of
studies.
2
.2 Synthesis of ligand (HL)
17–19
The 4-(aryl)thiosemicarbazides were prepared from the corre-
The design and synthesis of chemosensors for selective
recognition and sensing of a particular anion is an area of
5
4
sponding aryl amines by the published procedure. The thio-
semicarbazones were prepared by reuxing (2 h) a methanolic
solution of thiosemicarbazide with pyruvic aldehyde in 2 : 1
molar proportion.
20,21
immense importance.
Sensors that can detect and sense
biologically important anions in aqueous environments and
under physiological pH are of special signicance owing to their
Yield: ꢄ80–90%.
22
potential applications in biological eld. Among various
1
2
.2.1 Ligand 1 (L H
2
). Anal. calc. for: C17
H
18
N
6
S
2
: C, 55.11;
anions, recognition of phosphates, pyrophosphate, nucleotides,
H, 4.90; N, 22.69. Found: C, 55.19; H, 4.97; N, 22.73%. ESI-MS,
23,24
and nucleosides is important in this regard.
Among many
+
+
1
m/z: 371 [M + H] , 393 [M + Na] (Fig. S1 in ESI†); H NMR
DMSO-d ) (d ppm): 12.12 (1H, s), 10.78 (1H, s), 10.23 (1H, s),
phosphate anions, adenosine triphosphate (ATP) is most
important as a molecular currency of intracellular energy
(
1
6
0.04 (1H, s), 10.04 (1H, s), 7.55 (4H, m), 7.38 (4H, m), 7.22 (2H,
25
transfer.
ꢀ
1
m) 2.30 (3H, s) (Fig. S2 & S3 in ESI†); selected IR bands (cm ):
296(nNH), 3152 (nNH), 1597 (nC]N), 755 (nC]S) (Fig. S13 in ESI†).
Studies on the interaction of small molecules with DNA
continue to attract considerable attention due to their impor-
3
2
2
.2.2 Ligand 2 (L H
2 22 6 2
). Anal. calc. for C19H N S : C, 57.26;
26–31
tance in cancer therapy and molecular biology.
Over the last
H, 5.56; N, 21.09. Found: C, 57.18; H, 5.51; N, 21.05%. ESI-MS,
few decades, transition metal complexes that are capable of
binding DNA under physiological conditions are of interest in
+
1
m/z: 399 [M + H] (Fig. S4 in ESI†); H NMR (DMSO-d
2.06 (1H, s), 10.71 (1H, s), 10.14 (1H, s), 9.96 (1H, s), 7.84(1H,
s), 7.38 (4H, m), 7.15 (4H, m), 2.31(3H, s), 2.28 (6H, s) (Fig. S5 &
6
) (d ppm):
1
32–36
the development of metal-based anticancer agents.
These
complexes are used in DNA-footprinting studies, as sequence
ꢀ1
S6 in ESI†); selected IR bands (cm ): 3292 (nNH), 3156 (nNH),
specic DNA binding agents, diagnostic agents in medicinal
1596 (sh, nC]N), 730 (nC]S) (Fig. S14 in ESI†).
37–42
applications and for genomic research.
On the other hand, over the years, much effort has been
2
.3 Synthesis of the complexes
devoted to the development of metal complexes as models for
43
1
metallo-enzymes catalyzing phosphate ester hydrolysis.
A
2.3.1 Synthesis of [Zn (L ) ]$2H O (1). To a vigorously
4
4
2
number of zinc enzymes with multinuclear sites are known to stirred methanolic suspension (15 ml) of the thio-
4
4–53
1
be responsible for the hydrolysis of the phosphate esters.
semicarbazone ligand (L H
2
) (370 mg, 1 mmol), Et
COO) $2H
3
N (202 mg, 2
O (219 mg, 1
Herein we report the synthesis, characterization, and DNA mmol) was added. A solution of Zn(CH
3
2
2
binding property of two neutral tetranuclear and binuclear mmol) in methanol (10 ml) was added dropwise to the above
zinc(II) complexes which can be used to bind ATP in aqueous solution. The solution turned orange. The mixture was stirred at
medium at physiological pH. These two zinc complexes effec- room temperature for three hours and then ltered. The orange
tively hydrolyses p-nitrophenylphosphate to p-nitrophenolate. residue was washed with methanol and then dried over fused
The propensity of these complexes to bind to CT (calf-thymus) calcium chloride. The ltrate was allowed to evaporate slowly at
DNA has been investigated with UV-vis spectroscopic titration room temperature when orange colored square shaped shiny
1
and their intrinsic binding constants, K
b
, with CT-DNA have crystals of complex [Zn
4
(L )
4
]$2H
2
O (1), suitable for X-ray
been determined. Competitive binding studies with ethidium diffraction studies, appeared aer 4 days. Yield: 365 mg
bromide (EB), one of the most widely used intercalative agents (84%). Anal. calc. for C H N S O Zn : C, 46.10; H, 3.87; N,
6
8
68 24
8
2
4
and uorescence probes for DNA structure, have been used in 18.98. Found: C, 46.05; H, 3.80; N, 19.95%. ESI-MS, m/z: 432.91
1
+
1
the study of the interaction of the complexes with CT-DNA in [ZnL ] (Fig. S7 in ESI†); H NMR (DMSO-d ) (d ppm): 9.67 (1H,
6
order to investigate a potential intercalative binding mode.
s), 9.53 (1H, s), 7.86 (1H, s), 7.79 (4H, dd), 7.25 (4H, m), 6.94 (2H,
dd), 2.25 (3H, s) (Fig. S8 & S9 in ESI†); electronic spectrum in
ꢀ
1
ꢀ1
DMF solution
l
max/nm (3max/M
cm ): 343(25 690),
2
. Experimental
ꢀ
1
4
1
60(39 847). Selected IR bands (cm ): 3290(nNH), 1600(nC]N),
2.1. Materials and methods
545(nC]N) (Fig. S13 in ESI†).
2
Pyruvic aldehyde, Zn(CH COO)
3
2
$2H
2
O were purchased from
2.3.2 Synthesis of [Zn
2
(L )
2
] $2DMF (2). This was synthe-
Aldrich. All other chemicals and solvents were of reagent grade sized following a similar procedure to that of 1. Yield: 392 mg
and used as such. Solvents for spectroscopic and cyclic vol- (85%). Anal. calc. for C44 Zn : C, 49.39; H, 5.09; N,
tammetry were of HPLC grade obtained from Merck or Aldrich. 18.33. Found: C, 49.50; H, 4.98; N, 18.09 ESI-MS, m/z: 461.07
H
54
N
14
O
2
S
4
2
2
+
1
Elemental analyses were performed on a PerkinElmer 2400 C, [ZnL ] (Fig. S10 in ESI†); H NMR (DMSO-d ): d(ppm): 9.58 (1H,
6
H, N analyzer. Infrared spectra were recorded as KBr pellets on s), 9.43 (1H, s), 7.82 (1H, s), 7.66 (4H, m), 7.32 (4H, m), 2.27
a JASCO FT-IR-460 spectrophotometer. UV-vis spectra were (6H,s), 2.23 (3H, s) (Fig.s S11 & S12 in ESI†). Electronic spectrum
1
ꢀ1
ꢀ1
recorded using a JASCO V-530 UV-vis spectrophotometer.
H
in DMF solution lmax/nm (3max/M cm ): 342 (9814), 462
12736 | RSC Adv., 2020, 10, 12735–12746
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ꢀ
1
(
15 964). Selected IR bands (cm ): 3151(nNH), 2952(nNH), 2.6 Catalytic hydrolysis of 4-nitrophenyl phosphate
1
589(nC]N) (Fig. S14 in ESI†). Single crystals suitable for X-ray
In order to study the phosphatase activity of the complexes,
structural analysis were obtained from the slow evaporation of
the ltrate.
ꢀ4
10
M solutions of 1 and 2 in DMF were treated with 100 equiv.
of 4-nitrophenyl phosphate in DMF–water(1 : 1) mixture under
aerobic conditions at room temperature. The absorbance versus
wavelength (wavelength scan) of these solutions was recorded at
regular time intervals of 2 min in the wavelength range of 300–
2
.4 UV-vis titrations for anion sensing
Stock solutions of the Zn(II) complexes were prepared (1 ꢂ 10
M) in CH CN : H
buffer. The solution of the guest anion was prepared (2 ꢂ 10
ꢀ5
O (9 : 1, v/v) at pH 7.4 by using 20 mM HEPES 600 nm. To determine the dependence of the rate on the
3
2
ꢀ4
substrate concentration and various kinetic parameters,
ꢀ
5
M) in CH CN : H
O (1 : 1, v/v) at pH 7.4 by using 20 mM HEPES a 10 M solution of complex was treated with 10, 20, 30, 40, 50
buffer. The spectroscopic titrations were carried out by incre- and 100 equiv. of substrate. The reactions were followed spec-
3
2
ꢀ4
mental addition of 0.02 ml (2 ꢂ 10 M) of anion solutions to trophotometrically by monitoring the increase in the absor-
ꢀ5
the solution of the complexes 1 and 2 (1 ꢂ 10 M).
bance at 427 nm (4-nitrophenolate band maximum) as
a function of time (time scan).
The kinetics for the hydrolysis of the substrate NPP was
determined by the initial rate method at 25 C. The concen-
tration of the substrate NPP was always kept at least 10 times
larger than that of the complex and the increase of the 4-
2
.5 DNA-binding experiments
ꢃ
2.5.1 Absorption spectral studies. The binding experi-
ments of the complexes (1 and 2) with CT-DNA were carried out
in Tris–HCl buffer (50 mM, pH 7.4). A solution of CT-DNA in the
buffer gave a ratio of UV absorbance at 260 and 280 nm of about
nitrophenol concentration was determined at 429 and 427 nm,
ꢀ5
respectively for the two complexes. For this purpose, 10
M
1.8–1.9 : 1, indicating that the DNA was sufficiently free from
solution of a complex was treated with the substrate solution
having concentration ranging between 10-fold and 100-fold
than that of the complex. Each kinetic experiment at a given
substrate concentration was repeated thrice and average of the
three values are taken. The dependence of the initial rate on the
concentration of the substrates was monitored spectrophoto-
metrically at the respective wavelengths. The initial rate method
follows a rst-order dependence on complex concentration
since it showed saturation kinetics at higher substrate
concentrations. For this reason, a treatment based on the
Michaelis–Menten model was applied. The values of the
55
protein. The DNA concentration per nucleotide and poly-
nucleotide concentration were determined by absorption spec-
troscopy using the molar extinction coefficient (6600 M cm
at 260 nm. The intrinsic binding constant K for the interac-
tion of these metal (II) complexes with DNA has been calculated
from the absorption spectral titration data. The intrinsic
ꢀ
1
ꢀ1
)
56
b
5
7
binding constant K
b
was determined from,
½
DNAꢅ
½DNAꢅ
1
¼
þ
À
Á
3
a
ꢀ 3
f
3
b
ꢀ 3
f
Kb 3b ꢀ 3f
Michaelis binding constant (K ), maximum velocity (Vmax) and
M
The ‘apparent’ extinction coefficient (3
a
) was obtained by
b
correspond to the
rate constant for the dissociation of the substrate (i.e., turnover
number, kcat) were calculated for each complex by non-linear
curve tting using Michaelis–Menten equation:
calculating Aobs/[Zn]. The symbols 3 and 3
f
extinction coefficients of free (unbound) and the fully bound
complexes. A plot of [DNA]/(3 ) versus [DNA] will give a slope
ꢀ 3
/(3 ꢀ 3 ) and an intercept of 1/K (3 ꢀ 3 ). Therefore K can be
a
f
V
max þ ½Sꢅ
1
b
f
b
b
f
b
V
0
¼
58
KM þ ½Sꢅ
calculated from the ratio of the slope and intercept.
.5.2 Fluorescence spectral studies. The tetra and binu-
2
clear zinc(II) complexes do not exhibit luminescence, either in
DMF or in the presence of calf thymus DNA. Therefore the
binding of Zn(II) complexes to calf thymus DNA has been
studied by competitive binding experiments. Ethidium bromide
2
.7 X-ray crystallography
Data for both the complexes were collected on a Burker APEX
diffractometer at 293(2) K, using a graphite-monochromated
Mo–Ka radiation. The data were solved by direct method and
(
EB) is well known to show uorescence when bound to DNA
2
64
59
rened by full matrix least squares on F using SHELX 97. The
non-hydrogen atoms were rened with anisotropic displace-
ment parameters. All hydrogen atoms were placed at calculated
positions and rened as riding atoms using isotropic
displacement parameters. Corrections for Lorentz polarization
effects and semiempirical absorptions were applied. A summary
of the crystallographic data and the renement details are
summarized in Table 1. Important bond distances and bond
angles are collected in Table 2.
due to intercalative binding with DNA. The relative binding of
the complexes with CT-DNA were studied using EB-bound CT-
DNA solution in Tris–HCl buffer (pH 7.4). The uorescence
spectra were recorded at room temperature with excitation at
520 nm and emission at 610 nm. This experiment was carried
out by titrating the complexes with EB-DNA solution containing
ꢀ
6
ꢀ6
60
5
ꢂ 10 M EB and 5 ꢂ 10 M DNA. The competitive binding
of the zinc(II) complex to CT-DNA results in the displacement of
the bound EB. This causes a decrease in the emission intensity
due to uorescence quenching of the free EB by the solvent
61–63
2.8 Theoretical methods
molecules.
These spectroscopic features suggest that there
exists an interaction between the zinc(II) complexes and DNA
molecules.
2.8.1 DFT calculations. The geometries of the complexes
used in this study were optimized with a density functional
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ꢀ
ꢃ
Table 1 Crystal data and refinement details of complexes 1 and 2
Table 2 Selected bond distances (A) and angles ( ) for 1 & 2
Formula
C
68
H
64
N
24
O
2
S
8
Zn
4
C
44
H
54
N
14
O
2
S
4
Zn
2
1
Formula weight3
Crystal size(mm )
Temperature
Crystal system
Space group
1767.48
1069.99
ꢀ
0.22 ꢂ 0.20 ꢂ 0.10 0.26 ꢂ 0.20 ꢂ 0.12 Bond distances (A)
293(2) K
Triclinic
P1ꢁ
10.454(3)
10.621(4)
19.648(6)
75.017(8)
82.443(8)
67.822(8)
1.503
273(2) K
Monoclinic
P2 /c
1
21.767(4)
17.713(3)
13.738(2)
90
103.454(4)
90
1.380
4
1.144
2224
81 426
8614
5368
0.0958
0.0752, 0.1343
0.1391, 0.1675
8614/4/635
1.137
Zn1–N3
Zn1–N4
Zn1–S2
Zn1–S1
Zn1–S3
2.115(8)
2.124(8)
2.330(3)
2.347(3)
2.545(3)
Zn2–N9
Zn2–N10
Zn2–S3
Zn2–S4
Zn2–S4
2.090(8)
2.104(8)
2.374(3)
2.381(3)
2.599(3)
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
ꢃ
ꢃ
a ( )
Bond angles ( )
N3–Zn1–N4
N3–Zn1–S2
N4–Zn1–S2
N3–Zn1–S1
N4–Zn1–S1
S2–Zn1–S1
N3–Zn1–S3
N4–Zn1–S3
S2–Zn1–S3
S1–Zn1–S3
ꢃ
b ( )
73.8(4)
146.4(2)
80.5(3)
N9–Zn2–N10
N9–Zn2–S3
N10–Zn2–S3
N9–Zn2–S4
N10–Zn2–S4
S3–Zn2–S4
N9–Zn2–S4
N10–Zn2–S4
S3–Zn2–S4
S4–Zn2–S4
74.6(3)
80.8(3)
ꢃ
g ( )
d
Z
cal
148.8(2)
154.4(3)
80.6(2)
119.97(10)
93.8(2)
99.5(2)
101.02(9)
96.18(8)
1
80.3(3)
ꢀ1
m(mm
F(000)
)
1.490
902
12 001
3952
2508
0.0737
0.0610, 0.1617
0.1023, 0.1932
3952/0/480
1.011
152.4(3)
118.73(10)
103.5(2)
94.3(2)
99.58(10)
100.97(10)
Total reections
Unique reections
Observed data [I >2s(I)]
R(int)
R
R
1
, wR
, wR
2
[I >2s(I)]
(all data)
2
1
2
Data/restraints/parameters2
ꢀ
Bond distances (A)
Zn1–N10
Zn1–N9
Goodness-of-t (GOF) on F
2.093(6)
2.120(6)
2.322(2)
2.447(2)
2.454(2)
Zn2–N3
Zn2–N4
Zn2–S1
Zn2–S2
Zn2–S3
2.094(5)
2.113(5)
2.4468(19)
2.316(2)
2.454(2)
Zn1–S4
Zn1–S3
Zn1–S1
3
theory (DFT) method by using the all-electron Dmol program
code of Accelrys, Inc.
UM06-L/DNP+ levels of theory, using the COSMO polarizable
continuum model to mimic the water solvent effect. UM06-L is
a local meta hybrid functional recommended for application in
65–67
The calculations were optimized at the
ꢃ
Bond angles ( )
N10–Zn1–N9
N10–Zn1–S4
N9–Zn1–S4
N10–Zn1–S3
74.4(2)
N3–Zn2–N4
N3–Zn2–S2
N4– Zn2– S2
N3–Zn2–S1
N4–Zn2–S1
S2–Zn2–S1
N3–Zn2–S3
N4–Zn2–S3
S2–Zn2–S3
S1–Zn2–S3
74.9(2)
81.65(18)
148.91(17)
147.50(18)
78.24(17)
115.98(7)
105.95(17)
100.75(18)
104.71(8)
95.99(6)
80.67(16)
147.36(17)
147.40(16)
78.40(16)
114.84(7)
107.01(17)
100.86(16)
106.80(8)
96.00(6)
67
organometallic chemistry, using double numerical with
polarization and diffuse (DNP+) basis set and a global orbital N9–Zn1–S3
ꢀ
68
S4–Zn1–S3
N10–Zn1–S1
N9–Zn1–S1
S4–Zn1–S1
S3–Zn1–S1
cut off of 4.4 A.
2
.8.2 Molecular dynamics calculation
A sequence of twelve base pairs of the oligonucleotide, d
GCGCGGAAGCGC] like a model of calf thymus DNA was
[
2
chosen and constructed in the double-helix B-DNA conforma-
tion by Discover Studio 4.0 of Accelrys inc. To create the inter-
calation pocket in the starting DNA structure, the torsion angles
a–x of the sugar backbone were modied. The complex was
(
oligonucleotide and the 1 complex) was restrained with a force
ꢀ
1
ꢀ2
constant of 1000 kJ mol
1
nm , gradually relaxed to
ꢀ1
ꢀ2
th
th
00 kJ mol nm . Aerwards, a 100 ps equilibration step
intercalated in the major groove between 6 and 7 base pairs.
75
(
NVT) was carried out using the modied Berendsen thermo-
stat to increase the temperature of the system at 300 K. A second
00 ps equilibration step was performed using the Parrinello–
We have used amber tools 12.0 (ref. 69) to create all input les to
70,71
run the molecular dynamics simulations in Gromacs
so-
1
ware package. Molecular dynamics were performed using the
amber14ipq Field to describe all parameters of the dodecamer.
Partial charges for the Zn-complex were obtained using the
restrained electrostatic t (RESP) scheme. Electrostatic poten-
tials were computed using the ab initio HF/6-31G(d) calculations
76
Rahman pressure coupling model to equilibrate the pressure
of the system at 1 atm. Aer the NVT and NPT steps, we run 500
ns molecular dynamic, enough to obtain well equilibrated MD
trajectories of each system.
72
using the Gaussian03 package. All parameters of these mole-
cules were taken from the general amber force eld (GAFF)
3
. Results and discussion
73
when we use the antechamber soware. Finally, all systems
+
3.1 Synthesis of complexes
were neutralized with the addition of Na ions and then were
completed with a tetrahedral box of TIP3P waters to a depth of The Schiff base ligands used in this study were prepared
74
1
.0 nanometre on each side of the solutes.
Preliminary energies minimizations were run with the semicarbazide derivatives with pyruvic aldehyde in 2 : 1 molar
steepest descent algorithm, during which the system ratio. The Zn(II) complexes were obtained in moderately good
following same method by stirring appropriate thio-
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yield by reaction of the thiosemicarbazone Schiff bases with
Zn(OAc) $2H O in presence of triethyl amine in 1 : 1 : 2 molar
2
2
proportion in methanol at room temperature (Scheme 1). The
ligands were characterized by elemental analysis, mass, IR and
1
1
H-NMR spectroscopy. For the ligand L H
2
tetranuclear Zn(II)
2
complex was obtained, whereas for the ligand L H
2
binuclear
Zn(II) complex was obtained. The solution stability of the
complexes were ensured by measuring ESI-MS, NMR and UV-vis
spectra of the complexes 8–10 hours aer dissolution when the
spectral data remain unchanged.
3.2 Description of X-ray crystal structure
X-ray crystal structure determination of complex 1 (Fig. 1 and 2)
shows that though Zn : ligand stoichiometry is 1 : 1, the
molecular unit is tetranuclear. The two central Zn(II) ions are
bridged by two thiolato sulfur atoms in axial–equatorial mode,
while each of the two terminal Zn(II) ions are connected to
a central Zn(II) by a single thiolato bridge. Each of the four Zn(II)
1
Fig. 1 ORTEP diagram (50% probability level) of [Zn
4
(L )
4
2
]$2H O (1).
2 3
ions is in a square pyramidal N S -coordination environment,
the equatorial position being occupied by one dideprotonated
tetradentate ligand, and the axial position is occupied by
a sulfur atom of a ligand attached to a neighbouring Zn(II) atom.
Both the thiolato sulfur atoms belonging to the ligand attached
to each of the central Zn(II) atoms participate in bridging while
The structure of the binuclear complex is similar to the two
doubly bridged central Zn(II) atoms of the tetranuclear complex
with the terminal zinc centers being stripped off (Fig. 2). The
ꢀ
ꢀ
Zn/Zn distance is 3.250(1) A compared to 3.330(2) A for the
central Zn(II) atoms in the tetranuclear complex. The C–S bind
the sulfur atoms coordinated to the terminal Zn(II) atoms do not
ꢀ
distances lie in the range 1.739(8)–1.779(8) A, with the bridging
1
participate in bridging. The tetranuclear complex [Zn (L ) ]$
4
4
thiolate having slightly longer bond length. The Zn–N distances
are almost identical in both the complexes, while the Zn–S
distances are on an average slightly shorter in the binuclear
complex.
2
2H O (1) results from the pairing of two dinuclear units related
by crystallographic centre of inversion, by two thiolato bridges.
ꢀ
The distance between two central Zn(II) atom is 3.330(2) A while
the distance between the terminal and its adjacent Zn(II) atoms
ꢀ
is 3$716(2) A. The C–S bond distance lying between 1.734(10)–
1
3.3 Electronic spectra
ꢀ
.776(10) A indicates the bis-thiosemicarbazone ligand binds
The electronic spectra of the ligands and the complexes are
depicted in Fig. 3. The free ligands show two bands, a strong
sharp band at 350 nm and a very weak broad band at 460 nm.
These bands are probably p / p* (higher energy band) or n /
p* (lower energy band) in origin. In the complexes there is
a strong transition at 460 nm. Extensive delocalization on
deprotonation followed by complexation lowers the energy of
through thiolate-like than thione-like. Sum of the different
angles between the terminal metal centre and the donor atoms
ꢃ
in its equatorial plane is 353.33(10) , while that for the central
ꢃ
metal center is 355.973(10) , indicating the planarity of the
tetradentate ligand. The coordination polyhedrons around
Zn(II) centers may be described as axially elongated square
pyramid with s parameter being 0.09–0.10. The apical site of the
coordination polyhedron is occupied by a bridging thiolato S-
ꢀ
atom with Zn–S distance 2.545(3)–2.599(3) A which is consid-
ꢀ
erable longer than the Zn–S bond length (2.330(3)–2.381(3) A)
found in the basal plane.
Scheme 1 Schematic representation of the template synthesis of the
Zn(II) complexes.
2
Fig. 2 ORTEP diagram (50% probability level) of [Zn
2
(L )
2
]$2H
2
O (2).
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the LUMO which is a p*-orbital. The 460 nm band can have its
origin in a new p / p* transition or a LLCT transition from
thiolate pp to imine p* orbital. The 350 nm band remains
almost unaltered in complex 1 but in complex 2 it now appears
as a shoulder.
3
.4 ATP binding study
.4.1 Absorption spectra study of zinc(II) complexes to ATP.
In order to study binding selectivity of complexes 1and 2, UV-vis
titrations have been carried out at pH 7.4 in CH CN–H O (9 : 1)
3
3
2
ꢀ5
at a concentration of 10 M by adding salts of different anions.
Fig. 4 and 5 display the notable changes in the UV-vis spectrum
ꢀ
5
ꢀ
ꢀ
ꢀ
ꢀ
of 1 and 2 (10 M) on the addition of AcO , NO , F , Cl ,
3
ꢀ
2ꢀ
2ꢀ
H PO , HPO4 , P O
and ATP ions. Spectral changes were
2
4
2
7
observed only when ATP is added to the solution. Moreover, the
colour of the solutions 1 and 2 changed from yellow to colour-
less. However, no detectable spectral responses and colour
ꢀ
changes were observed even aer adding large amount of AcO ,
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
2ꢀ
NO
3
, F , Cl , H
2
PO
4
, HPO
4
and P
2
O
7
ions. These results
suggest that 1 and 2 can distinguish ATP from other anions.
To explore further about the applicability of 1 and 2 for ATP
sensing, the UV-vis titrations were performed at pH 7.4 in
CH CN–H O (9 : 1). The presence of ATP resulted in the
3
2
signicant increase of intensity of the absorbance band at
43 nm while the absorbance band at 450 nm decreases and the
peak at 239 nm shied to 260 nm with an increase in intensity
for 1 (Fig. 4b). Upon the addition of ATP to 2, the absorbance at
3
Fig. 4 (a) UV-vis absorption changes of 1 upon addition of various
anions at pH 7.4 (0.01 M HEPES) in CH
vis absorption titration spectra of 1 with ATP at pH 7.4 (0.01 M HEPES)
in CH CN–H O (9 : 1) medium.
3 2
CN–H 0 (9 : 1) medium. (b) UV-
261 nm increases while the absorption at 450 nm gradually
3
2
decreases. Moreover, a new absorption band appears at 349 nm,
and its absorbance gradually increases with the addition of ATP
(
Fig. 5b). The competitive binding ability of ATP by the
3
.5 DNA binding studies
complexes in presence of three fold excess of other biologically
important anions is shown in Fig. S16 in ESI.† It is clear from
these two gures that both the complexes show high selectivity
towards ATP.
3.5.1 Absorption spectra. Electronic absorption spectros-
copy is used for examining the binding modes of metal
complexes with DNA. Hypochromic or bathochromic shi of
the absorption band occurs due to strong stacking interaction
between the aromatic chromophore of the complex and the
adjacent base pairs of DNA. The interaction of the zinc(II)
complexes 1 and 2 in DMF solutions with CT-DNA was investi-
gated by UV-vis titrations. Absorption titration experiments of
the Zn(II) complexes in Tris-buffer (pH 7.4) were performed
using a xed complex concentration to which increments of
a DNA stock solution were added. The absorption band of
complex 1 at 346 nm is red shied and that at 460 nm is blue
shied with decrease of absorption intensity (Fig. 6a), while the
absorption band of complex 2 at 277 nm and 463 nm both are
blue shied with decrease of absorption intensity (Fig. 6b). The
binding constant values for 1 and 2 were calculated as 2.10
The association constant (K
a
) with ATP, as determined using
a spectrophotometric titration method, for complexes 1 and 2 is
4
ꢀ1
2
found to be 2.0(ꢁ0.07) ꢂ 10 M (R ¼ 0.988) and 7.1(ꢁ0.05) ꢂ
3
ꢀ1
2
1
0 M (R ¼ 0.979) respectively by using the Benesi–Hilde-
brand equation (Fig S17†). The calculated detection limits at 6.7
mM and 1.7 mM suggest that the complexes 1 and 2 are poten-
tially very good sensors of ATP (Fig S18†).
6
2
6
2
(
ꢁ0.07) ꢂ 10 (R ¼ 0.986) and 1.11(ꢁ0.04) ꢂ 10 (R ¼ 0.99),
respectively (Fig. S18 and Table S1 in ESI†). The Gibb's free
energy (DG) was calculated from eqn (1)
DG ¼ ꢀRT ln K
(1)
ꢀ
1
ꢀ1
where R is general gas constant (8.314 J K mol ) and T is the
temperature (298 K). The Gibb's free energy (DG) values for the 1
Fig. 3 UV-visible spectra of the ligands and the complexes in DMF at
298 K.
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Fig. 5 (a) UV-vis absorption changes of 2 upon addition of various
anions at pH 7.4 (0.01 M HEPES) in CH
vis absorption titration spectra of 2 with ATP at pH 7.4 (0.01 M HEPES)
in CH CN–H O (9 : 1)medium.
3 2
CN–H O (9 : 1) medium (b) UV-
ꢀ5
Fig. 6 Absorption spectra of (a) complex 1 (b) complex 2 (10 M) with
increasing amounts of DNA at pH 7.4.
3
2
ꢀ
1
The emission spectra were monitored by keeping the exci-
tation at 520 nm and the emission was monitored in the range
of 540–690 nm. The emission was observed at 611 nm. The
and 2 are ꢀ36.07 and ꢀ34.46 kJ mol
respectively. The
moderately high negative values of DG indicate signicant
interactions of these compounds with DNA.
quenching constant K was determined from the classical
3
.5.2 Fluorescence spectral studies. Complexes 1 and 2
SV
77
Stern–Volmer equation:
show no uorescence in DMF solutions both in presence as well
as absence of DNA. So, to study the interaction between DNA
and Zn(II) complexes we have chosen ethidium bromide(EB) as
a uorescence probe for DNA. Since the emission intensity of EB
in buffer medium is low, but it is enhanced by its stacking
interaction between adjacent DNA base pairs, so, the competi-
tive DNA binding of the complexes has been studied by moni-
toring changes in the emission intensity of ethidium bromide
I0
I
¼
1 þ KSV½Qꢅ
where I and I are the uorescence intensities in the absence
0
and presence of complexes, respectively, KSV is the linear Stern–
Volmer quenching constant, [Q] is the concentration of the
Zn(II) (quencher)complexes. The slope of the straight line gives
the value of KSV and the quenching constant value is
(
EB) bound to CT-DNA as a function of added complex
4
3
ꢀ1
1
(
.09(ꢁ0.007) ꢂ 10 and 5.87(ꢁ0.09) ꢂ 10
M
respectively
concentration to get a proof for the binding of the complex to
DNA via intercalation. When the complex was added to DNA
pretreated with EB {[DNA]/[EB] ¼ 1 : 1} at pH 7.4, the DNA-
induced emission intensity of EB decreased (Fig. 7). Addition
of a second DNA binding molecule would quench the EB
emission by either accepting an excited state electron from EB
or replacing the DNA-bound EB (if it binds to DNA more
strongly than EB).
Table 3). The decrease in the uorescence intensity thus proves
the partial replacement of EB bound to DNA by complexes 1 and
. The apparent DNA binding constant (Kapp) values of the
2
complexes were obtained from the uorescence spectral
measurement. The Kapp values were obtained from the equa-
tion: Kapp ꢂ [complex]50 ¼ KEB ꢂ [EB], where Kapp is the
apparent binding constant of the complex studied, [complex]50
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Fig. 8 UV-vis spectral changes of complexes 1 (a), 2 (b) in DMF upon
addition of 100 fold excess of NPP, observed at fixed intervals of time.
ꢃ
The reactions were carried out in DMF at 25 C under aerobic
conditions and were monitored by means of UV-vis spectros-
copy. The time dependent spectral changes of the complexes 1
and 2 upon addition of NPP in DMF are shown in Fig. 8, which
clearly demonstrate the gradual increment of the concentration
Fig. 7 Emission spectra of EB bound DNA with increasing amounts of of 4-nitro phenol (lmax ¼ 400 nm) with time (Fig. 8). These
the (a) complex 1 and (b) complex 2. Arrow shows the intensity
changes upon increasing the complex concentration.
results suggest that both the complexes are active in catalyzing
the hydrolysis of PNPP.
The enzyme kinetics data are listed in Table 4 and the kinetic
plots are given in Fig. 9 and 10. The kinetic study indicated that
complex 1 exhibits more than ve times greater catalytic activity
is the concentration of the complex at 50% quenching of DNA-
bound ethidium bromide emission intensity, KEB is the binding
than complex 2. Control experiments with PNPP only and PNPP
7
ꢀ1
constant of ethidium bromide (KEB ¼ 1$0 ꢂ 10 M ), and [EB]
mixed with zinc acetate solution shows negligible hydrolysis
Fig. S21†), conrming the efficacy of the complexes 1 and 2 as
catalysts for the model phosphate ester hydrolysis reaction.
ꢀ
6
78
is the concentration of ethidium bromide (5 ꢂ 10 ). The
(
apparent binding constant (K ) value was found to be
app
5
5
5
.41(ꢁ0.01) ꢂ 10 and 3.93(ꢁ0.02) ꢂ 10 for complexes 1 and 2
respectively.
3.7 Theoretical calculations
We have divided the theoretical study into two different parts to
analyze interesting aspects of the title compounds. First, we
3.6 Kinetics of the hydrolysis of the phosphate ester PNPP
The phosphate ester hydrolysis ability of the complexes was have analyzed the binding mechanism of the complex 1 to DNA
evaluated using PNPP (4-nitrophenyl phosphate) as substrate. by means of molecular dynamics simulations. Second, the
Table 3 DNA-binding data for the complexes
Electronic spectra
Fluorescence spectra
max (nm)
ꢀ
1
Complex
lmax (nm)
Change in absorption
K
b
(M
)
l
K
SV
K
app
6
6
4
5
5
1
2
460
463
Hypochromism
Hypochromism
2.10(ꢁ0.07) ꢂ 10
1.11(ꢁ0.04) ꢂ 10
611
611
1.09(ꢁ0.007) ꢂ 10
5.41(ꢁ0.01) ꢂ 10
3.93(ꢁ0.02) ꢂ 10
3
5.87(ꢁ0.09) ꢂ 10
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Table 4 Kinetic parameters obtained from Michaelis–Menten plots for NPP hydrolysis by the zinc complexes
ꢀ1
2
ꢀ1
Complex
Wavelength (nm)
Vmax (M min
)
K
M
(M)
R
k
cat (h
)
ꢀ
ꢀ
5
6
ꢀ4
ꢀ4
1
2
429
427
3.54(ꢁ0.67) ꢂ 10
6.25(ꢁ0.11) ꢂ 10
6.08(ꢁ0.22) ꢂ 10
2.81(ꢁ0.13) ꢂ 10
0.947
0.997
212 (ꢁ5)
38(ꢁ2)
interactions with one phenyl and formaldehyde thio-
semicarbazone groups with the nitrogenous bases of the
pocket. On the other hand, the other phenyl is orientated
outside the double helix. It is preserving this conformation for
the remaining time of the simulation. In our case, the DNA
oligonucleotide does not exhibit a signicant mobility, espe-
cially the central nitrogen bases.
An analysis of the statistical structures within the MD was
carried out, nding three different bonding types between the
Zn(II) complex 1 and the nucleobases: p–p stacking, metal–
nucleobase bonding and hydrogen bonding interactions. Using
the radial distribution function (g(r)), we calculate the complex–
nitrogen base average distances throughout the simulation
time. Fig. 12 and 13 represent these set of bonding between the
complex and the bases around it.
Fig. 9 Michaelis–Menten plot for hydrolysis of PNPP by complex 1.
The four nucleobases in the pocket present p–p stacking
binding geometries and energies of the ATP and related anions with some fraction of the complex intercalated. These interac-
to complex 1 have been computed using DFT calculations in tions do not have the same intensity level. The cytosine base
solution to rationalize its selectivity.
.8 Molecular dynamics studies
presents p–p stacking with a phenyl group with an average
ꢀ
distance of 3.5 A (yellow line in Fig. 13). This distance is in good
ꢀ
3
agreement with the interplanar distance of 3.4–3.6 A of benzene
dimer. The remaining nucleobases (G, A, T) present similar
average distances with the different thiosemicarbazone groups
in the complex (blue arrows in Fig. 12 and grey, red and blue
solid lines in Fig. 13, respectively). The guanine intensity in the
radial distribution function is slightly higher than the intensity
of the other nucleobases. This fact establishes that p–p stack-
ing interactions are the most general in the pocket of the calf
thymus DNA. Jointly, the guanine base presents two maximum
Molecular dynamics (MD) simulations of the DNA oligonucle-
otide has been used to investigate the intercalative binding of
the complex 1 to DNA. The MD simulations of complex 1 like
a model were conducted up to 500 ns in order to investigate if
the metal complex stays in the intercalation site (Fig. 11). The
complex was intercalated initially in the major grove. The
nitrogenous bases sequence pair chosen around the complex
were: G–C and A–T. Aer the rst 75 ns of the simulation time,
the complex nds the ideal orientation with respect to the
nitrogenous bases. As we can see in the Fig. 12 one part of the
complex intercalated perfectly within DNA, where there are
ꢀ
ꢀ
intensity picks on a 2.6 A and 2.9 A average distances (dis-
continues green and blue lines). This maximum in the function
corresponds to the average distance of N3 group of guanine and
0
O4 of the sugar of the guanine. Interestingly, only this base
Fig. 11 RMSD obtained for the calf thymus DNA model and complex 1
up to 500 ns of MD simulations. RMSD of 6.070 ꢁ 1.050 ꢀA .
Fig. 10 Michaelis–Menten plot for hydrolysis of PNPP by complex 2.
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Fig. 14 (a–c) DFT optimized geometries of the complexes of 1 with
several anions and ATP. (d) indicates the alternative mode of binding of
ꢀ
ATP. Distances in A. The binding energies are also indicated.
1
Fig. 12 View of the intercalation scheme of [Zn(L )] complex between
G–C (6th and 7th residues, respectively) and A–T bases in the MD
simulation. p–p stacking interactions are indicated with arrows.
Fig. 14. It can be observed that the ATP complexes are more
favorable than those with the other phosphorous inorganic
anions, in agreement with the experimental results. The
binding mode of the ATP is governed by two types of interac-
tions, rst, two strong Zn(II)/O interactions (coordination
bonds) x the geometry and explain the large binding energies
obtained for the complexes. Moreover, the ATP presents addi-
tional interactions that explain its higher affinity to compound
shows bonding with the Zn in the complex. Finally, a strong and
ꢀ
enduring hydrogen bond (2.0 A average distance) in the MD
0
simulation between the O4 sugar of the adenine and H26 of the
ligand complex is established.
The MD simulation demonstrates that the complex 1 is
a good intercalator of DNA. The pocket design has been chosen
in order to simulate the different bases within the DNA struc-
ture, nding an ideal orientation without needing a specic
repetitive sequence in the oligonucleotide. Guanine base has
a greater ability to interact with the complex through the Zn and
p–p stacking and we assume that the presence of this nucleo-
base is necessary for the complex to interleave.
1. In fact, we have found two possible orientations for the
ribose–adenine fragment that are almost isoenergetic and
present different ancillary interaction. First, the adenine forms
a stacking interaction with one phenyl ring of the organic ligand
(
Fig. 14(c)) and, second, one hydroxyl group of the sugar
establishes an OH/p interaction with the organic ligand (Fig.
4(d)).
1
4. Conclusions
3.9 DFT study of the ATP binding to compound 1
We have studied the energetic and geometric features of A tetranuclear and a binuclear complex of two tetradentate thio-
complexes of compound 1 with ATP and also other phosphate semicarbazone ligands were synthesized, characterized and their
2
ꢀ
4ꢀ
anions (HPO
4
and P
2
O
7
) for comparison purposes. The sensing, DNA binding and catalytic properties were evaluated. The
optimized complexes and binding energies are summarized in two zinc complexes can be used as ATP sensors at biological pH.
The DNA binding experiment through UV-vis, uorescence titra-
tions suggest that the interaction between the complex and DNA is
intercalative and the result suggest that complex 1 is more efficient
than complex 2 to bind DNA at biological pH. The intercalation of
compound 1 to DNA has been further demonstrated by means of
molecular dynamics simulations. Both the complexes catalyze the
hydrolysis of 4-nitrophenylphosphate to 4-nitrophenol.
Conflicts of interest
The authors declare no competing nancial interest.
Acknowledgements
Fig. 13 Radial distribution functions of principal interactions between
ZnL complex and DNA. p–p stacking, metal bonding and hydrogen
bonding are represented by solid, discontinues and points lines
respectively. Average distances throughout the all simulation time.
1
PA thanks University Grants Commission (New Delhi) and BG
thanks IIEST, Shibpur for their research fellowships. SKC
thanks DST (West Bengal) for partial funding of this study.
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(
4
f) M. S. Han and D. H. Kim, Angew. Chem., Int. Ed., 2002,
1, 3809–3811; (g) A. Ojida, M. Yoshifumi, W. Jirarut,
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