Phosphatase activity and DNA binding studies of dinuclear phenoxo-bridged zinc(II) complexes with an N,N,O-donor ligand and halide ions in a rare cis-configuration

Article abstract of DOI:10.1016/j.poly.2017.03.037

Three new dinuclear zinc(II) complexes of the types [Zn₂(L)₂(Cl)₂]·2(CH₃OH) (1) and [Zn₂(L)₂(X)₂] (HL?=?2-(((2-(dimethylamino)ethyl)amino)methyl)phenol; X?=?Br for 2 and I for 3)

Full text of DOI:10.1016/j.poly.2017.03.037

Accepted Manuscript
Phosphatase activity and DNA binding studies of dinuclear phenoxo-bridged
zinc(II) complexes with an N,N,O-donor ligand and halide ions in a rare cis-
configuration
Monika Sheoran, Kishalay Bhar, Tanveer A. Khan, Anuj K. Sharma, Sunil G.
Naik
PII:
S0277-5387(17)30233-4
DOI:
http://dx.doi.org/10.1016/j.poly.2017.03.037
POLY 12552
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
22 December 2016
17 March 2017
19 March 2017
Please cite this article as: M. Sheoran, K. Bhar, T.A. Khan, A.K. Sharma, S.G. Naik, Phosphatase activity and DNA
binding studies of dinuclear phenoxo-bridged zinc(II) complexes with an N,N,O-donor ligand and halide ions in a
rare cis-configuration, Polyhedron (2017), doi: http://dx.doi.org/10.1016/j.poly.2017.03.037
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Phosphatase activity and DNA binding studies of dinuclear phenoxo-
bridged zinc(II) complexes with an N,N,O-donor ligand and halide ions in a
rare cis-configuration
a
a
a
a
a
Monika Sheoran , Kishalay Bhar , Tanveer A. Khan , Anuj K. Sharma and Sunil G. Naik *
a
Department of Chemistry, School of Chemical Sciences and Pharmacy, Central University of
Rajasthan, NH-8, Bandarsindri, Ajmer Distt. Rajasthan, INDIA 305817
________________________________________________________________
Abstract
Three new dinuclear zinc(II) complexes of the types [Zn
2
(L)
2
(Cl)
2
].2(CH
3
OH) (1) and
[
Zn (L) (X) ] (HL = 2-((2-(dimethylamino)ethyl)amino)methyl)phenol; X = Br for 2 and I
2
2
2
for 3), having a rare cis-configuration of the halide ions, were isolated using the one-pot
synthesis of the building components in appropriate molar ratios and characterized. X-ray
structural studies reveal that 1-3 contain a discrete dinuclear unit bridged by two phenoxo
oxygen atoms of the deprotonated Schiff base ligand (HL) and adopt an overall rare cis-
configuration where two halide ions are on the same side of the Zn-Zn axis and the ligand
occupies the other side. Each Zn(II) centre in 1-3 has a distorted square pyramidal geometry
2 2
with a ZnO N X chromophore (X = Cl for 1; Br for 2; I for 3). The initial rate values for the
hydrolysis of 4-nitrophenylphosphate to 4-nitrophenolate by the complexes 1-3 are
-1
respectively 590, 334 and 705 min . The complexes show an external binding propensity to
2
2
2
−1
calf thymus DNA with binding constant values 9.45 x 10 , 7.06 x 10 and 6.51 x 10 M for
, 2 and 3 respectively.
1
•
Keywords: Dinuclear zinc(II) halides; Phenoxo bridge; Schiff base; Phosphatase activity;
DNA binding
_
__________________________________________________________________________
*
Corresponding author.
sunilnaik@curaj.ac.in
1

1
. Introduction
Metal-ion catalyzed reactions have gained a lot of importance in the past few years,
with zinc containing metalloenzymes constituting by far the widest category [1,2]. The Zn(II)
active site is present in a number of fundamental enzyme classes, like transferases,
hydrolases, oxidoreductases, lyases, isomerases and ligases [3-9]. The hydrolysis of
phosphate esters is of particular interest because it is involved in biosynthesis energy
transduction, regulation of protein function and control of secondary messengers [10,11].
Among the versatile range of enzymatic behaviour of the zinc(II) ion [12-15], phosphatase
activity is an important function of various functional mimics of phosphoesterases. Various
dinuclear Zn(II) complexes [16-22] have been studied over time as phosphate ester models,
taking into account their extraordinary Lewis acidity, nucleophile generation, redox rigidity,
leaving group stabilization and physiological relevancy [23-25]. In order to develop new anti-
cancer drugs, the binding mechanism of the complex with DNA [26-28] should be studied.
The intercalative mode is the most important mode in which transition metal complexes can
intercalate between the pair-bases of double helix DNA, forming π-π overlapping
interactions. It is the interaction that changes the DNA conventional behaviour and so the
transition metal complexes possess a very broad application in the field of bio-inorganic
chemistry. Of late, Schiff base bridged dinuclear Zn(II) complexes have gained lot of
significance owing to their good DNA interaction behaviour [18,29-32]. To explore the
chemistry of such phosphatase activity and DNA interaction, herein we have synthesised and
characterised three new dinuclear zinc(II) halide complexes containing a tridentate Schiff
base, 2-((2-(dimethylamino)ethyl)amino)methyl)phenol (HL, Scheme 1).
Scheme 1. The tridentate Schiff base, 2-((2-(dimethylamino)ethyl)amino)methyl)phenol (HL).
2

2
. Experimental
2
.1. Materials
All the chemicals used in the present study were obtained from different commercial
sources. Solvents were distilled before use according to standard procedures. The anhydrous
zinc halide salts (ZnCl , ZnBr ) were obtained from CDH, India. ZnI was synthesized by a
2
2
2
standard procedure using Zn metal and iodine. Water used in the physical measurements was
Milli-Q grade. The (p-nitrophenyl) phosphate (PNPP) used for the catalytic studies was
purchased from Spectrochem, India and re-crystallized from ethanol/water.
2
.2. Physical measurements
Elemental analyses were performed on a Perkin–Elmer 2400
CHNS elemental analyzer. Infrared spectra were recorded at room temperature using a Perkin
-1
1
Elmer FTIR spectrometer in the 4000-400 cm range. The H NMR spectra were recorded in
CDCl and DMSO-d solvent on a Bruker-500 MHz spectrometer at 25°C. UV-Vis spectra
and kinetic studies were performed at room temperature on an Agilent Technologies Cary
00 UV-Vis spectrophotometer equipped with multiple cell-holders. Fluorescence
measurements were done on a Perkin Elmer LS55 fluorescence spectrophotometer at 25°C.
3
6
1
2
.3. X-ray crystallography
Crystallographic data of compounds 1-3 were collected on a Bruker APEX-II CCD
diffractometer at 296 K (for 1) and 100 K (for 2 and 3) using graphite monochromated Mo
Kα radiation (λ = 0.71073 Å). For unit cell determination, the single crystal was exposed to
X-rays for 10 s in three sets of frames. The detector frames were integrated by use of the
program SAINT [33] and multi-scan absorption corrections were performed using the
SADABS program [34]. The structures were solved by direct methods and refined by full-
2
matrix least-squares methods based on F using SHELXS-97 [35] and SHELXL-97 [35]. All
the non-hydrogen atoms were refined with anisotropic displacement parameters, whereas
hydrogen atoms were placed in calculated positions when possible and given isotropic U
values 1.2 times that of the atom to which they are bonded. The NH hydrogen atoms of the
ligands in complex 2 were not located. The crystal of compound 1 is a twin with twin law
0
10 100 00-1 and BASF 0.01174. In the final difference Fourier maps, the residual maximum
-3
-3
and minimum were 0.221 and -0.287 e Å for 1, 1.627 and -1.730 e Å for 2 and 1.676 and
-
3
-
1.219 e Å for 3. Materials for publication were prepared using SHELXTL [35], PLATON
3

[
36] and Mercury [37] programs. A summary of the crystallographic data and structure
determination parameters are given in Table 1.
2
2
.4. Synthesis
.4.1. 2-((2-(dimethylamino)ethyl)amino)methyl)phenol(HL)
The Schiff base (HL) was synthesized using a reported procedure [38]. A solution
of salicylaldehyde (1 g, 8.18 mmol) in methanol was refluxed with N,N-
dimethylethylenediamine (0.72 g, 8.18 mmol) for 3 h. The reaction mixture was cooled to
4
room temperature and then NaBH (0.9 g, 23.7 mmol) was added at 0°C. The reaction
mixture was stirred for a further 30 minutes and HCl was added to adjust the pH to 1 or 2
initially, and followed by addition of NaOH to attain pH 12. The resulting mixture was
3
extracted with CHCl and dried over anhydrous sodium sulfate. On evaporating the
solvent, a yellow solid was obtained (Yield: 1.51 g, 95%). Elemental analysis calcd. for
1
C
11
H
18
N
2
O: C, 68.01; H, 9.34; N, 14.42; found: C, 68.15; H, 9.40; N, 14.55% . H NMR
(
500MHz, CDCl
CH ), 2.4 (2H, t, CH
58.35, 128.60, 128.32, 122.0, 118.87, 116.33, 58.71, 52.41, 45.71, 45.40. C (DEPT)
NMR (500 MHz, CDCl , 298K) δ, ppm: 128.60, 128.32, 122.0, 118.87, 116.33, 58.71,
2.41, 45.71, 45.40. FTIR (KBr, cm ): ν(N-H) 3010, ν(C-N) 1606. UV-vis, λmax (nm)
3
, 298K) δ, ppm: 6.7-7.5 (4H, aromatic), 3.9 (2H, s, CH
2
), 2.7 (2H, t,
1
3
2
2 3 3
), 2.23 (6H, s, CH ). C NMR (500 MHz, CDCl , 298K) δ, ppm:
1
3
1
3
-
1
5
3
-1
-1
3
(
ε
max, dm mol cm ): 277 (2.8×10 ).
.4.2. [Zn (L) (Cl) ].2(CH OH) (1)
A solution of HL (1 g, 5.15 mmol.) in methanol (20 mL) was stirred with NEt
g, 5.15 mmol) at room temperature for 15 minutes. A methanolic solution (10 mL) of ZnCl
0.70 g, 5.15 mmol) was added to the above solution, with stirring. The resulting white solid
2
2
2
2
3
3
(0.52
2
(
that formed was filtered and dried (Yield: 1.30 g, 86%). Elemental analysis calcd. for
1
C
24
H
42Cl
2
N
4
O
4
Zn
500MHz, DMSO-d
12H, CH ). FTIR (KBr, cm ): ν(N-H) 3008, ν(C-N) 1600. UV-vis, λmax (nm) (εmax, dm mol
2
: C, 44.19; H, 6.49; N, 8.59; found: C, 44.10; H, 6.52; N, 8.63%. H NMR
(
6
, 298K) δ, ppm: 6.5-7.3 (8H, aromatic), 2.38-3.86 (12H, aliphatic), 2.19
-1
3
-
(
3
1
-1
4
2
cm ): 283 (6.1×10 ), 371 (7.01×10 ).
.4.3.[Zn (L) (Br) ] (2)
Complex 2 was prepared by stirring a solution of HL (0.59 g, 3.06 mmol) and ZnBr
0.69 g, 3.06 mmol) in methanol for 30 minutes. The resulting white solid was filtered and
2
2
2
2
2
(
dried (Yield: 0.87 g, 84%). Elemental analysis calcd. for C22
H
34Br
2
N
4
O
2
Zn
2
: C, 39.02; H,
, 298K) δ,
1
5
.06; N, 8.27; found: C, 39.11; H, 5.08; N, 8.32%. H NMR (500MHz, DMSO-d
6
4

-1
ppm: 6.5-7.3 (8H, aromatic), 2.20 (12H, CH
ν(N-H), 3002, ν(C-N), 1600.UV-vis, λmax (nm) (εmax, dm mol cm ): 282 (6.2×10 ).
.4.4.[Zn (L) (I) ] (3)
3
), 2.38-3.95 (12H, aliphatic). FTIR (KBr, cm ):
3
-1
-1
4
2
2
2
2
Complex 3 was prepared by the same procedure as that of 2 by using anhydrous ZnI
2
(
0.90 g, 2.85 mmol) as the metal salt. A white coloured solid was filtered and dried (Yield:
0
.9 g, 90%). Elemental analysis calcd. for C22
H
34
I
2
N
4
O
2
Zn
2
: C, 34.26; H, 4.44; N, 7.27;
, 298K) δ, ppm: 6.3-7.1
1
found: C, 34.20; H, 4.52; N, 7.32%. H NMR (500MHz, DMSO-d
6
-1
(
8H, aromatic), 2.26 (12H, CH
3
), 2.49-4.13 (12H, aliphatic). FTIR (KBr,cm ): ν(N-H) 3002,
3
-1
-1
4
ν(C-N) 1600.UV-vis, λmax (nm) (εmax, dm mol cm ): 284 (6.5×10 ).
2
.5. Hydrolysis of phosphate ester
Disodium (4-nitrophenyl)phosphatehexahydrate (4-NPP) was used as the substrate to
study the Phosphatase activity. All experiments were done in a 97.5% DMF/H
2
O mixture
[
17,39,40]. Solutions of the zinc complexes and substrate 4-NPP were freshly prepared and
the total volume maintained was 3 mL. The kinetic data was collected after 2 h of the reaction
when 2% of the reaction was complete. The rate of hydrolysis of 4-NPP in the presence of
zinc complexes 1-3 was measured by an initial rate method, following the increase in
-1
-1
absorption at 423 nm due to the formation of the 4-nitrophenolate ion (ε, 18500 M cm ) at
5 °C [39]. The kinetic experiments were performed both at variable substrate or complex
2
concentration, keeping either the complex or the substrate concentration constant. The study
was comprised of 5 sets of experiments having the complex concentration as 0.05 mmol and
the substrate concentration as 0.5 (10 equiv.), 0.7 (14 equiv.), 1.0 (20 equiv), 1.2 (24 equiv.)
and 1.5 (30 equiv.) mmol. The stock solutions of 2.5 mM of the complexes and substrate
2
were prepared in a DMF:H O mixture. The reactions were performed by injecting 60 µL of
the complex and different volumes of the substrate (0.6, 0.84, 1.2, 1.44 and 1.8 mL) in a
cuvette and making the final volume to 3 mL with solvent. The visible absorption increase
was recorded for a total period of 2 h, at intervals of 5 min. All the recordings were
performed at least twice and average values were taken for the data. The reactions were
corrected for the degree of ionization of 4-nitrophenolate at 25 °C using the molar extinction
coefficient for 4-nitrophenolate at 423 nm [40]. The final absorbance values for each set were
obtained after 72 h.
2
.6. Control experiments
5

Control experiments were performed for the zinc salts and the ligand to identify any
individual effects on the hydrolysis of 4-NPP. There was no noticeable change in the results.
2
.7. Fluorescence spectroscopy method
The intrinsic fluorescence measurements of DNA with the zinc complexes 1-3 were
done on a Perkin Elmer LS55 Fluorescence spectrophotometer at room temperature. The
concentrations of DNA and ethidium bromide (EB) were kept constant at 37 µM and 10.5
µM, respectively in all the experiments and the complex concentration was varied from 0 to
4
30 µM. The excitation and emission slit widths were fixed at 15 and 20 nm, respectively.
The emission spectra were recorded in the wavelength range λem= 490-800 nm by exciting
the DNA at the wavelength λex = 480 nm. Stock solutions (2 mM) of thte zinc complexes
were prepared in a 95% H
2
O/DMSO mixture using Tris-10mM/EDTA 1 mM (T10E ) buffer.
1
3
. Results and discussion
3
.1. Syntheses and spectroscopic studies
Complexes 1-3 were isolated as white solids by mixing a 1:1 molar ratio of the
zinc(II) halides and the Schiff base (HL) in the presence of triethyl amine in MeOH at
room temperature. All the reactions were reproducible, as was evident from repetitive
spectral behaviours. Details of sequential reactions are summarized in Scheme 2.
H
X
N
O
Zn
NH
OH
ZnX
2
N
N
Zn
X
NEt3
MeOH
RT
O
N
N
H
(
HL)
Complexes (X = Cl, 1; Br, 2 and I, 3)
Scheme 2. Synthesis of the zinc(II) complexes 1-3.
The compounds were characterized using FTIR, UV-Vis and NMR spectroscopy.
The spectroscopic results are in good agreement with the formulations 1-3. All the
6

compounds are stable over a long period of time in powdery or crystalline form. 1-3 are
soluble in a wide range of common non-aqueous solvents, such as acetonitrile,
1
dimethylformamide and dimethylsulfoxide. The H NMR spectrum of HL in CDCl
3
shows four signals in the aliphatic region, consisting of two singlets and two triplets. In
the aromatic region (δ 6.7-7.5 ppm), four signals are observed (Supporting Information,
1
3
13
Fig. S1) for HL. The ligand was further characterized by C NMR and C (DEPT) NMR
spectra in CDCl , which showed the presence of aromatic carbon signals at δ 158.35-
16.33 ppm in addition to CH
3
1
2
3
- and –CH signals at δ 58.1-45.40 ppm (Supporting
1
Information, Figs. S2 and S3). The H NMR spectra of the metal complexes 1-3 taken in
DMSO-d have a similar type of spectrum as HL, with slightly shifted values (Supporting
6
Information, Figs. S4-S6). On comparing the spectra of the ligand and diamagnetic
zinc(II) complexes, it was found that there is not much difference because the coordinated
and uncoordinated groups have same kind of magnetic environment. Thus, from these
NMR results it appears that the solution state structural formulations are in good
agreement with their crystalline form. In DMF solution, the ligand (HL) exhibits an
intraligand charge transfer band at 277 nm. The corresponding complexes 1-3 in DMF
show a strong ligand-based [39,41] transition at ca. 280 nm (Supporting Information, Fig.
S7). In addition, complex 1 displays a low energy ligand to metal charge transfer band
-
II
[
17] at 371 nm, which is probably due to a PhO →Zn transition or to a mixture of the
-
II
former and a Cl →Zn transition. In the FTIR spectra, the free Schiff base (HL) and
-
1
complexes 1-3 show weak to medium stretches in the 3000-3010 cm region,
-
1
corresponding to ν(N-H), and a strong band at ca. 1600 cm which is attributed to ν(C-N)
stretching [42] (Supporting Information, Fig. S8- S11).
3
.2. Crystal structures
Single crystal X-ray diffraction studies revealed that compounds
(L) (Cl) ].2(CH OH) (1), [Zn (L) (Br) ] (2) and [Zn (L) (I) ] (3) contain discrete
[
Zn
2
2
2
3
2
2
2
2
2
2
dinuclear units formed by two Zn(II) ions [Zn(1) and Zn(1a) (a = -x,1-y,z) for 1 and Zn(1)
and Zn(2) for 2 and 3], bridged by the two phenoxo oxygen atoms [O(1) and O(1a) for 1 and
O(1) and O(2) for 2 and 3] of the deprotonated Schiff base ligands (L). Complex 1 has an
inversion center and crystallises in the orthorhombic system with the space group Aba2,
whereas complexes 2 and 3 are located on general positions and crystallise in the triclinic
system with the space group P-1. Each Zn(II) center is penta-coordinated by two µO,O
-
7

bridging phenoxo O-atoms, two N atoms of the amine groups of the deprotonated Schiff base
ligand and one terminal halide anion (Figs. 1-3). The ZnO X chromophores (X = Cl for 1;
2
N
2
X = Br for 2 and I for 3) may be described as distorted square pyramids (SP), and their
Addison parameter [43] τ is 0.14 for Zn1 and Zn1a in 1, 0.038 for Zn1 and 0.12 for Zn2 in 2
and 0.086 for Zn1 and 0.064 for Zn2 in 3. The two amine N atoms (N1, N2 for Zn1 and N1a,
N2a for Zn1a in 1 and N1, N2 for Zn1 and N3, N4 for Zn2 in 2 and 3) along with two µO,O
-
bridging phenoxo O atoms (O1 and O1a in 1; O1 and O2 in 2 and 3) form an equatorial
plane, whereas the terminal halide anions (Cl1 for Zn1 and Cl1a for Zn1a in 1, Br2 for Zn1
and Br4 for Zn2 in 2, I1 for Zn1 and I2 for Zn2 in 3) occupy the axial positions. It is
interesting to note that 1-3 adopt an overall rare cis-configuration (Figs. 1-3), where two
halide ions (Cl1 and Cl1a in 1, Br2 and Br4 in 2, I1 and I2 in 3) are on the same side of the
Zn-Zn axis and the ligand (HL) occupies the other side. To the best of our knowledge only
2 2
four such dinuclear complexes [30,45] have been reported so far. The Zn O bridging core in
the dinuclear units of 1-3 is asymmetric in nature, with two different Zn-O distances (Table
2
4
2
) which is consistent with similar diphenolato-bridged dinuclear zinc complexes [17,30,44-
7]. The differences in the two Zn-O distances is found to be 0.020 Å in 1, 0.095, 0.074 Å in
2 2
and 0.107, 0.068 Å in 3, indicating that the Zn O moiety in 2 and 3 is less symmetrical,
whilst in 1 it is symmetrical. The cis-oriented Zn-X distances, along with Zn-N(amine)
lengths (Table 2), are consistent with the literature data [17,30,44-47]. In dinuclear 1-3, the
two zinc(II) centers are 3.217, 3.220 and 3.209 Å apart from each other, respectively.
It is well known that weak non-covalent bonds, such as C-H...X (X = O, N, S,
halogens), C-H...π, anion...π interactions and π... π-stacking, though weaker than classical
hydrogen bonds, play an important role in the conformation, crystal packing, supramolecular
assembly and physicochemical properties. Such non-covalent interactions are at the core of
most chemical and biological processes and hence knowledge of their nature, strength,
occurrence and consequences is of paramount importance. Weak inter-molecular C-H...Cl,
O-H...Cl, C-H...O and N-H...O interactions (Table 3) in 1 propagate along the (100)-plane,
giving rise to a 2D zigzag sheet-like crystalline architecture (Fig. 4). On the other hand, weak
C-H...Br (in 2) and N-H...I, C-H...I (in 3) interactions combine individual dinuclear units
along the a-axis, forming 1D chains (Table 3, Fig. 5).
3
.3. Phosphatase activity and kinetic study
8

The phosphatase activity of complexes 1-3 was studied using the disodium salt of (4-
nitrophenyl) phosphate hexahydrate. The hydrolytic tendency of 1-3 was determined
spectrophotometrically by monitoring the evolution of 4-nitrophenolate (λmax = 423 nm)
through a wavelength scan from 200 to 800 nm in aqueous DMF [40]. The change in spectral
behaviour of 1 is shown in Fig.6, as a representative case, and those of the other complexes (2
and 3) are given in the Supporting Information (Figs. S12 and S13).
The kinetic behaviour of complexes 1-3 was studied by the initial slope method,
following the rate of increase in absorption of the band corresponding to the formation of 4-
nitrophenolate at 423 nm. The plot of log[A
∞
∞
t
/A -A ] values versus time gave the initial rate
-
1
constants, V (M min ) for the zinc complexes (Fig. 7 for 1). The dependence of the rate
constants on five different substrate concentrations gave first order kinetics at lower
concentrations, but deviates from unity at higher concentrations (Fig. 8 for 1). The kinetic
parameters (Vmax, K , Kcat) for the catalyzed reactions of the zinc complexes were
M
determined from the Lineweaver-Burk plot of 1/V versus 1/[S], as per the Michaelis Menten
approach of enzymatic kinetics. Similar plots for complexes 2 and 3 are given in the
Supporting Information (Figs. S14 and S15). All the kinetic parameters are given in Table 4.
Control experiments were performed to determine any role of the ligand (HL) and zinc(II)
halide salts in the catalytic reaction. The results (Supporting Information, Figs. S16 and S17)
indicate no appreciable change. The catalytic activity order of the complexes, 3 > 1 > 2,
shows a direct correspondence to the Zn...Zn separation (3.217 Å for 1, 3.220 Å for 2 and
3
.209 Å for 3), indicating the shorter Zn...Zn distance is better suited to accommodate the
phosphate group.
3
.4. DNA binding study
To understand the binding of the zinc complexes 1-3 with DNA and the fluorescence
quenching, 0-430 µM of the zinc complexes were successively added to DNA (37 µM)
solutions containing 10.5 µM ethidium bromide (EB) in T10 buffer. The samples were
E
1
excited at 480nm and the emission spectra were observed at 594 nm. EB strongly intercalates
between the DNA base pairs and gives intense fluorescence [30]. Representative emission
spectra of EB bound to DNA in the absence and presence of 1 are shown in Fig.9.
There is a steady decrease in the intensity of the emission maxima upon addition of
complex 1, leading to saturation levels. The binding constant was calculated using the Stern-
Volmer equation [48].
9

I
o
/I = 1 + KSV[R]
and I are the fluorescence intensities in the absence and presence of the zinc
complexes respectively, KSV is the Stern-Volmer quenching constant, [R] is the concentration
of the quencher (complexes). The ratio of the slope to the intercept in the plot of I /I vs [R]
where I
o
o
gives the value of KSV (Fig.10). For complexes 2 and 3, the results are shown in the
Supporting Information (Figs. S18-S21). The Stern-Volmer constant Ksv for 1, 2 and 3 are
2
2
2
9
.45 × 10 , 7.06 × 10 , 6.51 × 10 respectively, indicating weak external binding to CT-DNA.
These results show that 1 has a high binding constant with CT-DNA as compared with the
binding constants of 2 and 3.
4
. Conclusion
Three new dinuclear zinc(II) halide complexes (1-3) containing a tridentate
N,N,O-donor Schiff base and having a rare cis-configuration of the halide ions were
synthesised and characterized using analytical and spectroscopic methods. Single crystal X-
ray studies reveal a phenoxo bridged dinuclear nature of the complexes with the rare cis-
configuration, where the terminal halide ligands are on the same side of the Zn-Zn axis and
the Schiff bases are on the other side. The complexes show moderate catalytic activity
towards the hydrolysis of the disodium salt of (4-nitrophenyl)phosphate hexahydrate and
weak external binding to CT-DNA. We are now active in extending such work using other
NNO-donor ligands and zinc(II) halides.
Conflict of interest
Authors declare that there are no conflicts of interests.
Acknowledgements
Financial support from the CSIR [Grant No. 01(2840/16/EMR-II)] and DST-SERB
(
Grant No. EMR/2015/000764), New Delhi, India, and the Central University of Rajasthan is
gratefully acknowledged. MS and TAK are grateful to the UGC and KB is grateful to DST-
SERB, New Delhi, India for fellowships. Instrumental facilities at the Department of
Chemistry, Central University of Rajasthan are also acknowledged. AKS thanks the DST-
INSPIRE program for research grant IFA-13, CH-97. We are thankful to the Department of
Chemistry, IIT, Jodhpur for the X-ray crystallographic facility.
Appendix A. Supplementary materials
1
0

CCDC 1522603, 1522604 and 1522605 contain the supplementary crystallographic
data for 1-3, respectively. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Additionally, the
NMR, Mass, UV-Vis and FTIR spectra of the ligand (HL) and complexes 1-3, the graphs of
phophatase activity and DNA binding studies of complexes 2 and 3 are submitted.
References
1
2
3
4
5
6
7
8
9
1
1
1
1
. D.E. Wilcox, Chem. Rev. 96 (1996) 2435.
. L.J. Daumann, G. Schenk, D.L. Ollis, L.R. Gahan, Dalton Trans. 43 (2014) 910.
. B.L. Vallee, D.S. Auld, Acc. Chem. Res. 26 (1993) 543.
. M. Hambidge, N. Krebs, J. Pediatr. 135 (1999) 661.
. G.A. Eby, J. Antimicrob. Chemother. 40 (1997) 483.
. A. Gadomski, J. Am. Med. Assoc. 279 (1998) 1999.
. M.L. Cleveland Macknin, Clin. J. Med. 66 (1999) 27.
. D.G. Barceloux. J. Toxicol. Clin. Toxicol. 37 (1999) 279.
. D.R. Williams, Coord. Chem. Rev. 186 (1999) 177.
0. R.C.H. Jin SeogSeo, Daniel Williams, Jik Chin, J. Am. Chem. Soc. 120 (1998) 9943.
1. F.H. Westheimer, Science 235 (1987) 4793.
2. M.J. Jedrjezas, P. Setlow, Chem. Rev. 101 (2001) 607.
3. M. Subat, K. Woinaroschy, C. Gerstl. B. Sarkar, W. Kaim, B. Konig, Inorg. Chem. 47
(
2008) 4661.
1
1
1
4. J. Wetson, Chem. Rev. 105 (2005) 2151.
5. M.A.R. Raycroft, C. Tony Liu, R. S. Brown. Inorg. Chem. 51 (2012) 3846.
6. P. Chakraborty, J. Adhikary, R Sanyal, A. Khan, K. Manna, S. Dey, E. Zangrando, A.
Bauzá, A. Frontera, D. Das, Inorg. Chim. Acta 421 (2014) 364.
1
7. (a) P. Kundu, P. Chakraborty, J. Adhikary, T. Chattopadhyay, R.C. Fischer, F.A.
Mautner, D. Das, Polyhedron 85 (2015) 320; (b) R. Sanyal, P. Chakraborty, E.
Zangrando, D. Das, Polyhedron 97 (2015) 55.
1
1
2
8. S. Anbu, S. Kamalraj, B. Varghese, J. Muthumary, M. Kandaswamy, Inorg. Chem. 51
(
2012) 5580 and references therein.
9. K. Abe, J. Izumi, M. Ohba, T Yokoyama, H. Okawa. Bull. Chem. Soc. Jpn. 74 (2001)
5.
0. S. Brooker, T.C. Davidson, Chem. Commun. (1997) 2007.
8
1
1

2
1. (a) H. Arora, S.K. Barman, F. Lloret, R.N. Mukherjee, Inorg. Chem. 51 (2012) 5539;
b) K. Naik, A. Navrekar, D.G. Kokare, A. Kotian, V. Kamat, V.K. Revankar, J. Mol.
(
Struct. 1125 (2016) 671.
22. O. Iranzo, A.Y. Kovalevsky, J.R. Morrow, J.P. Richard, J. Am. Chem. Soc. 125
(
2003) 1988.
2
2
2
3. E. Kimura, T. Koike, Adv. Inorg. Chem. 44 (1997) 229.
4. J.E. Coleman, Curr. Opin. Chem. Biol. 2 (1998) 222.
5. B. Bauer-Siebenlist, F. Meyer, E. Farkas, D. Vidovic, S. Dechert, Chem. Eur. J. 11
(
2005) 4349.
2
6. (a) C. Metcalfe, J.A. Thomas, Chem. Soc. Rev. 32 (2003) 215; (b) C.X. Zhang, S.J
Lippard, Curr. Opin. Chem. Biol. 7 (2003) 481; (c) C. Liu, M. Wang, T. Zhang, H.
Sun, Coord. Chem. Rev. 248 (2004) 147.
2
2
2
7. P. CA Bruijnincx, P.J Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197.
8. A.M. Pizarro, P.J Sadler, Biochimie 91 (2009) 1198.
9. X. Sheng, X. Guo, X.-M. Lu, G.-Y. Lu, Y. Shao, F. Liu, Q. Xu, Bioconjugate Chem.
1
9 (2008) 490.
30. K. Ghosh, P. Kumar, N. Tyagi, U. P. Singh, Inorg. Chem. 49 (2010) 7614.
3
1. (a) M. Patel, M. Chhasatia, P. Parmar, Eur. J. Med. Chem. 45 (2010) 439; (b) Z.
Kazemi, H.A. Rudbari, V. Mirkhani, M. Sahihi, M. Moghadam, S. Tangestaninejad, I.
Mohammadpoor-Baltork, J. Mol. Struct. 1096 (2015) 110.
32. U.M. Rafi, D. Mahendiran, A.K.Haleel, R. P. Nankar, M. Doble, A.K. Rahiman, New
J. Chem. 40 (2016) 2451.
3
3. SAINT PLUS, v.6.01, Bruker Analytical X-ray, Madison, WI, 1999.
4. SADABS v.2.01, Bruker/Siemens Area Detector Absorption Correction
Program,Bruker AXS, Madison, Wisconsin, USA, 1998.
3
3
3
3
5. G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112.
6. A.L. Spek, Acta Crystallogr. D65 (2009) 148.
7. C.F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M.
Towler, J. Van de Streek, J. ApplCrystallogr. 39 (2006) 453.
38. A. Biswas, M.G.B. Drew, C. Diaz, A. Bauzá, A. Frontera, A. Ghosh, Dalton Trans.41
(
2012) 12200.
9. R. Sanyal, A. Guha, T. Ghosh, T.K. Mondal, E. Zangrando, D. Das, Inorg. Chem. 53
2014) 85.
3
(
1
2

4
4
4
0. M. Subat, K. Woinaroschy, S. Anthofer, B. Malterer, B. Konig, Inorg. Chem. 46
2007) 4336.
1. A.B.P. Lever, Inorganic Electronic Spectroscopy, second ed., Elsevier, New York,
984.
(
1
2. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds, Part B, fifth ed., John Wiley & Sons, New Jersey, 2009.
43. W. Addision, T. N. Rao, J. Reedijik, J. V. Rijn, G. C. Verschoor, J. Chem. Soc.,
Dalton Trans. (1984) 1349.
4
4
4
4
4. H.-Y. Chen, C.-C. Lin, Acta Crystallogr., E62 (2006) m1693.
5. A. Mukherjee, F. Lloret, R. Mukherjee, Inorg. Chem. 47 (2008)4471.
6. S. Bosch, P. Comba, L. R. Gaham, G. Schenk, Inorg. Chem,53 (2014) 9036.
7. R. Sanyal, X. Zhang, P. Kundu, T. Chattopadhyay, F. A. Mautner, D. Das, Inorg.
Chem, 54 (2015) 2315.
48. Y.-L. Song, Y.-T. Li, Z.-Y. Wu, J. Inorg. Biochem. 101 (2007) 233.
1
3

Tables
Table 1. Crystallographic data for 1-3
Crystal parameters
1
2
3
CCDC No.
Formula
Weight
Crystal system
Space group
a/Å
b/Å
c/Å
α/°
β/°
1522603
1522604
1522605
C H I N O Zn
22 34 2 4 2 2
769.05
Triclinic
P-1
C
24
H42Cl
2
N
4
O
4
Zn
2
C
22
H32Br
2
N
4
O
2
Zn
2
652.30
Orthorhombic
Aba2
17.2388(9)
17.4159(11)
9.9579(5)
90
90
90
2989.7(3)
0.71073
1.449
675.07
Triclinic
P-1
7.6106(8)
8.0004(7)
13.0995(15)
13.7770(15)
93.327(8)
92.662(7)
105.768(7)
1316.8(3)
0.71073
1.703
13.0702(12)
13.7450(13)
91.570(4)
94.751(4)
105.286(4)
1379.8(2)
0.71073
1.856
γ/°
V/Å
3
λ/Å
-3
σcalcd/gm cm
Z
4
2
2
T/K
µ/mm
296(2)
1.818
100(2)
4.881
100(2)
4.000
-
1
F(000)
ranges/°
h/k/l
1360.0
4.702 to 28.411
-23,23/-23,23/-13,13 -8,8/-15,15/-16,16
38199
3740
676
752
1.484 to 24.878
4.342 to 27.996
-10,10/-17,17/-18,17
24392
Reflections collected
Independent reflections
16991
4418
6578
Data/restraints/parameters 3740/2/172
4418/0/291
1.063
6578/2/301
1.091
2
1.080
Goodness-of-fit on F
Final R indices [I>2(I)]
R1 = 0.0190 and
wR2 = 0.0521
R1 = 0.0204 and
wR2 = 0.0526
R1 = 0.0788 and
wR2 = 0.1924
R1 = 0.1182 and
wR2 = 0.2279
R1 = 0.0312 and
wR2 = 0.0934
R1 = 0.0337 and
wR2 = 0.0960
R indices (all data)
-
3
Largest peak and hole/eÅ
Weighting scheme: R = F
0.221 and -0.287
1.627 and -1.730
1.676 and -1.219
2
2 2
2 2 1/2
o
-F
c
/F
o
, wR = [w(F
o
-F
c
) /w(F ) ]
o
1
4

Table 2. Selected bond distances (Å) and bond angles (°) for 1-3
1
3
Zn1-O1a
Zn1-O1
2.0298(12)
.0528(11)
Zn1-O1
Zn1-O2
2.0922(19)
1.993(2)
2
Zn1-N1
Zn1-N2
Zn1-Cl1
O1-Zn1a
O1a-Zn1-N2
O1-Zn1-N1
O1-Zn1-O1a
Zn1-O1-Zn1a
N1-Zn1-N2
O1-Zn1-N2
O1a-Zn1-N1
2.1835(18)
2.162(2)
Zn1-N1
Zn1-N2
Zn1-I1
Zn2-O1
Zn2-O2
Zn2-N3
Zn2-N4
Zn2-I2
2.138(2)
2.196(2)
2.5915(4)
1.984(2)
2.062(2)
2.164(3)
2.196(3)
2.6148(4)
75.32(8)
147.79(9)
2.2751(7)
2.0298(12)
139.70(9)
148.00(8)
75.98(5)
104.00(5)
81.14(8)
93.91(7)
O1-Zn1-O2
O1-Zn1-N2
O2-Zn1-N1
87.45(7)
1
42.46(9)
85.82(8)
6.53(8)
82.36(9)
6.19(8)
2
O1-Zn1-N1
O2-Zn1-N2
Zn1-O(2)
1.992(8)
9
Zn1-O(1)
Zn1-N(1)
2.082(7)
2.148(9)
N1-Zn1-N2
O1-Zn2-O2
7
Zn1-N(2)
Zn1-BR2
Zn2-O(1)
Zn2-O(2)
Zn2-N(4)
Zn2-N(3)
Zn2-BR4
O(2)-Zn1-O(1)
O(2)-Zn1-N(1)
O(1)-Zn1-N(1)
O(2)-Zn1-N(2)
O(1)-Zn1-N(2)
N(1)-Zn1-N(2)
O(1)-Zn2-O(2)
O(1)-Zn2-N(4)
O(2)-Zn2-N(4)
O(1)-Zn2-N(3)
O(2)-Zn2-N(3)
N(4)-Zn2-N(3)
2.182(8)
2.4028(17)
1.987(8)
2.067(7)
2.139(10)
2.215(8)
2.4268(16)
75.0(3)
144.9(3)
85.9(3)
97.1(3)
147.2(3)
83.2(3)
75.4(3)
O1-Zn2-N3
O2-Zn2-N4
O1-Zn2-N4
O2-Zn2-N3
N3-Zn2-N4
141.15(9)
145.21(9)
93.97(9)
85.62(9)
81.69(10)
139.9(3)
86.2(3)
95.3(3)
147.2(3)
81.0(3)
Symmetry code: a = -x, -y+1, z
1
5

Table 3. Hydrogen bonds donor/acceptor scheme (Å, °) for 1-3
Compound D-H···A
D-H H···A
0.97 2.83
0.97 2.55
0.86 2.55
D···A
D-H···A Symmetry codes
1
C(1)-H(13)...Cl(1)
3.788(3)
3.496(5)
171
167
-x, 1/2-y, 1/2+z
-1/2+x, 1-y, 1/2+z
1/2-x,-1/2+y,z
1/2+x,1-y,1/2+z
-1+x, y, z
C(10)-H(11)...O(2)
N(1)-H(111)...O(2)
3.228(4) 140.9
3.234(3) 146
3.641(13) 133
3.639(2) 129
O(2)-H(112)...Cl(1) 0.82 2.52
2
3
C(11)-H(11B)...Br(2)
N(1)-H(111)...I(1)
C(2)-H(6)...I(1)
0.99 2.89
0.86 3.04
0.99 3.09
-x,1-y,1-z
3.832(3)
132.4
-1+x, y, z
Table 4. Kinetic parameters calculated from Michaelis-Menten plot for 1-3
-2
-1
-3
-1
Complex
V
max × 10 (M min )
K
M
× 10 (M)
K
cat (min )
1
2
3
2.95
1.67
3.52
5.5
3.44
6.36
590
334
705
1
6

Figures
Fig. 1 An ORTEP diagram of the individual unit in 1 with the atom numbering scheme and
5
0% thermal ellipsoid probability (H atoms are omitted for clarity).
Fig. 2 Dinuclear unit in 2 (ORTEP; 50% probability ellipsoids). H atoms are omitted for
clarity.
1
7

Fig. 3 Molecular structure of 3 (ORTEP; 50% probability ellipsoids). H atoms are omitted for
clarity.
Fig. 4 Perspective view of the 2D sheet structure in 1 formed through inter-molecular
hydrogen bonds along the (100)-plane.
1
8

(
a)
(
b)
Fig. 5 Perspective view of different 1D chain structures (a) in 2 and (b) in 3 formed through
inter-molecular C-H...X (X = Br in 2 and I in 3) hydrogen bonds along the a-axis.
1
9

0.5
0.4
0.3
0.2
0.1
0.0
4-NPP
2
5
hours
min
400
500
Wavelength (nm)
Fig. 6 Wavelength scan for the hydrolysis of 4-NPP in the absence and presence of 1
(
substrate:catalyst = 20:1) in 97.5 % DMF at intervals of 5 minutes for 2 hours. [4-NPP] = 1
-3
-3
×
10 M, [Complex] = 0.05 × 10 M.
0.5
0.4
0.3
0.2
0.1
0.0
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0
20
40
60
80
100
120
time (min)
0
20
40
60
80
100
120
Time (min)
Fig. 7 Absorption vs time plot for complex 1 at 423 nm (least-squares fit of second
2
order, R = 0.997). Inset shows the plot of log[A
∞
/(A
∞
-A )] vs. time (R = 0.997).
t
2
0

0
0
0
0
0
0
0
0
.007
.006
.005
.004
.003
.002
.001
.000
4
4
3
3
2
2
1
1
50
00
50
00
50
00
50
00
5
0
600
800
1000 1200 1400 1600 1800 2000 2200
-
1
1/[S] (M
)
0
.0000
0.0005
0.0010
0.0015
[
Substrate] (M)
2
Fig. 8 Plot of enzymatic kinetics for 1 (Rate vs [Substrate], R = 0.980). Inset shows the
Lineweaver-Burk plot (1/V vs 1/[S]) having the intercept = 33.8677, slope = 0.18737 and R =
0
.971.
800
600
400
200
0
550
600
650
700
Wavelength
Fig. 9 Fluorescence emission profile associated with the binding of 1 with 37µM DNA at 298
K.
2
1

1
.40
.35
.30
.25
.20
.15
.10
.05
.00
1
1
1
1
1
1
1
1
0.95
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
[Complex] M
Fig. 10 Plot of I˳/I vs [complex] for the fluorescence quenching curves of EB bound to DNA
by 1 (R = 0.995)
2
2

Phosphatase activity and DNA binding studies of dinuclear phenoxo-
bridged zinc(II) complexes with an N,N,O-donor ligand and halide ions in a
rare cis-configuration
a
a
a
a
a
Monika Sheoran , KishalayBhar , Tanveer A. Khan , Anuj K. Sharma and Sunil G. Naik *
Department of Chemistry, School of Chemical Sciences and Pharmacy, Central University of
Rajasthan, NH-8, Bandarsindri, Ajmer Distt. Rajasthan, 305817
Graphical Abstract (Pictogram)
2
3

Phosphatase activity and DNA binding studies of dinuclear phenoxo-
bridged zinc(II) complexes with an N,N,O-donor ligand and halide ions in a
rare cis-configuration
a
a
a
a
a
Monika Sheoran , Kishalay Bhar , Tanveer A. Khan , Anuj K. Sharma and Sunil G. Naik *
Department of Chemistry, School of Chemical Sciences and Pharmacy, Central University of
Rajasthan, NH-8, Bandarsindri, Ajmer Distt. Rajasthan, 305817
Graphical Abstract (Synopsis)
A tridentate NNO Schiff base ligand (HL) and its Zn(II) complexes of the types
[
2 2 2 3 2 2 2
Zn (L) (Cl)] .2(CH OH) (1)and [Zn (L) (X)] [X = Br (2), I (3)] have been synthesized and
characterized by different spectroscopic techniques. The molecular structures of the three
complexes were determined by single crystal X-ray crystallography. The initial rate values
for the hydrolysis of 4-nitrophenylphosphate to 4-nitrophenolate by complexes 1-3 are
-1
respectively 590, 334 and 705 min . The complexes show a weak external binding
2
2
propensity to calf thymus DNA with binding constant values 9.45 × 10 , 7.06 × 10 , 6.51 ×
2
−1
1
0 M for 1, 2 and 3 respectively.
2
4

Reviewer #4: The authors have made significant corrections and improvements to the overall
manuscript, however there are still some corrections to be made in terms of the
crystallography of this paper.
I would recommend this paper for publication once the following corrections and
considerations have been made.
Have the authors check for missed symmetry on PLATON for structures 2 and 3?
Reply: Yes, we have checked for missed symmetry on PLATON for structures 2 and 3. It
shows ‘no obvious change in symmetry needed’.
1
. The chemical formula for 1 in the cif, contains an incomplete CH3OH moiety. Please can
the authors correct this and the overall chemical formula.
Reply: Now we have corrected the formula of complex 1 by adding H-atoms to CH3OH
moiety and NH- groups of ligands.
2
. On page 3 line 58, while the H atoms have not been found (on all the NH groups), they
need to still be included in the overall structure (where possible) and its formulas using the
riding models (or fixed models if necessary). Corrections to this will need to be updated in
the final CIF files.
Reply: Now for complexes 1 and 3 we have added the H-atoms to NH- groups of ligand and
refined accordingly. But for complex 2 we are unable to locate such H-atoms. The corrected
cifs are also submitted to CCDC.
3
. The authors would also do well to note that the relatively low quality of data possibly due
to the high-temperature of data collection does not provide a solid basis for detailed
discussions of structural details and interactions.
Reply: According to honourable reviewer’s suggestion we have modified the following
structural part.
For example P8 line 37, 'In 1-3, Zn (II) centres show considerable deviations…' at this level
of structural solutions and refinements, the comparisons and deviations are not necessarily
considerable mainly due to the significant variation in the quality of the structure solution and
refinement details.
It would be best to simply note the range of deviation if absolutely necessary.
Reply: According to honourable reviewer’s suggestion we have omitted “P8 line 37, 'In 1-3,
Zn (II) centres show considerable deviations” this description from structural details.
2
5

4
. Similarly the deviation in the Zn-O distances, as discussed on P8, Line 54, 'more
unsymmetrical' - should be 'more asymmetrical' or 'less symmetrical', although in 1, this is
symmetrical.
Reply: According to honourable reviewer’s suggestion we have also modified the discussed
on deviation in the Zn-O distances in P8, Line 54, as 'less symmetrical, although in 1, this is
symmetrical’.
5
. Could the authors also consider the following in their discussion of the 'relationship
between structure and catalytic activity'.
Reply: According to honourable reviewer’s suggestion we have also modified this section as
“
The catalytic activity order of complexes 3>1>2 shows direct correspondence to the Zn...Zn
separation (3.217 Å for 1, 3.220 Å for 2 and 3.209 Å for 3) indicating shorter Zn...Zn
distance is better suited to accommodate the phosphate group.”
2
6