Molecular hybridization approach of bio-potent Cu II /ZnII complexes derived

from N, O donor bidentate imine scaffolds: Synthesis, spectral, human serum
albumin binding, antioxidant and antibacterial studies

Mohammad Shakir, Summaiya Hanif, Md. Fazle Alam, Hina Younus

PII: S1011-1344(16)30626-1
DOI: doi:10.1016/j.jphotobiol.2016.10.006
Reference: JPB 10605

To appear in:

Received date: 1 August 2016

Accepted date: 6 October 2016

Please cite this article as: Mohammad Shakir, Summaiya Hanif, Md. Fazle
Alam, Hina Younus, Molecular hybridization approach of bio-potent CuII /ZnII
complexes derived from N, O donor bidentate imine scaffolds: Synthesis, spec-
tral, human serum albumin binding, antioxidant and antibacterial studies, (2016),
doi:10.1016/j.jphotobiol.2016.10.006

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 ACCEPTED MANUSCRIPT

Molecular hybridization approach of bio-potent CuII/ZnII complexes derived from N, O

donor bidentate imine scaffolds: Synthesis, spectral, Human Serum Albumin binding,

antioxidant and antibacterial studies

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Mohammad Shakira*, Summaiya Hanifa, Md. Fazle Alamb and Hina Younusb

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a*

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Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India.

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b
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.

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*
To whom correspondence should be addressed

Email: shakir078@yahoo.com
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Phone No. +91-9837430035
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Abstract

Novel bio-relevant monometallic Schiff base complexes of the type, [Cu(L1)2] (1),

[Zn(L1)2].2H2O (2), [Cu(L2)2].2H2O (3) and [Zn(L2)2].H2O (4) [L1 = (E)-3-(((3-chloro-4-

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hydroxyphenyl)imino)methyl)naphthalen-2-ol and L2 = (E)-2-chloro-4-((1-(5-chloro-2-

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hydroxyphenyl)ethylidene)amino)phenol] were synthesized and characterized. A comparative

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account of analytical, spectroscopic (FT-IR, 1H and 13
C NMR, Mass, UV–vis and EPR),

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thermal (TGA/DTA), XRD and SEM studies revealed a correlation between the structure and

function of these biologically active molecular entities. HSA (Human serum albumin)

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binding profiles of the metal complexes (1-4) were monitored using biophysical techniques
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viz., absorbance, fluorescence, circular dichromism (CD) and foster resonance energy

transfer (FRET). The intrinsic binding constant (Kb) demonstrated substantial binding

propensity of L1 linked complexes (1 and 2) in comparison to L2 complexes (3 and 4)

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suggesting L1 to be more bio-active pharmacophore due to higher planarity and conjugation

as compared to L2 ligand. The outcome of fluorescence study revealed static quenching

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mechanism on the basis of the quenching of HSA by the complexes (1–4). However,
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modifications in the secondary structure of HSA by complexes (1–4) inferred via CD

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measurements which revealed the enhancement of α-helicity (67.47 % to 69.20%) with the

preference order of 1>2>3>4. Furthermore, in-vitro antibacterial study against different

bacteria and antioxidant activities against DPPH• and superoxide radical (O2-•) at variable

concenterations outspread discernible bio-potencies of the metal complexes as compared to

free ligand scaffolds due to the chelation effect.

Keywords: Schiff base; metal complexes; spectroscopic studies; Human Serum Albumin;

antioxidant

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1. Introduction

The alchemy of complexes provides massive prospects for the designing of bioactive

scaffolds due to the variety of different accessible metals which have capability to modify the

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structural reactivity of the complexes by their ligand sphere [1, 2]. The transition metal

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complexes containing Schiff base scaffolds have gained momentous attention amongst

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inorganic chemists over the years as Schiff base chromophore acquires substantial

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anharmonic phonons which afford extensive contributions to their coordination ability with

various kinds of ligands in view of their structural variability, stability and biocompatibility

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[3-5]. It is well known that chelation effect enhances the mode of action of drug and
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efficiency of a therapeutic agent in metal complexes [6]. The therapeutical efficiency of

complexes also depends on the properties of the metal ions and the donor sequence of the
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ligands as different ligands revealed different biological implications [7]. Among the
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transition metals, copper is a physiologically significant endogenous metal center which

carried out a crucial role in Cu(II)/Cu(I) redox couple reactions that are quite substantial in
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different life processes as copper is found to be an integral cofactor of several enzymes

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engrossed in different oxidative metabolism. Consequently, the diverse structural aspects of

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copper (II) complexes and their synergistic activities with drugs have been the focal theme

for the large number of researchers [8]. Likewise, zinc also authenticated its vital role for

numerous cell processes and is a major regulatory ion in the metabolism of cells [9]. It has

also been revealed that zinc (II) complexes exhibit diverse bio-potent activities viz.,

antibacterial [10], anti-inflammatory [11] and antioxidant [12].

Recently, privileged structure activity conception has been materialized as a triumphant

approach for the new-fangled drug discovery in the field of medicinal chemistry [13]. The

molecular hybridization (MH) approach has become a potent strategy for the rational design

of such ligands or prototypes which depend on the recognition of pharmacophoric sub-units

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in the molecular framework and directed the design of new hybrid architectures that maintain

pre-selected characteristics of the original templates [14] and generate a new hybrid

compound with improved affinity and efficiency as compared to the parent drugs. In view of

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this rational approach, hybrid pharmacophore ligands derived from 2-

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hydroxynaphthaldehyde/5-chloro-2-hydroxyacetophenone and 4-amino-2-chloro phenol have

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been undertaken as Schiff bases resulted from aromatic aldehydes/ketones with ortho

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substituted hydroxyl group have immensely aroused interest among Chemists because of

their capability to act as bidentate ligands for the transition metal ions [15-18]. In literature,

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bidentate ligands possessing imine groups hold great promise as modulators in the
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construction of transition metal centers [19-22]. The pharmacological efficiency of Schiff

bases based on aminophenol derivatives have been the subject of indubitable interest because
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of their wide biological applications [23, 24]. Schiff-bases, in particular 2-hydroxy-1-

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napthaldehyde scaffold have been comprehensively studied due to their broad spectrum

applications in the medicinal field as they form stable complexes with metal ions because of
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the presence of a hydroxyl group at their ortho position which may participate in bonding
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with the metal ion due to deprotonation [15]. Zhou and co-workers have drawn considerable
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attention toward hydroxyl-substituted Schiff-bases due to their good free radical scavenging

activities. However, metal complexes derived from 2-hydroxyacetophenone and aminophenol

scaffold have not been addressed seriously in spite of their prospective metal chelating

properties and applicabilities [16, 25, 26]. In view of the above criteria, the strategies and

rational design based upon the selection of these scaffolds stalk out the fact that complexes

obtained from aromatic Schiff bases containing aromatic motif would consequently augment

lipophilicity of drug which is of paramount significant to the ongoing drug researches and

developments. An outlook of these developments in metallodrugs, Schiff base complexes

based upon bioactive scaffolds kindle immense efforts for a better prospective of

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understanding structural features in order to gain more insights on the exact relationship

between their specific chemical structures and the absolute biological data. [27].

It is noteworthy to consider that factors responsible for the pharmaceutical action of

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metallodrugs and their specific delivery to its target cells may be attained by the appropriate

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usage of targeting groups or by tuning the physical and chemical traits of the drug or drug

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carrier such as molecular size and hydrophobicity. It is well established that various types of

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macromolecules worked as carrier molecules where Human serum albumin (HSA) is one of

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the significant carrier protein as literature revealed that research in this field explored that

albumins from blood plasma can interact with different compounds such as Schiff base
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ligands [28, 29, 30] and other Cu(II), Ni(II) and Zn(II) complexes [31-36]. The interactions of

protein with drug formulate a stable protein drug–complex which in turn makes an impact on
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the concentration, distribution and metabolism of the drug in the blood stream. The
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distribution of drugs is usually directed by HSA as mostly drugs circulate in plasma and

attain the targeted tissues via binding to HSA. Therefore, binding of drugs to HSA turned into
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an essential determinant of pharmacokinetics restricting the unbound concentration and

affecting distribution and elimination [37]. Thus, subsequently make it a suitable candidate
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for drug delivery. So far most of the researches have focussed on DNA studies of metal

complexes particularly based on ortho-hydroxyl Schiff base ligand scaffold but scare reports

have been taken which addressed the complex - protein binding studies based on bioactive

hydroxyl substituted Schiff bases [38, 39].

Keeping in view of the above facts, we report the synthesis and characterization of Schiff

base ligands (L1 and L2) based upon the molecular hybrid pharmacophore rational approach

[4,40], via condensation reaction between two bioactive scaffolds, 4-amino-2-

chloroaminophenol and 2-hydroxynaphthaldehyde or 5-chloro-2-hydroxy acetophenone,

respectively and their corresponding Cu(II) and Zn(II) complexes. The biological

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significance in terms of their structure activity relationship was assessed through antioxidant

and antibacterial activities. Furthermore, HSA binding study of the complexes was also

carried out in order to investigate their interactions by employing electronic absorption,

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fluorescence quenching (assessment number binding sites and binding mode), circular

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dichroism and FRET spectroscopic studies.

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2. Experimental section

2.1. Materials

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All chemicals and solvents were procured commercially. The microanalyses were recorded
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on Perkin Elmer 2400 CHN elemental analyser where as IR spectra (KBr pellets, 4000-400

cm-1) were obtained from Perkin Elmer – 2400 spectrometer. The molar conductivity

measurements of complexes (10-4 M DMSO solution) were recorded at room temperature on

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a Systronic type 302 conductivity bridge equilibrated at 25±0.01◦C while magnetic

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measurements of complexes were analysed by using Faraday balance at room temperature.

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The 1H and 13
C NMR spectra were performed in DMSO- d6 using Bruker Avance II 400
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NMR spectrometer and JEOL 400 spectrometer, respectively. EPR spectra of complexes
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were analysed at room temperature using DPPH as standard (g=2.0036) at 9.167 GHz on a

ES-DVT4 Spectrometer. The mass spectra were obtained using a WATERS Q-TOF premier

mass spectrometer. The UV-vis spectra of the compounds (10-4 M DMSO solution, 200-800

nm) were recorded on a Pye-Unican 8800 spectrophotometer. Thermal analyses (TGA/DTA)

of complexes were quantified under N2 atmosphere at a heating rate of 20°C /minute on a

Schimadzu Thermal using alumina as reference. However, presence of metal ion in the

complexes was evaluated volumetrically [40]. The XRD patterns of complexes were recorded

on a X’Pert PRO Diffractometer (10◦≤2Ɵ≤80◦) at room temperature using CuKα

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monochromatic radiation (λ=1.54060A◦) with voltage of 40KV and current of 30mA while

SEM micrographs were taken on JEOL JSM-6510LV Scanning Electron Microscope.

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2.2. Synthesis of Schiff base ligand, (E)-3-(((3-chloro-4-

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hydroxyphenyl)imino)methyl)naphthalen-2-ol, L1

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To a stirring methanolic solution (20 ml) of 2-hydroxynaphthaldehyde (2 mmol), a

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methanolic solution (20 ml) of 4-amino-2-chloro phenol (2 mmol) was added dropwise. The

content of resulting mixture was refluxed for 2 hours leading to the isolation of

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microcrystalline solid product. The product was filtered and washed with methanol and then
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finally dried under vacuo over anhydrous calcium chloride (Scheme 1).

Ligand (L1) : Yield: 73%, Colour: yellow, m.p. > 300 ◦C, Anal. Calc. for C17H12NO2Cl (%)
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C, 68.58; H, 4.06; N, 4.70. Found: C, 68.54; H, 4.03; N, 4.62. IR (KBr, cm -1) 1630 (HC=N),
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3437 (OH), 1318 (C-O). ESI–MS (m/z+), 298.89.

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2.3. Synthesis of Schiff base ligand, (E)-2-chloro-4-((1-(5-chloro-2-

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hydroxyphenyl)ethylidene)amino)phenol, L2
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A 2 mmol hot methanolic (20 ml) solution of 5-chloro-2-hydroxyacetophenone was added in

a drop wise manner to a hot methanolic (20 ml) solution of 4-amino-2-chloro phenol (2

mmol). The resultant reaction mixture was refluxed for 3 hours leading to the isolation of

microcrystalline solid product. The product was filtered and washed with methanol and then

finally dried under vacuo over anhydrous calcium chloride. (Scheme 2).

Ligand (L2) : Yield, 73%, Colour: brown, m.p. > 300 ◦C, Anal. Calc. for C14H11NO2Cl2 (%)

C, 56.77; H, 3.74; N, 4.72. Found: C, 56.71; H, 3.70; N, 4.68 IR (KBr, cm-1): 1622 (HC=N),

3424 (OH), 1324 (C-O). ESI–MS (m/z+), 297.40.

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2.4. Synthesis of [Cu(L1)2], [Cu(L2)2].2H2O, [Zn(L1)2].2H2O and [Zn(L2)2].H2O

complexes

To a 1.0 mmol solution of metal salt (MCl2.nH2O; M = Cu and Zn, n = 2 and 6) dissolved in

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15 ml of methanol, a 2.0 mmol solution of ligand, L1 / L2 in 25 ml methanol was added

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slowly. The resulting mixture was stirred under reflux for 4-5 h resulting in the isolation of

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microcrystalline solid product. The product was filtered and washed with methanol and then

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finally dried under vacuo over anhydrous calcium chloride. (Scheme 1 and 2).

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[Cu(L1)2] complex : Yield: 73%, Colour: dark green, m.p. > 300 ◦C, Anal. Calc. for

CuC34H22N2O4Cl2 (%) C, 62.15; H, 3.37; N, 4.26. Found: C, 62.10; H, 3.32; N, 4.21 IR (KBr,
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cm-1): 1610 (HC=N), 1310 (C-O), 508 (M-N), 426 (M-O). Molar Conductance (1 x 10-
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M, DMSO): ˄m (15.21 mol−1 cm2 ohm−1). ESI–MS (m/z+), 657.28.
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[Cu(L2)2].2H2O complex : Yield: 73%, Colour: green, m.p. > 300 ◦C, Anal. Calc. for
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CuC28H24N2O6Cl4 (%) C, 48.74; H, 3.50; N, 4.05. Found: C, 48.68; H, 3.45; N, 4.01. IR

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(KBr, cm-1): 1602 (HC=N), 1312 (C-O), 510 (M-N), 434 (M-O). Molar Conductance (1
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x 10-3M, DMSO): ˄m (12.15 mol−1 cm2 ohm−1). ESI–MS (m/z+), 654.48.

[Zn(L1)2].2H2O complex : Yield: 73%, Colour: light yellow, m.p. > 300 ◦C, Anal. Calc. for
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ZnC34H26N2O6Cl2 (%) C, 58.76; H, 3.77; N, 4.02. Found: C, 58.70; H, 3.71; N, 4.00 IR (KBr,

cm-1) 1617 (HC=N), 1307 (C-O), 505 (M-N), 432 (M-O). Molar Conductance (1 x 10-
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M, DMSO): ˄m (9.25 mol−1 cm2 ohm−1). ESI–MS (m/z+), 660.42.

[Zn(L2)2].H2O complex : Yield: 73%, Colour: light buff, m.p. > 300 ◦C, Anal. Calc. for

ZnC28H22N2O5Cl4 (%) C, 49.91; H, 3.29; N, 4.15. Found: C, 49.89; H, 3.24; N, 4.10 IR (KBr,

cm-1): 1609 (HC=N), 1309 (C-O), 512 (M-N), 436 (M-O). Molar Conductance (1 x 10-
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M, DMSO): ˄m (8.12 mol−1 cm2 ohm−1). ESI–MS (m/z+), 657.35.

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2.4. UV- Visible Spectroscopy

Interaction of complexes with HSA was recorded by using UV–1800 Shimadzu

spectrophotometer equipped with peltier temperature programmer-1 (PTP-1). The absorbance

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titration experiments were conducted by keeping the constant concentration of HSA (5µM) in

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20 mM phosphate buffer at pH 7.4 with varying concentration of complexes (0-100µM) and

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the spectra were recorded in the range of 240-400 nm.

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2.5. Fluorescence quenching experiments

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The fluorescence quenching measurements were performed on Shimadzu 5301 PC
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fluorescence spectrophotometer equipped with temperature control system. The excitation

and emission slit widths were set at 3 mm and 3 mm, respectively. The titration of the
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complexes (0–50 µM) to HSA (5 µM) solution in 20 mM of phosphate buffer at pH 7.4 were
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recorded on a dual-path length fluorescence cuvette (10 x 3.5 mm). Intrinsic fluorescence

intensity was measured by exciting HSA at 280 nm and the corresponding emission spectra
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were recorded in the range of 300–450 nm. The decrease in fluorescence intensities were
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analysed according to the Stern–Volmer equation (1):

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𝑭𝟎 /𝑭 = 𝑲𝒔𝒗[𝑸] + 𝟏 = 𝒌𝒒𝝉𝟎 [𝑸] + 𝟏 (1)

Where F0 and F were the fluorescence intensities in absence and presence of quencher

(complex), Ksv is the Stern–Volmer quenching constant, kq is the bimolecular rate constant

of the quenching reaction and 𝜏0 is the average integral fluorescence life time of tryptophan

(~ 10-8 s). The binding sites and the binding constant were obtained from modified Stern–

Volmer equation (2):

log (F0/F-1) = log Kb +n log [Q] ------------(2)

Where Kb is the binding constant and n is the number of binding sites.

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2.6. Fluorescence resonance energy transfer (FRET)

The absorption spectra of each complex (3µM) and the fluorescence spectra of HSA (3µM)

were scanned in similar way as described in ‘UV- visible’ and ‘fluorescence quenching’

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section in the range of 300-400 nm. If the complex (acceptor) and HSA (donor) spectra

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overlap each other then donor–acceptor pair will be considered within Forster distance so that

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the transfer of energy might be taken place. [41]. The efficiency of energy transfer (E) is

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calculated using the following equation (3)

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𝑭 𝟎 𝑹𝟔
𝑬 = 𝟏 − 𝑭 = 𝑹𝟔 +𝒓 𝟔
----------------- (3)
𝟎 𝟎
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Where, F0 and F are the fluorescence intensities of HSA in the absence and presence of

complex, respectively while r is the distance between donor and acceptor and R0 is the critical
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distance at which transfer efficiency equals to 50% which can be calculated from the
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following equation:
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𝑹𝟔𝟎 = 𝟖. 𝟕𝟗 𝑿 𝟏𝟎−𝟐𝟓 𝑲𝟐 𝒏−𝟒 ∅𝑱 ------------ (4)

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where K2 is the orientation factor related to the geometry of the donor - acceptor of dipole, n
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is the refractive index of the medium, ∅ is the fluorescence quantum yield of the donor in the

absence of an acceptor and J is the degree of spectral overlap of the donor emission and the

acceptor absorption [41] which can be evaluated by integrating the overlap spectral area

between 300 to 400 nm by using the given equation:

∞ 𝑭(𝝀)𝜺𝝀𝟒 𝒅𝝀
𝑱 = ∫𝟎 ∞ ------------------ (5)
∫𝟎 𝑭(𝝀)𝒅𝝀

Where F (λ) is the fluorescence intensity of the fluorescence donor at wavelength (λ) and 𝜀(λ

is the molar extinction coefficient of the acceptor at wavelength (λ). The values of K2, ∅ and

n were taken as 2/3, 0.118 and 1.336, respectively.

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2.7. CD spectra measurements

The CD spectra were recorded on Jasco spectropolarimeter (J-815, Jasco International Co.

Ltd., Tokyo, Japan) equipped with a Peltiertype temperature controller (PTC-423S/15)

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attached to a water bath. Prior to start experiment, instrument was calibrated with (+)-10-

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camphorsulfonic acid. The spectra were measured at 25 0C using a scan speed of 50 nm/min

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and response time of 1s. Far-UV CD spectra of different HSA- complex at molar ratios of

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1:0, 1:5, 1:10 and 1:15 were measured in 0.1 cm cell in the range of 190-250 nm. Appropriate

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blanks were subtracted from all the spectra. The helical content of each HSA - Schiff base

complex was calculated using online K2D and later smoothen by using the Savitzky–Golay
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method with 25 convolution width. HSA concenteration used for far-UV CD was taken as 5

µM.
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2.8. Antioxidant activity

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2.8.1. DPPH assay

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The scavenging data of compounds were evaluated using standard 2,2-diphenyl-1-

picrylhydrazyl (DPPH) method [42]. Different concenterations (0-150 µg/mL) of compounds

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and standard control (ascorbic acid) were taken in different tubes and volume of each tube

was adjusted to 100 µL by DMF and then 5 mL of methanolic DPPH (0.01 mM) solution was

mixed to each tube and kept for incubation in dark for a period of 30 min at 25 0C. Then, the

absorbance at 517 nm was measured by spectrophotometer. The lowering of the absorbance

of the reacting mixture indicated the higher inhibitory scavenging effect. The capability to

scavenge the DPPH radical was calculated using the following equation:

𝑨𝟎 −𝑨𝟏
DPPH• scavenging effect (%) =( ) 𝑿 𝟏𝟎𝟎
𝑨𝟎

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Where A0 is the absorbance in the absence of the tested compound and A 1 is the absorbance

of the tested compound.

2.8.2. SOD mimetic assay

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SOD mimetic activity of the compounds was measured spectrophotometically by using PMS-

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NADH-NBT system [43]. Superoxide radicals are generated in PMS-NADH systems by

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oxidation of NADH which react with nitrobluetetrazolium (NBT) to form blue colour

formazane. The reaction mixture consisted of 20 mM sodium phosphate buffer at pH 8.2,

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PMS (1.9 µM), NBT (184 µM), NADH (205 µM) and different concentration (0-150 µg/mL)

of compounds. The reaction was started with the subsequent addition of PMS and then the
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absorbance at 560 nm was continuously monitored for the period of 5 min as an index of

NBT reduction using a double beam Shimadzu spectrophotometer. Each test was performed
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in triplicate manner. The percentage of inhibition was calculated by using following formula.
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𝑨𝟎 −𝑨𝟏
Percent (%) inhibition = ( ) 𝑿 𝟏𝟎𝟎
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𝑨𝟎
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Where, A0 and A1 are the absorbance of control and test sample at 560 nm, respectively.
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2.9. In-vitro antibacterial assay

The antibacterial activity of compounds was carried out against Staphylococcus aureus

(MTCC-3610), Listeria monocytogenes (MTCC-1143) Escherichia coli (ATCC-25922) and

Salmonella typhimurium (MTCC-3216) using disc diffusion method [44]. A standard

inoculums (1 - 2 x 107cfu/ml, 0.5 McFarland standards) was introduced on the surface of

sterile agar plates and a sterile glass spreader was used for even distribution of the inoculums.

The discs measuring of about 6 mm in diameter were set up using Whatman No. 1 filter paper

and sterilized by dry heat at 140 ◦C for 1 h. The sterile discs previously soaked in a known

concentration of the test compounds were placed in the nutrient agar medium. Solvent and

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growth controls were kept. The reference drug, Ciprofloxacin (30 µg) was used as positive

control while the disk poured in DMSO was used as negative control. The plates were

overturned and incubated for 24 h at 37◦C. The susceptibility was determined on the basis of

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the diameter of the zone of inhibition against different bacterial strains. Further, zones of

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inhibition were assessed and compared with the controls.

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3. Results and Discussion

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Novel monometallic complexes of the type, [Cu(L1)2] (1), [Zn(L1)2].2H2O (2),

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[Cu(L2)2].2H2O (3) and [Zn(L2)2].H2O (4) were synthesized by interacting metal with an

appropriate ligand frameworks, L1 and L2 (Scheme 1 and 2). All four complexes (1–4) were
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found to be stable at room temperature and easily dissolve in DMSO. The physico-chemical

data of the complexes compliment with the proposed formulae with 1:2 metal to ligand
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stoichiometry. The lower molar conductance values (11 – 16 Ω−1 cm2 mol−1) of the
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complexes in DMSO (1 × 10−4 M) were too low to account for any dissociative ions in the

complexes which indicated their non-electrolytic nature [45]. The proposed structures were
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formulated on the basis of their elemental, IR, NMR (1H and 13

C), Mass, UV- visible, EPR

and thermal data (TGA/ DTA) while crystallite size and morphology of the complexes were
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employed by XRD and SEM analysis. Furthermore, HSA binding affinity of the complexes

deduced by various spectroscopic studies which quantified various binding parameters (Kb,

Ksv, Kq and n). In addition, radical scavenging effect against DPPH• and superoxide radical

(O2-•) and antibacterial activity of the compounds were also evaluated on the basis of their

structural aspects.

3.1. IR Spectra

Comparative study of IR spectral data provides considerable information regarding the

formation of Schiff base compounds. The spectral data of the ligands (L1 and L2) exhibit a

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strong imine peak in the region 1622-1630 cm-1 confirming the formation of proposed Schiff

base ligand frameworks [46]. The spectra exhibit broad band in the region 3424-3437 cm-1

assignable to (OH) stretching vibration while medium intensity band in the region 1318-

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1324 cm-1 may ascribed to (C-O) phenolic vibration [47, 48]. The characteristic medium to

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strong intensity bands in the region 1440-1480, 1080-1100 and 768-798cm-1 may

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unambiguously be assigned to aromatic ring vibrations[49] whereas medium intensity band in

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the region 2926-2941 and 3025-3121 cm-1 may be ascribed to (C-H) aliphatic and (C-H)

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aromatic vibrations, respectively [50]. However, characteristic imine band was found to be

shifted to lower wavenumber in metal complexes indicating the coordination of imine

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nitrogen to metal ion [51]. This is further substantiated by the appearance of band in the

region 505-512 cm-1 attributable to (M-N) vibration [52]. The disappearance of prominent
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(OH) band in the region 3424-3437 cm-1 indicates the co-ordination of phenolic oxygen
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after deprotonation [53] which is supported by the lowering of phenolic (C-O) band,
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suggesting the coordination of phenolate oxygen to metal ion [54]. This is further
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corroborated by the emergence of low intensity band in the region 426-436 cm-1 which may

be assigned to (M-O) vibration [55]. A new medium intensity broad band in the region
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3320-3345 cm-1 may ascribed to (OH) of lattice water molecule(s) in the complexes (2, 3

and 4) [55]. However, bands associated with aromatic ring vibrations appeared at their

expected positions.

3.2. 1H NMR

The 1H NMR spectra of L1 and L2 (Fig. 1a and b) exhibited multiplets in the region δ 6.85-

8.22 ppm and 6.26-7.75 ppm which may be assigned to aromatic ring protons of L1 and L2

frameworks, respectively [56]. The appearance of singlet at 8.24 and 3.47 ppm in the spectra

of L1 and L2 may ascribed to azomethine proton (-CH=N-) and methyl protons, respectively

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[15, 57]. Moreover, a singlet appeared at 16.01 and 12.63 ppm may reasonably be assigned to

hydroxyl group of 2-hydroxynaphthaldehyde and 2-hydroxy-5-chloro acetophenone moieties

in ligand frameworks, respectively whereas the resonance signals at 9.57 and 9.56 ppm

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correspond to hydroxyl group of 4-amino-2- chlorophenol moiety in both ligand frameworks

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[58-60]. A comparison of 1H NMR spectrum of complex (1) with that of free ligand, L1

R
displayed a downfield shift in resonance signal of azomethine proton which appeared at 8.44

SC
ppm suggesting the coordination of azomethine nitrogen to Zn(II) ion (Fig. 2a). However,

resonance signals at 16.03 and 12.03 ppm corresponding to hydroxyl group in free ligands

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were found to be absent in both 2 and 4 complexes, indicating the deprotonation of hydroxyl
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group of naphthaldehyde/ acetophenone moiety of ligand frameworks whereas signals

corresponding to aromatic ring protons in L1 and L2 underwent downfield shift further

D

support the coordination of ligand frameworks (Fig. 2a and b) [61].

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3.3. 13C NMR

P

13
The C NMR spectra of Schiff base ligands, L1 and L2 (Fig. 3a and b) exhibited sharp
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aromatic signals at 108.48, 120.13, 126.59, 127.79, 128.88, 130.01, 134.71, 135.78, 156.65,

159.75 ppm and 18.44, 118.01, 121.81, 132.51, 133.73, 155.85, 158.98 ppm, respectively
AC

assignable to the carbons of naphaldehyde and acetophenone moieties [62] whereas signals at

116.13, 122.81, 122.96, 123.13, 150.95, 153.71 and 114.43, 120.21, 120.99, 124.54, 147.73,

152.71 ppm may be assigned to aminophenol moiety of both ligand frameworks, respectively.

The characteristic signals associated with imine carbons of L1 and L2 were found at 162.95

and 161.77 ppm, respectively [62] However, these imine signals undergo downfield shift in

both the complexes (2 and 4), indicating the coordination of ligands, L1 and L2 via imine

nitrogen whereas the signals of other aromatic carbons displayed a slight change from their

estimated positions, invoking the involvement of phenolic oxygen of ligand frameworks in

the coordination (Fig. 4a and b) [63].

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3.4. Mass spectral studies

The molecular ion peaks of ligands, L1 and L2 and their complexes (1-4) appeared at m/z

298.89, 297.40, 657.28, 660.42, 654.28 and 657.35, respectively which substantiate their

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proposed molecular formulae. The representative spectra of ligands, L1 and L2 and their

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complexes (2 and 4) are presented in Supplementary Information, Fig. S1 (a-d).

R
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3.5. Electronic spectra and magnetic moment

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The band positions in the electronic spectra and magnetic moment values were recorded at

room temperature in order to ascertain the stereochemistry of Cu(II) ion in the complexes 1
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and 3 (Table 1). The electronic spectra of ligands, L1 and L2 exhibited high intensity

absorption bands at 268 and 333 nm; 270 and 330 nm, respectively were assigned to
D

intraligand π-π* and n- π* transitions, respectively associated with aromatic rings and
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azomethine chromophore transitions [64]. However, the positions of these bands in both the
P

Cu(II) complexes were found to be shifted to higher wavelength suggesting the coordination
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of the ligand frameworks to Cu ion. The proposed square planar geometry in the complexes 1

and 3 was inferred from absorption broad bands at 615 and 612 nm, respectively which may
AC

reasonably be assigned to 2B1g→2A1g transition [65]. The observed magnetic moment values

of 1.83 and 1.87 BM for the complexes 1 and 3 further corroborated the square planar

environment around Cu (II) ion [64].

3.6. EPR spectrum

The EPR spectra of complexes (1 and 3) were recorded at room temperature (Supplementary

Information, Fig. S2 a and b). The ‘g’ tensor values of complexes could be used to determine

the ground state of the ion. The analysis of the spectra exhibited g|| =2.1808 and 2.1680, g┴ =

2.0452 and 2.0381 and gav= 2.0912 and 2.0789, respectively computed from the expression,

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g2av =1/3(g2|| + 2g2┴). The trend (g|| > g┴> 2.04) for the complexes indicate that the unpaired

electron is localized in the dx2-y2 orbital of the Cu(II) ion with 2B1g spectroscopic state [65].

The observed g values were found to be consistent with the square planar geometry [66]. The

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deviation of gav from that of free electron (2.0023) is due to covalence character as per

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Kivelson and Neiman [67]. The geometric parameter (G) is the measure of exchange

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interaction and is evaluated by using Kneubuhl’s method, G= g|| -2/ g┴ -2 [68] which comes

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out to be 4.50 and 4.40 for the 1 and 3 complexes, respectively indicated that the exchange

coupling effects are not operative[69].

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3.7. Thermal analysis (TGA/DTA)

TGA/ DTA analysis was carried out in order to inspect the correlations between the different
D

degradation steps of the complexes with their corresponding weight losses showing their
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thermal stability and thermal behaviour. The TGA and DTA curves of the complexes (1-4)

were investigated in the temperature range of 25-800◦C and the corresponding percentage of
P
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weight losses are calculated (Supplementary Information, Fig. S3 a and b). The TGA curves

of complexes (2, 3 and 4) displayed weight losses (obs = 5.15, 5.19 and 2.60 %; calc = 5.18,
AC

5.21 and 2.67%) in the temperature range of 80-265 ◦C which correspond to the elimination

of lattice water molecule(s) as supported by DTA peak in the temperature range of 95-100 ◦C

[70]. However in complex (1) no visible change in TGA graph has been noticed in this

temperature range which is further authenticated by the absence of DTA [71]. While the

complexes (1 and 2) exhibited weight loss (obs = 46.49 and 44.19 %; calc = 46.62 and 44.26

%) and (obs = 38.63 and 36.58 %; calc = 38.83 and 36.71 %) in the temperature range of

290-500 ◦C and 520-680 ◦C attributable to the degradation of naphthalene part of L1 scaffold

at imine bond and loss of remaining organic moieties of the ligand framework, respectively.

These decomposition steps further supported by the appearance of endothermic DTA peak in

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the temperature range of 350-380 ◦C and exothermic DTA peak in the temperature range of

550-600 ◦C, respectively. However, in case of complexes (3 and 4), the weight losses (obs =

80.84 and 82.81 %; calc = 80.92 and 82.87 %) in the temperature range of 300-660 ◦C may

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reasonably be due to the simultaneous expulsion of acetophenone moiety of L2 scaffold at

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imine bond alongwith the remaining organic moieties of the ligand framework. The presence

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of exothermic DTA peak in the temperature range of 600-620 ◦C further confirmed this

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decomposition step in the complexes (3 and 4). Moreover, horizontal curves beyond 700 ◦C

in all the thermograms indicated no further weight loss which implying respective metal

oxide as the final product [72].

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3.8. X- Ray diffraction study
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The X-ray powder diffraction studies of the complexes were carried out in order to determine
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the structural information of polycrystalline complexes because all the efforts have failed to

isolate suitable single crystal for X-ray crystallography. The powder analysis of metal
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complex (1-4) showed well defined patterns substantiating the crystalline nature of the
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complexes (Fig. 5). The X- ray diffraction of complex 1 exhibited peaks at 19.85, 22.45 and
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37.88 ascribed to (111), (200) and (311) crystal planes (Space group: Fm3m, JCPDS: 01-

1241) whereas complex 3 exhibited peaks at 20.48, 22.82 and 31.37 assigned to (111), (200)

and (220) crystal planes (Space group: Fm3m, JCPDS: 01-1242), respectively characteristic

of cubic arrangement of Cu atoms. However, complex 2 demonstrated peaks at 20.26, 25.86,

30.23, 35.01, 36.10, 42.25, 49.49 and 53.72 assigned to (101), (102), (110), (112), (201),

(203), (006) and (300) crystal planes (Space group: P63/mmc, JCPDS: 01-1244) while

complex 4 depicted fine indexed peaks at 20.83, 23.64 and 31.96 assigned to (101), (102) and

(110) crystal planes (Space group: P63/mmc, JCPDS: 01-1238), respectively characteristic of

hexagonal arrangement of Zn atoms. The different lattice parameters obtained for the

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complexes (1-4) are mentioned in Table 2. Moreover, the average crystallite size of the

complexes was calculated using the Debye Scherrer’s formula [73]:

D = 0.9λ/β.cos Ɵ

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Where D is the particle size, constant 0.9 is the shape factor, λ is taken as the wavelength (X-

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ray) of CuKα radiation (1.5406 A◦), Ɵ is the Bragg diffraction angle and β is the full width at

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half maximum (FWHM) value of Ɵ. The crystallite size of the metal complexes, 1-4 were

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found to be 12.93, 14.38, 27.18 and 24.56 nm, respectively.

4.0. Human Serum Binding (HSA) Studies NU

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HSA is an important physiological transporter of the essential metal ions especially Cu2+ and

Zn2+ in the bloodstream. It is documented that Schiff base complexes exhibit significant
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interaction with HSA [38, 39, 74] therefore, methodical analyses were carried out to
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investigate the interaction of HSA with complexes using different spectral studies.
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4.1. Absorption spectral studies

AC

The interaction of HSA with complexes (1-4) was examined by titrating the complexes with

constant concentration of HSA (Fig. 6). The spectra indicated the concomitant enhancement

of the intraligand band around 278 nm which is attributed to “hyperchromism”, suggesting

that aromatic acid residues originally buried in a hydrophobic cavity in HSA were exposed to

a certain degree of an aqueous milieu [74]. This consequently leads to the inflection in the

absorption profile because of the effect of polar solvent (water) alongwith the perturbations

which occurred in the micro-environment of the polypeptide backbone of the HSA [63]. The

observed trend of hyperchromatic effect 1> 2> 3> 4 demonstrated that L1 linked metal

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complexes more profoundly interact with HSA as compared to L2 linked complexes

suggesting greater biopotency, planarity and conjugation of L1 ligand framework.

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4.2. Fluorescence quenching studies

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Fluorescence spectroscopy is a valuable technique used to investigate the interaction of

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complex with HSA. The aromatic amino acid residues like tryptophan, tyrosine and

phenylalanine contribute significantly towards the intrinsic flourogenic properties of a protein

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[75] which attracts many protein biochemists to use it as an endogenous probe in order to

scrutinize the alterations due to ligand- protein interaction [76].

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HSA exhibits a strong fluorescence emission at 340 nm due to the emission from the single
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tryptophan residue (Trp-214). Upon addition of each complex (1, 2, 3, and 4) upto 50 µM,
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fluorescence intensity decreases by 43.99%, 32.91%, 30.84% and 13.92%, respectively (Fig.

7). The strong quenching of HSA fluorescence indicated that the complex interacts with HSA
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which may change the microenvironment around Tryptophan residue. The decrease in
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fluorescence intensity upon addition of complex was evaluated by Stern- Volmer plot (Fig.
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8). There is a linear agreement with the ratio of F0/F and molar concentration of complex in

Stern- Volmer plot. The slope of Stern- Volmer equation provides the Ksv values of each

complex with HSA. The values of Ksv of each complex (1-4) were found to be 1.43 x 104,

9.92 x 103, 9.74 x 103 and 3.17 x 103, respectively illustrating proficient quenching of L1

based complexes especially in complex (1). The quenching may be of dynamic or static type,

dynamic quenching refers to a process when the fluorophore and the quencher come into

contact during the transient existence of the exited state while static quenching refers to a

fluorophore–quencher complex formation [77]. From the result (Table 3) it is found that kq

(2.50 x 1012 , 1; 1.73 x 1012 , 2; 1.70 x 1012 , 3 and 5.55 x 1011 M-1 S-1, 4) of each metal

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complex was more than that of maximum scatter collision quenching constant, i.e, 2 x 1010

mol-1 sec-1 which clearly indicate static type of quenching.

For the determination of Kb and n, modified Stern- Volmer Plot was discussed in equation 2

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in experimental section. The binding equilibrium plots for the fluorescence quenching of

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HSA by complexes (1–4) at room temperature were shown in Fig. 9. The values of Kb (9.86 x

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104, 1; 1.03 x 104, 2; 3.23 x 103, 3; and 1.58 x 103, 4) and n (binding site) were calculated by

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the intercepts and slopes, respectively of the linear fitting plots of log (F0-F)/F versus log [Q]

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as shown in Fig. 9 and their respective data were displayed in Table 3. The optimum Kb

values of complexes (1-4) were consistent with the above results and revealed relatively
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higher binding propensity of L1 containing complexes in comparison to L2 based complexes

which evidently formulated them potent avid binder of HSA. Moreover, it is noted that the Kb
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value in the range of 104–106 M−1 is satisfactory for any drug carriers in blood [78].
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The results revealed that L1 linked complexes were more profoundly interact with HSA as

compared to complexes of L2 scaffold. This is due to the fact that L1 constitutes a

P
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naphthaldehyde moiety in its ligand framework which is more bioactive pharmacophore than

acetophenone moiety as it contains two fused rings of benzene that consequently results in
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higher ring planarity and greater conjugation (π- π interactions) to the system in comparison

to acetophenone comprised L2 scaffold. Such π- π interactions would improve better

understanding of mechanisms by which complex (drug) exert its activity [79]. Hence, these

properties altogether assist in anchoring more quenching behaviour of L1 linked metal

complexes.

4.3. Energy transfer between complexes (1–4) and HSA

FRET is an effective study used to demonstrate the overlap spectra of HSA (emission

spectra) and complex (absorption spectra) with molar ratio HSA: complex (donor: acceptor)

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as 1 (Fig. 10). According to FRET theory, energy transfer depend upon three aspects such as

strong quantum yield of donor, profound overlap of emission and absorption spectra and the

distance (r) between the acceptor and the donor which should be within 7 nm.

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The values of E, J, R0 and r were evaluated by using various equations as discussed in

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experimental FRET section (Table S1). The value (r) of each complex is found to be much

R
lower than 7 nm with order of 0.5R0 < r < 2.0R0 which warrants the probability of transfer of

SC
non-radiation energy from HSA to complex [80]. However, critical distance R0 of each

complex is lower than that of respective, r which indicates that the static quenching is more

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likely to be responsible for fluorescence quenching [81]. The short distance values calculated
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by this method between bound complex and tryptophan residue suggested considerable

interaction between the complex and HSA.

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4.4. Circular Dichromism spectral analysis

Circular dichroism is a spectroscopic technique which is extensively used to study the

P
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interaction affinity and binding mode of complexes with biomolecules [82]. The effects of

complexes on the conformation of HSA are displayed in Fig. 11. The CD spectrum of HSA in
AC

the absence of complex has two negative peaks in far UV region centered at 208 nm and 222

nm characteristic of a protein rich in α-helices due to n→π* and π→π* transfer, respectively

[75]. The binding of complex with HSA leads to increase both negative lamina peak i.e., 208

nm and 222 nm. This shows that helicity of HSA increased on addition of complex within the

range of 67.47 - 69.20%. The maximum increase in helicity was noticed for complex (1) with

an order of intensity 1>2>3>4. Hence, CD spectra clearly revealed that the interaction of

complex with HSA tempted modification in secondary structure which is substantiated with

the results of other biophysical probes and thus subsequently support their binding propensity

with HSA.

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4.5. Antioxidant studies

It is reported that many reactive oxygen species (ROS) like superoxide anion, hydroxyl anion

and hydrogen peroxide formed during the biochemical process responsible for detrimental

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effects on the body system [43]. It is reported that ortho- hydroxyl Schiff base derivatives

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exhibit great effects on scavenging the production of free radical and thus lead to potentially

R
effectual drugs [15]. The antioxidant results of the compounds using DPPH and SOD

SC
mimetic at different concenterations are shown in Table 4; Fig. 12 and 13.

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4.5.1. DPPH radical scavenging study

The comparative inhibitory effects of compounds were assessed in the standard reaction
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condition (Fig. 12). The data revealed that the complexes exhibited a significant scavenging

effect in comparison to their respective ligands with relative order as 1> 2 > 3> 4> L1> L2.
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4.5.2. SOD mimetic

P

Several studies reported that transition metal complexes with N, O donor bidentate ligands
CE

show their enhanced reactivity toward dioxygen [83]. The percent inhibitory effect of
AC

compounds based upon superoxide radial (O2-•) was examined and compared with standard

(Fig. 13). The results showed that complexes (1-4) showed better inhibitory effect as

compared to their ligand scaffolds because of the synergistic combination of metal ion and

ligand. However, among all the complexes, Cu complexes (1 and 3) elicited exemplanary

results in comparison to the standard similar to that reported earlier [84].

The overall scavenging results processed by DPPH and SOD mimetics experiments revealed

that chelation effect demonstrates metal complexes (1-4) as efficient antioxidants in

comparison to their respective free ligands scaffolds [85]. This may be explained in terms of

coordination environment due to their structural features as they possess coordination sites

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like azomethine nitrogen (C=N) and hydroxyl group (OH). These coordination sites can

participate in antioxidant process via donation of an electron or hydrogen [86]. However,

antioxidant data revealed that L1 based complexes have shown better scavenging activity

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whereas complexes of L2 exhibited moderate activity in comparison to their standards. It is

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given in the literature that the redox properties of metal ions in the complexes have great

R
influence on the scavenging effect as it depends on number of factors like size of chelate ring,

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axial ligation and extent of unsaturation in the whole system of chelate ring [87].

4.6. Antibacterial study NU

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The advancement of new bacterial resistant strains to current antibiotics becomes a grave

concern in health related issues instigating the exploration of new chemical scaffolds as
D

potential bactericides. The antibacterial data evidently showed that all the compounds elicited
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different degree of inhibition (Supplementary Information, Table S2 and Fig. S5). Moreover,
P

overall assessment of antibacterial results showed that the complexes (1-4) displayed
CE

momentous activity as compared to their free ligand scaffolds.

It is well accounted that complexes based on Schiff bases act as bio-relevant chemical
AC

scaffolds against pathogens because of the existence of imine functional group which imports

in elucidating the mechanism of transamination and resamination reaction in biological

systems. The enhanced antibacterial activity of the complexes in comparison to their ligands

under identical experimental conditions may be explained by Tweedy's chelation theory [88].

The chelation effect significantly reduced the metal ion polarity because of partial sharing of

its positive charge with donor groups and also due to the electron delocalization over the

whole chelate ring system which in turn enhances the lipophilic character of the central metal

atom that subsequently favours its permeation through the lipid layer of the cell membrane.

The variation in the activity of compounds against different bacteria depends either on the

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impermeability of the cells of the microorganisms or on the differences in the ribosome of

microbial cells. Moreover, mode of action of the complexes may also engage in the

formulation of a hydrogen bond via nitrogen atom of azomethine group specifically with the

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active centers of the cellular constituents intruding with the normal cell process. Although

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chelation effect itself play a dominant role in deciding the antibacterial effect of the

R
complexes but concurrently other factors like size, dipole moment, solubility, sites of

SC
coordination, redox properties of metal ion, bond length distance between the ligand and

metal, geometry of complexes, pharmacokinetic, steric, hydrophobicity and concenteration

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also have substantial affect on the antibacterial activity [89, 90].
MA
5. Conclusion
D

The present study reports the design and synthesis of bio-efficient monometallic Schiff base
TE

complexes of the type, [Cu(L1)2] (1), [Zn(L1)2].2H2O (2), [Cu(L2)2].2H2O (3) and

[Zn(L2)2].H2O(4) complexes [L1 = (E)-3-(((3-chloro-4-

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hydroxyphenyl)imino)methyl)naphthalen-2-ol and L2 = (E)-2-chloro-4-((1-(5-chloro-2-

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hydroxyphenyl)ethylidene)amino)phenol]. The synthesized ligands and their complexes were

AC

characterised by analytical and spectral techniques while thermal, morphological and

crystalline nature was deduced by TGA/DTA, SEM and XRD techniques. In-vitro HSA

binding profiles of complexes (1–4) were assessed by absorption, fluorescence, FRET and

CD measurements. The biophysical experimental results of the complexes with HSA follow

the order 1 > 2 > 3 > 4, warranting them as avid binder with HSA. In addition, scavenging

and antibacterial studies demonstrated that metal complexes show considerably superior

scavenging and antibacterial activities in comparison to their free ligands due to the chelation

effect. These studies suggested a significant synergistic interaction between bio-potent ligand

framework and metal ion in evaluating their interaction with HSA at the molecular level. The

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aforesaid studies endorse these Schiff base compounds for in vivo exploration which may

enlighten new perspectives into the experimental pharmacological assays on the basis of

conventional molecular hybridization approach.

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Acknowledgements

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Authors are indepted to Punjab University for XRD, ESI-Mass, NMR spectra, IIT Bombay

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for providing CHN analysis. We also express our gratitude to Department of Chemistry and

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USIF, A.M.U. Aligarh for providing EPR, TGA/DTA and SEM analysis. The author,

Summaiya Hanif is thankful to University Grants Commission (UGC) for financial

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assistance. The financial grant from DRS-II, FIST and PURSE to the Department of
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Chemistry is also appreciated and acknowledged.
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Figures Caption

Fig. 1. 1H NMR spectra of Schiff base ligands, L1 (a) and L2 (b).

Fig. 2. 1H NMR spectra of Zn(II), 2 (a) and Zn(II), 4 (b) complexes.

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Fig. 3. 13CNMR spectra of Schiff base ligands, L1 (a) and L2 (b).

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Fig. 4. 13CNMR spectra of Zn(II), 2 (a) and Zn(II), 4 (b) complexes.

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Fig. 5. XRD patterns of Schiff base metal complexes (1-4).

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Fig. 6. UV absorption spectra of the HSA–complexes (1-4) conjugate system obtained at

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constant concenteration of HSA (5 µM) in phosphate buffer (20 Mm) at pH 7.4 with variable

concenteration of complexes (0-100 µM). The change in intensity upon increasing

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concentration of the complexes is shown by the arrows.

Fig. 7. The fluorescence quenching spectra of HSA (5 µM) with variable concentrations of
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complexes (1-4) at the excitation wavelength (280 nm) in phosphate buffer (20 mM) at pH
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7.4. The fluorescence quenching upon increasing concentration of complexes (1–4) are
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shown by the arrows.

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Fig. 8. The plots (Stern–Volmer) displaying HSA tryptophan quenching by complexes (1-4)

at room temperature (pH 7.40, λex = 280 nm, λem = 340 nm).
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Fig. 9. The fluorescence titration of complexes (1–4) to HSA are shown by the plots of Log

[(F0 - F)/F] vs. Log [Q] at room temperature.

Fig. 10. Spectral overlap between the absorption of (1–4) complexes (3 µM) and the

normalized emission of HSA (3 µM).

Fig. 11. CD spectra of different HSA-Schiff base complexes (1-4) at molar ratios of 1:0, 1:5,

1:10 and 1: 15 at room temperature.

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Fig. 12. Concenteration dependent curves in scavenging the DPPH radical by the Schiff base

ligands, L1 and L2 and their Cu(II),1; Zn(II), 2; Cu(II), 3 and Zn(II), 4 complexes using

Ascorbic acid as Standard.

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Fig. 13. Superoxide (O2-•) scavenging effect of Schiff base ligands, L1 and L2 and their

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Cu(II),1; Zn(II), 2; Cu(II), 3 and Zn(II), 4 complexes using BHA as standard.

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Schemes caption

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Scheme 1: Schematic representation of Schiff base ligand, L1 and its [Cu(L1)2] and

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[Zn(L1)2].2H2O complexes.
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Scheme 2: Schematic representation of Schiff base ligand, L2 and its [Cu(L2)2].2H2O and

[Zn(L2)2].H2O complexes.
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Tables Caption
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Table 1. Magnetic moment and Electronic absorption data of Schiff base ligand, L1 and
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L2 and their [Cu(L1)2] and [Cu(L2)2].2H2O complexes with their band assignments.
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Table 2. XRD parameters of Cu(II), 1; Zn(II), 2; Cu(II), 3 and Zn(II), 4 complexes.

Table 3. Fluorescence binding parameter of HSA with different Schiff base complexes (1-4).

Table 4. The radical scavenging activity of Schiff base ligands (L1 and L2) and their metal

complexes (1-4).

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Supplementary Figure and Table captions

Fig. S1. Mass spectra of Schiff base ligands L1 (a) and L2 (b) and their Zn(L1)2].2H2O (c) and

[Zn(L2)2].H2O (d) complexes.

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Fig. S2. EPR spectra of [Cu(L1)2] (a) and [Cu(L2)2].2H2O] (b) complexes at room

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temperature.

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Fig. S3. TGA (a) and DTA (b) curves of metal complexes (1-4).

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Fig. S4. SEM images of Schiff base ligands, L1 and L2 and their complexes (1-4).

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Fig. S5. Zone of inhibition (in mm) of ligands L1 and L2 and their complexes examined

against S. Aureus, L. Monocytogenes, E. coli and S. typhimurium.

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Table S1. Different parameters of energy transfer between complex and HSA.

Table S2.The zone of inhibition (mm) of ligands, L1 and L2 and their metal complexes (1–4)
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screened against gram negative and gram positive bacteria.

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Table 1

Magnetic moment and Electronic absorption data of Schiff base ligand, L1 and L2 and their

[Cu(L1)2] and [Cu(L2)2].2H2O complexes with their band assignments.

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Compounds Magnetic Band Band Assignments Geometry

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moment positions

(nm)

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L1 - 268 π - π* -

333 n - π*
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L2 - 270 π - π* -

n - π*
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330
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[Cu(L1)2] 1.83 274 π - π* Square planar

344 n - π*
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2
615 B1g→2A1g
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[Cu(L2)2].2H2O 1.87 278 π - π* Square planar

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348 n - π*
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610 B1g→2A1g

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Table 2

XRD parameters of Cu(II), 1; Zn(II), 2; Cu(II), 3 and Zn(II), 4 complexes.

Parameter Complex 1 Complex 2 Complex 3 Complex 4

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Formula [CuC34H22N2O4Cl2] [ZnC34H26N2O6Cl2] [CuC28H24N2O6Cl4] [ZnC28H22N2O5Cl4]

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Temperature 298 298 298 298

(K)

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Wavelength 1.540598 1.540598 1.540598 1.540598

Radiation Cu-Kα Cu-Kα Cu-Kα Cu-Kα

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Crystal System Cubic Hexagonal Cubic Hexagonal

Space group Fm3m P63/mmc Fm3m P63/mmc

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Lattice Face-centered (F) Primitive (P) Face-centered (F) Primitive (P)

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Unit Cell Dimensions

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a(A◦) 3.607 2.670 3.597 2.659

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b(A◦) 3.607 2.670 3.597 2.659

c(A◦) 3.607 4.966 3.597 4.935

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α◦ 90 90 90 90

β◦ 90 90 90 90

δ◦ 90 120 90 120

2Ɵ 20-80◦C 20-80◦C 10-80◦C 10-80◦C

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Table 3

Fluorescence binding parameter of HSA with different Schiff base complexes (1-4).

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Complexes Ksv (M-1) kq (M-1 S-1) Kb (M-1) n

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Complex 1 1.43 x 104 2.50 x 1012 9.86 x 104 1.19
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Complex 2 9.92 x 103 1.73 x 1012 1.03 x 104 1.01

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Complex 3 9.74 x 103 1.70 x 1012 3.23 x 103 0.89

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Complex 4 3.17 x 103 5.55 x 1011 1.58 x 103 0.93

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Table 4

The radical scavenging activity of Schiff base ligands (L1 and L2) and their metal complexes

(1-4).

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IC50 (µg/mL)

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Compounds

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DPPH Assay SOD Mimetics

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Control 83.75 ± 1.83 76.91 ± 3.12
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Complex 1 93.08 ± 2.17 96.02 ± 1.87

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Complex 2 131.8 ± 1.29 110.6 ± 2.43

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Complex 3 240.2 ± 1.31 142.0 ± 4.11

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Complex 4 215.6 ± 2.87 185.8 ± 3.09

L1 491.8 ± 4.65 403.6 ± 7.54

L2 635.1 ± 12.64 366.2 ± 3.66

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Fig. 1(a)
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Fig. 1. 1H NMR spectra of Schiff base ligands, L1 (a) and L2 (b).

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Fig. 2(a)
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Fig. 2. 1H NMR spectra of Zn(II), 2 (a) and Zn(II), 4 (b) complexes.

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Fig. 3(a)
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(b)

Fig. 3. 13CNMR spectra of Schiff base ligands, L1 (a) and L2 (b).

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Fig. 4(a)
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(b)

Fig. 4. 13CNMR spectra of Zn(II), 2 (a) and Zn(II), 4 (b) complexes.

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Fig. 5. XRD patterns of Schiff base metal complexes (1-4).
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Fig. 6. UV absorption spectra of the HSA–complexes (1-4) conjugate system obtained at

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constant concenteration of HSA (5 µM) in phosphate buffer (20 Mm) at pH 7.4 with variable
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concenteration of complexes (0-100 µM). The change in intensity upon increasing

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concentration of the complexes is shown by the arrows.

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Fig. 7. The fluorescence quenching spectra of HSA (5 µM) with variable concentrations of

complexes (1-4) at the excitation wavelength (280 nm) in phosphate buffer (20 mM) at pH
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7.4. The fluorescence quenching upon increasing concentration of complexes (1–4) are
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shown by the arrows.

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Fig. 8. The plots (Stern–Volmer) displaying HSA tryptophan quenching by complexes (1-4)
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at room temperature (pH 7.40, λex = 280 nm, λem = 340 nm).
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Fig. 9. The fluorescence titration of complexes (1–4) to HSA are shown by the plots of Log
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[(F0 - F)/F] vs. Log [Q] at room temperature.

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Fig. 10. Spectral overlap between the absorption of (1–4) complexes (3

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µM) and the normalized emission of HSA (3 µM).

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Fig. 11. CD spectra of different HSA-Schiff base complexes (1-4) at molar

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ratios of 1:0, 1:5, 1:10 and 1: 15 at room temperature.

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Fig. 12. Concenteration dependent curves in scavenging the DPPH radical by the Schiff base

ligands, L1 and L2 and their Cu(II),1; Zn(II), 2; Cu(II), 3 and Zn(II), 4 complexes using

Ascorbic acid as Standard.

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Fig. 13. Superoxide (O2-•) scavenging effect of Schiff base ligands, L1 and L2 and their

Cu(II),1; Zn(II), 2; Cu(II), 3 and Zn(II), 4 complexes using BHA as standard.

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Scheme 1: Schematic representation of Schiff base ligand, L1

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and its [Cu(L1)2] and [Zn(L1)2].2H2O complexes.

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Scheme 2: Schematic representation of Schiff base ligand, L2 and

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its [Cu(L2)2].2H2O and [Zn(L2)2].H2O complexes.

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Graphical Abstract

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Highlights
 Synthesis of CuII/ZnII complexes based on N, O donor bidentate imine scaffolds.

 Validated by analytical, spectroscopic and X- ray diffraction studies.

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 HSA binding profiles of metal complexes using biophysical studies.

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 In- vitro antioxidant and antibacterial activities.

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