A comparative analyses of bioactive Cu(II) complexes using Hirshfeld surface and density functional theory (DFT) methods: DNA binding studies, cleavage and antibiofilm activities

Article abstract of DOI:10.1016/j.ica.2016.08.011

Two mononuclear Copper(II) complexes, [Cu(dip)₂] (1) and [Cu(dimp)₂] (2) have been synthesized and characterized thoroughly by various spectroscopic techniques and single crystal X-ray diffraction analysis. Dispersion corrected B3LYP/DFT calculations have been carried out to elucidate the tentative assignments of N-Cu-N/O-Cu-O/O-Cu-N and other significant vibrational modes. Additionally, quantitative analysis of intra and intermolecular non-covalent interactions were carried out using Hirshfeld surface calculations to explore H-bonding, C–H/π and Cu…H–C interactions. In vitro DNA binding studies of 1 and 2 were performed by employing optical spectroscopic techniques. The cleavage activity of 1 and 2 with pBR322 plasmid DNA was carried out by gel electrophoretic assay and it was observed that both 1 and 2 cleaved DNA by oxidative pathway via ROS species viz., O₂^?,¹O₂etc. Furthermore, antibiofilm activity of the complexes 1 and 2 was evaluated against E. Coli 25922 and it was found to interfere and inhibit the biofilm forming ability of E. Coli 25922.

Full text of DOI:10.1016/j.ica.2016.08.011

Accepted Manuscript
A comparative analyses of bioactive Cu(II) complexes using Hirshfeld surface
and Density Functional Theory (DFT) methods: DNA binding studies, Cleavage
and Antibiofilm activities
Mohammad Usman, Farukh Arjmand, Musheer Ahmad, Mohammad Shavez
Khan, Iqbal Ahmad, Sartaj Tabassum
PII:
S0020-1693(16)30463-7
http://dx.doi.org/10.1016/j.ica.2016.08.011
ICA 17205
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 June 2016
5 August 2016
6 August 2016
Please cite this article as: M. Usman, F. Arjmand, M. Ahmad, M.S. Khan, I. Ahmad, S. Tabassum, A comparative
analyses of bioactive Cu(II) complexes using Hirshfeld surface and Density Functional Theory (DFT) methods:
DNA binding studies, Cleavage and Antibiofilm activities, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/
10.1016/j.ica.2016.08.011
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A comparative analyses of bioactive Cu(II) complexes using Hirshfeld surface
and Density Functional Theory (DFT) methods: DNA binding studies,
Cleavage and Antibiofilm activities
Mohammad Usmana, Farukh Arjmanda, Musheer Ahmadb, Mohammad Shavez Khanc, Iqbal
Ahmadc and Sartaj Tabassuma*
aDepartment of Chemistry, Aligarh Muslim University, Aligarh, 202002, India.
bDepartment of Applied Chemistry, ZHCET, Aligarh Muslim University, Aligarh, 202002, India.
cDepartment of Agricultural Microbiology, Aligarh Muslim University, Aligarh, 202002, India.
*
E–mail address: tsartaj62@yahoo.com +91 9358255791, Corresponding author: Aligarh
Muslim University, Aligarh, UP–202002, India.
Keywords: Single crystal X-ray structure; DFT; Hirshfeld surface analyses; In vitro DNA
binding; pBR322 DNA cleavage; Antibiofilm activity.
Abbreviations
dip
[{(2,6-diisopropylphenyl)imino}methyl]phenol
[{(2,6-diisopropylphenyl)imino}methyl]-6-methoxyphenol
Calf thymus DNA
dimp
CT DNA
EB
Ethidium bromide
QM
Quantum mechanical
Abstract
Two mononuclear Copper(II) complexes, [Cu(dip)2] (1) and [Cu(dimp)2] (2) have been
synthesized and characterized thoroughly by various spectroscopic techniques and single crystal
X-ray diffraction analysis. Dispersion corrected B3LYP/DFT calculations have been carried out
to elucidate the tentative assignments of N-Cu-N/O-Cu-O/O-Cu-N and other significant
vibrational modes. Additionally, quantitative analysis of intra and intermolecular non-covalent
interactions were carried out using Hirshfeld surface calculations to explore H-bonding, C-H/π
and Cu…H-C interactions. In vitro DNA binding studies of 1 and 2 were performed by
employing optical spectroscopic techniques. The cleavage activity of 1 and 2 with pBR322
plasmid DNA was carried out by gel electrophoretic assay and it was observed that both 1 and 2

·-
cleaved DNA by oxidative pathway via ROS species viz., O2 , 1O2 etc. Furthermore, antibiofilm
activity of the complexes 1 and 2 was evaluated against E. Coli 25922 and it was found to
interfere and inhibit the biofilm forming ability of E. Coli 25922.
1. Introduction
Biofilm formation by pathogenic bacteria is considered a major step for establishing successful
colonization on implanted medical devices, stents, catheter and endocarditis or lung infections in
hospitals [1]. These biofilms being tolerant to both antibiotics and immune system are therefore
difficult to treat and result in severe pathological conditions for host [2]. There is therefore, a
huge demand of efficacious therapeutic molecules for combating bacterial biofilms, and new
innovative strategies are required for the development of novel, potent as well as less toxic
antibacterial agents [3].
In recent past, much attention has been focused for the development of DNA targeted
antimicrobials [4] as previous literature reveals that DNA is an important primary cellular target
for most of the therapeutic drugs in clinical use [5]. Considering the role of DNA (both
extracellular and intracellular) in the development and maturation of biofilms [6], DNA targeting
molecules can be considered as potential antibiofilm agents. Therapeutic agents exert their
cytotoxic and antibacterial effect by binding to DNA, thereby cleaving the DNA and inhibiting
the growth of the cells, preventing further aggregation or uncontrolled division of cells leading to
colonization [7].
Currently, tremendous efforts are directed for the design and synthesis of metal-based
therapeutics and much focus is on the development of DNA- targeted drug candidates involving
the interactions with specific domain of DNA helix preferably by non-covalent mode (viz.,
intercalation, groove binding and surface interaction with negatively charged oxygen atoms of

the phosphate linkage) [8]. Among the metal ions utilized in drug design, copper being an
endogenous bio-essential element plays a pivotal role in the functioning of several
metalloenzymes and proteins [9]. Due to its high redox activity, copper has potential to catalyze
the generation of reactive oxygen species (ROS), interrupt the redox equilibrium of cell and also
intervene in the redox mediated cellular signaling pathways [10]. It is speculated that ROS are
capable of inducing DNA strand scission and altering various cellular component like proteins,
lipids and nucleic acids resulting in oxidative or hydrolytic damage of cells [11]. Moreover, the
design of ligand framework has a significant impact on tuning and modulating the specificity of
tailored drug candidate towards the biological target. Therefore, the coordination between copper
ion and pharmacophore scaffold provides a high potency, specificity and efficacy to the drug
entity for their biological target [12]. Among the several organic ligands, our interest stems in
Schiff bases derived from o-vanillin and salicylaldehyde, as their metal complexes are known to
exhibit diverse biological applications viz., antitumor, anti-inflammatory, antiviral, antibacterial
and cell imaging agents [13].
Molecular self-assembly through weak non-covalent forces is the hallmark of biological systems.
Amongst a number of non-covalent forces viz., H-bonding, C…H, C-H/π, π…π, cation/anion…π,
lone-pair…π, metal…H interactions etc. have been utilized in directing molecular assembly for the
synchronization of several biochemical processes [14]. These non-covalent interactions have
been demonstrated to play a key role in physicochemical properties, molecular recognition, drug
receptor interaction, antigen-antibody recognition, enzymatic inhibition and protein folding [15].
Herein, we present the fully characterized Cu(II) complexes 1 and 2, derived from Schiff base
ligands 2-(((2,6-diisopropylphenyl)imino)methyl)-6-methylphenol,
dip,
and
2-[((2,6-
diisopropylphenyl)imino)methyl)-6-methoxyphenol, dimp, structure elucidation of complexes

was done by single crystal X-ray diffraction analysis. Density Functional Theory (DFT)
methods were employed to calculate electronic structure, HOMO-LUMO gap, atomic charges
and vibrational frequencies of metal-ligand linkages. The antibiofilm activity against E. Coli
25922, DNA binding profile and DNA cleavage experiment with pBR322 DNA was carried out
for both the complexes 1 and 2.
2. Experimental
2.1. Materials
All reagents were commercially available and used as supplied without further purification.
Copper nitrate trihydrate (Merck), o-vanillin (Sigma-Aldrich), 2, 6-Diisopropyl-aniline (Merck)
and Calf thymus DNA (CT DNA) (Sigma–Aldrich), supercoiled plasmid DNA pBR322 (Genei)
were utilized as received.
2.2. Methods and instrumentation
Microanalyses for the copper complexes were performed using a CE–440 elemental analyzer
(Exeter Analytical Inc.). Infrared spectra were obtained (KBr disk, 400–4000 cm–1) on a Perkin–
Elmer Model 1320 spectrometer. EPR spectra of copper complexes was recorded at X-band
(9.167 GHz) at room temperature on JEOL FA-200 continuous-wave spectrophotometer using
Mn as standard (g=2.0036). Electronic spectra was recorded on PerkinElmer UV–vis
spectrophotometer. Fluorescence measurements were carried out on a RF–5301 PC
spectrofluorophotometer (Schimadzu). DNA cleavage experiments were performed with the help
of Axygen made electrophoresis supported by a Genie power supply with a potential range of
50–500 volts, visualized and photographed by a Vilber–INFINITY gel documentation system.
2.3. Synthesis of [Cu(dip)2] (1)

A methanolic solution of salicylaldehyde (10 ml, 4 mmol) was mixed with methanolic solution
of 2,6-diisopropylaniline (5 ml, 4 mmol). The mixture was stirred for 2h at 70 0C to give a clear
deep yellow solution. To the above reaction mixture, methanolic solution of Cu(NO3)2.3H2O (5
ml) was added which was refluxed for 4 h, until dark brown solution resulted. The completion of
reaction was monitored by TLC. After completion of the reaction, the mixture was reduced in
volume by heating on hot plate and filtered under hot condition. On slow evaporation of filtrate,
deep brown colored crystals were obtained after 6-7 days. The crystals obtained washed with
acetone and air dried. Yield 78%, M.P 180 0C. Anal. Calc. for [C40H48CuN2O4] (%): C, 70.20; H,
7.02; N, 4.09; Found: C, 70.26; H, 7.09; N, 4.04. FT-IR (KBr pellet) 1605 cm-1 (s, C=N); 2870
cm-1 (s, CH3); 3057 cm-1 (s, Ar-CH). UV-vis (1×10-4 M, MeOH, λmax nm): 242, 272, 368, 590
nm. Molar conductance: ʌM (1 × 10-3 M, DMSO): 1.53 Ω-1 cm2 mol-1 (non-electrolyte).
2.4. Synthesis of [Cu(dimp)2] (2)
Yield 82%, M.P 163 0C. Anal. Calc. for [C38H44CuN2O2] (%): C, 73.11; H, 7.10; N, 4.49; Found:
C, 73.19; H, 7.21; N, 4.61. FT-IR (KBr pellet) 1608 cm-1 (s, C=N); 2869 cm-1 (s, CH3); 3061 cm-
1 (s, Ar-CH). UV-vis (1×10-4 M, MeOH, λmax nm): 238, 284, 378, 595 nm. Molar conductance:
ʌM (1 × 10-3 M, DMSO): 1.42 Ω-1 cm2 mol-1 (non-electrolyte).
2.5. Description of X–ray Crystal structure
Single crystal X–ray data of complex 1 and 2 was collected at 100 K on a Bruker SMART APEX
CCD diffractometer using graphite monochromatic MoKα radiation (λ = 0.71073 Å). The linear
absorption coefficients, scattering factors for the atoms and the anomalous dispersion corrections
were referred from the International Tables for X–ray Crystallography. The data integration and
reduction were worked out with SAINT software. Empirical absorption correction was applied to

the collected reflections with SADABS program [16] and the space group was determined using
XPREP. The structure was solved by direct methods using SHELXTL–97 and refined on F2 by
full–matrix least–squares using the SHELXTL–97 programme package [17]. Only a few H atoms
could be located in the difference Fourier maps in the structure. The rest were placed at
calculated positions using idealized geometries (riding model) and assigned fixed isotropic
displacement parameters. All non–H atoms were refined anisotropically. Several DFIX
commands were used for fixing some bond distances in complex 1 and 2. The crystal and
refinement data are collected in Table 1. Selective bond distances and angles are given in Table
S1 and S2.
Table 1. Crystal and Structure Refinement Data for complexes 1 and 2.
Parameters
CCDC No.
Formula
1
2
971306
C38H44CuN2O2
624.30
Triclinic
P–1
7.910(5)
10.199(5)
11.138(5)
113.824(5)
97.275(5)
97.646(5)
798.4(7)
1
1006580
C40H48CuN2O4
684.34
Triclinic
P–1
10.899(5)
15.981(5)
22.961(5)
97.395(5)
103.101(5)
108.274(5)
3611(2)
4
Fw (g mol–1)
Crystal System
Space Group
a (Å)
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
U (Å3)
Z
ρcalc (g/cm3)
µ (mm–1)
1.298
0.720
1.259
0.647
F(000)
331
1452
Crystal size (mm)
Temp (K)
Measured reflns
Unique reflns
θ Range (deg)/completeness (%)
GOFa
0.26 × 0.19 × 0.16
100
4256
0.25 × 0.18 × 0.15
100
18205
2577
8646
2.04 to 25.50/0.972 0.93 to 25.00/ 0.977
1.154
R1 = 0.0529
1.132
R1 = 0.0805
Final Rb indices

wR2 = 0.1335
R1 = 0.0678
wR2 = 0.2626
R1 = 0.1220
wR2 = 0.3013
[I>2σ(I)]
Rb indices
(all data)
wR2 = 0.1907
2
aGoF is defined as {Σ[w(F0 –Fc2)]/(n–P)}1/2 where n is the number of data and p is the number of
2
2 2
parameters. bR = {Σ||F0|–|Fc||/Σ|F0|, wR2 = {Σ w(F0 –Fc2)2 / Σ w(F0 ) }1/2 .
2.6. Theoretical Calculations
The density functional theory (DFT) calculations were done with the ORCA 3.0.1 computational
package [18]. The geometry optimization was carried out by hybrid B3LYP functional [19] using
the Aldrich’s def2–TZVP basis set for copper atom and def2–SVP basis set for C, H, O, N atoms
[20]. The optimized structure was further re-calculated using def2–TZVP basis set for all atoms
to calculate the HOMO and LUMO energies. The initial geometry was taken from the single-
crystal X-ray data and subjected to optimization. To accelerate the calculations we utilized the
resolution of identity (RI) approximation with the decontracted auxiliary def2-SVP/J and def2–
TZV/J Coulomb fitting basis sets and the chain–of–spheres (RIJCOSX) approximation to exact
exchange as implemented in ORCA [21]. Furthermore, analytical vibrational frequency
calculations were performed on the optimized geometries of complexes 1 and 2 using same basis
set parameters. The scaling factor 0.9572 for Cu(II) complexes have been applied on the
wavenumber regions from 500-4000 cm-1 for a better correlation with experimental spectra.
Hirshfeld surface were mapped using Crystal Explorer [22] software using crystal structure
coordinates of CIF files.
The molecular docking studies have been performed by using AutodockVina version 1.1.2 [23].
All rotatable bonds within the ligand were allowed to rotate freely and receptor was considered
rigid. The crystal structure of the B–DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA)

was retrieved from the protein data bank (http://www.rcsb.org./pdb). Visualization of minimum
energy favorable docked poses has been performed using Discovery studio 4.1 and PyMol [24].
2.7. DNA Binding and cleavage Experiments
DNA binding experiments include absorption spectral traces, emission spectroscopy and circular
dichroism conformed to the standard methods and practices previously adopted by our laboratory
[25] whereas DNA cleavage was performed by the standard protocol as described previously [26].
While measuring the absorption spectra an equal amount of DNA was added to both the
compound solutions and the reference solution to eliminate the absorbance of the CT–DNA itself,
and Tris–HCl buffer was subtracted through base line correction. All the experiments involving
interaction of the complexes with CT–DNA were performed in double distilled buffer containing
tris(hydroxymethyl)aminomethane and adjusted to pH 7.3 with hydrochloric acid. Solution of CT–
DNA in buffer gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.9:1 indicating that DNA
was sufficiently free of protein. The DNA concentration per nucleotide was determined by
absorption spectroscopy with the molar absorption coefficient 6600 M–1cm–1 at 260 nm.
2.8. Biofilm inhibition Assay
Minimum inhibitory concentration (MIC) of compound was determined against standard strain
of E. Coli and Sub-MICs were selected for the assessment of anti-biofilm activity. Antibiofilm
potential of Sub MICs of the complex 1 was measured using the polyvinyl chloride biofilm
formation assay [27] Briefly, overnight (treated and untreated) culture of E. coli 25922 were
visualized for biofilm formation by staining with 0.1% crystal violet solution. The 96-well
microtiter plates were rinsed to remove planktonic cells, and the surface-attached cells were then
quantified by solubilizing the dye in ethanol and measuring the absorbance at OD 470. Further

biofilm formed on glass coverslip were visualized under light microscope and under scanning
electron microscope using briefly modified method as described earlier [28].
3. Result and discussion
The in situ condensation reaction of the o-vanillin/salicylaldehyde and 2,6-diisopropylaniline in
1:1 molar ratio resulted in the deep yellow colored ligand (dip and dimp) and subsequently its
mononuclear copper complexes 1 and 2 were synthesized with Cu(NO3)2.3H2O. Both the
complexes were characterized by elemental analysis, UV-vis, EPR and FTIR and single crystal
X-ray studies. Complexes are air-stable and miscible in MeOH, DMF, MeCN and DMSO.
Complexes 1 and 2 were predesigned to validate their recognition with specific domain of DNA
helix and for bacterial bioflim inhibition activity.
3.1. Structure description
Crystal structures of complexes 1 and 2 were solved by single crystal X-ray diffraction analysis
at 100 K. Note that the crystal framework of 1 and 2 have been already reported in the literature
[29] but the Space group and crystal lattice parameters of our crystal systems are totally different
due to some experimental conditions specially temperature and measured parameters.
Single crystal X-ray study showed that the complex 1 crystallized in the triclinic crystal system.
Fig. 1a depicts the ORTEP structure of complex 1 and Tables S1 show the selected bond lengths
and angles. The structure was successfully solved and converged in the space group P–1. The
asymmetric unit consists of half Cu(II) ion (sit on the special position having two-fold axis of
symmetry) and one Schiff base ligands dip. The complex 1 showed square planar CuO2N2
coordination geometry with ligation from two nitrogen atoms (Cu–N = 2.015(3) Å) and two
oxygen atoms (Cu–O = 1.876(3) Å) from ligands dip. All the Cu–O and Cu–N bond distances

are within the range reported for square planar Cu(II) complexes [30]. 2D supramolecular
architecture is depicted in Fig. S1.
Complex 2 also crystallized in the triclinic crystal system with space group P–1. However, the
asymmetric unit consists of two molecules and each molecule contains one Cu(II) ion and two
Schiff base ligands dimp. Fig. 1b depicts the ORTEP structure of complex 1 and Tables S2 show
the selected bond lengths and angles. The complex 2 exhibited distorted square planar CuO2N2
coordination geometry with ligation from two nitrogen atoms (Cu–N = 1.970(6) – 1.991(5) Å)
and two oxygen atoms (Cu–O = 1.875(4) – 1.875(4) Å) from two different ligands moieties. All
the Cu–O and Cu–N bond distances are within the range reported for distorted square planar
Cu(II) complexes [31]. Contrary to 1, it shows some deviation from orthogonality which could
be attributed to the presence of steric crowding at both phenyl rings of the Schiff base ligand.
The steric hindrance caused by isopropyl groups is many folds stronger than that caused by
methoxy moieties, therefore the orthogonality could not be achieved. 2D supramolecular
structure (Fig. S2) was generated which was stabilized by multipoint weak H–bonding and
strong -CH– π aromatic stacking arrangement between two neighboring phenyl moieties as
depicted in Fig. S3.

Fig. 1. X−ray molecular structure of the (a) complex 1 and (b) complex 2, along with labeling of
atoms. Thermal ellipsoids were shown at 50% probability. Hydrogen atoms have been omitted
for clarity.
3.2. Spectral characterization
The structure of complexes 1 and 2 was also validated by other spectroscopic techniques viz
EPR, UV-vis and FT-IR spectroscopy.
EPR studies of polycrystalline samples of complexes 1 and 2 recorded at room temperature (Fig.
S4) exhibited g|| and g⊥ values of 2.12-2.09 and 2.06-2.06, respectivily, while gav values was
2
2
determined to be 2.08-2.07 from the relation gav = 1/3(g||2+2g⊥ ). These calculated data are in
agreement with an orbitally non-degenerate state for four-coordinate Cu(II) complexes . The
value of g|| > g⊥ > 2.0023 revealed that the unpaired electron was most likely to be localized in
2
the dx2 - ground state, and a square based geometry proposed for the complexes [32]. The
y
experimental g|| values for complexes were less than 2.3, in agreement with the covalent
character of the M-L bond [33].
The ESI mass spectrum of complexes 1 revealed a characteristic molecular ion peak at m/z 624.8
attributed to the [C35H44CuN2O2 + H+] moiety. Fragmentation peaks at m/z 279.2, 180.9 were

observed for [C19H22NO + 2H+] and [C7H5CuNO + H+], respectively (Fig. S5). For complex 2,
molecular ion peak at m/z 685.2 attributed to [C40H48CuN2O4 + H+] moiety and fragmentation
peak at m/z 312.1 observed for [C20H24NO2 + 2H+] species ( Fig. S6).
The electronic spectra of complex 1 was recorded in MeOH revealed two well resolved
absorption peaks at 242 and 272 nm corresponding to π–π* intraligand transition; a broad
absorption maxima at 368 nm was attributed to LMCT transition (Fig. S7). A very weak and
broad d-d transition envelop observed at 590 nm in the visible region was consistent with square
planar environment around the Cu(II) center [34]. In complex 2, bands were observed at 238,
284 nm (π–π*) and broad band around 378 nm (LMCT). A low intensity shoulder band was also
observed at 595 nm adjacent to d-d band, consistent with the distorted square planar geometry of
Cu(II) ion.
3.3. Density functional theory
The gas phase structure optimization of copper complexes are performed at B3LYP/DFT level
and calculated bond lengths, angles and Mulliken charges are given in Table S1, S2 and S3. The
calculated bond lengths are longer than the experimental data because the former were optimized
in the gas phase and latter were in tight crystal lattice.
3.3.2. FT-IR spectroscopy vs. vibrational mode analyses
The proper assignment of metal framework vibrations in far-IR region was challenging due to
the higher mixture of the different internal coordinates that take part in the normal modes.
Therefore, DFT calculations were undertaken to provide better insight in the involvement of
vibrational dynamics of metal-based drug entities. The calculations predict all 3N-6 vibrational
mods with real frequencies, which ranges from 35 cm-1 to about 3212 cm-1. DFT results revealed
the several types of metallic core dynamics (a) Out-of-plane vibrations and (b) In-plane

vibrations. Various type of out-of-plane vibrations predicted below 350 cm-1 and In-plane
vibrations predicted above 350 cm-1. The experimental and simulated spectra of complexes 1 and
2 are shown in Fig.S8-S15. Slight variation have been observed in simulated spectra because
experimental values contained both harmonic and anharmonic vibrational frequencies while
calculated one depicts only harmonic vibrations. The observed as well as theoretical vibrational
modes correspond to in plane bending, out of plane rocking, stretching and in-plane rocking are
depicted in Table 2 and S4. Some animated images of significant modes of vibration with
displacement vectors have also been included in supplementary material.
Table 2. Selected experimental Far-IR and DFT calculated spectra for complexes 1-2,
(wavenumber in cm-1).
Complex 1
Experimental
Complex 2
Experimental
DFT
DFT
Assignment
ϒN-Cu-N
ϒO-Cu-O
ϒO-Cu-O
ϒO-Cu-O
53.27
60.43
113.42
149.35
204.11
218.06
263.27
292.07
330.45
350.74
60.17
103.77
156.37
205.82
229.69
251.38
291.11
324.08
340.27
369.21
405.29
440.12
472.25
524.67
582.04
618.12
61.13
119.50
157.12
205.81
230.22
266.82
292.89
325.62
345.58
372.12
407.21
445.42
477.03
538.6
102.19
142.33
203.02
219.02
259.18
290.09
328.82
345.31
ϒN-Cu-N
ϒO-Cu-O, ϒN-Cu-N, τCH3
νO-Cu-O, δN-Cu-N
πO-Cu-N
ϒN-Cu-N, ϒN=CH
ϒO-C(φ)- C(φ)-O,
δO-Cu-O, δN-Cu-N
δO-Cu-N
πO-Cu-O, πN-Cu-N
ρO-Cu-N
aνO-Cu-O
sνN-Cu-N, sνO-Cu-O
402.19
433.42
464.27
524.07
589.24
617.08
405.02
437.37
470.81
533.87
595.12
620.98
590.18
626.12
sν, symmetrical stretching; aν, anti-symmetrical stretching; δ, in-plane bending; ϒ, out-of-plane bending; ρ, in-plane
rocking; π, out-of-plane rocking.

3.3.3. Frontier molecular orbital analysis
Frontier molecular orbitals (HOMO and LUMO) play an important role to exemplify the
chemical reactivity, kinetic stability and electrical transport properties of the molecule. Recently,
the HOMO-LUMO energy gap has been used to verify the bioactivity from intramolecular
charge transfer (ICT) and was also correlated with various biological activities like antibacterial,
antioxidant and DNA binding studies [35]. A small gap of HOMO-LUMO of molecule was
associated with a noteworthy degree of intramolecular charge transfer (ICT) from the electron-
donor groups to the electron-acceptor groups through a π-conjugated path. A large HOMO-
LUMO gap implies high stability of the molecule in the sense of its lower reactivity in chemical
and biochemical reactions [36]. Systems having high EHOMO are good electron donors while
those having low ELUMO are good electron acceptors. The calculated energy gap between HOMO
and LUMO of complexes 1 and 2 were found to be -3.84 and -3.61 eV (Fig. 2). Previous
literature reports have proven the fact that higher HOMO energy of DNA and lower LUMO
energy of the interacting molecule reflect much stronger binding propensity, because “electronic
charge density” facilitates more easily from the HOMO of the DNA molecule to the LUMO of
the interacting molecule and consequently, a stronger interaction between DNA and the
interacting molecules takes place.

Fig. 2. Diagrammatic representation of Frontier Molecular Orbitals for complex 1 and 2.
3.4. Hirshfeld surface analyses
The Hirshfeld surfaces of complexes 1 and 2 are illustrated in Fig. S16, depicting surfaces that
have been mapped over a dnorm range of -0.5 to 1.5 Å, shape index (-1.0 to 1.0 Å) and curvedness
(-4.0 to 0.4 Å). The parameter dnorm displayed a surface with a red-white-blue color scheme,
whereas deep red spots highlighted show shorter contacts viz., hydrogen bonding. The white
areas represent contacts around the van der Waals separation like H…H contacts and the blue
regions are devoid of such close contacts. On the Hirshfeld surface mapped with shape index
function, one can examine both red regions corresponding to C-H…π interactions as well as
‘bow-tie patterns’ which indicated the presence of aromatic stacking (π…π) interactions. The

curvedness surface indicated the electron density of surface curves around the molecular
interactions. The fingerprint plots of complexes 1 and 2 are presented in Fig. S17 and S18, can
be disintegrated to highlight particular atom-pair close contacts. This itemization allows parting
of contributions from different interaction types, which overlap in the full fingerprint. For
complex 1, the proportions of H…H, C…H and C…C interactions comprises 80%, 18.1% and
1.9% of the total Hirshfeld surface for each molecule, respectively. In the case of complex 2, the
proportions of H…H, O…H, C…H, O…C and C-H…Cu (anagostic) interactions encompasses
78.5%, 5.4%, 14.8%, 0.6% and 0.5% of the total Hirshfeld surface for each molecule,
respectively [37].
3.5. DNA Binding studies
The binding propensity of complex 1 and 2 with CT−DNA was studied using electronic
absorption spectroscopy. Upon successive additions of CT-DNA (0.0-2.0×10-4 M) to a fixed
concentration of complexes 1 and 2 (6.67×10-5 M), a concomitant increase in the absorption
intensity at the interaligand absorption bands (272 nm for 1 and 284 nm for 2) with a blue shift
of 2-6 nm and a decrease in intensity at the LMCT band (368 for 1 and 378 nm for 2) with no
shift was observed (Fig. 3).
The observed strong hyperchromic shift (53% and 64% in complexes 1 and 2, respectively) and
weak hypochromic shift (12% and 32% in complexes 1 and 2, respectively) could be attributed
to electrostatic mode of interaction of complexes 1 and 2 with partial intercalation [38]. The
observed hyperchromism is due to external contact with DNA while hypochromism could be
attributed to the base pair stacking interaction between the aromatic chromophore ligand moiety
of the complexes 1 and 2 and CT-DNA while the substituted isopropyl groups on position-2, 6 of
the Schiff base ligand could favor the hydrophobic contacts within the interior of DNA helical

sites. Further, quantification of the binding strength of complexes 1 and 2 towards the CT-DNA
were ascertained by the intrinsic binding constant, Kb values calculated using the Wolfe-Shimer
equation [39]. Kb values for complexes 1 and 2 were found to be 2.15( 0.09) × 104 and
5.63( 0.12) × 104 M-1, respectively. For explaining the greater binding affinity of the complex 2
we utilizes the results of frontier molecular orbital and Hirshfeld surface analysis. As we know
that lower LUMO energy of the complex, greater will be the binding affinity and complex 2 has
lower LUMO energy (3.61 eV) as compare to complex 1 (3.84 eV). Additionally, molecular
docking studies also confirmed the greater binding affinity of complex 2 (section 3.8).
Fig. 3. UV−vis spectra of (a) complex 1 and (b) complex 2,in Tris−HCl buffer at pH 7.2 upon
addition CT−DNA, [DNA] = 0.00−1.8 x 10−4 M, [Complexes 1-2] = 1.0 x 10−4 M.
Arrow indicates change in absorbance with increasing concentration of CT−DNA.
Fluorescence studies were carried out to get further insights into the binding event of complexes
1 and 2. No luminescence was observed for the complexes 1 and 2 at room temperature in

aqueous solution, in any organic solvent, or in the presence of CT-DNA. So, the binding of
complexes 1 and 2 with DNA cannot be directly predicted through the emission spectra.
Therefore, interaction of complex 1 and 2 with DNA were evaluated by a competitive binding
experiment using ethidium bromide (EB) as a probe. EB is a phenanthridine fluorescence dye,
which emits intense fluorescence in the presence of DNA at 595 nm (λem) due to its strong
intercalation between the adjacent base pairs [40]. In our experiments, it was observed that upon
successive addition of 1 and 2 to EB bound DNA system, emission intensity decreased
progressively (Fig. S19), indicative of competition between EB and complexes 1 and 2 towards
CT-DNA binding.
A decrease in the emission intensities was attributed to the displacement of EB from EB-DNA
system, leading to the energy/electron transfer from the guanine base of DNA to MLCT of the
complexes 1 and 2, suggesting their strong competition for the intercalative binding site [41].
Since EB was not completely displaced, partial intercalation in addition to the electrostatic mode
of interaction cannot be ruled out. Furthermore, Fluorescence quenching extent was determined
qualitatively by using the Stern-Volmer equation [42]. The Ksv value for complexes 1 and 2
were found to be 4.93( 0.07) × 104 M-1 and 5.03( 0.11) × 104 M-1, respectively.
The precondition of good DNA binding agents was fulfilled by drug entities 1 and 2 as
confirmed by UV-vis titration and florescence quenching experiments, another quite sensitive
optical spectroscopy viz., circular dichroic technique was carried out to capture the
conformational changes during drug – DNA interactions. The CD spectra of CT DNA consists of
a positive band at 280 nm due to base-pair π - π stacking and a negative band at 245 nm due to
right handed helicity which is characteristic of DNA in the right B-form [43]. An intercalative
mode of interaction enhances the intensity of both bands while no or less perturbation is

attributed for groove and electrostatic binding. Upon the addition of complexes 1 and 2, the
positive band decreased in intensity with a concomitant red shift to 280 nm while negative band
(245 nm) increased in intensity (Fig. S20), suggested that complexes 1 and 2 could unwind the
DNA helix and lead to the loss of helicity. The complex 2 registered more decrease in the CD
band intensity than 1 at the same concentration which implies that complex 2 is more effective in
perturbing the secondary structure of DNA.
3.6. DNA cleavage studies
To assess the DNA cleavage ability of complexes 1 and 2, supercoiled pBR322 DNA was
incubated with varying concentration of complexes in 5 mM Tris – HCl/50 mM NaCl buffer at
pH 7.2 for 1 h in the absence of any reducing agent. Upon successive addition of complexes 1
and 2, supercoiled form (SC Form I) of DNA was gradually converted into slower – moving
nicked circular form (NC Form II) (Lane 2 – 4 for 1 and 7-10 for 2). When the concentration of
complex 1-2 reached to 25 µM (Lane 6 for 1 and 11 for 2), the Form I was completely converted
into Form II without the formation of linear circular form (LC Form III), which migrates in
between SC and NC form (Fig. 4) revealing single strand DNA cleavage [44].
The mechanistic cleavage activity of complexes 1 and 2 was analyzed in presence of activators
viz. H2O2, ascorbate (ASC), 3-mercaptopropionic acid (MPA) and glutathione (GSH). The
cleavage activity was significantly enhanced by these activators in the order H2O2 > GSH >
MPA > ASC (Fig. S21). Furthermore, to elucidate the DNA cleavage pathway, reactions of
complexes 1 and 2 were carried out in presence of ROS scavengers viz., hydroxyl radical
scavenger (DMSO and EtOH), singlet oxygen quencher (NaN3) and super oxide scavenger
(SOD) (Fig. S22). It was observed that DMSO and EtOH did not show any apparent inhibition of
the DNA strand scission, ruling out the possibility of involvement of diffusible hydroxyl radicals

in the cleavage reaction. However, in presence of NaN3, significant inhibition was observed
which revealed that ROS species viz., singlet oxygen, 1O2 was responsible for the DNA cleavage
process via. oxidative pathway [45].
To further confirm the binding of the complexes 1 and 2 with the DNA helix, the cleavage
reactions with minor groove binder, DAPI and major groove binder, methyl green (MG) were
carried out (Fig. S21). There was no apparent inhibition of DNA damage with methyl green in
presence of complexes 1 and 2, while significant inhibition of DNA cleavage in presence of
DAPI was observed implicating the binding of the complexes to DNA in the minor groove of
DNA double helix. These results were also substantiated by molecular docking experiments.
Fig. 4. Gel electrophoresis assay of complexes 1-2 with pBR322. (a) Cleavage of pBR322 plasmid
DNA (300ng) by complex 1 in 50 mM Tris–HCl/NaCl buffer (pH, 7.3) after 40 min exposure
time at different concentration; Lane 1: DNA control; Lane 2: 5 µM 1 + DNA; Lane 3: 10 µM
1 + DNA; Lane 4: 15 µΜ 1 + DNA; Lane 5: 20 µM 1 + DNA; Lane 6: 25 µM 1 + DNA. (b)
Lane 7: 5 µM 2 + DNA; Lane 8: 10 µM 1 + DNA; Lane 9: 15 µΜ 1 + DNA; Lane 10: 20 µM
1 + DNA; Lane 11: 25 µM 1 + DNA.
3.8. Molecular docking
Since complexes 1 and 2 possess good DNA binding and significant cleaving activity, therefore
molecular docking study was employed to investigate the preferable binding site of copper
complexes to DNA helix. Moreover, molecular docking is a virtual computational method which
provides the deeper insight of binding affinity and involvement of non-covalent interactions
responsible for binding [46]. An energetically favorable docked pose obtained from the

molecular docking of complexes
1
and
2
with a DNA duplex of sequence
d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) is shown in Fig. 10. Docking results
revealed that DNA- complexes 1 and 2 were stabilized by forming non-covalent interactions
mainly electrostatic and hydrophobic. The evaluation of docked model showed that complexes 1
and 2 snugly fitted in the minor groove (Fig 10 and S23). Additionally, the flexible alkyl arms of
complexes 1 and 2 anchored the hydrophobic interior surface of DNA and derived various
hydrophobic contacts. Relative binding energy of the docked structures of 1 and 2 was found to
be -4.8 kcal/mol and -5.2 kcal/mol, respectively. Therefore, we can conclude that there is a good
agreement between the experimental DNA binding results and molecular modelling calculations.
Fig. 5. Molecular docked model of (a) complex 1 and (b) complex 2 with the minor groove of
DNA.

3.9. Bacterial anti-biofilm activity
The antibacterial activity of the complexes 1 and 2 was demonstrated by minimal inhibitory
concentration (MIC) and was found to be 175 µg-ml-1 and 100 µg-ml-1 against gram negative
bacteria E. coli 25922, respectively. MIC of the complexes was determined to select the sub-
MICs to access the biofilm inhibitory effect of complexes against the test bacteria. Significant
inhibition in biofilm formation was observed in test bacterial strain when tested in the presence
of sub-MICs (12.5-50 µg-ml-1) of complexes. At the highest tested concentration (50 µg-ml-1),
complexes 1 and 2 exhibited 47% and 65% reduction, respectively in biofilm formation (Table
S5). The activity of copper complexes against biofilm was reasonably good at their sub-
inhibitory concentrations, however, complex 2 showed comparatively better effects. Light
microscopy and SEM analysis showed the formation of well-developed biofilm by untreated
control, whereas the bacterial strain treated with the complexes 1 and 2 (50 µg-ml-1) developed
poor biofilm (Fig 6).
Earlier literature reports have shown that copper complexes exhibit antibiofilm activity against
gram positive and gram negative bacteria [47]. Mechanistic insight of the antibiofilm activity of
copper ions in the complexes suggested different mode of actions albeit, more prominently by
DNA destruction, as extracellular and intracellular genetic materials play a pivotal role during
the various stages of biofilm development and maturation [48]. It is implicated that DNA
cleavage and membrane permeability play a critical role in determining the antibiofilm effect of
different complexes [49]. Since bacterial plasmids are necessary for maintaining biofilm integrity
and conferring the gene resistance among the biofilm community via horizontal gene transfer

[50], therefore the proven cleaving activity of complexes 1 and 2 could also be responsible for
the inhibition of biofilms.
In complex 2, enhanced antibiofilm effect could be attributed to the ligand, o-vanillin- a well-
known membrane active metabolite, that affects the ion balance and normal respiratory
functioning and ultimately leads to improved permeability with additional antibacterial effects
specifically against E. coli [51]. Thus, the inhibition of biofilm forming ability of tested bacteria
by these complexes is a coordinated affair, where each moiety in its own capacity and overall
tailored metal-based drug entities induce the cumulative effect affecting the molecular machinery
of bacterium. The present study highlighted the anti-infective potential of copper therapeutic
agents based primarily on DNA damage and cleavage properties of copper chelated with
bioactive organic ligand scaffold.
Fig. 6. Microscopic images of E. coli 25922 biofilm in the presence and absence of sub-MICs
Complexes. (a) Light microscopic image of untreated control, (b) treated with 50 µg/ml of
complex 1, (c) treated with 50 µg/ml of complex 2, (d) Scanning electron microscopic image of
untreated control and (e) treated with 50 µg/ml of complex 1 and (f) treated with 50 µg/ml of
complex 2.

Conclusion
Copper-based therapeutic drug entities, [Cu(dip)2] (1) and [Cu(dimp)2] (2) have been
synthesized and characterized thoroughly by single crystal X-ray diffraction analysis and other
spectroscopic techniques. The crystal structure and Hirshfeld surface analysis revealed the
molecular assembly of complexes 1 and 2 stabilized through H…H, C…H and H…H, O…H, C…H,
O…C and rare C-H…Cu anagostic type of non-covalent forces, respectively. The biological
relevance of various low energy out-of-plane vibrational mode of copper skeletal have been
assigned to below 200 cm-1 range of wavenumber. In vitro DNA binding profile of complexes 1
and 2 revealed the electrostatic mode of binding with partial intercalation in minor groove
region. Additionally, molecular docking studies have been performed to corroborate the trend of
DNA binding site and affinity of the complexes 1 and 2 which is in good agreement with the
experimental findings. The complexes 1 and 2 having the ability of DNA cleavage through an
oxidative mechanism induced by reactive oxygen species (ROS). Additionally, antibiofilm
activity of complexes 1 and 2 was evaluated against E. coli 25922. The complex 2 showed 65%
reduction in biofilm formation of E. coli 25922 at 50 µg/ml while complex 1 exhibited 47%,
which could be due to the extracellular DNA binding ability of the complexes. Thus, the results
of anti-biofilm activity experiment validated that copper complexes is a potent drug entity and
could be employed against bacterial biofilm infections.
Acknowledgements
The authors are grateful to SAIF Panjab University, Chandigarh, for providing ESI–MS and
elemental analysis facility. We are also thankful to Department of Chemistry and USIF, Aligarh
Muslim University for providing the FT-IR, UV-vis, EPR and SEM facility. The author
(Mohammad Usman) sincerely acknowledges financial support from University Grants

Commission (UGC), New Delhi, for providing the fellowship and the Department of Chemistry,
AMU through UGC assisted DRS–SAP, DST -FIST and DST PURSE Programme.
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DNA minor groove targeted copper(II) complexes explored as an antibiofilm active agents
against E. Coli 25922.