Dicyanamide-interlaced assembly of Zn(II)-schiff-base complexes derived from salicylaldimino type compartmental ligands: Syntheses, crystal structures, FMO, ESP, TD-DFT, fluorescence lifetime, in vitro antibacterial and anti-biofilm properties

Article abstract of DOI:10.1016/j.ica.2019.02.022

Two new dicyanamide-interlaced tetranuclear Zn(II)-Schiff-base complexes [Zn₂(L^OMe)(μ_-dca-κN¹κN⁵)(μ-dca-κN¹)]₂ (1) and [Zn₂(L^OEt)(μ_-dca-κN^1<

Full text of DOI:10.1016/j.ica.2019.02.022

Accepted Manuscript
Research paper
Dicyanamide-interlaced assembly of Zn(II)-Schiff-base complexes derived
from salicylaldimino type compartmental ligands: syntheses, crystal structures,
FMO, ESP,TD-DFT, fluorescence lifetime, in vitro antibacterial and anti-bio-
film properties
Dhrubajyoti Majumdar, Dhiraj Das, S.S. Sreejith, Sourav Das, Jayanta Kumar
Biswas, Monojit Mondal, Debarati Ghosh, Kalipada Bankura, Dipankar Mishra
PII:
S0020-1693(18)31904-2
https://doi.org/10.1016/j.ica.2019.02.022
ICA 18785
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 December 2018
18 February 2019
19 February 2019
Please cite this article as: D. Majumdar, D. Das, S.S. Sreejith, S. Das, J. Kumar Biswas, M. Mondal, D. Ghosh, K.
Bankura, D. Mishra, Dicyanamide-interlaced assembly of Zn(II)-Schiff-base complexes derived from
salicylaldimino type compartmental ligands: syntheses, crystal structures, FMO, ESP,TD-DFT, fluorescence
lifetime, in vitro antibacterial and anti-biofilm properties, Inorganica Chimica Acta (2019), doi: https://doi.org/
10.1016/j.ica.2019.02.022
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1
Dicyanamide-interlaced assembly of Zn(II)-Schiff-base complexes derived from
salicylaldimino type compartmental ligands: syntheses, crystal structures, FMO, ESP,TD-
DFT, fluorescence lifetime, in vitro antibacterial and anti-biofilm properties
Dhrubajyoti Majumdara,b, Dhiraj Dasc, S. S. Sreejithd, Sourav Dase, Jayanta Kumar Biswasf, Monojit
Mondalf, Debarati Ghoshf, Kalipada Bankura a, Dipankar Mishra a,*
aDepartment of Chemistry, Tamralipta Mahavidyalaya, Tamluk-721636, West Bengal, India
bDepartment of Applied Chemistry, Indian Institute of Technology (Indian School of Mines),
Dhanbad, Jharkhand 826004, India
cDepartment of Chemical Sciences, IISER, Mohali, Manauli, PO 140306, India
dDepartment of Applied Chemistry, Cochin University of Science and Technology, Kochi,
682022, Kerala, India
eDepartment of Chemistry; Institute of Infrastructure Technology Research and Management;
Near Khokhara Circle, Maninagar East, Ahmedabad-380026, Gujarat, India
fDepartment of Ecological Studies & International Centre for Ecological Engineering, University
of Kalyani, Kalyani 741235, West Bengal, India
AUTHOR EMAIL ADDRESS: dmishra.ic@gmail.com

2
ABSTRACT
Two new dicyanamide-interlaced tetranuclear Zn(II)-Schiff-base complexes [Zn2(LOMe)(µ-dca-
κN1κN5)(µ-dca-κN1)]2 (1) and [Zn2(LOEt)(µ-dca-κN1κN5)(µ-dca-κN1)]2 (2) were synthesized by
using salicylaldimino type Schiff bases (H2LOMe) and (H2LOEt) respectively. Schiff base ligands
and the complexes were characterized by elemental analyses, powder X-ray diffraction, FT-IR,
1
FT-Raman, H NMR, 13C NMR, UV-Vis, TGA and fluorescence spectroscopy. Dicyanamide
modulated complexes were structurally characterized by single crystal X-ray diffraction studies.
X-ray crystal structure divulges that the two complexes are iso-structural. In both the complexes,
the Zn1 metal centers fulfill 5-coordinated distorted square pyramidal geometry having ZnN3O2
chromospheres where Schiff bases are mainly trapped in their complete deprotonated dianionic
forms[L]2-, whereas Zn2 metal center attained distorted octahedral geometry. In both complexes
two asymmetric units are connected by double μ1,5-dicyanamide ion thus forming Zn4-nuclear
metal complex. B3LYP/def2-TZVP level of theory (DFT) successfully applied in both
complexes. The complexes (1-2) exhibit intraligand (π→π*) fluorescence in DMSO solvent with
lifetimes in the range 0.66-0.82 ns. In vitro antibacterial, membrane damage assay and anti-
biofilm properties of both complexes are evaluated against some important Gram-positive and
Gram-negative bacterial strains. Finally, the UV-Vis experimental spectral findings are well
rationalized with the electronic distribution of HOMO-LUMO through TD-DFT level of
calculations.
Keywords: Schiff base, Dicyanamide, tetranuclear Zn(II), TD-DFT, Antibacterial, Fluorescence

3
1. Introduction
The coordination chemistry is largely explored to design and synthesis of polynuclear and
supramolecular structure in last few decades [1a-b]. Proper selection of ligands in conjunction
-
with different ancillary co-ligands, like pseudo-halide (SCN-/N3 /NCO-/CN-), dicyanamide
-
[N(CN)2 ] anion is the key factors for construction of coordination compounds of such
architectures and topologies [1c-4].The flexible dicyanamide pseudo-halide anion exhibit diverse
coordination motifs through amide and nitrile nitrogen atoms when coordinated with particular
transition metal ions [5] to extend varieties of molecular architectures from discrete, di-nuclear to
multi-dimensional etc. [6-9].Till date different dimensional network structures of dicyanamide
ligand involving complexes have emerged in literature [10-25] but terminal monodentate
coordination mode of dicyanamide (µ1-M) is truly rare owing to its long chain structure and
negative (–ve) charge delocalization over the symmetrical molecule [26].The design and
syntheses of such Zn(II) metal template complexes involving dicyanamide pseudo-halide ligands
are drawing much more aesthetic attention not only due to different structural varieties observed
in the metal-organic frameworks or porous coordination polymers but also their important
emerging applications like catalysis [27], magnetism [28], light emission, electron-transport
processes [29], material science, optoelectronic devices etc. viz. Zn(II) salen-type Schiff base
complexes are always excellent electroluminescent materials [30] and in supramolecular
chemistry such Zn(II) complexes have been widely used in sensors and in host-guest chemistry.
Thus significant number of research works was imposed on supramolecular Zn(II)-Schiff-base
complexes [31a-n].From biological standpoint, zinc is the most important second abundant trace
metal after iron and play several important roles in enzyme catalysis, apoptosis,
neurotransmission [32a-c]. Further, Zn(II) metal ion, a d10 electronic system may adopt

4
versatile coordination number with different type of molecular architectures and topologies that
may be produced in presence of suitable ligands framework .
In the last few years, we have explored photoluminescent, DFT, TD-DFT, thermal, in vitro
antibacterial, membrane damage assay and anti-biofilm properties of Zn(II)/Cd(II) metal ion
complexes in presence of [N2O4] donor-type salicylaldimino ligands (H2LOMe/H2LOEt
[H2LOMe=N,N’-bis(3-methoxysalicylidenimino)-1,3-diaminopropane,H2LOEt=N,N’-bis(3-
)
ethoxysalicylidenimino)-1,3-diaminopropane] and potential pseudo-halide anions [SCN]-, [N3]-
-
or anions like Cl-, NO3 etc. Therefore, the only outcome of previous works is that pseudo-
halides [SCN]-/[N3]- linked metal complexes exhibit strong photoluminescence, antimicrobial
and anti-biofilm properties [33a-g]. In search of new luminescent Zn(II)-Schiff-base complexes,
we have been motivated to frame another two closely resembling less explored salen-type
ligands (H2LOMe/H2LOEt).Thus a target oriented complexing reaction was carried out in presence
of Zn(II) metal template, Schiff-base ligands and dicyanamide pseudo-halide leading to the
formation of complexes (1-2).
In this work, we report the syntheses, characterizations, crystal structures, FMO, ESP, TD-DFT,
fluorescence life-time, thermal, in vitro antibacterial and anti-biofilm properties of tetranuclear
Zn(II) dicyanamide modulated Schiff base complexes.

5
2. Experimental section
2.1. Materials and physical measurements
All the reagents were of analytical grade and used as purchased without further purification.
Elemental analyses were performed on a Perkin-Elmer 2400 II analyzer. IR spectra were
recorded as KBr pellets within the range 4000–400 cm-1on a Perkin–Elmer spectrum RX 1.
Raman spectra were analyzed by using BRUKER RFS 27 between 4000-50 cm-1. UV-Visible
spectra (200-1100nm) in DMSO were performed with a Hitachi model U-3501
spectrophotometer. Fluorescence spectra in DMSO were measured by using Perkin-Elmer
LS50B Spectrofluorimeter model at room temperature (298K). Fluorescence lifetime
measurements were recorded by using JOBIN-VYON M/S Fluorimeter. Thermogravimetric
analyses (TGA) of both complexes were carried out on a TGA-5OH analyzer from ambient
temperature to 700°C at a temperature rate of 10°C/min in a flowing 30ml/min under
environment of nitrogen atmosphere using a platinum cell. X-ray powder diffraction
measurements were carried out using BRUKER AXS, GERMANY X-ray diffractometer model
using radiation Cu K-alpha-1. Quantum yield (Φ) have been determined using popular equation
(1) where quinine sulfate is used as the secondary standard (Φ = 0.57 in water) [34]
S AS (Abs) nS2


R  -----------------------------------------------------------------------------------(1)
R AR (Abs)S nR2
According to equation (1), A terms denote the fluorescence area under the curve; Abs denotes
absorbance; n is the refractive index of the medium; Φ is the fluorescence quantum yield; and
subscripts S and R denote parameters for the studied sample and reference respectively.

6
2.2. X-ray crystallography
The good quality crystal data of complexes (1-2) were collected on a Bruker SMART CCD [35]
diffractometer using MoKα radiation atλ = 0.71073 Å. Different common programs were
operated for collection of crystal data of complex 1, 2 e.g. SMART program used for collecting
frames of data, indexing reflections, and determining lattice parameters, SAINT [36] for
integration of the intensity of reflections and scaling, SADAB[37] for absorption correction, and
popular SHELXTL for space group and structure determination and least-squares refinements on
F2. The crystal structure of complex 1, 2 was fully solved and refined by full-matrix least-
squares methods against F2 by using the program SHELXL-2014[38]and Olex-2 software [39].
For that reason all the non-hydrogen atoms were refined with anisotropic displacement
parameters. Hydrogen positions were fixed at calculated positions and refined isotropically.
Crystallographic diagrams of different molecules were constructed for 1, 2 with the aid of latest
Diamond software [40]. In this context a detailed summary of the crystallographic data and full
crystal structure refinement parameters for complexes (1-2) are clearly presented in Table 1.
Crystallographic data (excluding structure factors) for both complexes have been deposited with
the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 1876204-
1876205. Copies of the data can be obtained, free of charge, on application to CCDC, 12
UnionRoad, Cambridge CB2 1EZ, U.K.:http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi, e-
mail:data_request@ccdc.cam.ac.uk, or fax: +44 1223 336033.

7
Table 1 Crystal data and full structure refinement parameters of 1 and 2
Formula
M/g
C46H40N16O8Zn4 (1)
1206.52
C50H48N16O8Zn4 (2)
1262.52
Crystal system
Triclinic
Triclinic
Space group
P-1
P-1
a/Å
9.5908(14)
9.4196(19)
b/Å
c/Å
11.3039(17)
12.838(2)
94.164(8)
1277.3(3)
11.815(2)
13.394(3)
93.183(11)
1360.8(5)
β (°)
V/Å3
Z
1
1
ρc/g cm-3
1.5684
1.541
μ/mm-1
1.924
1.809
F(000)
Cryst size (mm3)
613.4464
644
0.2× 0.2 × 0.1
0.2× 0.2 × 0.1
θ range (deg)
0.9817
0.998
Limiting indices
-12 ≤ h ≤ 12
-15 ≤ k ≤ 13
0≤ l ≤ 17
-11 ≤ h ≤ 11
-14≤ k ≤ 14
-14≤ l ≤ 15
Reflns collected
Ind reflns
18562
6319[Rint = 0.1242, Rsigma
0.0831]
11007
4774 [Rint = 0.0291, Rsigma
0.0435]
=
=
Completeness to θ (%)
98.17
99.8
Refinement method
Full-matrix-block least-
squares on F2
Full-matrix-block least-squares on
F2
Data/restraints/
parameters
6319/0/295
4774/0/301
Goodness-of-fit on F2
Final R indices
[I > 2θ(I)]
1.0314
1.049
R1 = 0.0854
wR2 = 0.2546
R1 = 0.0727
wR2 = 0.1886
R indices (all data)
R1 = 0.1254
R1 = 0.1057
wR2 = 0.3011
wR2 = 0.2120
Largest diff. peak and
hole(e ·Å-3)
1.8426 and -1.1660
0.981 and -0.970

8
CCDC
1876204
1876205
2.3. Computational methodologies
DFT calculations were done using Becke's three-parameter hybrid exchange functional including
[41] the Lee-Yange-Parr [42] nonlocal correlation functional (B3LYP) by employing ORCA
3.0.3 software package [43-44]. The triple zeta valence basis set with one set of polarization
function TZVP basis sets [42] was used.To accelerate the calculations, we utilized the resolution
of identity (RI) approximation with the decontracted auxiliary def2eTZV/J Coulomb fitting basis
sets and the chain-of-spheres (RIJCOSX) approximation to exact exchange as implemented in
ORCA [45]. The orbital composition were found out using Mulliken method by using multiwfn
program [46].The graphics of the results were plotted using Avogadro software [47a].The choice
of the TZVP basis set was made on the known fact that it reduces the basis set superposition
error (BSSE) to negligible in the calculation of systems with noncovalent interactions.The Time
Dependent-Density Functional Theory (TD-DFT) calculation were performed for complexes (1-
2) by using Gaussian 09 [47b] at DFT/B3LYP level using 6-31G* basis set for C, H, O and N
atoms and common LANL2DZ [47c-d] basis set and an effective core potential was used for Zn
atoms. The effect of the solvent was modeled with the polarized continuum model (PCM)
[47e].The crystallographic coordinates have been considered for the DFT calculation. All the
generated molecular orbitals (MOs) at the time of TD-DFT calculations were commonly
visualized using software Gauss view 5.0.
2.4. Antibacterial screening
The antibacterial potentials of the selected two tetranuclear Zn(II) dicyanamide complexes were
tested in vitro against bacterial strains belonging to Gram-positive (Enterococcus gallinarum

9
MTCC 7049 and Staphylococcus haemolyticus MTCC 3383) and Gram-negative (Enterobacter
aerogenes MTCC 111 and Proteus vulgaris MTCC 744) categories, which were obtained from
Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology,
Chandigarh, India.
The assessments of antibacterial potential and minimum inhibitory concentrations (MIC) of the
complexes were performed against the selected strains following standard protocol
recommended by Clinical and Laboratory Standards Institute (CLSI) [48]. The susceptibility of
the bacterial strains to those complexes was ascertained by agar well diffusion method and the
potentiality was expressed by growth inhibition zone diameter (mm) measurement [49]. At mid
log phase the representative Gram-positive (E. gallinarum) and Gram-negative (P. vulgaris)
bacterial strains were subjected to the complexes at their respective MICs, and respective time-
kill curves were drawn [49]. The biofilm removal efficiencies of (1-2) were assessed against
Gram-positive (E. gallinarum) and Gram-negative (P. vulgaris) bacterial strains following
standard protocol [50a-b].
Membrane damage assay was performed on a Gram-positive (E. gallinarum) and a Gram-
negative (P. vulgaris) bacterial strain to test one of the plausible modes of antimicrobial action of
the complexes tested on affecting bacterial cell membrane disintegration. Membrane damage was
evaluated by estimating the amount of nucleic acids released out of the bacterial cells [51, 52a].
Overnight cultures of the both bacterial strains were subjected to centrifugation at 8000 rpm for 5
min. The cell pellet was washed three times by phosphate buffer saline (PBS, pH 7.4) and
resuspended in the liquid media. The specific optical density (0.7) of the selected bacterial
suspension was fixed at 600 nm, and disseminated into different conical flasks. The individual
concentration of the tested complexes was set at their respective MIC relevant against the

10
bacterial strains. The set devoid of any complexes was maintained as control. On 30 min interval
sample (2 mL) was taken and filtered with 0.2 µm syringe filter. The filtrate was measured on a
UV-Vis spectrophotometer at 260 nm, and the amount of released nucleic acids was quantified.
2.5. Synthesis of the ligands
Two Schiff base ligands (H2LOMe and H2LOEt) were synthesized by the common condensation
process of 1 mmol (0.152g) 3-methoxysalicylaldehyde or (0.1662g) 3-ethoxysalicylaldehyde
with 0.5 mmol (0.0371g) 1,2-propanediamine (Pn) in (50 mL) of methanol for ca. 1 h (Scheme
1).The dark orange yellow solution was directly used for tetranuclear Zn(II) dicyanamide
complex formation.
Scheme1. Syntheses of Schiff base ligands
2.6. Synthesis of [Zn2(LOMe)(µ-dca-κN1κN5)(µ-dca-κN1)]2 (1)
To the methanolic solution (25 mL) of zinc acetate dihydrate (0.2195g, 1 mmol), dark orange
yellow solution of Schiff base (H2LOMe) was added directly followed by mixing a methanolic
solution (5 mL) of sodium dicyanamide (0.0891g, 1 mmol) and the overall solution mixture was

11
0
refluxed for 1 hour at 60 C. Acetonitrile (CH3CN) (5 mL) was then added into this refluxed
solution. Yellowsolution was immediately cooled at room temperature and constant
stirringcontinued for 1 h. Finally, the light-yellow filtrate was kept for crystallization by slow
evaporation at normal room temperature. After 4 days yellow colored single crystal suitable for
X-ray crystallography was obtained. Crystals were isolated by filtration and air dried. Yield:
0.365 g (68%), Anal. Calc. for C46H40N16O8Zn4: C, 45.79; H, 3.34; N, 18.58.Found: C, 44.9; H,
3.5; N, 18.35 %. IR (KBr cm-1) selected bands: ν(C꞊N), 1634 vs, ν(C≡N), 2284 m, 2221 m, 2176
vs, FT-Raman (cm-1) selected bands: ν(C꞊N), 1644 vs, ν(C≡N), 2280 vs, 2228 s, 2173 w.
2.7. Synthesis of [Zn2(LOEt)(µ-dca-κN1κN5)(µ-dca-κN1)]2 (2)
Complex 2 was prepared by the identical procedure as 1 except Schiff base solution used is
(H2LOEt). Yield: 0.368 g (68.75%), Anal. Calc. for C50H48N16O8Zn4: C, 47.64; H, 3.68; N, 17.78.
Found: C, 47.35; H, 3.45; N, 17.58%. IR (KBr cm-1) selected bands: ν(C꞊N) 1633 vs, ν(C≡N)
2282 m, 2223 s, 2175 vs, FT-Raman (cm-1) selected bands: ν(C꞊N) 1631 vs, ν(C꞊N) 2283 w,
2240 m, 2129 m.
3.Results and discussion
3.1. Synthetic methodology
The tetradentate Schiff-base ligands were synthesized using the literature method [52b]. The
tetra nuclear Zn(II) metal complexes ( 1 and 2) were synthesized by stirring Zn(OAc)2.2H2O
with Schiff base ligands in methanol followed by slow addition of methanolic solution of
[N(CN)2]-. in 1:1:1 molar ratio (Scheme 2). Complexes were successfully isolated in crystalline
form from slow evaporation of CH3OH-CH3CN mixture. Metal assisted (M+2) versatile binding
ability of Schiff base ligands and dicyanamide anions are presented in Scheme S1 and S2. The

12
obtained complexes were air stable, yellow colored, soluble in common organic solvents.
Complexes are characterized by elemental analyses, UV-Vis, IR, Raman, powder X-ray
diffraction, thermal, fluorescence and X-ray crystallography. Schiff base ligands are tetradentate
in nature but in context of our synthesis they are well behaved as ‘pendant ligands’. A large
number of ‘pendant ligands’ are used in a variety of different chemical applications [53a-e]. Till
date, using identical ligands and dicyanamide anions four zinc metal complexes have emerged in
literature [54]. Hence, at least to the best of our knowledge this specific type of Schiff-base
ligands has never been used to synthesize zinc dicyanamide-interlaced assembly.
Scheme2. Synthetic scheme for complexes (1-2)
3.2. Spectral characterizations
The characteristic imines (C=N) stretching vibration of Schiff base ligands are found to be 1628,
1631 cm-1 respectively [55a-b] (Fig.S1, Fig.S3, Fig.S5A). The above stretching vibration bands
are shifted to 1634, 1633 cm-1 in complexes (1-2) [55c].The dicyanamide anion [N(CN)2]-
showed three sharp strong characteristic band preferably in the region 2300-2170 cm-1[56].Two
medium intensity bands (at 2284, 2221 cm-1in 1 and 2282, 2223 cm-1 in 2) and a strong intensity

13
band (at 2176 cm-1 in 1 and 2175 cm-1 in 2) are indicative of the presence of dicyanamide in
these two complexes. FT-IR spectra with other reported Zn(II) dicyanamide complexes were
compared in TableS2.FT-Raman spectral data of some reported Zn(II) dicyanamide complexes
were further consider to established the bridging mode of [N(CN)2]- anions shown in Table S3.In
addition, aliphatic C-H stretching resonances for Zn(II) dicyanamide complexes were observed
within the range 2934-2835 cm-1and 2924-2873 cm-1[57a]. Ar-O stretching frequencies in 1-2
observed near at 1250-1222 cm-1and 1244-1218 cm-1 which is similar to the other reported salen-
type ligands [57b].
3.3. UV-Vis spectra
UV–Visible spectral bands near at 283 and 334 nm of Schiff base ligands are assigned due to
π→π* and n→π* electronic transitions (Fig.S2). Both complexes in DMSO solvent exhibits
ligand–based transition near at 286, 368 nmand 282, 368 nm presumably due to π→π* or n→π*
type of transitions [58, 59]. Since Zn(II) metal have filled d10 configuration hence no metal
centric d-d transition band was observed in the complex spectrums.
3.4 X-ray powder diffraction
The phase purity of complexes (1-2) was ensured by X-ray powder diffraction pattern. The X-ray
powder diffraction pattern for both tetranuclear complexes was recorded experimentally after
considering effectively 2θ scan from 40 to 500. The experimental powder X-ray diffraction
pattern of two complexes agree very well with the patterns simulated from single-crystal
diffraction data (cif file) obtained from Mercury software (Fig. S4). Hence the study of X-ray
powder diffraction is further established the phase purity of the bulk crystal samples of Zn4-
nuclear metal complexes. Thus, powder X-ray diffraction patterns of (1-2) positively confirmed
that dicyanamide modulated complexes are completely isostructural.

14
4. Description of the X-ray crystal structures
4.1. [Zn2(LOMe)(µ-dca-κN1κN5)(µ-dca-κN1)]2 (1) and [Zn2(LOEt)(µ-dca-κN1κN5)(µ-dca-κN1)]2 (2)
The solid-state structures of the complexes have been confirmed by single-crystal X-ray
̅
diffraction and both the complexes crystallize in triclinic space group P1. The complexes are
tetranuclear homometallic in nature and isostructural. Full crystallometric parameters for
complexes (1-2) are summarized in Table S1. In connection of the structural aspects of
homometallic tetranuclear complexes, herein we have only described in details the structure of
complex 1 as a representative example to show the common structural features of two
complexes. The asymmetric unit (Fig.1) contains two Zinc metal ions, one Schiff base ligand
fully deprotonic forms [LOMe]2-or [LOEt]2-and two dicyanamide ions that is equivalent to basic
structural core [Zn2(μ2-O)2]2+. The ligand has two binding sites, which is chelated with the two
zinc ions. The crystal structure analysis of 1 divulges that Zn1 center in ZnN3O2chromophoric
environment adopted distorted square pyramidal geometry (Fig.2) and Addition tau (τ)
parameter [60] is 0.04 (ideal value of tau for square pyramidal geometry is 0). The equatorial
positions are occupied by two imine nitrogens (N1 and N2) and two phenolic oxygens. The
apical position is occupied by the nitrogen atom (N8) of dicyanamide ion. Zn1 is displaced
towards axial N8/N7 atom by 0.623 and 0.641 Å, respectively in 1 and 2 from their respective
mean equatorial planes. The Zn2 center is hexa-coordinated (Fig.2) and adopted distorted
octahedral geometry. The equatorial plane is occupied by two phenolic oxygens (O1 and O3) and
two methoxy oxygens (O2 and O4). The axial positions are occupied by nitrogen atoms (N3 and
N6) of two dicyanamide ions. Selected some major important bond lengths and angles of 1-2 are
given in Table 2.The above bond distances are totally comparable to other reported Zn(II)
dicyanamide complexes (Table S4).The bond distances between Zn2 and oxygen atoms of

15
alkoxy groups are quite longer {Zn2-O2 = 2.631Å, Zn2-O4 = 2.781 Å in 1, Zn2-O3 = 2.798 Å,
Zn2-O4 = 2.722 Å in 2}, as this site is open in nature causing steric hindrance by methyl groups
[61].The Zn1 and Zn2 are bridged by phenolic oxygens (O1/O1 and O3/O2) and the distances
are 3.085 Å and 3.068 Å in 1 and 2, respectively.The distance between Zn 1 and Zn 2 metal
centers is 3.085 Åthat is comparable to other Zn—Zn separation of double phenoxo bridged Zn2
complexes[62a-b].One dicyanamide ion act as a monodentate ligand via µ1-dca bridging mode
and coordinated to the Zn2, whereas the other one connects Zn1 and Zn2 centers via µ1,5-dca
bridging and the distance between two centers is 7.973 Å and 8.111 Å in 1 and 2, respectively.
Thus, two asymmetric units are connected by double μ1,5-dicyanamide ion, [Zn1(NC-N-CN)
Zn2]2 and formed a discrete dimeric Zn4-nuclear metal complex 1 (Fig.2) and for complex 2
(Fig.4). In the solid-state, they exhibit hydrogen bonding interactions and developed network
structure. The N5 atom of dicyanamide ion is hydrogen bonded in a bifurcated way with imine
hydrogen and methine hydrogen and N4 is hydrogen bonded with hydrogen of the methoxy
group and generated a 2-dimensional network (2D) (Fig.3). Packing diagram of complex 1 is
clearly shown in Fig.S5B. In complex 2, the solid-state structure shows C-H····π interactions and
generated a 1-dimensional network (1D) (Fig.5). The effective strong steric effect by ethyl side
chain in complex 2 as compared to 1 prevents further association of the molecules through
dicyanamide co-ligand resulting 1D dimensional network [61]. In the structural aspect,
architectures and bridging propensity of dicyanamide anion of complex 1 and 2 has been
compared with different complexes coordinated to identical salen-type ligands (Table S5).

16
Table 2 Selected bond distances and angles for 1 and 2
Bond lengths (Å)
Value (Å)
Bond angles (°)
Value (°)
1
Zn1-N1
Zn1-N2
Zn1-N8
Zn1-O1
Zn1-O3
Zn2-N6
Zn2-N3
2
2.032(1)
2.049(8)
1.999(7)
2..001(6)
2.022(6)
1.957(6)
1.942(8)
N1-Zn1-N2
O1-Zn1-O3
O3-Zn1-N2
O1-Zn1-O3
N2-Zn1-N8
N2-Zn2-N6
N1-Zn1-N8
81.90(3)
78.20(2)
142.8(2)
78.20(2)
108.4(2)
115.9(2)
113.2(2)
Zn1-N1
Zn1-N2
Zn1-N7
Zn1-O1
Zn1-O2
Zn2-N3
2.09(1)
2.049(8)
1.991(7)
2.016(6)
2.026(6)
1.948(9)
N1-Zn1-N2
O1-Zn1-O2
O2-Zn2-N6
O2-Zn2-N3
O1-Zn2-N3
N2-Zn1-N7
80.50(4)
78.90(2)
112.2(3)
114.10(3)
113.10(3)
114.7(3)

17
1(a)
2(b)
Fig.1. ORTEP view of asymmetric unit for complex 1(a) and for complex 2(b)
Fig.2.Dimeric unit of complex 1

18
Fig.3.2D hydrogen bonded network of complex 1
Fig.4.Dimeric unit of complex 2

19
Fig.5.1D hydrogen bonded network of complex 2
4.2. Frontier molecular orbital (FMO) analysis
The two complexes were optimised in the gaseous phase at B3LYP/TZVP level of theory. The
optimised geometry was found to be of minimum energy via carrying out frequency calculations
of the optimised structures which returned no imaginary frequency. The value of HOMO
(highest occupied molecular orbital) dictates the ability of a molecule to donate electron while
the electron accepting nature is decided by LUMO (lowest unoccupied molecular orbital) value
and from the computed values suggest that complex 1 is more kinetically stable and chemically
inert than complex 2 (Fig.6). From the plot itself it is clear that the HOMO of 1 is mainly
concentrated on methoxy oxygen (15.64%, p type) and the two phenyl rings on either side
(58.49%, π-type) while in the case of LUMO, methoxy group is not at all involved and it is
concentrated on chelate rings and phenyl rings (91.32%, π*type). In the case of 2, the HOMO is
fully on theN(CN)2 coligand and LUMO is having heavy contribution from both metallocycles
and phenyl rings (67.63%, π* type). The global chemical reactivity descriptors associated with 1
and 2 are given in Table S6.

20
Fig.6.FMO with the associated energies computed at B3LYP/TZVP level of theory
4.3. Electrostatic Potentials (ESP) analysis
Electrostatic potential maps were mapped on electron density iso-surface between +50 kcal/mol
to-50 kcal/mol reveals a positive charge on metal centre on both the complexes. With complex 2,
has a relatively high positive charge compared to complex 1 (Fig.7). It is worth noting that, in
both the complexes the higher positive charge is near the area in the proximity of azomethine
hydrogen (+43.1kcal/mol and+44.6 kcal/mol for 1 and 2) respectively which is common in these
classes of compounds [63]. Phenyl rings in the two compounds have a value ca.-23 kcal/mol
while the donor oxygen atoms in both the compounds have comparatively high value with an
average of about -36 kcal/mol. The highest negative potential is near the coligand (dca) in both
the complexes with the highest value for 1. This different but opposite potentials between co-
ligand (dicyanamide) and metal centre (Zn) results in the formation of dimeric structures in the

21
packing diagrams of both the structures of the complexes and account for the stability of their
solid structures.
Fig.7. ESP mapped over the electron density isosurface of 1 and 2 computed at B3LYP/TZVP
depicting the nucleophilic and electrophilic regions
4.4. TD-DFT analysis
The computational TD-DFT study of both tetranuclear Zn(II) dicyanamide complexes exhibits
electronic transitions that are mainly due to intra-ligand charge transfer transition.The calculated
absorption maxima for complex 1 arises at 357.90 nm (Experimental ~368 nm) with oscillator
strength, f = 0.0161. In case of complex 2, it shows calculated peak at 368.88 nm (Experimental
~368 nm) that arises mainly due to intra-ligand transitions with oscillator strength, f = 0.0595.
All the HOMOs in both tetranuclear Zn(II) dicyanamide complexes are mainly consist of π-
orbitals only and the LUMOs are consist of ligand π*-orbitals only.TD-DFT related all possible
orbital electronic transitions are clearly submitted in Table S7. Further, TD-DFT generated all
possible MOs are lucidly presented in Fig.S6.

22
4.5. Fluorescence and lifetime studies
Metal complexes fluorescence behaviors are extremely important due to their plenty of
application such as chemical sensors, in photochemistry and also in electroluminescent displays
[64a]. The fluorescence behavior of Schiff base ligands (H2LOMe)/(H2LOEt) and the complexes
were recorded in DMSO solvent at room temperature (Table 3). The comparable fluorescence
spectra are presented in Fig.8 revealing strongly that each Schiff base ligand is practically weak
fluorescence with respect to either complex 1 or 2 but both complexes are highly fluorescent
active. Upon photo excitation at 333 nm, both Schiff bases exhibits a fluorescent emission
maximum centered at 441nm and 445 nm. Complexes 1, 2 upon photo excitation at wavelength
370 nm shows fluorescence maxima with the emission peak near at 483 nm and 485 nm.
Emission spectra of Zn4 dicyanamide complexes and ligands are similar with other reported
Schiff bases e.g. L0H2, L1H2, L2H2, L3H2, L4H2 etc. [64b] and Zn(II) dicyanamide complexes
(Table S7). C. E. Burrow et. al, has reported an emission spectrum of mixed metal [Zn2Tb3]
complex of salen type ligand [65]. Apart from, quantum yield (ɸ) value of Schiff base ligands
and complexes have been determined (Table3). Schiff bases quantum yield (ɸ)values are
comparable with other reported salen-type ligands L1H2, L2H2, L0H2, L3H2, L4H2etc [66a-b].The
strong fluorescence behavior of Zn(II)-Schiff-base complexes compared to ligands may be due to
the reason that d10 candid metal is difficult or hard to oxidize or reduce. Hence the enhance of
fluorescence behavior in both complexes may presumably attributed due to the increased in
conformation rigidity of the Schiff base ligands impartedvia chelation effect with Zn(II) metal
ion. Therefore, the fluorescence may be assigned mainly due to the intraligand (π→π*) transition
or L→M charge transfer. Such type of fluorescence emission is called CHEF [67-70] but in case
of quenching of fluorescence concept this is referred as ‘Chelation enhancement of Quenching

23
effect’(CHEQ) [71]. The individual decay profiles of Schiff base ligands and the complexes have
been lucidly generated in Fig.S7. The average fluorescence decay life times <τav> have been
determined for ligands and the complexes using the following the literature method in Equation
S1. The nature of decay profile of Schiff base ligands can be best fitted to tri-exponential manner
[72] (Fig.S7) whereas for tetranuclear Zn(II) complexes it will be best fitted to bi-exponential
nature (with acceptable comparable χ2 values). Decay profile are comparable to other reported
Zn(II)-salen complexes [73a-c]. According to Table 3, it reveals that the average fluorescence
lifetime for Zn4-nuclear metal complexes are smaller than free salen-type ligands. Therefore,
fluorescence lifetime analysis further divulges that excited states of both complexes are unstable
with respect to Schiff base ligands. The bi-exponential decay profiles for both complexes are
best explained as due to competition of different excited states that is π→π* excited states
involved in the emission features for both complexes and the preferably CT character that
quenches the fluorescence [74a].
Table 3 Photoluminescence and time resolved photoluminescence decay of Schiff base ligands
and complexes (1-2)
Compounds λex (nm) λem (nm)
(ɸ)
τ1[ns]
τ2[ns]
τ3[ns] <τ>[ns]
χ2
(H2LOMe
)
333
333
370
370
441
445
483
489
0.00353
2.78
0.73
9.44
2.35
1.21
(17.4%) (81.5%) (1.1%)
2.77 0.75 9.41
(17.4%) (81.5%) (1.1%)
(H2LOEt
)
0.00351
0.00209
0.01445
2.34
1.20
1.24
1.23
0.58
(99.8%)
0.61
5.54
(0.2%)
4.93
-
1
2
0.66
0.82
-
(99.4%)
(0.6%)

24
30000
25000
20000
15000
10000
5000
0
Ligand1
Ligand2
Complex1
Complex2
450
500
550
600
650
Wavelength(nm)
Fig.8.Ligand centered emission spectra of Schiff bases and complexes (1-2)
4.6. Thermogravimetric analysis
Thermal characteristics of the complexes were studied using TG-DTG techniques under nitrogen
atmosphere. In complex 1, an initial disintegration within the μ1,5-dicyanamide linker unit occurs
in the temperature range 225-385°C which corresponds to a mass loss of about 4.98 % and is in
good agreement with the calculated value (5.06 %) Fig.S8 (a). The powder sample obtained after
this run was investigated with IR spectral study which clearly shows the disappearance of
common dicyanamide FT-IR spectral peaks but Schiff base ligand stretching frequencies are still
present [74b]. This is followed by almost complete decomposition of the dimeric complex (95.21
%) in the narrow temperature range 397-404°C. Unlike complex 1, complex 2 undergoes well-
defined thermal multi-decomposition pattern Fig.S8 (b). In the temperature range 100-287 °C,

25
there is a mass loss of about 6.84 % (calculated value 6.57 %) which may be due to the loss of
the two μ1,5-dicyanamide linker units linking the dimeric complex. This is further proved by a
major weight loss of about 21.24 % (calculated value 21.61 %) between 285-528 °C which
marks the initiation of the thermal disintegration of the complex after the removal of
dicyanamide linking units. This may be due to the decomposition of the Schiff base ligand units
in one of the complexes of the dimer. The thermal decomposition of the complex continues at
higher temperatures. Even though the thermal disintegration pattern is different for the two
complexes, they have similar thermal stability up to temperatures around 400 °C (except
dicyanamide unit).
4.7. Analysis of in vitro antibacterial, membrane damage assay and anti-biofilm properties
Complexes and respective Schiff base ligands (H2LOMe/H2LOEt) exhibited antibacterial potency
against the four selected bacterial strains. The selected both complexes showed high antibacterial
activity against both Gram-positive and Gram-negative bacterial strains which is evident from
their respective MIC values and growth inhibition zone diameters whereas Zn4 dicyanamide
complexes had the lower MIC (µg/mL) values compared to their respective salen-type ligands
implying greater antimicrobial efficiencies shown in Table 4. In our previous paper [52a]. it has
been shown that both complexes have higher growth inhibition zone diameter (mm) than the free
Schiff-base ligands (H2LOMe/H2LOEt) (Table 4). With the lowest MIC (µg mL-1) and highest
growth inhibition zone diameter (mm) both the complexes were found to be most effective
against P. vulgaris among the tested strains. In contrary, the complexes had the maximum MIC
(µg mL-1) and minimum growth inhibition zone diameter (mm) against E. gallinarum among
tested bacterial strains showing their least efficacy against the bacterial strain.

26
Subjected to the challenge of complexes (1-2) at MIC levels both bacterial growths suffered an
inhibition. The time-kill curves (Fig.9) of the bacterial strains (E. gallinarum and P. vulgaris)
showed distinct treatment differences in the bacterial growth inhibition pattern in the following
order: complex 2 > complex 1 (LSD test; p < 0.05). Biofilm removal study undertaken against E.
gallinarum and P. vulgaris (Fig.10) showed that both the complexes were more effective in
removing biofilm produced by Gram-negative bacteria (P. vulgaris) compared to that of Gram-
positive bacteria (E. gallinarum) (Fig.11). The complex 2 had better biofilm removal efficiency
for the tested bacterial strains.
Membrane damage assay with the complexes against Gram-positive (E. gallinarum) and a Gram -
negative (P. vulgaris) bacterial strain (Fig.11) presented promising potential for damaging
bacterial cell membrane. Both complexes showed higher efficacy against Gram-negative (P.
vulgaris) bacteria than the Gram-positive (E. gallinarum) one. The quantum of nucleic acids
released differed significantly ((LSD test; p < 0.05) between the complexes (complex 2 >
complex 1). Based on this signature of bacterial membrane disintegration of complex 2 was
proved more powerful than complex 1.
Table 4 Minimum inhibitory concentration (MIC; µg/mL) and bacterial growth inhibition zone
diameter (mm) of Schiff base ligands (H2LOMe/ H2LOEt) against the selected Gram-positive and
Gram-negative bacterial strains [52a]
Bacteria
(H2LOMe
)
(H2LOEt
)
MIC
(µg/ML)
550
Growth Inhibition
Zone (mm)
MIC
(µg/ML)
500
Growth inhibition
zone (mm)
B. subtilis
4.13±0.28
4.35±0.51

27
E. gallinarum
P. vulgaris
500
350
450
5.89±0.31
7.94±0.22
6.67±0.41
450
300
400
5.13±0.62
8.29±0.42
7.68±0.19
E. aerogenes
Table 4 Minimum inhibitory concentration (MIC; µg/mL) and bacterial growth inhibition zone
diameter (mm) of complex 1 and 2 against the selected Gram-positive and Gram-negative
bacterial strains. All values of growth inhibition zone diameters have been expressed as mean ±
standard deviation
Bacterial
strains
Complex 1
Complex 2
(X)
(Y)
MIC
Growth
inhibition
zone (mm)
MIC
Growth
inhibition
zone (mm)
(µg/mL)
(µg/ML)
S. haemolyticus
E. gallinarum
P. vulgaris
270
300
240
250
7.80 ± 0.49
7.20 ± 0.40
13.60 ± 0.80
13.20 ± 0.75
240
250
200
230
9.2 ± 0.97
8.4 ± 0.49
14.4 ± 0.49
14.00 ± 0.63
E. aerogenes

28
Fig.9.Time-kill curves of the selected bacterial strains as challenged by the tested chemical
complex 1 and complex 2, (a) E. gallinarum, (b) P. vulgaris. All data were taken in triplicate,
and error bars represent standard deviation
Fig.10.Biofilm removal efficiency of the tested complexes 1 & 2 against E. gallinarum and P.
vulgaris. All data were taken in triplicate and error bars represent standard deviation

29
Fig.11. Membrane damage potential of the complexes against selected bacterial strains: (a)
Gram-positive (E. gallinarum); (b) Gram-negative (P. vulgaris). All data were taken in triplicate
and error bars shows standard deviation
5. Conclusion
This paper primarily describes the syntheses and molecular architectures of Zn(II) dicyanamide
complexes utilizing (N2O2) donor type Schiff base ligands. Schiff bases are potentially trapped
by Zn(II) metal after strong deprotonic actions [L]-2. In both complexes, dicyanamide pseudo-
halide linker plays crucial role showing fascinating µ1,5 and µ1 dual bridging modes to form
polymeric metal-organic frameworks and thus to attain metal-organic framework thermal
stability. The photophysical properties of zinc metal complexes and that of the ligands have been
determined in DMSO solvent with a comparative approach to search mainly the origin of
emission spectra. The fluorescence behaviors of both complexes are predominantly ligand
centered. Fluorescence lifetime study further confirmed that the importance of excited state
stabilities of Schiff base ligands is greater than Zn4-nuclear metal complexes. Both complexes
exhibited in vitro antibacterial and anti-biofilm properties against some important Gram-positive