Synthesis, structure, polyphenol oxidase mimicking and bactericidal activity of a zinc-schiff base complex

Article abstract of DOI:10.1016/j.poly.2020.114933

Focusing on the important biological functions of metallo-enzymes and metallo-therapeutics in living world, this research work demonstrates the synthesis, crystal structure, supramolecular architecture, 4-methylcatechol oxidation and bactericidal activity

Full text of DOI:10.1016/j.poly.2020.114933

Journal Pre-proofs
Synthesis, Structure, Polyphenol Oxidase Mimicking and Bactericidal Activi‐
ty of a Zinc-Schiff Base Complex
Shreya Mahato, Nishith Meheta, K Muddukrishnaiah, Mayank Joshi,
Madhusudan Shit, Angshuman Roy Choudhury, Bhaskar Biswas
PII:
S0277-5387(20)30590-8
DOI:
Reference:
https://doi.org/10.1016/j.poly.2020.114933
POLY 114933
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Polyhedron
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9 September 2020
18 November 2020
Please cite this article as: S. Mahato, N. Meheta, K. Muddukrishnaiah, M. Joshi, M. Shit, A. Roy Choudhury, B.
Biswas, Synthesis, Structure, Polyphenol Oxidase Mimicking and Bactericidal Activity of a Zinc-Schiff Base
Complex, Polyhedron (2020), doi: https://doi.org/10.1016/j.poly.2020.114933
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Synthesis, Structure, Polyphenol Oxidase Mimicking and Bactericidal
Activity of a Zinc-Schiff Base Complex
Shreya Mahato,a Nishith Meheta,a K Muddukrishnaiah,b Mayank Joshi,c Madhusudan
Shit,d Angshuman Roy Choudhury,c and Bhaskar Biswasa,*
aDepartment of Chemistry, University of North Bengal, Darjeeling-734013, India
bDepartment of Pharmaceutical Technology, Anna University, BIT Campus Thiruchirappalli,
India
cDepartment of Chemical Sciences, Indian Institute of Science Education and Research,
Mohali, Sector 81, Knowledge City, S. A. S. Nagar, Manauli PO, Mohali, Punjab 140306,
India
dDepartment of Chemistry, Dinabandhu Andrews College, Kolkata 700084, India
Focusing on the important biological functions of metallo-enzymes and metallo-therapeutics
in living world, this research work demonstrates the synthesis, crystal structure,
supramolecular architecture, 4-methylcatechol oxidation and bactericidal activity of an
interesting zinc-Schiff base complex, [Zn(HL)2Cl2] (1), [Schiff base (HL) = 2-(2-
methoxybenzylideneamino)phenol]. Crystal structure analysis of the zinc-Schiff base reveals
that zinc centre exists in a distorted tetrahedral geometry. The Schiff base adopts three donor
centres, however it gets protonated to exist in a zwitter ionic form and behaves as a
monodentate coordinator in 1. This zinc-Schiff base complex has been examined towards the
bio-mimetic oxidation of 4-methylcatechol (4-MC) in methanol and portrays its good efficacy
with good turnover number, 1.45×103 h–1. Electro-chemical study, electron paramagnetic
resonance analysis and electrospray ionization mass spectrometry results for the zinc-Schiff
base complex in presence of 4-MC ensures that the catalytic reaction undergoes through
enzyme-substrate binding, and generation of radical in the course of catalysis drives the
catalytic oxidation of 4-MC. Antibacterial study has also been performed against few clinical
pathogens (Bacillus SP, Enterococcus, and E.coli). Scanning electron microscope and EDAX
analysis for the pathogen with little dose of zinc complex confirms the destruction of bacterial
cell membrane with 1.44% occurrence of zinc in the selected zone of inhibition area. This
observation holds a great promise to develop future antibacterial agent.

2
Keywords: Zinc(II); Elecrochemical analysis; 4-methylcatechol oxidation; Schiff base; X-ray
structure; Bactericidal activity
*Corresponding author. E-mail: bhaskarbiswas@nbu.ac.in / icbbiswas@gmail.com
1. Introduction
In the field of coordination chemistry, Schiff base, a unique class of compounds has been
emerged as a widely used chelator towards the development of metal complexes [1-7]. The
newly prepared Schiff bases and their coordination compounds hold a great promise to
scientific community for their novel properties in designing efficient catalysts, smart magnetic
materials, important therapeutics, opto-electronic devices, bio-mimetic models and so on [8-
14]. Among the different 3d elements, zinc is an indispensable bio-metal in living system
which has significant functions in various biological processes [15]. Most captivatingly, zinc-
Schiff-base complexes have been well recognized for their catalytic, opto-electronic and
therapeutic properties [13,14,16-21]. Zinc ion in association with Schiff base exhibits
diversified coordination geometries [22,23] and interesting supramolecular architectures [24].
Added to this, zinc-Schiff base complexes also provide promising potency towards the
resistance of microbial activity and consider as one of the suitable candidates for the discovery
of potent metallo-therapeutics [25-27].
Polyphenol oxidase (PPO) consists of a group of copper containing proteins which
monitors the browning of wide range of natural fruits, vegetables, and sea-food products
through the enzymatic oxidation of phenolics and lead to the development of brown pigments
[28,29]. PPO catalyzes the hydroxylation of monophenol (monophenolase activity) following
the subsequent oxidation of diphenol to quinones (diphenolase activity) which promptly
convert to brown coloured relatively insoluble polymers (melanins) [30]. This development of
brown colour catalyzed by PPO has received paramount attention in food industry as it
provides significant information about nutritional quality and, thereby, produced a crucial
economic impact on the food-processing industry [31]. Scientific literatures exhibit that the
rate of metallo-enzyme catalysed browning of natural fruits as well as vegetables
fundamentally depends on concentrations of phenolic substrate, pH, temperature, and specific
activity of the PPO [28-31]. It is also well documented that catalytic oxidation of phenols
actually produces biologically important o-quinones which exhibits facile redox reactions for
its higher chemical activity. The o-quinone also plays various biological functions likely to

3
develop antibiotics, defensive secretions, and pigmentation [32]. Melanin pigment, a
polymerise form of o-quinone protects all the living organisms from UV-light of sun [33]. In
the context, with an aim to study the bactericidal activity and catalytic fate towards the bio-
mimetic oxidation of 4-MC, we have designed, synthesized, and structurally characterized of
an interesting zinc-Schiff base complex and studied its bactericidal and bio-mimicking
activity.
2. Experimental
2.1. Preparation of the Schiff base and zinc-Schiff base complex
(a) Chemicals, solvents and starting materials
Highly pure o-anisidine (Sigma Aldrich, USA), salicylaldehyde (Sigma Aldrich, USA)
and zinc chloride hexahydrate (Merck, India) were purchased from the respective outlets. All
the chemicals and solvents were of analytical grade and used as received without further
purification.
(b) Synthesis of the Schiff base and zinc-Schiff base complex
The synthetic details and spectroscopic characterization of this Schiff base, HL was
recently reported by our group [34].
The zinc-Schiff base complex was synthesized by addition of solid zinc(II) chloride
hexahydrate (0.244 g, 1 mmol) to aqueous-methanolic solution of HL (0.454 g, 2 mmol). The
yellow coloured solution of Schiff base was turned to bright yellow solution. After that, the
reaction mixture was kept for slow stirring for ~30 mins and set in open atmosphere for slow
evaporation. Bright yellow coloured single crystals of zinc-Schiff base were separated out
after 10-12 days. The crystalline compound was washed with hexane and dried over silica gel.
Finally, different spectroscopic methods were employed to establish the molecular
composition of zinc-Schiff base and the results are summarized as follows.
Yield of 1: 0.415 g (~63.5% based on metal salt) Anal. Calc. for C28H26N2O4Zn (1): C, 56.92;
H, 4.44; N, 4.74; Found: C, 56.95; H, 4.41; N, 4.80. IR (KBr pellet, cm-1; Fig. S1): 3571, 3488
(C=N), 1623,1609 (C=N); UV-Vis (1×10–4 M, λmax(abs), nm, MeOH; Fig. S2): 230, 269, 344;
1H NMR (δ ppm, 400 Mz, DMSO-d6; Fig. S3) δ = 13.79 (s, 2H), 8.94 (s, 2H), 7.41-6.92 (Ar-
13
H, 16H), 3.84 (t, 6H) ppm. C NMR (400 MHz, DMSO-d6; Fig. S4): 192.26 (HC=NH+),
163.03, 161.22 (HC=N), 153.09 (Ar-N=C), 136.81, 133.54, 132.90, 129.74, 128. 65, 119.81,

4
119.64, 117.68, 117.16, 114.53, 114.53, 112.68 (Ar-C), 56.23, 55.66 (-OCH3). ESI-Ms (m/z,
MeOH; Fig. S5): [Zn(HL)(CH3OH)2+H], 310.14 (Theoretical m/z 310.10).
2.2. Physical measurements
FT-IR spectra of the Schiff base and zinc-Schiff base were recorded with a FTIR-
8400S SHIMADZU spectrophotometer (Shimadzu, Kyoto, Japan) from 400 to 3600 cm–1 with
1
KBr pellet. H and 13C NMR spectra of the Schiff base ligand (HL) were measured on a
Bruker Advance 400 MHz spectrometer (Bruker, Massachusetts, USA) in CDCl3 at 298 K.
Steady-state absorption and other electronic bands were recorded with a JASCO V-730 UV-
Vis spectrophotometer (Jasco, Tokyo, Japan). Electrospray ionization (ESI) mass spectrum
zinc-Schiff base was recorded using a Q-tof-micro quadruple mass spectrometer. Elemental
analyses were performed on a Perkin Elmer 2400 CHN microanalyser (Perkin Elmer,
Waltham, USA). X-band EPR spectrum was recorded with a Magnettech GmbH MiniScope
MS400 spectrometer (equipped with temperature controller TC H03, Magnettech, Berlin,
Germany).
2.3. Crystal structure determination and refinement
A Rigaku XtaLABmini diffractometer equipped with Mercury 375R (2×2 bin mode)
CCD detector was employed to collect the X-ray diffraction data for zinc-Schiff base. The
data were collected with graphite monochromated Mo-Kα radiation (λ=0.71073 Å) at 298 K
using  scans. The data were reduced using CrysAlisPro 1.171.39.35c [35] and the space
group determination was done using Olex2. The structure was resolved by dual space method
using SHELXT-2015 [36] and refined by full-matrix least-squares procedures using the
SHELXL-2015 [37] software package through OLEX2 suite [38]. All hydrogen atoms were
geometrically fixed.
2.4. Hirshfeld surface analysis of zinc-Schiff base complex
Hirshfeld surfaces and 2D fingerprint plots for the zinc-Schiff base complex were
generated employing Crystal Explorer 17.5 [39] progam to understand the involvement of
intermolecular interactions in crystal packing. The function, dnorm is used to find out the ratio
of the distances of any surface point to the nearest interior (di) and exterior (de) atom and the

5
van der Waals radii of the atoms [40,41]. The normalized contact distance (dnorm) is expressed
di  rvdw de  r vdw
as
i
e
dnorm


rvdw
r vdw
i
e
...(1)
Where, revdW and rivdW denote the corresponding van der Waals radii of atoms. The details of
analysis can be obtained elsewhere [39-41].
2.5. 4-methylcatechol oxidation study
The oxidation of 4-methylcatechol (4-MC) was carried out by addition of 1×10−4
M
solution of zinc complex with 1×10−2 M of 4-MC solution in methanol. The course of catalysis
was performed under aerobic conditions. The wavelength scans of catalytic oxidation were
monitored through a UV-Vis spectrophometer with an interval of 8 minutes for 2h from 200-
700 nm [10,11,17-20,42-44].
Kinetic experiments were performed with a spectrophotometer and kinetic parameters were
analysed to understand the course of 4-MC oxidation by this zinc-Schiff base in MeOH [17-
20]. 0.04 mL 1×10–4 M of zinc-Schiff base solution was treated with 2 mL of 4-MC with a
variation of its concentration from 1×10–3 M to 1×10–2 M in order to achieve the final
concentration of zinc-Schiff base as 1×10–4 M. The catalytic conversion of 4-MC was
monitored with the progress of time at 409 nm (time scan) in MeOH [42-46]. All the kinetic
experiments were performed in triplicate.
The extraction of purified oxidation product of PPO has remained difficult because of
irreversible binding of high phenolic content in purification steps. Different scientists showed
different way of extraction for catechol oxidation product, o-quinone. Suyama and group [45]
extracted the oxidation product of 4-MC as 4-methyl-o-quinone (4MQ) in treatment with n-
butylamine under oxygen saturated environment. In general, amines preferably attack the
oxidized catechols by either Michael-type addition or through formation of a Schiff base [46].
In 2018, we were successfully isolated the single crystals of 3,5-di-tert-butyl-o-quinone in
association with the hydrogen bonded catechol in non-aqueous medium [14]. In this 4-MC
oxidation, the red coloured product was isolated employing column chromatography with n-
hexane-diethyl ether solvent mixture (9:1, v/v). The identification of the product was
confirmed by a comparison of spectral data between the extracted compound and reported

6
compound. 1H NMR data for 4-methyl-o-quinone, (CDCl3, 400 MHz,) H: 6.76 (m, 1H), 6.64
(d, 1H), 6.52 (d, 1H), 2.18 (s, 3H).
2.6. Electro-chemical analysis
BASi Epsilon-EC was employed to carry out the for cyclic voltammetric experiments in
CH2Cl2 solutions containing 0.2 M tetrabutylammonium hexafluorophosphate as supporting
electrolyte. The BASi platinum working electrode, platinum auxiliary electrode, Ag/AgCl
reference electrode were used for the measurements.
2.7. Clinical bacterial cultures and culture media
The antimicrobial property of the zinc-Schiff base was examined against few clinical
pathogenic bacteria like Bacillus SP, Enterococcus and E. coli. Clinical Microbial cultures
were procured from government medical college from Tiruchirappalli, Tamil Nadu. Muller-
Hinton agar media of Himedia Pvt. Bombay, India was used for the media for the microbial
test. The antibacterial activity was evaluated by using the Himedia zone reader.
2.7.1. Inoculums preparation
The collected clinical pathogens, Bacillus SP, Enterococcus and E.coli were inoculated
individually in 5 mL of sterile nutrient broth (NB) media and incubated at 37°C for 24h.
Thereafter, 200 µL of the fresh culture of organisms was dispensed into 30 mL sterile nutrient
broth and incubated 24 h to standardize the bacterial culture to 108 CFU/ml (colony forming
units).
2.7.2. Agar well diffusion method (Kirby-Bauer method)
The bactericidal activity of the synthesized zinc-Schiff base compound and a standard
marketed drug (Amikacin, 100 mg/2mL) was studied initially by using agar well plate method
[47]. Bacillus SP, Enterococcus and E.coli inoculums were prepared by using sterile nutrient
broth media. Mueller Hinton agar double strength media were made by autoclaving 760 mg in
100 mL. Standardized inoculums inoculate the test microorganisms on the Mueller Hinton
agar plates by using sterile cotton swabs. Four 8 mm diameter agar wells were prepared using
sterile cork-borer, and 100µL (50 mg/mL) zinc-Schiff base complex and Amikacin 5 µL (10
mg/mL) were placed on agar well using micropipette under aseptic conditions. Sterile water
used as a negative control. Agar plates were incubated for 30 min in the refrigerator to diffuse
the formulation into the agar, and finally, plates were incubated at 37˚ C for 24 h.
Antibacterial activity was evaluated by using the Himedia zone reader.

7
Preparation of stock solutions for MIC
Weight of the powder (mg) =
Volume of solution (mL) × Concentration (mg/L)
The potency of powder (mg/g)
2.7.3 Determination of MIC and MBC for zinc-Schiff base against clinical bacillus SP
The method of micro-dilution was used to establish the antibacterial potential of the
zinc-Schiff base and respective controls. A spectrophotometer (OD595 = 0.22) equivalent to
108 CFU/mL was used to fix the bacterial cultures to 0.22 optical density at 595 nm. Different
concentrations of zinc-Schiff base (100, 50, 25, 12.5, 6.25 mg/mL) and standard drug (50, 25,
12.5, 6.25, 3.125, 1.5625 mg/mL) were added in 2 mL MIC tubes as the respective controls.
100 μL of the zinc-Schiff base solutions were added to each MIC test tube. MIC tubes were
incubated overnight at 37 °C for 24 h.
2.7.4 Antimicrobial activity of zinc-Schiff base with field emission scanning electronic
microscope (FESEM) and energy dispersive X-ray analysis (EDAX)
The area showing an inhibition against the clinical Bacillus SP pathogen on the MHA plate
was removed by cutting agar, fixed by soaking in 2 % glutaraldehyde in cacodylate buffer 0.1
M for 60 min [48]. The samples were washed with 0.1 M cacodylate buffer and added in
triplicate for successive 60 min wash. Samples were placed in a freshly prepared cold
cacodylate buffer for transportation into the laboratory for FE-SEM and EDAX.
3. Results and discussion
3.1. Synthesis and formulation of the Schiff base (HL) and zinc-Schiff base complex
The Schiff base was obtained from the condensation between salicylaldehyde with o-
anisidine under reflux (Scheme 1). The synthetic route of zinc-Schiff base is presented in
Scheme 1. The zinc complex was synthesized by portionwise addition of hydrated zinc(II)
chloride to HL in aq.-methanol medium with slow thermal agitation on a magnetic stirrer. The
mole ratio between zinc chloride and HL was varied to examine the variation in molecular
composition, although the molecular formulation was determined as [Zn(HL)2Cl2] for every
reaction. The single crystals of zinc-Schiff base were obtained employing slow evaporation
technique at room temperature. The zinc complex was soluble in methanol, chloroform etc.

8
Scheme 1. Synthetic procedure of 1
3.2. Description of crystal structure and Hirshfeld surface analysis of zinc complex
Crystal structure analysis reveals that the zinc-Schiff base complex crystallizes in a
triclinic system with P1 space group. The thermal ellipsoids of the zinc complex are shown in
Fig. 1. The crystal structure refinement parameter for this zinc-Schiff base complex is
presented in Table 1. The metal centric bond distances and bond angles are presented in Table
2. The crystal structure of zinc-Schiff base complex looks very interesting in which two
azomethine-Ns of the HL abstract two protons from phenolic-OH and lead to zwitter ionic
form of the Schiff base. This phenomenon prohibits the chelation property of the Schiff base
ligand and thereby HL behaves as a simple monodentate ligand instead of tridentate chelating
ligand towards Zn(II) ion. The zinc centre in the crystal structure exists in distorted tetrahedral
coordination geometry. The Schiff base may be considered as a zwitter ion since it contains
equal number of positively- and negatively-charged functional groups. This zwitter ionic form
of HL facilitates the mono-coordination of phenoxo-O to ZnCl2 leading to a tetrahedral
structure. Two HL units in association with two chlorides coordinate with Zn(II) ion to form
tetracoordinate zinc complex. The methoxy group attached to phenyl ring in the Schiff base
remains non-reactive in the complex formation. The formation of tetrahedral geometry was
further evident from the values of metal centric bond angles [Cl1-Zn1-Cl2, 124.59(5)º; Cl1-
Zn1-O2, 104.69(9)º; Cl1-Zn1-O3, 104.42(10)º; Cl2-Zn1-O2, 103.97(10)º; Cl2-Zn1-O3;
103.73(9)º; O2-Zn1-O3, 116.31(12)º]. The average bond angle value around zinc(II) centre
was estimated as 109. 61º which deviates little from ideal tetrahedral geometry.
Hirshfeld surface analysis of zinc-Schiff base was carried out using dnorm calculation
with Crystal Explorer software (Fig. S6). Red highlighted areas represent dnorm area as well as
supramolecular interactions of 1 with its neighbouring zinc complex units. The elemental
contribution of each element with % share in close interaction with others is expressed in
Table S1. In the dnorm, blue areas are showing the engagement of π…π interactions between

9
phenyl centroid of the Schiff base while red area highlights intermolecular C-H…Cl
interactions and displays in Fingerprint plots (Fig. S7). Self-assembled architecture for the
zinc complex displayed important C-H…Cl interactions which help to develop a 3D
supramolecular structure (Fig. S8). Moreover, π…π interactions between the phenyl centroids
of the HL provide additional strength to the 3D architecture (Fig. S9, Table S2). Previously, a
crystal structure of zinc complex [ZnLCl2] containing a different Schiff base, L = 4-methyl-
2,6-di[(S)-(+)-1-phenylethylimino methyl]phenol}was reported by Das et al. [24] Although
we found the CIF of that structure in SI file; however we didn’t find the CCDC number of the
CIF in the published paper as well as in CCDC. That zinc(II) complex crystallized in
orthorhombic system with P212121 space group and produced supramolecular helices through
C-H…Cl interaction [24]. In that case, a N,O,N type tridentate Schiff base was
monoprotonated to one azomethine-N and behaves as a bidentate chelator towards Zn(II) ion.
However, in our case, the Schiff base is a O,N,O-type ligand which exists in a zwitter ionic
form and act as a monodentate ligand towards Zn(II) ion. The zinc complex crystallizes in
triclinic system with P1 space group and forms a 3D architecture employing C-H…Cl and π…π
interactions in the crystalline phase (Fig. S8, Fig. S9, Table S2). The reported value for C-
H…Cl hydrogen bond by Das et al. [24] was larger (3.665 Å in length) than that of the value in
the self-assembled architecture in our synthetic zinc complex. The values of C-H…Cl H-
bonding are of comparable range with the reported values [49,50].
3.3. Solution behaviour of the zinc-Schiff base complex
The zinc-Schiff base shows good solubility in methanol (MeOH). The zinc complex
exhibits characteristic electronic transitions at 230, 269 and 344 nm which are very close to
the electronic transitions of the Schiff base (230, 270 and 346 nm). The electronic spectra for
HL and zinc complex are displayed in Fig. S2. The electronic bands of zinc(II) complex at
230 and 269 nm are assignable to n→π* and π→π* transitions of azomethine origin [14,17-
20] and the optical band at 344 nm may be attributed as intra-ligand electronic transition [17-
20,51].
3.4. 4-methylcatechol oxidation study of the zinc-Schiff base complex
The catalytic oxidation of 4-methylcatechol (4-MC) was studied by addition of catalytic
amount of zinc-Schiff base to 100 fold of 4-MC under aerobic atmosphere at 25°C (Scheme
2).

10
Scheme 2. Catalytic oxidation by polyphenol oxidase (PPO)
The changes of absorbance upon addition of zinc complex to 4-MC solution were
observed with a UV–Vis spectrophotometer. The nature of spectral change in the course of
catalysis was monitored for 1h with 8 min interval (Fig. 2). Upon addition of 1×10-4 M zinc-
Schiff base solution to 1×10-3 M solution of 4-MC in MeOH, a new electronic band at 409 nm
with a red shift of ~16 nm was developed. This development of the electronic band at 409 nm
was a definite signature for the production of 4-methyl-o-benzoquinone species in solution
(Fig. 2) [14,18,20,42-44]. Controlled experiment was also performed in presence of catalytic
amount of HL under identical reaction set up. No significant changes of electronic bands have
been observed for 2h in the course of catalysis. This phenomenon accounts on about the non-
functional activity of HL in the catalytic oxidation of 4-methylcatechol.
The kinetics for the catalytic oxidation of 4-MC was studied following Michaelis–
Menten model of saturation kinetics and the method of initial rates was applied to find out the
kinetic parameter.
The Michaelis–Menten equation is expressed as:
V
max [S]
V =
………(2)
K +
[S]
M
Where, V indicates the velocity of reaction (rate of the reaction), Km expresses the Michaelis–
Menten constant, Vmax presents the maximum reaction velocity, and [S] is the
substrate concentration. The growth of 4-methyl-o-benzoquinone species was monitored at
409 nm with the function of time [14,18,20,34,35,41]. The profile of kinetics was plotted rate
constants vs. substrate concentration (Fig. S10). The values of kinetics parameters were
determined as Vmax(MS-1) = 4.0310-3; KM = 1.3810-3 [Std. Error for Vmax (MS-1) = 4.8110-
4; Std. Error for Km (M)= 5.5310-4]. A comparison of KM values towards the oxidation of 4-
MC by the zinc-Schiff base as well as in natural sources is summarized in Table 3 [52,53].
The zinc-Schiff base catalyzed the oxidation of 4-MC under aerobic atmosphere with high
efficacy, kcat/KM = 1.05×106 h-1.

11
3.5. Mechanistic studies on 4-methylcatechol oxidation
The electrochemical potentials of the zinc-Schiff base in absence and presence of 4-MC
were studied with cyclic voltammetry in CH2Cl2 at 295 K. The redox potential data
referenced to ferrocenium/ferrocene (Fc+/Fc) couple are summarized in Table S3. The
cyclic voltammograms of zinc complex and its reaction mixture with 4-MC are
illustrated in Fig. 3. In the cyclic voltammogram of zinc-Schiff base, two reversible
anodic peaks at 0.064 V and 0.421 V were developed for the oxidation of two
phenoxide ion in two Schiff base ligands which are assignable to phenoxide/phenoxide
radical (O−/O•−) redox couple in the solution. The zinc-Schiff base didn’t exhibit any
cathodic peak indicating the stable +2 oxidation state of zinc ion. The electrochemical
potential plots of the reaction mixture of zinc complex with 4-MC exhibits one
reversible anodic wave at -0.55 V and one irreversible cathodic peak at -1.33 V. The
appearance of anodic peak is due to oxidation of 4-methylcatechol to 4-methyl-o-
benzosemiquinone (cat/sq•−) and the irreversible cathodic wave is attributed to the
reduction of Zn2+ to Zn (Zn2+/Zn). Thus, electrochemical analysis ensures the active
participation of zinc-Schiff base and facilitates the oxidation process of catechol to
benzo-semiquinone radical formation. Therefore, zinc complex exhibits metal mediated
polyphenol oxidase activity through the radical pathway.
EPR studies were further investigated to consolidate the electrochemical observations in the
course of catalytic oxidation of 4-MC in CH2Cl2. To unveil the mechanistic insights, we have
recorded the EPR spectrum of the zinc-Schiff base in presence of 4-MC in CH2Cl2 medium
(Fig. 4). The EPR spectrum of zinc complex in presence of 4-MC at room temperature
produced a characteristic signal for the development of organic radical at g ca 2.001 which
strongly suggests that the course of catalysis was driven by generation of radical species (Fig.
4). The g value for oxidised 3,5-ditertbutylcatechol was previously reported as 2.0051 in 10-1
M Bu4NPF6 [14,54,55]. Scheme 3 presents the plausible mechanistic pathway for the course
of catalytic oxidation of 4-MC.

12
Scheme 3. Plausible mechanism for the catalytic oxidation of 4-MC
Therefore, based on the experimental outcomes of spectrophotometric titration,
electrochemical analysis and electron paramagnetic measurement it may be portrayed that 4-
MC initially binds with zinc-Schiff base (enzyme) complex to generate enzyme-substrate (ES)
adduct with concurrent development of benzo-semiquinone radical. The zinc centre thereby
supplies electron to antibonding molecular orbital of oxygen molecule to facilitate the
production of o-benzoquinone product with returning back to ES along with the production of
hydrogen peroxide. Noteworthy, if the course of 4-MC oxidation runs for longer time, there is
every possibility to progress polymerisation reaction of 4-MC. To the best of our knowledge,
our synthetic zinc-Schiff base complex will be the first zinc-Schiff base complex which
exhibits 4-MC oxidation activity in scientific literature.
3.6. Antibacterial activity
The study of the antibacterial activity of zinc-Schiff base was initially determined by the
method of well diffusion against clinical Bacillus SP, Enterococcus, and E.coli. The results of

13
the inhibition zone diameters shown in Table S4. The bactericidal efficacy of the zinc-Schiff
base on Bacillus SP Minimum was evaluated in terms of the estimation of minimum
inhibitory Concentration (MIC) and minimum bacterial concentration (MBC) (Table S5). The
MIC and MBC values were obtained as 50 and 25 mg/mL respectively against Bacillus SP
strain.
According to scientific literatures the mechanism for the destruction of cell membranes of
microbial species is well established. It is experimentally proved that the generation of
reactive oxygen species (ROS) remains the driving force to cause oxidative stress and leads to
the damage of cell membrane and DNA. This phenomenon actually destroys
microbial/bacterial species and prevents the growth of microbes effectively (Scheme 4).
Scheme 4. Zinc-Schiff base induced cell membrane destruction mechanism for Bacillus SP
In the context of evaluation of bactericidal activity of this zinc-Schiff base against few
pathogenic bacteria, Zn2+ ion interacts with the enzyme, amino acids and proteins in bacteria.
The specific interaction between zinc ion and cell membrane through electrostatic forces
destroy the plasma membrane and cause leakage of intracellular material (Scheme 4). Herein,
zinc ion prefers stronger interactions with the amino acids and proteins in bacteria and
decomposition of coordination environment of the Zn(II) ion covered by two units of Schiff
bases is actually occurred. Though the Schiff base contains three donor centres and should be
behaved as a tridentate chelator, however in reality the Schiff base acts as a simple
monodentate ligand towards Zn(II) ion. Self-protonation of the Schiff base diminishes its
chelation property and prefers stronger interactions with the amino acids / proteins in bacteria.

14
In our previous work, we studied the bactericidal effect of a mononuclear copper(II)-Schiff
base complex containing this same Schiff base [10]. The comparison of MIC values clearly
indicates that this zinc complex shows an excellent activity compared to that of the copper(II)-
Schiff base complex. Previously, Keypour et al. and Das et al. synthesized different metal
complexes using polydentate Schiff base ligands and studied their antibacterial effect towards
different gram positive and gram negative bacteria [56,57]. Their zinc-Schiff base compounds
developed good inhibition zone and MIC values against the bacterial species compared to our
synthesized zinc-Schiff base complex (Table S4 and Table S5). We also studied the
antibacterial effect of Amikacin under identical experimental conditions. Amikacin is a
medically recommended antibiotic used for a number of bacterial infections. Truly, this zinc-
Schiff base exhibits little activity compared to that of Amikacin (Table S4 and Table S5).
Most strikingly, we were able to detect the presence of 1.44% zinc in the chosen zone of
inhibition and it was confirmed by energy dispersive X-ray (EDAX) analysis which portrayed
the area in a quantitative manner (Fig.S11). This is a rare observation in the study of
bactericidal activity and portrays a real promise for the future development of antibacterial
agents. Fig. 5 displays the electron microscope scan image which shows the morphological
changes in the multiplication of clinical bacillus SP at zone inhibition site and a clear
indication of destruction of the bacterial cell membrane.
4. Conclusions
This present study reports the synthesis, crystal structure, supramolecular architecture,
bactericidal activity and bio-mimics of 4-MC oxidation of a newly developed zinc-Schiff base
complex, [Zn(HL)2Cl2]. Crystal structure analysis reveals that Zn(II) centre adopts a distorted
tetrahedral geometry. More captivatingly, the tridentate chelator actually exists in zwitter ionic
form and behaves a monodentate ligand towards zinc ion. The zinc complex exhibits good
bio-mimicking activity towards the oxidation of 4-MC with turnover number, 1.45×103 h–1.
The electrochemical potential analysis of the zinc complex in presence and absence of 4-MC
ensures the production of 4-methyl-o-benzosemiquinone (cat/sq•−) with an active involvement
of zinc(II) centre to facilitate the formation of benzo-semiquinone radical. Further, EPR
studies confirm the generation of radical species in the course of catalytic oxidation of 4-MC.
To the best of our knowledge, our synthetic zinc-Schiff base complex will be the first report to
exhibit 4-MC oxidation activity in scientific literature. The scanning electron microscopy
images indicate the destruction of bacterial cell membrane and energy dispersive X-ray

15
spectrum confirmed the existence of 1.44% zinc in the cell membrane. The incorporation of
zinc by the bacterial cell will certainly bring some new light in designing future antimicrobial
agents of good promise.
Supplementary data
Supplementary crystallographic data are available free of charge from The Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336033; E-mail:
deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk) upon request, quoting
deposition number CCDC 2020846. Experimental information such FT-IR, UV-Vis, 1H & 13C
NMR, ESI mass spectra, rate vs. [substrate] plot, Hirshfeld surface plot, bond distance & bond
angle parameters, redox potential data etc are given here.
Acknowledgement
BB thanks SERB, India for financial support under the TEACHERS ASSOCIATESHIP for
RESEARCH EXCELLENCE (TAR/000473/2018).
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19
Table 1. Crystallographic data and structure refinement parameters for zinc-Schiff base
Parameters
1
Empirical formula
C28H26Cl2N2O4Zn
Formula weight
590.78
298
Temperature (K)
Crystal system
Triclinic
P-1
Space group
a (Å)
9.2904(4)
10.6979(4)
14.9283(4)
94.357(3)
97.159(3)
114.451(4)
1326.67(10)
2
b (Å)
c (Å)
α
β
γ
Volume (Å3)
Z
ρ (gcm–3)
μ (mm–1)
F (000)
Rint
1.479
1.164
608
0.083
θ ranges (º)
2.4-32.9
Number of unique reflections
Total number of reflections
Final R indices (R1 and wR2)
Largest peak and hole (eA˚-3)
9032
24064
0.0603, 0.1919
0.67, -0.60

20
Table 2. Bond angles and bond distances value of zinc-Schiff base complex
Bond distances (Å)
Zn1-O2
Zn1-O3
1.981(3)
1.979(3)
Zn1-Cl1
Zn1-Cl2
2.2344(15)
2.2369(15)
Bond angles (º)
Cl1-Zn1-Cl2
Cl1-Zn1-O2
Cl1-Zn1 -O3
124.59(5)
104.69(9)
104.42(10)
Cl2-Zn1-O2
Cl2-Zn1-O3
O2-Zn1-O3
103.97(10)
103.73(9)
116.31(12)
Table 3. Comparison of KM values for catalytic oxidation of 4-MC in natural sources and by
zinc-Schiff base complex (4-Methylcatechol = 4-MC)
Source
Substrate with
higher affinity
Km
(mM)
Optimum
pH
Optimum
Temperature
(ºC)
References
Apple
(cv.Amasya)
Apricot
4-MC
Catechol
4-MC
3.1
34.0
7
15
[53]
5–5.5
6
25
25
[52]
[53]
Artichoke
4-MC
Catechol
Catechol
10.2
12.4
8.5
Banana
(cv.Anamur)
Cherry
7
30
[53]
4-MC
Catechol
4-MC
4-MC
4-MC
4-MC
4-MC
4-MC
4-MC
4.5
7
[52]
[53]
Cucumber
Eggplant
Litchi
5–6.5
7.4
[53]
10
70
35
[53]
Longan
6.5
[53]
Olive
5.5–7.5
5
[53]
Peach
[52]
Plum
4–5.5
5.5
[52]
1.3810-3
[Zn(HL)2Cl2]
27
This work

21
Figure 1. X-ray structure of the zinc-Schiff base complex with 30% ellipsoid probability
Figure 2. Rise of new electronic band at 409 nm after treatment of zinc complex to 4-MC in
MeOH with a time interval of 8 mins. Inset: Time vs Absorbance plot at defined wavelength

22
Figure 3. Left: Cyclic voltammogram of the zinc complex in anhydrous DCM medium;
Right: Cyclic voltammogram of zinc complex in presence of 4-MC under molecular oxygen
atmosphere in anhydrous DCM in CH2Cl2 (0.20 M [N(n−Bu)4]PF6) at 295 K.
Figure 4. X-band EPR spectrum of 4-MC in presence zinc complex after 10 mins.

23
A
B
Figure 5. Electron microscope scans showing morphological changes in pathogenic bacteria.
A: Zinc complex induced destruction of the bacterial cell membrane (MIC) and B:
morphological change of bacterial cell (MBC)
Shreya Mahato: Conceptualization, Formal analysis, Methodology, Investigation; Nishith
Meheta: Formal analysis, Visualization; K Muddukrishnaiah: Solution preparation and
study for antimicrobial activity; Mayank Joshi: Data analysis; Madhusudan Shit:
Electrochemical analysis; Angshuman Roy Choudhury: X-ray structure analysis; Bhaskar
Biswas: Writing-Reviewing and Editing, Supervision.
Synthesis, Structure, Polyphenol Oxidase Mimicking and Bactericidal
Activity of a Zinc-Schiff Base
Shreya Mahato,a Nishith Meheta,a K Muddukrishnaiah,b Mayank Joshi,c Madhusudan
Shit,d Angshuman Roy Choudhury,c and Bhaskar Biswasa,*

24
aDepartment of Chemistry, University of North Bengal, Darjeeling-734013, India
bDepartment of Pharmaceutical Technology, Anna University, BIT Campus Thiruchirappalli,
India
cDepartment of Chemical Sciences, Indian Institute of Science Education and Research,
Mohali, Sector 81, Knowledge City, S. A. S. Nagar, Manauli PO, Mohali, Punjab 140306,
India
dDepartment of Chemistry, Dinabandhu Andrews College, Kolkata 700084, India
This research work demonstrates the synthesis, crystal structure, supramolecular architecture,
4-methylcatechol oxidation and bactericidal activity of a newly designed zinc complex
containing a protonated Schiff base of zwitter ion type.
Highlights



Synthesis and crystal structure of a tetrahedral zinc-Schiff base complex
Schiff base gets protonated to exhibit monodentate behaviour
The zinc complex exhibits good efficiency towards the bio-mimetic oxidation of 4-
methylcatechol

Radical driven catalytic activity has been observed.

25

Shows good bactericidal activity with 1.44% presence of zinc in cell membrane of bacteria