Antibacterial and catecholase activities of Co(III) and Ni(II) Schiff base complexes

Article abstract of DOI:10.1016/j.poly.2016.01.015

X-ray structural analyses of two newly synthesized Schiff base-complexes, [Co(L)₃] (1) and [Ni(L)₂] (2) [HL = 2-((E)-(2-pyridine-2-pyridin-2-ylthio)ethylimino)methyl)phenol], reveal the bidentate chelation behavior of the ligand HL w

Full text of DOI:10.1016/j.poly.2016.01.015

Accepted Manuscript
Antibacterial and catecholase activities of Co(III) and Ni(II) Schiff base com-
plexes
Ayon Kanti Ghosh, Merry Mitra, Aafreen Fathima, Hareram Yadav,
Angshuman Roy Choudhury, Balachandran Unni Nair, Rajarshi Ghosh
PII:
S0277-5387(16)00019-X
http://dx.doi.org/10.1016/j.poly.2016.01.015
POLY 11777
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
29 September 2015
7 January 2016
Please cite this article as: A.K. Ghosh, M. Mitra, A. Fathima, H. Yadav, A.R. Choudhury, B.U. Nair, R. Ghosh,
Antibacterial and catecholase activities of Co(III) and Ni(II) Schiff base complexes, Polyhedron (2016), doi: http://
dx.doi.org/10.1016/j.poly.2016.01.015
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Antibacterial and catecholase activities of Co(III) and Ni(II) Schiff base complexes
Ayon Kanti Ghosha, Merry Mitraa, Aafreen Fathimab, Hareram Yadavc,
Angshuman Roy Choudhuryc, Balachandran Unni Nairb,*, Rajarshi Ghosha,*
aDepartment of Chemistry, The University of Burdwan, Burdwan 713 104, India
bChemical Laboratory, Central Leather Research Institute, Adyar, Chennai 600
020, India
cDepartment of Chemical Sciences, Indian Institute of Science Education &
Research, Mohali, Sector 81, S.A.S. Nagar, Knowledge City, Manauli PO, Mohali
140 306, India
Abstract
X-ray structural analyses of two newly synthesized Schiff base-complexes,
[Co(L)3] (1) and [Ni(L)2] (2) [HL = 2-((E)-(2-pyridine-2-pyridin-2-
ylthio)ethylimino)methyl)phenol], reveal the bidentate chelation behavior of the
ligand HL with octahedral and square planar geometries at the metal centers in 1
and 2, respectively. Both the compounds show antibacterial activity as well as
catecholase activity. The antibacterial activities of the compounds were
investigated on gram positive and gram negative bacteria; compound 2 was found
to be better in terms of antibacterial activity than 1 against both the bacteria.

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Moreover, both compounds catalyse the oxidation of 3,5-ditertiarybutylcatechol in
methanol in the presence of molecular oxygen with first order reaction kinetics and
turn over numbers 3.48 × 103 h-1 (1) and 2.68 × 103 h-1 (2).
Keyword: Cobalt, Nickel, Schiff base, Antibacterial activity, Catecholase activity
____________
*Corresponding author, E. mail: rghosh@chem.buruniv.ac.in
1. Introduction
The synthesis and characterization of coordination complexes are presently of
increased interest because of their potential application in catalysis [1], adsorption
[2], storage [3], magnetism [4], molecular recognition [5], fluorescence [6], sensors
[7], etc. Judicious choice of the metal ions and ligands often makes molecules
multifunctional instead of showing a single property [8]. Transition metals having
different geometries and oxidation numbers, and synthetically easy accessible
Schiff bases with varied denticities may be used for this purpose. As organic
imines are successful intermediates in synthesizing different biologically active
organic molecules and pharmaceutical substances [9], here we have synthesized
and characterized one nickel(II) and one cobalt(III) complex of a newly
synthesized (N,S,O) donor Schiff base ligand, 2-((E)-(2-pyridine-2-pyridin-2-

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ylthio)ethylimino)methyl)phenol (HL), having one imine group, though in each
complex the ligand binds with the metal ions using its (N,O) donor sites.
Coordination compound models of the active sites of enzymes, which are
capable of processing molecular oxygen under ambient conditions, have received a
great deal of attention [10] as they can serve as efficient catalysts for synthetic
transformations of industrial importance [11]. Hence modelling coordination
compounds of metalloenzymes with oxidase (oxygenase) activity in particular are
very important for the development of bioinspired catalysts for oxidation reactions
[12]. In parallel, designing synthetic coordination compounds with better
antimicrobial activity than the commercially available antibiotics is also of
immense importance in medicinal chemistry [13,14].
In this present endeavor, our newly synthesized Schiff base-complexes [Co(L)3]
(1) and [Ni(L)2] (2) show more than one function, such as antimicrobial and
catecholase activity. In addition to the activity of the complexes against microbes,
each of these serves as models for the plant enzyme catecholase and hence
catalyses the oxidation of 3,5-ditertiarybutyl catechol (3,5-DTBC) to 3,5-
ditertiarybutyl quinone (3,5-DTBQ) in the presence of aerial oxygen.
2. Experimental
2.1. Materials

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2-Mercaptopyridine, 2-chloroethylamine and salicyaldehyde were purchased
from Sigma Aldrich and used as received. Cobalt(II) acetate tetrahydrate and
nickel(II) acetate tetrahydrate were purchased from E. Merck India. Solvents were
of reagent grade and used without further purification.
The gram positive bacteria Bacillus subtilis was received from the
biotechnology department of Indian Institute of Technology Madras (IITM) and
E.coli MTCC 40 gram negative bacteria was procured from the Microbial type
culture collection (MTCC), Chandigarh, India. Growth media, such as Nutrient
agar and Nutrient broth, were obtained from Hi-Media (Mumbai, India). The
antibiotic discs and sterile filter paper discs were obtained from Hi-Media
(Mumbai, India). All other chemicals used were of analytical grade procured from
SD Fine chemicals, India.
2.2. Physical measurements
Elemental analyses (carbon, hydrogen and nitrogen) were performed on a
Perkin-Elmer 2400 CHNS/O elemental analyzer. UV-VIS and IR spectra (KBr
discs, 4000-300 cm-1) were recorded using a Shimadzu UV-VIS 2450
spectrophotometer and Perkin-Elmer FT-IR model RX1 spectrometer, respectively.
2.3. Zone of inhibition by the Agar diffusion method

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The antibacterial activity was measured by the Kirby Bauer disc diffusion
method [15] against gram positive bacteria (Bacillus Subtilis) and gram negative
bacteria (E.coli MTCC 40). An overnight culture adjusted to 0.5 Mac Farland
turbidity was used for swabbing nutrient agar plates (comprising of 3 g/l meat
extract, 5 g/l peptic digest of animal tissue and 15 g/l of agar) to do a lawn culture
of the test bacterium. Then using sterile forceps, plain filter paper discs and
antibiotic loaded discs were placed onto the agar plates. 20 µl of varying
concentrations (0.1, 1 and 2.5 µg/disc) of test sample were loaded over the filter
paper discs. The plates were incubated at 37º C for 24 h and observed for the zone
of inhibition around the discs. Commercial antibiotics such as Ampicillin and
Cephotaxime were used as standards for comparison.
2.4. Minimum Inhibitory Concentration (MIC)
The MIC was carried out at different concentrations ranging from 100, 50,
25 to 1.56 µg/ml using nutrient broth (Comprising of 5 g/l peptone, 1.5 g/l beef
extract, 1.5 g/l yeast extract, 5g/l NaCl) by the broth dilution method [16]. The
tubes containing the growth medium were serially diluted with the test sample,
followed by the addition of a bacterial inoculum of 0.5 Mac Farland turbidity. The
tubes were incubated at 37º C for 24 h and the MIC’s were recorded as the tube
showing no visible growth after comparing with the positive controls.

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2.5.Minimum Bactericidal Concentration (MBC)
The MBC (Minimum Bactericidal Concentration) was determined by spot
inoculating a nutrient agar plate with the same tubes used for the MIC
measurements, having varying concentrations of the test sample. The nutrient agar
plates were incubated at 37 °C for 24 h and the MBC was recorded as the lowest
concentration of the test sample which did not produce any bacterial growth on the
nutrient agar medium [17].
2.6.Time kill assay
A time kill assay for the test sample was carried out by the modified plating
technique [18]. To tubes containing 8 ml of nutrient broth, 1 ml of the test sample
of MIC and 2 x MIC concentration was incorporated, followed by 1 ml of bacterial
inoculum, so that the overall volume of the tubes was 10 ml. The tubes, along with
the controls, were incubated at 37 °C with moderate shaking in an orbital shaker.
Samples were collected after 0, 4, 8 and 12 h incubation to determine the cfu/ml by
the plate count technique.
2.7.Synthesis of HL

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The ligand’s synthesis is depicted in Scheme 1. 2-Mercaptopyridine (8
mmol, 0.88 g) was added gradually to a sodium ethoxide solution with continuous
stirring. Then 2-chloroethylamine (8 mmol, 0.63 g) was mixed slowly to this
reaction mixture and the mixture was stirred for about 1 hour. During this stirring
precipitation of sodium chloride confirms the formation of 2-(pyridin-2-
1
ylthio)ethanamine (Scheme 1). This product was characterized by H NMR.
Sodium chloride was separated by filtration. Addition of salicyaldehyde (8 mmol,
0.97 g) to this solution of 2-(pyridin-2-ylthio)ethanamine with a four hour reflux
gives the ligand HL.
Scheme 1
Yield (2-(pyridin-2-ylthio)ethanamine): 0.98 g (80%). Anal cal. for
C7H10N2S: C, 54.51; H, 6.54; N, 18.17; Found: C, 54.01; H, 6.21; N, 18.20%. IR
(KBr pellet, cm-1): 756, 1557, 1576. UV-Vis (λ, nm, MeOH): 248 (ε = 6.51 × 103),
287 (ε = 4.81 × 103), 359 (ε = 1.05 × 103) 1H NMR δ (ppm): 2.11 (bs, 2H), 2.99 (t,
J = 6.4 Hz, 2H), 3.28 (t, J = 6.4 Hz, 2H), 4.50 (bs, 2H), 6.95-6.98 (m, 1H), 7.18 (d,

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J = 8.0 Hz, 1H), 7.44-7.48 (m, 1H), 8.40 (d, J = 4.8 Hz, 1H) (Fig. S1, Supporting
Information).
Yield (HL): 1.15 g (70%). Anal cal. for C14H14N2SO: C, 65.08; H, 5.46; N,
10.84; Found: C, 65.01; H, 5.11; N, 10.91%. IR (KBr pellet, cm-1): 757, 1557,
1574, 1626. UV-Vis (λ, nm, MeOH): 213 (ε = 1.70 × 104), 251 (ε = 1.23 × 104),
1
289 (ε = 4.60 × 103), 402 (ε = 5.57 × 102). H NMR δ (ppm): 3.51 (t, J = 6.4 Hz,
2H), 3.91 (td, J = 6.4, 0.8 Hz, 2H), 6.85 (td, J = 7.6, 0.8 Hz, 1H), 6.94-6.98 (m,
2H), 7.16 (dd, J = 8.4, 0.8 Hz, 1H), 7.23 (dd, J = 7.6, 1.6 Hz, 2H), 7.27-7.31 (m,
1H), 7.44 (td, J = 7.6, 1.6 Hz, 1H), 8.34 (s, 1H), 8.41-8.43 (m, 1H), 13.32 (bs, 1H)
(Fig. S2, Supporting Information)
2.8.Synthesis of compound 1
Solid cobalt(II) acetate tetrahydrate (0.1 mmol, 0.02 g) was slowly added to
a stirring solution of HL (0.1 mmol, 0.02 g) in ethanol. The mixture was stirred
with a little warming until all the cobalt(II) salt was dissolved. The brown colour of
the ligand solution intensified on dissolution of the metal salt. Brown coloured X-
ray quality crystals of 1 appeared after keeping the solution in the open air after a
week or so.
Yield: 0.04 g (60%). Anal cal. for C42H39N6S3O3Co: C, 60.71; H, 4.73; N,
10.11; Found: C, 60.59; H, 4.68; N, 10.21%. IR (KBr pellet, cm-1): 750, 1576,
1609. UV-Vis (λ, nm, MeOH): 250, 390, 600.

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2.9.Synthesis of Compound 2
An ethanolic solution of nickel(II) acetate tetrahydrate (0.1 mmol, 0.02 g)
was added dropwise to the ligand HL (0.1 mmol, 0.25 g) in ethanol with gentle
stirring. The brown coloured ligand solution changed to light green. The reaction
mixture was kept in the open air. After 2 days green coloured block shaped crystals
of 2 appeared.
Yield: 0.0315 g (55%). Anal cal. for C28H26N4S2O2Ni: C, 58.66; H, 4.57; N,
9.77; Found: C, 58.50; H, 4.51; N, 9.91%. IR (KBr pellet, cm-1): 3452, 1608, 756.
UV-Vis (λ, nm, MeOH): 218, 244, 372, 610.
2.10.X-ray diffraction study
Single crystal X-ray diffraction data were collected using a
RigakuXtaLABmini diffractometer equipped with a Mercury CCD detector. The
data were collected with graphite monochromated Mo-Kα radiation (λ = 0.71073
Å) at 100 K using ω scans. The data were reduced using Crystal Clear suite 2.0
[19] and the space group determination was done using Olex2 [20]. The structure
was resolved by direct methods and refined by full-matrix least-squares procedures
using the SHELXL [21] package with the OLEX2 suite. The crystal data and

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refinement details are listed in Table 1. All the figures have been generated using
ORTEP-32 [22].
3. Results and discussion
3.1. Synthesis of the ligand and the complexes
The ligand HL was synthesized in two steps. Initially 2-mercaptopyridine
was refluxed with 2-chloroethylamine in dry ethanol in the presence of sodium
ethoxide to produce 2-(pyridin-2-ylthio)ethanamine. The organic product was
extracted and the solvent was evaporated as a yellow solid. This was characterized
using spectroscopic tools. 2-(Pyridin-2-ylthio)ethanamine was further refluxed
with salicyalidehyde to synthesize HL. In the IR spectrum of HL, the band at 756
cm-1 was assigned as a υC-S stretching frequency, whereas bands at 1557 and 1576
cm-1 are due to asymmetric υC=N frequencies. The 1H NMR spectrum of 2-(pyridin-
2-ylthio)ethanamine in CDCl3 showed the characteristic peaks of the protons. The
signals of the amine protons of 2-(pyridin-2-ylthio)ethanamine were at the δ 2.11
ppm (bs, 2H). Two triplet signals for the four protons of the two methylene groups
were observed at δ 2.99 ppm (t, J = 6.4 Hz, 2H) for one methylene group α to the
NH2 group and δ 3.28 ppm (t, J = 6.4 Hz, 2H) for that β to the amine group. The
signals of the three aromatic protons were found at different δ values, namely 6.95-
6.98 (m, 1H), 7.18 (d, J = 8.0 Hz, 1H) and 7.44-7.48 ppm (m, 1H). One doublet

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signal in the deshielded region, at δ 8.48 ppm (d, J = 4.8 Hz, 1H) is attributable to
1
the proton in an ortho position to the pyridine N atom. The H NMR spectrum of
HL in CDCl3 also showed the characteristic peaks of the protons. The two peaks at
δ 3.51 ppm (t, J = 6.4 Hz, 2H) for one methylene group α to the NH2 group and δ
3.91 ppm (td, J = 6.4 , 0.8 Hz, 2H) for another β to the amine group were shifted
downfield after condensation. The imine proton was confirmed by a peak
appearing at δ 8.34 ppm (s, 1H). The peaks for the aromatic protons in the ortho
position to the pyridine N atom were observed at δ 8.41-8.43 ppm (m, 1H). The
disappearance of the amine peak at δ 2.11, the simultaneous downfield shift of two
methylene peaks and the formation of an imine proton peak at δ 8.34 ppm proves
the formation of the imine bond.
Compounds 1 and 2 were synthesized in good yield mixing the metal(II)
acetates with the ligand HL in methanol at room temperature. In the IR spectra of
the complexes the bands at ~1576-1609 cm-1 were attributed to the asymmetric
υC=N stretching and the band at ~750 cm-1 was detected for the υC-S stretching.
3.2. X-ray structures
Thermal plots of 1 and 2 are given in Figs. 1 and 2, respectively. From the
bond angle and bond distance data (Table 2), the geometry around the Co(III) ion
can be described as a distorted octahedron with a meridional conformation. Four of

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the three coordination sites in the horizontal plane of the octahedron are occupied
by the imine nitrogen atoms (N1, N2 and N3) of the Schiff base. The remaining
position in the octahedral horizontal plane is occupied by the phenoxo oxygen
atom (O1) of the ligand framework. The other two phenoxo oxygen atoms (O2 and
O3) are placed in the axial positions. The bond distances in the horizontal plane are
in the range 1.882(3)-1.962(3) Å. The dihedral angle between the planes
containing the two rings of each ligand in 1 comprising the atoms N4-C10-C11-
C14-C13-C12 and C7-C6-C2-C3-C4-C5 is 41.30°, that of N6-C38-C39-C40-C41-
C42 and C35-C31-C30-C32-C33-C34 is 42.36° and that of N5-C24-C28-C25-C26-
C27 and C21-C16-C19-C17-C20-C18 is 53.97°.
In the same way, considering the bond angle and bond distance data (Table
3), the coordination environment around the Ni(II) ion is best described as a
distorted square planar geometry with the imine nitrogen atoms (N1 and N1') and
phenoxo oxygen atoms (O1 and O1') in mutually trans positions. The dihedral
angle between the planes containing the two rings of each ligand in 2 comprising
the atoms N2-C10-C11-C12-C14-C13 and C1-C6-C5-C3-C2-C4 is 81.87°.
3.3. Antibacterial activity
The antibacterial activity of the ligand (HL) and compound 1 was measured
by the Kirby Bauer disc diffusion method against gram positive bacteria (Bacillus

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Subtilis) and gram negative bacteria (E.coli MTCC 40). The concentrations of the
compounds tested were 0.1, 1 and 2.5 µg/disc. For standards, two commercial
antibiotics were used, namely Cephotaxime – Ce10 and Ampicillin – AMP10
(Tables 4 and 5).
The zone of inhibitions for the maximum concentration (2.5 µg/disc) tested
against Bacillus subtilis and E. coli were found to be 13 and 12 mm, respectively
for HL and 12 and 10 mm, respectively for 1. The respective minimum inhibitory
concentration (MIC) results for Bacillus subtilis and E.coli were found to be 50
and 100 µg/ml. The minimum bacterial concentration (MBC) results for both HL
and 1 for E.coli were undetermined as the concentrations tested against E.coli were
not able to inhibit the growth of the bacteria even at 100 µg/ml, whereas for
Bacillus subtilis the MBC was found to be 100 µg/ml.
From the rate of kill assay results (for 1) it could be concluded that the
compound does not possess profound bactericidal activity for Bacillus subtilis and
E.coli, even at 2 × MIC concentration. The time kill assay data is presented in
terms of log10 cfu/ml in Table 6. The difference in the antibacterial activity might
be due to the type and composition of the bacterial cell wall as well as membrane
permeabilization, which is why upon comparison the compound shows fair activity
against gram positive bacteria (Bacillus subtilis) and was not active against gram
negative bacteria (E.coli) [23].

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The antibacterial activity of compound 2 was studied by the Agar disc
diffusion method and it was found that out of the three concentrations tested the
compound showed better inhibition at a concentration of 2.5 µg/ 20 µl for Bacillus
subtilis, with an inhibition zone of 15.5 mm, as compared to E.coli, which showed
10.25 mm as the zone of inhibition for the same concentration tested (Table 7). To
determine the minimum amount of sample required to inhibit the bacterial growth,
the MIC was measured and it was found that out of the various concentrations
tested (100, 50, 25, 12.5, 6.25, 3.12, 1.56 µg/ml), the growth of Bacillus subtilis
was inhibited at 12.5 µg/ ml, whereas the growth of E.coli was inhibited at 25
µg/ml. The MBC values were found to be 25 µg/ml for Bacillus subtilis and 50
µg/ml for E.coli.
In this study compound 2 was found to be more active towards gram positive
bacteria, Bacillus subtilis. The reason for this might be differences in the bacterial
cell wall composition and its thickness, as gram negative bacteria have an extra
layer of lipopolysaccharide (LPS) [24]. The permeability barrier of the cell
membranes also plays an important role in allowing the penetration of the
antibacterial agent [23]. From the rate of kill assay results it could be concluded,
after comparing the reduction in viable cell count, that the compound was found to
be bactericidal for Bacillus subtilis at 2 × MIC concentration (25 µg/ml), showing
no growth or colony formation. For E. coli the compound showed no significant

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bactericidal activity at the MIC (25 µg/ml) or at 2 × MIC (50 µg/ml) concentration.
The time kill assay data is presented in terms of log10 cfu/ml in Table 8. The
difference in the antibacterial activity might be due to the type and composition of
the bacterial cell wall as well as membrane permeabilization which is why the
compound shows better activity against gram positive bacteria (Bacillus subtilis)
than gram negative bacteria (E.coli) [23].
3.4.Catecholase activity of 1 and 2: spectrophotometric study
For the study of the catecholase activity of complexes 1 and 2, 3,5-DTBC,
with two bulky t-butyl substituents on the ring and a low quinone-catechol
reduction potential, has been chosen as the substrate. This makes it easily
oxidizable to the corresponding o-quinone, 3,5-DTBQ, which is highly stable and
shows a maximum absorption at 401 nm in methanol. Solutions of 1 and 2 were
treated with 100 equivalents of 3,5-DTBC under aerobic conditions. Repetitive
UV-Vis spectral scans for both complexes were recorded in pure MeOH (Figs. 3
and 4). Spectral bands at 250, 390 and 600, and 218, 244, 372 and 610 nm
appeared in the electronic spectra of complexes 1 and 2, respectively whereas 3,5-
DTBC shows a single band at 282 nm. After addition of 3,5-DTBC, the time
dependent spectral scans show a very smooth growth of the quinone band at 401
nm, as reported by Krebs et al [25], concomitant with a decrease in the

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characteristic 282 nm band for 3,5-DTBC, which indicates the formation of the
respective quinone derivative, 3,5-DTBQ, that was subsequently purified by
column chromatography. The product was isolated in high yield (71.1 and 75.8%
for 1 and 2, respectively) by slow evaporation of the eluant and was identified by
1
1H NMR spectroscopy (Figs. S3 and S4; Supporting information). H NMR
(CDCl3, 400 MHz) for 1, δH (ppm): 1.22 (s, 9H), 1.26 (s, 9H), 6.21 (d, J = 2.4 Hz,
1H), 6.93 (d, J = 2.0 Hz, 1H). For 2, δH (ppm): 1.16 (s, 9H), 1.20 (s, 9H), 6.15 (d, J
= 2.4 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H).
To find out the comparative reaction velocity between 3,5-DTBC and the
complexes, the reaction kinetics between the complexes and 3,5-DTBC was
studied by observing the time dependent change in absorbance at a wavelength of
401 nm, which is characteristic of 3,5-DTBQ in methanol. The colour of the
solution gradually turned deep brown, indicative of the gradual conversion of 3,5-
DTBC to 3,5-DTBQ. The difference in absorbance ∆A at 401 nm, was plotted
against time to obtain the velocity for that particular catalyst to substrate
concentration ratio (Figs. 5 and 6). An overall first-order catalytic reaction was
observed in the slowest rate-determining step, with velocities 2.54 × 10-3 and 2.77
× 10-3 min-1 for 1 and 2 respectively. Both the reactions are zero order with respect
to O2 (Figs. S5 and S6; Supporting information). The overall rate constants (kobs)

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are 0.02341 min-1 for 1 and 0.02345 min-1 for 2 (Figs. S7 and S8; Supporting
information).
3.5. Enzyme kinetics study
Enzymatic
kinetic
experiments
were
performed
UV-Vis
spectrophotometrically, thermostated at 25 °C, using complex 1 and 2 with the
substrate 3,5-DTBC in MeOH. 0.04 ml of each complex solution, with a constant
concentration of 1 × 10-4 M, was added to 2 ml of 3,5-DTBC of a particular
concentration (varying its concentration from 1 × 10-3 to 1 × 10-2 M) to achieve an
ultimate concentration of the complex of 1 × 10-4 M. The conversion of 3,-5-DTBC
to 3,5-DTBQ was monitored with time at a wavelength of 401 nm for solutions in
MeOH. The rate for each concentration of the substrate was determined by the
initial rate method.
The rate versus concentration of substrate data were analyzed on the basis of
the Michaelis-Menten approach of enzymatic kinetics to get the Lineweaver-Burk
(double reciprocal) plot as well as the values of various kinetic parameters, namely
Vmax, KM and Kcat. The observed rate vs. [substrate] plot in methanol solution, as
well as the Lineweaver-Burk plot, is given in Figs. 7 and 8 for 1 and 2 respectively.
The kinetic parameters are listed in Table 9. The turnover numbers (Kcat) are 3.48 ×
103 (1) and 2.68 × 103 h-1 (2).

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3.6. Mechanism of catecholase activity
In order to obtain a mechanistic inference of the catecholase activity and to
get an idea about the complex-substrate intermediate, we recorded the ESI-MS
spectrum (Figs. S9a and S9b; Supporting Information) of a 1:100 mixture of
complex 1 and 3,5-DTBC. The protonated complex [Co(L)3H]+ exhibits a peak at
m/z = 831. From the ESI-MS analysis we can predict that there is a detachment of
a ligand moiety from the complex, which is evident from an m/z peak at 574 for
the species [Co(L)2]+. Further detachment of the ligand is confirmed by a peak at
m/z = 316 due to the formation of [Co(L)]2+. The peak at m/z = 243 can be
assigned to a sodium aggregate of quinone [3,5-DTBQ-Na]+. The intermediate
Co(II) species [Co(L)2(dtbq)2] (Scheme S1, Supporting Information) is identified
by a peak at m/z = 1015. The Co(III) complex is reduced to Co(II) in the
intermediate state and 3,5-DTBC is oxidised to quinone in the presence of
molecular oxygen.
Similarly, we recorded the ESI-MS spectrum (Figs. S10a and S10b;
Supporting Information) of a 1:100 mixture of complex 2 and 3,5-DTBC. The
signal at m/z = 259 is due to the formation of the protonated ligand [(HL)H]+. The
protonated complex [Ni(L)2H]+ exhibits a peak at m/z = 573. The peak at m/z =
243 can be assigned to a sodium aggregate of quinone [3,5-DTBQ-Na]+. We can

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also predict here that there is a detachment of a ligand moiety from the complex
[Ni(L)2]. This is evident from the m/z peak at 315 for the species [Ni(L)]+.
Formation of a sodium aggregate of the intermediate species [Ni(L)(DTBC)Na]
(Scheme S2; Supporting Information) is identified by a peak at m/z = 558. The
molecular oxygen that takes part in both oxidation processes for complexes 1 and 2
is converted to H2O2. The liberated H2O2 is identified and characterized
spectrophotometrically (Fig. S11; Supporting Information) [26].
4. Conclusions
We have designed and synthesized two new compounds, [Co(L)3] (1) and
[Ni(L)2] (2), which show antibacterial and catecholase activities. The antibacterial
activities of both compounds, along with the ligand, were investigated on gram
positive (Bacillus subtilis) and gram negative bacteria (E.coli). Compound 2 was
found to be more active against Bacillus subtilis than E.coli, whereas compound 1
showed fair activity against Bacillus subtilis and was inactive against E.coli. The
overall comparison of the compounds based on antibacterial activity suggests
compound 1 is more active against both the bacteria than compound 2. In parallel,
the compounds show activity in catalyzing the oxidation of 3,5-
ditertiarybutylcatechol to 3,5-ditertiarybutylquinone in methanol solvent in the
presence of molecular oxygen. The reactions follow first order kinetics with

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ꢀ
reaction rates of 2.54 × 10-3 and 2.77 × 10-3 min-1, respectively. From the
Michaelis-Menten reaction kinetics, the turnover numbers are calculated as 3.48 ×
103 (1) and 2.68 × 103 h-1 (2).
5. Supplementary data
CCDC 1415640 (1) and 1415641 (2) contain the supplementary
crystallographic data for 1 and 2. This data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
6. Acknowledgements
RG sincerely thanks the Department of Science & Technology, Government
of West Bengal [No. 781(Sanc.)/ST/P/S&T/4G-4/2013 dated 04-12-2014] for
financial assistance. AKG and MM thank CSIR, New Delhi and The University of
Burdwan, respectively for their fellowships.
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Table 1. Crystal data and structure refinement parameters of 1 and 2
Emperical formula
Formula weight
T (K)
C42H39N6O3S3Co (1)
830.90
C28H26N4O2S2Ni (2)
573.38
100
100
Wavelenght (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
0.71073
0.71073
Monoclinic
P21/n
Monoclinic
P21/c
10.870(3)
25.355(5)
14.332(3)
5.1294 (8)
9.9200 (17)
24.111 (4)
b (Å)
c (Å)

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ꢀ
α (˚)
90
90
β (˚)
102.257(7)
95.124 (10)
90
γ (˚)
90
V (Å3)
3859.8(14)
1222.0 (4)
4
Z
4
Dcalc (Mg m-3)
1.430
1.558
Absorption coefficient (mm-1) 0.656
1.001
F(000)
1728
596
Crystal size (mm3)
0.1 0.1 × 0.05
0.2 0.2 × 0.2
Theta range for data collection 3.018-27.546
(˚)
3.270-27.486
Index ranges
-14 ≤ h ≤ 13, -32 ≤ k
-6 ≤ h ≤ 6, -12 ≤ k
≤12,
≤24, -18 ≤ l ≤18
-31 ≤ l ≤31
10477
Reflections collected
24548
Independent reflections
8815 [Rint= 0.0751]
2802 [Rint=
0.0969]
Completeness of theta
Absorption correction
99.0 % [θ=25.242]
99.5 % [θ=25.242]
Multi-scan
Multi-scan

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ꢀ
Tmax and Tmin
1.000 and 0.897
Full matrix
1.000 and 0.796
Full matrix
Refinement method
Data/restrains/parameters
Goodness-of fit (GOF) F2
Final R index [l >2σ(l)]
8815/0/ 496
2802/0/ 169
1.049
1.069
R1 = 0.0685 and
wR2 = 0.1381
R1 = 0.1112 and
wR2 = 0.1680
0.768, -0.572
R1 = 0.0644 and
wR2 = 0.1253
R1 = 0.1040 and
wR2 = 0.1564
1.202, -0.488
R index (all data)
Largest difference between
peak and hole (e Å-3)
Table 2. Bond lengths (Å) and bond angles (˚) data of 1
Bond lengths
Co(1)-O(1)
1.882(3)
1.892(3)
1.890(3)
Co(1)-N(1)
Co(1)-N(2)
Co(1)-N(3)
1.962(3)
1.949(3)
1.950(3)
Co(1)-O(2)
Co(1)-O(3)
Bond angles
O(3)-Co(1)-O(2)
O(3)-Co(1)-N(2)
171.98(11)
93.80(12)
O(1)-Co(1)-O(2)
O(1)-Co(1)-N(2)
86.75(12)
171.29(12)

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ꢀ
O(3)-Co(1)-N(1)
86.19(12)
92.74(13)
91.26(12)
87.22(12)
93.70(13)
89.16(12)
O(1)-Co(1)-N(1)
O(1)-Co(1)-N(3)
N(2)-Co(1)-N(1)
N(2)-Co(1)-N(3)
N(3)-Co(1)-N(1)
O1 Co1 N2
94.10(12)
84.18(12)
94.27(13)
87.49(13)
178.00(13)
171.29(12)
O(3)-Co(1)-N(3)
O(2)-Co(1)-N(2)
O(2)-Co(1)-N(1)
O(2)-Co(1)-N(3)
O(1)-Co(1)-O(3)
Table 3. Bond lengths (Å) and bond angles (˚) data of 2
Bond lengths
Ni(1)-O(1)
Ni(1)-N(1)
1.823(3)
1.925(4)
Ni(1)-O(1')
Ni(1)-N(1')
1.823(3)
1.925(4)
Bond angles
O(1)-Ni(1)-N(1)
O(1')-Ni(1)-N(1')
O(1)-Ni(1)-N(1)
92.90(15)
92.90(15)
87.10(15)
O(1')-Ni(1)-N(1') 87.10(15)
O(1)-Ni(1)-O(1') 180.0
N(1)-Ni(1)-N(1') 180.0
Table 4. Zone of inhibition by the disc diffusion method for HL

29ꢀ
ꢀ
Bacteria
Zone of Inhibition (mm)
Cephotaxime
Ampicillin
HL
(10 µg / Disc)
(10 µg / Disc)
2.5 µg/
Disc
1µg/
Disc
0.1 µg/
Disc
13 1.6 12 1.4
27 1.8
18 0.7
Bacillus
Subtilis
12
1.1
E.coli
12 1
10 1.2
24 0.8
10 0.5
10
1.4
MIC (Minimum Inhibitory Concentration)
Concentrations tested: 100, 50, 25, 12.5, 6.25, 3.12, 1.56 µg / ml
Bacillus subtilis - 50 µg/ml, E.coli - 100 µg/ml
MBC (Minimum Bactericidal Concentration)
Concentrations tested: 100, 50, 25, 12.5, 6.25, 3.12, 1.56 µg / ml
Bacillus subtilis - 100 µg/ml; E.coli - > 100 µg/ml