Synthesis and structure of a cobalt(III) complex containing pendant Schiff base ligand: Exploration of its catechol oxidase and phenoxazinone synthase like activity

Article abstract of DOI:10.1016/j.ica.2018.05.025

A new cobalt(III) complex, [Co(L-κ-N,N,O)(L-κ-N,O)(NCS)]·0.5H₂O, with a pendant Schiff base {HL = 2((2-morpholinoethylimino)methyl)-6-ethoxyphenol} has been synthesized and characterized by elemental and several spectral analyses. Single crystal X-ray diffraction studies confirmed its structure. Extended supra-molecular assemblies were generated in the complex through weak noncovalent interactions. The complex was found to exhibit catechol oxidase and phenoxazinone synthase mimicking activity.

Full text of DOI:10.1016/j.ica.2018.05.025

Accepted Manuscript
Research paper
Synthesis and structure of a cobalt(III) complex containing pendant Schiff base
ligand: Exploration of its catechol oxidase and phenoxazinone synthase like
activity
Kousik Ghosh, Michael G.B. Drew, Shouvik Chattopadhyay
PII:
S0020-1693(17)31935-7
https://doi.org/10.1016/j.ica.2018.05.025
ICA 18276
DOI:
Reference:
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Inorganica Chimica Acta
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Revised Date:
Accepted Date:
27 December 2017
14 May 2018
19 May 2018
Please cite this article as: K. Ghosh, M.G.B. Drew, S. Chattopadhyay, Synthesis and structure of a cobalt(III)
complex containing pendant Schiff base ligand: Exploration of its catechol oxidase and phenoxazinone synthase
like activity, Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/j.ica.2018.05.025
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Synthesis and structure of a cobalt(III) complex containing pendant
Schiff base ligand: Exploration of its catechol oxidase and
phenoxazinone synthase like activity
[a]
[b]
[a]
Kousik Ghosh, Michael G. B. Drew, Shouvik Chattopadhyay*
[a]
Department of Chemistry, Inorganic Section, Jadavpur University, Kolkata 700 032, India
e-mail: shouvik.chem@gmail.com Tel. +91-33- 2414-2941
[b]
School of Chemistry, The University of Reading, P.O. Box 224, Whiteknights, Reading RG6
6
AD, UK. E-mail: m.g.b.drew@reading.ac.uk
Abstract
A new cobalt(III) complex, [Co(L--N,N,O)(L--N,O)(NCS)]·0.5H O, with a pendant
2
Schiff base {HL =2((2-morpholinoethylimino)methyl)-6-ethoxyphenol} has been synthesized
and characterized by elemental and several spectral analyses. Single crystal X-ray diffraction
studies confirmed its structure. Extended supra-molecular assemblies were generated in the
complex through weak noncovalent interactions. The complex was found to exhibit catechol
oxidase and phenoxazinone synthase mimicking activity.
Keywords: Cobalt(III); Pendant Schiff base; Crystal structure; catechol oxidase mimicking
activity; Phenoxazinone synthase mimicking activity.
1
. Introduction

The efficiency and selectivity of copper containing metalloenzymes in processing
molecular dioxygen have lead synthetic inorganic chemists and coordination chemists to
investigate the structural and catalytic properties of these enzymes [1-7]. Two such important
copper containing metalloenzymes are catechol oxidase and phenoxazinone synthase, both of
which have important roles in the bio-degradation of aromatic molecules [8-10]. Catechol
oxidase catalyzes the oxidation of catechols into o-quinones, whereas phenoxazinone synthase
catalyzes the oxidative coupling of o-aminophenol derivatives to form the 2-
aminophenoxazinone chromophore, with concomitant reduction of molecular dioxygen to water
in each case [11-13]. To investigate the ability of transition metal complexes to mimic the
activity of these enzymes is an active area of research at the interface of chemistry and biology
[
14-18]. It is to be noted here that many transition metal complexes have already been
synthesized and their ability to be used as functional models of these metalloenzymes have been
examined extensively [19-21].
We prepared a series of mononuclear cobalt(III) Schiff base complexes with pseudo-
halide co-ligand and explored their catalytic activity towards the oxidation of 3,5-di-
tertbutylcatechol (DTBC) and o-aminophenol (OAPH) {catechol oxidase and phenoxazinone
synthase mimicking activities} [22,23]. It was observed that bis-(Schiff base ligand) complexes
were inactive towards the above catalysis. In the present work, our primary aim is to develop a
new bis-(Schiff base ligand) cobalt(III) complex, which may have the potential to mimic the
functions of these oxidase enzymes. For this purpose, a tridentate Schiff base, HL [2((2-
morpholinoethylimino)methyl)-6-ethoxyphenol], has been used to prepare a new cobalt(III)
complex, [Co(L--N,N,O)(L--N,O)(NCS)]·0.5H O. The complex is attractive in the sense that
2
one molecule of the Schiff base is exhibiting tridentate bonding mode and the other molecule of

it is showing bidentate bonding mode keeping a pendant side arm. The structure of the complex
has been confirmed by single crystal X-ray crystallography. The complex exhibits catechol
oxidase and phenoxazinone synthase mimicking activities.
2
. Experimental Section
All chemicals and solvents used in this work were commercially available and used
without further purification. Cobalt(II) acetate tetrahydrate and sodium thiocyanate were
purchased from Mark, India. All other chemicals and solvents were purchased from Sigma-
Aldrich.
2
2
.1. Preparations
.1.1. Preparation of [Co(L--N,N,O)(L--N,O)(NCS)]·0.5H O
2
A methanol solution (20 ml) of 4-(2-aminoethyl)morpholine (0.13 mL, 1 mmol) and 3-
ethoxysalicyldehyde (166 mg, 1 mmol) was refluxed for ca. 1 h to prepare the tridentate Schiff
base ligand, HL. A methanol solution (5 ml) of cobalt(II) acetate tetrahydrate (1 mmol, 249 mg)
was then added to it, followed by the addition of a methanol solution (5 ml) of sodium
thiocyanate (1 mmol, 81 mg) with constant stirring. The stirring was continued for additional 2 h.
Dark brown single crystals, suitable for X-ray diffraction, were started to separate after ca. 7
days.
Yield: 286 mg (~42%); based on cobalt(III). Anal. Calc. for C H CoN O S (FW =
3
1
43
5
6.5
6
80.69): C, 54.65; H, 6.17; N, 10.28%. Found: C, 54.58; H, 6.12; N, 10.34%. ESI-MS (positive
+
-1
ion mode, acetonitrile) m/z: 613.1965 [Co-(L) ] . FT-IR (KBr, cm ): 1599, 1624, 1646, (υC=N);
2
3
-1
-1
2
097 (υSCN); 2862-2967 (υC-H); 3440 (υO-H). UV-Vis, max (nm), [εmax (dm mol cm )]

2
3
3
4
(
CH CN), 690 (1.81 x 10 ), 457 (1.03 × 10 ), 396 (6.28 × 10 ), 262 (5.06 × 10 ). Magnetic
3
moment: 0.086 BM.
2
.2. Physical measurements
Elemental analysis (carbon, hydrogen and nitrogen) was carried out on a Perkin-Elmer
40C elemental analyzer. Infrared spectrum was measured with a PerkinElmer Spectrum Two
2
-1
FTIR spectrophotometer in the range of 4500-500 cm . Electronic spectra in acetonitrile
medium (900-200 nm) was recorded in a PerkinElmer Lambda 35 UV-Vis spectrophotometer.
The powder XRD data was collected on a Bruker D8 Advance X-ray diffractometer using copper
K radiation (λ = 1.548 Å) generated at 40 kV and 40 mA. 1-D Lynxeye detector (5º < 2θ > 50º)
α
was used at ambient conditions. The magnetic susceptibility measurement was performed with a
magnetic susceptibility balance, made by Sherwood Scientific, Cambridge, UK at room
temperature (300 K). The corrected magnetic susceptibility,  , is calculated using the relation:
m
m = meas -  . Diamagnetic susceptibilities,  were calculated using Pascal's constants [24].
D
D
1
/2
Effective magnetic moments were calculated using the formula,  = 2.828(χ T) , where χ
eff
m
m
was the corrected molar susceptibility. The instrument was calibrated with metallic nickel.
Electrospray ionization mass spectra were recorded using Waters QTOF Micro YA263 mass
analyzer. Electron paramagnetic resonance (EPR) spectra were recorded in standard quartz EPR
tubes using JEOL JES-FA200 X-band spectrometer.
2
.3. X-ray crystallography and Hirshfeld Surface Analysis
X-ray diffraction intensities of a suitable single crystal were picked and mounted on glass
fibre, and diffraction intensities were measured at 150 K with an Oxford Diffraction X Calibur
diffractometer equipped with molybdenum K radiation (λ = 0.71073 Å), generated at 50 kV, 40
α

mA. Crysalis software was used for data collection and reduction [25]. The structure was solved
2
by direct method and refined by full-matrix least squares on F using the SHELXL-2016/6
package [26,27]. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were
placed in their geometrically idealized positions and constrained to ride on their parent atoms.
The programs used included PLATON [28], DIAMOND [29], ORTEP [30] and MERCURY
[
31]. A lattice water molecule present in the unit cell has been refined with occupancy 0.5.
Hydrogen atoms bonded to water oxygen atom could not be located and was not included into
the refinement. Details of crystallographic and refinement data are given in Table 1.
Crystal Explorer [32] was used to calculate Hirshfeld surfaces [33,34] and associated 2D
fingerprint plots [35-37]. The more details of Hirshfeld surface analysis could be found in
supplementary information part.
2
.4. Catalytic activity and kinetic study
The catechol oxidase and phenoxazinone synthase mimicking activity of the complex are
monitored spectrophotometrically. The more experimental details of catalytic activity and kinetic
study are given in supplementary information part.
3
. Results and discussion
3
.1. Synthesis
The tridentate Schiff base ligand, HL, was prepared by facile condensation of 4-(2-
aminoethyl)morpholine with 3-ethoxysalicyldehyde in methanol following the literature method
38-41]. The ligand was not isolated and the yellow coloured methanol solution of the ligand was
[
then reacted with a methanol solution of cobalt(II) acetate tetrahydrate. Addition of sodium
thiocyanate into the solution produced a mononuclear Schiff base complex, [Co(L--N,N,O)(L-


-N,O)(NCS)]·0.5H O. Cobalt(II) was converted into cobalt(III) by aerial oxidation in presence
2
of (strong field) Schiff base ligand, as observed in many previous cases [42-44]. Use of
anaerobic condition prevents the formation of this complex. The formation of the complex is
shown in Scheme 1.
Scheme 1: Synthetic route to the complex.
3
.2. Crystal structure description and Hirshfeld surface analysis
Single crystal X-ray analysis reveals that the cobalt(III) Schiff base complex has 2:1
ligand to metal stoichiometry. The complex crystallizes in triclinic system with P space group.
A perspective view of the complex together with the selective atom numbering scheme is shown
in Figure 1. Structure of the complex consists of a hexa-coordinated cobalt(III) in a distorted
octahedral geometry. The central cobalt(III) is coordinated by one amine nitrogen atom, N(22),
two imine nitrogen atoms, N(19) and N(39), and two phenoxo oxygen atoms, O(11) and O(31),
-
from two deprotonated pendent Schiff base ligand, (L ). The thiocyanate nitrogen atom, N(1),
occupied the sixth coordination site of cobalt(III) to complete its distorted octahedral geometry.
The equatorial plane of the octahedron is constructed by amine nitrogen atom, N(22), imine
nitrogen atoms, N(19) and N(39), and phenoxo oxygen atom, O(11). Whereas phenoxo oxygen
atoms, O(31) and the thiocyanate nitrogen atom, N(1) coordinate axially. One interesting
observation is that some of the equatorial bond lengths are larger than that of axial bond lengths.

This happens possibly due to the steric congestion around the metal center. The most attractive
structural feature of the complex is bidentate binding mode of a tridentate Schiff base. This
makes the Schiff base to remain as pendant. Relevant bond lengths and angles are summarized in
Tables 2 and 3, respectively. The bond angles deviate from the ideal values of 90 for the cis
angles and 180 for the trans angles, which clearly indicate the distortion from octahedral
geometry. The Co(III)-Namine bond length is slightly longer than the Co(III)-Nimine bond length,
as were also observed in similar previously reported complexes [45,46]. The Co(III)-Nimine bond
length in tridentate bonding Schiff base is somewhat shorter than Co-Nimine bond length in
bidentate bonding Schiff base. The saturated five-membered chelate ring, [Co(1)-N(19)-C(20)-
C(21)-N(22)], has an envelope conformation with puckering parameters [47,48] Q = 0.437(5) Å
and ϕ = 278.50(5)°. The N(19)-Co(1)-N(22) angle is 86.39(16)º and is characteristic of five
membered chelate rings [49,50]. A comparative study of geometrical parameters of investigated
complex with some similar type mononuclear cobalt(III) complexes has also been done. It is
evident from the study that as the steric congestion increases around the metal center, the
saturated chelate rings prefer envelope conformation. All these complexes are gathered in Table
4
.

Figure 1: Perspective view of the complex with selective atom numbering scheme. The
hydrogen atoms are omitted for clarity.
The complex shows significant C-H interactions. The hydrogen atoms, H(13B) and
H(14), attached to ethoxy carbon atom, C(132), and phenyl carbon atom, C(14), are participated
in inter-molecular C-H interaction with the symmetry related (2-x,2-y,2-z) phenyl rings, Cg6
[
C(12)-C(13)-C(14)-C(15)-C(16)-C(17)] and Cg7 [C(32)-C(33)-C(34)-C(35)-C(36)-C(37)],
respectively. Other hydrogen atom, H(33D), attached to the carbon atom, C(333), is involved in
another inter-molecular C-H interaction with the symmetry related (x,-1+y,z) phenyl ring,
Cg6 [C(12)-C(13)-C(14)-C(15)-C(16)-C(17)]. Because of these types of inter-molecular C-H
interactions, a 1D array has been formed in crystal packing of the complex (Figure 2). Geometric
features of the C-H interactions are given in Table 5.

Figure 2: C-H···π interactions in the complex. Only relevant hydrogen atoms are shown for
clarity.
The nature of intermolecular interactions and packing modes of complex in the
crystalline state may be examined by Hirshfeld surface analysis, which provides a visual picture
of intermolecular interactions and of molecular shapes in crystalline environment. The Hirshfeld
surface of the complex is exemplified in Figure 3, by showing dnorm, shape-index and curvedness
surface. To visualize the molecular moiety, transparent mapped surfaces are shown. The
dominant interactions are between oxygen and hydrogen and between sulphur and hydrogen.
Red spots on the dnorm surface indicate these interactions (Figure 3). Additionally, the 2D
fingerprint plots (Figure 4) illustrate the difference between the intermolecular interaction
patterns and the relative contributions (in percentage) [64-66] for the major intermolecular
interactions associated with the complex. In the 2D fingerprint plot (Figure 4) intermolecular
interactions appear as distinct spikes.

Figure 3: Hirshfeld surfaces mapped with dnorm (left-side), shape index (middle) and curvedness
right-side).
(
Figure 4: Fingerprint plot: Full and resolved into H···H, C⋯H / H⋯C, N···H / H···N, O···H /
H···O and S···H / H···S contact contributed to the total Hirshfeld Surface area of the complex.
3
.3. IR and electronic spectroscopy

The IR and electronic spectra of the complex are in well agreement with its crystal
structure. The condensation of the free amine group is conformed from the IR spectrum of the
complex as no band due to υN-H was observed. The band corresponding to azomethine (C=N)
-1
-1
group is distinct and occur at 1624 cm [51-53]. The appearance of a strong band at 2097 cm
indicates the presence of N-coordinated thiocyanate in the complex [54,55]. The bands in the
-1
range of 2925-2940 cm due to alkyl C-H stretching vibrations are routinely noticed [56,57].
The UV-Vis absorption spectrum of the complex in acetonitrile shows two d-d transition
bands at 690 and 457 nm, as expected for a low spin cobalt(III) complex in octahedral geometry
[
58,59]. The intense absorption bands below 400 nm may be assigned to ligand to metal charge
transfer bands (LMCT) and intra-ligand -* / n-* transitions [60-62]. IR, UV and electrospray
ionization mass spectra (ESI-MS positive) of the complex are given in Supplementary
information.
The complex is diamagnetic as expected for low-spin cobalt(III) complex [63].
3
.4. X-ray diffraction of powdered material
The purity of the bulk material can easily be cross-checked by comparing the patterns of
experimental and simulated X-ray diffraction of powdered material. In our case, the experimental
powder X-ray diffraction and simulated powder XRD patterns from single crystal X-ray
diffraction are in excellent concurrence. The simulated pattern was calculated from the single
crystal structural data (cif file) using the CCDC MERCURY software. Experimental and
simulated powder XRD patterns of the complex are given in Figure S6 (Supplementary
information).

3
.5. Catalytic oxidation of 3,5-DTBC and OAPH (Catechol oxidase and phenoxazinone synthase
mimicking activity)
Catalytic properties of the complex for the oxidation of 3,5-DTBC to 3,5-DTBQ and
OAPH to 2-aminophenoxazine-3-one with molecular dioxygen as the oxidant at room
temperature were explored. The key focus of this experimental measurement was to check
catechol oxidase and phenoxazinone synthase mimicking activity of the complex. The catalytic
activities were monitored in acetonitrile medium because the complex, substrates and their
products are highly soluble in acetonitrile. The catalytic reactions were carried out in absence of
any supplementary base to minimize the possibility of oxidation of substrates by molecular
dioxygen. The progress of the reactions was followed spectrophotometrically.
Spectrophotometric scans revealed a gradual increase in intensity of the absorption band at ~ 400
nm (characteristic for 3,5-DTBQ) or ~ 433 nm (characteristic for phenoxazinone chromophore)
for catechol oxidase or phenoxazinone synthase like activities respectively. The resulting
solutions were also monitored spectrophotometrically after 24 hour which show the formation of
3
,5-DTBQ or 2-aminophenoxazine-3-one respectively as the sole product. These results clearly
indicate that the complex is active towards both catechol oxidase and phenoxazinone synthase
mimicking activities. Blank experiments without catalyst for the oxidation of 3,5-DTBC and
OAPH were also performed under identical conditions and in each case, any significant growth
of the absorption bands around 400 or 433 nm could not be observed. The molar extinction
3
4
coefficient value of 3,5-DTBQ and 2-aminophenoxazine-3-one are 8.630x10 and 1.093x10 ,
respectively at 400 nm and at 433 nm in acetonitrile medium. The time dependent spectral
growth for catalytic oxidations of 3,5-DTBC and OAPH are shown in Figure 6.The detail kinetic
studies were performed to understand the extent of the catalytic efficiency.

Figure 6: The time dependent spectral growth of 3,5-DTBQ (a) and 2-aminophenoxazine-3-one
-2
-4
(
b), upon addition of 10 M substrate to 10 M of complex. The time interval between each
reading of the catalytic cycles is 5 min.
3
.6. Kinetic investigation through several enzyme kinetic plots
Biochemical reactions involving a single substrate are often assumed to follow
Michaelis-Menten kinetics, which is an important tool for treating this type of saturation rate
dependency on substrate concentration. In this model, following equation (eq.1) is used to relate
the rate of enzymatic reactions, V to the concentration of substrate, S.
V =
∙∙∙(1)
Here, Vmax represents the maximum rate achieved by the system, at saturating substrate
concentration. K is the Michaelis constant and it is equal to the substrate concentration at which
M
the reaction rate is half of Vmax
.
Linearization of Michaelis-Menten equation produces a double reciprocal Lineweaver-
Burk plot, which is used to analyze a variety of kinetic parameters, e.g. Vmax and K . The
M
Lineweaver-Burk Equation may be represented by equation 2.

+
∙∙∙(2)
On the other hand, rearrangement of Michaelis-Menten equation gives Hanes equation
(eq. 3). In this case, the ratio of the substrate concentration (S) to the reaction rate (V) is plotted
against substrate concentration (S).
=
+
∙∙∙(3)
Eadie-Hofstee equation (eq. 4) is sometimes used in biochemistry for a graphical
representation of enzyme kinetics. Here reaction rate (V) is plotted as a function of the ratio
between reaction rate (V) and substrate concentration (S):
−
∙∙∙(4)
All these enzyme kinetic plots are used to evaluate several kinetic parameters, including
turnover number (k ) and specificity constant (k /K ) for both catechol oxidase and
cat
cat
M
phenoxazinone synthase mimicking activity of the complex. In enzyme kinetics, turnover
number (also termed as k ) is defined as the maximum number of catalytic conversions of
cat
substrate molecules per unit time that a single catalytic site will execute for a specific enzyme
concentration. The specificity constant (also termed as k /K ratio) is a measure of how
cat
M
efficiently a catalyst converts substrates into products. This ratio is a useful index for measuring
the substrate specificity of catalyst. The higher the specificity constant, the more the enzyme
prefers that substrate. Figures 7 and 8 represent the Michaelis-Menten plot, Lineweaver-Burk
plot, Hanes-Woolf plot and Eadie-Hofstee plot for catalytic oxidation of 3,5-DTBC and OAPH.
Tables 6 and 7 contain kinetic parameters for catechol oxidase and phenoxazinone synthase
mimicking activities, respectively.

Figure 7: Michaelis-Menten plot (a), Lineweaver-Burk plot (b), Hanes-Woolf plot (c) and
Eadie-Hofstee plot (d) for catalytic oxidation of 3,5-DTBC.

Figure 8: Michaelis-Menten plot (a), Lineweaver-Burk plot (b), Hanes-Woolf plot (c) and
Eadie-Hofstee plot (d) for catalytic oxidation of OAPH.
3
.7. Origin and mechanistic pathway of catalytic activity
At this stage, it is very clear that the complex is reactive towards the aerial oxidation of
,5-DTBC and OAPH. So as to confirm the involvement of molecular dioxygen in the catalytic
3
-4
oxidation of 3,5-DTBC and OAPH, electronic spectra of a mixture of a 10 M solution of the
complex with large excess of substrates (3,5-DTBC or OAPH) were recorded under anaerobic
atmosphere and no significant spectral growths for 3,5-DTBQ or 2-aminophenoxazine-3-one

were observed up to 24 h of mixing. Based on the above results, tentative catalytic cycles for the
formation of the quinone and phenoxazinone chromophores are shown in Scheme 2 and 3,
respectively.
Scheme 2: Plausible mechanistic pathway for the aerial oxidation of 3,5-DTBC.

Scheme 3: Proposed mechanistic pathway for the aerial oxidation of o-aminophenol.
Several factors may affect the catalytic activity, such as the variable oxidation state of the
metal, coordination geometry around the metal center, metal-metal distance, ligand flexibility,
exogenous bridging ligand etc. In several previous reports where mechanistic considerations of
these types of catalytic oxidation are available, it is established that the oxidation reaction
proceeds through involvement of molecular dioxygen [67-69]. At initial step, the substrate is

coordinated with the complex, by replacing monodentate thiocyanate co-ligand. This complex
substrate intermediate degrades in the rate determining step by the production of substrate
radical, which subsequently yields respective quinone or phenoxazinone chromophore in
presence of molecular dioxygen via several oxidative dehydrogenation processes, as shown in
Scheme 2 and 3. In our previous reports, we have also observed similar mechanistic pathway for
the catalytic oxidations of 3,5-DTBC and OAPH [22,23,70].
The electrospray ionization mass spectra (ESI-MS positive) of 1 : 50 mixture of the
complex and substrate (3,5-DTBC for catechol oxidase mimicking activity and OAPH for
phenoxazinone synthase mimicking activity) were recorded after 10 minutes of mixing in
acetonitrile medium in order to get an insight into the possible mechanistic pathways. ESI-MS
positive spectra of catalytic oxidation of 3,5-DTBC and OAPH are given as Supplementary
Figures S4 and S5, respectively. For catalytic oxidation of 3,5-DTBC, the mass spectrum
exhibits a peak at m/z = 243.1187 corresponding to 3,5-DTBQ-sodium aggregate [3,5-DTBQ-
+
Na] , confirming the catechol oxidase mimicking activity of the complex. Similarly, for catalytic
oxidation of OAPH, the mass spectrum exhibits a peak at m/z = 279.2084 which can be assigned
+
to the sodium adduct of 2-aminophenoxazine-3-one, [2-aminophenoxazine-3-one-Na-CH CN] .
3
This peak clearly confirms the phenoxazinone synthase mimicking activity of the complex. Peak
+
observed at m/z ~ 613 arises due to the loss of monodentate thiocyanate co-ligand [Co-(L) ] ,
2
which is the initial step of catalysis. This peak is also observed in the mass spectral analysis of
the pure complex (given in Supplementary information, Figure S3). So it is clear that
monodentate thiocyanate co-ligand can easily be replaced. The mechanistic pathways shown in
Schemes 2 and 3 are therefore justified by the mass spectral analysis.

Electron paramagnetic resonance spectroscopy is a useful tool to monitor the generation
of substrate radical during the catalytic cycles. To confirmed the formation of substrate radicals,
EPR measurements have been performed on the reaction mixture of the complex and 3,5-DTBC
and o-aminophenol separately. Mixture solutions (complex and substrate) shows EPR signal
around 1.998 (g value) with a peak-to-peak line width of ca. 3400 G. The g value of the signal is
closed to 2.0023 (value for a free electron), [71] this value is characteristic for organic radicals,
such as a phenoxyl-type radical [72, 73]. Therefore it can be concluded that the catalytic cycles
passed through a radical pathway, as shown in the Schemes 2 and 3.
Figure 9: EPR spectrum m obtained from the acetonitrile solution of the complex after
addition of 3,5-DTBC, recorded at 77K.
A few similar type mononuclear cobalt(III) complexes showing catechol oxidase and
phenoxazinone synthase mimicking activity already exist in literature. These previously reported

complexes exhibiting catechol oxidase and phenoxazinone synthase mimicking activity are
gathered in Tables 8 and 9, respectively. As the turnover number (k ) value of investigated
cat
complex lies in between the previously reported complexes turnover number value, it can be
considered as moderate catalyst.
4
. Conclusion
In conclusion, a cobalt(III) complex with a pendant Schiff base ligand has been
synthesized and characterized by elemental and several spectral analyses. Crystal structure
determination shows that a tridentate Schiff base is acting as a bidentate ligand, keeping the
remaining donor site pendant. Furthermore, the solid state structure of the complex shows
participation of the organic ligand in several supramolecular interactions involving ethoxy arms
and aromatic rings. The complex is found to be active towards catechol oxidase and
phenoxazinone synthase mimicking activity. The mimicking activity was assessed by following
conventional Michaelis-Menten enzymatic kinetics. Kinetic studies of the catalytic cycles have
been performed in detail using a variety of enzyme kinetics plots to calculate different kinetic
parameters, including the specificity constant.
Acknowledgement
K.G. thanks UGC, India, for awarding a Senior Research Fellowship.
Appendix A. Supplementary data
CCDC 1570018 contains the supplementary crystallographic data of the complex. 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.
(
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Table 1: Crystal data and refinement details of the complex.
Formula
C H CoN O S
31 43 5 6.5
Formula Weight
Crystal System
Space group
680.69
Triclinic
P
a(Å)
b(Å)
c(Å)
α(°)
β(°)
γ(°)
9.6870(9)
10.359(1)
17.3597(15)
101.621(7)
96.025(8)
107.117(8)
1.40
3
d(calc) [g/cm ]
-1
µ [ mm ]
0.652
F(000)
716
Total Reflections
Unique Reflections
Observed data[I > 2 σ(I)]
No of parameters
R(int)
11392
8986
6008
403
0.065
R1, wR2(all data)
R1, wR2([I > 2 σ(I)]
0.1277, 0.2572
0.0932, 0.2352