Synthesis and structural characterization of a cobalt(III) complex with an (N,S,O) donor Schiff base ligand: Catechol oxidase and phenoxazinone synthase activities

Article abstract of DOI:10.1016/j.poly.2018.08.021

A mononuclear Co(III) complex [CoL₂]Cl (1) [HL = (E)-2-((3-(2-hydroxyethylthio)propylimino)methyl)phenol] was synthesized and its X-ray crystallographic characterization revealed a distorted octahedral geometry around the metal centre. 1 shows catechol oxidase as well as phenoxazinone synthase activities in different solvents. The turn over numbers for the catechol oxidase activity were 1.18 × 10² h^?1 in MeOH and 1.80 × 10³ h^?1 in MeCN, and that of phenoxazinone synthase activity were 9.72 × 10³ h^?1 in MeOH.

Full text of DOI:10.1016/j.poly.2018.08.021

Accepted Manuscript
Synthesis and structural characterization of a cobalt(III) complex with an
(N,S,O) donor Schiff base ligand: catechol oxidase and phenoxazinone synthase
activities
Ayon Kanti Ghosh, Chandra Shekhar Purohit, Rajarshi Ghosh
PII:
S0277-5387(18)30486-8
https://doi.org/10.1016/j.poly.2018.08.021
POLY 13344
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 July 2018
5 August 2018
7 August 2018
Please cite this article as: A. Kanti Ghosh, C. Shekhar Purohit, R. Ghosh, Synthesis and structural characterization
of a cobalt(III) complex with an (N,S,O) donor Schiff base ligand: catechol oxidase and phenoxazinone synthase
activities, Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.08.021
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1
Synthesis and structural characterization of a cobalt(III) complex with an
(N,S,O) donor Schiff base ligand: catechol oxidase and phenoxazinone
synthase activities
Ayon Kanti Ghosha, Chandra Shekhar Purohitb, Rajarshi Ghosha,*
aDepartment of Chemistry, The University of Burdwan, Burdwan 713 104, India
bSchool of Chemical Sciences, National Institute of Science Education & Research,
Bhubaneswar, Orissa, 751 005, India
Abstract
A
mononuclear Co(III) complex [CoL2]Cl (1) [HL
=
(E)-2-((3-(2-
hydroxyethylthio)propylimino)methyl)phenol] was synthesized and its X-ray crystallographic
characterization revealed a distorted octahedral geometry around the metal centre. 1 shows
catechol oxidase as well as phenoxazinone synthase activities in different solvents. The turn
over numbers for the catechol oxidase activity were 1.18 × 102 h-1 in MeOH and 1.80 × 103 h-
1 in MeCN, and that of phenoxazinone synthase activity were 9.72 × 103 h-1 in MeOH.
Keywords: Cobalt, Schiff base, catechol oxidase activity, phenoxazinone synthase activity
____________
*Corresponding author, E. mail: rghosh@chem.buruniv.ac.in

2
1. Introduction
Oxidation of organic molecules with molecular oxygen at mild conditions is of utmost
importance for industrial and synthetic processes both from economic and environmental
aspects [1]. Metalloproteins in nature can easily activate the kinetically inert triplet aerial
oxygen to its active singlet form and use that for oxidising different organic substrates [2].
Metal containing active sites of those metalloproteins are now being mimicked in the form of
small molecules to oxidise same/similar substrates in laboratory. Catachol oxidase, a copper
containing metalloprotein having a dinuclear hydroxobridged Cu(II) active site [3], catalyses
the oxidation of catechol to quinone (Scheme 1) in plant cell in presence of aerial oxygen. A
number of reports with small dinuclear Cu(II) complexes as catechol oxidase mimic are there
in literature [4-7]. Not only copper(II) but also some non-copper transition metals like
manganese [8], nickel [9], iron [10], cobalt [11], zinc [12], vanadium [13] etc. are found to
show catechol oxidase activity. Phenoxazinone synthase, a multicopper oxidase which is
found in the bacterium Streptomyces antibioticus, is responsible for six electron oxidative
coupling of two molecules of the o-aminophenol derivative, 4-methyl-3-hydroxyanthraniloyl
pentapeptide to phenoxazinone chromophore (Scheme 2). In this case also along with Cu(II)
[14] different non-copper complexes [15] catalyze the catechol oxidase and/or phenoxazinone
synthase activities.
In continuation to our continued interest with Co(III) [11(b), 11(e), 15(f)] here we report
the synthesis and X-ray structural characterization of a mononuclear cobalt(III) complex with
(N,S,O)
donor
Schiff
base
ligand
[CoL2]Cl
[HL
=
(E)-2-((3-(2-
hydroxyethylthio)propylimino)methyl)phenol] (1) which did catalyse aerial oxidation of 3,5-
di-tert-butyl catechol (3,5-DTBC) and o-aminophenol (OAPH) separately to corresponding
quinone and the phenoxazinone chromophore, respectively. catechol oxidase- and/or
phenoxazinone synthase-active metal complexes with (N,S,O) donor ligands are rare [16].

3
The catalytic results are found in methanol and acetonitrile solvents and in each case reaction
rates and turn over numbers were measured. The Michealis-Menten kinetics in each solvent
of each activity were measured.
2. Experimental
2.1. Materials
High purity 2-mercaptoethanol, 3-chloropropylamine, salicylaldehyde and cobalt(II)
chloride hexahydrate were purchased from Sigma Aldrich, India and used as received.
Ethanol, methanol and acetonitrile were used as reagent grade solvents. 3,5-DTBC and 2-
aminophenol were purchased from Sigma Aldrich, India and from Loba Chemie, India,
respectively.
2.2. Physical measurement
Elemental analyses (carbon, hydrogen and nitrogen) were performed on Perkin-Elmer
2400 CHNS/O elemental analyzer. UV-VIS and IR spectra (KBr discs, 4000-500 cm-1) were
recorded using a Shimadzu UV-VIS 1800 spectrophotometer and Shimadzu FT-IR model IR
Prestige-21 spectrometer, respectively. NMR experiment was carried out in Bruker 400 MHz
NMR spectrometer and mass spectral study was done in Thermo Exactive plus high
resolution mass spectrometer.
2.3. Synthesis of HL
2-Mercaptoethanol (8 mmol, 0.625 g) was added gradually in the ice cold sodium
ethoxide solution which was prepared by the addition of metallic sodium in dry ethanol with
continuous stirring. 3-chloropropylamine (8 mmol, 1.04 g) was then mixed slowly to this
reaction mixture which was stirred for about 1 hour. During this stirring sodium chloride was
precipitated that confirms the formation of the 2-(3-aminopropylthio)ethanol. Sodium

4
chloride which was produced as by product was separated by filtration. Salicyaldehyde (8
mmol, 0.97 g) was added to this solution of 2-(3-aminopropylthio)ethanol and this mixture
was refluxed for 4 hours. This resulted the ligand HL. The synthesis of ligand (HL) is
schematically shown in Scheme 3. Yield, 1.434 g (75% based on aldehyde). Anal cal. for
1
C12H17NO2S (HL): C, 60.22; H, 7.16; N, 5.85 Found: C, 60.12; H, 7.23; N, 5.89. H NMR δ
(ppm): 2.17 (q, J = 6.4 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H), 2.73 (t, J = 6.0 Hz, 2H), 3.62 (t, J =
6.0 Hz, 2H), 3.90 (t, J = 6.0 Hz, 2H), 6.85-6.96 (m, 2H), 7.24-7.33 (m, 2H), 13.28 (bs, 1H).
2.4. Synthesis of 1
Ethanolic solution of cobalt(II) chloride hexahydrate (0.1 mmol, 0.024 g) was slowly
added to the hot ethanolic solution of HL (0.2 mmol, 0.048 g) with continuous stirring for 4
hours (Scheme 4). The brown colour of the ligand solution was changed to reddish brown on
dissolving the metal salt. This reaction mixture was kept in open air for slow evaporation.
After about 6 weeks reddish brown coloured block shaped crystals appeared. Yield, 0.0398 g
(70% based on metal salt). Anal cal. for C24H32N2O4S2ClCo (1): C, 50.48; H, 5.65; N, 4.91
Found: C, 50.51; H, 5.68; N, 4.89.
2.5. X-ray diffraction study
X-ray quality single crystals of 1 were mounted on Bruker AXS KAPPA APEX II
diffractometer using Mo-Kα radiation (λ = 0.71073 Å) at 296(2) K. Identification of the
space group P21/c for 1 were based on systematically absent reflections. Out of 9131 total
reflections 2698 with I >2r(I) were used for structure solutions. The direct methods were used
for structure solution, and the structure solution and refinement were based on |F2|. All non-
hydrogen and hydrogen atoms were refined with anisotropic displacement parameters and
were placed in calculated positions when possible and given isotropic U values 1.2 times that
of the atom to which they are bonded, respectively. The maximum and minimum peak

5
heights at 0.607 and -1.101 eÅ-3 for 1 with no chemical significance were shown by final
differences Fourier map. SHELXL-2014 [17] and ORTEP-32 [18] were used to carry out all
calculations. The crystal data and data collection parameters are listed in Table 1.
3. Results and discussion
3.1. Synthesis and formulation
The two-step synthesis of the organic ligand HL was carried out, firstly, by abstracting
proton from the 2-mercaptoethanol by in situ built sodium ethoxide in dry ethanol. The
ethanolic solution of 3-chloropropylamine was added dropwise with the mixture and stirred
for an hour to get 2-(3-aminopropylthio)ethanol. As a result precipitated sodium chloride was
filtered off. Next to this, treatment of salicyaldehyde with this reaction mixture and
consequent stirring for four hours or so leads to the final compound solution which on
evaporation of the solvent in rotary evaporator resulted the solid brown coloured ligand HL
1
(Scheme 3). The HNMR spectrum of HL in CDCl3 showed the characteristic peaks of the
protons (Figs. S1 and S2; supporting information). The quintet signal for the two methylene
protons which are  to C-S bond and  to imine bond of HL appeared at  2.17 (q, J = 6.4 Hz,
2H). The two triplet signals for four protons of the two methyl groups were found at  2.63 (t,
J = 7.2 Hz, 2H) for one methylene group α to C-S bond and  2.73 (t, J = 6.0 Hz, 2H) for
another methylene group  to the OH group. The signal appeared at  3.62 (t, J = 6.0 Hz, 2H)
due to the methylene group which is α to the imine bond. A triplet signal appears at  3.90 (t,
J = 6.0 Hz, 2H) for another methylene group α to the OH group. The signals of the four
aromatic protons were appeared at the range between δ 6.85 ppm to δ 7.33 ppm (m, 4H). In
IR spectrum some characteristic frequencies are at 2926 cm-1 is due to O-H bond vibration,
1629 due to imine bond stretching and 754 cm-1 for C-S bond stretching [19]. Absorption
bands of the ligand were found at 401, 313, 254, 214 (π-π*) nm in MeOH solution in the 200-

6
800 nm range. The IR bands for complex 1 at 1595 cm-1 was attributed to the asymmetric
ῡ(C=N), band at 1062 cm-1 was due to the metal-oxo bond stretching and band at 752 cm-1
was detected as ῡ(C-S) stretching [19]. Absorption bands of the complex in MeOH solution
displays the spectral pattern of the complex. The absorption band at 223, 260 nm is due to the
(π - π*) transition from the ligand side and band at 394 nm for 1A1g  1T1g transition and 555
nm due to 1A1g  1T2g transition of octahedral cobalt(III) centre [20].
3.2. X-ray structure
The thermal plot of 1 which is a centrosymmetric mononuclear cobalt(III) complex is
presented in Fig. 1. Considering bond angle bond distance data (Table 2) the cobalt(III)
centre is found to be hexa coordinated and the geometry around the cobalt(III) is distorted
octahedral. Again from the bond angle-bond distance parameters, phenolic oxygens (O1 and
O1i) and imine nitrogens (N1 and N1i) of the ligand are in the square plane and thio ether
sulfurs (S1 and S1i) from the ligand network are situated at the axial position. The bond
distance range in the basal plane is 1.889(3) to 2.2776(10) Å (Table 2) which are consistent
with some of the reported cobalt(III) complexes [11a, 11b, 11d, 11e].
3.3. Catechol oxidase activity of 1: spectrophotometric study
3,5-DTBC was taken as a substrate for the investigation of catechol oxidase activity of 1.
The reason for this choice was that it (3,5-DTBC) could easily be oxidised to respective 3,5-
di-tert-butylquinone (3,5-DTBQ) which is electrochemically stable having low quinone-
catechol reduction potential. The formation of quinone was studied by repetitive UV-Vis
scanning for 6 hours by mixing 1 (1 × 10-4 M) and 100 equivalent of 3,5-DTBC separately in
methanol (Fig. 2) and acetonitrile (Fig. 3) media. Pure 3,5-DTBC shows bands at 282 nm
(inset to Fig. 2) and 278 nm (inset to Fig. 3) respectively in methanol and acetonitrile.

7
Characteristic peak of the oxidation product 3,5-DTBQ appears at around 400 nm in both the
solvents [21]. The newly generated 3,5-DTBQ was purified by column chromatography
(yield = 68%) and characterized by measuring its melting point at 110˚C [22].
During the measurement of rate of this reaction it was found out that absorbances at
~400 nm in both methanol and acetonitrile were time dependent. The gradual conversion of
colour from light yellow to deep brown of the mixture of 1 and 3,5-DTBC in both the
solvents in air is indicative of the slow conversion of 3,5-DTBC to 3,5-DTBQ. The
absorbance differences at ~400 nm were plotted against time to calculate the initial rate in
methanol (Fig. 4) and acetonitrile (Fig. 5), respectively. In both the solvents first order
kinetics were found with initial rate 5.08 × 10-3 min-1 (methanol) and 3.25 × 10-5 min-1
(acetonitrile).
3.3.1. Enzyme kinetics study
Spectrophotometric measurements thermostated at 25°C with 1 and the substrate 3,5-
DTBC were used to collect the enzyme kinetic data in both methanol and acetonitrile. 0.04
ml 1 × 10-4 M solution of 1 was added to 2 ml of 3,5-DTBC of concentration varying from 1
× 10-3 M to 1 × 10-2 M. 3,5-DTBC to 3,5-DTBQ conversion was monitored with time at the
wavelength of ~400 nm in methanol and in acetonitrile solvents. Initial rate method was used
to determine the rate for each concentration of the substrate.
The rate versus substrate concentration data were found to follow the Michaelis-Menten
approach of enzyme kinetics to get the double reciprocal Lineweaver-Burk plot and the
values of the Vmax (maximum velocity), KM (Michaelis-Menten constant) and Kcat (Turn over
number). The observed rate vs. [substrate] plot along with Lineweaver-Burk plot in methanol
and acetonitrile solutions are given respectively in Figs. 6 and 7. The Kcat values (turnover
numbers) are calculated as 1.18 × 102 h-1 in methanol and 1.80 × 103 h-1 in acetonitrile (Table

8
3) whereas the highest Kcat reported till date, to the best of our knowledge, is 1.32 × 106 h-1
[23].
3.3.2. Mechanism of catechol oxidase activity
The catalytic cycle of the process is given in Scheme 5. Probably, in the first step, the 1:1
adduct of catechol and the cobalt complex is formed. HRMS spectrum of 1 in methanol and
of a 1:100 mixture of 1 and 3,5-DTBC in methanol solvent were recorded. The peak at m/z =
243.56 can be assigned to sodium aggregate of quinone [3,5-DTBQ]-Na+ (Fig. S3;
Supporting information). The complex 1 exhibits a peak at m/z = 572.65 (Fig. S4; Supporting
information). The formation of a complex-substrate adduct intermediate species (III, Scheme
5) is identified by the peak at m/z = 517.22 (Fig. S5; Supporting information). The catalytic
process is shown in Scheme 4 and the each reaction intermediates (II-V, Scheme 5) are
identified by HRMS (Figs. S6 to S8; Supporting information). The catechol derivative, 3,5-
DTBC gets oxidised to quinone in presence of aerial oxygen. The aerial oxygen that oxidises
3,5-DTBC to 3,5-DTBQ in this process is converted to H2O2. H2O2 thus liberated was
identified and characterized spectrophotometrically (S1; Supporting information) [24].
3.4. Phenoxazinone synthase activity of 1: Spectrophotometric study
In case of phenoxazinone synthase activity OAPH was used as the substrate. 1
catalyzed the aerial oxidation of OAPH to the corresponding 2-aminophenoxazinone-3-one.
Repetitive UV-Vis scan of the mixture of methanolic solution of 1 and 100 equivalent of
OAPH was recorded. The plot for the representative scan is given in Fig. 8. OAPH absorbs at
232 nm and 286 nm in methanol (inset to Fig. 8). The characteristic peak of 2-
aminophenoxazine-3-one (APX) grows smoothly at 427 nm in MeOH [14a].
To monitor the reaction kinetics and find out the initial rate between OAPH and 1, time
dependent change of absorbance for the catalytic reaction was performed at 427 nm for 60

9
min in the above solvent. The difference in absorbance at 427 nm is plotted against time in
minute in methanol solvent. The plot is given in Fig. 9. The initial rate for the particular
catalyst and substrate is obtained from the slope. The initial rate is found to be 2.50 × 10-3
min-1 in MeOH.
3.4.1. Enzyme kinetics study
UV-Vis spectrophotometric measurements thermostated at 25°C were used for the
enzymatic kinetic experiments with the complex and the substrate (OAPH) in MeOH. 0.04
ml of complex solution (with a constant concentration of 1 × 10-4 M) was added to 2 ml of
OAPH of a particular concentration varying from 1 × 10-3 M to 1 × 10-2 M to make the final
concentration of the complex as 1 × 10-4 M. The conversion of OAPH to APX was monitored
with time at a wavelength of 427 nm. Initial rate method was used to determine the rate for
each concentration of the substrate by the initial rate method.
The rate versus concentration of substrate data were used (on the basis of Michaelis-
Menten approach of enzymatic kinetics) to get the Lineweaver-Burk (double reciprocal) plot
as well as the values of the various kinetic parameters Vmax, KM and Kcat in MeOH (Table 4).
Fig. 10 demonstrates the plot in MeOH and the turnover number (Kcat) is found to be 9.72 ×
103 h-1 in that solvent.
3.4.2. Mechanism of phenoxazinone synthase activity
The sodium aggregate of APX was also identified by HRMS at m/z value 235.54 (Fig.
S9; Supporting information). To get the reaction intermediate that is 1-OAPH adduct, HRMS
study was carried out with m/z value 440.11 (OAPH-1) (Fig. S10; Supporting information).
This adduct generates 2-amoinophenoxazine-3-one through some intermediate reaction with

10
molecular oxygen which get reduced to H2O2. H2O2 was detected spectrophotometrically (S1;
Supporting information) [24].
4. Conclusion
Synthesis and X-ray crystallographic characterization of a new cobalt(III) complex 1
with an (N,S,O) donor Schiff base were reported. Catalytic behaviour of 1 towards aerial
oxidation of 3,5-di-tert-butyl catechol to its corresponding quione and o-aminophenol to
phenoxazinone chromophore separately. The catalyst 1 shows better turn over numbers (Kcat)
in methanol and acetonitrile than that of the other reported cobalt complexes [11(a), (d)].
Similarly Kcat values for phenoxazinone synthase activity by 1 in methanol is also higher than
some of the reported ones [15(a), (d), (e)].
5. Supplementary data
CCDC 1846518 contains the supplementary crystallographic data for 1. 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.
Acknowledgement
RG is thankful to Department of Science & Technology, Government of West Bengal
[No. 781(Sanc.)/ST/P/S&T/4G-4/2013 dated 04-12-2014], India for financial assistance.
AKG thanks CSIR, New Delhi for his fellowship. Different infrastructural facilities at our
university provided by PURSE programme, DST, Govt of India is gratefully acknowledged.

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H. R. Yadav, C. S. Purohit, A. R. Choudhury, R. Ghosh, Polyhedron, 111 (2015)
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14
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RSC Adv., 6 (2016) 58831; (b) M. R. Maurya, S. Sikarwar, T. Joseph, S. B. Halligudi
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Dalton Trans. 46 (2017) 1249.
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55A (2016) 681.
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15
[21] F. Zippel, F. Ahlers, R. Werner, W. Haase, H.-F. Nolting, B. Krebs, Inorg. Chem., 35
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Green and Co. Ltd, London, 1961. p. 366.

16
Table 1. Crystal data and structure refinement parameters for 1
Emperical formula
Formula weight
T (K)
C24H32N2O4S2ClCo
571.04
296(2)
Wavelenght (Å)
Crystal system
Space group
0.71073
Monoclinic
P21/c
Unit cell dimensions
A (Å)
9.3982(11)
17.578(3)
8.4820(10)
110.654(8)
1311.2(3)
2
B (Å)
C (Å)
β (˚)
V(Å3)
Z
Dcalc (mg m-3)
Absorption coefficient (mm-1)
F(000)
1.536
1.052
630
Crystal size (mm3)
Theta range for data collection (˚)
Index ranges
0.87 0.42 × 0.22
2.316 - 26.453
-11 ≤ h ≤ 11, -21 ≤ k ≤19, -10 ≤ l ≤10
9131
Reflections collected
Independent reflections
Completeness of theta
Absorption correction
2698 [Rint= 0.0371]
99.9 % [θ=25.242]
Multi-scan

17
Tmax and Tmin
0.7454 and 0.4620
Full matrix
Refinement method
Data/restrains/parameters
Goodness-of fit (GOF) F2
Final R index [l >2σ(l)]
R index (all data)
2698/0/ 164
1.068
R1 = 0.0587 and wR2 = 0.1682
R1 = 0.0704 and wR2 = 0.1772
0.607, -1.101
Largest difference between peak and hole (e Å-3)
2
2 2
2
R = |Fo|  |Fc||/|Fo, wR = [w(Fo Fc2)2/w(Fo ) ]1/2, calcd w = 1/[2(Fo ) + (0.1311P)2 +
2
9.1582P]; where P = (Fo + 2Fc2)/3.
Table 2. Bond lengths [Å] and angles [°] for 1
Bond lengths
Co(1)-O(1)
1.889(3)
1.953(3)
2.2776(10)
Co(1)-N(1)
Co(1)-S(1)
Bond angles
N(1)-Co(1)-O(1)
O(1)-Co(1)-S(1)
N(1)-Co(1)-S(1)
N(1)-Co(1′)-N(1′)
S(1)-Co(1′)-S(1′)
O(1)-Co(1′)-O(1′)
91.40(13)
91.61(9)
89.89(10)
180.0
180.0
180.0
Symmetry transformation: 'x, y, z', '-x, y+1/2, -z+1/2', '-x, -y, -z', 'x, -y-1/2, z-1/2'

18
Table 3. Kinetic parameters for the oxidation of 3,5-DTBC catalyzed by 1
Solvents
Vmax (M s-1) Standard
error
KM (M)
Standard
error
Kcat (h-1)
MeOH
MeCN
3.28 × 10-6
5.00 × 10-5
5.62 × 10-7 1.8 × 10-3
7.64 × 10-6 2.36 × 10-3
7.50 × 10-4
1.50 × 10-4
1.18 × 102
1.80 × 103
Table 4. Kinetic parameters for the oxidation of OAPH catalyzed by 1
Solvents Vmax (M s-1)
MeOH
2.70 × 10-4
Standard error KM (M)
1.05 × 10-4 1.22 × 10-2
Standard error Kcat (h-1)
2.04 × 10-3 9.72 × 103

19
Fig. 1  ORTEP of 1 with 30% ellipsoid probability (H atoms are omitted for clarity)
Fig. 2  Change in spectral pattern of 1 after addition of 3,5-DTBC and allowing the reaction
for 6 h in MeOH [inset: spectrum of pure 3,5-DTBC in MeOH]

20
Fig. 3  Change in spectral pattern of 1 after addition of 3,5-DTBC and allowing the reaction
for 6 h in MeCN [inset: spectrum of pure 3,5-DTBC in MeCN]
Fig. 4  Plot of the difference in absorbance (ΔA) vs time to evaluate the rate of catalysis
of 3,5-DTBC by 1 in MeOH

21
Fig. 5  Plot of the difference in absorbance (ΔA) vs time to evaluate the rate of catalysis of
3,5-DTBC by 1 in MeCN
Fig. 6  Plot of rate vs. [substrate] (3,5-DTBC) in presence of 1 in MeOH [inset:
Lineweaver-Burk plot]

22
Fig. 7  Plot of rate vs. [substrate] (3,5-DTBC) in presence of 1 in MeCN [inset: Lineweaver-
Burk plot]
Fig. 8  Change in spectral pattern of 1 after addition of OAPH and allowing the reaction for
6 h in MeOH [inset: spectrum of pure OAPH in MeOH]

23
Fig. 9  Plot of the difference in absorbance (ΔA) vs time to evaluate the rate of catalysis of
OAPH by 1 in MeOH
Fig. 10  Plot of rate vs. [substrate] (OAPH) in presence of 1 in MeOH [inset: Lineweaver-
Burk plot]

24
Scheme 1. Catechol oxidase activity
Scheme 2. Phenoxazinone synthase activity
Scheme 3. Synthesis of ligand HL
Scheme 4. Synthesis of 1

25
Scheme 5. Proposed catalytic cycle for catechol oxidase activity

26