Catecholase and phenoxazinone synthase activities of a ferromagnetically coupled tetranuclear Cu(II) complex

Article abstract of DOI:10.1039/c6ra11433d

A crystallographically characterized tetranuclear Cu(ii) complex [CuII4(L)₄] (1) [H₂L = N-(2-hydroxyethyl)-3-methoxysalicylaldimine] is found to show overall ferromagnetic exchange coupling. Complex (1) mimics the catalytic activity of the plant enzyme catechol oxidase by oxidising 3,5-di-tert-butylcatechol to its corresponding quinone in methanol and dichloromethane medium in the presence of aerial oxygen. The reaction follows Michaelis-Menten enzymatic reaction kinetics with turnover numbers (K_cat) 6.99 × 10³ and 1.85 × 10³ h^-1 in methanol and dichloromethane, respectively. 1 is also phenoxazinone synthase active in methanol medium with a turnover number of 1.21 × 10⁵ h^-1.

Full text of DOI:10.1039/c6ra11433d

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Catecholase and phenoxazinone synthase activities
of a ferromagnetically coupled tetranuclear Cu(II)
complex†
Cite this: RSC Adv., 2016, 6, 58831
Merry Mitra,a Tanay Kundu,‡b Gurpreet Kaur,§c Gyaneswar Sharma,d
c
d
a
*
Angshuman Roy Choudhury, Yogesh Singh and Rajarshi Ghosh
A crystallographically characterized tetranuclear Cu(II) complex [CuI4I(L)4] (1) [H2L ¼ N-(2-hydroxyethyl)-3-
methoxysalicylaldimine] is found to show overall ferromagnetic exchange coupling. Complex (1) mimics
the catalytic activity of the plant enzyme catechol oxidase by oxidising 3,5-di-tert-butylcatechol to its
corresponding quinone in methanol and dichloromethane medium in the presence of aerial oxygen. The
reaction follows Michaelis–Menten enzymatic reaction kinetics with turnover numbers (Kcat) 6.99 ꢀ 103
and 1.85 ꢀ 103 hꢁ1 in methanol and dichloromethane, respectively. 1 is also phenoxazinone synthase
Received 3rd May 2016
Accepted 13th June 2016
DOI: 10.1039/c6ra11433d
www.rsc.org/advances
active in methanol medium with a turnover number of 1.21 ꢀ 105 hꢁ1
.
metalloenzymes.7 Catechol oxidase, the plant enzyme which
converts catechol to quinone coupled with the 2e/2H+ reduction
of oxygen to water in the presence of aerial oxygen, (Scheme 1)
has a dinuclear hydroxobridged antiferromagnetically coupled
Cu(II) active site.8–10 They may have important physiological
roles in photosynthesis, ower colouration and plant disease
resistance.8 As soon as the crystal structure of the active site of
catecholase was published, a good number of copper complexes
with different nuclearity appeared in the literature.5c,11–14
Phenoxazinone synthase, another multicopper oxidase,
naturally produced by Streptomyces antibioticus which has been
cloned and over-expressed in S. lividians is responsible for the
six electron oxidative coupling of two molecules of an o-ami-
nophenol (OAPH) derivative, 4-methyl-3-hydroxyanthraniloyl
pentapeptide, to form the phenoxazinone chromophore15
(Scheme 2) of the antineoplastic agent actinomycin D which is
clinically used for the treatment of Wilm's tumor, gestational
choriocarcinoma and other tumors. Few recent reports are
found in modeling the phenoxazinone synthase.16
Introduction
High nuclearity transition-metal complexes are of current
interest for their possible utility in molecular magnetism1 with
special emphasis on single molecule magnets.2 The study of
spin–spin interactions between paramagnetic metal centres has
become one of the most important research areas in inorganic
as well as coordination chemistry. Copper has long been the
metal of choice for assembling high nuclearity clusters3,4
primarily based on ligands with oxygen along with nitrogen
donor atoms.
Enzymes that are capable of processing molecular oxygen
under ambient conditions are being paid considerable atten-
tion now-a-days because complexes modeling their active site5
may serve as efficient, mild catalysts to carry out synthetic
organic transformations of industrial importance.6 Dinuclear
copper complexes play an important role in biological
aDepartment of Chemistry, The University of Burdwan, Burdwan 713 104, India.
E-mail: rghosh@chem.buruniv.ac.in
bPhysical/Materials Chemistry Division, National Chemical Laboratory, Pune 411 008,
In the present report we have synthesized a tetranuclear
copper(II) complex [Cu4II(L)4] (1) [H2L ¼ N-(2-hydroxyethyl)-3-
methoxysalicylaldimine] which acts as a model for catechol
oxidase and phenoxazinone synthase, and shows overall
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
dDepartment of Physical Sciences, Indian Institute of Science Education & Research,
Mohali, Sector 81, S.A.S. Nagar, Knowledge City, Manauli PO, Mohali 140 306, India
† Electronic supplementary information (ESI) available: UV-Vis, NMR, ESI-MS,
HRMS and EPR data and are reported here. CCDC 1455999. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c6ra11433d
´
‡ Present address: Institut Lavoisier, UMR CNRS 8180, Universite de Versailles
Saint-Quentin-en-Yvelines, 45 avenue des Etats-Unis, 78035 Versailles cedex,
France.
§ Present address: Department of Chemistry, DAV University, Jalandhar, Punjab,
144 012, India.
Scheme 1 Catecholase activity.
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Table 1 Crystal data and structure refinement parameters for 1
Empirical formula
Formula weight
T (K)
Wavelength (A)
Crystal system
Space group
C40H44N4O12Cu4
1068.00
100.0(2)
0.71073
Triclinic
ꢀ
ꢁ
P1
Unit cell dimensions
ꢀ
a (A)
11.9179(14)
12.1517(14)
15.1464(18)
102.667(2)
92.928(2)
100.128(1)
2097.6(4)
2
ꢀ
b (A)
ꢀ
c (A)
a (ꢂ)
b (ꢂ)
g (ꢂ)
ꢀ3
V (A )
Scheme 2 Phenoxazinone synthase activity.
Z
Dcalc (mg mꢁ3
)
1.691
2.071
1092.0
Absorption coefficient (mmꢁ1
F(000)
)
ferromagnetic exchange. The mechanistic aspect of this activity
is also tried to be unraveled with experimental support. The X-
ray structure similar, but not identical to 1 is found in recent
literature.17
Crystal size (mm3)
0.2 ꢀ 0.1 ꢀ 0.1
Theta range for data collection (ꢂ)
Index ranges
1.384–25.027
ꢁ14 # h # 13, ꢁ14 # k # 14,
ꢁ18 # l # 17
Reections collected
Independent reections
Completeness to theta
Absorption correction
Tmax and Tmin
21 470
7373 [Rint ¼ 0.0248]
Results and discussion
Synthesis and formulation
0.994
Multi-scan
0.7455 and 0.7030
Full-matrix least-squares on F2
7373/0/573
Complex 1 was synthesized by addition of solid Cu(II) acetate
dihydrate into the dichloromethane–acetonitrile mixture solu-
tion of the ligand H2L.
Renement method
Data/restraints/parameters
Goodness-of-t (GOF) on F2
Final R indices [l > 2s(l)]
R indices (all data)
Largest difference in
peak and hole (e A
1.047
R1 ¼ 0.0272, wR2 ¼ 0.0680
R1 ¼ 0.0345, wR2 ¼ 0.0644
0.629, ꢁ0.324
The reaction is scheme is here:
ꢀꢁ3
2Cu2(CH3COO)4$2H2O + 4H2L /
)
[CuI4I(L)4] + 8CH3COOH + 4H2O
another ligand molecule in the complex. From bond angles and
distances data (Table 2), it appears that each copper(II) centre is
in distorted square planar geometry. The distortion occurs due
to puckered structure of the ligand.
The complex was soluble in methanol and was characterized
by spectroscopy, ESI-mass spectrometry and single-crystal X-ray
crystallography. In the IR spectra, relatively intense peaks
around 1590–1600 cmꢁ1 due to the C]N stretching frequency
appears the complex.
Magnetism
Crystallography
The c vs. T data are shown in Fig. 2. To extract the nature and
Single crystal X-ray crystallographic analysis of 1 was carried out magnitude of magnetic interactions in the system we have
on a Bruker SMART APEX II CCD diffractometer using Mo-Ka analyzed the c data using the Curie–Weiss model for which the
ꢀ
radiation (l ¼ 0.71073 A). Diffraction data was collected at 100.0 susceptibility in the paramagnetic state is given by the expres-
ꢁ
(2) K and was identied as P1 space groups. The crystal data and sion c ¼ c0 + C/(T ꢁ q), where c0 is the temperature indepen-
renement details are listed in Table 1. The structure was solved dent contribution, C is the Curie constant which depends on the
by direct methods, and the structure solution and renement effective magnetic moment, and q is the Weiss temperature, the
were based on |F|2. All calculations were carried out using sign and magnitude of which will give the nature of the
SHELXL-97 (ref. 18) and were rened using SHELSL-97.19 All the magnetic exchange interactions. For small c0, this will lead to 1/
gures have been generated using ORTEP-32.20
c being linear. The 1/c vs. T data are shown in the inset (a) of
Crystal structure of 1 is shown in Fig. 1 while selected bond Fig. 2. It can be seen that at high temperatures, the behavior is
angles and distances are given in Table 2. The crystal structure linear as expected. The high temperature data between T ¼ 200
of the compound reveals that it is a tetranuclear Cu(II) complex K to 300 K data were t to the above Curie–Weiss expression.
with tetracoordination at each Cu centre. Each metal centre is The parameters obtained from the t are c0 ¼ 1.17(3) ꢀ 10ꢁ4
coordinated with an imine, a phenoxo and one sharing alkoxo cm3 per Cu mol, C ¼ 0.435(7) cm3 K per Cu mol, and q ¼ 16.6 K.
group from a single ligand backbone and the fourth coordina- From C, the effective moment meff can be estimated using the
tion site is satised by another sharing alkoxo group from expression C ¼ NAmeff2/3kBT, where NA is the Avogadro's number
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Fig. 2 Plots of c vs. T for 1; (a) 1/c vs. T plot; (b) cT vs. T plot showing
value of q ¼ 16.6 K indicating moderate ferromagnetic coupling
between the Cu ions.
is shown in inset (b) of Fig. 2. cT, which is roughly the effective
moment, is more or less T-independent for higher temperatures
but increases dramatically as the temperature is lowered
towards T ¼ q. Normally, the system would undergo long-ranged
ferromagnetic transition at this temperature. However, our
system is more complex. It can be seen from inset (a) of Fig. 2
that the 1/c data deviate from the Curie–Weiss t at lower
temperatures. The 1/c becomes higher than expected from the
t, which in turn means that the c becomes lower than expected
for purely ferromagnetic interactions. This implies the presence
of weak antiferromagnetic interactions which show up at much
Fig. 1 An ORTEP of [Cu4(L)4] (1) with atom numbering scheme and
20% probability ellipsoids for all non-hydrogen atoms.
ꢂ
ꢀ
Table 2 Bond lengths [A] and angles [ ] for 1
Bond lengths
Cu(1)–O(7)
Cu(1)–O(8)
Cu(1)–O(11)
Cu(1)–N(4)
Cu(2)–O(1)
Cu(2)–O(8)
Cu(2)–O(9)
Cu(2)–N(1)
1.9457(17)
1.9851(18)
1.9056(19)
1.941(2)
1.9748(17)
1.9551(16)
1.9116(17)
1.927(2)
Cu(3)–O(2)
Cu(3)–O(5)
Cu(3)–O(7)
Cu(3)–N(2)
Cu(4)–O(1)
Cu(4)–O(2)
Cu(4)–O(3)
Cu(4)–N(3)
1.9536(16)
1.8936(18)
1.9651(17)
1.936(2)
1.9406(15)
1.9763(17) lower temperatures. Evidence for this can also be seen in the cT
1.9005(18)
1.936(2)
data which passes over a sharp maximum around T ¼ q before
decreasing sharply for lower temperatures. Thus, our magnetic
study reveals very complex magnetic interactions in this system
with both ferromagnetic and antiferromagnetic interactions of
different magnitudes present.
We propose that within a Cu4 tetramer we have ferromag-
netic exchange, while the inter-tetramer exchange is small and
antiferromagnetic. With this preliminary information about the
magnetic exchange interactions in the system we have analyzed
the full c vs. T data using an isotropic Heisenberg spin Hamil-
tonian given by,
Bond angles
O(7)–Cu(1)–O(8)
O(7)–Cu(1)–O(11)
O(8)–Cu(1)–O(11)
N(4)–Cu(1)–O(7)
N(4)–Cu(1)–O(8)
N(4)–Cu(1)–O(11)
O(1)–Cu(2)–O(8)
O(1)–Cu(2)–O(9)
O(8)–Cu(2)–O(9)
N(1)–Cu(2)–O(1)
N(1)–Cu(2)–O(8)
N(1)–Cu(2)–O(9)
O(2)–Cu(3)–O(7)
O(2)–Cu(3)–O(5)
87.53(7)
94.06(8)
172.83(8)
169.00(8)
84.05(8)
93.45(9)
86.16(7)
177.72(7)
95.69(7)
83.41(8)
167.97(8)
94.88(8)
86.14(7)
96.10(7)
O(5)–Cu(3)–O(7)
N(2)–Cu(3)–O(2)
N(2)–Cu(3)–O(5)
N(2)–Cu(3)–O(7)
O(1)–Cu(4)–O(2)
O(1)–Cu(4)–O(3)
O(2)–Cu(4)–O(3)
N(3)–Cu(4)–O(1)
N(3)–Cu(4)–O(2)
N(3)–Cu(4)–O(3)
Cu(1)–O(7)–Cu(3)
Cu(3)–O(2)–Cu(4)
Cu(4)–O(1)–Cu(2)
Cu(2)–O(8)–Cu(1)
176.91(7)
165.51(8)
94.14(8)
84.04(8)
86.70(7)
94.94(7)
172.58(7)
169.25(8)
84.02(8)
93.59(8)
108.58(8)
104.60(8)
108.14(8)
104.53(8)
H ¼ ꢁJ1(S1S2 + S2S3 + S3S4 + S1S4)
where J1 is the ferromagnetic exchange between the spins Si
within one tetramer. Using the substitutions
SA ¼ S1 + S3, SB ¼ S2 + S4, and ST ¼ SA + SB,
and kB is the Boltzmann constant. This gives meff ¼ 1.86(2) mB.
The value expected for S ¼ 1/2 magnetic moments is meff ¼ 1.73
mB assuming a g-factor of g ¼ 2. A value g ¼ 2.1 is enough to
where ST is the spin of the complete molecule, the Hamiltonian
can be rewritten as
explain our slightly enhanced meff
.
2
2
H ¼ ꢁJ1(S1 + S3)(S2 + S4) ¼ ꢁJ1SASB ¼ ꢁJ1(ST ꢁ SA ꢁ SB2)/2
The eigen values E(ST, SA, SB) are then given by,
RSC Adv., 2016, 6, 58831–58838 | 58833
Let us now turn to the nature of the magnetic exchange
interactions between these S ¼ 1/2 Cu moments. The value q ¼
16.6 K indicate moderate ferromagnetic coupling between the
Cu ions. Evidence for this can also be seen in the cT data which
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E(ST, SA, SB) ¼ ꢁJ1[ST(ST + 1) ꢁ SA(SA + 1) ꢁ SB(SB + 1)]/2
where SA ¼ 0 and 1, SB ¼ 0 and 1, and ST ¼ 0, 1, and 2. The
magnetic susceptibility c for a collection of Cu4-tetramers,
which can then be calculated using the Van Vleck equation,
comes out to be
2
3
J1
2T
2J1
2T
ꢀ
ꢁ
2NAmB2g2
2 þ e þ 5e
4
5
c0 ¼
:
J1
2T
2J1
2T
kBT
7 þ 3e þ 6e
Assuming that the interactions between tetramers acts as
a mean-eld, the susceptibility of interacting tetramers can
then be written down as
ꢀ
ꢁ
c0
c ¼
;
1 þ lc0
Fig. 3 Change in spectral pattern of complex 1 after reaction with 3,5-
DTBC, observing the reaction for 6 h in methanol; inset: spectrum of
pure 3,5-DTBC in methanol. This repetitive spectra were obtained in 9
min interval at room temperature. The peak for the coloured product
(3,5-DTBQ) appears at ꢃ390 nm which gradually shifts to 401 nm.22
where l is the exchange coupling which can be given in terms of
the inter-tetramer exchange J2 as l ¼ zJ2/3C, where z ¼ 6 is the
number of nearest neighbor tetramers and C is the Curie
constant for S ¼ 1/2. The experimental c data were t by the
above theoretical expression. The t, shown in Fig. 2 as the red
curve through the data gave the values J1 ¼ 33.0 (4) K (i.e. 22.9
cmꢁ1) and J2 ¼ ꢁ1.0 (7) K (i.e. ꢁ0.6 cmꢁ1). The error factor came
out to be R ¼ 1.31 ꢀ 10ꢁ5. Similar exchange interactions are
found in some of the recent literature.17,21
concentration ratio (Fig. 4). A rst-order catalytic reaction is
observed, with rate 0.0211 minꢁ1
.
The catecholase activity of complex 1 was similarly studied in
DCM media. Similar to that of MeOH, in DCM also 3,5-DTBQ
shows maximum absorption at ꢃ390 nm which shis to 401 nm
nally (Fig. S2; ESI†). 3,5-DTBQ obtained, was puried by
column chromatography and characterized by determining its
melting point (ꢃ110 ꢂC) which agreed well with that reported in
literature.23
The reaction kinetics was studied by observing the time
dependent change in absorbance at a wavelength of 401 nm for
catalysis in dichloromethane. The difference in absorbance DA
at these particular wavelengths, were plotted against time to
Catecholase activity
Spectrophotometric studies. The catecholase activity study
was carried out using the substrate 3,5-di-tert-butyl catechol
(3,5-DTBC) having two bulky t-butyl substituents on the ring
and low quinone–catechol reduction potential. This makes it
easily oxidized to the corresponding ortho-quinone derivative
3,5-di-tert-butyl quinone (3,5-DTBQ) which is highly stable and
shows a maximum absorption at 401 nm in methanol.11 Spec-
tral bands at 666, 374, 278 and 234 nm appear in the electronic
spectrum of complex 1, whereas 3,5-DTBC shows a single band
at 282 nm. Upon treatment of methanolic solution of 1 into 100
equivalents of 3,5-DTBC under aerobic conditions, the repeti-
tive UV-Vis spectral scan was recorded (Fig. 3). The colourless
solution gradually turned deep brown which indicates conver-
sion of 3,5-DTBC to 3,5-DTBQ. In UV-Vis spectrophotometer,
aer this addition, the spectral scan shows very smooth
increase of a quinone band at around ꢃ390 nm which shis
ultimately to 401 nm, as reported by Krebs et al.22 The same
reaction was carried out by scaling up the reagents and 3,5-
DTBQ was puried by column chromatography. It was then
isolated and identied by H1 NMR spectroscopy (Fig. S1; ESI†).
H1 NMR (CDCl3, 400 MHz): dH ¼ 1.16 (s, 9H), 1.20 (s, 9H), 6.15
(d, J ¼ 2.4 Hz, 1H), 6.86 (d, J ¼ 2.4 Hz, 1H).
The time dependent change in absorbance at a wavelength of
401 nm, characteristic of 3,5-DTBQ in methanol, was observed
for 25 min to comprehend the reaction kinetics and nd out the
reaction rate between 3,5-DTBC and 1. The difference in
absorbance DA at 401 nm, was plotted against time to obtain the
Fig. 4 A plot of the difference in absorbance (DA) vs. time to evaluate
rate/velocity for that particular catalyst to substrate the rate of catalysis of 3,5-DTBC by 1 in methanol.
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obtain the rate of the reaction. A rst-order catalytic reaction complex 1 and 3,5-DTBC of mixing them together (Fig. S5b;
with rate 5.95 ꢀ 10ꢁ3 minꢁ1 is observed in DCM (Fig. S3; ESI†). ESI†). The signal at m/z ¼ 196 is due to the formation of the
Enzyme kinetic study. Kinetic experiments were performed protonated ligand [H2L]–H+. The peak at m/z ¼ 243 can be
spectrophotometrically with complex 1 and the substrate 3,5- assigned to sodium aggregate of quinone [3,5-DTBQ]–Na+. The
DTBC in methanol and dichloromethane, thermostated at 20 protonated complex [Cu4(L)4]–H+ exhibits a peak at m/z ¼ 1027.
ꢂC. 0.04 ml of the complex solution, with a constant concen- The catalytic process is shown in Scheme 3. The formation of an
tration of 1 ꢀ 10ꢁ4 M, was added to 2 ml of 3,5-DTBC of complex-substrate adduct intermediate species is identied by
a particular concentration (varying its concentration from 1 ꢀ the peak at m/z ¼ 477. The semiquinone is identied from EPR
10ꢁ3 M to 1 ꢀ 10ꢁ2 M) to achieve the nal concentration of the spectrum (Fig. S6; ESI†). The catechol derivative, 3,5-DTBC gets
complex as 1 ꢀ 10ꢁ4 M. The conversion of 3,5-DTBC to 3,5- oxidised to quinone in presence of oxygen. The aerial oxygen
DTBQ was monitored with time at a wavelength of 401 nm for that oxidises 3,5-DTBC to 3,5-DTBQ in this process is converted
solutions in MeOH and DCM. The rate for each concentration of to H2O2. H2O2 thus liberated was identied and characterized
the substrate was determined by the initial rate method.
The rate versus concentration of substrate data were analyzed
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. The observed rate vs. [substrate] plot as well as Lineweaver–
Burk plot in methanol and dichloromethane solutions are given
in Fig. 5 and S4 (ESI†) respectively. The kinetic parameters are
listed in Table 3. The turnover numbers (Kcat) are 6.99 ꢀ 103 and
1.85 ꢀ 103 hꢁ1 in MeOH and DCM, respectively.
spectrophotometrically (S1; ESI†).24
Phenoxazinone synthase activity
Spectrophotometric studies. The phenoxazinone synthase
activity study was carried out using the substrate o-amino-
phenol (OAPH). OAPH shows bands at 286 and 232 nm in pure
methanol. Upon treatment of methanolic solution of 1 into 100
equivalents of OAPH under aerobic conditions, the repetitive
Reaction mechanism. The catalytic process follows a two-
step mechanistic pathway. The rst step is the rate deter-
mining step. Probably, in this step, the 1 : 1 adduct of catechol
and the copper complex is formed. To obtain a mechanistic
inference of the catecholase activity and to get an idea about the
complex-substrate intermediate, we recorded an ESI-MS spec-
trum of 1 in methanol (Fig. S5a; ESI†) and of a 1 : 100 mixture of
Fig. 5 Plot of rate vs. [substrate] (3,5-DTBC) in presence of 1 in MeOH;
inset: Lineweaver–Burk plot.
Scheme 3 Catalytic cycle for catecholase activity by 1.
Table 3 Kinetic parameters for the oxidation of 3,5-DTBC catalyzed by 1
Solvent
Vmax (M sꢁ1
)
Std. error
KM (M)
Std. error
Kcat (hꢁ1
)
MeOH
DCM
1.94 ꢀ 10ꢁ4
5.14 ꢀ 10ꢁ5
6.94 ꢀ 10ꢁ5
3.37 ꢀ 10ꢁ6
5.39 ꢀ 10ꢁ3
4.04 ꢀ 10ꢁ4
2.86 ꢀ 10ꢁ3
1.04 ꢀ 10ꢁ4
6.99 ꢀ 103
1.85 ꢀ 103
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UV-Vis spectral scan was recorded (Fig. 6). The colourless
solution gradually turned deep brown which indicates conver-
sion of OAPH to 2-aminophenoxazine-3-one (APX). The spectral
scan shows very smooth growing of APX band at 427 nm.16 APX
was identied by HRMS with m/z ¼ 213 (Fig. S7; ESI†). The time
dependent change in absorbance at a wavelength of 427 nm,
characteristic of APX in methanol, was observed for 25 min to
comprehend the reaction kinetics and nd out the reaction rate
between OAPH and 1. The difference in absorbance DA at 427
nm, was plotted against time to obtain the initial rate for that
particular catalyst to substrate concentration ratio (Fig. 7). A
rst-order catalytic reaction is observed, with initial rate 0.0188
minꢁ1
.
Enzyme kinetic study. Enzymatic kinetic experiments were
performed UV-Vis spectrophotometrically thermostated at 25 ꢂC
with complex 1 and the substrate OAPH in MeOH. 0.04 ml of the
Fig. 8 Plot of rate vs. [substrate] (OAPH) in presence of 1 in MeOH;
inset: Lineweaver–Burk plot.
Table 4 Kinetic parameters for the oxidative coupling of OAPH
catalyzed by 1
Solvent Vmax (M sꢁ1) Std. error
KM (M)
Std. error
Kcat (hꢁ1
)
MeOH 3.36 ꢀ 10ꢁ3 2.11 ꢀ 10ꢁ4 2.89 ꢀ 10ꢁ3 2.39 ꢀ 10ꢁ4 1.21 ꢀ 105
complex solution, with a constant concentration of 1 ꢀ 10ꢁ4 M,
was added to 2 ml of OAPH of a particular concentration
(varying its concentration from 1 ꢀ 10ꢁ3 M to 1 ꢀ 10ꢁ2 M) to
achieve the nal concentration of the complex as 1 ꢀ 10ꢁ4 M.
The conversion of OAPH to APX was monitored with time at
a wavelength of 427 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 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. The observed rate vs. [substrate]
plot in methanol solution as well as Lineweaver–Burk plot is
given in Fig. 8. The kinetic parameters are listed in Table 4. The
turnover number (Kcat) is 1.21 ꢀ 105 hꢁ1 in methanol.
Fig. 6 Change in spectral pattern of complex 1 in MeOH after reaction
with OAPH, observing the reaction for 6 h; inset: spectrum of pure
OAPH in methanol.
Experimental
Materials and methods
High purity o-vanillin (Aldrich, UK), 2-aminoethanol (Aldrich,
UK), copper(II) acetate dihydrate (Aldrich, UK), 3,5-di-tert-
butylcatechol (Aldrich, UK), o-aminophenol (Aldrich, UK) and
all other solvents were purchased from the respective concerns
and used as received. Solvents were dried according to standard
procedure and distilled prior to use.
The ligand H2L was prepared using a reported procedure.3a
O-vanillin (0.3043 g, 2 mmol) was heated under reux with
ethanol amine (0.1222 g, 2 mmol) in 30 ml dehydrated ethanol.
Fig. 7 A plot of the change in absorbance with time to evaluate the
initial rate of the catalytic oxidation of OAPH by 1.
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Aer 2 h, the reaction solution was evaporated under reduced catecholase activity than the recent reported ones, though, to
pressure to yield a yellow coloured solid, which was dried under the best of our knowledge, the most active catalyst11a reported so
vacuum and stored over CaCl2 for subsequent use.
far, exhibits a turnover number of 3.24 ꢀ 104 hꢁ1. It is also
For catecholase activity study, 1 ꢀ 10ꢁ4 mol dmꢁ3 solution of phenoxazinone synthase active in methanol medium with
1 (0.0011 g) was treated with 1 ꢀ 10ꢁ2 mol dmꢁ3 (100 equiva- a turnover number of 1.21 ꢀ 105 hꢁ1. So comparing all these
lents) of 3,5-DTBC (0.0222 g) under aerobic conditions.
data it can be concluded that the reported complex (1) is quite
For phenoxazinone synthase activity study, 1 ꢀ 10ꢁ4 mol an efficient catalyst and has an appreciable turnover rates in
dmꢁ3 solution of 1 (0.0011 g) was treated with 1 ꢀ 10ꢁ2 mol various solvents.
dmꢁ3 (100 equivalents) of OAPH (0.0109 g) under aerobic
conditions.
Acknowledgements
RG sincerely thanks Department of Science & Technology,
Government of West Bengal [No. 781(Sanc.)/ST/P/S&T/4G-4/
2013 dated 04-12-2014], India for nancial assistance. MM
thanks The University of Burdwan for her fellowship. We extend
our gratitude to Dr R. Banerjee, Physical/Materials Chemistry
Division, National Chemical Laboratory, Pune 411 008, India for
X-ray crystallographic studies.
General procedure for synthesis of 1
Compound 1 was prepared by addition of solid Cu2(OAc)4$2H2O
(0.0498 g, 0.25 mmol) into a stirring solution of H2L (0.0244 g,
0.125 mmol) in dichloromethane–acetonitrile mixture (15 ml).
The resulting deep green coloured solution was kept in open air
for slow evaporation. Aer 8–10 days, the deep green crystals of
1 was collected, washed with hexane and dried in vacuo over
silica gel indicator.
Notes and references
Yield: (based on metal salt) 0.0562 g (84.25%). Anal. calc. for
C
40H44N4O12Cu4 (1): C, 44.94; H, 4.15; N, 5.24; found: C, 39.97;
´
`
1 (a) J. Hernandez-Gil, N. Ovejak, S. Ferrer, F. Lloret and
H, 3.84; N, 3.82. Selected IR bands (KBr pellet, cmꢁ1): 3464 (s),
1637 (s), 1606 (s), 1473 (s) 1440 (s). UV-Vis (l, nm): 666, 374, 278,
234.
˜
A. Castineiras, Inorg. Chem., 2013, 52, 2289; (b)
R. C. Poulten, M. J. Page, A. G. Algarra, J. J. Le Roy,
´
I. Lopez, E. Carter, A. Llobet, S. A. Macgregor,
M. F. Mahon, D. M. Murphy, M. Murugesu and
M. K. Whittlesey, J. Am. Chem. Soc., 2013, 135, 13640; (c)
Physical measurements
Elemental analyses (carbon, hydrogen and nitrogen) were per-
formed on a Perkin-Elmer 2400 CHNS/O elemental analyzer.
UV-Vis and IR spectra (KBr discs, 4000–300 cmꢁ1) were recorded
using a Shimadzu UV-Vis 2450 spectrophotometer and Perkin-
Elmer FT-IR model RX1 spectrometer, respectively. The H1
NMR spectral data were collected in CDCl3 on a Bruker 400 MHz
spectrometer. Mass spectrometric data were collected on
Waters Q-TOF-MICRO MS system using electron spray ionisa-
tion (ESI) techniques. Magnetic susceptibility c versus temper-
ature T measurements were performed on powder samples in
an applied magnetic eld of H ¼ 1 T, using the VSM option of
a Quantum Design PPMS.
´
A. Aliev, M. Huve, S. Colis, M. Colmont, A. Dinia and
´
O. Mentre, Angew. Chem., Int. Ed., 2012, 124, 9527; (d)
A. Mukherjee, R. Raghunathan, M. K. Saha, M. Nethaji,
S. Ramasesha and A. R. Chakravarty, Chem.–Eur. J., 2005,
11, 3087.
2 (a) S. K. Langley, N. F. Chilton, B. Moubaraki and
K. S. Murray, Inorg. Chem., 2013, 52, 7183; (b) A. Masello,
K. A. Abboud, W. Wernsdorfer and G. Christou, Inorg.
Chem., 2013, 52, 10414; (c) S.-D. Jiang, B.-W. Wang, G. Su,
Z.-M. Wang and S. Gao, Angew. Chem., Int. Ed., 2010, 122,
7610.
3 (a) Y. Nishida and S. Kida, J. Chem. Soc., Dalton Trans., 1986,
2633; (b) W. Mazurek, B. J. Kennedy, K. S. Murray,
M. J. O'connor, J. R. Rodgers, M. R. Snow, A. G. Wedd and
P. R. Zwack, Inorg. Chem., 1985, 24, 3258; (c) K. Geetha,
M. Nethaji, N. Y. Vasanthacharya and A. R. Chakravarty, J.
Coord. Chem., 1999, 47, 77.
4 (a) R. D. Harcourt, F. L. Skrezenek and R. G. A. R. Maclagan,
J. Am. Chem. Soc., 1986, 108, 5403; (b) M. Kato and Y. Muto,
Coord. Chem. Rev., 1988, 92, 45; (c) M. Melnik, Coord. Chem.
Rev., 1982, 42, 259; (d) S.-F. Si, J.-K. Tang, D.-Z. Liao,
Z.-H. Jiang and S.-P. Yan, Inorg. Chem. Commun., 2002, 5, 76.
5 (a) S. Majumder, S. Mondal, P. Lemoine and S. Mohanta,
Dalton Trans., 2013, 42, 4561; (b) S. Mukherjee,
T. Weyhermuller, E. Bothe, K. Wieghardt and
P. Chaudhuri, Dalton Trans., 2004, 3842; (c) S. Mandal,
J. Mukherjee, F. Lloret and R. N. Mukherjee, Inorg. Chem.,
2012, 51, 13148.
Conclusions
Synthesis and crystallographic characterization of a tetranu-
clear Cu(II) Schiff base complex is reported here. The variable
temperature magnetic measurement shows an overall ferro-
magnetic along with a weak antiferromagnetic interaction in
the complex. The compound shows catecholase activity in
methanol and dichloromethane solvent with turnover numbers
6.99 ꢀ 103 and 1.85 ꢀ 103 hꢁ1, respectively, which are much
higher than those reported in recent times.11b–d,24b Mohanta
et al. reported the turnover numbers 39 hꢁ1, 40 hꢁ1, and 48 hꢁ1
in DMF, and 167 hꢁ1 and 215 hꢁ1 in MeCN for Cu(II) complex-
es.11b In a recent report by Rajak et al., two Cu(II) complexes
show a turnover rate of about 29 and 37 hꢁ1
.
11c The same group
reported a dicopper complex with turnover number of 26 hꢁ1
.
11d
A turnover rate of 28 hꢁ1 of a Cu(II) complex is reported by Neves
et al.24b The above results indicate that 1 is an effective model for
6 (a) S. Friedle, E. Reisner and S. J. Lippard, Chem. Soc. Rev.,
2010, 39, 2768; (b) P. C. A. Bruijnincx, G. van Koten and
This journal is © The Royal Society of Chemistry 2016
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Paper
R. J. M. Klein Gebbink, Chem. Soc. Rev., 2008, 37, 2716; (c)
S. I. Chan and S. S.-F. Yu, Acc. Chem. Res., 2008, 41, 969.
D. Floner, O. Lavastrec and F. Geneste, New J. Chem., 2012,
36, 1828.
7 I. Bertini, H. H. Gray, S. J. Lippard and J. S. Valentine, 14 E. Mijangos, J. Reedijk and L. Gasque, Dalton Trans., 2008,
Bioinorganic chemistry, Viva Books Pvt. Ltd., New Delhi, 1998. 1857.
8 C. Gerdemann, C. Eicken and B. Krebs, Acc. Chem. Res., 2002, 15 A. W. Smith, A. Camara-Artigas, M. Wang, J. P. Allen and
35, 183. W. A. Francisco, Biochemistry, 2006, 45, 4378.
9 E. I. Solomon, U. M. Sundaram and T. E. Machonkin, Chem. 16 (a) A. Panja, M. Shyamal, A. Saha and T. K. Mandal, Dalton
Rev., 1996, 96, 2563.
Trans., 2014, 43, 5443; (b) A. Panja, Dalton Trans., 2014, 43,
¨
10 (a) T. Klabunde, C. Eicken, J. C. Sacchettini and B. Krebs,
Nat. Struct. Biol., 1998, 5, 1084; (b) C. Eicken, F. Zippel,
7760; (c) C. Mukherjee, T. Weyhermuller, E. Bothe and
P. Chaudhuri, C. R. Chim., 2007, 10, 313; (d) C. Mukherjee,
¨
¨
K. Buldt-Karentzopoulos and B. Krebs, FEBS Lett., 1998,
T. Weyhermuller, E. Bothe, E. Rentschler and
¨
436, 293; (c) A. Rompel, H. Fischer, D. Meiwes, K. Buldt-
P. Chaudhuri, Inorg. Chem., 2007, 46, 9895.
Karentzopoulos, R. Dillinger, F. Tuczek, H. Witzel and 17 E. Gungor, H. Kara, E. Colacio and A. J. Mota, Eur. J. Inorg.
B. Krebs, JBIC, J. Biol. Inorg. Chem., 1999, 4, 56. Chem., 2014, 1552.
11 (a) K. S. Banu, T. Chattopadhyay, A. Banerjee, 18 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr.,
S. Bhattacharya, E. Suresh, M. Nethaji, E. Zangrando and 2008, 64, 112.
D. Das, Inorg. Chem., 2008, 47, 7083; (b) S. Majumder, 19 O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard
˜
S. Sarkar, S. Sasmal, E. Carolina Sanudo and
S. S. Mohanta, Inorg. Chem., 2011, 50, 7540; (c) A. Banerjee, 20 L. J. Farrugia, ORTEP-32 for Windows, University of Glasgow,
S. Sarkar, D. Chopra, E. Colacio and K. K. Rajak, Inorg. Scotland, 1998.
Chem., 2008, 47, 4023; (d) A. Banerjee, R. Singh, E. Colacio 21 (a) L. Salmon, P. Thuery, E. Riviere, J.-P. Costes, A. J. Mota
and H. Puschmann, J. Appl. Crystallogr., 2009, 42, 339.
´
`
and K. K. Rajak, Eur. J. Inorg. Chem., 2009, 277.
and M. Ephritikhine, New J. Chem., 2014, 38, 1306; (b)
B. K. Babu, A. R. Biju, S. Sunkari, M. V. Rajasekharan and
J.-P. Tuchagues, Eur. J. Inorg. Chem., 2013, 1444; (c)
12 (a) K. Selmeczi, M. Reglier, M. Giorgi and G. Speier, Coord.
Chem. Rev., 2003, 245, 191; (b) M. C. Mimmi, M. Gullotti,
L. Santagostini, A. Saladino, L. Casella, E. Monzani and
R. Pagliarin, J. Mol. Catal. A: Chem., 2003, 204–205, 381; (c)
C. T. Yang, M. Vetrichelvan, X. Yang, B. Moubaraki,
K. S. Murray and J. J. Vittal, Dalton Trans., 2004, 113; (d)
J. Reim and B. Krebs, J. Chem. Soc., Dalton Trans., 1997,
`
R. Papadakis, E. Riviere, M. Giorgi, H. Jamet, P. Rousselot-
´
Pailley, M. Reglier, A. J. Simaan and T. Tron, Inorg. Chem.,
2013, 52, 5824; (d) S. Mukherjee and P. S. Mukherjee,
Dalton Trans., 2013, 42, 4019; (e) M. Sutradhar,
M. V. Kirillova, M. F. C. G. da Silva, C.-M. Liu and
A. J. L. Pombeiro, Dalton Trans., 2013, 42, 16578.
´
´
´
37934; (e) L. Gonzalez-Sebastian, V. M. Ugalde-Saldıvar,
E. Mijangos, M. R. Mendoza-Quijano, L. Ortiz-Frade and 22 F. Zippel, F. Ahlers, R. Werner, W. Haase, H.-F. Nolting and
L. Gasque, J. Inorg. Biochem., 2010, 104, 1112; (f) L. Gasque, B. Krebs, Inorg. Chem., 1996, 35, 3409.
V. M. Ugalde-Saldıvar, I. Membrillo, J. Olguın, E. Mijangos, 23 S. Tsuruya, S.-I. Yanai and M. Masai, Inorg. Chem., 1986, 25,
´
´
`
´
S. Bernes and I. Gonzalez, J. Inorg. Biochem., 2008, 102, 1227.
141.
13 (a) A. L. Abuhijleh, J. Pollitte and C. Woods, Inorg. Chim. 24 (a) A. I. Vogel, Textbook of quantitative inorganic analysis,
Acta, 1994, 215, 131; (b) A. L. Abuhijleh, C. Woods,
E. Bogas and G. L. Guenniou, Inorg. Chim. Acta, 1992, 195,
67; (c) M. R. Malachowski, M. G. Davidson and
J. N. Hoffman, Inorg. Chim. Acta, 1989, 157, 91; (d)
M. R. Malachowski and M. G. Davidson, Inorg. Chim. Acta,
1989, 162, 199; (e) R. Marion, N. M. Saleh, N. Le Poul,
Green and Co. Ltd., London, 1961, 3rd end, p. 366,
Longmans; (b) A. Neves, L. M. Rossi, A. J. Bortoluzzi,
B. Szpoganicz, C. Wiezbicki and E. Schwingel, Inorg.
Chem., 2002, 41, 1788; (c) E. Monzani, L. Quinti, A. Perotti,
L. Casella, M. Gullotti, L. Randaccio, S. Geremia,
G. Nardin, P. Faleschini and G. Tabbi, Inorg. Chem., 1998,
37, 553.
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