Mannich base Cu(II) complexes as biomimetic oxidative catalyst

Article abstract of DOI:10.1016/j.jinorgbio.2019.03.023

Galactose Oxidase (GOase) and catechol oxidase (COase) are the metalloenzymes of copper having monomeric and dimeric sites of coordination, respectively. This paper summarizes the results of our studies on the structural, spectral and catalytic properties of new mononuclear copper (II) complexes [CuL(OAc)] (1), and [CuL₂] (2), (HL = 2,4?dichloro?6?{[(2′?dimethyl?aminoethyl)methylamino]methyl}?phenol) which can mimic the functionalities of the metalloenzymes GOase and COase. The structure of the compounds has been elucidated by X-ray crystallography and the mimicked Cu(II) catalysts were further characterized by EPR. These mimicked models were used for GOase and COase catalysis. The GOase catalytic results were identified by GC–MS and, analyzed by HPLC at room temperature. The conversion of benzyl alcohol to benzaldehyde were significant in presence of a strong base, Bu₄NOMe in comparison to the neutral medium. Apart from that, despite of being monomeric in nature, both the homogeneous catalysts are very prone to participate in COase mimicking oxidation reaction. Nevertheless, during COase catalysis, complex 1 was found to convert 3,5?ditertarybutyl catechol (3,5-DTBC) to 3,5?ditertarybutyl quinone (3,5-DTBQ) having greater rate constant, k_cat or turn over number (TON) value over complex 2. The generation of reactive intermediates during COase catalysis were accounted by electrospray ionization mass spectrometry (ESI-MS). Through mechanistic approach, we found that H₂O₂ is the byproduct for both the GOase and COase catalysis, thus, confirming the generation of reactive oxygen species during catalysis. Notably, complex 1 having mono-ligand coordinating atmosphere has superior catalytic activity for both cases in comparison to complex 2, that is having di-ligand environment.

Full text of DOI:10.1016/j.jinorgbio.2019.03.023

Accepted Manuscript
Mannich base Cu(II) complexes as biomimetic oxidative catalyst
Bidyut Kumar Kundu, Rishi Ranjan, Attreyee Mukherjee, Shaikh
M. Mobin, Suman Mukhopadhyay
PII:
S0162-0134(19)30048-0
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.03.023
JIB 10683
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
22 January 2019
23 March 2019
28 March 2019
Please cite this article as: B.K. Kundu, R. Ranjan, A. Mukherjee, et al., Mannich base
Cu(II) complexes as biomimetic oxidative catalyst, Journal of Inorganic Biochemistry,
https://doi.org/10.1016/j.jinorgbio.2019.03.023
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ACCEPTED MANUSCRIPT
Mannich base Cu(II) complexes as biomimetic oxidative catalyst
a
a
b
a
Bidyut Kumar Kundu, Rishi Ranjan, Attreyee Mukherjee, Shaikh M. Mobin,
a, c,
Suman Mukhopadhyay
*
a
Discipline of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore,
Khandwa Road, Simrol, Indore 453552, India.
b
Department of Chemistry, Ananda Mohan College, Kolkata 700009, India
c
Discipline of Biosciences and Biomedical Engineering, School of Engineering, Indian
Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India.
*
E-mail: suman@iiti.ac.in; Fax: +91 731 2361; Tel: +91 731 2438 735
Abstract
Galactose Oxidase (GOase) and catechol oxidase (COase) are the metalloenzymes of copper
having monomeric and dimeric sites of coordination, respectively. This paper summarizes the
results of our studies on the structural, spectral and catalytic properties of new mononuclear
copper (II) complexes [CuL(OAc)] (1), and [CuL ] (2), (HL = 2,4-dichloro-6-{[(2'-dimethyl-
2
aminoethyl)methylamino]methyl}-phenol) which can mimic the functionalities of the
metalloenzymes GOase and COase. The structure of the compounds has been elucidated by
X-ray crystallography and the mimicked Cu(II) catalysts were further characterized by
EPR. These mimicked models were used for GOase and COase catalysis. The GOase
catalytic results were identified by GC-MS and, analyzed by HPLC at room temperature. The
conversion of benzyl alcohol to benzaldehyde were significant in presence of a strong base,
Bu NOMe in comparison to the neutral medium. Apart from that, despite of being
4
monomeric in nature, both the homogeneous catalysts are very prone to participate in COase
mimicking oxidation reaction. Nevertheless, during COase catalysis, complex 1 was found to
convert 3,5-ditertarybutyl catechol (3,5-DTBC) to 3,5-ditertarybutyl quinone (3,5-DTBQ)
having greater rate constant, k or turn over number (TON) value over complex 2. The
cat
generation of reactive intermediates during COase catalysis were accounted by electrospray
ionization mass spectrometry (ESI-MS). Through mechanistic approach, we found that H O
2
2
is the byproduct for both the GOase and COase catalysis, thus, confirming the generation of
reactive oxygen species during catalysis. Notably, complex 1 having mono-ligand
coordinating atmosphere has superior catalytic activity for both cases in comparison to
complex 2, that is having di-ligand environment.
Introduction
Enzymatic reactions have motivated a lot of researchers to plan some small molecule
imitates which can perform the function of the enzymes under various catalytic conditions.[1,
2
] Among which metalloenzymes is a class of efficient biological catalysts showing high
1

ACCEPTED MANUSCRIPT
catalytic conversion of organic substrates where metal ion cofactor attached to the amino
acids side chain in its active site(s) plays the most crucial role.[3] Copper having significant
redox properties can be found in many metalloenzyme, which allows it to participate in
various oxidation reactions.[4, 5] Cu(II) containing metalloenzymes are basically surrounded
by the coordinate-covalent bonds with–N/–O donor centres mostly from the side chains of
amino acids.[5] However, mimicking structural and functional properties of any
metalloenzyme, by synthesized transition metal coordination complexes, is of great interest
for past few decades.[6, 7] Yet, complicated synthetic procedures of suitable mimics, stability
in homogeneous conditions, lower solubility of catalysts in organic or aqueous-buffer media,
less conversion efficiency of catalytic substrates, and extreme reaction conditions are the
prominent challenges, which need to be explored for further improvement.
Galactose Oxidase (GOase) is a mononuclear type II copper fungal metalloenzyme,
-
which is efficient for two e transfer oxidation of primary alcohols to aldehydes.[8-10] Its
active site consists of two oxygen atom of tyrosine (Tyr272 and Tyr495) and two nitrogen
atom of histidine (His496 and His581) protein residues to coordinate with Cu(II) center along
with a labile water molecule (pH~7) to form a distorted square pyramidal geometry as
depicted in Scheme 1.[11, 12] Sometimes water can be replaced by an acetate group at lower
pH~3.5. Furthermore, this enzyme is capable of dioxygen reduction to hydrogen peroxide
under aerobic condition.[2, 13] Therefore, to activate aerial oxygen by using laboratory
synthesized transition metal complexes is an important phenomena towards green oxidation
based on bio-inspired perception. Furthermore the importance of oxidation of primary alcohol
into aldehyde lies in the fact that aromatic aldehydes are widely used for a flavor in
chocolates, chewing gums, and ice creams, etc.[14, 15] Moreover, these can be easily
oxidized to acids, which have appreciable water solubility, and can act as a preservative in
many drinks.[16] The design of N O based mononuclear Cu(II) based GOase enzyme is thus
2
2
too important to understand to follow the mechanistic pathways of original enzyme. Hence,
researchers have focused to the synthesis of salen based ligand (N O ), or N O model as
2
2
3
GOase analogues in recent past.[17-19] Despite of being functionally similar, the mechanistic
approach of GOase catalysis needs to be explored further for better understanding and
improved activity.
2

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Scheme 1. Structural and functional model of galactose oxidase (GOase), and catechol
oxidase (COase)
Apart from that, as described in Scheme 1, catechol oxidase (COase) is a type III di-
copper plant or fungi cofactor surrounded by six histidine residues.[20-22] This is well
known for the reversible binding of molecular oxygen at ambient conditions. It oxidises ortho
di-phenols into ortho quinones along with reduction of O to H O.[23] Ripening of fruits or
2
2
vegetables is the primary cause of quinone formation by COase catalyst.[24, 25] The
production of quinone based compounds can be used to generate potent anticancer agents,
scavenging agent of sulphides, mercaptans, and cyanides, and can inhibit proteases or bone
marrow function. However, there are numerous reports on the several transition metals based
small molecule organic mimics of COase, but many of those are either less active towards
catalysis, or lack of mechanistic evidence.[26-29]
Although, lots of Schiff base complexes based on –N/–O donors have been
synthesized over the years to mimic the functional properties of metalloenzymes[30-32] but
the stability of them are always in question in fully aqueous-buffer media as the imine double
bond is prone to hydrolysis. In comparison to Schiff base, which is capable of electrophilic
substitution for its imine bond, Mannich base compounds which are not easily hydrolysable
in aqueous and non-aqueous solvents are more potent candidates. In spite of lower conversion
efficiency with respect to original enzyme, laboratory synthesized catalysts can show more
susceptibility towards higher temperature, and large solvent scope.
Herein, we have synthesized two mononuclear Cu(II) Mannich base
complexes to check the galctose oxidase (GOase) and catechol oxidase (COase) mimicking
activity.
Complex
CuL(OAc)
(1)
[HL
=
2,4-dichloro-6-{[(2'-dimethyl
3

ACCEPTED MANUSCRIPT
aminoethyl)methylamino] methyl}-phenol] with N O site of coordination can act as proper
2
2
GOase and COase mimics. Moreover, a di-ligand based octahedral complex [CuL ] (2) have
2
been also been synthesized to compare the catalytic activity with that of 1.
Experimental Section
Kindly consult to the electronic supplementary for the details instrumentation.
HPLC analysis to monitor GOase mimicking activity
The method for recording high performance liquid chromatography (HPLC) was
taken as described earlier.[33] Kindly consult to the supporting information for the details
elaboration.
Synthesis and characterization
Synthesis of 2,4-dichloro-6-{[(2'-dimethyl aminoethyl)methylamino]methyl}-phenol, (HL)
An improved synthetic method has been applied for synthesizing the novel Mannich
base ligand, HL.[34] The detailed synthetic procedure is described in Section S05 of ESI†.
Synthesis of 2,4-dichloro-6-{[(2'-dimethylaminoethyl)methylamino]methyl}-phenolcupric
acetate, CuL(OAc) (1)
A solution of HL (0.14 g; 0.5 mmol), triethylamine (76 μL; 0.5 mmol), and
Cu(OAc) . H O (0.1 g; 0.5 mmol) in MeOH (15 mL) was stirred under refluxing for 4 h. The
2
2
dark greenish microcrystalline solid was collected by filtration. Several methods have been
employed to obtain single crystals of the compound. However, in almost all cases the
diffraction pattern in very weak and remains non-reportable. Yield: 0.204 g (∼86%). Anal.
Calcd (%) for C H Cl CuN O : C, 42.17; H, 5.06; N, 7.02; Found (%): C, 42.20; H, 5.08;
1
4
20
2
2
3
−
1
N, 6.98; FTIR (in KBr, ʋ in cm ): 1663, 1460 {ʋ
(s)}; 3640–3610 {ʋC-O, (s, sh)}; 3000–
C=O,
2
{
850 {ʋC-H, (m)}; 1250–1020 {ʋ
(m)} (Figure S2). ESI-MS in HPLC MeOH (+, m/z):
C-N,
+
+
C H Cl CuN O } : 399.8(100%), {C H Cl CuN O} : 340.0 (Figure S3b); UV-Vis in
14
20
2
2
3
14 20
2
2
HPLC MeOH: (λmax, nm): 440.
Synthesis of bis-[2,4-dichloro-6-{[(2'-dimethylaminoethyl)methylamino]methyl}-phenol]
cuprate, CuL (2)
2
Complex 2 have also been synthesized as described above. The respective ligand
(0.27 g; 1.0 mmol), was dissolved in 20 mL of MeOH followed by addition of triethylamine
(
152 μL; 1.0 mmol) and Cu(OAc) . H O (0.1 g; 0.5 mmol). The formation of the complex
2
2
was immediately indicated by a change in colour. After stirring the mixture for 3 h at room
temperature, the mixture was filtered through a filter paper and the solvent was removed in
vacuo. Crystals suitable for X-ray diffraction analysis were obtained through slow vapour
diffusion process by di-iso-propyl ether into CH Cl -CH CN (1:1) mixture of complex.
2
2
3
Yield: 0.342 g (∼91%). Anal. Calcd (%) for C H Cl CuN O : C, 46.80; H, 5.56; N, 9.10;
2
4
34
4
4
2
−
1
Found (%): C, 46.76; H, 5.61; N, 9.15; FTIR (KBr ʋ in cm ): 3100–3000 {ʋ
(s); 3000–
C-H,
2
850 {ʋC-H, (m)},1580-1459 {ʋC-C, (s)}; 1250–1020 {ʋC-N, (m)} (Figure S2). ESI-MS in
4

ACCEPTED MANUSCRIPT
+
HPLC MeOH (+, m/z): {C H Cl CuN O } : 616.1(100%) (Figure S3c); UV-Vis in HPLC
24
34
4
4
2
MeOH: (λmax, nm): 444.
Where, the above abbreviations define; (s) = strong; (m) = medium, and (sh) = sharp band
Results and discussion
Synthesis of compounds
Both the compounds were isolated as solid crystalline form (Scheme S1). Ligand and
1
13
its complexes have been characterized by several analytical techniques viz. H, and C NMR,
FTIR, electrospray ionization mass spectrometry (ESI-MS), thermogravemetric analysis
(TGA), and cyclic voltammetry (CV). To obtain different metal complexes ligand to metal
ratio have been varied. Apart from the reported NMR peaks, the molecular ion peak of ligand
appears at 277.1, followed by the formation of stable iminium ion with mass of 115.1 in the
first step of Mannich reaction. Complex 1 has shown molecular ion peak at 399.8, whereas
that of complex 2 is 616.1. An additional peak at 340.0 in complex 1 comes for the easily
+
dissociable labile acetate group to form stable [CuL] . FTIR spectrum of complex 1 indicates
-1
the stretching frequencies at 1663, 1460 cm due to the asymmetric and symmetric stretching
of acetate group,[35] which is absent in the complex 2. Complex 2 possesses some common
-1
weak bands in the range of 1250–1020 cm assigned for ʋ bond.[36] Thermogravimetry
C-N
profile suggests that 58.2% weight loss is happening for the release of iminium moeity at 228
°
C for HL (Figure S4). Moreover, complex 2 is more stable than that of complex 1 and both
showing considerable first degradation around 246 °C. Furthermore, wavelength maxima
obtained at 445 and 439 nm for complex 1 and 2 is responsible for the metal-to-ligand charge
transfer (MLCT) transition.[37]
The electrochemical behaviour of the Mannich base phenolic ligand and its
complexes were investigated by cyclic voltammetry analysis using Bu NPF as the
4
6
+
supporting electrolyte in dichloromethane. The redox potentials are quoted against Ag /AgCl
electrode couple (Figure S5, Table S1). Upon oxidation of HL, an anodic peak at E = +0.86
pa
V was observed along with a highest cathodic peak, E = +0.71 V providing quasi-reversible
pa
behaviour of phenoxyl moiety.[38, 39] Quasi-reversible peaks are also observed for
complexes 1 and 2 for metal centered redox behaviour whereas ligand centered peaks become
less prominent. Oxidation potential ranges of complexes 1 and 2 from + 0.89 to + 1.20 V
suggest that it requires an external oxidant for oxidation process.[40] Moreover, the solution
and solid state electron paramagnetic resonance (EPR) spectra of both the copper complexes
were recorded at 298 K and 77 K, respectively. Both the A
greater than that of complex 2 (Figure 1). A less intense noisy spectra was observer for 2
having larger A and g values (A = 54 gauss, and g = 2.141). As depicted in Figure 1, the
solid phase EPR signals at liquid nitrogen temperature having g at 2.086, and g at 2.335 for
complex 1, respectively, ascribed to the unpaired electron of Cu (d system), results in a
paramagnetic system. Similarly, g at 2.074 and g at 2.345 in case of complex 2 signifies the
‖ ⊥
and g values of complex 1 is
⊥
‖
⊥
‖
⊥
‖
II
9
⊥
‖
paramagnetic nature of distorted octahedral geometry.[41]
5

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less
intense
a
b
A = 71 G
‖
A = 54 G
┴
more
intense
broad
sharp
A = 80 G
‖
A = 43 G
┴
Figure 1. (a) to (c) represents the EPR spectra of homogeneous catalysts at room temperature
in CH Cl ; where, (a) merged plot of the complexes 1 and 2, (b) Stacking diagram showing
2
2
noisy peaks of complex 2, (c) ‘g’ parameters of 1 and 2, and (d) solid phase EPR spectra of 1
and 2 and their corresponding ‘g’ values at 77 K.
Single crystal X-ray Diffraction Study (XRD)
Ligand, HL and complex 2 have been characterized by X-ray crystallography. These two
compounds are monoclinic in nature having space group of P 21/c. Figure 2 represents the
ORTEP diagram of HL and complex 2 having 50% thermal probability of ellipsoids. The
structure refinement parameters are summarized in Table S2 and the selective bond angles
and bond lengths are presented in Table S3. There is a prominent intramolecular hydrogen
bond in ligand HL between O1-H101…N2 (Figure S6). There are two crystallographically
independent copper ions and both of them show octahedrally coordinated geometry which are
6

ACCEPTED MANUSCRIPT
fulfilled by four N atom of ligand and two phenoxide O atom. Two molecules of complex 2
may interact with each other via. C-H…π (C23 of Cu(2) and centroid of Cu(1)) force of
attraction (Figure S6). Those molecules also form intermolecular hydrogen bond between
Cl(1)…H(17)-C, which can be further extended to form a 1D or 2D polymeric sheet (Figure
2
c).[42, 43] There are not many non-covalent interactions observed between the individual
molecules. All the bond lengths and angles are in good agreement with earlier reports.[23,
6] Furthermore, geometry optimized structure of complex 1 was elucidated by density
3
functional theory (DFT) (using similar basis set as used in our previous reports[37, 44]), and
the corresponding structure along with bond lengths and bond angles around the central metal
is depicted in Figure S7.
(a)
(b)
1
D
2D
(c)
Figure 2. ORTEP view with 50% probability of ellipsoids showing geometric displacement
and partial atomic labels for (a) HL, (b) CuL , and (c) polymeric extension through
2
intermolecular H-bonding.
Galactose oxidase (GOase) catalysis
Galactose oxidase mimicked aerial oxidation of benzyl alcohol
-4
A 2.5 × 10 M solution of complexes used as GOase mimicked catalyst to oxidize
-3
-1
substituted benzyl alcohol (substrate concentration range of 10 × 10 - 2.5 × 10 M) in
CH Cl . 0.8 equivalent tetrabutylammoniummethoxide, n-Bu NOMe (TBAM) was added at
2
2
4
the initial stage of catalysis to abstract primary alcoholic proton. After that, the whole mixture
was stirred at room temperature in air. The solution was diluted as required for acquisition of
the electronic spectra using a blank with the catalyst as well as standard substrates. HPLC and
7

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gas chromatography (GC) were performed for quantitative determination of the concentration
of major oxidized product.
Caution! The solvents used for this catalysis should be free from any aliphatic or aromatic
primary -OH functional group.
Change in electronic spectra during GOase catalysis
Benzyl alcohol has been taken as a substrate for alcohol oxidation, with a λmax
(
wavelength maxima) of 256 nm (π→π* transition).[37] In CH Cl solution, the mononuclear
2 2
-
complex 1 gives a strong d-d band at 685 nm having molar extinction coefficient (ꞓ in Lmol
1
-1
2
cm ) of 4.9 × 10 (inset representation of Figure 3a). As describes above, the catalytic
mixture was subjected to perform UV-Vis analysis after 5 hours of interval. The generation
of a new peak at 290 nm is assigned to the formation of benzaldehyde as oxidized product,
which was further confirmed by a blank experiment (inset plot of Figure 3a). It shows that
optical density at 290 nm, responsible for the oxidized product is greater for catalyst 1 with
respect to catalyst 2, signifying the better catalytic activity of complex 1 (Figure 3a). Figure
3
2
b is the representation of time dependent catalysis by most active catalyst 1. Absorbance at
90 nm increases with increasing the duration of catalysis, which was further plotted as
shown in the inset (a) of Figure 3b. A plot of molar extinction coefficient is shown in Figure
S8a, resulting a nonlinear fitted saturated equilibrium curve after 20 h of catalysis (Table S4).
Figure 3. Electronic spectra of the complexes and the catalytic mixture in CH Cl at 25 °C:
2
2
(
a) Changes in the UV-Vis spectrum during the course for reaction with benzyl alcohol (0.4
-4
M) by complex 1 and 2 (2.5 × 10 M), in 5 hrs (color significance: black = catalysis by
CuL(OAc), red = catalysis by CuL , inset: absorption profile of reference samples). (b)
2
Gradual increment in O.D. value during benzyl alcohol oxidation by catalyst 1 (0-20 hrs,
absorbance vs. time profile is on the inset plot).
Monitoring the oxidation of substituted benzyl alcohol
Room temperature catalytic oxidation of benzyl alcohol to benzaldehyde and/ or
benzoic acid were monitored by taking 0.125 M of each substrate in 10 mL of
-3
dichloromethane by fixing the catalyst concentration of 2.5 x 10 M. The results were
analysed via. HPLC analysis at the wavelength maximum of 254 nm and 290 nm,
8

ACCEPTED MANUSCRIPT
respectively. One equivalent of strong base, tetrabutylammoniummethoxide (TBAM) has
been used in each oxidation reaction because alcoholic proton does not get easily dissociated
in presence of a weak base. As summarized in Table 1, benzyl alcohol gets converted of
9
3.43% and 92.80% in 8 hrs by using catalyst 1 and 2, respectively to produce benzaldehyde
as sole product. This oxidized product is 100% selective. A percentage conversion up to 3-
5
% have increased within 12 hrs of catalysis.
Although, we varied a series of substrate to get the proper substitutional effect, but
these are not following the regular trends depending upon the electronic properties of the
functional group attached to the benzyl alcohol. However, chloro- substituted benzyl alcohol
has been found to show better conversion rate with respect to bromo substitution. This might
be attributed to the greater electron withdrawing effect of chloro group over bromo.
Moreover, electron donating methyl and isopropyl-substituted substrates also provide a high
percentage conversion within 12 hrs.
Table 1. Percentage conversion of substituted benzyl alcohol by homogeneous catalysts 1
and 2
Entry
Substrates to
check GOase
activity
Time Conversion
Time
(h)
Conversion
(%)
(h)
(%)
1
2
1
2
0
0
0
0
0
1
2
3
4
5
08
08
12
08
08
93.43 92.80
90.14 84.38
64.11 68.39
66.83 62.63
65.68 63.42
12
16
20
16
12
98.56 95.34
94.39 92.00
84.55 70.20
78.29 76.35
85.72 81.56
0
0
6
7
08
12
86.71 79.09
62.7 66.78
12
20
93.45 91.30
71.34 70.53
Figure 4 represents the product(s) formed after catalysis in 8 hrs. Pure references of
substrate and product were taken to get the value of retention time before analysing the
catalytic mixture. Apart from producing aldehydes, substituted benzyl alcohols have also
given the corresponding benzoic acid. Furthermore, the selectivity of benzaldehyde decreases
with increase in time. Apart from that, variation in the amount of base is directly related to
9

ACCEPTED MANUSCRIPT
the percent conversion of oxidized products. As shown in Figure S8, upon increasing the
amount of base increment in the percentage conversion has been observed. This reach to
maximum (42.26% within 3.5 hrs) when the ratio of substrate: base is 1:1 (Table S5).
Although 0.8 equivalent of base is very effective to covert 41.93% in 3.5 hrs. Hence, it seems
that one equivalent of base is required to abstract one proton of primary aryl alcohols.
Furthermore, a table having competitive kinetic parameters on GOase catalysis have been
summarized in Table S6, which shows that the complex 1 is having greater K value of 2.8 ×
cat
-3
-1
-4 -1
1
0 s over complex 2 having same of 4.2 × 10 s .
Figure 4. HPLC interpretation of some oxidation reactions after catalysis (‘Ref.’ signifies the
standard sample).
Mechanism of action for GOase like activity
Y. Wang et al.[40] elaborated the formation of active catalyst under 1 atm of O2
(Scheme 2). Later on, the proposed mechanism of GOase mimicked was theoretically
investigated by F. Himo et al.[45] According to that mechanism of original enzyme, it
is presumed that substrate must be binding the metal center replacing the exogenous
water or acetate in the equatorial position (Scheme 1 and 2). Although both the
homogeneous catalysts contributed for the significant conversion, yet, we have chosen
the catalyst 1 to describe the probable GOase like mechanism, as it contains one
easily removable acetate group in its coordination sphere. In the very first step of this
catalysis proton abstraction happens for the primary alcohol in presence of a strong
base, though in original enzyme it is presumed that the axial tyrosine ligand is
1
0

ACCEPTED MANUSCRIPT
involved in proton abstraction. In the second step, the deprotonated form of the
substrate prefers for a nucleophilic attack to the copper centre by displacing labile
acetate moiety attached to copper center (Scheme 2 and Scheme S2). Next step is
regarding the hydrogen atom transfer from substrate to the phenolic O-atom.
generating a radical intermediate to favour the electron transfer from substrate to the
metal centre with a decrease its oxidation state from Cu(II)→Cu(I), with the
concomitant generation of the oxidized product (i.e.; aldehyde). Moreover, the
intermediate Cu(I) complex is very unstable and subjected to get back its oxidized
form with the reduction of aerial oxygen producing hydrogen peroxide (H O ) in the
2
2
medium in the process.
Scheme 2. Plausible mechanism for GOase mimicking catalysis by catalyst 1
Catecholase activity (COase) studies
Catechol oxidase mimicked aerial oxidation of 3,5-DTBC
3
,5-di-tert-butylcatechol (3,5-DTBC) has taken as the substrate to explore the COase
like activity of the synthesized Cu(II) complexes. It is already reported that any kind of ortho
diol can act as the substrate for the COase mimicking activity.[46] Moreover, the bulkiness of
t-butyl substituents in the ring helps to decrease the reduction potential of catechol.[1] The
−
4
working concentration range of 3,5-DTBC have been prepared from 50 × 10 M to 350 ×
−
4
-4
1
0
M upon fixing the catalyst strength of 4.0 × 10 M in methanol solvent. The time
ο
dependent oxidation reaction was monitored later on by UV-Vis spectroscopy at 25 C. The
standard solution of 3,5-DTBC, and 3,5-ditertarybutyl quinone (3,5-DTBQ) shows the
absorption band maxima at 350 nm, and 400 nm, respectively. The rapid oxidation of 3,5-
DTBC with respect to time, generates a new band at 400 nm. This is due to the formation of
1
1

ACCEPTED MANUSCRIPT
the oxidized product, 3,5-DTBQ (Figure S10).[23] Furthermore, the slope values of each
concentration dependent experiment were evaluated by using kinetic software of UV-Vis
spectrometer.
To understand the kinetic aspect of COase catalysis, the rate constant for a catalyst
was determined by plotting the observed rates (slope values) vs. substrate concentrations. The
data were interpreted by utilizing the Michaelis–Menten equation of enzyme kinetic.[47]
Further, the linearization of Lineweaver−Burk plots gives the value of Michaelis−Menten
constant (K ) and maximum initial rate (Vmax) (Figure S11).[48] The turnover number (TON
m
or k ) of catalysts were obtained by dividing V
value with the concentration of the
cat
max
corresponding complexes with respect to time. As summarized in Table 2, complex 1 and 2 is
5
4
-1
having the TON values of 1.976 × 10 , and 1.039 × 10 h , respectively. These values are
quite high in comparison to the previously reported analogous.[49-51] Moreover, the TON
parameters of natural metalloenzymes have been compared with our laboratory synthesized
complexes, and the corresponding values are summarized in Tables S6. Results suggest that
catalyst 1 is more active over catalyst 2. This might be due to the mono-ligand coordination,
which is leading to the favorable substrate attachment as shown in Scheme 3.
Table 2. Summary of various kinetic parameters of catecholase like activity of catalyst 1, and
4
-1
2
. Compared with that of ∼1 × 10 h of the most efficient catalyst reported till date[52] said
by K. S. Banu et al.[53]
Complex/ Cat. Conc.
Solvent λmax Vmax (M K (M)
Standard
Error (%)
Kcat / TON
m
-1
-1
Catalyst
(in M)
(nm)
min )
(h )
-
4
4
-2
5
1
4.0 × 10
MeOH
MeOH
440
1.3174 0.25306
1.8 × 10
1.976 × 10
-
-3
4
2
4.0 × 10
444 0.06928 0.04302
4.6 × 10
1.039 × 10
Mechanism of action for COase like activity
To get an insight into the possible mechanism of COase like activity, complex 1 has
been chosen due to its greater activity. We have explored the mechanism by ESI-mass
spectrometry, as described as reported in earlier cases to investigate the probable complex-
substrate intermediate formation during catalysis.[54] ESI-MS positive spectrum of a 1:100
equivalent ratio of catalyst 1 and 3,5-DTBC recorded after 5 min of mixing exhibits two
major peaks at +, m/z = 243.4 and 463.6 respectively, along with two small peaks at 560.5 &
1
143.9 (Scheme 3 and Figure 5). The former two peaks correspond to the quinone-sodium
+
+
aggregates [(3,5-DTBQ) + Na] and [(3,5-DTBQ) + Na] .[55] The later smaller peaks at
2
5
73.4 & 1143.9 might be attributed to the formation of monomeric and dimeric complex-
substrate aggregate (Scheme 3).[53] Notably, ESI-MS peak at 1143.9 is the signature of the
original enzyme kinetic mechanism, where di-copper involves during COase mimicking
catalysis. At the end of oxidation, the colour of the solution turned into brown, which gives a
d-d transition band in UV-visible spectrum due to Cu(II) complex. Apart from that, the
reduction of molecular oxygen took place resulting the formation of hydrogen peroxide in the
reaction medium, which has been be elaborated further.
1
2

ACCEPTED MANUSCRIPT
Scheme 3. Probable catalytic cycle of oxidation of 3,5-DTBC by complex 1. Calculated mass
of intermediates and product(s) are given in Figure 5.
Mass (560)
=
559 + H
Mass (243)
220 + Na
=
1
3

ACCEPTED MANUSCRIPT
Figure 5. Evidence of the formation of dimeric ‘substrate-catalyst’ bounded intermediate as
described in Scheme 3.
Detection of hydrogen peroxide during GOase and COase catalysis
It is also important to note that, at the end of catalysis (GOase or COase mimicked)
the dioxygen of atmosphere is reduced to H O . Hence, for the qualitative or quantitative
2
2
detection of reduced product(s) (H O or H O), evolved during both catalytic reactions, a
2
2
2
modified iodometric method was successfully employed.[23, 56] Though, H O can be
2
2
determined amperometrically by direct oxidation or by reduction of mediators but we have
followed a simple electronic spectroscopy for the detection.[57] The duration of collecting
the catalytic mixture for GOase as well as COase mimicking catalysis were 12 hrs and 1 h,
respectively.
Reaction mixtures for the determination of hydrogen peroxide were prepared as per
method described in our previous literature.[25] It describes that after completion of kinetic
experiments, an equal volume of water (say 10 mL) was added to the reaction mixture, and
oxidized products (viz benzaldehyde or quinone) were extracted with 3 × 10 mL
-3
-1
dichloromethane. After that, the aqueous layer was acidified with H SO (5.0 × 10 molL )
2
4
to pH~2 (checked by pH-meter) to stop further oxidation. Then 1 mL of a 10% aqueous
solution of KI was poured into the mixture followed by the addition of 3 drops of a 3%
solution of ammonium molybdate.
-
The I ions may further oxidised to I in the presence of H O as shown according to
2
2
2
-
+
the iodometric reaction: H O + 2I + 2H → 2H O + I (aq.). An excess of iodide ions react
with molecular iodine to produce highly stable tri-iodide ion (I ) by following a reaction: I
aq.) + I → I . The rate of reaction is slow but increases with increasing concentrations of
2
2
2
2
-
3
2
-
-
(
3
acid. However, upon addition of an ammonium molybdate solution the reaction can
-
immediately cease. A characteristic I band at λ = 353 nm is depicted in the Figure 6a-b.
3
max
Figure 6a represents the two kind of absorption maxima at 290 nm and 353 nm for GOase
catalysis, respectively. Among which, the formation of benzaldehyde in the range of 290 nm
is predominant which is a consequence of better solubility of benzaldehyde in aqueous
[58]
medium.
Hence, all the above experiments hereby support the radical mechanism
pathways for GOase or COase mimicking catalysis. Furthermore, the electronic spectra of the
reference solution of substrates and products for GOase, and COase catalysis have been
depicted in Figure 6c-d.
1
4

ACCEPTED MANUSCRIPT
GOase
COase
Catalysis
Catalysis
(
a)
(b)
(
d)
(
c)
Figure 6. Detection of hydrogen peroxide in the catalytic reaction: (a) formation of H O
2
2
(
λmax = 353 nm) as well as PhCHO (λmax = 290 nm) during GOase catalysis, (b) H O
2
2
production during COase like activity monitored by UV-Vis spectra of the characteristic
absorption band at λmax = 353 nm, (c) and (d) represents the electronic spectra of the reference
solution of substrates and products for GOase, and COase catalysis, respectively.
Conclusions
In summary, the structure of Mannich base ligand and complex 2 were elucidated by
single crystal X-ray crystallography, whereas geometry optimized model of complex 1 has
been derived theoretically. Considering GOase liked catalysis, benzyl alcohol selectively
produce only benzaldehyde by a percentage conversion of 93.43% and 92.80% in 8 hrs with
respect to catalyst 1 and 2, respectively. Furthermore, extremely high TON values of 1.976 ×
5
4
1
0 , and 1.039 × 10 per hour have been observed for complex 1 and 2, respectively. Despite
of being catalytically active, catalyst 1 follows a regular M-M kinetic mechanism, which
resembles the mechanistic pathways of original enzyme. Apart from that, the labile nature of
acetate group attached to the central copper atom of complex 1 might be responsible for the
better catalytic activity for both the GOase and COase mimicking catalysis.
Acknowledgement
This work is supported by DST India under Indo-Portuguese joint collaborative project
scheme (Grant Number INT/PORTUGAL/P-04/2013). We thank IIT Indore for proving the
1
5

ACCEPTED MANUSCRIPT
infrastructure, and financial support. The author is grateful to Dr. Auprba K. Das for his kind
help in HPLC. We also express thanks to SIC, IIT Indore for the experimental facilities, and
structure interpretation.
Supplementary data
CCDC 1882115 and 1455095 encompass the complementary crystallographic information for
HL, and 2, respectively. These information can be acquired free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic
Data Center, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
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Mannich base Cu(II) complexes as biomimetic oxidative catalyst
a
a
b
a
Bidyut Kumar Kundu, Rishi Ranjan, Attreyee Mukherjee, Shaikh M. Mobin,
a, c,
Suman Mukhopadhyay
*
a
Discipline of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore,
Khandwa Road, Simrol, Indore 453552, India.
b
Department of Chemistry, Ananda Mohan College, Kolkata 700009, India
c
Discipline of Biosciences and Biomedical Engineering, School of Engineering, Indian
Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India.
*
E-mail: suman@iiti.ac.in; Fax: +91 731 2361; Tel: +91 731 2438 735
Graphical Abstract
Representing the formation of active catalyst from [CuL(OAc)] (HL = 2,4-dichloro-6-{[(2'-
dimethyl-aminoethyl)methylamino]methyl}-phenol) during galactose oxidase (GOase)
mimicking catalysis by utilizing 1 atm of O as oxidant via. superoxide generation process.
2
1
8

ACCEPTED MANUSCRIPT
Mannich base Cu(II) complexes as biomimetic oxidative catalyst
a
a
b
a
Bidyut Kumar Kundu, Rishi Ranjan, Attreyee Mukherjee, Shaikh M. Mobin,
a, c,
Suman Mukhopadhyay
*
a
Discipline of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore,
Khandwa Road, Simrol, Indore 453552, India.
b
Department of Chemistry, Ananda Mohan College, Kolkata 700009, India
c
Discipline of Biosciences and Biomedical Engineering, School of Engineering, Indian
Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India.
*
E-mail: suman@iiti.ac.in; Fax: +91 731 2361; Tel: +91 731 2438 735
Highlights
Syntheses of Mannich base Cu(II) complexes as metalloenzyme mimics
Galactose Oxidase (GOase) and catechol oxidase (COase) based catalysis
The generation of reactive oxygen species during catalysis
Reduction of aerial oxygen to form hydrogen peroxide
Mechanistic aspects of GOase and COase via. electrospray ionization mass spectrometry
1
9