Inorganic-organic covalent hybrid of polyoxometalate-calixarene: Synthesis, characterization and enzyme mimetic activity

Article abstract of DOI:10.1016/j.ica.2018.08.034

The inorganic-organic hybrid based on covalently attached trilacunary phosphotungstate and calixarene conjugate (POM-Calix hybrid) has been synthesized via facile click chemistry approach. The characterization studies showed that both the moieties are present together in the hybrid upon covalent modification. The morphology of the hybrid was studied by SEM, TEM and AFM analyses suggesting spherical shaped nanoparticles of 40–60 nm size for the hybrid. The POM-Calix hybrid was successfully employed to demonstrate peroxidase-like activity for the oxidation of the model substrate, viz., o-phenylenediamine (OPD), for the enzyme. The activity of the POM-Calix hybrid was ～170% greater than that exhibited by simple POM and this is mainly attributed to the introduction of hydrophobic character by the covalently attached calixarene conjugate and the hydrophobicity is supported by the contact angle measurement. From the kinetic studies, the Michaelis constants, K_m and V_max were estimated to be 2.55 mM and 0.756 × 10^?8 M s^?1, respectively. It was observed that, the POM-Calix hybrid facilitates the formation of [rad]OH radicals when treated with H₂O₂ which eventually results in the oxidation of the substrate. The POM-Calix hybrid exhibits excellent enzyme-like activity over a wide pH range, which would enable its bio-applications at physiological conditions.

Full text of DOI:10.1016/j.ica.2018.08.034

Accepted Manuscript
Research paper
Inorganic-organic covalent hybrid of polyoxometalate-calixarene: Synthesis,
characterization and enzyme mimetic activity
Nilesh Narkhede, Bhawna Uttam, Chebrolu Pulla Rao
PII:
S0020-1693(18)31084-3
https://doi.org/10.1016/j.ica.2018.08.034
ICA 18434
DOI:
Reference:
Inorganica Chimica Acta
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23 August 2018
23 August 2018
Please cite this article as: N. Narkhede, B. Uttam, C.P. Rao, Inorganic-organic covalent hybrid of polyoxometalate-
calixarene: Synthesis, characterization and enzyme mimetic activity, Inorganica Chimica Acta (2018), doi: https://
doi.org/10.1016/j.ica.2018.08.034
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Inorganic-organic covalent hybrid of polyoxometalate-calixarene:
Synthesis, characterization and enzyme mimetic activity
Nilesh Narkhede, Bhawna Uttam and Chebrolu Pulla Rao*
Bioinorganic Laboratory, Department of Chemistry, Indian Institute of Technology
Bombay, Powai, Mumbai-400 076, India, Email: cprao@iitb.ac.in
Abstract: The inorganic-organic hybrid based on covalently attached trilacunary
phosphotungstate and calixarene conjugate (POM-Calix hybrid) has been synthesized via
facile click chemistry approach. The characterization studies showed that both the moieties
are present together in the hybrid upon covalent modification. The morphology of the hybrid
was studied by SEM, TEM and AFM analyses suggesting spherical shaped nanoparticles of
40-60 nm size for the hybrid. The POM-Calix hybrid was successfully employed to
demonstrate peroxidase-like activity for the oxidation of the model substrate, viz., o-
phenylenediamine (OPD), for the enzyme. The activity of the POM-Calix hybrid was ~170%
greater than that exhibited by simple POM and this is mainly attributed to the introduction of
hydrophobic character by the covalently attached calixarene conjugate and the
hydrophobicity is supported by the contact angle measurement. From the kinetic studies, the
Michaelis constants, Km and Vmax were estimated to be 2.55 mM and 0.756 x 10-8 M sec-1,
respectively. It was observed that, the POM-Calix hybrid facilitates the formation of ·OH
radicals when treated with H2O2 which eventually results in the oxidation of the substrate.
The POM-Calix hybrid exhibits excellent enzyme-like activity over a wide pH range, which
would enable its bio-applications at physiological conditions.
Keywords: Polyoxometalate-calixarene hybrid, peroxidase-like activity, detection of ·OH
radical, o-phenylenediamine oxidation, thiol-ene click reaction
1

Introduction
Polyoxometalates (POMs) is a large family of well-defined anionic metal-oxygen clusters
containing Group V and VI metals in their highest oxidation states. The chemistry of POMs
has attracted much attention due to their oxidative nature as well as the acid-base reactivity
and thereby exhibit variety of catalytic applications that extends into medicine and materials
[1-5]. An added advantage of the POMs is their easy tunability of both these properties at
atomic or molecular levels [6-7]. This can be done by removal of one, two or three metal
octahedrons from the parent ones leading to the formation of mono-, di- or tri-lacunary POMs
[8]. The organic derivatization of POMs is a contemporary topic of the family of anionic
metal-oxo clusters where only few such class II hybrids are known in which both the
precursors are connected through covalent bonding [9-16]. The route for the synthesis
mainly includes the attachment of the hybrid POM to the inorganic mesoporous silica via an
alkyl side chain (-SiR-Y where Y=SH, Cl, NH2). Thus, the organic domains connected to
POM nano-scaffolds can be explored to design new functional molecules and materials. In
particular, POM-appended inorganic-organic hybrids can direct the supramolecular
organization leading to functional nanostructures and or nanoreactors.
Calixarenes (Calix) have been extensively studied and utilized as building blocks owing to
the fact that it has a hydrophobic cavity and that it can be easily modified by attaching
functional groups at both the lower and upper rims [17-18]. Therefore, the combination of
calixarene with POMs is expected to give structural diversity that would lead to
multifunctional assemblies owing to their diverse natures and possible synergetic effects.
Literature study shows that there are limited reports on such POM-Calix hybrids with non-
covalent interactions [19-23]. To our knowledge, there is no such report where POM is being
covalently modified by a calixarene. Recently, our group contributed to the immobilization
of organic calixarene conjugates on to the inorganic surfaces, such as, silica and gold in order
2

to gain their dual advantages [24]. In continuation to our efforts towards the formation of
such inorganic-organic covalent hybrids, here in we report the synthesis and characterization
of POM-Calix hybrid formed by covalent modification of a tetra-derivatized trilacunary
phosphotungstate with the allyl calixarene conjugate and demonstrated its peroxidise like
enzyme mimetic activity.
Experimental section
Materials and methods
The chemicals used in this work were of A.R. grade. (3-Mercaptopropyl)trimethoxysilane
(MPTMS) and o-phenylenediamine (OPD) were purchased from Sigma-Aldrich. All other
reagents and solvents were purchased from Merck and used as received unless specified. The
Milli-Q water is used in the present study. The organic precursors were characterized by
1
31
13
ESI-MS by using Q-TOF instrument in positive ion mode and H, P and C NMR spectra
by Avance III-400 Bruker. The FT-IR spectra of all the samples were measured in KBr in
4000-400 cm-1 using Perkin-Elmer spectrometer. TGA analysis of the precursors as well as
the hybrid was carried out using a Mettler Toledo Star SW 7.01 under nitrogen atmosphere.
The TEM analysis was carried on PHILIPS TEM model-CM 200 working at an accelerating
voltage 200 kV. XPS analysis was performed on Kratos Analytical (AXIS Supra model).
The SEM images were taken on JSM-7600F field emission gun scanning electron
microscope.
Synthesis of Na8H[A-PW9O34]*11H2O (1). The sodium salt of trilacunary phosphotungstate
was synthesized following the literature procedure [25] as given in the supporting
information S01. The isolated yield is 88%. FTIR (KBr): 1058 (s), 1015 (m), 938 (s), 885
(m), 818 (s), 746 (s, broad) cm-1. ICP-AES elemental analysis (%): W =63.10, P = 0.96, Na=
6.88 and the calculated elemental analysis (%): W = 63.3, P = 1.19, Na= 7.04).
3

Synthesis and characterization of (Bu4N)3[A-PW9O34(HS(CH2)3SiO)3(Si(CH2)3SH)] (2).
The synthesis of thiol functionalized polyoxometalate was carried out following the reported
procedure [26] as given in supporting information S01. The isolated yield is 51%. FTIR
(KBr): 2564 w [SH], 1481 m, 1382 m, 1342 m, 1302 m, 1256 m, 1239 m, 1156 m, 1118 vs,
1047 m, 1013 m, 974 vs, 952 vs, 869 vs, 813 vs, 726 vs, 600 w, 578 w, 534 m, 507 m, 486 w,
440 w cm-1. Negative ion ESI-MS: 898.09 for [M3-] and is expected at m/z = 897.70 and the
spectrum is given in supporting information Fig. S1.
Synthesis and characterization of allyl conjugated calixarene (3). Synthesis of allyl
calixarene conjugate was carried out by following our recently reported procedure [24] as
given in supporting information S01. The isolated yield is 91%. 1H NMR: (400 MHz,
CDCl3) δ: 10.15 (s, 4H), 6.84 (s, 8H), 5.89 (ddt, J=17, 10.5, 6.8 Hz, 4H), 5.04 (br. d, J=17.0
Hz, 4H), 5.03 (br. d, J=17.0 Hz, 4H), 4.19 (br. d, J=9.0 Hz, 4H), 3.45 (br. d, J=9.0 Hz, 4H),
13
3.18 (d, J=6.8 Hz, 8H). C NMR (CDCl3, 400MHz, δ ppm): 31.81 (Ar-CH2-Ar), 39.34 (Ar-
CH2), 115.59, 128.20 (-CH=CH2) 128.98, 133.47, 137.61, 147.07 (Ar-C). ESI-MS: 623.25
[M+K]. The elemental analysis obtained experimentally corresponds to, C = 79.36% and H =
6.75%, and the calculated one along with one molecule of water corresponds to, C = 79.70%,
H = 6.68% (supporting information Fig. S2-S4).
Synthesis and characterization of POM-Calix hybrid (4). A 54.5 mg (0.1 mmol) of 3 was
added to the solution containing 341.8 mg (0.1 mmol) of 2 in 15 mL dry acetonitrile. To this,
10 mg of azobisisobutyronitrile (AIBN) was added immediately and the mixture was refluxed
under N2 atmosphere for 60 h in order to ensure complete conversion. The reaction mixture
was then concentrated to 3 mL and the product was isolated by the addition of water and
filtered through medium frit. The product, 4 was washed with 30 mL each of water, ethanol
followed by diethyl ether and dried under vacuum. The isolated yield is 85%. FTIR (KBr):
2955 vs, 2930 vs, 2872 vs, 1670 s, 1625 s, 1468 vs, 1386 w, 1291 w, 1233 w, 1121 vs, 1088
4

vs, 1033 s, 966 s, 930 s, 865 s, 810 s, 781 w, 736 m. 693 m, 631 m, 604 m, 577 w, 544 w,
503 m, 455 w, 428 w cm-1. The ICP-AES elemental analysis of 4 (%) yielded, W = 47.6 and
P = 0.52, while the calculated analysis (%) with 3 water molecules is, W = 47.2 and P = 0.69.
13C MAS NMR (δ, ppm): 30.68 (Ar-CH2-Ar), 37.11 (Ar-CH2), 120.28 to 145.98 (Ar-C),
12.63, 18.70, 22.77 and 56.51 (Bu4N). 31P MAS NMR (δ, ppm): -18.18. The detailed
characterization by 31P NMR, C MAS-NMR, MALDI, AFM, SEM and TEM can be found
13
under results and discussion section.
Peroxidase-like activity of the hybrid. In order to investigate the peroxidase-like activity of
the hybrid 4, catalytic oxidation of substrate, o-phynelenediamine (OPD) in the presence of
H2O2 was carried out. The oxidation of OPD produces 2,3-diaminophenazine (DAP), which
has yellowish orange colour with its absorption maximum at 415 nm. The stock solutions of
0.1 M OPD and 0.5 M H2O2 was prepared in PBS buffer while 0.5 mM of POM-Calix hybrid
was prepared in acetonitrile. In a typical colorimetric experiment, 0.6 to 10 mM of OPD was
added to the PBS buffer at pH 7.4 containing 100 mM H2O2 and 10 µM of 4. The progress of
OPD oxidation was monitored by UV−Vis spectroscopy by following the band at 415 nm.
The Michaelis-Menten constant was calculated using the Lineweaver-Burk plot. The
formation of DAP was confirmed by its [M+H]+ peak at m/z = 211 in ESI-mass spectra and
the corresponding spectra are given in the supporting information as Fig. S5.
Detection of hydroxyl radicals: Terephthalic acid (TA) was used as fluorescent probe for
tracking the hydroxyl radicals. A 25 mM Na2TA was prepared using TA and NaOH. The
fixed volume of Na2TA (75 µL) was added into a mixture of 3 mL PBS (pH 7.4) containing
10 µM of 4 and 1 mM H2O2. After 24 h incubation in the dark, the resulting solution was
analysed by fluorescence measurement of the emission band having a maximum at 425 nm at
an excitation wavelength of 315 nm.
5

Results and discussions
A thiolated tri-lacunary phosphotungstate (2) was modified covalently by an allyl calixarene
(3) to result in POM-Calix hybrid, 4 via thiol-ene click reaction as shown in Scheme 1. The
hybrid 4 was characterized by a variety of spectroscopy and microscopy techniques.
3-
HS
Si
SH
3
SH
3
SH
3
3
Si
O
O
O
SH
Si
Si
H2O, CH3CN
TBABr, HCl
H2CO Si OCH3
H3CO
1
3-
2
OH
HO
OH
OH
3
3
3
3
CH3CN, Reflux
S
S
S
S
3
3
3
3
Thiol- Ene
Click Reaction
Si
O
O
Si
Si
O
Si
HO
OH OH
OH
3
4
Scheme 1. Synthesis of inorganic-organic hybrid of polyoxometalate, 4 containing calixarene
via thiol-ene click reaction starting from trilacunary phosphotungstate.
Characterization of POM-Calix hybrid (4): The XPS clearly showed the presence of all
the elements expected for 4, viz., C, N, O, P, Si and W (Supporting information Fig. S6).
The TGA curves for 4 in comparison with its precursors, viz., 1, 2 and 3 are shown in Fig. 1a.
o
The compound 1 showed 2.2% weight loss up to 100 C due to adsorbed water and 7.8%
o
weight loss up to 250 C due to the loss of water of crystallization corresponding to 11 water
o
molecules. The thiol functionalized compound 2 showed 26% weight loss up to 600 C due
to the combustion of the counter tetrabutylammonium (TBA) ions and other alkyl chains
present in it. The compound 4 showed 1.4% decrease in weight due to the loss of three water
molecules from the POM-Calix hybrid. The hybrid, 4 showed 8.3% greater weight loss at
6

o
700 C as compared to 2 and this is attributable to the loss of the calixarene conjugate. This
weight loss corresponds to ~14.3% of calixarene content present in the POM-Calix hybrid, 4
which agrees well with the calculated value of 14.5%.
3
1
2
4
(c)
(b)
(d)
(a)
100
75
Experimental
4
2
1
Simulated
3990
4005
4020
1
2
3
4
50
2000 4000 6000 8000
200 400 600 800
T, oC
0
20
40
2θo
60
80
4000 3000 2000 1000
υ, cm-1
m/z
Fig. 1. (a) TGA curves and (b) FTIR spectra for 1, 2, 3 and 4. (c) MALDI-TOF for 4 using
DTCB matrix. (d) XRD patterns of 1, 2 and 4.
The FTIR spectra of 1, 2, 3 and 4 are comparatively shown in Fig. 1b. The unmodified
lacunary polyoxometalate (1) showed characteristic bands at 1058 and 1015 cm-1 due to
νas(P-O), 938 cm-1 due to νas(W=O), and 885, 818 and 746 cm-1 due to νas(W-O-W). The
presence of these peaks in the hybrid materials 2 and 4 suggests the retention of the Keggin
ion structure of 1 even after functionalization. The alkylthiol functionalization carried out on
1 leading to 2 is gauged from the presence of new vibrational bands observed in 1060 to 1170
cm-1 due to the stretching vibrations of Si-O and Si-C groups. The 2 further shows a
stretching vibrational band for S-H group at 2564 cm-1. The peak assignment for 1 and 2,
agrees well with that reported in the literature [26-27]. The coupling between 2 and 3 leading
to 4 is supported by the disappearance of this S-H stretching vibrational band. The broad
band observed in the region of 3696-2498 cm-1 for water in case of 1 was disappeared when
this is thiol functionalized to give compound 2, and this eventually appeared in the region
3735-3042 cm-1 after attaching the calixarene conjugate as in 4 with significant reduction in
the H- bonding as evidenced from the decreased band width of νOH. The Fig. 1c shows the
MALDI-TOF spectrum for 4 taken in 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-
7

enylidene]malononitrile (DTCB) as matrix. The peak observed at m/z = 4002.6 corresponds
to the molecular ion peak [M+H]+. The simulated isotopic peak pattern agrees well with the
observed envelope of peaks (inset Fig. 1c) due to the presence of different isotopes arising
from W, P and Si. Fig. 1d shows the XRD patterns of 1, 2 and 4. It can be noted that the
trilacunary phosphotungstate, 1 is crystalline in nature which upon alkyl-thiol
functionalization, loses its crystallinity to some extent and this is further maintained even
when it is covalently connected to the calix-conjugate to give hybrid, 4. This is further
supported by the lattice fringes observed from HRTEM of 4 (Fig. 3d).
13
The C MAS-NMR spectra of compounds 2, 3 and 4 are given in Fig. 2a. The presence of
13C chemical shifts in the region, δ = 120-150 ppm in 4 supports the presence of calixarene
based aromatic carbons in the hybrid material which were absent in its precursor compound
2. The ene carbon signals of allyl moiety were observed in δ = 110-120 ppm in case of 3 and
these are absent in case of 4 as expected since the thiol function is added to the double bond.
31
The P NMR spectrum of 1 (inset in Fig. 2b) shows a signal at -10.8 ppm corresponding to
the central P atom of the trilacunary phosphotungstate suggesting that the 1 is intact. This
has been shifted to -18.18 ppm due to the covalent attachment of the calixarene conjugate to
POM. All these results support the presence of the integral POM unit in the hybrid, 4. The
chemical shift agrees well with that reported in the literature [28].
-10.8
(a)
(b)
3
4
1
0
-5 -10 -15 -20
δ, ppm
4
2
100
0
0
-100
-200
200 150 100
50
δ, ppm
δ, ppm
Fig. 2. (a) 13C MAS- NMR spectra of 2, 3 and 4. (b) 31P NMR spectrum of 4.
8

The morphological features of the hybrid 4 were studied by AFM, SEM and TEM analyses.
AFM of 4 showed spherical morphology with particle size of 50-60 nm (Fig. 3 a,b). This was
further supported by SEM which also showed spherical particles of the size 40-50 nm (Fig.
3c). The crystallinity of the POM is evident from HRTEM (Fig. 3d) where the lattice spacing
supports the crystalline POM in the hybrid.
(b)
(a)
(c)
(d)
Fig. 3. Micrographs for 4. (a,b) are AFM images, (c) is SEM image (100 nm scale), and (d) is
TEM image (2 nm scale).
Peroxidase-like activity of polyoxometalate-allyl calixarene hybrid (4):
NH2
NH2
NH2
N
H2O2
Catalyst
NH2
N
2,3-diaminophenazine
(DAP)
o-Phynelediamine
(OPD)
Scheme 2. Peroxidase like activity for o-phenylenediamine (OPD) oxidation to 2,3-
diaminophenazine (DAP).
The activity of the POM-Calix hybrid 4, was evaluated for the oxidation of OPD to DAP by
using H2O2 in PBS buffer (pH 7.4) as per that given in Scheme 2. The oxidised product,
9

DAP is yellow and has absorption λmax at 415 nm and the progress of the reaction has been
monitored by following this band.
The progress of the reaction as a function of time can be seen in Fig. 4a. There is a gradual
increase in the intensity of the band at 415 nm corresponding to the oxidised product (DAP).
This indicates that the POM-Calix hybrid exhibits peroxidase-like activity. The control
experiments showed that OPD alone in presence of H2O2 does not show any significant
oxidation up to 14 min of the reaction time (Fig. 4b). The control reaction for the oxidation
of OPD in presence of H2O2 and 1 showed increase in the absorbance at 415 nm indicating
that the inorganic POM also showed peroxidase like activity as expected. However, the
reaction in presence of POM-Calix hybrid, 4 showed maximum activity demonstrating 170%
increase in the oxidised OPD (i.e., DAP) formation when compared to the simple inorganic
POM. The increase in the activity was further evidenced from the TOF values evaluated for
1 and 4 which comes out to be 0.036 min-1 and 0.0567 min-1, respectively derived from the
data obtained after 14 min of the reaction under similar reaction conditions. While the simple
POM is highly hydrophilic, its inorganic-organic covalent hybrid, 4 shows a contact angle of
75° showing a definite decrease in the hydrophilicity since the allylCalix (3) itself is
hydrophobic and shows a contact angle of 130o (supporting information Fig. S7). All this is
due to the introduction of the calixarene conjugate on to POM covalently in the hybrid, 4. It
is known that the calixarene conjugates are well suited for the recognition of aromatic
molecules [29-30]. This property is responsible for increased interaction of OPD with the
inorganic-organic hybrid, 4. The results of UV-Vis spectral data were supported by the
observed colour changes (inset Fig. 4b) yielding a colourless mixture after 14 min for the
reaction without the catalyst, and this is pale yellow when 1 is used as catalyst and dark
yellow when 4 is used as the catalyst. The POM-Calix hybrid, 4 interestingly exhibited
excellent catalytic activity over a large pH range (Fig. 4c). This suggests that the present
10

catalytic system can be used under physiological conditions unlike most of the reported
catalytic systems [31-33] which showed only little activity at pH 7.4. This is due to the
covalent modification of POM by calix-conjugate to result in inorganic-organic hybrid, 4.
The stability of POM-Calix hybrid, 4 has been studied under different pH conditions by using
UV-Vis spectroscopy and the results are shown in the Fig. 4d. All the UV-Vis spectra of 4
showed two bands between 200-320 nm having maxima at 206 and 267 nm which is
unaffected under different pH conditions suggesting that the POM-Calix hybrid is stable
under these conditions.
(a)
(b)
0.2
0.1
0.0
0.15
0.10
0.05
0.00
(c)
(d)
0.9
1
4
100
pH 3
pH 4
14 Min
0 Min
75
50
25
0
pH 6
pH 7.4
pH 9.5
0.6
0.3
0.0
4
1
Blank
300
400
500
600
0
3
6
9
12 15
200
400
600
800
2
4
6
8
10
Time, min
λ, nm
λ, nm
pH
Fig. 4. (a) UV-Vis spectra for OPD oxidation with respect to time using 4 as catalyst.
Reaction conditions: OPD = 10 mM, H2O2 = 100 mM and 4 = 10 µM. (b) Control
experiments using 1, 4 and blank (without catalyst). The inset shows the change in the colour
of the reaction mixture after 14 mins. Reaction condition: OPD = 10 mM, H2O2 = 100 mM
and 1 or 4 = 10 µM. (c) Effect of pH on OPD oxidation by using 10 µM of 4, 10 mM of
OPD and 100 mM of H2O2. (d) UV-Vis spectra of POM-Calix hybrid, 4 under different pH
conditions.
The steady-state kinetic parameters were evaluated by changing the concentrations of OPD
and that of H2O2. From the corresponding absorption-time plot, the initial velocity was
estimated by the equation, Vinitial = slopeinitial/ɛOPD. The concentration of the oxidized product
was estimated by Lambert-Beer law using εOPD of 16700 M-1 cm-1 for the absorption band at
415 nm. The plot of reaction velocity as a function of the substrate concentration shows
11

typical Michaelis-Menten saturation curve (Fig. 5a,c). The reaction rates increased gradually
with increasing substrate concentration until reaching a maximum rate, indicating saturation
in the reactivity of 4. The Michaelis-Menten parameters, Km and Vmax, were evaluated from
the double-reciprocal Lineweaver-Burk plot (Fig. 5b,d) to yield 2.55 mM and 0.756 x 10-8 M
Sec-1 in case of OPD and 707.8 mM and 3.21 x 10-8 M Sec-1 in case of H2O2, respectively.
0.7
(b)
(a)
8.0
6.0
4.0
2.0
0.0
0.5
0.3
0.1
y = 3.3797x+ 1.3229
0
2
4
6
8
10 12
-0.5
0
0.5
1
1.5
2
[OPD], mM
1/[OPD]
5.0
(c)
(d)
0.8
0.6
0.4
0.2
4.0
3.0
2.0
1.0
0.0
y = 220.35x+ 0.3109
0.004 0.014
5
30
55
80 105
-0.006
[H2O2] mM
1/[H2O2]
Fig 5. (a) and (c) Steady state kinetics (b) and (d) double-reciprocal plots for the calculation
of enzyme kinetic parameters by the Michaelis-Menten equation for 4 using varying
concentrations of OPD and H2O2. Reaction conditions: For (a & b) OPD = 0.67 to 10 mM,
H2O2 = 100 mM and 4 = 10 µM and for (c & d) OPD = 15 mM, H2O2 = 20-100 mM and 4 =
10 µM.
(b)
(a)
100
TA+ H O + 4
(a) TA+ H2O2+ 4
(b) TA+ H2O2+ 1
(c) TA+ H2O2
2
2
TA+ H O + 1
450
2
2
75
50
25
0
TA+ H O
2
2
TA+ 4
TA
(d) TA+ 4
300
150
0
(e) TA
a
b
c
d
e
375
450
525
600
λ, nm
Sample
12

Fig. 6. (a) Fluorescence spectra for detection of ·OH radicals with different controls (λex =
315 nm). (b) Relative fluorescence intensity of HTA at 425 nm for different controls.
A possible mechanism of peroxidase-like activity exhibited by 4 originates from its catalytic
ability towards the decomposition of H2O2 to generate hydroxyl radical (·OH) [33].
Terephthalic acid (TA) was used as a fluorescence probe for detecting ·OH because it
captures these radicals and generate 2-hydroxy terephthalic acid (HTA), which emits ~425
nm when excited at 315 nm [33]. As shown in Fig. 6a, the control, i.e., only TA, and TA in
presence of 4 do not show significant intensity for HTA. The control experiment for TA in
presence of H2O2 showed increase in the intensity to some extent suggesting the formation of
·OH. The TA in presence of H2O2 and 4 showed remarkable increase in the intensity by
200% when compared to the TA in presence of H2O2 and 1. The two-fold increase observed
in the concentration of ·OH can be correlated with the ~1.7-fold increase observed in the
peroxidase-like activity of the POM-Calix hybrid, 4 (Fig. 6b). This supports that the covalent
attachment of the Calix conjugate resulting in the hybrid POM facilitates the formation of
·OH and hence exhibited increased activity for the oxidation of OPD.
Conclusion and comparisons
A new class of inorganic-organic hybrid comprising of polyoxometalate and calixarene was
synthesized by thiol-ene click reaction between thiolated polyoxometalate and allyl-
calixarene conjugate for the first time. The POM-Calix hybrid, 4 was characterized by
several spectroscopy and microscopy methods. The TGA showed 14.3 wt% of the calixarene
content in the hybrid, 4 which agrees with the calculated value of 14.5%. The FTIR analysis
of the hybrid, 4 showed all the characteristic bands, viz., νas(P-O), νas(W=O), and νas(W-O-
W) corresponding to the trilacunary phosphotungstate suggesting that the polyoxometalate is
intact even after the formation of the hybrid. Further, the appearance of the bands
13

corresponding to the -CH stretching and disappearance of -SH stretching supported
successful attachment of calixarene conjugate to the POM-Calix hybrid. The MALDI of
hybrid, 4 showed the presence of molecular ion peak with isotopic envelope pattern matching
with that of the experimental one. The 13C MAS-NMR spectra showed the presence of
aromatic and aliphatic carbon resonances arising from the calixarene conjugate in addition to
the carbons from the tetra-butyl ammonium cations. The disappearance of allyl carbon peaks
(100-110 ppm) supports the covalent modification of the conjugate onto the POM surface.
The 31P NMR showed single peak attributed to the central phosphorus atom of the POM unit.
All this suggests the presence of intact polyoxometalate in the hybrid, 4. The morphology of
the hybrid, 4 was studied by AFM, SEM and TEM which showed spherical particles of 40-60
nm size. The combination of all these analyses supports the successful formation of the
POM-Calix hybrid.
The synthesized POM-Calix hybrid, 4 was employed successfully for the enzyme mimetic
activity for the peroxidase substrate, OPD using H2O2 as oxidant. The activity of the hybrid
was monitored by following the gradual increase in the product (DAP) formed which is
evident from the increase observed in the intensity of the band at 415 nm in the UV-Vis
spectra. The control experiments showed that in the absence of the catalyst, the progress of
the reaction was very slow. The POM-Calix hybrid, 4 showed ~170% increase in the
peroxidase activity when compared to the activity of inorganic polyoxometalate, 1. This can
be correlated with the increased hydrophobicity of the hybrid due to the presence of the calix-
conjugate as obtained from the contact angle measurements. The POM-Calix hybrid showed
excellent peroxidase like activity over a wide range of pH, such as, 2.5-7.4 which includes
the physiological pH, and hence would facilitate its biological and medical applications.
From the kinetic experiments, the Michaelis constants of Km and Vmax were obtained as 2.55
mM and 0.756 x 10-8 M sec-1 respectively. The peroxidase-like activity of POM-Calix
14

hybrid, 4 arises from the H2O2 decomposition into ·OH and this was identified by the
remarkable increase observed in the fluoresce intensity of HTA when TA was treated with 4
in presence of H2O2. Hence, our findings provide strong evidence for the POM-Calix hybrid,
4 as useful peroxidase mimetic catalyst which may find possible applications in biosensing
and biotechnology.
Acknowledgements. CPR thanks the financial support of SERB project
{EMR/2014/000985}
and
DST/SERB
for
JC
Bose
National
Fellowship
{SB/S2/JCB/066/2015} and IIT Bombay for Chair Professorship. NN acknowledges SERB
for the award of National Postdoctoral Fellowship (PDF/2016/000211) and BU acknowledges
CSIR for the award of Senior Research Fellowship {09/087(0835)/2015-EMR-I}. We
acknowledge the services provided by the central facilities of IIT Bombay (SEM, AFM,
TEM, TGA, XPS, MALDI, ICP-AES). We thank Ms. Aekta Upadhyay and Mr. Rahul Nag
for helping with measuring the SEM & TEM micrographs respectively.
Appendix A. Supplementary data. The experimental section and spectral data for synthesis
of 3 can be found in the supporting information. Supplementary data associated with this
article can be found, in the online version, at http://dx.doi.org/xxx/xxx.
References
[1] C. L. Hill, C. M. Prosser-McCartha, Coord. Chem. Rev.143 (1995) 407-55.
[2] S. -S.Wang, G. -Y. Yang, Chem. Rev. 115 (2015) 4893- 4962.
[3] Y. -F. Song, R. Tsunashima, Chem. Soc. Rev. 41 (2012) 7384-7402.
[4] F. Lei, H. Gao, M. Yan, S. Li, X. Li, Z. Dai, S. Li, Small 11 (2015) 2938-2945.
[5] J. T. Rhule, C. L. Hill, D. A. Judd, Chem. Rev. 98 (1998) 327-357.
[6] P. Yang, H. Li, T. Ma, F. Haso, T. Liu, L. Fan, Z. Lin, C. Hu, U. Kortz, Chem. Eur. J.
24 (2018) 2466-2473.
15

[7] A. Patel, N. Narkhede, S. Singh, S. Pathan, Catal. Rev. 58 (2016) 337-370.
[8] M. T. Pope, Heteropoly and Isopoly Oxometalates; Springer-Verlag:ꢀ Berlin, (1983).
[9] V. Dufaud, F. Lefebvre, Materials 3 (2010) 682-703.
[10] R. Zhang, C. Yang, J. Mater. Chem., 18 (2008) 2691-2703.
[11] X. Luoa, C. Yang, Phys. Chem. Chem. Phys. 13 (2011) 7892-7902.
[12] M. Carraro, G. Bergamini, M. Di Lauro, G. Modugno, M. Baroncini, P. Ceroni, M.
Bonchio, Eur. J. Inorg. Chem. (2016) 3405-3410.
[13] B. Matt, S. Renaudineau, L. -M. Chamoreau, C. Afonso, G. Izzet, A. Proust, J. Org.
Chem. 76 (2011) 3107-3112.
[14] D. Mercier, S. Boujday, C. Annabi, R. Villanneau, C. M. Pradier, A. Proust, J. Phys.
Chem. C, 116 (2012) 13217-13224.
[15] C. R. Mayer, I. Fournier, R. Thouvenot, Chem. Eur. J. 6 (2000) 105-110.
[16] I. Bar-Nahum, H. Cohen, R. Neumann, Inorg. Chem. 42 (2003) 3677-3684.
[17] R. Joseph, C. P. Rao, Chem. Rev. 111 (2011) 4658-4702.
[18] M. Song, Z. Sun, C. Han, D. Tian, H. Li, J. S. Kim, Chem.- Asian J. 9 (2014) 2344-
2357.
[19] Y. Bi, S. Duab, W. Liao, Chem. Commun. 47 (2011) 4724-4726.
[20] X. -M. Zhou, W. Chen, Y. F. Song, Eur. J. Inorg. Chem. (2014) 812-817.
[21] T. N. Ursale, I. Silaghi-Dumitrescua, R. Grecu, L. Silaghi-Dumitrescu, N. Popovici, E.
-J. Popovici, J. Optoelectron. Adv. M. 6 (20040 471-476.
[22] Y. Ishii, Y. Takenaka, K. Konishi, Angew. Chem. 116 (2004) 2756-2759.
[23] S. Meninno, A. Parrella, G. Brancatelli, S. Geremia, C. Gaeta, C. Talotta, P. Neri, A.
Lattanzi, Org. Lett. 17 (2015) 5100-5103.
[24] N. Narkhede, B. Uttam, R. Kandi, C. P. Rao, ACS Omega, 3 (2018) 229-239.
16

[25] R. Massart, R. Contant, J. M. Fruchart. J. P. Ciabrini, M. Fournier, Inorg. Chem. (1977)
2916-2921.
[26] S. Aoki, T. Kurashina, Y. Kasahara, T. Nishijima K. Nomiya, Dalton Trans. 40 (2011)
1243-1253.
[27] W. J. Randall, M. W. Droege, N. Mizuno, K. Nomiya, T. J. R. Weakley, R. G. Finkey,
Inorg. Synth. 31 (1997), 167-184.
[28] A. Mazeaud, N. Ammari, F. Robert, R. Thowenot, Angew. Chem. Int. Ed. Engl. 38
(1996) 1961-1964.
[29] P. Leyton, S. Sanchez-Cortes, J. V. Garcia-Ramos, C. Domingo, M. Campos-Vallette,
C. Saitz, R. E. Clavijo, J. Phys. Chem. B, 108 (2004) 17484-17490.
[30] A. K. Bandela, S. Patrudu, C. P. Rao, Chem. Eur. J. 21 (2015) 13364-13374.
[31] Z. Ma, Y. Qiu, H. Yang, Y. Huang, J. Liu, Y. Lu, C. Zhang, P. Hu, ACS Appl. Mater.
Interfaces, 7 (2015) 22036-22045.
[32] J. Wang, D. Han, X. Wang, B. Qi, M. Zhao, Biosens. Bioelectron. 36 (2012) 18-21.
[33] T. M. Chen, J. Xiao, G. W. Yang, RSC Adv., 6 (2016) 70124-70132.
17

Highlights
•
•
•
The synthesis and characterization of covalently attached POM-Calix hybrid has been
demonstrated for the first time.
The POM-Calix covalent hybrid was characterized by spectroscopy and microscopy
techniques.
The POM-Calix hybrid has been shown to act as peroxidase mimetic catalyst under wide
range of pH including 2.5 to 7.4.
•
•
The POM-Calix hybrid showed ~170% better activity as compared to simple POM.
The mechanism pathway for the peroxidation reaction via hydroxyl radical has also been
demonstrated.
18

Graphical Abstract
Synopsis:
The polyoxometalate-calixarene covalent hybrid has been synthesized,
characterized and has been demonstrated as catalyst for the peroxidase like enzyme activity.
19