Paraoxonase Mimic by a Nanoreactor Aggregate Containing Benzimidazolium Calix and l-Histidine: Demonstration of the Acetylcholine Esterase Activity

Article abstract of DOI:10.1002/chem.202004944

An anion-mediated preorganization approach was used to design and synthesize the benzimidazolium-based calix compound R1?2 ClO₄^?. X-ray crystallography analysis revealed that the hydrogen-bonding interactions between the benzimidazol

Full text of DOI:10.1002/chem.202004944

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&
Organophosphate Removal
Paraoxonase Mimic by a Nanoreactor Aggregate Containing
Benzimidazolium Calix and l-Histidine: Demonstration of the
Acetylcholine Esterase Activity
Amanpreet Singh,[a] Sanjeev Saini,[a] Mayank,[a] Navneet Kaur,*[b] Ajnesh Singh,[c]
Narinder Singh,*[a] and Doo Ok Jang*[d]
Abstract: An anion-mediated preorganization approach was
used to design and synthesize the benzimidazolium-based
calix compound R1·2ClO4À. X-ray crystallography analysis re-
vealed that the hydrogen-bonding interactions between the
benzimidazolium cations and N,N-dimethylformamide (DMF)
nanoreactor could detoxify paraoxon in 30 min. l-His played
a vital role in this process. Paraoxonase is a well-known
enzyme used for pesticide degradation. The Ellman’s reagent
was used to determine the percentage inhibition of the
acetylcholinesterase (AChE) activity in the presence of the
nanoreactor. The results indicated that the nanoreactor in-
hibited AChE inhibition.
À
helped R1·2ClO4 encapsulate DMF molecule(s). A nanoreac-
À
tor, with R1·2ClO4 and l-histidine (l-His) as the compo-
nents, was fabricated by using a neutralization method. The
Introduction
tivity is inhibited when its active site, serine, binds to the OPs
(Scheme 1). The OPs that bind irreversibly to AChE through
serine residues are more harmful than carbamate-based pesti-
cides that bind reversibly to a target enzyme.[18–23]
The increasing global hunger index has put the agriculture
sector under pressure to produce more crops. However, in-
creased crop production cannot be achieved without the use
of pesticides.[1–3] There are numerous pesticides that do not de-
grade even after a considerable time has passed since its appli-
cation and that cause serious health concerns.[4–7] The com-
monly known non-degradable pesticides are the organophos-
phates (OPs) and carbamates, which are the most prevalent
toxic chemicals.[8–12] Acetylcholinesterase (AChE) hydrolyzes
acetylcholine into choline, which plays a key role in maintain-
ing the proper functioning of the peripheral cholinergic
system. The inhibition of AChE activity induces the accumula-
tion of acetylcholine, thereby resulting in the disruption of me-
tabolism, which can eventually cause death.[13–17] The AChE ac-
Moreover, the OPs are employed as chemical warfare agents
(CWAs) and are the major components of the nerve gases.[24,25]
Therefore, to reduce pesticide poisoning (for sustainable devel-
opment) and produce antidotes for CWAs, it is desirable to de-
velop a highly efficient catalyst that can rapidly hydrolyze the
OPs and detoxify them.[26–28] The hydrolysis of the OPs affords
compounds incapable of phosphorylating the serine residue of
AChE.
The complete removal of OPs from the environment and, ul-
timately, from food is challenging because most OPs are stable
over a wide pH range.[29,30] The enzyme paraoxonase is well-
known for its ability to hydrolyze various OPs. Paraoxonase
possesses two histidine residues (His 115 and 134) in its struc-
ture, which are involved in proton transfer. The asparagine resi-
due present in this enzyme stabilizes the intermediates at the
active site.[31–33] However, the isolation of such enzymes is chal-
lenging. Hence, they cannot be used for the large-scale degra-
dation of pesticides.
[a] A. Singh, S. Saini, Mayank, Dr. N. Singh
Department of Chemistry, Indian Institute of Technology Ropar
Roopnagar, Punjab 140001 (India)
E-mail: nsingh@iitrpr.ac.in
[b] Dr. N. Kaur
Department of Chemistry, Panjab University, Chandigarh 160014 (India)
E-mail: navneetkaur@pu.ac.in
[c] A. Singh
Department of Applied Sciences & Humanities
Jawaharlal Nehru Govt. Eng. College, Sundernagar 175018 (India)
[d] Dr. D. O. Jang
Department of Chemistry, Yonsei University
Wonju 26493 (Republic of Korea)
E-mail: dojang@yonsei.ac.kr
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.202004944.
Scheme 1. Schematic representation of AChE inhibition by OPs.
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Although metal-based catalysts that can hydrolyze OPs have
been reported, the use of expensive metals,[34–36] reduced effi-
ciency of the catalysts (through hydration of the metal center
in aqueous media), and poor biocompatibility limit their practi-
cal application. Farha and co-workers developed a zirconium-
based metal–organic framework, which encapsulates OPs and
hydrolyzes them to produce non-toxic phosphates.[26] It was re-
ported that the catalyst system should bear highly active sub-
stituents attached to the metal complex to function efficiently
in an aqueous medium. A nickel complex and a copper-based
polymer that can sense OPs have also been reported.[37] Al-
though these compounds could detect OPs, the metal com-
plexes dissociated when they interacted with the OPs. This
eventually led to low catalytic efficiencies (for the degradation
of OPs). Furthermore, the mechanism of catalytic degradation
of CWAs has been explained by an aggregation-based deacti-
vation mechanism.[38]
Scheme 2. Synthetic scheme for preparing benzimidazolium-based calix
À
R1·2ClO4
.
Herein, we have explored the ionic interactions between
anionic surfactants and benzimidazolium-based organic cations
to develop micelle-type aggregates for use as catalysts. We hy-
pothesized that the ionic liquid (IL)-based catalysts could po-
tentially overcome the limitations posed by metal-based cata-
lysts toward the hydrolysis of OPs as the imidazolium moieties
in the ILs exhibit high phosphate ion affinities.[39–45]
benzimidazole) validated the formation of compound 2. The
calix R1·2XÀ was subsequently prepared by using the anion-in-
duced preorganization approach. The anions are known to
induce the formation of a particular isomer of the reactants,
leading to the exclusive production of the desired product.[51,52]
Compound 2 exists in different steric orientations, all of which
can potentially lead to the formation of different products. In
such a scenario, the isolation of the products becomes chal-
lenging. Therefore, tetrabutylammonium (TBA) halides (TBAXs)
were added to the reaction mixture to reorganize compound 2
for the exclusive formation of one calix compound. Among all
the TBAXs used for the reaction, the use of TBACl resulted in
the maximum yield of the calix R1·2XÀ compound. This is be-
cause the cavity formed by the organized benzimidazole-
based dipodal compound 2 could suitably bind to the chloride
anion through hydrogen bonding.[53] To further validate the
role of the chloride ion in the synthesis of the calix R1·2ClÀ,
the reaction was performed by using different concentrations
of TBACl. The progress of the reaction is presented in Figure 1.
Herein, we have developed a strategy for the in situ devel-
opment of a nanoreactor, from benzimidazolium-based cation-
ic calix and l-histidine (l-His) units, having all the essential cat-
alyst parameters for the degradation of OPs. An anion-induced
preorganization approach was used to synthesize the benzimi-
dazolium-based calix unit. The cationic benzimidazolium
moiety could interact with the l-His residue via ionic interac-
tions and form a self-assembled nanoreactor. Both the units of
the nanoreactor play crucial roles in facilitating the catalytic ac-
tivity of the nanoreactor toward the hydrolysis of OPs. The
benzimidazolium ring is known to hydrogen bond with elec-
tron-rich species and is widely used to activate electrophiles
that participate in nucleophilic substitution reactions. l-His par-
ticipates in enzyme catalysis and activates nucleophiles by ab-
stracting protons.[46] The l-His residue of paraoxonase plays a
crucial role during pesticide degradation.[31] Although ILs have
been prevalently used in catalysis,[47–50] to the best of our
knowledge, this is the first report of an aggregate nanoreactor
containing benzimidazolium calix and l-His units that can de-
grade harmful pesticides.
Results and Discussion
À
Synthesis and characterization of the calix R1·2ClO4
The nucleophilic substitution reaction between compound 1[50]
and benzimidazole, in the presence of K2CO3, afforded com-
pound 2 (Scheme 2). The structure of compound 2 was con-
1
firmed by using H NMR spectroscopy and 13C NMR spectrosco-
py techniques. The 1H NMR spectrum exhibited a singlet at
4.5 ppm (corresponding to the four aliphatic methylene pro-
tons), indicating the attachment of the benzimidazole moiety
to the mesitylene unit. Another singlet at 10.7 ppm (CÀH,
Figure 1. Variation in the yields of the calix R1·2ClÀ versus time at different
chloride ion concentrations.
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The Calix R1·2ClÀ was obtained in 90% yield when five equiva-
lents of TBACl were used, and the reaction was allowed to pro-
ceed for 300 min. In the absence of TBACl, the calix R1·2ClÀ
compound was obtained in 20% yield. Interestingly, with five
equivalents of the chloride ions, the desired product separated
out as a white solid in acetonitrile. Side products were not
À
formed. The chloride ions were replaced by ClO4 ions to grow
crystals suitable for carrying out single-crystal X-ray diffraction
(XRD) experiments. The calix R1·2ClÀ was dissolved in H2O/
DMF (9:1, v/v) and 10 equivalents of TBAClO4 were added to
the solution. After boiling, the solution was cooled down to
room temperature to yield white crystals of the calix
R1·2ClO4À, which were suitable for single-crystal XRD analysis.
Figure 2. (A) ORTEP diagram and the atom numbering scheme for the calix
R1·2ClO4À with thermal ellipsoids drawn at the 40% probability level (for
clarity, the disordered ClO4 and solvent molecules of crystallization are not
shown). (B) Packing image of calix R1·2ClO4À viewed down the b-axis.
À
Nanoreactor fabrication: in situ formation of aggregates
from ionic conjugates formed of the benzimidazolium calix
R1 and l-His
À
Structural description of the calix R1·2ClO4
À
The ionic conjugates of the benzimidazolium calix R1 and l-
The crystal structure analysis revealed that the calix R1·2ClO4
À
His were prepared by replacing the ClO4 ions in the calix
molecule crystallized in the tetragonal crystal system (space
À
R1·2ClO4 with hydroxides.[54] Subsequently, the hydroxide
group: I41/acd, Table 1). The crystal consisted of one calix mol-
ions were replaced with l-His (Figure 3A).[55,56] The resulting l-
His anions formed an anionic conjugate with the benzimidazo-
lium cation. The intermolecular hydrogen bonding l-His resi-
dues of the aggregates and the blockage of the hydrophilic
À
ecule, two ClO4 counterions, and two DMF molecules of crys-
À
tallization. The packing of calix R1·2ClO4 has not been exten-
sively discussed as the ClO4 and DMF molecules were disor-
À
dered. Selected bond lengths and angles are presented in
Table S1 (in the Supporting Information). The ORTEP diagram
À
and the atom numbering scheme for the calix R1·2ClO4 is
shown in Figure 2A. The packing diagram of the calix
À
R1·2ClO4 is shown in Figure 2B, which reveals that the mesi-
tylene rings of the two calix molecules are oriented in a face-
to-face manner, whereas the benzimidazolium rings remain
outside.
À
Table 1. Crystal data and refinement parameters of the calix R1·2ClO4
.
-
Parameters
R1·2ClO4
empirical formula
formula weight
T [8C]
C42H52Cl2N6O10
871.80
20(2)
crystal system
space group
a [Š]
b [Š]
c [Š]
tetragonal
I41/acd
20.5860(9)
20.5860(9)
40.0136(16)
90
a [8]
b [8]
90
g [8]
90
V [Š3]
16957.1(12)
16
1.366
Z
Dc [MgmÀ3
]
m [mmÀ1
]
0.218
reflections collected
data/restraints/parameters
unique reflections, [Rint
GOF=Sall
236852
4175/0/332
4175 [0.0714]
1.040
]
final R indices
R1, wR2 [I>2sI]
R1, wR2 (all data)
D1max/D1min [Š3]
0.0779, 0.2193
0.0888, 0.2270
1.017/À1.077
Figure 3. (A) Formation of self-assembled aggregates from the ionic conju-
gate of the calix R1·2ClO4À and l-His. AFM images of the aggregates:
(B) spherical aggregates; (C) amplitude error image of spherical aggregates;
(D) TEM image of aggregates (100 nm); (E) 3D view of spherical aggregates.
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core of the ionic conjugate (by the hydrophobic aromatic rings
of the benzimidazolium moiety, leading to reduced solubility),
can potentially explain the formation of the aggregates. Inter-
estingly, when two equivalents of l-His were added to a clear
solution of the calix R1·2OHÀ, the solution became turbid. The
turbidity was due to the formation of aggregates, as observed
by dynamic light scattering (DLS) experiments (Figure S1 in the
Supporting Information). It was reported that the aggregates
were used as sensors and catalysts.[57–61] High-resolution
images of the aggregates were recorded by using the atomic
force microscopy (AFM) technique to investigate the sizes and
morphologies of the aggregates (Figure 3B,C). The images re-
vealed the aggregation of spherical particles having a radius of
172 nm. A transmission electron microscopy (TEM) image was
also obtained with the size of aggregates of about 100 nm
(Figure 3D).
Scheme 3. Degradation of paraoxon in the presence of the fabricated cata-
lytic nanoreactor using benzimidazolium-based calix R1·2HOÀ (0.1 equiva-
lent) and a cocatalyst (0.1 equivalent).
lysts, substrates, and byproducts exhibited an absorbance
maxima that was >380 nm. Therefore, they could not poten-
tially interfere with the degradation of paraoxon. Catalytic
studies revealed that neither the calix R1·2OHÀ nor the cocata-
lyst could independently facilitate significant conversion of
paraoxon to p-nitrophenol. However, the self-assembled aggre-
gates facilitated the conversion of paraoxon (>80% efficiency)
to the less-toxic products in 25 min (Figure 4A,B). The calibra-
tion plot (absorbance intensity vs. time, Figure 4C) was linear.
The calculated half-life (t1/2) of the catalyst was 6.9 min (Fig-
ure S6 in the Supporting Information). The catalytic activities of
the calix R1·2OHÀ were examined in the presence of other co-
catalysts. The percentage of paraoxon degradation was signifi-
cantly high when l-His was used as the cocatalyst (compared
with other cocatalysts such as triethylamine, diethylamine, as-
paragine, piperidine, and imidazole, Figure S7 in the Support-
ing Information). The turnover number (Kcat) was calculated at
different catalyst equivalents. The value of Kcat increased con-
tinuously when the catalyst amount was ꢀ0.1 equivalent. A
stable Kcat value was reached at higher catalyst concentrations
(Figure S8 in the Supporting Information). The degradation of
OPs, such as paraoxone methyl, parathion, and parathion
methyl was studied by using the nanoreactor. Similar catalytic
activities were observed in the reaction involving paraoxon
(Figure 5).
The properties of the self-assembled aggregates formed
À
from the ionic conjugates of the calix R1·2ClO4 and l-His
were investigated by using the diffusion-ordered NMR spec-
troscopy (DOSY) technique. DOSY allows non-invasive detec-
tion of self-aggregation in the solution phase. The diffusion co-
efficient of the solute species can also be estimated by using
À
this technique. The DOSY spectrum of R1·2ClO4 was recorded
and the diffusion coefficient (D) was determined to be 1.55”
10À10 m2 sÀ1 (Figures S2 and S3 in the Supporting Information).
After the addition of an adequate amount of l-His, the DOSY
spectrum was recorded again. Analysis of the spectrum re-
vealed that upon the addition of l-His, the value of the diffu-
sion coefficient (D) decreased to 1.35”10À10 m2 sÀ1 (Figures S4
and S5 in the Supporting Information). The results confirmed
aggregation.
Degradation of paraoxon
It is known that AChE reacts with OPs via the l-His residue,
which otherwise does not react with the OPs.[62,63] Various
supramolecular interactions helped encapsulate the OPs in the
cavity of AChE. These interactions enhanced the reactivity of
the OPs by increasing the electrophilicity of the phosphate
center.
Raushel et al. reported the role of phosphodiesterase in the
hydrolysis of OPs.[64,65] Phosphodiesterase is a zinc-containing
enzyme that hydrolyzes the PÀO bond. The binding of OPs to
the metal ions facilitates a nucleophilic attack on the phospho-
rus center, which favors the formation of a weak PÀO bond, re-
sulting in the eventual hydrolysis of the PÀO bond. To date,
zinc and zirconium complexes have been used for the hydroly-
sis of PÀO bonds in phosphodiesters.[66–68] We have attempted
to replace these metal ions with organic cationic receptors.
Initially, the conversion of paraoxon to p-nitrophenol was
achieved by using the calix R1·2OHÀ as a catalyst in the pres-
ence of a cocatalyst (Scheme 3). The ionic conjugates of the
calix R1·2OHÀ and/or the cocatalysts (0.1 equivalent) were dis-
persed in an aqueous medium, following which paraoxon was
added to the solution. The degradation of paraoxon was moni-
tored by recording the absorbance spectra of the solution at
412 nm (lmax of p-nitrophenol). None of the catalysts, cocata-
Figure 4. (A) Chart of the degradation of paraoxon at different catalytic con-
ditions. (B) Change in the absorbance spectra of the solution of paraoxon
À
(50 mm) and calix R1·2ClO4 (0.1 equivalent) in the presence of l-His
(0.1 equivalent) with respect to time. (C) A calibration plot of the variation in
absorbance with time.
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oxon split into two peaks in the presence of the calix unit, con-
firming the interactive association between paraoxon and the
calix unit in the catalytic system, which results in the hydrolysis
of paraoxon.
To determine the binding affinity of the phosphate esters for
cyclic bis-cations, 1H NMR titration was performed. Diethyl
chlorophosphate was chosen instead of aromatic organophos-
phate to eliminate the interference caused by aromatic pro-
tons. The imidazolium proton (CÀH), which is highly acidic
compared with other protons, readily interacts with organo-
phosphate. Upon the addition of a diethylchlorophosphate,
the singlet at 7.82 ppm (corresponding to the imidazolium CÀ
H proton) shifted to 9.74 ppm (Figure S10 in the Supporting In-
formation). The binding constant was calculated to be 4.36”
104 mÀ1 (Figure S11 in the Supporting Information).[69]
Figure 5. Percent degradation of different OPs in the presence of the nano-
reactor. OPs (50 mm) were used with 0.1 equivalent (5 mm) of the nanoreac-
tor. Reaction time: 25 min.
Quantitative investigation of AChE activity
The Ellman’s assay was performed to investigate the percent-
age of inhibition of AChE in the presence of the fabricated cat-
alyst (Scheme 5). Instead of paraoxon, diethylchlorophosphate
and diethylcyanophosphate (both devoid of nitro functionali-
ties), that exhibit similar reactivities, were used for the studies.
The Ellman’s reagent, DTNB (5,5’-dithiobis-(2-nitrobenzoic acid),
a chromogen), contains an SÀS bond and an aromatic nitro
group. The reagent reacts with a free thiol group to cleave the
SÀS bond, releasing 5-thio-2-nitrobenzoic acid (TNB), which ex-
hibits a strong absorbance band at 412 nm. The concentration
of the free thiol group was monitored by the ultraviolet-visible
(UV/Vis) absorption spectroscopy technique. A solution of the
OPs (50 mm), such as diethylchlorophophate and diethylcyano-
phophate, in the presence of the nanoreactor (0.1 equivalent)
and the self-assembled aggregates, was stirred for 30 min. At
every 2 min, an aliquot of the solution was collected and incu-
Hypothesized mechanism of paraoxon degradation
A hypothesized mechanism for the degradation of paraoxon is
presented in Scheme 4. The single-crystal structure of the calix
À
R1·2ClO4 reveals that the cavity formed by the calix can en-
capsulate the paraoxon molecule through hydrogen bonding,
leading to the enhanced electrophilic character of the phos-
phates. l-His acts as a base, which increases the nucleophilicity
of water, thus facilitating the reaction. The conversion of para-
oxon to diethyl phosphate increases the hydrophilicity of the
cavity, resulting in the removal of diethyl phosphate from the
benzimidazolium cavity. This creates a vacant site for another
catalytic cycle. The 31P NMR spectrum of paraoxon was record-
ed in the presence of the catalytic system to elucidate the
nature of the interaction present between the calix unit and
paraoxon (Figure S9 in the Supporting Information). The
31P NMR signal corresponding to the phosphorous unit in para-
Scheme 4. Hypothesized mechanism of the degradation of paraoxon by the
nanoreactor, the self-assembled aggregates of the ionic conjugate of the
calix R1·2OHÀ and l-His.
Scheme 5. AChE inhibition resistance owing to the hydrolysis of OPs and
the principal reactions of Ellman’s assay for the colorimetric determination
of AChE activity.
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bated with a known concentration of AChE. Following this, the
solution was incubated with acetylthiocholine. Although some
amounts of AChE were inhibited by the OPs, the activated
AChE catalyzed the hydrolysis of acetylthiocholine. The AChE
activity was evaluated by monitoring the concentration of thio-
choline released during the hydrolysis reaction (using Ellman’s
reagent). The concentration of the released thiocholine should
potentially be directly proportional to the efficiency of the cat-
alyst. AChE inhibition was plotted with respect to time. The
nanoreactor detoxicated the OPs within 30 min (Figure 6). It
has been reported that b-cyclodextrin-oxime derivatives de-
toxicated OPs in the presence of 500 equivalents of the cata-
lyst. A comparable activity was achieved with 0.1 equivalent of
the fabricated catalyst reported herein.[70] It is worth mention-
ing that the calix R1·2OHÀ or l-His moieties did not individual-
ly exhibit significant AChE inhibition abilities (Figures S12 and
S13 in the Supporting Information).
ed catalytic system is metal-free and less expensive compared
with metal-based catalysts.
Experimental Section
General
All chemicals were purchased from Sigma–Aldrich Chemical Co.
and used without further purification. The AChE enzyme, from
Streptomyces diastatochromogenes, was purchased from Sigma–Al-
drich. The ultraviolet-visible (UV/Vis) absorption spectra were re-
corded by using the Shimadzu UV-2600 spectrophotometer. For re-
1
cording the H NMR and 13C NMR spectra, a JEOL spectrometer op-
erating at 400 and 100 MHz, respectively, was used. The chemical
shifts are expressed in ppm. Elemental analyses were conducted
by using Fisons CHNS analyzers. The single-crystal X-ray diffraction
(XRD) data were recorded at room temperature by using a Bruker
X8 APEXIII KAPPA charge-coupled device (CCD) diffractometer. The
intensities were measured by using graphite-monochromatized
MoKa radiation (l=0.71073 Š). Scanning electron microscopy
images were recorded with a JEOL JSM-6610LV instrument (volt-
age: 15 kV). AFM experiments were performed by using a Bruker
AXS instrument (Model: Multimode 8). The particle size of the
nanoparticles was determined by DLS experiments, employing the
external probe feature of the Metrohm Microtrac Ultra Nanotrac
particle size analyzer.
Synthesis of compound 1[71]
A
solution of mesitylene (12 g, 0.10 mol), paraformaldehyde
(6.15 g, 0.20 mol), and HBr (40 mL, 31 wt% HBr/acetic acid solution)
in glacial acetic acid (50 mL) was stirred at 508C. After 3 h, distilled
water (100 mL) was added to the reaction mixture, following which
a white solid precipitated out. The reaction mixture was filtered,
and the solid was collected. Subsequently, it was washed with
water and air-dried to obtain compound 1 in 95% yield (29 g).
1H NMR (400 MHz, CDCl3): d=2.37 (s, 6H, CH3), 2.43 (s, 3H, CH3),
4.55 (s, 4H, CH2), 6.89 ppm (s, 1H, ArH); 13C NMR (100 MHz, CDCl3):
d=14.8, 19.6, 31.1, 130.8, 132.7, 137.3, 138.2 ppm; elemental analy-
sis calcd (%) for C11H14Br2: C 43.17, H 4.61, Br 52.22; found: C 43.06,
H 4.53.
Figure 6. AChE inhibitory activity of diethylchlorophosphate and diethylcya-
nophosphate in the presence of the nanoreactor: OPs (50 mm) were used
with 0.1 equivalent (5 mm) of the nanoreactor in a phosphate buffer (0.1m,
pH 7.8) at 378C.
Synthesis of compound 2
A solution of anhydrous K2CO3 (414 mg, 3 mmol) and benzimid-
azole (236 mg, 2 mmol) was stirred in dry acetonitrile (100 mL) at
808C for 15 min. Subsequently, compound 1 (3.6 mg, 1 mmol) was
added to the reaction mixture and the mixture was stirred for 10 h
at 808C. The progress of the reaction was monitored by using the
thin-layer chromatography technique. The solvent was evaporated
and the obtained residue was purified by column chromatography
on silica gel (eluent: n-hexane/EtOAc, 9:1) to obtain compound 2
in 88% yield (334 mg). 1H NMR (400 MHz, [D6]DMSO): d=2.13 (s,
3H, CH3), 2.30 (s, 6H, CH3), 5.46 (s, 4H, CH2), 7.15 (s, 1H, ArH), 7.20–
7.23 (q, J=8 Hz, 4H, bimiH), 7.45–7.47 (t, J=8 Hz, 2H, bimiH),
7.65–7.67 (q, J=8 Hz, 2H, bimiH), 7.78 ppm (s, 2H, 2-bimiH); ele-
mental analysis calcd (%) for C25H24N4: C 78.92, H 6.36, N 14.73;
found C 78.84, H 6.30, N 14.66.
Conclusion
A nanoreactor, formed of aggregates of an anionic conjugate
of benzimidazolium calix and l-His residues, was developed.
Because of the rigid cyclic structure (ionic in nature), the fabri-
cated catalyst was highly stable. Furthermore, it was highly
soluble in aqueous media. The catalyst could reversibly hydro-
gen bond to OPs and activate the electrophilic site of the
phosphates, allowing a nucleophilic attack. The nanoreactor
could degrade paraoxon and reactive CWAs with high efficien-
cy. The developed nanoreactor could efficiently detoxify OPs
and resist the inhibition of AChE. The catalytic activities of
metal-based catalysts, toward the hydrolysis of numerous OPs,
decrease in aqueous media. However, the new catalyst report-
ed herein could be efficiently used for the degradation of OPs
in aqueous media. Therefore, it can be used to detoxify orga-
nophosphates present in agricultural wastewater. The fabricat-
Synthesis of calix R1·2ClÀ
A solution of compound 1 (303 mg, 1 mmol), 2 (380 mg, 1 mmol),
and tetrabutylammonium chloride (TBACl, 1.4 g, 5 mmol) in dry
acetonitrile (50 mL) was heated at reflux for 5 h under an argon at-
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mosphere. Evaporation of the solvent afforded R1·2ClÀ in 90%
yield as a solid compound (615 mg). H NMR (400 MHz, [D6]DMSO):
were recorded after a short interval. The rate of hydrolysis was de-
termined from the plot of ln(c) vs. time.
1
d=1.60 (s, 6H), 2.39 (s, 12H), 5.54 (d, J=12 Hz, 4H), 5.73 (d, J=
12 Hz, 4H), 7.20 (s, 2H), 7.64 (s, 2H), 7.80 (q, J=4 Hz, 4H),
8.27 ppm (q, J=4 Hz, 4H); 13C NMR (100 MHz, [D6]DMSO): d=15.5,
20.5, 46.1, 114.6, 127.7, 127.9, 132.3, 132.9, 138.3, 138.7, 140.8 ppm;
HRMS (ESI): m/z calcd for (C36H38N4)2+: 263.1543 [M]2+; found:
263.1502.
Analysis of AChE activity
The Ellman’s solution was prepared by dissolving acetylthiocholine
iodide (200 mg, 0.7 mmol), 5,5’-dithiobis-(2-nitrobenzoic acid)
(DTNB; 100 mg, 0.175 mmol), and NaHCO3 (50 mg) in a phosphate
buffer (25 mL, 0.1m, pH 7.8). The solution was diluted 10-fold. The
activity of the residual AChE was determined by using a microplate
reader (infinite M200PRO, TECAN).
À
Synthesis of calix R1·2ClO4
The calix R1·2ClÀ (606 mg, 1 mmol) was dissolved in 30 mL of a
mixed solvent system (H2O/DMF, 9:1, v/v) and tetrabutylammoni-
um perchlorate (TBAClO4, 1.7 g, 5 mmol) was added tÀo the solu-
tion. The mixture was heated at reflux for 4 h. R1·2ClO4 separated
out as crystals when the reaction mixture was cooled down to
room temperature. Elemental analysis calcd (%) for C36H38N4Cl2O8:
C 59.59, H 5.28, N 7.72; found: C 59.43, H 5.20, N 7.61.
Acknowledgments
This work was supported by a research grant from the SERB
Project (Project No. EMR/2017/003438). A.S. is thankful to IIT
Ropar for the institute’s postdoctoral fellowship.
À
XRD analysis of the calix R1·2ClO4
Conflict of interest
À
The XRD data of the calix R1·2ClO4 were recorded with a Bruker
D8 Venture PHOTON 100 CMOS diffractometer at 208C using
mirror monochromatized MoKa radiation (l=0.71073 Š). Diffraction
patterns were collected at a detector distance of 50 mm from
where the crystal was positioned (counting time: 10 s). Data reduc-
tion and multi-scan absorption were conducted by using the APEX
The authors declare no conflict of interest.
Keywords: acetylcholine esterase · calix · nanoreactors
organophosphates · paraoxonase · pesticides
·
À
II program suite (Bruker, 2007). The structure of the calix R1·2ClO4
compound was solved by using the SIR-92 program and full-matrix
least-square refinement was performed by using the SHELXL-97
program.[72] All non-hydrogen atoms were refined anisotropically.
The hydrogen atoms of the CÀH groups were assigned isotropic
parameters the values of which were 1.2 times greater than those
of the corresponding non-hydrogen atoms to which they were at-
tached. All other calculations were performed by using the WinGX
and PARST programs. Molecular diagrams were drawn by using DI-
AMOND. The final R-values, together with the selected refinement
details, are given in Table 1. Deposition number 1555450 contains
the supplementary crystallographic data for this paper. These data
are provided free of charge by the joint Cambridge Crystallograph-
ic Data Centre and Fachinformationszentrum Karlsruhe Access
Structures service.
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Fabrication of the nanoreactor
À
A solution of the calix R1·2ClO4 (724 mg, 1 mol) and NaOH
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Chem. Eur. J. 2021, 27, 5737 –5744
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Manuscript received: November 13, 2020
Accepted manuscript online: December 22, 2020
Version of record online: March 3, 2021
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