Metal-Organocatalyst for Detoxification of Phosphorothioate Pesticides: Demonstration of Acetylcholine Esterase Activity

Article abstract of DOI:10.1021/acs.inorgchem.9b00770

In recent years, transition metal complexes have been developed for catalytical degradation of a phosphate ester bond, particularly in RNA and DNA; however, less consideration has been given for development of complexes for the degradation of a phosphorot

Full text of DOI:10.1021/acs.inorgchem.9b00770

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Metal−Organocatalyst for Detoxification of Phosphorothioate
Pesticides: Demonstration of Acetylcholine Esterase Activity
Amanpreet Singh,† Pushap Raj,† Ajnesh Singh,‡ Jan J. Dubowski,§ Navneet Kaur,*,∥
and Narinder Singh*,†
†Department of Chemistry, Indian Institute of Technology Ropar, Punjab 140001, India
‡Department of Applied Sciences and Humanities, Jawaharlal Nehru Govt. Engineering College, Sundernagar, Mandi (H.P.),
175018, India
§Laboratory for Quantum Semiconductors and Photo-based Biotechnology, Interdisciplinary Institute for Technological Innovation
(3IT), CNRS UMI-3463, Department of Electrical and Computer Engineering, Universite de Sherbrooke, 3000 Boulevard de
́
l’Universite, Sherbrooke, QC J1K 0A5, Canada
∥Department of Chemistry, Panjab University, Chandigarh, 160014, India
S
* Supporting Information
ABSTRACT: In recent years, transition metal complexes have been
developed for catalytical degradation of a phosphate ester bond, particularly
in RNA and DNA; however, less consideration has been given for
development of complexes for the degradation of a phosphorothioate bond,
as they are the foremost used pesticides in the environment and are toxic to
human beings. In this context, we have developed copper complexes of
benzimidazolium based ligands for catalytical degradation of a series of
organophosphates (parathion, paraoxon, methyl-parathion) at ambient
conditions. The copper complexes (assigned as N1−N3) were characterized
using single X-ray crystallography which revealed that all three complexes
are mononuclear and distorted square planner in geometry. Further, the
solution state studies of the prepared complexes were carried out using UV−
visible absorption, fluorescence spectroscopy, and cyclic voltametry. The
complexes N1 and N2 have benzimidazolium ionic liquid as base attached
with two 2-mercapto-benzimidazole pods, whereas complex N3 contains a nonionic ligand. The synthesized copper complexes
were evaluated for their catalytic activity for degradation of organophosphates. It is interesting that the complex containing the
ionic ligand efficiently degrades phosphorothioate pesticides, whereas complex N3 was not found to be appropriate for
degradation due to a weaker conversion rate. The organophosphate degradation studies were monitored by recording
absorbance spectra of parathion in the presence of catalyst, i.e., copper complexes with respect to time. The parathion was
hydrolyzed into para-nitrophenol and diethyl thiophosphate. Moreover, to analyze the inhibition activity of the pesticides
toward acetylcholine esterase enzyme in the presence of prepared metal complexes, Ellman’s assay was performed and revealed
that, within 20 min, the inhibition of acetylcholine esterase enzyme decreases by up to 13%.
not react, whereas the serine residue of acetylcholinesterase
protein efficiently binds with phosphorothioate. It means that
supramolecular interaction30−32 of organophosphates with
acetylcholinesterase also plays an important role in the
hydrolysis process. Such kind of interaction increases the
electrophilicity of the phosphate center which results in the
increased rate of reaction between the serine residue and OPs.
Various enzymes such as organophosphorus hydrolase (OPH),
organophosphate-degrading enzyme from agrobacterium ra-
diobacter (OpdA), methyl parathion hydrolase (MPH), and
glycerophosphodiesterase (GpdQ) are known for phosphate
ester hydrolysis that are having high catalytic efficacy due to
INTRODUCTION
■
Phosphorothioates are used as pesticides to increase the
cultivation in the agriculture sector; their major disrepute is
found as chemical warfare agents.1−8 The recent use of
organophosphate (OP) compounds as chemical warfare agents
in the Syria war makes constraint to the world. Therefore, the
catalyst for degradation of these super toxic chemicals at
environment conditions is gaining remarkable interest.9−18
Phosphorothioates are neurotoxins that bind irreversibly with
acetylcholinesterase enzyme and inhibit an important enzy-
metic conversion from acetylcholine to choline which is a
necessary neurotransmitter.19−23 It is well documented that
phosphorothioates bind with acetylcholinesterase enzyme
through the hydroxyl group of serine amino acid present in
the enzyme.22−29 It is interesting that serine in free state does
Received: March 18, 2019
© XXXX American Chemical Society
A
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their tendency to interact with a substrate through supra-
molecular interactions.33−38 Therefore, to achieve a high rate
of hydrolysis using an artificial or synthetic catalyst, the strong
interaction between substrate and catalyst is required.39,40 For
this purpose, ionic liquids are promising candidates, as these
compounds are well-known for catalysis through activating the
carbonyl carbon.41−46 To develop the catalyst for degradation
of pesticides, ionic and other noncovalent interactions should
be strong enough to increase the electrophilicity of the
phosphorus center of OPs.47 This enhancement of electro-
philic character will favor the addition of water and removal of
the withdrawing group. Moreover, the interaction of organo-
phosphates with metal ions favors the hydrolysis process. For
this purpose, various metal oxides were developed with their
own limitations such as a process being stoichiometric, not
catalytic. Also, the metal oxides have less possibility of
tunability to increase the reaction rate. Recently, Farha et al.
have developed a porous zirconium organic framework for
encapsulation and degradation of organophosphates.10,48−51
These porous materials efficiently degrade the phosphorothi-
onate into less toxic byproducts. Our group has also developed
some metal complexes and metal organic polymers for sensing
and degradation of pesticides.52−55 These metal complexes
were capable of detecting pesticides by fluorescence spectros-
copy; however, they could not proceed for a degradation
process. Previously, we developed a copper-organic two-
dimensional polymer of benzimidazolium zwitterion that
catalyzes the degradation process of OPs. The self-degradation
of such a framework by reacting with free phosphate ion was its
major disadvantage. Dissociation of the complex causes
removal of the zwitterion ligand by the cation displacement
assay which results in heightening of the fluorescence intensity.
The process of degradation was stoichiometric, not catalytic.
From this work, we have taken a lead that the combination of
benzimidazolium zwitterion and the copper ion is proficient to
detoxify OPs. In another work, ionic conjugates of
benzimidazolium dipodal and ionic surfactants were developed
for the same purpose.2 All of these work only to decontaminate
the OPs in an aqueous medium. However, an antidote for its
detoxification by reactivation of acetylcholine esterase is still
our goal. As acetylcholine esterase binds strongly with OPs; it
is not easy to extract OPs from the active site of the enzyme.
This could be achieved with some organocatalysts, as well as
metal complexes. Here, we took both catalytic centers into
consideration by making a complex of benzimidazolium with
metal. By putting one step forward toward this direction, we
have developed a copper(II) ion complex of benzimidazolium
based on a dipodal receptor that was further explored for
catalytic degradation reactions. The advantage of this nitrogen
donor ligand involved stronger binding affinity toward
copper(II) ion which restricts extraction of copper by
phosphate derivatives.56 Moreover, a benzimidazolium unit
attached with a dipodal receptor provides another catalytic site
which enhances the hydrolysis process. To confirm the
involvement of the benzimidazolium unit, another complex
(N3) was synthesized, having a similar geometry and the same
metal ion, except for the benzimidazolium unit, which was
replaced by a nonionic molecule. It is interesting that complex
(N3) showed a negligible catalytic activity as compared to N1
and N2. The kinetics of the degradation process was analyzed
using UV−visible absorption spectroscopy.
EXPERIMENTAL SECTION
■
Material. Copper perchlorate, copper chloride, potassium
carbonate, 2,2′-bis(bromomethyl)-1,1′-biphenyl, 2-mercaptobenzimi-
dazole, 2-mercaptobenzothiazole, benzimidazole, and dibromoethane
were purchased from Sigma-Aldrich and used without further
purification. Due to their explosive nature, the perchlorate salts may
be harmful upon ingestion or absorption through the skin. These
compounds may cause irritation to the skin, respiratory tract, and
eyes. Hence, proper protection and care should be taken while using
them. All the organophosphate pesticides were bought from Sigma-
Aldrich and used as received. Caution! Due to the high toxicity of
pesticides, precautions were taken as suggested in the Sigma-Aldrich safety
data sheet. Milli-Q water was used to prepare all the aqueous
solutions. Acetonitrile and deuterated solvents were purchased from
Merck and Sigma-Aldrich, respectively.
1
Methods. The H NMR, 13C NMR, and 31P NMR spectra of
prepared compounds were noted on a Jeol Instrument operating at
1
400 MHz for H NMR, at 100 MHz for 13C NMR, and at 160 MHz
for 31P NMR. The chemical shifts were recorded in ppm relative to
tetramethylsilane as an internal reference. A PerkinElmer L55
Spectrophotometer was used to conduct the fluorescence measure-
ments at room temperature with a fixed scanning speed and emission
slit width (10 nm). UV−visible absorption titrations were performed
using a Shimadzu spectrophotometer UV-2600. The concentration of
the degraded product was calculated using gas chromatography from
Shimadzu (GC-2010). The pH measurement was carried out on the
ME/962P instrument. A Fisons CHN analyzer was used for elemental
analyses. The cyclic voltammetry studies were carried out on a
Metrohm instrument.
Evaluation of Photophysical Properties. All spectroscopic
experiments were performed at 25 °C in the pure aqueous medium.
The stock solutions of ligand L1 (10 μM) and copper salts (1 mM)
were prepared in distilled water, whereas the solution of the L2 ligand
(10 μM) was prepared in DMSO:water (20:80) which was further
diluted as per the need of experiments. The change in absorbance and
fluorescence intensity was observed with stepwise addition of the
copper salt solution. Binding studies were carried out in an aqueous
solution buffered at pH 8.0 using Tris buffer. The sample was
prepared in a volumetric flask and sonicated for 5 min to attain an
equilibrium before recording the spectra. All the experiments were
repeated for three times, and errors in experiments were calculated
using a standard deviation method.
Cyclic Voltammetry. The aqueous stock solutions of complexes
N1 and N2 at a concentration of 10 μM were prepared in Tris buffer
at pH 8, which was further diluted as per the requirement of
experiments. Due to the insolubility of complex N3 in water, its
solution was prepared in 70:30 water/DMSO. Tetrabutylammonium
iodide was used as electrolyte. The current was measured by varying
the potential in the range from 0.55 to −1.1 V. The cyclic volumetric
graph was recorded by varying the scan rate from 50 to 250 mV s−1.
Method for Calculation of the Rate of Hydrolysis. The
solutions of phosphorothioates were prepared in water up to 1 mM
concentrations which were further diluted as per the need of the
experiment. To the aqueous solution of complex N1 and N2 (both
are 10 μM) buffered at pH 8.0 using Tris buffer was added parathion
(100 μM). The solutions were shaken vigorously before recording the
absorbance spectra that was recorded after a short interval of time.
The concentration of the degraded product was determined from the
absorbance intensity that was further used to analyze the binding
constant. The effect of substrate concentration and pH on the rate of
hydrolysis was evaluated. Another catalytic parameter such as kcat and
catalytic efficiency was determined using a Lineweaver−Burk plot.
Acetylcholine Esterase Activity Analysis. The residual AChE
activity was determined on a microplate reader infinite M200PRO
(TECAN). AChE activities were monitored to control activity,
whereas the data are represented as % of control. We have used
diethylchlorophosphate and diethylcyanophosphate as enzyme
inhibitor rather than parathion or paraoxon, because of their
interference in absorbance intensity at 412 nm in the Ellman assay.
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Scheme 1. Synthesis Scheme of Compound 1, Ligands L1 and L2
Scheme 2. Chemical Structure of Complexes N1, N2, and N3
Ellman solution for the detoxification assays of phosphorothioate by
metal complexes was prepared by dissolving acetylthiocholine iodide
(200 mg, 0.7 mmol), 5,5′-dithiobis(2-nitrobenzoic acid) (100 mg,
0.25 mmol), and sodium bicarbonate (50 mg) in 25 mL of Tris buffer
(pH 8). A 10-fold dilution of this solution was then carried out.
X-ray Structure Determination. The diffraction data for all the
copper complexes were collected on a Bruker D8 Venture Photon 100
CMOS diffractometer at 293 K using mirror-monochromatized Mo−
Kα radiation. The diffraction spots were measured using a counting
time of 10 s, and the crystals were positioned at 50 mm from the
CCD. The APEX II program was used for data reduction and
multiscan absorption. The structures were solved by the direct
method with the SIR97 program and refined using SHELXL-97.
Anisotropic thermal parameters were used for all non-H atoms. All
other calculations were performed using WinGX and PARST. The
ORTEP diagrams were drawn using ORTEP3 and OLEX2. The
related crystallographic information can be obtained from the
Cambridge Crystallographic Data center (CCDC) (CCDC Nos.
1818792−1818794).
CH3OH which gave colorless crystals (3.58 g). Yield = 86%. 1H NMR
(400 MHz, DMSO-d6) δ (ppm) = 4.04 (t, J = 8.0 Hz, 4H, CH2), 5.01
(t, J = 8.0 Hz, 4H, CH2), 7.69 (d, 2H, ArH), 8.17 (d, 2H, ArH), 10.07
(s, 1H, CH); 13C NMR (100 MHz, DMSO-d6) δ (ppm) = 31.6, 48.7,
114.4, 127.5, 131.3, 143.9. Anal. Calcd for C11H13Br3N2: C, 31.99; H,
3.17; N, 6.78. Found: C, 31.95; H, 3.18; N, 6.81.
Synthesis of Ligand L1. Compound 1 (410 mg, 1 mmol) was
dissolved in dry CH3CN and heated at 80 °C for 30 min. 2-
Mercaptobenzimidazole (300 mg, 2 mmol) was added to the
saturated solution of compound 1, and the resulting solution was
refluxed for 8 h. The reaction was monitored using TLC; after 8 h, the
product was separated out as a white powder which was washed with
hot chloroform. The resulting white powder constitutes clean ligand
L1. Yield = 85%. 1H NMR (400 MHz, DMSO-d6 δ (ppm) = 3.99 (t, J
= 8.0 Hz, 4H, CH2), 4.96 (t, J = 8.0 Hz, 4H, CH2), 7.03 (dd, J = 8.0
Hz, 2H, ArH), 7.30 (d, J = 8.0 Hz, 4H, ArH), 7.54−7.51 (m, 6H,
ArH, NH), 8.04 (d, J = 8.0 Hz, 2H, ArH), 10.09 (s, 1H, CH). 13C
NMR (100 MHz, DMSO-d6) δ (ppm) = 149.1, 143.6, 136.2, 131.3,
127.1, 126.6, 123.9, 122.7, 114.9, 114.1, 113.9, 109.9, 46.9, 31.7. Anal.
Calcd for C25H23BrN6S2: C, 54.44; H, 4.20; N, 15.24, Found: C,
54.48; H, 4.18; N, 15.22.
Synthesis of Compound 1. Compound 1 was synthesized using
our previously developed procedure (Scheme 1).57 In brief, the
benzimidazole (2.36 g, 20 mmol) and dibromoethane (17.12 mL, 200
mmol) were first dissolved in acetonitrile (100 mL), and the resulting
solution was refluxed for 24 h. On completion of the reaction, the
volatiles were evaporated using a rotary evaporator. The resulting
crude mixture was washed with CHCl3 and finally recrystallized in
Synthesis of Ligand L2. Ligand L2 was synthesized using a
similar method as that adopted for ligand L1. In a round-bottom flask,
2,2′-bis(bromomethyl)-1,1′-biphenyl (340 mg, 1 mmol) was dis-
solved in 50 mL of acetonitrile. To the saturated solution was added
300 mg of 2-mercaptobenzothiazole (2 mmol), and resulting mixture
C
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was refluxed for 8 h. The product was separated out as a light-yellow
colored compound which filtered out and washed with diethyl ether.
The resulting yellow compound constitutes pure ligand L2. Yield =
was refluxed for 8 h. After that, the product was separated out
as a light-yellow powder and characterized using 1H NMR and
13C NMR (Figures S1−S6). The proton signals due to the
methylene group split into two doublets which confirms the
formation of the product. Complex N3 was synthesized by
reacting L2 with copper chloride in methanol and charac-
terized using single crystal X-ray diffraction.
Crystal Structure. Complexes N1 and N2 crystallized in a
triclinic crystal system with space group P21/n, whereas N3
̅
crystallized in a monoclinic crystal system with space group P1.
1
89%. H NMR (400 MHz, DMSO-d6) δ (ppm) = 4.25 (d, J = 12.0
Hz, 2H, CH2), 4.45 (d, J = 12.0 Hz, 2H, CH2), 7.21 (t, 2H, ArH),
7.29−7.33 (m, 6H, ArH), 7.38 (t, J = 8.0 Hz, 2H, ArH), 7.59 (t, J =
8.0 Hz, 2H, ArH), 7.75 (d, J = 8.0 Hz, 2H, ArH), 7.89 (d, J = 8.0 Hz,
2H, ArH), 13C NMR (100 MHz, DMSO-d6) δ (ppm) = 166.2, 152.9,
140.2, 135.1, 134.3, 130.6, 130.5, 128.9, 128.3, 126.9, 125.1, 122.3,
121.7, 35.7. Anal. Calcd for C25H23BrN6S2: C, 65.59; H, 3.93; N, 5.46.
Found: C, 65.61; H, 3.89; N, 5.49.
Synthesis of Copper Complex N1. In a round-bottom flask,
copper perchlorate (89 mg, 0.5 mmol) was added to the saturated
solution of ligand L1 (275 mg, 0.5 mmol) in distilled water, and the
mixture was heated at 60 °C for 20 min. The blue crystals were
obtained after 10 h, which were found to be suitable for single crystal
data collection. Yield = 55%. Anal. Calcd for C25H25Br2ClCuN6O5S2:
C, 36.96; H, 3.10; N, 10.34 Found: C, 37.04; H, 2.95; N, 10.35.
Synthesis of Copper Complex N2. Complex N2 was
synthesized using a similar method as that for N1; copper(II)
chloride was used instead of copper perchlorate. Blue colored crystals
were grown after 12 h which were found to be suitable for single
crystal data collection. Yield = 64%. Anal. Calcd for C25H25Cl3Cu-
N6O1S2: C, 45.53; H, 3.82; N, 12.74 Found: C, 45.45; H 3.50; N,
12.66.
The complexes have a copper center with distorted square
planar geometry, where halide ions are at trans positions
compensating the dipositive charges on the metal ion (Figure
1). In complex N1, copper(II) ion is coordinated to dipodal
Synthesis of Copper Complex N3. Complex N3 was
synthesized by reacting copper chloride (50 mg, 0.5 mmol) and
ligand (100 mg) L2 in methanol. The resulting mixture was refluxed
for 1 h and cooled down to room temperature. Blue crystals grew after
36 h in 60% yield which were suitable for single crystal data collection.
Yield = 58%. Anal. Calcd for C28H20Cl2CuN2S4: C, 51.97; H, 3.12; N,
4.33. Found: C, 51.56; H, 2.97; N, 4.28.
Figure 1. Single crystal structure of copper complexes N1, N2, and
N3, showing intramolecular hydrogen bonding.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Metal Complexes.
Ligand L1 was synthesized using our previously reported
method. Earlier, we constructed fluorescence aggregates of L1
by interacting it with anionic surfactants that were further
explored as a fluorescence sensor mimicing chemical warfare
agents (diethyl chlorophosphate). It was observed that ligand
L1 interacts with diethyl chlorophosphate (DCP) through
hydrogen bonding and efficiently degrades it in 1 h. Here, we
have designed complexes N1 and N2 in such a way that the
benzimidazolium unit is still free to interact with organo-
phosphate derivatives through hydrogen bonding (Scheme 2).
Such kinds of pincer ligands based complexes are well-known
for catalytic applications.58−60 Along with the insertion of
copper ion in the pocket of the dipodal receptor, this will
provide ionic interaction to organophosphate which enhances
the electrophilicity of the phosphorus center and increases the
rate of the hydrolysis process. Keeping this in mind, complexes
N1 and N2 were synthesized by reacting ligand L1 in distilled
water with copper perchlorate and copper chloride, respec-
tively. The mixture was heated for 20 min and cool down to
room temperature. It was interesting that, in the case of N1,
the counterion (bromide) present in L1 coordinated with
copper metal ion, whereas perchlorate ion interacted with the
benzimidazole ring through hydrogen bonding.
ligand L1 through nitrogen atoms of the benzimidazole
moiety, whereas the other two sites are occupied by halide
ions. It is interesting to note that, in complex N1, copper
perchlorate was used; however, the bromide ions (counterion
of L1) got coordinated to the copper(II) center. This may be
due to the stronger binding affinity of copper ion toward
bromide than perchlorate in the particular complex (N1) or
the conducive geometrical constrains that favors the
coordination of bromide ion to the metal center. Also, one
of the coordinated bromide ions is interacting with the
benzimidazolium moiety through hydrogen bonding, which
slightly weakens its coordination with copper ion comparative
to another bromide which is evident from the Cu−Br bond
distances (Table S2). In complex N2, two chloride ions and
one ligand L1 are completing the coordination sphere of
Cu(II), whereas one chloride ion is present as a counterion.
Similar to complexes N1 and N2, complex N3 also has a
distorted square planar geometry. The structure analyses have
revealed that, in complexes N1 and N2, crystal lattices are
stabilized by the interplay of hydrogen bonding interaction of
the type N−H···O/Cl, C−H···O, and O−H···Cl (hydrogen
bonding parameters are given in Tables S5 and S6). No
significant hydrogen bonding is observed in complex N3, and
this may be one of the reasons for poor solubility of N3 in
water. The final R values along with selected refinement details
and bond parameters are given in Tables S1−S6. The ORTEP
diagrams together with the atom numbering scheme of
compounds N1−N3 with 40% probability thermal ellipsoids
are shown in Figures S24−S26.
First, we checked the catalytic activity of complexes N1 and
N2. After that, to confirm the role of the benzimidazolium
moiety in the catalytic process, another complex (N3) was
synthesized with a similar geometry; the only difference was a
nonionic bridge instead of the benzimidazolium moiety.
Ligand L2 was synthesized by reacting 2 equiv of 2-
mercaptobenzothiazole with 1 equiv of 2,2′-bis-
(bromomethyl)-1,1′-biphenyl in dry acetonitrile. The mixture
Photophysical Properties of Copper Complexes. The
UV−visible absorbance spectra of ligand L1, recorded in an
aqueous medium (pH = 8), show two absorbance bands at 248
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Figure 2. (A) Change in the UV−visible absorption spectra of ligand L1 (pH = 8.0) upon successive addition of copper chloride (0−8 μM). (B)
The nonlinear relationship between the copper ion concentration and absorbance ratio (A326/A227). (C) Cyclic voltammetry profile of complex N2
(10 μM) at different scan rates.
and 325 nm, respectively, for π−π* and n−π* transitions
(Figure S7). The successive addition of copper perchlorate to
the solution of L1 leads to a significant change in the
absorbance spectra (Figure S8). The absorbance intensity at
248 nm (π−π* transition) increases, whereas the absorbance
at 325 nm (n−π* transition) decreases. Moreover, a new band
at 380 nm appears due to charge transfer between the ligand to
metal ion. Similarly, absorbance spectra of L1 were logged by
sequential addition of copper chloride. The absorbance
intensity due to the n−π* transition at 326 nm decreases,
whereas absorbance maxima corresponding to the π−π*
transition showed enhancement in intensity. It is worth to
remark here that the charge transfer band at 380 nm causes a
small enhancement in absorbance intensity, which revealed the
poor charge transfer ability of complex N2 in comparison to
that of complex N1 (Figure 2). The fluorescence spectra of
ligands L1 and L2 have been recorded in the same solvent
system as used for UV−visible absorption spectroscopy.
Ligand L1 was showing strong emission at 405 nm (λex =
325 nm), which was quenched by addition of copper
perchlorate and copper bromide. However, both complexes
N1 and N2 were showing a similar behavior in the absorbance
profile. Emission intensity of both complexes is almost similar.
At the same concentration of copper salt, complex N1 showed
a similar behavior toward fluorescence quenching as the N2
complex (Figure S11). Complex N2 has shown weaker charge
transfer transition as compared to N1 that was attributed to a
comparatively stronger LMCT (ligand to metal charge
transfer) in the N1 complex.
Due to insolubility of ligand L2 in water, photophysical
studies were carried out in DMSO:water (30:70). Like ligand
L1, it also shows two absorbance bands at 240 and 315 nm,
respectively, due to π−π* and n−π* transitions (Table 1).
Titration was performed by consecutive addition of copper
chloride to L2 solution (Figures S12 and S13). It was found
that copper ions significantly change the absorbance profile of
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efficiency of the benzimidazolium based catalyst, we
synthesized their copper complexes. Particularly, copper ion
was chosen due to its biocompatibility and affinity to interact
with phosphates. To probe the efficiency of our catalyst for the
catalytic destruction of the phosphate ester bond, we
investigated the ability of the catalyst to degrade the OPs.
Ten equivalents of tested organophosphates was added to the
10 μM solution of complex N1. The mixture was stirred for 20
min, and after every 2 min, samples were collected for
recording the UV−visible absorption spectra. The increase in
absorbance intensity at 402 nm can be used to determine the
rate of hydrolysis. This enhancement in absorbance intensity is
due to release of para-nitrophenol. In the case of parathion and
paraoxon, the lone pair of electrons on phenolic oxygen shifted
toward phosphorus, whereas, upon hydrolysis, the para-
nitrophenol will release, which results in delocalization of the
electron pair inside the aromatic ring; consequently, the
absorbance at 402 nm increases.63 Also, background reactions
were carried out without using a catalyst under identical
conditions.
Table 1. Photophysical Properties of Metal Complexes and
Ligands
λabs (nm)
λem (nm)
λex (nm)
L1
L2
N1
N2
N3
248, 325
240, 315
248, 325, 380
248, 325, 380
240, 315, 380
405
390
405
405
390
248
240
248
248
240
ligand L2. The absorbance intensity at 240 nm increases,
whereas, at 315 nm, it decreases, and a new weak band appears
at 380 nm due to the weak charge transfer transition. The
emission profile of ligand L2 was recorded at an excitation
wavelength of 315 nm which exhibited emission maxima at 390
nm (Figure S14). Fluorescence intensity got quenched as the
concentration of copper ions increased. The nonlinear
regression between copper ion concentration and emission
intensity at 390 nm is showing a linear decrease in fluorescence
intensity (Figure S15). This decrease in fluorescence can be
explained based on the open shell effect of copper ion.
As indicated in Figure 3, the plot of time dependent
absorbance at 402 nm shows enhancement. Absorbance
increased up to 20 min; after that, it becomes constant,
which suggests that hydrolysis occurred within 20 min.
Furthermore, the effect of substrate concentration on the
rate of hydrolysis using the copper complexes as catalyst was
investigated by the Michaelis−Menten method (Figure S20).
The graph of concentration of substrate vs rate of hydrolysis
showed the linear increase up to the concentration of 400 μM,
whereas, at higher concentrations, the curve attained saturation
that indicated the formation of complex−phosphate inter-
mediates. To calculate the efficiency of the catalysts, a
Lineweaver−Burk plot has been constructed (Figure S21).
The plot between the inverse of pesticide concentration and
inverse of rate shows a linear increase. The Michaelis constants
KM and Vmax have been calculated for intercept of the
Lineweaver−Burk plot and are listed in Table 3. Among all the
metal complexes, complex N2 shows a better catalytic
efficiency, whereas complex N3 has poor catalytic efficiency,
suggesting involvement of an ionic benzimidazolium unit in
the catalytic process. Therefore, it is a kind of dual catalytic
system, made up from organocatalyst and metal ion. Similarly,
we have calculated the rate parameters for hydrolysis of
different pesticides using complex N2, which showed a similar
trend for paraoxon, parathion, and methyl-parathion. The
effect of pH on the hydrolysis process was also monitored,
which indicated that maximum catalytic efficiency was
achieved at pH 8, while, in acidic condition, catalytic efficiency
decreased (Figures S22 and S23). Moreover, the catalytic
efficiency of N2 is slightly better than that of N1, which
indicated that complex N2 may have a greater affinity to
interact with organophosphate species in the reversible
pathway.
Electrochemical Studies. The electrochemical studies of
prepared complexes were carried out using cyclic voltammetry.
The current was measured by varying the potential in the range
from 0.55 to −1.1 V. The electrochemical measurements for
complexes N1 and N2 were recorded at a constant pH of 8.0
in water, whereas, due to the insolubility of complex N3 in
water, studies were carried out in DMSO:water (30:70).
Complex N1 was showing a cathodic peak at 0.43 V, whereas
the anodic peak was observed at −0.60 V (Figure S16). To
further investigate the electrochemical process, the cyclic
voltammetry graph was recorded by varying the scan rate from
50 to 250 mV s−1. The ratio of cathodic and anodic current
(Ipc/Ipa) was greater than 1, which revealed that the process
was quasi-reversible. Moreover, that ΔEp increases with the
increase in scan rate also validates the observation.
Coulometric experiment by potentiometeric exhaustive elec-
trolysis revealed the consumption of one electron in the redox
couple of all the copper complexes. Similarly, electrochemical
measurement of complex N2 discovered the redox process as
quasi-reversible. However, complex N2 showed a compara-
tively lower value of current for the same concentration
(Figure 2), which might be due to more ionic strength of
perchlorate ion as compared to bromide ion. The one electron
quasi-reversible reduction of the complexes has attributed to
the Cu+2/Cu+ redox couple. Similarly, redox parameters for
complex N3 were determined as listed in Table 2 (Figure S17).
Table 2. Result of Electrochemical Measurements of Copper
Complexes
S. no.
metal complex
Epc (V)
Epa (V)
E1/2 (V)
ΔE (mV)
1
2
3
N1
N2
N3
0.32
0.43
0.47
−0.60
−0.60
−0.84
−0.14
−0.08
−0.18
920
1030
1310
The kinetics parameters for catalyst N2, for hydrolysis of
different pesticides such as parathion, paraoxon, and methyl-
parathion as shown in Table 3, reveal that phosphorothioate
pesticides are hydrolyzed with a higher rate than paraoxon.
This can be explained on the basis of the higher affinity of
sulfur to coordinate with copper ion, which enhances the
efficiency of a catalyst. Moreover, the catalytic activity of
previously reported catalysts has been compared with the
current work which shows an advantage over previous work in
terms of high kcat and aqueous solubility.55,62,65−74
Degradation of Phosphorothioate Using N1, N2, and
N3 Copper Complexes. The transition metal complexes are
known for their phosphatase activity.61−64 However, few pincer
ligand based complexes are reported for detoxification of
pesticides. In our previous studies, it is clear that the
benzimidazolium ring plays a significant role in detoxification
of organophoshates. Therefore, to increase the catalytic
F
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Figure 3. (A) Change in absorbance spectra on conversion of parathion to para-nitrophenol using catalyst N2. (B) Change in absorbance intensity
at 402 nm with the progress of the degradation process. (C) Enzymatic kinetic plot. (D) Lineweaver−Burk plot.
Table 3. First-Order Rate Parameters for Hydrolysis of Different Pesticides As Obtained by the Michaelis−Menten Treatment
a
of Complexes
pesticides
parathion
complex
Vmax (M s−1
)
Km (M)
kcat (s−1
)
k
cat/Km (M−1 s−1
)
N1
N2
N3
N1
N2
N3
N1
N2
N3
5.4 × 10−5
6.6 × 10−5
9.3 × 10−6
5.3 × 10−5
6.2 × 10−5
9.4 × 10−6
5.3 × 10−5
6.4 × 10−5
9.2 × 10−6
9.99 × 10−2
9.43 × 10−2
1.01 × 10−1
1.07 × 10−1
9.32 × 10−2
9.92 × 10−2
1.02 × 10−1
9.30 × 10−2
1.05 × 10−1
5.45 0.02
6.67 0.04
0.93 0.08
5.33 0.03
6.20 0.05
0.94 0.07
5.38 0.03
6.49 0.06
0.92 0.09
54.6 0.18
70.7 0.21
9.2 0.10
49.6 0.17
66.5 0.20
9.4 0.10
52.7 0.17
70.1 0.19
8.7 0.09
paraoxon
methyl-parathion
a
Note: All the experiments were repeated three times, and the average value of data has been taken to calculate the catalytic parameter.
31P NMR spectra were used to investigate the mechanism of
solution of methyl-parathion was changed to yellow after 25
min, which is attributed to the formation of para-nitrophenol.
Therefore, it can be predicted that methyl-parathion is
hydrolyzed into dimethyl thiophosphate and para-nitrophenol.
Plausible Mechanism of Degradation. As shown in the
crystal structure of complex N1, copper is in a distorted square
planar geometry, with two bromide ions covalently attached to
a copper ion. It is worth to mention that both bromide ions are
not in the same coordination with the copper ion. One of the
bromide ions is interacting with benzimidazolium hydrogen
through hydrogen bonding. The organophosphate can occupy
the fifth coordination site of copper ion through the reversible
pathway, in such a way that acidic hydrogen of the
hydrolysis reaction of methyl-parathion in the presence of
catalyst N2 (Figure S27A). 31P NMR spectra obtained in the
successive time periods of reaction exhibit two species: the
signal at δ = 65.46 ppm corresponds to methyl-parathion
which is slightly upfield shifted (δ = 63.58 ppm) in the
presence of N2 catalyst. The signal at δ = 63.58 ppm
disappears during the reaction, and a new signal at δ = 61.80
ppm appears which can be assigned to the hydrolyzed product,
dimethyl thiohydrogen phosphate [(CH3O)2(OH)PS].75,76
Furthermore, the photographic images of the hydrolysis
reaction were recorded with regular intervals of time as in
Figure S27B. The investigation reveals that the colorless
G
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Scheme 3. Plausible Mechanism of Degradation of Organophosphate Based Pesticides
benzimidazolium cation can interact with organophosphate
through hydrogen bonding (Scheme 3). Both of these
interactions enhance the electrophilicity of the phosphorus
center and therefore will enhance the rate of hydrolysis.66
Here, two catalytic centers (benzimidazolium as organocatalyst
and copper ion as metal based catalyst) work synergistically to
enhance the catalytic activity for hydrolysis of pesticides. To
investigate further the role of the benzimidazolium moiety in
the catalytic process, an additional metal complex (N3) was
prepared with a similar binding environment for copper ion;
however, the nonionic biphenyl ring replaced the benzimida-
zolium ring. The prepared copper complex of the nonionic
ligand was also explored for degradation of pesticides;
however, it showed a negligible activity in comparison to
that of complex N1. Moreover, the ability of ligand L1 for the
hydrolysis of pesticide was examined, which revealed that it did
not catalyze the hydrolysis of the pesticide (Table S9).
Although, we have carried out all catalytic studies in the
solution phase, but these noncovalent interactions play an
important role in the catalytic activity of complexes.66 It was
revealed from the solid state structure analyses that, in complex
N2, a water molecule interacts with the benzimidazole
hydrogen (N-H) and chloride ion through hydrogen bonding,
which enhances the nucleophilicity of water toward the
nucleophilic substitution reaction at the phosphorus center.
Histidine plays a catalytic role in various enzymatic reactions
by interacting with nucleophilic species through hydrogen
bonding.39 Therefore, it is believed that noncovalent
interactions are responsible for the catalytic activity of
complexes in the present study. In complex N1, a similar
type of hydrogen bonding interaction is observed with water
molecules and perchlorate ions. Due to the absence of any
counterion in complex N3, hydrogen bonding with a solvent
molecule and anion is absent. Although, other interactions,
such as C−H···Cl and C−H···π, are involved in supramolecular
packing of investigated complexes. These interactions do not
affect the catalytic activity of complexes; thus the absence of
such a kind of interaction in complex N3 could be another
reason for the lack of a catalytic affinity toward the hydrolysis
of organophosphate species.
Supramolecular interactions involved in the solid state
structure were investigated further to determine the mecha-
nism of degradation of organophosphates (Figure 4).
Quantitative Investigation of Acetylcholine Esterase
Activity. Quantitative investigation of acetylcholine esterase
activity has been demonstrated using Ellman’s assay which
makes use of Ellman’s reagent (dithiobisnitro-benzoate,
DTNB), a highly reactive reagent having a S−S bond that
breaks quickly by reacting with a free thiol group and gives the
intense yellow color of thiobisnitro-benzoate ion (Scheme 4).
In this way, hydrolysis of acetylthiocholine into thiocholine
could be monitored using Ellman’s assay by measuring the
characteristic absorption band at λmax = 412 nm due to
generation of a thiobisnitro-benzoate ion.29,77 Because para-
thion will cause interference due to its byproduct (para-
nitrophenol), we used diethyl chlorophosphate as a pesticide
that has a similar reactivity as parathion and releases byproduct
Figure 4. Noncovalent interactions involved in supramolecular
packing of complexes N1, N2, and N3.
H
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Scheme 4. Complete Scheme for Colorimetric Determination of Cholinesterase Activities: Ellman’s Assay
that did not cause any interference in region 412 nm. Diethyl
chlorophosphate (100 μM) reacted with complex N1 (10 μM)
for 20 min; after every 2 min, the sample was collected from
the reaction mixture. To the collected sample (80 μL) was
added a solution of acetylcholine esterase enzyme, followed by
addition of Ellman solution (10 μL). All the collected samples
were put in a 96-well plate, and the absorbance intensity at 412
nm was recorded using a TECAN plate reader.
The increase in absorbance at 412 nm is attributed to the
free thiol group in the solution and was used to monitor the
hydrolysis of acetylthiocholine. It was observed that, in the
presence of a catalyst, the concentration of free thiol decreases
up to 13% within 20 min (Figure 5). Therefore, our catalyst is
efficient toward detoxification of organophosphates in a very
short span of time. It is worth to be mentioned here that N2
complex also effectively decreases the inhibition of acetylcho-
line esterase enzyme in the presence of organophosphate,
whereas complex N3 was not found to be effective to reduce
the inhibition of enzyme (Figures S28 and S29).
CONCLUSION
■
Figure 5. Detoxification of diethyl chlorophosphate and diethyl
cyanophosphate by complex N2. Data are given as the mean of two
assays.
We have synthesized novel copper(II) complexes having a
benzimidazolium unit as a secondary catalytic site along with
the primary catalytic site of copper ion. Complexes N1 and N2
coordinate with organophosphate species in a reversible
pathway, which enhances the electrophilicity of phosphorus
and favors the hydrolysis process. The rate of hydrolysis of
first-order kinetics was measured using UV−visible absorption
spectroscopy by monitoring the absorption at 405 nm. The
prepared catalyst has various advantages over existing methods,
such as a high rate of hydrolysis, working in the pure aqueous
medium, and a better catalytic efficiency. To demonstrate
further the acetylcholine esterase activity, Ellman’s reagent was
used to monitor the hydrolysis of acetylthiocholine. The
inhibition of acetylcholine esterase enzyme due to pesticide
decreased up to 13% within 20 min.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b00770.
■
S
UV−visible absorption, emission, H NMR, 13C NMR,
bond angles, and bond length of complexes (PDF)
1
Accession Codes
CCDC 1818792−1818794 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
I
DOI: 10.1021/acs.inorgchem.9b00770
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Article
(11) Lum, K. T.; Huebner, H. J.; Li, Y.; Phillips, T. D.; Raushel, F.
M. Organophosphate Nerve Agent Toxicity in Hydra Attenuata.
Chem. Res. Toxicol. 2003, 16, 953−957.
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(12) Liu, Y.; Bonizzoni, M. A Supramolecular Sensing Array for
Qualitative and Quantitative Analysis of Organophosphates in Water.
J. Am. Chem. Soc. 2014, 136, 14223−14229.
AUTHOR INFORMATION
Corresponding Authors
■
(13) Totten, R. K.; Kim, Y. S.; Weston, M. H.; Farha, O. K.; Hupp, J.
T.; Nguyen, S. T. Enhanced Catalytic Activity through the Tuning of
Micropore Environment and Supercritical CO2 Processing: Al-
(Porphyrin)-Based Porous Organic Polymers for the Degradation of
a Nerve Agent Simulant. J. Am. Chem. Soc. 2013, 135, 11720−11723.
(14) Pinto, G.; Mazzone, G.; Russo, N.; Toscano, M. Trimethyl-
phosphate and Dimethylphosphate Hydrolysis by Binuclear CdII,
MnII, and ZnII − FeII Promiscuous Organophosphate- Degrading
Enzyme : Reaction Mechanisms. Chem. - Eur. J. 2017, 23, 13742−
13753.
(15) Moon, S.; Proussaloglou, E.; Peterson, G. W.; Decoste, J. B.;
Hall, M. G.; Howarth, A. J.; Hupp, J. T.; Farha, O. K. Detoxification of
Chemical Warfare Agents Using a Zr 6 -Based Metal − Organic
Framework/Polymer Mixture. Chem. - Eur. J. 2016, 22, 14864−
14868.
*E-mail: nsingh@iitrpr.ac.in. Tel: +91-1881242176 (N.S.).
*E-mail: navneetkaur@pu.ac.in. Phone: +91 9815245098
(N.K.).
ORCID
Amanpreet Singh: 0000-0002-1461-4607
Jan J. Dubowski: 0000-0003-0022-527X
Narinder Singh: 0000-0002-8794-8157
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a research grant from the joint
Indo-Canada project sponsored by DBT-New Delhi and IC-
IMPACTS Canada. A.S. and P.R. are thankful to the Indian
Institute of Technology, Ropar, for the Director-postdoc
fellowship.
(16) Woo, D. J.; Obendorf, S. K. MgO-Embedded Fibre-Based
Substrate as an Effective Sorbent for Toxic Organophosphates. RSC
Adv. 2014, 4, 15727.
(17) Braatz, A. D.; Antonio, M. R.; Nilsson, M. Structural Study of
Complexes Formed by Acidic and Neutral Organophosphorus
Reagents. Dalt. Trans. 2017, 46, 1194−1206.
REFERENCES
■
(18) Gupta, S. K.; Dar, A. A.; Rajeshkumar, T.; Kuppuswamy, S.;
Langley, S. K.; Murray, K. S.; Rajaraman, G.; Murugavel, R. Discrete
(1) Kim, K.; Tsay, O. G.; Atwood, D. A.; Churchill, D. G.
Destruction and Detection of Chemical Warfare Agents. Chem. Rev.
2011, 111, 5345−5403.
{GdIII M} (M = GdIII or CoII) Pentanuclear Complexes: A New
4
Class of Metal-Organophosphate Molecular Coolers. Dalt. Trans.
2015, 44, 5961−5965.
(2) Singh, A.; Raj, P.; Singh, N. Benzimidazolium-Based Self-
Assembled Fluorescent Aggregates for Sensing and Catalytic
Degradation of Diethylchlorophosphate. ACS Appl. Mater. Interfaces
2016, 8, 28641−28651.
(3) Sambrook, M. R.; Hiscock, J. R.; Cook, A.; Green, a C.; Holden,
I.; Vincent, J. C.; Gale, P. A. Hydrogen Bond-Mediated Recognition
of the Chemical Warfare Agent Soman (GD). Chem. Commun. 2012,
48, 5605−5607.
(4) Yao, J.; Fu, Y.; Xu, W.; Fan, T.; Gao, Y.; He, Q.; Zhu, D.; Cao,
H.; Cheng, J. Concise and Efficient Fluorescent Probe via an
Intromolecular Charge Transfer for the Chemical Warfare Agent
Mimic Diethylchlorophosphate Vapor Detection. Anal. Chem. 2016,
88, 2497−2501.
(5) Klet, R. C.; Liu, Y.; Wang, T. C.; Hupp, T.; Farha, O. K.
Evaluation of Brønsted Acidity and Proton Topology in Zr- and Hf-
Based Metal−organic Frameworks Using Potentiometric Acid−base
Titration. J. Mater. Chem. A 2016, 4, 1479−1485.
(6) Le, Q. A. T.; Chang, R.; Kim, Y. H. Rational Design of
Paraoxonase 1 (PON1) for the Efficient Hydrolysis of Organo-
phosphates. Chem. Commun. 2015, 51, 14536−14539.
(7) Diaz de Grenu, B.; Moreno, D.; Torroba, T.; Berg, A.; Gunnars,
J.; Nilsson, T.; Nyman, R.; Persson, M.; Pettersson, J.; Eklind, I.;
Wasterby, P. Fluorescent Discrimination between Traces of Chemical
Warfare Agents and Their Mimics. J. Am. Chem. Soc. 2014, 136,
4125−4128.
(8) Bhat, G. A.; Maqbool, R.; Dar, A. A.; Ul Hussain, M.; Murugavel,
R. Selective Formation of Discrete versus Polymeric Copper
Organophosphates: DNA Cleavage and Cytotoxic Activity. Dalt.
Trans. 2017, 46, 13409−13420.
(9) Viveros, L.; Paliwal, S.; McCrae, D.; Wild, J.; Simonian, A. A
Fluorescence-Based Biosensor for the Detection of Organophosphate
Pesticides and Chemical Warfare Agents. Sens. Actuators, B 2006, 115,
150−157.
(10) Mondloch, J. E.; Katz, M. J.; Isley, W. C., III; Ghosh, P.; Liao,
P.; Bury, W.; Wagner, G. W.; Hall, M. G.; Decoste, J. B.; Peterson, G.
W.; Snurr, R. Q.; Cramer, C. J.; Hupp, J. T.; Farha, O. K. Destruction
of Chemical Warfare Agents Using Metal−organic Frameworks. Nat.
Mater. 2015, 14, 512−516.
(19) Singh, A. K.; Flounders, A. W.; Volponi, J. V.; Ashley, C. S.;
Wally, K.; Schoeniger, J. S. Development of Sensors for Direct
Detection of Organophosphates. Part I: Immobilization, Character-
ization and Stabilization of Acetylcholinesterase and Organo-
phosphate Hydrolase on Silica Supports. Biosens. Bioelectron. 1999,
14, 703−713.
(20) Liu, G.; Lin, Y. Biosensor Based on Self-Assembling
Acetylcholinesterase on Carbon Nanotubes for Flow Injection/
amperometric Detection of Organophosphate Pesticides and Nerve
Agents. Anal. Chem. 2006, 78, 835−843.
(21) Tormos, J. R.; Wiley, K. L.; Wang, Y.; Fournier, D.; Masson, P.;
Nachon, F.; Quinn, D. M. Accumulation of Tetrahedral Intermediates
in Cholinesterase Catalysis: A Secondary Isotope Effect Study. J. Am.
Chem. Soc. 2010, 132, 17751−17759.
(22) Liu, D.; Chen, W.; Wei, J.; Li, X.; Wang, Z.; Jiang, X. A Highly
Sensitive, Dual-Readout Assay Based on Gold Nanoparticles for
Organophosphorus and Carbamate Pesticides. Anal. Chem. 2012, 84,
4185−4191.
̈
(23) Fenzl, C.; Genslein, C.; Zopfl, A.; Baeumner, A. J.; Hirsch, T. A
Photonic Crystal Based Sensing Scheme for Acetylcholine and
Acetylcholinesterase Inhibitors. J. Mater. Chem. B 2015, 3, 2089−
2095.
(24) Qian, S.; Lin, H. Colorimetric Sensor Array for Detection and
Identification of Organophosphorus and Carbamate Pesticides. Anal.
Chem. 2015, 87, 5395−5400.
(25) Zhu, X.; Li, B.; Yang, J.; Li, Y.; Zhao, W.; Shi, J.; Gu, J. Effective
Adsorption and Enhanced Removal of Organophosphorus Pesticides
from Aqueous Solution by Zr-Based MOFs of UiO-67. ACS Appl.
Mater. Interfaces 2015, 7, 223−231.
(26) Li, H.; Li, J.; Xu, Q.; Hu, X. Poly(3-hexylthiophene)/TiO2
Nanoparticle-Functionalized Electrodes for Visible Light and Low
Potential Photoelectrochemical Sensing of Organophosphorus
Pesticide Chlopyrifos. Anal. Chem. 2011, 83, 9681−9686.
(27) de Koning, M. C.; Joosen, M. J. A.; Worek, F.; Nachon, F.; van
Grol, M.; Klaassen, S. D.; Alkema, D. P. W.; Wille, T.; de Bruijn, H.
M. Application of the Ugi Multicomponent Reaction in the Synthesis
J
DOI: 10.1021/acs.inorgchem.9b00770
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
of Reactivators of Nerve Agent Inhibited Acetylcholinesterase. J. Med.
Chem. 2017, 60, 9376−9392.
(28) Liao, S.; Han, W.; Ding, H.; Xie, D.; Tan, H.; Yang, S.; Wu, Z.;
Shen, G.; Yu, R. Modulated Dye Retention for the Signal-on
Fluorometric Determination of Acetylcholinesterase Inhibitor. Anal.
Chem. 2013, 85, 4968−4973.
(44) Ghosh, A. K.; Brindisi, M. Organic Carbamates in Drug Design
and Medicinal Chemistry. J. Med. Chem. 2015, 58, 2895−2940.
(45) Sit, R. K.; Fokin, V. V.; Amitai, G.; Sharpless, K. B.; Taylor, P.;
Radic, Z. Imidazole Aldoximes Effective in Assisting Butyrylcholines-
terase Catalysis of Organophosphate Detoxification. J. Med. Chem.
2014, 57, 1378−1389.
(29) Lei, C.; Wang, Z.; Nie, Z.; Deng, H.; Hu, H.; Huang, Y.; Yao, S.
Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free
Detection of copper(II) Ion and Acetylcholinesterase Activity. Anal.
Chem. 2015, 87, 1974−1980.
(46) Rajendiran, S.; Park, K.; Lee, K.; Yoon, S. Ionic-Liquid-Based
Heterogeneous Covalent Triazine Framework Cobalt Catalyst for the
Direct Synthesis of Methyl 3-Hydroxybutyrate from Propylene Oxide.
Inorg. Chem. 2017, 56, 7270−7277.
(30) Purohit, C. S.; Mishra, A. K.; Verma, S. Four-Stranded
Coordination Helices Containing Silver−Adenine (Purine) Metal-
laquartets. Inorg. Chem. 2007, 46, 8493−8495.
(47) Pavez, P.; Millan, D.; Morales, J. I.; Castro, E. A.; Lopez A, C.;
Santos, J. G. Mechanisms of Degradation of Paraoxon in Different
Ionic Liquids. J. Org. Chem. 2013, 78, 9670−9676.
(31) Mishra, A. K.; Verma, S. Interconnected Trimeric, Pentameric,
and Hexameric Metallacycles in a Singular Silver - Adenine
Framework. Inorg. Chem. 2010, 49, 8012−8016.
(32) Pandey, M. D.; Mishra, A. K.; Chandrasekhar, V.; Verma, S.
Silver-Guided Excimer Emission in an Adenine - Pyrene Conjugate :
Fluorescence Lifetime and Crystal Studies. Inorg. Chem. 2010, 49,
2020−2022.
(33) Hadler, K. S.; Mitic, N.; Yip, S. H.-C.; Gahan, L. R; Ollis, D. L.;
Schenk, G.; Larrabee, J. A. Electronic Structure Analysis of the
Dinuclear Metal Center in the Bioremediator Glycerophosphodiester-
ase (GpdQ) from Enterobacter Aerogenes. Inorg. Chem. 2010, 48,
2727−2734.
(34) Hadler, K. S.; Tanifum, E. A.; Yip, S. H.-C.; Mitic, N.; Guddat,
L. W.; Jackson, C. J.; Gahan, L. R.; Nguyen, K.; Carr, P. D.; Ollis, D.
L.; Hengge, A. C.; Larrabee, J. A.; Schenk, G. Substrate-Promoted
Formation of a Catalytically Competent Binuclear Center and
Regulation of Reactivity in a Glycerophosphodiesterase from
Enterobacter Aerogenes. J. Am. Chem. Soc. 2008, 130, 14129−14138.
(35) Pedroso, M. M.; Ely, F.; Mitic, N.; Carpenter, M. C.; Gahan, L.
R.; Wilcox, D. E.; Larrabee, J. L.; Ollis, D. L.; Schenk, G. Comparative
Investigation of the Reaction Mechanisms of the Organophosphate-
Degrading Phosphotriesterases from Agrobacterium Radiobacter
(OpdA) and Pseudomonas Diminuta (OPH). J. Biol. Inorg. Chem.
2014, 19, 1263−1275.
(36) Ng, T.; Gahan, L. R.; Schenk, G.; Ollis, D. L. Altering the
Substrate Specificity of Methyl Parathion Hydrolase with Directed
Evolution. Arch. Biochem. Biophys. 2015, 573, 59−68.
(37) Schenk, G.; Mateen, I.; Ng, T.-K.; Pedroso, M. M.; Mitic, N.;
Jafelicci, M.; Marques, R. F.C.; Gahan, L. R.; Ollis, D. L.
Organophosphate-Degrading Metallohydrolases : Structure and Func-
tion of Potent Catalysts for Applications in Bioremediation. Coord.
Chem. Rev. 2016, 317, 122−131.
(48) Totten, R. K.; Weston, M. H.; Park, J. K.; Farha, O. K.; Hupp, J.
T.; Nguyen, S. T. Catalytic Solvolytic and Hydrolytic Degradation of
Toxic Methyl Paraoxon with La(catecholate)-Functionalized Porous
Organic Polymers. ACS Catal. 2013, 3, 1454−1459.
(49) Bromberg, L.; Creasy, W. R.; McGarvey, D. J.; Wilusz, E.;
Hatton, T. A. Nucleophilic Polymers and Gels in Hydrolytic
Degradation of Chemical Warfare Agents. ACS Appl. Mater. Interfaces
2015, 7, 22001−22011.
(50) Islamoglu, T.; Atilgan, A.; Moon, S.-Y.; Peterson, G. W.;
DeCoste, J. B.; Hall, M.; Hupp, J. T.; Farha, O. K. Cerium(IV) vs
Zirconium(IV) Based Metal−Organic Frameworks for Detoxification
of a Nerve Agent. Chem. Mater. 2017, 29, 2672−2676.
(51) Li, P.; Moon, S. Y.; Guelta, M. A.; Lin, L.; Gomez-Gualdron, D.
A.; Snurr, R. Q.; Harvey, S. P.; Hupp, J. T.; Farha, O. K. Nanosizing a
Metal-Organic Framework Enzyme Carrier for Accelerating Nerve
Agent Hydrolysis. ACS Nano 2016, 10, 9174−9182.
(52) Singh, A.; Singh, J.; Singh, N.; Jang, D. O. A Benzimidazolium-
Based Mixed Organic−inorganic Polymer of Cu(II) Ions for Highly
Selective Sensing of Phosphates in Water: Applications for Detection
of Harmful Organophosphates. Tetrahedron 2015, 71, 6143−6147.
(53) Raj, P.; Singh, A.; Kaur, K.; Aree, T.; Singh, A.; Singh, N.
Fluorescent Chemosensors for Selective and Sensitive Detection of
Phosmet/Chlorpyrifos with Octahedral Ni2+ Complexes. Inorg. Chem.
2016, 55, 4874−4883.
(54) Raj, P.; Singh, A.; Singh, A.; Singh, N. Syntheses, Crystal
Structures and Photophysical Properties of Cu(II) Complexes: Fine
Tuning of a Coordination Sphere for Selective Binding of
Azamethiphos. Dalt. Trans. 2017, 46, 985−994.
(55) Daumann, L. J.; Schenk, G.; Ollis, D. L.; Gahan, L. R.
Spectroscopic and Mechanistic Studies of Dinuclear Metallohydro-
lases and Their Biomimetic Complexes. Dalt. Trans. 2014, 43, 910−
928.
(38) Hadler, K. S.; Mitic, N.; Ely, F.; Hanson, G. R.; Gahan, L. R;
Larrabee, J. A.; Ollis, D. L.; Schenk, G. Structural Flexibility Enhances
the Reactivity of the Bioremediator Glycerophosphodiesterase by
Fine-Tuning Its Mechanism of Hydrolysis. J. Am. Chem. Soc. 2009,
131, 11900−11908.
(39) Sharma, U. K.; Sharma, N.; Kumar, R.; Kumar, R.; Sinha, A. K.
Biocatalytic Promiscuity of Lipase in Chemoselective Oxidation of
Aryl Alcohols/Acetates : A Unique Synergism of CAL-B and [hmim]
Br for the Metal-Free H2O2 Activation. Org. Lett. 2009, 11, 4846−
4848.
(56) Sendzik, M.; Pushie, M. J.; Stefaniak, E.; Haas, K. L. Structure
and Affinity of Cu(I) Bound to Human Serum Albumin. Inorg. Chem.
2017, 56, 15057−15065.
(57) Singh, A.; Singh, A.; Singh, N.; Jang, D. O. A Benzimidazolium-
Based Organic Trication: A Selective Fluorescent Sensor for
Detecting Cysteine in Water. RSC Adv. 2015, 5, 72084−72089.
(58) Chen, L.; Khadivi, A.; Singh, M.; Jurss, J. W. Synthesis of a
Pentadentate, Polypyrazine Ligand and Its Application in Cobalt-
Catalyzed Hydrogen Production. Inorg. Chem. Front. 2017, 4, 1649−
1653.
(40) Zhao, Y.; Li, Z.; Sharma, U. K.; Sharma, N.; Song, G.; Van der
Eycken, E. V. Copper-Catalyzed Alkylarylation of Activated Alkenes
Using Isocyanides as the Alkyl Source : an efficient radical access to
3,3-dialkylated oxindoles. Chemchomm 2016, 52, 6395−6398.
(41) Sharma, H.; Kaur, N.; Singh, N.; Jang, D. O. Synergetic
Catalytic Effect of Ionic Liquids and ZnO Nanoparticles on the
Selective Synthesis of 1,2-Disubstituted Benzimidazoles Using a Ball-
Milling Technique. Green Chem. 2015, 17, 4263−4270.
(42) Zhang, Q.; Zhang, S.; Deng, Y. Recent Advances in Ionic
Liquid Catalysis. Green Chem. 2011, 13, 2619−2637.
(59) Bauer, G.; Hu, X. Recent Developments of Iron Pincer
Complexes for Catalytic Applications. Inorg. Chem. Front. 2016, 3,
741−765.
(60) DeMott, J. C.; Dekarske, J. R.; McCulloch, B. J.; Ozerov, O. V.
Cyclometallation of the NNN Pincer Ligand in Complexes of
Platinum. Inorg. Chem. Front. 2015, 2, 912−916.
(61) Sanyal, R.; Guha, A.; Ghosh, T.; Mondal, T. K.; Zangrando, E.;
Das, D. Influence of the Coordination Environment of zinc(II)
Complexes of Designed Mannich Ligands on Phosphatase Activity: A
Combined Experimental and Theoretical Study. Inorg. Chem. 2014,
53, 85−96.
(43) Mehnert, C. P.; Mozeleski, E. J.; Cook, R. A. Supported Ionic
Liquid Catalysis Investigated for Hydrogenation Reactions. Chem.
Commun. 2002, 24, 3010−3011.
(62) Sanyal, R.; Zhang, X.; Kundu, P.; Chattopadhyay, T.; Zhao, C.;
Mautner, F. A.; Das, D. Mechanistic Implications in the Phosphatase
K
DOI: 10.1021/acs.inorgchem.9b00770
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Activity of Mannich-Based Dinuclear Zinc Complexes with
Theoretical Modeling. Inorg. Chem. 2015, 54, 2315−2324.
(63) Arora, H.; Barman, S. K.; Lloret, F.; Mukherjee, R. Isostructural
Dinuclear Phenoxo-/acetato-Bridged manganese(II), cobalt(II), and
zinc(II) Complexes with Labile Sites: Kinetics of Transesterification
of 2-Hydroxypropyl-P-Nitrophenylphosphate. Inorg. Chem. 2012, 51,
5539−5553.
(77) Le Provost, R.; Wille, T.; Louise, L.; Masurier, N.; Muller, S.;
Reiter, G.; Renard, P.-Y.; Lafont, O.; Worek, F.; Estour, F. Optimized
Strategies to Synthesize B -Cyclodextrin-Oxime Conjugates as a New
Generation of Organophosphate Scavengers. Org. Biomol. Chem.
2011, 9, 3026−3032.
(64) Esteves, L. F.; Rey, N. A.; Dos Santos, H. F.; Costa, L. A. S.
Theoretical Proposal for the Whole Phosphate Diester Hydrolysis
Mechanism Promoted by a Catalytic Promiscuous Dinuclear
Copper(II) Complex. Inorg. Chem. 2016, 55, 2806−2818.
(65) Osorio, R. E. H. M. B.; Peralta, R. A.; Bortoluzzi, A. J.; de
Almeida, V. R.; Szpoganicz, B.; Fischer, F. L.; Terenzi, H.; Mangrich,
A. S.; Mantovani, K. M.; Ferreira, D. E. C.; Rocha, W. R.; Haase, W.;
Tomkowicz, Z.; Anjos, A. d.; Neves, A. Synthesis, Magnetostructural
Correlation, and Catalytic Promiscuity of Unsymmetric Dinuclear
Copper(II) Complexes: Models for Catechol Oxidases and Hydro-
lases. Inorg. Chem. 2012, 51, 1569−1589.
(66) Camargo, T. P.; Neves, A.; Peralta, R. A.; Chaves, C.; Maia, E.
C. P.; Lizarazo-Jaimes, E. H.; Gomes, D. A.; Bortolotto, T.; Norberto,
D. R.; Terenzi, H.; Tierney, D. L.; Schenk, G. Second-Sphere Effects
in Dinuclear FeIIIZnII Hydrolase Biomimetics: Tuning Binding and
Reactivity Properties. Inorg. Chem. 2018, 57, 187−203.
(67) Osorio, R. E.H.M.B.; Neves, A.; Camargo, T. P.; Mireski, S. L.;
Bortoluzzi, A. J.; Castellano, E. E.; Haase, W.; Tomkowicz, Z.
Synthesis, Structure, Magnetism, and Hydrolase and Catecholase
Activity of a New Trinuclear Copper (II) Complex. Inorg. Chim. Acta
2015, 435, 153−158.
(68) Peralta, R. A.; Bortoluzzi, A. J.; de Souza, B.; Jovito, R.; Xavier,
F. R.; Couto, R. A. A.; Casellato, A.; Nome, F.; Dick, A.; Gahan, L..
R.; Schenk, G.; Hanson, G. R.; de Paula, F. C. S.; Pereira-Maia, E. C.;
de P. Machado, S.; Severino, P. C.; Pich, C.; Bortolotto, T.; Terenzi,
H.; Castellano, E. E.; Neves, A.; Riley, M. J. Electronic Structure and
Spectro-Structural Correlations of FeIIIZnII Biomimetics for Purple
Acid Phosphatases : Relevance to DNA Cleavage and Cytotoxic
Activity. Inorg. Chem. 2010, 49, 11421−11438.
(69) dos Santos Silva, G. A.; Amorim, A. L.; Souza, B. d.; Gabriel, P.;
Terenzi, H.; Nordlander, E.; Neves, A.; Peralta, R. A. Synthesis and
Characterization of FeIII(μ-OH)ZnII Complexes: Effects of a Second
Coordination Sphere and Increase in the Chelate Ring Size on the
Hydrolysis of a Phosphate Diester and DNA. Dalt. Trans. 2017, 46,
11380−11394.
(70) Jarenmark, M.; Csapo, E.; Singh, J.; Wockel, S.; Farkas, E.;
Meyer, F.; Haukka, M.; Nordlander, E. Unsymmetrical Dizinc
Complexes as Models for the Active Sites of Phosphohydrolases.
Dalton Trans. 2010, 39, 8183−8194.
(71) Jarenmark, M.; Haukka, M.; Demeshko, S.; Tuczek, F.;
Zuppiroli, L.; Meyer, F.; Nordlander, E. Synthesis, Characterization,
and Reactivity Studies of Heterodinuclear Complexes Modeling
Active Sites in Purple Acid Phospatases. Inorg. Chem. 2011, 50, 3866−
3887.
(72) Roberts, A. E.; Schenk, G.; Gahan, L. R. A Heterodinuclear Fe
III Zn II Complex as a Mimic for Purple Acid Phosphatase with Site-
Specific Zn II Binding. Eur. J. Inorg. Chem. 2015, 2015, 3076−3086.
(73) Daumann, L. J.; Gahan, L. R.; Comba, P.; Schenk, G.
Cadmium(II) Complexes: Mimics of Organophosphate Pesticide
Degrading Enzymes and Metallo- β -Lactamases. Inorg. Chem. 2012,
51, 7669−7681.
(74) Daumann, L. J.; Comba, P.; Larrabee, J. A.; Schenk, G.;
Stranger, R.; Cavigliasso, G.; Gahan, L. R. Synthesis, Magnetic
Properties, and Phosphoesterase Activity of Dinuclear Cobalt(II)
Complexes. Inorg. Chem. 2013, 52, 2029−2043.
(75) Seger, M. R.; Maciel, G. E. NMR Investigation of the Behavior
of an Organothiophosphate Pesticide, Methyl Parathion, Sorbed on
Clays. Environ. Sci. Technol. 2006, 40, 552−558.
(76) Silva, V. B.; Nascimento, L. L. Q.; Nunes, M. C.; Campos, R.
B.; Oliveira, A. R. M.; Orth, E. S. Puzzling Reaction of Imidazole with
Methyl Parathion: P=S versus P=O Mechanistic Shift Dilemma in
Organophosphates. Chem. - Eur. J. 2019, 25, 817−822.
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