Molecular recognition of organophosphorus compounds in water and inhibition of their toxicity to acetylcholinesterase

Article abstract of DOI:10.1039/c9cc04603h

Molecular tubes with hydrogen bonding donors in their deep hydrophobic cavities are able to selectively bind organophosphorus compounds in water through hydrogen bonding and the hydrophobic effect. They can also be used as a fluorescent sensor for nerve agent simulants and as an inhibitor to reduce the toxicity of paraoxon to acetylcholinesterase.

Full text of DOI:10.1039/c9cc04603h

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DOI: 10.1039/C9CC04603H
Journal Name
COMMUNICATION
Molecular Recognition of Organophosphorus Compounds in
Water and Inhibition of Their Toxicity to Acetylcholinesterase
Received 00th January 20xx,
Accepted 00th January 20xx
Wei-Er Liu,a,b Zhao Chen, Liu-Pan Yang, Ho Yu Au-Yeung,* and Wei Jiang *
b
b
,a
,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
The molecular tubes with hydrogen bonding donors in their deep
hydrophobic cavity are able to selectively bind organophosphorus
compounds in water through hydrogen bonding and the
hydrophobic effect. They can also be used as a fluorescent sensor
for nerve agent simulants and to reduce the toxicity of paraoxon
to acetylcholinesterase.
Organophosphorus compounds1 are commonly used as
pesticides and nerve agents. They have been listed by the
United States Environmental Protection Agency as acute,
2
highly toxic agents to bees, wildlife, and human beings. The
widely-accepted mechanism of organophosphates’ toxicity is
through irreversibly binding and blocking acetylcholinesterase
(
AChE).3 This leads to the accumulation of acetylcholine,
resulting in dangerous effects such as paralysis and
convulsions. There are expensive and nonportable analytical
4
methods for detecting toxic organophosphorus compounds.
However, a cheaper and portable method relying on
spectroscopy is still urgently needed.
Many reaction-based detection methods have been
explored.5 But
a
supramolecular method based on
noncovalent interactions would be more appealing even for
6
7
the catalytic destruction or the recognition-based removal of
toxic organophosphorus compounds. This has been explored
with many macrocyclic receptors. For example, deep
5
c
6a,8
6a,9
cavitands, cyclodextrins,
baskets7 have been reported to be able to encapsulate some
of the nerve agent simulants. This recognition ability was
further used for their catalytic destruction and removal from
calixarenes,
and molecular Fig. 1 (a) Cartoon representation of organophosphorus compound and the molecular
,10
tube; (b) Chemical structures of molecular tubes 1a and 1b and organophosphorus
compounds involved in this research.
water. Structurally, toxic organophosphorus compounds, and hydrophobic side chains (Fig. 1a, left). In general, the
including nerve agents, feature both polar phosphate groups aforementioned macrocyclic receptors are designed to
encapsulate these organophosphorus compounds mainly
through the hydrophobic effect. Thus, the recognition
selectivity relies only on the size and shape complementarity
between the guests and the cavities. A more hydrophobic
molecule with similar size and shape will surely compete for
the hydrophobic cavity, which may significantly interfere with
a. Department of Chemistry, The University of Hong Kong, Hong Kong, China.
E-mail: hoyuay@hku.hk
b.
Shenzhen Grubbs Institute and Department of Chemistry, Southern University of
Science and Technology, Shenzhen, 518055, China. E-mail:
jiangw@sustech.edu.cn
Electronic Supplementary Information (ESI) available: Experimental procedures
and all the NMR spectra. See DOI: 10.1039/x0xx00000x

ChemComm
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DOI: 10.1039/C9CC04603H
1
Fig. 3 Energy minimized structures of (a) 3@1a and (b) 3@1b computed at the
Semiempirical PM06 level of theory by Spartan’14 (Wavefunction, Inc.). The
peripheral feet were shortened to methyl groups for convenience.
Fig. 2 H NMR spectra (400 MHz, D
2
O, 0.5 mM, 298 K) of (a) 2, (c) 1b, and (b) their
equimolar mixture.
the sensing or inhibit the catalytic destruction or removal of
the organophosphates.
shielding effect of the four naphthalenes of 1b. Similar binding
behaviour was observed for other organophosphorus
With these aforementioned receptors containing only a
hydrophobic cavity, the head phosphate groups with hydrogen
bonding acceptors are not appropriately addressed in the
recognition. An ideal receptor would interact with the
phosphate group and the hydrophobic side chain through
hydrogen bonding and the hydrophobic effect, respectively. In
this way, a much higher selectivity and higher binding affinity
may be achieved. This would be good for high-selectivity
sensing and destruction/removal, and reduce the probability
of false response.
1
31
compounds by using H NMR (Figs. S1-S13) and P NMR (Fig.
S14) spectra. Job’s plot and molar ratio titration plot (Fig. S15)
support the binding to be in a 1:1 stoichiometry. The
association constants of all the organophosphorus compounds
2
with 1a and 1b have been determined in D O by NMR
titrations (Figs. S16-S41) and are listed in Table 1.
Generally speaking, the anti-configured molecular tube 1b
is a much better host than 1a for guests 2 – 5; while 1a binds
more strongly than 1b to 7 and 8. With increasing the
hydrophobicity of the phosphonates, the association constants
become higher. These molecular tubes can not only bind to
model organophosphorus compounds, such as 2, 3, 6, and 7,
but also show high binding affinities to real pesticides, such as
acephate (4), dimethoate (5), and paraoxon (8). The binding
Recently, we reported a pair of endo-functionalized
molecular tubes (1a and 1b, Fig. 1b) which contain hydrogen
1
1
bonding donors in their hydrophobic cavities.
These
molecular tubes are able to selectively recognize highly
hydrophilic molecules in water by using hydrogen bonding and
the hydrophobic effect. The cavity features of the molecular
tubes are rather complementary to organophosphorus
compounds (Fig. 1a, right). Therefore, we wondered whether
these molecular tubes can recognize organophosphorus
compounds in water. Herein, we report that the molecular
tubes not only can strongly bind organophosphorus
compounds (Fig. 1c) in water, but also work as a fluorescent
sensor and even inhibit the toxicity of paraoxon to AChE.
Organophosphate 2 is the simplest organophosphorus
compound and only contains the phosphate head group and
minimal hydrophobic side chains (methyl groups). We started
with 2 to test the ability of molecular tubes 1a and 1b in
binding organophosphorus compounds in water. As shown in
Fig. 2, the protons of 2 undergo obvious upfield shift in the 1:1
mixture with 1b when compared to free 2. This suggests that 2
should be bound inside the cavity and thus experiences the
4
-1
constants reach 10 M for 7 and 8.
The binding affinities of the molecular tubes are generally
higher than those of the aforementioned molecular receptors.
Cyclodextrins show the best binding to diphenyl
-
1 8
methylphosphonate with K
a
= 700 M ; Badjić’s molecular
-
1
7b
-1
baskets have a binding constant of 447 M to 2 , 154 M to
1
0a
-1
7b
3
,
and 8891 M to 7. The molecular baskets and 1b are
comparable in the binding to 2, but the association constant of
molecular tubes to guests 3 and 7 are significantly higher.
Both hydrogen bonding and the hydrophobic effect are
known to operate in the molecular recognition of these
1
1c
molecular tubes. But further experiments were performed
to confirm their roles in the recognition of these organo-
phosphorus compounds. Firstly, 1b was titrated with 3 in 9:1
2 2
H O/D O (Fig. S42). NH protons of the host are visible in this
solvent and were observed to shift upfield with adding more 3.
This suggests that hydrogen bonds between 1b and 3 are
slightly weaker than those between 1b and the encapsulated
-1
Table 1 Association constants (M ) of 1a and 1b with organophosphorus compounds
in water at 25 °C as determined by NMR titrations (400 MHz, D
2
O)a
1
1c
water. Thus, the hydrophobic effect through releasing the
1
2
2
31±7
340±70
6
3
4
-
5
“high-energy” cavity water is the major driving force; but
after the guest is encapsulated in the cavity, hydrogen bonding
should also operate and contribute to the binding. This is
supported by molecular computation. As shown in Fig. 3,
hydrogen bonds are indeed formed between the oxygen
atoms of 3 and the NH protons of the hosts. Only one
hydrogen bond is formed in the case of 1a; while two
hydrogen bonds are observed for 1b. This may be due to the
b
1
1
a
b
530±20
1500±60
7
45±6
280±30
130±20
8
1
1
a
b
1500±200
1400±200
15000±600
3800±300
28000±2000
850±10
a
b
The concentration of the hosts is fixed at 0.2 mM. The association constant is
too small (< 10 M ) to be determined.
-1

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DOI: 10.1039/C9CC04603H
-5
Fig. 5 Fluorescence spectra of 1a (1.0×10 M) when titrated with 7 (0-0.019 mM) in
o
deionized H
2
O at 25 C. Inset: the LOD and the detection range of 7.
fluorescence of the molecular tubes. Binding of 6 (Fig. S44) and
7
1
(Fig. 5) however significantly enhances the fluorescence of
a. The limit of detection (LOD, 3δ/slope) and the detection
range for 7 were determined to be 2.3 μmol/L and 2.3-5
μmol/L, respectively. Consequently, these molecular tubes
may be used as a turn-on fluorescent sensor for certain toxic
organophosphorus compounds such as nerve agents.
Nerve agents and some organophosphorus compounds are
toxic to human and animals because they inhibit the function
of AChE by covantly linking to the serine residue (Fig. 6). Thus,
AChE losts its function in cleaving acetylcholine into choline
and acetate. Accumulation of acetylcholine in the body causes
the symptons of paralysis and convulsions, and eventually
leads to death.
Fig. 4 ITC titration plots (heat rate versus time and heat versus guest/host ratio) of 1a
with 7 in H O at 298 K.
2
twisted geometry in 1a which is not favourable to form two
hydrogen bonds with the phosphonates. This may also explain
why 1b is a better receptor for small organophosphorus
compounds. But 1a has a more well-defined hydrophobic
cavity and thus binds better to the organophosphorus
compounds with a large hydrophobic group.
Isothermal titration calorimetry (ITC) experiments were
performed for the binding of 1a to 7 (Fig. 4). The binding is
largely driven by enthalpic contribution (ΔH = −29.6 kJ/mol)
with an unfavourable entropy (−TΔS = 8.1 kJ/mol). This is in
drastic contrast to other macrocyclic receptors. For example,
A known method to eliminate the toxicity of nerve agents is
to use butyrylcholinesterase or paraoxonase-1 enzymes as
bioscavengers to covalently trap the nerve agents, followed by
1
3
1
0
their removal from the bloodstream . However, this method
requires significant amount of the enzymes and is thus very
expensive. By using the efficient binding of 1a to paraoxon 8,
we wondered whether 1a can be used as an noncovalent
scavenger to prevent the toxicity of paraoxon 8 to AChE.
the binding of Badjić’s molecular baskets is dominated by
entropic contribution with minor or even unfavourable
enthalpic contribution (for example, for guest 7, ΔH = −4.4
1
0c
kJ/mol; −TΔS = −14.4 kJ/mol) . A classic hydrophobic effect
was invoked to explain this. In our case, the large enthalpic
contribution may be originated from the release of “high-
1
4
By following the literature procedure, in vitro experiments
o
1
2
at 37 C were performed to test the effect of 1a in preventing
energy” cavity water and hydrogen bonding between the
host and the guest shielded in the hydrophobic cavity.
Consequently, these results support that hydrogen bonding
and the hydrophobic effect are cooperatively the driving
forces for the selective binding of the molecular tubes to
organophosphorus compounds.
the toxicity of paraoxon (8) to AChE (Figs. 6, 7, and S45-S51).
The bioactivity of acetylcholinesterase (AChE) under several
The efficient binding of the molecular tubes to the toxic
pesticides (4 and 5) and paraoxon (8) encouraged us to test
their application as a fluorescent sensor and as an inhibitor to
protect the function of AChE.
The molecular tubes are fluorescent because they contain
four naphthalenes and often show fluorescent enhancements
upon guest binding.11 We have demonstrated that they can be
used as a fluorescent sensor for the detection of persistent
1
1a,11g
environmental contaminants in water
and for monitoring
1
1f
the hydrolysis kinetics of nonfluorescent esters. However,
addition of most of these organophosphorus compounds does Fig. 6 The toxicity mechanism of paraoxon 8 to AChE (top) and the inhibition
mechanism of 1a to the toxicity of paraoxon 8 (bottom).
not obviously change (1 - 5) or quenches (8, Fig. S43) the

ChemComm
Page 4 of 5
pesticides. Consequently, these molecular tubes should be
3
useful in combating chemical warfare agents.
View Article Online
This research was financially supported by the National
DOI: 10.1039/C9CC04603H
Natural Science Foundation of China (Nos. 21772083,
2
1822104), the Shenzhen Science and Technology Innovation
Committee (Nos. JCYJ20180504165810828 and KQJSCX
0170728162528382), and the Shenzhen Nobel Prize Scientists
2
Laboratory Project (C17783101). We thank SUSTech-MCPC for
instrumental assistance.
Conflicts of interest
There are no conflicts to declare.
o
Fig. 7 In vitro enzymatic experiments (37 C, UV-vis absorption monitored at 412nm)
to demonstrate the prevention of paraoxon’s toxicity to AChE by 1a. a) AChE +
substrate + indicator; b) 1 eq. 1a + AChE + substrate + indicator; c) 5 eq. 1a + AChE +
substrate + indicator + 1 eq. paraoxon; d) 1 eq. 1a + AChE + substrate + indicator + 1
eq. paraoxon; e) AChE + substrate + indicator + 1 eq. paraoxon; f) 1 eq. 1a + substrate
Notes and references
+
indicator. Substrate: AICI; indicator: DTNB.
1
2
3
R. C. Gupta, Toxicology of Organophosphate and Carbamate
Compounds, Elsevier Academic Press, New York, 2006.
https://www.epa.gov/sites/production/files/documents/rmpp_6thed_c
h5_organophosphates.pdf
conditions were detected with the help of the indicator 5,5'-
dithiobis(2-nitrobenzoic acid) (DTNB) and the substrate
acetylthiocholine iodide (AICI). Figs. 7a, 7b, and 7f show that
one equivalent of molecular tube 1a has little influence on the
hydrolysis of AICI by AChE. But the existence of one equivalent
of paraoxon 8 completely shut down the activity of AChE (Fig.
Y. J. Jang, K. Kim, O. G. Tsay, D. A. Atwood and D. G. Churchill, Chem. Rev.,
2015, 115, PR1.
4
5
S. S. Aleksenko, P. Gareil and A. R. Timerbaev, Analyst, 2011, 136, 4103.
(a) T. J. Dale and J. Rebek Jr, J. Am. Chem. Soc., 2006, 128, 4500; (b) T. J. Dale
and J. Rebek Jr., Angew. Chem., Int. Ed., 2009, 48, 7850; (c) D.
Ajami and J. Rebek Jr., Org. Biomol. Chem., 2013, 11, 3936; (d) X. Sun and E.
V. Anslyn, Angew. Chem. Int. Ed., 2017, 56, 9522; (e) X. Sun, S. D.
Dahlhauser and E. V. Anslyn, J. Am. Chem. Soc., 2017, 139, 4635; (f) S.-
W. Zhang, T. M. Swager, J. Am. Chem. Soc., 2003, 125, 3420.
a) M. Zengerle, F. Braundhuber, C. Schneider, F. Worek, G. Reiter and S.
Kubik, Beilstein J. Org. Chem., 2011, 7, 1543; (b) C. Schneider, A. Bierwisch, M.
Koller, F. Worek and S. Kubik, Angew. Chem., Int. Ed., 2016, 55, 12668.
7
e). In the presence of one equivalent of 1a, the function of
AChE can be restored to some extent even when adding
paraoxon 8 (Figs. 6 and 7d). But this is only effective when
adding 1a before 8 (Figs. S52-54). This is in line with the fact
that 8 irreversibly blocks to the catalytic site of AChE.
Increasing the amount of 1a to five equivalents does not
significantly improve the restoration (Fig. 7c). This may be due
6
7
(a) S. E. Border, R. Z. Pavlovic, Z. Lei and J. D. Badjić, J. Am. Chem.
Soc., 2017, 139, 18496; (b) S. E. Border, R. Z. Pavlovic, Z. Lei, M. J.
Gunther, H. Wang, H. Cui and J. D. Badjić, Chem. Commun., 2019,
-1
a
to the following reasons: 1a can also bind AICl (K = 7300 M ,
Figs. S55-S57) and thus the existence of a large amount of 1a
would also inhibit the hydrolysis of AICl by AChE. Nevertheless,
AICl will be slowly released from the cavity of 1a and undergo
hydrolysis catalysed by AChE. Indeed, the hydrolysis kinetics
did not reach a plateau even after 160 min (Fig. 7c) when all
other experiments have been finished.
55, 1987.
8
9
B.-L. Poh and W. Saenger, Spectrochim. Acta., 1983, 39, 305.
C. Tudisco, P. Betti, A. Motta, R. Pinalli, L. Bombaci, E. Dalcanale and G. G.
Condorelli, Langmuir, 2012, 28, 1782.
1
0 (a) Y. Ruan, H. A. Taha, R. J. Yoder, V. Maslak, C. M. Hadad and
J. D. Badji
Ruan, J. D. Brown, J. Gallucci, V. Maslak, C. M. Hadad and J. D.
Badji , J. Am. Chem. Soc., 2013, 135, 14964; (c) S. Chen, Y. Ruan,
J. D. Brown, C. M. Hadad and J. D. Badji , J. Am. Chem. Soc.,
014, 136, 17337.
ć, J. Phys. Chem. B, 2013, 117, 3240; (b) S. Chen, Y.
ć
In summary, we report that the molecular tubes with
hydrogen bonding donors in their deep hydrophobic cavity can
ć
2
strongly bind organophosphorus compounds in water by using 11 (a) G.-B. Huang, S.-H. Wang, H. Ke, L.-P. Yang and W. Jiang, J.
hydrogen bonding and the hydrophobic effect. These
organophosphorus compounds include the toxic pesticides
dimethoate, acephate and paraoxon. The binding affinities are
generally stronger than other molecular receptors only with a
non-functionalized hydrophobic cavity. The binding is
enthapically driven, in contrast to the dominated entropic
contribution for other molecular receptors. In addition, the
molecular tubes show fluorescent enhancement to certain
organophosphorus compounds, permitting their use as
fluorescent sensors to toxic organophosphorus compounds
such as nerve agents. Finally, the in vitro enzymatic
experiments suggest that the molecular tubes may be used as
a non-covalent scavenger to reduce the toxicity of paraoxon to
Am. Chem. Soc., 2016, 138, 14550; (b) L.-L. Wang, Z. Chen, W.-E.
Liu, H. Ke, S.-H. Wang and W. Jiang, J. Am. Chem. Soc., 2017,
1
39, 8436; (c) H. Yao, H. Ke, X. Zhang, S.-J. Pan, M.-S. Li, L. P.
Yang, G. Schreckenbach and W. Jiang, J. Am. Chem. Soc., 2018,
40, 13466; (d) G.-B. Huang, W.-E. Liu, A. Valkonen, H. Yao, K.
1
Rissanen and W. Jiang, Chin. Chem. Lett., 2018, 29, 91; (e) H. Ke, L.-P.
Yang, M. Xie, Z. Chen, H. Yao and W. Jiang, Nat. Chem., 2019,
1
1, 470; (f) L.-M. Bai, H. Zhou, W.-E. Liu, H. Chai, L.-P. Yang, W.
Yan, W. Zhang, H.-H. Yang and W. Jiang. Chem.
Commun. 2019, 55, 3128; (g) L.-M. Bai, H. Yao, L.-P. Yang, W. Zhang
and W. Jiang. Chin. Chem. Lett. 2019, 30, 881.
2 (a) F. Biedermann, W. M. Nau and H.-J. Schneider, Angew. Chem. Int.
Ed., 2014, 53, 11158; (b) P. S. Cremer, A. H. Flood, B. C. Gibb and D. L.
Mobley, Nat. Chem., 2018, 10, 8.
1
1
3 Nachon, X. Brazzolotto, M. Trovaslet and P. Masson, Chem.-Biol. Interact.,
2013, 206, 536.
acetylcholinesterase. This may be extended to other toxic 14 G. L. Ellman, K. D. Courtney, V. Andres Jr. and R. M. Featherstone.
Biochem. Parmacol., 1961, 7, 88.
organophosphorus compounds, such as nerve agents and

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C9CC04603H
The endo-functionalized molecular tubes are able to recognize toxic organophosphorus compounds in water. They can
be used as their fluorescent sensor and as an inhibitor to reduce the toxicity of paraoxon to acetylcholinesterase.