Novel and selective detection of Tabun mimics

Article abstract of DOI:10.1039/c4cc02689f

Detection of nerve agent-related molecules based on BODIPY-salicylaldehyde oxime conjugation was studied. Fluorescence intensity of the B-SAL-OXIME species increases in the presence of DECP, whereas it decreases in the presence of DCP and DEMP (limit of detection = 997 nM). Benzonitrile formation in the novel fluorescent B-SAL-OXIME system was elucidated using model substrates. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02689f

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Novel and selective detection of Tabun mimics†
Yoon Jeong Jang,a Olga G. Tsay,a Dhiraj P. Murale,a Jeong A. Jeong,a Aviv Segevb
Cite this: Chem. Commun., 2014,
50, 7531
and David G. Churchill*a
Received 11th April 2014,
Accepted 22nd May 2014
DOI: 10.1039/c4cc02689f
www.rsc.org/chemcomm
Detection of nerve agent-related molecules based on BODIPY– but approaches for detection of Tabun (GA) selectively over other
salicylaldehyde oxime conjugation was studied. Fluorescence intensity nerve agents are still scarce or non-existent.1 Recently, many
of the B–SAL–OXIME species increases in the presence of DECP, methods of detecting nerve agents continue to be developed and
whereas it decreases in the presence of DCP and DEMP (limit of optimized, which include ion mobility spectrometry, mass spectro-
detection = 997 nM). Benzonitrile formation in the novel fluorescent metry, NMR spectroscopy, and enzyme sensors.5 These methods
B–SAL–OXIME system was elucidated using model substrates.
provide good sensitivity, but do not afford convenient access to
appropriate selectivity and/or are not convenient and simplified
Organophosphorus chemicals include nerve agents and pesticides real-time methods for the ‘‘field.’’ Fluorescent and chromogenic
and are considered as being among the most toxic chemicals to sensing methods are becoming convenient and widely used for
living things.1 Nerve agents were developed to effectively harm detecting nerve agents because they are very sensitive, convenient,
people and decrease the military power of an opposing force.2 allow for detection in real-time and are easy to assess by the
Historically, nerve agents have been used during certain conflicts unassisted eye.6 Oximes (R1R2CQNOH) include aldoximes and
and/or have been stockpiled. The Chemical Weapons Convention ketoximes in which R1 is a hydrogen or another organic group.7
(CWC) has placed a ban on synthesizing, stockpiling and deploying Aldoximes have been used for the treatment of nerve agents.8 An
chemical warfare agents (CWAs) to prevent further casualties oxime is a ‘‘super-nucleophile’’ and is capable of attacking the
and reduce global dangers; however, recent news of stockpiles internal phosphorus at the phosphorylated serine–OH in AChE to
and utilization of nerve agents in the Syrian Arab Republic restore the serine–OH, and thus the function of AChE.9 BODIPY
(Ghouta) showed that there remains a clear and present danger species bearing oximes have recently been explored in chemo-
in the world regarding nerve agents.3 The toxic mechanism sensing (reactive oxygen species).10 Conjugates of (a) BODIPY(4,4-
involves phosphorylation and phosphonylation of the active difluoro-4-bora-3a,4a-diaza-s-indacene), a well-known fluorophore
serine–OH residues in synapses. Nerve agents bind covalently to class, and (b) the salicylaldehyde core, very widely used in transition
the acetylcholinesterase active site and block the breakdown of metal ligand synthetic designs, have recently been prepared.
acetylcholine in synapses. It causes an increase in acetylcholine Bodipy derivatives provide high quantum yield and possess
concentration and results in a neurological imbalance in the robust chemical properties and photostability and tunable
synapse.4 Tabun is a unique agent because of its nitrile group. solubility for use in bioimaging and chemosensing.11,12 Herein,
Tabun includes a P–CN group, while other nerve agents of the we extend our efforts using this conjugation for selective detection
G-series possess a P–F group. It has been suggested that this of nerve agents and their mimics.
chemical group may reveal unique chemistry in chemosensing,
In the present study, a new oxime-based Bodipy system (B–SAL–
OXIME) was synthesized and used as a fluorescent sensor for the
sensing and detection of nerve agent simulants, DCP (diethyl
chlorophosphate), DEMP (diethyl methylphosphonate), and DECP
(diethyl cyanophosphonate) (Fig. 1). Fluorescence emission
changes of B–SAL–OXIME (compound 6) with 0.1 M DCP, DEMP,
and DECP were studied; solutions were made by 1 Â 10À6 M in
0.1 mM, pH 7.4 HEPES buffer and 100 to 1200 Â 10À6 M of DCP,
DEMP and 2 Â 10À7 M in 0.1 mM, pH 7.4 HEPES buffer and 100 to
1200 Â 10À6 M of DECP. Fluorescence emission of B–SAL–OXIME
with DCP and DEMP decreased (Fig. 2 and Fig. S15a and b, ESI†),
a Molecular Logic Gate Laboratory, Department of Chemistry,
Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro,
Yuseong-gu, Daejeon, 305-701, Republic of Korea. E-mail: dchurchill@kaist.ac.kr;
Fax: +82 42-350-2810; Tel: +82 42-350-2845
b Department of Knowledge Service Engineering – Korea Advanced Institute of
Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701,
Republic of Korea. E-mail: aviv@kaist.edu; Fax: +82 42-350-1610;
Tel: +82 42-350-1614
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc02689f
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The ethoxy group played the role of the leaving group from DEMP to
give R = N–O–P(O)(OEt)Me (Scheme 1) which causes a decrease in
fluorescence intensity.14 The fluorescence intensity of B–SAL–OXIME
with DECP increases even in the presence of appreciable concentra-
tions of DCP and DEMP. Here, cyanide (CNÀ) was the leaving group
to give R = N–O–P(O)(Et)2. Compound 7 for this reaction could not
be found by HRMS or NMR spectra (1H and 31P).
The detection limit of B–SAL–OXIME with DECP was deter-
mined to be 92.2 mM for a linear fit; but, in the case of
nonlinear methods, the fitting that involves a lower LOD value
also possesses a higher R2 value (997 nM and R2 = 0.99) (see
Fig. S16, ESI† for comparative fittings).15 This LOD (linear fit)
value is not extraordinarily low. Therefore, as part of our future
aims we will continue to develop new systems that possess
novel modalities, or enhance newly discovered methods to tune the
sensitivity, e.g., conjugates with GNPs (gold nanoparticles).15,16
B–SAL–OXIME however does have enough sensitivity in the detec-
tion of nerve agent simulants, considering the respective, relevant
LD50 values (Table S1, ESI†).
To support the proposed pathway(s) (Scheme 1), a mechanistic
study was conducted through the use of model benzene derivatives
bearing the same substituents found in salicylaldehyde–oxime.
Phenol, benzaldehyde oxime and salicylaldehyde oxime underwent
respective reactions with DCP and DECP under basic conditions
(triethylamine) in acetonitrile (Table 1 and Fig. S2, ESI†); 1H NMR
and 31P NMR spectra were studied after purification. While kinetic
differences exist between the model system and the actual probe,
the model systems importantly help clarify the reactivity. 31P NMR
spectra results of DECP and DCP gave a singlet (d À20.5 and 5.0,
resp.) shifting to d À5.7 and À6.0, respectively, in the presence of
phenol (Fig. S12, ESI†). These data support that phenol with DECP
and DCP gives Ph–O–P(O)(OEt)2 functionality through nucleophilic
substitution. A singlet in the 31P NMR spectrum was found to be
À0.1 and À0.5 in the presence of benzaldehyde oxime (Fig. S13,
ESI†), and at 0.02 and 0.7 in the presence of salicylaldehyde
Fig. 1 Structures of probes and G-series chemical warfare nerve agents
and simulants (blue color–DECP–Tabun mimic).1
but with DECP, intensity increased at lexic = 499 nm, lemis = 508 nm
(Fig. 2a). Possible mechanisms of B–SAL–OXIME with DCP, DEMP
and DECP were proposed and relate to standard nucleophilic attack
pathways. ClÀ was a leaving group, departing from DCP, which
together with H+ from R = N–OH helps form R = N–O–P(O)(OEt)2
(Scheme 1) causing a decrease in fluorescence intensity (Scheme 1).13
Fig. 2 (a) Emission titration spectra of B–SAL–OXIME (2 Â 10À7 M in 0.1 mM,
pH 7.4 HEPES buffer) with DECP (100 to 1200 mM in acetonitrile) at lexic
=
499 nm, lemis = 508 nm. (b) Fluorescence intensity comparing bar graph among
B–SAL–OXIME (1 Â 10À6 M in 0.1 mM, pH 7.4 HEPES buffer) with 1200 mM DCP,
DEMP and DECP in acetonitrile at lexic = 499 nm, lemis = 508 nm.
Scheme 1 Proposed mechanism of B–SAL–OXIME with DCP, DEMP, and
DECP.
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Table 1 31P NMR spectra results of B–SAL–OXIME, phenol, benzaldehyde
oxime and salicyladehyde oxime with nerve agent simulants DECP and DCP
31P NMR shift (ppm)
Chemicals
Structure
DECP
DCP
Solvent (CDCl3)
À20.5
5.0
Phenol
Benzaldehyde oxime
Salicylaldehyde oxime
Fig. 3 Logic gate construct for B–SAL–OXIME with DCP, DEMP and
DECP according to fluorescence intensity.
Bodipy oxime
DEMP, and DECP, each with two different options of fluores-
cence intensity for 900 mM or lower.
Fluorescence change monitoring for compound 8 with metal
ions (Ag+, Ca2+, Cd+, Co2+, Cu2+, Fe2+, Fe3+, K+, Hg2+, Mg2+, Mn2+, Na+,
Pb2+, or Zn2+) shows that no interference exists except for Ag+ at
concentrations equimolar to that of the organophosphonate species.
A strong quenching event was found (99.7%, probe: 1 Â 10À6 M in
0.1 mM, pH 7.4 HEPES buffer lexic = 499 nm, lemis = 508 nm,
acetonitrile). Other trials including B–SAL–OXIME and Ag+ or probe
with DCP and Ag+ show no change (Fig. S16 and S17, ESI†).
In conclusion, herein we introduce a novel B–SAL–OXIME
probe for detecting chemical warfare nerve agent simulants. In
the most straight-forward manifestation, it can be implemented as
a fluorescent detection medium for the detection of DECP over DCP
and DEMP. Fluorescence intensity of B–SAL–OXIME increased
with DECP selectively, and decreased with DCP and DEMP
concentrations. Models were treated with DECP and DCP and
monitored by 1H NMR and 31P NMR spectroscopy to help interpret
spectra obtained after the reaction of the B–SAL–OXIME probe with
simulants. Through these model studies, B–SAL–OXIME was
found to be dehydrated to the nitrile and the OH bonds to
DECP leading to loss of HCl.
oxime (Fig. S14, ESI†) upon treatment with DECP and DCP,
respectively (Table 1). The 31P NMR spectrum of B–SAL–OXIME
with DECP reveals a singlet (d À7.1), supporting that DECP
is phenolate-bound in B–SAL–OXIME, and not bound to the
R = NOH group. The model study with salicylaldehyde oxime
revealed that dehydration occurs to give 2-hydroxyl-benzonitrile
with DECP and DCP; 31P NMR and 1H NMR spectroscopy
confirm this reaction (Fig. S14, ESI†).17 The dehydration of
oxime to nitrile occurs with DECP, in B–SAL–OXIME; the OH
group in B–SAL–OXIME was also attacked by DECP wherein
mass spectroscopy helps confirm this mechanism. The mass
spectrum of B–SAL–OXIME with DECP was observed at m/z
524.1696; compound 8 formulated as [C28H30O11P2Na]+ gives a
calculated value of 524.1693 (Fig. S7, ESI†).
We believe that the nitrogen of the oxime is the electron
donating group and the PET mechanism gives no strong
signalling for compounds 6 and 7. However, the cyano group
in compound 8 works as an electron-withdrawing group and
allows for strong fluorescence by inhibiting PET between the
donor–acceptor units of the dyad (Scheme 1).
To assist in recognition, a logic gating treatment18 was
invoked where data were interpreted in blocks of emission
intensity; these help form exclusive logic gate tiers of increasing
intensity (Fig. 3). Intensity of A is 0 to 50 nm, B 50 to 100 nm,
C 100 to 150 nm, D 150 to 200, E 200 to 250 nm, and F 250 to
500 nm. Each emission intensity block can be identified by a
combination of levels of B–SAL–OXIME from DCP, DEMP, and
DECP according to fluorescence intensity, using ‘‘AND,’’ ‘‘OR,’’ and
‘‘NOT’’ logic gates. For the 250–500 nm region, the concen-
tration of DECP is 700 mM or greater with two different levels of
fluorescence intensity. In the regions of B, C and D, the
concentration of DCP may be zero; for the E region, none of
the three agents may be zero. The high intensity gate for the
250–500 nm region (F) is a three-input gate based on levels of
DECP and no DCP and DEMP; the 100–250 nm zone is a four-
input gate; and the 0–50 nm gate is a six-input gate with DCP,
D.G.C. acknowledges research support from the National
Research Foundation (NRF) (Grant # 2011-0017280). Mr Hack
Soo and Ms Sung A Kim are acknowledged respectively for
facilitating the acquisition of NMR spectroscopic and MS data.
Notes and references
1 K. Kim, O. G. Tsay, D. A. Atwood and D. G. Churchill, Chem. Rev.,
2011, 111, 5345.
2 (a) T. Okumura, N. Takasu, S. Ishimatsu, S. Miyanoki, A. Mitsuhashi,
K. Kumada, K. Tanaka and S. Hinohara, Ann. Emerg. Med., 1996,
28, 129; (b) H. Okudera, H. Morita, T. Iwashita, T. Shibata, T. Otagiri,
S. Kobayashi and N. Yanagisawa, Am. J. Emerg. Med., 1997, 15, 527.
3 (a) S. W. Wiener and R. S. Hoffman, J. Intensive Care Med., 2004,
19, 22; (b) I. R. Kenyon, K. Gutschmidt and O. Cosivi, Toxicology,
2005, 214, 249; (c) C. E. H. Christopher, M. Blanchard and
M. B. D. Nikitin, Congressional Research Service, 2014.
4 W.-h. Wu, J.-j. Dong, X. Wang, J. Li, S.-h. Sui, G.-y. Chen, J.-w. Liu
and M. Zhang, Analyst, 2012, 137, 3224.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7531--7534 | 7533

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5 (a) G. R. Asbury, C. Wu, W. F. Siems and H. H. Hill, Anal. Chim. Acta,
2000, 404, 273; (b) T. J. Henderson, Anal. Chem., 2002, 74, 191;
(c) R. M. Black, R. J. Clarke, R. W. Read and M. T. J. Reid,
9 (a) F. Worek, P. Eyer, N. Aurbek, L. Szinicz and H. Thiermann, Toxicol.
Appl. Pharmacol., 2007, 219, 226; (b) O. G. Tsay, S. T. Manjare, H. Kim,
K. M. Lee, Y. S. Lee and D. G. Churchill, Inorg. Chem., 2013, 52, 10052.
J. Chromatogr. A, 1994, 662, 301; (d) A. Mulchandani, I. Kaneva 10 (a) M. Emrullahoglu, M. Ucuncu and E. Karakus, Chem. Commun.,
and W. Chen, Biotechnol. Bioeng., 1999, 63, 216; (e) S. Royo,
R. Martinez-Manez, F. Sancenon, A. M. Costero, M. Parra and
S. Gil, Chem. Commun., 2007, 4839.
2013, 49, 7836; (b) G. Cheng, J. Fan, W. Sun, K. Sui, X. Jin, J. Wang
and X. Peng, Analyst, 2013, 138, 6091.
11 G. Ulrich, R. Ziessel and A. Harriman, Angew. Chem., Int. Ed., 2008,
47, 1184.
6 (a) I. Walton, M. Davis, L. Munro, V. J. Catalano, P. J. Cragg,
M. T. Huggins and K. J. Wallace, Org. Lett., 2012, 14, 2686; (b) X. Wu, 12 C. Thivierge, J. Han, R. M. Jenkins and K. Burgess, J. Org. Chem.,
´
Z. Wu and S. Han, Chem. Commun., 2011, 47, 11468; (c) B. Dıaz de
2011, 76, 5219.
˜
Grenu, D. Moreno, T. Torroba, A. Berg, J. Gunnars, T. Nilsson, 13 I. Damljanovic, M. Vukicevic and R. D. Vukicevic, Monatsh. Chem.,
¨
R. Nyman, M. Persson, J. Pettersson, I. Eklind and P. Wasterby, J. Am.
2006, 137, 301.
Chem. Soc., 2014, 136, 4125; (d) M. Burnworth, S. J. Rowan and C. Weder, 14 J. J. Topczewski and D. M. Quinn, Org. Lett., 2013, 15, 1084.
Chem. – Eur. J., 2007, 13, 7828; (e) A. Wild, A. Winter, M. D. Hager and 15 A. Hakonen and N. Stromberg, Analyst, 2012, 137, 315.
U. S. Schubert, Chem. Commun., 2012, 48, 964; ( f ) D. Ajami and J. Rebek 16 (a) Y. J. Jang, D. P. Murale and D. G. Churchill, Analyst, 2014, 139, 1614;
Jr., Org. Biomol. Chem., 2013, 11, 3936.
7 J. Kassa, J. Z. Karasova, V. Sepsova, G. Kunesova, F. Caisberger,
M. Pohanka and J. Bajgar, Toxicol. Lett., 2011, 205, S128.
(b) A. Hakonen, Anal. Chem., 2009, 81, 4555; (c) A. Hakonen and
N. Stromberg, Chem. Commun., 2011, 47, 3433; (d) N. Stromberg and
A. Hakonen, Anal. Chim. Acta, 2011, 704, 139.
8 (a) F. Worek, H. Thiermann, L. Szinicz and P. Eyer, Biochem. 17 A. R. Sardarian, Z. Shahsavari-Fard, H. R. Shahsavari and
Pharmacol., 2004, 68, 2237; (b) J. Kassa, J. Toxicol., Clin. Toxicol.,
2002, 40, 803.
Z. Ebrahimi, Tetrahedron Lett., 2007, 48, 2639.
18 A. P. de Silva, Chem. – Asian J., 2011, 6, 750.
7534 | Chem. Commun., 2014, 50, 7531--7534
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