Chromo-fluorogenic detection of soman and its simulant by thiourea-based rhodamine probe

Article abstract of DOI:10.3390/molecules24050827

Here, we introduced a novel thiourea-based rhodamine compound as a chromo-fluorogenic indicator of nerve agent Soman and its simulant diethyl chlorophosphate (DCP). The synthesized probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT), which has a rhodamine core linked by a cyanophenyl thiosemicarbazide group, enabled a rapidly and highly sensitive response to DCP with clear fluorescence and color changes. The detection limit was as low as 2 × 10^?6 M. The sensing mechanism showed that opening of the spirolactam ring following the phosphorylation of thiosemicarbazides group formed a seven-membered heterocycle adduct, according to MS analysis and TD-DFT calculations. RB-CT exhibited high detecting selectivity for DCP, among other organophosphorus compounds. Moreover, two test kits were employed and successfully used to detect real nerve agent Soman in liquid and gas phase.

Full text of DOI:10.3390/molecules24050827

molecules
Article
Chromo-Fluorogenic Detection of Soman and Its
Simulant by Thiourea-Based Rhodamine Probe
Shengsong Li 1 , Yongchao Zheng
,†
1,2, ,†
* , Weiqiang Chen 3 , Meiling Zheng , He Zheng 1,2,
4
Zhe Zhang , Yan Cui , Jinyi Zhong 1,2,* and Chonglin Zhao
1
1,2
1,2,
*
1
Research Institute of Chemical Defense, Beijing 102205, China; dotasongge@163.com (S.L.);
fhyjyzh@126.com (H.Z.); loopifer@163.com (Z.Z.); tracypiscescy@163.com (Y.C.)
State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China
Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China;
chenwq7315@impcas.ac.cn
Laboratory of Organic NanoPhotonics and CAS Key Laboratory of Bio-Inspired Materials and Interfacial
Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China;
zhengmeiling@mail.ipc.ac.cn
2
3
4
*
Correspondence: zhengycfh@163.com (Y.Z.); linfzjy@163.com (J.Z.); zhaochonglin@126.com (C.Z.);
Tel.: +86-10-66758321(C.Z.)
†
These authors have contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Simone Morais
Received: 3 February 2019; Accepted: 18 February 2019; Published: 26 February 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Here, we introduced a novel thiourea-based rhodamine compound as a chromo-fluorogenic
indicator of nerve agent Soman and its simulant diethyl chlorophosphate (DCP). The synthesized
probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT), which has a rhodamine core
linked by a cyanophenyl thiosemicarbazide group, enabled a rapidly and highly sensitive response
−
6
to DCP with clear fluorescence and color changes. The detection limit was as low as 2
×
10 M.
The sensing mechanism showed that opening of the spirolactam ring following the phosphorylation
of thiosemicarbazides group formed a seven-membered heterocycle adduct, according to MS
analysis and TD-DFT calculations. RB-CT exhibited high detecting selectivity for DCP, among
other organophosphorus compounds. Moreover, two test kits were employed and successfully used
to detect real nerve agent Soman in liquid and gas phase.
Keywords: Nerve agents; Soman; Rhodamine B; Thiosemicarbazide; Chromo-fluorogenic
probe; Detection
1
. Introduction
Nerve agents are a class of highly toxic organophosphates, mainly including Tabun (GA), Sarin
(GB), Soman (GD), and VX [1] (Scheme 1). These compounds are extremely dangerous because
they are able to enter human body through respiration or penetration and inhibit the activity of the
acetylcholinesterase, then the accumulation of acetylcholine in the synapse causes neuromuscular
paralysis and eventually death [
odorless, and the possible use in terrorist attacks, the development of reliable and rapid detection
systems for nerve agents is highly desirable [ ]. In addition, rapidly discriminating the distribution
2]. Due to the easy production, high toxicity, being colorless and
3
of nerve agents in the contaminated areas can also provide effective guidance for subsequent
decontamination operations and further safety confirmation.
Molecules 2019, 24, 827; doi:10.3390/molecules24050827
www.mdpi.com/journal/molecules

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Scheme 1. Chemical structures of nerve-agents Sarin, Soman, Tabun, and VX, their simulant DCP, and
some organophosphorus compounds as potential interferences.
Several methodologies have been employed for the detection of nerve agents, including enzymatic
assays [4,5], interferometry [6], ion mobility spectroscopy [7], electrochemistry [8], micro-cantilevers [9,10],
and photonic crystals [11]. Nevertheless, these protocols usually have some drawbacks, such as
operation complexity, non-portability, difficulties in real-time monitoring, etc. As alternatives to these
procedures, some new approaches have been explored involving molecularly imprinted polymers [12],
nanoparticles [13], and chromo-fluorogenic probes [14–22]. Above all, chromo-fluorogenic probes
have recently gained increasing interest due to their cost-effectiveness, simplicity, and “naked-eye”
detection [23,24].
A typical chromo-fluorogenic probe is usually formed by two moieties: (1) a chromo-fluorogenic
reporter group, which translates the binding event into the change of color and fluorescence, mainly
containing rhodamine [22], fluorescein [15], boron dipyrromethene (BODIPY) [16
cyanine dye [18]; (2) a selective reactive group, which provides a reactive binding site for nucleophilic
attack, mainly containing hydroxyl [15 25], oxime [19], and amino groups [26]. Recently, thiourea
has been proved to be capable of reacting with nerve agents through hydrogen-bond interaction
between N-H protons of thiourea and phosphonate oxygen or hydrolyzed products [27 28]. It has
,17], azo [14,20], and
,
,
been also proven that the reaction of thiosemicarbazide group can induce opening of the spirolactam
ring in rhodamine B accompanied with color change and enhanced fluorescence [29]. Thus, the
thiosemicarbazide group is expected to become a reactive group bonding with rhodamine B core for
chromo-fluorogenic detection of nerve agents and simulants.
Herein, a rhodamine B based probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT)
was designed and synthesized, and as shown in Scheme 2, the electron-withdrawing cyanophenyl
group in the molecule could promote the intramolecular charge transfer (ICT) process to enhance

Molecules 2019, 24, 827
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activity of ring-opening reaction and strengthen the change of color. Spectra analysis and mechanism
study confirmed the probe was capable of the rapid detection of diethyl chlorophosphate (DCP, a
simulant of the Soman) with remarkable change of color and fluorescence through the phosphorylation
of the thiosemicarbazide group. Finally, the probe was also successfully used in the detection of GD in
both liquid and gas phase.
CN
CN
EtO
O
O
P
N
O S
NH
N
H
S
O
OEt
OEt
N
N
NH
P
Cl
N
O
N
N
O
N
Colorless
Fluorescence 'off'
Pink
Fluorescence 'on'
Scheme 2. Proposed mechanism of RB-CT with DCP.
2
. Results and Discussion
2
.1. Spectroscopic Properties
To demonstrate the response of RB-CT to nerve agents, absorption and fluorescence spectra
were first studied. DCP was used as low-toxic simulant because of its similar chemical structure
and reactivity with nerve agents (Tabun, Sarin and Soman). Et N (3 Vt%) was added in the CH CN
3
3
solution of RB-CT to avoid the interference of proton. As shown in Figure 1a, an obvious absorption
band at 560 nm was observed after the addition of DCP in the RB-CT solution. The change in
color from colorless to pink implied the spirolactam ring-opening reaction of RB-CT caused by DCP
(
Figure 1b). The thiosemicarbazide group of RB-CT is likely to react with DCP, resulting in a remarkable
enhancement of absorption intensity. The result highlights that RB-CT is of high potential as a
naked-eye probe for the detection of nerve agents.
Figure 1c presented the fluorescence spectra of RB-CT with increasing concentration of DCP; a
new fluorescent band at 583 nm appears and shows remarkable enhancement. The spectral changes
provided further evidence for the opening of the spirolactam ring, according to the changes in
absorption spectra. Moreover, the fluorescence intensity of RB-CT showed a good linear relationship
−
3
−3
at the concentration of DCP in the range of 0.1 × 10 –1.9 × 10 M (Figure 1d).
The limit of detection (LOD) was determined from the fluorescence spectral data, using the
equation K
the slope of the calibration curve. The limit of detection was found to be 2
high sensitivity to detect DCP by RB-CT. The sensing ability for the naked eye was evaluated by
immobilizing RB-CT on silica plates. After the plates were dipped in the CH CN solution of DCP
×
Sb1/S, where K = 3, Sb1 is the standard deviation of blank measurements and S is
−
6
×
10 M, indicating
3
(2–2000 ppm), clear enhancement of the fluorescence was observed by the naked eye in the range
of 20 and 2000 ppm (Figure S1), which demonstrates the LOD for the naked eye is as low as the
value calculated.

Molecules 2019, 24, 827
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−
5
−4
Figure 1.
(
a
) Absorption spectra of RB-CT (1.0
×
10 M) upon the addition of DCP (0.5
×
10 –8.0
×
−
4
10
M) in CH CN (3% Et N). ( ) The images of the RB-CT solution before and after the addition of
b
3
3
−
6
DCP under sun light (left) and UV light (right). (
λex = 540 nm, upon the addition of DCP (0.1
emission intensity of RB-CT at 583 nm.
c
) Fluorescence spectra of RB-CT (1.0
×
10 M) at
−
3
−3
×
10 –1.9
×
10 M) in CH CN (3% Et N). ( ) Plot of
d
3
3
2
.2. Reaction Kinetics Study
To investigate the reaction kinetics of RB-CT and DCP, time-dependent fluorescence change was
performed under different DCP concentrations at room temperature. As shown in Figure 2a, the
reaction was almost complete and the fluorescence intensity was saturated within 1200 s in all cases.
The kinetic constants were further examined following pseudo-first-order kinetic rate law as the clear
linear relationships between the change of fluorescence intensity and reaction time (Figure S2). The
observed rate constants kobs, half-life time t1/2, and the constant rates (k) were summarized in Table 1.
The average half-life time was about 280 s, less than 5 min. The short half-life times are the basis for
the rapid detection of nerve agents.

Molecules 2019, 24, 827
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−
6
Figure 2.
(
a
) Kinetic profiles of the fluorescence intensity at 583 nm of RB-CT ((1.0
×
10 M, CH CN,
3
−
4
−4
−4
−3
3
% Et N) after the addition of 4.0
×
10 M, 5.0
×
10 M, 6.0
×
10 M, and 1.2
×
10 M DCP.
3
(
b)The correlation between kobs and the concentration of DCP.
Table 1. Observed reaction rates (kobs), half-life time (t1/2), and the constant rates (k) for the reaction of
RB-CT ((1.0 × 10− M, CH CN, 3% Et N) and DCP with different concentrations.
6
3
3
The Concentrations
of DCP
× 10−
4
5 × 10−4
6 × 10−4
1.2 × 10−3
4
Kobs (s 1)
−
0.00244
284
0.00245
283
0.00246
282
0.00251
276
t1 (s)
/2
−
2
−1
)
8.645 × 10−6
k (M
S
2
.3. Mechanism Study
The absorption and fluorescence changes can be attributed to the opening of spirolactam ring
via nucleophilic reaction between RB-CT and DCP. An ESI analysis was employed to investigate the
possible products and a major peak at 708.3223 was observed, which indicated the phosphorylation
of RB-CT (Figure S3). The new singlet observed (
addition of RB-CT gives further evidence for the occurrence of phosphorylation reaction (Figure S4).
As the formation of a heterocycle in RB-CT/DCP adduct is a reasonable mechanism [30 31], DFT
δ
0.32) in the 31P NMR spectrum of DCP after
,
and TD-DFT calculations were used to confirm the structure of the adduct through optimizing the
geometries formed by nucleophilic attack with different nitrogen sites in the thiosemicarbazide group.
The proposed mechanism was presented in Scheme 2; a seven-membered heterocycle was proved to
be a favorable adduct structure owing to its relatively lower energy, which also agreed well with the
mass spectrometry result (Figure S5). The optimized structures of RB-CT and RB-CT/DCP adducts
were shown in Figure 3. The HOMO–LUMO energy gap (2.651 eV) of the adduct is less than that of
RB-CT (3.885 eV) and the electron densities in LUMO are more concentrated in the thiourea group
after the reaction. The calculated main contributing electronic transitions for S0
→
S1 energy state are
HOMO LUMO (2.14 eV/578 nm) and HOMO-1 LUMO+1 (2.34 eV/530 nm), which is consistent
→
→
with the absorption band at 560 nm obtained experimentally. The considerable difference in the energy
and electronic transition can shed light on the changes of absorption spectra and color.

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Figure 3. Energy optimized structures, HOMO-LUMO energy levels, and interfacial plots of the orbitals
of RB-CT and RB-CT/DCP adduct.
2
.4. Interferents
As organophosphorus pesticides often act as interferences to render false positives during the
detection of nerve agents, several conventional organophosphorus compounds were chosen as target
species to study the selectivity of RB-CT. As shown in Figure 4, the interferences did not cause any
obvious color and fluorescence changes in DCP, which indicated the selective detection of DCP can
be achieved by RB-CT, among other conventional organophosphorus compounds. It is noted that
although proton can induce the spirolactam ring opening of the rhodamine molecule [32], RB-CT
exhibited little changes of color and fluorescence intensity in the presence of hydrochloric acid. Thus,
the false-positive caused by proton can be effectively avoided under the detection condition with the
addition of 3% Et N, since the suitable detection pH range of the probe is 7.0 to 10.0 (Figure S6).
3

Molecules 2019, 24, 827
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−
5
Figure 4.
after the addition of different organophosphorus compounds (1.0
solution of RB-CT without Et N. (
(
a
) Relative fluorescence intensity of RB-CT (1
×
10 M, CH CN, 3% Et N) at 583 nm
3
3
3
−
×
10 M). Blank is the CH CN
3
b
) Relative color change of RB-CT after the addition of different
3
organophosphorus compounds under sunlight and UV light.
2
.5. Practical Application toward Real Nerve Agent
To confirm in situ and rapid sensing ability of RB-CT toward real nerve agents, we have
investigated the response characteristics of the probe to GD in both liquid and gas phase. Clear
color changes under sunlight and fluorescence enhancement irradiated by UV lamp were all observed
by the naked eye immediately in liquid and within 10 min in gas phase (40 ppm GD introduced in a
flask as an aerosol) (as shown in Figure 5). The remarkable visual differences did not fade or quench
after 24 h, which indicated the irreversible cyclization had occurred [33]. The above results illustrate
the potential application of RB-CT in the rapid and facile naked-eye detection of nerve agents in both
liquid and vapor phases.
−
10 M in CH CN, 3%
3
4
Figure 5.
(
a
) Color change of filter paper treated with RB-CT solution (1.0
) Color change of filter paper
10 M in CH CN, 3% Et N) and exposed to GD in gas phase under
×
Et N) and GD in liquid phase under sunlight (left) and UV light (right). (
b
3
−
4
treated with RB-CT solution (1.0
sunlight (left) and UV light (right).
×
3
3
3
. Materials and Methods
3
.1. Materials
All of the solvents were obtained from Beijing Chemical Reagent Company and used without
further purification. Rhodamine B, 4-cyanophenyl isothiocyanate and anhydrous magnesium sulfate
(
(
MgSO ) were purchased from Aladdin In. Co. (Los Angeles, CA, USA). Diethyl chlorophosphate
DCP) was purchased from Sigma Co. (Louis, MO, USA). Nerve agent GD (the purity is 80 %) was
4
provided by the Research Institute of Chemical Defense of China.

Molecules 2019, 24, 827
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3
.2. Measurements
The ESI mass analysis was performed using an Q Exactive HF-X Hybrid Quadrupole-Orbitrap
1
Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). The H NMR spectra were recorded
on AV 400 spectrometer (Bruker, Karlsruhe, Germany). Chemical shifts were expressed in parts per
millions (δ) downfield from the internal standard tetramethyl silane and were reported as s (singlet),
d (doublet), bs (broad singlet), t (triplet), and m (multiplet). Absorbance and fluorescence spectra
were recorded at room temperature with a Hitachi U-3900 UV-Visible spectrophotometer and F-4500
fluorescence spectrophotometer, respectively, using a fluorescence cell of 10 mm path. The excitation
wavelength was set to 540 nm (slit width 5 nm), and emission was monitored from 560–700 nm (slit
width 5 nm). Column chromatography was conducted over silica gel (mesh 100–200).
3
.3. Synthetic Procedures
As shown in Scheme 3, compound 1 was synthesized using the reported procedure [34].
Compound 2 was synthesized in a facile way.
◦
Scheme 3. Synthesis route of RB-TU. Reagents and conditions: (a) CH OH, hydrazine hydrate, 80 C,
3
6
h; (b) DMF, 4-cyanophenyl isothiocyanate, r.t, 12 h.
Synthesis of compound 1: In a 100 mL flask, 1 mL hydrazine hydrate was drop-wise added to a
solution of rhodamine B (1.67 mmol, 800 mg) in anhydrous CH OH (30 mL), the mixed solution was
3
then stirred and heated to 80 °C, and refluxed for 6 h. Pure water (30 mL) was added and the solvent
was extracted with EtOAc (3
and evaporated, orange solid compound 1 (356 mg, 48.8% yield) was obtained, and used without
×
60 mL), after the organic phase was dried with anhydrous MgSO4
further purification.
1
The H NMR (400 MHz, DMSO-d6)
δ 7.82 (d, J = 8.3 Hz, 1H), 7.58-7.48 (m, 2H), 7.04 (d, J = 8.0 Hz,
1
H), 6.41 (d, J = 17.1 Hz, 6H), 4.32 (s, 2H), 3.36 (d, J = 7.0 Hz, 8H), 1.14 (t, J = 6.9 Hz, 12H) (Figure S7).
Synthesis of compound 2(RB-TU probe): To a solution of compound 1 (0.22 mmol, 100 mg) in
.5 mL DMF, a solution of 4-cyanophenyl isothiocyanate (0.31 mmol,50 mg) in 1.5 mL DMF was added,
1
the reaction mixture was stirred for 12 h at room temperature. After the solution evaporated, the
residue was purified by flash chromatography (EtOAc: hexane = 1:10) to afford RB-TU probe (36 mg,
2
6.6% yield).
1
The H NMR (400 MHz, Chloroform-d)
δ 8.01 (d, J = 7.6 Hz, 1H), 7.72 - 7.57 (m, 3H), 7.44 (d,
J = 8.4 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.47 (d, J = 8.8 Hz, 2H), 6.42
d, J = 2.5 Hz, 2H), 6.29 (d, J = 2.6 Hz, 1H), 6.27 (d, J = 2.6 Hz, 1H), 3.32 (qd, J = 7.2, 2.5 Hz, 8H), 1.15 (t,
J = 7.0 Hz, 12H) (Figure S8).
HR-MS (C H N O S): Calcd.: 616.2620; Found: 617.2681(9.9 ppm) (Figure S9).
(
36
36
6
2
3
.4. Reaction Kinetics Study
−
6
The fluorescence spectra of RB-CT (1.0
×
10 M) were recorded after the addition of DCP in
−
4
−4
−4
−3
10 M over a 0–1200 s
the concentrations of 4.0
×
10 M, 5.0
×
10 M, 6.0
×
10 M, and 1.2
×

Molecules 2019, 24, 827
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incubation period at room temperature. The observed rate constants kobs and corresponding half-life
time t1/2 were determined according to Equations (1) and (2).
ln[(Fmax-Ft)/Fmax] = −kobs
t
(1)
(2)
t1/2 = ln2/kobs
where Fmax and F
t
are maximum fluorescence intensity obtained after the reaction was completed and
the fluorescence intensities at 583 nm at reaction time t.
3
.5. Computational Methods
Quantum chemical calculations were carried out by Gaussian 09 package [35]. Geometry was
optimized using density functional theory (DFT) functional B3LYP [36–38] and 6–31G (d) basis
set [39,40]. Time-dependent density functional theory (TD-DFT) calculation [41] was also performed
at the same level of theory. Considering the effects of the environment on the calculated energies,
solvation effects were considered throughout geometry optimizations via the self-consistent reaction
field (SCRF) and conductor-like polarizable continuum model (CPCM) method [42].
3
.6. Interferents
Selectivity and specificity tests were performed with different organophosphorus compounds
(OPs), i.e., dimethyl methylphosphonate (DMMP), triphenyl phosphate (TPP), trimethyl phosphate
(
TMP), acephate, isocarbophos, and dimethoate (Scheme 1). To 2 mL reagent solution of the probe
−
5
−1
10 M)
molecule (1.0
×
10 M) in CH CN containing 3% Et N, 20
µL stock solution of OPs (1.0
×
3
3
was added. The solutions were incubated at room temperature for 5 min and the fluorescence spectra
was recorded.
3
.7. The Preparation of Test Kits of Real Nerve Agents in Liquid and Gas Phase
The test kits toward GD in liquid and gas phase were produced by the following steps. For liquid
−
10 M, 3% Et N),
3
4
phase, a waterman filter paper was soaked in the CH CN solution of RB-CT (1.0
×
3
after the solvent was air-dried, a “GD” logo was patterned by GD. For gas phase, a solution of RB-CT
−
10 M, 3% Et N) was sprayed onto a filter paper to spell out “GD” first, after the
3
4
in CH CN (1.0
×
3
solvent was air-dried, the paper was exposed to GD vapor in a flask (40 ppm introduced as an aerosol)
for 10 mins. The LCt50 and LD50 of Soman are estimated to be 100 mg min/m and 300 mg/individual,
3
respectively [43].
Safety Note: Only highly qualified and experienced personnel should work with CWAs employed
here, as described in the experimental part below. Due to the high volatility and toxicity of the CWAs,
all of the experiments in this part were carried out inside a safety fume hood and operated under
efficient ventilation systems. To avoid any risk of CWA inhalation, respiratory protection of involved
personnel was provided by protective breathing masks equipped with combined NBC filters.
4
. Conclusions
In conclusion, we have designed and synthesized a novel probe N-(rhodamine
B)-lactam-2-(4-cyanophenyl) thiourea for chromogenic and fluorogenic detection of nerve agents.
The probe undergoes an irreversible opening-ring reaction following the form of a seven-membered
heterocycle adduct in the presence of DCP, accompanied by the obvious color change from colorless
to pink, which has been confirmed by MS analysis and TD-DFT calculations. The response is that
instantaneously half-life time is about 280 s at room temperature and the detection limit was found to
−
6
be 2
×
10 M. Furthermore, we have demonstrated that the probe is applicable for rapid and facile in
situ detection of GD with the naked eye in both liquid and vapor phase.

Molecules 2019, 24, 827
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Supplementary Materials: The following are available online. Figure S1: The limit of detection for naked eye.
Figure S2: Calibration plots by using fluorescence intensity of RB-CT (1 M) as a function of reaction time t in
µ
3
1
different DCP concentrations. Figure S3: ESI-MS spectrum of RB-CT/DCP adduct. Figure S4: P NMR spectra of
DCP and RB-CT with DCP in CD CN. Figure S5: HOMO-LUMO energy levels of TD-DFT optimized geometries
3
of probe and predicted RB-CT/DCP adducts. Figure S6: The plot of pH versus fluorescence intensity of free
1
1
RB-CT and RB-CT+DCP. Figure S7: H NMR Spectrum of Compound 1. Figures S8 and S9: H NMR and ESI-MS
Spectra of RB-CT.
Author Contributions: Y.Z. and W.C. conceived the idea and designed the experiments; S.L. and Y.Z. performed
the synthesis, characterization, and co-wrote the manuscript draft; S.L., H.Z., Y.C., and Z.Z. discussed the data
and the mechanisms; M.Z. and C.Z. contributed to spectra analysis; C.Z., J.Z., M.Z., and W.C. edited and revised
the manuscript; Y.Z. and M.Z. assisted in DFT and TD-DFT calculation; Authors S.L. and Y.Z. contributed equally.
All authors discussed the results and commented on the manuscript.
Funding: This research was funded by the National Key Research and Development Program of China (Grant No.
2
017YFC0108500).
Acknowledgments: The authors thank the financial support of the National Key Research and Development
Program of China (Grant No. Grant No. 2017YFC0108500). The authors would like to thank Prof. Dr. Shi-Lu
Chen for valuable guidance and discussion about the quantum chemistry calculation, and Dr. Xiang-Xi Meng for
discussions in the synthesis and purification process of RB-CT.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Szinicz, L. History of chemical and biological warfare agents. Toxicology 2005, 214, 167–181. [CrossRef]
PubMed]
Gunderson, C.H.; Lehmann, C.R.; Sidell, F.R.; Jabbari, B. Nerve agents: A review. Neurology 1992, 42, 946–950.
CrossRef] [PubMed]
Lei, Z.; Yang, Y. A concise colorimetric and fluorimetric probe for sarin related threats designed via the
covalent-assembly" approach. J. Am. Chem. Soc. 2014, 136, 6594–6597. [CrossRef] [PubMed]
[
[
"
Russell, R.J.; Pishko, M.V.; Simonian, A.L.; Wild, J.R. Poly(ethylene glycol) hydrogel-encapsulated
fluorophore-enzyme conjugates for direct detection of organophosphorus neurotoxins. Anal. Chem. 1999, 71,
4
909–4912. [CrossRef] [PubMed]
5.
6.
7.
Russell, A.J.; Berberich, J.A.; Drevon, G.F.; Koepsel, R.R. Biomaterials for mediation of chemical and biological
warfare agents. Annu. Rev. Biomed. Eng. 2003, 5, 1–27. [CrossRef] [PubMed]
Kim, S.; Cho, B.; Sohn, H. Detection of nerve agent stimulants based on photoluminescent porous silicon
interferometer. Nanoscale Res. Lett. 2012, 7, 527. [CrossRef] [PubMed]
Sorribes-Soriano, A.; de la Guardia, M.; Esteve-Turrillas, F.A.; Armenta, S. Trace analysis by ion mobility
spectrometry: From conventional to smart sample preconcentration methods. A review. Anal. Chim. Acta
2
018, 1026, 37–50. [CrossRef] [PubMed]
8
.
Maresova, E.; Tomecek, D.; Fitl, P.; Vlcek, J.; Novotny, M.; Fiser, L.; Havlova, S.; Hozak, P.; Tudor, A.;
Glennon, T.; et al. Textile chemiresistors with sensitive layers based on polymer ionic liquids: Applicability
for detection of toxic gases and chemical warfare agents. Sens. Actuator B-Chem. 2018, 266, 830–840.
[CrossRef]
9
1
.
Walker, J.P.; Kimble, K.W.; Asher, S.A. Photonic crystal sensor for organophosphate nerve agents utilizing
the organophosphorus hydrolase enzyme. Anal. Bioanal Chem. 2007, 389, 2115–2124. [CrossRef] [PubMed]
0. He, W.;
Liu, Z.;
Du, X.;
Jiang, Y.;
Xiao, D. Analytical application of
poly{methyl[3-(2-hydroxy-3,4-difluoro)phenyl]propyl siloxane} as a QCM coating for DMMP detection.
Talanta 2008, 76, 698–702. [CrossRef] [PubMed]
11. Kittle, J.D.; Fisher, B.P.; Esparza, A.J.; Morey, A.M.; Iacono, S.T. Sensing Chemical Warfare Agent Simulants
via Photonic Crystals of the Morpho didius Butterfly. ACS Omega 2017, 2, 8301–8307. [CrossRef] [PubMed]
12. Roy, K.S.; Mazumder, A.; Goud, D.R.; Dubey, D.K. A simplistic designing of molecularly imprinted polymers
for derivative of nerve agents marker using P-31{H-1}NMR. Eur. Polym. J. 2018, 98, 105–115. [CrossRef]
13. Nanda Kumar, D.; Satija, J.; Chandrasekaran, N.; Mukherjee, A. Acetylcholinesterase-based inhibition
screening through in situ synthesis of gold nanoparticles: Application for detection of nerve agent simulant.
J. Mol. Liq. 2018, 249, 623–628. [CrossRef]

Molecules 2019, 24, 827
11 of 12
1
1
1
1
1
4. Costero, A.M.; Parra, M.; Gil, S.; Gotor, R.; Mancini, P.M.E.; Martinez-Manez, R.; Sancenon, F.; Royo, S.
Chromo-fluorogenic detection of nerve-agent mimics using triggered cyclization reactions in push-pull dyes.
Chem. Asian J. 2010, 5, 1573–1585. [CrossRef] [PubMed]
5. Goud, D.R.; Pardasani, D.; Tak, V.; Dubey, D.K. A highly selective visual detection of tabun mimic diethyl
cyanophosphate (DCNP): effective discrimination of DCNP from other nerve agent mimics. RSC Adv. 2014
,
4
, 24645. [CrossRef]
6. Barba-Bon, A.; Costero, A.M.; Gil, S.; Martinez-Manez, R.; Sancenon, F. Selective chromo-fluorogenic
detection of DFP (a Sarin and Soman mimic) and DCNP (a Tabun mimic) with a unique probe based on a
boron dipyrromethene (BODIPY) dye. Org. Biomol. Chem. 2014, 12, 8745–8751. [CrossRef] [PubMed]
7. Kim, Y.; Jang, Y.J.; Lee, D.; Kim, B.-S.; Churchill, D.G. Real nerve agent study assessing pyridyl reactivity:
Selective fluorogenic and colorimetric detection of Soman and simulant. Sens. Actuator B-Chem. 2017, 238,
1
45–149. [CrossRef]
8. Ali, S.S.; Gangopadhyay, A.; Maiti, K.; Mondal, S.; Pramanik, A.K.; Guria, U.N.; Uddin, M.R.; Mandal, S.;
Mandal, D.; Mahapatra, A.K. A chromogenic and ratiometric fluorogenic probe for rapid detection of a nerve
agent simulant DCP based on a hybrid hydroxynaphthalene-hemicyanine dye. Org. Biomol. Chem. 2017, 15,
5
959–5967. [CrossRef] [PubMed]
1
2
2
9. Cai, Y.-C.; Li, C.; Song, Q.-H. Selective and visual detection of a nerve agent mimic by phosphorylation and
protonation of quinolin oximes. J. Mater. Chem. C 2017, 5, 7337–7343. [CrossRef]
0. Royo, S.; Costero, A.M.; Parra, M.; Gil, S.; Martinez-Manez, R.; Sancenon, F. Chromogenic, specific detection
of the nerve-agent mimic DCNP (a tabun mimic). Chemistry 2011, 17, 6931–6934. [CrossRef] [PubMed]
1. Pascual, L.; Sayed, S.E.; Martínez-Máñez, R.; Costero, A.M.; Gil, S.; Gaviña, P.; Sancenón, F.
Acetylcholinesterase-Capped Mesoporous Silica Nanoparticles That Open in the Presence of
Diisopropylfluorophosphate (a Sarin or Soman Simulant). Org. Lett. 2016, 18, 5548–5551. [CrossRef]
[PubMed]
2
2
2
2
2. Sarkar, H.S.; Ghosh, A.; Das, S.; Maiti, P.K.; Maitra, S.; Mandal, S.; Sahoo, P. Visualisation of DCP, a nerve
agent mimic, in Catfish brain by a simple chemosensor. Sci. Rep. 2018, 8, 3402. [CrossRef] [PubMed]
3. Chen, L.; Wu, D.; Yoon, J. Recent Advances in the Development of Chromophore-Based Chemosensors for
Nerve Agents and Phosgene. ACS Sens. 2018, 3, 27–43. [CrossRef] [PubMed]
4. Royo, S.; Martínez-Máñez, R.; Sancenón, F.; Costero, A.M.; Parra, M.; Gil, S. Chromogenic and fluorogenic
reagents for chemical warfare nerve agents’ detection. Chem. Commun. 2007, 4839–4847. [CrossRef]
5. El Sayed, S.; Pascual, L.; Agostini, A.; Martinez-Manez, R.; Sancenon, F.; Costero, A.M.; Parra, M.; Gil, S. A
Chromogenic Probe for the Selective Recognition of Sarin and Soman Mimic DFP. ChemistryOpen 2014, 3,
1
42–145. [CrossRef] [PubMed]
2
6. Khan, M.S.J.; Wang, Y.-W.; Senge, M.O.; Peng, Y. Sensitive fluorescence on-off probes for the fast detection of
a chemical warfare agent mimic. J. Hazard Mater. 2018, 342, 10–19. [CrossRef] [PubMed]
2
7. Ha, S.; Lee, M.; Seo, H.O.; Song, S.G.; Kim, K.S.; Park, C.H.; Kim, I.H.; Kim, Y.D.; Song, C. Structural Effect of
Thioureas on the Detection of Chemical Warfare Agent Simulants. ACS Sens. 2017, 2, 1146–1151. [CrossRef]
[PubMed]
2
2
8. Kumar, V.; Rana, H.; Raviraju, G.; Garg, P.; Baghel, A.; Gupta, A.K. Chromogenic and fluorogenic multianalyte
detection with a tuned receptor: refining selectivity for toxic anions and nerve agents. RSC Adv. 2016, 6,
5
9648–59656. [CrossRef]
9. Yang, Y.K.; Keunjeong Yook, A.; Tae, J. A Rhodamine-Based Fluorescent and Colorimetric Chemodosimeter
for the Rapid Detection of Hg2+ Ions in Aqueous Media. J. Am. Chem. Soc. 2005, 127, 16760–16761. [CrossRef]
[PubMed]
3
0. Goswami, S.; Manna, A.; Paul, S. Rapid ‘naked eye’ response of DCP, a nerve agent simulant: from molecules
to low-cost devices for both liquid and vapour phase detection. RSC Adv. 2014, 4, 21984–21988. [CrossRef]
1. So, H.-S.; Angupillai, S.; Son, Y.-A. Prompt liquid-phase visual detection and low-cost vapor-phase detection
of DCP, a chemical warfare agent mimic. Sens. Actuators B: Chem. 2016, 235, 447–456. [CrossRef]
3
3
3
2. Ramette, R.W.; Sandell, E.B. Rhodamine B Equilibria. J. Am. Chem. Soc. 1956, 78, 4872–4878. [CrossRef]
3. Gotor, R.; Gaviña, P.; Ochando, L.E.; Chulvi, K.; Lorente, A.; Martínez-Máñez, R.; Costero, A.M. BODIPY
dyes functionalized with 2-(2-dimethylaminophenyl)ethanol moieties as selective OFF–ON fluorescent
chemodosimeters for the nerve agent mimics DCNP and DFP. RSC Adv. 2014, 4, 15975–15982. [CrossRef]

Molecules 2019, 24, 827
12 of 12
3
3
3
3
3
3
4. Yang, X.-F.; Guo, X.-Q.; Zhao, Y.-B. Development of a novel rhodamine-type fluorescent probe to determine
peroxynitrite. Talanta 2002, 57, 883–890. [CrossRef]
5. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G.A.; et al. Gaussian 09, Revision D. 01; Gaussian. Inc.: Wallingford, CT, USA, 2009.
6. Lee, C.; Yang, W.; Parr, R.G. Development of the Colle-Salvetti correlation-energy formula into a functional
of the electron density. Phys. Rev. B 1988, 37, 785–789. [CrossRef]
7. Becke, A.D. A new mixing of Hartree–Fock and local density-functional theories. J. Chem. Phys. 1993, 98,
1
372–1377. [CrossRef]
8. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98,
648–5652. [CrossRef]
9. Hehre, W.J.; Ditchfield, R.; Pople, J.A. Self—Consistent Molecular Orbital Methods. XII. Further Extensions
of Gaussian—Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 1972
6, 2257–2261. [CrossRef]
0. Francl, M.M.; Pietro, W.J.; Hehre, W.J.; Binkley, J.S.; Gordon, M.S.; DeFrees, D.J.; Pople, J.A. Self-consistent
molecular orbital methods. XXIII. A polarization-type basis set for second-row elements. J. Chem. Phys. 1982
7, 3654–3665. [CrossRef]
5
,
5
4
,
7
41. Bauernschmitt, R.; Ahlrichs, R. Treatment of electronic excitations within the adiabatic approximation of
time dependent density functional theory. Chem. Phys. Lett. 1996, 256, 454–464. [CrossRef]
42. Ming, W.W.; Wiberg, K.B.; Frisch, M.J. Solvent effects. 3. Tautomeric Equilibria of Formamide and 2-pyridone
in the Gas Phase and Solution: an ab Initio SCRF Study. J. Am. Chem. Soc. 1992, 114, 1645–1652. [CrossRef]
43. Sadik, O.A.; Land, W.H., Jr.; Wang, J. Targeting Chemical and Biological Warfare Agents at the Molecular
Level. Electroanalysis 2003, 15, 1149–1159. [CrossRef]
Sample Availability: Samples of compounds 1–2 are available from the authors.
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