Ratiometric sensing of nerve agent mimic DCP through in situ benzisoxazole formation

Article abstract of DOI:10.1016/j.dyepig.2019.107585

In recent year syria rejuvenate interest in chemical warfare agents (CWAs) for terrorist attacks. Nerve agents are the most scandalous chemicals and can cause death of a person owing to the inhibition of acetyl cholinesterase activity. In this article, we

Full text of DOI:10.1016/j.dyepig.2019.107585

Accepted Manuscript
Ratiometric sensing of nerve agent mimic DCP through in situ benzisoxazole
formation
Syed Samim Ali, Ankita Gangopadhyay, Ajoy Kumar Pramanik, Uday Narayan Guria,
Sandip Kumar Samanta, Ajit Kumar Mahapatra
PII:
S0143-7208(19)30143-3
DOI:
https://doi.org/10.1016/j.dyepig.2019.107585
Article Number: 107585
Reference:
DYPI 107585
To appear in: Dyes and Pigments
Received Date: 18 January 2019
Revised Date: 14 February 2019
Accepted Date: 24 May 2019
Please cite this article as: Ali SS, Gangopadhyay A, Pramanik AK, Guria UN, Samanta SK, Mahapatra
AK, Ratiometric sensing of nerve agent mimic DCP through in situ benzisoxazole formation, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107585.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Ratiometric sensing of nerve agent mimic DCP through in situ
Benzisoxazole formation
Syed Samim Ali, a Ankita Gangopadhyay, a Ajoy Kumar Pramanik,a Uday Narayan Guria,a
Sandip Kumar Samanta a and Ajit Kumar Mahapatra*a
aDepartment of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur,
Howrah – 711103, India.
*Corresponding author: Tel.: +91 33 2668 4561; fax: +91 33 26684564;
E-mail: mahapatra574@gmail.com, akmahapatra@rediffmail.com
DCP
H
O
O
N
OH
N
N
N

ACCEPTED MANUSCRIPT
Ratiometric sensing of nerve agent mimic DCP through in situ Benzisoxazole
formation
Syed Samim Ali, a Ankita Gangopadhyay, a Ajoy Kumar Pramanik,a Uday Narayan Guria,a Sandip
Kumar Samanta a and Ajit Kumar Mahapatra*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Keywords: Ratiometric probe; chemical warfare agents; vapor phase detection in filter paper
strip; detection of DCP in soil sample; DFT/TDDFT calculations; cellular imaging; crystal
structure analysis.
Abstract
In recent year syria rejuvenate interest in chemical warfare agents (CWAs) for terrorist attacks.
Nerve agents are the most scandalous chemicals and can cause death of a person owing to the
inhibition of acetyl cholinesterase activity. In this article, we report a colorometric and
ratiometric fluorescent probe 4-diphenylamino-2-hydroxy benzaldehyde oxime (TPOD) that
could be efficient of instant and real-time recording of DCP vapour. Upon reaction with DCP,
bathochromic shift from ~463 to ~532 nm was observed due to the inhibition of PET from
oxime-hydroxyl to the triphenyl amine moiety and cyclization to the benzisoxazole. The
transformation from oxime to benzisoxazole was confirmed by NMR and HRMS data. The probe
TPOD can be applied on a portable and cost effective test kits where in presence of DCP vapor
dramatic color and fluorescence change were observed within 30 secs. Furthermore, the probe
can efficiently detect DCP in presence of other nerve agents and electrophile and the detection
limit is 0.14 µM. Moreover to evaluate the sensing mechanism we have prepared a control
compound TPD whose crystal structure and non covalent interactions have been incorporated.
To achieve the on-site application of TPOD we have performed an experiment in soil sample to
detect trace amount of DCP. Moreover the probe can be useful in cellular imaging of DCP
containing bio-samples. Thus the probe TPOD is an auspicious, instant and on site monitoring
ratiometric fluorescent probe for selective DCP detection in presence of other interfering nerve
agents.
1. Introduction
Organophosphorous compounds (OPs) include agricultural pesticides, herbicides and chemical
warfare agents (CWA) i.e; nerve agents and are regarded as being the most poisonous chemicals
to living things ever discovered or synthesized [1-3]. However, the substantial use of these
compounds in agricultural industry around the world has generated the poisoning of thousands of
humans. In 1936, during synthesis of pesticides, the first nerve agent (Tabun, GA) was

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discovered as a by-product [4-6]. OPs, such as sarin, soman, and tabun have also been utilized as
nerve agents in chemical warfare [7-8]. So, the potential exposure to chemical warfare agents
(CWAs) due to terrorist and military activity has thus become a serious threat to our soldiers and
civilians [9-12]. It is evident that the primary toxicity of nerve agents comes from the inhibition
of acetylcholine esterases (AChE) by reaction with reactive phosphate group of OPs with the
hydroxyl groups of AChE, that prevents the decomposition of acetylcholine. As a result, rapid
death of a human, paralysis of central nervous system and organ failure may occur [13,14].
Therefore, a reliable, facile and selective methods are required to resolve the problem. Various
sensing methods for nerve agents have been reported [15-17], such as mass spectroscopy/gas
chromatography [18-21], electrochemical sensor [22,23], enzymatic sensor [24-26], and
interferometry [27-29]. However, these methods have some limitations, such as limited
selectivity, low sensitivity, slow response, lack of specificity, operational complexity, non-
portability, costly and inconvenient for real-time detection and monitoring [30-32]. So, it is
highly appealing to develop a probe which can show distinct changes both in color and
fluorescence in the presence of OPs. In this direction, fluorescent chemosensors have attracted
significant attention owing to their portability, low cost, highly sensitivity, and easy operation in
recent years [33-35]. Generally, diethylchlorophosphate (DCP) is claimed as a nerve-agent
mimic owing to its low toxicity but similar reactivity of NAs and hence, a cheap and easy
method is most desired for DCP detection.
In this article, we report a new ratiometric chemosensor based on an o-hydroxyl substituted
triphenylaminoaldehyde oxime group that can detect nerve agent simulant diethyl
chlorophosphate (DCP) very rapidly and selectively in aqueous acetonitrile solution. It comes to
light that by deprotonation of an oxime, supernucleophile oximate is formed and is capable of
attacking the strong electrophilic organophosphates and accordingly it has been incorporated into
sensor system to detect nerve agents [36, 37]. In addition, the oxime group is a well-known
medical antidotal treatment for nerve agent exposure [38-41] because “supernucleophile” oxime
is capable to restore the serine–OH from the phosphorylated serine–OH in AChE by the
attacking at internal phosphorus (serine-O-P to serine-OH), and thus the function of AChE
reactivates [42-47]. Besides, the o-hydroxy group of the chemosensor enhance the rate of
cyclization to generates the benzisoxazole product with a greatly increased sensitivity [48-51].
Moreover, for sensing a target analyte a ratiometric method offers an alternative approach to
overcome the disadvantage of intensity based calculations by integrated correction of two
emission bands. Finally, ratiometric probe can be employed to detect liquid and gaseous nerve
agents [52-55]. Hence, we obtained a concise colorimetric and ratiometric fluorescent probe,
bearing an electron donating amine group and rate enhancing o-hydroxyl functionality in the
structure, that could realize fast response and is capable of real-time and onsite recording of DCP
vapor. A naked-eye detection limit down to 0.14 µM level is remarkably achieved, which makes
it a highly optimistic fluorescent sensor for a simple, rapid and low-cost detection for DCP . We
have reported another derivative (TPD) which is devoid of the ortho-hydroxyl group. In this
molecule the characteristic spectrophotometric properties was observed but upon interaction with

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DCP appreciable change in absorbance and fluorescence were not observed to the same extent.
This proves the participation of ortho-OH in the chemodosimetric reaction mechanism. However
the compound TPD has been treated as a control compound whose crystal structure (Fig.8) and
analytical data (ESI†) have been recorded.
2. Experimental Section
2.1. Chemicals and physical quantification
All the chemicals were obtained from sigma-aldrich and were used without further purification.
13
For NMR spectra, TMS was used as an internal standard and DMSO-d6 as solvent. 1H and C
NMR spectra were measured on a Brucker 400 MHz instrument. Chemical shifts and 1H–1H and
1H–C coupling constants are expressed in δ ppm and in Hz units. Mass spectra and fluorescence
spectra were done on a Waters QTOF Micro YA 263 mass spectrometer and Perkin Elmer
Model LS 55 spectrophotometer.
2.2. Structure optimization study
All calculations for density functional theory (DFT) and Time-dependent density functional
theory have been performed using Gaussian 09 program. Optimization of structures have been
done using the B3LYP/6-31G (d, p) level of theory. TDDFT calculation has also been carried out
at the same level.
2.3. Calculation of detection limit
The detection limit of TPOD was calculated on the basis of absorption and fluorescence
titration. The absorption and fluorescence spectrum of TPOD was measured 10 times, and to get
the slope, the ratio of the absorption and fluorescence intensity at 405 nm and 532 nm were
plotted as a concentration of DCP. Detection limit was calculated from following equation:
Detection limit =3Sb1/S where S is the slope of the calibration curve and Sbl is the standard
deviation of the blank measurement.
2.4. Method of solution preparation for UV−Vis titration
Stock solution of TPOD and TPD (0.4 µM) was prepared in CH3CN/H2O (1:1v/ v). All
experiments were performed in CH3CN/H2O solution (CH3CN/H2O = 4:6 v/v, 10 mM HEPES
buffer, pH = 7.4). During titration each time 2 ml solution of TPOD (8 µM) was charged in a
quartz optical cell (optical path length= 1 cm). By using a micropipette slowly the stock solutions
of analyte (8 µM) were put in the quartz optical cell.
2.5. Method of solution preparation for fluorescence titration
Stock solution of TPOD and TPD (0.4 µM) was prepared in CH3CN/H2O (4:6 v/ v). All
experiments were performed in CH3CN/H2O solution (CH3CN/H2O = 4:6 v/v, 10 mM HEPES

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buffer, pH = 7.4). During titration each time 2 ml solution of TPOD (8 µM) was charged in a
quartz optical cell (optical path length= 1 cm). By using a micropipette slowly the stock solutions
of analyte (8 µM) were put in the quartz optical cell.
2.6. Synthetic procedures
2.6.1. Preparation of compound 1
Phosphorous Oxychloride (POCl3) (3 ml) and DMF (3 ml) were placed in a two neck round
bottomed flask, Then the flask was dipped in an ice bath, the stirrer was started, and 3-
methoxytriphenylamine (500 mg, 1.81 mmol) dissolved in DMF (1 ml), was added to the POCl3
and DMF mixtures over a period of eight minutes. After completing the addition the mixture was
again stirred for 15 minutes. Then the ice bath was separated and again the mixture was allowed
to heat at 55-60°C for 6 hr., cooled to the room temperature and poured into ice water. The
aqueous part was extricated four times with ethyl acetate. Then organic layer was collected in a
beaker and dried over sodium sulfate. Organic solvents were removed in rotary evaporator under
reduced pressure. Finally the residue was purified by column chromatography using ethyl acetate
in hexane (10% v/v) to give pure 3-methoxytriphenylaminoaldehyde. Light yellow solid, (320
mg, 58.3%), mp: 150-160oC, MS (LCMS): (m/z, %): 321.2 [(Compound 1+H2O), 100 %];
1
Calculated for C20H17NO2: 303.12. H-NMR (DMSO-d6, 400 MHz): δ (ppm) 10.01 (s, 1H), 7.47
(d, J = 8.4 Hz, 1H,), 7.32 (t, 4H), 7.05 (m, 6H), 6.96 (s, 1H), 3.39 (s, 3H).
2.6.2. Preparation of compound 2
BBr3 (1 ml, 1.47 mmol) was added to the solution of compound 1 (300 mg, 0.98 mmol) in
CH2Cl2 (2 ml) at 0 °C under a nitrogen atmosphere. The mixture was allowed to stir at 0 °C for
0.5 h and at room temperature for 3 h. Then the reaction mixture was cooled to 0 °C, and
carefully MeOH (100 mL) was added. The solvent was removed in rotary evaporator and the
residue was added to MeOH (100 mL). This process was continued three times. The residue was
diluted with organic solvent DCM, the precipitate was filtered and washed with DCM to
give compound 2 (160 mg, 56.4%) as a yellow solid, mp >180 °C. MS (LCMS): (m/z, %): 313.2
[(Compound 2+H++Na), 100 %]; Calculated for C19H15NO2: 289.32. 1H NMR (400 MHz,
DMSO-d6,): δ (ppm) 10.75 (s, 1H), 9.29 (s, 1H), 7.45 (d, J = 8 Hz, 1H), 7.37 (m, 1H), 7.25
(d, J = 8.4 Hz, 3H), 7.11 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8 Hz, 4H), 6.97 (d, J = 8 Hz, 2H).
2.6.3. Preparation of compound TPOD
NH2OH-HCl (28 mg, 0.40 mmol) and K2CO3 (71 mg, 0.51 mmol) were added to the solution of
compound 2 (100 mg, 0.34 mmol) in 5 mL CH3OH. The mixture was stirred at room
temperature over 5 h, then 30 mL water was added and separated four times with 50 mL EtOAc.
The organic part was dried over anhydrous Na2SO4 and concentrated to dryness in a rotary
evaporator under reduced pressure. Finally the residue was refined by column chromatography
(Hexane/EtOAc, 1:2, v/v) to yield TPOD (60 mg, 58%) as a white solid. mp: 130-140oC, MS

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(LCMS): (m/z, %): 327.10 [(TPOD+ Na), 100 %]; Calculated for C19H16N2O2: 304.34. 1H NMR
(400 MHz, DMSO-d6), d: 11.01 (s, 1H), 9.36 (s,1H,), 8.20 (s, 1H), 8.06 (s, 1H), 7.54 (m, 1H),
7.35(t, 1H), 7.13 (t, 2H), 7.04 (t, 2H), 6.56 (t, 2H).; 13C NMR (100 MHz, DMSO-d6), δ (ppm)
162.15, 151.55, 146.53, 134.99, 133.62, 129.16, 118.32, 117.12, 115.43.
2.6.4. Preparation of compound TPD
NH2OH-HCl (30 mg, 0.43 mmol) and K2CO3 (75 mg, 0.54 mmol) were added to the solution of
3 (100 mg, 0.36 mmol) in 5 mL CH3OH. The mixture was stirred at room temperature over 5 h,
then 30 mL water was added and separated four times with 50 mL EtOAc. The organic phase
was dried over anhydrous Na2SO4 and concentrated to dryness in a rotary evaporator under
reduced pressure. Finally the residue was refined by column chromatography (Hexane/EtOAc,
1:2, v/v) to yield TPD (65 mg, 63%) as a light green solid. mp: 140-150oC, MS (LCMS): (m/z,
%): 289.3 [(TPD+H+), 100 %]; Calculated for C19H16N2O: 288.34.
2.6.5. Preparation of compound TPODP complex
In a small round bottomed flask TPOD (20 mg, 0.06 mmol) was taken in 2 ml acetonitrile and to
this mixture DCP (20.7 mg, 0.06 mmol) was added dropwise. After the completion of the
reaction acetonitrile was evaporated in rotary evaporator. To get pure compound purification was
done by column chromatography (Hexane: EtOAc = 9:1(v/v)). Finally we got pure product as a
light yellow solid. MS (LCMS): (m/z, %): 304.1269 [TPODP] + H2O, 100 %]; Calculated for
C19H14N2O: 286.1106. 1H-NMR (CDCl3, 400 MHz): δ (ppm) 8.03 (s, 1H), 7.45(d, J=8.4 Hz,
1H), 7.29 (m, 4H), 7.04 (m, 6H), 6.91 (d, J=8.4 Hz, 2H). 13C-NMR (DMSO-d6, 100 MHz): δ
(ppm) 162.14, 151.55, 146.53, 134.99, 133.62, 129.16, 118.32, 117.12, 115.43.
3. Results and discussion
3.1. Synthesis and characterization
Following Vilsmeier-Haack synthesis procedure we first prepared 4 – diphenylamino - 2-
methoxy benzaldehyde (Compound1) then demethylation of the Compound1 was carried out
using BBr3 in dichloromethane [56-61]. Finally probe TPOD has been synthesized with the
reaction of hydroxyl amine in methanol. We confirmed the structural identification of the probe
1
13
TPOD by mass spectroscopy, H NMR and C NMR (ESI†). We first observed the spectral
response of TPOD towards DCP in 4:6 v/v CH3CN–H2O in HEPES buffer solution. When we
added DCP to TPOD rapidly ratiometric color change from cyan to green was observed in
fluorescence lamp.The reaction is highly dependent on solvent system and the efficient changes
was observed at pH 7.4 in CH3CN–H2O (4:6 v/v). Thus the probe is applicable to detect DCP
under the change in relative humidity.

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OH
OCH3
OH
CHO
CHO
N
c
b
OH
N
N
N
1
2
TPOD
a
OCH3
CHO
N
OH
c
N
N
N
TPD
3
Scheme 1. Reagents and Conditions: (a) DMF, POCl3, Heat 80oC, 6 hrs (b) BBr3, CH2Cl2, r.t, 5
hrs (c) NH2OH, K2CO3,MeOH, r.t, 5 hrs.
3.2. Color and fluorescence response of TPOD towards DCP
Inspection of the absorption spectrum of TPOD upon treatment with DCP (Fig.1a) shows that a
new band appears at ~405 nm. The band at ~329 nm and ~ 358 nm were gradually decreased and
an isosbestic point at ~ 379 nm was appeared. Thus an increase in the long wavelength band and
decrease in the short-wavelength band with an isosbestic point at ~379 nm shows that the
sensing reaction is a single and promising converted process. The images displayed in inset of
Figure 1a showed that the addition of DCP to the aqueous CH3CN solution of TPOD causes a
distinct color change from colorless to yellow. Figure 1b shows the change of absorption ratio
with successive addition of DCP (0-10 equiv).
As shown in Figure 2a, fluorescence spectra of 1 µM probe TPOD solution upon addition of
different amount of DCP (0-10 equiv) exhibits a gradual change in the detection mode in a
ratiometric way when excited at 379 nm (ϵ379 = 11× 104 M-1 cm-1). Initially it shows a band at ~
463 nm (ϕ = 0.005) which is gradually decreases and a new peak at ~ 532 nm (ϕ = 0.23)
gradually increases with the addition of DCP and saturation came after 10 equiv addition of
DCP. The interaction of TPOD and DCP was clearly investigated by the appearance of an
isoemissive point at ~ 491 nm. In fact the interaction is clearly visible through the color change
from cyan to green. Based on the Job’s plot (Fig. S17†) it has been proved that the stoichiometry
of the formation of compound TPODP between probe TPOD and DCP is 1:1. The ratio of
fluorescence intensities of probe TPOD at ~ 463 and ~ 532 nm (I463/ I532) showed a momentous
change from 0.782 to 37.05 upon charged with DCP (Fig. 2a), a ~48 fold inflation in the
ratiometric emission. Limit of detection (LOD) of TPOD towards DCP was calculated (Fig.
S15† and S16†) in terms of the formula (3σ/k) and are 0.14 µM and 0.23µM from absorption and
fluorescence data through fitting a straight line. Now the plots of fluorescence intensities ratio vs
concentration of DCP has been shown in Fig. 2b. In case of our control compound (TPD) the
change in the spectra (Fig. S13† and S14†) was observed only due to the inhibition of PET from

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the oxime-OH as a result of DCP binding. In probe TPOD the spectral response is facilitated by
the cyclization brought about by the ortho-OH group.
3.3. Interference study of TPOD in presence of active electrophiles
To assess the selectivity of TPOD for DCP, we performed the UV-Vis and fluorescence
response of TPOD to some active electrophiles and nerve agents [62-65], such as thionyl
chloride (SOCl2), acetyl chloride (CH3COCl), phosphorous oxychloride (POCl3), oxalyl chloride
(COCl)2, triethyl phosphate (TEP), diisopropyl fluorophosphates (DFP), tributyl phosphate
(TBP), diethylcyano phosphonate (DCNP) and dimethylmethyl phosphonate (DMMP). As
shown in Figure 3a and 3b, among DCP and these interfering substances, only DCP induced an
obvious ratiometric naked eye color change from colorless to yellow and a ratiometric
fluorescence change from cyan to green was observed. But the fluorescence of the TPOD probe
remained undisturbed or was only slightly increased in presence of various interfering agents
(Figure S18†). The probe molecule TPOD has no response with DCNP and DFP because of
having worse leaving aptitude of cyanide and fluoride. Although DCNP, DFP, SOCl2, POCl3 etc
can react with the –OH group, but they have no impact on the fluorescence of TPOD due to the
unique intramolecular cyclization process that is reliant upon the properties of Cl- as a good
leaving group and this sensing event is successful for DCP.
3.4. Time dependent fluorescence investigation
Next we studied response time of TPOD at different amount of DCP. We investigated the
fluorescence response of TPOD over a (0- 1 min) incubation time at the different concentration
range of DCP (0- 10 equiv). The fluorescence intensity at ~ 532 nm increases and at ~ 463 nm
decreases with increasing concentration of DCP and at higher concentration of DCP promoted
the reaction to complete within a min. The change of fluorescence with time graph (Fig. 4a)
explored that the reaction of the TPOD and DCP could be accomplished within a minute. As
expected, with increasing the concentration of DCP the fluorescence intensity at ~532 nm
increases and higher concentrations of DCP showed a quick response, and the color and
fluorescence changes are easily perceptible to the naked eye and in UV lamp under ~379 nm
irradiation. The pseudo-first order rate constant (kobs) calculated for the DCP-prompted
̶ Ft)/Fmax] = ̶
cyclization is k/ = 0.149s-1 (Fig. 4b). Following the eqn ln[(Fmax k/t [66, 67], Where
Fmax and Ft are the fluorescence intensities at 532 nm at a maximum value acquired after the
reaction is terminated and at time t and k/ is the observed pseudo-first-order rate constant, the
rate constant k/ was calculated.
3.5. Computational Studies
To explain optical responses of probe TPOD upon reaction with DCP, density functional theory
(DFT) and time dependent density functional theory (TDDFT) calculations were performed
using the B3LYP/6-31G(d, p) level of Gaussian 09 program [68, 69]. Energy optimized
structures molecular orbital of HOMO and LUMO for compound TPOD and TPODP were

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shown in Fig. 5. For TPOD, the electron density in the HOMO was diffused over the whole
molecule but in LUMO the electron density was mainly accumulated on the oxime group
attached phenyl ring. As for TPODP, the electron density in HOMO and LUMO orbitals was
mostly spread over the benzisoxazole moiety. From Fig. 5 we found that the energy gap for the
TPODP (3.749 eV) was diminished as compared to the probe TPOD (4.074 eV) (Table S3†),
indicating the formation of cyclized product.
Now the experimentally observed UV−Vis spectra were compared with the TDDFT of probes
TPOD and TPODP. It is noteworthy that computationally found data to have compatibility with
the experimental data. TDDFT calculations showed absorption band at ~348 and ~305 nm
corresponds to the electronic transition from HOMO→LUMO (69.81%) (f = 0.6493) and
HOMO→LUMO+2 (58.48%) (f = 0.2069) energy states respectively for probe TPOD (Table
S2†) and absorption band at ~371 and ~402 nm corresponds to the electronic transition from
HOMO-1→LUMO+1(61.45%) (f = 0.1076) and HOMO→LUMO (71.68%) (f = 0.3641) energy
states respectively for probe TPODP. These values are concurrent with the absorbance bands of
the two probes TPOD and TPODP thus the theoretical study supports the experimentally
observed absorption bands.
3.6. Reaction path of DCP sensing
To validate the reaction mechanism, when DCP was added to the acetonitrile solution of TPOD
(procedure is given in experimental section) the oxime hydroxyl group get phosphorylated due to
its extreme nucleophilicity and then intramolecular nucleophilic attack from phenolic-OH to the
oxime nitrogen leads to the formation of benzisoxazole derivative TPODP that has been
characterized by HRMS (Fig. S8†) and NMR spectra (Fig. S9†).
H
O
O
P
H
OH
OEt
OEt
O
O
N
N
DCP
N
N
TPOD
O
N
O
P
+
N
OEt
HO
OEt
TPODP
Scheme 2 Plausible reaction mechanism
The structure of TPODP and probable mechanism has been shown in Scheme 2. In case of
control compound after reaction with DCP, only the oxime hydroxyl get phosphorylated and
TPDP was formed [70, 71]. The mass spectra of TPDP has been given in supporting
information.

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3.7. Recognition of DCP vapor in a sealed vial
We have studied the detection of DCP in gaseous state with probe TPOD immersed filter paper.
A small slice of filter paper test kit was dipped in a CH3CN solution contained probe TPOD (1
µM) and 10 mM HEPES buffer. Then the probe-loaded filter paper was kept in a sealed vial
containing DCP (10 equiv) in CH3CN solution (Fig. 6). As time proceeded (0-30 sec) in day light
filter paper turned from colorless to yellow and in UV lamp cyan color had almost disappeared
and green color was appeared. This result implies that our probe TPOD may serve as a
prospective test kit for on-spot detection of DCP vapour.
3.8. Detection of DCP in soil sample
We explored our probe to confirm the presence of DCP in soil samples. In a beaker 20 µL of
DCP was spiked in 6.0 g of soil. Then the spiked soil samples were permitted to stand for 2
hours and separated using chloroform. The chloroform part was evaporate in rotary evaporator
and the residual part was dissolved in CH3CN. Finally the dissolved part was treated with 20 µM
TPOD solution. Immediately green color was appeared with the spiked sample, while the blank
did not give any response (Fig. 7). As Our probe shows ratiometric color change in presence of
DCP, so it would not have any practical problem in field trial method.
3.9. Cellular imaging
To further investigate the potential application of TPOD for DCP in biological samples, confocal
microscopy imaging inside Vero 76 cells (Vero 76, ATCC no. CRL-1587) were performed.
Firstly, we have checked the cytotoxicity of probe TPOD by MTT assays in Vero 76 cells (Fig.
S19†). The result suggesting that low micromolar concentrations of TPOD were almost free of
toxicity to the Vero 76 cells and thus this sensor is suitable in the cell imaging.
Firstly Vero 76 cells incubated with probe TPOD (10
0.5%DMSO exhibited blue intracellular fluorescence (Fig. 8B) indicating that TPOD is cell permeable.
Interestingly when the cells were incubated with 10 M of TPOD for 20 min and further exposed with
DCP (40 M) for another 5min, blue fluorescence was completely replaced with green fluorescence after
µM) for 20 min at 37 °C in PBS buffer with
µ
µ
5 min (Fig. 8D). Here the bright field images did not show any morphological changes that suggested that
Vero 76 cells were viable. These results demonstrate that the probe had effective cellular permeability and
possess high resolution in cell imaging. Moreover the ratiometric changes can easily be observed by little
changes in DCP levels.
3.10. Single crystal x-ray analysis of TPD
A shinny and X-ray quality single crystal of green colored probe TPD was grown by gentle
evaporation of solvent from a solution in MeOH–CH3CN (1:1) at room temperature with
ambient condition over few days. The probe crystallizes as monoclinic system with space group

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P21/n and CCDC entry no. is 1870349. A summary of the crystallographic parameters and
structure refinement details of the probe are given in Table S4†. All calculations were carried out
using SHELXL-97 [72,73], ORTEP-32 [74], and PLATON-99 [75] programs. The molecular
structure of TPD (Fig. 9) is stabilized by a head to tail type cyclic intermolecular hydrogen
bonding which is dimeric in nature. In addition to hydrogen bonding, each dimer alternately
repeats along b-axis and generates a 1-dimensional supramolecular association (Fig. 10) through
a aromatic C ̶ H....π interaction. Each molecule simultaneously participates in ring O—H
(donor).....imine ˗ N (acceptor) hydrogen bonding in the oxime part of the molecule forming a
2
chain of centro symmetric rings with single R2 (6) motif. The hydrogen bonding interaction of
O1—H1…..N2 with donor ˗H.....Acceptor distances of 2.799(4) Å to the nearest unit.
Supramolecular framework is attained under alternative C
the repeating molecule along b-axis (ac plane) and this C
nature with C H …Cg (2) distance of 2.73(Å).
̶
H....π interaction incorporating with
̶
H....π interaction are aromatic in
̶
4. Conclusion
In summary, we have developed a ratiometric fluorescent probe for instant, selective and
sensitive detection of DCP vapour. In TPOD inhibition of PET process from oxime-hydroxyl to
the triphenyl amine moiety and cyclization to the benzisoxazole are responsible for ratiometric
color change from cyan to green. We have developed a control compound TPD and compared
spectral responses with TPOD. In the crystal structure of TPD we have studied C ̶ H....π and H-
bonding interactions. Probe TPOD can detect DCP in vapour phase as well as in liquid phase
and detection limit of the probe calculated from absorption and fluorescence data are 0.14µM
and 0.23µM. Practical observations are well correlated with theoretical calculation by DFT and
TDDFT. Furthermore, the probe can be employed to detect trace amounts DCP (10−50 ppm) in
soil sample and used in ratiometric fluorescent cellular imaging, which make it an efficient probe
for on-site visual detection of environmental DCP gas. We believe that rapidity and selectivity
made our present probe highly appealing and multifaceted.
Acknowledgements
We are delighted to acknowledge CSIR-India [Project No. 02(0334)/18/EMR-ll] for financial
assistance. SSA would like to give special thanks to UGC-MANF (Student ID: WES-67786),
New Delhi for the research grant.
Author Information
aDepartment of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur,
Howrah-711103, West Bengal, India, Email: akmahapatra@rediffmail.com, Fax:
+913326684564
†Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/b000000x/

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Figure Captions
Fig. 1 (a) Absorption spectra of TPOD (1µM) in CH3CN–H2O (4:6 v/v) in presence of DCP (40
µM) and 10 mM HEPES buffer, pH 7.4, at 25oC. (b) Plot of absorption ratio change and
concentration of DCP.
Fig. 2 (a) Fluorescence spectra of TPOD (1µM) in CH3CN–H2O (4:6 v/v) in presence of DCP
(40 µM) and 10 mM HEPES buffer, pH 7.4, at 25oC, λex = 379 nm. (b) Plot of fluorescence ratio
change and concentration of DCP.
Fig. 3 (a) Relative absorption spectra of TPOD (1µM) in CH3CN-H2O (4:6 v/v, 10 mM HEPES
o
buffer, pH 7.4 at 25 C) in presence of DCP (10 equiv) and 50 equiv of CH3COCl, SOCl2,
POCl3, (COCl)2, TEP, DFP, TBP, DCNP and DMMP. (b) Relative fluorescence spectra of of
TPOD (1µM) in CH3CN-H2O (4:6 v/v, 10 mM HEPES buffer, , pH 7.4 at 25 oC) in presence of
DCP (10 equiv) and 50 equiv of CH3COCl, SOCl2, POCl3,(COCl)2, TEP, DFP, TBP, DCNP and
DMMP.
Fig.4 (a) Time dependent fluorescence enhancement of TPOD (1 µM) with increasing
concentration of DCP (0- 10 equiv) in CH3CN-H2O (10 mM HEPES buffer, 4:6 v/v, pH 7.4 at
25oC) solution, λex = 379 nm; λem = 532 nm. (b) Pseudo first order rate constant plot of TPOD
(1 µM) in presence of DCP (40 µM).
Fig.5 Energy minimized structure and HOMO-LUMO energy gap of TPOD and TPODP.
Fig.6 Photograph of color and fluorescence response of TPOD upon exposure to DCP (0-10
equiv) for 0-30 sec. Photographs were taken in day light and in UV-lamp.
Fig.7 Fluorescence spectra of free TPOD, Unspiked sample and Spiked sample.
Fig.8 Confocal microscopy imaging of TPOD in Vero 76 cells (Vero 76, ATCC no. CRL-1587):
(A) Bright field image of the cells stained with probe TPOD at 1.0×10-6 M, (B) Image scan of
the probe TPOD at 1.0×10-6 M concentration for 20 min at 37 oC, (C) Merged images of B and
A, (D) Bright field image of the cells treated with TPOD at 1.0×10-6 M and DCP at
concentration 4.0×10-5 M, (E) Image scan of the cells treated with probe TPOD at 1.0×10-6 M
for 20 min and further incubated with DCP at concentration 4.0×10-5 M for 5 min; and green
fluorescence signal was detected. (F) Merged images of D and E. λex= 379 nm, λem= 460- 540
nm. Scale bar, 100 µm.
Fig.9 Ortep diagram of TPD, showing 35% thermal ellipsoids probability level for non-H atoms
with atom numbering scheme.
Fig.10 The crystal packing diagram of TPD shows head to tail O-H…N hydrogen bonded cyclic
dimmer and each dimer constitutes a 1-D supramolecular network involving C
interaction viewed along the b-axis (H atoms not involved are omitted in for clarity).
̶ H....π

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Fig. 1
b)
a)
TPOD+
DCP
TPOD
Fig. 2
a)
b)
Fig. 3
a)
b)

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Fig. 4
b)
a)
Fig. 5
-1.160 eV
LUMO
-1.268 eV
LUMO
E=3.740
E=4.074
HOMO
-5.017 eV
HOMO
-5.234 eV
TPOD
TPODP

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Fig. 6
Sec
0
15
0
15
30
30
Day light
UV-lamp
Fig. 7
Spiked sample + TPOD
TPOD alone
Unspiked sample +TPOD
Fig. 8
Bright field
Merged
Em: 460-540 nm
A)
D)
B)
E)
C)
F)

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Fig. 9
Fig. 10

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Ratiometric sensing of nerve agent mimic DCP through in situ Benzisoxazole
formation
Syed Samim Ali, a Ankita Gangopadhyay, a Ajoy Kumar Pramanik,a Uday Narayan Guria,a Sandip
Kumar Samanta a and Ajit Kumar Mahapatra*a
aDepartment of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur,
Howrah – 711103, India.
*Corresponding author: Tel.: +91 33 2668 4561; fax: +91 33 26684564;
E-mail: mahapatra574@gmail.com, akmahapatra@rediffmail.com
Highlights
1. 4-diphenylamino-2-hydroxy benzaldehyde oxime has been synthesized for the ratiometric
detection of DCP.
2. Ratiometric color and fluorescence change were achieved due to cyclization and PET
mechanism.
3. The probe TPOD can be utilised in-field detection of DCP by using a portable filter paper
strip.
4. Probe TPOD has an exceptional selectivity towards DCP over other nerve agents and active
electrophiles.
5. The probe TPOD can able to detect DCP in soil sample and biological sample also.
6. Significance of ortho hydroxyl group in TPOD could be justified by comparing with a probe
(TPD) which does not contain that functionality.