A unique dual sensor for the detection of DCNP (nerve agent mimic) and Cd2+ in water

Article abstract of DOI:10.1039/c9nj03327k

Both DCNP (nerve gas Tabun mimic) and Cd²⁺ are extremely toxic to mankind. We have focused our research on quantitative estimation of DCNP and Cd²⁺ in given water samples. A carbazole-pyrrole conjugate CPC has been designed and synthesized which possesses a unique characteristic feature of detecting two hazardous analytes DCNP and Cd²⁺ by giving two different optical responses i.e. visual color changes and fluorescence "turn-on" respectively. The PET based chemosensor CPC produces a new compound CPC-1 and a highly fluorescent CPC-Cd²⁺ complex upon addition of DCNP and Cd²⁺, respectively. The detection limit of CPC for DCNP and Cd²⁺ has been estimated to be 7.75 nM and 0.27 μM, respectively, at pH 7.0. Quantum chemical calculations have been executed to demonstrate the electronic properties and the mechanism of the receptor-donor interactions. The concentrations of DCNP and Cd²⁺ ions in various water samples have been estimated with the help of standard absorbance and fluorescence curves.

Full text of DOI:10.1039/c9nj03327k

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A unique dual sensor for the detection of DCNP
(nerve agent mimic) and Cd2+ in water†
Cite this: New J. Chem., 2019,
43, 16968
Ayndrila Ghosh, ‡ Sujoy Das, ‡ Saurodeep Mandal
and Prithidipa Sahoo
*
Both DCNP (nerve gas Tabun mimic) and Cd2+ are extremely toxic to mankind. We have focused our
research on quantitative estimation of DCNP and Cd2+ in given water samples. A carbazole–pyrrole con-
jugate CPC has been designed and synthesized which possesses a unique characteristic feature of
detecting two hazardous analytes DCNP and Cd2+ by giving two different optical responses i.e. visual
color changes and fluorescence ‘‘turn-on’’ respectively. The PET based chemosensor CPC produces a
new compound CPC-1 and a highly fluorescent CPC–Cd2+ complex upon addition of DCNP and Cd2+
,
respectively. The detection limit of CPC for DCNP and Cd2+ has been estimated to be 7.75 nM and
0.27 mM, respectively, at pH 7.0. Quantum chemical calculations have been executed to demonstrate
the electronic properties and the mechanism of the receptor–donor interactions. The concentrations of
DCNP and Cd2+ ions in various water samples have been estimated with the help of standard absorbance
and fluorescence curves.
Received 26th June 2019,
Accepted 10th October 2019
DOI: 10.1039/c9nj03327k
rsc.li/njc
of these nerve agents. By satisfying the characteristics of a
practical and real time sensing system, in recent years,
Introduction
Toxicity, the word that interprets the degree of damage of an chemosensors14–22 have become superior to almost all conven-
organism caused by a chemical substance or a particular tional sensing methods. This is why, we have streamed our
mixture of substances, has been a very well-known concept to research in this field and designed a new chemosensing probe,
human civilization for many years. Effects of toxicants, whether carbazole–pyrrole conjugate (CPC), that not only can accomplish
it is chemical, physical or pathogens, are dose dependent; even a selective, rapid naked eye detection of the tabun mimic –
pure water can act as a toxicant when taken in too high a dose diethylcyanophosphonate (DCNP) – in the nanomolar range by
by biological systems.1 In the performing stage of chemical forming a new compound CPC-1 but also has been applied to
toxicants, nowadays, various analogues of organophosphate quantify the DCNP concentration in various water samples
appear to be promising candidates as nerve agents. These nerve (performance comparison Table S1, ESI†).
agents disrupt the function of the acetylcholinesterase enzyme
Surprisingly, it has been observed that when CPC detects the
which catalyzes the neurotransmitter, acetylcholine. This dis- presence of DCNP by a colorimetric change from colorless to
ruption results in an effective paralysis of muscles including strong yellow, it is also able to give a vivid fluorescence ‘‘turn-
the heart muscle and the muscles used for breathing2 finally on’’ response to the exposure of cadmium (Cd2+) ions, a heavy
leading to death.3,4 Among all the nerve agents i.e. G-series, metal toxicant. The vigorous use of cadmium in phosphate
V-series, carbamates, Novichok agents and insecticides, the fertilizers, rechargeable nickel–cadmium batteries, silver–cadmium
G-series nerve gases – tabun (GA), sarin (GB), and soman batteries, artists’ colors, chalk pastels, smelting and refining of
(GD) – claim the most attention due to their usage as warfare metals, electroplating, television phosphors, QLED TVs, semicon-
agents.5–9 From their debut at the terrorist attack in the Tokyo ductors, photoconductors, quantum dots, control rods of nuclear
subway in 1995 to the recent blast on 4 April 2017 in Khan reactors, the paint used on our house-hold crockeries, plastics and
Shaykhun, a city of Syria, the fatal destructive power of nerve even in jewellery makes it important in agricultural and industrial
gases has been exhibited again and again.10–13 This dreadful areas respectively.23–26 But this very same metal ion, i.e. Cd2+, owing
threat to mankind forced researchers to focus on the detection to a long half-life of 15–20 hours in the human body, can exert an
extremely toxic effect through a strong bond formation with
sulphur, resulting in the displacement of essential metal ions such
Department of Chemistry, Siksha Bhavana, Visva-Bharati University, Santiniketan,
as Zn2+ and Ca2+ from the binding sites of certain enzymes.27–33
731235, India. E-mail: prithidipa@hotmail.com
Cadmium is readily accumulated in the cells, kidneys, liver, lungs
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
and stomach with ensuing physical disorders, e.g. headache,
c9nj03327k
‡ These authors contributed equally to this work.
dizziness, chest pain, fever, pulmonary oedema, tracheo-bronchitis,
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Fig. 1 (a) UV-vis absorption spectra of CPC (20 mM) in acetonitrile–water
(1 : 6, v/v) and 10 mM phosphate buffer (pH 7.0), upon addition of DCNP.
(b) Pseudo first-order kinetics plot for the reaction of CPC with DCNP.
Scheme 1 Visual and fluorescence colour changes among CPC, CPC-1
and the CPC–Cd2+ complex.
stability (Fig. S7, ESI†). A time dependent absorbance intensity
graph shows the formation of CPC-1 from CPC within 5 seconds
(Fig. S8, ESI†). The pseudo-ꢁfi1rst-order rate constant (k0) has been
calculated to be k0 = 1.26 s (Fig. 1b) according to eqn (1):
osteomalacia, proximal renal tubular dysfunction and carcinogenic
effects.34–38 According to the International Agency for Research on
Cancer, the Environmental Protection Agency (EPA) and the
European Chemical Agency, Cd2+ has been classified as a human
carcinogen and the WHO (World Health Organization) has fixed
the limit of Cd2+ to be 3 ppb in drinking water.39–42 So, in the
field of toxicology, cadmium(II) has attracted immense research
interest.
ln[(Amax ꢁ At)/Amax] = ꢁk0t
(1)
where At and Amax are the absorbance at 417 nm at time ‘t’ and
the maximum value obtained after the reaction is complete,
respectively, and k0 is the observed pseudo-first order rate
constant.
Our probe CPC can selectively sense Cd2+ along with DCNP
at the very same time both qualitatively and quantitatively
(Scheme 1). CPC was synthesized with 70% yield through a
simple one-pot reaction using just two reagents: 3-amino-9-
ethylcarbazole and pyrrole-2-carboxaldehyde. The structure of
the probe was deduced by 1H NMR, 13C NMR and mass spectral
studies (Fig. S1–S3, ESI†).
Spectral behaviour of CPC–Cd2+ with CPC
The absorbance study of CPC with Cd2+ showed a gradual
decrease in absorbance at 304 and 344 nm (Fig. 2a) due to
the termination of photoinduced electron transfer (PET) from
the relatively high energy non-bonding electron pair on the
pyrrole nitrogen to the fluorophore – the carbazole moiety.
Here, the absorbance plot signifies a different mode of attach-
ment between CPC and the cadmium(II) ion from that of DCNP.
Fluorimetric titration reveals 17-fold hike in fluorescence intensity
at 435 nm (lex = 380 nm) after sequential addition of Cd2+ solution
(up to 5 equiv.) to a 20 mM probe solution (Fig. 2b). Probably a
non-covalent interaction between the cadmium ion and the imine
nitrogen in CPC initiates a chelation enhanced fluorescence
(CHEF) effect and the non-fluorescent probe exhibits a strong blue
‘‘turn-on’’ fluorescence response.
Results and discussion
To establish the interactive properties of probe CPC towards its
two target analytes i.e. DCNP and Cd2+, a number of analytical
and spectral techniques such as UV-vis, fluorescence, 1H NMR,
13C NMR, 31P NMR titration studies and TD-DFT calculations
have been exhaustively carried out and explained.
The binding affinity of the CPC probe towards Cd2+ has been
measured through linear regression analysis and the value is
2.83 ꢀ 104 Mꢁ1 (Fig. S9, ESI†). From the detection limit calcula-
tion taking the linear range of 0–3.5 mM, it was found that CPC
Spectral behaviour of CPC-1 with CPC
UV-vis titration has been performed in acetonitrile–water (1 : 6, v/v),
buffered with 10 mM phosphate buffer (pH 7.0). The successive
addition of 5 equiv. of DCNP (40, 80 and 100 mM) to CPC (20 mM)
lowers the absorbance at 344 nm while at 417 nm the absorbance
is increased significantly with a clear isosbestic point at 375 nm.
This phenomenon is probably due to the attachment of a phos-
phonate group to probe CPC resulting in a new compound CPC-1
which exhibits an intramolecular charge transfer (ICT) from the
carbazole to pyrrole moiety (Fig. 1a). The formation of CPC-1
from CPC is justified by its high association constant value
(44.9 ꢀ 105 Mꢁ1) (Fig. S4, ESI†). The detection limit of CPC for
DCNP is 7.75 nM (Fig. S5, ESI†) where the standard curve is in
the concentration range of 0–0.06 mM. Job’s plot analysis reveals
a 1 : 1 interaction stoichiometry between CPC and DCNP (Fig. S6,
ESI†). The product, CPC-1, is quite stable in a wide range of pH
from pH 6 to pH 14 though an acidic environment disturbs the
Fig. 2 (a) UV-vis absorption spectra of CPC (20 mM) in acetonitrile–water
(1 : 6, v/v) and 10 mM phosphate buffer (pH 7.0), upon addition of Cd2+
.
(b) Fluorescence emission spectra (lex = 380 nm) of CPC (20 mM) in aceto-
nitrile–water (1 : 6, v/v), neutral pH, with 5 equiv. of Cd2+
.
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can detect Cd2+ at the limit concentration of 0.27 mM (Fig. S10,
ESI†). The plot of fluorescence intensity as a function of different
host/guest ratios shows a stoichiometric ratio of 1 : 1 for the
CPC–Cd2+complexation (Fig. S11, ESI†) and a plot of fluores-
cence intensity as a function of time confirms that a maximum
of 30 seconds are required to complete this complexation
(Fig. S12, ESI†). As the CPC–Cd2+ complex is base sensitive
(Fig. S13, ESI†), all the spectral studies were performed in a
buffered solution of pH 7.0.
Selectivity of CPC
When the selectivity of CPC was tested, the colorimetric observa-
tion clarified that only the addition of DCNP to the probe could
give the strong yellow coloration from the colorless solution
of CPC whereas other nerve agent simulants, ROS, RNS, gaso-
transmitters and metal cations (Fe2+, Cu2+, Zn2+, Cd2+, Pb2+, Ni2+,
Ag2+, Hg2+, As3+, Mg2+ and Ca2+) including cadmium ions failed
to do that. Interestingly, under a UV lamp the fluorescence turn
on response of CPC–Cd2+ has been observed while the above said
guests do not interfere the inherent non-fluorescent character of
CPC (Fig. 3).
Fig. 5 Partial 1H NMR titration [400 MHz] of CPC in DMSO-d6 at 25 1C
and the corresponding changes after the gradual addition of one equiv. of
DCNP in D2O from (i) only CPC, (ii) CPC + 0.5 equiv. of DCNP, (iii) CPC +
1 equiv. of DCNP.
NMR titration
NMR titration studies have been done to elucidate the inter-
actions of CPC with DCNP and Cd2+. In 1H NMR titration, the
disappearance of the characteristic proton peak of the –NH func-
tional group in the pyrrole moiety at d 11.78 ppm after addition of
1 equiv. of DCNP in the probe solution justifies the formation of
the new compound, CPC-1. A downfield shift of carbazole protons
indicate the electron transition from the carbazole to pyrrole moiety
due to the formation of CPC-1 where the phosphonate of DCNP
abstracts electrons from carbazole through pyrrole nitrogen of CPC
(Fig. 5). The gradual shift of the imine proton (Ha) from 8.54 ppm
to 8.72 ppm and the proton–carbon coupling acquired from HMQC
spectra of both CPC and CPC-1 also corroborate the above electro-
nic transition (Fig. S14 and S15, ESI†).
These practical observations have also been long-established
from the comparative absorbance and fluorescence studies
done with the above mentioned analyte solutions (Fig. 4).
From further study of 31P NMR titration, the appearance of a
new peak at d ꢁ13.42 ppm also confirms the formation of the
new compound (Fig. 6).
On the other hand, in the CPC–Cd2+ complex, upon gradual
addition of 1 equiv. of Cd2+ to CPC, there is a downfield shift of the
pyrrole-NHb of about 0.17 ppm (Fig. 7) and the aromatic protons of
both carbazole and pyrrole have also shifted downfield which
indicates the chelation of the Cd(II) with the probe, CPC. Here,
we also observe the gradual downfield shift of imine proton Hc,
evidencing the strong chelation in the CPC–Cd2+ complex.
Fig. 3 Naked eye and fluorescence response of CPC (20 mM) to various
guest molecules (100 mM). From left to right: (1) only CPC, CPC with
(2) DCP, (3) DCNP, (4) NO, (5) CO, (6) H2S, (7) Fe2+, (8) Cu2+, (9) Cd2+
,
(10) Zn2+ and (11) Hg2+ in acetonitrile–water (1 : 6 v/v, pH 7.0, 10 mM
phosphate buffer) solution.
Mass spectral titration studies
Partial mass (HRMS) spectra have also been recorded for
the reaction mixtures of CPC with both DCNP and Cd2+ which
give new peaks at m/z: 423.17 and at m/z: 455.07 specifying
the formation of CPC-1 and CPC–Cd2+, respectively (Fig. S16
and S17, ESI†). These spectra also verify the 1 : 1 stoichiometric
interaction between the probe and the analytes.
Fig. 4 (a) Comparative absorbance diagram of CPC with various nerve
agent mimics, ROS, RNS and gasotransmitters added into acetonitrile–
water (1 : 6, v/v) and 10 mM phosphate buffer (pH 7.0). (b) Comparative
fluorescence diagram of CPC with different metal ions in acetonitrile–
water (1 : 6, v/v) and 10 mM phosphate buffer (pH 7.0).
Theoretical analysis
Ground state optimization of the structures and the time
dependent density functional theory (TDDFT) calculations for
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Fig. 8 Optimized structures of the trans- and cis-isomer of CPC.
resembles the enhanced absorbance at 417 nm obtained
experimentally.
On complexation with Cd2+, a sharp electronic transition
( f = 0.5608) from the S0 to the S2 state has occurred at 403 nm
which is comparable to the excitation wavelength for the
fluorimetric titration i.e. 380 nm. The HOMO–LUMO energy
gap of CPC is higher than that of the CPC-1 and CPC–Cd2+
complex by 0.20 eV and 0.28 eV of energy respectively (Fig. 9).
This theoretical study connects all the experimental findings.
Fig. 6 31P NMR titration [400 MHz] of (i) DCNP and (ii) CPC-1 in DMSO-d6 at
25 1C showing the corresponding changes after formation of the new product.
Plausible mechanism and explanation
All the theoretical and experimental findings directed us to
propose a schematic pathway by which probe CPC can detect
DCNP and Cd2+ at the same time (Scheme 2). In the case of
DCNP, the lone pair on pyrrole nitrogen attacks the phosphorous
centre of DCNP, forcing the adjacent cyanide group to leave
with the subsequent formation of CPC-1 via deprotonation
(Scheme S1, ESI†). The occurrence of PET from pyrrole to
carbazole in CPC quenches the fluorescence of carbazole and
makes the probe colorless but in the newly formed CPC-1,
the presence of the electron withdrawing phosphonate group
initiates the electron flow from the carbazole to pyrrole moiety
thus leading to an intramolecular charge transfer (ICT) and
develops the characteristic yellow colour of CPC-1.
In contrast, chelation of Cd2+ with CPC increases the rigidity
of the complex CPC–Cd2+ by restricting the free rotation of the
probe. Consequently, PET is terminated and the CPC–Cd2+
complex becomes highly fluorescent through the initiation of
the CHEF effect. So, it is obvious that different modes of
binding of these two analytes with CPC are the key reason for
Fig. 7 Partial 1H NMR titration [400 MHz] of CPC in DMSO-d6 at 25 1C and
the corresponding changes after the gradual addition of one equiv. of Cd2+
from (i) only CPC, (ii) CPC + 0.5 equiv. of Cd2+, (iii) CPC + 1 equiv. of Cd2+
.
the probe with both products, i.e. CPC-1 and the CPC–Cd2+ attainment of two different optical signals (Scheme 2).
complex, have been studied using the hybrid exchange–correlation
B3LYP functional (Becke, three parameter, Lee–Yang–Parr) and
6-311G** basis set implemented in the Gaussian 09 program
(Fig. S18, ESI†).43 The CPCM (Conductor like Polarizable Continuum
Model) solvent model was used for solvent (H2O) effect incorpora-
tion. In Tables S2–S4 (ESI†), all the energies of the optimized
structures, vertical electronic transitions, i.e., the calculated lmax
,
orbital transition and oscillator strength ( f ) are listed.
Although a cis–trans free rotation is possible in probe CPC,
theoretical calculation shows that the trans-isomer is more
favourable with about 3.64 kcal molꢁ1 lower energy compared
to the cis-isomer (Fig. 8 and Table S2, ESI†). The major transi-
tion in CPC at 366 nm (S0 - S1, f = 0.8193) is very close to that
observed at 344 nm experimentally. After addition of DCNP, the
new absorbance peak observed at 390 nm (S0 - S1, f = 0.8338)
Fig. 9 Orbital distributions of the HOMO and LUMO in CPC, CPC-1 and
the CPC–Cd2+ complex.
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Table 1 Determination of DCNP in water samples
Sample
Added (nM) Found (nM) Recovery (%)
Ground water (tap water)
10
15
20
9.95
14.27
19.84
99.5
95.1
99.2
Stagnant water (pond water) 10
9.41
14.35
19.09
94.1
95.6
95.4
15
20
Marine water (sea water)
10
15
20
9.09
13.99
19.00
90.9
93.2
95.0
Table 2 Determination of Cd2+ in water samples
Scheme 2 Proposed scheme for simultaneous detection of DCNP and
Cd2+ by CPC.
Sample
Added (mM) Found (mM) Recovery (%)
Ground water (tap water)
0.5
1.0
1.5
0.515
1.030
1.530
103.0
103.0
102.0
Quantitative analysis
To evaluate the quantification ability and the practical utility of
our probe CPC, various water samples, e.g., stagnant water,
ground water and marine water, were used, as water is the most
accumulated and important liquid in biosystems. Water bodies
are the ultimate dumping station of all wastes or toxic materials.
In fact, toxicants dumped in soil can contaminate the ground
water of that particular area. We took tap water and pond water
from Santiniketan, India, and sea water from Haldia Port area,
Midnapore, India, for this quantitative study. 10, 15 and 20 nM
of DCNP was added in each sample of volume 2 mL and
absorbance changes were measured. The DCNP concentration
was calculated using the standard curve of absorbance titra-
tion studies (Fig. 10). For all the samples the analyte recoveries
were greater than 90% (Table 1 and Fig. S19, ESI†).
Stagnant water (pond water) 0.5
0.555
1.090
1.620
111.0
109.0
108.0
1.0
1.5
Marine water (sea water)
0.5
1.0
1.5
0.510
1.04
1.530
102.0
104.0
102.0
Surprisingly it was noticed that for all the samples, recov-
eries of Cd2+ calculated were greater than 100%. This finding
confirmed the presence of the heavy metal toxicant, Cd2+, in
water of the areas from where the water samples were taken
(Table 2 and Fig. S20, ESI†).
Vapour phase detection
Fig. 11 Vapour phase detection of DCNP by CPC.
CPC has high ability to detect DCNP not only in the solution
phase but also in the vapour phase. We executed the detection
experiment of DCNP in the vapour phase using our probe by a dip
stick method. The experiment was conducted with the help of a
cellulose paper strip which we had soaked in the probe solution
and dangled in a stoppered conical flask containing DCNP
solution in it.§ After 5 minutes it was observed that the colorless
paper strip became yellow in color declaring the strong binding
affinity of CPC to DCNP even in its vapour phase (Fig. 11).
§ All the experiments done with DCNP were carried out in a sealed tube or a
stoppered conical flask because of the toxic by-product HCN produced during the
formation of the CPC-1 compound. The solution in the reaction vessel was diluted
100 times by adding water and then treated with sodium hypochlorite solution
(bleach) while the pH was maintained at greater than 10.0. The mixture was
monitored with the help of a potassium iodide paper strip by checking whether it
turned dark blue. If it turned, this confirmed the presence of excess bleach which
converted the amenable cyanide to much less toxic cyanate ions (–CNO). These
cyanate ions cannot reconvert to cyanide and moreover these will be dissociated
Fig. 10 Estimation of concentration of DCNP and Cd2+ in tap water
samples using the standard graph. All the values are an average of three
replicate measurements with standard deviation.
into ammonia and carbon dioxide by certain bacteria present in soil. 1H NMR, 13
C
NMR, and MS spectra, additional spectroscopic data, supplementary fluorescence
images, and theoretical calculation tables are given in the ESI.†
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Experimental section
Materials and instruments
287.14 (M + H+, 100%). Anal. calcd forC19H17N3: C, 79.41;
H, 5.96; N, 14.62; O, 9.57; found: C, 79.39; H, 5.92; N, 14.39.
3-Amino-9-ethyl carbazole, pyrrole-2-carboxaldehyde, cadmium,
iron, copper, mercury (all as metal perchlorate salts), diethylcyano-
phosphonate, and diethyl chlorophosphate were purchased from
Sigma-Aldrich Pvt. Ltd (India). Materials were obtained from
commercial suppliers and were used without further purification.
Solvents were dried maintaining the conditions of standard
procedures. Water used in all respective experiments was double
distilled water. 1H, 13C and 31P NMR spectra were recorded on a
Bruker 400 MHz instrument. For NMR spectra and for NMR
titration DMSO-d6, D2O were used as the solvent using trimethyl-
silane as an internal standard. Chemical shifts are expressed in
d ppm units and 1H–1H and 1H–C coupling constants in Hz.
The following abbreviations are used to describe spin multi-
Conclusion
To summarize, we have successfully developed a unique PET
based chemosensor, CPC, which can easily and rapidly detect
two deadly toxic analytes, e.g. DCNP which is a nerve agent
mimic and widely used metal toxicant Cd2+, at the very same
time by showing different optical signals, i.e. visual changes for
DCNP and fluorescence turn on for Cd2+. The ability of estimation
of guest concentrations even in the nano-molar range in water
samples makes CPC efficient as a simple, less time consuming
and cost-effective practical sensing system to estimate DCNP and
Cd2+ in in vitro and in vivo samples. In addition, CPC could also be
used as a potential tool to detect the presence of the nerve gas
mimic DCNP in the vapour phase.
1
plicities in H NMR spectra: s = singlet; d = doublet; t = triplet;
m = multiplet. The mass spectrum (HRMS) was recorded on
a micromass Q-TOF Microt instrument using acetonitrile as a
solvent. Fluorescence spectra were recorded on a PerkinElmer
Model LS55 spectrophotometer. UV spectra were recorded on a
SHIMADZU UV-3101PC spectrophotometer. Elemental analysis
of the compounds was carried out on a PerkinElmer 2400 series
CHNS/O Analyzer.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Synthesis of CPC
PS acknowledges UGC, India, for awarding her a start-up grant
[Project file no. F.30-7/2014 (BSR)]. AG and SD are sincerely
thankful to UGC and CSIR, India, respectively, for research
fellowships.
3-Amino-9-ethyl carbazole (0.42 g, 2 mmol) was dissolved in
MeOH (15 mL). Pyrrole-2-carboxaldehyde (0.19 g, 2 mmol) was
added to the solution and set to reflux for 12 hours (Scheme 3).
The solvent was removed by evaporation. Water (20 mL)
was added to the resultant mixture and extracted with CHCl3
(20 mL ꢀ 2). The combined organic phase was washed twice
with water, treated with brine and dried over Na2SO4. The
product was purified through column chromatography using
chloroform and ethyl acetate in 15 : 1 ratio as the eluent. After
solvent evaporation under reduced pressure, a chocolate brown
coloured solid product was obtained (0.40 g, 70% yield).
1H NMR (400 MHz, DMSO-d6): d (ppm) = 11.78 (s, 1H), 8.54
(s, 1H), 8.186–8.167 (d, J = 7.6 Hz, 1H), 8.060 (s, 1H), 7.622–
7.583 (t, J = 15.6 Hz, 2H), 7.472–7.413 (m, J = 23.6 Hz, 2H),
7.210–7.172 (t, J = 15.2 Hz, 1H), 7.077 (s, 1H), 6.763 (s, 1H),
6.248 (s, 1H), 4.468–4.416 (m, J = 20.8 Hz, 2H), 1.339–1.303
(t, J = 14.4 Hz, 3H). 13C NMR (400 MHz, CDCl3): d (ppm) =
148.91, 140.56, 126.35, 123.26, 122.76, 121.02, 120.39, 119.11,
111.94, 110.41, 109.96, 109.68, 40.62, 40.42, 39.79, 39.58, 39.37,
37.53, 14.20. HRMS: anal. calcd for C19H17N3: 287.14; found:
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Scheme 3 Synthesis of the CPC probe.
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