Chromogenic detection of nerve agent mimics

Article abstract of DOI:10.1039/b811247a

A new chromogenic protocol for the selective detection of nerve agent mimics is reported. The Royal Society of Chemistry.

Full text of DOI:10.1039/b811247a

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Chromogenic detection of nerve agent mimicsw
Ana M. Costero,*a Salvador Gil,a Margarita Parra,a Pedro M. E. Mancini,a
b
a
b
´ ´ ´ ´
Ramon Martınez-Manez,* Felix Sancenon and Santiago Royo
´ ˜
Received (in Cambridge, UK) 2nd July 2008, Accepted 2nd September 2008
First published as an Advance Article on the web 14th October 2008
DOI: 10.1039/b811247a
A new chromogenic protocol for the selective detection of nerve
agent mimics is reported.
Remaining with the versatility of the last approach and at the
same time being interested in the design of colorimetric probes we
envisioned that the coupling of the dimethylaminophenyl donor
group with an acceptor moiety (A) could be a suitable approach
for the design of tailor-made colorimetric probes for nerve agents’
detection. We expected that the conversion of the tertiary amine I
to the quaternary ammonium III upon reaction with certain
organophosphorus (OP) substrates would dramatically change
the electronic donor properties of the nitrogen atom thus resulting
in a decrease of the push–pull character of the dye and in a colour
modulation.
The current rise in international concern over criminal terrorist
attacks via chemical warfare (CW) agents has resulted in an
increasing interest in the detection of these lethal chemicals.
Among CW species, nerve agents are extremely dangerous and
their high toxicity and ease of production underscore the need to
detect these deadly chemicals via quick and reliable procedures. A
number of detection systems have been developed, most of them
based on enzymatic and physical methodologies.1 However, these
usually show limitations such as low selectivity, lack of portability
and a certain complexity in their use. An alternative to these
classical methods that has been gaining interest in recent years is
the development of fluorogenic or chromogenic chemosensors or
reagents.2 Reported paradigms involve PET-based fluorescent
probes,3 colorimetric probes with oximate-containing deriva-
tives,4 molecular imprinting polymers,5 nanoparticles,6 carbon
nanotubes,7 porous silicon8 or displacement-like assays.9 Most
of these reported protocols rely on fluorescence changes and as
far as we know chromogenic systems for nerve agent detection in
aqueous solution or as vapour are rare. Some reported colori-
metric examples employ enzymatic methods and are based on the
inhibition of the activity of the enzyme acetylcholine esterase.10
Despite these interesting examples, colorimetric detection is par-
ticularly appealing as it requires low-cost and widely used in-
strumentation and offers the possibility to detect ‘‘with the naked
eye’’ target analytes in rapid semi-quantitative assays. Following
our interest in using supramolecular-inspired concepts for the
development of chromogenic protocols, we report herein the
design of colorimetric probes for nerve agents.
As a proof-of-the-concept and using 2-(2-(dimethylamino)-
phenyl)ethanol (DAPE) as a building block we prepared the
chromoreactand 1 (see Scheme 2).z The starting compound
DAPE was synthesized from 2-nitrotoluene that was in a
first step converted into 2-(2-nitrophenyl)ethanol following a
previously described method.11 Then, a reductive amination
in the presence of formaldehyde gives rise to 2-(2-(dimethyl-
amino)phenyl)ethanol.12 The new probe 1 was prepared from
the reaction of DAPE with the diazonium salt of 4-nitroaniline
using known procedures for the preparation of azo dyes.13
As a first step, the reactivity of 1 was tested with diethyl
chlorophosphate (DCP), diisopropyl fluorophosphate (DFP)
and diethyl cyanophosphate (DCNP) in acetonitrile. These
organophosphates have been widely used as simulants as they
display a reactivity similar to that of nerve agents such as
Tabun, Sarin and Soman, yet they lack their toxicity. Aceto-
nitrile solutions of chromoreactand 1 display an absorption
spectrum typical of azo dyes with an intense absorption band
(log e 44) at 410 nm. This yellow band has a charge-transfer
character due to the presence of an electron donor anilinium
group and a nitrophenyl moiety acting as electron acceptor.
Upon addition of 100 equiv. of DCP to acetonitrile solutions
of chromoreactand 1 a hypsochromic shift of 85 nm (Fig. 1)
was observed that resulted in a modulation from yellow to
colourless. This shift to shorter wavelengths is consistent with
the intramolecular cyclization process; i.e. the N,N-dimethyl-
anilino moiety drastically reduces its donor character
Traditionally the development of colorimetric probes usually
relies on the advanced design of two components: (i) a tailor-made
binding or reactive site and (ii) a colorimetric event. For the
former we selected the suitable properties of 2-(2-(dimethylamino)-
phenyl)ethanol derivatives (see Scheme 1). This group contains a
nucleophile, the hydroxyl moiety, which is known to undergo
acylation reactions with phosphonate substrates to form inter-
mediate II that suffers a rapid intramolecular N-alkylation to yield
III, a quaternary ammonium salt.3
a
´
´
´
Departamento de Quımica Organica, Facultad de Ciencias Quımicas,
Universitat de Valencia, 46100 Burjassot Valencia, Spain.
E-mail: ana.costero@uv.es
b
´
´
Instituto de Quımica Molecular Aplicada, Departamento de Quımica,
Universidad Politecnica de Valencia, Camino de Vera s/n, 46022
´
Scheme 1 Colorimetric sensing protocol. A 2-(2-(dimethylamino)-
phenyl)ethanol (DAPE) group acts as donor in charge-transfer dyes
such as I containing an acceptor group (A). In III there is a drastic
reduction of the donor ability of the amine group resulting in a
remarkable weakening of the charge-transfer (CT) band.
Valencia, Spain. E-mail: rmaez@qim.upv.es
w Electronic supplementary information (ESI) available: 1H-NMR
and 13C-NMR spectra of receptor 1 and 1H-NMR spectrum of
receptor 2. See DOI: 10.1039/b811247a
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Fig. 2 Chemical structures of the warfare agent simulants.
Scheme 2 Transformation of the azo dye 1 to the corresponding
ammonium derivative via acylation with a phosphate substrate and
further intramolecular cyclization.
pseudo first-order kinetic conditions with an excess of the
corresponding simulant was studied. As has been suggested for
similar systems, the reaction of the alcohol with the phosphate
derivatives to form the phosphate ester intermediate is slow
relative to the intramolecular cyclization.3 Monitoring the
absorbance intensity at 410 nm and plotting ln [(A0 ꢂ A)/A]
versus time (where A0 is the final absorbance and A is the
absorbance at a given time) allowed the determination of the
rate constants (k) with values of 0.13 sꢂ1, 0.061 sꢂ1 and
0.20 sꢂ1 for DCP, DCNP and DFP, respectively. Therefore
the half-life was calculated (t1/2 = ln 2/k) to be approximately
5.3 s, 11.4 s and 3.5 s for DCP, DCNP and DFP, respectively.
Fig. 3 shows the absorbance decrease of the visible band of dye
1 on reaction with DCP.
Fig. 1 Normalized absorption spectra of: (a) chromoreactand 1
(acetonitrile, 1.0 ꢁ 10ꢂ5 mol dmꢂ3), (b) 1 with 100 equiv. of DCP
and (c) the cyclization product obtained on reaction of 1 with
p-toluenesulfonyl chloride (see text). The inset photographs show the
colour of probe 1 and the bleaching observed upon addition of DCP.
Motivated by these favourable features we took a step forward
towards the potential use of 1 for in situ sensing and rapid
screening applications. For this purpose the molecular
probe 1 was adsorbed on silica gel. First, a solution of 1
(1 ꢁ 10ꢂ5 mol dmꢂ3) and MES (5 ꢁ 10ꢂ2 mol dmꢂ3) in
acetonitrile–water (1 : 3) was prepared. Then, 2 mL of this
solution were adsorbed on silica gel (500 mg) and air-dried, finally
resulting in a light orange solid. In a first test, the coloured
support containing 1 was placed in a 1000 mL flask that
contained ca. 5 ppm of DCP vapour (obtained by evaporation
of a solution of the simulant into the flask volume). A clear
change from orange–yellow to colourless was observed for the
sensor-loaded silica gel.
(vide ante). A similar behaviour was observed in the presence
of DFP and DCNP. For instance, a detection limit of
1.0 ꢁ 10ꢂ4 mol dmꢂ3 for DCNP and DCP was found.
Additionally, in order to assess the mechanism involved in
the observed chromogenic response, compound 2 was also
prepared following a different synthetic route involving the
reaction of chromoreactand 1 with p-toluenesulfonyl chloride
in the presence of sodium carbonate in acetonitrile. This gives
the corresponding tosylated derivative that suffered a sponta-
neous cyclization to give 2. The formation of 2 is easily
detected in the 1H-NMR spectra of the final product
that shows one triplet centered at 4.36 ppm indicative of a
methylene subunit located near the quaternary ammonium.
Also, a significant long-range 1H–13C coupling between this
signal and those 13C signals corresponding to the methyls
(54 ppm) and to the aromatic carbon (149 ppm) attached to
the N,N-dimethylanilinium fragment points toward the
unambiguous formation of the cyclized product. It was
unambiguously confirmed that the product obtained by tosyla-
tion was the same as that obtained from the reaction of 1 with
the DCP simulant (see Fig. 1 for the UV spectrum), indicating
that the reaction in Scheme 1 is responsible for the colour
modulation.
In a second test, the orange–yellow coloured silica was
placed on a column and a water–acetonitrile 75 : 25 v/v solution
containing DCP buffered at pH 7 (CDCP = 1.0 ꢁ 10ꢂ3 mol dmꢂ3
,
MES 5.0 ꢁ 10ꢂ2 mol dmꢂ3) was passed through. As the silica
comes into contact with the sample solution it becomes colourless
(see Fig. 4). In both tests the response time was a few seconds. The
bleaching of the coloured silica support containing 1 in the
presence of vapours or mixed aqueous solutions of DCP is
consistent with the reaction shown in Scheme 2.
In a further step, the reactivity of 1 with the organophos-
phorus (OP) compounds shown in Fig. 2 was studied in mixed
water–acetonitrile 75 : 25 v/v mixtures buffered at pH 7 (MES
5.0 ꢁ 10ꢂ2 mol dmꢂ3). As stated above, a selective bleaching
was found in the presence of DCP, DFP and DCNP, whereas
solutions stay orange–yellow for the remaining OP species (no
reaction was observed even after a long time). In order to
achieve a better understanding of the reaction, the reactivity
between 1 and DCP, DCNP or DFP in acetonitrile under
Fig. 3 Absorption changes at 410 nm upon reaction of 1 with DCP.
Inset: first-order kinetic plot.
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room temperature, then it was neutralized with Na2CO3 and extracted
with CH2Cl2. The dried (MgSO4) organic phases were evaporated to
give a dark oil that was dissolved in ethyl acetate. Upon addition of
hexane, a dark red precipitate appeared, identified as 1 (1.2 g, 63%),
mp 98–100 1C. 1H NMR (CDCl3, 400 MHz) d 8.36 (d, J = 9.0 Hz,
2H), 7.98 (d, J = 9.0 Hz, 2H), 7.85 (d, J = 8.1 Hz, 1H), 7.81 (s, 1H),
7.26 (d, J = 8.1 Hz, 1H), 3.95 (t, J = 5.8 Hz, 2H), 3.10 (t, J = 5.8 Hz,
2H), 2.83 (s, 6H) ppm; 13C{1H} NMR (CDCl3, 100 MHz): d 156.7,
155.9, 148.8, 148.4, 135.4, 125.4, 124.7, 123.9, 123.2, 119.9, 63.8, 44.6,
35.8 ppm; IR (neat) 2948, 2873, 1591, 1517, 1345, 1099, 1039, 858 and
827 cmꢂ1. HRMS-EI m/z 314.1371 [M+; calcd. for C16H18N4O3:
314.1379].
Fig. 4 Left: a glass tube containing silica gel with dispersed 1.
Right: silica gel containing 1 after the addition of water–acetonitrile
75 : 25 v/v mixtures containing DCP buffered at pH 7 (CDCP = 1.0 ꢁ
10ꢂ3 mol dmꢂ3, MES 5.0 ꢁ 10ꢂ2 mol dmꢂ3).
Synthesis of 2: To a stirred mixture of 1 (63 mg, 0.2 mmol) and K2CO3
(56 mg, 0.4 mmol) in acetonitrile (1 mL), tosyl chloride (36 mg,
0.2 mmol) in acetonitrile (0.5 mL) was slowly added. After stirring
at room temperature for 6 h, toluene (5 mL) was added. The formed
precipitate was filtered to give 2 (as its tosylate salt) (46 mg, 0.1 mmol)
as a white solid, mp 4350 1C. 1H NMR (CD3OD, 400 MHz) d 8.46
(d, J = 8.8 Hz, 2H), 8.15 (m, 4H), 8.02 (d, J = 8.7 Hz, 1H), 7.72 (d,
J = 7.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 4.36 (t, J = 7.2 Hz, 2H),
3.65 (s, 6H), 3.59 (t, J = 7.2 Hz, 2H) ppm; 13C{1H} NMR (CDCl3,
100 MHz): d 155.2, 153.8, 149.0, 142.1, 140.4, 128.4, 125.6, 124.9,
124.6, 123.6, 120.3, 118.2, 68.5, 54.0, 26.6, 19.9 ppm; IR (neat) 3082,
2961, 1628, 1398, 1381, 1127, 1007, 831, 701, and 664 cmꢂ1. HRMS-EI
m/z 297.1355 [M+; calcd. for C16H17N4O2: 297.1352].
In summary, a new chromoreactand for the colorimetric
sensing of nerve agent simulants has been prepared. This
chromoreactand displays an intramolecular cyclization reac-
tion coupled with a colour change upon interaction with
certain nerve agent simulants. The facts that the probe retains
its signalling abilities upon adsorption on silica and displays
colour modulations to nerve agent simulants as both vapours
or in mixed aqueous solution make this method a suitable
alternative to other systems used in the detection of toxic nerve
agents. It is noteworthy that the chemosensor 1 reacts only
with DCP, DFP and DCNP that show close chemical struc-
tures to Sarin, Soman and Tabun, whereas 1 remains silent in
the presence of other organophosphorus derivatives such as
OP-2–5. Additionally, the approach is highly modular in
concept bearing in mind that a number of acceptor groups
can be anchored to the 2-(2-(dimethylamino)phenyl)ethanol
donor moiety (see Scheme 1) pointing towards the potential
preparation of a range of colorimetric probes for the detection
of nerve agents based on push–pull chromophores.
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z Synthesis of 2-(2-N,N-dimethylaminophenyl)ethanol: 2-nitrotoluene
(9.2 g, 67.2 mmol), sodium phenoxide (0.06 g, 0.56 mmol) and
paraformaldehyde (95%) (0.9 g, 25 mmol) in DMSO (20 mL) were
heated under stirring at 60–70 1C for 1 h. The cold reaction mixture
was poured into water (20 mL) and extracted with ethyl ether. The
combined organic phases were washed (aq. NaCl), dried (MgSO4),
evaporated and the residue was distilled (Kugelrohr, 160 1C,
0.2 mmHg) to yield 2-(2-nitrophenyl)ethanol (4.3 g, 37%). Spectral
data were in agreement with those described in the literature.8 Then,
2-(2-nitrophenyl)ethanol (3.30 g, 19.7 mmol), formaldehyde (37%)
(4.83 mL, 32.3 mmol), abs. ethanol (200 ml) and Pd/C (10%) (480 mg)
were placed under an H2 atmosphere until the uptake of hydrogen
ceased. After filtration through Celite the solvent was evaporated and
the residue was dissolved in chloroform and extracted with aq. HCl
(1 M, 2 ꢁ 50 mL). The aqueous phase was basified with sat. Na2CO3
and extracted with chloroform. After drying (MgSO4) the solvent was
evaporated to give 2-(2-N,N-dimethylaminophenyl)ethanol (2.85 g,
84%) as a light yellow oil.
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1H NMR (CDCl3, 400 MHz) d 7.3–7.0 (m, 4H), 5.3 (br. s, 1H), 3.85
(t, J = 11.6 Hz, 2H), 3.01 (t, J = 11.6 Hz, 2H), 2.70 (s, 6H) ppm;
13C{1H} NMR (CDCl3, 100 MHz): d 152.1, 136.0, 131.1, 127.6, 124.9,
120.0, 64.2, 44.9, 36.0 ppm; IR (neat) 3367, 3059, 3020, 2937, 2360,
2826, 2785, 1597, 1492, 1451, 1046, 945, 768 and 749 cmꢂ1. HRMS-EI
m/z 165.1153 [M+; calcd for C10H15NO: 165.1154].
Synthesis of 1: 4-Nitroaniline (0.836 g, 6.05 mmol), conc. sulfuric acid
(1.25 mL, 23 mmol) and water (8 mL) were stirred and slightly heated
until the 4-nitroaniline was completely dissolved and then the mixture
was placed in an ice-bath (0–5 1C) for 10 min before a NaNO2 (0.418 g,
6.05 mmol) solution in water (4 mL) was added dropwise. After
stirring for an additional 10 min at 0–5 1C, a solution of the amine
synthesised above (1.0 g, 6.05 mmol), conc. HCl (1.3 mL, 15.1 mmol)
and water (4 mL) was added dropwise during 15 min. The resulting
orange solution was stirred for 15 min in an ice-bath and 15 min at
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This journal is The Royal Society of Chemistry 2008
6004 | Chem. Commun., 2008, 6002–6004