Detection of chemical warfare simulants by phosphorylation of a coumarin oximate

Article abstract of DOI:10.1039/b609861d

The detection of chemical warfare simulants is attained by the PET mechanism that gives an "off-on" fluorescent response with a half-life of approximately 50 ms upon phosphorylation of a reactive oximate functionality; the X-ray crystal structure of the oximate was also obtained and is discussed. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b609861d

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Detection of chemical warfare simulants by phosphorylation of a
coumarin oximate{
Karl J. Wallace,a Ruth I. Fagbemi,a Frantz J. Folmer-Andersen,a Jeroni Morey,b Vincent M. Lyntha and
Eric V. Anslyn*a
Received (in Cambridge, UK) 11th July 2006, Accepted 11th August 2006
First published as an Advance Article on the web 1st September 2006
DOI: 10.1039/b609861d
direction is obtained.12 Even though colorimetric systems have one
The detection of chemical warfare simulants is attained by the
particular advantage, i.e., the response can be seen with the naked
eye, a particular disadvantage is the sensitivity of the system. The
Centers for Disease Control and Prevention (CDC) has established
the concentration that is Immediately Dangerous to Life or
Health, IDLH value, of sarin and soman to be 0.1 mg m23
(1.7 ppm vapor). At these concentrations a colorimetric approach
is not sensitive enough. However, it is well known that fluorescence
allows for very high sensitivity, particularly when the signal is
turned ‘‘on’’ as demonstrated by both Rebek and Swager.4,9
Our design approach was to use coumarin aldehyde 110,13 which
is simply converted to an oxime functionality. Reacting 1 with
hydroxylamine hydrochloride and sodium hydroxide gave the
desired oxime 2a in respectable yield (Scheme 1). The analogous
methyloxime (2b) was also synthesized to act as a control.
Both compounds were characterised by NMR and HRMS
spectroscopy and all the spectroscopic data agreed with the
proposed compounds.
PET mechanism that gives an ‘‘off–on’’ fluorescent response
with a half-life of approximately 50 ms upon phosphorylation
of a reactive oximate functionality; the X-ray crystal structure
of the oximate was also obtained and is discussed.
The current rise in international concern over criminal terrorist
attacks using chemical warfare (CW) agents has brought about the
need for reliable and affordable detection of toxic gases. For
example, in March 1995 the Aum Shinrikyo sect killed 12 and
injured nearly 6000 people when sarin gas was released in a Tokyo
subway. Therefore, there has been a significant interest in the
decontamination1,2 and detection3,4 of CW agents. One approach
that has been studied uses chromogenic detector reagents, which
directly bind to, or react with, a target nerve agent, causing a
modulation in the absorbance spectra.5–8
Recently, Rebek developed a fluorescent sensor based on a PET
quenching mechanism of a fluorophore that upon cyclization
produced a strong fluorescent signal.9 A very similar concept was
also developed by Timothy Swager in 2003. He created a
fluorescent indicator with a dramatic spectral change created in
response to the cyclization of a flexible chromophore. Upon
exposure to a sarin surrogate (diisopropylfluorophosphate, DFP)
the system creates a fluorophore, causing an ‘‘off–on’’ response in
the micromolar concentration range.4a In both of these recent
examples a fluorescent signal is turned ‘‘on’’, which is a highly
desirable property. However, both systems use an alcohol as a
nucleophile, and the half-life for the reaction of an alcohol with
DFP (let alone that anticipated with sarin/soman) approaches an
hour.4b Due to the intrinsically slow rate of the reaction of alcohols
with phosphorus(V)-fluoride groups, we decided to explore a
fluorophore containing an oxime moiety, 2a. Upon deprotonation
an oximate ion, which is a well-known supernucleophile, is formed.
The fluorophore of choice was a coumarin, as Timothy Glass has
previously shown that an increase in fluorescence is observed for
the detection of amines via the reversible formation of imines in situ
with aldehyde 1.10,11
In order to observe whether the fluorophore 2a induced a color
change in the UV-Vis spectra, a set of experiments was carried out
by titrating aliquots of DFP{ (6.0 mol dm23 in DMSO) into a
2.5 6 1025 mol dm23 solution of 2a in DMSO. It is well known
that strong bases such as NaOH can react with DFP to form the
less toxic hydrolysis product.1 This is a common procedure for the
decontamination of CW agents. To avoid this, a nitrogen–
phosphorus super-base was used: the P4-t-Bu base
(
DMSOpKBH+ 5 30.25). This base is approximately six orders
of magnitude stronger than DBU (MeCNpKBH+ 5 24.3) but is
non-nucleophilic.
The initial UV-Vis solution of oxime 2a (2.5 6 1025 mol dm23
)
in DMSO showed a broad band at lmax 5 409 nm. On the
addition of two equivalents of the P4 base a band at lmax 5 443 nm,
assigned to the g–p* transition, appears.14 This shift in the red
direction is consistent with an anionic species. Upon the gradual
addition of aliquots of DFP the band is hypsochromically shifted
to lmax 5 409 nm through a pseudo isosbestic point at 425 nm
We have previously shown that a simple colorimetric response is
obtained when an oximate moiety is attached to a benzene core.
Upon phosphorylation with DFP a UV-Vis shift in the blue
aDepartment of Chemistry and Biochemistry, The University of Texas at
Austin, Austin, TX, USA. E-mail: anslyn@ccwf.cc.utexas.edu;
Fax: (512) 477 7911; Tel: (512) 471 0068
bDepartament de Qu´ımica, Universitat de les Illes Balears, 07122, Palma
de Mallorca, Spain. E-mail: jeroni.morey@uib.es
{ Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b609861d
Scheme 1 Preparation of oximes 2a and 2b.
3886 | Chem. Commun., 2006, 3886–3888
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(Fig. 1 Top). This can be explained by the experimental procedure.
The isosbestic point is not perfect because the conjugate base of
compound 2a is diluted upon the addition of DFP throughout the
titration. In order to keep the concentration of 2a constant we
need to titrate with a solution of 2a and DFP, but due to the
instantaneous and irreversible reaction between these two entities
this is not possible. A control experiment was carried out
analogous to the experiment described above. A solution of 2b
(2.5 6 1025 mol dm23) in DMSO was prepared, and on addition
of DFP no shift in the UV-Vis absorption was observed. This
confirms that the oximate phosphorylates the DFP (see supple-
mentary material).
It is imperative that the detection of CW agents is achieved
quickly. It was observed that upon the addition of an aliquot of
DFP there is an immediate change in the UV-Vis or fluorescence
spectrum, suggesting that the rate of the reaction is very fast. Stop-
flow kinetics experiments were carried out by watching the turn
‘‘on’’ of the fluorescence signal upon the addition of DFP. A 2.5 6
1025 mol dm23 solution of 2a and a 1.25 6 1024 mol dm23
solution of DFP were prepared and placed in the stop-flow
apparatus. Equal volumes of the solutions were mixed together
and the reaction was monitored for 1 second. The fluorescence
intensity increased upon mixing, in good agreement with the
fluorescence studies described above. Pseudo first-order kinetics
were used and by monitoring the fluorescence intensity at
various times one can calculate the rate of the reaction by plotting
ln(Ao/(Ao 2 P)) versus time (Fig. 2 inset), where Ao is the final
fluorescence intensity and P is the fluorescence intensity at each
time interval measured. The rate constant k (slope) was calculated
to be 1410 s21. Therefore the half-life (tK 5 ln(2)/k) was calculated
to be approximately 50 ms.
Fluorescence studies on compounds 2a and 2b were carried out
by preparing a 0.5 6 1026 mol dm23 solution in DMSO with a
50 fold excess of P4 base, and titrating small aliquots of DFP. The
fluorescence signal of 2a with the P4 base alone is weak. This is a
result of the high-energy lone pair of the oximate anion quenching
the fluorescence via a PET mechanism. Yet, upon phosphorylation
by DFP, the energies of these orbitals are lowered, reducing the
PET quenching effect, and the fluorescence is turned ‘‘on’’ (Fig. 1
Bottom). Compounds 1 and 2b are also highly fluorescent. This is
in good agreement with the PET mechanism, as neither compound
has a negative charge to quench the fluorescence. On addition of
DFP to 2b the signal remains unchanged (see supplementary
material).
As an aside to the goal of this project, X-ray quality crystals of
the coumarin oxime 2a were obtained (Fig. 3). There are two
independent molecules in the asymmetric unit and each forms a
hydrogen bonding chain (vide infra).§ The oxime functional group
in our system has been used as the reactive site for phosphorylation
upon deprotonation. However, another facet of the oxime moiety
is its use as a building block to control the aggregation of
molecules via intermolecular interactions to form arrays of infinite
networks. The ideal way to tailor supramolecular structures is
readily achieved when interactions are directional and are held
together by complementary functional groups that form strong
intermolecular bonds. Crystal engineering has extensively used
carboxyl,15 amide16 and alcohol17 functional groups to build
elaborate architectures. Recently, the oxime moiety has been the
functional group of interest in supramolecular chemistry, with
respect to crystal engineering.18 A recent study carried out by
Motherwell et al. predicts the binding motifs of oximes using
known structures of oximes in the CSD.19 Typical binding motifs
are dimers, trimers, tetramers and catemers (infinite chains). The
formation of these motifs is dependent on the accessibility of the
nitrogen atom surface towards a hydrogen atom. This is dependent
Fig. 1 (Top) UV-Vis absorption spectra of 2a on addition of aliquots of
DFP into a 2.5 6 1025 mol dm23 DMSO solution with 2 equivalent of P4
base. (Bottom) Fluorescence spectra of 2a 0.5 6 1026 mol dm23 in
DMSO (excitation 410 nm) as a function of added DFP.
Fig. 2 Fluorescence increase upon phosphorylation. Inset, first-order
kinetic plot of ln(Ao/Ao 2 P) versus time.
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Fig. 3 The crystal structure of 2a (molecule A) and view illustrating a portion of the H-bonding array observed for molecule 2a. Most hydrogen atoms
…
have been removed for clarity. Dashed lines are indicative of H-bonding interactions. The geometries of these interactions are: O14–H14 O11 (related by
…
…
…
˚
˚
x, 3/2 2 y, K + z); O O 2.673(4) A, H O 1.73(6) A, O–H O 170(6)u.
§ Crystal data for 2a. C18H24N2O3, M = 316.39, monoclinic, a 5 14.9783(4),
˚
on the configuration of the NLC double bond with respect to the
OH group, and as a consequence these stereoisomeric forms have
a detrimental effect on the binding motif. The structure of 2a
shows that the accessibility of the nitrogen atom is hindered by the
bulky butyl chain in the 4 position of the coumarin molecule,
thereby forcing the trans isomer to adopt catemer formation
producing a single hydrogen bond interaction between the OH
group of one coumarin molecule and the carbonyl functionality of
an adjacent coumarin molecule (Fig. 3).
b 5 16.9346(6), c 5 13.5481(6) A, a 5 90, b 5 99.484(2), c 5 90u, U =
3
˚
˚
3389.5(2) A , T = 117(2) K, space group P21/c, Z = 8, l 0.71073 A. Density
(calculated) 1.240 Mg m23, m 0.085 mm21, F(000) 1360, crystal size 0.27 6
0.20 6 0.04 mm, 3.01 to 25.00u, reflections collected 10984, independent
reflections 5895 [Rint 5 0.1388], R1 5 0.0786, wR2 5 0.1143. R indices (all
data). R1 5 0.2382, wR2 5 0.1414. Extinction coefficient 2.6(3) 6 1026
.
Largest diff. peak and hole 0.558 and 20.251 e A23. CCDC 614391. For
˚
crystallographic data in CIF or other electronic format see DOI: 10.1039/
b609861d
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The authors would like to thank K. A. Johnson for the use of
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this project which was sponsored by the Army Research
Laboratory and the Welch Foundation and was accomplished
under Cooperative Agreement Number W911NF-04-2-0043.
The views and conclusions contained in this document are those
of the authors and should not be interpreted as representing the
official policies, either expressed or implied, of the Army Research
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{ DFP is notoriously acidic, containing trace amounts of HF even when
bought fresh from Aldrich. To avoid acid interfering with our system DFP
solution was stored over piperidinomethyl polystyrene prior to use.
3888 | Chem. Commun., 2006, 3886–3888
This journal is ß The Royal Society of Chemistry 2006