Colorimetric detection of chemical warfare simulants

Article abstract of DOI:10.1039/b506100h

Two simple chromogenic indicators (4) and (5), containing different supernucleophilic moieties, have been synthesized. Upon phosphorylation with two chemical warfare agent (CWA) simulants, a hypsochromic shift of approximately 50 nm is observed in an NaOH

Full text of DOI:10.1039/b506100h

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P A P E R
Colorimetric detection of chemical warfare simulantsw
Karl J. Wallace,a Jeroni Morey,*b Vincent M. Lyncha and Eric V. Anslyn*a
a Department of Chemistry and Biochemistry, The University of Texas at Austin, 24th and
Speedway, Welch 5.201, Austin, TX 78712-1167, USA. E-mail: anslyn@ccwf.cc.utexas.edu;
Fax: þ1 (512) 471 7791; Tel: þ1 (512) 471 0068
b
´
Departament de Quımica, Universitat de les Illes Balears, 07122 Palma de Mallorca, Spain.
E-mail: jeroni.morey@uib.es
Received (in St Louis, MO, USA) 3rd May 2005, Accepted 31st August 2005
First published as an Advance Article on the web 22nd September 2005
Two simple chromogenic indicators (4) and (5), containing different supernucleophilic moieties, have been
synthesized. Upon phosphorylation with two chemical warfare agent (CWA) simulants, a hypsochromic shift
of approximately 50 nm is observed in an NaOH–DMSO solution. The oximate supernucleophile was found
to be a better supernucleophile than the hydrazone moiety. Two X-ray crystal structures were obtained from
unexpected synthetic side products obtained in this study. These will also be discussed.
anticipated with sarin/soman) is quite slow (half-life approach-
ing one hour).
Herein, we report the development of two simple organic
molecules as indicators for phospho-fluorides: an oxime (4)
and a hydrazone (5).
Introduction
It is well established that many organophosphorus compounds
are powerful neurotoxic agents that inhibit acetylcholinesterase
(AchE) by the process of phosphorylation.1,2 A particularly
dangerous class of organophosphorus compounds are the
phosphoryl fluoride containing species. Two such species are
the Chemical Warfare Agents (CWAs) sarin (isopropyl methyl-
phosphonofluoridate), and soman (pinacolyl methylphospho-
nofluoridate), referred to as GB and GD agents, respectively.
There has been significant interest in the decontamination3,4
and detection5–7 of CWAs over the last five decades. One
approach uses chromogenic detector reagents, which directly
bind to, or react with, a target nerve agent, causing a modula-
tion in the UV-Vis wavelength.8–10
Results and discussion
Design criteria
Our approach was to create a very simple colorimetric system
that contains a highly nucleophilic moiety, known as a super-
nucleophile. A supernucleophile is a reactive species whereby
an atom containing an unshared electron pair, typically a
nitrogen or oxygen atom, is adjacent to the nucleophilic center.
This increases the nucleophilicity of the reactive center, a
phenomena commonly known as the a-effect.11 Two such
supernucleophiles are oximate (RNOꢀ) functionalities and
hydrazone (RNNH2) moieties. Both the oximate and hydra-
zone reactive sites can react with the phosphorus(V) center of
the CWA simulant. For the molecule to act as a colorimetric
system, a UV-Vis absorbance spectral change needs to be
observed. A typical functional group that is an ideal UV-Vis
‘handle’ is the nitro moiety.
One common CWA simulant is diisopropyl fluorophosphate
(DFP). A less toxic simulant that is also often used is diethyl
chlorophosphate (DCP). Recently, Swager introduced a fluor-
escent indicator that undergoes a dramatic spectral change in
response to the cyclization of a flexible chromophore. A rigid
and highly conjugated fluorophore is created on the addition of
DFP, causing an ‘off–on’ response in the micromolar concen-
tration range.6 Yet, the system uses an alcohol as a nucleophile,
and hence the rate of reaction with DFP (let alone that
Synthesis
Commercially available 4-acetamidobenzaldehyde was reacted
with fuming nitric acid (>90%). The nitro group is introduced
into the meta position in relation to the aldehyde functionality
and the ortho position in relation to the amide group of the
benzene ring, forming 4-acetamido-3-nitrobenzaldehyde (2) in
moderate yield, typically 50%. The inclusion of the nitro group
w Electronic supplementary information (ESI) available: UV-Vis spec-
tra of 5. See http://dx.doi.org/10.1039/b506100h
T h i s j o u r n a l i s & T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 5
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 4 6 9 – 1 4 7 4
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Scheme 1 The preparation of 4-amino-3-nitrobenzaldoxime (4) and 4-hydrazonomethyl-2-nitrophenylamine (5): (a) fuming HNO3 (490%); (b)
HCl; (c) NH2OH ꢂ HCl, NaOH; (d) H2NNH2 ꢂ H2O, cat p-TsOH.
is clearly apparent in the appearance of the NO2 symmetric and
antisymmetric stretches at 1458 and 1377 cmꢀ1, respectively, in
the IR spectrum. Addition of hydrochloric acid to 2 subse-
quently produces 4-amino-3-nitrobenzaldehyde (3) in 75%
yield. Compound 3 is converted to the oxime 4 by reacting 3
with hydroxylamine hydrochloride and sodium hydroxide.
Compound 3 was also treated with hydrazine hydrate, with a
catalytic amount of p-toluenesulfonic acid, to produce 4-hy-
drazonomethyl-2-nitrophenylamine (5), Scheme 1.12 All com-
pounds were fully characterized using 1H and 13C NMR,
HRMS, IR and UV-Vis spectroscopy, which are in agreement
with the proposed structures.
over time, the oximate–DCP complex is unstable. It is well
documented that keto-oximes undergo a Beckmann rearrange-
ment to form secondary amides. However, aldo-oximes have
been shown to undergo dehydration to form nitrile species. In
this particular example, 4-amino-3-nitrobenzonitrile (6), was
evident as a minor product, from the 1H NMR spectra and the
[M]1 peak observed at 163 in the mass spectra, Scheme 2.13
Further evidence to support the existence of the oxime–DXP
adduct was seen in the 31P NMR spectra. A DCP solution of
DMSO-d6 and D2O–NaOH was prepared and the 31P NMR
spectrum recorded (dP ¼ 0.64 ppm). On addition of 1 equiva-
lent of oxime 4 the phosphorus signal shifted to dP ¼ 1.82 ppm;
the same trend is also observed for DFP.
Solution studies
UV-Vis studies
Due to the high toxicity of CWAs sarin and soman, model
compounds DCP and DFP were used in this study. However,
phosphoryl–chloride bonds are significantly weaker than phos-
phoryl–fluoride bonds (326 and 490 kJ molꢀ1, respectively),
and therefore a direct comparison of DCP with CWA must
take these bond strengths into consideration. In solution, it is
well documented that DFP and DCP are unstable to basic
medium over a prolonged period of time. The Sigma-Aldrich
company states that the half-life of DFP at pH ¼ 7.5 is only
one hour; therefore, all samples were prepared as fresh solu-
tions, before each study.
Experiments were carried out by titrating aliquots of DCP and
DFP into a solution of 4, (3 ꢁ 10ꢀ4 mol dmꢀ3 in a 1 : 1 mixture
of DMSO–NaOH at room temperature). In both cases, the
initial UV-Vis spectra showed a broad band at lmax ¼ 461 nm,
A base is required to deprotonate the oxime functionality to
1
form the oximate anion. A set of H NMR and mass spectro-
scopy experiments where carried out to determine whether or
not 4 is susceptible to nucleophilic attack by the phosphorus(V)
center. A sample of 4 was prepared by dissolving 4 in a 1 : 1
mixture of (DMSO-d6)–(D2O–NaOH, 1 mol dmꢀ3) solution in
an NMR tube and the 1H NMR spectrum was recorded. Three
signals appear at d 7.05, 7.75 and 8.08 ppm, assigned to the
aromatic protons on the benzene core of 4. Upon addition of
1.1 equivalents of DCP, three new sets of signals immediately
appeared in a more downfield region of the NMR spectrum
and were assigned to the oximate–DCP complex. The signals
assigned to the ethyl protons in DCP appear at d 1.47 and 3.30
ppm, and on the addition of the oximate, two sets of new
signals appear at d 1.11 and 3.84 ppm. The sample tube was left
to stand for 24 h and the 1H NMR spectrum was recorded
again. A third set of aromatic signals appeared in the spectrum
at d 8.44, 7.32 and 6.96 ppm. The mass spectrum of the NMR
sample clearly showed the existence of three components. The
appearance of the third set of signals after 24 h suggested that,
Scheme 2 Proposed mechanism for the nucleophilic attack of 4
undergoing dehydration to form 4-amino-3-nitrobenzonitrile (6).
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strates.11 A hydrazone moiety was tabulated to be more
nucleophilic than a simple oximate anion when treated with
the same substrate. Therefore, it was anticipated that hydra-
zone 5 would react with the simulants in the same fashion
(eliminating the use of NaOH) at the nucleophilic site.
Solutions of 5 were prepared in CHCl3, MeOH, and DMSO,
with the lmax values at 425 nm, 432 nm and 461 nm, respec-
tively. On addition of DFP a broad absorbance at lmax at 353
nm grows in over time. However, the new species was identified
as the protonated form of 5 and not the hydrazone–DFP
adduct. This was due to a small amount of acid in the DFP
solution and solvent (as CHCl3 contains small traces of HCl).
Protonation of compound 5 was confirmed by preparing a
1.5 ꢁ 10ꢀ4 mol dmꢀ3 solution in MeOH to which 1 mL of 2 M
HCl was added to the UV cell. This spectrum confirmed the
appearance of the band at 350 nm, (see ESIw). The acidity
problem was overcome by preparing a buffered solution of
p-toluenesulfonic acid and Hunigs base 5.0 ꢁ 10ꢀ4 mol dmꢀ3 in
¨
DMSO. The initial pH was recorded at 9.95 and the lmax is at
445 nm for 5. On the addition of 50 equivalents of DFP, an
insignificant shift of 5 nm is observed in the blue direction and
the final pH was recorded at 9.63. This small shift is attributed
to the small change in concentration on the addition of DFP. It
is interesting to note that 5 does not react with DFP in basic
solution but 4, when fully deprotonated reacts with the simu-
lants. This is the opposite of the findings by Klopman, suggest-
ing that 4 is a better supernucleophile than 5 under the
conditions employed and with the substrates used herein.
Solid-state studies
It has been demonstrated that the oxime (4) can undergo
phosphorylation with excess base in solution to form the
oximate anion. An attempt was made to isolate the oximate
salt by treating 4 with tetrabutylammonium hydroxide in a
1
1 mol dmꢀ1 methanol solution. Unfortunately, the H NMR
Fig. 1 The UV-Vis titration spectra of 4 upon addition of DCP (top)
and DFP (bottom) in NaOH–DMSO solution (3 ꢁ 10ꢀ4 mol dmꢀ3).
spectrum of the isolated solid did not agree with the proposed
structure. The integration of the aromatic signals was found to
be half that of the CH2 groups in the tetrabutylammonium
cation, suggesting the formation of a dimer (7), and not the
proposed oximate salt. This was confirmed by single crystal
X-ray crystallography. Tetrabutylammonium hydroxide is
probably deprotonating the oxime in solution. However, it
is interesting to note that the isolated product is identified as
the oximate–oxime dimer. It is unclear whether the excess of
tetrabutylammonium hydroxide leads to only half the oxime
molecules being deprotonated or if a proton is ‘‘picked’’ up
during the recrystallization that subsequently forms the more
stable oximate–oxime dimer.
assigned to the n–p* transition.14 Upon the gradual addition of
DCP, the absorbance intensity at 461 nm decreased with the
appearance of another band hypsochromically shifted to 410
nm through an isosbestic point, 431 nm, Fig. 1. The same trend
is observed with the addition of aliquots of DFP to 4. Here a
decrease in absorbance intensity at 461 nm is accompanied by
another band increasing at 413 nm through an isosbestic point
at 437 nm.
It is well known that strong bases such as NaOH can react
with DFP, to form the less toxic hydrolysis product.3 To
confirm the result proposed in Scheme 2, a stronger but a far
less nucleophilic base was used. A nitrogen–phosphorous
super-base was chosen. The phosphazene base P4-tBu solution
X-Ray crystal structure of oxime–oximate dimer (7)
(
DMSOpKBH þ ¼ 30.25) is approximately two orders of magni-
Crystals of compound 7 were grown by slow diffusion of
diethyl ether into DMSO to form long orange needles, Fig.
2a. The oximate–oxime dimer observed in the solid-state is
consistent with the 1H NMR spectrum obtained for 7 in
DMSO-d6 solution. Oximate–oxime dimers are common in
the literature and have been extensively studied by Maurin.15–17
Interestingly, the intermolecular hydrogen bond lengths
observed in 7 between O(30)ꢂ ꢂ ꢂO(3) are shorter than the typical
intermolecular hydrogen bond length found in Maurin’s sys-
tem, presumably due to the electron withdrawing ability of the
nitro group making the O(30) hydrogen more acidic, thereby
forming a stronger hydrogen bond with an oxime molecule.
The crystal packing is dominated by the extensive hydrogen
bonding network, which forms linear polymeric chains, Fig.
3a, with a combination of strong intramolecular hydrogen
bonds [O(2)ꢂ ꢂ ꢂHN(2) and N(10)ꢂ ꢂ ꢂHN(20) 2.654(3) and
2.658(3)], and weaker intermolecular hydrogen bonds,
[O(10)ꢂ ꢂ ꢂN(2) 4.202(3) and N(20)ꢂ ꢂ ꢂO(2) 3.296(3)]. The long
tude stronger than DBU (MeCNpKBH þ ¼ 24.3) but less
nucleophilic. On the addition of the P4 base to a solution of
the oxime 4, an intense band at 530 nm is observed. On
addition of DFP the band is again hypsochromically shifted
to 417 nm, analogous to the NaOH experiment. This is
consistent evidence that the oximate supernucleophile is phos-
phorylating the DFP.
In 1970 Klopman tabulated data showing relative rate
constants of various supernucleophiles with different sub-
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 4 6 9 – 1 4 7 4
1471

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bridging hydrogen atom. To date over 100 structures in the
crystallographic database show Oꢂ ꢂ ꢂO interactions with dis-
tances shorter than 2.83 A. The overall structure is also
stabilized due to weak CHꢂ ꢂ ꢂON interactions. The second
oxygen atom on the nitro group is hydrogen bonded to two
CH groups in a bifurcated fashion to the adjacent strand. A
bifurcated motif of this nature is commonly found in crystal
structures and is one of five hydrogen bonding patterns
commonly found in weak CHꢂ ꢂ ꢂO hydrogen bonding patterns
that involve nitro groups as acceptor moieties as demonstrated
by Desiraju.18 The stability of the complex demonstrates the
importance of weak CH hydrogen bonding interactions found
in hydrogen bonded polymeric structures.
The strong hydrogen bonding network observed in the X-ray
structure is also supported by the solid-state infrared spectrum
(Nujol mull). The n(OH) functionality in the oximate–oxime
dimer 7 (N–Oꢂ ꢂ ꢂH–O–N) displays a single n(OH) stretch at
3270 cmꢀ1 This is observed at a higher wavenumber than the
neutral oxime 4, (n(OH) stretch at 3193 cmꢀ1). This bath-
ochromic shift is consistent with strong hydrogen bonding
motifs. Which is in good agreement with the short bond lengths
obtained from the X-ray diffraction studies, and is commonly
found in other oximate–oxime dimers.19 A strong single ab-
sorption band at 1377 cmꢀ1 for n(NO) is observed in the IR
spectrum of 7, which is consistent for aromatic nitrosoamines
that have a trans geometry, again consistent with the crystal
structure.
Fig. 2 (A) Compound 7 showing the formation of the oximate–oxime
dimer. Selected hydrogen bond length, O(30)ꢂ ꢂ ꢂO(3) 2.509(2) A. (B)
Compound 8, selected hydrogen bond lengths, N12–H12Aꢂ ꢂ ꢂO1A,
Nꢂ ꢂ ꢂO 2.840(4) A, Hꢂ ꢂ ꢂO 1.98(4) A. N12–H12Aꢂ ꢂ ꢂO11A, Nꢂ ꢂ ꢂO
2.657(4) A, Hꢂ ꢂ ꢂO 2.037(3) A. Hydrogen atoms removed for clarity.
X-Ray crystal structure of E,E azine (8)
hydrogen bonding interactions are a consequence of the nitro
groups on adjacent molecules being in close proximity to one
another (the Oꢂ ꢂ ꢂO interaction observed at 2.83 A). This is a
surprisingly short distance for an Oꢂ ꢂ ꢂO interaction without a
The condensation reaction between hydrazone hydrochloride
and the aldehyde 3 in a 1 : 1 ratio produced the desired
hydrazone. It is well known that an azine is the product of
two equivalents of a carbonyl group reacting with one equiva-
lent of hydrazone hydrochloride. The characterization of 5 was
in good agreement with the proposed structure. However, a
DMSO solution yielded X-ray quality crystals that proved to
be an azine (compound 8), Fig. 2b. Glaser et al. have carried
out an extensive study on E,E configured para-substituted
acetophenone azines.20–22 They have synthesized a large num-
ber of highly polar azine molecules with varying degrees of
dipole moment, depending on the functional groups substi-
tuted on the benzene core. These highly polar organic mole-
cules show diverse optical and electronic properties that are
important in the design of non-linear optic (NOL) materials.23
The azine functionality in 8 is planar, the torsion angles
between N1A–N1–C2–C3 and N1–C2–C3–C4 are 178.8(3)1
176.7(3)1, respectively. The planarity extends across the entire
molecule. The amino groups are also in plane with the aro-
matic ring system. The amines torsion angle is 179.8(4)1 (N12–
C6–C7–C8). The nitro groups have a slight twist with a torsion
angle of 173.3(3)1 (C6–C5–N9–O11). Interestingly, virtually all
of the compounds studied by Glaser et al. show degrees of
twisting of the phenyl groups around the azine moiety.24
However, all of their systems have one substituent in the para
position, whereas our system has a nitro group ortho to the
amine functionality. The planarity of the system is a conse-
quence of the intramolecular hydrogen bonding interaction
between the amine moiety and the nitro group, 2.037(3) A.
The bond lengths for N N, C2 N1, C5–N9 and C6–N12
Q
Q
are 1.411(5), 1.285(4), 1.435(4) and 1.338(4) A, respectively,
and fall in the normal range. However, the Cipso–Cazine bond
length is 1.453 A. This seems to be shorter than any bond
length previously published in these systems. This can be
attributed to the bis-substituted benzene core. The crystal
packing of 8 shows classical off-centered face-to-face p–p
stacking interactions at a distance of 3.57 A, which is typical
for such interactions.
Fig. 3 The crystal packing of (A) 7, showing the hydrogen bonding
polymer of oximate–oxime dimer molecules. Selected bond lengths,
N(20)ꢂ ꢂ ꢂO(3) 2.871(3) A, N(20)ꢂ ꢂ ꢂO(1) 2.658(3) A and N(2)ꢂ ꢂ ꢂO(2)
2.654(3) A. The tetrabutylammonium counter ion has been removed
for clarity. (B) A chemical diagram highlighting the hydrogen bonding
in the crystal structure.
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1
mp 191–192 1C. H NMR: (DMSO-d6, J/Hz, d/ppm): 7.12 (d,
1H, J ¼ 8.5, ArH), 7.81 (dd, 1H, J ¼ 2.0, 8.9, ArH), 8.18 (s br,
2H, NH), disappears on D2O shake, 8.56 (d, 1H, J ¼ 2.0, ArH),
9.76 (s, 1H, CHO). 13C NMR: (DMSO, d/ppm): 119.89,
124.56, 129.70, 132.45, 132.74, 149.82, 189.63. IR (Nujol mull,
Conclusion
Two simple chromogenic compounds containing supernucleo-
phile moieties have been attached to a benzene scaffold.
Compound 4 can be fully deprotonated and undergoes phos-
phorylation with DCP and DFP in solution. We are confident
that the base does not react with DCP or DFP as a strong and
highly hindered phosphazene base was used to confirm phos-
phorylation. Unfortunately, we were unable to synthesize the
salt of the oxime, as a dimer seems to be the more stable
species. This was confirmed by X-ray crystal analysis. Cur-
rently, there are attempts to prepare fluorescent oxime indica-
tors for CWA detection and these will be published in due
course.
n/cmꢀ1): 3436, 3319 s (NH), 1686 s (C O, aldehyde), 1463 s
Q
(asymmetric), 1407 s (symmetric) (NO2). CI-MS: m/z ¼ [M þ
H]1 167, CI-HRMS: calcd for C7H7N2O3 [M þ H]1167.046,
found 167.046.
4-Amino-3-nitrobenzaldoxime (4). A suspension of 4-amino-
3-nitrobenzaldehyde (3) (800 mg, 4.8 mmol) in ethanol (15 mL)
was added to a solution of hydroxylamine hydrochloride (670
mg, 9.6 mmol) and sodium hydroxide (386 mg, 9.6 mmol) in
water (10 mL) and stirred at room temperature for 7 h. The
solution was adjusted to pH 6 by the addition of glacial acetic
acid and placed into an ice bath for 1 h. The yellow solid was
filtered, washed once with a mixture of glacial acetic acid and
water (50 mL, 10 : 1) and recrystallized from ethanol–water.
Yield (600 mg, 68%), mp 207 1C. 1H NMR: (DMSO-d6, J/Hz,
d/ppm): 7.05 (d, 1H, J ¼ 8.90, ArH), 7.70 (s br, 3H, NH2, OH),
disappears on D2O shake, 8.13 (d, 1H, J ¼ 2.0, ArH), 11.03 (s,
1H, oxime). 13C NMR: (DMSO, d/ppm): 119.88, 120.80,
124.43, 129.69, 132.20, 146.20, 146.74. UV-Vis (DMSO,
l ¼ 461 nm) CI-MS: m/z ¼ [M þ H]1 182. CI-HRMS: calcd
for C7H8N3O3 [M þ H]1 182.057, found 182.057.
Experimental
General techniques
1H and 13C NMR spectra were recorded on a Varian Unity
Plus 300 spectrometer in DMSO-d6. Chemical shifts are re-
ported in parts per million (d) downfield from tetramethyl-
silane (0 ppm) as the internal standard and coupling constants
(J) are recorded in hertz (Hz). The multiplicities in the 1H
NMR spectra are reported as (br) broad, (s) singlet, (d)
doublet, (dd) doublet of doublets, (ddd) doublet of doublet
of doublets, (t) triplet, (sp) septet and (m) multiplet. All spectra
are recorded at ambient temperatures. UV-Vis experiments
were performed on a Beckman DU-70 UV-Vis spectrometer.
Low and high resolution mass spectra were measured with a
Finnigan TSQ70 and VG Analytical ZAB2-E instruments,
respectively. IR spectra were recorded on a Perkin-Elmer
Paragon 100 Fourier transform IR spectrometer as Nujol
mulls, and the peaks of interest were reported in wavenumbers
(cmꢀ1) and described as weak (w), medium (m) and strong (s).
4-Hydrazonomethyl-2-nitrophenylamine (5). 4-Amino-3-ni-
trobenzaldehyde (2 g, 12 mmol) was dissolved in 96% ethanol
(60 mL), to which hydrazine hydrate (3.52 mmol, 72 mmol)
and a catalytic amount of p-toluenesulfonic acid (23 mg, 0.12
mmol) were added. The reaction mixture was heated to 70–80
1C for 4 h (during this time period a precipitate formed). The
reaction was cooled to 5–10 1C and left to stand at that
temperature for a further 1 h. The orange solid was filtered
and washed twice with diethyl ether (20 mL) and dried in the
air for 24 h. Yield (1.5 g, 75%). 1H NMR: (DMSO-d6, J/Hz, d/
ppm): 6.59 (s br, 2H, NH2), disappears on D2O shake, 7.02 (d,
1H, J ¼ 8.8, ArH), 7.52 (s br, 2H, NH), disappears on D2O
shake, 7.67 (m, 2H, ArH), 7.95 (d, 1H, J ¼ 2.0, imine). 13C
NMR: (DMSO, d/ppm): 119.72, 121.90, 124.56, 126.76,
132.05, 137.38, 145.79. IR (Nujol mull n/cmꢀ1): 3399, 3280,
3147 s (NH2), 1341 s (asymmetric), 1419 s (symmetric) (NO2).
UV-Vis (CHCl3, l ¼ 425 nm; MeOH, l ¼ 432 nm; DMSO, l ¼
461 nm). CI-MS: m/z ¼ [M]1 180, [M þ H]1 181 [2M þ H]1
361. CI-HRMS: calcd for C7H7N2O3 [M þ H]1 181.072, found
181.072.
General spectroscopic methods
Solutions of 4 and 5 (3 ꢁ 10ꢀ4 mol dmꢀ3) were prepared in a
1 mol dmꢀ1 NaOH–DMSO solution. Aliquots of DFP or DCP
(1 mL), were added to a 1 mL UV-Vis cell. The UV-Vis
spectrum was recorded after each addition.
4-Acetamido-3-nitrobenzaldehyde (2). 4-Acetamidobenzalde-
hyde (17 g, 104 mmol) was added slowly to a cold solution
(0–5 1C) of fuming nitric acid (44 ml, 940 mmol) and stirred for
2 h. The dark brown solution was then poured into cold water
(860 mL). The resulting solid was filtered off and triturated
with cold H2O (430 mL) for a further 1 h. The solid was filtered
and then recrystallized from 2-propanol (250 mL), washed with
cold 2-propanol (2 ꢁ 50 mL) and dried in the air for 24 h. Yield
4-Amino-3-nitrobenzaldoxime tetrabutylammonium salt (7).
Tetrabutylammonium hydroxide, 1.0 M solution in methanol
(21 mL) was added to a methanolic solution (10 mL) of 4-
amino-3-nitrobenzaldoxime (3) (0.104 g, 0.574 mmol) and
stirred for 7 h. During this time a red precipitate was formed,
which was filtered and washed with petroleum-ether (5 ꢁ 5
mL) and dried in air for 24 h. Yield (0.247 g, 57.6%). 1H NMR:
(DMSO-d6, J/Hz, d/ppm): 0.929 (t, 12H, J ¼ 7.4, CH3), 1.30 (q,
8H, J ¼ 7.4, CH2), 1.56 (m, 8H, CH2) 3.16 (m, 8H, CH3) 6.87
(d, 2H, J ¼ 9.0. ArH), 7.49 (dd, 2H, J ¼ 2.0, 9.2, ArH), 7.86 (d,
2H J ¼ 2.0, ArH), 7.91 (s, 2H, aldehyde). 13C NMR: (DMSO,
d/ppm): 13.38, 19.11, 22.97, 57.45, 120.78, 122.98, 123.773,
128.66, 130.38, 145.22, 149.89.
1
(11.3 g, 52%), mp 155 1C. H NMR: (CHCl3, J/Hz, d/ppm):
2.37 (s, 3H, CH3), 8.16 (dd, 1H, J ¼ 2.0, 8.7, ArH), 8.73 (d, 1H,
J ¼ 2.0, ArH), 9.03 (d, 1H, J ¼ 8.7, ArH), 9.99 (s, 1H, CHO),
10.62 (s br, 1H, NH), disappears on a D2O shake. 13C NMR:
(CHCl3, d/ppm): 25.71, 122.17, 127.90, 130.82, 135.44, 135.79,
139.35, 169.17, 188.74. IR (Nujol mull, n/cmꢀ1): 3340 s (NH),
1710 s (C O, aldehyde), 1690 s (C O, amide), 1458, 1377 s
Q
Q
(NO2). CI-MS: m/z ¼ [M þ H]1 209. CI-HRMS: calcd for
C9H9N2O4, [M þ H]1, 209.056, found, 209.057.
4-Amino-3-nitrobenzaldehyde (3). 4-Acetamido-3-nitroben-
zaldehyde (2) (5 g, 24 mmol) was added to concentrated HCl
(25 mL). The suspension was stirred for 2 h at 75 1C. Cold
water (130 mL) was then added to the suspension and stirred
for a further 1 h. The resulting solid was filtered and triturated
with cold water (130 mL) containing NaHCO3 (5.6 g, 67 mmol)
for 1 h. The solid was filtered, washed twice with cold water (50
mL) and crystallized from 2-propanol (250 mL), filtered and
washed with cold 2-propanol (2 ꢁ 50 mL). Yield (3.0 g, 75%),
X-Ray crystallography for (7). X-Ray experimental for
(C7H7N3O3)(C7H6N3O3)ꢀ(C16H36N)1: crystals grew as long
needles by vapor diffusion of ether into a DMSO solution of
the complex. The data crystal had approximate dimensions;
0.50 ꢁ 0.11 ꢁ 0.08 mm. The data were collected on a Nonius
Kappa CCD diffractometer using a graphite monochromator
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 4 6 9 – 1 4 7 4
1473

View Article Online
SHELXL-97.27 The hydrogen atoms on carbon were calcu-
lated in ideal positions with isotropic displacement parameters
set to 1.2 ꢁ Ueq of the attached atom (1.5 ꢁ Ueq for methyl
hydrogen atoms). The hydrogen atoms on the amino nitrogen,
N12, were observed in a DF map and refined with isotropic
displacement parameters.
Table 1 Crystallographic data and refinement parameters for com-
pounds 7 and 8
Compound
7
8
Chemical formula
M
C
30H49N7O6
C18H24N6O6S2
242.28
Triclinic
603.76
Monoclinic
C2/c
Crystal system
Space group
a (A)
ꢀ
P1
Acknowledgements
39.7560(6)
8.4865(2)
21.6058(4)
90.00
6.814(1)
8.759(1)
11.015(1)
67.536(2)
76.657(2)
79.036(2)
587.32(12)
153(2)
J. M. thanks the MECD for a ‘‘Salvador de Madariaga’’
sabbatical grant. The authors would also like to thank Intel-
ligent Optical System (IOS) for additional financial support
and Dr Steve Cordero for helpful discussions.
b (A)
c (A)
a (1)
b (1)
116.6960(10)
90.00
g (1)
U (eA3)
T (K)
Z
6512.5(2)
153(2)
References
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8
0.087
1
0.272
m(MoKa) (mmꢀ1
)
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Reflections measured
Unique reflections
Rint
24895
3527
2307
1596
0.1318
0.0620
0.1010
0.1725
0.1261
0.0000
0.0583
0.1219
0.0956
0.1393
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wR2 (all data)
with MoKa radiation (l ¼ 0.71073 A). A total of 418 frames of
data were collected using o-scans with a scan range of 1.11 and
a counting time of 76 seconds per frame. The data were
collected at 153 K using an Oxford Cryostream low tempera-
ture device. Details of crystal data, data collection and struc-
ture refinement can be seen in the ESI,z and Table 1. Data
reduction was performed using DENZO-SMN.25 The structure
was solved by direct methods using SIR9726 and refined by full-
matrix least-squares on F2 with anisotropic displacement
parameters for the non-H atoms using SHELXL-97.27 The
hydrogen atoms on carbon were calculated in ideal positions
with isotropic displacement parameters set to 1.2 ꢁ Ueq of the
attached atom (1.5 ꢁ Ueq for methyl hydrogen atoms). The
hydrogen atoms on nitrogen and oxygen were observed in a DF
map and refined with isotropic displacement parameters. A
portion of an n-butyl side chain was disordered.
8
9
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X-Ray crystallography for (8). X-Ray experimental for
C14H12N6O4 ꢂ 2C2H6SO: crystals grew as small prisms by slow
evaporation from DMSO. The data crystal was a prism that
had approximate dimensions; 0.20 ꢁ 0.15 ꢁ 0.06 mm. The data
were collected on a Nonius Kappa CCD diffractometer using a
graphite monochromator with MoKa radiation (l ¼ 0.71073
A). A total of 324 frames of data were collected using o-scans
with a scan range of 0.71 and a counting time of 214 seconds
per frame. The data was collected at 153 K using an Oxford
Cryostream low temperature device. Details of crystal data,
data collection and structure refinement are listed in Table 1.z
Data reduction was performed using DENZO-SMN.25 The
structure was solved by direct methods using SIR9726 and
refined by full-matrix least-squares on F2 with anisotropic
displacement parameters for the non-H atoms using
structures, University of Gottingen, Germany, 1997.
¨
z CCDC reference numbers 280696–280697. See http://dx.doi.org/
10.1039/b506100h for crystallographic data in CIF or other electronic
format.
1474
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 4 6 9 – 1 4 7 4