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related V-type nerve agents are also detoxified. The rates of
detoxification of the methyl phosphonates in the presence of
a were almost equally fast, while the ethyl phosphonates VE
2
and i-propyl-VE are detoxified significantly slower. In the
case of 2b, the V-type nerve agents with linear alkyl groups in
the side chains are detoxified less efficiently than VX, and
going from the methyl phosphonates to the ethyl phospho-
nates causes another drop in activity. Equimolar mixtures of
the sulfanilic acid 3a and the calixarenes 1 or 2c proved to be
inactive, thus showing that the covalent linkage between the
calixarene unit and the substituent in 2a is required for
effective detoxification.
1
Figure 4. Sections of the H NMR spectra of a) VX (2.93 mm) in
phosphate buffer (0.1m in D O, pD 7.81) and b) of a mixture of 2c
2
For comparison, the effects of the prepared compounds on
the detoxification of soman (GD) were also assessed. Table 1
shows that the O-methylated hydroxamic acid 2c also does
not mediate the detoxification of GD, which suggests that the
free hydroxamic OH group is required for the reaction. The
carboxylate group in 2d is presumably not sufficiently
nucleophilic to have an effect. All the other compounds
detoxify GD to some extent, irrespective of whether they
contain a calixarene moiety or not. The half-lives of GD
detoxifications by 2a and 3a or by 2b and 3b are very similar.
Thus, the detoxification of the neutral GD does not seem to
benefit from the calixarene ring. By contrast, this ring is
essential for the detoxification of the cationic V-type nerve
agents, as none of the sulfanilic acid derivatives exhibited
notable activity. The lack of activity of the simple hydroxamic
acids is consistent with the higher stability of phosphono-
thioates with respect to GD (the rate constants of sponta-
and VX in a 2:1 molar ratio. Characteristic shifts of the isopropyl,
ethyl, and phosphonate methyl signals of VX are indicated.
geometries are preferred, with the cationic ammonium group
located inside the calixarene cavity. As a consequence, the
phosphorus atom of the nerve agent should be preferentially
oriented near the hydroxamic acid group of the scavenger,
thus facilitating reaction. An NMR titration under the same
conditions yielded a binding constant logK of 4.11 Æ 0.12 for
a
the complex between 2c and VX, thus confirming the
appreciable affinity of the sulfonatocalix[4]arene for posi-
tively charged nerve agents in water.
Efficient complexation of the nerve agents could, there-
fore, explain the detoxification activities of the calixarene-
based scavengers. The different activities observed for 2a, 2b,
and 2e may be related to differences in the nucleophilicities
of their substituents or to structural aspects of the complexes
formed. The former is less likely because benzohydroxamic
acid and corresponding pyridine derivatives do not typically
À3
À1
neous hydrolysis are 6.3 ꢀ 10 min
for GD and 5.2 ꢀ
À5
À1
1
0
min for VX under the chosen conditions). The ability
of 2a and 2b to overcompensate this stability difference and
allow them to detoxify V-type nerve agents even faster than
GD, could, therefore, be an indication that the predicted
interactions between the calixarenes and cationic OPs indeed
facilitate detoxification.
exhibit large differences in their pK values or rates of
a
[
5a,b,12a]
reaction in the presence of OPs.
We therefore attribute
the higher activity of 2a with respect to 2b, and also the
different rates with which the investigated nerve agents are
detoxified, to structural effects of the respective complexes.
The loss of activity when moving the hydroxamic acid from
the 3- to the 4-position of the aromatic substituent may
likewise be caused by the inability of the hydroxamic acid in
the VX complex with 2e to efficiently reach the phosphorous
atom of the nerve agent.
To test this assumption, binding studies were performed
by using VX and the inactive calixarene derivative 2c as
binding partners. Qualitative information about complex
1
formation was obtained by comparing the H NMR spectrum
of VX with that of VX in the presence of 2 equiv of 2c in
phosphate buffer (0.1m in D O, pD 7.81). Phosphate buffer
2
was used to avoid signals from organic buffer molecules in the
H NMR spectra. Figure 4 shows that the presence of 2c has
a pronounced effect on the resonances of the VX protons (for
the effect of complexation on the receptor signals, see
Figure S4). All the VX protons are shielded, with the
strongest upfield shift of 1.3 ppm observed for the signals of
Information about the pathways underlying the detoxifi-
cation was obtained by following the reactions between VX
1
3
1
and calixarenes 2a and 2b by P NMR spectroscopy and
3
1
mass spectrometry. P NMR spectroscopy showed that the
spontaneous hydrolysis of VX (2.93 mm) in Tris-HCl buffer
(0.1m, pH 7.40) at 378C is associated with a progressive
decrease in the VX signal. Concomitantly, signals appeared
showing the formation of ethyl methylphosphonic acid
(EMPA) and the toxic metabolite of VX, S-(2-(diisopropyl-
amino)ethyl) methylphosphonothioate (EA-2192). About
25% of the VX was hydrolyzed after 24 h under these
conditions, with the EMPA/EA-2192 ratio amounting to 5.7:1.
Incubating VX (2.93 mm) in Tris-HCl buffer (0.1m,
pH 7.40) at 378C with 1 equiv of 2a led to a notable 78%
drop in VX concentration within the first hour of the
experiment and a drop of 94% when using 2 equiv of 2a,
thus confirming the high detoxification ability of this scav-
the isopropyl-CH groups. The shifts become smaller as the
3
distance of the respective proton from the diisopropylamino
group increases, but effects are even evident for the reso-
nances of the ethyl-CH and P-CH protons. Another notable
3
3
feature is the splitting of the isopropyl-CH signal into two
3
doublets, which indicates that the stabilization of the tetrahe-
dral ammonium group of VX upon complexation causes the
isopropyl-CH groups to become diastereotopic.
3
1
The profound signal shifts observed in the H NMR
spectrum are clear indications for the binding of VX to 2c.
Moreover, the extents of the shifts suggest that complex
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3
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