Highly efficient cyclosarin degradation mediated by a β-cyclodextrin derivative containing an oxime-derived substituent

Article abstract of DOI:10.3762/bjoc.7.182

The potential of appropriately substituted cyclodextrins to act as scavengers for neurotoxic organophosphonates under physiological conditions was evaluated. To this end, a series of derivatives containing substituents with an aldoxime or a ketoxime moiety along the narrow opening of the β-cyclodextrin cavity was synthesized, and the ability of these compounds to reduce the inhibitory effect of the neurotoxic organophosphonate cyclosarin on its key target, acetylcholinesterase, was assessed in vitro. All compounds exhibited a larger effect than native β-cyclodextrin, and characteristic differences were noted. These differences in activity were correlated with the structural and electronic parameters of the substituents. In addition, the relatively strong effect of the cyclodextrin derivatives on cyclosarin degradation and, in particular, the observed enantioselectivity are good indications that noncovalent interactions between the cyclodextrin ring and the substrate, presumably involving the inclusion of the cyclohexyl moiety of cyclosarin into the cyclodextrin cavity, contribute to the mode of action. Among the nine compounds investigated, one exhibited remarkable activity, completely preventing acetylcholinesterase inhibition by the (-)-enantiomer of cyclosarin within seconds under the conditions of the assay. Thus, these investigations demonstrate that decoration of cyclodextrins with appropriate substituents represents a promising approach for the development of scavengers able to detoxify highly toxic nerve agents.

Full text of DOI:10.3762/bjoc.7.182

Highly efficient cyclosarin degradation
mediated by a β-cyclodextrin derivative
containing an oxime-derived substituent
Michael Zengerle1, Florian Brandhuber2, Christian Schneider1,
Franz Worek2, Georg Reiter2 and Stefan Kubik*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2011, 7, 1543–1554.
1Fachbereich Chemie - Organische Chemie, Technische Universität
Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern,
Germany, Fax: +49-631-205-3921 and 2Institut für Pharmakologie
und Toxikologie der Bundeswehr, Neuherbergstraße 11, D-80937
München, Germany
doi:10.3762/bjoc.7.182
Received: 28 September 2011
Accepted: 08 November 2011
Published: 22 November 2011
Email:
This article is part of the Thematic Series "Supramolecular chemistry II".
Guest Editor: C. A. Schalley
Stefan Kubik* - kubik@chemie.uni-kl.de
* Corresponding author
© 2011 Zengerle et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
acetylcholinesterase; cyclodextrins; cyclosarin; neurotoxic
organophosphonates; oximes
Abstract
The potential of appropriately substituted cyclodextrins to act as scavengers for neurotoxic organophosphonates under physiolog-
ical conditions was evaluated. To this end, a series of derivatives containing substituents with an aldoxime or a ketoxime moiety
along the narrow opening of the β-cyclodextrin cavity was synthesized, and the ability of these compounds to reduce the inhibitory
effect of the neurotoxic organophosphonate cyclosarin on its key target, acetylcholinesterase, was assessed in vitro. All compounds
exhibited a larger effect than native β-cyclodextrin, and characteristic differences were noted. These differences in activity were
correlated with the structural and electronic parameters of the substituents. In addition, the relatively strong effect of the cyclodex-
trin derivatives on cyclosarin degradation and, in particular, the observed enantioselectivity are good indications that noncovalent
interactions between the cyclodextrin ring and the substrate, presumably involving the inclusion of the cyclohexyl moiety of
cyclosarin into the cyclodextrin cavity, contribute to the mode of action. Among the nine compounds investigated, one exhibited
remarkable activity, completely preventing acetylcholinesterase inhibition by the (−)-enantiomer of cyclosarin within seconds under
the conditions of the assay. Thus, these investigations demonstrate that decoration of cyclodextrins with appropriate substituents
represents a promising approach for the development of scavengers able to detoxify highly toxic nerve agents.
Introduction
Cyclodextrins, cyclic oligosaccharides composed of α-1,4- easy availability, their ability to include organic nonpolar mole-
linked D-glucose units, represent one of the most important cules into the cavity made up by the cyclically arranged glucose
classes of host systems in supramolecular chemistry [1]. Their units in aqueous solution, their predictable and controllable
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binding properties, and their relatively straightforward chem- distinct subunits each of which has a characteristic function
ical modification have made cyclodextrins indispensable tools (Figure 1):
in applications such as sensing [2], nanotechnology [3,4],
polymer chemistry [5-8], medicinal chemistry [9,10], food
chemistry [11], and others. Importantly, the scope of cyclodex-
trins goes beyond molecular recognition since the recognition
event can in some cases be coupled with the chemical transfor-
mation of a substrate. This property was already realized in
1959 when it was shown that native cyclodextrins accelerate the
cleavage of some acetic acid esters [12]. Subsequent work then
established cyclodextrins containing appropriate substituents or
dimeric cyclodextrins as a potent class of enzyme mimics
[13,14]. Interestingly, it was also demonstrated relatively early
in the field of cyclodextrin chemistry that native cyclodextrins
are able to accelerate the cleavage of phosphates and phospho-
nates [15-18], including the highly neurotoxic organophospho-
nates (OP) sarin and soman [19-21]. While α-cyclodextrin, the
cyclodextrin containing six anhydroglucose units along the ring,
was shown to be most effective for sarin [17,22], the larger
β-cyclodextrin with the seven-membered ring was demon-
strated to also mediate soman degradation [19,20]. Moreover,
the cyclodextrins were shown to act enantioselectively, being
more effective for the more toxic (R)-(−)-enantiomer of sarin,
for example [17,22]. Surprisingly, this work has largely been
overlooked despite the fact that it provided strong indications
for the potential applicability of cyclodextrins for the detoxifi-
cation of chemical warfare agents. Only very recently has the
use of cyclodextrins to induce degradation of neurotoxic OPs
been addressed again. These investigations showed that
β-cyclodextrin derivatives with a substituent along the wider
rim of the cavity, bearing a nucleophilic group in the form of an
iodosylbenzoate [23-25] or an oxime [26], efficiently react with
paraoxon, cyclosarin (GF), and tabun thus reducing the
inhibitory effects of these OPs on the key target of OP toxicity,
acetylcholinesterase (AChE). Moreover, the results indicate that
the mode of action of these cyclodextrin derivatives involves
the formation of an inclusion complex with the OP.
• A cyclodextrin ring, which forms the basis of each com-
pound. Complex formation between this subunit and the
substrate should bring the P-atom of the substrate into
spatial proximity with the substituent on the ring, thus
facilitating the attack by the reactive group on the
substituent. The type of cyclodextrin in this subunit (α, β,
γ) controls the substrate affinity.
• The linking unit between the cyclodextrin ring and the
reactive part of the substituent. This group should be
chosen to allow straightforward synthetic access to the
cyclodextrin derivatives, ideally allowing the synthesis
to proceed in a modular fashion.
• The reactive unit bearing a functional group that should
be able to specifically cleave the P–X bond on the sub-
strate. In the case of GF (Scheme 1), for example, the
most labile P–F bond is the one that is most prone to
cleavage. In general, the functional group contains a suit-
able nucleophilic center, with α-effect nucleophiles
possessing particularly promising activities [27,28].
Accordingly, the first step of OP deactivation is expected
to consist of the phosphonylation of this nucleophilic
group, similar to the phosphonylation of the serine
residue in the active site of AChE during OP-mediated
inhibition of this enzyme. If the compound thus formed
is hydrolytically unstable, it will be cleaved in the
aqueous medium, releasing the original reactive unit and
allowing it to mediate another reaction.
The question thus arises as to whether suitable cyclodextrin
derivatives could also be used in vivo as antidotes against OP
poisonings. Such compounds should be able to act as scav-
engers by rapidly decomposing the OP into nontoxic byprod-
ucts before inhibition of AChE occurs. Catalytic action of the
scavenger is desirable, reducing the necessary dose of the drug,
but is not required if the toxicity is low. Since the amount
of data currently available is too low to assess whether this
approach has a realistic prospect of success, we initiated a
research program aimed at the synthesis of a large number of
structurally diverse cyclodextrin derivatives and the evaluation
of their effect on OP degradation. In terms of structure, these
compounds follow a common design principle, involving three
Figure 1: Schematic representation of the general structural design of
the investigated cyclodextrin derivatives (CD = cyclodextrin ring,
L = linking unit, Nu = reactive nucleophilic unit).
Scheme 1: Structure of cyclosarin (GF).
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Here, we describe our first results in this project, involving a Results and Discussion
series of β-cyclodextrin derivatives with substituents on the pri- Synthesis. The cyclodextrin derivatives investigated in this
mary face of the cyclodextrin ring, containing oximes as study are shown in Scheme 2. All compounds derive from the
nucleophilic groups. Oximes are well-known antidotes for the seven-membered β-cyclodextrin and contain the substituent in
treatment of OP poisonings. Their mode of action involves reac- the 6-position of a glucose unit, i.e., along the narrower rim of
tivation of the OP-inhibited acetylcholinesterase [29], yet the cyclodextrin cavity.
previous work has also indicated that certain oximes are able to
cleave OPs directly [30]. We show that some of our cyclodex- These products were prepared by following two different
trin derivatives efficiently reduce GF concentrations in solution routes. Cyclodextrins 1a–e derive from mono-6-(p-tolylsul-
under physiological conditions within seconds, thus preventing fonyl)-β-cyclodextrin (3), which can be expediently prepared
the OP from inhibiting AChE. The observed correlation of from β-cyclodextrin and p-tolylsulfonyl chloride [31]. Reaction
structural and electronic parameters of the cyclodextrin deriva- of 3 with an appropriate nucleophile then afforded the function-
tives with their activity strongly indicates that the interaction alized products (Scheme 3, route A). Compound 1a was
between the cyclodextrin ring and the substrate plays a decisive prepared by the reaction of 3 with 3-(aminomethyl)benzalde-
role in the mode of action.
hyde oxime (5) (Scheme 4), the latter of which was synthesized
Scheme 2: Cyclodextrin derivatives tested as potential GF scavengers.
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Beilstein J. Org. Chem. 2011, 7, 1543–1554.
Scheme 3: General strategies used for the preparation of the investigated cyclodextrin derivatives.
from isophtalaldehyde, as shown in Scheme 5. Compounds
1b–e were obtained from 3 and the corresponding pyridine
aldoximes or ketoximes (Scheme 4) all of which are easily
accessible from the commercially available aldehydes or
ketones by reaction with hydroxylamine [32]. Attempts to also
prepare the analogous derivative with the oxime group in the
2-position of the pyridinium ring, by reaction of 3 with
2-formylpyridine oxime or 2-acetylpyridine oxime, unfortu-
nately failed to produce the desired products.
Scheme 5: Synthesis of 3-(aminomethyl)benzaldehyde oxime (5).
Reagents and conditions: i. NaBH4, EtOH, 1 h, 25 °C; ii. HBr/HOAc,
2 h, 25 °C, 38% (2 steps); iii. NaN3, EtOH, 4 h, reflux, 94%; iv.
NH2OH·HCl, NEt3, EtOH, 1.5 h, 25 °C, 87%; v. PPh3, H2O/MeOH,
18 h, 25 °C, 80%.
The cyclodextrin derivatives 2a–d contain 1,4-disubstituted
1,2,3-triazole moieties as the linking units. Accordingly, they
were prepared by copper(I)-catalyzed azide–alkyne cycload-
Scheme 4: Reaction conditions used for the preparation of cyclodex-
trin derivatives 1a–e from tosylate 3.
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Beilstein J. Org. Chem. 2011, 7, 1543–1554.
dition (“click-reaction”) [33] from mono-6-azido-6-deoxy-β-
cyclodextrin (4) and a functionalized alkyne (Scheme 3, route
B). Conjugations by copper(I)-catalyzed azide–alkyne cycload-
ditions have become popular in many different fields of chem-
istry [33,34], including cyclodextrin chemistry [35-41]. The
alkyne 6 required for the synthesis of 2a was obtained from
propargyl bromide and 3-formylpyridine oxime (Scheme 6),
and those for 2b, 2c, and 2d, following the routes shown in
Scheme 7. Reaction of these alkynes with 4 in the presence of
copper(II) sulfate, sodium ascorbate and tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]amine (TBTA) [42] afforded the
corresponding coupled products (Scheme 8).
Scheme 6: Synthesis of 3-((hydroxyimino)methyl)-1-(prop-2-ynyl)pyri-
dinium bromide (6). Reagents and conditions: i. KI, DMF/toluene, 3 h,
25 °C, 39%.
All functionalized cyclodextrins 1a–e and 2a–d were purified
by preparative HPLC. To this end, a universal method was
developed that involves the use of a reversed-phase C18 column
and a gradient of a binary solvent mixture, acetonitrile/0.025%
aqueous ammonia. All products were thus obtained in analyti-
cally pure form, which is necessary to ensure that the subse-
Scheme 8: Reaction conditions used for the preparation of cyclodex-
trin derivatives 2a–d from azide 4.
quent evaluation of activity yields reliable results. Elemental respective cyclodextrin derivative at 37.0 °C in an aqueous
analyses and NMR spectroscopy indicated that the pyridinium- TRIS-HCl buffer (pH 7.40). Aliquots of this solution were
containing cyclodextrins do not contain a counterion and that added to a solution containing human acetylcholinesterase
they therefore most probably represent the betaine forms (hAChE, EC 3.1.1.7), acetylthiocholine (ATCh) and 5,5'-
depicted in Scheme 2.
dithiobis-(2-nitrobenzoic acid) (DTNB) immediately after
mixing and after 30 and 60 min. The inhibitory effect of GF on
Qualitative assay. In a first step, the ability of the prepared the enzyme was then quantified photometrically by following
cyclodextrin derivatives to reduce GF concentration in solution the rate of formation of the 2-nitro-5-thiobenzoate dianion
and, as a consequence, the inhibitory effect of this OP on AChE (Ellman assay) [44,45]. A first order rate constant k1 was
was estimated by using a fully automated high-throughput derived from these curves by nonlinear regression analysis,
screening assay recently developed for the characterization of which is a measure of the extent of enzyme inhibition: The
potential nerve agent detoxifying materials [43]. This test larger the value of k1 the stronger the inhibitory effect of the
involves incubation of the nerve agent with an excess of a nerve agent on the enzyme activity. By relating k1 to k1ref, the
Scheme 7: Syntheses of 6-ethynyl-formylpyridine oximes (7a–c). Reagents and conditions: i. CuI, (PPh3)2PdCl2, DPEphos, NEt3, 18–20 h, 25 °C,
71-87%; ii. NH2OH, H2O, 30 min, 80 °C; iii. TBAF, THF, 1 h, 0 °C, 68–83%.
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Beilstein J. Org. Chem. 2011, 7, 1543–1554.
rate constant determined in the absence of the cyclodextrin, and pyridine ring can most probably be attributed to the higher
to k1native, the rate constant determined in a preliminary assay in acidity of the aldoxime proton in pyridinium aldoximes, for
the absence of both cyclodextrin and GF, one obtains example. For reference, the pKa of the aldoxime proton in
(k1ref−k1)/(k1ref−k1native)·100% = Δk1, a term which correlates 3-formylpyridine oxime amounts to 10.36 and that of the
with the activity of the cyclodextrin. If the extent of enzyme corresponding proton in the 1-methiodide of 3-formylpyridine
inhibition is the same in the absence and the presence of the oxime to 9.22. Correspondingly, the pKa of the aldoxime proton
cyclodextrin (k1ref = k1) the cyclodextrin is inactive and in 4-formylpyridine oxime (9.99) also decreases by more than
Δk1 = 0%. If, however, the nerve agent is decomposed faster in one order of magnitude to 8.57 upon methylation of the ring
the presence of the cyclodextrin than during the background nitrogen [46]. Thus, oximes on pyridinium rings are deproto-
reaction, k1 becomes smaller with respect to k1ref until it nated more easily, which renders them more nucleophilic.
approaches the value of k1native. As a consequence Δk1
increases up to 100% for full enzymatic activity (k1 = k1native). The influence of structural effects on the activity becomes
The results of this assay obtained for cyclodextrin derivatives evident when comparing 1b and 2a, both of which have the
1a–e and 2a–d are summarized in Figure 2.
oxime moiety in the same position on the aromatic ring. The
significantly larger activity of 1b is an indication that the posi-
Figure 2 shows that even native β-cyclodextrin exhibits a tioning of the nucleophilic group closer to the cavity of the
substantial activity, consistent with the results of previous cyclodextrin facilitates the reaction with GF. Interestingly, even
investigations [19,20]. The reduction of AChE inhibition is small structural variations such as shifting the substituent on the
clearly visible after 30 min, and the enzyme is fully active when pyridinium ring from the 3- into the 4-position (1b versus 1c) or
GF was incubated with β-cyclodextrin for 1 h prior to the replacing the aldoxime with a ketoxime (1b versus 1d) are
Ellman assay. Notably, all substituted cyclodextrin derivatives associated with a significant loss of activity. While the latter
already exhibit an effect in the first measurement, thus clearly effect is presumably due to the lower acidity of ketoximes with
demonstrating the enhancement of activity caused by the respect to aldoximes by ca. one order of magnitude [32], the
substituents. While this effect is small for most cyclodextrins, it higher activity of 1b with respect to 1c is more likely to be a
is significant for 1d, and the activity of 1b is so high that no structural effect, since on the basis of the pKa values of
enzyme inhibition is observable even in the first measurement. 1-methiodides of 3-formylpyridine oxime (9.22) and
4-formylpyridine oxime (8.57) one would expect the opposite
The correlation between the type of substituent on the cyclodex- trend.
trin and the reduction of AChE inhibition, which is evidenced in
Figure 2, indicates that the activity of these cyclodextrin deriva- The pronounced activity of the investigated cyclodextrin deriva-
tives depends sensitively on the structural parameters of the tives, in particular of 1b, also indicates that noncovalent interac-
substituents in combination with the electronic properties. The tions, most probably the inclusion of the apolar cyclohexyl
generally larger activity of the pyridinium derivatives such as moiety of GF into the cyclodextrin cavity, are an important
1b–e and 2a with respect to compounds containing a neutral aspect of the mode of action. Assuming that the affinity of GF
Figure 2: Diagram summarizing the observed Δk1 values for cyclodextrins 1a–e and 2a–d. For comparison, the results obtained for native
β-cyclodextrin are also included in the diagram. Large bars indicate low enzyme inhibition and, consequently, high activity of the corresponding
cyclodextrin. The results are averages over three independent measurements.
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to β-cyclodextrin in water is similar to that of cyclohexanol containing the nerve agent was incubated at 37.0 °C and an
(687 M−1 at 30 °C) [47,48], and that it is approximately inde- aliquot was taken to determine the initial concentration of GF,
pendent of the type of substituent, ca. 25% of the substrate c0(GF). After the addition of a thermostatted cyclodextrin solu-
molecules are estimated to reside in the cyclodextrin cavity tion, aliquots were taken at defined intervals. These samples
under the conditions of the assay (500 μM cyclodextrin and were immediately extracted with hexane and subjected to
1 μM GF). This value presumably represents a lower limit, GC–MS analysis by using d11-GF (GF with a perdeuterated
because hydrogen-bonding interactions between the OH-groups cyclohexyl residue) as the internal standard. The use of a chiral
along the rim of the cyclodextrin cavity and the P=O group of stationary phase allowed independent evaluation of the effect of
the substrate can cause the GFcomplex to be more stable than the cyclodextrin on both GF enantiomers. The results of these
that of cyclohexanol. However, it indicates that complexation of measurements are shown in Figure 3.
only part of the total substrate amount could ensure efficient
conversion, if the oxime group of the cyclodextrin can ap- One important aspect that becomes immediately apparent when
proach the phosphonate moiety of the substrate in the complex, looking at the graphs in Figure 3 is that the cyclodextrins act
and if complexation/decomplexation kinetics are fast as is enantioselectively, with the (−)-enantiomer of GF being the one
usually the case for cyclodextrin complexes [49]. The remark- that disappears faster, independent of the cyclodextrin deriva-
able high activity observed for 1b prompted us to evaluate in tive used. This result, which is consistent with those of previous
more detail the rate with which GF is degraded in the presence investigations [22,23], is another indication of the involvement
of this compound. For comparison, the less active isomer of 1b, of the chiral cyclodextrin residue in the reaction. The top left
4-substituted derivative 1c, and the triazole-linked derivative 2a graph shows that under the standard conditions of this measure-
were also included in this study.
ment (−)-GF is consumed so quickly, with conversion being
complete after ca. 5 s, that no reliable kinetic analysis could be
Quantitative assay. The kinetic parameters for the reduction of performed. Decomposition of the corresponding (+)-enan-
GF concentration in solution mediated by 1b, 1c, and 2a were tiomer is slower, but also complete after ca. 4 min. In order to
determined as described previously for other cyclodextrin follow the rate with which (−)-GF disappears, the concentra-
derivatives [23]. Briefly, a buffered solution (pH 7.40) tion of cyclodextrin during incubation with GF was reduced to
Figure 3: Time-dependent decrease of GF concentration in the presence of 1b (top row), 1c (middle row), and 2a (bottom row). The measurements
were performed at different concentrations of cyclodextrin (CD) and nerve agent, which are indicated at the top of each column. The data points
(means ± SD, n = 2) denote the experimental results normalized to the initial concentration of GF, c0(GF). The curves show the results of fitting the
data points to a first-order rate reaction. Disappearance of the (−)-enantiomer of GF is shown in red and that of the (+)-enantiomer in blue.
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Beilstein J. Org. Chem. 2011, 7, 1543–1554.
50 μM. As expected, this modification of conditions caused a thermodynamics of the reaction could be obtained by following
reduced rate of conversion for both enantiomers, presumably the dependency of the rate of reaction on the GF concentration
because the amount of GF molecules bound inside the (Michaelis–Menten kinetics). These measurements are,
cyclodextrin cavity decreases by a factor of ca. 10. Performing however, demanding in light of the complexity of the currently
the analysis at 50 μM of cyclodextrin and 10 μM of GF does used kinetic assay and have therefore not yet been performed.
not have a large effect on the rate of reaction, which is consis-
tent with the fact that increasing the GF concentration does not Conclusion
shift the complex equilibrium to a large extent. It should be These investigations demonstrate that appropriately substituted
noted that this assay currently does not allow us to ascertain cyclodextrin derivatives can efficiently reduce the inhibitory
whether the action of the cyclodextrins is stoichiometric or effect of GF on AChE under physiological conditions. The
catalytic, because the cyclodextrin derivatives were used in relatively strong effect of the cyclodextrin derivatives on GF
excess with respect to GF.
degradation and, in particular, the observed enantioselectivity
are good indications for noncovalent interactions between the
Similar trends were observed for the other cyclodextrin deriva- cyclodextrin ring and the substrate, presumably involving the
tives, although their overall rates of conversion were consis- inclusion of the cyclohexyl moiety of cyclosarin into the
tently lower than those of 1b. Quantitative information in terms cyclodextrin cavity, which contribute to the mode of action. In
of the observed rate constants kobs and half-lives t1/2was addition, the correlation between structural parameters and
obtained by fitting the experimentally obtained decay curves to activity can be rationalized on the basis of the distance of the
a first order rate reaction and subtracting the effect of spontan- substituents from the cyclodextrin cavity, where OP binding
eous GF hydrolysis under these conditions (1.5·10−4 s−1) [23]. presumably takes place, and the expected nucleophilicity of the
The results thus obtained are summarized in Table 1.
oxime groups of the substituents. With 1b a compound could be
identified that, to the best of our knowledge, currently repre-
Comparison of these rate constants with ones obtained previ- sents the most active cyclodextrin derivative mediating GF de-
ously under similar conditions for native β-cyclodextrin and gradation in solution. These results make us optimistic that
2-O-(carboxy-iodosobenzyl)-β-cyclodextrin leads to the conclu- substituted cyclodextrins represent a very promising platform
sion that the substituted cyclodextrins investigated here enhance for the development of scavengers for highly toxic organophos-
the GF decomposition by at least one order of magnitude more phonates, including also ones that are more persistent than GF,
efficiently than native β-cyclodextrin does [23]. Degradation of such as tabun or VX. Work in this context is ongoing in our
(+)-GF is ca. twice as fast in the presence of 1b than in the group.
presence of 2-O-(carboxy-iodosobenzyl)-β-cyclodextrin.
Because of the relatively low enantioselectivity observed for Experimental
2-O-(carboxy-iodosobenzyl)-β-cyclodextrin, the activity of General details. The synthesized compounds were character-
cyclodextrin 1b is estimated to be at least one order of magni- ized as follows: Melting points, Müller SPM-X 300; NMR,
tude higher. More detailed information about the kinetics and Bruker Avance 600, Bruker DPX 400; MALDI-TOF-MS,
Table 1: Kinetic constants determined for GF degradation mediated by cyclodextrins 1b, 1c, and 2a (kobs in s−1, t1/2 in s).
(−)-GF
(+)-GF
(−)-GF
(+)-GF
cyclodextrin concentration
cyclodextrin
1 μM
10 μM
kobs
t1/2
n.d.a
n.d.a
1.6·10−2
43
1b
1c
2a
kobs
t1/2
1.8·10−2
39
3.1·10−3
211
500 μM
kobs
t1/2
6.1·10−3
114
1.2·10−3
583
kobs
t1/2
1.5·10−2
46
6.5·10−4
1071
9.6·10−3
72
4.7·10−4
1465
1b
1c
50 μM
kobs
t1/2
6.6·10−4
1053
1.8·10−4
3857
5.9·10−4
1179
1.7·10−4
4143
ano reliable rate constant and half-life could be determined.
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Bruker Ultraflex TOF/TOF; ESI–MS, Bruker Esquire 3000; IR, maintained at 37.0 °C. Further aliquots of the test mixture were
FT-IR System Spectrum BX, Perkin-Elmer; elemental analysis, taken after 30 min and 60 min and treated analogously. The rate
Elementar vario Micro cube. All chemicals, unless other stated, constants k1 were determined by nonlinear regression analysis
are commercially available and were used without further of the resulting curves by using Prism 5.0 (GraphPad Software,
purification. Cyclosarin and deuterated cyclosarin (d11- San Diego, CA, USA) as described previously [43]. All results
cyclosarin, d11-GF) (>98% by GC–MS, 1H NMR, and 31P shown are mean values of n = 3 experiments. The value for
NMR) were made available by the German Ministry of Defense k1native was determined in independent experiments to be
(All experiments with cyclosarin were performed at the Institut 8.56·10−5 s−1.
für Pharmakologie und Toxikologie der Bundeswehr). Hemo-
globin-free erythrocyte ghosts as a source for human erythro- Quantitative assay. The quantitative assay was performed in a
cyte acetylcholinesterase (AChE, EC 3.1.1.7) were prepared stirred 2.0 ml cryo vial (Wheaton Science Products, Millville,
according to the procedure of Dodge et al. [50] with minor NJ, USA) positioned in a temperature controlled water bath.
modifications [51]. AChE activity was adjusted to 4000 U/l by Buffer (1850 µL, TRIS-HCl 0.1 M, pH 7.40) containing GF
dilution with phosphate buffer (0.1 M, pH 7.40). Aliquots were (1.1 µM) was incubated at 37.0 °C and an aliquot (50 µL) was
stored at a temperature of −80 °C. Prior to use, ghosts were removed to determine the initial concentration of GF, c0(GF)
homogenized with a Sonoplus HD 2070 ultrasonic homogenator (Scheme 10). After preheated cyclodextrin solution (200 µL,
(Bandelin electronic, Berlin, Germany) twice for 5 s with a 20 s final concentration 500 µM) was added, aliquots (50 µL) were
interval to achieve a homogeneous matrix. For the preparative taken at defined time points. These samples were transferred
HPLC the following conditions were used: HPLC, Dionex into a tube containing sodium formate buffer (20 µL, 300 µM,
UltiMate 3000; column, Thermo Fisher, BetaBasic-18, pH 3.5) and a solution of the internal standard d11-GF in
250 × 21.2 mm, 5 μm particle size; flow, 12 mL/min; eluent, 2-propanol (2.5 µL, 10 µg/mL). Afterward, ice-cold hexane
aqueous: 0.025% aqueous ammonia, organic: acetonitrile; for (500 µL) was immediately added, and the mixture was shaken
the separation of neutral compounds (1a, 2b–2d) the following vigorously and stored on ice. Each sample was centrifuged as
gradient was used: 0–6 min, 0% organic; 6–27 min, linear soon as possible (10.000 rpm, 10 min, 4 °C), and the organic
increase of organic to 40%; 27–33 min, 40% organic; layer was removed immediately and transferred to GC vials
33–39 min, linear decrease to 0% organic; 39–45 min, 0% for analysis. The experimental results were normalized to the
organic; charged compounds (1b–1e, 2a) were purified by initial concentration of GF in the absence of cyclodextrin,
employing a slightly different gradient: 0–6 min, 0% organic; c0(GF). The kinetic constants kobs were determined by, first,
6–22 min, linear increase to 15% organic; 22–25 min, linear nonlinear regression analysis of the resulting decay curves
increase to 50% organic; 25–30 min, 50% organic; 30–37 min, (using GraphPad Prism 5.0, San Diego, CA, USA) on the basis
linear decrease to 0% organic; 37–45 min, 0% organic.
of the following equation: c(+/-)GF,t = c(+/-)GF,0*·exp(−kobs·t), in
which c(+/-)GF,t denotes the concentration of (+)-GF or (−)-GF
Qualitative assay. The qualitative test was performed by using at a certain time t and c(+/-)GF,0* the concentration of (+)-GF or
a Tecan Freedom EVO liquid handling system (Männedorf, (−)-GFimmediately after cyclodextrin addition, followed by
Switzerland) [43]. A solution of cyclodextrin (500 µM) and GF subtracting the effect of spontaneous GF hydrolysis
(1 µM) was prepared and incubated in TRIS-HCl buffer (0.1 M, (1.5·10−4 s−1) [23]. All data shown are the mean values of n = 2
pH 7.40) at 37.0 °C (Scheme 9). Immediately after mixing, a experiments.
sample (25 µL) was transferred to a measuring plate prefilled
with buffer (2400 µL, 0.1 M TRIS-HCl, pH 7.40 and 0.3 mM Quantification of GF enantiomers by PCI MS. Quantifica-
DTNB) and human AChE (10 µL) and preheated to 37.0 °C. tion of GF enantiomers was performed by GC–MS, as
ATCh (50 µL, 49.7 mM) was added immediately, the described before but with slight modifications [52]. In brief,
microplate was transferred to a photometer and the absorption a gas chromatographic system 6890N coupled with a mass
was measured at 436 nm for 30 min while the temperature was spectrometer detector 5973 with positive chemical ionization
Scheme 9: Schematic protocol for the qualitative assay.
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Beilstein J. Org. Chem. 2011, 7, 1543–1554.
Scheme 10: Schematic protocol of the quantitative assay.
(PCI) (Agilent Technologies, Waldbronn, Germany), large Acknowledgements
volume injection (LVI), and a GAMMA DEXTM 225 This research was supported by a contract of the German
GC column (30 m × 0.25 mm, 0.25 µm film thickness, Armed Forces (E/UR3G/9G127/9A803). The generous funding
Sigma–Aldrich Chemie, Taufkirchen, Germany) was used is gratefully acknowledged.
(Table 2). CI quantification was performed by applying
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