Optimized strategies to synthesize β-cyclodextrin-oxime conjugates as a new generation of organophosphate scavengers

Article abstract of DOI:10.1039/c0ob00931h

A new generation of organophosphate (OP) scavengers was obtained by synthesis of β-cyclodextrin-oxime derivatives 8-12. Selective monosubstitution of β-cyclodextrin was the main difficulty in order to access these compounds, because reaction onto the olig

Full text of DOI:10.1039/c0ob00931h

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PAPER
Optimized strategies to synthesize b-cyclodextrin-oxime conjugates as a new
generation of organophosphate scavengers†
Romain Le Provost,a,b Timo Wille,c Ludivine Louise,a,d Nicolas Masurier,a,b Susanne Mu¨ller,c Georg Reiter,c
Pierre-Yves Renard,a,d Olivier Lafont,b Franz Worekc and Franc¸ois Estour*a,b
Received 25th October 2010, Accepted 27th January 2011
DOI: 10.1039/c0ob00931h
A new generation of organophosphate (OP) scavengers was obtained by synthesis of
b-cyclodextrin-oxime derivatives 8–12. Selective monosubstitution of b-cyclodextrin was the main
difficulty in order to access these compounds, because reaction onto the oligosaccharide was closely
related to the nature of the incoming group. For this purpose, non-conventional activation conditions
were also evaluated. Intermediates 5 and 7 were then obtained with the better yields under ultrasounds
irradiation. Finally, the desired compounds 8–10 were obtained from 5–7 in high purity by desilylation
using potassium fluoride. Quaternarisation of compounds 8 and 9 was carried out. OP hydrolytic
activity of compounds 8–12 was evaluated against cyclosarin (GF) and VX. None of the tested
compounds was active against VX, but these five cyclodextrin derivatives detoxified GF, and the most
active scavengers 10 and 11 allowed an almost complete hydrolysis of GF within 10 min. Even more
fascinating is the fact that compounds 9 and 10 were able to hydrolyze enantioselectively GF.
irreversible acetylcholinesterase (AChE) inhibitors.1 This enzyme
Introduction
plays a key role in the central and peripheral cholinergic system
Organophosphorus compounds (OP), such as pest control agents
and intoxication by OPs can cause life-threatening cholinergic
signs and symptoms.2 The development of new scavengers to
prevent phosphylation of cholinesterases by these poisons is of
paramount importance,3 not only because poisoning by OP pest
control agents is considered to cause more than 200,000 fatalities
yearly,4 but also since the Matsumoto and Tokyo subway strikes
with sarin underlined the reality of a terrorist threat.5 In order
to gain access to efficient antidotes, it is necessary to develop
compounds which will be able to detoxify OP compounds by rapid
binding and hydrolysis. The purpose of this work is to design a
new generation of scavengers exhibiting this activity. Recently,
functionalized cyclo-maltoheptaose (b-CD) derivatives bearing a
reactive moiety against OPs were developed as artificial enzymes.6
This oligosaccharide can form inclusion complexes with various
organic substrates, and in particular with organophosphorus
poisons.7 The OP, once “trapped” in the internal cavity of the b-CD
can moreover be subjected to attack by a reactive group branched
on one of the hydroxyl groups. The interest of this strategy is to
take advantage of the OP binding into the oligosaccharide to force
a reactive positioning of the OP towards a nucleophilic function.
In order to effectively hydrolyse OPs to nontoxic metabolites, the
nucleophile must specifically cleave the P-X bond (X = F, S, CN,
N, depending on the nerve agent).
or chemical warfare nerve agents (Fig. 1) sarin, cyclosarin, soman,
tabun (G-type nerve agents) or exceedingly toxic VX act as
Fig. 1 Structures of chemical warfare agents.
aUMR 6014 & FR 3038 CNRS, rue Tesnie`re, 76821, Mont-Saint-Aignan
Cedex, France. E-mail: francois.estour@univ-rouen.fr; Fax: (+) 33-235-
522-971
bUniversite´ de Rouen, UFR de Me´decine-Pharmacie, Laboratoire de Phar-
macochimie, 22 boulevard Gambetta, 76183, Rouen Cedex 1, France
cBundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
dUniversite´ de Rouen, Place Emile Blondel, 76821, Mont-Saint-Aignan,
France
a-Nucleophilic groups have the ability to realize this cleavage,
and oximes seem to be the most suitable.8 Although pyridi-
naldoximes and pyridinium-aldoximes such as pralidoxime are
† Electronic supplementary information (ESI) available: Experimental
procedures, NMR data, identification of regioisomers, NMR spectra for
representative products. See DOI: 10.1039/c0ob00931h
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Table 1 Experimental details of coupling methods between b-CD and
mainly known for their effects in reactivating OP-inhibited
acetylcholinesterases,7 some data concerning a direct reaction
of such oximes with nerve agents have already been published,9
but the obtained maximal reaction velocities are too low to be
applied for an in vivo detoxification.10 Thus, introduction of a
2-pyridinaldoxime backbone on b-CD was also a challenge to
access to a new generation of organophosphate scavengers. To
modify the orientation of the nucleophilic group toward the
internal oligosaccharidic cavity, the reactive group could also be
grafted on the three distinct positions of b-CD, i.e. positions 2,
3 (upper rim of b-CD torus) and 6 (lower rim) of the glucose
moieties. The secondary side of cyclodextrins is stated to be
catalytically more efficient,11,12 and therefore, modifications of
this face are believed to produce valuable derivatives for enzyme
mimics. If formation of an inclusion complex by the cavity of
cyclodextrins played a very prominent role in determining the
reaction site for the incoming group, the acidity of hydroxyl groups
was also an important parameter. The basicity of hydroxyl groups
are in the order 6 > 3 > 2. This feature has been exploited by using
a strong base under anhydrous conditions for selective substitution
at the position 2.
electrophiles
Deprotonation step
(time/temperature)
Substitution step
(time/temperature)
Method
USa
MWb
A
B
C
D
—
—
+
—
—
—
+
14 h/r.t.
8 h/r.t.
14 h/70 ◦
1 h/r.t.
C
8 h/70 ◦
C
3 h/r.t. to 45 ◦
30 min/70 ◦
C
—
5 min/70 ◦
C
C
a US = Ultrasounds; b MW = Microwaves
Table 2 Influence of method use on the substitution rates
Total substitu- Polysubstitu- Monosubstitution
Reagent Method tion yield (%) tion yield (%) yield at O-2 (%)
3
2
1
A
B
C
D
A
B
23
24
36
25
29
7
16
25
5
2
3
<1
2
0
0
0
0
0
18
18
30
20
29
7
16
25
5
C
D
Aa
Ab
C
Results and discussion
3
14
14
0
0
0
3
14
14
D
To prepare these scavengers, the synthetic strategy consists in
monosubstituting b-CD with a suitable bromomethyl pyridine
derivative. The key step is then the regioselective monosubstitution
of the oligosaccharidic moiety by the tert-butyldiphenylsilyl (TB-
DPS) protected 4, 5 or 6-bromomethyl pyridinaldoxime motives
1–3.‡ The total substitution yields were deceptively low using
the optimized monosubstitution method on the 2 position of
the sugar we previously published in the case of benzyl halides,13
because an unavoidable deprotection of the TBDPS group under
these experimental conditions was observed, and led to poly-
addition side products. These conditions, optimized for other
benzyl bromides, had thus to be modified. Different coupling
reactions between b-CD 4 and electrophiles 1–3 were carried out
with dimethylsulfoxide as the solvent, and under basic conditions
(Scheme 1).
a The time of substitution was decreased to 90 min. b The time of
substitution was decreased to 45 min.
substitute b-CD.14 This strong base was then tested here. In order
to further improve the efficiency of the b-CD substitution, different
activation processes were also evaluated (Methods A–D, Table 1),
including ultrasounds or microwaves irradiation.15,16 These non-
conventional conditions already allowed a tosylation of one of the
alcohols at O-6 or O-2 of CDs.16 They were used here to directly
introduce the desired functional group in the position 2 of one
glucose unit of the native b-CD (Table 2).
In the case of compound 3, heat or microwaves activation has
no influence, but, sonication allowed for a substantial increase
(+ 50%) of the substitution yield. The strong shockwave generated
from the collapsing bubbles by sonication seemed to improve
the substitution efficiency. Whatever the method A-D used, the
2-monosubstituted derivative 7 was the main isolated product, but
a small amount of another regioisomer was also detected. High
intensity ultrasounds irradiation led also to the best selectivity
in favour of the monosubstitution against the polysubstitution
observed as a side reaction. As already proved, this technique was
also very advantageous in terms of reaction times for the synthesis
of highly valuable CD derivatives.16 In this case, the substitution
time was considerably shortened down to three hours instead of
eight hours with methods A and B. Altogether, for compound
3, under ultrasounds activation, an acceptable 30% yield of O-2
monosubstitution product was cleanly isolated.
Scheme 1 Coupling reactions using methods A–D.
For compound 2, the best isolated yield (29%) was obtained
using sodium hydride in dimethylsulfoxide at room temperature.
Microwaves also gave a satisfactory substitution yield of 25%. On
the other hand, activation through an increase of the temperature
to 70 C led to a decrease to 7% yield. Sonication did not allow
in this case a satisfactory result, because the substitution yield
The hydroxyl groups at the position 2 being the most acidic,
sodium hydride has also been successfully used to selectively
◦
‡ The corresponding procedures for the syntheses of compounds 1–3 are
included in the ESI.†
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reached only 16%. Contrary to what we observed with reagent
3, the 2-modified b-CD 6 was the only isolated regioisomer.
For electrophile 1 which proved to be a more unstable reagent
compared to compounds 2 and 3, standard methods only allowed
isolation of the desired compound 5 with a particularly low yield,
the best result leading to 5% monosubstitution yield by the method
A. In this case, only activation through the use of ultrasounds
or microwaves allowed an increase of the substitution yield to
14%. All these results showed that the monosubstitution reaction
onto b-CD is a tricky and hardly predictable reaction, since the
substitution pattern and global yield are closely related to the
nature of the incoming group. In some cases, non conventional
activations such as ultrasounds or microwaves irradiation were
then interesting alternative methods to lead efficiently in a short
time to the desired monosubstituted b-CD. Moreover, ultrasounds
or microwaves did not affect significantly the regioselectivity, when
a mixture of regioisomers was obtained by classical methods.
Identification of 2-monosubstituted b-CD regioisomers was
carried out using a previously described methodology.17,§ Once
the protected b-CD-oximes were obtained in a sufficient yield,
deprotection of compounds 5–7 was carried out using potassium
fluoride to give cleanly the corresponding b-CD-oxime conjugates
8–10 (Scheme 2).
Scheme 3 Quaternarization of the pyridine nitrogen atom in compounds
8 and 9.
assay (remaining AChE cholinesterase activity after treatment
with the nerve agent with or without pre-treatment with the
scavenger).
The efficiency of 8–12 to detoxify nerve agents was tested with
a single GF and VX concentration, i.e. 1 mM (Fig. 2). Hereby,
these cyclodextrins (500 mM) were able to degrade GF in the
order 10 = 11 > 9 > 8 > 12. With 10 and 11 an almost complete
GF detoxification was achieved within 10 min and the five tested
cyclodextrins were able to degrade GF within 40 min completely.
After a pre-treatment with free pyridine-2-aldoxime methiodide
(pralidoxime) itself used as the same concentration than the
scavengers 8–12, no relevant degradation of GF was observed
after 40 min.¶ These kinetic assays showed the interest to graft an
oxime derivative on b-CD to improve GF-hydrolyzing efficiency.
Based on the structure of compounds 8–10, results proved that
hydrolytic activity on GF was regiocontrolled. It had been already
the case with b-CD-iodosobenzoate conjugates,6a but the fast
detoxification of GF was obtained here when the linker between
b-CD and the pyridinic moiety was in meta-position of the oxime
function and just beside the cyclic nitrogen atom. Even if the linker
was in meta-position of the oxime group, compound 8 showed
the weakest activity. An intermediate activity was obtained with
compound 9, when the linker was in position 5¢ of the aromatic
ring. Although compound 10 was tested in a mixture with another
regioisomer (respectively in an 80/20 ratio), hydrolytic assays
seemed to indicate that the oxime function and nitrogen atom of
Scheme 2 Deprotection of the oxime function.
In order to increase the nucleophilic efficiency of the oximate
function against organophosphorus compounds at a physiological
pH, quaternarization of the pyridine nitrogen atom was also
accomplished to access a nucleophilic reactive group similar to
pralidoxime. Different experimental conditions tested with com-
pound 10 by varying the amount of alkylating agent, temperatures
and reaction times, did not allow the access to the corresponding
quaternary derivative.
Even if alkylation was attempted with compound 3 itself
before substitution by b-CD, it was not possible to introduce a
methyl group on the nitrogen atom situated between two bulky
groups. On the other hand, methylation of pyridinic nitrogen
was successfully performed with a large excess of methyl iodide
in DMF without trace of base from compound 8 and 9 to give
respectively derivatives 11 and 12 (Scheme 3).
In a first set of experiments the ability of cyclodextrin derivatives
to detoxify cyclosarin (GF) and VX was tested with a biological
Fig. 2 Detoxification of cyclosarin (GF; 1 mM) by 8, 9, 10, 11, and 12
(500 mM). Data are given as means SD of two assays.
§ Accurate NMR structural determination of compound 9 is detailed in
the Electronic Supplementary Information† as a specific and representative
example.
¶ Kinetic course of the direct reaction of GF (1 mM) and 2-PAM (500 mM)
is included in the ESI.†
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Table 3 Kinetic constants (min-1) for the hydrolysis of GF enantiomers
by 9 and 10
Compound
(-)GF
(+)GF
9
10
0.0656
0.0925
0.0497
0.0422
Fig. 4 Spontaneous, 9 and 10 catalyzed hydrolysis of cyclosarin.
Fig. 3 Detoxification of cyclosarin (GF) and VX by 10 (top) and 9
(bottom). Data are given as means SD of two assays.
the ring should be both in the surroundings of GF the upper rim of
b-CD. The higher activity obtained with compound 11 compared
to those of compound 8 provided evidence that quaternarization
of pyridinic nitrogen atom increased hydrolysis rate (through a
decrease of the oxime pKa). In the case of compound 12, the
decrease of the detoxification efficacy of GF was certainly due to
the relative steric hindrance caused by the methyl group, which
modifies the relative positioning of the oxime towards the nerve
agent. None of the tested compounds had any detoxifying effect
with VX (data shown in Fig. 4 in the case of 9 and 10). This
surprising result can be explained by a different (a non reactive)
binding mode of VX in the b-CD as compared to GF.
In addition, at the given conditions, i.e. 500 mM cyclodextrins, 9
and 10 were able to detoxify GF in a concentration-dependent
manner (Fig. 3). With both compounds 1 mM cyclosarin was
totally detoxified within 20 min. The results obtained from the
biological assay were further confirmed by a GC-MS quantitative
analysis. The direct degradation of GF (100 mM) by 9 and
10 (500 mM) was then tested by a pH-stat procedure (Fig. 4).
Incubation of GF with 9 or 10 resulted in a rapid degradation
of the fluorophosphonate with a half-time of 15.9 and 20.9 min,
respectively, and was faster than spontaneous hydrolysis of GF
(t12 = 74 min).
Finally, the stereoselective hydrolysis of cyclosarin (100 mM)
by 9 and 10 (500 mM) was investigated by GC-MS. The results
showed a moderate enantioselective hydrolysis of GF. With both
compounds the degradation of the more toxic enantiomer (-)GF
was faster (Fig. 5). However, the difference in the hydrolysis of
both GF enantiomers was more pronounced with 10 (Table 3). As
Fig. 5 Stereoselective hydrolysis of cyclosarin by 9 and 10.
observed with native b-CD,18 these results indicate an enantiose-
lective effect of the investigated cyclodextrin derivatives which is
reversed as compared with in vivo enzymatic degradation.11
In the design of OP scavengers, b-CD was chosen for its ability
to bind a guest molecule of OP. The fact that we observed an enan-
tioselective hydrolysis of cyclosarin by compounds 9 and 10 further
proves that b-CD is involved in the hydrolysis phenomenon, and
thus that b-CD acted then as a substrate binding site. It is generally
assumed that the lipophilic part of the organophosphate molecule
is taken up into the cyclodextrin central cavity.19 Nevertheless, the
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hydroxyl groups on the outer surface of the cyclic oligosaccharide
are also able to form a complex with lipophilic compounds,20 and
a mixture of inclusion and non-inclusion complexes can coexist in
aqueous solutions.21 For this purpose, further experiments would
be required to characterize the exact type of bonding between
GF and the new b-CD-oxime conjugates, but such experiments
are complicated by the fast kinetics of GF detoxification. They
could only be undertaken with a stable analogue of GF under
the conditions used for kinetic assays, thus not reflecting the
precise GF complexation mode. Yet whatever the bonding process
involved, a close interaction occurs, since b-CD induces an
enantioselective effect, even if the substrate enantioselectivity is
moderate. Further modifications of the b-CD scaffold will have to
be investigated in order to improve the chiral recognition of the
substrate.
and reagents were purchased from commercial sources and used
without further purification.
General procedures for substitution of b-cyclodextrin
Methods A and B. To a solution of b-cyclodextrin (0.5 g,
0.44 mmol, previously dried for 24 h under vacuum at 80 ◦C)
in 2.5 mL of dry DMSO, was added NaH (60% in mineral oil,
18 mg, 0.44 mmol) and the mixture was stirred under argon
for 14 h at room temperature (method A) or at 70 ◦C (method
B). A solution of bromomethyl-2-pyridinealdoxime (1–3) (0.2 g,
0.44 mmol) in 1.5 mL of dry DMSO was then added and
the reaction mixture was stirred for 8 h at room temperature
(method A) and at 70 ◦C (method B). Acetone (250 mL) was
then added and the crude product precipitated. After filtration,
the solid residue was chromatographed on silica gel; solvent: ethyl
acetate/isopropanol/water (12 : 7 : 4 to 8 : 7 : 4, v/v/v).
Conclusions
Methods C and D. To a solution of b-cyclodextrin (0.5 g,
0.44 mmol, previously dried for 24 h under vacuum at 80 ◦C)
in 2.5 mL of dry DMSO, was added NaH (60% in mineral oil,
18 mg, 0.44 mmol) and the mixture was stirred under argon for
1 h at room temperature (method C) and for 5 min at 70 ◦C
(method D). A solution of bromomethyl-2-pyridinealdoxime (1–
3) (0.2 g, 0.44 mmol) in 1.5 mL of dry DMSO was then added
and the reaction mixture was stirred for 3 h at room temperature
(method C) and for 30 min at 70 ◦C (method D). Acetone (250 mL)
was added and the crude product precipitated. After filtration, the
solid residue was chromatographed on silica gel; solvent: ethyl
acetate/isopropanol/water (12 : 7 : 4 to 8 : 7 : 4, v/v/v).
Different b-CD derivatives monosubstituted by a 2-pyridin-
aldoxime moiety at O-2 were prepared. Selective mono-
substitution reaction onto b-CD with functionalized and bulky
aromatic groups was tricky and this key step needed refined
strategies so as to obtain such compounds bearing different
pyridinaldoximes on the upper rim of the oligosaccharidic torus.
Functionalisation efficiency was closely related to the nature of the
incoming group. The most important and pivotal factor was then
the relative stability of the silyl protecting group of the electrophile.
In this regard, the use of non conventional activation such as
ultrasounds or microwaves irradiation constituted in some cases
an interesting alternative to access the desired substitution at the
O-2 position. All the synthezised oligosaccharidic compounds
were able to detoxify the highly deadly chemical warfare agent
GF, and hydrolysis rates of this OP were dependent on the
position of the linker between b-CD and the reactive moiety.
Even more fascinating is the fact that two scavengers were able
to hydrolize enantioselectively GF. In the design of OP scavengers,
b-CD has been chosen for its ability to bind a molecule of OP
and further studies will be developed to explore structure–activity
relationships.
Mono-2-O-({2-[({[tert-butyldiphenylsilyl]oxy}imino)methyl] pyr-
idin-4-yl}methyl)-cyclo-maltoheptaose 5. mp > 260 ◦C; IR (KBr)
n
max/cm-1 3390 (OH), 2930 (CH), 1655 (CN); 1H NMR (300 MHz,
DMSO-d6): d = 8.61 (d, J = 5 Hz, 1H; CH-Py), 8.56 (s, 1H, CH-
aldoxime), 7.72–7.68 (m, 4H, CH-Ph), 7.64 (s, 1H, CH-Py), 7.54
(d, J = 5 Hz, 1H; CH-Py), 7.48–7.40 (m, 6H, CH-Ph), 5.97–5.66
(m, 13H, OH-2, OH-3), 5.05 (d, J = 3 Hz, 1H, H-1¢), 4.85–4.80 (m,
8H, H-1, CH2-Py), 4.52–4.45 (m, 7H, OH-6), 3.88 (t, J = 9 Hz, 1H,
H-3¢), 3.68–3.30 (m, 41H, 15H2O), 1.11 (s, 9H, CH3); 13C NMR
(75 MHz, DMSO-d6): d = 155.6, 150.7, 149.8, 148.3, 135.0, 132.6,
130.0, 127.9, 122.9, 118.8, 101.9, 101.6, 99.8, 82.0, 81.5, 80.4, 73.0,
72.3, 72.0, 71.8, 71.0, 59.8, 26.8, 19.0; MS ES+: 1508 (M+H)+,
1530 (M+Na)+; HMRS calcd for C65H94N2O36NaSi: 1529.5253,
found: 1529.5289.
Experimental
General
NMR spectra were recorded on either a Bruker Advance 300
instrument or a Bruker Avance DMX 500 spectrometer. Chem-
ical shift (d) values are given in ppm and coupling constants
(J) are given in Hertz. DMSO-d6, H2O-d6 and CDCl3 with
an isotopic purity of 99.8% were purchased from Cambridge
Isotope Laboratories Inc. Reactions were monitored by thin-layer
chromatography (TLC) on a plate of silica gel 60 F254 (E. Merck,
Darmstadt, Germany). Developed plates were visualised using
ultraviolet light and/or by dipping the plates into a solution of
malonic acid (1 g), aniline (1 mL) and orthophosphoric acid at 85%
(3 mL) in ethanol (200 mL) followed by heating with a heat gun.
Column chromatographies were performed on silica gel 60 (0.063–
0.200 mm, E. Merck). MW-promoted reactions were carried out
in a professional oven, CEM Discover. All sonochemical reactions
were carried out with S120H reactor, Elmasonic. All solvents
Mono-2-O-({6-[({[tert-butyldiphenylsilyl]oxy}imino)methyl] pyr-
idin-3-yl}methyl)-cyclo-maltoheptaose 6. mp > 260 ◦C; IR (KBr)
n
max/cm-1 3400 (OH), 2930 (CH), 1655 (CN); 1H NMR (300 MHz,
(300 MHz, DMSO-d6): d = 8.65 (s, 1H, CH-Py), 8.56 (s, 1H, CH-
aldoxime), 7.86 (d, 1H, J = 6 Hz, CH-Py), 7.72–7.65 (m, 5H,
CH-Py, CH-Ph), 7.49–7.41 (m, 6H, CH-Ph), 5.97–5.67 (m, 13H,
OH-2, OH-3), 5.02 (s, 1H, H-1¢), 4.87–4.80 (m, 8H, CH2-Py, H-1),
4.58–4.45 (s, 7H, OH-6), 3.85 (t, J = 9 Hz, 1H; H-3¢), 3.70–3.25 (m,
41H, 14H2O), 1.10 (s, 9H; CH3); 13C NMR (75 MHz, DMSO-d6):
d = 155.4, 150.2, 149.2, 136.6, 135.0, 132.6, 130.0, 128.9, 127.9,
120.4, 101.9, 101.6, 99.9, 82.0, 81.5, 80.1, 73.0, 72.3, 72.0, 71.7,
70.2, 59.8, 26.8, 18.9; MS ES+: 1508 (M+H)+, 1530 (M+Na)+;
HMRS calcd for C65H94N2O36NaSi: 1529.5253, found: 1529.5295.
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81.4, 80.4, 73.4, 73.0, 72.9, 72.4, 72.0, 71.8, 71.7, 59.9; HMRS
calcd for C49H76N2O36Na: 1291.4075, found: 1291.4049.
Mono-2-O-({6-[({[tert-butyldiphenylsilyl]oxy}imino)methyl] pyr-
idine-2-yl}methyl)-cyclo-maltoheptaose 7. mp > 260 ◦C; IR
1
(KBr) nmax/cm-1 3400 (OH), 2930 (CH), 1650 (CN); H NMR
General procedure for the preparation of compounds 11 and
12. To a solution of cyclodextrin derivative 8 or 9 (0.201 mg,
0.158 mmol) in 1.8 mL of dry DMF, was added iodomethane
(0.39 mL, 6.32 mmol). The mixture was stirred for 14 h at
50 ◦C. After cooling to room temperature, the product was
precipitated with acetone (30 mL) and filtered to obtain the desired
compound as a powder.
(300 MHz, DMSO-d6): d = 8.54 (s, 1H; CH-aldoxime), 7.86 (t,
1H, J = 7.71 Hz, CH-Py), 7.71–7.61 (m, 4H, CH-Ph), 7.64–7.58
(m, 2H, CH-Py), 7.48–7.44 (m, 6H, CH-Ph), 5.97–5.70 (m, 13H,
OH-2, OH-3), 5.12 (d, 1H, J = 3.00 Hz, H-1¢), 4.91 (s, 2H, CH2-Py),
4.86–4.82 (m, 6H, H1), 4.68–4.48 (m, 7H, OH-6), 3.90 (t, 1H, J =
9 Hz, H-3¢), 3.70–3.25 (m, 41H, 20H2O), 1.10 ppm (s, 9H; CH3); 13C
NMR (75 MHz, DMSO-d6): d = 158.4, 155.4, 150.1, 137.8, 135.0,
132.6, 130.0, 127.9, 119.3, 101.9, 81.8, 81.5, 81.4, 81.3, 81.2, 80.5,
73.0, 72.3, 72.0, 71.8, 59.9, 26.86, 18.9; MS ES+: 1508 (M+H)+,
1530 (M+Na)+; HMRS calcd for C65H94N2O36NaSi: 1529.5253,
found: 1529.5262.
Mono-2-O-({2-[(hydroxyimino)mehtyl]-1-methylpyridinium-4-
yl}methyl)-cyclomalto heptaose iodide 11. IR (KBr) nmax/cm-1
3370 (OH), 2930 (CH), 1655 (CN); 1H NMR (300 MHz, DMSO-
d6): d = 8.92 (d, 1H, J = 6 Hz, CH-Py), 8.66 (s, 1H, CH-aldoxime),
8.35 (s, 1H, CH-Py), 8.08 (d, 1H, J = 6 Hz, CH-Py), 5.98–5.66 (m,
13H; OH-2, OH-3), 5.17–5.05 (m, 3H, H-1¢, CH2-Py), 4.85–4.82
(m, 6H, H-1), 4.50–4.45 (m, 7H, OH-6), 4.33 (s, 3H, CH3), 3.90
(t, 1H, J = 9 Hz, H-3¢), 3.60–3.25 (m, 41H, 23H2O); 13C NMR
(75 MHz, DMSO-d6): d = 158.0, 146.7, 146.1, 141.7, 124.2, 121.6,
101.9, 101.5, 99.2, 81.5, 81.0, 80.8, 73.0, 72.2, 72.0, 71.6, 69.7,
59.9, 45.8; MS ES+: 1284 (M)+; HMRS calcd for C50H79N2O36:
1283.4413, found: 1283.4425.
General procedure for the preparation of compounds 8–10. To
a solution of cyclodextrin derivative (5–7) (0.22 mg, 0.146 mmol)
in 1.3 mL of water and 6.3 mL of methanol, was added KF·8H2O
(21 mg, 0.219 mmol). The reaction mixture was stirred for 12 h at
room temperature. Methanol was distilled under reduced pressure.
Water (5 mL) was added to the mixture. The solution was then
washed with CH2Cl2 (3 ¥ 50 mL) and the aqueous layer was
concentrated to give the desired product.
Mono-2-O-({6-[(hydroxyimino)mehtyl]-1-methylpyridinium-3-
yl}methyl)-cyclomalto-heptaose iodide 12. IR (KBr) nmax/cm-1
3380 (OH), 2930 (CH), 1655 (CN); 1H NMR (300 MHz, DMSO-
d6): d = 9.06 (s, 1H, CH-Py), 8.66 (s, 1H, CH-aldoxime), 8.47 (d,
1H, J = 8 Hz, CH-Py), 8.39 (d, 1H, J = 8 Hz, CH-Py), 5.99–5.55
(m, 13H; OH-2, OH-3), 5.13 (s, 1H, H-1¢), 4.97 (s, 2H, CH2-Py),
4.85–4.82 (m, 6H, H-1), 4.66–4.47 (m, 7H, OH-6), 4.34 (s, 3H,
CH3), 3.89 (t, 1H, J = 8 Hz, H-3¢), 3.65–3.30 (m, 41H, 8H2O);
13C NMR (75 MHz, DMSO-d6): d = 146.3, 144.9, 143.2, 141.6,
138.6, 124.3, 101.9, 101.4, 99.3, 81.6, 81.5, 81.2, 80.9, 80.4, 79.4,
73.0, 72.3, 72.0, 71.6, 68.3, 59.8, 46.3; MS ES+: 1284 (M)+; HMRS
calcd for C50H79N2O36: 1283.4413, found: 1283.4376.
Mono-2-O-({2-[(hydroxyimino)methyl]pyridin-4-yl}methyl)-cycl-
omaltoheptaose 8. mp > 260 ◦C; IR (KBr) nmax/cm-1 3300 (OH),
2930 (CH), 1655 (CN); H NMR (300 MHz, DMSO-d6): d =
1
8.54 (d, 1H, J = 5 Hz, CH-Py), 8.07 (s, 1H, CH-aldoxime), 7.79
(s, 1H, CH-Py), 7.42 (d, 1H, J = 5 Hz, CH-Py), 6.05–5.60 (m,
13H; OH-2, OH-3), 5.06 (s, 1H, H-1¢), 4.85–4.82 (m, 8H, H-1,
CH2-Py), 4.52–4.46 (m, 7H, OH-6), 3.88 (t, 1H, J = 9 Hz, H-3¢),
3.60–3.25 (m, 41H, 6H2O); 13C NMR (75 MHz, DMSO-d6): d =
152.0, 149.4, 148.9, 147.7, 122.2, 117.9, 101.8, 101.6, 99.7, 81.9,
81.5, 80.4, 73.0, 72.3, 71.9, 71.7, 71.1, 59.8; HMRS calcd for
C49H76N2O36Na: 1291.4075, found: 1291.4070.
Mono-2-O-({6-[(hydroxyimino)methyl]pyridin-3-yl}methyl)-cycl-
omaltoheptaose 9. mp > 260 ◦C; IR (KBr) nmax/cm-1 3400 (OH),
2930 (CH), 1635 (CN); H NMR (500 MHz, D2O): d = 8.49 (s,
Procedure for evaluation of the detoxification of cyclosarin and VX
1
Cyclosarin (GF) and VX (1 or 100 mM) were incubated with
cyclodextrin derivates (500 mM) in TRIS HCl (0.1 M) buffer at
pH 7.4 and 37 ◦C for up to 50 min. At different time points
samples were taken and diluted to a nerve agent concentration
of 500 nM with TRIS buffer. Afterwards, 10 ml of the solution
were incubated with 90 ml AChE (erythrocyte ghosts) for 3 min
at 37 ◦C. Then, a 10 ml aliquot was taken and the AChE
activity was measured spectrophotometrically (412 nm) with
a modified Ellman assay22 to determine the residual AChE
activity. The assay mixture (3.16 ml) contained 0.45 mM of
acetylthiocholine iodide (ATCh) as substrate and 0.3 mM of 5,5¢-
dithio-bis-2-nitrobenzoic acid (DTNB) as chromogen in 0.1 M
tris-(hydroxymethyl)aminomethane (TRIS) HCl (pH 7.4). AChE
activities were referred to control activity and data are given as %
of control. Data were corrected for spontaneous degradation of
GF and VX.
1H, CH-Py), 8.14 (s, 1H, CH-aldoxime), 8.03 (d, 1H, J = 4 Hz,
CH-Py), 7.85 (d, 1H, J = 4 Hz, CH-Py), 5.08 (d, 1H, J = 1 Hz,
H-1¢), 5.02–4.93 (m, 7H; H-1, CH2-Py), 4.89 (d, 1H, J = 7 Hz,
CH2-Py), 4.04 (t, 1H, J = 5 Hz, H-3¢), 3.85–3.50 (m, 40H), 3.40
(d, 1H, J = 6 Hz, H-2¢); 13C NMR (125.75 MHz, D2O): d = 151.5,
149.4 (CH-aldoxime), 148.2 (CH-Py), 137.3 (CH-Py), 134.0,
120.6 (CH-Py), 102.1–101.6 (6C, C-1), 100.2 (C’-1), 81.6–80.6
(7C, C-4), 78.9 (C’-2), 73.3–73.2 (6C, C-3), 72.8 (C¢-3), 72.1–71.7
(13C, C-2,C-5), 69.9 (CH2-Py), 60.4–60.0 (7C, C-6); HMRS calcd
for C49H76N2O36Na: 1291.4075, found: 1291.4069.
Mono-2-O-({6-[(hydroxyimino)methyl]pyridin-2-yl}methyl)-cycl-
omaltoheptaose 10. mp > 260 ◦C; IR (KBr) nmax/cm-1 3400
1
(OH), 2930 (CH), 1635(CN); H NMR (300 MHz, DMSO-d6):
d = 11.68 (s, 1H; OH-aldoxime), 8.09 (s, 1H, CH-aldoxime), 7.86
(t, 1H, J = 8 Hz, CH-Py), 7.70 (d, 1H, J = 8 Hz, CH-Py), 7.54
(d, 1H; J = 8 Hz, CH-Py), 5.99–5.68 (m, 13H; OH-2, OH-3), 5.09
(d, 1H, J = 3 Hz, H-1¢), 4.88 (s, 2H, CH2-Py), 4.82 (s, 6H, H-1),
4.49–4.47 (m, 7H, OH-6), 3.89 (t, J = 9 Hz, 1H; H-3¢), 3.70–
3.22 (m, 41H, 7H2O); 13C NMR (75 MHz, DMSO-d6): d = 157.8,
151.4, 148.7, 137.5, 121.6, 118.5, 101.8, 101.5, 100.0, 81.8, 81.5,
General procedure for kinetic evaluation of cyclosarin hydrolysis
by compounds 9 and 10
The hydrolysis of GF (100 mM) by compounds 9 and 10 (500 mM)
was initially tested with pH-Stat using 155 mM NaCl at pH 7.4
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and 37 ◦C. For the determination of stereoselective kinetics of
cyclosarin hydrolysis by these cyclodextrin derivatives TRIS buffer
(0.1 M) was used as incubation medium (pH 7.4 and 37 ◦C).
GF enantiomers were determined by chiral GC-MS as described
previously with minor changes.23
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Acknowledgements
The authors are grateful to M. Wiltzsch for his skilful and
engaged technical assistance. They thank Dr A. Romieu for helpful
compound analysis in mass spectrometry and C. Martin for excel-
lent technical assistance. De´le´gation Ge´ne´rale pour l’Armement
(DGA, French Departement of Defense Procurement Agency) is
gratefully acknowledged for PhD research grants for RLP, LL and
NM, Re´gion Haute Normandie (Crunch programm) and Agence
Nationale pour la Recherche (ANR – detoxneuro BLAN06-
1_134538) are gratefully acknowledged for financial support.
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