Inclusion of the insecticide fenitrothion in dimethylated-β- cyclodextrin: Unusual guest disorder in the solid state and efficient retardation of the hydrolysis rate of the complexed guest in alkaline solution

Article abstract of DOI:10.3762/bjoc.9.14

An anhydrous 1:1 crystalline inclusion complex between the organophosphorus insecticide fenitrothion [O,O-dimethyl O-(3- methyl-4-nitrophenyl) phosphorothioate] and the host compound heptakis(2,6-di-O-methyl)-β- cyclodextrin (DIMEB) was prepared and its structure elucidated by single-crystal X-ray diffraction. This revealed two independent host molecules in the asymmetric unit. In one of these, the cavity is occupied by two disordered guest components (distinguishable as rotamers with respect to the P-OAr bond) while in the other, three distinct guest components with site-occupancies 0.44, 0.29 and 0.27 appear, the last having a reversed orientation relative to all the other components. Kinetic studies of the alkaline hydrolysis of fenitrothion in the presence of DIMEB showed a remarkable reduction of 84% in the rate of this reaction relative to that for the free substrate, a value exceeding those previously attained with the native hosts, β- and γ-cyclodextrin, and fully methylated β-cyclodextrin.

Full text of DOI:10.3762/bjoc.9.14

Inclusion of the insecticide fenitrothion in dimethyl-
ated-β-cyclodextrin: unusual guest disorder in the
solid state and efficient retardation of the hydrolysis
rate of the complexed guest in alkaline solution
Dyanne L. Cruickshank1, Natalia M. Rougier2, Raquel V. Vico2,
Susan A. Bourne1, Elba I. Buján*2, Mino R. Caira*1 and Rita H. de Rossi*2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 106–117.
1Department of Chemistry, University of Cape Town, Rondebosch
7701, South Africa and 2Instituto de Investigaciones en Físico
Química de Córdoba (INFIQC-CONICET), Departamento de Química
Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba, Ciudad Universitaria, X5000HUA, Córdoba, Argentina
doi:10.3762/bjoc.9.14
Received: 31 August 2012
Accepted: 12 December 2012
Published: 17 January 2013
Email:
This article is part of the Thematic Series "Superstructures with
cyclodextrins: Chemistry and applications".
Elba I. Buján* - elba@fcq.unc.edu.ar; Mino R. Caira* -
Mino.Caira@uct.ac.za; Rita H. de Rossi* - ritah@fcq.unc.edu.ar
Guest Editor: H. Ritter
* Corresponding author
© 2013 Cruickshank et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
crystal structure; cyclodextrin; fenitrothion; hydrolysis; inclusion
complex
Abstract
An anhydrous 1:1 crystalline inclusion complex between the organophosphorus insecticide fenitrothion [O,O-dimethyl O-(3-
methyl-4-nitrophenyl)phosphorothioate] and the host compound heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) was prepared
and its structure elucidated by single-crystal X-ray diffraction. This revealed two independent host molecules in the asymmetric
unit. In one of these, the cavity is occupied by two disordered guest components (distinguishable as rotamers with respect to the
P–OAr bond) while in the other, three distinct guest components with site-occupancies 0.44, 0.29 and 0.27 appear, the last having a
reversed orientation relative to all the other components. Kinetic studies of the alkaline hydrolysis of fenitrothion in the presence of
DIMEB showed a remarkable reduction of 84% in the rate of this reaction relative to that for the free substrate, a value exceeding
those previously attained with the native hosts, β- and γ-cyclodextrin, and fully methylated β-cyclodextrin.
Introduction
Whereas cyclodextrins (CDs) have been employed for many theless, very significant advantages of encapsulating agrochem-
years in the pharmaceutical industry to modify drug-delivery icals such as pesticides (insecticides, herbicides, fungicides) in
properties, the application of CD technology to the improve- CDs may be gained [1], including, e.g., the conversion of toxic
ment of agrochemicals is a more recent innovation [1,2]. Never- volatile liquids into solids, more localised pesticide application
106

Beilstein J. Org. Chem. 2013, 9, 106–117.
to improve delivery and reduce wastage, and stabilisation of the association in the case of α-CD and insoluble complex forma-
included pesticide against undesired degradation reactions.
tion in the case of the host TRIMEA [9], whereas for β-CD,
γ-CD and TRIMEB the association constants for the inclusion
Fenitrothion [O,O-dimethyl O-(3-methyl-4-nitrophenyl)phos- complexes of 1 had the respective values 417, 99 and 511 M−1
phorothioate] (1, Figure 1) is an organophosphorus insecticide [6], the hydrolytic decomposition of 1 being significantly
and acaricide [3]. It is effective against a wide range of pests retarded by all three CDs.
that damage forests and various crops and it can also be used in
the form of a residual contact spray to control flies, mosquitoes The present study relates to the interaction between 1 and
and cockroaches. Fenitrothion has also been employed in anti- heptakis(2,6-di-O-methyl)-β-CD (DIMEB), the latter molecule
malarial programmes, where spraying of houses and animal having properties intermediate between those of the native and
shelters with this insecticide over extended periods signifi- fully methylated counterparts [10]. The preparation and
cantly reduced the incidence and prevalence of the disease [4]. physicochemical characterization of the crystalline inclusion
complex DIMEB·1 are presented, this complex crystallizing in a
novel arrangement and showing severe guest disorder, which
was eventually resolved and successfully modelled. In addition,
the results of a kinetic study of the hydrolytic degradation of 1
in the presence of DIMEB are reported and compared with
those observed for the other CDs under the same reaction condi-
tions.
Figure 1: Chemical structure of fenitrothion (1).
Results and Discussion
In our recent reports on the interaction between CDs and the Thermal analysis
organophosphorus insecticide 1 we gave an account of the Figure 2 shows representative thermogravimetric (TGA) and
X-ray crystal structures and thermal decomposition profiles of differential scanning calorimetric (DSC) traces for the novel
two solid inclusion complexes between permethylated α- and inclusion complex DIMEB·1. Three reproducible thermal
β-cyclodextrins, hexakis(2,3,6-tri-O-methyl)-α-CD (TRIMEA) events occur in the DSC trace, the first of which is a small
and heptakis(2,3,6-tri-O-methyl)-β-CD (TRIMEB) and the endotherm at 132 °C, which we interpret as a minor solid–solid
guest [5]. We have also described the results of solution-based phase change, based on supporting hot stage microscopic
kinetic studies aimed at determining the effect of the presence (HSM) observations that show the initially colourless crystals
of various CDs on the rate of alkaline hydrolysis of fenitrothion turning opaque in this temperature region. A significantly larger
in aqueous solution containing 2% dioxane at 25 °C [6]. A crys- endotherm follows at 158 °C accompanying the major phase of
talline inclusion complex (TRIMEA)2·1 was isolated and shown guest loss, while the broad exotherm at ~230 °C is interpreted
by X-ray analysis to contain a novel capsule formed by head-to- as indicating the onset of host decomposition. The latter event is
head contact of the secondary rims of two TRIMEA molecules also confirmed by HSM showing the opaque crystals turning
[5]. The encapsulated fenitrothion molecule showed minor brown in the temperature region following guest loss. In the TG
disorder in the form of two rotamers (with respect to the P–OAr trace, major host decomposition is seen to occur above 300 °C.
bond). In contrast, with the host TRIMEB, a monomeric inclu-
sion complex TRIMEB·1 was obtained, the guest molecule
being statistically disordered over two positions [5]. In both
crystalline complexes, however, the sensitive phosphate ester
moiety was found to be buried deep within the CD cavity and
we suggested that if this feature were to be obtained in solution,
it would account for earlier observations that the rate of alka-
line degradation of the ester is retarded in the presence of CDs
[7,8].
Subsequently, a study of the kinetics of hydrolysis of the insec-
ticide 1 in aqueous solution containing 2% dioxane at 25 °C and
in the presence of the native cyclodextrins α-CD, β-CD, γ-CD
Figure 2: Representative TGA (top) and DSC (bottom) traces for
DIMEB·1.
as well as the permethylated derivatives TRIMEA and
TRIMEB, was performed [6]. This revealed weak host–guest
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Beilstein J. Org. Chem. 2013, 9, 106–117.
Complete loss of the guest from the inclusion complex resulted
in an estimated 17.9 ± 0.3% (n = 2) mass loss, which is in good
agreement with the theoretical value of 17.2% for a 1:1
host–guest complex. The complex stoichiometry deduced from
thermal analysis was subsequently confirmed by NMR spec-
troscopy, as described below.
Table 2: Crystal data and data-collection parameters.
Compound
DIMEB·1
Chemical formula
Formula weight / (g mol−1)
Crystal system
Space group
A / (Å)
C56H98O35·C9H12O5NPS
1608.57
Monoclinic
P21
NMR spectroscopy
19.626(2)
15.049(2)
26.854(3)
90
From the 1H NMR spectrum of the solid complex dissolved in
CDCl3, a 1:1 DIMEB·1 stoichiometry was calculated. Table 1
lists the relevant host and guest proton integrations, showing
however, that a small excess of DIMEB, cocrystallized with the
complex, was present in the sample.
B / (Å)
C / (Å)
α / (°)
β / (°)
96.451(2)
90
γ / (°)
V / (Å3)
7881.0(15)
4
Z
Fenitrothion is known to decompose at elevated temperature
[11]. Since the complex crystals had been isolated at 60 °C, the
possibility that the fenitrothion may have degraded thermally
was checked by 31P NMR spectroscopy. Pure, uncomplexed
fenitrothion dissolved in CDCl3 shows a single peak at
65.60 ppm. The complex crystals dissolved in CDCl3 also
showed the presence of only one signal at 65.47 ppm
confirming the structural integrity of the included insecticide
molecule.
Dc / (Mg m−3)
μ[MoKα] (mm−1)
F(000)
1.356
0.156
3432
Temperature of data collection /
(K)
173(2)
Crystal size / (mm)
0.46 × 0.35 × 0.26
Range scanned θ/ (°)
Index ranges ±h, ±k, ±l
Φ and ω scan angles / (°)
Total no. of frames
2.04–28.39
−26, 26; −20, 19; −35, 35
0.5
2494
Crystal to detector distance /
(mm)
The 31P NMR spectrum of a solution of 1 (2 mM) in 2%
dioxane-d8/D2O gave a single peak at 65.14 ppm, while in the
same solution but in the presence of DIMEB (2 mM) the signal
appears at 66.82 ppm, in agreement with data reported for 1 in
water [6,12-14]. The shift observed indicates that a complex is
formed.
50.00
Total no. of reflections collected 125785
No. of independent reflections
No. of reflections with I > 2σ(I)
Rint
20368
17090
0.0383
No. of refined parameters
No. of least-squares restraints
Goodness-of-fit, S
R1 [I > 2σ(I)]
1636
75
1.019
The 1H NMR spectrum shows that the protons H3 and H5 of
the cyclodextrin rings that are located inside the cavity are
slightly shifted downfield, confirming the inclusion of the guest
into the cavity.
0.0770
No. of reflections omitted
wR on F2
34
0.2129
Weighting scheme parameters
(Δ/σ)mean
a = 0.144 and b = 7.338
<0.001
X-ray analysis
Δρ excursions (eÅ−3)
CCDC no.
1.30 and −0.98
898328
Table 2 lists the crystal data and data-collection parameters for
the complex. No DIMEB-containing crystals with similar unit
cell dimensions and the same space group as those of the
Table 1: Integrals of the NMR signals for the protons of solid complex DIMEB·1 dissolved in CDCl3 used to confirm the complex stoichiometry.
[Theoretical number of
protons](DIMEB·1)
δ (ppm)
Experimental integration of
peaks
Experimental peak integral/theoretical proton
number
[7 × H1] (DIMEB)
[7 × H2] (DIMEB)
[3 × CH] (Ar-CH3, 1)
4.952
3.271
2.621
9.02
8.47
1.3
1.2
1
3.00a
aReference integral.
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Beilstein J. Org. Chem. 2013, 9, 106–117.
DIMEB·1 crystals were identified in the Cambridge Crystallo- level of guest disorder occurring in this complex, despite the
graphic Database [15], which necessitated its ab initio struc- low temperature of the data collection. Figure 4 shows the
tural solution by using direct methods. Density considerations structures and atomic numbering of the two independent host
indicated an asymmetric unit containing two complex mole- molecule A and B, whose conformational parameters are listed
cules. In addition, prior to structural solution, a special feature in Table 3.
was evident from the X-ray diffraction pattern, namely the
general alternation of strong and weak intensities for the recip-
rocal lattice levels hkl with h even and odd, respectively. This
immediately indicated that the unit cell contents at x, y, z and
x + 1/2, y, z are quite similar and that the correct direct-methods
solution should be consistent with this prediction.
This pseudo-translational symmetry was indeed a feature of the
structural solution and is illustrated in Figure 3, where it relates
the two complex units A and B in the crystal asymmetric unit of
DIMEB·1.
Figure 4: The host molecules in the asymmetric unit of DIMEB·1 with
the labelling of both residues and atoms indicated. H atoms are
omitted for clarity.
The parameters in Table 3 that define the host structures include
commonly employed descriptors of the O4-heptagon of each
cyclodextrin molecule as well as the inclinations of the indi-
vidual dimethylglucose residues (A1–A7, B1–B7, headed Res
in Table 3) relative to the mean plane of the O4 atoms. These
parameters are defined as follows: l, the distance of each O4
atom from the centroid of the O4-polygon; D, the glycosidic
O4···O4′ distance; Φ the O4···O4′···O4′′ angle; d, the
O4···O4′···O4′′···O4′′′ torsion angle; α, the deviation of each O4
atom from the O4 mean plane; D3, the O2···O3′ intramolecular
hydrogen bond distance; tilt angle τ1, the angle between the six
atoms of the glucopyranose ring (C1, C2, C3, C4, C5, O5) and
the line orthogonal to the O4 mean plane; tilt angle τ2 between
Figure 3: The asymmetric unit in DIMEB•1 viewed along [010] (top)
and [100] (bottom). H atoms are omitted for clarity.
the O4 plane and the mean plane through atoms O4, C4, C1 and
O4′ of a given glucose ring.
All the dimethylglucose rings of host molecules A and B adopt
the 4C1 chair conformation. On their primary sides, with few
exceptions, the values of the torsion angles ω (O5–C5–C6–O6)
indicate (−)-gauche conformations, with the C6–O6 bonds thus
directed away from the centres of the respective cavities. This
While the gross structures of the independent complex units
appear similar, there are obvious differences in the conforma-
tions of the two independent host molecules, and in particular,
the images of the respective guest molecules are very different,
as described further below. This is due to an unusually high
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Beilstein J. Org. Chem. 2013, 9, 106–117.
Table 3: Conformational parameters of the host molecules.a
Res
l (Å)
D (Å)
Φ (°)
d (°)
α (Å)
D3 (Å)
τ1 (°)
τ2 (°)
A1
A2
A3
A4
A5
A6
A7
5.18
4.74
5.09
5.20
4.97
4.80
5.11
4.35
4.51
4.32
4.34
4.40
4.37
4.22
123.6
135.9
125.2
125.7
128.7
132.3
126.8
−11.0
0.3
−0.240
0.122
2.864
2.849
3.049
2.831
2.744
2.715
2.954
18.5
14.7
6.1
18.1
19.7
6.6
7.8
0.169
0.4
−0.171
−0.134
0.268
19.8
15.9
8.8
19.1
17.0
12.6
12.3
−13.5
9.5
5.5
−0.014
11.3
B1
B2
B3
B4
B5
B6
5.31
4.65
5.03
5.33
4.97
4.72
4.42
4.56
4.23
4.32
4.47
4.42
121.1
137.7
127.4
123.1
129.4
134.0
−6.5
4.7
−0.078
0.147
0.018
−0.137
0.009
0.174
2.991
2.925
2.906
2.841
2.796
2.755
15.5
14.5
13.9
17.4
10.1
9.9
16.5
17.0
13.4
17.5
11.5
11.9
3.3
−2.6
−6.8
10.7
B7
5.18
4.16
126.5
−2.5
−0.132
2.887
2.0
4.1
aThe mean e.s.d.s for α, D3, τ1 and τ2 are 0.003 Å, 0.005 Å, 0.1° and 0.2° respectively.
results in relatively open cavities, contrary to the usual situa- Details of the guest disorder within the host A cavity are illus-
tion in inclusion complexes of the fully methylated host trated in Figure 5.
analogue TRIMEB, where the primary methoxy groups typi-
cally act as a “lid”, sealing that side of the macrocycle. In the Here, a single molecule of fenitrothion occupies the cavity,
DIMEB·1 complex, there is thus no distinct “boundary” sepa- being present as two rotamers whose phosphorothioate groups
rating the guest molecules within the cavity of host molecule A are related by a rotation of 126° around the O11–P12 bond. The
from those within host molecule B.
phosphorothioate group of each disordered component of the
guest 1 is located near the primary side of the host molecule.
The tilt angles (τ1 and τ2) are all positive indicating that each
ring has its primary side tipped towards the centre of the cavity. Instead, within the cavity of host molecule B, as many as three
The slight ellipticity in the host conformations, reflected in the disordered guest components (B, C, E) were observed and
values of l, D and Φ (Table 3) is a compromise between the ten- modelled, as shown in Figure 6. These disordered components
dency for “roundness” (induced by the intramolecular O2···O3′ (B, C, E) are shown in green, blue and pink, respectively, in the
hydrogen bonds with lengths D3) and slight distortion due to stereoview of Figure 6.
guest inclusion. The O4 mean planes of host molecules A and B
are slightly offset from one another but are nearly parallel The major disordered guest components B and C, with respec-
(interplanar angle 2.65(2)°).
tive site-occupancy factors (sofs) 0.44 and 0.29, adopt the same
Figure 5: The rotamers of 1 occupying the cavity of host molecule A. Common atoms have labels with suffix A, while in the component on the right
the alternative sulfur and methoxy group positions are labelled with suffix D. These disordered guest components (labelled A, D) have site occupan-
cies 0.70 and 0.30, respectively.
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Beilstein J. Org. Chem. 2013, 9, 106–117.
Host molecules A and B have slightly different geometries (e.g.,
extents of ellipticity), as indicated by the parameters in Table 3.
More detailed analysis shows that these differences are attribut-
able to their accommodation of different assemblies of disor-
dered guest components.
An analysis of hydrogen bonding in the crystal of DIMEB·1
revealed several types of interactions. These can be summarised
as follows: (a) For the two independent host molecules A and B,
a total of 14 intramolecular H-bonds O2n···H–O3(n−1) charac-
teristic of the DIMEB molecule (O···O distances in the range
2.715(5)–3.049(5) Å), linking contiguous glucose residues and
thus inducing a “round” host conformation (parameter D3 in
Table 3); (b) intramolecular H-bonds C6–H6···O5′/O6′ and
C7–H7···O3′, which also contribute to stabilisation of the host
conformation; and (c) 10 unique host–host intermolecular
C–H···O hydrogen bonds that stabilise the host framework.
Figure 6: Stereoview of the three disordered guest components that
occupy the cavity of host molecule B. Guest components B (green)
and C (blue) have similar orientations while component E (pink) adopts
the opposite orientation.
All guest molecules are fully encapsulated by the host mole-
orientation, their phosphorothioate groups being located near cules and there are no close contacts between the disordered
the primary side of host molecule B (analogous to the situation guest molecules and host molecules. Figure 7 shows space-
in host molecule A). However, the third guest component E, filling diagrams that illustrate representative modes of guest
with the lowest sof (0.27), adopts the opposite orientation to inclusion.
those of components B and C. This low sof indicates that the
guest orientations adopted by the other four disordered compo- Crystal packing is shown in Figure 8. The complex units stack
nents (A–D) is the preferred one in the solid state.
head-to-tail in infinite columns parallel to the x-direction.
Figure 7: Space-filling diagrams showing the relative orientations of guest molecules within the cavities of the independent DIMEB molecules. Side-
views of the complexes and cross-sectional views showing guest molecules (a) A and B, and (b) guest molecules A and E, encapsulated by their
respective host molecules.
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Beilstein J. Org. Chem. 2013, 9, 106–117.
Figure 8: Packing diagrams of the DIMEB·1 structure, viewed along [100] (left) and [010] (right). The symmetry related molecules are distinguished
by the reverse green and black host and guest molecules. For clarity, only guest molecules A and B are shown and the hydrogen atoms have been
omitted.
Owing to the twofold screw axis parallel to the b-axis, the adja- The ICD spectrum of fenitrothion in the 200–300 nm wave-
cent columns are antiparallel. The slight ellipticity of the host length range in the presence of β-CD was reported in the litera-
molecules and the extension of the primary methoxy groups ture [21] and the spectrum shows a negative peak at ≈220 nm
into the interstitial sites assist in the close packing of the com- and a positive peak at ≈280 nm. This spectrum was measured
plex units.
for a basic solution and in the paper it is not stated what time
had elapsed between the preparation of the solution and the
running of the spectrum. We suspect that the spectrum reported
UV–vis and ICD studies
Spectroscopic methods are frequently used to determine associ- corresponds to that of the hydrolysis product, namely 3-methyl-
ation constants between cyclodextrins and different guests [16]. 4-nitrophenol 2. We carried out measurement of the ICD spec-
We attempted to determine the binding constant of 1 with trum of 2 in a 3% dioxane–water solution, and the spectrum
DIMEB by UV–vis spectroscopy; however, when we recorded obtained was similar to that reported by Kamiya et al. [21] for
the spectra of 1 in the presence of DIMEB, only a small fenitrothion (1).
hypsochromic shift was noted and the change in absorbance
was not large enough for an accurate determination of the It is remarkable that the sign of the peaks in the ICD spectrum
binding constant.
of 1 in the presence of TRIMEB is inverted when compared
with the spectra of β-CD and DIMEB with 1. As suggested
The induced circular dichroism (ICD) spectrum of DIMEB and previously [6,8,22], the change in the sign of an ICD spectrum
1 is shown in Figure 9a, where the spectrum recorded for β-CD may indicate extrusion of the guest or a change in orientation of
and TRIMEB with 1 is also shown for comparison.
the guest in the cavity.
It can be seen that the native β-CD and DIMEB show a nega- There are two possible modes of inclusion of the insecticide
tive and a positive peak (Figure 9a). The negative peaks appear molecule in the cavity of a CD, either with the phosphoro-
at 320 and 324 nm and the positive ones at 260 and 265 nm for thioate moiety (type A) or with the aromatic moiety (type B)
β-CD and DIMEB, respectively. The positive peak in the inside the cavity [6]. For most of the esters having an aromatic
circular dichroism spectrum is slightly displaced from the group it is proposed that this residue is included in the cavity of
maximum of the UV–vis spectrum as was observed in other the CDs. This was suggested for parathion, methyl parathion,
cases where cyclodextrin was the chiral component [17-20]. and paraoxon [17,21,23]. Theoretical calculation of the ener-
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Beilstein J. Org. Chem. 2013, 9, 106–117.
from that in the complex with TRIMEB. It appears that the
presence of OH in the rim of the cavity has an important role in
determining the orientation of the included guest molecule.
Based on the fact that the ICD spectra of p-nitrophenol and
fenitrothion are similar, Kamiya [21] suggested that the inclu-
sion of both compounds should be similar. This is not the case
under our reaction conditions; therefore we think that the type A
complex is predominant for β-CD and DIMEB.
Kinetic studies
The hydrolysis reaction of fenitrothion takes place with P–O
bond fission as shown in Scheme 1 [12] and it was studied in
the presence of constant HO− and variable DIMEB concentra-
tions, as reported previously with other CDs [6].
In Figure 10 a plot of the observed pseudo-first-order rate
constants kobs as a function of the concentration of DIMEB is
Figure 9: Induced circular dichroism (a) and UV–vis (b) spectra of 1 in
the presence of β-CD (10 mM, green), DIMEB (2.5 mM, pink) and
TRIMEB (10 mM, blue).
gies of the two types of complexes in the gas phase indicates
that there is no significant difference in their values [24].
Single-crystal X-ray analysis shows that for β-CD and
TRIMEB, complexes of type A are formed while for DIMEB
(see above) in the unit cell a small percentage of the inclusion
complexes exhibit the type B orientation.
Figure 10: Plot of kobs versus [DIMEB] for the hydrolysis reaction of
fenitrothion with HO– at different concentrations of DIMEB. T = 25 °C,
[NaOH] = 0.5 M, ionic strength = 1 M (NaCl). Solvent: water with 2%
1,4-dioxane. The line was drawn by using the data calculated for k0, k2
and K2 in Equation 1.
By comparing the ICD spectra of the three complexes it seems
that, in solution, the fenitrothion molecule adopts the same
orientation in the β-CD and DIMEB cavities, which is different
Scheme 1: Reaction of fenitrothion in basic media.
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Beilstein J. Org. Chem. 2013, 9, 106–117.
shown. The highest concentration of DIMEB used was It is remarkable that the kinetic data obtained for β-CD are in
0.005 M. At a cyclodextrin concentration higher than this, much better agreement.
precipitation of the DIMEB occurred under the conditions of
our studies (ionic strength 1 M, NaOH 0.5 M aqueous solution The large difference in the inhibitory effect when compared to
with 2% 1,4-dioxane). This was confirmed by performing an the native CDs may be due to the methyl groups present on
experiment with DIMEB at a concentration of 0.01 M without DIMEB and TRIMEB, which elongate the cavity of the CD
the addition of substrate; a day later there was a solid precipi- molecules and provide a slightly larger pocket in which the
tate at the bottom of the flask, which was confirmed by fenitrothion molecules can situate themselves. Furthermore, the
1H NMR spectroscopy to be DIMEB.
methyl groups have the ability to block the entering hydroxide
ions, thereby preventing nucleophilic attack at the phosphorus
The reaction of 1 with NaOH in the presence of DIMEB may atom. From the X-ray crystal structures of the CD complexes of
take place as shown in Scheme 2 [7,8], where k0 and k2 repre- fenitrothion with TRIMEB [6] and DIMEB (see above), it is
sent the reactions of the free substrate and of the substrate clear that the major components of the fenitrothion molecules
complexed with ionized cyclodextrin (DIMEB–), respectively have their reactive phosphorothioate moieties positioned at the
[25]. The observed rate constant for Scheme 2 is shown in narrower primary rims of the cyclodextrin cavities. The stronger
Equation 1.
inhibition of fenitrothion hydrolysis in the presence of DIMEB
in comparison with TRIMEB may indicate that the phosphoro-
thioate unit is more protected from nucleophilic attack when it
is in the cavity of DIMEB. A DIMEB molecule has a more
rounded shape when compared to TRIMEB, due to the
intramolecular O2···H–O3' hydrogen bonds that link neigh-
bouring methylglucose residues. As a result, there is more avail-
able space in the cavity of a DIMEB molecule than in that of a
TRIMEB molecule, and therefore the guest fenitrothion can
penetrate further into the cavity.
Scheme 2: Mechanism of the hydrolysis reaction of 1 mediated by
DIMEB.
Conclusion
(1)
Single-crystal X-ray analysis of the 1:1 DIMEB fenitrothion
inclusion complex revealed that the mode of inclusion of the
disordered guest molecules is predominantly of type A (phos-
The values of the rate and equilibrium constants were obtained phorothioate group near the host primary side). ICD spectra of
by treating the kinetic data as previously published [6] and they the DIMEB and β-CD complexes indicated that analogous
are (1.20 ± 0.01) × 10–3 s–1, (0.24 ± 0.04) M–1 s–1 and (1.69 ± type A inclusion prevails in solution. The significantly higher
0.09) × 103 M–1, for k0[HO–], k2K2[HO–], and K2, respectively. association constant reported here for fenitrothion complexa-
From these data k2 = 0.14 × 10–3 s–1 can be obtained and this is tion with DIMEB in aqueous solution compared with previous
a measure of the rate of the reaction taking place within the data for its complexation with β-CD and TRIMEB results in
DIMEB cavity.
more effective protection of the insecticide molecule from
hydrolytic degradation.
The association constant determined for DIMEB and 1, viz.
(1.69 ± 0.9) × 103 M–1 is significantly higher than the values Kinetic data of the type established in this and previous studies
previously measured for complexation of 1 with β-CD and that we have undertaken, give indications of which type of
TRIMEB, namely 417 and 511 M–1 [6]. Furthermore, the inhi- cyclodextrin may be best suited in an agrochemical formulation
bition effect as measured by the ratio k0/k2 is also significantly to ensure resistance of sensitive bioactive compounds to chem-
higher, namely 7.5, 8.6 and 5.0 for TRIMEB, DIMEB and ical degradation, thus maximising their intended biological
β-CD, respectively, indicating that DIMEB is more effective in effect. Thermodynamic data, such as complex association
terms of protecting the substrate against the reaction with the constants, are also relevant parameters, especially in the context
nucleophile. The value of K2 obtained by other authors under of CD-mediated remedial treatment of soils that are contami-
similar but not equal reaction conditions is 189 M–1 [21]. Part nated by persistent pesticides. As regards fenitrothion, a periph-
of the discrepancy with our results can be attributed to the eral but important advantage of its inclusion in CDs is the
different reaction conditions, but we think that the main differ- significant reduction in its volatility (and hence toxicity) by
ence may come from the sample of DIMEB used in each work. conversion of the liquid pesticide into a solid phase.
114

Beilstein J. Org. Chem. 2013, 9, 106–117.
Experimental
positions (sof = 0.69) while C8 was shared. All ordered host
The compounds used in this study were purified as indicated atoms were refined anisotropically, except atoms C7A3, C7A7,
before [6].
C8B4 and C8B5, which had reasonable isotropic thermal para-
meters but unacceptable thermal ellipsoids when refined
anisotropically.
Crystal preparation and X-ray diffraction
analysis
Fenitrothion (1, 20 mg, ~0.072 mmol) was added to a saturated The host hydrogen atoms were placed by using a riding model
aqueous solution of DIMEB (96 mg, 0.072 mmol) at 20 °C. and were refined isotropically with thermal displacement para-
Continuous stirring for 6 h, as well as several heating and meters 1.2 times those of their parent atoms. For the hydroxy
cooling cycles between 20 and 60 °C led to formation of a clear groups on each methyl glucose unit a hydrogen-bond searching
solution, which was then filtered into a clean vial, capped and model (AFIX 83) was used to place the hydrogen atoms in
placed in an oven at 60 °C. Colourless single crystals of the geometrically reasonable hydrogen-bonding positions.
complex DIMEB·1 appeared over a 24 h period.
However, this procedure was not suitable for all the hydroxy
groups, and thus, distance restraints were applied between the
Collection of intensity data was performed on a Bruker KAPPA hydrogen atom of the hydroxy group and the hydrogen-bond-
APEX II DUO diffractometer with the crystal cooled in a acceptor oxygen atom on the adjacent glucopyranose unit. This
constant stream of nitrogen vapour (Oxford Cryostream, UK). was necessary for hydroxy groups on methyl glucose units A4,
Upon inspection of the reciprocal lattice layers with the A5, A6, A7, B6 and B1.
program LAYER [26], the X-ray diffraction pattern revealed
Laue symmetry 2/m, corresponding to the monoclinic crystal When both host molecules had been located, the first guest
system. The intensity-weighted reciprocal lattice also revealed molecule that resulted from the initial SHELXD solution was
alternation in intensity of the layer lines perpendicular to a*, modelled, followed by another in the second crystallographi-
from which it was deduced that the structural motif at x, y, z cally independent DIMEB molecule. After refinement, the
repeats itself at approximately x + ½, y, z. The space group P21 thermal displacement parameters of the guest in host molecule
was identified from the reflection conditions hkl: none; h0l: A were reasonable, with the exception of those of the atoms
none; 0k0: k = 2n (the alternative, centric space group P21/m belonging to the O,O-dimethyl phosphorothioate moiety. The
was eliminated since it could not accommodate the chiral two disordered guest molecules were found to be rotamers that
cyclodextrin host). Owing to the alternating strong and weak result from rotation of substituents around the O11–P12 bond.
reflections it was possible to deduce the presence of two Distance restraints were applied to atoms of the phosphoro-
DIMEB molecules in the asymmetric unit.
thioate moiety and an AFIX 66 instruction was applied to the
phenyl group, which constrained the ring as a rigid hexagon.
As no isostructural DIMEB complex could be found in the The common atoms of the guest molecule were labelled A
literature, the program SHELXD [27] was employed to solve while the second sulfur and methoxy group were labelled D.
the structure by ab initio direct methods. The initial structural The refined values of the sofs of the two rotamers were 0.70
solution (correlation coefficient = 84.9) revealed most of the and 0.30. All the ordered atoms of guest molecule A were
nonhydrogen host atoms of the two DIMEB molecules and a refined anisotropically, with the exception of atoms O9A, O10A
partial guest molecule in one of the DIMEB host cavities. and C17A.
Before the first refinement in SHELXH-97 [28] the carbon and
oxygen atoms of the host were correctly assigned, as were the The first disordered guest component molecule modelled in host
atoms of the first guest molecule. The remaining host atoms molecule B had many additional high electron-density peaks
were located in the initial and subsequent difference Fourier surrounding it and the entire molecule had unacceptably high
maps. The methyl glucose moieties of two independent CD thermal displacement parameters. There was an abnormally
molecules in the asymmetric unit were labelled A1–A7 and large peak situated close to the phenyl ring but it was not in the
B1–B7.
plane of the phenyl group. Two additional hexagonal rings were
discerned in the difference Fourier map, suggesting two other
One primary methoxy group per host molecule was disordered guest-molecule positions. After many attempts at modelling the
over two positions (on glucose residues B1 and A4). In the case disorder, three disordered components within host molecule B
of the primary methoxy group on methyl glucose unit B1, all were evident. The relative population of each was unknown and
three atoms (C6, O6 and C8) were disordered and the major required an analysis of the initial electron-density peak heights
component refined with a sof of 0.53, while methyl glucose unit for the phosphorus atoms of each component. Guest compo-
A4 contained only atom O6, which was disordered over two nents B, C and E had initial phosphorus electron-density peaks
115

Beilstein J. Org. Chem. 2013, 9, 106–117.
of 4.66, 3.07 and 2.86 eÅ−3, respectively, which resulted in Nacional de Córdoba are gratefully acknowledged. This work
fixed sof values of 0.44, 0.29 and 0.27 being applied to the three was carried out as part of a bilateral cooperation project
disordered components. Owing to the excessive and unusual supported by the NRF (South Africa) and MINCyT (Argentina).
type of disorder of the guests present in this particular structure, The assistance of Dr. Gloria Bonetto with the NMR experi-
many distance restraints and three AFIX 66 commands were ments is greatly appreciated. N.M.R. is a grateful recipient of a
applied to maintain reasonable geometries. Global isotropic fellowship from CONICET, and R.V.V., E.I.B. and R.H.deR
thermal displacement parameters were assigned to each compo- are members of the research career, CONICET (Argentina).
nent and these refined to values of 0.10, 0.07 and 0.13 Å2 for
References
1. Lucas-Abellán, C.; Gabladón-Hernández, J. A.; Penalva, J.;
guest molecules B, C and E, respectively. The guest hydrogen
atoms were placed in idealised positions in a riding model with
Fortea, M. I.; Nuñez-Delicado, E. J. Agric. Food Chem. 2008, 56,
isotropic thermal displacement parameters in the range 1.2–1.5
8081–8085. doi:10.1021/jf8015046
times those of their parent atoms.
2. Morillo, E. Application of Cyclodextrins in Agrochemistry. In
Cyclodextrins and their Complexes: Chemistry, Analytical Chemistry,
CCDC 898328 contains the supplementary crystallographic data
Applications; Dodziuk, H., Ed.; Wiley-VCH: Weinheim, Germany, 2006;
for this paper. These data can be obtained free of charge at
http://www.ccdc.cam.ac.uk/products/csd/request/ [or from the
Cambridge Crystallographic Data Centre (CCDC), 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033;
email: deposit@ccdc.cam.ac.uk].
pp 459–466.
3. http://pmep.cce.cornell.edu/profiles/extoxnet/dienochlor-glyphosate/feni
trothion-ext.html. (accessed Aug 16, 2012).
4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395585/ (accessed Aug
16, 2012).
5. Cruickshank, D.; Rougier, N. M.; Vico, R. V.; de Rossi, R. H.;
Buján, E. I.; Bourne, S. A.; Caira, M. R. Carbohydr. Res. 2010, 345,
141–147. doi:10.1016/j.carres.2009.10.023
Thermal analysis
6. Rougier, N. M.; Cruickshank, D. L.; Vico, R. V.; Bourne, S. A.;
Caira, M. R.; Buján, E. I.; de Rossi, R. H. Carbohydr. Res. 2011, 346,
322–327. doi:10.1016/j.carres.2010.06.016
Mass loss on heating the crystals of DIMEB·1 was measured on
a TA-Q500 Thermogravimetric Analyzer, TA instruments,
equipped with Universal Analysis 2000 software. The differen-
tial scanning calorimetric trace was recorded on a DSC-Q200
calorimeter, TA instruments. For both techniques, a heating rate
of 10 K min−1 and dry nitrogen purge gas flowing at a rate of
30 mL min−1 were employed. Crystals were transferred to an
open alumina or aluminium crucible in the case of TG measure-
ments and closed vented aluminium pans for DSC measure-
ments. Sample masses were between 0.5 and 2.0 mg for DSC
measurements and between 2 and 8 mg for the TG runs.
7. Vico, R. V.; Buján, E. I.; de Rossi, R. H. J. Phys. Org. Chem. 2002, 15,
858–862. doi:10.1002/poc.560
8. Vico, R. V.; de Rossi, R. H.; Buján, E. I. J. Phys. Org. Chem. 2009, 22,
691–702. doi:10.1002/poc.1502
9. In later work from our labs (unpublished), we succeeded in measuring
the kinetics of the hydrolysis reaction of 1 in the presence of TRIMEA
and found that in solution complexes of stoichiometry 1:1 and 1:2
(guest–host) were also formed and the reaction was strongly retarded.
10.Frömming, K.-H.; Szejtli, J. Cyclodextrins in Pharmacy; Kluwer
Academic Press: Dordrecht, 1994; pp 25–27.
11.Fukuto, T. R.; Hornig, E. O.; Metcalf, R. L. J. Agric. Food Chem. 1964,
12, 169–171. doi:10.1021/jf60132a020
Kinetic procedures
The kinetic procedures were described previously [6].
12.Rougier, N. M.; Vico, R. V.; de Rossi, R. H.; Buján, E. I. J. Org. Chem.
2010, 75, 3427–3436. doi:10.1021/jo100541y
13.Balakrishnan, V. K.; Han, X.; vanLoon, G. W.; Dust, J. M.; Toullec, J.;
Buncel, E. Langmuir 2004, 20, 6586–6593. doi:10.1021/la049572d
14.Han, X.; Balakrishnan, V. K.; vanLoon, G. W.; Buncel, E. Langmuir
2006, 22, 9009–9017. doi:10.1021/la060641t
The DIMEB molecule has hydroxy groups that can be deproto-
nated by hydroxide ions in basic media; therefore, when calcu-
lating the NaOH concentration required for preparation of the
solutions it is necessary to take this fact into account. Since we
did not find values for the pKa of DIMEB in the literature, we
used the pKa value of β-CD, viz. 12.20 [29,30].
15.Cambridge Structural Database and Cambridge Structural Database
system, Version 5.32; Cambridge Crystallographic Data Centre,
University Chemical Laboratory: Cambridge, England, 2012.
16.Connors, K. A. Binding Constants. The Measurement of Molecular
Complex Stability; John Wiley & Sons: New York, 1987; pp 139–216.
17.Kawamura, M.; Higashi, M. J. Inclusion Phenom. Macrocyclic Chem.
2005, 51, 11–15. doi:10.1007/s10847-004-5390-9
Acknowledgements
This material is based upon work supported by the National
Research Foundation under UID 75868. M.R.C. and D.L.C.
express their thanks to the NRF and the University of Cape
Town for financial assistance.
18.Shimizu, H.; Kaito, A.; Hatano, M. Bull. Chem. Soc. Jpn. 1979, 52,
2678–2684. doi:10.1246/bcsj.52.2678
19.Shimizu, H.; Kaito, A.; Hatano, M. Bull. Chem. Soc. Jpn. 1981, 54,
513–519. doi:10.1246/bcsj.54.513
20.Kamiya, M.; Mitsuhashi, S.; Makino, M.; Yoshioka, H. J. Phys. Chem.
1992, 96, 95–99. doi:10.1021/j100180a021
Financial assistance from CONICET, FONCYT, MINCyT-
Córdoba (Argentina) and support from the Universidad
116

Beilstein J. Org. Chem. 2013, 9, 106–117.
21.Kamiya, M.; Nakamura, K. Pestic. Sci. 1994, 41, 305–309.
doi:10.1002/ps.2780410404
22.Hamai, S.; Ikeda, T.; Nakamura, A.; Ikeda, H.; Ueno, A.; Toda, F.
J. Am. Chem. Soc. 1992, 114, 6012–6016. doi:10.1021/ja00041a017
23.Kamiya, M.; Nakamura, K.; Sasaki, C. Chemosphere 1995, 30,
653–660. doi:10.1016/0045-6535(94)00431-S
24.Coscarello, E. N.; Barbiric, D. A.; Castro, E. A.; Vico, R. V.; Buján, E. I.;
de Rossi, R. H. J. Struct. Chem. 2009, 50, 671–679.
doi:10.1007/s10947-009-0103-2
25.Under the condition of our study all the DIMEB present is expected to
be ionized.
26.Barbour, L. J. J. Appl. Crystallogr. 1999, 32, 351–352.
doi:10.1107/S0021889898012667
27.Sheldrick, G. M. In Direct Methods for Solving Macromolecular
Structures; Fortier, S., Ed.; Kluwer Academic Publishers: Dordrecht,
1998; pp 401–411.
28.Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112–122.
doi:10.1107/S0108767307043930
29.Gelb, R. I.; Schwartz, L. M.; Bradshaw, J. J.; Laufer, D. A.
Bioorg. Chem. 1980, 9, 299–304. doi:10.1016/0045-2068(80)90039-5
30.Maeztu, R.; Tardajos, G.; González-Gaitano, G.
J. Inclusion Phenom. Macrocyclic Chem. 2011, 69, 361–367.
doi:10.1007/s10847-010-9753-0
In this publication the value of the pKa of β-CD is reported as 12.36, but
recalculating all the data using this value does not change the results
significantly.
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