Complexes of peracetylated cyclodextrin in a non-aqueous aprotic medium: the role of residual water

Article abstract of DOI:10.1039/c5cp02379c

This paper describes the interaction between aromatic esters and peracetylated cyclodextrins (CDs) studied by NMR spectroscopy in deuterochloroform (CDCl₃). The observed chemical shift changes highlight the existence of interactions between an aromatic alkyl ester, water and peracetylated CDs. In some cases, substituent chemical shift determination was influenced by the low water content of CDCl₃ and/or the host molecule. Higher CD concentrations resulted in water signal drifts in all studied cases. It was not possible to obtain a completely dry sample of peracetyl γCD: ～1 mol of water remained and the water signal showed reversed movement, with respect to the other two CD analogues, upon increasing host concentration. The estimated 1 : 1 stability constants for the water : peracetyl CD complexes are in the 50-150 M^-1 range in CDCl₃, but show a relatively large calculation error. The calculated 1 : 1 stability constants for the peracetyl CD : ester complexes are also in this range, but 1 : 2 and 2 : 1 complex compositions are also possible. Overall, our results highlight dynamic aspects of water nanoconfined in a highly hydrophobic environment, thus mimicking biological recognition where a few water molecules often play a pivotal role.

Full text of DOI:10.1039/c5cp02379c

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Complexes of peracetylated cyclodextrin in a
non-aqueous aprotic medium: the role of residual
Cite this: Phys.Chem.Chem.Phys.,
water†
2
015, 17, 17380
a
a
a
b
Laszlo Jicsinszky,* Katia Martina, Marina Caporaso, Pedro Cintas,
c
a
Andrea Zanichelli and Giancarlo Cravotto*
This paper describes the interaction between aromatic esters and peracetylated cyclodextrins (CDs)
studied by NMR spectroscopy in deuterochloroform (CDCl ). The observed chemical shift changes
highlight the existence of interactions between an aromatic alkyl ester, water and peracetylated CDs. In
some cases, substituent chemical shift determination was influenced by the low water content of CDCl
3
3
and/or the host molecule. Higher CD concentrations resulted in water signal drifts in all studied cases.
It was not possible to obtain a completely dry sample of peracetyl gCD: B1 mol of water remained and
the water signal showed reversed movement, with respect to the other two CD analogues, upon
increasing host concentration. The estimated 1 : 1 stability constants for the water : peracetyl CD
À1
Received 23rd April 2015,
Accepted 29th May 2015
complexes are in the 50–150 M range in CDCl
3
, but show a relatively large calculation error. The
calculated 1 : 1 stability constants for the peracetyl CD : ester complexes are also in this range, but 1 : 2
and 2 : 1 complex compositions are also possible. Overall, our results highlight dynamic aspects of water
nanoconfined in a highly hydrophobic environment, thus mimicking biological recognition where a few
water molecules often play a pivotal role.
DOI: 10.1039/c5cp02379c
www.rsc.org/pccp
symmetric structures are frozen in the crystalline state only,
in solutions a dynamic equilibrium exists between association
1
. Introduction
Cyclic maltooligosaccharides, commonly known as cyclodextrins and dissociation. Native CDs, except bCD, are easily soluble in
CDs), are macrocycles of a(1 - 4) linked anhydroglucopyrano- water, while apolar guests easily displace the water molecules
(
4
side units in a C1 conformation. In practice, the 6-, 7- and in the cavity via complexation resulting in a thermodynamically
-membered versions are known as a-, b-, and g-cyclodextrins more favorable state. Inclusion complexes and the sandwich-
aCD, bCD, and gCD), respectively, and have all been well like molecular associations are also often stabilized by hydro-
studied. Their low commercial price, particularly when bought gen bonds.
8
(
in bulk, means that they are used worldwide in a variety of fields.
Once all hydroxyls are substituted, particularly as alkyl ethers
The circular hydrogen bonds between the secondary hydroxyl or esters, the CD lipophilicity profile is completely reversed and
groups on adjacent rings leads to a rigid truncated cone shape, the cavity becomes more hydrophilic than the outer surfaces,
where the hydroxyls form a highly hydrophilic rim and the while substitution also enlarges CD cavity dimensions. However,
glycosidic oxygens, together with a number of methine groups, substituted oxygens, particularly glycosidic oxygens, are still able
create a considerably less hydrophilic environment inside the to form hydrogen bonds with appropriate hydrogen bond donors
cavity. However, long-term inclusion normally occurs and rigid and lipophilic interactions become dominant, particularly at the
edges of the enhanced cavities. It is also generally accepted that
some smaller substituents, such as methyl or acetyl group, can
a
Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin,
considerably deform the structures in the absence of a guest, as
Via P. Giuria, 9, 10125, Turin, Italy. E-mail: ljicsinszky@gmail.com,
1
2
has been described for peracetylated b- and -g-cyclodextrins
(PAcbCD and PAcgCD, respectively). The crystal structures of
giancarlo.cravotto@unito.it
b
Departamento de Qu ´ı mica Org a´ nica e Inorg a´ nica, Facultad de Ciencias-UEX,
Avda. de Elvas s/n, E-06006 Badajoz, Spain
3
4
peracetyl-a-cyclodextrin (PAcaCD), and probably -bCD, are
somehow symmetrical and stabilized by crystal water, although
it is also true that these substituted CDs rarely reproduce the
usual truncated cone structure of unsubstituted CDs. The more
flexible structures obtained by breaking hydrogen bonds or
c
Resilia SRL, Via Milano 201 - 21017, Samarate, VA, Italy
1
†
Electronic supplementary information (ESI) available: H NMR spectra of pure
compounds and complexes at low, middle, and high peracetyl CD molar ratios,
tables of proton chemical shifts, and figures of calculated concentrations and
chemical shifts. See DOI: 10.1039/c5cp02379c
17380 | Phys. Chem. Chem. Phys., 2015, 17, 17380--17390
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even completely losing the hydrogen-bond belt mean that the chiral auxiliary and one, or other, of the enantiomers in diastereo-
glucopyranose rings are able to rotate around the glycosidic isomeric complexes. Both native and modified CDs are effectively
linkages. In fact, some isolated units have been found to distort used as chiral selectors in the majority of enantioseparation
4
7,11
from the usual C1 chair conformation to skew or almost techniques.
1
1
completely inverted C4 conformers. The larger macrocycle
Although proton NMR measurements are the most common
in gCD derivatives allows for greater flexibility and results in ways to investigate molecular associations, carbon NMR can also
2
the most distorted crystal structures, as reported by Caira et al.
be used, but it is less feasible due to the less natural abundancy
1
3
Their solid-state structures have been described as elliptically of NMR active C and longer relaxation time of carbon. Other
distorted macrocycles where glucose units undergo significant geometrical parameters, such as dihedral angles, can be deter-
rotation around the glycosidic connections and deform to mined using empirical relationships to analyze vicinal coupling
create twisted conformers. Derivatization may not only create constant dependence on dihedral angles.
an enlarged cavity but can also lead to the self-inclusion of resolution NMR equipments also enable the scientists to study the
functional groups. effects of complexation on vicinal coupling constants. However,
4
12,13
State-of-the-art high
NMR techniques are currently the leading non-destructive this effect is usually considerably less intense and more com-
methods for studying complexation processes in solution. pound dependent than simple chemical shift analyses.
Although various approaches have been formulated, two principal
The enantioselective inclusion phenomena of lipophilic CD
objectives are at the heart of this NMR titration based method. derivatives were reported in the pioneering work of K o¨ nig
1
4
These two methodologies require two conflicting approximations. et al., who described the interaction between (R,S)-methyl-2-
Determination of complex stoichiometry needs the sum of molar chloropropionate and heptakis(3-O-acetyl-2,6-di-O-pentyl)-bCD
1
5–17
concentrations, a boundary condition for the most commonly (Lipodex D). NMR spectra recorded in cyclohexane-d12
used Job’s plot method, to be constant. Calculation of complex indicated that the guest enantiomeric methine proton displayed
stability constants, however, usually requires guest concentration very high differentiation. Complexation shifts were in accordance
being held constant in order to eliminate concentration depen- with GC retention data and a molecular dynamics simulation
5,6
dent chemical shift drifts. In both cases, the equation for based chiral recognition rational was devised. Numerous
measured chemical shift (dobs), as a weighted average of the publications that used regiospecific acetylated CDs, mainly per-
1
5–17
chemical shifts of the uncomplexed (dfree) and complexed (dbound
species, is combined with the equation for the association con-
)
(6-TBDMS-2,3-di-O-acetyl) CDs, then appeared.
While H-3 and H-5 protons are the most heavily involved in
stant according to complex stoichiometry. A comprehensive the inclusion processes in unsubstituted CDs, hydroxyl group
review by Uccello-Barretta et al. traces the development and substitution increases the number of moieties to be investi-
historical milestones in NMR technology’s study of cyclodextrin gated. In addition, the resulting spectra sometimes show very
7
complexes.
complex chemical shift patterns which are due to the normally
Several exhaustive reviews have been dedicated to the use of distorted structures in the un-complexed states discussed
5
,8–10
NMR methods on cyclodextrins and their complexes,
while above. However, the spectra of unsubstituted and persubstituted
11
Chankvetadze has published a fundamental review on enantio- CDs are much simpler than those of the partially derivatized
discrimination studies. He also pointed out that the multiple compounds.
forces involved in analyte–CD interactions mean that combined
Persubstituted versions are the simplest of the acetylated CD
molecular modeling-spectroscopy approaches are required if we derivatives to prepare. They can be easily produced even on
are to shed more light on the molecular basis of chiral recognition a ten-hundred-kg scale and are available as fine chemicals
processes.
as well. These CDs are highly lipophilic compounds and are
The presence of self-association phenomena can also be soluble in various organic solvents such as methylene chloride,
evaluated by analyzing the NMR spectra of the pure component chloroform, toluene, ethyl acetate and acetone, for example. In
in progressively diluted solutions; the self-association constant toluene, they form inclusion complexes, which precipitate after
can be determined when chemical shifts depend on concen- dissolution of the CD. They are chemically inert and the acetyl
tration. Preliminary information on intermolecular interactions groups cause the cavity of the cyclic structure to widen. The
can be obtained directly from an analysis of the complexation steric stress inherent in these macrocycles makes it possible for
shifts (Dd = dobs À dfree), measured for all the species nuclei, in anhydrous acids or Lewis acids to cleave the glycosidic bonds,
1
H NMR spectra. However, the most important conformational while acetyl groups can usually be cleaved under basic conditions,
analysis method when defining binding geometry is the detec- like in the majority of organic esters.
5
tion of dipolar interactions (NOEs). This method furnishes
Although peracetylated CDs were among the first CDs to
1
8
information on the spatial proximity between nuclei and, there- be prepared, their complexation properties have only been
fore, highlights proximity constraints between nuclei that are sporadically studied. However, the discovery that even polar
either located inside the same molecule (intramolecular dipolar compounds, such as organic alkali metal salts, can be com-
interactions) or belong to a different one (intermolecular dipolar plexed by PAcbCD, despite the cavity’s reversed polarity, is not
interactions). This technique is also an important tool in chiral novel. This same phenomenon was also observed in perace-
discrimination research, as intermolecular dipolar interactions tylated 3,6-anhydro-CDs. However it remained but a curiosity
make it possible to define the relative stereochemistry of the despite the possible advantages it may offer to the routinely
1
9
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2
0,21
used analytical method, TLC.
Despite some attempts made similar to the symmetry perturbations detected in CDs bearing
to use these complexation properties in capillary GC, the benzoyl, at least on the secondary sites. These distortions were
unfavorably high melting point of peracetyl CDs (B235 1C, explained by the loss of the strong hydrogen-bond network
B198 1C and B248 1C for peracetyl-a-, -b- and -gCD, respec- on the secondary sites of the adjacent units and the need to
tively) has been an obstacle to the wider penetration of these minimize the repulsive steric interactions between the bulk
2
2
derivatives into the analytical repertoire.
substituents. Glucopyranoside units are found in an intermediate
Enhanced lipophilicity and widened cavities would appear to conformation, as they are between the two chairs. These ring
4
make peracetylated CD use in the controlled release of hydrophilic deformations affect some units only and the undistorted
drug obvious. Their potential has been known for 25 years,
C
1
2
3
chairs were simultaneously detected. The truncated cone shape is
however after the initial advantageous attempts, only a few heavily perturbed because of these distortions and the polarity
publications can be found. Some enantioseparations, both on differences between the outside and the inside of the cavity can
2
4–28
analytical and preparative scales,
have also been published be affected significantly, as can the solubility of derivatized CDs
and a couple of solid complexes have been prepared using and their complexation properties. Similar conformations have
2
9–31
1
various techniques.
The investigation of soluble complexes in solution, parti-
cularly in apolar solvents, is rare and only a few examples are (2-ethylhexyl)benzene-1,2-dicarboxylate (our model compound is
been found in both CDCl
3 3
and CD OD.
Complex formation between a structural analogue, 1,4-bis-
4
,19,24,32
available in technical papers.
Guest molecules in those 1,4-dicarboxylate), and perbenzoylated bCD has been investi-
3
4
cases also carry some polar components, apart from a species gated by Yu et al., who concluded that the host–guest complex
studied in a recent analytical impacted publication by Kopytin was stabilized by p–p interactions between the aromatic ester
et al. The effect of aCD peracylation on its molecular structure and the perbenzoylated bCD.
2
2
3
has been investigated using single crystal X-ray analysis, and
In this paper, we report our studies on the interaction
only revealed the presence of at least two associated water mole- between a branched alkyl chain ester of terephthalic acid and
cules in the solid state, despite the crystalline complex being peracetylated CDs. The presence of incompletely and possibly
prepared from aqueous 2-propanol. Crystals of a heptakis(2,3,6- inhomogeneously dried products – due either to the drying
tri-O-acetyl)-bCD methanol complex, as well as other peracylated procedure or the adsorption of small amounts of water from the
CDs, were found to contain elliptically distorted molecules with air – is to be expected when bulk quantities are handled. It is worth
4
non-planar, boat-shaped structures. Steric interactions between pointing out that polar solvent molecules can be retained within
adjacent acyl chains in these molecules result in the severe the inner cavity of native CDs. Molecular dynamics evidence
tilting of the glucose units relative to the glycosidic O(4) planes. that the average residence time for residual DMSO lies in the
Additionally, significant acyl residue self-inclusion was found nanosecond range, which is at least one order of magnitude
3
5
in those peracylated molecules. This self-inclusion means that the longer than the one found for water. Molecular dynamics was
guest inclusion, occurring in native host molecules, is unfavorable also used to study the solvent polarity effect on the formation of
both in the solid state and in solution. The structure of fully CD dimers and emphasized the importance of hydrogen bonds
acetylated gCD also showed extensive macrocyclic distortion, in in the formation. Although, it is also true that in the simulation
this case accompanied by the self-inclusion of three acetyl the authors used solvents too, in which the native CDs are
residues, while the complexation of alcohols occurred together practically insoluble, and/or form complexes. Losing the strong
2
with water molecules. A unique feature of the structure is the hydrogen bonds by the substitution of the electrostatic and
3
6
partitioning, by these residues, of the cavity region into two lipophilic interactions become dominant. Having noticed
distinct sub-cavities that accommodate water and ethanol the difficulties of water removal from the lipophilic CDs, the
molecules. This unusual type of solvent dual inclusion was possible effect of water on solution complexation in the non-
3
discovered thanks to powder X-ray diffraction experiments on polar solvent, CDCl , was also investigated.
PAcgCD and a series of solvents, such as acetone, n-propanol
2
and ethanol.
The complete assignment of peracetylated bCD signals has 2. Experimental
been carried out by Uccello-Barretta et al. who have also deter-
2
.1. Materials
mined proximity constraints between PAcbCD protons using
1
Acetic anhydride, methanol, FeCl Á6H O and methylene chloride
3
2
2
3 3
D ROESY analysis in CDCl and CD OD. It was also found
were purchased from Sigma-Aldrich, while a-, b- and gCDs were
generous gifts from Roquette, France. CDCl is a product of
that H-3, H-2 and H-6a,6b protons correlate with the carbonyl
groups on the same sites which, in turn, correlate with the
respective methyl protons. The proton J-coupling patterns were
found to be similar to those in the unsubstituted systems. The
acetyl on the O2-site in the distorted glucopyranose ring(s) is in
proximity to H-5 and hence pseudo axial, making H-2 pseudo
equatorial, on the same site. The same extent of average distor-
3
EurIsotop (D007H). Racemic 1,4-bis(2-ethylhexyl)benzene-1,4-
dicarboxylate was obtained from Giganplast, Italy.
2.2. Methods
1
2.2.1. H NMR measurements. Water-saturated CDCl
3
was
tion can also be found in alkylated, benzylated and benzoylated prepared by shaking CDCl
CDs. The stereochemical distortions found in solution are and then allowed to separate overnight. Molar ratios of water in
3
(10 ml) and water (2 ml) for 5 min
3
3
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the CDCl
3
solutions of peracetyl CDs were calculated from the and finally ethanol free chloroform (2 Â 20 ml) were dried at
integrals of the anomeric proton signals of cyclodextrin and the 80 1C at 2 mbar.
water protons.
NMR stock solutions were prepared in CDCl at fixed 16–25 Æ
3
3
. Results and discussion
1
mM concentrations (except for water complexation experi-
3.1. Peracetyl cyclodextrin–water systems
ments) and the guest was added via a microliter syringe. In
order to determine apparent complex stability constants, CD The X-ray structure of peracetylated aCD contains two moles of
3
concentrations were kept almost constant to avoid concentration water as reported by Harata. One of these water molecules is
dependent shift drifts. The dried peracetylated CDs were weighed found inside the CD cavity at the primary rim, which is not
and added gradually to the water saturated CDCl
3
solution in the surprising considering that the acetyl groups introduced to the
aqueous experiments.
macrocycle reversed its lipophilicity and that carbonyl oxygens
NMR spectra were recorded on a Bruker Avance 300 MHz at can stabilize the water in an apolar environment via hydrogen
2
2
2.6 Æ 0.1 1C. Residual chloroform of CDCl was used as an bonding. Harata, unlike Caira with PAcgCD, was able to prepare
3
internal standard and chemical shifts were calibrated to the pure water–PAcaCD associates although he crystallized PAcaCD
residual CHCl
3
(dH = 7.260). Chemical shifts (d) are given in ppm. from 80% aqueous 2-propanol.
To reduce the possible errors, pure components were always
NMR measurements show that even dried peracetylated CDs
recorded at the beginning of the experiments in each series. All (Scheme 1a) contain water. The weight percentage content of
experiments of the same run had been recorded on same days water is 1–3% in the air-dried products, which is in the 1–2 molar
without interruption, using 64 transients, in a 4496.40 Hz equivalent range of peracetylated CDs due to the ca. 95–130-fold
1
spectral width. The H NMR data were processed using an difference in molecular weight between peracetylated CDs and
42
ACD/NMR Processor, using a 4-fold zero filling and Lorentzian– water. It was found that simple heat assisted drying did not
Gaussian window function (LB = À0.3, GF = 0.3) prior to Fourier usually result in water-free materials. It was possible to com-
transformations.
pletely dry peracetylated a- and bCDs by azeotropic distillations.
However, a relatively large amount of organic solvents used
means that this method is not feasible for drying in bulk quantities.
2.3. Synthesis
2.3.1. Peracetylated cyclodextrins. Dried CDs (10 mmol, PAcgCD showed B1 mol residual water after treatment. Air-dried
9
.7–13 g) were acetylated in acetic anhydride using FeCl
3
Á6H
2
3
O
compounds were used to study the effects of CD concentration
on water chemical shifts for the NMR titrations of 1,4-bis(2-
7
as a catalyst (10 mol%), according to the published method,
and recrystallized twice from MeOH prior to use. The obtained ethylhexyl)benzene-1,4-dicarboxylate (Scheme 1b). It was also
materials, PAcaCD (16 g, 94%), PAcbCD (16 g, 80%) and PAcgCD found that the water content did not considerably affect NMR
(
20 g, 88%), were attempted to dry as described in the next analyses, except for PAcgCD which produced overlapping water
section. and guest methine proton signals. The water signals smoothly
.3.2. Drying of peracetylated cyclodextrins. The CD deri- drifted with increasing guest concentration and finally moved
vatives were subjected to further drying to remove most of the away from the methine signal.
water present by means of the following procedures: (a) room From the experimental data of 2-ethylhexyl benzoate, the
temperature, air, 3 days; (b) 80 1C, air, 3 days; (c) 80 1C, vacuum, methyl and some methylene protons from the guest molecule
in the presence of P and KOH, 3 days; (d) peracetyl CD (10 g) could be tentatively assigned, which enabled us to use their
2
3
8
2 5
O
was dissolved in methylene chloride (10 ml) and dried with chemical shifts for our determination of complex composition
sodium sulfate (1 g), filtered and rotoevaporated. The solid was and calculation of apparent complex stability constants. H-6
dried at 80 1C for 3 days. (e) From peracetyl CD (2 g) by azeo- protons practically gave single signals in PAcaCD, while the
tropic distillations: toluene (2 Â 10 ml), then methanol (3 Â 20 ml), methylene protons were split and well separated in both PAcbCD
Scheme 1 Structures of host and guest and labels for NMR identification.
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Fig. 2 Water proton chemical shift differences at almost identical molar
Fig. 1 Movement of water proton signals upon increasing CD concentration.
concentrations but different peracetylated CD molar fractions.
and PAcgCD (H-6a and H-6b). Overlapping signals were also
observed in the H-5 protons of peracetyl-aCD, the H-6a protons
of peracetyl b- and g-CDs and the ester methylene protons of the
guest. Despite these overlapping signals, the chemical shifts of
H-5, H-6a and the ester methylene protons were determined,
but with relatively large errors. In worst cases, the methyl and
methylene protons of the 2-ethylhexyl moiety, the most separated
signals were used instead of the multiplet chemical shifts.
Although, this method is not absolutely correct because it assumes
that the coupling constants remain constant upon the noncovalent
interaction of the molecules. Changes in coupling constants may
39
also be indicative of complexation, but now they are in the range
of experimental errors at the NMR frequency used.
Fig. 3 Job’s plot-like graph of water proton shift differences as a function
of water molar fraction.
3
.1.1. Water protons. CDCl was saturated with water and
3
the concentrations of the peracetylated CDs were gradually
increased. Constant downfield shifts were observed in the water
protons with increasing CD molar concentration, as seen in weaknesses
However, Job’s plot-like chart can be created despite these
8
–10,40
(Fig. 3). When Job’s plot is created at a non-
Fig. 1. The curves follow saturation-like curves. However, it is constant sum of moles, the extremity is shifted to higher host
necessary to note that the last point overlaps the acetyl protons molar fractions.
and so an exact shift cannot be determined. It was therefore set
as being equal to the acetyl protons and the uncertain points fit almost equimolar association between water and peracetylated
into the trends well. CDs. This is also confirmed by the nonlinear curve fitting and
Water proton chemical shift differences (Dd) show larger stability constant calculations.
Considering this effect we concluded the existence of an
deviances at almost identical peracetylated CD molar concen-
3.1.2. Cyclodextrin protons. A tentative assignment of the
trations, but at different molar fractions. This is probably due acetyl methyl protons of peracetyl-a- and -gCDs is based on the
to experimental weaknesses, such as uncertainties in weighing assignment of peracetyl-bCD published by Uccello-Barretta
1
and changing sample concentrations upon the transfer of solu- et al. Although it appears that the acetyl protons are involved
tions to the tubes. There are enough points to obtain a trend for in the complexation of water and the phthalate ester (see later),
PAcaCD, but the measurements still show large deviations, as unambiguous assignments are not vital at the secondary rim
seen in Fig. 2.
of CDs. CD chemical shifts show small changes with CD
Water signals moved and finally overlapped with the acetyl molar concentration without any recognizable trends. These
signals of the CD derivative and this fact prevented the exact changes in all protons are in the range of 0.003–0.005 ppm over
À1
À1
determination of the water content at high CD/water ratios. 0.03 M , and almost twice that of below 0.002 M . Concentration
This also means that the absence or presence of water cannot dependent curves seem to be linear when plotted against
be seen at high peracetyl CD–water molar ratios. When water chemical shift changes as a function of CD concentration but
was replaced with deuterium oxide, the HDO signals moved for molar fractions the quadratic curves usually fit better. Some
away from the acetyl proton signals, in PAcaCD and PAcbCD, demonstrative examples are seen in Fig. 4. These results
but not in PAcgCD (see the ESI†). The water content of CDCl3 indicate the existence of dynamic changes in CD structures in
(
r0.01%) is in the same molar range as the CDs. Owing to chloroform that are triggered by complexed water. This is
ambiguous water content in the peracetylated CDs, the water in accordance with the Uccello-Barretta et al. publication on
content of the test solutions and the water content of ‘‘absolute’’ peracetylated bCD.
1
3
CDCl , the boundary conditions of Job’s method (constant sum
In PAcaCD, methylene (H-6) protons are indistinguishable at
of molar concentrations) could not be fulfilled.
low CD/high water concentrations. As the CD/water ratio decreased,
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Table
1
Calculated 1 : 1 complex stability constants for peracetyl
CD–water systems in CDCl
3
Estimated
uncomplexed
water shift
Estimated
complexed
water shift
À1
a
K11 [M
]
Peracetyl-aCD
Peracetyl-bCD
Peracetyl-gCD
114.3 Æ 31.2
94.2 Æ 14.1
58.2 Æ 9.6
1.494 Æ 0.029
1.523 Æ 0.015
1.545 Æ 0.032
2.125 Æ 0.036
2.061 Æ 0.014
2.234 Æ 0.068
a
In ppm, measured value: 1.556 Æ 0.005 ppm at B0.005 M water
concentration.
3
Only the 1 : 1 peracetyl CD–water complexes in CDCl gave
results that fulfill acceptance criteria. The calculated stability
constants are tabulated in Table 1. Calculated concentrations and
chemical shift are given in the ESI,† Fig. S3.1 and S3.2. Values for
1
:2 and 2:1 complexes were in the range of thousands. However,
both their relative errors and poor estimations of uncomplexed
water shifts suggest a much lower probability that such asso-
ciations form.
Fig. 4 Plots of selected chemical shift differences vs. molar concen-
tration and vs. molar fraction of peracetylated CDs (a = alpha, b = beta,
g = gamma CD, numbers refer to the appropriate proton as shown in
Scheme 1a).
3.2. 1,4-Bis(2-ethylhexyl)benzene-1,4-dicarboxylate complex
The two-arm structure of the ester allows two CDs to interact
with the guest molecule. Possible association modes are depicted
in Scheme 2, where the additional rings represent the macrocycle
being enlarged by the acetyl substituents. The free rotation of the
ester moiety means that the longer alkyl chain can also approach
closer to the macrocycle cavity, while the coordination of the
second CD is sterically more hindered in this case. The molecular
dimensions of the peracetyl-gCD allow for deeper intrusion into
the cyclodextrin cavity and even the complete cross is geometri-
cally allowed.
3.2.1. Water protons. The water signal showed a downfield
movement with increasing guest concentration in the case
of PAcaCD and PAcbCD, while the opposite movement was
detected in the PAcgCD analogue. This would appear to suggest
that water is partially, but continuously, excluded from PAcgCD,
while two other CDs may participate in a three component
complexation process with CD, water molecules and the guest.
In all cases, the maximal chemical shift changes were at around
Fig. 5 Split of methylene protons in PAcaCD when increasing the CD : water
molar ratio.
at first shoulder appeared only then the signal was divided into 0.1 ppm, while water signals finally overlapped with the CH
two overlapping peaks, as seen in Fig. 5. signals of the ester in PAcgCD. The dried PAcgCD still con-
CD : water molar ratio values were found to be at around tained equimolar water, which together with the water content
0
.4 in our samples. The high chemical shift drift in the mM of the CDCl was a competitor for the guest molecule as seen
3
concentration range indicated the presence of a strong inter- in Fig. 6.
action, according to the theoretical complex calculations by
A section of proton spectra is shown in Fig. 7 in order to
5
Fielding. The apparent 1 : 1 complex stability constants are in demonstrate water proton movements as a function of the
1
2
À1
the range of 10 –10 M
.
host : guest ratio.
The a priori determination of complex stoichiometry is not
Calculation of complex stability constants for water in our
necessary if we use the nonlinear curve fitting procedure. three-component systems (neglecting the solvent) gave very
6,41
WinEQNMR2
is a suitable program for calculating 1 : 1, 1 : 2 high errors in all cases, as can be concluded from Table 2.
and 2 : 1 complex stability constants from NMR data. Though this The peracetylated cyclodextrins contain water and in the com-
program cannot calculate higher host:guest stoichiometries, how- plexation experiments, the water concentrations varied in a very
ever the peracetyl-a- and -gCD X-ray data suggest that these com- tight range. Additionally, in the case of PAcgCD due to the water
positions are sufficient for the calculations. The goodness of signal overlaps with the methine signal of the organic guest
parameters obtained from a nonlinear curve fitting is always and its chemical shifts are uncertain and integration is not
challenging, particularly when the experiment number is limited. satisfactory. For the PAcaCD case overlapping water and Ac3
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Scheme 2 Possible structures for various peracetylated CDs and 1,4-bis(2-ethylhexyl)benzene-1,4-dicarboxylate complexes.
Fig. 6 Water proton signal movement as a function of the molar ratio.
methyl signals cause similar difficulties. Because the estimated
stability constants are out of acceptance, the complexed/free
water ratio cannot be well estimated. From Fig. 6 it can be
concluded that the guest molecule cannot exclude water from
PAcaCD and PAbCD, the lower limit of 1 : 1 apparent stability
Fig. 7 Water proton signal movement upon increasing the guest molecule
molar ratio. Top: PerAcaCD, middle: PerAcbCD, bottom: PerAcgCD. Guest
À1
constant is at around 100 M , while for the PAgCD system the
À1
upper limit is around 200 M . These values are in good molar ratio increases from bottom to top in each spectrum; some curves
agreement with the values of Table 3 for PAca-and -bCDs.
.2.2. Cyclodextrin protons. While the protons outside the
cavity (H-1, H-2, H-4, and often H-6) usually show negligible
have been removed to improve visibility.
3
shift changes in natural or partially substituted CDs due to apolar environment. The wider cavities allow for interaction
their more rigid structures, in peracetylated CDs these protons with larger molecules, e.g. those that contain branched alkyl
also reflect structural changes upon complexation. At the same chains or structural isomers of aromatic compounds.
time, the widened cavity can reduce the environmental changes
Almost all CD protons moved, but usually without charac-
in protons close to the cavity. Hydrophobic substituents, like teristic extremities in Job’s plots, upon changing the concen-
methyl or acetyl or generally, alkyl- and alkylcarbonyl groups, tration of the host molecule. It is assumed that these missing
virtually inverse the polarity of the CD cavity due to their apolar extremities are due to the opposite movement or the large
character and the cavities favor the more polar groups in an distances between interacting and non-interacting moieties.
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Table 2 Calculated 1 : 1 complex stability constants for peracetyl CD–
water-1,4-bis(2-ethylhexyl)benzene-1,4-dicarboxylate systems in CDCl
3
Estimated
K11 [M ] (based on Table 3)
À1
À1
K11 [M
]
Peracetyl-aCD
Peracetyl-bCD
Peracetyl-gCD
4.1 Æ 38.2
6.2 Æ 4.6
493 (lower limit)
4111 (lower limit)
o182 (upper limit)
34.2 Æ 37.6
The reproducibility of chemical shifts at identical concentra-
tions was typically in the 0.001–0.004 range. Although PAcaCD
H-5 protons overlap the ester methylene protons, it was possi-
ble to determine individual chemical shifts in most cases. This
was not true for PAcbCD and PAcgCD where the H-6a protons
completely overlapped the ester methylene protons at a higher
CD molar ratio and it was impossible to determine the indivi-
dual chemical shifts.
Fig. 8 Job’s plot of CD acetyl methyl protons.
that both alkyl chains can be complexed at low guest molar
Although the acetyl protons are in huge excess in the CDs ratios giving 2 : 1 host : guest associations. The 1 : 1 complex
and their methyl groups can be very far from each other, the became dominant upon increasing guest concentration, as is
outcome of their shift changes are above experimental errors, illustrated in Scheme 2. The branched alkyl chain can penetrate
as can be seen in their Job’s plots, Fig. 8. The curves show large deeper into the CD cavity allowing the more polar carbonyl
fluctuations, at ester molar fractions of around 0.25–0.3, 0.45–0.5 group(s) to interact with the similar CD groups. Unfortunately,
and 0.67, while definite minima – corresponding to 2 : 1 (or 3 : 1, the guest molecule’s most sensitive proton signals, the ester
due to inherent uncertainty in Job’s plot), 1 : 1 and 1 : 2 host : guest methylene and branch methine proton, overlap with some CD
ratios – can be found in the case of PAcaCD and PAcbCD. Only one signals and also with water signals, in the case of PAcgCD.
minimum is found for PAcgCD at B0.3 guest molar fraction. The Additionally, the host methine group’s chiral center brings new
acetyl groups are far from the cavity and the observed chemical complexities as a potential enantiomer-selective complexation.
shift drifts can give information about the host and guest inter- Chemical shift determinations are ambiguous as a consequence
action centers. In the case of PAcaCD, the secondary acetyl sub- of these bad constellations and, as such, conclusions should be
stituents show an almost equal but smaller interaction than the handled carefully. Furthermore, CD Job’s plots do not comple-
primary rim substituent, where the 0.25–0.3 molar ratio minimum tely confirm the a priori assumed association stoichiometry.
is missing. An interaction between the host and guest is therefore Job’s plots of ester methylene and the methine protons can be
assumed to exist at the secondary rim, while the extremities point seen in Fig. 9.
to a secondary rim interaction for 1 : 1, and a head-to-head associa-
The aromatic region of the guest molecule is the only part of
tion for the 2 : 1 host : guest ratio. PAcbCD shows almost equal the molecule which has no signals that overlap with CD and
participation from the O(3)- and O(6) acetyl groups in complexa- water protons. Job’s plot created from the data given in Fig. 10
tion, while the largest differences can be found in all PAcgCD acetyl is not absolutely clear, however the signal shape of the aromatic
groups which affect both sides of the host molecule. The dimen- protons shows essential changes upon host : guest molar ratio
sions of both molecules allow the guest to pass through the CD changes, as is demonstrated in Fig. 11. The large extremities
cavity in the latter case. This can also be confirmed by the at the B0.3 molar ratio of the guest suggest 2 : 1 CD : ester
migration of water protons in the opposite direction suggesting associations for PAcaCD and PAcbCD. In the PAcgCD case,
that the ester is able to move water from a relatively polar where water can be more readily excluded, a minimum can be
environment to a less polar one. NOESY and/or ROESY experi- found at around the 1 : 1 molar ratio. Water exclusion by the
ments may have given a more realistic picture, however these host is also confirmed by the higher complex stability constants
experiments failed due to the high number of almost chemically seen in Table 3. The reasonable 1 : 2 host guest ratio also
equivalent protons combined with the chirality of the ester.
supports the idea that the terephthalate ester can pass through
3.2.3. Guest protons. The phthalate ester has two arms that the PAcgCD cavity. The weak minima, which rather seem to be
are potential targets for complexation. It is a priori assumed shoulders in the other two cases, suggest the existence of the
Table 3 Calculated stability constants from the aromatic proton shifts of different complex models (numbers after K shows the host : guest molar ratio)
a
À1
b
À1
b
MÀ2
c
À1
c
À2
K11 [M
]
K11 [M
]
K12 [
]
K11 [M
]
K21 [M ]
Peracetyl-aCD
Peracetyl-bCD
Peracetyl-gCD
96.7 Æ 3.4
114.8 Æ 3.0
187.4 Æ 3.7
8.6 Æ 1.3
28.3 Æ 1.6
68.1 Æ 1.7
272 Æ 75.6
51.3 Æ 2.0
28.2 Æ 0.9
37.7 Æ 1.5
49.8 Æ 1.3
404.3 Æ 0.0
25.4 Æ 10.5
190.6 Æ 102.7
277.9 Æ 129
a
b
c
The results are from the 1 : 1 (host : guest) curve fitting procedure. The results are from the 1 : 2 (host : guest) curve fitting procedure. The
results are from the 2 : 1 (host : guest) curve fitting procedure.
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Fig. 9 Job’s plot of guest methine and ester methylene protons.
The calculated stability constants show that 1 : 1 and 2 : 1
complexes are the most probable associations in the case of
PAcaCD, while 1:1 and both 1:2 and 2:1 associations for PAcbCD
and PAcgCD are reasonable but in these cases, the 1 : 1 association
is the most probable. These results are also in accordance with the
molecular dimensions of CDs. The large error suggests that
the 1 : 2 complex, for PAcaCD, and 1 : 2 and 2 : 1 complexes, for
PAcgCD, are less adequate models for the association by hand-
ling the peracetyl CD–water as one unit.
Fig. 10 Job’s plot of aromatic protons.
Similar calculations for the CD protons pointed to the glucose
1
: 1 and 1 : 2 host : guest associations for the PAcgCD and 1 : 1 units participating differently in the complexation and showed
and 2 : 1 for the other two CDs. Only loose associations can be considerable geometrical changes upon association. Data for the
5
made from all figures. According to the literature, artificial calculations can be found in the ESI.†
complex simulations gave the apparent complex stability con-
À1
stants to be in the 10–100 M range. To calculate the stability
constants, the three-component systems were reduced to two
4
. Conclusions
components as water was neglected and the multicomponent
6
,41
curve fitting procedure from WinEQNMR2
was used.
Water interacts strongly with the polar moieties of peracetyl CDs.
The stability constants calculated from the aromatic protons The water content of peracetylated CDs can control the complexa-
of the guest are summarized in Table 3. Simulated concentration tion of apolar organic molecules. Although the limited solubility
curves and chemical shifts can be found in the ESI,† Fig. S3.3 of water in chloroform results in very low water content in the
and S3.4. Calculations for other than ester aromatic protons failed solvent, this residual amount of water is still in the equimolar
to give reasonable results, which is partially the consequence of range, as compared to the low dissolved CD concentration.
the relatively large error in the chemical shifts of the overlapped Complete water removal from the peracetylated CDs is difficult
signals in the complex concentration range. An additional source and far from feasibility in bulk quantities. Approximately equi-
of the failed calculations can be found from the fact that the two molar amounts of water remained in peracetyl-gCD even after
identical arms of the guest the 1 :1, 1 :2 and 2:1 host:guest ratios azeotropic removal, which was the only successful method for
are practically indistinguishable in the alkyl region.
a- and bCDs. The water signal at higher CD : water molar ratios
Fig. 11 Aromatic protons at low, medium, and high guest ratios (black: no CD, red: low, green: middle, blue: high peracetyl CD : guest molar ratio,
respectively).
17388 | Phys. Chem. Chem. Phys., 2015, 17, 17380--17390
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overlapped with the acetyl methyl signals. Complexation means
that the water content of chloroform can be considerably
higher than when without peracetylated CDs.
9 B. Chankvetadze, G. Endresz and G. Blaschke, Chem. Soc.
Rev., 1996, 25, 141–153.
10 H. J. Schneider, F. Hacket and V. R u¨ diger, Chem. Rev., 1998,
98, 1755–1785.
Host and guest proton signals, and often water signals,
overlap which made the determination of the chemical shifts 11 B. Chankvetadze, Chem. Soc. Rev., 2004, 33, 337–347.
of the interacting moieties difficult and prone to large relative 12 M. Karplus, J. Am. Chem. Soc., 1963, 85, 2870–2871.
errors. Structural changes in CDs upon interaction with the 13 C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1973,
guest molecule affect all CD protons, regardless of their spatial
interactions in the complexation process. The water proton 14 J. E. H. K o¨ hler, M. Hohla, M. Richters and W. K o¨ nig, Angew.
NMR signal showed different shift directions upon increasing
Chem., Int. Ed. Engl., 1992, 31, 319–320.
the aromatic ester molar ratio: a downfield shift in the cases 15 T. K ¨o pke, H. G. Schmarr and A. Mosandl, Flavour Fragrance
95, 2333–2344.
of PAcaCD and PAcbCD and an upfield shift in the case of
PAcgCD.
J., 1992, 7, 205–211.
16 I. Abe, N. Fujimoto and T. Nakahara, J. Chromatogr. A, 1994,
676, 469–473.
The interaction between apolar CD derivatives and polar (water)
or apolar guests (1,4-bis(2-ethylhexyl)benzene-1,4-dicarboxylate) 17 C. Bicchi, V. Manzin, A. D’Amato and P. Rubiolo, Flavour
was confirmed via NMR. The peracetyl CD–water complexes are
Fragrance J., 1995, 10, 127–137.
dominantly found in a 1 : 1 molar ratio with moderate complex 18 K. Freudenberg and R. Jacobi, Justus Liebigs Ann. Chem.,
stability constants. Both peracetyl-a- and -bCDs seem to form three
1935, 518, 102–108.
component complexes with 1 : 1, 1 : 2, and 2 : 1 molecular ratios 19 M. Komiyama, H. Yamamoto and H. Hirai, Chem. Lett.,
between CDs and esters, while the guest can exclude water from
1984, 1081–1084.
the peracetyl-gCD cavity by forming dominantly 1 : 1 molecular 20 C. Baudin, B. Perly and A. Gadelle, EP Pat., 787744 A1
associations.
970806, 1997.
2
2
1 C. Baudin, F. Tardy, J.-P. Dalbiez, C. Jankowski, C. Fajolles,
G. Leclair, B. Amekraz, B. Perly and L. Mauclaire, Carbohydr.
Res., 2005, 340, 131–138.
2 K. A. Kopytin, S. Y. Kudryashov and L. A. Onuchak, Russ.
J. Phys. Chem. A, 2012, 86, 147–150.
Acknowledgements
This research was kindly funded by the European Community’s
Seventh Framework Program through the EU project MAPSYN: 23 K. Matsubara, T. Kuriki, H. Arima, K. Wakamatsu, T. Irie and
Microwave, Acoustic and Plasma Syntheses, under grant agree-
ment No. CP-IP 309376. P. Cintas thanks the Ministry of
K. Uekama, Minutes of the 5th International Symposium on
Cyclodextrins, Paris, 1990, p. 491.
Economy and Competitiveness (Project CTQ2013-44787-P) and 24 M. Thunhorst and U. Holzgrabe, Magn. Reson. Chem., 1998,
the Junta de Extremadura-FEDER (Grant GR15022) for financial
36, 211–216.
support. Financial support from Resilia SRL (Samarate, Italy) is 25 J. Zmitek, D. Fercej-Temeljotov, K. Verhnjak, S. Kotnik and
acknowledged. The authors acknowledge the assistance and
M. Kovacic, EP Pat., 578231 A1, 1994.
suggestions of Orsolya T o¨ ke (MTA-TKK, Budapest, Hungary) in 26 T. Kitagawa, A. Okamoto, T. Kanai, K. Shibayama, T. Aoki
and S. Yamakawa, EP Pat., 970936, 2000.
Cyclolab Ltd, Budapest, Hungary) in manuscript preparation. 27 L. Kim, A.-D. Stancu, E. Diacu, H.-J. Buschmann and
´
NMR experiments, and the invaluable help of Eva Fenyvesi
(
L. Mutihac, Supramol. Chem., 2009, 21, 131–134.
8 E. Diacu, L. Mutihac, E. Ruse and M. M. Ceausescu,
J. Inclusion Phenom. Macrocyclic Chem., 2011, 71, 339–342.
9 Z. P. Onuchak, L. A. Burmatnova, T. S. Spiryaeva, E. A.
Kuraeva and Y. G. Belousova, Russ. J. Phys. Chem. A, 2012,
86, 1308–1317.
2
2
References
1
G. Uccello-Barretta, G. Sicoli, F. Balzano and P. Salvadori,
Carbohydr. Res., 2003, 338, 1103–1107.
2
M. R. Caira, G. Bettinetti, M. Sorrenti, L. Catenacci, 30 C. M. Buchanan, D. W. Dixon, R. J. Offermann, J. Szejtli,
D. Cruickshank and K. Davies, Chem. Commun., 2007,
221–1223.
K. Harata, Chem. Lett., 1998, 589–590.
L. Szente and M. Vikmon, Minutes of the 10th International
Symposium on Cyclodextrins, Michigan, 2000, p. 586.
31 M. Y. Lee, H. S. Ganapathy and K. T. Lim, J. Phys. Chem.
Solids, 2010, 71, 630–633.
1
3
4
M. A n˜ ibarro, K. Gessler, I. Uson, G. M. Sheldrick, K. Harata,
K. Uekama, F. Hirayama, Y. Abe and W. Saenger, J. Am. 32 G. Gattuso, C. Gargiulli and M. F. Parisi, Int. J. Mol. Sci.,
Chem. Soc., 2001, 123, 11854–11862.
2007, 8, 1052–1063.
5
6
7
L. Fielding, Tetrahedron, 2000, 56, 6151–6170.
M. J. Hynes, J. Chem. Soc., Dalton Trans., 1993, 311–312.
33 G. Uccello-Barretta, F. Balzano, A. Cuzzola, R. Menicagli and
P. Salvadori, Eur. J. Org. Chem., 2000, 449–453.
G. Uccello-Barretta, L. Vanni and F. Balzano, J. Chromatogr. A, 34 B. Z. Yu, J. W. Chung and S.-Y. Kwak, Environ. Sci. Technol.,
010, 1217, 928–940. 2008, 42, 7522–7527.
H. Dodziuk, W. Kozminski and A. Ejchart, Chirality, 2004, 35 J. Rodriguez, D. Hern ´a n Rico, L. Domenianni and D. Laria,
2
8
16, 90–105.
J. Phys. Chem. B, 2008, 112, 7522–7529.
This journal is ©the Owner Societies 2015
Phys. Chem. Chem. Phys., 2015, 17, 17380--17390 | 17389

View Article Online
PCCP
Paper
3
6 H. Zhang, T. Tan, W. Feng and D. van der Spoel, J. Phys. 40 S. Lee, D.-H. Yi and S. Jung, Bull. Korean Chem. Soc., 2004,
Chem. B, 2012, 116, 12684–12693. 25, 216–220.
7 L. Jicsinszky and J. Szejtli, Minutes of the 6th Inter- 41 Current version of WinEQNMR (v2.0) can be obtained free of
3
national Symposium on Cyclodextrins, Chicago, 1992,
p. 96.
8 2-Ethylhexyl benzoate: SDBS No. 17041HSP-43-349, http://
sdbs.db.aist.go.jp. (Accessed on September, 2014).
9 S. Bekiroglu, L. Kenne and C. Sandstr o¨ m, J. Org. Chem.,
charge from Michel J. Hynes, National University of Ireland,
Department of Chemistry, Galway, Ireland; michael.j.hynes@
nuigalway.ie.
3
3
42 ACD/NMR Processor Academic Edition Release 12.00 product
version 12.01 (Build 39104), Advanced Chemistry Development
Inc. (www.acdlabs.com).
2003, 68, 1671–1678.
17390 | Phys. Chem. Chem. Phys., 2015, 17, 17380--17390
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