Crystallographic, UV spectroscopic and computational studies of the inclusion complex of α-cyclodextrin with p -aminobenzoic acid

Article abstract of DOI:10.1080/10610278.2012.658392

Crystal structure of the cyclomaltohexaose (α-cyclodextrin, α-CD) inclusion complex with p-aminobenzoic acid (pABA) has been determined by X-ray diffraction. The host:guest stoichiometry is 1:1. The pABA molecule is included in the cavity with its axis coincident with the axis of α-CD; the benzoic group is inserted in the cavity, while the amino group sticks out from the cavity. Four water molecules are located near the cavity rims and in interstices between the molecules of α-CD participating in a dense network of intermolecular hydrogen bonds. UV-visible spectroscopy was applied to estimate the stability constant (K_c) at different temperatures on the basis of the Benesi-Hildebrand equation. This allowed calculation of complexation ΔH°_c and ΔS° _c on the basis of the Van't Hoff equation. The results are in good agreement with the values obtained by other methods in the literature. Phase-solubility profiles indicate that the solubility of pABA is significantly increased in the presence of α-CD at different pH values, and it was classified as AL-type, indicating a 1:1 stoichiometric inclusion complex in solution. A theoretical investigation has also been carried out on the α-CD-pABA systems in order to search for other stable complexes. PM6 semi-empirical calculations were made to investigate equilibrium geometries of inclusion complexes formed between α-CD and neutral, anionic, cationic and zwitterionic forms of pABA. Two possible orientations were considered (A, with the carboxylic end inside the cavity and B, with the amino group inside the cavity). Preference between A and B orientations of each α-CD-pABA form results from different H-bond interaction patterns.

Full text of DOI:10.1080/10610278.2012.658392

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Crystallographic, UV spectroscopic and computational
studies of the inclusion complex of α-cyclodextrin with
p-aminobenzoic acid
Kossay Elasaad a b , Bernadette Norberg a & Johan Wouters a
a Department of Chemistry , University of Namur , rue de Bruxelles, 61, B-5000 , Namur ,
Belgium
b Ministry of Higher Education in Syrian Arab Republic , 9251, Mezzeh Gamarik Square,
Damascus , Syria
Published online: 03 Apr 2012.
To cite this article: Kossay Elasaad , Bernadette Norberg & Johan Wouters (2012) Crystallographic, UV spectroscopic and
computational studies of the inclusion complex of α-cyclodextrin with p-aminobenzoic acid, Supramolecular Chemistry, 24:5,
312-324, DOI: 10.1080/10610278.2012.658392
To link to this article: http://dx.doi.org/10.1080/10610278.2012.658392
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Supramolecular Chemistry
Vol. 24, No. 5, May 2012, 312–324
Crystallographic, UV spectroscopic and computational studies of the inclusion complex of
a-cyclodextrin with p-aminobenzoic acid
Kossay Elasaada,b*, Bernadette Norberga and Johan Woutersa
b
aDepartment of Chemistry, University of Namur, rue de Bruxelles, 61, B-5000 Namur, Belgium; Ministry of Higher Education
in Syrian Arab Republic, 9251, Mezzeh Gamarik Square, Damascus, Syria
(Received 17 October 2011; final version received 5 January 2012)
Crystal structure of the cyclomaltohexaose (a-cyclodextrin, a-CD) inclusion complex with p-aminobenzoic acid (pABA)
has been determined by X-ray diffraction. The host:guest stoichiometry is 1:1. The pABA molecule is included in the cavity
with its axis coincident with the axis of a-CD; the benzoic group is inserted in the cavity, while the amino group sticks out
from the cavity. Four water molecules are located near the cavity rims and in interstices between the molecules of a-CD
participating in a dense network of intermolecular hydrogen bonds. UV–visible spectroscopy was applied to estimate the
stability constant (Kc) at different temperatures on the basis of the Benesi–Hildebrand equation. This allowed calculation of
complexation DH8c and DSc8 on the basis of the Van’t Hoff equation. The results are in good agreement with the values
obtained by other methods in the literature. Phase-solubility profiles indicate that the solubility of pABA is significantly
increased in the presence of a-CD at different pH values, and it was classified as AL-type, indicating a 1:1 stoichiometric
inclusion complex in solution. A theoretical investigation has also been carried out on the a-CD-pABA systems in order to
search for other stable complexes. PM6 semi-empirical calculations were made to investigate equilibrium geometries of
inclusion complexes formed between a-CD and neutral, anionic, cationic and zwitterionic forms of pABA. Two possible
orientations were considered (A, with the carboxylic end inside the cavity and B, with the amino group inside the cavity).
Preference between A and B orientations of each a-CD-pABA form results from different H-bond interaction patterns.
Keywords: cyclodextrin; para-aminobenzoic acid; crystal structure; inclusion complex; host–guest complex
1
Introduction
unwanted side effects, which can limit its industrial
applications. For instance, dermatological side effects and
pABA capability to damage DNA after UVirradiation were
observed (6–8). To reduce these unwanted effects and to
increase the aqueous solubility of the compound, the use of
ABA forms encapsulated in CDs is an attractive goal.
Cyclomaltooligosaccharides (CDs) are cyclic oligo-
saccharides with a-(1 ! 4)n-linked glucose units, where
n ¼ 6 (a-CD), n ¼ 7 (b-CD) or n ¼ 8 (g-CD). CDs have a
truncated conical structure (a torus) with the primary
hydroxyl groups at the narrow side (head) and the
secondary hydroxyl groups at the wide side (tail). CDs
form inclusion complexes with a variety of molecules (9).
Several factors are recognised to be responsible for the
formation of inclusion complexes of CDs with suitable
guest molecules: van der Waals forces and hydrogen bonds,
as well as hydrophobic and dipole–dipole interactions
(10). Crystal structures of a-CD complexes with various
guests have been reported (11), suggesting that a para-
substituted phenol is a suitable guest for a-CD. Crystal
structures of a-CD complexes with p-hydroxybenzoic acid
( p-COOHC6H4OH), p-nitrophenol ( p-NO2C6H4–OH), p-
iodophenol and p-fluorophenol have been successfully
determined (12–14). A number of studies were carried out
In pharmaceutical practice, poor water solubility of drugs
is a well-known problem. At present, about 40% of the
drugs in the development pipelines and up to 60% of the
compounds coming directly from synthesis are poorly
soluble (1). To act on target receptors, drugs must
generally be dissolved in the physiological fluid and
thereafter absorbed through entrance ports. Various
methods have been used to increase the dissolution rate
including micronisation, modification of the physico-
chemical properties of the drug, addition of water-soluble
polymers or complex formation with cyclodextrins (CDs).
Aminobenzoic acids are benzoic acid derivatives
bearing an amino group in ortho, meta or para position of
the aromatic ring. Among these structural isomers, only p-
aminobenzoic acid (pABA) is of biological importance.
pABA, known as vitamin H1, belongs to B-group vitamins.
It is widely distributed in nature, participates in important
biochemical processes (e.g. biosynthesis of folic acid) and
reduces the mutagenicity of chemical mutagens (2).
Moreover, pABA is used in the synthesis of some medicines
(e.g. Novocain, 4-aminosalicylic acid) and as UV filter in
sunscreen cosmetics (3–5). However, pABA can cause
*Corresponding author. Email: kelasaad@fundp.ac.be
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10610278.2012.658392
http://www.tandfonline.com

Supramolecular Chemistry
313
on the inclusion complex of pABA with a-CD in solution
using calorimetry, circular dichroism, fluorescence, 1H
NMR, fluorimetry and theoretical methods (15–23). To the
best of our knowledge, no crystal structure of inclusion
complex of pABA and a-CD has been reported so far.
In this study, the crystal structure of the a-CD and
pABA inclusioncomplexhas beendetermined, UV–visible
spectroscopy has been applied to determine the stability
constant (Kc) and thermodynamic parameters (DH8c and
DS8c) of pABA complexation with a-CD and a theoretical
investigation has been carried out on different a-CD-pABA
systems in order to evaluate the relative stability of other
stable complexes involving different forms of pABA.
pABA (0.1 mmol) in ethanol (2.5 ml) at 658C. Both
solutions were mixed and stirred at 658C for 6 h. Then the
mixture was stored at room temperature. Colourless
crystals suitable for X-ray data collection were obtained by
slow evaporation after 1 week.
2.2.2 X-ray diffraction experiment
X-ray diffraction experiment was carried out on an Oxford
˚
Gemini R Ultra using Mo Ka radiation (l ¼ 0.71073 A)
operating at 50 kV, 30 mA. A single crystal of the complex
of a-CD-pABAwas mounted on the head of the four-circle
kappa goniometer. A total of 17,075 unique reflections
were measured in the u-range of 3.28–32.578. Data
reduction was carried out with the CrysAlisPro programme
(24). Crystal data collection and refinement details are
listed in Table 1. The structure belongs to the orthorhombic
P212121 space group with unit cell dimensions
2
Experimental
2.1 Materials
pABA (analytical reagent grade) and a-CD were purchased
from Sigma-Aldrich (GmbH P.O. 1120, 89552 Steinheim,
Germany). Other reagents were of analytical reagent grade
and double distilled water was used throughout. Buffer
solutions at pH ¼ 1.5, 3.5 and 6.0 were prepared using
phosphoric acid (85%) and its mono and disodium salts.
˚
a ¼ 13.4823(3), b ¼ 15.5091(2) and c ¼ 24.8256(4) A.
2.2.3 Structure solution and refinement
The structure of the complex was determined by direct
methods and refined using full-matrix least-squares based on
F 2 with the programme SHELX (25), based on 775
parameters and 2 restraints. The refinement converged to
R1 ¼ 0.0554 for 9586 F0 . 4s(F0) and0.1022forall17,075
data. Four water molecules (two ordered and two disordered
2.2 X-ray diffraction analysis
2.2.1 Crystallisation
Crystals of a-CD in complex with pABA were prepared by
dissolving a-CD (0.1 mmol) in water (2.5 ml) at 658C and
Table 1. Crystallographic data.
Chemical formula
C36H60O36.C7H7NO2.4H2O
1120.00
Formula weight (g mol21
Temperature (K)
)
293(2)
0.71073
˚
Wavelength (A)
Crystal system
Space group
Orthorhombic
P212121
13.4823(3)
15.5091(2)
24.8256(4)
90.000
90.000
90.000
˚
a (A)
˚
b (A)
˚
c (A)
A (8)
b (8)
g (8)
3
˚
Volume (A )
Z
Crystal size (mm)
5190.99(16)
4
0.50 £ 0.30 £ 0.18
Calculated density (g cm23
)
1.498
0.13
Absorption coefficient (mm21
F(0 0 0)
)
2468
3.28–32.57
Theta range for data collection (8)
Limiting indices
Reflections collected/unique
R (int)
220 # h # 17, 213 # k # 23, 237 # l # 33
37,378/17,075
0.0293
17075/2/778
0.9
Full-matrix least-squares on F 2
0.0553
Data/restraints/parameters
Goodness-of-fit (S) on F 2
Refinement method
Final R indices (for 9586 with F20 . 4s(F02))
Final R indices (all 17,075 data)
0.1021
0.37 and 20.50
3
Largest difference peak and hole (e/A )
˚

314
K. Elasaad et al.
over two positions) were located in difference Fourier maps.
All non-hydrogen atoms were anisotropically refined.
Secondary hydroxyl groups were refined with two alternate
conformations. Hydrogen atoms were calculated at ideal
positions and refined using a riding model except those
attached to some hydroxyl groups and on the two ordered
watermoleculesthatwerelocatedindifferenceFouriermaps.
2.5 Computational methods
2.5.1 Ab initio geometry optimisation
Ab initio energy minimisation was performed on pABA in
different ionisation forms (neutral/zwitterionic, protonated
and deprotonated) to better approach geometry of the
different protonation states. Calculation was done with the
Gaussian09 (27) programme in the ground state using the 6-
311G (5d, 7f) standard basis set and the B3LYP functional
density method. Resulting geometries were compared with
the crystal structure data retrieved from the CSD.
2.2.4 Statistical search in the Cambridge structure
database
A statistical survey of the Cambridge structural database
(CSD) was performed searching for aromatic rings
substituted by a carboxylic (ZCOOH), or carboxylate
(ZCOO2) group, and in its para position an amino (ZNH2),
or ammonium (2NHþ3 ) group. The statistical procedures
usedtheVISTAsoftwareassociatedwithCSD/2011package
(11). In this study, we focused on the CZO and Car–C bond
lengthsin carboxylic orcarboxylategroupsandontheCar–N
bond length in amino or ammonium groups. The results were
compared with the values in the crystal structure to establish
the protonation state of pABA in the complex.
2.5.2 Semi-empirical simulation of complexes
Starting geometry for the a-CD-pABA inclusion complex
was based on the crystal structure obtained in this work. In
the first step, all water molecules were removed. Two
possible orientations for pABA inside the a-CD cavity were
simulated: in orientation A, the carboxylic group (COOH) of
pABA is located inside the a-CD cavity while orientation B
corresponds to the amino group (NH2) inside the cavity
(Figure 1). Orientation B was generated by exchanging the
carboxylic and amino groups from the starting X-ray crystal
structure by means of the builder module (Vega ZZ (28)).
The final structures were saved as PDB file in order to
prepare a suitable format file (OpenBabelGUI (29)) to start
semi-empirical PM6 calculation (MOPAC2009 (30)). The
input structures were edited to adjust the protonation state of
pABA: cationic form (extra H on the amino group and
charge ¼ þ1), anionic form (no H on the carboxylic group
and charge ¼ 21), neutral form (protonated carboxylic
groupandunprotonatedamine, charge ¼ 0)andzwitterionic
form (unprotonated carboxylic group and protonated amine,
charge ¼ 0)). The systems were energy optimised using the
PM6 method to locate low energy structures of complexes.
2.3 UV–visible absorption spectroscopy
UV–visible absorption spectra were recorded with a
UVIKON XS spectrophotometer (Bio-TEK, Milano, Italy)
at 1, 5, 10, 15, 20 and 258C using 1 cm quartz cuvettes.
Temperature was controlled by a cooling jacket Julabo
bath circulation thermostat (model F30-HC/8 Julabo
Labortechnik, Seelback, Germany) connected to the
spectrophotometer. All solutions were prepared in distilled
water. The concentration of pABA was held constant at
5 £ 1025 M. The concentration of a-CD varied from 0 to
5 £ 1023 M. The pH was controlled by 0.5 M phosphate
buffers. Samples were incubated inside the spectropho-
tometer for 30 min at the working temperature before
recording the spectra.
2.5.3 Effects of solvation on simulation of complexes
In order to study the influence of solvation on the overall
stability of the complexes, all the previous starting
structures (orientations A or B with the different
2.4 Phase-solubility diagram
˚
protonation states for pABA) were placed in a 10 A
Phase-solubility diagrams allow evaluation of the affinity
between a-CD and pABA in water. Phase-solubility studies
were carried out according to the method reported by
Higuchi and Connors (26). pABA, in an amount that
exceeded its solubility, was taken into a tube in which 10 ml
of buffer (pH 6.0, 3.5 and 1.5) containing various
concentrations of a-CD (0–25 mmol) was added. These
tubes were sealed and shaken at 258C for 72 h. This amount
of time is considered sufficient to reach equilibrium.
Subsequently, aliquots were filtered immediately through
Whatman filter paper and diluted. A portion of the sample
was analysed by UV spectrophotometer to determine the
solubility of pABA by means of pABA calibration curves.
sphere containing 66 water molecules, and the same
procedure of energy minimisation (PM6 level) was applied
on those solvated systems.
3
Results and discussion
3.1 Crystal structure
The structures of the host and guest molecules are shown
in Figure 2. Gn denotes the nth glucosidic residue of the a-
CD. In our crystal structure, a-CD forms a 1:1 inclusion
complex with pABA (Figure 3).

Supramolecular Chemistry
315
Figure 1. Two possible orientations of pABA (A and B) in a-CD (top), and pABA forms at different pH values (bottom).
3.1.1 Geometry of the host molecule
out the cavity. In G3 and G5, O6n is disordered in two sites
with occupancy 60:40% in G3 and 75:25% in G5. A
gauche-trans orientation is adopted for the second site and
All glucose residues are in the normal 4C1 chair
conformation, and the overall a-CD molecules show
distorted hexagon geometry. The diagonal distance
measured between the glycosidic oxygen atoms (O4n) is
O41· · ·O44 ¼ 8.289(2), O42· · ·O45 ¼ 8.221(2), and
O6n points inside the cavity. Four ordered water molecules
are situated outside the cavity at the borders of the toroid
rims and in interstices between a-CD molecules. Two
water molecules, W2 and W4, are disordered in two sites
with occupancy 50:50% and 60:40%, respectively. Table 3
lists the hydrogen bonds in this complex. No direct
hydrogen bonds were found between the host and its guest.
˚
O43· · ·O46 ¼ 8.931(2) A. A similar distorted structure
was observed for other a-CD complexes with phenyl
derivatives (31). Selected geometric parameters for the a-
CD host molecule are listed in Table 2.
The glycosidic O4n atoms lie in a plane within
˚
0.0752 A, the O4n· · ·O4(nþ1) distances varying between
4.103(2) and 4.468(2) A, and the values of the angles
˚
3.1.2 Geometry of the guest molecule
between O4(n21)· · ·O4n· · ·O4(nþ1) differing significantly
from 1208, the ideal value for an angle in a regular
hexagon. Indeed, these values range from 113.71(4)8 to
123.95(5)8, denoting that the cavity is distorted due to
inclusion. The annular shape of a-CD is stabilised by
inter-glucose hydrogen bonds connecting the secondary
hydroxyl groups O3n and O2(nþ1) of the neighbouring
glucosidic units (Table 2) (average O3n· · ·O2(nþ1) distance
2.849(2) A, ranging from 2.708(2) to 2.984(2) A). The
orientation of the C6n–O6n bond is described by torsion
angles C4n–C5n–C6n–O6n and O5n–C5n–C6n–O6n, listed
in Table 2. Most of the primary hydroxyl groups (major
disordered site) adopt the gauche–gauche conformation
(mean torsion angles C4n–C5n–C6n–O6n and O5n–C5n–
C6n–O6n 49.9(3)8 and 271.4(3)8, respectively) and point
In the crystal structure, the anionic form of pABA is
observed in the inclusion complex with a-CD. Indeed, no
extra electron density peak is observed in difference
Fourier maps close neither to the COO2 nor to the NH2
groups that could account for an extra H atom.
Deprotonation of the carboxylic function is further
established by similar values for the two CZO bond
˚
lengths (1.255(3) and 1.275(3) A). The carboxylate and the
˚
˚
benzene rings are coplanar with torsion angles: O1–C7–
C1–C2 ¼ 20.1(5)8, O1–C7–C1–C6 ¼ 2178.4 (3)8, O2–
C7–C1–C2 ¼ 177.2(3)8 and O2–C7–C1–C6 ¼ 21.2(5)8.
Non-hydrogen atoms in pABA lie in a plane within
˚
0.0343 A, favouring electronic delocalisation and leading
˚
to a short CAr –C7 distance equal to 1.473(4) A.
Delocalisation is prolonged to the nitrogen atom with a

316
K. Elasaad et al.
Figure 3. Axial view of the X-ray structure of the 1:1 host–
guest inclusion complex of a-CD and pABA. Water molecules
are shown with red light colour. Hydrogen atoms were omitted
for clarity.
the supplementary data S1) confirms that the mean CZO
bond lengths in Ar–COO2 fragments (1035 fragments
˚
retrieved) are 1.255(^0.022) and 1.253(^0.028) A, and
˚
the mean of CAr–Ccarb bond lengths is 1.509(^0.021) A in
case of the anionic forms (deprotonated). For Ar–COOH
neutral forms (1259 fragments retrieved), the bonds differ
significantly with C ¼ O and CZOH mean bond lengths of
˚
1.227(^0.025) and 1.302(^0.025) A, respectively, and
˚
the mean of CAr–Ccarb bond lengths is 1.485(^0.018) A.
The mean of CAr–N bond lengths in Ar–NH2 fragments
˚
(1696 fragments retrieved) is 1.396(^0.037) A, and
the mean of valence angles around N is CAr
–
NZH1 ¼ 115.1(^6.5)8, CAr–NZH2 ¼ 115.1(^6.2)8 and
H1 –NZH2 ¼ 115.3(^8.4)8. In Ar–NHþ3 fragments
(509 fragments retrieved), the mean of CAr–N bond
˚
length is 1.465(^0.014) A, and the mean valence
angles around N are CAr–NZH1 ¼ 110.157(^2.596)8,
C
Ar –NZH2 ¼ 109.906(^3.195)8, CAr –NZH3 ¼ 110.0
(^2.6)8, H1–NZH2 ¼ 108.8(^4.4)8, H1–NZH3 ¼ 108.8
(^4.4)8 and H2–NZH3 ¼ 108.6(^5.8)8. The bond lengths
˚
in our crystal pABA molecule are 1.255(3) and 1.275(3)A
˚
for CZO in carboxylic group, 1.395(3) A for CAr–N and
˚
1.472(4) A for CAr–C, in good agreement with the values
associated with an anionic form of pABA.
This is further confirmed by geometries optimised ab
initio (B3LYP-6-311G (5d, 7f)) and with the semi-
empirical PM6 method for the different protonation states
of pABA: neutral, cationic, anionic and zwitterionic form
(Table 4). According to the values coming from the
theoretical studies and taking into account the experimen-
tal values, it is clear that the anionic form of pABA must be
adopted in our crystal structure. This conclusion opens the
question of the type and position of the counter cation. No
extra electronic density was observed in difference Fourier
maps that could correspond to a cation. So we suggest that
Figure 2. Structure and numbering scheme of a-CD and p-
aminobenzoic acid. Only numbering of one glycosidic residue
(Gn) of the a-CD is presented.
˚
short CAr–N distance equal to 1.398(3) A, and a marked
sp2 hybridisation character of the nitrogen atom with
valence angles: C4–N1–H1N1 ¼ 104.2(3.2)8, C4–N1–
H2N1 ¼ 108.7(3.7)8 and H1N1–N1–H2N1 ¼ 129.4(4.6)8.
A general search in the CSD (11) of aromatic
compounds containing a carboxylic or amino group (see

Supramolecular Chemistry
317
Table 2. a-CD macrocyclic characteristics.
a
d
DO 2 O4
˚
(A)
w b
˚
(A)
d c
˚
(A)
DO · · ·O2
Torsion angle (8)
Torsion angle (8)
O5n2C5n2C6n2O6n
4
3
˚
(A)
Residue
C4n2C5n2C6n2O6n
G1
G2
G3
G4
G5
G6
4.374 (2)
4.194 (2)
4.105 (2)
4.468 (2)
4.171 (2)
4.149 (2)
122.2 (1)
123.9 (1)
113.7 (1)
122.8 (1)
122.3(1)
114.6 (1)
20.098(1)
0.050(1)
0.056(1)
20.113(1)
0.064(1)
0.040(1)
2.821(3)
2.708(2)
2.804(2)
2.985(2)
2.883(3)
2.883(3)
42.9(3)
49.7(3)
51.5(12) 175.9(15)
52.4 (2)
66.2(5)–153.3(17)
46.3(3)
278.6(3)
70.5(3)
273.0(13) 59.7(23)
269.9(3)
255.0(4) 84.6(28)
275.6(3)
*
a Distance between atoms O4n· · ·O4(nþ1)
.
b Angles between atoms O4(n21)· · ·O4n· · ·O4(nþ1)
Deviations (A) from the least-squares optimum plan of the six O4n atoms.
.
c
˚
d Intramolecular hydrogen-bond distance between O3n· · ·O2(nþ1)
.
one of the water molecules could be protonated to serve as
a counter cation of anionic pABA.
side as shown in Figure 3. The molecular plane of pABA
forms an angle of 87.20(3)8 with the plane made by the six
O4n atoms of the a-CD. The mass centre of the aromatic
˚
ring is 1.23 A below the mass centre of the O4n glycosidic
3.1.3 Host–guest interaction
atoms (see supplementary data S2). The corresponding
values obtained from the b-CD-pABA inclusion complex
In the crystal structure, pABA is deeply inserted into the
cavity of a-CD, with the amino group protruding from the
wide side of the cavity and the carboxyl group at the narrow
˚
are 72.38 and 1.64 A (23). As a consequence, the pABA
molecule better fits the a-CD cavity than the b-CD one, in
8
˚
Table 3. Hydrogen bonds in a-CD–pABA complex with H· · ·A , r(A) þ2.0 A and DHA .110 .
,DHA8
˚
˚
d(H..A) A
˚
d(D..A) A
D–H
A
d(D–H) A
O21–H21O
O21–H21O
O22–H22O
O23–H23O
O24–H24O
O25–H25O
O25–H25O
O26–H26O
O26–H26O
O31–H31O
O32–H32O
O33–H33O
O34–H34O
O35–H35O
O36–H36O
O61–H61O
O62–H62O
O62–H62O
O63A–H63A
O63B–H63B
O64–H64O
O65A–H65A
O65B–H65B
O66–H66O
N1–H1N1
O36
O46
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.82
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.820
0.81(5)
0.95(5)
0.856(19)
0.856(19)
0.819(18)
1.041(19)
1.041(19)
1.024(19)
1.024(19)
2.12
2.30
1.97
1.95
2.03
2.19
2.33
2.02
2.34
2.00
1.91
2.03
1.89
2.11
2.05
2.14
2.04
2.63
2.19
2.33
2.30
2.07
2.52
2.23
2.21(5)
2.09(5)
2.26(3)
2.48(5)
2.28(5)
1.73(4)
2.50(3)
1.49(5)
1.70(3)
155.6
113.8
163.8
167.8
156.4
164.8
113.7
158.6
119.8
174.6
166.8
133.6
170.7
157.0
176.1
125.4
172.3
126.0
155.0
127.3
127.7
166.5
144.2
133.1
137(5)
171(5)
157(5)
109(4)
125(4)
2.601(6)
3.462(6)
2.248(9)
2.675(8)
2.883(3)
2.740(3)
2.769(2)
2.755(3)
2.804(2)
2.985(2)
2.762(2)
2.801(6)
2.839(9)
2.821(3)
2.708(2)
2.666(2)
2.698(2)
2.885(3)
2.872(3)
2.701(3)
2.857(4)
3.179(9)
2.956(4)
2.899(10)
2.872(3)
2.872(4)
3.220(9)
2.853(3)
2.861(3)
3.032(3)
3.063(3)
2.875(4)
2.837(10)
138(5)
O24 [x 2 1/2, 2y þ 1/2, 2z ]
N1 [x 2 1/2, 2y þ 1/2, 2z ]
O33
O34
O44
O4WA [x þ 1, y, z ]
O4WB [x þ 1, y, z ]
O22
O23
O35 [x 2 1/2, 2y þ 1/2, 2z ]
O32 [x þ 1/2, 2y þ 1/2, 2z ]
O26
O64 [2x þ 3/2, 2y þ 1, z þ 1/2]
O33 [2x þ 1/2, 2y þ 1, z þ 1/2]
O65A [x 2 1, y, z]
O4WB [2x, y þ 1/2, 2z þ 1/2]
O65A [x 2 1/2, 2y þ 3/2, 2z ]
O2WB
O36 [2x þ 3/2, 2y þ 1, z 2 1/2]
O1W [x þ 1, y, z]
O21 [2x þ 1, y þ 1/2, 2z þ 1/2]
O34 [2x þ 3/2, 2y þ 1, z þ 1/2]
O66 [2x þ 1, y 2 1/2, 2z þ 1/2]
O25 [x 2 1/2, 2y þ 1/2, 2z ]
O52
N1–H2N1
O1W–H1W1
O1W–H1W1
O1W–H2W1
O3W–H1W3
O3W–H1W3
O3W–H2W3
O3W–H2W3
O65A [x 2 1, y, z]
O4WB [2x, y þ 1/2, 2z þ 1/2]
O2 [2x þ 1, y 2 1/2, 2z þ 1/2]
O1 [2x þ 1, y 2 1/2, 2z þ 1/2]
O2WB [2x þ 1, y 2 1/2, 2z þ 1/2]
O2WA [2x þ 1, y 2 1/2, 2z þ 1/2]
152(4)
126(4)
158(5)
Notes: A refers to the first occupied site of disordered O6n; B refers to the second occupied site of disordered O6n
.

318
K. Elasaad et al.
Table 4. The bond lengths in carboxylic (or carboxylate) and amino (or ammonium) groups in different pABA forms resulting from Ab
initio and PM6 minimisation.
˚
C7–O1 (A)
˚
C7–O2 (A)
˚
C4–N1 (A)
˚
C1–C7 (A)
pABA (crystal structure)
Anionic-pABA
1.2554 (31)
1.2753 (35)
1.3946 (33)
1.4725 (40)
Ab-initio
PM6
Ab-initio
PM6
Ab-initio
PM6
Ab-initio
PM6
1.280
1.240
1.240
1.210
1.280
1.230
1.230
1.200
1.280
1.250
1.390
1.390
1.280
1.230
1.370
1.370
1.410
1.440
1.380
1.390
1.510
1.500
1.510
1.500
1.530
1.550
1.460
1.460
1.540
1.560
1.490
1.500
Neutral-pABA
Zwitterionic-pABA
Protonated-pABA
agreement with the results of studies in solution (16). It is
noteworthy that the location of the aromatic ring is similar
in other a-CDs complexes with aromatic guests (12–14,
34–37). This is probably due to the fact that this position is
sterically favourable for the aromatic ring.
stabilised by intra-layers hydrogen bonds formed between
hydroxyl groups, bythehydrogen bondsformed betweenthe
guests and hosts from different layers and by those formed
between a-CDs molecules (see supplementary data S3).
Host–guest interactions play a crucial role in
determining the orientation of guest molecules in the
cavity of a-CD (32). In the case of the pABA-a-CD
complex, the guest is held in the host cavity mainly by Van
der Waals contacts (with a-CD) and hydrogen bonds
(mediated through water molecules).
3.2 UV-spectroscopy
Absorption spectra (between 200 and 350 nm) of pABA in
the presence of increasing concentrations of a-CD are
shown in Figure 5. In this wavelength range, pABA exhibits
two maxima (284 and 222 nm) at pH ¼ 3.5. The addition of
increasing amounts of a-CD to the aqueous pABA solution
resulted in a small bathochromic shift and a significant
increase in the pABA absorption. These results confirm that
pABA forms an inclusion complex with a-CD in solution.
Inclusion of the pABA molecule into the macrocyclic
cavity is accompanied by changes in its environment,
causing changes in the absorption and a bathochromic shift.
In order to determine the stoichiometry and stability
constant of the inclusion complex, the dependence of the
pABA absorbance with respect to the a-CD concentration
was analysed using the Benesi–Hildebrand Equation (43).
Distinct equations are expected for a 1:1 (Equation (1)) or
a 1:2 complex (Equation (2)) between pABA and a-CD.
Selected observed hydrogen bonds in the complex are
listed in Table 3. The bonds listed in the table are those for
which the distance between the acceptor and the hydrogen
atom is smaller than the radius of the acceptor atom plus
˚
2.0 A, and the angle between the donor atom, the hydrogen
and the acceptor atom is larger than 1108.
No direct hydrogen bond is observed between a-CD and
its included guest molecule. Indirect hydrogen bonds further
stabilisepABAinsidea-CDviawatermolecules.Additional
hydrogen bonds involve the amino and carboxylic groups of
pABA and the hydroxyl groups of adjacent a-CD molecules
(see supplementary data S3 and Table 3).
D1ꢀKð1:1ÞꢀCpABAꢀ½Ca2CD
1 þ Kð1:1ÞꢀCpABA
ꢁ
;
A ¼ A0 þ
A ¼ A0 þ
ð1Þ
ð2Þ
3.1.4 Crystal packing
a-CD molecules are arranged in the crystal cell nearly
parallel to its ac plane, forming a molecular layer (Figure 4).
The least-squares plane through the six O4n atoms forms an
angle of 5.78 with respect to the ac plane. This crystal
arrangement is different from the cage-type structure (37–
41) since both ends of the cavity are open to the space
between the layers. The a-CD molecules, which lie in the
next layer, are slipped so that the overlap of the annular
apertures is quite small. Therefore, in this arrangement,
a-CD molecules do not form a continuous channel such as
the one found in other channel type structures (33, 34, 42).
The guest molecules are situated nearly parallel to the bc
plane. The empty space between the a-CD molecules is
filled with four water molecules. Crystal packing is further
2
D1ꢀKð2:1ÞꢀCpABAꢀ½Ca2CD
1 þ Kð2:1ÞꢀCpABA
ꢁ
;
where A and A0 are the absorption of pABA in the presence
and absence of a-CD, respectively; D1 5 1CD.pABA 2 1
pABA is the difference in the molar absorptivities between
free and complexed pABA; CpABA and Ca-CD are the initial
concentrations of pABA and a-CD, respectively; Kc is the
stability constant of the complex.
These equations can be rewritten in more useful forms
(Equations (3) and (4)), which show a linear relationship
between ð1=DAÞ and ð1=ðCaꢂCDÞÞ in the case of a 1:1
complex.

Supramolecular Chemistry
319
Figure 4. Drawing of the packing of the complex, viewed along a-axis (a) and b-axis (b).
1
1
1
¼
ꢀ
2
1
1
1
DA D1 £ Kð2:1Þ £ CpABA
½Ca2CD
ꢁ
¼
ꢀ
DA D1 £ Kð1:1Þ £ CpABA ½Ca2CD
ꢁ
1
þ
:
ð4Þ
1
D1 £ CpABA
þ
;
ð3Þ
D1 £ CpABA
In the case of pABA, the stability constants of the
complex were determined by measuring the change in the
maximum absorption of pABA (at ,280 and ,220 nm),

320
K. Elasaad et al.
Figure 5. Absorption spectra of pABA: (a) 12.5 £ 1025 at pH ¼ 1.5, (b) 5 £ 1025 M at pH ¼ 3.5, (c) 5 £ 1025 M at pH ¼ 6.0, in the
absence and the presence of different concentrations of a-CD.
DA, in the presence of increasing concentrations of a-CD.
The dependency of DA on the a-CD concentration is
shown in Figure 6 (see also supplementary data S4). The
data could be fitted to Equation (3) corresponding to a 1:1
stoichiometry. From these data, complexation constant,
Kc, of pABA by a-CD is estimated to be 2319.4 ^ 241.0
and 596.0 ^ 80.5 M21 at pH ¼ 3.5 and 1.5, respectively,
at T ¼ 258C. Values of 1400 ^ 35 and 1000 ^ 148 M21

Supramolecular Chemistry
321
Figure 6. Dependence of DAmax of pABA solutions (5 £ 1025 M at pH ¼ 3.5) on the a-CD concentration. Benesi–Hildebrand plots
(linear relationship between 1/DA and 1/Ca-CD) assuming formation of a 1:1 complex between pABA and a-CD give access to stability
constants of the complex.
were obtained at pH ¼ 3.5 and 2.0 (T ¼ 208C), respect-
ively, by fluorescence method (44).
(1) partial breaking of the hydration shells of the solutes;
(2) exclusion of cavity-bound water molecules that are
considered as ‘enthalpy-rich’ (45);
Repeating the same procedure at different temperatures
(4, 10, 15, 20 and 258C) showed that the stability constant
increases with decreasing temperature (Table 5). The use of
the Van’t Hoff equation gives direct access to DH8 and DS8
of complex formation (Figure 7 and Table 6) (see also
supplementarydata S5). ThedatapresentedinTables5and6
indicate that the complex formation of pABA with a-CD is
exothermic. Thus, the complex of pABA with a-CD is
enthalpy–entropy stabilised. Enthalpy and entropy values
includethecontributionsfromthe followingmainprocesses:
(3) conformational changes (e.g. deviation from
symmetry and restriction of conformational flexibility);
(4) direct binding due to non-covalent interactions (van
der Waals interactions, H-bonding, hydrophobic effects
and electrostatic interactions) (45);
(5) hydration of complex.
The observed small DH8 value (Table 6) may be a
result of the important role of hydrophobic interactions

322
K. Elasaad et al.
Table 5. Stability constants K (M21) for pABA-a-CD complex at pH ¼ 3.5 and different temperatures.
48C
108C
158C
208C
258C
K(lmax ¼ 222 nm) M21
K(lmax ¼ 284 nm) M21
3155.4 ^ 298.3
2880.2 ^ 188.3
2972.7 ^ 317.3
2686.1 ^ 121.4
2854.9 ^ 317.5
2505.2 ^ 155.6
2698.3 ^ 314.6
2414.7 ^ 232.0
2503.2 ^ 272.2
2319.4 ^ 241.0
and dehydration of the solutes occurring upon penetration
of the pABA molecule into the hydrophobic cavity of a-
CD. This happens when the guest molecule is deeply
inserted into the CD cavity, as can be seen from the crystal
structure of the complex.
Phase-solubility diagrams for the complex formation
between pABA and a-CD are presented (see also
supplementary data S6). Our data indicate that the
solubility of pABA in water depends on the pH of the
solution: it is higher for the charged (protonated
(pH ¼ 1.5) and unprotonated (pH ¼ 6)) forms:
S0 ¼ 0.145, 0.030 and 0.170 M21 at pH ¼ 1.5, 3.5 and
6.0, respectively. The phase-solubility diagrams show that
the aqueous solubility of pABA increases linearly as a
function of the a-CD concentration.
Positive entropy changes (Table 6) underline the
extensive dehydration accompanied by the release of
water molecules from the solvation shells of reagents into
the bulk aqueous solution. Furthermore, hydrophobic
interactions of the aromatic ring of pABA with the apolar
cavity of CD can play a significant role in the
complexation process, resulting in a positive DS8 (45).
The linear host–guest correlation with a slope of ,1
confirms the formation of a 1:1 complex in solution with
respect to a-CD concentration.
The increase in solubility of pABA resulting from
inclusion in a-CD is more important at pH 3.5 (compare
slopes). This study shows that the complex between pABA
and a-CD is more stable at pH ¼ 3.5, with an apparent
stability constant, K(1:1), obtained from the slope of the
3.3 Phase-solubility diagrams
An apparent stability constant (Kc) can be calculated from
the phase-solubility diagrams using Equation (5), accord-
ing to the hypothesis of a 1:1 stoichiometric of the
complex (26).
solubility
diagrams,
of
736.9 ^ 132.4
and
1710.7 ^ 219.6 M21 at 25 and 48C, respectively.
slope
K
¼
:
ð5Þ
ð1:1Þ
S0ð1 2 slopeÞ
3.4 Semi-empirical simulation of complexes
The slope was obtained from the initial straight line
portion of the plot of the pABA solubility against the a-
CD concentration, and S0 is the equilibrium solubility of
pABA in water.
pABA can exist in three forms (neutral, anion and cation)
according to the pH of the environment (Figure 1). Ab
initio (B3LYP-6-311G (5d,7f)) calculations have been
applied to optimise the geometry of the different
protonation forms of pABA alone. The results were used
as a complement to crystal structures retrieved from the
CSD to analyse the crystal structure of this guest in a-CD
(see Section 3.1.2). Similar results have been obtained
using the PM6 semi-empirical method. Based on those
calculations, the most stable form is the protonated state
(EB3LYP ¼ 2476.394
A.U.
(212963.16 eV);
EPM6 ¼ 21721.625 eV) followed by the neutral and
zwitterionic states (EB3LYP ¼ 2476.170 (212957.06 eV)
and 2476.064 A.U. (12954.18 eV); EPM6 ¼ 21715.149
and 21712.645 eV, respectively). The anionic form is the
less stable (EB3LYP ¼ 2475.604 A.U. (12941.66 eV);
EPM6 ¼ 21703.001 eV). Interestingly, it is this last form
Table 6. DH8 and DS8 of complex formation, calculated from
UV-spectroscopy data at pH ¼ 3.5.
Figure 7. Van’t Hoff analysis (dependence of Ln(K) of pABA-
a-CD complex formation on the temperature (1/T) at pH ¼ 3.5
(K values determined from data at lmax ¼ 284) give access to
thermodynamic parameters DH8 and DS8 for the 1:1 complex
formation of pABA with a-CD.
lmax ¼ 222 nm
lmax ¼ 284 nm
DH8 (kJ mol21
DS8 (J K21 mol21
)
–8.69 ^ 0.88
35.9 ^ 3.1
–6.25 ^ 0.31
43.5 ^ 1.1
)

Supramolecular Chemistry
323
that is observed in the crystal structure of the inclusion
complex with a-CD, illustrating the fact that stabilisation
effects resulting from complexation by the CD do affect
differently the distinct protonation forms of pABA.
Therefore, different structures of a-CD-pABA
inclusion complexes have been simulated. Two possible
orientations of pABA inside the a-CD cavity have been
retained, one (orientation A) with the carboxylic group of
pABA located inside the a-CD cavity and the other
(orientation B) corresponding to the amino group inside
the cavity. For both orientations, the different protonation
states of pABA (protonated, anionic, neutral or zwitter-
ionic) were considered. The effect of solvation on the
overall stability of the complexes was also approached.
Energy minimisation of the different complexes was
performed (PM6 level).
of the CD, with the amino group protruding from the wide
side of the cavity and the (unprotonated) carboxylate at the
narrow side. The crystal structure corresponds to the
anionic form of pABA. The pABA guest is held in the host
cavity mainly by Van der Waals contacts and hydrogen
bonds. Spectroscopic studies of the interactions of pABA
with a-CD demonstrate the insertion of pABA into the CD
cavity in solution, with the formation of a 1:1 inclusion
complex with stability constant 2319.4 ^ 241.0 M21 at
258C and pH ¼ 3.5.The phase-solubility studies confirm
the formation of a 1:1 pABA-a-CD inclusion complex.
The solubility of pABA in water is markedly enhanced by
complexation with a-CD. The calculations carried out
with the PM6 method show that inclusion complexes of
different pABA protonation states with a-CD lead to
stable structures and this confirms experimental obser-
vations. During this theoretical study, orientation A
(carboxylic group inside) was more favourable than
orientation B (amino group inside), in agreement with our
crystal structure.
The optimised geometries were reasonable when water
molecules were included in the calculation. In the absence
of explicit water molecules, the in vacuum optimised
complexes with neutral pABA (orientation A) or
zwitterionic pABA (orientation B) converged to distorted
geometries. This is in agreement with a similar study
performed on inclusion complexes of pABA and b-CD
(46). All the other systems converged to chemically
reasonable complexes, suggesting that the PM6 method is
well adapted to study the pABA-CD systems.
Supplementary data
Tables of atomic coordinates, bond lengths and bond
angles have been deposited with the Cambridge Crystal-
lographic Data Center, CCDC Nos. 845857 for the a-CD–
pABA complex. These data may be obtained free of
charge, on request, from The Director, Cambridge
Crystallographic Data Center, 12 Union Road, Cambridge,
CB2lEZ, UK (fax: þ44-1223-336033; e-mail: deposit@
ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
In particular, the PM6 method reproduced the crystal
structure rather well. Indeed, the enthalpy of formation of
pABA included in a-CD was the most negative with the
anionic form of pABA, in orientation
A
(Hf(PM6) ¼ 24599.39 kcal) compared to the anionic form
of the guest in orientation B (Hf(PM6) ¼ 24583.40 kcal).
This result is in excellent agreement with the crystal
structure reported in this study. The PM6 method suggests
that complexation of the anionic form is favoured
compared to the other protonation states of pABA
(Hf(PM6) ¼ 24599.39, 24519.80, 24481.46 and
24349.56 kcal) for complexes with anionic pABA2,
neutral pABA, zwitterionic pABAþ/ 2 and cationic
pABAþ forms, in orientation A. Similar calculations
performed on complexes including pABA and b-CD (46)
led to different results. In that work, orientation B was
more stable but, in agreement with our results, the complex
with anionic pABA was favoured.
Additional data are available in the online supplemen-
tary information associated with this paper.
Acknowledgements
This study was supported by the Ministry of Higher Education in
Syria in collaboration with chemistry biological structural
laboratory (CBS), at the University of Namur (FUNDP) –
Belgium.
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