Rapid analysis of drug binding to β-cyclodextrin: Part II substituents effect on physicochemical and co-conformational stability of drug/cyclodextrin complex

Article abstract of DOI:10.1039/c3ra47010e

In the pharmaceutical industry, the most common application of cyclodextrins (CyDs) is to enhance the solubility, stability, safety and bioavailability of drug molecules. Although, there have been many reports on the complexation of drugs with cyclodextrins, to date, CyDs interaction with drugs is not well understood. The purpose of this work is to show the successful applications of previous work employing a novel, versatile, compact, low-volume, routine apparatus, which consists of a pump, flask and a rotating cell holder for the simultaneous measurements of absorbance and circular dichroism (CD) that allows for the concurrent use of four different pathlengths for binding studies. To show the effect of substituents on drug/CyD binding, benzoic acid as an important precursor for the synthesis of many other organic substances and benzoic acid derivatives were used in this study. Also, an effective novel method for binding titration was employed. The pK_a, binding constants, stoichiometry and structural co-conformations of benzoic acid and derivatives/β-CyD complexes were elucidated and determined with accuracy. Substituents type and location show a significant effect on the extent of binding and physicochemical properties of the binding complex. The system proved efficiency, ability to be used routinely and the analysis time was reduced significantly to less than one fourth of the total analysis time used in conventional methods, with possible automation for high-throughput analysis.

Full text of DOI:10.1039/c3ra47010e

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Rapid analysis of drug binding to b-cyclodextrin:
part II substituents effect on physicochemical and
co-conformational stability of drug/cyclodextrin
complex
Cite this: RSC Adv., 2014, 4, 6624
*
Ali Aboel Dahab
In the pharmaceutical industry, the most common application of cyclodextrins (CyDs) is to enhance the
solubility, stability, safety and bioavailability of drug molecules. Although, there have been many reports
on the complexation of drugs with cyclodextrins, to date, CyDs interaction with drugs is not well
understood. The purpose of this work is to show the successful applications of previous work employing
a novel, versatile, compact, low-volume, routine apparatus, which consists of a pump, flask and a
rotating cell holder for the simultaneous measurements of absorbance and circular dichroism (CD) that
allows for the concurrent use of four different pathlengths for binding studies. To show the effect of
substituents on drug/CyD binding, benzoic acid as an important precursor for the synthesis of many
other organic substances and benzoic acid derivatives were used in this study. Also, an effective novel
method for binding titration was employed. The pKa, binding constants, stoichiometry and structural co-
conformations of benzoic acid and derivatives/b-CyD complexes were elucidated and determined with
accuracy. Substituents type and location show a significant effect on the extent of binding and
physicochemical properties of the binding complex. The system proved efficiency, ability to be used
routinely and the analysis time was reduced significantly to less than one fourth of the total analysis time
used in conventional methods, with possible automation for high-throughput analysis.
Received 25th November 2013
Accepted 17th December 2013
DOI: 10.1039/c3ra47010e
www.rsc.org/advances
stability.5,6 For example, cyclodextrins can be used to reduce
gastrointestinal drug irritation, convert liquid drugs into
Introduction
microcrystalline or amorphous powder, and prevent drug–drug
and drug–excipient interactions.7 Moreover, the encapsulation
of drugs by cyclodextrins can be useful since the host molecules
may act as concentration-regulating agents, decreasing the
adverse side effects at the organ or cellular level and improving
the optimal dose of the drug and its frequency of administra-
tion.8 Their doughnut-shaped structure, with hydrophilic
external faces and hydrophobic inner surface, makes them the
most important simple organic compounds capable of forming
noncovalently bonded inclusion complexes with a wide variety
of drug molecules in aqueous solution. The resulting host–
guest systems become more soluble and stable, the stability
being a result of the concurrence of different contributions,
such as van der Waals interactions, hydrophobic effect, solvent
reorganization, and hydrogen bonding.9 This special molecular
arrangement of CyDs also accounts for the variety of benecial
effects cyclodextrins have on proteins, which is widely used in
pharmacological applications.10 Although inclusion complexes
of drug/CyD can assume higher order complexes (e.g. 2 : 1 drug/
CyD), the formation of an inclusion complex oen has a stoi-
chiometry of 1 : 1 interaction, which is illustrated in Fig. 1.
Nevertheless, the extent of drug binding to cyclodextrin
Molecular recognition is a term used to describe noncovalent
interactions between molecules. The eld encompasses both
theory and experiment of host–guest chemistry and supramo-
lecular chemistry.1 Whether it is sequestration, sensing, crys-
tallization, catalysis, solubility, self-assembly, distribution
ratios in immiscible solvents, or drug–receptor interactions, the
nature and strength of interactions between molecules creates
many of the phenomena of contemporary interest in chemistry,
biology, and materials science. Molecular encapsulation of
drugs by cyclodextrins (CyDs) is probably the most widely used
method to overcome problems associated with drugs, such as
poor solubility and unwanted side effects.2,3 The potential guest
list for molecular encapsulation in cyclodextrins is quite varied
and includes such compounds as straight or branched chain
aliphatics, aldehydes, ketones, alcohols, organic acids, fatty
acids, aromatics, gases, and polar compounds such as halo-
gens, oxyacids and amines.4 Cyclodextrins also are used as
complexing agents to increase drug bioavailability and
Pharmaceutical Sciences Research Division, King's College London, 150 Stamford
Street, London SE1 9NH, UK. E-mail: ali.aboel_dahab@kcl.ac.uk; aliadahab@
yahoo.co.UK; Fax: +44 (0)2078484462; Tel: +44 (0)2078483944; +44 (0)7950698740
6624 | RSC Adv., 2014, 4, 6624–6637
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three major absorption bands with the characteristics: 1Lb (3275
1
1
¼ 100–1000), La (3210 ¼ 10 000), E (3195 ¼ 50 000).24 Conven-
tionally, this is likely to be undertaken with three separate scans
in three different cells; changes in concentration may have
implications for solution studies particularly those involving
concentration-dependent equilibria such as binding investiga-
tions.25 Therefore, in binding studies, the use of conventional
methods is time consuming and prone to errors due to manual
withdrawal and addition.
Fig. 1 Binding of a drug with a cyclodextrin to form a 1 : 1 inclusion
complex.
This article describes applications of a novel four-ow cell
system that has been recently introduced by us26 for rapid
(binding constant, K) presents a key issue in the whole binding binding analysis using the simultaneous measurement of
process, it is particularly important in therapeutics, where the absorption and circular dichroism.27 The binding of b-CyD as a
pharmacological effect of a drug is directly related to its nature function of pH to benzoic acid and benzoic acid derivatives as
(meaning in free or complexed form).11 The extent of binding important precursors for the synthesis of many other organic
varies between drugs and so as the stability of the drug/CyD substances (e.g. antimicrobial drugs) were chosen as an
complex, depending on various factors such as physicochemical example to study the effect of substituents on binding interac-
and conformational properties of the drugs used. The physi- tions and on the stability of drug/b-CyD complex. The new
cochemical properties of drugs, including their complexation system was used and the analysis time was reduced to less than
ability, may be greatly affected by the properties of the substit- one fourth of that for conventional methods. Using an effective
uents such as their type, number, and position on the parent approach for binding titration, the extent of binding, stoichi-
drug molecule. In addition, the pH of the medium i.e. the ometry and the change of physicochemical properties are esti-
ionisation state of the guest compound has a signicant effect mated with accuracy. The mathematical analysis of the
on the thermodynamic parameters of the (drug/CyD) complex- spectroscopic variations in binding titrations and the equations
ation reaction. To date, the inuence of pH has not been used to estimate the binding constant are explained. Also, the
investigated in detail yet, only a few articles are devoted to this co-conformation of b-CyD and these molecules are assigned
problem, using indirect methods such as potentiometry12 and and elucidated according to spectroscopic changes upon
phase-solubility studies.13 However, experimentally determined complexation. The results show the applicability and efficiency
values of some spectroscopic variables, e.g., uorescence of the new system in determining the impact of substituents on
quantum yield,14,15 absorption coefficient,16,17 chemical shi18,19 physicochemical and co-conformational stability of drug/
and induced circular dichroism (ICD)16 can be used to monitor cyclodextrin complex.
such reactions.
Optical activity measurements, circular dichroism (CD) in
particular, offer a convenient way of monitoring absolute
Materials and methods
Materials
stereochemistry and shape in solution at relatively low
concentration, mainly with respect to solution variables such as
temperature, concentration, pH, and additives. However, when
dealing with achiral drugs, which is the case in this study,
induced circular dichroism (ICD) can be used for monitoring
drug–ligand interactions.20 On the other hand, measurements
of absorption alongside CD measurements are important and
oen necessary. The UV/Vis absorption spectrum of an organic
compound is normally composed of only a few broad spectral
NaOH and HCl were purchased from BDH, UK, benzoic acid,
and benzoic acid derivatives from Sigma-Aldrich Co. LLC., UK.
Water was distilled in-house at the pharmacy department,
King's College London.
Methods
Spectroscopic measurements and data analysis. CD and UV
features, where pathlength/concentration/analysis and wave- spectra were measured simultaneously using a Jasco J720
length combinations are chosen to optimise measurements. spectrometer from Jasco Corporation, Tokyo, Japan. Spectral
Circular dichroism spectroscopy has oen been used to study data were extracted as described by Aboel Dahab et al.,27 in
molecular binding,21 and CD spectra are more generally ana- particular, the absorbance spectra were obtained from the
lysed for molecular structure information, conformation and photomultiplier HV measurements. Weighing was done with a
conguration.22,23 It is desirable to have a complete CD spec- microbalance (Mettler Toledo MT5) from Mettler-Toledo Ltd.,
trum covering the whole spectral range, however, the selection UK and pH measurements were made with Thermo-Electron
rules for ordinary absorption and CD are different, e.g. prom- Russell pH combination electrode (KCMAW11) connected to a
inent features in a CD spectrum such as n–p* of CO2H may not Mettler-Toledo meter (Corning pH 105). As the temperature
be apparent in the ordinary absorption. Therefore, to measure a factor was not an issue in this experiment, all experiments were
complete CD spectrum while keeping the ordinary absorption performed at 22 ꢀC. Data was analysed using the relevant
in the optimum range (A ¼ 0.5 to 1.5), measurements have to be equation with both visual tting in the Math So, Math Cad
made in stages with different pathlengths and/or concentra- computer program and Levenberg–Marquardt non-linear
tions. Thus, typically the aromatic phenyl chromophore has equation tting in the Microcal Origin computer program.
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The binding of drugs to cyclodextrin as a function of pH
potentially requires the consideration of four equilibria:
Titrations. The production of a strain-free, variable path-
length cell with precise control of pathlength may be either
imprecise or requiring large solution volumes. An alternative
approach is to employ four separate ow cells connected in
series in a ow system involving a pump and a titration ask
(Fig. 2). Full details of the new system have been explained
previously by the author.26
The advantages of this system over the conventional
methods are that in addition to faster analysis time, it helps
avoiding solution loss and concentration errors during the
manual transfer of measurement solution from one cell to
another particularly small pathlength cells e.g. 0.05 or 0.01 cm
cells. Also, it provides efficient method for mixing of solutions
with variable speeds that can be controlled by the operating
pump. This is predominantly important when a lot of stirring is
required as it is the case in binding and pH titration especially
when the solutions involved are sensitive to mechanical aggre-
gation and precipitation e.g. protein28 or when dealing with
extremely volatile compounds29 which can lead to concentration
variations.
The pH titrations were performed to determine the pKa
values for both the free and the bound drugs to conclude the
change of physicochemical properties upon binding with b-
cyclodextrin.
For pH values < pH 3.0, 10.0 ml aliquots of 3.0 M HCl need to
be added; between pH 3.0 and pH 10.0, 10.0 ml aliquots of 0.1 M
NaOH/HCl suffice; above pH 10.0, 3.0 M NaOH is required.
Appropriate volumes of drug solutions of appropriate concen-
trations were added to the titration ask and titrated with the
addition of 10 ml aliquots of NaOH/HCl leaving the drug
concentration effectively constant. The total volume was ꢁ2.01–
2.1 ml which was sufficient to ensure good mixing by the pump.
The solution was mixed thoroughly using the pump and spectra
were measured in the normal way at each pH interval with the
cell carousel being rotated to change the cell (pathlength).
Binding titrations. The main principle of drug binding
studies is to keep the concentration of the monitored species
constant. Using the new system, while the pH is monitored at all
times, the titration can start by measurement of a free drug
solution (solution A) and then increase the host (b-CyD)
concentration gradually using a solution containing the same
drug concentration with excess of host concentration e.g. 1 : 15
respectively (solution B), then, pushing the equilibrium to
completion with solution A, initially in the titration apparatus
Fig. 2 Schematic diagram showing the practical principles of the
setup for the new system.
Fig. 3 Schematic diagram showing the most effective ways in binding titration.
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titrated with solution B. This is achieved by withdrawing a
certain amount of solution A, then adding the same amount
from solution B and measuring aer each addition (Fig. 3).
Alternatively, the titration can start from the fully bound state
solution B and progressively reducing the host concentration,
however, in this case it is not possible to achieve a completely
zero host concentration.
As the pKas for these compounds are between 2.0 and 7.0, pH
2.0 was chosen as the lower limit and pH 7.0 as the higher limit to
ensure achieving the two state ionisation required for this study.
Binding constant (K). Mathematical analysis to deduce the
equation for the binding constant is based on the binding of a
single guest (ligand) to a single host site, which can adequately
be described by the standard formalism:
Results and discussion
Benzoic acid (BA)
To show the application of the new system in binding studies,
particularly the effect of substituents on the binding process, BA
was chosen as the parent molecule. The pH titration of BA was
performed using the new setup and the pH dependent UV
absorption spectra of BA in H2O are presented in Fig. 4a. Critical
inspection of the UV spectrum shows evidence of the three
1
1
absorption systems, Lb, CT, and La for both the ionised and
unionised forms of BA. For the unionised form, the bands are
positioned at ꢁ283, 230 and ꢁ212 nm respectively. For the
ionised form, the bands are positioned at ꢁ277, 224 and ꢁ209
nm respectively. The results are in agreement with reported
values for COOH substituted benzene.30 Substitution of benzene
with polar groups containing unshared electrons shis the
absorption bands to a longer wavelength and also intensies
them due to the interaction of the unshared electrons with the
benzene nucleus (p–p conjugation).31 The vibronic components
associated with the 1Lb band between ꢁ290 nm and 260 nm are
not cleanly resolved. However, the unionised form shows three
peaks; a smaller peak representing the 0–0 origin at ꢁ283 nm
(35 336 cmꢂ1), one at ꢁ280 nm (35 714 cmꢂ1, 378 cmꢂ1
component) and a third at ꢁ273 nm (36 630 cmꢂ1, 916 cmꢂ1
component). At pH 7.0 (ionised form), the peaks are weaker,
slightly sharper and blue shied to ꢁ277 nm and 269.8 nm. The
CT bands of the unionised and ionised forms of benzoic acid
host (H) + guest (G) # [host:guest complex]
At equilibrium the complex concentration (HG) and free host
and guest concentrations (Hf and Gf respectively) are related by:
Hf + Gf # HG, HT ¼ Hf + HG and GT ¼ Gf + HG,
where GT and HT are the total guest concentration and the total
host concentration respectively.
However, based upon Beer's law:
Aobs ¼ AH + AG + AHG
(1)
(2)
f
f
1
have 3230 ꢁ 11 500 and 3224 ꢁ 8950 respectively. The La bands
¯
Aobs ¼ 3HHf + 3GGf + 3HGHG
have 3212 ꢁ 6100 and 3209 ꢁ 3000 for the unionised and the
ionised forms respectively. Good isosbestic points were
observed and plotting A274 against pH gave pKa ¼ 4.3 (Fig. 5a),
which is in close agreement with reported values.32
where Aobs is the total observed absorbance, 3H and 3G are the
extinction coefficients of the host and the guest respectively,
then:
b-CyD titration of BA at pH 2.0 was performed as
described earlier starting from a 0.001 M BA only solution
and adding appropriate volumes of the 1 : 15 (molar ratio)
¯
Aobs ¼ 3HHT + 3GGT + (3HG ꢂ 3H ꢂ 3G)HG
(3)
Combining standard solution equilibria equations for BA/b-CyD solution. BA is an achiral molecule, which does
solving for the complex concentration, HG as a function of HT, not have a CD spectrum. However, in the chiral environment
and substitution of Beer's law equations gives:26
of b-CyD, CD is induced. UV absorbance and ICD
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8
<
9
=
2
4
3
5
2
2
½ðKassHTÞ þ ðKassGTÞ þ 1ꢃ ꢂ ½ðKassHTÞ þ ðKassGTÞ þ 1ꢃ ꢂ 4Kass ðHTGTÞ
2Kass
Aobs ¼ ½3HHT þ 3GGTꢃ þ ð3HG ꢂ 3H ꢂ 3GÞ
(4)
:
;
where HG is now given by equation with only the negative of the spectra of BA binding to b-CyD at pH 2.0 are presented in
root giving realistic values.
Fig. 4d and e.
¯
In eqn (4), Aobs is the dependent variable depending upon
There is an apparent increase in the absorption intensity of
three spectroscopic constants (the extinction coefficients of the the 1Lb band with a ꢁ1 nm wavelength red shi upon addition
original species G, H and HG), the association constant (Kass
)
of b-CyD indicating hydrogen bonding between BA–COOH and
and the original (total) guest and host concentrations. To obtain b-CyD. The CT band shows a decrease in absorption intensity
a value for the binding constant eqn (4) was entered in the upon addition of b-CyD with no wavelength shi. The vibronic
Origin program. A plot of Aobs against GT (or HT) should give a components of the 1Lb band became more dened with
¯
rectangular hyperbola with only a particular value of Kass giving increasing b-CyD concentration, which indicates that the ring
a good t to the experimental data.
system of BA is in the nonpolar environment of the b-CyD cavity.
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Fig. 4 UV and CD spectra of BA (0.001 M) in the free and the bound form at pH 2.0 and pH 7.0 in 1.0 and 0.1 cm (a) pH dependent UV spectra of
BA in the free form, (b and c) pH titration spectra of BA/b-CyD complex, UV and ICD respectively (d and e) b-CyD effect on UV and ICD spectra
respectively at pH 2.0 (f and g) b-CyD effect on UV and ICD spectra respectively at pH 7.0.
The ICD band corresponding to the 1Lb is bisignate with a band shows a decrease in intensity and there is no wavelength
crossing point from negative to positive at ꢁ270 nm which lies shi with increasing b-CyD concentration due to the loss of
approximately in the centre of the 1Lb absorption band. The hydrogen bonding indicating that BA-COOꢂ does not partici-
intensity increases with increasing b-CyD concentration to a pate as a hydrogen bond acceptor most probably due to solva-
point where further addition has no effect on the ICD spectra. tion effect at high pH conrming that hydrogen bonding of
ICD can be either positive or negative depending on the co- COOH group is the main force contributing to the stability of
conformation of the binding complex (alignment of the tran- the binding complex. Also, the vibrational structure is not as
sition moments of BA inside or outside the cavity of b-CyD).33 well dened as it is at pH 2.0 suggesting that the ring system is
The bisignate nature of the ICD signal is explained by the not immersed inside the nonpolar environment of the b-CyD
presence of two conformers resulting in the alignment of two cavity, or the molecule is in and out of the cavity to minimise its
transition moments with the axis of b-CyD. The major positive net dipole moments by taking a more stable co-conformation.
ICD signal near 230 nm corresponds to the phenyl chromo- The absorption CT band shows an increased intensity with
phore transition aligned slightly parallel to the axis, which is increasing b-CyD concentration this implies that there is an
most probably due to restricted co-conformation enforced by interaction most probably hydrophobic interaction between the
hydrogen bonding with the secondary hydroxyl rim. The other ring system and b-CyD as a result of being in close proximity to
transition moment between the ring system and the COOH the b-CyD cavity. On the other hand, the ICD associated with the
group is aligned orthogonally to the axis giving rise to negative 1Lb and CT bands is very weak with the disappearance of
ICD, and thus at pH 2.0 (unionised form) BA is adopting a bisignate characteristic of the 1Lb band suggesting that the loss
slightly tilted lateral co-conformation (Fig. 5f) which gives the of hydrogen bonding gave the molecule more freedom and
bisignate ICD (Fig. 4e).34 These results are in close agreement resulted in the presence of only one conformer, which is not
with structural assignments produced by NMR and crystallog- immersed deeply in the b-CyD cavity adopting unstable tilted
raphy.35,36 The binding constant (K) at pH 2.0 was determined by frontal co-conformation as shown in Fig. 5f. The binding curve
plotting the value of A274 and DA274 against b-CyD concentration produced by plotting the values of DA254 against b-CyD
(Fig. 5c) and the analysis of the binding curve produced a mean concentration (Fig. 5d) produced a very small value of K ¼ 0.53
K ¼ 5.63 ꢄ 102 Mꢂ1
.
ꢄ 102 Mꢂ1 conrming that the ionised form of BA does not form
At pH 7.0, the UV and CD spectra of the BA/b-CyD titration a stable complex with b-CyD. Analysis of the binding curve
1
are presented in Fig. 4f and g respectively. The absorption Lb (Fig. 5e) show a stoichiometry of 1 : 1 for BA/b-CyD binding
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Fig. 5 Titration curves of BA in H2O, in 1.0 and 0.1 cm cell (a) pH titration curve of free BA (b) pH titration curve of BA/b-CyD complex (c) binding
curves at pH 2.0 (d) binding curves at pH 7.0 (e) analysis of binding curve showing 1 : 1 stoichiometry of BA/b-CyD complex (f) possible co-
conformation of BA/b-CyD complex at pH 2.0 and pH 7.0.
1
complex. The pH titration of the bound form of BA at 1 : 15 and 229 nm at pH 7.0, however, the La band is positioned at
molar ratio (BA/b-CyD) (Fig. 5b and c) conrms the results ꢁ217 nm at both pHs. A plot of A283 against pH (Fig. 7a) gave a
obtained previously. As expected, the ICD collapses going from value of pKa ¼ 3.8. This value is considerably smaller than the
pH 2.0 to pH 7.0, however, plotting the values of A274 and DA274
,
pKa values of BA (pKa ¼ 4.3), which is due to the effect of Cl at
against pH (Fig. 5b) produces a mean pKa ¼ 5.0, which is the meta position on the CO2ꢂ group making it less susceptible
signicantly higher than the pKa of the free form of BA, this to the solvent as a result of charge dispersal and a change in
increase in pKa value, or decrease in BA acidity suggests that the electron density distribution in the CO2H group resulting in
CO2H resonance is disrupted by hydrogen bonding with b-CyD; easier loss of H upon titration. In general, substitution of
also the co-conformation or steric effect on entropy makes benzene by auxochromes (nonchromophoric groups, usually
CO2H less exposed to the solvent molecules resulting in the containing unshared electrons such as –Cl, –NR, –OH), has
decreased acidity.
varying effects on the absorption spectra, with the position of
the substitution is of great importance to the developed spectra.
At pH 2.0, the binding titration of 0.001 M m-Cl-BA with a
solution of m-Cl-BA/b-CyD (1 : 15) was performed in the same
m-Chlorobenzoic acid (m-Cl-BA)
The pH-dependent UV-absorption spectra of free m-Cl-BA with way using 1.0 and 0.1 cm cells and the resulting UV and ICD
two different pathlengths (0.1, 1.0 cm) are shown in Fig. 6a. The spectra are shown in Fig. 6d and e. The 1Lb absorption is slightly
1Lb, CT, and 1La bands show a red shi compared to BA with the decreased and red shied by ꢁ2 nm with increasing b-CyD
biggest shi of ꢁ8 and ꢁ7 nm in the 1Lb band at pH 2.0 and pH concentration. The vibrational structure becomes more dened
7.0 respectively. The CT band is positioned at 232 nm at pH 2.0, with increasing b-CyD concentration, which can be seen in the
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Fig. 6 UV and CD spectra of m-Cl-BA (0.001 M) in the free and the bound form at pH 2.0 and pH 7.0 in 1.0 and 0.1 cm (a) pH dependent UV
spectra of m-Cl-BA in the free form, (b and c) pH titration spectra of m-Cl-BA/b-CyD complex, UV and ICD respectively (d and e) b-CyD effect on
UV and ICD spectra respectively at pH 2.0 (f and g) b-CyD effect on UV and ICD spectra respectively at pH 7.0.
vibronic components at 292 and 285 nm as they became sharper and shows three peaks at ꢁ285, 277 and ꢁ268 nm, indicating
upon increasing b-CyD concentration than it is the case with BA. that the ring system is inside the cavity and being stabilised
This indicates that the ring system is immersed deeper inside mainly by Cl and hydrophobic interactions. Unlike the ionised
the nonpolar cavity of b-CyD. The ICD shows optical activity very form of BA, the ionised form of m-Cl-BA exhibits a bisignate ICD
similar to benzoic acid, it is bisignate with a crossing point from signal with a crossover point at ꢁ273 nm and two vibronic
negative to positive at 268 nm. However, the ICD of the 1Lb band components at ꢁ285 and ꢁ277 nm, which indicate the effect of
is signicantly more intense than that of BA, indicating the m-Cl on the conformational stability of the binding complex,
effect of Cl on the charge distribution and the alignment of the forcing the molecule to adopt two conformers at different
transition moments within the b-CyD cavity. On the other hand, energy states. The binding curves of A285 and DA285 against b-
the addition of b-CyD does not cause any wavelength shi of the CyD concentration produced a value of K ¼ 1.29 ꢄ 102 (Fig. 7d).
1
CT or the La band. Below 210 nm, the UV and CD spectra are The relatively small value of K indicates that the main contrib-
1
dominated by the Ba transition. However, the spectra are dis- utor to binding stability is H-bonding and therefore, the ionised
torted due to the high absorbance value, thus measurements of form of m-Cl-BA complex is less stable. The effect of pH change
this transition under these conditions are not reliable. The on the UV absorption and ICD spectra of the bound form of m-
binding curves of m-Cl-BA/b-CyD interaction (Fig. 7c) are Cl-BA is presented in Fig. 6b and c. Good isosbestic points are
produced by plotting the values of A233 and DA233 against b-CyD observed indicating a two states system (unionised/ionised).
concentration. The mean value obtained was K ¼ 4.03 ꢄ 103 The ICD intensity decreases going from pH 2.0 to pH 7.0 with
Mꢂ1 which is signicantly higher than that of BA (ꢁ10 fold isosbestic points at ꢁ258 and ꢁ214 nm. The ICD at these
increase) as result of m-Cl-substitution.
wavelengths is equal for both ionised and unionised form
At pH 7.0, the effect of b-CyD titration on the UV and ICD suggesting the same alignment of these transitions with b-CyD
spectra of the ionised form of m-Cl-BA are shown in Fig. 7f and g at both the ionised and non-ionised forms.
respectively. There is no signicant wavelength shi at this pH
Plotting the values of absorbance and ICD at A283, and DA283
compared to the free form of m-Cl-BA and the spectra are less against pH (Fig. 7b) gives a mean value of pKa ¼ 4.92, which is
intense, however, contrary to the non-ionised form, the inten- signicantly higher than that of the free acid (pKa ¼ 3.8). This
sity of the absorption bands increases with increasing b-CyD decrease in acidity upon inclusion in b-CyD signies the effect
1
concentration. Also, the Lb vibronic structure is well dened of b-CyD interaction with COOH and Clꢂ on the charge
6630 | RSC Adv., 2014, 4, 6624–6637
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Fig. 7 Titration curves of m-chlorobenzoic acid in H2O, in 1.0 and 0.1 cm cell (a) pH titration curve of free m-chlorobenzoic acid (b) pH titration
curve of m-chlorobenzoic acid/b-CyD complex (c) binding curves at pH 2.0 (d) binding curves at pH 7.0 (e) analysis of binding curve showing 1 : 1
stoichiometry of m-Cl-BA/b-CyD complex (f) possible co-conformation of m-Cl-BA/b-CyD complex at pH 2.0 and pH 7.0.
distribution around the H of the carboxyl group making it less the addition of b-CyD accompanied by ꢁ1 nm red shi of
susceptible to the solvent resulting in a higher pKa value. both the 1Lb and CT bands. The red shi and change in
Analysis of the binding curve (Fig. 7e) conrms a 1 : 1 stoichi- intensity of the 1Lb band suggests hydrogen bonding of the
ometry of the m-Cl-BA/b-CyD complex. The results conrm that carboxyl group; however the red shi of the CT band indicates
the Cl substitution at the meta position has made a signicant that a lower energy transition has occurred as a result of binding
contribution to binding stability at both pHs.
to b-CyD. There is no signicant change in either intensity or
position of the La band located at 214 nm. The ICD of the CT
1
transition matches the absorption wavelength and its intensity
increases with increasing b-CyD concentration. However, the
p-Chlorobenzoic acid (p-Cl-BA)
The effect of substitution position can be emphasised by using ICD of the p-Cl-BA 1Lb band at ꢁ283 nm is more complex than
p-Cl-BA to show the effect of Cl in the para position on binding its counterpart in BA. It is again bisignate with a crossover point
to b-CyD. The pH dependent absorption spectra of the free form at ꢁ280.5 nm, nevertheless, the 1Lb ICD is considerably weaker
of p-Cl-BA (results not shown) shows in comparison to BA, no than the BA counterpart due to the different angle of the tran-
wavelength shi in the 1Lb (282 nm), however, the 1La (214 nm) sition moment alignment inside the b-CyD cavity. The CT
band is red shied by ꢁ4 nm at both pH 2.0 and pH 7.0. The CT absorption and ICD bands increase in intensity with increasing
band shows a signicant red shi of ꢁ12 nm at both pHs b-CyD concentration. However, the ICD signal at the lower
indicating the inductive effect of p-Cl on the ring system. Plot- wavelength range is also bisignate, going from positive to
ting A274 and A217 nm (results not shown) against pH gave a negative ICD with a crossing point at ꢁ214 nm and a negative
value of pKa ¼ 4.2, which is slightly lower than pKa of BA and maximum at ꢁ208 nm most probably associated with steric
signicantly higher than pKa of m-Cl-BA as a result of eld effect effect resulting from p-Cl forcing the molecule into a negative
of Clꢂ on the CO2H group. The absorbance and ICD spectra of p- co-conformation with increase in intensity and a shi towards
Cl-BA titration with b-CyD at pH 2.0 are shown in Fig. 8a and b higher energy (shorter wavelength) upon increase of b-CyD
respectively. The UV spectra exhibit a decrease in intensity with concentration. The binding curves produced by plotting the
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Fig. 8 b-CyD titration of p-Cl-BA (0.001 M) at pH 2.0 and pH 7.0 in 1.0 and 0.1 cm (a) effect of b-CyD on UV spectra (b) ICD spectra (c) binding
curves at pH 2.0 (d) binding curves at pH 7.0 (e) possible co-conformation of p-Cl-BA/b-CyD complex at pH 2.0 and pH 7.0.
values of DA242 and DA207 against b-CyD concentration are
Plotting A270 and DA237 against b-CyD concentration (Fig. 8d)
shown in Fig. 8c. The mean value of K ¼ 2.47 ꢄ 102 Mꢂ1 indi- gave mean K ¼ 2.11 ꢄ 102 Mꢂ1, which is slightly less than the K
cates a relatively low binding at pH 2.0 in comparison to m-Cl- value at pH 2.0 (K ¼ 2.47 ꢄ 102 Mꢂ1). In the same way, the pH
BA, conrming the signicant effect of substituent position on titration of bound form of p-Cl-BA was performed and plotting
the stability of the binding complex.
absorbance and ICD values against pH gave pKa ¼ 3.83, which is
At pH 7.0, where p-Cl-BA is completely ionised; the resulting less than the pKa of the free form of p-Cl-BA. The increased
absorption and CD spectra are similar to those at pH 2.0 (results acidity of p-Cl-BA in the bound form is mostly due to immersion
not shown) As at pH 2.0, the 1Lb absorbance decreases in of the molecule in the b-CyD cavity and the polarized local
intensity and exhibits a blue shi of 1 nm with increasing b-CyD environment around the COOH, which made it more suscep-
concentration. At pH 7.0, more of the 1Lb vibronic structure of p- tible to the solvent, in addition to the eld effect of p-Cl that
Cl-BA bound to b-CyD is revealed due to the immersion of the might have become closer to the CO2H in the environment of b-
ring system into the b-CyD cavity. Collectively, the intensity of CyD due to changes in the molecule conformation resulting in
the ICD at pH 7.0 is almost as half as that at pH 2.0 and the stabilising the COOꢂ and the easier loss of the H ion. According
negative ICD band at ꢁ208 nm is not clearly dened. ICD is to the resulting ICD, the p-Cl-BA molecule seems to assume a
ꢂ
angle dependent and the loss of hydrogen bonding in the CO2
tilted frontal co-conformation with the Cl going in rst and
gave more freedom to the molecule in the cavity, which resulted deep into the cavity (Fig. 8e). This assumption is in close
in a change of the angle of co-conformation with b-CyD and a agreement with results obtained from NMR analysis, which
lower intensity ICD.
state that the center of the aromatic ring of the drug molecule
6632 | RSC Adv., 2014, 4, 6624–6637
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˚
was considered to be located in the cavity 1.2 A inside from the ꢁ206 nm and 207 nm respectively, both with 3 ¼ ꢁ8100. There
H-5 plane of b-cyclodextrin.37 However, the bisignate nature of are pronounced shoulders at ꢁ215 nm (3 ¼ 3600) and 218 nm (3
the ICD suggests that the molecule attempts to minimise its net ¼ 4500) for the unionised and ionised form respectively. This
dipole moment in the nonpolar environment of the b-CyD (low indicates another transition due to intramolecular electron/
dielectric) by adopting different conformers resulting in the charge transfer to the p-system of the aromatic ring, which can
overlapping of a shorter wavelength (ꢁ208 nm) negative ICD be conrmed by the notably increased intensity and red shi of
with a positive ICD and another negative but small ICD at the ionised form. Plotting various absorbance values against pH
higher wavelengths due to the angle of alignment of these gave a mean pKa ¼ 4.35, which is in agreement with literature
transition moments within the b-CyD environment. The data values.38
suggests that although p-Cl-BA has less binding affinity than
The titration of PAA with b-CyD at pH 2.0 showed no
both BA and m-Cl-BA, the conformational equilibrium of p-Cl- signicant change in the far UV region (results not shown).
BA is more sensitive to the b-CyD environment than BA or m-Cl- Then again, in the 300–230 nm range, the 1Lb UV band (Fig. 9a)
BA. This conrms that the location of the substituents can is effectively unchanged with the addition of b-CyD and there is
1
cause a signicant difference to the binding complex.
no evidence of ne structure ICD associated with the Lb tran-
sition (Fig. 9b) indicating that the ring system is not in the
nonpolar environment of b-CyD. However, there is a slight
decrease in absorbance running from 260 nm to 235 nm
accompanied by a weak positive ICD as a result of the inclusion
of PAA in the dissymmetric environment of b-CyD. The relatively
small change in the ICD at 260–230 region implies that any
Phenylacetic acid (PAA)
PAA shows the effect of introducing a saturated methylene
group between the two chromophores (CO2H–ring system) on
binding affinity. Due to solubility constraints, the titration of
PAA was performed in a 2.0 cm cell for the wavelength range binding is most probably due to weak hydrogen bonding
(300–230 nm) and a 1.0 cm cell for the range 260–190 nm. The between the CO2H moiety and b-CyD outer rim. The binding
pH titration (spectra not shown) showed good isosbestic points, curve obtained by plotting DA240 against b-CyD concentration
which validate the reliability of the titration and the existence of (Fig. 9c) produced a value of K ¼ 1.15 ꢄ 102 Mꢂ1, which is very
a two state system (unionised and ionised). The vibronic low and indicative of non specic binding in agreement with
structure is apparent in the symmetry forbidden weak 1Lb band,
the low ICD results. At pH 7.0, phenylacetic acid is ionised, the
b-CyD titration showed no signicant effect on the UV spectra
with the 0–0 component at ꢁ267 nm and at ꢁ268 nm for the
and there is effectively no evidence of ICD (spectra not shown).
unionised and ionised form respectively. In the far UV region,
the 1La transition band of the unionised and ionised form is at However, the absorbance intensity of all transitions shows very
Fig. 9 b-CyD titration of PPA (0.001 M), pH 2.0, in H2O, 2.0 cm.
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slight increase with increasing b-CyD concentration opposite to exibility and prevents it from deeper penetration resulting in a
the case at pH 2.0. The results indicate that there is apparently lower K value, which signies the effect of isobutyl group on the
no PAA/b-CyD binding at pH 7.0 conrming that at pH 2.0, the binding interaction. This can be conrmed by the increased
weak binding is due to weak forces mainly hydrogen bonding. acidity upon complexation (Table 1) and a co-conformation,
So far, there are no reports that show any binding of the ionised which has led to stabilisation of the ionised form and easier loss
form of PAA to b-CyD. The binding of PAA to b-CyD indicates of the hydrogen ion. On the other hand, ASA contains a
that introducing a saturated methylene group between the two substituted phenyl ester group which is a nucleophilic center
chromophores may cause a loss of exibility resulting in a poor susceptible to hydrolysis and other acyl transferring reactions.42
t within the cavity, which signicantly reduces the binding It can undergo acid-catalyzed ester hydrolysis, as well as a base-
affinity.
promoted ester hydrolysis and the formation of salicylic acid as
The pH titration of PAA/b-CyD at a molar ratio of 1 : 14 a hydrolysis product. The results showed evidence of ASA acetyl
showed no signicant ICD as a result of phenylacetic acid/b-CyD hydrolysis in the 1.0 cm pathlength range (320–230 nm) as a
interaction (results are not shown). Nevertheless, plotting A260 peak was appearing at ꢁ300 nm with time and increasing pH as
and A235 against pH gave a mean pKa ¼ 4.6, which is slightly a result of salicylic acid presence in the solution. Binding
higher than the pKa for the free form of PAA (pKa ¼ 4.35). The titration at pH 7.0, the results (not shown) showed no ICD upon
slight increase in the pKa value indicates that PAA is slightly less complexation with b-CyD indicating that the ionised form of
acidic in the presence of b-CyD. The change in pKa again ASA adopts no co-conformation which could induce optical
conrms that there is interaction between PAA–CO2H group activity suggesting that the ionized ASA does not form
and b-CyD, albeit weak and only at acid pH. This weak inter- complexes with the b-CyD.43 However, at pH 2.0, in comparison
action made the CO2H slightly resistant to the solvent and to the unbound or free ASA, again the UV spectra show a small
decreased the acidity by ꢁ0.25 pH units.
red shi of the 1Lb band of ꢁ1 nm and increased intensity with
Similarly, the effect of substituents of other compounds on increasing b-CyD concentration, as evidence of hydrogen
binding interaction was studied using the new system.26 These bonding. At pH 2.0, the unionised form of ASA showed a posi-
compounds included S-(+)ibuprofen (S-Ibu) as the pharmaco- tive ICD and the bisignate nature has disappeared signifying the
logically active component of the racemic mixture, aspirin effect of the acetyl group on the conformation of ASA in the b-
(acetyl salicylic acid, ASA) and salicylic acid (SA), which are CyD environment. The major ICD signal is near 230 nm, which
important type of drugs (NSAIDs) and are one of the most correlates with the CT band, it corresponds to the phenyl
commonly used drugs that are sold over the counter. The results chromophore transitions and is most probably due to restricted
for all compounds are summarised in Table 1. In the case of S- co-conformation enforced by the acetyl group and the forma-
Ibu, which is a chiral molecule, the introduction of an isobutyl tion of hydrogen bond with the secondary hydroxyl rim, and
group has shied all transitions to a longer wavelength (a red- thus, adopting a tilted lateral co-conformation with the phenyl
shi) with the phenyl related transition 3264 ¼ ꢁ380 (spectra not ring slightly immersed inside the cavity, which gives the positive
shown). At pH 7.0, the change in the UV absorbance and CD of ICD. The binding constant at pH 2.0 was determined; K ¼ 1.49
S-Ibu with increasing b-CyD concentration suggests an inter- ꢄ 102 Mꢂ1, which is in close agreement with reported values.44
action between both compounds and thus a concomitant The b-CyD titration gives two isosbestic points in the UV spectra
complex formation at this pH. The 1Lb band shows two iso- near 300 and 260 nm indicative of a two state free/bound 1 : 1
sbestic points at 280 nm and 272.6 nm, which indicates a 1 : 1 stoichiometry of the ASA/b-CyD inclusion complex. The pH
stoichiometry of S-Ibu/b-CyD complex.39 At pH 7.0, the ionised titration of the bound form of ASA gave a pKa ¼ 4.14, which is
form of S-Ibu is not restricted by hydrogen bonding to the b-CyD signicantly higher than the pH of the free form (pKa ¼ 3.6).
rim and the molecule has more freedom to immerse deeper into This suggests that the CO2H resonance is disrupted by
the cavity. The CD spectra are negative in sign and they increase hydrogen bonding with b-CyD; also the co-conformation or
in intensity with increasing b-CyD concentration. The increase steric effect on entropy makes the hydrogen less exposed to the
or the ICD resulting from interaction with b-CyD is negative and solvent molecules resulting in the decreased acidity. In addi-
more intense suggesting a frontal co-conformational inclusion. tion, the UV band at ꢁ300 nm that was apparent in the pH
The presence of the isobutyl group may be a cause of deeper titration of the free form has disappeared or signicantly
penetration into the cavity, because the affinity of the isobutyl decreased indicating a decelerating effect of b-CyD binding on
group for the b-CyD cavity is favorable since the inner walls of b- hydrolysis. This is more likely as a result of shielding of the ester
CyD are considerably hydrophobic, this has been substantiated group due to steric hindrance by b-CyD.
in literature by interaction energy analysis and force eld
In the same way, salicylic acid (SA) pH titration gave a mean
calculations.40,41 Also, the vibrational ne structure does not pKa ¼ 2.8, which is in agreement with literature values.45,46 The
1
exhibit any change suggesting that the ring is far from the non- protonated form of SA exhibited the three transitions Lb, CT
polar part of the cavity which leads to conclude that the isobutyl and 1La at ꢁ303 nm, 238 nm and ꢁ218 nm respectively.
group is inside the cavity near the nonpolar end, whereas it is However, at pH 7.0, the ionised form three transitions were at
highly unlikely for the polar CO2H group to be near the ꢁ300 nm, ꢁ230 nm and ꢁ205 nm. Titration of SA with b-CyD at
nonpolar part of the cavity. The K value of the ionised S-Ibu is pH 7.0 showed no induced CD, this suggests that SAꢂ ion does
considerably higher than that of the unionised form (Table 1), not bind to b-CyD, or most probably the structural co-confor-
implying that hydrogen bonding restricts the molecule mation does not induce any CD, however, it was reported in
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Table 1 Summary of the results of the binding studies using the new system showing substituents effect on binding constants and pKa
K (ꢄ 102 Mꢂ1
)
pKa
Compound
BA
Substituent
Ionised
Non-ionised
5.63
Free form
Bound form
5.00
KCyD:HA/KCyD:Aꢂ
0.53
1.29
4.30
3.80
10.62
m-Cl-BA
p-Cl-BA
40.03
2.47
4.90
3.83
31.03
1.17
2.11
N/A
4.20
4.35
PAA
1.15
4.60
4.60
N/A
(+)-(S)-Ibu
27.37
13.17
5.20
0.48
ASA
SA
N/A
1.49
4.14
3.60
2.80
4.14
3.40
N/A
1.05
3.94
literature that the salicylate ion, albeit weakly binding (Table 1), The CT and 1La transitions have positive ICD at ꢁ240 and ꢁ205
it does bind to b-CyD.47
nm respectively. The bisignate feature most probably suggests
Therefore, it is reasonable to ascribe binding of the ionised two different conformers of SA at the corresponding wave-
form to weak forces e.g. hydrophobic interaction. Moreover, lengths with a slightly tilted frontal co-conformation of SA/b-
when the molecule is in the ionised form it is acting as both CyD inclusion complex, where the 1Lb transition is orthogonally
hydrogen bond donor and acceptor at the same time due to the aligned in the b-CyD cavity giving a small negative ICD and the
loss of intramolecular hydrogen bonding between OH group CT and 1La transitions are in a slightly tilted parallel alignment
and CO2H group upon ionisation leaving the ortho-OH group as giving a small positive ICD. The results indicate that the H on
a possible hydrogen bond donor and COOꢂ as hydrogen bond the CO2H group on SA is again the main contributor, which
acceptor, which makes it unsusceptible to the environment of b- interacts with the nucleophilic OH of b-CyD. The pH titration of
CyD, therefore the 1Lb band exhibits no shi and no ICD the bound form of SA gave a mean value of pKa ¼ 3.4, suggesting
observed. The titration of SA with b-CyD at pH 2.0 showed a red loss of the resonance effect due to interaction with b-CyD. In
shi of the 1Lb absorption band by ꢁ5.0 nm, indicating addition, conformational change may cause the loss of intra-
hydrogen bonding.
molecular hydrogen bonding in SA, which contribute to the low
Again, the ICD of the 1Lb band is bisignate with a crossover acidity of SA making it harder to lose the H upon NaOH addition
point from negative to positive at ꢁ270 nm similar to that of BA. and consequently decreased acidity. In comparison to the
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Table 2 The wavelength ratios of 1Lb/CT and Lb/1La at pH 2.0
Paper
1
important when the amount of available samples is limited. It is
an efficient mixing apparatus in pH and binding titration
particularly with small volumes, also, the variable speed pump
is very useful when gentle mixing is required as it is the case in
protein studies to avoid mechanical aggregation. In general, the
analysis time is signicantly reduced to #1/4 of total analysis
time used in a one-cell system. Moreover, this device can readily
be automated for routine high throughput analysis. However,
the only disadvantage was that aer rinsing the cells, they were
not completely empty. The amount of water or rinse solution
le in the cell was #100 ml, which was readily accounted for in
the calculation. This problem was solved by rinsing the cells
with ethanol solution and then attaching the cell tubing to a
vacuum to make sure that the cells are completely empty.
The charge on the guest molecule, its structure and substit-
uents are of great importance in regulating the stability of the
guest/b-CyD complexes; hence the bioavailability of drugs. What
is apparently generally neglected is that the CO2H group is more
Compound
1Lb (nm)
CT (nm)
1La (nm)
1Lb/CT
1Lb/1La
BA
274
283
276
258
265
278
300
230
232
240
218
225
230
238
212
217
214
206
212
211
218
1.19
1.21
1.15
1.18
1.18
1.20
1.26
1.29
1.30
1.28
1.25
1.25
1.31
1.37
m-Cl-BA
p-Cl-BA
PAA
S-Ibu
ASA
SA
bound form of BA, the increased pKa of the bound form of SA as
well as the binding constant (K ¼ 4.14 ꢄ 10 2 ꢅ 16 Mꢂ1, which is
in close agreement with reported values44) are slightly less
indicating the effect of OH group on binding stability.
The binding constants as an indication of the stability of the
inclusion complexes of these compounds in the protonated
form are in this order, m-Cl-BA > S-Ibu > BA > SA > p-Cl-BA > ASA
> PAA. Table 1 show the different K values for these compounds
and the ratios between KCD:HA/KCD:Aꢂ for each compound, which
is characteristic of the guest/b-CyD complex and must then be
constant. The CO2H group when involved in an intramolecular
charge transfer, makes a substantial contribution to the ground
state,30 but their predominant contributions are to the excited
state, resulting in a bathochromic shi accompanied by
hyperchromism. Moreover, according to the extensive studies
by Doub and Vandenbelt48 on the effects of substituents on the
1La band of benzene, they found that the ratio of the wave-
lengths of the 1Lb band and the 1La band is about 1.25 for most
mono-substituted benzenes including benzoic acid. The results
show that the ratio between the 1Lb band and the 1La band
assigned here is about 1.25, 1.25 and 1.29 for PAA, S-Ibu and BA
respectively. The ratio between the 1Lb band and the CT band is
between ꢁ1.15 and 1.26 for all compounds.
ꢂ
likely to participate in H-bonded structures than CO2 particu-
larly if it is an H-donor. Changes in the UV and/or CD spectra
upon addition of b-CyD have provided evidence of the signicant
effects of substituents on binding interaction. Also, the change in
the guest environment is evident in the vibronic structure of the
molecules upon complexation. The change in spectroscopic
variables (UV, CD and ICD) have provided good quantitative
measures for substituent effects on pKa, binding constant and
stoichiometry. Disubstituted benzenes can be the subject of a
future work as another application of the new device. Drug
binding to Cyclodextrin is not well understood, however, the
results undoubtedly helped to assign possible structural co-
conformations for the drug/b-CyD inclusion complex increasing
our understanding of drug/cyclodextrin interactions.
Acknowledgements
The values of these ratios for all the compounds at pH 2.0
based on lmax (Table 2) are in close agreement with the
assumption that these bands are due to intramolecular charge
transfer.
Many thanks Dr Dhia El-Hag, molecular biophysics group at
King's College London and Applied Photo Physics (APL) Ltd. for
their support.
Notes and references
Conclusion
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6636 | RSC Adv., 2014, 4, 6624–6637
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