Thermodynamics of the interaction of γ-cyclodextrin and tauro- and glyco-conjugated bile salts

Article abstract of DOI:10.1007/s10847-012-0165-1

The structural differences in the interaction between natural γ-cyclodextrin and bile salts common in rat, dog and man was were investigated by ¹H-ROESY and ¹³C NMR and molecular modeling and the thermodynamic parameters of the reaction by isothermal titration calorimetry. The γ-cyclodextrin was selected based upon its frequent use in drug formulation as excipients to facilitate the solubilisation of drug substances with low aqueous solubility upon oral administration. The NMR studies and the molecular modeling demonstrated an interaction with inclusion of the C-ring of the steroid body of the bile salt and partly inclusion of the B and D ring. A large variation was observed in the stability constants among the investigated bile salt. The variations in the enthalpic and entropic contributions to the overall Gibbs free energy and consequently the stability constants revealed structural differences between the bile salts, where bile salts with a hydroxyl group on C12 has a weaker interaction than the bile salts without the hydroxyl group. Based upon the theoretical calculations of the available surface area the differences observed in the entropic contribution seems to be mainly driven by dehydration effects.

Full text of DOI:10.1007/s10847-012-0165-1

J Incl Phenom Macrocycl Chem (2013) 75:223–233
DOI 10.1007/s10847-012-0165-1
ORIGINAL ARTICLE
Thermodynamics of the interaction of c-cyclodextrin
and tauro- and glyco-conjugated bile salts
•
Rene Holm Christian Schonbeck
•
´
Sune Askjær Peter Westh
¨
•
Received: 1 December 2011 / Accepted: 27 April 2012 / Published online: 9 June 2012
Ó Springer Science+Business Media B.V. 2012
Abstract The structural differences in the interaction
between natural c-cyclodextrin and bile salts common in
rat, dog and man was were investigated by 1H-ROESY and
13C NMR and molecular modeling and the thermodynamic
parameters of the reaction by isothermal titration calo-
rimetry. The c-cyclodextrin was selected based upon its
frequent use in drug formulation as excipients to facilitate
the solubilisation of drug substances with low aqueous
solubility upon oral administration. The NMR studies and
the molecular modeling demonstrated an interaction with
inclusion of the C-ring of the steroid body of the bile salt
and partly inclusion of the B and D ring. A large variation
was observed in the stability constants among the investi-
gated bile salt. The variations in the enthalpic and entropic
contributions to the overall Gibbs free energy and conse-
quently the stability constants revealed structural differ-
ences between the bile salts, where bile salts with a
hydroxyl group on C12 has a weaker interaction than the
bile salts without the hydroxyl group. Based upon the
theoretical calculations of the available surface area the
differences observed in the entropic contribution seems to
be mainly driven by dehydration effects.
Keywords Isothermal titration calorimetry ꢀ Bile salts ꢀ
Complexation ꢀ Cyclodextrin ꢀ Gamma-cyclodextrin ꢀ
Molecular modelling
Introduction
Cyclodextrins (CDs) are cyclic macromolecules consisting
of glucose units linked together with a-1,4-glycosidic
bonds, which creates a shape of a truncated cone, with
cavities of different size depending on the number of gly-
copyranose units. The most frequently investigated CDs
consist of six, seven, or eight units of a-D-(?)-glucopyra-
nose, denoted a, b, or c-CDs, respectively [1]. CDs are
capable of including smaller molecules into their cavity
region by non-covalent interactions with the inside of the
CD; primarily through hydrophobic interactions. In the
pharmaceutical field this phenomenon is utilized to enhance
the solubility of drugs, in order to enhance the bioavail-
ability of compounds with low aqueous solubility admin-
istered both orally and parentally (reviewed by e.g. [1–8]).
The oral bioavailability of both a-, b-, and cCDs is very
low [8], therefore only the free form of the drug, which is
in equilibrium with the molecular complex, is available for
absorption. For compounds with a high CD stability con-
stant, the bioavailability of the compound may therefore be
reduced if excess CD are coadministered [9]. The release
of the compound from the complex is based upon simple
dilution effects upon ingestion according to the law of mass
action, but also displacement of the drug molecule from the
CD cavity by compounds present within the gastrointesti-
nal tract [10]. Of the compounds within the gastrointestinal
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-012-0165-1) contains supplementary
material, which is available to authorized users.
¨
R. Holm (&) ꢀ C. Schonbeck
Preformulation, H. Lundbeck A/S, Ottiliavej 9, 2500 Valby,
Denmark
e-mail: rhol@lundbeck.com
¨
C. Schonbeck ꢀ P. Westh
NSM, Research Unit for Functional Biomaterials, Roskilde
University, Universitetsvej 1, 4000 Roskilde, Denmark
S. Askjær
Computational Chemistry, H. Lundbeck A/S, Ottilavej 9,
2500 Valby, Denmark
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J Incl Phenom Macrocycl Chem (2013) 75:223–233
Fig. 1 Chemical structure of
investigate bile salts
tract the bile salts have been demonstrated to have an
important role in the displacement from the CD cavity [11].
It is therefore of importance to understand the interaction
between CDs and bile salts better to facilitate a better and
more rational drug formulation process in oral formulations
utilizing CDs.
binding to cCD have only been investigated for TC. The
purpose of the current study was therefore to investigate the
differences in binding thermodynamics and mode of binding
of bile salts present in man, rat and dog to natural cCD by
ITC, 1H-ROESY and 13C NMR and molecular modeling to
increase the understanding of the interaction and to facilitate
rational drug formulation with cCDs.
CDs are a-1,4 oligomers of glucose and are therefore
susceptible to hydrolysis by amylase. However, only the
larger cCD is readily broken down enzymatically, possibly
due to the high curvature of the smaller CDs [12, 13]. Modern
drug design approaches are based on combinational chem-
istry and quantitative structure–activity relationship, which
generates a vast numbers of new potent compounds, how-
ever, unfortunately often endowed with high molecular
weights, high octanol/water partition coefficients (log P) and
low water solubilities [14]. This possible enzymatic hydro-
lysis of cCD in the intestine combined with a larger cavity
capable of complexing larger compounds when compared to
the classically used bCDs makes cCDs of increasing interest
for pharmaceutical applications. The interaction between
some bile salts and cCD have been investigated by NMR [15,
16], affinity capilary electrophoresis [17], ITC [18], and
phase solubility studies [19]. According to Alvaro et al. [20]
six different bile salts dominate in the intestine of man, rat
and dog. These are, taurocholate (TC), taurodeoxycholate
(TDC), taurochenodeoxycholate (TCDC), glycocholate
(GC), glycodeoxycholate (GDC) and glycochenodeoxych-
olate (GCDC), see Fig. 1, but the thermodynamics of
Materials and methods
Chemicals
Taurocholate (2-[3a,7a,12a-trihydroxy-24-oxo-5b-cholan-
24-yl) amino]ethanesulfonic acid), taurochenodeoxycho-
late (2-([3a,7a-dihydroxy-24-oxo-5b-cholan-24-yl]amino)
ethanesulfonic acid), taurodeoxycholate (2-([3a,12a-dihy-
droxy-24-oxo-5b-cholan-24-yl]amino)ethanesulfonic acid),
glycocholate (3a,7a,12a-trihydroxy-5b-cholan-24-oic acid
N-(carboxymethyl)amide), glycodeoxycholate (3a,12a-
dihydroxy-5b-cholan-24-oic acid N-(carboxymethyl)
amide) and glycochenodeoxycholate (3a,7a-dihydroxy-24-
oxo-5b-cholan-24-oic acid N-(carboxymethyl)amide) and
cCD was all purchased from SigmaAldrich (St. Louis,
USA) and used as provided. Sodium phosphate and D2O
were obtained from SigmaAldrich. The water used in the
experiments was obtained from a Millipore purification
system.
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NMR
225
[27–33] in combination with a Generalized Born solvation
model as implemented in MOE, which also included a
parameterized model hydrogen bonds like the ones that can
be seen for the host–guest systems evaluated in the present
work. The change in the non-polar and hydrophobic
solvation energy term is approximated by the change in
water accessible hydrophobic surface area between the free
cyclodextrin and bile salt and their mutual complex,
1D 1H and 13C spectra and 2D HSQC, HMBC and ROESY
spectra of 10 mM cCD mixed with 10 mM GC, GDC or
GCDC in D2O, as well as 1D 1H and 13C spectra of 10 mM
cCD, were recorded at 25 °C on a Bruker Avance-600
NMR spectrometer operating at 14.1 Tesla and equipped
with a cryogenic cooled probe. We have previously
recorded and assigned the spectra of the free BSs [21] and
the spectra of the bound BSs were assigned from the
recorded HSQC and HMBC spectra. 13C complexation
induced shifts (CIS) were calculated by subtracting the
chemical shifts of the bound BSs from the free BSs.
2
˚
DASAapolar, times a factor, bapolar, of 0.0025 kJ/mol A
2
(0.006 kcal/mol A ) [22].
˚
Different conformers within 12.5 kJ/mol (3 kcal/mol) of
the global energy minimum were retained and subse-
quently docked into crystal structures of the cyclodextrin.
The cCD molecule was obtained from the crystal structure
with the Cambridge Structural Database code KUTKOZ.
The structures were prepared for molecular docking by
removal of the co-crystallized ligand molecule and manual
inspection of the bond definitions and atom types. For each
of the bile salt/cCD complexes 500 docking solutions were
generated using MOE. Each complex was energy mini-
mized and duplicates, defined as those having a root mean
Molecular modeling
The free energy of the interaction between the bile salts
and the cCD were calculated with a physics based molec-
ular mechanics method using the harmonic oscillator
approximation to integrate the relevant energy wells of the
potential energy surfaces of the joint CD-bile salt and the
free species.
˚
squared distance \0.05 A, was removed. The computa-
The overall change in potential energy includes the
anharmonic energy potentials for the non-bonding inter-
actions and torsions, why the method takes the anharmonic
effects of potential energy functions for the non-bonded
interactions into consideration in the calculation of the
force constants of the vibrations for each conformational
energy minima. The calculations were carried out with a
simplified implementation of the second-generation Mining
Minima method, also denoted MS [22, 23], locally
implemented in the MOE molecular mechanics platform
[24], as previously described in detail [25]. The simplifi-
cation in the model lead to exclusion of the hard degrees of
freedom like bond stretching and angle bending and
assumes harmonic energy wells. The fundament for this
simplification is the previously reported insignificant
entropy loss compared to the loss of the soft degrees of
freedom, being the restriction imposed on the torsion
angles and the loss of external rotational and translational
freedom of the ligand upon binding [26]. The Boltzmann
weighting of the individual chemical potentials gives a
probability distribution describing the likelihood of a given
complex conformation. In addition the method allows for a
calculation of the total Gibbs free energy of binding when
the computed chemical potential of the free cyclodextrin
and bile salt are subtracted from the total chemical
potential of the corresponding complex calculated over the
ensemble of probability weighted complexes:
tions of the chemical potential for the free bile salts and the
bile salts in complex with the cCD were performed over the
ensembles of the unique conformers of these species.
Because of the complications arising from the cyclic and
symmetrical structure of the cCD molecule the change in
entropy for this species was ignored and only the change in
conformational energy was included in the calculation of
the Gibbs free energy of binding. The solvation energy
correction due to the hydrophobic effect was calculated as
b
apolar. DASAapolar across the Boltzmann weighted con-
formation ensembles of the different species and summed
with the chemical potentials to yield the total change in the
calculated Gibbs free energy of binding.
Isothermal titration calorimetry
Microcalorimetric titrations were performed at 25 °C and
atmospheric pressure in an aqueous 50 mM phosphate
buffer solution, pH 7.0, using a MicroCal VP-ITC titration
microcalorimeter (MicroCal, Northhampton, MA, USA).
The isothermal titration calorimeter was calibrated elec-
tronically. The calorimetric cells was loaded with 0.9 mM
solution of the bile salts and titrated with twenty-five 10 ll
aliquots of 13.5 mM CD solution with 200s separation. All
the solutions were degassed using a ThermoVac (Micro-
Cal, Northhampton, MA, USA) before the titration exper-
iment. The bile salt concentration was selected to ensure
measurements below the critical micelle concentration
(cmc), which for bile salts have been reported in the range
of 2–10 mM [16, 18, 34–38]. This ensured that the
obtained enthalpy change represented the complex
DG ¼ lcomplex ꢁ lcyclodextrin ꢁ lbile salt
ð1Þ
The ensembles of the free bile salts were generated by
conformational analysis using the MMFF94s force field
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J Incl Phenom Macrocycl Chem (2013) 75:223–233
formation without contributions from a concomitant dis-
solution of micellar aggregates. The ITC data were ana-
lysed by standard binding models and a non-linear
regression routine implemented in the ORIGIN software
(version 7.0). In all cases, the simplest model was used,
which assumes one class of equal independent binding sites
and provides the binding constant (Ks), binding enthalpy
(DH) and the stoichiometry (n). This model accounted well
for the observed enthalpograms. The standard free energy
(DG°) and the standard entropy (DS°) were calculated from
the standard relations to:
study the interaction between bile salts and modified or
natural bCDs [40–45]. Those nuclei that take part in the
complexation experience a change in environment, which
can be measured as a change in their NMR chemical shifts,
induced by the complexation. The nuclei that are not part
of the binding site are not expected to show significant CIS.
These techniques thus have the potential to determine the
specific mode of interaction of the CD and guest. Cabrer
et al. [15] have previously investigated cCD and bile salts
1
with H-ROESY NMR where the interactions of GC, TC,
GDC and TDC was studied. Their work did not identify
differences caused by the bile salt conjugation. Therefore
ROESY and CIS were only determined for the glycocon-
jugates: GC, GDC and GCDC in the present study.
CIS of all 13C nuclei in cCD and the BSs GC, GDC and
GCDC are presented in Table 1. Most 13C nuclei on the
BSs experience significant CIS, meaning that most parts of
the BS molecules are affected by the complexation. There
DGꢂ ¼ ꢁRT ln KS ¼ DHꢂ ꢁ TDSꢂ
ð2Þ
where R is the gas constant and T is the absolute temper-
ature. The heat of dilution was estimated from the heat
change at high CD concentrations, where practically all
bile salt had been bound, and subtracted from the raw data
prior to non-linear regression analysis.
Table 1 Complexation induced shifts (ppm, Dd) in 13C NMR signals
induced by complexation of bile salts with cCD measured in D2O
solutions
Results and discussion
The inclusion of bile salts by CDs may potentially affect
the oral availability. Drugs complexed to CD are dependent
on displacement of the drug from the CD cavity by bile
salts when a drug is coadministratered with CDs. The
propensity for such displacement to occur may be evalu-
ated by the stability constants for complexes of cCD and
the relevant glyco- and tauroconjugated bile salts. This
study presents a collective thermodynamic and structural
description of the interaction between the six major bile
salts present in the intestine of rats, dogs and humans [20]
and cCD. Further, the experimental setup and the bile salts
used here are the same as those in our earlier work [25],
consequently it follows that the results may form the basis
for a comparative analysis, which can elucidate interrela-
tionships of chemical modification and differences between
bCD and cCD and the binding of bile salt.
13C nr.
CIS (ppm)a
GC
GDC
GCDC
1
0.11
0.22
0.15
0.59
0.22
0.40
2
3
-0.10
0.02
-0.27
0.21
-0.19
-0.05
-0.14
0.63
4
5
-0.03
0.63
-0.10
0.33
6
7
-0.28
0.53
0.71
-0.62
0.58
8
0.65
9
0.29
0.12
0.50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
26
27
a
0.07
0.22
0.13
0.99
1.00
0.82
-0.17
0.27
-0.68
0.28
1.09
0.37
0.23
0.12
0.36
NMR and molecular modeling of the inclusion
complexes
1.02
0.89
0.68
0.16
-0.06
0.44
0.29
0.77
1.68
The interaction between bile salts and cCD have previously
been reported to be a 1:1 complex [15, 17, 18], which is in
accordance with the findings in the present study. In order
to study the structure of these 1:1 complexes two experi-
mental techniques are employed, ROESY NMR and
Complexation Induced Shifts (CIS) based upon changes in
the 13C NMR spectra. ROESY NMR is a two dimensional
NMR technique, in which cross peaks of protons with
0.79
0.65
0.31
0.78
0.72
0.77
0.59
0.22
0.43
0.82
0.51
0.48
0.70
0.33
0.75
0.80
0.34
0.75
-0.94
0.09
-0.71
0.10
-0.78
0.11
˚
intra- and inter-molecular distances of \3-4 A can be
observed [39]. CIS is another measure of where the inter-
-0.13
0.22
-0.05
actions take place, which have previously been used to
CIS = d (Bound bile salt) - d (free bile salt)
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227
are important differences between the CIS induced by
complexation with cCD and those previously reported for
complexation with bCD [21, 25, 40]. The CIS of nuclei at
the A- and B-rings and part of the C ring (nuclei 1-
11 ? 19) are larger when the BSs are complexed with cCD
than with bCD. In contrast to this, the nuclei at the D-ring
and the side chain exhibit smaller CIS when complexed
with cCD compared to bCD. These observations indicate
that cCD tend to encroach further onto the steroid body of
the BSs than bCD, which mainly binds to the D-ring and
the side chain [21, 25, 40].
GCDC are included slightly deeper than GC. In addition to
these crosspeaks, all spectra show a large number of
intermolecular crosspeaks between H3 and H5 and various
protons on the steroid body of the BS. H5 primarily
interacts with protons on the C- and D-rings and H3 also
interacts with protons on the A-ring, except in the case of
GC where it is not possible to identify interactions between
H3 and the A-ring. H6, which is located at the primary rim
of the CD, interacts with P18, P21 and P23 and supports
the conclusion that the BS side chain exits the CD from the
secondary rim. The suggested complex structures are
similar to the complexes formed with bCD but cCD pri-
marily resides on the steroid body whereas bCD is
restricted to the sidechain, the D-ring and part of the
C-ring. It is likely that the cCD complexes are less
restricted and allows cCD to move back and forth on the
steroid body, which leads to the interactions with several of
the rings in the steroid body of the bile salts.
1
A partial H-ROESY NMR spectra for the interaction
between GCDC and cCD is presented in Fig. 2, while the
two other spetra can be found in the supporting informa-
tion. The conclusions derived from 13C NMR were sup-
ported by the ROESY spectra. In all spectra the strongest
intermolecular crosspeaks were observed between the in-
tracavity CD protons, H3 and H5, and the BS protons on
the methyl groups P18, P19 and P21. H3 has the strongest
interaction with P19 but does not seem to interact with P18
and P21 of GDC and GCDC. Only in the complex with GC
an interaction between H3 and P18 is observed. In all
spectra crosspeaks between H5 and P18, P19 and P21 are
observed but in the complexes with GDC and GCDC the
crosspeaks with P19 are stronger than in the GC complex,
which on the contrary show stronger interactions between
H5 and P21. These observations show that deep inclusion
complexes are formed with all bile salts but GDC and
The modelling showed the interaction included the C
ring of the steroid skeleton of the bile salt completely and
the B and D ring partly, see Fig. 3. This observation is in
accordance with the binding suggested by Cabrer et al.
[15]. However, for TC/GC and TDC/GDC the modelling
also suggested a different orientation with the conjugation
of the bile salt out of the CD from both the primary side,
see Fig. 4. This was neither supported by the chemical
shifts observed in the 13C NMR nor the 1H-ROESY NMR.
As there is a lack of experimental data supporting the
Fig. 2 Partial ROESY NMR
spectrum of GCDC and cCD.
The region of mostly bile salt
chemical shift is depicted at the
x-axis and the region of CD
chemical shift at the y-axis. BS
protons are denoted with ‘‘P’’
and Cd protons with ‘‘H’’
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Fig. 3 Most stable conformation for complexes with cCD and a GCDC and b GC. cCD are presented as the stick model and the bile salts as the
space filling model
Fig. 4 Inclusion of GC with cCD in the two suggested orientations.
a The most stable orientation with exit of the bile salt conjugation
from the secondary ring; and b the less stable orientation with exit of
the bile salt conjugation from the primary ring. CD are presented as
the stick model and the bile salts as the space filling model.
Independent of orientation the molecular modeling suggest that cCDs
make complexes with bile salt where the bile salt D- and B-rings are
almost entirely included in the cyclodextrin interior, whereas the
C-ring is entirely included
modelling on this point, the conclusions should be kept
with this precaution on mind. However, the possible ori-
entations predicted by the modelling could be interpretated
as an indication of a less restricted interaction than the
similar interaction with bCD, in that bile salt can be ori-
ented in both directions in the CD cavity.
obtained through the ITC experiments for the six different
complexes are presented in Table 2 together with the
available literature data measure by the same method. The
affinity for TDC and TCDC was slightly lower than its
glycol-conjugated counterparts, GDC and GCDC, whereas
no difference was seen between TC and GC.
The stability constant for the binding of the two trihy-
droxy bile salts TC and GC to cCD was just over 5,000 M-1,
see Table 2. The stability constant for TDC and GDC was
significantly higher and even higher for TCDC/GCDC. The
strength of the stability constant for TCDC/GCDC is at a
size, where it could be considered to isolate crystals of the
Stability constants
ITC was used to study the interaction of the six bile salts
and cCD at 25 °C. A typical thermogram following an ITC
titration is presented in Fig. 5. The stability constants
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229
salts and cCD to occur with full inclusion of the bile salts
C-ring. TCDC/GCDC lack a hydroxyl group on C12,
making this interaction easier and hence stronger than the
two other bile salt classes, which has a hydroxyl group in
this position. Comparing the chemical structure of TC/GC
with TDC/GDC reveals a hydroxyl group less on the B-ring
of the latter. As both NMR and modeling demonstrated
interactions with the B ring, a hydroxyl group there appears
to obstruct the interaction.
The ranking and relative size of the stability constants is
similar to what is obtained for the binding of the same bile
salts to bCD [25, 46]. This is despite that Holm et al. [25]
demonstrated complexation on the bile salts D-ring when
interacting with bCD by ROESY NMR and molecular
modeling, whereas the interaction with cCD occurs on the
D-, C- and B-ring. GDC/TDC has a higher stability con-
stant to cCD than to bCD, probably do to a different
inclusion into the larger cavity of the cCD, but still the
binding strengths of the two different CDs must be con-
sidered relatively similar, which despite the different
binding positions, could indicate common driving forces in
the interaction with bile salts, e.g. similar hydration and
electrostatic effects [47].
Fig. 5 Calorimetric titration of TC with cCD in 50 mM phosphate
buffer (pH 7.0) at 25 °C. Raw data for sequential 10 ll injections of
cCD solution (10 mM) into the bile salt solution (1 mM). Heats of
reaction as obtained from the integration of the calorimetric traces
Enthalpy and entropy changes
The interaction was exothermic in all cases (Table 2), and
the size of the enthalpy contribution was in good agreement
with the previous reported data for TDC [18]. As discussed
in the previous section the stability constants were highly
affected by the position of the hydroxyl groups on the bile
salts. This may be further elucidated by the enthalpy and
entropy data as these are generally more sensitive to details
in the mode of interaction than the stability constants, due
to the characteristic enthalpy–entropy compensation, which
tends to hide effects on the level of the free energy changes
(or binding constants). Thus, the entropic contribution to
complex for X-ray investigations, which could be a topic for
future studies. The obtained stability constants was in range
with previously published results generated by different
techniques [15–17, 19] verifying the results obtained in the
present work. The binding strengths of the different kinds of
bile salts show that the number and positions of hydroxyl
groups on the steroid body of the bile salts has a strong
influence on the complex stabilities, as previously discussed
by Holm and coworkers [17]. Both the NMR studies and the
modeling demonstrated the interaction between the bile
Table 2 Complex stability constant (K), standard free energy (DG°), enthalpy (DH°) and entropy changes (DS°) of 1:1 inclusion complexes of
bile salts with cCD
Guest
K (M-1
)
DG° (kJ mol-1
)
DH° (kJ mol-1
)
DS° (J K-1 mol-1
)
TC
5,060 201
5,030 303
13,300 889
12,078a
-21.15 0.10
-21.13 0.15
-23.54 0.17
-23.3a
-13.03 0.38
-13.13 0.36
-10.00 0.25
-7.3a
27.19 1.60
26.83 1.40
45.40 1.39
53.7a
GC
TDC
GDC
14,400 721
83,567 2098
95,067 1137
-23.73 0.13
-28.09 0.06
-28.41 0.03
-10.38 0.15
-14.01 0.04
-13.90 0.05
44.79 0.84
47.25 0.21
48.67 0.27
TCDC
GCDC
Values are given as mean range from three different experiments in 50 mM phosphate buffer (pH 7.0) at 298.15°K (mean SD, n = 3). Data
given in italic are literature data for comparison
a
Literature data determined by isothermal titration calorimetry from Ref. [18]
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the complex formation is about twice as large for the
TCDC/GCDC and TDC/GDC compared to TC/GC
(Table 2).The two former are dihydroxy bile salts while the
latter is a trihydroxy bile salt. Also the enthalpic contri-
bution was different. One way to analyse these differences
is through a so-called compensation plot, which shows the
enthalpy change as a function of the entropy change. It
appears from Fig. 6a that the four weakly-binding bile salts
(the cholates and deoxycholates) fall on a line (dashed line,
R2 = 0.992). A linear correlation of DS and DH in a
homologous series is normally considered as an indication
that the members of the series share a single source of
additivity [48]. In the current context, this suggests that
TC, GC, TDC and GDC bind to cCD with a common
mechanism, but with different contributions to DS and DH
dictated by the detailed chemical structure of the ligand.
Conversely, Fig. 5 suggests that TCDC/GCDC show
a qualitatively different mode of interaction, which was
not strongly supported by the structural analysis nor the
1H-ROESY NMR, but could be a reflection of the lacking
hydroxyl group on C12.
Comparing the present data for cCD with the results for
bCD and the same bile salts [25] shows differences in the
thermodynamics. The interaction for bCD is mostly
enthalpy driven with a relative large entropic penalty.
Conversely for cCD we observe a favourable entropic
contribution to the complexation. For both b- and cCD,
inclusion of the bile salt expels water from the cavity,
which is thought to contribute positively to entropy [49],
under the assumption that water is more ordered in the
cavity than in the bulk. This would cause a larger increase
in entropy in the case of cCD with a larger cavity. The
higher enthalpic contribution for bCD could also originate
from the smaller cavity size of bCD, which will in general
give shorter interaction distances and hence lower energy.
However, this narrower fit will also lower the entropy, by
locking the complex tighter contributing to the unfavour-
able entropies of the bCD complexes. Similar comparison
to the interaction between bile salts and aCD is not pos-
sible, as this complexation is very weak [15, 18, 50, 51]. In
Fig. 6b the compensation plot includes data for both b- and
cCDs interacting with bile salts. As mentioned above,
placement on the same line is an indication of a single
source of additivity [48]. It has been suggested that the
slope and the intercept of the enthalpy–entropy compen-
sation plot can be related to the degree of conformational
change and the extent of desolvation induced upon com-
plexation, respectively [52–55]. Figure 6b suggests a rel-
ative large intercept with the x-axis for both classes of CDs
and bile salts. This large intercept indicates an extensive
desolvation effect in the interaction between the investi-
gated species, i.e. a common driving force for the two CDs
in the interaction.
Differences in the buried polar and hydrophobic surface
areas are also reflected in the binding affinities. The theo-
retically calculated ASA for the bile salts and the bile salt-
CD complex are presented in Table 3. Reduction of ASA
for the hydrophobic parts (ASAapolar) of the molecules is
perceived as thermodynamically favorable, i.e. the larger
the reduction the stronger the (hydrophobic) attraction. In
contrast to this, reduction of ASA for the polar components
is thermodynamically unfavorable, i.e. the smaller reduc-
tion the better. This correlation between stability and
changes in polar and nonpolar surface area is indeed found
here. Thus, the most stable complexes (TCDC and GCDC,
Table 2) show the largest DASAapolar and the smallest
DASApolar (Table 3). The opposite is found for the most
weakly bound bile salts (TC and GC). The differences
observed in the changes in the two ASA values for the
interaction between the different bile salts and cCD
therefore indicate that the variations in DS may, at least
partly, be ascribed to differences in the hydration effects.
The difference in the entropy changes could also be a
reflection of the changes in water accessible surface area
Fig. 6 Compensation plot of the individual data points showing the
enthalpy change as a function of the entropy change for a) the
investigated cCD complexes and b) the cCD and bCD complexes
from Holm et al. [25]. The bile salts are indicated by labels in the plot.
The dashed line is a linear plot to the results (see main text for details)
123

J Incl Phenom Macrocycl Chem (2013) 75:223–233
231
2
Table 3 Theoretical calculated accessible surface area (ASA) for water to the surface of the bile salt or bile salt-CD complex (in A ) calculated
˚
˚
using a probe radius of 1.4 A
2
˚
A
Free species
ASAApolar
In complex
ASAApolar
Changea
ASAPolar
ASAPolar
DASAApolar
DASAPolar
TC
479.7
466.2
503.1
488.4
503.8
489.5
509.0
274.2
240.6
248.0
214.4
249.1
215.3
869.6
541.2
529.5
542.9
536.8
541.0
525.8
-
979.4
935.2
959.3
928.5
985.0
941.0
-
-447.5
-445.7
-469.3
-460.6
-471.9
-472.7
-
-164.4
-175.1
-158.3
-155.5
-133.7
-143.8
-
GC
TDC
GDC
TCDC
GCDC
c-CD
Numbers are summed from the Boltzmann weighted conformational ensembles of each species. ASAtotal: Water accessible surface area of all
atoms; ASAApolar: Water accessible surface area of all hydrophobic (|qi| \ 0.2) atoms and ASAPolar: Water accessible surface area of all polar
(|qi| C0.2) atoms, where qi denote the partial charge of atom i calculated using MMFF in MOE
a
The change in ASA was calculated by subtracting both ASA from the bile salt and cCD from ASA obtained for the entire complex
ASAhyd, respectively. This in general indicates more
favorable entropy for the interaction between bile salts and
cCD when compared to the interaction with bCD, which is
in accordance with the experimental results reported for the
two different systems. This systematic difference between
the two classes of CDs combined with the NMR and the
ITC data clearly suggest that the insertion of the B, C and
D ring of the bile salt molecule leads to a more effective
dehydration of the apolar part of the bile salt molecule and
therefore a more favorable DS when interacting with cCD
Conclusions
In conclusion, this study has presented 1:1 stability con-
Fig. 7 Correlation between TDS° and non-polar dehydrated surface
stants and thermodynamic parameters for the binding of six
area of the complexes, the black line shows the best fit to the data
biologically relevant glyco- and tauro-conjugated bile salts
1
to the natural cCD. H-ROESY NMR experiments, sup-
ported by molecular modeling, demonstrated the interac-
(ASA) when the complex forms. When the water is
tion to include the C-ring of the bile salt completely into
transferred from the hydration shell of a non-polar moiety
the complex and the B- and D-ring partly irrespectively of
to the bulk a strong positive contribution to DS is observed.
Therefore, the higher values of DS for TCDC, GCDC, TDC
and GDC could also reflect a more pronounced hydro-
the bile salt type.
ITC was successfully applied for the investigation of the
bile salt–CD interactions. All of the investigated bile salts
phobic interaction (dehydration of more non-polar ASA)
had a significant affinity for cCD. The complexation was
found to be driven by both enthalpy and entropy for all the
for these complexes. A linear fit of TDS plotted against
DASAapolar, see Fig. 7, has a slope of -241 32 J/mol/
bile salts, which was in contrast to reported data on bCD.
2
2
˚
˚
A . Costas et al. [55] have calculated that each A of a non-
polar molecule that is hydrated contributes with -231 J/
mol to TDS. The similarity of these two values thus sug-
gests that differences in DASAapolar between the complexes
are the main contributor to the differences in DS.
This is attributed to the larger non-polar surface area
dehydrated upon complexation with the larger cCD. The
presence of hydroxyl groups at position C12 strongly
affected the affinity of the bile salts for the CDs and lead to
a lower stability constant.
When the changes in ASA for cCD from the present
study is compared to similar calculations for bCD [25] a
smaller and higher change is observed for ASApolar and
Acknowledgments Erik L. Rasmussen is acknowledged for skil-
fulled technical help with the ITC measurements.
123

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J Incl Phenom Macrocycl Chem (2013) 75:223–233
complexation with tauro- and glyco-conjugated bile salts. Lang-
muir 27, 5832–5841 (2011)
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