Molecular recognition thermodynamics and structural elucidation of interactions between steroids and bridged bis(β-cyclodextrin)s

Article abstract of DOI:10.1021/jo049143k

A series of bridged bis(β-cyclodextrin(CD))s (2-7) were synthesized, i.e., bridged bis(β-CD)s 2 and 3 bearing binaphthyl or biquinoline tethers and bridged bis(β-CD)s 4-7 possessing dithiobis(benzoyl) tether, and their complex stability constants (K_S), enthalpy (ΔH°), and entropy changes (ΔS°) for the 1:2 inclusion complexation with representative steroids, deoxycholate, cholate, glycocholate, and taurocholate, have been determined in an aqueous phosphate buffer solution of pH 7.20 at 298.15 K by means of titration microcalorimetry. The original conformations of bridged bis(β-cyclodextrin)s were investigated by circular dichroism and ¹H ROESY spectroscopy. Structures of the inclusion complexes between steroids and bridged bis(β-CD)s in solution were elucidated by 2D NMR experiments, indicating that anionic groups of two steroid molecules penetrate, respectively, into the two hydrophobic CD cavities in one 6,6′-bridged bis(β-CD) molecule from the secondary rim to give a 1:2 binding mode upon inclusion complexation. The results obtained from titration microcalorimetry and 2D NMR experiments jointly demonstrate that bridged bis(β-CD)s 2, 3 and 5-7 tethered by protonated amino group possessing different substituted groups can enhance not only the molecular binding ability toward steroids by electrostatic interaction but also molecular selectivity. Thermodynamically, the resulting 1:2 bis(β-CD)-steroid complexes are formed by an enthalpy-driven process, accompanied by smaller entropy loss. The increased complex stability mainly results from enthalpy gain, accompanied by large conformational change and extensive desolvation effects for the 1:2 inclusion complexation between bis(β-CD)s and steroids.

Full text of DOI:10.1021/jo049143k

Molecu la r Recogn ition Th er m od yn a m ics a n d Str u ctu r a l
Elu cid a tion of In ter a ction s betw een Ster oid s a n d Br id ged
Bis(â-cyclod extr in )s
Yu Liu,* Ying-Wei Yang, En-Cui Yang, and Xu-Dong Guan
Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry,
Nankai University, Tianjin 300071, P. R. China
yuliu@public.tpt.tj.cn
Received May 20, 2004
A series of bridged bis(â-cyclodextrin(CD))s (2-7) were synthesized, i.e., bridged bis(â-CD)s 2 and
3 bearing binaphthyl or biquinoline tethers and bridged bis(â-CD)s 4-7 possessing dithiobis(benzoyl)
tether, and their complex stability constants (KS), enthalpy (∆H°), and entropy changes (∆S°) for
the 1:2 inclusion complexation with representative steroids, deoxycholate, cholate, glycocholate,
and taurocholate, have been determined in an aqueous phosphate buffer solution of pH 7.20 at
298.15 K by means of titration microcalorimetry. The original conformations of bridged bis(â-
1
cyclodextrin)s were investigated by circular dichroism and H ROESY spectroscopy. Structures of
the inclusion complexes between steroids and bridged bis(â-CD)s in solution were elucidated by
2D NMR experiments, indicating that anionic groups of two steroid molecules penetrate,
respectively, into the two hydrophobic CD cavities in one 6,6′-bridged bis(â-CD) molecule from the
secondary rim to give a 1:2 binding mode upon inclusion complexation. The results obtained from
titration microcalorimetry and 2D NMR experiments jointly demonstrate that bridged bis(â-CD)s
2, 3 and 5-7 tethered by protonated amino group possessing different substituted groups can
enhance not only the molecular binding ability toward steroids by electrostatic interaction but also
molecular selectivity. Thermodynamically, the resulting 1:2 bis(â-CD)-steroid complexes are formed
by an enthalpy-driven process, accompanied by smaller entropy loss. The increased complex stability
mainly results from enthalpy gain, accompanied by large conformational change and extensive
desolvation effects for the 1:2 inclusion complexation between bis(â-CD)s and steroids.
In tr od u ction
cavities located in close vicinity. Our recent investigation
results demonstrated that a paclitaxel and a bridged bis-
(â-CD) containing an appropriate spacer could form a 1:2
inclusion complex through the binding of each cavity of
the bis(â-CD) with one paclitaxel molecule, and this
noncooperative binding mode had been successfully ap-
plied onto the solubilization increase of paclitaxel.9 In
Bridged bis(â-cyclodextrin(CD))s, as a very important
family of CD derivatives, have been known to alter
significantly the molecular binding ability and selectivity
toward a variety of guests in comparison with parent
â-CDs through the cooperative binding of a single model
substrate by two hydrophobic cavities located in a closely
vicinity1 and therefore provide an excellent model system
mimicking the substrate-specific interaction of enzymes.2
Consequently, a wide variety of bridged bis(â-CD)s have
been synthesized in order to examine and compare the
molecular binding abilities of native â-CD and bridged
bis(â-CD)s and also to gain insights into factors governing
the inclusion complexation phenomena between the host
bis(â-CD)s and guest molecules.1-8 On the other hand,
the inclusion complexation of bridged bis(â-CD)s toward
guest molecules does not always give the cooperative 1:1
binding of a single guest molecule by the two hydrophobic
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* To whom correspondence should be addressed. Tel: +86-022-
23503625. Fax: +86-022-23504853.
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118, 257. (c) French, R. R.; Wirz, J .; Woggon, W.-D. Helv. Chim. Acta
1998, 81, 1521. (d) R. Breslow, B. Zhang, J . Am. Chem. Soc. 1996,
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10.1021/jo049143k CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/02/2004
6590
J . Org. Chem. 2004, 69, 6590-6602

Interactions between Steroids and Bridged Bis(â-cyclodextrin)s
CHART 1
particluar, in our present studies on the inclusion com-
plexation of 6,6′-bridged bis(â-CD)s with anionic biologi-
cal amphiphilic surfactant-like compounds, steroids, we
observed a particular complexation mode. Anionic groups
of two steroid molecules penetrated respectively into the
two hydrophobic CD cavities in one 6,6′-bridged bis(â-
CD) molecule from the secondary rim to give a 1:2 bis-
(â-CD)-guest binding mode, instead of the cooperative
binding of a guest molecule by the two hydrophobic
cavities located in a close vicinity. According to recent
reports, this inclusion complexation is significant for the
understanding of the multiple-recognition mechanism of
CDs. Recently, Kano and Kuroda et al. reported the
inclusion complexation of CDs and charged porphyrins,
demonstrating that the anionic porphyrins penetrated
into the hydrophobic CD cavity from the secondary side,
and thermodynamically, strong van der Waals interac-
tions and dehydration from CD played an important role
in those systems.10 Tato and co-workers11 studied the
complex geometry of â-CD and its derivatives in D2O by
ROESY experiments, which exhibit different binding
modes for the inclusion complexation with steroids and
showing that the steroid molecule can enter into the
hydrophobic CD cavity from the secondary side with the
carboxylate group (tail). More recently, we investigated
the 1:1 inclusion complexation of aminated mono-â-CDs
with representative steroid molecules, demonstrating
that steroid molecules can enter into aminated mono-
modified â-CDs that possess different substituted groups
from the secondary side by the cooperation of electrostatic
interaction and van der Waals interaction and, therefore,
enhance the molecular binding ability toward steroids.12
However, to the best of our knowledge, the mechanism
and thermodynamic origin of this 1:2 bis(â-CD)-guest
inclusion complexation between bridged bis(â-CD)s and
guest molecules have rarely been thoroughly investi-
gated, despite its importance on the understanding of the
multiple-recognition mechanism of CDs.
(10) (a) Kano, K.; Nishiyabu, R.; Asada, T.; Kuroda, Y. J . Am. Chem.
Soc. 2002, 124, 9937. (b) Sasaki, K.; Nakagawa, H.; Zhang, X.; Sakurai,
S.; Kano, K.; Kuroda, Y. Chem. Commun. 2004, 408.
(11) (a) Cabrer, P. R.; Alvarez-Parrilla, E.; Meijide, F.; Seijas, J . A.;
Rodr´ıguez Nu´n˜ez, E.; Va´zquez Tato, J . Langmuir 1985, 1, 5, 5489. (b)
Singh, A. P.; Cabrer, P. R.; AÄ lvarez-Parrilla, E.; Meijide, F.; Va´zquez
Tato, J . J . Inclusion Phenom. Macro. Chem. 1999, 35, 335. (c) Cabrer,
P. R.; AÄ lvarez-Parrilla, E.; Al-Soufi, W.; Meijide, F.; Rodr´ıguez Nu´n˜ez,
E.; Va´zquez Tato, J . Supramol. Chem. 2003, 15, 33.
(8) (a) Chiu, S.-H.; Myles, D. C.; Garrell, R. L.; Stoddart, J . F. J .
Org. Chem. 2000, 65, 2792. (b) Petter, R. C.; Sikorski, C. T.; Waldeck,
D. H. J . Am. Chem. Soc. 1991, 113, 2325. (c) Sikorski, C. T.; Petter, R.
C. Tetrahedron Lett. 1994, 35, 4275. (d) Hishiya, T.; Asanuma, H.;
Komiyama, M. J . Am. Chem. Soc. 2002, 124, 570. (e) Sallas, F.;
Marsura, A.; Petot, V.; Pinte´r, I.; Kova´cs, J .; J icsinszky, L. Helv. Chim.
Acta 1998, 81, 632.
(9) Liu, Y.; Chen, G.-S.; Li, L.; Zhang, H.-Y.; Gao, D.-X.; Yuan, Y.-J .
J . Med. Chem. 2003, 46, 4634.
(12) Liu, Y.; Yang, Y.-W.; Cao, R.; Song, S.-H.; Zhang, H.-X.; Wang,
L.-H. J . Phys. Chem. B 2003, 107, 14130.
J . Org. Chem, Vol. 69, No. 20, 2004 6591

Liu et al.
F IGURE 1. Calorimetric titrations of host 2 with CA (left) and GCA (right) in a phosphate buffer solution of pH 7.2 at 25 °C.
(a) Raw data for sequential 10 µL injections of CD solution (2.01 mM) into steroid solution (0.13 and 0.287 mM). (b) Heats of
reaction as obtained from the integration of the calorimetric traces.
solution of pH 7.20 for spectral measurements and microcalo-
rimetric titrations.
CHART 2
NMR Mea su r em en ts. All of the 1H NMR experiments were
carried out in deuterium oxide at 298.2 (0.1 K. Two-
dimensional rotating frame nuclear Overhauser effect spec-
troscopy (ROESY) was performed on a Bruker AV600 spec-
trometer at 600.13 MHz. A Bruker standard sequence with
water suppression (roesyphpr) was necessary to remove the
signal of residual HOD and to be able to observe weak
intermolecular NOE interactions. The data consisted of eight
scans collected over 2048 complex points and for a spectral
width of 6127 Hz. A mixing time of 200 ms, a repetition delay
of 1.5 s, an acquisition time of 0.167 s, and a 90° pulse width
of 7.95 µs at -3 dB power attenuation were used. The data
were zero-filled to 1024 × 1024 points and processed by
applying a π/2 shifted Q-sine window in both dimensions.
Small cross-peaks were neglected when their magnitude was
close to that of noise.
In this paper, we report the syntheses of bridged bis-
(â-CD)s 2-7 with different spacers (Chart 1) and their
molecular recognition thermodynamics and solution struc-
tures upon inclusion complexation with representative
steroids (Chart 2). It is our special interest to examine
the main factors governing the above 1:2 bis(â-CD)-guest
inclusion complexation, which will serve our further
understanding of this recently developing, but thermo-
dynamically less investigated, area of bridged bis(â-CD).
Syn th esis of Bis(â-CD) 2. (R)-(+)-2,2′-Diaminoacetylm-
ethylene chloride-1,1′-binaphthyl was prepared by the reac-
tion of acetyl chloride with (R)-(+)-2,2′-diamino-1,1′-binaphthyl
in acetonitrile in 86% yield. Then (R)-(+)-2,2′-diaminoacetyl
chloride-1,1′-binaphthyl (0.2 g) was added to a solution of
6-ethylenediamine-â-CD (1.2 g) in 40 mL of dry DMF con-
taining triethylamine (5 mL) with magnetic stirring under N2
protection. The solution was stirred at room temperature for
24 h and then was allowed to warm and react at 80 °C for 3
days. The reaction mixture was poured into acetone to give a
brown precipitate. The crude solid product was collected by
filtration and then purified by Sephadex G-25 column chro-
matography with deionizated water as eluent to give brown
Exp er im en ta l Section
Ma ter ia ls. â-CD of reagent grade was recrystallized twice
from water and dried in vacuo at 100 °C for 24 h prior to use.
N,N-Dimethylformamide (DMF) and pyridine were dried over
calcium hydride for 2 days and then distilled under a reduced
pressure prior to use. All chemicals, i.e., dicyclohexylcarbodi-
imide (DCC) and triethylamine, were commercially available
and used without further purification. All steroid guests, i.e.,
deoxycholate (DCA), cholate (CA), glycocholate (GCA), and
taurocholate (TCA), were purchased from Sigma and used as
received. Mono[6-O-(p-toluenesulfonyl)]-â-CD (6-OTs-â-CD)
was prepared by the reaction of â-CD with p-toluenesulfonyl
chloride in aqueous alkaline solution.13 Bridged bis(â-CD)s 3,
5, 6, and 7 were prepared according to our reported pro-
cedures.3c,14 (R)-(+)-2,2′-Diamino-1,1′-binaphthyl was synthe-
sized by the literature method.15 Disodium hydrogen phos-
phate and sodium dihydrogen phosphate were dissolved in
distilled, deionized water to make a 0.1 M phosphate buffer
1
product (400 mg) in 29% yield. H NMR (D2O, TMS, ppm): δ
(13) Petter, R. C.; Salek, J . S.; Sikorski, C. T.; Kumaravel, G.; Lin,
F.-T. J . Am. Chem. Soc. 1990, 112, 3860.
(14) (a) Liu, Y.; Yang, Y.-W.; Song, Y.; Zhang, H.-Y.; Ding, F.; Wada,
T.; Inoue, Y. ChemBioChem 2004, 5, 868. (b) Liu, Y.; Yang, Y.-W.; Chen,
Y.; Ding, F. Unpublished work. (c) Liu, Y.; Yang, Y.-W.; Chen, Y.; Zou,
H.-X. Unpublished work.
(15) (a) Miyano, S.; Nawa, M.; Mori, A.; Nashimoto, H. Bull. Chem.
Soc. J pn. 1984, 57, 2171. (b) Tan, D.-M.; Wu, J .-A.; Wang, B.; Xu, Z.-
L. Chin. J . Org. Chem. 2001, 21, 64 (in Chinese).
6592 J . Org. Chem., Vol. 69, No. 20, 2004

Interactions between Steroids and Bridged Bis(â-cyclodextrin)s
F IGURE 2. (a) Heat effects of dilution (I) and of complexation (II) of 6 with DCA for each injection during titration
microcalorimetric experiment. (b) “Net” heat effect obtained by subtracting the heat of dilution from the heat of reaction, which
was analyzed by computer simulation using the “one set of binding sites” model.
2.5-2.9 (m, 8H); 2.9-3.8 (m, 88H); 4.9 (d, 14H); 6.8-8.1 (m,
12H). 13C NMR (D2O, ppm): δ 164.9, 132.1, 128.1, 124.6, 101.9,
83.4, 81.2, 72.5, 69.6, 60.3, 47.5, 48.9, 47.8, 30.3. Anal. Calcd
for C112H170O70N6‚14H2O: C, 45.25; H, 6.71; N, 2.83. Found:
C, 45.26; H, 6.70; N, 2.67.
In each run, a phosphate buffer solution of the host molecule
in a 0.250 mL syringe was sequentially injected into the
calorimeter sample cell containing a buffer solution of steroid
guests with stirring at 300 rpm. The sample cell volume was
1.4227 mL in all experiments. Each titration experiment was
composed of 25 successive injections (10 µL per injection).
Steroid solutions were applied at a concentration of the range
between 0.10 and 0.52 mM, which is below their critical micelle
concentration (CMC). (The CMC of the steroids employed here
are >1 mM17) Typical titration curves are shown in Figure 1.
Each addition of CD into the sample cell gave rise to a heat of
reaction, caused by the formation of inclusion complexes
between steroid molecules and CDs. The heats of reaction
decrease after each injection of host CD because less and less
steroid molecules are available to form inclusion complexes.
A control experiment was performed to determine the heat
of dilution by injecting a host buffer solution into a pure buffer
solution containing no steroid molecules. The dilution enthalpy
was subtracted from the apparent enthalpy obtained in each
titration run, and the net reaction enthalpy was analyzed by
using the “one set of binding sites” model for bridged bis(â-
CD)s 2-7, as exemplified in Figure 2 for the complexation of
DCA with bridged bis(â-CD) 6. This model will work for any
number of sites N if all sites have the same KS and ∆H°. In
this case, the total heat Q was fitted via a nolinear least-
squares minimization method to the total CD concentration
in cell (Mt) using the following equation:
Syn th esis of Bis(â-CD) 4. Native â-CD (3.0 g) and 25 mL
of dry pyridine was added to a solution of DMF (100 mL)
containing 2,2′-dithiobis(benzoic acid) (0.4 g) and dicyclohexy-
lcarbodiimide (DCC) (0.44 g) in the presence of molecular
sieves 4 Å. The resultant mixture was stirred for 12 h in an
ice bath and another 48 h at room temperature. The precipitate
was removed by filtration, and the filtrate was evaporated to
dryness under reduced pressure. The residue was dissolved
in 25 mL of hot water and then poured into 400 mL of acetone.
The precipitate formed was collected by filtration to afford a
yellow powder, which was purified on a column of Sephadex
G-25 to give 0.33 g (10% yield) of 6,6′-[2,2′-dithiobis[2-(ben-
zoylamino)ethylamino]]-bridged bis(â-CD) 4 as a light yellow
solid. 1H NMR (D2O, TMS, ppm): δ 2.64-3.01 (m, 8H); 3.27-
3.79 (m, 84H); 4.87 (d, 14H); 6.72-7.8 (m, Ar 8H). 13C NMR
(D2O, ppm): δ 177.0, 163.1, 135.8, 120.6, 118.7, 104.5, 83.8,
75.7, 74.7, 74.4, 62.9. Anal. Calcd for C98H146O72S2‚22H2O: C,
40.08; H, 6.52; S, 2.18. Found: C, 39.87; H, 6.98; S, 2.15. IR
(KBr): νmax/cm-1 3335, 2927, 2063, 1658, 1422, 1333, 1230,
1249, 1203, 1156, 1079, 1029, 945, 856, 757, 707, 577, 529.
Micr oca lor im etr ic Titr a tion . The microcalorimetric titra-
tions were performed at 25 °C under atmospheric pressure in
an aqueous phosphate buffer solution of pH 7.20 by using a
Microcal VP-ITC titration microcalorimeter, which allows us
to simultaneously determine the enthalpy and equilibrium con-
stant from a single titration curve. The instrument was cali-
brated chemically by performing the complexation reaction of
â-CD with cyclohexanol, which gave thermodynamic param-
eters in good agreement with the literature data.16 All solutions
were degassed and thermostated using a ThermoVac accessory
before the titration experiment and titrations were performed
below the critical micelle concentration of the steroid molecules.
NXt∆HVo
Q )
2
2
Mt
Mt
4Mt
1
1
1 +
+
-
1 +
+
-
(1)
(
)
[
]
NXt NKSXt
NXt NKSXt
NXt
x
Where N is the number of binding sites of CD, Xt is the total
concentration of steroids in cell and Vo is the cell volume. The
(17) (a) Collo, A.; Meijide, F.; Rodriguez Nunez, E.; Vazquez Tato,
J . J . Pharm. Sci. 1996, 85, 9. (b) Cooper, A.; Nutley, M. A.; Camilleri,
P. Anal. Chem. 1998, 70, 5024.
(16) Rekharsky, M. V.; Schwarz, F. P.; Tewari, Y. B.; Goldberg, R.
N.; Tanaka, M.; Yamashoji, Y. J . Phys. Chem. 1994, 98, 4098.
J . Org. Chem, Vol. 69, No. 20, 2004 6593

Liu et al.
TABLE 1. Com p lex Sta bility Con sta n ts (KS) a n d Sta n d a r d En th a lp ies (∆H°) a n d En tr op y Ch a n ges (T∆S°) for th e
In clu sion Com p lexa tion of Ster oid Gu ests w ith Na tive â-CD 1 a n d Br id ged Bis(â-CD)s 2-7 in a P h osp h a te Bu ffer
Solu tion of p H 7.20 a t T ) 298.1 K
hosta
guestb
Nc
KS/M-1
-∆H°/kJ ‚mol-1
T∆S°/kJ ‚mol-1
1
DCA
CA
GCA
TCA
DCA
CA
GCA
TCA
DCA
CA
GCA
TCA
DCA
CA
GCA
TCA
DCA
CA
GCA
TCA
DCA
CA
GCA
TCA
DCA
CA
GCA
TCA
1
1
1
1
2
2.02
2.05
2
2.01
2
2
2
2
2.09
2
2
2
2.13
2
2
2.02
2
2
2
2
2.14
2
2
4844 ( 16
4068 ( 84
2394 ( 69
2293 ( 13
5504 ( 50
7351 ( 48
5936 ( 58
3058 ( 38
8372 ( 71
5559 ( 38
2979 ( 15
4441 ( 31
7900 ( 32
5039 ( 32
4262 ( 27
1975 ( 10
8912 ( 37
10700 ( 98
5689 ( 38
2762 ( 36
11150 ( 130
9899 ( 36
4061 ( 22
2502 ( 15
10325 ( 245
6196 ( 134
2891 ( 25
2189 ( 18
25.8 ( 0.0
23.0 ( 0.5
23.0 ( 0.1
23.8 ( 0.1
42.7 ( 0.2
33.0 ( 0.1
15.1 ( 0.2
24.5 ( 0.2
48.1 ( 0.2
49.3 ( 0.4
18.1 ( 0.1
19.7 ( 0.1
31.6 ( 0.1
28.2 ( 0.1
21.5 ( 0.1
22.0 ( 0.1
38.1 ( 0.1
30.6 ( 0.2
22.7 ( 0.1
37.3 ( 0.1
39.9 ( 0.2
37.5 ( 0.1
23.5 ( 0.2
20.2 ( 0.2
39.4 ( 0.6
39.3 ( 0.5
23.3 ( 0.1
20.0 ( 0.1
-4.76 ( 0.0
-2.38 ( 0.5
-3.7 ( 0.1
-4.59 ( 0.1
-21.4 ( 0.2
-10.9 ( 0.1
6.4 ( 0.2
2
3
4
5
6
7
-4.6 ( 0.2
-25.7 ( 0.2
-27.9 ( 0.4
4.2 ( 0.1
1.1 ( 0.1
-9.4 ( 0.1
-7.1 ( 0.1
-0.8 ( 0.1
-3.2 ( 0.1
-15.6 ( 0.1
-7.6 ( 0.2
-1.3 ( 0.1
-17.6 ( 0.1
-16.8 ( 0.2
-14.7 ( 0.1
-2.9 ( 0.2
-0.8 ( 0.2
-16.5 ( 0.7
-17.6 ( 0.5
-3.5 ( 0.1
-0.9 ( 0.1
a
b
[Host] ) 1.97-2.06 mM. [Guest] ) 0.12-0.30 mM. c Stoichiometry given by fitting program or fixed.
value of Q above can be calculated (for any designated values
of N, K, and ∆H) at the end of the ith injection and designated
Q(i). Then the correct expression for the heat released, ∆Q(i),
from the ith injection is
dVi
Q(i) + Q(i - 1)
∆Q(i) ) Q(i) +
- Q(i - 1) (2)
[
]
<ital>Vo
2
where dVi is the volume of titrant added to the solution. Along
with obtaining of KS and ∆H° in this fitting program, the N
value in eq 1 can also be obtained, which represents the
numbers of steroids bound to one CD molecule.
If a CD molecule has sites with two different values of KS
and/or ∆H°, then the model with “two sets of binding sites”
must be used. The calculations of the present system were also
performed by using the two sets of binding site models as well
as the sequential binding model in addition to the one set of
binding site model. However, the parameters calculated by the
latter two binding models gave large uncertainties, and the
quality of fit was not improved. Therefore, two independent
titration experiments were performed to afford self-consistent
parameters by fitting with one set of binding sites model, and
the averaged values were reported in Table 1.
F IGURE 3. Circular dichroism spectra of bis(â-CD)s 3-7 in
an aqueous phosphate buffer solution of pH ) 7.20 at 25 °C.
The ORIGIN software (Microcal), used for the calculation
of the binding constant (KS) and standard molar reaction en-
thalpy (∆H°) from the titration curve, gave the relevant stan-
dard derivation on the basis of the scatter of data points in a
single titration experiment. The binding stoichiometry was also
given as a parameter when fitting the binding isotherm (panel
b in Figure 2). Knowledge of the binding constant (KS) and
molar reaction enthalpy (∆H°) enabled the calculation of the
standard free energy of binding (∆G°) and entropy change
(∆S°), according to
Multiple independent titration runs (N ) 2-4) were per-
formed to afford self-consistent thermodynamic parameters,
and the averaged values are reported in Table 1. The reported
uncertainties in the thermodynamic parameters reported for
host-guest complexation are the standard deviations of the
mean values unless stated otherwise.
Resu lts a n d Discu ssion
Syn th esis. As illustrated in Scheme 1, the (R)-(+)-2,2′-
diamino-1,1′-binaphthyl, which was synthesized accord-
ing to the reported procedure, was converted to (R)-(+)-
2,2′-diaminoacetyl chloride-1,1′-binaphthyl and then
∆G° ) -RT ln KS ) ∆H° - T∆S°
(1)
where R is the gas constant and T is the absolute temperature.
6594 J . Org. Chem., Vol. 69, No. 20, 2004

Interactions between Steroids and Bridged Bis(â-cyclodextrin)s
SCHEME 1
reacted with 6-ethylenediamine-â-CD to give the final
product 2 with a chiral tether moiety in a moderate yield.
The bridged bis(â-CD) 4 was synthesized in moderate
yield by the reaction of 2,2′-dithiobis(benzoic acid) with
native â-CD, while bis(â-CD)s 5-7 were synthesized from
the corresponding mono[6-oligo(ethylenediamino)-6-deoxy]-
â-CD. For the syntheses of bis(â-CD)s 4-7, caution
should be exercised to keep the mixture anhydrous and
at low temperature during the reaction, particularly at
the initial stage, for a smooth and clean reaction without
undesirable products.
absence of guest, which indicates that there exist differ-
ent degrees of interaction between the aromatic tether
and two chiral cavities of the bridged bis-CDs.
The circular dichroism spectrum of bridged bis(â-CD)
3 exhibits two negative Cotton effect peaks with moderate
intensities at 225 nm (∆ꢀ ) -3.83 M-1 cm-1) and 283
nm (∆ꢀ ) -2.22 M-1 cm-1), which may be assigned to
the 1La and 1Lb bands, respectively. The sign of the
induced Cotton effect enables us to elucidate the confor-
mation of biquinoline unit in bridged bis(â-CD) 3. The
1
negative Cotton effects observed for the 1La and Lb bands
Or igin a l Con for m a tion of Br id ged Bis(â-CD)s. It
has been amply demonstrated that the inclusion of a
chromophoric achiral guest/moiety in a chiral host such
as CDs produces induced circular dichroism (ICD) signals
at the wavelengths at which the guest chromophore has
absorbance.18 To confirm the original conformation of the
bridged bis(â-CD)s possessing chromophoric achiral teth-
ers in an aqueous solution, their ICD spectra were
measured at 25 °C. As can be seen from Figure 3, hosts
3-7 display different or similar ICD spectra in the
indicate that the biquinoline unit is shallowly included
into the two CD cavities of bridged bis(â-CD) 3 in the
longitudinal direction, which is in good agreement with
the 2D NMR spectral study.3c
The circular dichroism spectrum of bridged bis(â-CD)
4 possessing a relatively rigid tether does not show any
(18) (a) Connors, K. A. Chem. Rev. 1997, 97, 1325. (b) Rekharsky,
M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875. (c) Zhdanov, Y. A.;
Alekseev, Y. E.; Kompantseva, E. V.; Vergeichik, E. N. Russ. Chem.
Rev. 1992, 61, 563.
J . Org. Chem, Vol. 69, No. 20, 2004 6595

Liu et al.
significant ICD signal, indicating that the chromophoric
achiral tether is far away from the CD cavities. Mean-
while, bridged bis(â-CD) 5 possessing the same chro-
mophoric group but a longer tether as compared with
bis(â-CD) 4 displays a very weak positive Cotton effect
peaks at 290 nm (∆ꢀ ) 0.10 M-1 cm-1) for the 1Lb
transition and a relatively strong positive Cotton effect
1
at 232 nm (∆ꢀ ) 1.04 M-1 cm-1) for the La transition of
phenyl group in 2,2′-dithiobisbenzoic group. As a higher
homologue of host 5, host 6 gives a similar circular
dichroism sign; that is, two positive Cotton effect peaks
with moderate intensities at 234 nm (∆ꢀ ) 0.95 M-1 cm-1
)
and 296 nm (∆ꢀ ) 0.21 M-1 cm-1), which may be assigned
to the 1La and 1Lb bands, respectively. Host 7 also displays
1
1
two weak positive Cotton effect peaks for the La and Lb
bands at 278 nm (∆ꢀ ) 1.05 M-1 cm-1) and 232 (∆ꢀ )
0.25 M-1 cm-1). According to the generally accepted
empirical rule,19 the sign of ICD signal depends on the
orientation of the transition dipole moment of the chro-
mophore with respect to the dipole moment of the CD.
The electronic transition parallel to the CD axis gives a
positive ICD signal, whereas the perpendicular transition
gives a negative signal. However, the situation is re-
versed for a guest located just outside the CD cavity.
Therefore, we can deduce that the phenyl chromophores
in 2,2′-dithiobisbenzoic group of 5 and 6 and 4,4′-
dithiobisbenzoic group of 7 penetrate deeply or shallowly
into the CD cavities.
2D NMR spectroscopy has recently become an essential
method in the study of the conformation of the CD
derivatives and the interaction between host CDs and
guest molecules, since one can conclude that two protons
are closely located in space if an NOE cross-peak is
detected between the relevant protons in the NOESY or
ROESY spectrum. Therefore, it is possible to estimate
the orientation of the tether moiety in the CD cavity
using the assigned NOE correlations. If the tether moiety
is self-included in the CD cavity, the NOE correlations
between the protons of the tether moiety and the H3/H5
protons of the CD should be observed. To this end, to
obtain further evidence about the initial geometry of the
self-included model of the bridged bis(â-CD)s with dif-
ferent tether groups, 2D NMR spectroscopy experiments
of bridged bis(â-CD)s 2-7 have been performed in neutral
D2O solution on a Bruker AV600 spectrometer following
the circular dichroism experiments. It is worthy to note
that the concentration of the hosts in the ROESY
experiments is closer to that in the calorimetric titration
experiments as compared with the low concentration
used in the above CD experiments.
F IGURE 4. ROESY spectra of (a) bridged bis(â-CD) 5 and
(b) bridged bis(â-CD) 7 with a mixing time of 200 ms at 298.1
K.
The representative spectra obtained for bridged bis(â-
CD)s 5 and 7 are shown in parts a and b, respectively, of
Figure 4. Both of them give appreciable NOE correlations
between the protons of the tether moiety and the H3/H5
protons of the CD cavity. As can be seen from Figure 4a,
the NOE cross-peaks (peaks A) between H3 and H5 of
CD cavity and Ha/Hc of the aromatic protons and that
between H3 and Hb are observed, suggesting that the
Ha-Hb-Hc side of the tether phenyl group is included
into the CD cavity. Meantime, NOE cross-peaks between
Hd of the tether phenyl group and H3/H5 of the CD cavity
are not observed, indicating that the aromatic moiety of
the CD tether is not entirely but partially self-included
into the hydrophobic cavity from the secondary side of
the CD. Furthermore, those cross-peaks (peaks B) be-
tween the ethylamino protons He and Hf of the CD tether
and H5 of CD cavity are also clearly observed. We thus
conclude that both the ethylamino tether of bridged CD
and the tether phenyl group are included into the CD
cavity. On the other hand, in the ROESY spectrum of
bridged bis(â-CD) 7, not only the cross-peaks (peaks A
and B) between Hb of the phenyl moiety and He of the
tether ethylamino group with both H3 and H5 of CD
(19) (a) Kajtar, M.; Horvath, T. C.; Kuthi, E.; Szejtli, J . Acta Chim.
Acad. Sci. Hung. 1982, 110, 327. (b) M. Kodaka, J . Am. Chem. Soc.
1993, 115, 3702. (c) Bright, F. V. Catena, G. C. Anal. Chem. 1989, 61,
905. (d) Harata, K.; Uedaira, H. Bull. Chem. Soc. J pn. 1975, 48, 375.
6596 J . Org. Chem., Vol. 69, No. 20, 2004

Interactions between Steroids and Bridged Bis(â-cyclodextrin)s
spectra of the steroids show the intramolecular NOE
correlations among the steroid protons, which will help
us easily recognize the NOE correlations between host
and guest from the ROESY spectra of bis(â-CD)-steroid
complexes (see the Supporting Information). As shown
in Figure 6, the spectrum for the resulting complex of
CA-5 exhibits clear NOE cross-peaks (peaks A) between
the side-chain protons (P-21) and H3, H5 of CD. Mean-
while, the cross-peaks B, C, and D correspond to the
interactions of the protons at D-ring of CA with H3 and/
or H5 of CD cavity, showing that the D-ring of CA is
accommodated in the cavity. It is also clear that P18 of
CA (peaks B) only interacts with H3 of CD and the
interactions (peaks C) of P15, 16 of CA with H3 of CD
are slightly stronger than those with H5. From the above
information, it is deduced that CA enters the CD cavity
of 5 from the second side of CD with the side chain and
D-ring. The ROESY experiments suggest that the side
chain with the negative carboxylate group of CA moves
toward the positive protonated amino group of 5. The
close examination of the ROESY spectra (see the Sup-
porting Information) of the resulting complexes CA-7,
CA-2, DCA-5, DCA-7, DCA-2, and GCA-2 consistently
demonstrate the similar binding mode with the complex
CA-5, with only a slight degree of difference in the depth
of guest insertion. Since the above-mentioned microcalo-
rimetric titration results indicate that two molecules of
steroids are included ino one bis-CD molecule, we propose
the possible 1:2 bis(â-CD)-steroid binding modes (Figure
7).
F IGURE 5. Plausible molecular structures of bridged bis(â-
CD)s 5 and 7 deduced from the ROESY spectra.
cavity, but also those between Ha/Hf of the tether and
CDs H3 are found, as can be seen from Figure 4b. Noting
that the interactions of Ha/He and H3 are stronger than
those with H5, we can further conclude that the tether
phenyl group and ethylamino group are cooperatively
self-included into the CD cavity from the secondary rim
of CD. The self-included binding modes of hosts 5 and 7
obtained from the ROESY experiments are in excellent
agreement with the results obtained from the previous
circular dichroism experiments. Based on the above facts,
we propose the following two self-inclusion binding modes
of bridged bis(â-CD)s 5 and 7 (Figure 5)
Bin d in g Stoich iom etr y. Several stoichiometric ratios
between CDs and guests, such as 1:2, 2:1, and 2:2, have
been reported,1-8,11 but the most common ratio has
hitherto shown to be 1:1. The stoichiometric ratios (N
value) that we observed from the binding patterns for
the titrations of steroids with bridged bis(â-CD)s 2-7 fell
within the range of ∼1.8-2.1:1. This clearly indicates
that the majority of the inclusion complexes have a 1:2
stoichiometry of steroids and bridged bis(â-CD)s. The 1:2
binding modes for the inclusion complexation of bridged
bis(â-CD)s with steroids have also been investigated by
ROESY experiments, which further verifies the above
microcalorimetric experiment results.
Bin d in g Mod es. Previous studies on the complexation
of steroids with CDs demonstrated that the steroid body
could enter into the inner cavity of â-CD or monomodified
â-CDs by the side of the secondary hydroxyl groups, with
the side chain folded or unfolded toward the steroid body,
to give different binding stabilities upon complexation
with CDs.11,12 In the present study on the inclusion
complexation of steroids and bridged bis(â-CD)s 2-7,
aminated bridged bis(â-CD)s 2, 3, and 5-7 showed larger
stability constants than the native â-CyD 1 and the
neutral bridged bis(â-CyD) 4, which does not have amino
groups in the tether. To further investigate the mecha-
nism of the enhanced binding ability and the binding
mode of interactions between bridged bis(â-CD)s 2-7 and
steroids as well as to establish the correlation between
the conformation of the resulting complexes and the
thermodynamics obtained for bridged bis(â-CD)s 2-7
tethered with different groups, the ROESY experiments
of bridged bis(â-CD)s 2-7 were performed in the presence
of CA/DCA/GCA in D2O to elucidate the complex struc-
ture in an aqueous solution. The spectra obtained are
shown in Figure 6 and Figures S3-S11 (seeing the
Supporting Information), respectively.
Com p lexa tion of Ster oid s by â-CD 1 a n d Br id ged
Bis(â-CD) 4. As can be seen from Table 1, native â-CD
1 and bridged bis(â-CD) 4 show relatively lower binding
ability upon inclusion complexation with steroids as
compared with aminated bridged bis(â-CD)s 2, 3, and
5-7. Even so, bridged bis(â-CD) 4, possessing a relatively
short and rigid tether without amino groups, still gives
an enhanced binding ability upon complexation with
steroids, except TCA, when compared its one single unit
of cavity with that of native â-CD 1. One possible reason
is that although the spacers of bridged bis(â-CD) 4 cannot
interact directly with the steroid molecules during the
independent identical 1:2 binding process due to the
relatively shallow inclusion of steroid body in the CD
cavity, they can still change the host-guest binding
abilities to some extent by affecting the conformation of
bis(â-CD) and/or the penetration depth of the guest into
the CD cavity. Thermodynamically, the enthalpy changes
for the inclusion complexation of bridged bis(â-CD) 4 with
DCA and CA are more negative than that of native â-CD
1 (-∆∆H° ca. 5 kJ ‚mol-1), resulting in the relatively
stronger binding. On the other hand, the enthalpy change
for the complexation of 4 with DCA (31.6 ( 0.1 kJ mol-1
)
is higher than that with CA (28.2 ( 0.1 kJ mol-1), which
directly contributes to the increased complex stability.
Indeed, the binding ability (KS ) 7900 ( 32) of 4 with
DCA is higher than that with CA (KS ) 5039 ( 32), which
is similar to that of 1. It is reasonable that, possessing
the more hydrophobic structure due to the absence of C-7
hydroxyl group as compared with CA, DCA is easier to
bind into the â-CD cavity than CA, which should lead to
the more favorable van der Waals interactions. Therefore,
the stronger interaction between 4 and DCA gives the
larger negative enthalpic change and directly leads to the
The notations used are Hn for CD protons and Pn for
steroids protons, where n is the carbon number indicated
in Chart 2. In the control experiments, the ROESY
J . Org. Chem, Vol. 69, No. 20, 2004 6597

Liu et al.
F IGURE 6. ROESY spectrum of CA and host 5 with a mixing time of 200 ms at 298 K.
relatively larger complex stability constant. It is worthy
to note that bridged bis(â-CD) 4 enhances the guest
selectivity for GCA/TCA pair up to 2.16, as compared
with the nearly nonselectivity toward GCA/TCA by native
â-CD.
Com p lexa tion of CA a n d DCA by Br id ged Bis(â-
CD)s 2, 3, a n d 5-7 P ossessin g Am in a ted Teth er s.
As indicated above from the circular dichroism spectra
and ROESY experiments, the spacer of hosts 2, 3, and
5-7 self-includes, more or less, into the CD cavity. Upon
complexation with steroid, these bridged bis(CD)s exhibit
outspread conformation, with their tether out of the CD
cavity. These ICD and ROESY results indicate that
steroid molecules penetrate into the CD cavity of ami-
6598 J . Org. Chem., Vol. 69, No. 20, 2004

Interactions between Steroids and Bridged Bis(â-cyclodextrin)s
F IGURE 7. Possible binding modes of bridged bis(â-CD)-steroid complexes.
nated bridged bis(â-CD)s from the secondary side with
the steroid side chain, which possesses the negative
carboxylate group, moving toward the positive protonated
amino group of CD tethers. It is also demonstrated that
large conformation changes of aminated bridged bis(â-
CD)s occur in the complexation process. The electrostatic
interaction between protonated amino group of hosts 2,
3, and 5-7 and the negative charged carboxyl or sulfonic
acid group of the steroids must play an important role
in the binding behavior and should be responsible for the
different thermodynamic behavior. Herein, two families
of bridged bis(â-CD)s possessing spacers with different
structure and flexibility, i.e., bridged bis(â-CD)s 2 and 3
bearing binaphthyl or biquinoline tethers and bridged
bis(â-CD)s 4-7 possessing dithiobis(benzoyl) tether, are
taken as excellent hosts binding with steroids in order
to quantitatively investigate the aminated spacer effects
of bridged bis(â-CD)s and host-guest electrostatic inter-
actions upon complexation with steroids. We had dem-
onstrated that monoaminated â-CDs, possessing positive
charge at pH 7.2, could enhance the binding ability
toward negatively charged steroid molecules through the
additional electrostatic interactions in the opposite charged
host-guest complexation.12 In the present study, the
bridged bis(â-CD)s possess positive charged 6,6′-amino
groups at pH 7.2 and, therefore, should also enhance the
binding ability toward negatively charged steroid mol-
ecules, as compared with neutral bridged bis(â-CD) 4,
through the additional electrostatic interactions upon
inclusion complexation with steroid molecules. Anionic
groups of the two steroid molecules penetrated, respec-
tively, into the two hydrophobic CD cavities in one 6,6′-
bridged bis(â-CD) molecule from the secondary rim to
give a 1:2 binding mode. Therefore, the positive charged
spacer of bridged bis(â-CD)s can act as a charged cap
covering the CD cavity and further participate in the
inclusion complexation toward guest steroids.
freedoms upon complexation.20 Meanwhile, all the com-
plexation of aminated bridged bis(â-CD)s toward DCA
and CA give more negative enthalpy changes (-∆∆H° )
6.5-16.5 kJ ‚mol-1 and 2.4-21.1 kJ ‚mol-1) as compared
with that of neutral bridged bis(â-CD)s 4, validating the
contribution of the attractive electrostatic interactions
between positively charged protonated amino group of
â-CD tethers and negatively charged carboxylate group
of DCA and CA. Our previous study21 also indicated that
the effective electrostatic interaction between host and
guest usually led to a more exothermic reaction enthalpy.
In deed, the disturbance and rearrangement of the
originally well-optimized host-guest van der Waals
interaction in the â-CD cavity caused by the modification
of CD and the complexation of guest is also another main
factor governing the degree of enthalpy changes upon the
host-guest complexation. Accompanied with the more
exothermic reaction enthalpies, the inclusion complex-
ation of DCA and CA by aminated bridged bis(â-CD)s
exhibits more unfavorable ∆S° compared to that for
neutral bridged bis(â-CD) 4 and native â-CD 1, which
possibly originates from the conformation fixation of host
and guest and the rigid complex formation upon com-
plexation. As a result, the additional attractive electro-
static interactions evidently enhance the molecular bind-
ing ability of bridged bis(â-CD)s except for 2-DCA pair
and alter the molecular selectivity toward CA and DCA.
Thermodynamically, the enhanced binding ability is
mainly driven by the experimentally observed enthalpic
increases despite of the more unfavorable entropy changes.
In another word, the additional electrostatic interactions
between positively charged protonated amino group of
CD tethers and negatively charged carboxylate group of
DCA and CA influence and control the binding ability of
the host bis-CDs.
Com p lexa tion of GCA a n d TCA by Br id ged Bis-
(â-CD)s 2, 3, a n d 5-7 P ossessin g Am in a ted Teth er .
Bridged bis(â-CD)s 2, 3, and 5-7 give the lower binding
ability upon complexation with GCA and TCA as com-
pared with the complexation with CA and DCA, which
is similar with the complexation of â-CD 1 and bridged
As can be seen from Table 1, thermodynamically, the
binding behavior of DCA and CA by CDs 1-7 is entirely
driven by favorable enthalpy changes with accompanying
unfavorable entropy changes (∆H° < 0; T∆S° < 0), which
are attributed to the predominant contribution of the van
der Waals interaction arising from the size/shape-fit and
geometrical complement between host and guest and the
accompanying decreases in translational and structural
(20) (a) Rekharsky, M. V.; Inoue, Y. J . Am. Chem. Soc. 2002, 124,
813. (b) Rekharsky, M.; Inoue, Y. J . Am. Chem. Soc. 2000, 122, 10949.
(21) (a) Inoue, Y.; Liu, Y.; Tong, L.-H.; Shen, B.-J .; J in, D.-S. J . Am.
Chem. Soc. 1993, 115, 10637. (b) Inoue, Y.; Hakushi, T.; Liu, Y.; Tong,
L.-H.; Shen, B.-J .; J in. D.-S. J . Am. Chem. Soc. 1993, 115, 475.
J . Org. Chem, Vol. 69, No. 20, 2004 6599

Liu et al.
attracted electrostatic interactions, bridged bis(â-CD) 2
gives strong binding ability (examining one cavity’s
binding ability) toward GCA up to 5936 ( 58 and
enhances the binding ability toward GCA as compared
with those of bridged bis(â-CD) 4 (4262 ( 27 M-1) and
native â-CD 1 (2394 ( 69 M-1). This mainly contributes
to the favorable entropy changes (6.4 ( 0.2 kJ ‚mol-1),
showing that there must exist relative larger conforma-
tion changes and more extensive solvent-desolvating
effect during the complexation process.
Bin d in g Ability a n d Molecu la r Selectivity To-
w a r d Ster oid s. To visualize and easily discuss the
molecular binding behavior and selectivity between CDs
and steroid molecules, the changing profiles of the
complex stability constants (KS) as well as the standard
enthalpy (∆H°) and entropy changes (T∆S°) are plotted
in Figures 8 and 9, respectively.
Native â-CD 1 and bridged bis(â-CD) 4 afford relatively
small binding constants probably due to the weak van
der Waals, the hydrophobic interactions, and the host
structure features, as discussed previously. However,
possessing positively charged amino tethers, the ami-
nated bridged bis(â-CD)s 2, 3, and 5-7 exhibit the
enhanced molecular binding ability through the coopera-
tive electrostatic, van der Waals interactions, etc. Ex-
perimental data indicate that the additional attractive
electrostatic interactions play an important role in their
complexation.
It is also interesting to compare the obtained “host
selectivity” sequence for each steroid. The binding con-
stant for the complexation of each steroid by neutral
bridged bis(â-CD) 4 and aminated bridged bis(â-CD)s
2-3 and 5-7 increases in the following order:
F IGURE 8. Complex stability constants (KS) upon inclusion
complexation of steroids with â-CD 1 and bridged bis(â-CD)s
2-7 in phosphate aqueous buffer solution at 298 K.
bis(â-CD) 4. The universal decreased binding ability
toward GCA and TCA must relate to the structure
differences from CA and DCA. GCA and TCA possess the
same steroid skeleton with CA, but GCA and TCA are
the resulting compounds of the conjugation of chololic
acid with glycine and taurine, respectively. Thus, the
more polar side chains at C23 for GCA and TCA remark-
ably affect their binding thermodynamics. The results
from the ROESY experiments indicate that the repre-
sentative 2-GCA system (Figure S3 in the Supporting
Information) exhibits the main binding mode: GCA
enters the interior of CD with their “tail” first and the
D-ring is shallowly included in the CD cavity. The NOE
cross-peak of the interactions for P19 of GCA and H3 of
CD is also observed despite the very weak intensity,
suggesting that another binding mode should also exist
in the 2-GCA system, that is, GCA enters the interior
of CD with their A-ring (OH). Significantly, due to the
mostly existed former binding mode caused by the
DCA: 2 < 4 < 3 < 5 < 7 < 6
CA: 4 < 3 < 7 < 2 < 6 < 5
GCA: 7 < 3 < 6 < 4 < 5 < 2
TCA: 4 < 7 < 6 < 5 < 2 < 3
As can be seen from Table 1 and Figure 8, aminated
bridged bis(â-CD)s 2, 3, and 5-7 show mostly enhanced
F IGURE 9. Standard enthalpy (∆H°) and entropy changes (T∆S°) for the 1:1 inclusion complexation of â-CD 1 and the 2:1
inclusion complexation of bridged bis(â-CD)s 2-7 in phosphate aqueous buffer solution at 298 K.
6600 J . Org. Chem., Vol. 69, No. 20, 2004

Interactions between Steroids and Bridged Bis(â-cyclodextrin)s
binding ability toward steroid molecules as compared
with neutral bridged bis(â-CD) 4, except for 2-DCA,
7-GCA, 3-GCA, and 6-GCA pairs. For instance, the KS
values for the inclusion complexation of aminated bridged
bis(â-CD)s 2, 3, and 5-7 with CA are enhanced by factors
of 1.46 for 2, 1.10 for 3, 2.12 for 5, 1.96 for 6, and 1.23
for 7, respectively, as compared with that for bridged bis-
(â-CD) 4. The differential reaction enthalpy and entropy
changes calculated by using the data presented in Table
1 reveal that nearly all the enthalpy changes (-2.4 kJ /
mol < ∆H°hosts 2,3,5,6,7 - ∆H°host 4 < -21.1 kJ /mol for CA)
are more remarkable than the entropy changes (-0.5
kJ ‚mol-1 < T∆S°hosts 2,3,5,6,7 - T∆S°host 4 < -20.8 kJ ‚mol-1
for CA). Therefore, the enthalpy changes apparently
control the enhanced binding ability of aminated bridged
bis(â-CD)s as compared to neutral bridged bis(â-CD).
On the other hand, hosts 3, 6, and 7 give stronger
binding ability toward DCA versus CA, which is the same
as that for host 4. Hosts 2 and 5 reverse this binding
selectivity, showing larger binding constants for CA than
DCA. Bridged bis(â-CD) 2 gives the largest guest selec-
tivity for CA/DCA up to 1.33. Meanwhile, bridged bis(â-
CD) 7 gives the largest guest selectivity for DCA/TCA
up to 4.7 (KS(7-DCA)/KS(7-TCA)) and 1.7 times guest selectiv-
ity for DCA/CA. Aminated bridged bis(â-CD) 6 gives 2.44
times selectivity for CA versus GCA, enhancing the
selectivity (1.18 for CA/GCA) of neutral bridged bis(â-
CD) 4. Thermodynamically, the complexation of GCA
with 6 gives ∆H° and T∆S° values comparable to those
for neutral bridged bis(â-CD) 4, and the enthalpic gain
(∆H°6 - ∆H°4 ) -2.0 kJ ‚mol-1) is well compensated by
the equally increasing entropic loss (T∆S°6 - T∆S°4 )
-2.1 kJ ‚mol-1). In contrast, the complexation of CA with
6 gives a larger enthalpic gain than that for neutral
bridged bis(â-CD) 4 (∆H°6 - ∆H°4 ) -9.3 kJ ‚mol-1),
which is not compensated by a relatively small entropic
loss (T∆S°6 - T∆S°4 ) -7.6 kJ ‚mol-1). As a consequence
of such opposite behavior of ∆H° and T∆S°, the negligible
molecular selectivity of â-CD (∆∆G° ) ∆G°TCA - ∆G°CA
) 1.2 kJ ‚mol-1) is substantially enhanced to give a ∆∆G°
value of 3.8 kJ ‚mol-1 for host 6.
F IGUR E 10. Enthalpy-entropy compensation plot for the
inclusion complexation of various steroids with bridged bis-
(â-CD)s 2-7 obtained in the present work in an aqueous buffer
solution at 298.15 K.
canceled by entropic loss. The positive intercept indicates
that the complex is stabilized even in the absence of
enthalpic contributions, as far as the T∆S term is
positive. In the present study, the T∆S values are plotted
against the ∆H values to give an excellent straight line
with a correlation coefficient of 0.99, as can be seen from
Figure 10, and the reporting limited bridged bis(CD)-
steroid systems presently examined has an intercept
T∆S0 ) 19.3 kJ ‚mol-1 and slope ) 0.94, respectively.
Previous reports on enthalpy-entropy compensation for
the complexation of 1070 guest molecules with natural
CDs gave a slope (R ) 0.88) and intercept (T∆S0 ) 12
kJ ‚mol-1). And our previous studies on enthalpy-entropy
compensation for the complexation of steroids with mono
aminated CDs gave a slope (R ) 0.93) and intercept
(T∆S0 ) 18.7 kJ ‚mol-1). From the above results, we can
see that the values we obtained are much larger than
those relative to the natural CDs and slightly larger than
those relative to the aminated mono-CDs, and in agree-
ment with those reported in the literature for complexes
formed by bis(CD)s with flexible tethers. This indicates
that the inclusion complexation of aminated bridged bis-
(â-CD)s with steroid occurs the larger conformational
change and extensive desolvation effect. Although the
analysis was performed with a limited number of data,
we have gained clear and sensible insights into the
factors and mechanism governing the inclusion complex-
ation of bis(â-CD)s in the present case. Work concerned
with extending the reliability of these thermodynamic
data is currently in progress.
En th a lp y-En tr op y Com p en sa tion . Enthalpy-en-
tropy compensation has often been observed empirically
in the kinetic and thermodynamic parameters deter-
mined for a wide variety of reactions and equilibria and
has long been an active topic in the chemical field.
Numerous experimental data in the original and review
articles indicate that the compensation enthalpy-entropy
relationship is a powerful tool to understand and even
to predict thermodynamic behavior.21,22
The linear ∆H-∆S relationship observed experimen-
tally leads to eq 3 and gives eq 4 when integrated.
Con clu sion s
T∆∆S ) R∆∆H
(3)
(4)
In summary, solution structural investigations on the
inclusion complex of bridged bis(â-CD)s and steroids
showed that anionic groups of two steroid molecules
penetrated respectively into the two hydrophobic CD
cavities in one bis(â-CD) molecule from the secondary rim
to exhibit 1:2 host-guest binding mode. From the struc-
tures of bis(â-CD)s and their complexes, it is noted that
the relative large conformation changes and self-inclusion
of the spacer must occur in the inclusion complexation
with steroid molecules, corresponding to the more nega-
tive entropy changes. Thermodynamically, the enhanced
T∆S ) R∆H + T∆S0
Thus, the slope (R) of the T∆S versus ∆H plot (eq 4)
indicates to what extent the enthalpic gain (∆∆H) is
(22) (a) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875. (b)
Grunwald, E. Thermodynamics of Molecular Species; Wiley-Iner-
science: New York, 1996. (c) Chen, R.-T. Correlation Analysis in
Coordination Chemistry; Anhui Educational Publishing: Hefei, 1995
(in Chinese). (d) Exner, O. Correlation Analysis of Chemical Data;
Plenum: New York, 1988. (e) Rekharsky, M. V.; Inoue, Y. J . Am. Chem.
Soc. 2000, 122, 4418.
J . Org. Chem, Vol. 69, No. 20, 2004 6601

Liu et al.
van der Waals interactions and additional attractive
electrostatic interactions between bis(â-CD) and steroid
led to a more exothermic reaction enthalpy, which
directly contributed to the increased inclusion complex
stability. Therefore, the current studies not only revealed
the enhanced binding ability and molecular selectivity
of steroid by bis(â-CD) but also established the correlation
between thermodynamic parameters and solution struc-
ture in the complexation of bridged bis(â-CD) and steroid,
which will further serve the understanding of the mo-
lecular multiple-recognition mechanism in supramolecu-
lar systems.
Ack n ow led gm en t. This work was supported by the
NNSF of China (Grant Nos. 90306009 and 20272028)
and the Natural Science Fund of Tianjin (Grant No.
043604411), which are gratefully acknowledged. We also
thank the reviewers for their highly valuable sugges-
tions regarding the revision.
Su p p or tin g In for m a tion Ava ila ble: ROESY spectra of
CA, DCA, CA-2, CA-7, DCA-2, DCA-5, DCA-7, and GCA-2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
J O049143K
6602 J . Org. Chem., Vol. 69, No. 20, 2004