Novel permethylated β-cyclodextrin derivatives appended with chromophores as efficient fluorescent sensors for the molecular recognition of bile salts

Article abstract of DOI:10.1021/jo071131m

(Figure Presented) Two novel permethylated β-cyclodextrin (PM-β-CD) derivatives, i.e., 6^I-O-(1-naphtholxy)-2 ^I,3¹-di-O-methylhexakis(2^II-VII,3 ^II-VII,6^II-VII-tri-O-methyl)-β-cyclodextrin(1)

Full text of DOI:10.1021/jo071131m

Novel Permethylated â-Cyclodextrin Derivatives Appended with
Chromophores as Efficient Fluorescent Sensors for the Molecular
Recognition of Bile Salts
Yu Liu,* Jun Shi, and Dong-Sheng Guo
Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai UniVersity,
Tianjin, 300071, People’s Republic of China
yuliu@nankai.edu.cn
ReceiVed May 29, 2007
Two novel permethylated â-cyclodextrin (PM-â-CD) derivatives, i.e., 6I-O-(1-naphtholxy)-2I,31-di-O-
methylhexakis(2II-VII,3II-VII,6II-VII-tri-O-methyl)-â-cyclodextrin (1) and 6I-O-(8-hydroxyquinoline)-2I,31-
di-O-methylhexakis(2II-VII,3II-VII,6II-VII- tri-O-methyl)-â-cyclodextrin (2), were synthesized in satisfactory
yields, and their inclusion modes, complex-induced fluorescent behaviors, binding ability, and selectivity
for bile salts of biological relevance (cholic acid sodium salt, CA; deoxycholic acid sodium salt, DCA;
glycochoic acid sodium salt, GCA; taurocholic acid sodium salt, TCA) were investigated by the circular
dichroism, 2D NMR, steady-state, and time-resolved fluorescent spectra. The results obtained from induced
circular dichroism and ROESY spectra show that the chromophore groups of 1 and 2 reside in the central
cavity of PM-â-CD, and are expelled to the region of narrow torus rim upon complexation with bile
guests, which presents the binding mode of cooperative inclusion. The transfer of the chromophore groups
from the central cavity to the more hydrophobic torus rim leads to the remarkable increase of fluorescent
intensities and longer fluorescent lifetimes of hosts 1 and 2 upon gradual addition of bile salts, which is
importantly distinct from the molecular recognition of the chromophore-modified â-CD species with
bile salts. Interestingly, hosts 1 and 2 present much stronger binding ability for bile guests than PM-â-
CD. Differing from native â-CD, all the PM-â-CDs are more prone to include bile salts with longer tails,
such as GCA and TCA. Their corresponding binding ability and molecular selectivity are closely discussed
from the viewpoints of difference of cavity size/shape between â-CD and PM-â-CD, effect of substituent
groups, and structures of bile guests, respectively.
Introduction
dimethylated CD, permethylated CD, random methylated CD,
and so on represent a particular family of CD derivatives that
are not only water-soluble but also ester-soluble.4,5 The multiple
modifications induce the cavity of CD to be more patulous and
In recent years, molecular recognition based on cyclodextrins
(CDs) and their derivatives has been extensively studied as a
significant topic in both chemistry and biology.1 Consequently,
much effort has been devoted to the synthesis of a wide variety
of CD derivatives to explore further their binding behaviors for
model substrates.2,3 In this context, methylated CDs, including
(2) (a) Kuwabara, T.; Nakamura, A.; Ueno, A.; Toda, F. J. Phys. Chem.
1994, 98, 6297-6303. (b) Corradini, R.; Dossena, A.; Galaverna, G.;
Marchelli, R.; Panagia, A.; Sartor, G. J. Org. Chem. 1997, 62, 6283-6289.
(c) Nowakowska, M.; Loukine, N.; Gravett, D. M.; Burke, N. A. D.; Guillet,
J. E. J. Am. Chem. Soc. 1997, 119, 4364-4368. (d) McAlpine, S. R.; Garcia-
Garibay, M. A. J. Am. Chem. Soc. 1998, 120, 4269-4275. (e) Rekharskey,
M.; Yamamura, H.; Kawai, M.; Inoue, Y. J. Am. Chem. Soc. 2001, 123,
5360-5361. (f) Kuwabara, T.; Aoyagi, T.; Takamura, M.; Matsushita, A.;
Nakamura, A.; Ueno, A. J. Org. Chem. 2002, 67, 720-725. (g) Park, J.
W.; Song, H. E.; Lee, S. Y. J. Phys. Chem. B 2002, 106, 5177-5183.
(1) (a) Szejtli, J. Chem. ReV. 1998, 98, 1743-1753. (b) Rekharsky, M.
V.; Inoue, Y. Chem. ReV. 1998, 98, 1875-1917. (c) Takahashi, K. Chem.
ReV. 1998, 98, 2013-2033. (d) Breslow, R.; Dong, S. D. Chem. ReV. 1998,
98, 1997-2011. (e) Liu, Y.; Chen, Y. Acc. Chem. Res. 2006, 39, 681-
691.
10.1021/jo071131m CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/03/2007
J. Org. Chem. 2007, 72, 8227-8234
8227

Liu et al.
flexible,6 and thereby endow methylated CDs many intrinsic
characteristics which cannot be owned by native CDs. As a
result, methylated CDs present much potential in applications
of chiral molecules even enantiomer separation and drug
delivery with more advantages than native CDs.7 Particularly,
once all the hydroxyl groups of CDs are substituted by
methyloxyls, which are called permethylated cyclodexytins
(PMCDs), not only the further alteration about cavity size and
shape appears, but also the most cavity hydrophobic region
inverses from the center to two torus rims of the cavity compared
with other methylated CDs and native CDs.6 Therefore, PMCDs
promise to exhibit much distinguishable inclusion complexation
behaviors for some special substrates during the course of
molecular recognition and separation.8-10 For example, PM-â-
CDs can afford binding constants up to 10-5 to 10-6 M-1 with
charged porphyrins in aqueous9d or aqueous/organic media.9b
However, investigations on the inclusion complexation behaviors
of the PMCDs, especially their functional derivatives, are still
very immature relative to those of CDs. Gelb and co-workers8
compared the binding abilities between native â-CD and
methylated â-CDs for a series of adamantanylammonium
derivatives. Kano and co-workers11 synthesized some PM-â-
CD derivatives and examined the inclusion mode and thermo-
dynamic parameters for complexation behavior of charged
porphyrins with these receptors.
SCHEME 1. Schematic Representation of the Formation of
Hosts 1 and 2
the CD framework not only provides additional binding sites
but the sidearms are also capable of acting as fluorescent sensors
with some organic guest molecules.
In the present work, we synthesized two novel PM-â-CD
derivatives (1 and 2) appended with naphthalene and quinoline
fluorophores, and further investigated their molecular binding
ability and selectivity for bile salts. Bile salts are classical
surfactant-like biological amphipathic compounds containing a
steroid skeleton which have distinctive detergent properties and
play a significant role in the metabolism and excretion of
cholesterol in mammals.15 Previously, research about molecular
recognitions of bile salts based on substituted CDs has been
widely performed.16,17 Possessing potential existent vivo adapt-
ability, the studies on the inclusion behaviors of PM-â-CDs with
bile salts seem to be more attractive. However, to the best of
our knowledge, the molecular recognition of bile salts by PM-
â-CD and its corresponding derivatives has never been studied
before. It is our special interest to compare the binding
mechanisms between â-CD and PM-â-CD with bile salts, and
further discuss how the substituted fluorescent groups affect the
inclusion behaviors of PM-â-CD derivatives and explore closely
their photophysical/photochemical behaviors upon complexation
with guests.
On the other hand, fluorescent spectra are a powerful tool
for studying the host-guest inclusion phenomena, which have
been widely employed in molecular recognition of CDs.12-14 It
is well-known that the introduction of fluorophore sidearms to
(3) (a) Liu, Y.; Zhang, Y.-M.; Qi, A. D.; Chen, R.-T.; Yamamoto, K.;
Wada, T.; Inoue, Y. J. Org. Chem. 1997, 62, 1826-1830. (b) Liu, Y.; You,
C.-C.; Wada, T.; Inoue, Y. J. Org. Chem. 1999, 64, 3630-3634. (c) Liu,
Y.; You, C.-C.; Wada, T.; Inoue, Y. J. Org. Chem. 1999, 64, 7781-7787.
(d) Liu, Y.; Li, B.; Wada, T.; Inoue, Y. Supramol. Chem. 1999, 10, 173-
184. (e) Liu, Y.; Li, L.; Li, X.-Y.; Zhang, H.-Y.; Wada, T.; Inoue, Y. J.
Org. Chem. 2003, 68, 3646-3657.
(4) Steiner, T.; Saenger, W. Angew. Chem., Int. Ed. 1998, 37, 3404-
3407.
(5) Harada, K. Chem. ReV. 1998, 98, 1803-1827.
(6) (a) Stefan, I.; Frieder, W. L, D. Starch/Sta¨rke 1996, 48, 225-232.
(b) Mino, R. C.; Susan, A. B.; Welcome, T. M.; Pamela, M. D. Chem.
Commun. 2004, 2216-2217.
(7) (a) Janusz, Z.; Danuta, S. J. Chromatogr. A 1988, 436, 381-390.
(b) Wilhelm, K.; Angela, K.; Wilhelm, M. J. High Resolut. Chromatogr.
1991, 14, 507-529. (c) Takashi, A.; Shinji, T.; Minoru, T. Anal. Chim.
Acta 2000, 410, 37-45. (d) Sigurdsson, H. H.; Stefa´nsson, E.; Gudmunds-
do´ttir, E.; Eysteinsson, T.; Thorsteinsdo´ttir, M.; Loftsson, T. J. Controlled
Release 2005, 102, 255-262.
(8) Gelb, R. I.; Schwartz, L. M. J. Inclusion Phenom. Mol. Recognit.
Chem. 1989, 7, 537-543.
Results and Discussion
(9) (a) Carofiglioa, T.; Fomasierb, R.; Lucchinic, V.; Rossob, C.;
Tonellatob, U. Tetrahedron Lett. 1996, 37, 8019-8022. (b) Kano, K.;
Nishiyabu, R.; Asada, T.; Kuroda, Y. J. Am. Chem. Soc. 2002, 124, 9937-
9944. (c) Kano, K.; Kitagishi, H.; Tamura, S.; Yamada, A. J. Am. Chem.
Soc. 2004, 126, 15202-15210. (d) Kano, K.; Nishiyabu, R.; Doi, R. J.
Org. Chem. 2005, 70, 3667-3673.
Conformations of Hosts and Binding Manners. The CD
cavity can provide a chiral microenvironment where achiral
(14) (a) Liu, Y.; Han, B.-H.; Sun, S.-X.; Wada, T.; Inoue, Y. J. Org.
Chem. 1999, 64, 1487-1493. (b) Liu, Y.; Han, B.-H.; Chen, Y.-T. J. Phys.
Chem. B 2002, 106, 4678-4687.
(15) Danielsson, H.; Sjo¨vall, J. Sterols and Bile Acids; Elsevier:
Amsterdam, The Netherlands, 1985; Chapter 13.
(10) Takashi, A.; Yudai, K.; Shinji, T.; Minoru, T. J. Chromatogr. A
1999, 845, 455-462.
(11) (a) Sasaki, K.; Nakagawa, H.; Zhang, X.; Sakurai, S.; Kano, K.;
Kuroda, Y. Chem. Commun. 2004, 408-409. (b) Kano, K.; Nishiyabu, R.;
Yamazaki, T.; Yamazaki, I. J. Am. Chem. Soc. 2003, 125, 10625-10634.
(c) Kano, K.; Kitagishi, H.; Dagallier, C.; Kodera, M.; Matsuo, T.; Hayashi,
T.; Hisaeda, Y.; Hirota, S. Inorg. Chem. 2006, 45, 4448-4460.
(12) (a) Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno, A.; Toda, F.;
Suzuki, I.; Osa. T. J. Am. Chem. Soc. 1993, 115, 5035-5040. (b) Ikeda,
H.; Nakamura, M.; Ise, N.; Oguma, N.; Nakamura, A.; Ikeda, T.; Toda, F.;
Ueno, A. J. Am. Chem. Soc. 1996, 118, 10980-10988. (c) Ueno, A.; Ikeda,
A.; Ikeda, H.; Ikeda, T.; Toda, F. J. Org. Chem. 1999, 64, 382-387. (d)
Nakashima, H.; Yoshida, N. Org. Lett. 2006, 8, 4997-5000.
(13) (a) Ken, S.; Masahiko, N.; Yasuhisa, K. Chem. Commun. 2001,
2630-2631. (b) Ikeda, H.; Lidaka, Y.; Ueno, A. Org. Lett. 2003, 5, 1625-
1627.
(16) (a) Tan, Z. J.; Zhu, X. X.; Brown, G. R. Langmuir 1994, 10, 1034-
1039. (b) Yim, C. T.; Zhu, X. X.; Brown, G. R. J. Phys. Chem. B 1999,
103, 597-602. (c) Singh, A. P.; Cabrer, P. R.; Alvarez-Parrilla, E.; Meijide,
F.; Va´zquez Tato, J. J. Inclusion Phenom. Macrocyclic Chem. 1999, 35,
335-348. (d) Alvarez-Parrilla, E.; Cabrer, P. R.; Singh, A. P.; Al-Soufi,
W.; Meijide, F.; Rodr´ıguez Nu´n˜ez, E.; Va´zquez Tato, J. Supramol. Chem.
2002, 14, 397-404. (e) Cabrer, P. R.; Alvarez-Parrilla, E.; Al-Soufi, W.;
Meijide, F.; Rodr´ıguez, Nu´n˜ez, E.; Va´zquez Tato, J. Supramol. Chem. 2003,
15, 33-43.
(17) (a) Liu, Y.; Yang, Y.-W.; Cao, R.; Song, S.-H.; Zhang, H.-X.; Wang,
L.-H. J. Phys. Chem. B 2003, 107, 14130-14139. (b) Wang, H.; Cao, R.;
Ke, C.-F.; Liu, Y.; Wada, T.; Inoue, Y. J. Org. Chem. 2005, 70, 8703-
8711.
8228 J. Org. Chem., Vol. 72, No. 22, 2007

â-Cyclodextrin DeriVatiVes Appended with Chromophores
FIGURE 2. Possible conformations of hosts 1 and 2 deduced from
the circular dichroism and 2D NMR spectra.
conformations of hosts 1 and 2 are illustrated in Figure 2.
Different from the native CD, PM-â-CD possesses many
methyloxyls instead of hydroxyls and a more extended cavity.
Its cavity is divided into two regions: the central cavity and
the additional torus rims. According to the aforementioned ICD
signals, the substituent chromophores (naphthalene and quino-
line) should be immersed into the chiral microenvironment of
the central cavity.
FIGURE 1. Circular dichroism spectra (a) and UV/vis absorption
spectra (b) of hosts 1 and 2 (2.0 × 10-4 mol‚dm-3) in aqueous solution
at 25 °C.
groups complexed by CDs show the induced circular dichroism
(ICD) signal(s) in corresponding transition band(s), and thereby,
circular dichroism spectrometry has become a convenient
method for the elucidation of the structure features of CD
derivatives and complexes.18,19 The circular dichroism spectra
and corresponding UV/vis absorption spectra of hosts 1 and 2
are shown in Figure 1. As can be seen, hosts 1 and 2 present
similar ICD signals, which implies that the conformations of
hosts 1 and 2 should be in a similar form. The circular dichroism
spectra of both hosts 1 and 2 show the strong positive cotton
effect peaks around 238 nm (host 1: ∆ꢀ ) 4.70 dm3‚mol-1‚cm-1;
host 2: ∆ꢀ ) 5.56 dm3‚mol-1‚cm-1), which should be attributed
to the La transition band of naphthalene and quinoline chro-
mophores. Moreover, a weak negative cotton effect peak is
found around 293 nm (∆ꢀ ) -0.40 dm3‚mol-1‚cm-1) in the
spectrum of host 1, which reflects the Lb transition band of the
naphthalene chromophore. Similarly, a weak negative ICD
signal is also presented in the spectrum of host 2. According to
the empirical rules on the ICD phenomena of CD complexes,18a,b
the positive signal of the La transition band and the negative
signal of the Lb transition band indicate that the substituent
chromophores of hosts 1 and 2 are located inside their cavities
with the La transition band parallel to the CD axis and the Lb
transition band upright to the CD axis.
The conformation of host 1 was further determined by the
2D ROESY NMR experiment. ROESY cross-peaks are indica-
tive of specific proximity relationships between adjacent protons
(the distance between protons is generally in the region of 4-5
Å).20 The obtained ROESY spectrum of host 1 is shown in
Figure S4 (Supporting Information). Although the spectrum
appears complicated and the peaks of protons of PM-â-CD
overlap each other, the cross-peaks between the inner PM-â-
CD protons and the naphthalene protons can be observed, which
also indicates that the appended naphthalene is self-included
into the PM-â-CD cavity. Therefore, combining the results of
circular dichroism and 2D NMR experiments, the possible
Furthermore, the 2D ROESY NMR experiment of complex
of 1 with CA was performed to investigate the binding geometry
between PM-â-CDs and bile salts. There are four hydrophobic
rings (A, B, C, and D) in each bile molecule. As reported
before,17,21 native CD and their derivatives can include bile
guests from their primary or secondary side, and either ring A
or ring D can be preferred to be included into the cavity. That
is to say, there are several possible binding manners during the
course of complexation between CDs and bile salts, which can
be well identified by 2D NMR spectroscopy. Moreover, for the
PM-â-CD cases, besides the CD protons of the inner cavity
(H3 and H5), the substituent methyl groups (CH3-2, CH3-3, and
CH3-6) can also provide valuable information about the complex
structures by analyzing the correlations between the protons of
methyls and guests. As shown in Figure 3, several cross-peaks
between PM-â-CD and CA protons appear in the 2D NMR
spectrum, suggesting that the CA guest exists in the cavity of
host 1. Peak C represents the correlation signals between CH3-2
protons of PM-â-CD and those of CA including the H21 of the
side chain and some protons of the ring D. Peak D shows the
weak correlation signals between H19 of CA and H5, H6, or
H3 of PM-â-CD. Furthermore, peak E exhibits the correlation
of H12 of CA and H3 of PM-â-CD as well as the correlation
signal between H7 of CA and H6 of PM-â-CD. It can be inferred
from these three cross-peaks that CA is deeply included into
the cavity of host 1 with its ring A in the region of the narrow
side and ring D in the region of the broad side. On the other
hand, the competitive inclusion of guests generally drives the
substituent group out of the cavity of CDs, especially for the
guest molecules with larger size/shape such as bile salts.17b
However, upon complexation with CA guest, the appended
naphthalene group in 1 is not entirely expelled out of the cavity
of PM-â-CD but is removed from the central cavity to the region
of the narrow torus rim. It can be clearly illustrated from Figure
3 (a1) that peak A shows the correlation signals between He,
Hf of naphthalene and H6, H6′ of PM-â-CD. Considering the
superposition of H3 and H6, H6′ peaks, the correlations between
H3 of PM-â-CD and naphthalene protons are also possible,
which indicates that the naphthalene group still resides in the
central cavity of host 1. However, the correlation signals
between other inner cavity proton H5 and any naphthalene
protons are not observed. Therefore, the hypothesis of the
naphthalene group in the central cavity should be excluded. All
(18) (a) Harata, K.; Uedaira, H. Bull. Chem. Soc. Jpn. 1975, 48, 375-
378. (b) Kajta´r, M.; Horvath-Toro, C.; Kuthi, E.; Szejtli, J. Acta Chim.
Acad. Sci. Hung. 1982, 110, 327-355. (c) Kodaka, M. J. Am. Chem. Soc.
1993, 115, 3702-3705.
(19) (a) Yang, S.-Y.; Green, M. M.; Schultz, G.; Jha, S. K.; Muller, A.
H. E. J. Am. Chem. Soc. 1997, 119, 12404-12405. (b) McAlpine, S. R.;
Garcia Garibay, M. A. J. Am. Chem. Soc. 1998, 120, 4269-4275. (c) Inoue,
Y.; Wada, T.; Sugahara, N.; Yamamoto, K.; Kimura, K.; Tong, L.-H.; Gao,
X.-M.; Hou, Z.-J.; Liu, Y. J. Org. Chem. 2000, 65, 8041-8050. (d)
Nakashima, H.; Takenaka, Y.; Higashi, M.; Yoshida, N. J. Chem. Soc.,
Perkin Trans. 2 2001, 2096-2103. (e) Takenaka, Y.; Higashi, M.; Yoshida,
N. J. Chem. Soc., Perkin Trans. 2 2002, 615-620.
(20) Schneider, H.-J.; Hacket, F.; Ru¨diger, V. Chem. ReV. 1998, 98,
1755-1785.
(21) Ollila, F.; Pentika¨inen, O. T.; Forss, S.; Johnson, M. S.; Slotte, J.
P. Langmuir 2001, 17, 7017-7111.
J. Org. Chem, Vol. 72, No. 22, 2007 8229

Liu et al.
FIGURE 3. (a) ROESY spectrum of complex of 1 (1.2 × 10-3 mol‚dm-3) with CA (2.3 × 10-3 mol‚dm-3) in D2O at 25 °C with a mixing time
of 300 ms and (b) possible binding mode of complex 1+CA.
8230 J. Org. Chem., Vol. 72, No. 22, 2007

â-Cyclodextrin DeriVatiVes Appended with Chromophores
CHART 1. Structures of the Guest Molecules Employed
the above information reveals that the CA guest is deeply
included into the PM-â-CD cavity from the secondary side with
the hydrophobic ring A, which is in accord with the binding
structure of methylated CD with bile salts reported before.21
Figure 3b shows the possible structure of the complex of host
1 with CA. The cooperative inclusion manner of both guest
molecule and substituent sidearm into the cavity is mainly
benefited from the extended framework of PM-â-CD.
cavity.17b Upon complexation with CA or DCA, the shorter
lifetime (τ1) almost maintains its original value, but the longer
lifetime (τ2 ) 20.13, 20.18 ns) is enhanced to a larger extent
than for host 1 itself. This result of fluorescent lifetime also
shows that the naphthaline sidearm in host 1 undergoes a transfer
to a more hydrophobic environment upon complexation with
bile salts.
The binding stoichiometry of the PM-â-CD derivatives 1 and
2 with bile salts was determined by the Job’s plot method. As
shown in Figure 6, the plot maximum point appears at a PM-
â-CD’s molar fraction of 0.5, which obviously indicates that a
1:1 inclusion complex is formed between host 1 and CA guest.
The same results were obtained in the other cases of the
inclusion complexation of PM-â-CDs with bile salts, which is
consistent with previous reports that native â-CD and their
monomodified derivatives always form a 1:1 inclusion complex
with bile salts.17
Fluorescent Behaviors and Spectral Titrations. For a
qualitative assessment of the inclusion complexation behaviors
of naphthaline or quinoline modified PM-â-CD derivatives, the
spectral titrations of hosts 1 and 2 with CA, DCA, GCA, and
TCA are performed at 25 °C in Tris-HCl aqueous buffer solution
at pH 7.2 by fluorescence spectroscopy. Figures 4 and 5 show
the typical spectral changes of 1 and 2 upon gradual addition
of GCA, respectively. As can be seen, the relative fluorescent
intensities of 1 and 2 present a continuous enhancement upon
the addition of GCA, which are distinct from most cases of
CDs appended with fluorescent groups.12a,b,14a In general, the
fluorescent intensities of sidearms of CD derivatives undergo
an obvious decrease when suitable guests are added because of
the competitive inclusion. That is, the preferred binding of guests
into the cavity of CDs excludes their own fluorescent sidearms
out of the cavity. And then the transfer of the fluorescent groups
from hydrophobic region to hydrophilic region makes their
fluorescence rationally be quenched. For the present PM-â-CD
cases, the naphthaline or quinoline group is not entirely out of
the cavity but moves from the central cavity to the more
hydrophobic region of the narrow torus rim, and thereby the
relative fluorescent intensities of hosts 1 and 2 are enhanced
dramatically upon complexation with bile salts. In other words,
PM-â-CDs can simultaneously accommodate the fluorescent
sidearms and bile salt guest into their extended cavities, which
is promised to favor multipoint cooperative binding.
Using 1:1 host/guest stoichiometry as elucidated before, we
could express the complexation of host (H) with the bile salt
guest (G) as eq 1, where KS represents complex formation
constant.
KS
H + G y z H‚G
(1)
In our fluorescence titration experiments, the host concentra-
tion was fixed, and the fluorescent intensity increased upon the
addition of guest molecule. Therefore, we defined ∆F as the
relative fluorescence intensity change, where ∆F ) F(with guest
molecule) - F(without guest molecule), which is assumed to
be proportional to the concentration of inclusion complex (H‚
G), i.e.
∆F ) R[H‚G]
(2)
The complexed fluorescent phenomena were further validated
by the measurements of fluorescent lifetime experiments for
host 1 in the absence and presence of CA or DCA (shown in
the Supporting Information). It implies that host 1 presents two
different lifetimes (τ1 ) 13.09 ns; τ2 ) 17.44 ns), which
indicates that the naphthaline group is located in two sorts of
environment of distinct hydrophobicity. As mentioned above,
the appended naphthaline chromophore can be self-included into
the cavity of PM-â-CD, and there should be a balance of
coexistence of both included and free naphthaline groups for
host 1. Thus, the shorter lifetime (τ1) is reasonably assigned to
the naphthaline chromophore that is exposed to the bulk aqueous
solution, and the longer lifetime (τ2) is assigned to the
naphthaline chromophore that is located in the hydrophobic
The proportionality coefficient R is taken as a sensitivity
factor for the fluorescence change upon inclusion complexation.
Thus, the effective complex formation constant (KS) can be
expressed by eq 3.
[H‚G]
[H]‚[G]
KS )
(3)
Colligated the eq 2 and eq 3, ∆F could be expressed as eq 4.
∆F ) {R([H]0 + [G]0 + 1/KS) (
R2([H] + [G] + 1/K )2 - 4R2[H] [G] }/2 (4)
x
0
0
S
0
0
J. Org. Chem, Vol. 72, No. 22, 2007 8231

Liu et al.
TABLE 1. Complex Stability Constants (KS) for the Inclusion
Complexation of Hosts 1 and 2 with Bile Salt Guests in Tris-HCl
Aqueous Buffer Solution (pH 7.2) at 25 °C
KS(HG)/M-1
â-CD 2,6-mâ-CD PM-â-CD
1
2
CA
4844a
2693b
6276b
1958b
2148b
61 ( 2c
910 ( 45d
3290 ( 175d
7460 ( 200d
DCA 4086a
GCA 2394a
TCA 2293a
774 ( 38c 4320 ( 210d
228 ( 10c 4340 ( 190d 10690 ( 340d
162 ( 9c
3820 ( 170d
8710 ( 270d
a Reference 17a. b Reference 21. c Tested by the ITC method. d Tested
by the fluorescence method.
of the stability constants obtained as well as the 1:1 stoichi-
ometry between host and guest molecules. When we repeated
the measurements, the KS values were reproducible within an
error of (5%.
FIGURE 4. (a) Fluorescence spectral changes of host 1 (2.0 × 10-6
mol‚dm-3) upon addition of GCA (0-1.0 × 10-3 mol‚dm-3 from a to
l) in 0.1 M tris buffer solution (pH 7.2) at 25 °C. (b) the nonlinear
least-squares analysis of the differential intensity (∆F) to calculate the
complex formation constant (KS). (λex ) 282.0 nm, λem ) 339.0 nm.)
Binding Ability and Selectivity. As discussed extensively
before, the hydrophobic and van de Waals interactions play a
large role in the inclusion complexation of cyclodextrins with
guests.1b,17 Moreover, the hydrogen bonds offered by the (narrow
and wide) rims’ hydroxyls of CDs with guests also help stabilize
the complexes to some extent.22 It was also well-documented
that the host-guest hydrogen-bonding interactions contribute
to stabilize the complexes during the course of complexation
of â-CD with bile salts besides the hydrophobic and van de
Waals interactions.17,23 For the PM-â-CD cases, all the hydroxyls
are methylated, and the loss of hydrogen bonds for the resulting
complexes is inevitable. Therefore, PM-â-CD only shows weak
complex stability constants (KS) to bile salts, which are much
lower than those of â-CD and 2,6-m-â-CD. In addition, the 1-2
orders of magnitude weaker binding ability of PM-â-CD to bile
salts than those of â-CD can also be reflected from two other
viewpoints. One is that the release of higher energy water
molecules in the cavity of â-CD upon complexation with guests
makes the inclusion complexation more favorable, which cannot
be obtained in the PM-â-CD cases because almost no water
molecule resides in the cavity of PM-â-CD.24 The other is that,
differing from â-CD, the shape of PM-â-CD’s cavity is pinched
to be elliptical, and so PM-â-CD possesses much stronger
binding ability to the planer-shape porphyrin guests as men-
tioned above. However, bile salts are tridimensional guests with
some columnar shape, and thereby PM-â-CD should need some
conformational adjustment to accommodate bile guests, which
is entropy-unfavorable for the inclusion complexation.
FIGURE 5. (a) Fluorescence spectral changes of host 2 (1.0 × 10-5
mol‚dm-3) upon addition of GCA (0-1.0 × 10-3 mol‚dm-3 from a to
j) in 0.1 M tris buffer solution (pH 7.2) at 25 °C. (b) The nonlinear
least-squares analysis of the differential intensity (∆F) to calculate the
complex formation constant (KS). (λex ) 300.0 nm, λem ) 398.0 nm.)
Interestingly, hosts 1 and 2 show much higher binding ability
to bile salts than PM-â-CD when the naphthalene (or quinoline)
sidearm is appended on it. As can be seen from Table 1, the KS
value for TCA is enhanced 23 times from PM-â-CD to host 1,
and it is enhanced 53 times from PM-â-CD to host 2. The
pronounced enhancement of complex stabilities for hosts 1 and
2 can be attributed to the cooperative complex interactions of
both the cavity of PM-â-CD and the chromophore sidearms.
According to the binding geometry deduced from the 2D NMR
experiment, the appended chromophore (naphthaline or quino-
line) and bile guest are simultaneously accommodated into the
FIGURE 6. Job’s plot of host 1/CA system at 339 nm. ([host 1]‚
[CA] ) 1 × 10-5 mol‚dm-1).
We obtained the complexation stability constant for each
host-guest complexation as shown in Table 1 using the
nonlinear least-squares curve-fitting method. Typical fitting
curve plots (Figures 4b and 5b) for titration of CA with host 1
and GCA with host 2 show excellent fits between the experi-
mental and calculated data. All the obtained R values of the
curve fitting plot are above 0.98, which supports the reliability
(22) (a) Tormo, L.; Organero, J. A.; Douhal, A. J. Phys. Chem. B 2005,
109, 17848-17854. (b) Douhal, A. Acc. Chem. Res. 2004, 37, 349-355.
(23) (a) Narita, M.; Koshizaka, S.; Hamada, F. J. Inclusion Phenom.
Mol. Recognit. Chem. 1999, 35, 605-619. (b) Liu, Y.; Li, L.; Chen, Y.;
Yu, L.; Fan, Z.; Ding, F. J. Phys. Chem. B 2005, 109, 4129-4134.
(24) (a) Grandeury, A.; Petit, S.; Gouhier, G.; Agassea, V.; Coquerela,
G. Tetrahedron: Asymmetry 2003, 14, 2143-2152. (b) Tsorteki, F.; Bethanis,
K.; Mentzafos, D. Carbohydr. Res. 2004, 339, 233-240.
8232 J. Org. Chem., Vol. 72, No. 22, 2007

â-Cyclodextrin DeriVatiVes Appended with Chromophores
cavity of PM-â-CD. The nearness between chromophore and
bile guest makes it possible that the appended sidearm provides
additional binding sites. Besides the hydrophobic and van de
Waals interactions between PM-â-CD’s cavity and bile salts,
there should be other noncovalent interactions between the
appended sidearm and bile guests to reinforce the complex
stabilities, such as C-H···π, nonconventional hydrogen bonds,
and so on. Upon complexation with bile guests, the naphthaline
(or quinoline) chromophore of host 1 (or 2) is not expelled out
of the cavity thoroughly, but is located at the narrow rim of
PM-â-CD. Therefore, hosts 1 and 2 appear more like a barrel
with a bottom, which can accommodate guest molecules more
immovable than PM-â-CD. Furthermore, it should be mentioned
that host 2 always forms more stable complexes with bile guests
than host 1. For example, the host selectivity of the 1/2 pair for
the CA guest is up to 3.6 times. This implies that the N atom
on the quinoline ring plays a crucial role during the course of
recognition of bile guests. The N atom possesses a lone electron
pair pointing out of the aromatic ring, which can act as a suitable
hydrogen-bonding acceptor to form hydrogen bonds with bile
guests. So, in comparison with host 1, the particular host-guest
interaction supplied by the N atom in host 2 makes it present a
stronger binding ability to bile guests.
weak binding ability for bile salts, while hosts 1 and 2 display
much stronger binding ability than PM-â-CD, with the aug-
mentation of 1-2 orders of magnitude. This mainly originates
from the cooperative binding manners of 1 (2) with bile guests.
The chromophore sidearms in 1 and 2 not only act as the
fluorescent probes but also play a great role as an additional
binding site together with the cavity of PM-â-CD during the
course of inclusion complexation. Finally, it is also well-
demonstrated that the family of PM-â-CD and its derivatives
with the extended cavity of two additional torus rims always
presents distinguishable binding modes, binding ability, and
molecular selectivity for special guest molecules in comparison
with native CD species.
Experimental Section
Materials. Both 1-naphthol and 8-hydroxylquinoline were com-
mercially available without further purification. Reagent Grade
â-CD was recrystallized twice from water and then dried in vacuum
at 95 °C for 24 h prior to use. N,N-Dimethylformamide (DMF)
was distilled under reduced pressure below 50 °C after being dried
over calcium dihydride for 2 days before use. All the other
chemicals used in reactions were reagent grade and were used
without further purification. Tris(hydroxymethyl)aminomethane and
hydrochloric acid were dissolved in deionized water to make a 0.1
M Tris-HCl aqueous buffer solution of pH 7.2, which was measured
by pH electrode for fluorescence spectral analysis.
As also can be seen from Table 1, â-CD presents the strongest
binding ability to CA guest among the four selected bile salts,
and the molecular selectivity for CA/GCA pairs is 2.0 times.
However, much different from â-CD, all the PM-â-CD deriva-
tives, PM-â-CD, hosts 1 and 2, present the weakest binding
ability to CA guest. It is ascribed that the cavity of PM-â-CD
possesses a broader hydrophobic region in comparison with
â-CD, and then PM-â-CD is more suitable to include bile guests
with longer tails (GCA and TCA) than â-CD. The molecular
selectivity for hosts 1 and 2 is reversed from that of â-CD, such
All the bile salts including Cholic acid sodium salt (CA),
deoxycholic acid sodium salt (DCA), glycochoic acid sodium salt
(GCA), and taurocholic acid sodium salt (TCA) were commercial
grade and were used without further purification.
Instruments. Circular dichroism and UV/vis spectra were
recorded in a conventional quartz cell (light path 10 mm) in aqueous
solution at 25 °C. Fluorescence spectra were recorded in a
conventional quartz cell (10 × 10 × 45 mm3) in 0.1 M Tris-HCl
buffer solution (pH 7.2) at 25 °C on a fluorescence spectrometer
with excitation and emission slits of 10 nm width. Fluorescence
lifetimes were recorded on a combined steady state and lifetime
spectrometer with a time resolution of 0.19 ns in 0.1 M Tris-HCl
buffer solution (pH 7.2) at 25 °C. A Nanosecond pulsed flash lamp
filled with hydrogen gas was employed as a pulsed light source.
Maximum counts of up to 10 000 were collected for each
measurement.
As the controlling experiments, the complex stability constants
(KS) of PM-â-CD with bile guests are determined by the method
of isothermal titration calorimetry (ITC) because there is no
chromophore in PM-â-CD to be detected by spectroscopy. The ITC
experiments were performed at atmospheric pressure in aqueous
phosphate buffer solution (pH 7.2) at 25 °C. The instrument is
calibrated chemically as we described before.17 All solutions were
degassed and thermostated before each titration run, and the
titrations were performed below the critical micelle concentration
(cmc) of four bile acid salts.21 In each run, a phosphate buffer
solution of host charged in a 250 µL syringe was sequentially
injected with stirring at 300 rpm into a buffer solution of bile acid
guest in the calorimeter’s sample cell. The sample volume was
1.4227 mL in all experiments. Each titration experiment was
composed of 29 successive injections (10 µL per injection). A
detailed experimental description and typical graphs of ITC titration
and fitting curve are provided in the Supporting Information.
Synthesis of 6I-O-(1-Naphtholxy)-2I,31-di-O-methylhexakis
(2II-VII,3II-VII,6II-VII-tri-O-methyl)-â-cyclodextrin (1). 1-Naphthol
(28.8 mg, 0.2mmol) dissolved in DMF (5 mL) and potassium
carbonate (320 mg, 2.0 mmol) was stirred under nitrogen atmo-
sphere for 1 h, then 6-OTs-PM-â-CD (160 mg, 0.1 mmol) was
added into the mixture. The reactant mixture was heated to 80 °C
and kept stirring under nitrogen atmosphere for 30 h. The precipitate
was removed by filtration, and the filtrate was evaporated to dryness
CA
CA
as, for host 1, KSGCA/KS ) 4.8, and for host 2, KSGCA/KS
) 3.3. As a result, host 2 affords the highest stability constant
up to 10 690 M-1 upon complexation with GCA guest.
Moreover, there are similar structures between CA and DCA
except for the difference of one hydroxyl in ring B. It is
attractive that DCA can be included more tightly by PM-â-
CD, hosts 1 and 2 than CA. Particularly, the molecular
selectivity of PM-â-CD for the DCA/CA pair is high, up to
12.7 times, although their KS values are not very high. One
reasonable explanation is that the absence of one hydroxyl in
ring B makes the whole framework of DCA more hydrophobic
than CA, and thereby DCA is more suitable to be immersed
into the cavity of PM-â-CDs. It also demonstrates that high
complex stability constant does not always imply high molecular
selectivity.
Conclusion
In summary, the binding behaviors of PM-â-CD and its
derivatives modified with fluorescent probes (1 and 2) with some
bile salts have been determined. According to the ICD and
ROESY experiments, the appended naphthaline (quinoline)
chromophore in host 1 (2) is proved to be self-included into
the central cavity of PM-â-CD. Upon complexation with bile
guests, the chromophores are not thoroughly expelled out of
the cavity but transfer from the original central cavity to the
region of the narrow torus rim. Therefore, both hosts 1 and 2
present the enhancement of complex-induced fluorescence
because the torus rims are more hydrophobic regions than the
central cavity for PM-â-CD. Furthermore, PM-â-CD only shows
J. Org. Chem, Vol. 72, No. 22, 2007 8233

Liu et al.
under reduced pressure. The crude product obtained was dissolved
in 2 mL of ethyl acetate and purified by chromatography over silica
gel, using ethyl acetate-methanol (8:1 v:v) as eluent. A pure sample
was obtained in a yield of 39%. UV/vis λmax (H2O)/nm 280 nm;
1H NMR (400 MHz, CD3Cl, ppm) δ 8.24 (d, Ar-H, 1H), 7.78 (d,
Ar-H, 1H), 7.56-7.39 (m, Ar-H, 4H), 6.83 (d, Ar-H, 1H), 5.13
(s, 7H, 1-H), 4.02-3.11 (m, 102H), 3.64 (21H, 3-OMe), 3.51 (21H,
2-OMe), 3.37 (18H, 6-OMe). ESI-MS m/z 1563.97 [M + Na+].
Anal. Calcd for C72H116O35: C 56.09, H 7.58. Found: C 55.84, H
7.82.
21H, 2-OMe), 3.37 (m, 18H, 6-OMe). ESI-MS m/z 1542.55 [M+].
Anal. Calcd for C71H115O35N: C 55.28, H 7.51, N 0.91 Found: C
56.25, H 7.43, N 0.88.
Acknowledgment. This work was supported by the 973
Program(2006CB932900),theNNSFC(20421202and20673061),
and the Special Fund for Doctoral Program from the Ministry
of Education of China (20050055004), which are gratefully
acknowledged.
Synthesisof6I-O-(8-Hydroxyquinoline)-2I,3I-di-O-methylhexakis-
(2II-VII,3II-VII,6II-VII-tri-O-methyl)-â-cyclodextrin (2). This syn-
thesis procedure was similar to that of compound 1. The crude
product obtained was dissolved in 2 mL of ethyl acetate and purified
by chromatography over silica gel, using ethyl acetate-methanol
(7:1 v:v) as eluent. A pure sample was acquired in a yield of 56%.
UV/vis λmax (H2O)/nm 238 nm, 293 nm; 1H NMR (400 MHz, CD3-
Cl, ppm) δ 8.87 (d, Ar-H, 1H), 8.12 (d, Ar-H, 1H), 7.91 (s, Ar-
H, 1H), 7.46-7.37 (m, Ar-H, 2H), 7.17 (d, Ar-H, 1H), 5.13 (s,
7H, 1-H), 4.02-3.11 (m, 102H), 3.64 (s, 21H, 3-OMe), 3.51 (s,
Supporting Information Available: General synthesis, 1H
NMR spectra for new compounds 1 and 2 and intermediate
compound 6-OTs-PM-â-CD, 2D ROESY NMR spectrum of host
1 in D2O, typical fluorescence lifetime spectra and experimental
data for host 1 in the absence and presence of CA or DCA, and
detailed ITC experimental description and typical graphic of titration
and fitting curve. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO071131M
8234 J. Org. Chem., Vol. 72, No. 22, 2007