Fluorescent anion sensor derived from cholic acid: The use of flexible side chain

Article abstract of DOI:10.1021/jo050913h

The fluorescent photoinduced electron transfer (PET) chemosensors 1-3 were synthesized from cholic acid. 1 and 2 containing amidothiourea groups as anion receptive sites demonstrated much higher affinity toward anions than 3 containing traditional thioure

Full text of DOI:10.1021/jo050913h

Fluorescent Anion Sensor Derived from Cholic Acid:
The Use of Flexible Side Chain
Lei Fang,†,‡ Wing-Hong Chan,*,† Yong-Bing He,‡ Daniel W. J. Kwong,† and Albert W. M. Lee†
Department of Chemistry and Central Laboratory of the Institute of Molecular Technology for Drug
Discovery and Synthesis, Hong Kong Baptist University, Hong Kong SAR, P. R. China, and Department
of Chemistry, Wuhan University, Hubei Province, P. R. China
whchan@hkbu.edu.hk
Received May 8, 2005
The fluorescent photoinduced electron transfer (PET) chemosensors 1-3 were synthesized from
cholic acid. 1 and 2 containing amidothiourea groups as anion receptive sites demonstrated much
higher affinity toward anions than 3 containing traditional thiourea H-bond donating group.
Comparative studies on their binding affinity toward carboxylates, dihydrogen phosphate, and
halides revealed that the amidothiourea moiety on the C17 side chain could work cooperatively
with H-bond donating groups on C7 and C12 to bind spherical halogen anions. An unexpected
specific fluorescence enhancement of 1 by coordinating bromide ion was observed.
Introduction
anions and synthetic difficulty in building selective
anionic receptive sites make the design and synthesis of
anion sensor intellectually more demanding and practi-
cally less predictable.1e Recently, neutral anion receptors
embracing hydrogen donor groups have been extensively
studied because of the absence of counteranion interfer-
ences and the nature of H-bond providing a directional
interaction.3 Notwithstanding these achievements, anion
receptors with a built-in signal transduction unit capable
of producing easily detectable signal after selective
binding are less common.4 Furthermore, to excel their
performance, anion sensors still need improvement in
their selectivity and sensitivity. In this article, we report
Anion sensing has attracted increasing attention from
various disciplines recently.1 Molecular recognition of
anionic species plays a pivotal role in a large number of
biological processes. However, practical detection of
anions has continued to be a more challenging issue than
the detection of cations, which has been fully developed
for almost 40 years.2 The variety of geometric shapes of
† Hong Kong Baptist University.
‡ Wuhan University.
(1) For reviews, see: (a) Martinez-Manez, R.; Sancenon, F. Chem.
Rev. 2003, 103, 4419-4476. (b) Supramolecular Chemistry of Anions;
Bianchi, A., Bowman-James, K., Garcia-Espana, E., Eds.; Wiley-
VCH: New York, 1997. (c) Bondy, C. R.; Loeb, S. J. Coord. Chem. Rev.
2003, 240, 77-99. (d) Suksai, C.; Tuntulani, T. Chem. Soc. Rev. 2003,
32, 192-202. (e) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001,
40, 486-516.
(3) (a) Pascal, R. A.; Spergel, J.; Engbersen, D. V. Tetrahedron Lett.
1986, 27, 4099-4102. (b) Kelley, T. R.; Kim, M. H. J. Am. Chem. Soc.
1994, 116, 7072-7080. (c) Antonisse, M. M. G.; Reinhoudt, D. N. Chem.
Commun. 1998, 443-448. (d) Chmielewski, M. J.; Charon, M.; Jurczak,
J. Org. Lett. 2004, 6, 3501-3504. (e) Ilioudis, C. A.; Tocher, D. A.; Steed,
J. W. J. Am. Chem. Soc. 2004, 126, 12395-12402. (f) Va´zquez, M.;
Fabbrizzi, L.; Taglietti, A.; Pedrido, R. M.; Gonza´lez-Noya, A. M.;
Bermejo, M. R. Angew. Chem., Int. Ed. 2004, 43, 1962-1965. (g) Choi,
K.; Hamilton, A. D. Coord. Chem. Rev. 2003, 240, 101-110. (h) Sessler,
J. L.; An, D.; Cho, W. S.; Lynch, V.; Marquez, M. Chem.-Eur. J. 2005,
11, 2001-2011. (i) Curiel, D.; Cowley, A.; Beer, P. D. Chem. Commun.
2005, 236-238.
(2) For selected recent progress in cation detection, see: (a) Yang,
R. H.; Chan, W. H.; Lee, A. W. M.; Xia, P. F.; Zhang, H. K.; Li, K. A.
J. Am. Chem. Soc. 2003, 125, 2884-2885. (b) Huang, H.; Wang, K.;
Tan, W.; An, D.; Yang, X.; Huang, S.; Zhai, Q.; Zhou, L.; Jin, Y. Angew.
Chem., Int. Ed. 2004, 43, 5635-5638. (c) Hanaoka, K.; Kikuchi, K.;
Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 12470-
12476. (d) Xu, Z.; Xiao, Y.; Qian, X.; Cui, J.; Cui, D. Org. Lett. 2005, 7,
889-892. (e) Chen, P.; Greenberg, B.; Taghavi, S.; Romano, C.; van
der Lelie, D.; He, C. Angew. Chem., Int. Ed. 2005, 44, 2715-2719.
10.1021/jo050913h CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/19/2005
7640
J. Org. Chem. 2005, 70, 7640-7646

Fluorescent Anion Sensor Derived from Cholic Acid
proximate C7 and C12 hydroxyl groups were function-
alized as carbamates serving as H-bond donors. Further-
more, to introduce the signal display element onto the
sensor scaffold, we placed anthracene moiety as a typical
emissive photoinduced electron transfer (PET) functional
unit on the molecule. Therefore, the “switch on-off”
property could be detected when the receptive binding
site interacts selectively with the guest molecules or
ions.7
Results and Discussion
On the basis of the aforementioned design strategy, 1
was obtained in moderate yield via a five-step synthetic
sequence. Its analogue 2, lacking the C7 and C12
carbamate groups, was assembled through a similar
procedure. Additionally, a typical C-3, 7, 12 tripodal
thiourea sensor 3 was also prepared for comparative
study. The synthetic routes are shown in Scheme 1.
Starting from commercially available starting material,
4 was treated with methyl acetate to protect the 3R-
hydroxyl group and carboxylic group of cholic acid.8 In
the presence of TMSCl, the 7R- and 12R-hydroxyl groups
of 4 reacted readily with phenylisocyanate to give 5 in
82% yield. Intending to prepare the carboxyl hydrazide,
we regenerated the 3R-hydroxyl group under basic condi-
tions, and the resultant hydroxyacid was then esterified
with methyl bromoacetate, affording 6. 6 was subse-
quently treated with excess hydrazine hydrate in metha-
nol to give 7 in 87% yield. The appendage of the
fluorescent moiety onto 7 was accomplished by its reac-
tion with 9-isothiocyanomethyl-anthracene to give the
adduct 1 in 54% yield. By a similar procedure, compound
2 was prepared in 32% overall yield as an analogue of 1
with hydroxyl groups on C-7 and C-12 being intact. On
the other hand, our synthetic route to 3 began with the
preparation of 3R-azido-methyl cholate 10 according to
the reported protocol developed by Davis’s group.5e After
conversion of the 7R- and 12R-hydroxyl groups of 10 to
biscarbamate by reacting with excess phenylisocyanate,
the azido group of resultant product 11 was reduced by
zinc powder to afford 3R-amino derivative. Treatment of
12 by 9-isothiocyanomethylanthracene in dichloromethane
at room temperature gave the corresponding thiourea 3
in 25% yield. All the new compounds and key intermedi-
ates were fully characterized by 1H NMR, 13C NMR, IR,
as well as HRMS.
FIGURE 1. Synthetic anion sensors 1, 2, and 3.
the facile synthesis of fluorescent anion sensors 1-3
(Figure 1) developed from cholic acid, which have strong
fluorescent response, high sensitivity, and specific selec-
tivity toward guest anions.
Davis made a seminal contribution in developing
cholate-based receptors, which promise to provide a
variety of excellent supramolecular properties.5 To obtain
high affinity and selectivity, the three axially oriented
functional groups on cholic acid (C3, C7, and C12) were
ingeniously assembled in such a way as to confer a
cooperative binding interaction to the guest anion. To the
best of our knowledge, in the literature, no attention has
been focused on mobilizing the flexible C17 side chain of
cholic acid as a chelating group. Examination of a
molecular model revealed that in cooperation with the
functionalized C7 and C12 pending ligating groups, the
template effect provided by a potential anion via three
point interactions may trigger the creation of an anionic
receptive site with the crucial participation of the C17
functionalized flexible side chain. Attempting to make
use of the flexible functional receptive site on C17 and
to investigate its potential cooperation effect with C7 and
C12 in designing sensor 1, we built an effective anion
receptive amidothiourea group on the C24 carboxyl group
of cholic acid.6 To demonstrate the focused chelating
ability of the flexible C17 side chain, the C3 hydroxyl
group was deliberately kept intact while the more
Since these sensors are designed as H-bond based
receptors, acetonitrile lacking the ability to form H-bond
with sensors was chosen as the solvent in the recognition
investigation. In fluorescence study, 1, 2, and 3 were
excited at 366 nm and gave out a characteristic emission
maximum at ca. 413 nm. All the fluorescent emissions
of 1, 2, and 3 can be quenched by different anions in the
form of tetraalkylammonium salts without significant
(4) (a) Nishizawa, S.; Kaneda, H, Uchida, T.; Teramae, N. J. Chem.
Soc., Perkin Trans. 2 1998, 2325-2327. (b) Miyaji, H.; Sato, W.; Sessler,
J. L. Angew. Chem., Int. Ed. 2000, 39, 1777-1780. (c) Wu, J. L.; He,
Y. B.; Zeng, Z. Y.; Wei, L. H.; Meng, L. Z.; Yang, T. X. Tetrahedron
2004, 60, 4309-4314. (d) Lee, D. H.; Kim, S. Y.; Hong, J. I. Angew.
Chem., Int. Ed. 2004, 43, 4777-4780. (e) Lee, J. Y.; Kim, S. K.; Jung,
J. H.; Kim, J. S. J. Org. Chem. 2005, 70, 1463-1466.
(5) (a) Davis, A. P.; Perry, J. J.; Williams, R. P. J. Am. Chem. Soc.
1997, 119, 1793-1794. (b) Ayling, A. J.; Perez-Payan, M. N.; Davis,
A. P. J. Am. Chem. Soc. 2001, 123, 12716-12717. (c) Siracus, L.;
Hurley, F. M.; Dresen, S.; Lawless, J.; Perez-Payan, M. N.; Davis, A.
P. Org. Lett. 2002, 4, 4639-4642. (d) Davis, A. P.; Joos, J. B. Coord.
Chem. Rev. 2003, 240, 143-156. (e) Lawless, L. J.; Blackburn, A. G.;
Ayling, A. J.; Perez-Payan, M. N.; Davis, A. P. J. Chem. Soc., Perkin
Trans. 1 2001, 1329-1341.
(6) (a) Wu, F. Y.; Li, Z.; Wen, Z. C.; Zhou, N.; Zhao, Y. F.; Jiang, Y.
B. Org. Lett. 2002, 4, 3203-3205. (b) Nie, L.; Li, Z.; Han, J.; Zhang,
X.; Yang, R.; Liu, W. X.; Wu, F. Y.; Xie, J. W.; Zhao, Y. F.; Jiang, Y. B.
J. Org. Chem. 2004, 69, 6449-6454. (c) Zeng, Z. Y.; He, Y. B.; Wu, J.
L.; Wei, L. H.; Liu, X.; Meng, L. Z.; Yang, X. Eur. J. Org. Chem. 2004,
2888-2893.
(7) (a) Gunnlaugsson, T.; Davis, A. P.; Glynn, M. Chem. Commun.
2001, 2556-2557. (b) Gunnlaugsson, T.; Davis, A. P.; O’Brien, J. E.;
Glynn, M. Org. Biomol. Chem. 2005, 3, 48-56.
(8) Davis, A. P.; Perez-Payan, M. N. Synlett 1999, S1, 991-993.
J. Org. Chem, Vol. 70, No. 19, 2005 7641

Fang et al.
SCHEME 1. Synthetic Route for 1, 2, and 3
change in their UV-visible absorption spectra (Figure
2a). The observed fluorescent quench could be attributed
to the typical guest-induced PET process.7a Upon anion
coordination with the sensor, the reduction potential of
binding moieties (i.e., thiourea or amidothiourea group)
would increase, leading to the enhancement of a PET
effect, which is responsible for the emission quenching
of compounds 1, 2, and 3.1a,7 Upon adding 5% water to
the work solution, all the fluorescent responses are
diminished because of water’s propensity to act as a
strong H-bond donating competitor.
Quantitative investigations of the binding behaviors
of 1, 2, and 3 were performed with different anions in
acetonitrile by means of titration fluorimetry. On the
basis of the change of fluorescent intensity in stepwise
adding guest anions, the complex association constants
(Ka) were calculated using nonlinear least-squares curve
fitting9 (Table 1). Excellent fitting (R > 0.99) of the
titration data points shown in Figure 3 show that 1, 2,
and 3 invariantly form 1:1 complexes with carboxylates,
dihydrogen phosphate, or bromide.6a,9,10 To bind different
carboxylates, the affinity of 1, 2, or 3 is correlated well
with their basicity. The sequence of the Ka values
(propionate > acetate > benzoate > lactate) reflects the
decreasing intrinsic basicity of the respective carboxy-
lates. Chloride was claimed to be a compatible anion that
can be fitted in the cholate cavity; however, in this study,
except for a slight fluorescence enhancement of 1, we
could not observe significant change in emission intensity
of the sensors upon adding excess amount of chloride.
Similar to the response of chloride, bromide can only
increase the fluorescent intensity of 1 without changing
emission spectra of 2 and 3. To make a complete study
on halide ions, iodide titrations were also conducted. It
was found that all the sensors exhibited no fluorescent
response to a moderate concentration of iodide (cI < 10-3
mol/L). However, when cI was higher than 10-3 mol/L,
the heavy atom effect of iodine became apparent and the
fluorescent intensities of 1, 2, and 3 were all quenched
by iodide via intersystem crossing.11
(9) Valeur, B.; Pouget, J.; Bourson, J.; Kaschke, M.; Ernsting, N. P.
J. Phys. Chem. 1992, 96, 6545-6549.
(10) Bernard, V. Molecular Fluorescence: Principles and Applica-
tions; Wiley-VCH: Weinheim, Germany, 2002.
7642 J. Org. Chem., Vol. 70, No. 19, 2005

Fluorescent Anion Sensor Derived from Cholic Acid
action with amidothiourea and thiourea H-bond donors.
The stoichiometrical ratio of sensor and F- was deter-
mined to be 2:1 by Job plot experiment when 1 or 2 was
titrated by fluoride (Figure 3) while a 1:1 ratio was found
in titration of 3 and F-. Such different binding behaviors
could be originated from the variation of the N-H bond
acidity of thiourea and amidothiourea. The more acidic
amidothiourea subunit tends to undergo an advanced
stage of proton transfer after typical H-bond addition.12
Such stepwise equilibria, illustrated by the equation
below where LH ) 1 or 2, consume two fluoride ions for
each amidothiourea-methylene anthracene moiety, giv-
ing rise to a substantial PET quenching.
r
[LH‚F]- + F- 98L- + [HF2]-
Comparison between the binding properties of 1 and
2 toward other different anions gave us a clear under-
standing on the nature of the binding. For trigonal planar
carboxylates and tetrahedral H2PO4-, their respective
binding constants with 1 and 2 are on the same magni-
tude, indicating that the H-bond interaction only occurs
on amidothiourea group, while for spherical anions such
as fluoride and bromide, the presence of the 7R- and 12R-
carbamate groups in 1 is apparently essential. Even the
second-order association constants of 1‚F- and 2‚F- are
too large to calculate; a much acuter response of 1 toward
fluoride in the titration curves indicates that 1 boasts
much higher affinity toward fluoride than 2 does (Figure
3a). Moreover, fluorescent intensity of 1 can be drastically
enhanced by bromide while such a phenomenon turns to
be marginalized on 2 under the same experimental
condition. It is clear that cooperation effect between the
C7, C12 carbamates and the amidothiourea on C17 side
chain led to the large difference between 1 and 2 in halide
binding ability. Although fluoride demonstrated a 2:1
coordination behavior, there is no doubt that the presence
of a multi H-bond donor in 1 would stabilize the forma-
tion of the initial [LH‚F]- complex and thus facilitate the
advanced deprotonation. For 1, the free rotating C17 side
chain donating H-bond could be assembled with the C7,
C12 carbamates under anionic template effect to achieve
more stable three point coordination13 without costing
much energy. Such assembly could only happen on
spherical halide anions on which negative charge is
distributed symmetrically on the surface, while for car-
FIGURE 2. Fluorescence response of 1 (5 µM) by adding (a)
TBA acetate and (b) TBA bromide in acetonitrile. λex ) 366
nm. Inset is the titration data point and the nonlinear least-
squares fitting curve.
To each of the tested anions, compounds 1 and 2
showed much larger binding constants than that of
compound 3, indicating that amidothiourea group has
higher H-bond donating capacity (stronger acidity) than
thiourea does. Reminiscent of the findings of others, the
intramolecular hydrogen bond present in 1 could enhance
the planarity of the amidothiourea group leading to a
stronger binding.6a,b Moreover, as one of the strongest
H-bond acceptors, F- exerts essentially a different inter-
-
boxylates and dihydrogen phosphate, the CO2- and PO2
subunits serving as directional H-bond acceptor can
hardly interact with additional H-bond donor after
TABLE 1. Association Constants (Ka) of Coordination between 1, 2, and 3 with Different Anions (M-1
anions
)
1
2
3
fluoride
chloride
bromide
iodide
a
a
(2.07 ( 0.38) × 104
negligible quenchingb
negligible quenchingb
8% enhancementb
(3.67 ( 0.38) × 103
no response
no response
no response when cI < 10-3 mol/L,
heavy atom quenching when cI > 10-3 mol/L
(3.11 ( 0.17) × 104
dihydrogen phosphate
propionate
acetate
benzoate
lactate
(2.72 ( 0.17) × 104
(5.59 ( 0.47) × 104
(4.13 ( 0.30) × 104
(6.12 ( 0.58) × 103
626 ( 20
(1.82 ( 0.32) × 103
(5.93 ( 1.24) × 104
(1.18 ( 0.12) × 104
475 ( 46
(1.60 ( 0.15) × 105
(7.69 ( 0.97) × 104
(7.10 ( 0.67) × 103
381 ( 46
no response
a The second-order association constant was too large to be calculated. b The associate constant could not be calculated precisely because
the signal change was too low to provide reliable data with tolerable error.
J. Org. Chem, Vol. 70, No. 19, 2005 7643

Fang et al.
FIGURE 3. (a) Fluorescence quench of 1 (5 µM) and 2 (5 µM) by titrating F-. (b) The Job plot of 1 and F- indicating 2:1 coordination
(x is the mole fraction of 1).
coordination with amidothiourea. This presumption was
further substantiated by 1H NMR study of 1 in the
presence and absence of different anions in CD3CN. The
N-H hydrogen bond signals of the free host 1 were
masked by the aromatic protons in 6.8-8.5 ppm. After
addition of 10 equiv of tetrabutylammonium acetate, only
two new peaks were found in the downfield (i.e., 11.3-
11.7 ppm), which were ascribed to anion-coordinated
amidothiourea N-H protons. In contrast, addition of 10
equiv of tetrabutylammonium chloride or bromide led to
the appearance of four new peaks in 8.7-10.1 ppm. These
new signals should be ascribed to the combination of
N-H signals of amidothiourea and carbamates that
underwent a cooperative binding of chloride or bromide.14
It is interesting to find that Br-, which is rarely
reported as a PET quencher, could enhance the emission
intensity of 1 for as much as 60% in acetonitrile (Figure
2b). But such phenomenon could not be observed on 3 or
on the analogue compound 2 under the same experimen-
FIGURE 4. Titration curves of 1 (5 µM) and Br- in different
solvents.
tal conditions. Moreover, the observed bromide-induced
fluorescent enhancement of 1 became much weaker when
the experiment was undertaken in other solvent such as
in charge than fluoride and oxygen anions; as a result,
they can hardly change the reduction potential of ami-
dothiourea even upon binding. The effect of chloride on
inducing quenching on 1 via PET may be comparable to
that of CH3CN, and thus only 8% emission enhancement
of 1 was detected upon binding with chloride. When CH3-
CN is replaced by the less negative bromide ion, the pre-
existed PET process due to the polar solvent will be
erased. As a result, an enhancement of fluorescence
intensity was observed.
CH2Cl2 and toluene (Figure 4). Conceivably, amidothio-
urea, being a powerful receptive site for anion, could
coordinate the polar CH3CN solvent molecules triggering
considerable PET fluorescent quenching on anthracene
moiety.15 We believe that compound 1 does coordinate
with Cl- and Br- via cooperative multi H-bond donors.
Upon addition of chloride or bromide, due to their
stronger binding affinity to 1 via multi H-bonding
interaction, they could readily break the CH3CN solvation
to form stable complexes with the host. It is noteworthy
that chloride and bromide anions are much less negative
Conclusion
(11) Shimizu, Y.; Azumi, T. J. Phys. Chem. 1982, 86, 22-26.
(12) (a) Boiocchi, M.; Boca, L. D.; Gomez, D. E.; Fabbrizzi, L.;
Licchelli, M.; Monzani, E. J. Am. Chem. Soc. 2004, 126, 16507-16514.
(b) Amendola, V.; Boiocchi, M.; Fabbrizzi, L.; Palchetti, A. Chem.-
Eur. J. 2005, 11, 120-127.
In summary, we have developed a novel efficient anion-
detecting system via an effective synthetic route to
construct H-bond donor group basing on cholic acid
scaffold. Extensive study of these synthetic sensors’
fluorescence response to different anions demonstrated
their excellent selectivity and affinity toward negatively
charged substrates. Rationally designed experiments
manifested the mechanism for fluorescent change of the
(13) Hettche, F.; Reiss, P.; Hoffmann, R. W. Chem.-Eur. J. 2002,
8, 4946-4956.
(14) For NMR spectra of 1 in the presence and absence of bromide
or acetate, see the Supporting Information.
(15) Okino, T.; Hoashi, Y.; Furukawa, T.; Xu, X.; Takemoto, Y. J.
Am. Chem. Soc. 2005, 127, 119-125.
7644 J. Org. Chem., Vol. 70, No. 19, 2005

Fluorescent Anion Sensor Derived from Cholic Acid
153.0, 174.6. FAB MS (m/z): [M + H] + ) 661.4. HRMS: calcd
chemosensor showing supramolecular cooperation among
the flexible C17 side chain, the C7, and the C12 axially
oriented arms of the molecule. The preorganized C3
functional site could be utilized to construct an additional
binding site. The design of ion pair chemosensors along
these tactics has been underway.
for C38H52N4O6, 683.3784; found, 683.3780.
Amidothiourea 1. A mixture of 7 (130 mg, 0.2 mmol) and
9-isothiocyanomethylanthracene (49 mg, 0.2 mmol) was stirred
in dry dichloromethane (12 mL) at room temperature for 48
h. Column chromatography (SiO2) eluted by the solvent of
hexane/ethyl acetate/dichloromethane (1:1.5:1) gave the pure
product 1 as a pale yellow solid (99 mg, 54%): mp ∼178-182
°C. 1H NMR (270 MHz, CDCl3): δ 0.56-0.60 (m, 6H), 0.92 (s,
3H), 3.47 (m, 1H), 4.89 (s, 1H), 4.96 (s, 1H), 5.52 (s, 2H), 6.96-
7.45 (m, 14H), 7.94 (d, 2H, J ) 7.8 Hz), 8.09 (d, 2H, J ) 8.3
Hz), 8.38 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 12.2, 17.3,
22.4, 22.7, 25.8, 26.7, 28.9, 29.7, 30.2, 31.4, 34.2, 34.4, 34.8,
37.9, 38.3, 40.8, 41.5, 43.3, 45.1, 46.8, 71.7, 76.0, 118.5, 119.1,
123.2, 123.4, 123.6, 125.2, 126.7, 128.129.0, 129.1, 130.3, 131.2,
138.2, 138.4, 153.3, 153.4, 170.8, 180.2. IR (KBr): 3287, 2950,
2868, 1726, 1705, 1600, 1533, 1443, 1313, 1225 cm-1. FAB MS
Experimental Section
Methyl 3r-Acetoxy-7r,12r-bis(phenylaminocarbonyl-
oxy) Cholate (5). To a solution of 4 (260 mg, 0.56 mmol) being
stirred in freshly distilled dry dichloromethane, a drop of
trimethylsilane chloride and redistilled phenylisocyanate (123
µL, 1.1 mmol) was added at room temperature. After being
stirred for 24 h, another 123 µL of phenylisocyanate was
injected to the solution. The solid was filtered out after 4 had
disappeared on TLC. Removing of solvent was followed by flash
column chromatography (SiO2) eluted by petroleum ether and
ethyl acetate (3:1) that gave the crude product. The product
was taken up by small amount of dichloromethane, and the
insoluble impurity was filtered out. Compound 5 was obtained
after removing of dichloromethane (322 mg, 82%): mp 115-
+
(m/z): [M + H] ) 910.5. MALDI TOF HRMS: calcd for
C54H63N5O6SNa, 932.4397; found, 932.4354.
Methoxycarbonylmethly Cholate (8). To a mixture of
cholic acid (613 mg, 1.5 mmol) and potassium carbonate (414
mg, 3 mmol) being stirred in dry DMF (20 mL), methyl
bromoacetate (156 µL, 1.7 mmol) was added by microsyringe.
After reacting at 60 °C for 15 h, the mixture was poured into
30 mL of saturated ammonium chloride solution and then
extracted by ethyl acetate (3 × 30 mL). Product 8 was obtained
as a white foam after drying and removal of organic solvent
1
118 °C. H NMR (270 MHz, CDCl3): δ 0.77 (s, 3H), 0.89 (d,
3H, J ) 6.2 Hz), 0.95 (s, 3H), 1.99 (s, 3H), 3.62 (s, 3H), 4.59
(m, 1H), 4.95 (s, 1H), 5.12 (s, 1H), 6.67 (br, 1H), 6.75 (br, 1H),
7.06-7.13 (m, 2H), 7.31-7.48 (m, 8H). 13C NMR (100 MHz,
CDCl3): δ 12.3, 17.5, 21.5, 22.5, 22.9, 25.8, 26.8, 27.1, 29.0,
30.7, 30.9, 31.5, 34.4, 34.6, 34.7, 37.9, 40.8, 43.6, 45.3, 47.4,
51.5, 71.9, 74.1, 118.6, 123.5, 129.0, 138.0, 152.9, 153.1, 170.3,
174.6. FAB MS (m/z): [M]+ ) 702.2; [M + H]+ 703.2. HRMS:
calcd for C41H54N2O8Na, 725.3777; found, 725.3775.
1
(700 mg, 97%): mp 108-111 °C. H NMR (400 MHz, CDCl3):
δ 0.67 (s, 3H), 0.88 (s, 3H), 0.99 (d, 3H, J ) 6.0 Hz), 3.43 (m,
1H), 3.76 (s, 3H), 3.84 (s, 1H), 3.96 (s, 1H), 4.61 (s, 2H). 13C
NMR (100 MHz, CDCl3): δ 12.5, 17.3, 22.5, 23.2, 26.4, 27.4,
28.2, 30.4, 30.7, 34.6, 34.7, 39.5, 39.6, 41.4, 41.7, 46.4, 47.0,
52.2, 60.4, 68.4, 71.9, 73.0, 173.6. FAB MS: m/z ) 481.7.
Methoxycarbonylmethyl 7r,12r-Bis(phenylaminocar-
bonyloxy) Cholate (6). 5 (250 mg, 0.35 mmol) was added
into a reflux solution of sodium hydroxide (99 mg, 1.8 mmol)
in 20 mL of methanol. After being stirred for 1 h, the mixture
was cooled to room temperature and stirred overnight. The
alkali solution was then neutralized by saturated ammonium
chloride solution. Removal of methanol under reduced pressure
was followed by extraction of aqueous solution by ethyl acetate.
The organic layer was dried, and the solvent was removed to
give deprotected bis-carbamate. The crude product was dis-
solved in dry DMF (15 mL) in the presence of potassium
carbonate (97 mg, 0.7 mmol). Methyl bromoacetate (33 µL, 0.35
mmol) was then added by microsyringe. After reacting at 70
°C for 15 h, the mixture was poured into 30 mL of saturated
ammonium chloride solution and then extracted by three
portions of ethyl acetate. Product 6 was obtained as white foam
after drying and removal of organic solvent (185 mg, 74%).
Cholic Hydrazide (9). The mixture of 8 (240 mg, 0.5 mmol)
and hydrazine monohydrate (0.5 mL, large excess) being
stirred in methanol (15 mL) was heated to 50 °C for 12 h.
Methanol was then removed, and the residue was purified by
flash column chromatography eluted by the mixed solvent of
CH3OH/CH2Cl2 (1:3) to get crude product 9 (162 mg, 77%). An
analytical sample was obtained by running another column
1
chromatography: mp 172-177 °C. H NMR (400 MHz, CD3-
OD): δ 0.70 (s, 3H), 0.91 (s, 3H), 1.02 (d, 3H, J ) 6.4 Hz), 3.36
(m, 1H), 3.79 (d, J ) 2.8 Hz, 1H), 3.94 (s, 1H). 13C NMR (100
MHz, CD3OD): δ 13.1, 17.7, 23.3, 24.3, 28.0, 28.8, 29.7, 31.3,
32.1, 33.2, 35.9, 36.0, 36.6, 37.0, 40.6, 41.1, 43.1, 43.3, 47.6,
48.1, 69.1, 73.0, 74.1, 176.0. IR (KBr): 3454, 2941, 2863, 1636,
1594, 1076 cm-1. TOF HRMS: calcd for C24H42N2O4Na,
445.3042; found, 445.3041.
1
Amidothiourea 2. A mixture of 9 (180 mg, 0.45 mmol) and
9-isothiocyanomethylanthracene (110 mg, 0.45 mmol) was
stirred in dry dichloromethane (12 mL) at room temperature
for 48 h. Column chromatography (SiO2) eluted by the solvent
of (CH2Cl2/methanol, 5:1) gave the pure product 2 as pale
yellow solid (130 mg, 43%): mp 185-190 °C. 1H NMR (400
MHz, CDCl3): δ 0.46 (s, 3H), 0.58-0.78 (m, 9H), 3.18 (m, 1H),
3.66 (s, 1H), 3.74 (s, 1H), 5.61 (s, 2H), 7.42-7.49 (m, 4H), 7.94
(d, 2H, J ) 7.6 Hz), 8.13-8.23 (m, 2H), 8.38 (s, 1H), 9.21 (br,
1H). 13C NMR (100 MHz, CDCl3): δ 12.1, 17.2, 18.0, 22.3, 22.6,
23.0, 26.1, 27.2, 27.6, 30.0, 30.5, 34.5, 34.8, 35.2, 39.3, 41.2,
41.6, 45.3, 46.0, 68.3, 71.6, 73.0, 122.2, 123.7, 124.1, 125.2,
126.7, 127.6, 128.2, 128.9, 130.6, 131.3, 146.5, 181.4. IR
mp 114-122 °C. H NMR (270 MHz, CDCl3): δ 0.78 (s, 3H),
0.86-0.93 (m, 9H), 3.52 (m, 1H), 3.73 (s, 3H), 4.57 (d, 2H, J )
2.4 Hz), 4.96 (s, 1H), 5.13 (s, 1H), 6.84 (br, 1H), 6.91 (br, 1H),
7.04-7.09 (m, 2H), 7.29-7.48 (m, 8H). 13C NMR (100 MHz,
CDCl3): δ 12.3, 14.1, 17.5, 22.6, 22.9, 26.0, 27.2, 27.7, 28.2,
29.4, 30.5, 31.1, 31.5, 34.6, 34.8, 35.4, 35.8, 37.9, 43.6, 45.4,
47.4, 52.2, 60.4, 66.5, 72.4, 118.6, 123.5, 129.0, 129.1, 138.0,
153.1, 168.4, 173.4. FAB MS (m/z): [M + H] + ) 718.3. MALDI
TOF HRMS: calcd for C41H54N2O9Na, 741.3727; found,
741.3703.
7r,12r-Bis(phenylaminocarbonyloxy) Cholic Hydrazide
(7). The stirring mixture of 6 (170 mg, 0.24 mmol) and
hydrazine (0.3 mL, large excess) monohydrate in methanol (10
mL) was heated to 50 °C for 12 h. Methanol was then removed,
and the residue was taken up by ethyl acetate. The organic
solution was first washed by saturated ammonium chloride
solution (3 × 30 mL) and then dried by sodium sulfate. Pure
product 7 was obtained after removal of organic solvent (137
mg, 87%): mp 170-175 °C. 1H NMR (270 MHz, CDCl3): δ 0.76
(s, 3H), 0.87-0.92 (m, 9H), 3.51 (m, 1H), 4.94 (s, 1H), 5.12 (s,
1H), 7.04-7.11 (m, 2H), 7.29-7.48 (m, 8H). 13C NMR (100
MHz, CDCl3): δ 12.3, 14.1, 17.6, 22.6, 22.9, 26.0, 27.2, 27.7,
28.2, 29.4, 30.7, 30.9, 31.2, 31.6, 34.7, 34.9, 35.4, 35.8, 37.9,
43.6, 45.4, 47.4, 51.5, 66.5, 72.5, 118.7, 123.5, 129.1, 138.0,
(KBr): 3414, 2926, 2863, 1684, 1644, 1525, 1446, 731 cm-1
.
FAB MS (m/z): [M + H]+ ) 672.2. MALDI TOF HRMS: calcd
for C40H53N3O4SNa, 694.3654; found, 694.3658.
Methyl 3r-Azido-7r,12r-bis(phenylaminocarbonyloxy)
Cholate (11). To a solution of 10 (223 mg 0.5 mmol) being
stirred in freshly distilled dry dichloromethane (12 mL), a drop
of trimethylsilane chloride and redistilled phenylisocyanate
(110 µL, 1 mmol) was added at room temperature. After being
stirred for 24 h, another 110 µL of phenylisocyanate was
injected to the solution. The solid was filtered out after 10 had
disappeared on TLC. Removal of solvent was followed by
J. Org. Chem, Vol. 70, No. 19, 2005 7645

Fang et al.
column chromatography (SiO2) that gave the crude product.
The product was taken up by a small amount of dichlo-
romethane, and the insoluble impurity was filtered out.
Compound 11 was obtained after removal of dichloromethane
(263 mg, 77%): mp 82-90 °C. 1H NMR (270 MHz, CDCl3): δ
0.78 (s, 3H), 0.89 (d, 3H, J ) 5.9 Hz), 0.95 (s, 3H), 3.18 (m,
1H), 4.95 (s, 1H), 5.13 (s, 1H), 6.66 (br, 1H), 6.73 (br, 1H), 7.07-
7.12 (m, 2H), 7.32-7.47 (m, 8H). 13C NMR (100 MHz, CDCl3):
δ 12.2, 17.5, 22.9, 24.6, 25.9, 27.2, 28.5, 29.7, 30.1, 30.7, 30.9,
31.1, 32.4, 34.7, 36.2, 37.9, 43.5, 47.4, 51.5, 58.2, 72.3, 118.6,
123.6, 129.0, 129.1, 137.8, 137.9, 153.0, 174.6. FAB MS: m/z
) 685.6 (M). MALDI TOF HRMS: calcd for C39H51N5O6Na,
708.3737; found, 708.3765.
153.8, 174.3. IR (KBr): 3376, 3266, 2947, 1727, 1537, 1218
cm-1. TOF HRMS: calcd for C55H64N4O6NaS, 931.4444; found,
931.4456.
Fluorescent Response Study. All the host compounds 1,
2, and 3 were prepared as stock solutions in acetonitrile for
∼2-5 × 10-4 mol/L. Except benzoate and lactate, whose
countercation were tetramethylammonium salt, all other
anions used in this report were in the form of tetrabutylam-
monium salt (fluoride used in this report was originally 75 wt
% water solution of tetrabutylammonium fluoride). They were
prepared to approximately 0.01 mol/L and ∼2-5 × 10-3 mol/L
of stock solutions in acetonitrile. The work solutions were
prepared by adding different volumes of anion solution to a
series of test tubes followed by dilution to 5 mL by acetonitrile.
Then, the same amount of stock solution of the host compound
was added into each of the test tubes. After being shaken for
several minutes, the work solutions could be measured im-
mediately.
Methyl 3r-Amino-7r,12r-bis (phenylaminocarbonyl-
oxy) Cholate (12). Activated zinc powder (400 mg, 6.2 mmol)
was added to a solution of 11 (220 mg, 0.32 mmol) in glacial
acetic acid (10 mL). The mixture was stirred vigorously for 24
h. Acetic acid was then completely removed under reduced
pressure by adding toluene several times. The residue, acetate
ammonium salt of 12, was dissolved by saturated sodium
chloride solution (10 mL). Basification of the solution by
excessive triethylamine and extraction of it by ethyl acetate
gave amine 12 as the pure product. After being dried by
sodium sulfate and removal of organic solvent, compound 12
was obtained as white foam (185 mg, 99%): mp 92-96 °C. 1H
NMR (400 MHz, CDCl3): δ 0.76 (s, 3H), 0.82-0.92 (m, 6H),
2.67 (m, 1H), 4.93 (s, 1H), 5.13 (s, 1H), 7.02-7.07 (m, 2H),
7.28-7.50 (m, 8H). 13C NMR (100 MHz, CDCl3): δ 12.3, 14.1,
17.6, 22.6, 22.9, 25.8, 27.2, 28.9, 30.7, 30.9, 31.6, 34.4, 34.7,
35.3, 38.0, 41.0, 43.5, 45.3, 47.4, 51.1, 51.5, 118.7, 123.1, 123.3,
1:1 Association constants of 1, 2, and 3 with anions are
calculated by nonlinear least-squares curve fitting using the
following equation in Origin 6.0:
Ilim/I0 - 1
CA
1
I/I0 ) 1 +
1 +
+
-
[
2
CH KsCH
2
CA
CA
CH
1
1 +
+
- 4
(
)
]
CH KsCH
x
where I0 is fluorescent intensity of host without anions, Ilim is
fluorescent intensity reaching a limitation by adding excessive
anions, CA is the concentration of anions added, and CH is the
concentration of host molecule.
129.0, 138.2, 138.5, 153.1, 174.6. FAB MS (m/z): [M + H]+
660.3.
)
By allowing 1/KaCH and Ilim/I0 to be floating parameters, we
can obtain the Ka and Ilim/I0 values by a nonlinear least-squares
analysis of I/I0 versus CA/CH.
Thiourea 3. 12 (180 mg, 0.27 mmol) and 9-isothiocyano-
methylanthracene (68 mg, 0.27 mmol) were dissolved in freshly
distilled dry dichloromethane (12 mL) and stirred for 20 h.
The reaction could not finish completely. The mixed product
was purified by column chromatography (SiO2) eluted by
dichloromethane first and then by dichloromethane/ethyl
acetate ) 8:1 to give 3 as a pale yellow powder (63 mg, 25%):
Acknowledgment. The work described in this ar-
ticle was fully supported by grants from the University
Grants Committee of the Hong Kong Special Adminis-
trative Region, China (AoE/P-10/01), and from the Hong
Kong Baptist University (FRG/03-04/II-15).
1
mp 129-133 °C. H NMR (400 MHz, CDCl3): δ 0.75 (s, 3H),
0.86 (s, 3H), 0.90 (d, 3H, J ) 6.4 Hz), 3.07 (m, 1H), 3.31 (s,
3H), 4.95 (s, 1H), 5.29 (s, 1H), 5.55 (dd, 2H, J1 ) 13.8 Hz, J2
) 5.4 Hz), 6.89-7.59 (m, 17H), 7.92 (d, 2H, J ) 11.6 Hz), 8.39
(s, 1H), 9.05 (br, 1H), 9.16 (br, 1H). 13C NMR (100 MHz,
CDCl3): δ 12.5, 17.6, 22.3, 22.8, 26.2, 27.1, 29.1, 30.4, 30.5,
30.9, 31.3, 33.8, 34.6, 35.1, 38.0, 40.9, 42.5, 43.6, 45.1, 47.1,
51.1, 52.8, 70.6, 75.2, 77.2, 118.2, 118.9, 122.8, 123.1, 125.3,
126.1, 127.1, 128.7, 128.8, 129.0, 130.4, 131.2, 138.7, 153.1,
Supporting Information Available: Compound charac-
terization spectra and fluorometric titration figures. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO050913H
7646 J. Org. Chem., Vol. 70, No. 19, 2005