Synthesis of cholate-based pyridinium receptor and its recognition toward L-tryptophan

Article abstract of DOI:10.1016/j.tet.2013.12.068

Four cholate-based pyridinium compounds were synthesized and their binding abilities toward unmodified amino acids were investigated by UV spectroscopy and fluorescence emission spectroscopy. Studies revealed that the recognition process involved hydrogen bonding, electrostatic force, and π-π interaction. The receptor 4a was found to recognize l-tryptophan specifically, and the complex was studied by ¹H NMR spectroscopy. The receptors 4b and 4c showed very little recognition ability toward l-tryptophan, indicating the important role of the benzyl group at pyridinium ring.

Full text of DOI:10.1016/j.tet.2013.12.068

Tetrahedron 70 (2014) 1223e1229
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of cholate-based pyridinium receptor and its recognition
toward
L
-tryptophan
*
Dazhi Li, Yunxu Yang , Chao Yang, Biwei Hu, Baolong Huo, Lingwei Xue, Aizhi Wang,
Feifei Yu
Department of Chemistry and Chemical Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing,
Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2013
Received in revised form 16 December 2013
Accepted 24 December 2013
Available online 31 December 2013
Four cholate-based pyridinium compounds were synthesized and their binding abilities toward un-
modified amino acids were investigated by UV spectroscopy and fluorescence emission spectroscopy.
Studies revealed that the recognition process involved hydrogen bonding, electrostatic force, and
pep
interaction. The receptor 4a was found to recognize L-tryptophan specifically, and the complex was
studied by 1H NMR spectroscopy. The receptors 4b and 4c showed very little recognition ability toward
L-
tryptophan, indicating the important role of the benzyl group at pyridinium ring.
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Molecular recognition
Hydrogen bonding
Conformation
Host
Amino acid
1. Introduction
the molecular structure of amine acid coexists with hydrogen bond
donor (eNH2) and hydrogen bond acceptor (eCOOꢀ). The carbox-
ylate in the molecule head also results in high negative charge
under physiological conditions. Taking the detailed structural in-
formation into consideration, we tried to design a water-soluble,
tweezer-like derivate of cholic acid for the recognition. Finally,
a polar benzyl pyridinium 4a featuring cholate groups that contains
backbones of concave hydrophilic face and convex hydrophobic
face, has been synthesized in our present work. The receptor would
provide positive center to bind with the negatively charged amine
acid via electrostatic interaction, and provide a benzyl group that
Among the numerous kinds of molecular recognition, amino
acid is one of the most preferred targets for molecular recognition
due to its central role in natural living systems. In trying to evaluate
the underlying intermolecular forces between amino acids and
other reciprocal artificial host compounds, many efforts have been
expended. It has been found that non-covalent forces, such as hy-
drogen bonding, pep stacking, and hydrophobic interactions pro-
vide the selectivity observed in recognition process.1 But most of
the investigated recognitions require derivation of amino acids,
such as methyl esterification2 and quaternization,3 due to the low
solubility in organic solvent. For this reason, neither under their
original forms involved in life process nor under appropriate
aqueous solution could be studied for most of amino acids.
could probably induce the
pep stacking with substrates. Mean-
while, three corresponding compounds 4b, 4c, and 6 were also
synthesized as model hosts to illustrate the integrant of benzyl
group and the importance of tweezer-like polycholate backbones
applied in multipoint interactions in our design.
In recent years, supramolecules containing cholate groups have
received much attention2d,4 for their good performance in molec-
ular recognition. Under changes of general environmental, the
cholate derivates are able to respond with conformational change,
which likely lead to molecular complementarity. Thus, the supra-
molecules can recognize the guests with its biding sites in a par-
ticular environment, such as specific pH, solvent polarity,
temperature etc. by one main conformation.5 On the other hand,
2. Results and discussion
2.1. Synthesis
3-Amino methyl cholate 2 was prepared from the start material
of cholic acid according to the literature procedures6 and then
reacted with 3,5-dinicotinic acid or 3-nicotinic acid under the
presence of benzotriazol-1-yloxytris(dimethylamino)phospho-
nium hexafluorophosphate (BOP) to give dinicotinamide 3 or
* Corresponding author. E-mail address: yxyang63@aliyun.com (Y. Yang).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.12.068

1224
D. Li et al. / Tetrahedron 70 (2014) 1223e1229
monomer 5. Hosts 4aec were obtained by quaternized 3 with
benzyl bromide, 9-(bromomethyl)-anthracene, and methyl iodide,
respectively. Nicotinamide 5 was quaternized with benzyl bromide
to give host 6.
2.2. Binding studies of 4a with amino acids
2.2.1. UVevis absorption and fluorescence binding studies of 4a with
amino acids. Nineteen
a-amino acids (as shown in Fig. 1, re-
spectively) were used to evaluate the binding properties of 4a in
water/ethanol (1/1, v/v, phosphate buffer solution (PBS), 5 mM,
pH¼7.2).4f,7 The fluorescence spectra were obtained by excitation of
4a at 247 nm. In order to make 4a fully conjugate with guests, amino
acids were added in tenfold excess to 4a. As shown in Fig. 1, 4a
showed an originally maximum absorption peak at 204 nm
(log ε¼4.49) and a minor broad peak at 341 nm (log ε¼3.88). After
addition of amino acids to the system of 4a, the main absorption
reduced and accompanied with a slightly red-shift at 204 nm; at
341 nm only a little decrease of the absorption occurred. But the
maximum absorption changes of 4a were quite obvious after adding
Fig. 2. The emission spectra of solution of 4a (5.0ꢁ10ꢀ5 M) under water/ethanol (1/1,
v/v) phosphate buffer solution (5 mM, pH¼7.2) upon addition of different amino acids
(5.0ꢁ10ꢀ4 M), lex¼247 nm.
L
-phe,
a significant red-shift (Dlmax¼34 nm for
dicated that the
L
-His,
L
-Tyr,
L
-Trp, which reduced to almost a half and gave
-Trp). These results in-
pep interaction truly plays an important role in the
L
intensities to the concentration of
and fluorescence titration experiments by the addition of in-
creasing amount of -Trp into the solution of 4a.
As shown in Fig. 3, In UVevis titration, the original main ab-
sorption of 4a at 204 nm reduced after -Trp was gradually added,
L-Trp, we carried out absorption
recognitionprocess between 4a and the substrate amino acids.8 With
the absorption results, the nineteen substrate amino acids could be
categorized into three groups: group I contains eleven amino acids,
which have small size and most of them are aliphatic amino acids;
L
L
and after 2 equiv excess, the absorption remained the same. Simi-
larly, in fluorescence titration, 4a exhibits strong emission spectra
Group II were
L
-Cys,
L-Met, L-Arg, and L-Lys; Group III possesses L-phe,
L
-His, -Tyr, and L
L
-Trp, all of which are aromatic amino acids.
(
lem¼410 nm) in the absence of
L
-Trp. However, the gradually ad-
dition of
L
-Trp (from 0.00 to 6.50ꢁ10ꢀ4 M) to the solution of 4a
would cause a fluorescence quenching and lead to a fluorescence
enhancement synchronously at 350 nm, as shown in Fig. 4.
Fig. 1. The UV spectra of 4a (5.00ꢁ10ꢀ5 M) in water/ethanol (1/1, v/v) phosphate buffer
solution (5 mM, pH¼7.2) upon addition of different amino acid (5.00ꢁ10ꢀ4 M), cor-
responding amino acid solution as reference.
Fig. 3. The UV spectra of 4a (5.0ꢁ10ꢀ5 M) in water/ethanol (1/1, v/v) phosphate buffer
solution (5 mM, pH¼7.2) upon addition of different amount of L-Trp (0.00, 2.50, 5.00,
7.50, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0ꢁ10ꢀ5 M); Inset: the difference of absorption
Similarly, in the fluorescence spectra, the addition of amino
acids to 4a would cause a fluorescence quenching under lex at
247 nm. The intensities of 4a with
group II of amino acids were relatively lower compared to other 16
amino acids. While, the fluorescence intensity change of 4a with
L-Cys and
L-Lys belonging to the
value (ꢀ A¼A0ꢀA) at 204 nm as a function of CL-Trp/C4a, corresponding L-Trp solution
D
as reference.
L-
Trp was the most obvious, which decreased by more than a half;
the spectrum had a slight blue-shift from 410 nm to 400 nm, as
shown in Fig. 2.
2.2.3. Binding constant and stoichiometry of 4a with
shown in Fig. 3, the UVevis absorption intensity of 4a at 204 nm
L
-Trp. As
Based on the above results, we could see that there is specific
was found decreased gradually with the increasing concentration
selectivity of 4a to
L-Trp by UVevis absorption and fluorescence
of
L
-Trp from 0.00 to 1.00ꢁ10ꢀ4 M and then was stable after
spectroscopy. Host 4a is a potential receptor for
L-Trp.
1.00ꢁ10ꢀ4 M. This phenomenon further confirmed the interaction
of 4a with
the excess added
at the insufficient addition of
L
-Trp and the formation of 1:2 complex of 4a/ -Trp after
L
2.2.2. UVevis and fluorescence titration of 4a with
L
-Trp. In order to
L
-Trp (a 1:1 complex would probably form at first
investigate the dependence of the absorption and fluorescence
L
-Trp4e). On the basis of the 1:2

D. Li et al. / Tetrahedron 70 (2014) 1223e1229
1225
Fig. 4. The emission spectra of 4a (5.0ꢁ10ꢀ5 M) in water/ethanol (1/1, v/v) phosphate
buffer solution (5 mM, pH¼7.2) upon addition of different amount of L-Trp (0.00, 0.250,
0.500, 0.750, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 3.00, 3.50, 4.00, 4.50, 5.00, 5.50, 6.00,
6.50ꢁ10ꢀ4 M), lex¼247 nm. Inset: at 406 nm, 1/(I0ꢀI) as a function of 1/C2
.
-Trp
L
stoichiometry, we calculated the value of the binding constant for
the complex according to Chen Y. L.,4e the equation was adopted as
follow:
1
I0 ꢀ I
1
1
¼
þ
KðI0 ꢀ I2Þ½Gꢂ2
I0 ꢀ I2
Where I0 and I is the fluorescence intensity of host solution and the
solution of host with guest; I2 is the fluorescence intensity of the
1:2 complex. [G] is the total concentration of guest. K is complex
constant. When the equation was applied, 1/(I0ꢀI) versus 1/[G]2
should give a straight line. The straight line (R2¼0.993) was dis-
played in Fig. 4 (inset), and K¼1.27ꢁ107 Mꢀ2
.
2.3. Binding studies of 4a using 1H NMR spectroscopy
Further binding studies between 4a and
detecting the change of proton magnetic resonance signal using 1H
NMR spectroscopy. -Trytophan sodium salt was added as guest in
two and half fold excess to 4a in CD3OD/D2O (5:1, v/v). With the
gradual addition of -Trp
-Trp to 4a, the 1H NMR signals of 4a and
L-Trp was carried out by
L
L
L
produced substantial movements with the protons on pyridinium
ring, benzyl, cholate arms, indole ring, and the amino acid side
chain. The titration spectra of 4a,
L
-Trp and the complex 4a/ -Trp,
L
which provided preliminary information of the interreactions on
the complex formation, were displayed in Fig. 5.
Initially, when 0.5fold
L-Trp was added to 4a solution, the overall
signals of 4a showed a little change as shown in Fig. 5C. The signals
0
0
0
0
intensity of C2 eH, C4 eH, and C9 eH diminished; the split of C5 eH,
0
0
C6 eH, and C8 eH changed from double peak to single peak; C12eH
changed from single peak to double peak; C7eH, C3eH displayed
a slight upfield shift; H23a, C21eH, C19eH changed from multiplet to
broad multiplet and also showed slight upfield shift. On the other
hand, the movements of the -Trp signals varied considerably. The
L
signals of CaeH, CdeH, CheH, CieH diminished, and CdeH, CheH,
CieH also showed downfield shift; CgeH changed from double peak
to broad multiplet, CkeH diminished following an obvious upfield
shift; the two double peaks of Hj1 disappeared and gave a multiplet;
the dd peaks of Hj2 diminished and displayed an obvious downfield
Fig. 5. The 1H NMR spectra of (A) 4a; (B) L-Trp; (C) 4aþ0.5 equiv L-Trp; (D)
4aþ1.0 equiv L-Trp; (E) 4aþ1.5 equiv L-Trp and (F) 4aþ2.5 equiv L-Trp in CD3OD/
D2O¼5/1 (v/v) at room temperature. The L-Trp was in the form of sodium salt.
shift. These results indeed demonstrated the formation of 4a/
complex and revealed the interactions via hydrogen bonding and
electrostatic interaction between 4a and -Trp.
L-Trp
L

1226
D. Li et al. / Tetrahedron 70 (2014) 1223e1229
Upon the addition of
4a (as shown in Fig. 5DeF), due to the classical
between indole ring of
L
-Trp (1.0 equiv, 1.5 equiv, and 2.5 equiv) to
interaction8,9
-Trp and pyridinium-benzyl group of 4a,
pep
L
0
0
the signals of C2 eH, C4 eH disappeared; the signals intensity of
0
0
0
0
0
C5 eH, C6 eH, C8 eH, and C9 eH diminished, and C9 eH splited into
0
two peaks as well (The split of C9 eH should also be contributed by
CHe
nium salt units and the carboxylate ions of
intensity on CaeH, CgeH, CdeH, CheH, CieH had been regaining
their proper position along with the addition of -Trp to 4a.
Moreover, owing to the multiple hydrogen bonding, hydrophobic
force, , and electrostatic interaction, the signals of C12eH,
21eH, and C19eH not only exhibited upfield shift but also splited,
p
conjugation, electrostatic interaction from positive ammo-
L-Trp); but the signals
L
pep
C
and finally to multiplet in C12eH, to double peak in C21eH; Fur-
thermore, CkeH changed back to their proper position from upfield
shift to downfield; Hj1, Hj2 changed back to their proper position
Fig. 6. Selectivity of 4a (4ꢁ10ꢀ5 M) to L-Trp (4ꢁ10ꢀ4 M) in water/ethanol (1/1, v/v)
phosphate buffer solution (5 mM, pH¼7.2) in the presence of other coexisting amino
acids (4ꢁ10ꢀ4 M).
from downfield shift with the addition of -Trp. It was also im-
L
portant to point out that, due to the continued multiple hydrogen
bonding on C12eOH, C7eOH, C3eNH, and the conformational
changes of the cholate arms, the 1H signals of C12eH, C7eH, C3eH,
and H23 showed a stable, sustained changes along with the addi-
behavior and conformation of cholates would be different. If 4a is
a
tion of
L-Trp to 4a.
placed in a polar solvent, the
of formation of hydrogen bonding, and the two
a
-faces will turn outside with the help
-faces will face to
b
2.4. Binding studies of host 4b, 4c, 6 with UVevis and fluo-
rescence spectroscopy
each other to create a micro hydrophobic cave environment. The
original constricted conformation of 4a, which the two ester groups
were folded into the hydrophobic cave,12 helped to reduce the
collision between 4a and solvent molecule. It could favor the en-
hancement of fluorescence intensity.
The biding investigations of 4b, 4c, 6 were carried out with -Trp
L
directly to illustrate the structure activity relation in recognition.
Unexpectedly, the UVevis absorption and the fluorescence of 4b
It was plausible to suppose that the electrostatic interaction
showed very little change upon addition of different amount of
L
-
force between 4a and
factor in recognition. The close approach of carboxylate group and
amine group units in -Trp may be driven by electrostatic, hydro-
gen bonding, and CHe interactions, which would contribute to
the overall binding energies of the complex. When -Trp carbox-
ylate ions closed to positive pyridinium ring driven by the elec-
trostatic force, -Trp would approach to 4a from its -face and lies
L
-Trp should be treated as the prime driving
Trp even though the maximum absorbance of 4b was greater than
that of 4a.10 What’s more, the 4b’s fluorescence intensity was 70%
lower than 4a. These hint that a larger size of anthracene ring
probably hindered the interaction of the recognition between 4b
and guests. On the other hand, 4c had 66% lower fluorescent in-
tensity than that of 4a and only showed a little fluorescent change
L
p
L
L
a
with the addition of
L
-Trp under maximum excitation wave-
in the cleft between benzyl group and cholate arms of 4a. Mean-
while, owing to the tweezer-like derivative 4a possesses hydroxyl
groups at each arms, it is able to form multiple hydrogen bonding
with the amino groups and carboxylate ions of L-Trp. Thus, the
hydrophobic cave in 4a would break down along with the complex
formation, and indeed, the quenching of fluorescence occurred
length;11a 6 has no fluorescence at all, although its UVevis ab-
sorption showed a similar change to 4a upon addition of different
amount of
teractions toward
rescence detection.
L
-Trp.11b Thus, compound 4b, 4c, and 6 should have in-
L-tryptophan, but they are not suitable for fluo-
with the addition of
decreased by more than a half and the emission spectra showed
a slight blue-shift from 410 to 400 nm after the addition of -Trp, as
L
-Trp to 4a. The fluorescence intensity of 4a
2.5. Selectivity of 4a to
isting amino acids
L-Trp in the presence of other coex-
L
shown in Fig. 2. This indicated that more energy consumption of
the excited state energy of 4a had happened with the energy
transmitting to guest molecules through resonance energy trans-
It is particularly important to evaluate the selectivity of 4a for
Trp over other amino acids. The interference of coexisting amino
acids to -Trp detection was investigated by fluorescence spectra.
As shown in Fig. 6, no significant fluorescence intensity changes of
L-
L
fer.13 The complex conformation of 4a/
-Trp is proposed in
L
Scheme 2.
4a/
L
-Trp solution could be observed in the presence of the coex-
Besides, the apparent changes of the 1H NMR signal on pyr-
idinium ring, phenyl, and indole rings supported and illustrated the
isting amino acids (4.00ꢁ10ꢀ5 M, 1.0 equiv to
L-Trp), indicating that
4a exhibits high selectivity to
L-Trp over other amino acids. These
electrostatic interaction, CHep, and pep interactions between host
results showed that 4a has good sensing selectivity to
L-Trp, which
and guest. What’s more, the 1H signals on C3, C7, and C12 clearly
demonstrated the formation of hydrogen bonding, and the changes
on CkeH, CjeH, C19eH, C21eH, and C23eH implied the conforma-
tional changes of cholate arms.
made its suitable of practical -Trp detection.
L
2.6. Sensing mechanism
Mono-cholate backbone 6 was used to estimate the arms’
function of cholate pyridinium salt, and 4b, 4c were used to esti-
mate the steric hindrance effect nearby the positive charge of
pyridinium units. The results indicated that 4b, 4c, and 6 had no
The strong evidence for the electrostatic interaction and mul-
tiple hydrogen bonds between 4a and -Trp came from the UVevis
L
absorption, fluorescence spectra, and 1H NMR analysis. Significant
changes on the absorption, fluorescence spectra of 4a can be well
selectivity or specificity binding capability to -Trp through fluo-
L
explained by the
with multiple hydrogen bond donors. Cholic acid has two faces:
face with three hydrophilic hydroxy groups and -face with three
hydrophobic methyl groups. Under different polar condition, the
L
-Trp-induced conformational change of backbone
rescence spectra. The structureeactivity relationship of 4aec and 6
further declared that both pyridinium-aryl unit and tweezer-like
cholate arms in 4a play very important roles in the selectivity/
a
-
b
sensitivity of -Trp over other amino acids.
L

D. Li et al. / Tetrahedron 70 (2014) 1223e1229
1227
Scheme 1. The synthesis routes of receptors.
Scheme 2. A proposed dynamic conformation of the complex of 4a/L-Trp in water/ethanol phosphate buffer solution (5 mM, pH¼7.2).
3. Conclusion
Instrument Co., Ltd). Infrared spectra were recorded on a Shimadzu
IR-8400S spectrophotometer. 1H NMR and 13C NMR spectra were
recorded by Bruker of 400 MHz on a Varian Gemin-400. ESI-TOF
Mass spectra were obtained on Bruker spectrometers. All UVevis
and fluorescence spectra in this work were recorded in Pgeneral
TU-1901 and Hitachi F-4500 fluorescence spectrometers. All pH
values were measured with a Model pHS-3C pH meter (Shanghai,
China).
In summary, we have designed a tweezer-like quaternary am-
monium salt derivative of cholate 4a, a highly sensitive and selec-
tive fluorescence receptor for -Trp over other a-amino acids in
ethanol aqueous solution. The appealing performance of the re-
ceptor was demonstrated to originate from the electrostatic and
multiple hydrogen bonding cooperative interactions, being syner-
getic with conformational change of cholate backbone along with
signal diminution. The structure recognition relationships showed
that pyridinium-aryl group and tweezers-like cholate arms play
L
4.1.3. Synthesis. The synthetic routes of 4aec and 6 were outlined
in Scheme 1, and the details were described below.
very important roles in the selectivity/sensitivity of -Trp.
L
4.1.3.1. Synthesis of methyl
3b-amino-7a, 12a-dihydroxy-5b-
cholan-24-oate hydrochloric salt 2. Methyl cholate was synthesized
firstly according to the literature,6a followed by sulfonylation with
methane sulfonyl chloride,6b then substitution by azido group.6b
Azido methyl cholate was reduced by triphenylphosphine,6c and
finally gave amino methyl cholate hydrochloric salt by addition of
concentrated hydrochloric acid at 0 ꢃC. The mixture was filtered to
give a white solid 2, overall yield: 70%; mp: w210 ꢃC (de-
4. Experimental
4.1. General
4.1.1. Materials. All reagents and solvents were commercially
available and were used without further purification unless oth-
erwise stated. The water used for investigation was distilled prior to
composition); [
a
]
D
25 þ25.0 (c 1.0, MeOH); IR (KBr, cmꢀ1) 3539, 3458,
use. Cholic acid was purchased from Alfa Aesar. All
a
-amino acids
3026, 2945, 2881, 1726, 1633, 1514, 1461, 1382, 1311, 1202, 1172,
were -configuration and were purchased from Beijing Chemical
L
1035; 1H NMR (DMSO-d6)
d 0.57 (s, 3H, 18-H), 0.86 (s, 3H, 19-H),
Regent Company. 9-(Bromomethyl)anthracene was prepared
according to literature14 from 9-methanolanthracene.
0.90 (d, J¼6.0 Hz, 3H, 21-H), 1.10e1.50 (m, 11H), 1.55e1.86 (m, 8H),
1.95e2.00 (m, 1H), 2.05e2.10 (m, 1H), 2.10e2.22 (m, 1H), 2.25e2.35
(m, 1H), 2.65 (t, J¼12.6 Hz, 1H), 3.56 (s, 3H, eCOOCH3), 3.60 (s, 1H,
7-H), 3.76 (s, 1H, 12-H), 4.19 (br s, 2H, eOH), 7.92 (s, 3H, eNHþ3 ).
4.1.2. Instruments. Optical rotations were measured at 25 ꢃC with
WZZ-3 automatic polarimeter (Shanghai Physical Optical

1228
D. Li et al. / Tetrahedron 70 (2014) 1223e1229
4.1.3.2. Synthesis of N,N0-dicholate-3,5-dinicotin-amide 3. A
a yellow solid, yield: 0.223 g, 100%. Rf: 0.27 (EA/EtOH, 3/1); mp:
25
similar method was adopted to synthesize the titled compound
according to literature.15 A mixture of 3,5-dinicotinic acid (0.084 g,
0.50 mmol), amino methyl cholate hydrochloric salt (0.458 g,
1.00 mmol), BOP (0.464 g, 1.05 mmol), DMF (5 mL), and TEA
(0.42 mL, 3.0 mmol) was stirred at room temperature for 24 h.
Then, the mixture was added into NaHCO3 (30 mL, 1 M) at 0 ꢃC. The
white precipitate was filtered and washed with water (100 mL),
dried in air at 50 ꢃC, purified with flash chromatography, yield:
196e197 ꢃC; [
a
]
þ32.4 (c 0.3, MeOH); IR (KBr, cmꢀ1) 3447, 2941,
D
2868, 1720, 1668, 1533, 1437, 1377, 1252, 1221, 1028, 851, 762, 669;
1H NMR (CD3OD)
d 0.72 (s, 6H,18-H), 1.01 (s, 12H,19, 21-H), 2.72 (td,
J1¼14.2 Hz, J2¼3.6 Hz, 2H), 3.49 (q, J¼6.8 Hz, 1H), 3.61 (q, J¼6.8 Hz,
1H), 3.64 (s, 6H, eCOOCH3), 3.82 (s, 2H, 7-H), 3.96 (s, 2H,12-H), 4.24
(s, 2H), 4.50 (s, 3H, NþeCH3), 9.14 (s, 1H, PyeH), 9.41 (s, 2H, PyeH).
4.1.3.6. Synthesis of compound 5. A mixture of nicotinic acid
(0.246 g, 1.00 mmol), amino methyl cholate hydrochloric salt 2
(0.458 g, 1.00 mmol), BOP (0.464 g, 1.05 mmol), DMF (5 mL), and
TEA (0.42 mL, 3.0 mmol) was stirred at room temperature for 24 h.
Then, the mixture was added into NaHCO3 (30 mL, 1 M) at 0 ꢃC. The
white precipitate was filtered and washed with water (100 mL),
dried in air at 50 ꢃC, purified with flash chromatography, yield:
0.482 g, 99%. Rf: 0.71 (EA/EtOH, 3/1); mp: 158e162 ꢃC; [
a
]
25 þ38.0
D
(c 1.0, MeOH); IR (KBr, cmꢀ1) 3443, 2939, 2868, 1724, 1649, 1528,
1437, 1377, 1270, 1038; 1H NMR (CD3OD)
d 0.72 (s, 6H, 18-H), 0.99 (s,
6H,19-H),1.01 (s, 6H, 21-H),1.25 (t, J¼7.2 Hz, 2H),1.95e2.10 (m, 7H),
2.20e2.40 (m, 7H), 2.69 (td, J1¼14.4 Hz, J2¼4.0 Hz, 2H), 3.60
(q, J¼7.2 Hz, 2H), 3.64 (s, 6H, eCOOCH3), 3.81 (s, 2H, 7-H), 3.96 (s,
2H, 12-H), 4.08 (q, J¼6.8 Hz, 2H), 4.20 (s, 2H), 8.45 (s, 1H, PyeH),
8.99 (d, J¼2.0 Hz, 2H, PyeH); ESI-TOF MS m/z: calcd for
[C57H87N3O10þNa]þ: 996.6289; found: 996.6133; calcd for
[4Mþ3Na]3þ: 1321.8442; found: 1321.8235.
0.479 g, 91%. Rf: 0.67 (ethyl acetate/ethanol, 3/1); mp117e120 ꢃC;
25
[a
]
þ34.9 (c 1.0, MeOH); IR (KBr, cmꢀ1) 3435, 2973, 2868, 1724,
D
1645, 1527, 1439, 1375, 1251, 1195, 1027; 1H NMR (CDCl3)
d 0.65 (s,
3H, 18-H), 0.93 (s, 6H, 19, 21-H), 1.06e1.10 (m, 2H), 2.63 (t, J¼14 Hz,
1H), 3.62 (s, 3H, eCOOCH3), 3.83 (s, 1H, 7-H), 3.95 (s, 1H, 12-H), 4.07
(d, J¼6.8 Hz, 1H), 4.27 (s, 1H), 6.43 (d, J¼4.8 Hz, 1H, eCONHe), 7.35
(s, 1H, PyeH), 8.06 (d, J¼6.8 Hz, 1H, PyeH), 8.66 (s, 1H, PyeH), 8.90
(s, 1H, PyeH); EI-TOF MS m/z: calcd for [C31H46N2O5]þ: 526.3406;
found: 526.3509.
4.1.3.3. Synthesis of compound 4a. A mixture of N,N0-dicholate-
3,5-dinicotinamide 3 (0.195 g, 0.200 mmol), BnBr (0.36 mL,
3.0 mmol) in acetonitrile (10 mL) was reflux for 5 h, and then the
solution was concentrated to half volume and added into ethyl
ether (15 mL). The white precipitate was collected and washed with
ether, yield: 0.220 g, 96%. Rf: 0.03 (EA/EtOH, 10/1); mp: 183e185 ꢃC;
4.1.3.7. Synthesis of compound 6. A mixture of 5 (0.105 g,
0.200 mmol), BnBr (0.36 mL, 3.0 mmol) in acetonitrile (10 mL) was
reflux for 5 h, and then the solution was concentrated to half vol-
ume and added into ethyl ether (15 mL). The white precipitate was
25
[
a]
þ51.1 (c 0.3, MeOH); IR (KBr, cmꢀ1) 3431, 2979, 2868, 1734,
D
1670, 1533, 1456, 1377, 1271, 1198, 1173, 1095, 1038; 1H NMR
(CD3OD)
d 0.71 (s, 6H, 18-H), 0.99 (s, 6H, 19-H), 1.01 (s, 6H, 21-H),
1.95e2.06 (m, 4H), 2.20e2.33 (m, 4H), 2.33e2.42 (m, 2H), 2.70 (td,
J1¼14.4 Hz, J2¼3.0 Hz, 2H), 3.47 (q, J¼7.2 Hz, 2H), 3.64 (s, 6H,
eCOOCH3), 3.81 (s, 2H, 7-H), 3.96 (s, 2H, 12-H), 4.22 (s, 2H), 5.96 (s,
2H, NþeCH2ePh), 7.49 (d, J¼3.6 Hz, 3H, benzyleH), 7.58 (d,
collected and washed with ether, yield: 0.135 g, 97%. Rf: 0.02 (EA/
25
EtOH, 10/1); mp: 139e143 ꢃC; [
a
]
þ21.3 (c 1.0, EtOH); IR (KBr,
D
cmꢀ1) 3420, 3063, 2937, 2868, 1734, 1663, 1539, 1497, 1456, 1375,
1198, 1177, 1028; 1H NMR (CDCl3)
d
0.65 (s, 3H, 18-H), 0.95 (d,
J¼4.0 Hz, 2H, benzyleH), 9.22 (s, 1H, PyeH), 9.48 (s, 2H, PyeH); 13
C
J¼5.6 Hz, 3H, 19-H), 0.98 (s, 3H, 21-H), 1.18 (t, J¼7.2 Hz, 1H), 3.45 (q,
J¼7.2 Hz, 1H, 3-H), 3.63 (s, 3H, eCOOCH3), 3.80 (s, 1H, 7-H), 3.95 (s,
1H, 12-H), 4.20 (s, 1H), 4.48 (s, 1H), 6.11 (s, 2H, NþeCH2ePh), 7.39 (s,
3H, benzyleH), 7.57 (s, 2H, benzyleH), 7.97 (s, 1H, PyeH), 8.65 (s,
1H, PyeH), 8.94 (s, 1H, PyeH), 8.98 (s, 1H, PyeH), 10.20 (s, 1H,
eCONHe); Anal. Calcd for C38H53N2O5Brþ2.5H2O: C, 61.38; H, 7.80;
N, 3.77; found: C, 61.86; H, 7.45; N, 3.76.
NMR (CD3OD):
d 14.26, 18.87, 24.63, 25.50, 26.55, 28.83, 29.93,
31.01, 33.14, 33.40, 33.50, 35.45, 36.67, 37.60, 38.03, 39.60, 42.21,
44.27, 48.82, 49.26, 53.27, 67.41, 70.34, 75.24, 131.81, 132.04, 132.53,
135.14, 137.71, 145.30, 148.17, 164.45, 177.77; Anal. Calcd for
C
64H94N3O10Brþ5H2O: C, 62.19; H, 8.42; N, 3.40; found: C, 62.34; H,
8.04; N, 3.37.
4.1.3.4. Synthesis of compound 4b. A mixture of 3 (0.292 g,
0.300 mmol), 9-(bromomethyl)-anthracene (0.095 g, 0.35 mmol) in
acetonitrile (5 mL) was reflux for 5 h until the reaction was com-
plete monitored by TLC. After cooling, the mixture was filtered and
the pale yellow solid was washed with cold MeCN (5 mL), yield:
4.2. UV and fluorescence titrations of 4a and L-Trp
3.00 mL of 4a (5.17ꢁ10ꢀ5 mol/L) phosphate buffer solution
(water/ethanol¼1/1, 0.005 M, pH¼7.2) was put in a test tube with
a cover, followed by addition of 100 mL different concentrated L-
0.283 g, 76%. Rf: 0.17 (EA/EtOH, 8/1); mp: 188e190 ꢃC; [
a]
25 þ47.3 (c
tryptophan solution. The UV spectra of 4a in the resulting solution
were recorded from 190 nm to 450 nm in 1 cm quartz cuvette. The
blank solution of 4a was used as reference. The fluorescence
emission spectra for 4a were obtained from 300 nm to 600 nm,
lex¼247 nm. The procedures for other compounds were similar.
D
0.3, MeOH); IR (KBr, cmꢀ1) 3431, 3053, 2938, 2867, 1724,1670, 1533,
1448, 1377, 1261, 1163, 1035; 1H NMR (CD3OD)
d 0.70 (s, 6H, 18-H),
0.93 (s, 6H, 19-H), 0.99 (d, J¼6.4 Hz, 6H, 21-H), 2.10e2.30 (m, 4H),
2.30e2.40 (m, 2H), 2.65 (td, J1¼13.6 Hz, J2¼3.8 Hz, 2H), 3.65 (s, 6H,
eCOOCH3), 3.78 (s, 2H, 7-H), 3.93 (s, 2H, 12-H), 4.10 (s, 2H), 7.01 (s,
2H, NþeCH2eAr), 7.58 (t, J¼7.4 Hz, 2H, AreH), 7.67 (t, J¼7.4 Hz, 2H,
AreH), 8.18 (d, J¼8.4 Hz, 2H, AreH), 8.38 (d, J¼8.4 Hz, 2H, AreH),
8.82 (s, 1H, AreH), 9.15(s, 1H, PyeH), 9.20 (s, 2H, PyeH). 13C NMR
4.3. Binding studies of host 4a with L
-Trp by 1H NMR
The test samples were prepared by dissolving 15 mg of 4a or
30 mg of
-Trp sodium in 0.5 mL CD3OD/D2O¼5/1 (v/v) (0.026 M for
4a and 0.27 M for -Trp) in 5 mm NMR tube. After the host and
guest solutions were measured individually, 25 L of above -Trp
(CD3OD)
d
¼14.27, 18.89, 24.68, 25.49, 29.92, 30.98, 33.14, 33.49,
L
36.65, 37.55, 38.01, 39.55, 42.17, 44.24, 48.80, 49.25, 53.28, 59.96,
70.30, 75.21, 122.29, 124.97, 128.22, 131.30, 132.30, 134.18, 134.48,
134.67, 137.54, 145.11, 147.18, 164.33, 177.74; Anal. Calcd for
L
m
L
sodium solution was added into the host solution at each time and
the mix was measured immediately at room temperature.
C
72H98N3O10Brþ3H2O: C, 66.51; H, 8.00; N, 3.23; found: C, 66.50; H,
7.80; N, 3.22.
Acknowledgements
4.1.3.5. Synthesis of compound 4c. MeI (2 mL) was added to the
solution of compound 3 (0.195 g, 0.200 mmol) in MeCN/MeOH
(5 mL/5 mL). The mixture was stirred at room temperature for 3
days, and then the solvent and excess MeI was removed to give
We thank the Science and Technology Innovation Foundation
for the College Students of Beijing (No. 13220055), the National
Natural Science Foundation of China (No. 20972015), and the

D. Li et al. / Tetrahedron 70 (2014) 1223e1229
1229
H. Y.; Wang, M.; Yu, H. M.; Yang, H.; Liu, Y. J. Org. Chem. 2006, 71, 6010e6019;
(e) Chen, Y. L.; Luo, J. T.; Zhu, X. X. J. Phys. Chem. B 2008, 112, 3402e3409; (f) Liu,
Y.; Zhao, Y. L.; Zhang, H. Y. Langmuir 2006, 22, 3434e3438; (g) Ariga, K.; Na-
kanishi, T.; Terasaka, Y.; Tsuji, H.; Sakai, D.; Kikuchi, J. I. Langmuir 2005, 21,
976e981; (h) Luo, J. T.; Chen, Y. L.; Zhu, X. X. Langmuir 2009, 18, 10913e10917;
(i) Ryu, E. H.; Cho, H. K.; Zhao, Y. Org. Lett. 2007, 25, 5147e5150; (j) Murakami,
Y.; Kikuchi, J.; Hayashida, O. Top. Curr. Chem. 1995, 175, 133e156; (k) Segura, M.;
Alcfizar, V.; Prados, P.; Mendoza, J. Tetrahedron 1997, 53, 13119e13128.
5. (a) Zhao, Y.; Ryu, E. H. J. Org. Chem. 2005, 70, 7585e7591; (b) Wallimann, P.;
Marti, T.; Frer, A.; Diederich, F. Chem. Rev. 1997, 97, 1567e1608.
6. (a) Gouin, S.; Zhu, X. X. Steroids 1996, 61, 664e669; (b) Pore, V. S.; Aher, N. G.;
Kumar, M.; Shukla, P. K. Tetrahedron 2006, 62, 11178e11186; (c) Ryu, E. H.; Yan,
J.; Zhong, Z. Q.; Zhao, Y. J. Org. Chem. 2006, 71, 7205e7213.
Natural Science Foundation of Beijing (No. 2112026) for financial
support.
Supplementary data
The UV and fluorescence titration spectra of 4b, 4c, and 6 with L-
Trp, the characterization spectra of compounds are provided as
supplementary data. Supplementary data related to this article can
be found at http://dx.doi.org/10.1016/j.tet.2013.12.068.
7. Liu, Y.; Zhang, Y. M.; Qi, A. D.; Chen, R. T.; Yamamoto, K.; Wada, T.; Inoue, Y. J.
Org. Chem. 1997, 62, 1826e1830.
References and notes
8. (a) Chen, H.; Maestre, M. F.; Fish, R. H. J. Am. Chem. Soc. 1995, 117, 3631e3632;
(b) Rebek, J. J.; Nemeth, D. J. Am. Chem. Soc. 1985, 107, 6738e6739.
9. Metzger, A.; Gloe, K.; Stephan, H.; Schmidtchen, F. P. J. Org. Chem. 1996, 61,
2051e2055.
10. See Supplementary data (S3e4).
11. (a) See Supplementary data (S6); (b) See Supplementary data (S7).
12. Li, Y.; Dias, J. R. Chem. Rev. 1997, 97, 283e304.
13. Xu, J. G.; Wang, Z. B. In Fluorimetry, 3rd ed.; Huang, H., Ed.; Science: Beijing,
China, 2006; pp 72e75; (in Chinese).
14. Jang, J. Y.; Lima, O. V.; Pei, Y.; Jiang, Z.; Chen, Z. G.; Yu, C. C.; Wang, J.; Zeng, X. C.;
Forsythe, E.; Tan, L. ACS Nano 2010, 7, 3773e3780.
15. Zhao, Y.; Zhong, Z. Q. J. Am. Chem. Soc. 2005, 127, 17894e17901.
1. Chen, H.; Ogo, S.; Fish, R. H. J. Am. Chem. Soc. 1996, 118, 4993e5001.
2. (a) Tsubaki, K.; Tanima, D.; Nuruzzaman, M.; Kusumoto, T.; Fuji, K.; Kawabata, T.
J. Org. Chem. 2005, 70, 4609e4616; (b) Tsubaki, K.; Nuruzzaman, M.; Kusumoto,
T.; Hayashi, N.; Wang, B. G.; Fuji, K. Org. Lett. 2001, 25, 4071e4073; (c) Alfonso,
I.; Burguete, M. I.; Galindo, F.; Luis, S. V.; Vigara, L. J. Org. Chem. 2009, 74,
6130e6142; (d) Zhao, H.; Hua, W. J. Org. Chem. 2000, 65, 2933e2938.
3. (a) Liu, S. Y.; Fang, L.; He, Y. B.; Chan, W. H.; Yeung, K. T.; Cheng, Y. K.; Yang, R. H.
Org. Lett. 2005, 26, 5825e5828; (b) Liu, S. Y.; He, Y. B.; Chan, W. H.; Lee, A. W. M.
Tetrahedron 2006, 62, 11687e11696.
4. (a) Chattopadhyay, P.; Pandey, P. S. Bioorg. Med. Chem. Lett. 2007, 17, 1553e1557;
(b) Liu, S. Y.; Law, K. Y.; He, Y. B.; Chan, W. H. Tetrahedron Lett. 2006, 47,
7857e7860; (c) Cui, H.; He, G.; Wang, H. Y.; Sun, X. H.; Liu, T. H.; Ding, L. P.;
Fang, Y. ACS Appl. Mater. Interfaces 2012, 4, 6935e6941; (d) Zhao, Y. L.; Zhang,