Binding of acetylcholine and quaternary ammonium compounds to a C 3-symmetric bowl-shaped tripeptide of 2-(3-aminophenoxy)propanoic acids acting as a ditopic receptor

Article abstract of DOI:10.1016/j.tetlet.2014.02.066

The new tripeptide reported here is composed of (R)-2-(3-aminophenoxy) propionic acid and is a bowl-shaped receptor that simultaneously binds both cations and anions of acetylcholine chloride and benzyltrimethylammonium compounds. An intriguing conformati

Full text of DOI:10.1016/j.tetlet.2014.02.066

Tetrahedron Letters 55 (2014) 2226–2229
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Binding of acetylcholine and quaternary ammonium compounds
to a C3-symmetric bowl-shaped tripeptide of 2-(3-aminophenoxy)
propanoic acids acting as a ditopic receptor
⇑
Motohiro Akazome , Norihiro Hamada, Koji Takagi, Daisuke Yagyu, Shoji Matsumoto
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoicho, Inageku, Chiba 263-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The new tripeptide reported here is composed of (R)-2-(3-aminophenoxy)propionic acid and is a bowl-
shaped receptor that simultaneously binds both cations and anions of acetylcholine chloride and benzylt-
rimethylammonium compounds. An intriguing conformational change of the host was observed in the
complexation of the ionic pair, where anion-induced flipping of the amide group on the macrocycle
occurred.
Received 20 January 2014
Revised 12 February 2014
Accepted 18 February 2014
Available online 25 February 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Tripeptide
Bowl-shaped structure
Ion-pair recognition
Quaternary ammonium compounds
Ion-pair receptors are of great interest in the field of host–guest
chemistry,1 and we have found that the small C3-symmetric bowl-
shaped tripeptide 1 has properties suitable for binding quaternary
ammonium compounds, including acetylcholine chloride. Since
the binding of acetylcholine has been attracting considerable inter-
est due to its biological importance as a neurotransmitter, we de-
other reason is that the benzene ring of 2-(3-aminophenoxy)prop-
anoic acid, which has two electron-donating groups (–NH– and –
OCsp3), seems to be an electron-enriched benzene ring suitable
for cation–p interaction. The third reason is that (S)-2-hydroxy-
propanoic acid is one of the (S)-2-hydroxyalkanoic acids easily
and enantiopurely prepared from natural amino acids via diazoti-
zation and displacement by water under acidic conditions (i.e.,
neighboring group participation reaction). Therefore several side
chains of natural amino acids can be applied to this tripeptide. In
the work reported here, we found the C3-symmetric bowl-shaped
tripeptide has an intriguing guest response using quaternary
ammonium compounds such as acetylcholine, and we elucidated
their binding properties.
signed a model system using the cation–
p interaction pointed
out by Dougherty.2 Many artificial acetylcholine receptors such
as calixarenes (including resorcinarenes),3 cavitands derived from
resorcinarenes,4 and other hosts5 have been reported, but the only
C3-symmetric peptide receptors were Kubik’s pioneer hosts: hexa-
peptides composed of 3-aminobenzoic acid (AB) and
a-amino
acids.6 When the Kubik host contained glutamic acid 5-isopropyl
ester [Glu(OiPr)-AB]3, the Ka for its complex with n-butyltrimethy-
The synthetic route of 1 is shown in Scheme 1. The Mitsunobu
reaction7 of methyl (S)-lactate and 3-nitrophenol afforded enatio-
pure methyl (R)-2-(3-nitrophenoxy)propanoate 4, which was
lammonium iodide was 300 MÀ1 6a
.
Substituting proline for glu-
tamic acid [yielding (Pro-AB)3] increased the association constant
by 2 orders of magnitude, to 21,100 MÀ1 6b
.
transformed into (R)-2-(3-nitrophenoxy)propanoic acid 5 and
Kubik’s findings with hexapeptides prompted us to introduce 2-
(3-aminophenoxy)propanoic acid instead of Kubik’s dipeptide unit
(N-3-aminobenzoylamino acid). The first reason that we use 2-(3-
aminophenoxy)propanoic acids is that molecular modeling of the
tripeptides of (R)-2-(3-aminophenoxy)propanoic acid shows a
proper cavity size and restricted conformation, which will reduce
the conformational entropy loss accompanying guest binding. An-
methyl (R)-2-(3-aminophenoxy)propanoate 6 by saponification
and hydrogenation, respectively. For the coupling reaction, we uti-
lized standard conditions with 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide (EDC) and 1-hydroxy-1H-benztriazole (HOBt).8
After reduction of the nitro group of 7, amino compound 8 was
coupled with 5 by the same conditions. After final reduction and
saponification of 9, the desirable cyclic tripeptide 1 was obtained
by the intramolecular coupling reaction of 11 using (ben-
zotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophos-
⇑
Corresponding author. Tel.: +81 43 290 3388; fax: +81 43 290 3401.
E-mail address: akazome@faculty.chiba-u.jp (M. Akazome).
phate (PyBOP) and 4-dimethylaminopyridine (DMAP).9
http://dx.doi.org/10.1016/j.tetlet.2014.02.066
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

M. Akazome et al. / Tetrahedron Letters 55 (2014) 2226–2229
2227
DEAD,
PPh3
Me
Me
+
OMe
OMe
HO
O2N
O
O2N
OH
87%
O
O
4
Me
LiOH
90%
OH
O2N
O
O
O
5
4
Me
H2, Pd-C
100%
OMe
H2N
O
6
O
Me
Me
OMe
N
O
EDC, HOBt
96%
H
O
O
5 + 6
7 X = NO2
H2, Pd-C
91%
8
X = NH2
X
O
Me
EDC, HOBt
89%
Me
OMe
n
N
H
O
5
8
+
O
O
n = 2
9 X = NO2
10X = NH2
H2, Pd-C
96%
X
Figure 2. Titration curve for up-field shifts of AChCl upon addition of 1.
Me
LiOH
PyBOP, DMAP
51%
H
OH
10
1
N
H
O
but also showed diastereotopic germinal coupling by forming a
complex with the chiral bowl of 1.
86%
O
n
11 n = 3
In addition, the association constants of the BnTriMA cation de-
pended on the counter anions and indicated that the anion binds
simultaneously. In 1H NMR spectra, NH of 1 caused down-field
shifting by complexation and the amplitude of the shift depended
on the counter anions. We therefore also estimated the association
constants of counter anions by reversed 1H NMR titration in CDCl3
(Method B, in which the guest solution was added to a solution of 1
and the down-field shift of the amide protons was measured by the
1H NMR titration curves). The association constant of chloride in
AChCl was 1640 MÀ1, which was estimated from nonlinear curve
fitting. Using BnTriMAX gave the association constants for Cl, Br,
and I anions that were 1284, 2725, and 2368 MÀ1, respectively.
In addition, we estimated the simple anion binding ability of 1
by Method B using tetrabutylammonium halide (TBAX), which is
bulky for the cavity of 1. Since the association constants of Cl, Br,
and I were respectively 252, 122, and 40 MÀ1. It is noteworthy that
these values were an order of magnitude smaller than those of
BnTriMAX. These results suggest the binding of cations in the cav-
ity of 1 facilitates the corresponding anion binding. Similar cooper-
ative effects were observed with the ditopic receptors reported by
Kubik6, Sessler–Schmidtchen–Gale,10 and Gibb.11
Scheme 1. Synthesis of compound 1.
First, the binding of acetylcholine chloride (AChCl) with 1 was
examined in CDCl3 by using 1H NMR spectra. In the presence of
AChCl, 1H NMR spectra of 1 showed only one third of the peaks
of the whole host structure with AChCl, which indicates rapid equi-
librium between free 1 and a host–guest complex (i.e., within the
NMR time-scale). The NMR titration experiment showed up-field
shift of guest protons (Fig. 2). The Job’s plot shows 1:1 complexa-
tion (see Fig. S1 in Supporting information). The shifts of the
methyl and methylene groups of the ammonium cation were
greater than the shift of the acetyl moiety, which suggests that
the ammonium cation is located in the bowl cavity throughout
the cation–p interaction. Therefore association constants of cations
were estimated from the up-field shift of cations by adding 1 to the
guest solution (Method A). Nonlinear curve fitting gave an associ-
ation constant of 1998 MÀ1 for the complex consisting of host 1
and AChCl. Host (1) also bound benzyltrimethylammoium (BnTr-
iMA) compounds with Ka = 705–1750 MÀ1 (Method A in Table 1).
During the NMR titration experiment, two protons of benzylic
methylene groups of BnTriMA cation not only shifted up-field
In a control experiment, where the association constants of
monomeric structure 2 (shown in Fig. 1) against chloride were esti-
mated, and they were 46 MÀ1 for BnTriMACl and 32 MÀ1 for TBACl.
No up-field shift of BnTriMA and TBA cations binding with 2 was
observed using Method A, however. Therefore the bowl-shaped
structure of tripeptide 1 is essential for the complexation of 1 with
cations.
We performed single-crystal X-ray analysis of 1 with AChCl and
elucidated the bowl-shaped structure with the cations as a guest
molecule (Fig. 3). As expected, the trimethylammonium part was
settled in the bowl-shaped cavity and surrounded by three ben-
zene rings with cation–p interaction via CH–p interaction. The
chloride anion was located in the bottom of bowl and bound with
an amide proton by a hydrogen bond. Water and ethanol mole-
cules, which came from chloroform solvent for crystallization,
bridged between the chloride and amide protons by two hydrogen
bonds. It is noteworthy that all three amide protons were directed
toward the bottom of the bowl structure and bound to anions
through hydrogen bonds. At the same time, we noticed that the
carbonyl oxygen of 1 was bound to the electro-deficient proton
Figure 1. Structures of hosts (1, 2, and 3) and a guest (AChCl).

2228
M. Akazome et al. / Tetrahedron Letters 55 (2014) 2226–2229
Table 1
To study the driving force for the complexation of AChCl with 1
in CDCl3 (Ka = 1998 MÀ1 at 298K,
D
G = À18.7 kJ molÀ1), we made a
Association constants (Ka) between
1 and various quaternary ammonium
compoundsa
van’t Hoff plot (see Supporting information) and found that
Guest
Cation binding Ka MÀ1
Anion binding Ka MÀ1
D
H = À17.5 kJ molÀ1 and
D
S = 4.0 J molÀ1 KÀ1. This means that
DG was gained from the enthalpy term, thus that the com-
(Method A)
(Method B)
94% of
AChCl
1998 213
1750 66
1680 87
705 28
—
1640 189
1284 216
2725 329
2368 454
252 12
plexation was caused by the cation–
the cooperative hydrogen bonding of the counter anion. Probably,
a small positive value of entropy change (6% of G) was gained
p and CH–O interactions and
BnTriMACl
BnTriMABr
BnTriMAI
TBACl
D
through the desolvation process of 1 with chloroform. In our previ-
ous Letter, a bowl-shaped ortho-substituted analog of 1 was crys-
tallized as a solvated structure with three chloroform molecules,
where one was located inside the cavity and another two mole-
cules were bound with 1 by weak CH–O hydrogen bonds.12
At this stage, we could not get crystal structure of guest free 1.
To estimate the preferred conformation, we performed a confor-
mational search for 1 by doing molecular mechanics calculations
with Monte Carlo minimization procedures. We did this using
the MMFF94s force field13 and GB/SA solvation treatment (chloro-
form)14 implemented in the MacroModel program.15 Figure 4
shows that the lowest-energy conformation of 1 is a C3-symmetric
bowl-shaped structure in which amide oxygen atoms are directed
toward the bottom of the bowl. The calculations revealed that
there are three other conformers within 7.8 kJ molÀ1: conformers
with higher energies of +5.6, +6.9, and +7.8 kJ molÀ1 (Fig. 4). We
could see that in each of those structures two coplanar arrange-
ments of the amide against the benzene ring are possible because
the amide group of 1 conjugates with the benzene ring. In fact, the
X-ray structure of 1 with acetylcholine chloride showed the guest
binding was accompanied by flipping motion of all the amides of 1.
Guest-induced relation of NOE’s on 1H NMR supported this con-
formational change of the amide group of 1 (Fig. 5). In addition, the
flipping of the amide groups of 1 caused the chemical shifts of the
benzene ring of 1 in 1H NMR spectra to move. Although Ha (in
Fig. 5) of free 1 is 6.7 ppm, the presence of 15 equiv amount of ACh-
Cl moved the peak to 7.4 ppm (down-field shift). This significant
change was caused by a deshielding effect from carbonyl groups
of the flipped amide group.
TBABr
TBAI
—
—
122
40
3
2
Ka values were determined by fitting 1H NMR spectroscopic titration curves.
Detail conditions are shown in Supporting Information.
a
Figure 3. Single-crystal X-ray structure of 1 with AChCl (AChCl shown by space
filling model).
To consider a binding effect of the chloride anion on this confor-
mational change of the amide group, we performed an additional
Figure 4. Molecular mechanics of 1.
of methyl groups in AChCl by CH–O interaction. The shortest dis-
tance between the carbonyl oxygen of 1 and the electron-deficient
proton of methyl group in AChCl was 2.60 Å, which is 0.3 Å shorter
than the sum of the van der Walls radii of H (1.20 Å) and O (1.70 Å).
A similar assist of electron-negative carbonyl oxygen atoms for the
binding of cationic guests has already been reported.5c,6b
Figure 5. NOE correlations of (a) free 1, (b) 1 with AChCl (1 equiv), (c) 1 with AChCl
and 3 (1 equiv) in CDCl3.

M. Akazome et al. / Tetrahedron Letters 55 (2014) 2226–2229
2229
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In this simultaneous binding of the ionic pair, anion-induced
flipping of the amide group of 1 occurred. This guest response is
a result of the flipping of the amide group causing dipole inversion
that increases electron density on the rim of the bowl cavity. In the
crystal structure, the amide oxygen atoms with negative charge are
located in the rim of the bowl and complementarily interact with
incoming ammonium cations. We conclude that this bowl-shaped
tripeptide (1), which is composed of (R)-2-(3-aminophenoxy)pro-
pionic acid, has the properties of ditopic receptors for acetylcholine
chloride and benzyltrimethylammoium compounds. The bowl-
shaped tripeptide is a candidate pepitidomimetic receptor for
molecular recognition of quaternary ammonium compounds.
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Supplementary data
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Crystallographic data for the structures in this Letter have been
deposited with the Cambridge Crystallographic Data Centre as sup-
plementary publication numbers CCDC 920352. These data can be
obtained free of charge from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 (0) 1223
336033 or e-mail: deposit@ccdc.cam.ac.uk.
Supplementary data associated with this article can be found, in
theonline version, at http://dx.doi.org/10.1016/j.tetlet.2014.02.066.
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