Hemicryptophanes with Improved Fluorescent Properties for the Selective Recognition of Acetylcholine over Choline

Article abstract of DOI:10.1021/acs.joc.0c00217

The synthesis of two new fluorescent hemicryptophanes is reported. They were found to be efficient and selective receptors for acetylcholine over choline. When compared to other hemicryptophane hosts previously reported for the selective recognition of ac

Full text of DOI:10.1021/acs.joc.0c00217

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Article
Hemicryptophanes with Improved Fluorescent Properties
for the Selective Recognition of Acetylcholine over Choline
Augustin Long, Elise Antonetti, Alberto Insuasty, Sandra Pinet, Isabelle
Gosse, Vincent Robert, Jean-Pierre Dutasta, and Alexandre Martinez
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00217 • Publication Date (Web): 06 Apr 2020
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Hemicryptophanes with Improved Fluorescent Properties for the
Selective Recognition of Acetylcholine over Choline
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Augustin Long,† Elise Antonetti,† Alberto Insuasty,† Sandra Pinet,§ Isabelle Gosse,§ Vincent Robert,
Jean-Pierre Dutasta,‡ and Alexandre Martinez*†
† Aix-Marseille Univ., CNRS, Centrale Marseille, iSm2, UMR 7113, 13397, Marseille, France
§ ISM, UMR 5255 CNRS, Bordeaux INP and Univ. Bordeaux, 351 cours de la Libération, F-33400 Talence, France.
 Laboratoire de Chimie Quantique Institut de Chimie, UMR CNRS 7177, Université de Strasbourg, 4, rue Blaise Pascal, F-
67070 Strasbourg, France.
‡ Laboratoire de Chimie, Ecole Normale Supérieure de Lyon, CNRS, UCBL, 46 allée d’Italie, F-69364 Lyon, France
Supporting Information Placeholder
(1.8%) and wavelength emission at 326 nm. We thus
decided to add additional conjugated units in order to
improve the fluorescent properties of the host without
altering the size and shape of the cavity, aiming at
preserving a good ACh>Ch selectivity. Herein we report
on the synthesis of the new hemicryptophanes 2 and 3
bearing fluorescent linkers (Figure 1). The fluorescent
properties have been improved when compared to our
previously reported cage 1, while the ACh/Ch selectivity
is retained.
ABSTRACT: The synthesis of two new fluorescent
hemicryptophanes is reported. They were found to be
efficient and selective receptors for acetylcholine over
choline. When compared to other hemicryptophane
hosts previously reported for the selective recognition of
acetylcholine, they display improved fluorescent
properties: their maximum emission wavelengths are red
shifted and the quantum yields are higher. NMR titration
experiments and DFT calculations support the results
obtained from fluorescence spectroscopy and give
insights into the interactions involved in the host/guest
complexes and into the selectivity for acetylcholine over
choline.
Figure 1. Structures of the hemicryptophane hosts (1-3)
and the guests used in this study.
RESULTS AND DISCUSSION
INTRODUCTION
Acetylcholine (ACh) is a neurotransmitter that plays a
crucial role in both the peripheral and central nervous
systems. For instance, ACh is involved in learning and
memory processes or in the transmission and regulation
of the nervous impulse in the synaptic key.1-4 Therefore,
the development of synthetic receptors for ACh has
O
OMe
NH
O
OMe
NH
MeO
O
OMe
MeO
O
OMe
O
O
Ph
NH
Ph
Ph
HN
NH
HN
O
O
O
O
O
O
1
2
arouse
a
considerable interest.5-13 In particular,
fluorescent supramolecular probes have been designed
to detect ACh as they allow for high sensitivity,
technical simplicity and fast response time, and are thus
convenient for optical imaging and analytical sensing.14-
O
N
O
O
OMe
MeO
Ph
O
OMe
O
Acetylcholine
NH
Ph
HN
NH
O
15
Although several fluorescent host compounds able to
Ph
OH
N
O
detect ACh have been reported,16-21 only few display
ACh/Choline (Ch) selectivity in favor of ACh.22-30
Recently, we reported the synthesis of the fluorescent
hemicryptophane 1, which is able to recognize ACh with
a selectivity KACh/KCh of 4.1 (Figure 1).31,32 However,
this host suffers from modest fluorescent properties due
to the naphthalene units included in the linkers of the
cage compound, which lead to a modest quantum yield
O
Choline
3
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Scheme 1. Synthesis of key intermediate 7 (a) and of hemicryptophanes 2 (b) and 3 (c).
Fluorescent hemicryptophanes 2 and 3 have been
two
doublets
obtained following the synthetic pathways shown in
Scheme
1.
Firstly,
the
tris(ethanamine)-
(CTV-NH2) is
(a)
cyclotriveratrylene derivative
7
synthesized according to our previously reported 4-steps
procedure: the reaction of vanillyl alcohol with
dibromoethane in EtOH followed by triple Friedel-
Crafts reaction in MeCN with Sc(OTf)3 as catalyst
affords CTV-Br 5 in 20 % yield.33 The three bromine
atoms are then substituted by azide functions, and a
subsequent Staudinger’s reaction gives CTV-NH2 7 with
15% overall yield.34 This key intermediate 7 was used
for the synthesis of cages 2 and 3. Secondly, 5-bromo-2-
hydroxybenzaldehyde reacts under classical Sonogashira
coupling conditions or Suzuki coupling conditions to
afford 9 and 12 in 66% and 68% yields, respectively.
Then, nucleophilic substitutions of the phenol unit of 9
and 12 on 1,3,5-tribromomethyl-benzene, using Cs2CO3
as a base, give the C3-symmetrical trialdehydes 10 and
13 in 96% and 92 % yields, respectively. Finally,
(b)
reductive aminations between CTV-NH2
7
and
trialdehydes 10 and 13 afford cages 2 and 3 in 40% and
24% yields respectively.
1H NMR spectra (Figure 2) show that both cages 2 and 3
exhibit the C3-symmetry usually observed for
hemicryptophane hosts in solution. Both present
characteristic signals of cages including a CTV moiety:
1
Figure 2. H NMR spectra (400 MHz, CDCl3, 298 K) of
hemicryptophanes 2 (a) and 3 (b).
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for the ArCH2 methylene bridges (A) around 4.8 and 3.5
observed, whereas cage 2 displays a decrease of the
fluorescence intensity upon progressive addition of guest
solutions. The expected higher rigidity of cage 2 might
account for this opposite behavior. Both hosts 2 and 3
exhibit binding constants for ACh and Ch in the same
range of magnitude than those of cage 1 (typically in the
range of 104 M–1, see Table 2), highlighting that the
introduction of fluorescent units in the linkers only
slightly affects the binding properties of the host,
probably because the size and shape of the cavity are
similar in all cases. The ACh/Ch selectivity is also
retained, with a KACh/KCh ratio of 2.8 and 4.4 for hosts 2
and 3 respectively.
1
2
3
4
5
6
7
8
ppm, the two singlets around 6.95 and 6.8 ppm for the
aromatic protons (C, D) and a singlet near 3.6 ppm for
the methoxy groups (B). The aromatic protons (K) and
the diastereotopic aliphatic protons (H) of the lower part
appear as a singlet at 7.0 ppm and an AB quartet around
4.3 ppm, respectively. The diastereotopic protons G of
the linkers give two AB doublets near 3.7 and 3.9 ppm,
whereas the protons F and E give slightly more
complicated pattern around 3.0 and 4.2 ppm
respectively. The aromatic protons J of the linkers are
also observed as multiplets between 7.2 and 7.6 ppm,
while the doublet around 6.6 ppm is characteristic of the
aromatic proton I.
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Table 2. Binding constants Ka (M–1) for the 1:1 complexes
formed between the different hosts and guests.
The absorption and emission properties of compounds 2
and 3 in DMSO containing 2% of water are shown in
Figure 3. The maximum emission wavelengths of 2 and
3 are in both cases red shifted compared to host 1: from
326 nm ( = 2.0 × 104 M-1 cm-1) for host 1 to 352 nm
( = 9.4 × 104 M-1 cm-1) and 334 nm ( = 5.1 × 104 M-1
cm-1) for hosts 2 and 3, respectively (Table 1). The
fluorescent quantum yields  are also improved when
compared to host 1: from 1.8% for cage 1 to 5.4% and
7.5% for hosts 2 and 3, respectively (Table 1).34
Substrate
Host
Ka (L.mol-1)a
2.4 × 104 ± 2.0%
1.2 × 104 ± 2.1%
KACh/KCh
1
2
-
-
O
N
O
ACh
3
1
2
3
4.4 × 104 ± 4.4%
5.9 × 103 ± 2.3%
4.3 × 103 ± 2.2%
1.0 × 104 ± 4.8%
-
4.1
2.8
4.4
N
OH
(a)
(b)
Ch
1000
800
800
600
400
200
0
0.3
0.2
0.1
0.2
0.1
a Ka was determined by fitting fluorescence titration curves
(DMSO + 2% H2O, 298 K) with Bindfit program.
600
400
200
1H NMR titration experiments were also performed to
give insight into these recognition processes. To a
solution of hosts 2 or 3 in a CDCl3/MeOH mixture
(95/5), was added a concentrated solution of ACh or Ch
in the same solvent mixture. Changes in the chemical
shifts of both, the cages and the guests, were observed
(Figures S30-S37), which is in agreement with the
expected fast host-guest exchange on the NMR time
scale that is usually observed with hemicryptophane
hosts. One can see that, by increasing the ACh or Ch
concentrations, both their (CH3)3N and CH2 protons are
downfield shifted. At the beginning of the titration
experiment the Ach or Ch guest is mostly encapsulated
in the host and undergoes the shielding effect of the
aromatic rings of hosts 2 and 3, leading to the upfield
chemical shifts observed for N(CH3)3 and CH2 protons
compared to that of the free guests in solution. The
successive additions of Ach or Ch guest increase the
percent of the free guests and results in an average
chemical shift shifted towards that of the free guest,
which explains the downfield shift observed for the
protons' guest during the titration experiments. This is
consistent with the encapsulation of the guests in the
heart of the cavities. For both hosts 2 and 3, the protons
A, H and J exhibit well-defined and sharp signals and
no overlapping with the signals of other protons occurs
during the titration experiments. Therefore, the
0
0
0
250
300
350
400
450
500
250
300
350
400
450
500
λ
(nm)
λ
(nm)
Figure 3. Absorption spectra (in blue) and emission spectra
(in red, exc = 290 nm): (a) hemicryptophane 2 (3.2 µM)
and (b) hemicryptophane 3 (4.7 µM) in solution in DMSO
+ 2% H2O at 298 K.
Table 1. Spectroscopic data of cages 1, 2 and 3 in DMSO
with 2% of water at 298 K.
푚푎푥
푚푎푥
휆
휆
푒푚
ε
Δλ
Host
푎푏푠
φf
(L.mol-1.cm-1)
2.0 × 104
(nm)
(nm)
288
294
274
(nm)
326
352
334
1
2
3
38
58
60
1.8%
5.4%
7.5%
9.4 × 104
5.1 × 104
We then studied the recognition of ACh and Ch by these
two new hosts 2 and 3. Additions of a solution of ACh
or Ch in DMSO + 2% H2O to hosts 2 and 3 induce
changes in fluorescence emission of the cages (Figure 4
for ACh and Figures S24 and S25 for Ch). However, the
fluorescence response of these compounds strongly
differs upon addition of the neurotransmitter guests.
Cage 3 shows the same behavior as that reported for
cage 1: an increase of the fluorescence intensity is
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variations of their chemical shifts against the guest/host
ratio were plotted and the resulting titration curves were
fitted with the Bindfit program, giving binding constants
Page 4 of 9
between (i) the oxygen of the C=O carbonyl of ACh and
the Ar-CH2 and N-H protons of the receptors and (ii) the
CH2 and C(O)-CH3 protons of ACh and the oxygen
atoms of the CTV unit can be observed (distances
between 2.2 and 2.5 Å). Thus, these DFT calculations
give insight into the origin of the ACh/Ch selectivity: as
Ch lacks the key ester function, these interactions
described above are not possible with this guest, leading
to a decrease of its affinity for the cavity of the
hemicryptophanes, when compared to ACh.
1
2
3
4
5
6
7
8
×
for ACh of 4.7
and for Ch of 6.49
103 L.mol-1 and 6.2
×
103 L.mol-1
×
102 L.mol-1 and 2.9
102 L.mol-
×
for host 2 and 3 respectively.35 The difference in the
solvent as well as the change in the counterions used for
NMR and fluorescent titration experiments could
account for the difference of the binding constants
measured by these two methods. Moreover, the changes
in the chemical shifts during the NMR titration
experiments are rather small, inducing higher errors in
the determination of the Ka values. However, these
NMR titration experiments nicely support the
fluorescence ones, highlighting the remarkable ACh/Ch
selectivity of this class of fluorescent hosts.
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(a)
(a)
(b)
(b)
(c)
(d)
Figure 4. Fluorescent emission spectra at 298 K (exc = 290
nm) in DMSO 2% H2O of solution of
Figure 5. DFT optimized structures of the encapsulated
acetylcholine guest within the cavity of the fluorescent
hemicryptophanes 2 (a) and 3 (b).
+
a
(a) hemicryptophane 2 (5.0 µM) and (c) hemicryptophane 3
(5.0 µM) upon progressive additions of a solution of ACh
(5.0 mM, counterion Cl–). Evolution of the relative
fluorescence intensity as a function of the concentration of
added ACh: (b) at 348 nm for host 2, and (d) at 338 nm for
host 3. Curves were fitted with the Bindfit program
(lines).35
CONCLUSION
In summary, the synthesis of two new hemicryptophane
cages 2 and 3 with improved fluorescent properties,
when compared to previously reported host 1, has been
described. These cages turn out to be efficient receptors
for neurotransmitter ACh displaying
a
marked
Then, DFT calculations were performed to grasp the
interactions involved in these recognition processes. The
selectivity for ACh over Ch. Whereas cage 3 behaves as
a turn ON receptor of ACh, a decrease of fluorescence is
observed when ACh is added to hemicryptophane 2.
Insights into the structure of the host/guest complexes
have been obtained from NMR titration experiments and
DFT calculations. Studies for longer-wavelength
fluorescence detection, and detection in water, using
hemicryptophanes, are underway.
optimized structures (Figure 5) show
a
partial
encapsulation of ACh in receptors 2 and 3. The
ammonium unit is located below the CTV moiety, in
agreement with the NMR titration experiments where
shielding of the (CH3)3N protons was observed in the
presence of the cages. The ammonium unit interacts
with both the aromatics of the CTV and that of the south
part by CH- interactions (C-H…Car distances between
2.35 and 2.62 Å). Furthermore, hydrogen bonding
EXPERIMENTAL SECTION
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1
General. Commercial grade solvents and starting
20/80); H NMR (400 MHz, CDCl3, 298 K) δ 10.52 (s,
1H), 8.05 (d, J = 2.2 Hz, 1H), 7.71 (dd, J = 8.6; 2.2 Hz,
1H), 7.56–7.50 (m, 3H), 7.36–7.33 (m, 3H), 7.05 (d, J =
8.7 Hz, 1H), 5.30 (s, 2H); 13C{1H} NMR (100 MHz,
CDCl3, 298 K) δ 188.6, 160.1, 138.6, 137.3, 132.4,
131.6, 128.4, 125.9, 125.2, 123.0, 116.8, 113.2, 89.5,
87.7, 70.2; IR  = 2922, 2854, 1682, 1592, 1496, 1268,
1220 cm-1; HRMS (ESI-TOF) m/z: [M+NH4]+ calcd for
C54H40NO6 798.2850; Found 798.2855; m.p. = 87 – 88
°C.
1
2
3
4
5
6
7
8
material were used without additional purification.
Chromatography and TLC were carry out with Merck 60
A (0.040 - 0.063 mm) silica gel and Merck silica gel 60
F254 plates respectively. A Büchi Melting Point B-545
was used for the determination of the melting points. A
Bruker Alpha Platinum ATR allowed for the recording
of the IR spectra. 1H NMR and 13C NMR were carry out
at 298 K on either a Bruker Avance III HD 400 MHz a
Bruker Avance III HD or 300 MHz spectrometer. The
protonated residual solvent signal was used as reference
for reporting the 1H NMR and 13C NMR chemical shifts
δ. HRMS were performed on a SYNAPT G2 HDMS
(Waters) mass spectrometer with API and spectra were
obtained with TOF analysis using two internal standards.
UV spectra were carry out on a Agilent Cary 60 UV-Vis
spectrophotometer and fluorescence spectra on a Jasco
FP-8600 spectrofluorometer. CTV-Br derivative 5,
CTV-NH2 7 and hemicryptophane 1 were synthesized
according to the previously reported procedure.32-34
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Synthesis of Compound 12. In a 100 mL round-bottom
flask, 5-bromo-2-hydroxybenzaldehyde 8 (1.01 g, 5.02
mmol) was dissolved in toluene (25 mL) and EtOH (5
mL) under argon atmosphere. Phenylboronic acid (1.25
g, 10.2 mmol), K2CO3 (1.75 g, 12.5 mmol) and
Pd(PPh3)4 (302 mg, 0.26 mmol) were added successively
under stirring. The reaction mixture was heated at 80 °C
(oil bath) for 2 h. Solvents were removed under reduced
pressure and the reaction mixture was extracted with
EtOAc (2 x 30 mL) and washed with a 1 M HCl solution
(2 x 30 mL). Organic layers were combined, washed,
dried over MgSO4 and the solvent was removed. The
crude was purified by silica gel column chromatography
with petroleum ether and EtOAc (98/2) as eluent to
afford 12 as a yellow powder (582 mg, 68%). Rf = 0.41
Synthesis of compound 9. 5-Bromo-2-hydroxy-
benzaldehyde 8 (1.61 g, 8.00 mmol), CuI (79 mg, 0.41
mmol) and Pd(PPh3)2Cl2 (282 mg, 0.19 mmol) were
added in a 100 mL round-bottom flask under argon
atmosphere. A 1/1 mixture of THF and Et3N (35 mL)
were added. Ethynylbenzene (1.75 mL, 15.9 mmol) was
added and the mixture was stirred under argon at 75 °C
(oil bath) during 24 h. The mixture was cooled to r.t. and
diluted with Et2O (50 mL) and water (50 mL). The
aqueous layer was acidified with 2M HCl solution (40
mL). The layers were separated and the aqueous phase
was extracted with Et2O (50 mL). Organic layers were
combined, washed with brine (60 mL) and dried over
MgSO4, filtered and concentrated under vacuum. The
crude was purified by silica gel column chromatography
with a gradient of petroleum ether and EtOAc (from
19/1 to 17/3) as eluent to afford 9 as a white powder
1
(PE/EtOAc: 95/5); H NMR (300 MHz, CDCl3, 298 K)
δ 11.00 (s, 1H), 9.98 (s, 1H), 7.80–7.75 (m, 2H), 7.59–
7.53 (m, 2H), 7.49–7.42 (m, 2H), 7.40–7.33 (m, 1H),
7.08 (d, J = 8.7 Hz, 1H); 13C{1H} NMR (75 MHz,
CDCl3, 298 K) δ 196.6, 161.0, 139.4, 135.7, 133.4,
131.9, 129.0, 128.8, 127.4, 126.8, 126.6, 120.8, 118.2;
IR  = 3070, 2932, 2876, 1660, 1591, 1491 cm-1; HRMS
(ESI-TOF) m/z: [M-H]- calcd for C13H9O2 197.0908;
Found 197.0907; m.p. = 93 – 94 °C.
Synthesis of compound 13. Compound 12 (274 mg,
1.38 mmol) and 1,3,5-tris(bromomethyl)benzene 11
(153 mg, 0.43 mmol) were dissolved in DMF (40 mL).
Cs2CO3 (612 mg, 1.88 mmol) was added and the
mixture was stirred at 55 °C (oil bath) for 16 h. The
solid obtained was filtered on a frit and washed with
cold ethanol and cold water to yield 13 as a slightly
yellow powder (287 mg, 92%). Rf = 0.16 (PE/CH2Cl2:
1
(1.18 g, 66%). Rf = 0.33 (PE/EtOAc: 95/5); H NMR
(400 MHz, CDCl3, 298 K) δ 11.14 (s, 1H), 9.92 (s, 1H),
7.79 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 8.6; 2.0 Hz, 1H),
7.57–7.52 (m, 2H), 7.41–7.36 (m, 3H), 7.02 (d, J = 8.6
Hz, 1H); 13C{1H} NMR (100 MHz, CDCl3, 298 K) δ
196.1, 161.4, 139.8, 136.9, 131.5, 128.4, 122.9, 120.5,
118.1, 115.3, 88.9, 87.5; IR  = 3054, 2924, 2856, 1656,
1594, 1494, 1142, 1123 cm-1; HRMS (ESI-TOF) m/z:
[M-H]- Calcd for C15H9O2 221.0608; Found 221.0608;
m.p. = 82 – 83 °C.
Synthesis of compound 10. In a 100 mL round-bottom
flask were dissolved 1,3,5-tris(bromomethyl)benzene 11
(201 mg, 0.563 mmol) and compound 9 (418 mg, 1.88
mmol) in DMF (30 mL). Cs2CO3 (770 mg, 2.36 mmol)
was added and the mixture was stirred at 55 °C (oil bath)
for 16 h. The mixture was diluted with EtOAc (100 mL),
washed with a 10% NaOH solution (4 x 50 mL), brine (2
x 40 mL) and dried over MgSO4. The solvent was
removed under reduce pressure to give 10 a slightly
orange solid (422 mg, 96%). Rf = 0.13 (PE/CH2Cl2:
1
20/80); H NMR (300 MHz, CDCl3, 298 K) δ 10.60 (s,
1H), 8.11 (d, J = 2.5 Hz, 1H), 7.78 (dd, J = 8.6; 2.5 Hz,
1H), 7.59–7.54 (m, 3H), 7.46–7.32 (m, 3H), 7.12 (d, J =
8.7 Hz, 1H), 5.33 (s, 2H); 13C{1H} NMR (75 MHz,
CDCl3, 298 K) δ 189.4, 160.0, 139.3, 137.6, 134.5,
134.3, 128.9, 127.4, 127.2, 126.7, 125.8, 125.4, 113.5,
70.3; IR  = 2932, 2857, 1688, 1595, 1491, 1260 cm-1;
HRMS (ESI-TOF) m/z: [M+NH4]+ Calcd for C48H40NO6
726.2850; Found 726.2852; m.p. = 114 – 116 °C.
Synthesis of hemicryptophane 2.In a 500 mL round-
bottom flask was dissolved CTV 7 (128 mg, 0.238
mmol) in a 1/1 mixture of CHCl3/MeOH (120 mL). A
solution of 10 (169 mg, 0.216 mmol) in the same
mixture of solvent (100 mL) was added dropwise under
stirring at r.t. and the reaction mixture was stirred at r.t.
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for 24 h. It was cooled to 0 °C and NaBH4 (290 mg, 7.67
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148.67, 146.87, 140.59, 137.28, 133.50, 133.02, 131.87,
128.97, 128.69, 126.89, 126.73, 126.65, 126.59, 116.54,
113.46, 112.21, 69.11, 69.03, 55.83, 49.51, 47.90, 36.44;
IR  = 3348, 2912, 1613, 1511, 1413, 1248, 1206, 1108
cm-1; HRMS (ESI-TOF) m/z: [M+2H]2+ Calcd for
C78H77N3O9 599.7824; Found 599.7823; m.p. = 187-189
°C.
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3
4
5
6
7
8
mmol) was added portionwise. The mixture was stirred
at r.t. for 4 h and solvents were evaporated. The crude
residue was dissolved in CHCl3 (50 mL) and washed
with a 10% NaOH solution (50 mL). Aqueous phase was
extracted with CHCl3 (50 mL) and combined organic
layers were washed with a 10% NaOH solution (3 x
40 mL), dried over MgSO4 and filtered. Organic solvent
was removed under reduced pressure to afford a
yellowish solid purified by silica gel column
chromatography with CHCl3/MeOH/Et3N as eluent
(gradient from 97/1/2 to 93/6/2). Hemicryptophane 2
was obtained as a white solid (110 mg, 40%). Rf = 0.27
(CHCl3/MeOH/Et3N: 95/3/2); λmax = 284 nm (DMSO +
2% H2O, ε = 94 000 L.mol-1.cm-1 ); 1H NMR (400 MHz,
CDCl3, 298 K) δ 7.49–7.46 (m, 2H), 7.40–7.37 (m, 1H),
7.31– 7.28 (m, 4H), 6.95 (s, 1H), 6.93 (s, 1H), 6.78 (s,
1H), 6.58 (d, J = 8.5 Hz, 1H), 4.75 (d, J = 13.6 Hz, 1H),
4.35 (d, J = 11.9 Hz, 1H), 4.26 (d, J = 12.0 Hz, 1H),
4.20–4.13 (m, 2H), 3.88 (d, J = 13.5 Hz, 1H), 3.67 (d, J
= 13.5 Hz, 1H), 3.62 (s, 3H), 3.53 (d, J = 13.9 Hz, 1H),
3.05–2.94 (m, 2H), 2.92–2.85 (m, 1H); 13C{1H} NMR
(100 MHz, CDCl3, 298 K) δ 156.47, 148.60, 146.87,
137.02, 133.52, 132.94, 131.88, 131.46, 128.27, 127.88,
126.97, 123.57, 116.39, 115.28, 113.47, 111.87, 89.28,
88.18, 69.17, 69.09, 55.84, 49.11, 47.78, 36.41; IR  =
3332, 2924, 1606, 1507, 1260, 1240, 1142 cm-1; HRMS
(ESI-TOF) m/z: [M+2H]2+ Calcd for C84H77N3O9
635.7824; Found 635.7819; m.p. = 169 – 171 °C.
Photophysic studies
The absorption spectrum of a solution in DMSO + 2%
H2O of each hemicryptophane was recorded between
265 nm and 500 nm using a quartz cuvette (1 cm x 1 cm
x 4.5 cm).
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The emission spectrum was recorded between 300 nm
and 550 nm. Emission spectra of 4 solutions of each
hemicryptophane in DMSO containing 2% H2O, with
absorbance lower than 0.1, were recorded, and the
average quantum yield was calculated with quinine
bisulfate in H2SO4 (0.5 M). All these solutions afforded
the same results. Spectra were recorded on a Jasco FP-
8600 spectrofluorometer equipped with a JASCO Peltier
cell holder ETC-815 to maintain the temperature at 25.0
± 0.2°C.
Fluorescence titrations.
A
solution of each
hemicryptophane (2.5 mL, 5.0 µM) in DMSO + 2% H2O
was taken into the quartz cuvette, and then certain
equivalents of a concentrated guest solution (5.0 mM) in
the same solvents were added stepwise with a
microsyringe. The final volume of the solution was
almost unchanged (2.5 mL) since very small volume of
guest solution was added. The solution was mixed and
incubated for 30 s before irradiation at 290 nm at 25 °C.
The corresponding emission values during titration were
then recorded.
Synthesis of hemicryptophane 3. In a 250 mL round-
bottom flask was added CTV 7 (99 mg, 0.184 mmol)
dissolved in a 1/1 mixture of CHCl3/MeOH (120 mL). A
solution of 13 (117 mg, 0.166 mmol) in the same
mixture of solvent (120 mL) was added dropwise under
stirring at r.t. and the reaction mixture was stirred at r.t.
overnight. NaBH4 (240 mg, 6.35 mmol) was added
portionwise at 0 °C and the mixture was stirred at r.t.
during 4 h. Solvents were evaporated, the crude residue
was dissolved in CHCl3 (30 mL) and washed with a 10%
NaOH solution (30 mL). Aqueous phase was extracted
twice with CHCl3 (2 x 30 mL) and combined organic
layers were washed with a 10% NaOH solution (3 x
25 mL) and dried over MgSO4. Organic solvent was
removed under reduced pressure to afford a white solid
purified by silica gel column chromatography
(CHCl3/MeOH/Et3N, gradient from 97/1/2 to 90/8/2).
Hemicryptophane 3 was obtained as a white solid
(48 mg, 24%). Rf = 0.31 (CHCl3/MeOH/Et3N: 95/3/2);
λmax = 274 nm (DMSO + 2% H2O, ε = 51 000 L.mol-
NMR titrations. A solution of hemicryptophane host 2
or 3 (1.0 × 10-3 M in CDCl3/CD3OD 95/5, 500 μL) was
titrated in NMR tubes with aliquots of a concentrated
solution (5.0 × 10-3 M in the same solvent) of picrate
salts of neurotransmitters. The shifts Δδ of the host’s
protons signals at 7.61 and 7.55 ppm were measured
after each addition and plotted as a function of the
substrate/receptor ratio ([S]/[R]). Association constant
Ka was obtained by nonlinear least-squares fitting of
these plots using Bindfit program from Thordarson’s
group.35
Computational method
.
In order to access
geometrical information upon the host–guest species,
full geometry optimizations were performed using
restricted Density Functional Theory (DFT) calculations.
A combination of BP86 functional and an all electron 6-
31G* basis set including polarization functions has
proven to be very satisfactory for similar issues.36
However, we checked using the hybrid B3LYP
functional that our results do not suffer from the
arbitrariness of the exchange correlation functional. All
calculations were carried out using the Gaussian 03 suite
of program.37
1
1.cm-1 ); H NMR (400 MHz, CDCl3, 298 K) δ 7.50–
7.43 (m, 3H), 7.39–7.33 (m, 2H), 7.33–7.28 (m, 2H),
6.97 (s, 1H), 6.95 (s, 1H), 6.79 (s, 1H), 6.65 (d, J = 8.5
Hz, 1H), 4.76 (d, J = 13.7 Hz, 1H), 4.36 (d, J = 12.1 Hz,
1H), 4.30–4.14 (m, 3H), 3.97 (d, J = 13.3 Hz, 1H), 3.78
(d, J = 13.2 Hz, 1H), 3.62 (s, 3H), 3.54 (d, J = 13.6 Hz,
1H), 3.14–3.05 (m, 1H), 2.98–2.90 (m, 1H), 2.20 (s,
1H); 13C{1H} NMR (100 MHz, CDCl3, 298 K) δ 155.93,
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Pradhan, T.; Jung, H. S.; Jang, J. H.; Kim, T. W.; Kang C.;
ASSOCIATED CONTENT
Kim, J. S. Chemical sensing of neurotransmitters. Chem. Soc. Rev.
2014, 43, 4684–4713.
16
1
2
Supporting information
Inouye, M.; Hashimoto K.; Isagawa, K. Nondestructive
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4
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6
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8
9
Detection of Acetylcholine in Protic Media: Artificial-Signaling
Acetylcholine Receptors. J. Am. Chem. Soc. 1994, 116, 5517–5518.
17 a) Tan, S.-D.; Chen, W.-H.; Satake, A.; Wang, B.; Xu Z.-L.;
Kobuke, Y. Tetracyanoresorcin[4]arene as a pH dependent artificial
acetylcholine receptor. Org. Biomol. Chem., 2004, 2, 2719–2721. b)
The Supporting Information is available free of charge on
the ACS Publications website.
1H and 13C NMR, mass and UV spectra, titration
experiments and cartesian coordiantes (PDF).
Koh, K. N.; Araki, K.;
Ikeda, A.; Otsuka H.; Shinkai, S.
Calixarene-Based Artificial-Signaling
Useful in Neutral Aqueous
Reinvestigation
Acetylcholine
of
AUTHOR INFORMATION
Receptors
Corresponding Author
(Water/Methanol) Solution. J. Am. Chem. Soc. 1996, 118, 755–758.
18 Liu, Y.; Perez, L.; Mettry, M.; Gill, A. D.; Byers, S. R.;
Easley, C. J.; Bardeen, C. J.; Zhong, W.; Hooley, R. J. Site selective
reading of epigenetic markers by a dual-mode synthetic receptor
array. Chem. Sci. 2017, 8, 3960-3970.
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* E-mail: alexandre.martinez@centrale-marseille.fr
Notes
19
Korbakov, N.; Timmerman, P.; Lidich, N.; Urbach, B.;
The authors declare no competing financial interest.
Sa’ar, A.; Yitzchaik, S. Acetylcholine Detection at Micromolar
Concentrations with the Use of an Artificial Receptor-Based
Fluorescence Switch. Langmuir 2008, 24, 2580–2587.
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SYNOPSIS TOC. The selective recognition of acetylcholine over choline was achieved with the synthesis of new
hemicryptophane hosts presenting improved fluorescence properties.
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