C3-triiodocyclotriveratrylene as a key intermediate to fluorescent probes: Application to selective choline recognition

Article abstract of DOI:10.1039/c1ob06231j

A new strategy to obtain fluorescent cyclotriveratrylene (CTV) probes is proposed. The key intermediate, a triiodo CTV, is prepared in 3 steps with 47% overall yield. The whole synthesis requires only one purification step. The potential of this triiodo C

Full text of DOI:10.1039/c1ob06231j

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Organic &
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PAPER
C -triiodocyclotriveratrylene as a key intermediate to fluorescent probes:
3
application to selective choline recognition†
a
b
a
c
c
d
Lisa Peyrard, Sabine Chierici, Sandra Pinet, Pinar Batat, Gediminas Jonusauskas, No e¨ l Pinaud,
e
a
Pierre Meyrand and Isabelle Gosse*
Received 22nd July 2011, Accepted 27th September 2011
DOI: 10.1039/c1ob06231j
A new strategy to obtain fluorescent cyclotriveratrylene (CTV) probes is proposed. The key
intermediate, a triiodo CTV, is prepared in 3 steps with 47% overall yield. The whole synthesis requires
only one purification step. The potential of this triiodo CTV as an intermediate is illustrated through
the synthesis of a fluorescent phosphorylated probe that is able to bind choline and acetylcholine in
pseudo-physiological conditions, with selectivity towards choline. As a consequence, this intermediate
should allow us to rapidly form a library of probes in order to highlight the most promising ones.
Introduction
Cyclotriveratrylenes (CTVs), cyclic trimers of veratrole, are
supramolecular bowl-shaped structures known for their ability
1–3
to complex with organic and organometallic guests. Thus,
there is a growing interest in the synthesis of modified CTVs
from their first development by A. Collet et al. in the early
4
1
980s. In our group, we are particularly interested in developing
CTVs as fluorescent probes, especially for acetylcholine (ACh)
and choline (Ch) imaging (Fig. 1). In this area, we recently
reported the first fluorescent CTV that is able to recognize similarly
5
ACh and Ch in aqueous media. Its fluorescence, due to an
intramolecular charge transfer between electron-withdrawing and
donating groups, located on the aromatic cores of the CTV, is
altered in the presence of the guest. The recognition is mainly due
to p–cation interactions between the CTV aryl groups and the
ammonium part of the guest. Additional ionic interactions can
a
Universit e´ de Bordeaux, Institut des Sciences Mol e´ culaires UMR CNRS
Fig. 1 Fluorescent CTVs for Ch and ACh detection: synthetic approach.
5
3
525, Groupe Nanosyst e` mes analytiques, Site ENSCBP, 16 Av. Pey Berland,
3607, Pessac, France. E-mail: gosse@enscbp.fr; Fax: +33 5 4000 2717;
Tel: +33 5 4000 6273
b
Universit e´ Joseph Fourier, D e´ partement de Chimie Mol e´ culaire UMR 5250,
BP 53 38041, Grenoble, France. E-mail: sabine.chierici@ujf-grenoble.fr;
Fax: +33 4 5652 0803; Tel: +33 4 5652 0804
Universit e´ de Bordeaux, Centre de Physique Mol e´ culaire Optique et
Hertzienne, UMR 5798, 351 cours de la lib e´ ration, 33400, Talence, France.
E-mail: pinar.batat@etu.u-bordeaux1.fr; Fax: +33 5 4000 6970; Tel: +33 5
also be induced by the substituents present on the aryl sub-units
of the CTV.
c
Other fluorescent probes are known to detect ACh and Ch, but
their principle of detection is based on the competitive recognition
6–8
4
000 6198
between the guest and a previously complexed fluorophore.
d
Universit e´ de Bordeaux, Institut des Sciences Mol e´ culaires UMR CNRS
When the guest is complexed by the non-fluorescent host, the
fluorophore is released and recovers its luminescence. Recently,
sub-micromolar concentrations of ACh have been detected using
5
525, Centre d’Etude Structurale et d’Analyse des Mol e´ cules Organiques,
51 cours de la lib e´ ration, 33400, Talence, France. E-mail: n.pinaud@ism.
3
u-bordeaux1.fr; Fax: +33 5 4000 2623; Tel: +33 5 4000 6992
e
7
Universit e´ de Bordeaux, Institut des Maladies Neurod e´ g e´ n e´ ratives UMR
this strategy. Nevertheless, with this method, information about
5
293, 351 cours de la lib e´ ration, 33405, Talence, France.E-mail: p.
the space-resolved migration of ACh is not available. As a
consequence these systems do not allow the imaging of ACh
or Ch. Hence, in order to highlight new molecular probes, the
development of a library of CTVs functionalized by diverse groups
meyrand@cnic.u-bordeaux1.fr; Fax: +33 5 5690 142; Tel: +33 5 4000 2562
†
Electronic supplementary information (ESI) available: NMR spectra
of all synthezised compounds, DOSY-NMR experiments, fluorescence
titration data. See DOI: 10.1039/c1ob06231j
This journal is © The Royal Society of Chemistry 2011
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is of interest to evaluate both their fluorescence and recognition
properties as well as their selectivity.
a bromo one prompted us to attempt this strategy. With the
iodine atom we expect to enhance the trimerization yield. As
a result, we focused our attention on an efficient synthesis of
compound 2. Several methods exist to introduce an iodine atom
on 3-hydroxy or 3-methoxy benzoic acid derivatives. Some of
However, only few CTVs with electron-withdrawing groups have
1
been synthesized to date. Indeed, CTVs are obtained by aromatic
electrophilic substitution (SEar) of 3,4-disubstituted benzyl alcohol
derivatives. The trimerization reaction (SEar) is well-known to be
efficient and regioselective when the substituents in positions 3 and
13
them are based on ortholithiation or aromatic electrophilic
14–17
substitution
reactions in the ortho-position of the hydroxy
4
3
are both donating groups. In this case, the substituent in position
should be especially strongly activating towards SEar, most
or methoxy groups. Others pass through the obtaining of a
18–20
diazonium salt.
Nevertheless, as the 3-hydroxy-4-iodobenzoic
commonly a methoxy group. On the contrary, trimerization is less
effective or even completely ineffective as soon as aromatic rings
are functionalized with electron-withdrawing groups. Therefore,
with the aim of preparing a library of new fluorescent CTVs
through a convergent and a minimum step synthesis, we focus
our attention on the synthesis of the key CTV intermediate 1
acid is commercially available, it was decided that it would be the
material of choice to prepare compound 2 (Scheme 1).
1,9
Thus, starting from 3-hydroxy-4-iodobenzoic acid, 2 was ob-
tained without purification in two steps in 94% overall yield.
After methylation withiodomethane, the methylester intermediate
was efficiently reduced using diisobutylaluminium hydride. Not
21
(
Fig. 1). This one presents in the ortho-position of the methoxy
only do these conditions prevent the deiodination observed
when LiAlH is used as the reductive agent but it also allows
group, an iodine atom, designed to be replaced by electron-
withdrawing groups via substitution reactions or more efficiently
by organometallic coupling reactions.
4
simultaneous attainment of the benzyl alcohol in 97% yield.
Indeed, in a previously reported synthesis of 2, the reduction of
the methyl ester through the corresponding acid chloride, requires
The synthesis of 1 was reported a few years ago, in five steps and
10
14
4
3% overall yield. The last step was a diazotization/iodination
three steps, reducing the yield to 72% of the alcohol.
sequence of a triamino CTV intermediate. In this communication,
we present an alternative route to access 1 in a minimum step
synthesis via the trimerization of the 4-iodo-3-methoxybenzyl
alcohol 2 (Fig. 1, Scheme 1). In addition, the potential of 1 as a key
intermediate towards new fluorescent CTV probes is illustrated
through the synthesis of the fluorescent phosphorylated CTV 4
which presents attractive recognition properties of ammonium
guests in pseudo-physiological medium (100 mM buffer solution
at physiological pH (Scheme 2)).
The last step was the trimerization of 2 in the presence of
phosphorus pentoxide in diethyl ether; the CTV 1 was obtained
with 50% yield. At this step, we had to carry out the only and easy
purification step of this synthesis. After filtration of the CTV 1 on
a silica gel plug and elution with dichloromethane, the iodinate
key intermediate was digested in a diethyl ether/CH
mixture.
2
2
Cl (95/5)
Thus, CTV 1 was efficiently obtained in only three steps with
a global yield of 47%. If this yield is similar to those previously
10
reported by A. Collet et al., the main advances of the synthesis
developed here lie in the minimum number of reaction and
purification steps.
Introduction of electron-withdrawing groups from the triiodo
CTV 1 was then investigated through the synthesis of the CTV 4
bearing phosphorylated groups (Scheme 2). Phosphonate groups
have been chosen, in a first attempt, for their binding properties
towards cations and their ability to improve the water solubility
of hydrophobic molecules. Indeed, in the fluorescent CTV probe
that we have previously described, phosphonate groups have
shown a good capability to bind ammonium parts of choline and
Scheme 1 Synthesis of CTV 1.
5
acetylcholine. In the CTV probe 4 prepared in the present study
and contrary to the former, phophorylated groups are directly
conjugated to the aromatic cores that might be advantageous for
the fluorescent properties in terms of charge transfer.
The synthesis of CTV 4 via the substitution of the iodine atoms
is described in Scheme 2. The phosphorylation of 1 using triethyl
22
Scheme 2 Functionalization of CTV 1.
phosphite in the presence of nickel chloride as the catalyst was
shown to be efficient, considering that three iodine atoms have to
be substituted. The tri(diethyl phosphonate) CTV 3 was obtained
with 68% yield. This intermediate was then dealkylated using
bromotrimethylsilane as the silylating reagent leading to CTV
4 with an 88% isolated yield.
The absorption and fluorescence properties of CTV 4 were
investigated in pseudo-physiological conditions (HEPES buffer
100 mM, pH = 7.5) (Fig. 2a). In such conditions, the new water-
soluble phosphorylated CTV 4 has an emission wavelength at 316
nm when excited at 295 nm. The fluorescent quantum yield f and
the time decay t of 4 were determined (f = 0.11 and t = 2.4 ns).
Results and discussion
23
As mentioned above, our strategy to prepare CTV 1 is based on
the trimerization via SEar of the 4-iodo-3-methoxybenzyl alcohol
2
(Fig. 1). The trimerization of the 4-bromo analogue has been
11,12
previously reported in the literature
with acceptable yields
in the range of 40%, showing that a weak electron-withdrawing
group does not deactivate the aromatic ring too much. The lower
electron-withdrawing effect of an iodo substituent compared to
8
490 | Org. Biomol. Chem., 2011, 9, 8489–8494
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Fig. 3 Selected region of the NOESY spectrum in D
2
O of CTV 4 in the
presence of an excess of Ch (mixing time 2 s).
guest, corresponding and confirming the formation of CTV4:guest
complexes (see ESI†).
As for the selectivity, we observed by fluorescence experiments,
a preference of CTV 4 for choline. It is noteworthy that only a few
examples of ammonium probes show some selectivity between Ch
25–28
and ACh.
With a KCh : KACh ratio of 2.8, our system has a
selectivity comparable to the best non-fluorescent selective probe
25
previously published in DMSO.
The structure/selectivity relationship is difficult to forecast
8,27,30
although some papers
highlight hydrophobicity and/or hy-
drogen bond interactions as critical elements. To understand, in
our case, the origin of the selectivity observed for choline, we
performed additional titration experiments (see ESI†).
Fig. 2 a) UV-Vis (solid line) and fluorescence (dashed line) spectra of
-5
1
0 M CTV 4 in 100 mM HEPES buffer pH 7.5; b) relative fluorescence
-5
Using tetramethylammonium (NMe ), trimethylpentylammo-
area of 4 (10 M) with increasing ammonium concentrations (0 to 0.16 M)
4
(
᭺) choline and (ꢀ) acetylcholine fitted with a 1 : 1 binding model.
nium (NMe
3
Pent) and methoxycholine (CholineOMe) (Fig. 1),
we expected to evaluate the influence of the guest chain on the
selectivity. Complexation constants of 48 ± 2 M (R = 0.994),
16.5 ± 2 M (R = 0.996), 18.5 ± 1 M (R = 0.997) were
respectively obtained. NMe is better recognized than all the
tested tetraalkylammonia except for Ch. This result indicates
that if charge–charge and/or p–cation interactions are mostly
responsible for the binding properties of the probe, additional
interactions have to be considered to explain the selectivity
observed for Ch. Polarity of the chain seems significant but is
-
1
2
Binding properties of 4, in the conditions described above, were
-
1
2
-1
2
studied. Titrations of CTV 4 with quaternary ammonium species
of biological interest such as ACh and Ch, present in the brain,
were achieved using fluorescence emission (Fig. 2b). Each titration
was done three times and led to reproducible results. An increase
of the fluorescence intensity of 15 and 23% for ACh and Ch
respectively was observed. In contrast, the fluorescence intensity
was unchanged after addition of salts such as sodium chloride,
or tetrapentylammonium in the same concentration range (results
not shown).
4
not the only factor since NMe Pent, ACh and CholineOMe have
3
similar Kass. In addition, the fact that CholineOMe is much less
recognized than Ch allows to envisage the existence of a hydrogen
bond between the hydroxyl group of the choline and one of the
phosphonate groups of the probe.
In order to confirm the presence of this hydrogen bond in the
CTV 4:Ch complex, molecular modelling was undertaken. Geom-
etry optimization in a periodic box containing water molecules
Assuming a 1 : 1 stoichiometry, the binding constants (Kass) were
24
-1
2
calculated. Binding constants of 23.5 ± 1 M (R = 0.998) for ACh
-
1
2
and 66 ± 4 M (R = 0.993) for Ch were found. The affinity of CTV
for Ch or ACh may seem weak compared to those described in
4
6,7
the literature, however, the binding constants of the previously
described systems are based on indirect detection methods and
most of them are not determined in highly concentrated salt
conditions.
Moreover, we confirmed by NMR experiments the formation
of a complex between CTV 4 and both Ch and ACh. Indeed,
on NOESY spectra, correlation spots were observed between
the methyl groups of the guest (Ch or ACh) and the aromatic
protons of CTV 4 (Fig. 3). Additional NMR diffusion experiments
+
using the MM molecular mechanics calculation was carried out.
31
A stable conformation of the 4:Ch structure (Fig. 4) showed
size complementarities between the bowl-shaped cavity and the
quaternary ammonium. The ammonium group is not located in
the middle of the cavity but is a little off-center. Ionic interactions
between the negative phosphonate groups and the ammonium
˚
part of Ch can occur (distances of 4.39, 4.46 and 4.94 A have been
(
DOSY), which have been particularly used in supramolecular
measured). It is also noteworthy that one hydrogen bond is possible
29
chemistry to investigate issues of structure in molecular assembly,
between one phosphonate of 4 and the OH group of the choline
◦
32
˚
(a distance H . . . O of 3.8 A and an angle of 110 are observed).
were also consistent with the binding of Ch and ACh by the
CTV 4. Besides the free CTV 4 and guest populations, we
observed a new population that enhances with the amount of the
This interaction is probably responsible for the selectivity of CTV
4 for choline.
This journal is © The Royal Society of Chemistry 2011
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experiments were performed using a frequency-tripled Nd-YAG
laser (355 nm, 8 ns pulse) for excitation. Right-angle detection
was used to collect the luminescence spectra using a gated CCD
camera and spectrograph at variable delays.
Synthesis
4-Iodo-3-methoxybenzyl
alcohol
(2). The
3-hydro-4-
iodobenzoic acid (1 g, 3.8 mmol) was dissolved in 50 mL
of dried acetone under a nitrogen atmosphere and potassium
carbonate (2.1 g, 15.2 mmol) was added. The reaction mixture
became white and cloudy. The iodomethane (1.42 mL, 22.8
mmol) was added slowly and the reaction mixture was warmed
◦
+
to 35 C under stirring overnight. Then, acetone was evaporated
Fig. 4 Stable conformation of the 4:Ch complex obtained by MM
molecular mechanics (cubic box of 56 A˚ containing 5833 water molecules).
under vacuum. The crude product was dissolved in 80 mL of ethyl
acetate and washed with distilled water until neutrality of the
aqueous phase was achieved. The organic phase was dried with
anhydrous magnesium sulfate and concentrated under vacuum.
The 4-iodo-3-methoxybenzoic acid methyl ester was obtained as a
bright-yellow oil (1.1 g, 98% yield). H NMR (400 MHz, CDCl
Water molecules and protons have been omitted for clarity.
Conclusion
To conclude, in this study we have developed a new route to prepare
-triiodocyclotriveratrylene compound 1 in three steps with
an overall yield of 47%. One of the major advantages of our
1
3
)
the C
3
d 7.83 (d, J = 8 Hz, 1H, Ar–H), 7.40 (d, J = 2 Hz, 1H, Ar–H),
.35 (dd, J = 8 Hz, 2 Hz, 1H, Ar–H), 3.93 (s, 3H, COOCH ), 3.90
) d 166.2 (COOCH ),
7
(
3
10
synthesis compared to those previously reported is that only
a simple filtration as step of purification is required. Through the
preparation of the phosphorylated CTV 4, this study also proved
the synthetic interest of 1 to access to fluorescent cyclotrivera-
trylenes bearing electron-withdrawing groups which are difficult
or not possible to obtain by electrophilic aromatic substitution
of corresponding benzyl alcohol derivatives. These CTVs are
of interest as fluorescent molecular probes especially to bind
biological ammonium species such as choline and acetylcholine. In
particular, as the excitation wavelength of CTV 4 is too low for in
vivo applications, it could be interesting to extend the conjugation
between acceptor and donor groups to increase its excitation
wavelength. Moreover it could also increase the hydrophobicity of
the probes and modulate the selectivity. We plan to synthesize such
more conjugated CTVs via carbon–carbon coupling reactions
from the intermediate 1 and thus rapidly obtaining a library of
probes to highlight the most promising ones.
13
s, 3H, OCH
3
). C NMR (100 MHz, CDCl
3
3
1
9
2
1
3
57.9 (CAr), 139.3 (CAr), 131.4 (CAr) 123.1 (CAr), 110.9 (CAr),
2.5 (CAr), 56.3 (COOCH
971, 2857, 1728, 1586, 1485, 1442, 1228, 1186, 1100, 1042,
013. HRMS (ESI): calc. C
14.9500.
The 4-iodo-3-methoxybenzoic acid methyl ester (2.4 g, 8.2
mmol) was dissolved in 40 mL of dried tetrahydrofuran under
an argon atmosphere. The mixture was cooled to -80 C before
the dropwise addition of diisobutylaluminium hydride (Dibal-H)
-1
3
), 52.2 (OCH
3
). IR (n cm ): 3021,
9
H
9
O I, Na adduct 314.9488, found
3
◦
(
41.1 mL of 1 M solution in hexane, 41.1 mmol). The mixture was
◦
stirred at room temperature overnight and then quenched at 0 C
by addition of 150 mL of 1 M HCl solution. The product was
extracted with dichloromethane (150 mL ¥ 3). All the combined
organic phases were washed with distilled water until neutrality of
the aqueous phase was achieved and then dried over anhydrous
magnesium sulfate. After concentration under vacuum, the 4-iodo-
3
-methoxybenzyl alcohol 2 was obtained as a bright-yellow oil
1
Experimental
(2.07 g, 96% yield). H NMR (400 MHz, CDCl
Hz, 1H, Ar–H), 6.81 (s, 1H, Ar–H), 6.64 (d, J = 8 Hz, 1H, Ar–
H), 4.59 (s, 2H, CH OH), 3.84 (s, 3H, OCH ), 2.44 (br, 1H, OH).
C NMR (100 MHz, CDCl ) d 158.2 (CAr), 143.0 (CAr), 139.4
Ar), 120.8 (CAr), 109.5 (CAr), 84.6 (CAr), 64.7 (CH OH), 56.4
). IR (n cm ): 3329, 2950, 2857, 1593, 1464, 1393, 1257,
IO , Na adduct 286.9539,
3
) d 7.68 (d, J = 8
General methods and materials
2
3
1
3
3
All commercially available reagents were used as received.
(
(
C
OCH
2
Dichloromethane was freshly distilled from CaH
Tetrahydrofuran was freshly distilled from sodium and benzophe-
none before use. NMe Pent and CholineOMe as the chloride
salts have been synthesized as previously reported. Acetone was
2
before use.
-
1
3
1
043, 1014. HRMS (ESI): calc. C
8
H
9
2
3
found 286.9539.
33
1
13
31
dried over MgSO
4
before use. H, C and P NMR spectra were
2,7,12-Triiodo-3,8,13-trimethoxy-10,15-dihydro-5H-tribenzo-
[a,d,g]cyclononene (1). A suspension of phosphorus pentoxide
recorded on a Bruker Ultrashield Avance 400 spectrometer. Chem-
ical shifts are reported using tetramethylsilane or the residual
P
2
5
O (5.75 g, 40.4 mmol) in 70 mL of dried diethyl ether was
1
13
solvent peak as internal reference for H or for C. Infrared were
determined on a Nicolet iS10 FT-IR spectrometer. Melting points
were measured using a Kofler Heizbank melting point bench
added under nitrogen to a vigorously stirred mixture of 4-iodo-3-
methoxybenzyl alcohol 2 (2.3 g, 9 mmol) in 2 mL of anhydrous
diethyl ether. The reaction mixture was warmed to reflux and the
stirring stopped, but the nitrogen conditions were maintained.
After 3 days the diethyl ether was evaporated. The crude product
was triturated with dichloromethane (100 mL) and filtered through
silica gel in a large, coarse-fritted glass funnel. The silica gel
was rinsed with dichloromethane (150 mL ¥ 4). The filtrate
(
model 7841). High resolution mass spectra were performed by
the CESAMO (Bordeaux, France) on a QStar Elite mass spec-
trometer (Applied Biosystems). UV-Vis spectra were recorded on
a CARY 100 scan spectrophotometer and fluorescence spectra on
a CARY Eclipse spectrophotometer. Time-resolved fluorescence
8
492 | Org. Biomol. Chem., 2011, 9, 8489–8494
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was concentrated under vacuum. The obtained solid was finally
Spectroscopic characterisations of CTV 4. All the measure-
ments were performed in HEPES 100 mM, pH = 7.5 solution,
digested with diethyl ether : dichloromethane (95 : 5) (10 mL ¥ 3)
◦
to give the CTV 1 as a white solid (524 mg, 50%). mp > 285 C
at 293 K. The molar extinction coefficient e(295 nm, HEPES 100 mM, pH = 7.5) =
1
2
-1
-1
(
decomposition). H NMR (400 MHz, CDCl
3
) d 7.72 (s, 3H, Ar–
), 3.86
). C NMR (100
) d 158.4 (CAr), 142.0 (CAr), 141.5 (CAr), 134.5 (CAr),
7995 ± 95 (R = 0.9991) M .cm was obtained in the range of
-
4
-7
H), 6.74 (s, 3H, Ar–H), 4.65 (d, J = 13.6 Hz, 3H, Ar–CH
2
10 –10 M. The data curve absorbance versus concentration was
fitted with a linear curve-fitting equation (with interception at 0
for x = 0) implemented within OriginꢀR . Emission spectra were
recorded with lex = 295 nm ± 5 nm. The fluorescence quantum
yield f = 0.11 was obtained by using Rhodamine 101 in ethanol
as a reference (fref = 0.92 ± 0.02).
1
3
(
s, 9H, OCH
3
), 3.58 (d, J = 13.6 Hz, 3H, Ar–CH
2
MHz, THF-d
1
3
(
8
-
1
13.3 (CAr), 84.4 (CAr), 56.9 (Ar–CH
010, 2914, 2839, 1585, 1448, 1470, 1431, 1249, 1039. HRMS
, Na adduct 760.8517, found 760.8535.
2
), 36.2 (COCH ). IR (n cm ):
3
ESI): calc. C24
H
21
O
I
3 3
Fluorescence titration experiments
Diethyl 12 - [bis(hydroxymethoxy)(methylene)phosphoranyl] - 7-
(
diethoxyphosphoryl)-3,8,13-trimethoxy-10,15-dihydro-5H-tribe-
Guest aliquots (0.5 M guest stock solution of Ch or ACh)
-
5
nzo[a,d,g]cyclononen-2-ylphosphonate (3). The CTV 1 (150 mg,
.2 mmol) was diluted in 6 mL of benzonitrile under argon atmo-
sphere. Nickel chloride (31.6 mg, 0.4 mmol) and triethylphosphite
were added to a 10 M solution of receptor CTV 4 in HEPES
100 mM, pH = 7.5 (2.0 mL) placed in the fluorimeter cell at
293 K. After each guest addition, the cell was carefully shaken
and allowed to equilibrate for 2 min before recording the emission
spectrum. The excitation wavelength was set at 295 nm ± 5 nm.
Intensity changes in the emission spectra of the CTV 4 were
monitored. The integration of the signal is done between 300 and
500 nm in order to obtain the area of the curve as a function
of the guest concentration. This curve can be analyzed using a
specifically written nonlinear least square curve-fitting program
implemented within OriginꢀR . The volume change due to the guest
addition is taken into account in the calculation. Assuming a 1 : 1
stoichiometry, the binding constants (Kass) were calculated.
0
(
660 mL, 3 mmol) were added, and the solution was allowed
◦
to react at 150 C overnight. The reaction mixture was cooled
down to room temperature. The crude solution was diluted in
5
ammonia solution. Then the organic phase was washed with
water until neutrality of the aqueous phase, dried over anhydrous
magnesium sulfate and concentrated under vacuum. The residue
was finally purified by chromatography on silica gel, eluted with
ethyl acetate : methanol 100 : 0 up to ethyl acetate : methanol,
0 mL of toluene and washed three times with a 5% aqueous
9
2
0 : 10. CTV 3 was obtained as a white solid (106 mg, 68%). mp
◦
1
45 C. H NMR (400 MHz, CDCl ) d 7.88 (d, JP–H = 15 Hz, 3H,
3
Ar–H), 6.96 (d, JP–H = 7 Hz, 3H, Ar–H), 4.78 (d, J = 13.6 Hz, 3H,
Ar–CH ), 4.07 (br, 12H, OCH CH ) 3.86 (s, 9H, OCH ), 3.75 (d,
J = 13.6 Hz, 3H, Ar–CH ), 1.30 (t, J = 7.2 Hz, 9H, OCH CH ),
.26 (t, J = 7.2 Hz, 9H, OCH ). P NMR (162 MHz, CDCl
d 16.8 (s). C NMR (100 MHz, CDCl ) d 160.2 (s, CAr), 146.2
s, CAr), 137.5 (s, JP–C = 8 Hz, CAr), 130.4 (d, JP–C = 15 Hz, CAr),
15.3 (d, JP–C = 187 Hz,CAr), 112.8 (d, JP–C = 10 Hz, CAr), 62.2
OCH CH ), 56.1(OCH ), 36.7 (Ar–CH ), 16.5 (OCH CH ). IR
n cm ): 2980, 2908, 1599, 1488, 1469, 1242, 1064, 1024. HRMS
ESI): calc. C36 , Na adduct 791.2485, found 791.2485.
Acknowledgements
2
2
3
3
2
2
3
We gratefully acknowledge the support of the ‘Minist e` re de la
Recherche’, the ‘Centre National de Recherche Scientifique’ and
the ‘R e´ gion Aquitaine’.
3
1
1
2
CH
3
3
)
1
3
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Notes and references
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1 A. Collet, J. P. Dutasta, B. Lozach and J. Canceill, Top. Curr. Chem.,
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993, 165, 103.
H
51
O
12
P
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2
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1
3
,8,13-Trimethoxy-7,12-diphosphono-10,15-dihydro-5H-tribe-
nzo[a,d,g]cyclononen-2-ylphosphonic acid (4). CTV 3 (50 mg,
.065 mmol) was diluted in 3 mL of dried dichloromethane, under
137.
5 M. Dumartin, C. Givelet, P. Meyrand, B. Bibal and I. Gosse, Org.
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0
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overnight. Then the dichloromethane was evaporated and 8 mL
of methanol were added to the crude mixture. The reaction mixture
was warmed to reflux. After 6 h, the solution was cooled to room
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1
Am. Chem. Soc., 1996, 118, 755; Y. J. Zhang, W. X. Ko and J. Xu, Chin.
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◦
1
7 N. Korbakov, P. Timmerman, N. Lidich, B. Urbach, A. Sa’ar and S.
mg, 88%). mp > 265 C (decomposition). H NMR (400 MHz,
O) d 7.78 (d, J = 15.2 Hz, 3H, Ar–H), 7.14 (d, J = 6.4 Hz, 3H,
Ar–H), 4.83 (d, J = 13.6 Hz, 3H, Ar–CH ), 3.85 (s, 9H, OCH ),
). P NMR (162 MHz, D O)
d 12.1 (s). C NMR (100 MHz, D O) d 159.6 (s, CAr), 145.2 (s,
Ar), 135.6 (d, JP–C = 7 Hz, CAr), 130.8 (d, JP–C = 13 Hz, CAr), 120.3
d, JP–C = 175 Hz, CAr), 112.8 (d, JP–C = 9 Hz, CAr), 55.7 (OCH ),
). IR (n cm ): 3372, 2940, 1595, 1491, 1463, 1260,
063, 989, 907. MALDI-MS: m/z for C24 , Na adduct,
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D
2
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3
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.78 (d, J = 13.6 Hz, 3H, Ar–CH
2
2
1
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2
C
(
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H
27
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