Synthesis and spectroscopic characterisation of fluorescent indicators for Na+ and K+ based on (di)pyridino crown ethers

Article abstract of DOI:10.1039/a901941c

The synthesis and the spectral and cation binding properties of a fluorescent indicator for Na⁺, caesium 5-{5-[3,6,9,12-tetraoxa-18-azabicyclo[12.3.1]octadeca-1(18),14,16-trien-16-yl]- 2-thienyl}isophthalate (SPYR), and of two fluorescent indic

Full text of DOI:10.1039/a901941c

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Synthesis and spectroscopic characterisation of fluorescent
indicators for Na؉ and K؉ based on (di)pyridino crown ethers
Abdellah Tahri, Els Cielen, Koen J. Van Aken, Georges J. Hoornaert, Frans C. De Schryver
and Noël Boens*
Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F,
3001 Heverlee, Belgium. E-mail: Noel.Boens@chem.kuleuven.ac.be
Received (in Cambridge) 11th March 1999, Accepted 21st May 1999
The synthesis and the spectral and cation binding properties of a fluorescent indicator for Naϩ, caesium 5-{5-
[3,6,9,12-tetraoxa-18-azabicyclo[12.3.1]octadeca-1(18),14,16-trien-16-yl]-2-thienyl}isophthalate (SPYR), and of
two fluorescent indicators for Kϩ, caesium 5-{5-[3,6,9,12,15-pentaoxa-21-azabicyclo[15.3.1]henicosa-1(21),17,19-
trien-19-yl]-2-thienyl}isophthalate (PPYR) and dicaesium 5-{5-[21-(5-{1-[3,5-di(caesiooxycarbonyl)phenyl]}-2-
thienyl)-3,6,14,17-tetraoxa-23,24-diazatricyclo[17.3.1.18,12]tetracosa-1(23),8(24),9,11,19,21-hexaene-10,21-diyl]-2-
thienyl}isophthalate (BIPY) are reported. To investigate the effect of an sp2-nitrogen in the aza-crown ether cavity
on the complex formation behaviour, a (bi)pyridino crown compound with an appropriate cavity for the sodium and
the potassium cation, respectively, is linked to an aryl thiophene fluorophore. New, versatile routes for the synthesis
of 4-substituted pyridino crown ethers and 4,4Ј-substituted sym-dipyridino crown ethers are described. The chelating
abilities of the indicators with monovalent cations have been studied in aqueous solution by their fluorescence
properties. Both the free and complexed forms of the indicators have an absorption maximum around 345 nm and
an emission maximum at 410 nm, displaying only a decrease in the fluorescence intensity upon cation binding. While
SPYR cannot discriminate between Naϩ and other monovalent cations, PPYR and BIPY show a peak selectivity for
Kϩ with a dissociation constant of 1.2 0.3 mM and 4.4 0.7 mM, respectively.
Cs+ -O2C
CO2-Cs+
Introduction
CO2-Cs+
The accurate measurement of free concentrations of biologic-
Cs+ -O2C
ally important ions and messengers, such as Kϩ, Naϩ, Mg2ϩ
,
Ca2ϩ and Hϩ, inside living tissues, is of tremendous impor-
tance in the fields of cell physiology and clinical medicine.1
Fluctuations in these concentrations are often fast and highly
localised, and are an essential link in biological signal trans-
duction. Optical probes are among the best tools for sensing
such concentrations, because they have an excellent signal-to-
noise ratio and are virtually instantaneous in their response
time.2 The development of fluorescent ion indicators has
become feasible, given the availability of ionophores that
selectively bind the desired ion in aqueous solutions with an
affinity that matches normal cytosolic levels (for Kϩ 50 mM <
Kd < 150 mM; for Naϩ 0 mM < Kd < 50 mM, where Kd is the
ground-state dissociation constant) or extracellular ion levels
(for Kϩ 0 mM < Kd < 20 mM; for Naϩ 120 mM < Kd < 450
mM).3,4 Fluorescent derivatives of such ionophores exhibit
a change, preferably a shift in the excitation and/or emission
spectrum, upon ion binding.5
S
S
N
N
N
O
O
O
O
O
O
O
O
n
1a n = 1 SPYR
1b n = 2 PPYR
S
CO2-Cs+
For the sodium and the potassium cations, the most popular
indicators for intracellular cation determination are sodium
binding benzofuran isophthalate (SBFI) and potassium bind-
ing benzofuran isophthalate (PBFI), respectively,6 consisting
of a diaza-crown ether with an appropriate cavity, linked to a
benzofuran fluorophore. Their spectral responses upon ion
binding permit excitation ratio measurements. In a previous
article, the synthesis and the photophysical studies of two new
fluorescent indicators for Naϩ and Kϩ based on benzocrown
ethers were reported.7 Both indicators only showed a change
in intensity upon ion binding and no corresponding spectral
shifts. To investigate the effect of the presence of an sp2 nitrogen
in the aza-crown cavity on the spectral response to ion binding,
we have developed a series of sodium and potassium indicators,
based on (di)pyridino containing crown derivatives (Fig. 1).
CO2- Cs+
2
BIPY
Fig. 1 Structures of the compounds SPYR, PPYR and BIPY syn-
thesised in this study. SPYR = sodium binding pyridine crown ether,
PPYR = potassium binding pyridine crown ether, BIPY = bispyridine
crown ether.
Taking into account the principles of molecular recognition,
a 15-crown-5 type crown ether was used as the chelating group
for the sodium ion, and an 18-crown-6 type for the potassium
ion. The sym-dipyridine crown unit in compound 2 (Fig. 1)
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748
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allows the attachment of two fluorophores; hence, an increase
in the absorption and the fluorescence intensity can be
expected. Macrocyclic ligands containing pyridino nitrogen
groups have not been studied extensively, because the incorpor-
ation of pyridino groups into unsubstituted crown rings (i.e.,
macrocyclic ligands bearing no ester functionalities) is difficult
synthetically.8 As it is known that introduction of a pyridino
unit into the 18-crown-6 ring produces only a minor decrease
in the stability of the complexes with Naϩ and Kϩ compared
to 18-crown-6, with selectivity among the cations remaining
essentially unaltered,9 it seemed worthwhile to use a pyridino
crown compound as the chelating moiety in a fluorescent in-
dicator. A thiophene derivative was chosen as the fluorophore,
which was linked to the 4-position of the pyridino moiety,
resulting in a 4-(2-thienyl)pyridine skeleton. Thienylpyridine
compounds have found wide applications in designing
fluorescent labeling reagents for HPLC and electrochromic
materials.10 Furthermore, the combination of the electron rich
nature of the thienyl ring and the electron deficient pyridine
centre results in nonlinear optical activity.11 For the indicators
described here, the thiophene group is linked further to a phenyl
ring, substituted with two carboxylate functionalities to ensure
water solubility. In this article, the syntheses of two indicators
with a mono-pyridino crown, SPYR 1a (caesium 5-{5-[3,6,9,12-
tetraoxa-18-azabicyclo[12.3.1]octadeca-1(18),14,16-trien-16-yl]-
2-thienyl}isophthalate) for Naϩ and PPYR 1b (caesium 5-{5-
[3,6,9,12,15-pentaoxa-21-azabicyclo[15.3.1]henicosa-1(21),17,
19-trien-19-yl]-2-thienyl}isophthalate) for Kϩ, and one sym-
dipyridino crown indicator, BIPY 2 (dicaesium 5-{5-[21-(5-{1-
[3,5-di(caesiooxycarbonyl)phenyl]}-2-thienyl)-3,6,14,17-tetra-
oxa-23,24-diazatricyclo[17.3.18,12]tetracosa-1(23),8(24),9,11,19,
21-hexaene-10,21-diyl]-2-thienyl}isophthalate) are presented.
Besides the syntheses of the indicators for Naϩ and Kϩ, the
spectral and cation binding properties of the indicators are
reported. From absorption and steady-state fluorescence
measurements, the position of the spectra, the molar extinction
coefficient (ε), the quantum yield of fluorescence (φf) of the free
and saturated forms of the indicators, as well as the dissociation
constant, Kd, in the ground state of the formed host–guest com-
plexes, and the selectivity vs. other cations were determined.
X and species n the n:1 ion–indicator complex. The global
reaction is determined by the composite dissociation constant
Kd (eqn. (2)).
If one assumes that the intermediate complexes (species 1–
(n Ϫ 1)) are present in concentrations low enough to give only a
negligible contribution to the fluorescence intensity, and that
the rate of ion binding in the excited state is negligible, the total
fluorescence signal due to excitation at λex and observed at λem
can be expressed by eqn. (3), where Fmin corresponds to the
[X]nFmax ϩ KdFmin
(3)
F =
Kd ϩ [X]n
fluorescence signal of the free form of the indicator, Fmax
denotes the fluorescence signal of the saturated form of the
n
indicator and Kd = ∏ Kdi. Fitting eqn. (3) to the fluorescence
i = 1
data F as a function of [X] yields values for Kd, n, Fmin, and
Fmax
.
Results
Synthesis
The syntheses of SPYR 1a, PPYR 1b and BIPY 2 are
approached in a convergent way to allow the attachment of
the thiophene-based fluorophore, which the three indicators
have in common, to the respective ionophores. In all cases,
the coupling between the fluorophore and the ionophore is
achieved via a Stille13 reaction between the halogenated
chelator and the stannylated fluorophore. Therefore, the avail-
ability of a halide-substituted ionophore is required. Although
the synthesis of unsubstituted (di)pyridino crown compounds
has long been known,8 no (di)pyridino crown ethers substituted
by a halide in the 4-position have, to our knowledge, been
synthesised yet. The conversion of the acyclic starting material
to the appropriately substituted pyridine analogue before
cyclisation is recommended, for Bradshaw and co-workers have
found that substitution of a 4-substituent on the pyridine ring
after cyclisation is unsuccessful.8
Synthesis of the monopyridine ionophores 10a,b. The chelating
groups of SPYR 1a and PPYR 1b were synthesised from
chelidamic acid (1,4-dihydro-4-oxopyridine-2,6-dicarboxylic
acid) 3, which was brominated and then esterified to yield
diethyl 4-bromopyridine-2,6-dicarboxylate 4, according to the
procedure described by Takalo and Kankare14 (Scheme 2).
Upon reduction with sodium borohydride,15,16 4-bromo-
pyridine-2,6-dimethanol 5 was obtained, which was converted
either into the corresponding ditosylate 6,17 or into dibromide
7 by addition of phosphorus tribromide.15 When toluene-p-
sulfonyl chloride was used as the tosylating agent, the mass
spectrum of compound 6 revealed a partial substitution of
the bromide in the 4-position by a chloride group, originating
from toluene-p-sulfonyl chloride. Therefore, toluene-p-sulfonyl
bromide was used, freshly prepared by oxidative bromination
of toluene-p-sulfonyl hydrazide.18 For the synthesis of the 4-
bromo-2,6-pyridino crown ethers 8a, two synthesis pathways
were investigated. In both cases, a solution of triethylene glycol
in THF was converted into the corresponding dialcoholate
using sodium hydride as a base, after which the dialcoholate
reacted with the pyridino precursor 6 or 7. When 4-bromo-2,6-
bis(bromomethyl)pyridine 7 was used under high dilution
conditions, much better yields (35%) were obtained than with 4-
bromo-2,6-bis[(p-tolylsulfonyl)methyl]pyridine 6 under normal
reaction conditions (15%). Therefore, compound 8b was syn-
thesised uniquely from the tribromide and could be isolated in
32%.
Theory
Determination of Kd from fluorimetric titration
The expression of the fluorescence signal as a function of the
ion concentration has been derived by Kowalczyk et al.12 for the
case of a 1:1 complex between a fluorescent indicator and a
cation. For a consecutive multiple binding model (Scheme 1),
[0][X]
[1]
(1.1)
(1.2)
0
1
•
•
•
X
1
2
•
•
•
Kd1
Kd2
=
=
+
[1][X]
[2]
+ X
•
•
•
[(n - 1)][X]
[n]
(n - 1)+
Kdn
Kd
=
(1.n)
X
n
[0] [X]n
[n]
n
=
= ΠKdi
0
+
nX
n
(2)
i = 1
Scheme 1
this expression can be expanded by taking into account the
formation of an n:1 ion–indicator complex. Consider a system
consisting of species 0 that can undergo a reversible reaction
with ion X to form species 1 (determined by the dissociation
constant Kd1), which in turn can react with another equivalent
of X, to yield species 2 (determined by the dissociation constant
Kd2), etc. (eqn. (1.1)–(1.n)). Species 0 represents the free form of
a fluorescent indicator, species 1 the 1:1 complex with a cation
Synthesis of the sym-dipyridyl ionophore 10. Few reports on
the synthesis of sym-dipyridyl-18-crown-6 compounds are
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J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748

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Br
Br
Br
N
OH
i
ii
N
N
N
HO2C
CO2H
Br
Br
O
O
O
O
O
3
7
i
O
OH
HO
N
9
Br
Br
ii
Br
N
CO2Et
EtO2C
N
10
OH
OH
4
5
Scheme 3 Reagents and conditions: i: a) 3.3 equiv. NaH, excess ethyl-
ene glycol, 15 min at rt, 30 min at 60 ЊC, b) 0 ЊC, 7, THF, 10 h at rt;
ii: a) 1.1 equiv. KH, THF, 0 ЊC, 11, b) THF, 7, 0 ЊC, rt overnight.
iv
iii
Reversal of the order of addition, i.e. addition of a solution of
potassium hydride in THF to a solution of the diol 9 in THF,
followed by diluted addition of the tribromide 7 to this mixture,
all caried out at 0 ЊC, improved the yield to 61%.
Br
Br
N
N
Br
Br
Synthesis of the indicators SPYR 1a, PPYR 1b and BIPY 2.
The next step comprises the coupling between both types of
crown compounds 8a,b and 10 and the fluorophore building
block. This synthon, dimethyl 5-(5-tributylstannyl-2-thienyl)-
isophthalate 14, was synthesised by a Stille coupling between 2-
(tributylstannyl)thiophene 11 and dimethyl 5-iodoisophthalate
12,7 followed by stannylation in the 5-position of the thiophene
nucleus of 13 with lithium 2,2,6,6-tetramethylpiperidine–
tributyltin chloride, as was described previously7 (Scheme 4).
The methyl esters of the fluorescent indicators 15a,b and 16
were obtained in good yields by refluxing a mixture of 1.2
equiv. of the fluorophore building block 14 (for 15a,b) or 2.4
equiv. of 14 (for 16) and 1 equiv. of the appropriate crown
compound 8a, 8b or 10 in anhydrous toluene under nitrogen
atmosphere in the presence of 1 mol% tetrakis(triphenyl-
phosphine)palladium. In the final reaction step, the esters 15a,b
and 16 were converted into the corresponding water soluble
forms. The free acid 17a was obtained by refluxing compound
15a overnight in dry THF with 2.5 equiv. potassium trimethyl-
silanolate.20 Since the free acid 17a showed a limited solubility
in aqueous solution at physiological pH (7.05), we preferred to
prepare directly the caesium salt,21 analogously to Minta and
Tsien, who used Csϩ as a truly inert background cation.6 Hence,
the caesium salts 1a,b and 2 were obtained from the corre-
sponding methyl esters 15a,b and 16 by reflux in methanol with
a large excess of caesium hydroxide and were used as such
for the fluorescence measurements. Comparison of the UV
spectra of the esters in methanol and the caesium salts in water
indicates that the fluorophore structure remains unaltered.
OTs
OTs
vi
6
7
v
O
O
O
N
Br
O
n
8a n = 1
8b n = 2
Scheme 2 Reagents and conditions: i: a) Br2, PBr3, hexane, 3 h at 90 ЊC,
b) CHCl3, EtOH; ii: NaBH4, EtOH, 2 h at rt, 15 h reflux; iii: PBr3, ether,
30 min at rt, overnight reflux; iv: a) 5, THF, 2.2 equiv. KOH, 15 min
at 0 ЊC, b) 2.1 equiv. toluene-p-sulfonyl bromide, THF, 5 h at 0 ЊC, 12 h
at rt; v: a) 1.1 equiv. HO(CH2CH2O)3H, 1.2 equiv. NaH, THF, 1 h
reflux, b) Ϫ78 ЊC, 6, THF, 2 days rt; vi: a) 1.1 equiv. NaH, THF, 0 ЊC,
HO(CH2CH2O)nH (n = 3 or 4), b) 200 cm3 THF, 7, 2 h at 0 ЊC, rt
overnight.
known in the literature.17,19 They are all based on a one-pot
reaction of two ethylene glycol units with two 2,6-disubstituted
pyridino building blocks and are characterised by yields not
exceeding 20%. Therefore, we built up the dipyridino con-
taining ligand 4,4Ј-dibromo-sym-dipyridyl-18-crown-6 10 in a
two-step reaction. This allows a fast and high yield formation
of the diol 9, without polymerisation as a side reaction. Thus,
4,4Ј-dibromo-sym-dipyridyl-18-crown-6 10 was synthesised
from 4-bromo-2,6-bis(bromomethyl)pyridine 7 (Scheme 3),
which was prepared as described above. By drop-wise addition
of this tribromide at 0 ЊC to a solution of excess ethylene glycol
in anhydrous THF in the presence of sodium hydride, com-
pound 9 was formed. Yet, upon removal of the ethylene glycol,
reaction at the 4-substituent of 9 occurred, due to the high
temperatures required. Therefore, compound 9 was isolated by
column chromatography in 95% yield. The acyclic precursor
9 was then cyclised to yield compound 10 by adding a second
4-bromo-2,6-bis(bromomethyl)pyridino unit 7 with potassium
hydride acting as a template. In a first attempt, the diol 9 was
added under high dilution conditions to a solution of 2 equiv.
potassium hydride in THF at room temperature, followed
by drop-wise addition of the tribromide 7 in THF. Due to a
side-reaction with the 4-bromo substituent of 7, the desired
dipyridino crown 10 could be isolated only in 14% yield.
Steady-state fluorescence
Steady-state fluorescence and absorption measurements were
performed on the caesium salts of the indicators in aqueous
solution at 20 ЊC and pH 7.05. The indicator concentrations
were of the order of (5–6) × 10Ϫ6 M, yielding an absorbance
per cm path length of approximately 0.1 at the absorption
maximum. A physiological pH of 7.05 was obtained by
buffering the solutions with 10Ϫ2 M MOPS (3-morpholino-
propanesulfonic acid), adjusted with tetramethylammonium
hydroxide.
SPYR 1a and PPYR 1b. The absorption spectra of SPYR
and PPYR show a small decrease in the absorbance with
increasing cation concentration, while the absorption maxima
remain located at 345 nm for SPYR and 342 nm for PPYR
(not shown). These decreases are reflected by small changes in
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748
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I
4'
3'
CO2Me
4
i
3
+
R
SnBu3
5'
2'
S
1'
5
S
2
MeO2C
CO2Me
6
1
CO2Me
11
12
13 R = H
14 R = SnBu3
ii
O
O
O
O
O
Br
N
N
Br
+
N
Br
iv
iii
+
O
O
O
n
10
8a n = 1
8b n = 2
RO2C
RO2C
RO2C
CO2R
S
O
N
O
S
O
O
15a n = 1 R = Me
15b n = 2 R = Me
n
N
O
O
O
vi
v (15a)
O
1a n = 1 R = Cs+
1b n = 2 R = Cs+
17a n = 1, R = H
N
S
CO2R
vi
2 R = Cs+
CO2R
16 R = Me
Scheme 4 Reagents and conditions: i: 2 mol% (MeCN)2PdCl2, DMF, air, rt; ii: a) 2,2,6,6-tetramethylpiperidine, BuLi, THF, 0 ЊC, b) 13, tributyltin
chloride, THF, 2 h at Ϫ78 ЊC, rt overnight; iii: 1.2 equiv. 14, anhydrous toluene, 8a or 8b, 1 mol% Pd(PPh3)4, 15 h reflux; iv: 2.4 equiv. 14, anhydrous
toluene, 10, 1 mol% Pd(PPh3)2, 15 h reflux; v: a) 2.5 equiv. KOSiMe3, THF, overnight reflux, b) HCl; vi: excess CsOH, MeOH, overnight reflux.
the molar extinction coefficients, which for SPYR are
(23.6 0.1) × 103 MϪ1 cmϪ1 for the free form of the indicator
and (20.8 0.2) × 103 MϪ1 cmϪ1 for the Naϩ saturated form.
The molar extinction coefficients of PPYR are somewhat lower:
(18.0 0.1) × 103 MϪ1 cmϪ1 and (17.2 0.3) × 103 MϪ1 cmϪ1
for the free and the Kϩ-bound form, respectively.
The ground-state dissociation constants, Kd, of the ion–
indicator complexes were determined by fitting eqn. (3) to the
fluorescence data F at λem = 410 nm and λex = 340 nm, yielding
additional values for n, Fmin, and Fmax. The non-linear fit for the
binding of Naϩ by SPYR, which is given in Fig. 4, yields a value
of 1.2 0.2 mM for Kd. n equals 1.05 0.04, indicating a 1:1
stoichiometry for the Naϩ–indicator complex. The Kd values for
the other monovalent cations with SPYR determined in this
way are summarised in Table 2.
Analysis of the data for PPYR in a similar way (Fig. 5) yields
a dissociation constant of 1.2 0.3 mM for Kϩ–PPYR and a
stoichiometry consistent with a 1:1 complex. The binding
properties of PPYR with the other monovalent cations are
given in Table 3.
The fluorescence excitation and emission spectra of SPYR
with Naϩ and PPYR with Kϩ are shown in Fig. 2 and Fig. 3,
respectively. Both indicators show an emission maximum at 410
nm; the excitation maxima are identical to those of absorption.
The relative decrease in the fluorescence intensity with increas-
ing concentrations of all cations is much more pronounced for
SPYR than for PPYR. The fluorescence intensity of SPYR
with Naϩ reaches a first plateau at 7 mM Naϩ, followed by a
further decrease, which can be attributed to an excited-state
reaction.12 The fluorescence quantum yields for the free and
bound form of SPYR are 0.73 and 0.63, respectively. PPYR is
saturated with Kϩ at 5 mM Kϩ, from whereon the intensity
reaches a constant value. The fluorescence quantum yields of
PPYR are even higher than those measured for SPYR (φf = 0.84
for the free and 0.80 for the Kϩ bound form). The spectral
properties of SPYR and PPYR are summarised in Table 1.
BIPY 2. The absorbance of the sym-dipyridino crown indi-
cator is rather insensitive to increasing cation concentration:
the extinction coefficients are (37.2 0.3) × 103 MϪ1 cmϪ1 for
the free form and (36.4 0.2) × 103 MϪ1 cmϪ1 for the Kϩ com-
plex. The changes in the emission and excitation spectra that
accompany Kϩ binding are shown in Fig. 6. The excitation
spectrum (with λmax = 344 nm) and the emission spectrum (with
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Table 1 Spectral properties of SPYR, PPYR and BIBY at pH 7.05 and 20 ЊC. For SPYR, the Naϩ complex is taken as the bound form, while for
PPYR and BIPY the Kϩ complex is considered
λmax/nm
ε/103 MϪ1 cmϪ1
φf
Indicator
excitation
emission
free
bound
free
bound
SPYR
PPYR
BIPY
345
342
344
410
410
410
23.6 0.1
18.0 0.1
37.2 0.3
20.8 0.2
17.2 0.3
36.4 0.2
0.73
0.84
0.35
0.63
0.80
0.13
Fig. 2 Fluorescence excitation (A) and emission (B) spectra of SPYR
as a function of [Naϩ] at 20 ЊC and pH 7.05.
Fig. 3 Fluorescence excitation (A) and emission (B) spectra of PPYR
as a function of [Kϩ]. The experimental conditions were as in Fig. 2.
a factor of 1.6, 3.6 times more strongly than Naϩ and 4.1 times
λmax = 410 nm) undergo a similar intensity decrease upon ion
complexation, but the effect is more pronounced for this indi-
cator than with the monopyridine derivatives. At elevated ion
concentrations, a second band at longer wavelengths shows up
in the emission spectrum, probably due to aggregate formation.
In order to obtain reproducible results, freshly prepared and
well stirred samples had to be used. Although enhanced fluor-
escence was expected for BIPY compared to SPYR and PPYR,
given the presence of two fluorophore moieties, much lower
quantum yields were observed: 0.35 for the free form, and 0.13
for BIPY saturated with Kϩ. The spectral properties of BIPY
are summarised in Table 1.
more strongly than Csϩ). BIPY also shows a high affinity for
the divalent cations Ca2ϩ and Mg2ϩ
.
Discussion
Synthesis
The synthesis of the indicators SPYR, PPYR and BIPY
involves the development of a synthetic route towards new 4-
(4Ј-) (di)halogenated (di)pyridino crown ethers as key inter-
mediates. In the 2,6-disubstituted pyridino building blocks a 4-
bromo substituent is introduced prior to cyclisation which takes
place under high-dilution conditions. By using a two-step reac-
tion instead of the known one-pot synthesis,17,19 the yield of
sym-dipyridino crown ethers could be raised to 60%. These
macrocyclic ionophores can be coupled in a Stille reaction to
the stannylated fluorophore to yield the desired fluorescent
indicators in good yields.
For the Kϩ–BIPY complex, a dissociation constant of 4.4
0.7 mM was determined by fitting eqn. (3) to the fluorescence
data at 410 nm (Fig. 7). The selectivity of BIPY vs. other
cations, like Kϩ, Liϩ, Csϩ, Mg2ϩ, and Ca2ϩ, can be calculated
from the ratio of their respective Kd values (Table 4). Com-
parison of the data for monovalent cations shows a peak
selectivity for Kϩ (Kϩ binds more strongly to BIPY than Liϩ by
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748
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Table 2 Binding properties of SPYR with the respective cations
determined by fitting eqn. (3) to the fluorescence data, with Fmax, Fmin
Table 4 Binding properties of BIPY with the respective cations
determined by fitting eqn. (3) to the fluorescence data, with Fmax, Fmin
Kd, and n as adjustable parameters. The experimental conditions were
,
,
Kd, and n as adjustable parameters. Since eqn. (3) allows direct fitting
for Kd and n, the errors on these values are reported. The dissociation
as in Table 2
constants were measured with 6.5 µM of the indicator and 10Ϫ2
M
MOPS at pH 7.05 buffered by (5–6) × 10Ϫ3 M tetramethylammonium
hydroxide. The emission intensity, originating from excitation at 340
nm, was recorded at 410 nm. No correction was made for the change in
ionic strength of the solutions
Ion
Kd/mM
n stoichiometry
Liϩ
7
16
4.4 0.7
18
4
2
3
1.0 0.1
1.0 0.1
0.98 0.04
1.1 0.1
1.1 0.1
1.0 0.1
Naϩ
Kϩ
Csϩ
Ca2ϩ
Mg2ϩ
5
1
Ion
Kd/mM
n stoichiometry
Liϩ
Naϩ
Kϩ
1.1 0.5
1.2 0.2
1.2 0.7
1.7 0.4
0.97 0.05
1.05 0.04
0.95 0.07
1.00 0.02
1.7 0.4
Csϩ
Table 3 Binding properties of PPYR with the respective cations
determined by fitting eqn. (3) to the fluorescence data, with Fmax, Fmin
,
Kd, and n as adjustable parameters. For Kϩ, the excitation intensity,
originating from emission at 410 nm, was recorded at 344 nm. For the
other cations, the experimental conditions were as in Table 2
Ion
Kd/mM
n stoichiometry
Liϩ
Naϩ
Kϩ
1.2 0.1
2.4 0.9
1.2 0.3
2.8 0.4
0.96 0.04
1.1 0.3
1.05 0.04
1.00 0.02
Csϩ
Fig. 4 Non-linear fit of eqn. (3) to the emission intensities of SPYR at
λem = 410 nm as a function of the Naϩ concentration. The excitation
wavelength was 340 nm and the other experimental conditions were as
in Fig. 2.
Fig. 6 Fluorescence excitation (A) and emission (B) spectra of BIPY
as a function of [Kϩ]. The experimental conditions were as in Fig. 2.
Fig. 5 Non-linear fit of eqn. (3) to the excitation intensities of PPYR
at λex = 344 nm as a function of the Kϩ concentration. The emission
wavelength was 410 nm and the other experimental conditions were as
in Fig. 2.
Fig. 7 Non-linear fit of eqn. (3) to the emission intensities of BIPY at
λem = 410 nm as a function of the Kϩ concentration. The excitation
wavelength was 340 nm and the other experimental conditions were as
in Fig. 2.
1744
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748

View Article Online
plexed form,8 the equilibrium conformation does not change
much upon complexation, thus causing little ligand deform-
ation. The weaker complex formation with the sym-dipyridyl
compound BIPY compared to PPYR might be attributed to
a higher degree of ligand deformation upon ion binding.27 This
hypothesis is supported by the more pronounced difference in
the shape of the spectra and the fluorescence quantum yields
for BIPY compared to PPYR and SPYR.
Spectral and cation binding properties
The most striking property of the mono-pyridino indicators
SPYR and PPYR is their excellent fluorescence quantum
yield, which in both cases lowers after ion binding. Although
for BIPY, a fluorescence enhancement was expected compared
to SPYR and PPYR, due to the presence of two fluorophores,
quantum yields are much smaller. Via time-resolved fluores-
cence measurements and global compartmental analysis, the
rate constants of the radiative and the non-radiative processes
can be determined to gain more insight into the mechanism of
the changes in the fluorescence intensity upon cation binding.28
Since the indicators display only a change in intensity upon
cation binding and no corresponding spectral shifts, they are
not suitable for ratiometric measurements at dual wavelengths.
While SPYR has an equal chelating power vs. all monovalent
cations, PPYR shows a two-fold selectivity for Kϩ over Naϩ,
whereas BIPY displays a similar 3.5-fold selectivity. This com-
pares favourably with PBFI, which shows a 2.6-fold selectivity
for Kϩ over Naϩ. Furthermore, BIPY gives rise to a more
pronounced response upon ion binding: a 40% decrease in the
fluorescence intensity between the free and the Kϩ saturated
form is observed, compared to a 15% decrease for PPYR with
Kϩ. The Kd value estimated for Kϩ–BIPY (4.4 0.7 mM) is
comparable to that for Kϩ–PBFI (5.1 mM), while Kd for Kϩ–
PPYR (1.2 0.3 mM) matches extracellular potassium levels
(0–20 mM). The lower fluorescence quantum yields of BIPY
compared to the monopyridine compounds still largely exceed
those of PBFI (0.024 for the free form and 0.072 for the Kϩ-
bound form, respectively).
Fig. 8 Comparison of the cation binding properties of the indicators
under study with 15C5 and 18C6 in water.
Basis for cation affinity and selectivity
From comparison of the binding behaviour of SPYR, PPYR
and BIPY, a peak selectivity22 is seen for the 18-crown-6 com-
pounds PPYR and BIPY, while the 15-crown-5 derivative
SPYR can hardly discriminate between the monovalent cations.
As Tables 3 and 4 point out, the Kd values for PPYR and BIPY
increase in the order Kϩ < Liϩ < Naϩ < Csϩ. For an optimal
complexation, the size of the cavity should be equal to the size
of the cation to be recognised, as is the case for Kϩ with 18-
crown-6.23,24 Both Liϩ and Naϩ have too small a radius to be
effectively accomodated within the 18-crown-6 cavity of PPYR
and BIPY. The unexpectedly strong complexation of Liϩ may
be explained by the high degree of solvatation of the lithium
ion compared to the other monovalent cations. This theory
was proposed by Valeur et al., who observed a similar strong
complexation of the aza crown indicator BOZ-crown (BOZ =
benzoxazinone) with Liϩ.24 By taking part in the complex
formation under its solvated form, Liϩ would fit better into the
18-crown-6 cavity. Csϩ, with an ion radius exceeding the crown
cavity radius, gives rise to a complex strongly destabilised by
cation–ligand repulsions as well as ligand deformations.25
Given the affinity of the respective indicators for Csϩ, com-
petition between the other monovalent cations and Csϩ, which
was used in preparing the caesium salts, can be expected. How-
ever, for an indicator to have optimal sensitivity for a given
cation X, it is required that 0.1 Kd ≤ [X] ≤ 10 Kd. In all samples
[Csϩ] = 5–6 × 10Ϫ5 M, a concentration which is well below 0.1
Kd. Therefore, interference with Csϩ is of minor importance.
Although the unsolvated sodium ion fits rather well into the
SPYR-cavity, an efficient complexation is prohibited by a
high solvatation energy.22 The high selectivity of BIPY for Ca2ϩ
and Mg2ϩ may be due to an enhanced affinity between the soft
pyridine nitrogen donors and more charge dense ions.26
Experimental
Materials and methods
When required, solvents and reagents were dried prior to use.
Tetrahydrofuran (THF) and toluene were distilled over sodium.
1H NMR spectra were recorded on a Bruker WM 250 or a
Bruker AMX 400 instrument. The spectra were measured using
deuteriochloroform (CDCl3) or deuterated DMSO as solvent.
1
The H and 13C chemical shifts are reported in ppm relative
to tetramethylsilane or the deuterated solvent as an internal
reference. The coupling constants J are given in Hz. Mass
spectra were run using a Kratos MS50TC instrument and a
DS90 data system. IR spectra were recorded on a Perkin-Elmer
1720 Fourier transform spectrometer. Melting points were
determined with a Reichert Thermovar apparatus and are
uncorrected.
Influence of the presence of a pyridino ring
The absorption measurements were performed on a Perkin-
Elmer Lambda 6 UV/VIS spectrophotometer. Corrected steady-
state excitation and emission spectra were recorded on a SPEX
Fluorolog 212. Fluorescence quantum yields of the free and
saturated forms of the indicators were determined using
quinine sulfate in 0.05 M sulfuric acid as reference. The
quantum yield of the reference was taken to be 0.54.29 The
samples were measured in Milli-Q water. No correction for the
refractive index was necessary.
In Fig. 8, the log Kd values of the ion complexes of SPYR,
PPYR and BIPY are compared with those of the corre-
sponding parent compounds 18C6 (18-crown-6) and 15C5
(15-crown-5). Although smaller fluctuations are observed for
PPYR and BIPY compared to 18C6, the three 18-membered
derivatives all follow the same tendency: Kd(Csϩ) ≥ Kd(Naϩ) >
Kd(Kϩ). The 15-membered compounds show hardly any selec-
tivity among monovalent cations. The increased stability of
the complexes of PPYR and SPYR compared to 18C6 and
15C5 is thought to be due to the stabilising presence of the
electron rich thiophene fluorophore, which may exert an
electron donating effect. Additionally, since the pyridine group
introduces organisation and rigidity both in the free and com-
Synthesis
Diethyl 4-bromopyridine-2,6-dicarboxylate 4. Compound 4
was prepared according to the procedure described by Takalo
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748
1745

View Article Online
and Kankare.14 Mp = 94–95 ЊC (lit:14 95–96 ЊC); νmax(KBr)/cmϪ1
3072–2980 (CH3, CH2), 1710 (CO), 1560 (pyridine), δH(CDCl3)
1.43 (6H, t, J 7.2, CH3), 4.48 (4H, q, J 7.2, CH2), 8.42 (2H,
s, Pyr-H); δC(CDCl3) 14.18 (CH3), 62.7 (CH2), 131 (CH),
residue was diluted with chloroform, washed with brine and
dried over MgSO4. After evaporation, the product was purified
on alumina (CHCl3–MeOH, 99:1). A viscous oil was obtained
(50 mg, 15%).
134.8 (CBr), 149.52 (C᎐N), 163.53 (CO); m/z 302 (Mϩ ϩ 1,
Method B. In a 500 cm3 three necked flask equipped with two
septa sodium hydride (198 mg, 3.3 mmol, 1.1 equiv.) was dis-
solved in anhydrous THF (50 cm3) under argon. After cooling
to 0 ЊC, triethylene glycol (0.4 cm3, 3 mmol) was added drop-
wise, followed by stirring for 1 h while the temperature was
kept constant. Then, THF (200 cm3) was added, whereupon
compound 7 (1.032 g, 3 mmol) dissolved in THF (50 cm3) was
added drop-wise at 0 ЊC over a period of 2 h. After stirring
overnight at room temperature, the formed precipitate was
filtered off and the filtrate was condensed by evaporation. The
obtained residue was dissolved in chloroform (100 cm3) and
water (200 cm3). The aqueous layer was extracted with chloro-
form (4 × 30 cm3) and the combined organic layers were washed
with brine and then dried over MgSO4. A viscous oil (348 mg,
35%) was obtained after column chromatography on alumina
(CHCl3–MeOH, 98:2); νmax(CHCl3)/cmϪ1 3020–2920 (CH2,
CH), 1570–1520 (pyridine), 1100 (C-O); δH(CDCl3) 3.48 (4H,
s, CH2), 3.47–3.6 (4H, m, CH2), 3.7–3.79 (4H, m, CH2), 4.66
(4H, s, CH2) 7.42 (2H, s, Pyr-H); δC(CDCl3) 70.34, 70.49,
ؒ
᎐
ϩ
9%), 256 (Mϩ Ϫ EtO, 12), 229 (M Ϫ C3H5O2, 100), 201
(C6H2BrNO2ϩ, 22), 76 (C5H2Nϩ, 31); Found: 300.9948, Calc.
for C11H12BrNO4: 300.9949.
ؒ
ؒ
[4-Bromo-6-(hydroxymethyl)-2-pyridyl]methanol 5. Com-
pound 5 was prepared according to the procedure described
by Takalo et al.15 and Lüning et al.16; mp = 163–164 ЊC (lit:15
162–164 ЊC); νmax(KBr)/cmϪ1 3350 (OH), 3090 (CH2), 1580
(pyridine); δH(DMSO-d6) 4.51 (4H, d, J 5.6, CH2), 5.49 (2H, t,
J 5.6, OH), 7.5 (2H, s, Pyr-H); δC(DMSO-d6) 63.62 (CH2),
121.03 (CH), 133.15 (CBr), 163.14 (C᎐N); m/z 217 (Mϩ , 22%),
ؒ
᎐
199 (Mϩ Ϫ H2O, 39), 170 (M Ϫ CH2O Ϫ OH, 27), 90
(C6H4Nϩ, 63), 76 (C5H2Nϩ, 22), 63 (C4HNϩ, 63), 51 (C3HNϩ,
100); Found: 216.9736, Calc. for C7H8BrNO2: 216.9738.
ϩ
ؒ
ؒ
4-Bromo-2,6-bis[(p-tolylsulfonyl)oxymethyl]pyridine 6. To a
suspension of the diol 5 (2.18 g, 10 mmol) in dry THF (80 cm3),
finely ground potassium hydroxide (1.45 g, 22 mmol, 2.2 equiv.)
was added. After stirring for 15 min at 0 ЊC under an argon
stream, toluene-p-sulfonyl bromide (4.94 g, 21 mmol, 2.1
equiv.) dissolved in THF (20 cm3) was added drop-wise. The
reaction mixture was then stirred for 5 h at 0 ЊC followed by
12 h at room temperature. The mixture was filtered on a glass
filter and then evaporated. The obtained residue was purified by
column chromatography on silica (CH2Cl2–hexane, 60:40)
yielding a white powder (4.2 g, 77%); mp = 110–111 ЊC
(CH2Cl2–hexane); νmax(KBr)/cmϪ1 3060 (CH2), 1570 (pyridine),
1370–1170 (-O-SO2), 670 (CBr); δH(CDCl3) 2.45 (6H, s, CH3),
5.01 (4H, s, CH2), 7.34 (4H, d, J 7.6, m-Ar-H), 7.4 (2H, s,
Pyr-H), 7.8 (4H, d, J 7.6, o-Ar-H); δC(CDCl3) 21.67 (CH3),
70.77, 73.75 (CH ), 124.66 (CH), 133.2 (CBr), 159.8 (C᎐N);
᎐
2
ϩ
m/z 332 (Mϩ, 72%), 288 [Mϩ Ϫ (CH2CH2O), 43], 244 [M
ؒ
ؒ
Ϫ
(CH2CH2O)2, 66], 200 [Mϩ Ϫ (CH2CH2O)3, 100], 184
ؒ
[Mϩ Ϫ (CH2CH2O)3 Ϫ O, 34]; Found: 331.0422, Calc. for
ؒ
C13H18BrNO4: 331.0419.
19-Bromo-3,6,9,12,15-pentaoxa-21-azabicyclo[15.3.1]-
henicosa-1(21),17,19-triene 8b. Compound 8b was prepared as
decribed in Method B for compound 8a from the tribromide
7 (344 mg, 1 mmol) and tetraethylene glycol (172 µl, 1 mmol,
1 equiv.). A very viscous oil (120 mg, 32%) was obtained after
column chromatography on alumina (CHCl3–MeOH, 98:2);
νmax(CHCl3)/cmϪ1 3015–2920 (CH2, CH), 1570–1520 (pyridine),
1100 (C-O); δH(CDCl3) 3.59 (8H, s, CH2), 3.66–3.69 (4H, m,
CH2), 3.73–3.76 (4H, m, CH2), 4.74 (s, 4H, CH2) 7.43 (2H,
s, Pyr-H); δC(CDCl3) 69.8, 70.4, 70.8, 71.2, 73.1 (CH2), 124.6
70.44 (CH2), 124.48 (CH(Pyr)), 128.03 (Co), 130 (Cm), 132.57
(C ), 134.45 (C-Br), 145.42 (C-S), 154.89 (C᎐N); m/z 525 (Mϩ
,
ؒ
᎐
p
4%), 370 (Mϩ Ϫ C7H7SO2, 5), 355 (M Ϫ C7H7SO3, 8), 155
(C7H7SO2ϩ, 15), 91 (C7H7ϩ, 100); Found: 524.9910, Calc. for
C21H20BrNO6S2: 524.9915.
ϩ
ؒ
ؒ
(CH), 133.5 (CBr), 159.7 (C᎐N); m/z 367 (Mϩ , 100%), 332
ؒ
᎐
[Mϩ Ϫ (CH2CH2O), 13], 297 (M Ϫ Br, 12), 288 [M
ϩ
ؒ
ϩ
ؒ
Ϫ
ؒ
4-Bromo-2,6-bis(bromomethyl)pyridine 7. The tribromo
derivative 7 was prepared from compound 5 as was described
by Takalo et al.15 To a suspension of the diol 5 (2.18 g,
10 mmol) in ether (100 cm3) at 0 ЊC, phosphorus tribromide
(4.07 g, 15 mmol) was added drop-wise, after which the mixture
was stirred at room temperature for 30 min and then refluxed
overnight. After cooling to room temperature, an aqueous
solution of 5% NaHCO3 was added gradually until neutral pH
was reached. The solution obtained was extracted with ether
(4 × 30 cm3) and the combined organic layers were con-
centrated. After filtering the residue on silica (CH2Cl2), white
crystals were obtained (2.63 g, 76%); mp = 131–132 ЊC (lit:15
128–129 ЊC) (CH2Cl2–hexane); νmax(KBr)/cmϪ1 3060 (CH2),
1560 (pyridine); δH(CDCl3) 4.64 (4H, s, CH2), 7.78 (2H, s,
(CH2CH2O)2, 3]; Found: 375.0683, Calc. for C15H22BrNO5:
375.0681.
4-Bromo-2,6-bis[(2-hydroxyethoxy)methyl]pyridine 9. To a
suspension of sodium hydride (304 mg, 12.6 mmol) ethylene
glycol (40 cm3) (freshly distilled) was added. The mixture was
stirred at room temperature for 15 min, followed by heating
at 60 ЊC for 30 min. After cooling to 0 ЊC, a solution of the
tribromide 7 (1.45 g, 4.2 mmol) in THF (5 cm3) was added
drop-wise. After stirring at room temperature for 10 h, the
mixture was poured into glacial brine (50 cm3), whereupon
ethyl acetate (20 cm3) was added. Subsequently, the product was
extracted with ethyl acetate (10 × 15 cm3) and dried over
MgSO4. The yellow oil obtained after evaporation was filtered
over alumina (CH2Cl2–MeOH, 90:10) yielding a white powder
(1.17 g, 91%); mp 62–63 ЊC (CH2Cl2–hexane); νmax(KBr)/cmϪ1
3058 (CH2), 1561 (pyridine); δH(CDCl3) 3.7–3.82 (8H, m,
Pyr-H); δC(CDCl3) 33.2 (CH2), 125.97 (CH), 133.11 (CBr),
ϩ
158.34 (C᎐N); m/z 341 (Mϩ , 10%), 262 (M Ϫ Br, 60), 183
ؒ
ؒ
᎐
(Mϩ Ϫ 2 × Br, 12), 104 (C6H4N , 63); Found: 340.8055, Calc.
ϩ
ؒ
for C7H6Br3N: 340.8050.
CH (O)), 4.28 (2H, t, J 5.87, OH), 4.64 (4H, s, CH C᎐N), 7.42
᎐
2 2
(2H, s, Pyr-H); δC(CDCl3) 61.47, 72.47, 73.13 (CH2), 123.45
(CH), 134.2 (CBr), 159.19 (C᎐N); m/z 306 (Mϩ , 18%), 245
ؒ
16-Bromo-3,6,9,12-tetraoxa-18-azabicyclo[12.3.1]octadeca-
1(18),14,16-triene 8a. Method A. In a three necked flask
equipped with a septum and a reflux condensor triethylene
glycol (165 mg, 1.1 mmol) in anhydrous THF (5 cm3) was added
to a solution of sodium hydride (36 mg, 1.2 mmol) in THF
(10 cm3) under argon. After refluxing the mixture for 1 h, the
solution was cooled to Ϫ78 ЊC and a solution of the ditosylate
6 (526 mg, 1 mmol) in THF (10 cm3) was added drop-wise.
After stirring at room temperature for 2 days, the precipitate
was filtered off and the filtrate was concentrated. The obtained
᎐
[Mϩ Ϫ (O(CH2)2OH), 100], 184 [M Ϫ (O(CH2)2OH)2, 19],
104 (C7H6Nϩ, 65); Found: 305.0258, Calc. for C11H16BrNO4:
305.0262.
ϩ
ؒ
ؒ
10,21-Dibromo-3,6,14,17-tetraoxa-23,24-diazatricyclo-
[17.3.1.18,12]tetracosa-1(23),8(24),9,11,19,21-hexaene 10. In a
three necked flask equipped with a septum and a stopcock,
potassium hydride (88 mg, 2.2 mmol, 1.1 equiv.) was dissolved
in anhydrous THF (20 cm3). After cooling to 0 ЊC, a solution of
1746
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748

View Article Online
ϩ
compound 9 (306 mg, 1 mmol) in THF (10 cm3) was added
drop-wise, followed by stirring at 0 ЊC for 30 min. Then, THF
(40 cm3) was added, whereupon a solution of the tribromide
7 (344 mg, 1 mmol) in THF (30 cm3) was added drop-wise at
0 ЊC over a period of 2 h. The reaction mixture was stirred
overnight at room temperature and subsequently poured into
water (50 cm3) and ether (30 cm3). The aqueous phase was
extracted with ether (4 × 10 cm3), and the combined organic
layers were washed with brine and then dried over MgSO4.
After column chromatography on alumina (CH2Cl2–MeOH,
98:2) white crystals were obtained (300 mg, 61%); mp
163–164 ЊC (CH2Cl2–hexane); νmax(KBr)/cmϪ1 2920 (CH2, CH),
1565–1440 (pyridine), 1100 (C-O), 680 (CBr); δH(CDCl3) 3.76
CHO, 29], 484 [Mϩ Ϫ ((CH2)2OCH2CHO), 22], 396 [M
Ϫ
ؒ
ؒ
[(CH2)2O]4H, 100], 381 [Mϩ Ϫ CH2[(CH2)2O)]4H, 76]; Found:
ؒ
571.1873, Calc. for C29H33NO9S: 571.1876.
Dimethyl
5-[5-(21-{5-[3,5-di(methoxycarbonyl)phenyl]-2-
thienyl}-3,6,14,17-tetraoxa-23,24-diazatricyclo[17.3.1.18,12]-
tetracosa-1(23),8(24),9,11,19,21-hexaen-10-yl)-2-thienyl]iso-
phthalate 16. To a solution of compound 14 (334 mg, 0.57
mmol, 1.2 equiv.) in anhydrous toluene (10 cm3) compound
10 (120 mg, 0.24 mmol) and Pd(PPh3)4 (6 mg, 1 mol%) were
added. The reaction mixture was refluxed during 15 h under an
argon stream. After cooling to room temperature, the solvent
was evaporated and the yellow residue was purified on alumina
(CHCl3–hexane, 70:30 then CHCl3–MeOH, 99.5:0.5). A
yellow powder was obtained, which was recrystallised from
hot CH2Cl2 followed by addition of cold hexane (443 mg, 60%);
mp 269–270 ЊC (CH2Cl2–hexane); νmax(KBr)/cmϪ1 2950 (CH3,
CH2, CH), 1720 (CO), 1600–1540 (pyridine), 1100 (C-O);
δH(CDCl3) 3.92 (8H, s, CH2), 3.99 (12H, s, CH3), 4.56 (8H, s,
(8H, s, CH (O)), 4.55 (8H, s, CH C᎐N), 7.43 (4H, s, Pyr-H);
᎐
2
2
δC(CDCl3) 70.22, 72.95 (CH2), 133.53 (CH), 133.64 (CBr),
ϩ
159.49 (C᎐N); m/z 489 (Mϩ , 15%), 459 (M Ϫ CH2O, 45), 445
ؒ
ؒ
᎐
[Mϩ Ϫ (O(CH2)2), 100], 409 (M Ϫ Br, 6), 199 (C7H6BrNO ,
85), 104 (C6H4Nϩ, 81), 77 (C5H3Nϩ, 76); Found: 485.9803,
Calc. for C18H20BrN2O4: 485.9793.
ϩ
ϩ
ؒ
ؒ
CH C᎐N), 7.27 (2H, d, J 3.6, 3Ј-H), 7.34 (4H, s, Pyr-H), 7.40
᎐
2
(2H, d, J 3.6, 4Ј-H), 8.20 (4H, d, J 1.6, 4-, 6-H), 8.40 (2H, t,
J 1.6, 2-H); δC(CDCl3) 52.52 (CH3), 70.06 (CH2O), 73.49
(CH C᎐N), 116.07 (Pyr-CH), 125.19, 126.31 (3Ј-, 4Ј-C), 129.26
Dimethyl 5-(2-thienyl)isophthalate 13. Compound 13 was
prepared from compound 12 according to the procedure
described by Cielen et al.7
᎐
2
(2-C), 128.89 (4-, 6-C), 131.13 (1-, 3-C), 134.28 (2Ј-C), 141.41,
141.78 (Pyr-C, 5-C), 142.89 (5Ј-C), 156.7 (C᎐N), 165.6 (CO),
Dimethyl
5-(5-tributylstannyl-2-thienyl)isophthalate
14.
᎐
ϩ
ϩ
m/z: 878 (Mϩ , 2%), 849 (M Ϫ CHO, 7), 835 [M Ϫ (CH2-
ؒ
ؒ
ؒ
Compound 14 was prepared from compound 13 according to
ϩ
CHO), 23], 396 (Mϩ Ϫ C25H25NO4S, 100), 381 (M Ϫ C26H27-
NO4S, 40); Found: 878.2184, Calc. for C46H42N2O12S2:
878.2179.
the procedure described by Cielen et al.7
ؒ
ؒ
Dimethyl 5-{5-[3,6,9,12-tetraoxa-18-azabicyclo[12.3.1]octa-
deca-1(18),14,16-trien-16-yl]-2-thienyl}isophthalate 15a. To a
solution of compound 14 (950 mg, 1.68 mmol, 1.2 equiv.) in
anhydrous toluene (20 cm3), compound 8a (465 mg, 1.4 mmol)
and Pd(PPh3)4 (16 mg, 1 mol%) were added. The reaction
mixture was refluxed during 15 h under an argon stream. After
cooling to room temperature, the solvent was evaporated and
the yellow residue was purified on alumina (CHCl3–hexane,
70:30 then CHCl3–MeOH, 99:1). The obtained yellow oil (1.1
g) was crystallised from hot CH2Cl2 followed by addition of
cold hexane. A white powder was obtained (443 mg, 60%); mp
115–117 ЊC (CH2Cl2–hexane); νmax(KBr)/cmϪ1 2860 (CH3, CH2,
CH), 1720 (CO), 1600–1560 (pyridine), 1125 (C-O); δH(CDCl3)
3.5 (4H, s, CH2), 3.61–3.64 (4H, m, CH2), 3.79–3.83 (4H, m,
CH2), 3.99 (6H, s, CH3), 4.73 (4H, s, CH2), 7.44 (2H, s, Pyr-H),
7.48 (1H, d, J 3.6, 3Ј-H), 7.53 (1H, d, J 3.6, 4Ј-H), 8.45 (2H,
s, 4-, 6-H), 8.59 (1H, s, 2-H); δC(CDCl3) 52.57 (CH3), 70.18,
70.54, 70.87 (CH2), 73.97 (CH2), 117.61 (Pyr-CH), 125.68,
126.48 (3Ј-, 4Ј-C), 129.69 (2-C), 130.51 (4-, 6-C), 131.48 (1-,
5-{5-[3,6,9,12-Tetraoxa-18-azabicyclo[12.3.1]octadeca-
1(18),14,16-trien-16-yl]-2-thienyl}isophthalic acid 17a. To a
suspension of the diester 15a (105 mg, 0.2 mmol) in anhydrous
THF (15 cm3), KOSiMe3 (64.2 mg, 0.5 mmol, 2.5 equiv.) was
added. The mixture was refluxed overnight, then filtered on a
glass filter, washed with ether, dissolved in water (10 cm3) and
acidified with an aqueous solution of hydrogen chloride (1 M)
until pH 4 was reacted. The obtained yellow precipitate was
filtered on a glass filter and washed with water. After drying in
a dessicator in the presence of P2O5, a yellow powder was
obtained (70 mg, 70%); mp 157–160 ЊC ; νmax(KBr)/cmϪ1 3000
(OH), 2900 (CH2, CH), 1711 (CO), 1600–1440 (pyridine), 1100
(C-O); δH(DMSO-d6) 3.37 (4H, s, CH2), 3.46–3.56 (4H, m,
CH2), 3.67–3.93 (4H, m, CH2), 4.73 (4H, s, CH2), 7.66 (2H,
s, 7Ј-, 11Ј-H), 7.86 (1H, d, J 3.8, 3Ј-H), 7.9 (1H, d, J 3.8, 4Ј-H),
8.41 (1H, s, 2-H), 8.42 (2H, s, 4-, 6-H); δC(DMSO-d6) 69.5,
69.7, 69.8 (CH2), 72.91 (CH2), 117.22 (Pyr-CH), 126.6, 127.93
(3Ј-, 4Ј-C), 129.1 (2-C), 129.4 (4-, 6-C), 132.57 (1-, 3-C), 133.85
3-C), 134.57 (2Ј-C), 141.62, 141.72 (Pyr-C), 143.35 (5Ј-C),
159.13 (C᎐N), 165.6 (C(O)), m/z: 527 (Mϩ , 10%), 496 (M
Ϫ
Ϫ
Ϫ
ϩ
ؒ
ؒ
ؒ
ؒ
(2Ј-C), 140.67, 140.7 (Pyr-C), 142.56 (5Ј-C), 158.87 (C᎐N),
᎐
᎐
ϩ
166.2 (CO), m/z: 499 (Mϩ , 5%), 470 (M Ϫ CHO, 15), 456
CH2OH, 16), 484 [Mϩ Ϫ ((CH2)2O), 29], 438 [Mϩ
ؒ
ؒ
ؒ
[Mϩ Ϫ (CH2)2O, 24], 410 [Mϩ Ϫ [(CH2)2O]2H, 28], 396
(CH2)2O)2, 29], 410 [Mϩ Ϫ (CH2)2O)2(CH2)2), 31], 396 [M
ϩ
ؒ
ؒ
ؒ
[Mϩ Ϫ O[(CH2)2O]2, 20], 382 [M Ϫ [(CH2)2O]2CHO, 50], 368
ϩ
(CH2)2O)3, 100], 381 [Mϩ Ϫ (CH2)2O)3CH2, 99] Found:
ؒ
ؒ
ؒ
[Mϩ Ϫ [(CH2)2O]2CH2CHO, 100], 353 [M Ϫ (CH2CH2O)3-
ϩ
ؒ
ؒ
527.1612, Calc. for C27H29NO8S: 527.1613.
CH2), 96]; Found: 499.1288, Calc. for C25H25NO8S: 499.1300.
Dimethyl 5-{5-[3,6,9,12,15-pentaoxa-21-azabicyclo[15.3.1]-
Caesium salts. 1a,b and 2 were prepared from compounds
15a,b and 16 according to the procedure described by Minta
and Tsien.21 A solution of ester (2.3 × 10Ϫ5 mol) and anhydrous
caesium hydroxide (2.3 × 10Ϫ4 mol, 10 equiv.) in methanol
(3 cm3) was refluxed overnight. After evaporation of the
methanol, the product was dissolved in water (100 cm3) and
used as such for the fluorescence measurements.
henicosa-1(21),17,19-trien-19-yl]-2-thienyl}isophthalate
15b.
Compound 15b was prepared as described for compound 15a,
starting from compounds 14 (216 mg, 0.38 mmol, 1.2 equiv.)
and 8b (120 mg, 0.32 mmol, 1 equiv.). A yellow powder was
obtained (115 mg, 63%); mp 123–124 ЊC; νmax(KBr)/cmϪ1 2860
(CH3, CH2, CH), 1720 (CO), 1600–1560 (pyridine), 1125 (C-O);
δH(CDCl3) 3.56–3.61 (8H, m, CH2), 3.62–3.7 (4H, m, CH2),
3.71–3.82 (4H, m, CH2), 3.98 (6H, s, CH3), 4.81 (4H, s, CH2),
7.47 (1H, d, J 3.9, 3Ј-H), 7.48 (2H, s, Pyr-H), 7.54 (1H, d, J 3.9,
4Ј-H), 8.45 (2H, d, J 1.5, 4-, 6-H), 8.6 (1H, t, J 1.5, 2-H);
δC(CDCl3) 52.5 (CH3), 69.8, 70.5, 70.67, 71.15 (CH2), 73.69
(CH2), 116.5 (Pyr-CH), 125.5, 126.4 (3Ј-, 4Ј-C), 129.66 (2-C),
130.53 (4-, 6-C), 131.48 (1-, 3-C), 134.64 (2Ј-C), 141.7, 141.95
Acknowledgements
The authors are indebted to Professor Dr S. Toppet and Ing. R.
De Boer for the NMR and mass spectra. E. C. is an Aspirant
and N. B. is an Onderzoeksdirecteur of the Fonds voor Weten-
schappelijk Onderzoek (FWO). A. T. and K. V. A. are post-
doctoral fellows at the K. U. Leuven. The continuing financial
(Pyr-C, 5-C), 143.25 (5Ј-C), 159.11 (C᎐N), 165.8 (CO), m/z: 571
᎐
ϩ
ϩ
(Mϩ , 23%), 528 (M Ϫ CH2CHO, 45), 512 [M Ϫ (OCH2-
ؒ
ؒ
ؒ
J. Chem. Soc., Perkin Trans. 2, 1999, 1739–1748
1747

View Article Online
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