Synthesis and spectroscopic characterisation of fluorescent indicators for Na+ and K+

Article abstract of DOI:10.1039/a802183j

The synthesis and the spectral and cation binding properties of new flourescent indicators for Na⁺, dicaesium 5-[5-(2,3,5,6,8,9,11,12-octahydro-1,4,7,10,13-benzopentaoxacyclopentadecin-15- yl)-2-thienyl]-isophthalate (Benzos), and for K⁺

Full text of DOI:10.1039/a802183j

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Synthesis and spectroscopic characterisation of fluorescent indicators
for Na؉ and K؉
Els Cielen, Abdellah Tahri, Katja Ver Heyen, Georges J. Hoornaert,
Frans C. De Schryver and Noël Boens*,†
Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F,
3001 Heverlee, Belgium
The synthesis and the spectral and cation binding properties of new fluorescent indicators for Na؉,
dicaesium 5-[5-(2,3,5,6,8,9,11,12-octahydro-1,4,7,10,13-benzopentaoxacyclopentadecin-15-yl)-2-thienyl]-
isophthalate (Benzos), and for K؉, dicaesium 5-[5-(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,13,16-benzo-
hexaoxacyclooctadecin-18-yl)-2-thienyl]isophthalate (Benzop), are reported. Both indicators consist of a
benzocrown ether with an appropriate cavity for the sodium and the potassium cation, respectively,
linked to an aryl thiophene fluorophore. In aqueous solution, the indicators have an absorption maximum
at 340 nm and an emission maximum at 430 nm. The chelating abilities of Benzos and Benzop with Na؉,
K؉, Li؉, Cs؉, Mg2؉ and Ca2؉ were studied by means of their fluorescence properties. Upon cation
binding, a change in the fluorescence intensity is observed, whereas the spectral distribution remains
unaltered. While Benzos cannot discriminate between Na؉ and other monovalent cations, Benzop
shows a three-fold selectivity for K؉ over Na؉.
Naϩ vs. other cations in aqueous solutions,13 an Naϩ indicator
Introduction
based on a 15-crown-5 derivative was developed, in accordance
Non-disruptive determination of intracellular cation concen-
trations, such as those of Naϩ, Kϩ, Ca2ϩ and Mg2ϩ, inside living
cells has, within a decade, become of great importance in cell
physiology and clinical medicine.1 Nearly all cells maintain a
large difference in ion concentrations between their interiors
(90–130 m for Kϩ, 5–30 m for Naϩ) and the extracellular
environment (1–10 m for Kϩ, 100–300 m for Naϩ).2 In the
past, the most commonly used techniques for measuring intra-
cellular sodium and potassium were flame photometry,3 ion
selective electrodes,4 atomic absorption spectroscopy5 and
nuclear magnetic resonance.6 Properly designed fluorescent
probes have proven to be the most suitable tools for sensing
such concentrations, because they are non-invasive, sensitive,
and their response is fast and highly localised.7
with the Naϩ indicators previously reported.14
Based on promising spectral properties of a model com-
pound, methyl 4-(4-methyl-5-phenyl-2-thienyl)benzoate,15 for
which the product of the molar extinction coefficient, ε, and the
quantum yield of fluorescence, φF, exceeds 103 Ϫ1 cmϪ1, a
thiophene-containing unit was chosen as the fluorophore. The
thiophene group is linked to a phenyl ring, substituted with two
carboxylate functionalities to ensure water solubility.
In this paper, we present the synthesis of new fluorescent
indicators for Naϩ, dicaesium 5-[5-(2,3,5,6,8,9,11,12-octa-
hydro-1,4,7,10,13-benzopentaoxacyclopentadecin-15-yl)-2-
thienyl]isophthalate (Benzos 1a), and for Kϩ, dicaesium 5-[5-
COO–Cs+
O
Successful indicators must fulfil a series of criteria in add-
ition to the chemically obvious ones of sensitivity and speci-
ficity.8 The major requirements for an indicator are: (i) an
affinity that matches normal cytosolic concentrations for the
ion in question (for Kϩ, 50 m < Kd < 150 m; for Naϩ, 0
m < Kd < 50 m, where Kd is the ground-state dissociation
constant), (ii) a selectivity for the desired ion over other ions
present inside the cytosol, and (iii) an easily detectable change
in the excitation and/or emission spectrum upon binding.
Although there has been a continuous development of fluor-
escent indicator technology, particularly for the determination
of intracellular Ca2ϩ,9 the indicators SBFI and PBFI (sodium-
and potassium-binding benzofuran isophthalate) for cytosolic
Naϩ and Kϩ, respectively, developed in 1989 by Tsien and co-
workers,10 are still the most frequently used.
To be useful in biological systems, synthetic cation receptors
must be able to bind the desired cation selectively in an aqueous
medium. Since a clear relationship exists between the cation
diameter and the cavity size of the crown compound11 on the
one hand, and the stability constants of the appropriate com-
plexes,12 on the other hand, an 18-crown-6 derivative has been
proposed as a selective ionophore for Kϩ. Although 15-crown-5
compounds as such are known to have a limited selectivity for
O
S
O
O
COO–Cs+
O
n
1a n = 1
1b n = 2
(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,13,16-benzohexa-
oxacyclooctadecin-18-yl)-2-thienyl]isophthalate (Benzop 1b).
Besides the synthesis of the indicators for Naϩ and Kϩ, the
spectral and cation binding properties of the indicators are
reported. From absorption and steady-state fluorescence meas-
urements, the position of the spectra, the molar extinction
coefficient, ε, the quantum yield of fluorescence, φF, of both
the free and saturated forms of the indicators, the dissociation
constant, Kd, in the ground state of the formed host:guest
complexes, and the selectivity vs. other ions were determined.
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.16 for the
case of a 1:1 complex between a fluorescent indicator and a
cation. For a consecutive, multiple binding model, this expres-
† Fax: ϩ-32-(0)16-327 990. E-mail: Noel.Boens@chem.kuleuven.ac.be
J. Chem. Soc., Perkin Trans. 2, 1998
1573

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sion 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
(determined by the dissociation constant Kd1) to form species 1,
which in turn can react with another equivalent of X (deter-
mined by the dissociation constant Kd2), to yield species 2, etc.
[eqns. (1.1)–(1.n)]. Species 0 represents the free form of a fluor-
escent indicator, species 1 the 1:1 complex with a cation X and
species n the n:1 ion:indicator complex. The global reaction is
determined by the composite dissociation constant Kd [eqn. (2)]
(Scheme 1).
To determine Kd and n via a Hill plot, the expression on the left
hand side of eqn. (7) is plotted as a function of log [X]. From
the slope of this linear graph, a value for n can be derived, while
the intersection with the abscissa corresponds to (log Kd)/n. In
some cases, it is difficult to obtain reliable values for the extreme
values Fmin and Fmax from the experimental fluorescence titra-
tion. Therefore, fitting eqn. (6) to the fluorescence data F may
yield values for Fmin, Fmax, Kd and n.
Results
Synthesis
The synthesis of both indicators 1a and 1b was carried out
following a convergent route (Scheme 2); in addition, for 1b, a
[0] [X]
0 ϩ X
1
Kd1
Kd2
=
=
(1.1)
(1.2)
[1]
NH2
I
I
[1] [X]
[2]
1 ϩ X
2
. .
. .
. .
.
i
ii
.
.
HO2C
CO2H HO2C
CO2H MeO2C
CO2Me
[(n Ϫ 1)] [X]
2
3
4
(n Ϫ 1) ϩ X
n
Kdn
=
(1.n)
(2)
[n]
n
[0] [X]n
[n]
0 ϩ nX
n
Kd =
= Π Kdi
i = 1
CO2Me
iv
CO2Me
iii
S
Br
S
Scheme 1
CO2Me
CO2Me
5
6
If one assumes that the intermediate complexes [species 1–
(n Ϫ 1)] are present in concentrations as low as to make only a
negligible contribution to the fluorescence intensity, the total
fluorescence signal due to excitation at λex and observed at λem
can be expressed by eqn. (3), where ε0(λex) and εn(λex) denote the
vi
v
CO2Me
F(λex, λem, [X]) = {a0(λem)ε0(λex)[0] ϩ
an(λem)εn(λex)[n]}ψ(λex, λem) (3)
Bu3Sn
S
CO2Me
7
molar extinction coefficients for species 0 and n, respectively.
ψ is a proportionality factor dependent on the impinging
flux of photons at λex and on instrumental properties of the
fluorimeter.
Assuming that the rate of ion binding in the excited state
is negligible, the coefficients a0(λem) and an(λem) are constant.
Scheme 2 Reagents and conditions: i: (a) 1 equiv. NaNO2, HCl,
0 ЊC, (b) 3.3 equiv. KI; ii: (a) SOCl2, reflux, (b) MeOH; iii: 2-
(tributylstannyl)thiophene, 2 mol% (MeCN)2PdCl2, DMF, air, rt; iv:
Br2, acetic acid, reflux; v: 1.5 equiv. bis(tributyltin), 1 mol% Pd(PPh3)4,
toluene, reflux; vi: (a) 2,2,6,6-tetramethylpiperidine, BunLi, THF, 0 ЊC,
(b) 5, tributyltin chloride, THF, 2 h at Ϫ78 ЊC, rt overnight
F [eqn. (3)] can be rewritten as a function of the product of
non-convergent reaction path (Scheme 4) was developed. In
both approaches, the main difficulty was to find an appropriate
method for the coupling of highly substituted (hetero)aromat-
ics. Thanks to important advances in the field of organotransi-
tion metal chemistry,17 several methods for carbon–carbon
coupling have been developed, among which palladium-
catalysed organotin reactions are the most general and power-
ful ones.
In the convergent route, the fluorophore synthon 7 was syn-
thesised independently (Scheme 2), after which it was attached
to the respective ionophores in a palladium-catalysed cross-
coupling reaction (Scheme 3). In the non-convergent route
(Scheme 4), the indicator 9b was gradually built up. The key
steps remained the carbon–carbon couplings, but they were per-
formed at different stages in the reaction sequence.
n
the dissociation constants, Kd = Π Kdi, the analytical con-
i = 1
centration of the fluorescent indicator, CA, and the ion concen-
tration, [X] [eqn. (4)]. The extreme values of F([X]) are
a0ε0Kd ϩ anεn[X]n
(4)
F([X]) =
ͩ
ͪ
CAψ
Kd ϩ [X]n
determined by eqns. (5a) and (5b), where Fmin corresponds to
Fmin = F([X]→0) = a0ε0CAψ
Fmax = F([X]→∞) = anεnCAψ
(5a)
(5b)
Convergent route. Design of the fluorophore.—The fluoro-
phore building block dimethyl 5-(5-tributylstannyl-2-thienyl)-
isophthalate 7 (Scheme 2) was synthesised starting from
5-aminoisophthalic acid 2, which was converted into 5-iodo-
isophthalic acid 3 by diazotization,18 followed by a Sandmeyer-
type reaction.19 The corresponding ester 420 was then coupled
at room temperature to 2-(tributylstannyl)thiophene in a cross-
coupling reaction under air in DMF, catalysed by 2 mol% of
the ligandless catalyst bis(acetonitrile)dichloropalladium()21
to give compound 5 in high yields. The conversion of dimethyl
5-(2-thienyl)isophthalate 5 to its stannyl derivative 7 was
achieved via two different routes.
the fluorescent signal of the free form of the indicator and Fmax
is the fluorescence signal of the saturated form of the indicator.
With these values, eqn. (4) can be rearranged to give eqn. (6).
[X]nFmax ϩ KdFmin
(6)
F =
Kd ϩ [X]n
Eqn. (6) can be linearised in the form of a Hill plot [eqn. (7)].
F Ϫ Fmin
(7)
log
= n log [X] Ϫ log Kd
Fmax Ϫ F
1574
J. Chem. Soc., Perkin Trans. 2, 1998

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In a first attempt, compound 5 was brominated22 and sub-
sequently converted into its stannyl analogue 7 by overnight
reflux in anhydrous toluene under argon in the presence of
1.5 equiv. bis(tributyltin) and 1 mol% tetrakis(triphenyl-
phosphine)palladium23 (step v of Scheme 2). The purification
was tedious, however, and, moreover, the yields were very low.
Therefore, the tin compound 7 was alternatively prepared from
compound 5 via a lithium intermediate (step vi of Scheme 2). A
sterically hindered base, lithium 2,2,6,6-tetramethylpiperidine,24
prepared in situ, was used to lithiate the 5-position of the thio-
phene nucleus in compound 5 without reaction on the ester
functionalities. Addition at Ϫ78 ЊC of this lithium derivative in
anhydrous THF to a mixture of compound 5 and tributyltin
chloride gave the desired fluorophore building block 7 in
moderate yields.
the fluorophore building block 7 and 1 equiv. of the appropriate
crown compound 8a or 8b in anhydrous toluene under nitrogen
atmosphere in the presence of 1 mol% tetrakis(triphenyl-
phosphine)palladium (Scheme 3). It is noteworthy that when
10 mol% of the catalyst was used, the yield decreased
significantly. From analysis of the crude reaction mixture, a
significant amount of a side product, originating from the
cross-coupling reaction of the tin compound 7 with a phenyl
ring of the triphenylphosphine ligand, was detected.
Non-convergent route. In the non-convergent approach to
synthesis of 9b (Scheme 4), 2-(tributylstannyl)thiophene 10 was
initially attached to the crown compound 8b in a cross-coupling
reaction similar to the one described above. The fluorophore
was built up gradually from compound 11. The thiophene
nucleus was brominated in the 2-position, as was done for com-
pound 6 (Scheme 2). The indicator 9b was then obtained alter-
natively by the palladium-catalysed coupling of the synthons 12
and 13. The latter was generated by reaction of dimethyl
5-iodoisophthalate 4 with bis(tributyltin) in conditions similar
to those described for the preparation of 7 from the brominated
precursor.
Palladium-catalysed cross-coupling reaction.—Both bromo
substituted crown compounds (8a for Naϩ and 8b for Kϩ,
Scheme 3) are commercially available. The fluorescent indi-
cators Benzos 9a for sodium and Benzop 9b for potassium were
obtained in good yields by refluxing a mixture of 1.2 equiv. of
Conversion to the water soluble form. The final reaction step
comprises the conversion of the esters 9a,b into the correspond-
ing water soluble forms, because the measurements are to be
performed in aqueous medium. The free acids 14a,b were
obtained by refluxing compounds 9a,b overnight in dry THF
with 2.5 equiv. potassium trimethylsilanolate.25 Since the free
acids 14a,b showed a limited solubility in aqueous solution at
physiological pH (7.05), we preferred to use the method of
Minta and Tsien26 to prepare directly their caesium salts. The
methyl esters 9a,b were converted into the corresponding
caesium salts 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 the same.
CO2R
O
O
O
Br
Bu3Sn
S
+
O
O
CO2R
7
n
8a n = 1
8b n = 2
4′ 3′
CO2R
O
O
4
O
5
6
3
i
ii
14a R = H, n = 1
14b R = H, n = 2
5′
2′
S
1′
O
2
O
1
iii
CO2R
n
1a R = Cs+, n = 1
1b R = Cs+, n = 2
9a R = Me, n = 1
9b R = Me, n = 2
Steady-state fluorescence
Steady-state fluorescence and absorption measurements were
performed on the caesium salts of the indicators in an aqueous
medium at 20 ЊC and pH 7.05. The indicator concentrations
Scheme 3 Reagents and conditions: i: 1 mol% Pd(PPh3)4, toluene, 15 h
reflux; ii: KOSiMe3, THF, overnight reflux; iii: 10 equiv. CsOH, MeOH,
overnight reflux
O
O
3′
2′
4′
5′
3
6
O
O
O
O
Br
O
O
O
O
2
4
i
S
1′
+
Bu3Sn
S
1
5
O
10
O
8b
11
O
O
SnBu3
O
O
Br
ii
S
+
O
O
MeO2C
CO2Me
13
12
iv
iii
I
O
O
CO2Me
O
O
O
S
MeO2C
CO2Me
O
4
CO2Me
9b
Scheme 4 Reagents and conditions: i: 10 mol% Pd(PPh3)4, toluene, 15 h reflux; ii: Br2, acetic acid, reflux; iii: 1.5 equiv. bis(tributyltin), 1 mol%
Pd(PPh3)4, toluene, reflux; iv: 1 mol% Pd(PPh3)4, toluene, 15 h reflux
J. Chem. Soc., Perkin Trans. 2, 1998
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Table 1 Binding properties of Benzos with the respective cations determined from fluorimetric Hill plots [eqn. (7)]. The dissociation constants were
measured with 5 µ of the indicator and 10Ϫ2  MOPS at a pH 7.05 buffered by (5–6) × 10Ϫ3  tetramethylammonium hydroxide. The fluorescence
emission spectra were measured as a function of the cation concentration. No correction was made for the change in ionic strength of the solution.
n
φF relative
to the free form
Saturation
value/
Ion
log Kd
Kd/m
Ksa/Ϫ1
stoichiometry
Liϩ
Ϫ1.5 0.3
Ϫ1.7 0.1
Ϫ1.8 0.2
Ϫ2.06 0.07
Ϫ2.1 0.2
Ϫ1.9 0.2
32.8
18.9
17.8
8.7
7.6
12.9
30.5
52.9
56.2
114.9
131.6
77.5
1.1 0.1
1.11 0.05
1.3 0.1
0.80 0.02
1.04 0.07
1.10 0.09
Decrease
Increase
Increase
Decrease
Decrease
Decrease
0.5
0.1
0.1
0.04
0.05
0.08
Naϩ
Kϩ
Csϩ
Mg2ϩ
Ca2ϩ
a Ks (= KdϪ1) represents the stability constant of the ion:indicator complex.
Fig. 2 Hill plot for the complex formation between Naϩ and Benzos
calculated from fluorescence emission spectra recorded for an extended
series of the solutions used in Fig. 1
in the fluorescence intensity occurs, which might be attributed
to an excited-state reaction.16
The ground-state dissociation constant, Kd, of the Naϩ :indi-
cator complex was determined from a Hill plot using fluor-
escence measurements at λex = 340 nm, λem = 430 nm, at 20 ЊC
and pH 7.05. The Hill plot for the binding of Naϩ by Benzos,
which is given in Fig. 2, yields a value of 18.9 m for Kd and a
slope of 1.11 0.05, indicating a 1:1 stoichiometry for the
Naϩ :indicator complex.
The fluorescence quantum yield, φF, is 0.009 for the free form
and 0.012 for the Naϩ bound form of Benzos.
The selectivity of Benzos vs. other cations, like Kϩ, Liϩ, Csϩ,
Mg2ϩ and Ca2ϩ, can be calculated from the ratio of their
respective Kd values (Table 1). From these values, it is clear that
the indicator shows a non-selective behaviour towards all
monovalent cations. Upon binding of Mg2ϩ and Ca2ϩ, the
indicator shows a particularly strong decrease in fluorescence
intensity. The characteristics of Benzos are summarised in
Table 1, while Fig. 3 shows the relative fluorescence responses
of the indicator towards the respective cations at the same
cation concentration ([X] = 30 m). The ionic strength was not
kept constant for all solutions.
Benzop. For Benzop, the measurements were done using the
same conditions as for Benzos. No shift in the absorption
maximum at 340 nm is observed upon cation binding, nor a
pronounced change in absorbance (figure not shown). The
molar extinction coefficient at 340 nm for the free form of
the indicator, ε0(340 nm), is (31 1) × 103 Ϫ1 cmϪ1, while the
correspondng value, εn(340 nm), for the Kϩ :indicator complex
is (29.1 0.6) × 103 Ϫ1 cmϪ1.
Fig. 1 Fluorescence excitation (A) and emission (B) spectra of Benzos
as a function of [Naϩ] at 20 ЊC and pH 7.05
were of the order of 5 × 10Ϫ6 , yielding an absorbance per cm
path length of approximately 0.1 at the absorption maximum.
A physiological pH was obtained by buffering the separate
solutions with 10Ϫ2  MOPS (3-morpholinopropanesulfonic
acid), adjusted with tetramethylammonium hydroxide.
Benzos. The absorption spectra of Benzos at various concen-
trations of the different cations show no pronounced change in
absorbance. The absorption maximum is situated at 340 nm
and the molar extinction coefficients at 340 nm of the free and
the Naϩ saturated forms of the indicator are (28.3 0.5) × 103

Ϫ1 cmϪ1 and (29.1 0.4) × 103 Ϫ1 cmϪ1, respectively.
The fluorescence excitation and emission spectra of Benzos
with Naϩ are shown in Fig. 1. The excitation maximum is situ-
ated at 340 nm and the emission maximum at 430 nm. An
increase in the fluorescence intensity is observed upon increas-
ing the concentration of Naϩ, while the position of the maxima
remains constant. The indicator is saturated at 100 m Naϩ,
where the fluorescence intensity reaches a plateau value. At
sodium concentrations above 250 m, a second slight increase
The fluorescence excitation and emission spectra of Benzop
with increasing concentrations of Kϩ are represented in Fig. 4.
Analogous to Benzos, no wavelength shift occurs upon complex
formation, but the spectral response on cation binding of
Benzop is more pronounced, as can be seen from Fig. 5, where
the relative fluorescence responses of the indicator towards the
cations studied are given.
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J. Chem. Soc., Perkin Trans. 2, 1998

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Fig. 3 Plot of the relative fluorescence responses F of Benzos upon
binding the respective cations X. All displayed spectra (except the free
form) are recorded at [X] = 30 m. The ionic strength was not kept the
same for all solutions. The other experimental conditions are the same
as in Fig. 1.
Fig. 5 Plot of the relative fluorescence responses F of Benzop upon
binding the respective cations X. All displayed spectra (except the free
form) are recorded at [X] = 30 m. The ionic strength was not kept the
same for all solutions. The other experimental conditions are the same
as in Fig. 4.
Fig. 6 Hill plot for the complex formation between Kϩ and Benzop
calculated from fluorescence emission spectra recorded for an extended
series of the solutions used in Fig. 4
Benzop is 0.005; for the Kϩ saturated form, φF equals 0.008.
The binding properties of Benzop are summarised in Table 2.
Discussion
Examination of the data obtained for Benzos clearly shows that
the indicator can hardly discriminate between Naϩ and the
other monovalent cations. The non-selective complexing
abilities of the indicator are consistent with earlier findings27
that the stability constants, Ks, for the complex formation in
aqueous solution of Naϩ and Kϩ with benzo-15-crown-5 are
nearly the same.
Expansion of the crown ether not only increases the Kϩ affin-
ity but also slightly decreases the Naϩ affinity. The potassium
indicator Benzop shows a selectivity of 3:1 for Kϩ over Naϩ,
this is two times better than the selectivity of PBFI for Kϩ vs.
Naϩ. The Kd of the Kϩ :Benzop complex is strongly dependent
on whether Naϩ is present, with a value of 5.54 m in the
absence of Naϩ and 26.9 m in solutions with [Naϩ] ϩ [Kϩ] =
135 m, approximating physiological ionic strength. A similar
dependence of the Kd value for the Kϩ :PBFI complex on the
presence of Naϩ was reported:10 a value of 5.1 m was found
for Kd in Naϩ free solutions, and 44 m in solutions with
combined Naϩ and Kϩ concentrations of 135 m. Since the
indicator has an affinity for both Naϩ and Kϩ, theoretically a
competitive binding model should be used in solutions contain-
ing Naϩ as well as Kϩ.
Fig. 4 Fluorescence excitation (A) and emission (B) spectra of Benzop
as a function of [Kϩ]. The experimental conditions are as in Fig. 1.
By means of a Hill plot (Fig. 6), the ground-state dissociation
constants, Kd, for the respective ion:indicator complexes were
determined. In the absence of Naϩ, a Kd value of 5.54 m for
the Kϩ :indicator complex is obtained and a slope of 0.96
0.05, indicating a 1:1 stoichiometry. The indicator is saturated
with Kϩ at 25 m. In solutions with a total Naϩ and Kϩ con-
centration of 135 m, the Kd value for the Kϩ :Benzop complex
equals 26.9 m.
The fluorescence quantum yield, φF, of the free form of
Since both indicators, Benzos and Benzop, only show a
J. Chem. Soc., Perkin Trans. 2, 1998
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Table 2 Binding properties of Benzop with the respective cations determined from fluorimetric titrations. The experimental conditions were the
same as in Table 1. A reliable Kd value for Ca2ϩ :Benzop could not be estimated from fluorescence measurements because no measurable change in
intensity was observed as a function of [Ca2ϩ]. In the fitting according to eqn. (6), Fmax, Fmin, Kd, and n are adjustable parameters.
n
φF relative
to the free form
Saturation
value/ 
Calculated
via eqn.
Ion
log Kd
Kd/m
Ksa/Ϫ1
stoichiometry
Liϩ
Ϫ1.4 0.1
Ϫ1.7 0.2
Ϫ2.3 0.1
Ϫ1.9 0.2
Ϫ2.5 0.1
Ϫ1.6 0.1
39.8
18.3
5.54
13.6
3.01
26.9
25.1
54.6
180.5
73.5
332.2
37.2
1.03 0.09
1.22 0.08
0.96 0.05
0.96 0.07
1.00 0.04
1.13 0.04
Decrease
Increase
Increase
Increase
Decrease
Increase
0.2
(7)
(6)
(6)
(7)
(6)
(7)
Naϩ
0.03
0.025
0.07
0.014
0.06
Kϩ
Csϩ
Mg2ϩ
[Naϩ] ϩ [Kϩ] = 0.1 
a Ks (= KdϪ1) represents the stability constant of the ion:indicator complex.
change in intensity upon cation binding and no corresponding
spectral shifts, they do not allow one to perform ratiometric
measurements at dual wavelengths. It is not yet understood why
the fluorescence intensity of the ion:indicator complex
increases for one ion and decreases for another ion relative to
that of the ion-free indicator (Tables 1 and 2, Figs. 3 and 5).
Benzop and Benzos both have reasonable molar extinction
coefficients, but rather low fluorescence quantum yields. While
the spectral response for Benzos is only minor (the fluorescence
intensity increases by one third between the free form and 10
m Naϩ), a more pronounced response was observed for
Benzop in the presence of Kϩ: the intensity increases by a factor
of 2 between 0 and 25 m Kϩ.
addition of methanol. Purification by column chromatography
(SiO2; CHCl3) yielded 4 as white crystals (54%); mp 102–103 ЊC
(lit.,20 100–102 ЊC); νmax(KBr)/cmϪ1 3080–3000 (CH3), 1730
(CO), 1570 (Ar); δH(CDCl3) 3.9 (6 H, s, CH3), 8.5 (2 H, d, J 1.5,
4-, 6-H), 8.58 (1 H, t, J 1.5, 2-H); δC(CDCl3) 52.57 (CH3),
93.4 (CI), 129.74 (2-C), 132.11 (1-, 3-C), 142.3 (4-, 6-CH),
164.8 (CO); m/z 320 (Mϩ, 100%), 289 (Mϩ Ϫ CH3O, 14),
277 (Mϩ Ϫ CO2, 16); Found: 319.9543, Calc. for C10H9IO4:
391.9545.
Dimethyl 5-(2-thienyl)isophthalate 5. Compound 5 was pre-
pared from compound 4 according to the procedure described
by Bumagin and Bumagina.21 To a mixture of compound 4 (5 g,
15.6 mmol) and 2-(tributylstannyl)thiophene (5.77 g, 15.6
mmol) in anhydrous DMF (50 cm3), (MeCN)2PdCl2 (81 mg,
0.31 mmol, 2%) was added. After stirring overnight at room
temperature, water (300 cm3) was added to the reaction mixture,
which was then extracted with chloroform (4 × 100 cm3). The
combined organic layers were washed with water (100 cm3), a
saturated potassium fluoride solution and again with water, and
then dried over MgSO4. After evaporation, the residue was
purified by column chromatography (SiO2; diethyl ether–
hexane, 1:1). A white powder was obtained (3.87 g, 90%); mp
133 ЊC; νmax(KBr)/cmϪ1 2955 (CH3, CH), 1720 (CO), 1560 (Ar);
δH(CDCl3) 3.95 (6 H, s, CH3), 7.11 (1 H, dd, J 3.65 and J 5.1,
4Ј-H), 7.34 (1 H, dd, J 1.1 and J 5.1, 5Ј-H), 7.43 (1 H, dd, J 1.1
and J 3.65, 3Ј-H), 8.42 (2 H, d, J 1.5, 4-, 6-H), 8.55 (1 H, t, J 1.5,
2-H), δC(CDCl3) 52.4 (CH3), 124.48 (3Ј-C), 126.09 (5Ј-C),
128.28 (4Ј-C), 129.13 (2-C), 130.66 (4-, 6-C), 131.3 (1-, 3-C),
135.26 (5-C), 142 (2Ј-C), 165.93 (CO); m/z 276 (Mϩ, 100%),
245 (Mϩ Ϫ CH3O, 87), 217 (Mϩ Ϫ CO2Me, 44), 202 [Mϩ Ϫ
C(OMe)2, 51]; Found: 276.0458, Calc. for C14H12O4S: 276.0456.
Dimethyl 5-(5-bromo-2-thienyl)isophthalate 6. Compound 6
was prepared from compound 5 according to the procedure
proposed by Gjøs and Gronowitz.22 To a stirred solution of 5
(2.6 g, 9.41 mmol) in acetic acid (18 cm3) was added drop-wise a
solution of bromine (1.5 g, 9.39 mmol) in acetic acid (15 cm3).
After complete addition, the mixture was refluxed for 5 h,
cooled down to room temperature and then poured into water
(80 cm3). After standing overnight, a precipitate was obtained
which was purified by column chromatography (SiO2; diethyl
ether–hexane, 2:1). A white powder was obtained (1.93 g,
58%); mp 158–161 ЊC; νmax(KBr)/cmϪ1 2950 (CH3, CH), 1720
(CO), 1560 (Ar), 750 (CBr); δH(CDCl3) 3.97 (6 H, s, CH3), 7.06
and 7.19 (2 H, 2 × d, J 3.93, 3Ј- and 4Ј-H), 8.32 (2 H, d, J 1.5,
4-, 6-H), 8.56 (1 H, t, J 1.5, 2-H); δC(CDCl3) 52.55 (CH3),
113.01 (CBr), 124.69 (3Ј-C), 129.49 (2-C), 130.7 (4-, 6-C),
131.15 (4Ј-C), 131.6 (1-, 3-C), 134.41 (5-C), 143.37 (2Ј-C),
165.79 (CO); m/z 354 (Mϩ, 100%), 323 (Mϩ Ϫ CH3O, 47), 295
(Mϩ Ϫ CO2Me, 15); Found: 353.9564, Calc. for C14H11BrO4S:
353.9561.
Experimental
Materials and methods
When required, solvents and reagents were dried prior to use.
Tetrahydrofuran (THF) and toluene were distilled over sodium.
4-Bromobenzo-15-crown-5 was purchased from Acros and
4-bromobenzo-18-crown-6 from Aldrich. Both compounds
1
were used without further purification. H NMR spectra were
recorded on a Bruker WM 250 or a Bruker AMX 400 instru-
ment. The spectra were measured using deuteriochloroform
(CDCl3) or deuterated DMSO as solvent, the 1H and 13C chem-
ical shifts are reported in ppm relative to tetramethylsilane or
the deuterated solvent as an internal reference and 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 trans-
form spectrometer. Melting points were determined with a
Reichert Thermovar apparatus and are uncorrected.
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  sulfuric acid as reference. The quan-
tum yield of the reference was taken to be 0.54.28 The samples
were measured in Milli-Q water. No correction for the refractive
index was necessary.
Synthesis
5-Iodoisophthalic acid 3. The diazonium salt of 2 was pre-
pared according to the procedure described by Delzenne and
Laridon.18 For the Sandmeyer-type reaction, the conditions
reported by Grahl19 were followed, yielding 5-iodoisophthalic
acid 3 (80%); mp 285–287 ЊC (lit.,19 288–289 ЊC); νmax(KBr)/
cmϪ1 3000–2700 (OH), 1700 (CO), 1560 (Ar); δH([2H6]DMSO)
8.4 (3 H, s, 2-, 4-, 6-H); δC([2H6]DMSO) 94.65 (CI), 129.07
(2-C), 133.06 (1-, 3-C), 141.37 (4-, 6-CH), 165.18 (CO); m/z 292
(Mϩ, 35%), 247 (Mϩ Ϫ CO2H, 5), 165 (Mϩ Ϫ I, 22), 127 (Iϩ,
Dimethyl
5-(5-tributylstannyl-2-thienyl)isophthalate
7.
Method A.—In a three-necked flask equipped with a septum
and a stopcock, 2,2,6,6-tetramethylpiperidine (3.36 g, 24 mmol)
was dissolved in anhydrous THF (50 cm3). After cooling to
0 ЊC, a solution of n-butyllithium (15 cm3, 24 mmol) was added
via a syringe. The slightly yellow mixture was stirred at room
ϩ
31), 74 (C6H2 , 78), 45 (CO2Hϩ, 100); Found: 291.9237, Calc.
for C8H5IO4: 291.9232.
Dimethyl 5-iodoisophthalate 4. 5-Iodoisophthalic acid 3 (24
mmol) was esterified by reflux in thionyl chloride followed by
1578
J. Chem. Soc., Perkin Trans. 2, 1998

View Online
temperature under argon for 1 h. This solution, containing the
lithiated anion, was then added dropwise to a mixture of the
thiophene 5 (5.52 g, 20 mmol) in THF (120 cm3) and tributyltin
chloride (7.82 g, 24 mmol) at Ϫ78 ЊC. The mixture was stirred
for 2 h at Ϫ78 ЊC and then overnight at room temperature.
Subsequently, it was poured into glacial brine (200 cm3) and
after addition of diethyl ether (100 cm3) was stirred for 15 min
at room temperature. The aqueous phase was extracted with
diethyl ether, whereupon the combined organic layers were
washed with brine. After evaporation, the crude product was
purified by column chromatography (Al2O3; hexane–AcOEt,
98:2), which yielded a reddish oil (4.66 g, 41%).
7.2 (1 H, d, J 8.25, Ar-H), 7.22 (1 H, d, J 3.8, 4Ј-H), 7.4 (1 H,
d, J 3.8, 3Ј-H), 8.43 (2 H, d, J 1.5, 4-, 6-H), 8.55 (1 H, t, J 1.5,
2-H); δC(CDCl3) 52.44 (CH3), 70.93, 70.83, 69.89, 69.47,
69.32 (CH2O), 112.32 and 114.55 (Ar-CH), 119.09 (3Ј-C),
123.35 (4Ј-C), 125.32 (Ar-CH), 127.54 (Ar-C), 128.93, 130.2,
131.32 (1-, 2-, 3-, 4-, 6-C), 135.25 (5Ј-C), 140.16 (5-C), 145.11
(2Ј-C), 149.28 and 149.33 (ArCO), 166 (CO); m/z 586 (Mϩ,
57%), 498 [Mϩ Ϫ (CH2CH2O)2, 9], 454 [Mϩ Ϫ (CH2CH2O)3, 6],
410 [Mϩ Ϫ (CH2CH2O)4, 100]; Found: 586.1871, Calc. for
C30H34O10S: 586.1872.
18-(2-Thienyl)-2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,13,
16-benzohexaoxacyclooctadecine 11. To a solution of 2-(tri-
butylstannyl)thiophene (1.21 g, 3.25 mmol, 1.3 equiv.) in
anhydrous toluene (15 cm3), 4-bromobenzo-18-crown-6 (1 g,
2.5 mmol) and Pd(PPh3)4 (278 mg, 10%) were added. The mix-
ture was refluxed for 15 h under nitrogen and then evaporated.
The black residue was purified by column chromatography
(Al2O3; hexane–CH2Cl2, 70:30 followed by CH2Cl2–MeOH,
99:1). The obtained white powder was recrystallised from hot
CH2Cl2 followed by addition of cold hexane (770 mg, 89%); mp
84–85 ЊC; νmax(KBr)/cmϪ1 2880 (CH2, CH), 1600, 1580, 1500
(Ar), 1120 (C-O); δH(CDCl3) (Scheme 4) 3.69 (4 H, s, CH2),
3.72–3.81 (8 H, m, CH2), 3.92–3.97 (4 H, m, CH2), 4.17–4.25
(4 H, m, CH2), 6.9 (1 H, d, J 8, Ar-H), 7.05 (1 H, dd, J 3.6 and
J 5, 4Ј-H), 7.1 (1 H, d, J 8, Ar-H), 7.15 (1 H, s, Ar-H), 7.2 (1 H,
d, J 5, 5Ј-H), 7.23 (1 H, d, J 3.6, 3Ј-H); δC(CDCl3) 70.9, 70.82,
69.72, 69.47, 69.35 (CH2O), 112 and 114.65 (Ar-CH), 119.27
(3Ј-C), 122.33 (Ar-CH), 124.02 (5Ј-C), 127.88 (4Ј-C), 128.18
(Ar-C), 144.37, 148.89, 149.25, (Ar-CO and 2Ј-C); m/z 394 (Mϩ,
100%), 350 [Mϩ Ϫ (CH2CH2O), 7], 306 [Mϩ Ϫ (CH2CH2O)2,
65], 262 [Mϩ Ϫ (CH2CH2O)3, 52], 218 [Mϩ Ϫ (CH2CH2O)4, 65];
Found: 394.1453, Calc. C20H26O6S: 394.1450.
Method B.—The procedure proposed here is based on the
method described by Azizian et al.23 To a solution of bis-
(tributyltin) (2.26 g, 3.9 mmol, 1.3 equiv.) in anhydrous toluene
(10 cm3) under argon, the brominated compound 6 (1.065 g,
3 mmol) and Pd(PPh3)4 (33.45 mg, 1 mol%) were added. After
refluxing for 48 h, the mixture was filtered and the filtrate was
evaporated. The yellow residue was purified by column chrom-
atography (Al2O3; hexane–EtOAc, 98:2); a colourless oil was
obtained (370 mg, 21%); νmax(CCl4)/cmϪ1 2960, 2930, 2875
(CH3, CH2, CH), 1730 (CO), 1600 (Ar); δH(CDCl3) 0.9–1.13
(9 H, m, CH3) 1.16–1.26 (6 H, m, CH2), 1.3–1.42 (6 H, m, CH2),
1.59–1.76 (6 H, m, CH2), 4 (6 H, s, CH3), 7.2 and 7.6 (2 H,
2 × d, J 3.3, 3Ј- and 4Ј-H), 8.46 (2 H, s, 4-, 6-H), 8.53 (1 H, s,
2-H); δC(CDCl3) 10.92 (CH2Sn), 13.62 (CH3), 27.23 (CH2),
28.94 (CH2), 52.36 (CH3), 125.6 (3Ј-C), 128.77 (4Ј-C), 130.66
(4-, 6-C), 131.22 (5Ј-C), 135.48 (1-, 3-C), 136.61 (2-C), 136.8
(5-C), 147.54 (2Ј-C), 166.08 (CO); m/z 566 (Mϩ, 20%), 509
ϩ
(Mϩ Ϫ butyl, 39), 290 [Sn(Bu)3 , 69], 276 [Mϩ Ϫ Sn(Bu)3, 60],
57 (butylϩ, 100); Found: 566.1507, Calc. for C26H38O4SSn:
566.1512.
Dimethyl 5-[5-(2,3,5,6,8,9,11,12-octahydro-1,4,7,10,13-benzo-
pentaoxacyclopentadecin-15-yl)-2-thienyl]isophthalate 9a. To a
solution of compound 7 (745 mg, 1.32 mmol, 1.2 equiv.) in
anhydrous toluene (20 cm3), 4-bromobenzo-15-crown-5 8a (382
mg, 1.1 mmol) and Pd(PPh3)4 (13 mg, 1%) were added. The
mixture was refluxed for 15 h under nitrogen. After evapor-
ation, the yellow residue was purified by column chrom-
atography (Al2O3; CHCl3–hexane, 70:30, then CHCl3–MeOH,
98:2). A yellow powder was obtained which was recrystallised
from hot CH2Cl2 followed by addition of cold hexane (490 mg,
82%); mp 145–146 ЊC; νmax(KBr)/cmϪ1 2950–2870 (CH3, CH2,
CH), 1730 (CO), 1600, 1560, 1510 (Ar), 1150 (C-O); δH(CDCl3)
(Scheme 3) 3.62 (8 H, s, CH2), 3.77–3.79 (4 H, m, CH2), 3.91
(6 H, s, CH3), 4.06–4.16 (4 H, m, CH2OAr), 6.97 (1 H, d, J 8.25,
Ar-H), 7.21 (1 H, dd, J 1.95 and J 8.25, Ar-H), 7.26 (1 H, d,
J 1.95, Ar-H), 7.48 and 7.69 (2 H, 2 × d, J 3.8, 3Ј- and 4Ј-H), 8.3
(2 H, d, J 1.45, 4-, 6-H), 8.33 (1 H, t, J 1.45, 2-H); δC(CDCl3)
52.44 (CH3), 71.13, 70.53, 69.58, 69.32, 69.12 (CH2O), 112.02
and 114.26 (Ar-CH), 119.01 (3Ј-C), 123.33 (4Ј-C), 125.28
(Ar-CH), 127.44 (Ar-C), 128.87, 130.08, 131.27 (1-, 2-, 3-, 4-,
6-C), 135.18 (5Ј-C), 140.06 (5-C), 145.06 (2Ј-C), 149.36 and
149.4 (ArC-O), 165.93 (CO); m/z 542 (Mϩ, 90%), 454 [Mϩ Ϫ
(CH2CH2O)2, 21], 410 [Mϩ Ϫ (CH2CH2O)3, 100]; Found:
542.1607, Calc. for C28H30O9S: 542.1610.
Dimethyl 5-[5-(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,13,
16-benzohexaoxacyclooctadecin-18-yl)-2-thienyl]isophthalate
9b. Method A.—Compound 9b was prepared from 4-bromo-
benzo-18-crown-6 8b (586 mg, 1.5 mmol) and compound 7
(1.02 g, 1.8 mmol, 1.2 equiv.) as described for 9a, and isolated as
a yellow powder (700 mg, 79%).
Method B.—Following the same procedure, starting from 12
(310 mg, 0.65 mmol), 13 (376 mg, 0.78 mmol, 1.2 equiv.) and
Pd(PPh3)4 (7 mg, 1%) in toluene (10 cm3), 9b was obtained in
80% yield (240 mg); mp 166–167 ЊC; νmax(KBr)/cmϪ1 2950,
2900, 2860 (CH3, CH2, CH), 1730 (CO), 1600, 1560, 1520 (Ar),
1150, 1120 (C-O); δH(CDCl3) 3.7 (4 H, s, CH2), 3.72–3.81 (8 H,
m, CH2), 3.93–3.97 (4 H, m, CH2), 3.98 (6 H, s, CH3), 4.19–4.25
(4 H, m, CH2), 6.9 (1 H, d, J 8.25, Ar-H), 7.16 (1 H, s, Ar-H),
18-(5-Bromo-2-thienyl)-2,3,5,6,8,9,11,12,14,15-decahydro-
1,4,7,10,13,16-benzohexaoxacyclooctadecine 12. This com-
pound was prepared as described for compound 6 starting from
compound 11 (394 mg, 1 mmol). A yellow oil was obtained (280
mg, 59%); νmax(CHCl3)/cmϪ1 2930–2880 (CH2, CH), 1600, 1580,
1500 (Ar), 1120 (C-O); δH(CDCl3) 3.69 (4 H, s, CH2), 3.7–3.79
(8 H, m, CH2), 3.9–3.96 (4 H, m, CH2), 4.13–4.24 (4 H, m,
CH2), 6.44 (1 H, s, Ar-H), 6.68 and 6.73 (2 H, 2 × d, J 8.45,
Ar-H), 6.88 and 7.36 (2 H, 2 × d, J 3.9, 3Ј-, 4Ј-H); δC(CDCl3)
69.22, 69.38, 70.75, 70.87 (CH2O), 112.16 (Ar-CH), 114.53
(CBr), 117.24 (Ar-CH), 119.39 (3Ј-C), 127.87 (Ar-CH), 130.66
(Ar-CH), 133.47 (4Ј-C), 148.14, 149.14, 149.46 (ArC-O and 2Ј-
C); m/z 472 (Mϩ, 32%), 384 [Mϩ Ϫ (CH2CH2O)2, 13], 340
[Mϩ Ϫ (CH2CH2O)3, 11], 296 [Mϩ Ϫ (CH2CH2O)4, 13], 176
ϩ
ϩ
[(CH2CH2O)4 , 20], 132 [(CH2CH2O)3 , 26], 86 (CH2CH2-
OCH᎐CHOϩ, 100); Found: 472.0549, Calc. for C H BrO S:
᎐
20 25
6
472.0555.
Dimethyl 5-(tributylstannyl)isophthalate 13. Compound 13
was prepared as described in method B for compound 7 starting
from compound 4 (960 mg, 3 mmol). A colourless oil was
obtained (650 mg, 45%); νmax(CCl4)/cmϪ1 2960, 2930, 2870
(CH2, CH3), 1730 (CO); δH(CDCl3) 0.95 (9 H, t, J 7.1, CH3),
1.15–1.21 (6 H, m, CH2), 1.35–1.47 (6 H, m, CH2), 1.57–1.69 (6
H, m, CH2Sn), 4.01 (6 H, s, CH3), 8.3 (2 H, d, J 1.5, 4-, 6-H), 8.6
(1 H, t, J 1.5, 2-H); δC(CDCl3) 9.76 (CH2Sn), 13.55 (CH3),
27.22–28.95 (CH2), 52.16 (CH3), 129.59 (CH2), 130.3 (1-, 3-C),
141.44 (4-, 6-CH), 143.4 (CSn), 166.7 (CO); m/z 484 (Mϩ, 28%),
ϩ
426 (Mϩ Ϫ butyl, 30), 290 (SnBu3 , 100); Found: 484.1632,
Calc. for C22H36O4Sn: 484.1635.
5-[5-(2,3,5,6,8,9,11,12-Octahydro-1,4,7,10,13-benzopentaoxa-
cyclopentadecin-15-yl)-2-thienyl]isophthalic acid 14a. The free
acid 14a was prepared from 9a following the procedure by
Laganis and Chenard25 (90%); mp 215–217 ЊC; νmax(KBr)/cmϪ1
3000 (OH), 2900–2870 (CH2, CH), 1720 (CO), 1600–1515 (Ar),
1100–1130 (C-O); δH([2H6]DMSO) 3.62 (8 H, s, CH2), 3.76 (4 H,
br s, CH2), 4.06–4.11 (4 H, m, CH2), 6.94 (1 H, d, J 8.35, 10Ј-H),
7.22 (1 H, d, J 8.35, 11Ј-H), 7.26 (1 H, s, 7Ј-H), 7.45 and 7.5
(2 H, 2 × d, J 3.4, 3Ј-H and 4Ј-H), 8.32 (2 H, s, 4-, 6-H), 8.37
J. Chem. Soc., Perkin Trans. 2, 1998
1579

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(1 H, s, 2-H); δC([2H6]DMSO) 69.87, 69.18, 68.29, 68.14
(CH2O), 110.74 (7Ј-C), 113.89 (10Ј-C), 118.19 (3Ј-C), 124.24
(4Ј-C), 125.65 (Ar-CH), 126.57 (6Ј-C), 128.38, 128.65, 133.8 (1-,
2-, 3-, 4-, 6-C), 133.9 (5Ј-C), 139.9 (5-C), 143.8, 148.26, 148.53
(ArC-O and 2Ј-C), 166.87 (CO); m/z 514 (Mϩ, 16), 470
[Mϩ Ϫ (CH2CH2O), 12], 382 [Mϩ Ϫ (CH2CH2O)3, 43], 338
[Mϩ Ϫ (CH2CH2O)4, 22], 45 (CO2Hϩ, 100); Found: 514.1300,
Calc. for C26H26O9S: 514.1297.
References
1 R. Y. Tsien, Am. J. Physiol. (Cell. Physiol. 32), 1992, 263, C723;
A. W. Czarnik, Fluorescent Chemosensors for Ion and Molecule
Recognition, ed. A. W. Czarnik, ACS Symposium Series,
Washington, 1993, p. 538.
2 J. Darnell, H. Lodish and D. Baltimore, Molecular Cell Biology,
Freeman, New York, 1986; M. N. Hughes, The Inorganic Chemistry
of Biological Processes, Wiley, London, 1972, p. 256.
3 I. P. Romanov, Lab. Delo, 1974, 7, 438.
5-[5-(2,3,5,6,8,9,11,12,14,15-Decahydro-1,4,7,10,13,16-
4 E. Dufau, H. Acker and D. Sylvester, Med. Prog. Technol., 1980, 7,
35.
benzohexaoxacyclooctadecin-18-yl)-2-thienyl]isophthalic
acid
14b. The free acid 14b was prepared from 9b as described for
14a (90%); mp 266–267 ЊC; νmax(KBr)/cmϪ1 3000 (OH), 2920–
2880 (CH2, CH), 1700 (CO), 1560–1520 (Ar), 1110 (C-O);
δH([2H6]DMSO) 3.52 (4 H, s, CH2), 3.56–3.59 (8 H, m, CH2),
3.77–3.91 (4 H, m, CH2), 4.1–4.19 (4 H, m, CH2), 7.23 (1 H,
d, J 8.5, Ar-H), 7.26 (1 H, s, Ar-H), 7.49–7.68 (2 H, 2 × d,
J 3.5, 3Ј-H and 4Ј-H), 7.98 (1 H, d, J 8.5, Ar-H), 8.36 (3 H, s, 2-,
4-, 6-H); δC([2H6]DMSO) 68.14, 68.2, 69.64, 69.74, 69.84
(CH2O), 110.2 (Ar-CH), 113.34 (Ar-CH), 118.04 (Ar-CH),
124.3 (4Ј-C), 126.01 (1-, 3-C), 126.21 (3Ј-C), 128.46 (2-C),
128.85 (4-, 6-C), 132.8 (Ar-C), 134.31 (5-C), 139.26 (5Ј-C),
144.06, 148.26, 148.46 (ArC-O and 2Ј-C), 166.52 (CO); m/z 558
(Mϩ, 21), 514 [Mϩ Ϫ (CH2CH2O), 8], 470 [Mϩ Ϫ (CH2CH2O)2,
5], 426 [Mϩ Ϫ (CH2CH2O)3, 5], 382 [Mϩ Ϫ (CH2CH2O)4, 83],
45 (CO2Hϩ, 100); Found: 558.1566, Calc. for C28H30O10S:
558.1559.
5 R. V. Smith and M. A. Nessen, J. Pharm. Sci., 1971, 60, 907.
6 D. Burstein and E. T. Fossel, Am. J. Physiol., 1987, 252, HI 138.
7 R. Y. Tsien, Annu. Rev. Neurosci., 1991, 12, 227. A. P. de Silva,
H. Q. N. Gunaratne, T. Gunnlaugson, A. J. M. Huxley, C. P. McCoy,
J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515.
8 J. R. Blinks, W. Wier, P. Hess and F. G. Prendergast, Prog. Biophys.
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Dicaesium
5-[5-(2,3,5,6,8,9,11,12-octahydro-1,4,7,10,13-
benzopentaoxacyclopentadecin-15-yl)-2-thienyl]isophthalate 1a.
The caesium salt 1a was prepared from 9a according to the
procedure described by Minta and Tsien.26 A solution of 9a (5.4
mg, 1 × 10Ϫ5 mol) and anhydrous caesium hydroxide (17 mg,
1 × 10Ϫ4 mol, 10 equiv.) in methanol (3 cm3) was refluxed over-
night. After evaporation of the methanol, the product was dis-
solved in water (100 cm3) and used as such for the fluorescence
measurements.
Dicaesium 5-[5-(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,
13,16-benzohexaoxacyclooctadecin-18-yl)-2-thienyl]isophthal-
ate 1b. The caesium salt 1b was prepared as described for 1a
starting from 9b (5.9 mg, 1 × 10Ϫ5 mol), dissolved in water (100
cm3) and used as such for the fluorescence measurements.
14 R. P. Haugland, Handbook of Fluorescent Probes and Research
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Acknowledgements
E. C. is indebted to Professor S. Gronowitz from the University
of Lund, Sweden, for offering her the opportunity to work with
his group during a short stay in 1996. The authors thank
Professor Y. Engelborghs from the K.U. Leuven for the use of
the fluorescence equipment. R. De Boer is thanked for taking
the mass spectra. E. C. is an Aspirant and N. B. is an Onder-
zoeksdirecteur of the Fonds voor Wetenschappelijk Onderzoek
(FWO). A. T. is a postdoctoral fellow at the K.U. Leuven.
K. V. is a predoctoral fellow of the Vlaams Instituut voor de
Bevordering van het Wetenschappelijk-Technologisch Onderzoek
in de Industrie (IWT). The continuing financial support of
Diensten voor Wetenschappelijke, Technologische en Culturele
Aangelegenheden (DWTC) through UIAP-4-11 is gratefully
acknowledged.
28 D. F. Eaton, Handbook of Organic Photochemistry, ed. J. C. Scaiano,
CRC Press, Boca Raton, FL, 1989, p. 231.
Paper 8/02183J
Received 19th March 1998
Accepted 30th April 1998
1580
J. Chem. Soc., Perkin Trans. 2, 1998