A highly K+-selective phenylaza-[18]crown-6-lariat-ether-based fluoroionophore and its application in the sensing of K+ ions with an optical sensor film and in cells

Article abstract of DOI:10.1002/chem.201302350

Herein, we report the synthesis of two phenylaza-[18]crown-6 lariat ethers with a coumarin fluorophore (1 and 2) and we reveal that compound 1 is an excellent probe for K⁺ ions under simulated physiological conditions. The presence of a 2-methoxyethoxy lariat group at the ortho position of the anilino moiety is crucial to the substantially increased stability of compounds 1 and 2 over their lariat-free phenylaza-[18]crown-6 ether analogues. Probe 1 shows a high K⁺/Na⁺ selectivity and a 2.5-fold fluorescence enhancement was observed in the presence of 100mM K⁺ ions. A fluorescent membrane sensor, which was prepared by incorporating probe 1 into a hydrogel, showed a fully reversible response, a response time of 150s, and a signal change of 7.8 % per 1mM K⁺ within the range 1-10mM K⁺. The membrane was easily fabricated (only a single sensing layer on a solid polyester support), yet no leaching was observed. Moreover, compound 1 rapidly permeated into cells, was cytocompatible, and was suitable for the fluorescent imaging of K⁺ ions on both the extracellular and intracellular levels. Copyright

Full text of DOI:10.1002/chem.201302350

FULL PAPER
DOI: 10.1002/chem.201302350
A Highly K+-Selective Phenylaza-[18]crown-6-Lariat-Ether-Based
Fluoroionophore and Its Application in the Sensing of K+ Ions with an
Optical Sensor Film and in Cells
Sandra Ast,[a, e] Thomas Schwarze,[a] Holger Mꢀller,[a] Aleksey Sukhanov,[b]
Stefanie Michaelis,[c] Joachim Wegener,[c] Otto S. Wolfbeis,[c] Thomas Kçrzdçrfer,[d]
Axel Dꢀrkop,*[c] and Hans-Jꢀrgen Holdt*[a]
Abstract: Herein, we report the synthe-
sis of two phenylaza-[18]crown-6 lariat
ethers with a coumarin fluorophore (1
and 2) and we reveal that compound
1 is an excellent probe for K+ ions
under simulated physiological condi-
tions. The presence of a 2-methoxye-
thoxy lariat group at the ortho position
of the anilino moiety is crucial to the
substantially increased stability of com-
pounds 1 and 2 over their lariat-free
phenylaza-[18]crown-6 ether analogues.
Probe 1 shows a high K+/Na+ selectivi-
ty and a 2.5-fold fluorescence enhance-
ment was observed in the presence of
100 mm K+ ions. A fluorescent mem-
brane sensor, which was prepared by
incorporating probe 1 into a hydrogel,
showed a fully reversible response, a
response time of 150 s, and a signal
change of 7.8% per 1 mmK+ within the
range 1–10 mmK+. The membrane was
easily fabricated (only a single sensing
layer on a solid polyester support), yet
no leaching was observed. Moreover,
compound 1 rapidly permeated into
cells, was cytocompatible, and was suit-
able for the fluorescent imaging of K+
ions on both the extracellular and in-
tracellular levels.
Keywords: crown compounds
·
fluorescence · gels · potassium · sen-
sors
Introduction
selectively distinguishing between extracellular K+ (4 mm)
and Na+ ions (140 mm) with a resolution of <0.1 mm.[3] Fur-
ther desirable features of a probe for K+ ions are its stability
and fast response, long-wavelength absorption (>400 nm)
and emission maxima (>490 nm), and no interference by
changes in pH value. A highly selective fluorescent molecu-
lar probe for potassium ions based on a calix[4]bisazacrown
that contained a boron-dipyrromethene fluorophore was in-
troduced by Valeur and co-workers, although this fluoroio-
nophore only worked in EtOH/water mixtures.[4] PBFI (po-
tassium-binding benzofuranisophthalate) is the most-popular
indicator for intracellular K+ ions.[5,6] PBFI incorporates a
diaza-[18]crown-6 ether as the recognition unit and, though
it lacks Na+/K+ selectivity, this probe is the only currently
commercially available K+ indicator.[7]
More-recent probes have overcome this problem of poor
K+/Na+ selectivity by employing a [2.2.3]-triazacryptand
(TAC) as a receptor unit.[8–14] The main drawback of these
extracellular probes is their extensive multistep syntheses.
Hence, there is still a demand for a simple, yet sensitive and
selective, probe for monitoring extracellular concentrations
of K+ ions (about 4 mm) against intracellular levels (about
150 mm).
Fluorescent molecular sensors for the recognition of alkali
and alkaline-earth metal ions have attracted extensive atten-
tion over the past few years[1,2] because of the increasing in-
terest in in vivo monitoring and in vitro diagnostics in rapid-
screening systems.[3] The measurement of K+ ions in blood
samples is of particular interest because of the challenge of
[a] Dr. S. Ast, Dr. T. Schwarze, H. Mꢀller, Prof. Dr. H.-J. Holdt
Institut fꢀr Chemie, Anorganische Chemie, Universitꢁt Potsdam
Karl-Liebknecht-Strasse 24–25, 14476 Golm (Germany)
Fax : (+49)331-977-5055
E-mail: holdt@chem.uni-potsdam.de
[b] Dr. A. Sukhanov
Tomsk Polytechnic University
Pr. Lenina 30, 634050 Tomsk (Russia)
[c] Dr. S. Michaelis, Prof. Dr. J. Wegener, Prof. Dr. O. S. Wolfbeis,
Dr. A. Dꢀrkop
Institut fꢀr Analytische Chemie, Chemo- und Biosensorik
Universitꢁt Regensburg, Universitꢁtsstrasse 31
93040 Regensburg (Germany)
E-mail: Axel.Duerkop@chemie.ur.de
[d] Prof. Dr. T. Kçrzdçrfer
Institut fꢀr Chemie, Theoretische Chemie
Universitꢁt Potsdam, Karl-Liebknecht-Strasse 24–25
14476 Golm (Germany)
Recently, we showed that the electronic conjugation of
1,2,3-triazol fluoroionophore within the signal-transduction
chain of phenylaza-[18]crown-6-1,2,3-triazol-1,4-diyl-coumar-
in yielded selective fluorescent probes for K+ ions under si-
mulated physiological conditions.[15–17] Unlike other probes
for K+ ions,[3,8–14] these probes were easily accessible in only
[e] Dr. S. Ast
School of Chemistry, University of Sydney
NSW 2006, Sydney (Australia)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302350.
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four steps. The estimated Kd values of these probes for K+
ions are about 260 mm, which is not suitable for monitoring
the physiological concentrations of K+ ions.
To decrease the Kd value of the receptor, we took advant-
age of the fact that the attachment of a lariat group signifi-
cantly increased the stability of complexes of monoazacrown
ethers with alkali-metal ions whilst retaining the flexibility
of the ionophores.[18] Further investigations on aza-
[18]crown-6 lariat ethers found that the strongest coordina-
tion of K+ ions was achieved by seven oxygen-donor
atoms.[19] This result indicated that the introduction of a
pendant 2-methoxyethoxy lariat group ortho to the trigger
anilino donor of the phenylaza-[18]crown-6 K+ receptor
should lead to a substantial decrease in the Kd value. The 2-
methoxyethoxy lariat unit is well-known. It was first intro-
duced by the group of Gokel[18] for the N-substitution of
aza-[18]crown-6 ether. Recent-
meated into cells, was cytocompatible, and was able to dif-
ferentiate between extracellular (about 5 mm) and intracel-
lular levels of K+ ions (about 150 mm).
Results and Discussion
Synthesis of molecular probes 1 and 2: The synthesis of the
new o-(2-methoxyethoxy)phenylaza-[18]crown-6 lariat ether
(4) started from commercially available 2-(2-methoxyethox-
y)aniline (Scheme 1); conversion into dihydroxy-aniline de-
rivative 3, followed by a macrocyclization step, yielded the
new K+ receptor 4. Benzaldehyde 5 served as a precursor of
alkyne 6 and was prepared through a Vilsmeyer reaction.[20]
The transformation of the aldehyde unit in compound 5 into
the terminal ethynyl unit in compound 6 was achieved by
ly, the same lariat group was
used for the functionalisation of
a [2.2.3] cryptand.[8] However,
the combination of the 2-me-
thoxyethoxy lariat group with a
simple phenlyaza-[18]crown-6
receptor for the selective com-
plexation of K+ ions has not
been reported before.
Herein, we report the synthe-
sis of a new K+-ion receptor, o-
(2-methoxyethoxy)phenylaza-
[18]crown-6 (4), its ethynyl and
azido derivatives (6 and 9, re-
spectively), and two phenylaza-
[18]crown-6-lariat-ether-based
fluoroionophores (1 and 2,
Scheme 1). Probes 1 and 2 are
1,4-disubstituted
1,2,3-triazol
constitutional isomers. Our
recent studies[15–17] revealed that
constitutional isomerism had a
fundamental influence on the
stability of K+ complexes and
on the K+/Na+ selectivity of
1,2,3-triazol fluoroionophores.
The stabilities of the K+ com-
plexes of probes 1 and 2 are
about tenfold higher than those
of their lariat-free analogues.
Probe 1 is highly K+/Na+ selec-
tive and allows the detection of
K+ ions within the concentra-
tion range 2–100 mm. Further-
more, probe 1 was also embed-
ded within a polymer matrix to
demonstrate its application as a
continuous sensor. Fluorescence
microscopy experiments re-
Scheme 1. Synthesis of probes 1 and 2: a) 2-chlorethanol, CaCO3, water, 608C, 90% yield; b) NaH, 1,17-dito-
syl-3,6,9,12,15-pentaoxaheptadecane, MeCN, reflux, 20% yield; c) POCl3, DMF, 27% yield; d) NaNO2, water,
glacial AcOH, RT, 23% yield; e) dimethyl-1-diazo-2-oxopropylphosphonate,[21] K2CO3, MeOH, 61% yield;
f) H2, 10% Pd/C, MeOH, 75 bar, >99%; g) NaNO2, NaN3, HCl, water, 08C to RT, 61% yield; h) CuSO4/Na
ascorbate, 3-ethynyl-7-diethylaminocoumarin,[25] THF/water, 608C, 73% yield; i) CuSO4/Na ascorbate, 3-azido-
vealed that probe 1 rapidly per- 7-diethylaminocoumarin,[24] THF/water, 608C, 93% yield.
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Sensing of K+ Ions with an Optical Sensor Film and in Cells
FULL PAPER
using dimethyl-1-diazo-2-oxopropylphosphonate.[21] Func-
tionalisation of compound 4 with an azide unit was carried
out through a mild nitration reaction. Subsequent reduction
into amine 8 and diazotization gave compound 9, ready for
the well-established CuI-catalyzed azide–alkyne cycloaddi-
tion (CuAAC) reaction.[22,23] Coupling of compound 6 and 9
with the respective coumarin-azide[24] or coumarine-
alkyne[25] gave molecular probes 1 and 2, respectively, in
good yields. Hence, the introduction of the lariat ether to
amend the Kd value of the iononphore could be achieved in
a small number (5 or 6) of synthetic steps.
The exposure of probe 1 to K+-free solutions that con-
tained various concentrations of Na+ ions (0–160 mm)
showed that the fluorescence of probe 1 remained almost
unaffected by the competing metal ion (Ff =0.062 at
160 mm Na+, Figure 2). This high K+/Na+ selectivity was fur-
Photophysical investigation under simulated physiological
conditions: The fluorescence-emission spectra of probe
1 under simulated physiological conditions were acquired as-
described in the Supporting Information at an excitation
wavelength of 420 nm. Uncomplexed 1 displayed an emis-
sion peak at 493 nm and a low quantum yield (Ff =0.062).
On increasing the K+-ion concentration within the physio-
logical range (2–160 mm), a 2.5-fold fluorescence enhance-
ment was observed (Figure 1a and Figure 2). The maximum
signal change was observed at 100 mmK+, with a Ff value of
0.184. In the low-K+-concentration range (1–10 mm), the
signal intensity increased by 3.5% per 1 mmK+.
Figure 2. Fluorescence-enhancement factors (If(max)/If0(max)
) for probes
1 and 2 (10 mm) under simulated physiological conditions (10 mm Tris-
buffer, pH 7.2) in the presence of various concentrations of K+ or Na+
ions; the ionic strength was maintained at 160 mm with choline chloride.
ther verified by using solutions that contained both K+ and
Na+ ions (Figure 1b and the Supporting Information, Fig-
ure S2). The enhancement by a factor of 2.4 at 100 mm K+
was only slightly lower than that in the experiment without
Na+ ions. The signal increased by a factor of 1.3 within the
clinical concentration range (1–10 mmK+), with a signal
change of 2.4% per 1 mmK+. Hence, the presence of Na+
ions has a negligible effect on the performance of probe
1 under simulated physiological conditions.
Dissociation constants were calculated from plots of fluo-
rescence intensities versus K+-ion concentration (see the
Supporting Information). The Kd value of probe 1 in Na+-
free solution was 26 mm and increased to about 29 mm (if
approximated) for the combined K+/Na+ solutions, again
demonstrating the high K+/Na+ selectivity of probe 1.
The K+/Na+ selectivity of probe 2 was not as high as that
observed for probe 1 (Figure 2).[26] Compound 2 complexed
both K+ and Na+ ions more strongly than compound 1. This
result was further supported by the lower Kd values for com-
pound 2 (compared to those for compound 1) in solutions
that contained either K+ ions alone or both K+ and Na+
ions (see the Supporting Information, Figure S3c,d). In
Na+-free solutions, the signal intensity of probe 2 increased
in the low-concentration range (1–10 mm) by 8.9% per
1 mmK+ (see the Supporting Information, Figure S2c,d).
Hence, the appended 2-methoxyethoxy lariat group in-
duced an approximately tenfold increase in the stability of
the K+ complexes of probes 1 and 2 over their lariat-free an-
alogues.[15] The Kd values of the K+ complexes of probes
1 and 2 in combined Na+/K+ solutions (about 29 mm) should
be useful for the determination of K+ concentrations within
Figure 1. Normalized fluorescence spectra of probe 1 (10 mm) to K+ in
the presence of various concentrations of ions under simulated physiolog-
ical conditions (10 mm Tris-buffer, pH 7.2); the ionic strength was main-
tained at 160 mm with a) choline chloride and b) various concentrations
of Na+ ions in combined K+/Na+ solutions.
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the range 2–100 mm. The sensitivity of probes 1 and 2 to-
wards change in pH value at various concentrations of K+
ions within the pH range 6.8–7.8 (see the Supporting Infor-
mation, Figure S4) was low, as shown by fluorescence in-
creases that remained largely unaffected by pH value. Thus,
probe 1 was selected for further imaging experiments in
NRK cells and for studies on its application as a K+-ion
sensor because it showed higher K+/Na+ selectivity than
probe 2 (see below).
The B3LYP/6-31G*-optimized geometries of compounds
1 and 2 (see the Supporting Information, Chapter 7, Fig-
ure S6) were similar to those of their lariat-free analogues.[15]
Thus, we assumed that the sensing mechanism of probes
1 and 2 also involved photoinduced electron transfer (PET)
processes on the basis of virtual spacers,[27] as discussed for
their lariat free analogues.[15]
image B1, see the Supporting Information, Figure S5). The
fluorescence image and the corresponding phase-contrast
micrograph (Figure 3, image A1) did not reveal any changes
in cell morphology, thus indicating that the probe was cyto-
compatible during the time period of incubation and obser-
vation.
After incubating the cells with Triton X-100, the cell
membranes became permeabilized and dissolved (Figure 3,
image A2), which finally led to cell death. Thus, the fluores-
cence image did not show any distinct cellular structures.
Rather, a low background fluorescence was observed, be-
cause the ionophore had been released into the buffer
(Figure 3, image B2). Even if the concentration of K+ ions
in the lysed cell sample was lowered to about 5 mm by dilu-
tion with Earle’s balanced salt solution, fluorescence was
still visible. Thus, one can clearly discriminate between in-
tracellular and extracellular concentrations of K+ ions by
fluorescence microscopy.
Imaging experiments in NRK cells: We also studied the
uptake of probe 1 by normal rat kidney (NRK) cells that
were grown in ordinary Petri dishes by using fluorescence
microscopy. The probe was allowed to distribute over the
cell body before the microscopic inspection. Figure 3,
image B1 shows a fluorescence image of a confluent NRK
Sensor applications: To use probe 1 in a sensor membrane
for the continuous monitoring of K+ concentration in flow-
ing samples, such as blood, we incorporated it into a poly-
mer matrix. We found that Hypan HN 80 hydrogel was a
convenient polymer for making stable membrane sensors.
Hypan is a polyacrylamide-co-polyacrylonitrile copolymer
that contains hard (polyacrylonitrile domains, lipophilic)
and soft blocks (polyacrylamide domains, hydrophilic).[28]
Moreover, Hypan can take-up water in fractions up to 80%
of its weight and has excellent ion permeability; this proper-
ty would allow cations to rapidly reach probe 1, which is
mostly located in the hard blocks. Thin layers of a sensor
cocktail (a solution of probe 1 and hydrogel in DMSO)
were spread over 125 mm dust-free transparent slices of an
ethylene-glycol-terephthalate polyester by knife coating.
Unlike other optical membranes for sensing K+ ions, this
membrane required neither a multilayered structure[8] nor
plasticisers or covalent binding of the indicator to one of the
polymers.[8,29] Hence, both the preparation of the cocktail
and the membrane sensor were significantly simplified. Cir-
cular sensor spots were punched out of the membrane and
exposed to solutions with various concentrations of K+ ions
(Figure 4) in a flow-through cell. The cell was linked to a
fluorimeter through a Y-shaped bifurcated optical fiber.
Embedding probe 1 into the membrane sensor shifted the
absorbance maximum by 3 nm to 423 nm, whilst the emis-
sion maximum was shifted by only 3 nm to shorter wave-
lengths. This result is typical for a luminophore on embed-
ding in a foil sensor with a more-lipophilic microenviron-
ment.[29] A distinct increase in fluorescence was found
(Figure 5) on increasing the concentration of K+ ions in the
sample flow (adjusted to an ionic strength of 150 mm with
NaCl). This increase was reversible, as demonstrated by the
time trace at decreasing concentrations of K+. The forward
response time (t90) was less than 150 s over the full range
and the backward response time (t90) to lower concentra-
tions of K+ was l70 s. This behavior was even more rapid
than found in earlier work for membranes based on co-ex-
Figure 3. Images of
a confluent NRK cell layer in buffer solution:
A) Phase-contrast micrographs and B) fluorescence images (1) after incu-
bation with probe 1 (30 min), (2) after incubation with probe 1 (30 min)
and subsequent permeabilization with Triton X-100, and (3) after incuba-
tion with buffer only.
monolayer after incubation with a solution of probe 1 for
30 min. The green fluorescence could be solely attributed to
the K+ complex with probe 1, because fluorescence images
without probe 1 did not show any cellular structures
(Figure 3, image B3). The endomembranes of the cells fluor-
esced brightly, whereas the cell nuclei remained dark, thus
indicating that the probe did not penetrate the nuclei. Fluo-
rescence intensity was not homogeneously distributed within
the cells, but was rather predominant within the hydropho-
bic membrane domains. A small band around the cell nu-
cleus, possibly the endoplasmic reticulum (ER), showed
higher fluorescence intensity compared to the more-periph-
eral endomembranes (for a magnified image of Figure 3,
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A calibration plot revealed that the membrane sensor
covered the clinically relevant range (1–10 mm). Herein, the
resolution, as determined by the equation: (DFÀF0)/F0)/
Dc(K+), was the best reported so far and the signal change
was about 7.8% per 1 mmK+. Interestingly, this result was
even better than that observed with more-complex mem-
brane sensors (three layers and the probe covalently bound
to the polymer)[8] and showed the improved performance of
probe 1. The detection limit (at 3s) for the determination of
K+ ions with the membrane sensor was 800 mmK+. Impor-
tantly, on the immobilization of probe 1 in the polymer
matrix, a clear discrimination between the average extracel-
lular (4 mm) and intracellular levels (150 mm) of K+ was pos-
sible, thus rendering probe 1 an attractive fluoroionophore
for applications in clinical diagnostic systems.
Figure 4. Application of probe 1 in a foil sensor.
Conclusion
In conclusion, herein, we have reported the facile synthesis
and application of K+-selective 1,2,3-triazol-fluoroiono-
phores 1 and 2, which featured a new o-(2-methoxyethoxy)-
phenylaza-[18]crown-6-lariat-ether receptor. Probe 1 per-
formed excellently under simulated physiological conditions
and exhibited high K+ selectivity over Na+. Probe 1 had a
low Kd value of about 29 mm in combined K+/Na+ solutions
and showed a 2.4% signal change per 1 mmK+. The K+/Na+
selectivity of constitutional isomer 2 was not as high as that
observed for probe 1, but, at low Na+ concentrations, K+
was detected with a greater fluorescence enhancement
(8.9% per 1 mmK+ within the range 1–10 mm). Probe 1 was
also incorporated into a membrane sensor that consisting of
a polymethane hydrogel and, in this form, it enabled the
continuous monitoring of physiological K+ levels with a
rapid response time. Moreover, cellular studies showed that
probe 1 was not damaging to cells. In summary, probe 1 is
well-suited for differentiating between extracellular (about
5 mm) and intracellular levels of K+ ions (about 150 mm) in
aqueous samples, if contained in foil sensors, or in cellular
experiments by using fluorescence microscopy.
Experimental Section
General methods and reagents: All commercially available chemicals
were used without further purification. Solvents were distilled prior use.
1H and 13C NMR spectra were recorded on 300 MHz and 600 MHz in-
struments, respectively; data are reported as follows: chemical shift (d, in
ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, qu=
quintet, sx=sextet, dd=doublet of doublets, m=multiplet), integration,
coupling constant (in Hz). ESI spectra were recorded on a Micromass Q-
TOF micro mass spectrometer in positive electrospray mode. IR spectra
were recorded on a Thermo Nicolet Nexus FTIR instrument. Air/water-
sensitive reactions were performed in oven-dried glassware under an
argon atmosphere. Column chromatography was performed on silica gel
(Merck; silica gel 60, 0.04–0.063 mesh).
Figure 5. Top: Response time, signal change, and reversibility of the
sensor membrane (that contained probe 1) in the presence of various
concentrations of K+ ions (10 mm Tris-buffer, pH 7.3; the ionic strength
was maintained at 150 mm with NaCl). Bottom: Calibration plot for the
determination of K+-ion concentration, as obtained from the sensor
membrane (excitation/emission at 423/472 nm).
traction.[29] No leaching of probe 1 out of the membrane
sensor was observed.
N,N-Bis(2-hydroxyethyl)-2-methoxyethoxyaniline (3): A mixture of 2-me-
thoxyethoxyaniline (21.9 g, 0.131 mol), 2-chloroethanol (52.77 g,
0.656 mol), and CaCO3 (18.37 g, 0.184 mol) in water (300 mL) was stirred
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A. Dꢀrkop, H.-J. Holdt et al.
for 6 days at 608C. After cooling to RT, Na2CO3 (75.0 g, 0.7 mol) was
added and the mixture was stirred for 40 min at 608C. Then, the resulting
solid was filtered off and the aqueous layer was saturated with NaCl and
extracted with methyl-tert-butyl ether (3ꢃ500 mL). The combined organ-
ic phases were dried over K2CO3, concentrated in vacuo, and the residue
was purified by column chromatography on silica gel (EtOAc) to give
75 MHz): d=148.39, 146.33, 139.08, 118.57, 115.65, 108.21, 70.77, 70.68,
70.64, 70.56, 70.52, 69.93, 67.92, 58.82, 52.83 ppm; MS (ESI+): m/z calcd
for C21H35N2O9: 459.23; found: 459.26.
N-(4-Amino-2-methoxyethoxyphenyl)aza-[18]crown-6 ether (8): 10% Pd/
C (30 mg) was added to a solution of compound 7 (0.22 g, 0.48 mmol) in
dry MeOH (30 mL). The mixture was hydrogenated in an autoclave for
16 h at 75 bar. The catalyst was removed by filtration through a bed of
Celite and the solvent was removed in vacuo to yield a colorless oil. This
compound quickly decomposed if exposed to air, thereby showing a color
change to dark purple. Therefore, no further purification was attempted
and compound 8 was directly used in the next step. Yield: 0.2 g, 97%;
MS (ESI+): m/z calcd for C21H37N2O7: 429.26; found: 429.20.
compound
3 as a
red oil (30.1 g, 90% yield). 1H NMR (CDCl3,
300 MHz): d=7.22 (dd, J=7.8, 1.6 Hz, 1H), 7.14–7.08 (m, 1H), 7.01–6.95
(m, 1H), 6.91 (dd, J=8.1, 1.2 Hz, 1H), 4.13–4.10 (m, 2H), 3.76–3.73 (m,
2H), 3.47 (t, J=5.3 Hz, 4H), 3.43 (s, 3H), 3.15 ppm (t, J=5.3 Hz, 4H);
13C NMR (CDCl3, 75 MHz): d=155.25, 139.28, 126.01, 125.51, 122.23,
113.33, 70.70, 67.80, 59.66, 58.93, 57.93 ppm; MS (ESI+): m/z calcd for
C13H22NO4: 256.15; found: 256.13.
N-(4-Azido-2-methoxyethoxyphenyl)aza-[18]crown-6 ether (9): A solu-
tion of compound 8 (0.2 g, 0.467 mmol) in 4m HCl (3.6 mL) was cooled
to 08C. A solution of NaNO2 (32 mg, 0.467 mmol) in water (1.8 mL) was
slowly added and the mixture was stirred at 08C for 10 min. Then, a solu-
tion of NaN3 (0.45 mg, 0.7 mmol) in water (1.8 mL) was added at 08C
and the mixture was stirred for 14 h at RT, neutralized with Li2CO3, and
extracted with CHCl3 (3ꢃ50 mL). The organic layers were combined,
dried over MgSO4, concentrated in vacuo, and the residue was purified
by column chromatography on silica gel (CHCl3/MeOH, 95/5 v/v) to give
compound 9 as a brown oil (0.13 g, 61% yield). 1H NMR (CDCl3,
300 MHz): d=6.89–6.59 (m, 3H), 4.13–4.10 (m, 2H), 3.77–3.56 (m, 26H),
3.43 ppm (s, 3H); 13C NMR (CDCl3, 75 MHz): d=146.06, 138.63, 121.84,
116.44, 111.74, 110.21, 72.49, 71.07, 70.53, 70.31, 70.25, 69.75, 68.06, 61.64,
59.05 ppm; MS (ESI+): m/z calcd for C21H35N4O7: 455.25; found: 455.25.
N-(2-Methoxyethoxyphenyl)aza-[18]crown-6 ether (4): NaH (80%, 5.2 g)
was added to a solution of compound 3 (17.86 g, 69.9 mmol) in dry
MeCN (510 mL) over a period of 1 h under an argon atmosphere. This
mixture was heated at reflux and a solution of 1,17-ditosyl-3,6,9,12,15-
pentaoxaheptadecane (35.16 g, 69.9 mmol) in dry MeCN (250 mL) was
added dropwise over a period of 4 h. Then, the mixture was heated at
reflux for 11 h and the resulting precipitate was filtered off. The solvent
was removed and the residue was purified by column chromatography on
silica gel (CHCl3/MeOH, 95:5 v/v) to yield compound 4 as a brown oil
1
(16.45 g, 57% yield). H NMR (CDCl3, 300 MHz): d=7.12–6.82 (m, 4H),
4.10–4.07 (m, 2H), 3.73–3.45 (m, 26H), 3.39 ppm (s, 3H); 13C NMR
(CDCl3, 75 MHz): d=152.04, 140.15, 121.96, 121.18, 121.14, 113.83, 71.02,
70.71, 70.59, 70.56, 70.27, 70.02, 67.54, 58.81, 52.63 ppm; MS (ESI+): m/z
calcd for C21H36NO7: 414.25; found: 414.16.
7-(Diethylamino)-3-{1-[3-(2-methoxyethoxy)-4-(1,4,7,10,13-pentaoxa-16-
azacyclooctadecan-16-yl)phenyl]-1H-1,2,3-triazol-4-yl)}-2H-chromen-2-
one (1): Compound 9 (228 mg, 0.5 mmol), 3-ethinyl-7-diethylamino-cou-
marin[25] (121 mg, 0.5 mmol), CuSO4·5H2O (6.3 mg, 5 mol%), and
sodium ascorbate (9.9 mg, 10 mol%) were dissolved in THF/water (3:1 v/
v, 15 mL). The reaction mixture was stirred at 608C for 24 h. Then, water
(15 mL) was added and the aqueous layer was extracted with CHCl3 (3ꢃ
15 mL), dried over MgSO4, and concentrated in vacuo. The crude product
was purified by column chromatography on silica gel (CHCl3/MeOH, 9:1
v/v) to give compound 1 as a yellow solid (254 mg, 73% yield). 1H NMR
(CDCl3, 300 MHz): d=8.60 (s, 1H), 8.58 (s, 1H), 7.35 (d, J=8.9 Hz, 1H),
7.29 (d, J=2.2 Hz, 1H), 7.21 (dd, J=2.2, 8.6 Hz, 1H), 7.11 (d, J=8.6 Hz,
1H), 6.57 (dd, J=2.4, 8.9 Hz, 1H), 6.47 (d, J=2.1 Hz, 1H), 4.17–4.14 (m,
2H), 3.75–3.72 (m, 2H), 3.70–3.31 (m, 31H), 1.16 ppm (t, J=7.0 Hz,
6H); 13C NMR (CDCl3, 75 MHz): d=160.42, 155.85, 151.96, 150.62,
141.99, 140.36, 138.30, 130.83, 129.32, 120.51, 120.18, 112.72, 110.38,
109.20, 108.48, 106.10, 96.85, 70.68, 70.64, 70.51, 70.46, 70.25, 69.78, 67.83,
58.75, 52.50, 44.67, 12.29 ppm; HRMS (ESI+): m/z calcd for
C36H50N5O9:696.3609; found: 696.3583.
N-(4-Formyl-2-methoxyethoxyphenyl)aza-[18]crown-6 ether (5): POCl3
(37 g, 241 mmol) was added to
a solution of compound 4 (9.98 g,
24.1 mmol) in dry DMF (60 mL) at À108C. The mixture was stirred for
12 h at RT, then heated at 708C and stirred for 1 h. The solution was
slowly poured over ice (250 mL), neutralized with Na2CO3, and extracted
with CH2Cl2 (3ꢃ250 mL). The combined organic phases were dried over
MgSO4, concentrated in vacuo, and the residue was purified by column
chromatography on silica gel (CHCl3/MeOH, 95/5 v/v) to yield com-
pound 5 as a yellow-brown oil (2.87 g, 27% yield). 1H NMR (CDCl3,
300 MHz): d=9.69 (s, 1H), 7.30 (dd, 1H, J=8.3 Hz, 1.8 Hz), 7.26 (d, 1H,
J=1.8 Hz), 6.93 (d, 1H, J=8.3 Hz), 4.11–4.08 (m, 2H), 3.71–3.55 (m,
26H), 3.36 ppm (s, 3H); 13C NMR (CDCl3, 75 MHz): d=190.14, 149.69,
146.08, 128.22, 126.73, 116.46, 111.01, 70.70, 70.61, 70.55, 70.48, 70.42,
69.95, 67.46, 58.65, 52.61 ppm; MS (ESI+): m/z calcd for C22H36NO8:
442.24; found: 442.25.
N-(4-Ethynyl-2-methoxyethoxyphenyl)aza-[18]crown-6 ether (6): Di-
methyl-1-diazo-2-oxopropylphosphonate[21] (522 mg, 2.7 mmol) was
added to a solution of compound 5 (1 g, 2.3 mmol) and K2CO3 (626 mg,
4.6 mmol) in dry MeOH (20 mL) and the mixture was stirred for 10 h at
RT. Then, the solvent was removed and the residue was diluted with
water (50 mL) and CH2Cl2 (50 mL). The organic layer was washed with
water (2ꢃ50 mL), dried over MgSO4, concentrated in vacuo, and the resi-
due was purified by column chromatography on silica gel (CHCl3/MeOH,
95/5 v/v) to yield compound 6 as a light-brown oil (603 mg, 61% yield).
1H NMR (CDCl3, 300 MHz): d=7.02 (dd, J=8.2 Hz, 1.8 Hz, 1H), 6.92–
6.89 (m, 2H), 4.07–4.04 (m, 2H), 3.72–3.47 (m, 26H), 3.38 (s, 3H),
2.98 ppm (s, 1H); 13C NMR (CDCl3, 75 MHz): d=150.57, 141.23, 125.66,
119.37, 116.92, 113.83, 84.09, 75.57, 70.84, 70.75, 70.63, 70.59, 70.36, 69.97,
67.61, 58.78, 52.45 ppm; MS (ESI+): m/z calcd for C23H36NO7: 438.25;
found: 438.21.
7-(Diethylamino)-3-{4-[3-(2-methoxyethoxy)-4-(1,4,7,10,13-pentaoxa-16-
azacyclooctadecan-16-yl)phenyl]-1H-1,2,3-triazol-1-yl}-2H-chromen-2-one
(2): Compound 6 (235 mg, 0.5 mmol), 3-azido-7-diethylamino-coumarin[24]
(139 mg, 0.5 mmol), CuSO4·5H2O (6.7 mg, 5 mol%), and sodium ascor-
bate (10.6 mg, 10 mol%) were dissolved in THF/water (3:1 v/v, 15 mL).
The reaction mixture was stirred at 608C for 24 h. Then, water (15 mL)
was added and the aqueous layer was extracted with CHCl3 (3ꢃ15 mL),
dried over MgSO4, and concentrated in vacuo. The crude product was pu-
rified by column chromatography on silica gel (CHCl3/MeOH, 9:1 v/v) to
give compound
2 as a
yellow solid (349 mg, 93% yield). 1H NMR
(CDCl3, 300 MHz): d=8.69 (s, 1H), 8.38 (s, 1H), 7.43–7.34 (m, 3H), 7.08
(d, J=8.2 Hz, 1H), 6.64 (dd, J=2.5, 8.9 Hz, 1H), 6.51 (d, J=2.2 Hz, 1H),
4.22–4.19 (m, 2H), 3.77–3.74 (m, 2H), 3.73–3.37 (m, 31H), 1.20 ppm (t,
J=7.0 Hz, 6H); 13C NMR (CDCl3, 75 MHz): d=156.82, 155.65,
151.97,151.44, 147.47, 140.18, 134.22, 129.86, 123.89, 120.62, 119.54,
118.78, 116.88, 111.01, 109.99, 107.03, 96.91, 70.96, 70.71, 70.56, 70.54,
70.32, 69.97, 67.64, 58.80, 52.70, 44.87, 12.32 ppm; HRMS (ESI+): m/z
calcd for C36H50N5O9: 696.3609; found: 696.3448.
N-(4-Nitro-2-methoxyethoxyphenyl)aza-[18]crown-6 ether (7): NaNO2
(0.81, 11.7 mmol) was added to
a solution of compound 4 (4.4 g,
10.65 mmol) in a mixture of water (340 mL) and glacial acetic acid
(34 mL) over a period of 10 min. Then, the reaction mixture was stirred
for 16 h at RT, neutralized with LiOH, and extracted with CH2Cl2 (3ꢃ
100 mL). The combined organic phases were dried over MgSO4, concen-
trated in vacuo, and the residue was purified by column chromatography
on silica gel (CHCl3/MeOH, 95/5 v/v) to yield compound 7 as a yellow
solid (1.1 g, 23% yield). 1H NMR (CDCl3, 300 MHz): d=7.80 (dd, J=
9.1, 2.5 Hz, 1H), 7.64 (d, J=2.5 Hz, 1H), 6.88 (d, J=9.1 Hz, 1H), 4.15–
4.12 (m, 2H), 3.75–3.58 (m, 26H), 3.40 ppm (s, 3H); 13C NMR (CDCl3,
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Sensing of K+ Ions with an Optical Sensor Film and in Cells
FULL PAPER
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Received: June 19, 2013
Published online: && &&, 0000
Chem. Eur. J. 2013, 00, 0 – 0
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!

A. Dꢀrkop, H.-J. Holdt et al.
The crown jewels: A new phenylaza-
[18]crown-6 lariat ether that contains a
coumarin fluorophore (1) is an excel-
lent fluorescent probe that shows high
selectivity and sensitivity for K+ ions
under simulated physiological condi-
tions and in cells (see scheme). A fluo-
rescent membrane sensor was pre-
pared by incorporating probe 1 into a
hydrogel, which showed a signal
Fluorescent Probes
S. Ast, T. Schwarze, H. Mꢀller,
A. Sukhanov, S. Michaelis, J. Wegener,
O. S. Wolfbeis, T. Kçrzdçrfer,
A. Dꢀrkop,* H.-J. Holdt* ... &&&& — &&&&
A Highly K+-Selective Phenylaza-
[18]crown-6-Lariat-Ether-Based Fluo-
roionophore and Its Application in the
Sensing of K+ Ions with an Optical
Sensor Film and in Cells
change of 7.8% per 1 mmK+ within
the range 1–10 mmK+.
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ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
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These are not the final page numbers!