Design of Na+-Selective Fluorescent Probes: A Systematic Study of the Na+-Complex Stability and the Na+/K+ Selectivity in Acetonitrile and Water

Article abstract of DOI:10.1002/chem.201605986

There is a tremendous demand for highly Na⁺-selective fluoroionophores to monitor the top analyte Na⁺ in life science. Here, we report a systematic route to develop highly Na⁺/K⁺ selective fluorescent probes. Thus, we synthesized a set of fluoroionophores 1, 3, 4, 5, 8 and 9 (see Scheme) to investigate the Na⁺/K⁺ selectivity and Na⁺- complex stability in CH₃CN and H₂O. These Na⁺-probes bear different 15-crown-5 moieties to bind Na⁺ stronger than K⁺. In the set of the diethylaminocoumarin-substituted fluoroionophores 1–5, the following trend of fluorescence quenching 1>3>2>4>5 in CH₃CN was observed. Therefore, the flexibility of the aza-15-crown-5 moieties in 1–4 determines the conjugation of the nitrogen lone pair with the aromatic ring. As a consequence, 1 showed in CH₃CN the highest Na⁺-induced fluorescence enhancement (FE) by a factor of 46.5 and a weaker K⁺ induced FE of 3.7. The Na⁺-complex stability of 1–4 in CH₃CN is enhanced in the following order of 2>4>3>1, assuming that the O-atom of the methoxy group in the ortho-position, as shown in 2, strengthened the Na⁺-complex formation. Furthermore, we found for the N-(o-methoxyphenyl)aza-15-crown-5 substituted fluoroionophores 2, 8 and 9 in H₂O, an enhanced Na⁺-complex stability in the following order 8>2>9 and an increased Na⁺/K⁺ selectivity in the reverse order 9>2>8. Notably, the Na⁺-induced FE of 8 (FEF=10.9), 2 (FEF=5.0) and 9 (FEF=2.0) showed a similar trend associated with a decreased K⁺-induced FE [8 (FEF=2.7)>2 (FEF=1.5)>9 (FEF=1.1)]. Here, the Na⁺-complex stability and Na⁺/K⁺ selectivity is also influenced by the fluorophore moiety. Thus, fluorescent probe 8 (K_d=48 mm) allows high-contrast, sensitive, and selective Na⁺ measurements over extracellular K⁺ levels. A higher Na⁺/K⁺ selectivity showed fluorescent probe 9, but also a higher K_d value of 223 mm. Therefore, 9 is a suitable tool to measure Na⁺ concentrations up to 300 mm at a fluorescence emission of 614 nm.

Full text of DOI:10.1002/chem.201605986

A Journal of
Accepted Article
Title: Design of Na+-Selective Fluorescent Probes - A Systematic
Study of the Na+-Complex Stability and the Na+/K+ Selectivity in
Acetonitrile and Water
Authors: Hans-Jürgen Holdt
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Design of Na+-Selective Fluorescent Probes – A Systematic Study
of the Na+-Complex Stability and the Na+/K+ Selectivity in
Acetonitrile and Water
Thomas Schwarze,[a] Holger Müller,[a] Darya Schmidt,[a] Janine Riemer[a] and Hans-Jürgen Holdt*[a]
Abstract: Overall, there is a tremendous demand for highly Na+-
selective fluoroionophores to monitor the top analyte Na+ in life
science. Herein, we report on a systematic route to develop highly
Na+/K+ selective fluorescent probes. Thus, we synthesized a set of
fluoroionophores 1, 3, 4, 5, 8 and 9 (cf. Scheme 1) to investigate the
Na+/K+ selectivity and Na+-complex stability in CH3CN and H2O.
These Na+-probes bearing different 15-crown-5 moieties to bind Na+
stronger than K+. In the set of the diethylaminocoumarin substituted
fluoroionophores 1-5 following trend of the fluorescence quenching 1
> 3 > 2 > 4 > 5 in CH3CN (cf. Table1) was observed. Therefore, the
flexibility of the aza-15-crown-5 moieties in 1-4 determines the
conjugation of the nitrogen lone pair with the aromatic ring. As a
contraction.[1a,b] In animal cells the Na+ concentration range
differs in the intra- (5-30 mM) and extracellular (100-150 mM)
space.[2] In both, K+ is the competitive cation and shows an
extra-(1-10 mM) and intracellular (100-150 mM) concentration
gradient contrary to Na+. Hence, for a reliable Na+ analyses in
vivo highly Na+/K+ selective tools were required. A favored
strategy to determine Na+ in vitro and vivo is based on the highly
sensitive fluorescence spectroscopy method. The non-
fluorescent Na+ is detected by fluorescent probes, which
containing an ion-binding moiety (ionophore) and a fluorescence
sensing unit (fluorophore). These fluorescent probes, so-called
fluoroionophores, show Na+-dependent fluorescence intensity
changes. Minta and Tsien designed the first fluoroionophore
SBFI (short for sodium-binding benzofuran isophthalate) for
intracellular Na+ levels (5-30 mM).[2] SBFI contains of a N,N’-
bis(o-methoxyphenyl)diaza-15-crown-5 moiety, which is the
most commonly used ionophore to detect intracellular Na+ levels.
Up to now, various fluoroionophores for a determination of
intracellular Na+ levels were reported.[3a-e] Nowadays, the
authors focused on the spectroscopic properties of the
fluorophore moiety.[3c-e] There, ratiometric red-fluorescent[3e] and
two photon excitable[3c,d] Na+-fluoroionophores were developed.
Further, to determine extracellular Na+ levels (100-140 mM), He
et al.[4a] and Gunnlaugsson et al.[4b] designed fluoroionophores
based on a N-(o-methoxyphenyl)aza-15-crown-5 ionophore. For
consequence,
1
showed in CH3CN the highest Na+ induced
fluorescence enhancement (FE) by a factor of 46.5 and a weaker K+
induced FE of 3.7. The Na+-complex stability of 1-4 in CH3CN is
enhanced in the following order 2 > 4 > 3 > 1, assuming that the O-
atom of the methoxy group in ortho position, as shown in 2,
strengthened the Na+-complex formation. Further, we found for the
N-(o-methoxyphenyl)aza-15-crown-5 substituted fluoroionophores 2,
8 and 9 in H2O an enhanced Na+-complex stability in the following
order 8 > 2 > 9 and an increased Na+/K+ selectivity in the reverse
order 9 > 2 > 8. Notable, the Na+ induced FE of 8 (FEF = 10.9), 2
(FEF = 5.0) and 9 (FEF = 2.0) showed a similar trend associated
with a decreased K+ induced FE (8 (FEF = 2.7) > 2 (FEF = 1.5) > 9
(FEF = 1.1)). Herein, the Na+-complex stability and Na+/K+ selectivity
is also influenced by the fluorophore moiety. Thus, fluorescent probe
8 (Kd = 48 mM) allows high-contrast, sensitive and selective Na+
the detection of higher Na+ levels >140 mM,
a
few
fluoroionophores were available.[5a-c] They contain as an
ionophore a N-methylacetate-aza-15-benzo-crown-5[5a] or a N-
phenylaza-15-crown-5[5c] moiety. A further Na+-selective binding
moiety is the nitrogen-free benzo-15-crown-5 ionophore, which
is part of Na+-fluoroionophores[6a,b], for instance, to determine
Na+ near micelle surfaces.[6b]
measurements over extracellular K+ levels.
selectivity showed fluorescent probe 9, but also a higher Kd value of
223 mM. Therefore, is
suitable tool to measure Na+
A
higher Na+/K+
9
a
concentrations up to 300 mM at a fluorescence emission of 614 nm.
All in all, there is rare number of fluoroionophores for a Na+
determination in vitro or in vivo. Therefore, tailor-made and
easily accessible Na+-fluoroionophores with a high Na+/K+
selectivity and with an appropriate dissociation constant to
detect Na+ in vitro and in vivo should be further developed.
Introduction
Sodium is one of the most important analytes in life science. It
plays a vital role by diverse physiological and pathological
processes, such as nerve conduction and muscle and heart
[a]
Dr. T. Schwarze, H. Müller, D. Schmidt, J. Riemer, Prof. Dr. H.-J.
Holdt
Institut für Chemie, Anorganische Chemie, Universität Potsdam
Karl-Liebknecht-Str. 24–25, 14476 Golm (Germany)
E-mail: holdt@uni-potsdam.de
Supporting information for this article is given via a link at the end of
the document.
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Scheme 1. Synthesis of Na+-responsive fluoroionophores 1, 3, 4, 5, 8 and 9: a) CuSO4/Na ascorbate, 3-azido-7-diethylaminocoumarin[10], THF/H2O, 60°C; b)
CuSO4/Na ascorbate, 10-azido-2,3,6,7-tetrahydro-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizin-11-one[10], THF/H2O, 60°C; c) CuSO4/Na ascorbate, 3-azido-8-
(dimethylamino)-2H-benzo[g]chromen-2-one[13], THF/H2O, 60°C.
Recently, we found that in CuAAC (short for Cu(I)-catalyzed
1,3-dipolar azide alkyne cycloaddition reaction) generated
fluoroionophores, the electronic conjugation of a N-phenylaza-
18-crown-6 and a 7-diethylaminocoumarin fluorophore through a
Results and Discussion
To study the Na+/K+ selectivity and the Na+-complex stability
in combination with the above-mentioned signal-transduction-
chain: anilino-triazole-coumarin, we introduced different 15-
membered crown ether derivatives as Na+-ionophores, which
are promising candidates for an exclusively binding of Na+ over
K+. Further, to investigate influences of flexibilities of aza-15-
crown-5 ionophores towards the fluorescence quenching in
these fluoroionophores and therefore for an effective sensing of
Na+ in CH3CN and in H2O, we synthesized fluorescent probes 1,
3, 4, 8 and 9. These fluorescent probes consisting of different
15-membered crown ionophores (cf. Scheme 1). The N-
phenylaza-15-crown-5 ionophore was introduced in 1.
1,2,3-triazol-1,4-diyl -linker results in
a
perfect signal
transduction chain for the sensing of K+ under simulated
physiological conditions, but not for physiological relevant K+
levels.[7a] Further, we attached in ortho position of the aniline unit
different alkoxy groups to enhance the K+ complex stability and
the K+/Na+ selectivity.[7b-d] In addition, we successfully
transferred the signal transduction chain to an exclusive Na+
sensing by introduction of a N-(o-methoxyphenyl)aza-15-crown-
5 ionophore as shown in fluoroionophore 2 (Scheme 1).[8]
Fluorescent probe 2 showed a high Na+/K+ selectivity when
embedded in a polymethacrylate hydrogel and a moderate
Na+/K+ selectivity under simulated physiological conditions.[8]
There, we reached a better Na+/K+ selectivity and a higher Na+-
induced fluorescence enhancement (FE), when the aniline donor
is connected to the position 4 of the triazole and the coumarin
acceptor to the position 1, as shown in fluorescent probe 2 (cf.
Scheme 1).
Fluorescent probes 2, 8, and
9 containing of a N-(o-
methoxyphenyl)aza-15-crown-5 ionophore. Further, 3 based on
a N,N’-bis(o-methoxyphenyl)diaza-15-crown-5 ionophore and 4
on a N-methylacetate-aza-15-benzo-crown-5 ionophore. For
comparison, we synthesized fluorescent probe 5 with a benzo-
15-crown-5, as the Na+-recognition unit. Further, to maintain the
above-mentioned signal transduction chain, we selected as
fluorophores, coumarin or -extended coumarin derivatives with
different fluorescence maxima max and fluorescence quantum
yields f in polar solvents. The introduction of the 2,3,6,7-
tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-
Herein, our superior goal is to synthesize easily accessible
and highly Na+/K+ selective fluorescent probes, which based on
a electron withdrawing 1,2,3-triazole unit to benefit of
selectivity advantage, as already shown for the highly K+/Na+
selective fluorescent probes[7a-d]
a
.
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one fluorophore (cf. Scheme 1, fluorescent probe 8) should
result in a slightly red-shifted max compared with the 7-
dietylaminocoumarin derivatives.[7c] A higher red-shifted max
should be observed with 9, where a -extended coumarin (8-
(dimethylamino)-2H-benzo[g]chromen-2-one) moiety is the
fluorophore (cf. Scheme 1).[9] Overall, we synthesized
fluorescent probes 1, 3, 4, 5, 8 and 9 with the same 1,4-triazole
linkage between the ionophore and the fluorophore, as shown in
fluorescent probe 2 (cf. Scheme 1), to take advantage of the
higher Na+/K+ selectivity and the higher Na+-induced
fluorescence enhancement (FE). Further, for comparison, we
synthesized the free aza-15-crown-5 fluorescent dye the 7-
(diethylamino)-3-(4-phenyl-1H-1,2,3-triazol-1-yl)-2H-chromen-2-
one (7) according to the literature[10] (see Scheme 1).
Syntheses of 1,2,3-triazol-1,4-diyl – fluoroionophores: To
built up 1, 3, 4, 5, 8 and 9, we synthesized according to the
literature the alkynes 10[11a], 13[11b] and 14[8] (cf. Scheme 1).
Further, alkynes 11 and 12 were synthesized from the
corresponding aldehydes[3b,5a] by using dimethyl-1-diazo-2-
oxopropylphosphonate.[12] Coupling of alkyne compounds 10-14
with the respective coumarin-azides[10,13] by using the well-
established CuAAC-reaction[14a,b], afforded fluoroionophores 1, 3,
4, 5, 8 and 9 (Scheme 1).
Spectroscopic properties of 1-9 in CH3CN: The UV/Vis
absorption spectra of fluoroionophores 1, 3, 4, 8 and 9 exhibit
two charge transfer (CT) bands in CH3CN (see Figures S1-S4,
S6 and S7), as already found for fluoroionophore 2.[8] The long-
wavelength CT absorption for the diethylaminocoumarin
derivatives 1-5 centered at around 410 nm and for 8 and 9 at
around 430 nm in CH3CN. The second CT absorption band,
which is typical for -conjugated anilino-1,2,3-triazol-1,4-diyl –
fluoroionophores, can be found for 1-4, 8 and 9 at around 290
nm in CH3CN corresponding to a CT from the nitrogen lone pair
of the aromatic ring (donor) to the 1,2,3-triazole acceptor unit.
Based on this band, it is possible to evaluate the conjugation of
the nitrogen lone pair with the aromatic ring. In the series of 1-4,
8 and 9, we found for 1 the most intense CT absorption at
around 290 nm (Figure 1a) with a extinction coefficient of 31000
Figure 1. UV/Vis absorption spectra of a) 1 (c = 5 10-5 M) and b) 2 (c = 5 10-5
M) in the presence of different Na+ concentrations in CH3CN.
Spectroscopic properties of 1-5, 8 and 9 + Na+ and K+ in
CH3CN: In the next step, we studied the influence of Na+ and K+
towards the UV/Vis absorption of 1-5, 8 and 9 in CH3CN. For 1-5,
8 and 9, we found no change of the long-wavelength absorption
in the presence of Na+ or K+ (cf. Figures S1a-S7a). In general for
1-4, 8 and 9 the complexation of Na+ and K+ can be seen from
the CT absorption at around 290 nm (see Figures S1a-S4a, S6a
and S7a). We observed for 1-4, 8 and 9 a decrease of the CT
band at 290 nm in the presence of Na+ and an increase of the
absorption at ~260 nm. However, for 1-4, 8 and 9 + K+, we
observed only a small change of the CT band (cf. Figure S1b-
S4b, S6b and S7b). The resulting UV/Vis absorption spectra of
1-4 + Na+ were similar to the anilino free reference compounds 5
and 7, respectively (cf. Figure S5a and S5b). Thus, Na+
decreases the donor ability of the anilino unit in 1-4, 8 and 9
which leads to a decrease of the CT absorption band. We found
for 1-4 + Na+ a different pronounced reduction of the CT band,
depending on the nature of the aza-15-crown-5 unit and on the
conjugation of the nitrogen lone pair with the aromatic ring. For 1,
we observed the strongest decrease of the CT band (see Figure
1a) and a smaller Na+-induced CT reduction at 290 nm for 2, 3
and 4, respectively (cf. Figures 1b, S3a and S4a). However, for
M
-1 cm-1. The fluoroionophores 2, 3, 4, 8 and 9 displays a CT
absorption at ~290 nm with a lower extinction coefficient (cf.
Figure 1a and 1b). The different aza-15-crown-5 units in 2, 3, 4,
8 and 9 are not so flexible as in 1, caused by the steric
hindrance of the substituent in ortho position as in 2, 3, 8 and 9
or by the directly in ortho position linked aza-15-crown-5 unit, as
shown in 4. As a consequence, in 2, 3, 4, 8 and 9 the C-N bond
between the aza-15-crown-5 unit and the aromatic ring is twisted
and this excludes a complete conjugation of the nitrogen lone
pair with the aromatic ring. Therefore, 1 posses a better electron
donor unit as 2, 3, 4, 8 and 9 caused by the more efficient
conjugation of the nitrogen lone pair with the aromatic ring.
Further, the reference compounds 5 and 7, without an anilino
unit, showed the typical coumarin CT absorption band at around
410 nm in CH3CN, but no CT absorption at around 290 nm (cf.
Figures S5a and S5b).
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2, 4, 8 and 9, we found at ~0.05 mM Na+ a complete decrease of
the short wavelength CT absorption and for 1 and 3 at a higher
Na+ concentration of ~5 mM. Thus, 2, 4, 8 and 9 formed more
stable Na+-complexes than 1 and 3 in CH3CN. Consequently,
the aza-15-crown-5 moiety and the alkoxy substituent in ortho
position were responsible for enhanced Na+-complex stabilities.
than that of the aza-15-crown-5-free fluorescent probe 7 (f =
0.702). The low f of probes 1-4 (cf. Table 1) suggests that a
photoinduced electron transfer (PET) takes place in 1-4, which
quenches the fluorescence, as already observed for the highly
K+/Na+ selective 1,2,3-triazol-fluoroionophores[7a-d]. There, we
investigated the influence of the isomeric triazole linkage
[7a,b]
towards the f.
Herein, the PET process is influenced by the
Table 1. Photophysical properties of 1-9 in CH3CN.[a]
ionophore moiety. In the series of 1-4, which are substituted with
the diethylaminocoumarin fluorophore, we observed following
trend of the f: 0.152 (4) > 0.130 (2) > 0.082 (3) > 0.014 (1), cf.
Table 1. As discussed vide supra, in 2, 3 and 4 the nitrogen lone
pair is not fully conjugated with the aromatic ring caused by the
steric hindrance of the substituent in ortho position as found for
2 and 3 or by the directly in ortho position linked aza-15-crown-5
unit, as shown in 4. In a more flexible arrangement, such as in 1,
the nitrogen lone pair of the anilino unit is mostly conjugated with
the phenyl ring and this acts as an efficient PET donor. Thus,
the anilino unit in 1 is a better PET donor than in 2, 3 and 4. In
addition to it, the low f value of 1 (0.014) is comparable with the
f value of the flexible diethylaminophenyl substituted 1,2,3-
triazol-coumarin derivative 6[7a] (0.017, see Scheme 1). In
contrast to 1, the benzo-15-crown-5 substituted fluoroionophore
5 exhibits a f value of 0.762, which is comparable with the f
value of the reference compound 7 (f = 0.702).
+
Na [d]
abs
f
f[b]
FEF[c]
Kd
[nm]
[nm]
[µM]
1
412
412
412
413
413
413
411
411
411
413
413
413
412
412
412
410
413
430
430
430
431
431
431
485
485
485
484
484
484
486
486
486
484
484
484
483
483
483
493
483
500
500
500
591
591
591
0.014
0.685
0.052
0.130
0.307
0.235
0.082
0.440
0.146
0.152
0.667
0.279
0.762
0.765
0.767
0.017
0.702
0.475
0.712
0.532
0.221
0.375
0.265
-
46.5
3.7
-
-
1 + Na+
1 + K+
2
867
1378
-
2 + Na+
2 + K+
3
2.2
1.4
-
5
16
-
3 + Na+
3 + K+
4
5.4
1.8
-
770
To substantiate the influence of the ionophore moieties
towards the fluorescence quenching in 1, 2 and 4, we measured
the redox properties of 1, 2, 4, 5 and 7 in CH3CN. The Rehm-
4454
-
12
33
-
Weller equation allows the estimation of the thermodynamic
4 + Na+
4 + K+
5
4.3
1.8
-
[15,16]
driving force for the PET process GPET
.
The
fluoroionophores 1, 2, 4, 5 and 7 have nearly the same reduction
potential ERed (-1.90 V) and the same energy difference between
the ground and the excited state E00 (2.77 eV), but they differ in
the oxidation potential EOx, caused by the different electron
donor capabilities of the ionophore moieties. We found for 1 a
EOx of 0.75 V, for 2 a 0.77 V and for 4 a 0.80 V, respectively.
Hence, we estimated for 1, 2 and 4 the following GPET values: -
0.22 eV, -0.20 eV and -0.17 eV, respectively. Herein, within 1, 2
5 + Na+
5 + K+
6
1.0
1.0
-
-
-
-
and
4 a reductive PET from the anilino donor to the
7
-
-
diethylaminocoumarin acceptor takes place, as indicated by
negative GPET values.[16] The more negative the GPET value,
the more likely the fluorescence quenching, as found in 1, 2 and
4 (cf. Table 1). In opposite, we found for 5 and 7 a EOx of 1.30 V
and 1.36 V, respectively. Therefore, we calculated positive
GPET values of +0.33 eV and +0.39 eV. Thus, in 5 (f = 0.762)
and 7 (f= 0.702) no reductive PET can take place, which
quenches the fluorescence. Hence, in this series 1-5, the
fluorescence is effectively quenched within 1.
8
-
-
8 + Na+
8 + K+
9
1.5
1.1
-
6
33
-
9 + Na+
9 + K+
1.6
1.2
7
15
Further, we observed in CH3CN a red-shift of max for 8 and 9
compared with the diethylaminocoumarin derivatives 1-4. The
[a] All data were measured in CH3CN in the absence and presence of
NaPF6 or KPF6, respectively. [b] Fluorescence quantum yield, (±
15)%. [c] Fluorescence enhancement factor, [FEF = I/I0], (± 0.2). [d]
Dissociation constants Kd for Na+-complexes.

max of 8 was found at 500 nm and of 9 at 591 nm, respectively.
Herein, the N-(o-methoxyphenyl)aza-15-crown-5 substituted
fluoroionophores 8 (f = 0.475) and 9 (f = 0.221) showed, as
also found for 2, high f values. This result confirms the influence
of the bulky methoxy group in ortho position towards the
fluorescence quenching process in 2, 8 and 9, as discussed vide
supra.
The fluorescence spectra of 1-7 showed emissions centered
at around 485 nm in CH3CN (cf. Table 1). Thus, the fluorophore
moiety adjusted max, but we found for probes 1-7 different
quantum yields f (cf. Table 1). Overall, the f of 1-4 were lower
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To validate the Na+ sensing capabilities of 1-5, 8 and 9 we
investigated the influence of Na+ and K+ on the fluorescence
intensity of 1-5, 8 and 9 in CH3CN. Initially, we performed
titration experiments with 1-5, 8 and 9 only in the presence of
Na+. Overall, for 1-4, 8 and 9 we observed a FE in the presence
of Na+ (representative for 1-4, 8 and 9 see Figure 2), but we
found for 5 + Na+ no change of the fluorescence intensity. In
summary, Figure 3a shows the titration curves of 1, 3 and 5 +
Na+ and Figure 3b of 2, 4, 8 and 9 + Na+ at max nm in CH3CN.
Herein, 1 showed the highest Na+-induced FE by a factor of 46.5.
The maximal FE of the N-phenylaza-15-crown-5 substituted
fluoroionophores 1 was reached in the presence of 5 mM Na+ (cf.
Figure 3a). For 1 + Na+, we determined a high f value of 0.685
(cf. Table 1). As mentioned above, the fluoroionophores 2, 3, 4,
8 and 9 exhibited higher f values as 1. Overall, the f values of
2, 3, 4, 8 and 9 + Na+ were higher than of the Na+-free solutions
(cf. Table 1). However, we observed smaller Na+-induced FE
factors for 2 (FEF = 2.2), 3 (FEF = 5.4), 4 (FEF = 4.3), 8 (FEF =
1.5) and 9 (FEF = 1.6). The maximal FE was reached for 2, 4, 8
and 9 at a Na+ concentration of 0.05 mM (see Figure 3b), but for
3 only at 5 mM Na+ (Figure 3a). The Na+ induced FEs of 1-4, 8
and 9 can be explained by an off switching of the PET
quenching process by Na+. We assume, that Na+ enhances the
oxidation potential of the ionophore moiety, as already observed
six donor atoms including five O-atoms and one N-atom. Thus,
the bulky methoxy group in 2, 8 and 9 prevents as mentioned
above a complete fluorescence quenching, but enhances the
Na+-complex stability. Noticeable, the diaza-15-crown-5 moiety
in 3 binds Na+, as well as the monoaza-15-crown-5 moiety in 1,
suggesting that the methoxy groups in ortho position were not
involved in the coordination sphere with Na+ in CH3CN. Overall,
the Na+-complex stability is influenced by the ionophore moiety
in CH3CN.
for several
-conjugated anilino-1,2,3-triazol-1,4-diyl
–
fluoroionophores for K+[7a-d] and
a
set of aza-15-crown-5
substituted Na+ fluoroionophores.[3c,4a,b,5c] Therefore, the GPET
value becomes more positive and the PET is more unlikely. This
results in an on switching of the fluorescence.
Figure 3. Fluorescence enhancement factors in the presence of Na+ for a) 1, 3,
and 5 and b) for 2, 4, 8 and 9 at max
.
In the next step, we investigated the influence of competing K+
ions on the fluorescence intensity of 1-4 in CH3CN. In all cases,
K+ enhances the fluorescence intensity of 1-4, but much weaker
than for Na+ (cf. Table 1 and Figure 4). As expected, 5 + K+
showed no change of the fluorescence.
Figure 2. Fluorescence spectra of 1 (c = 5 10-6 M, ex = 410 nm) in the
presence of different Na+ concentrations in CH3CN.
Overall, as required for an effective Na+ sensing, 1 showed a
moderate Na+/K+ selectivity, a high Na+ induced FE, a high f of
the Na+-complex and a Kd value of 867 µM in CH3CN.
To compare the Na+-complex stabilities of 1-4, 8 and 9 in
CH3CN, we determined the Kd values based on the Na+-induced
FEs.[13] The resulting Kd values of 1-4, 8 and 9 + Na+ can divided
into two groups. On the one hand, 1 and 3 + Na+ possess Kd
values of 867 µM and 770 µM and on the other hand 2, 3, 8 and
9 + Na+ showed Kd values of 5 µM, 12 µM, 6 µM and 7 µM,
respectively (cf. Table 1). Thus, we found for 2, 8 and 9 + Na+
the most stable Na+-complexes. In these Na+-complexes, the
aza-15-crown-5 is orientated orthogonal to the phenyl ring into
the direction of the methoxy group.[4a,b] Here, Na+ is bound via
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for 4, 8 and 9, in the presence of increasing Na+ concentrations.
Figure 6 diagrammed the titration curves of 4, 8 and 9 with Na+
at max, respectively. In all cases, we observed a Na+-induced FE,
but the maximal FE was reached at different Na+ concentrations.
Thus, for 4 at 500 mM NaCl with a FEF of 6.6, for 8 at 500 mM
NaCl with a FEF of 10.9 and for 9 at 1000 mM NaCl with a FEF
of 2.0. Further, we carried out titration experiments with 4, 8 and
9 in the presence of KCl. As expected, K+ showed a smaller
influence on the fluorescence of 4, 8 and 9. We observed for 4 +
500 mM K+ a FEF of 3.4, for 8 + 500 mM K+ a FEF of 2.7 and for
9 + 1000 mM K+ a FEF of 1.1. We found for 9 the highest Na+/K+
selectivity and for 4 a poor Na+/K+ selectivity.
Figure 4. Fluorescence enhancement factors of 1-5, 8 and 9 (black bars) at
max in the presence of maximal K+ (grey bars) and Na+ (light grey bars)
concentration (cf. Figure 3) in CH3CN.
Spectroscopic properties of 4, 8 and 9 + Na+ and + K+ in
H2O/DMSO (v/v 99/1) mixture: To ensure the solubility of 1, 3, 4,
8 and 9 in an aqueous milieu, we prepared solutions with
different fluoroionophore concentrations in the range of 510-5
M
to 10-6 M in a H2O/DMSO mixture of 99/1 (v/v). Herein, we only
found for 4, 8 and 9 a complete solubility in a H2O/DMSO
mixture of 99/1 (v/v) up to a fluoroionophore concentration of
~1.510-5 M (cf. Figures S22a-c). However, the fluorescent
probes 1 and 3 showed in a concentration range from 510-5
M
Figure 5. Fluorescence spectra of 8 (c = 10-5 M, ex = 440 nm) in the presence
of different Na+ concentrations in a H2O/DMSO (99/1 v/v) mixture with 10 mM
Tris buffer (pH = 7.2).
to 10-6 M no adequate solubility in various H2O/DMSO mixtures
(99/1, 9/1, 3/1 or 1/1 (v/v)).[13] Thus, the titration experiments
with NaCl and KCl were carried out only with 4, 8 and 9 (c = 10-5
M) under simulated physiological conditions (10 mM Tris-buffer,
pH 7.2, H2O/DMSO (v/v 99/1)). The fluorescence emission of the
diethylaminocoumarin substituted fluoroionophore 4 centered,
as also found for 2, at 500 nm. A slightly red-shifted max was
observed for 8 at 517 nm and a higher red-shifted max showed 9
at 613 nm. However, the f of 2 (f = 0.009) and 4 (f = 0.014)
were similar to each other. Herein, the fluorescence quenching
within the diethylaminocoumarin substituted fluoroionophores 2
and 4 is not only influenced by the ionophore moiety and
therefore by the PET process, as mentioned above in CH3CN,
but rather by H2O. H2O itself quenches the fluorescence of the
diethylaminocoumarin fluorophore, as already found for
diethylaminocoumarin derivatives[17] and for the reference
compound 7 (f = 0.106, cf. Table 1 and 2). Likewise, H2O
reduces also the f of 7-(diethylamino)-3-(4-phenyl-1H-1,2,3-
triazol-1-yl)-2H-chromen-2-one derivatives.[9] Thus, we found for
9 a very low f of 0.002. Furthermore, a higher f value of 0.025
Figure 6. Fluorescence enhancement factors of 4, 8, and 9 in the presence of
Na+ concentrations in a H2O/DMSO (99/1 v/v) mixture with 10 mM Tris buffer
(pH = 7.2).
showed
8
containing of
a
2,3,6,7-tetrahydro-1H,5H,11H-
pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one fluorophore. These
coumarin derivatives exhibited even in H2O high f values up to
0.95.[18] Concluding that in 8, the fluorescence is effectively
quenched by a PET process, as required for an effective Na+
sensing.
In the next step, we carried out titration experiments with 4, 8
and 9 in the presence of NaCl under simulated physiological
conditions. Figure 5 depicts the fluorescence of 8, representative
Again, the high Na+/K+ selectivity of 9 can be also seen from
the UV/Vis absorption spectra of 9 + Na+ and K+ in H2O. Here,
only Na+ reduces the CT band of 9 at around 300 nm (Figure
S8c). In contrast to 9 + K+, 4 and 8 showed also a K+-reduced
CT absorption at around 300 nm (cf. Figures S8a and S8b).
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Dissociation constants (Kd) of fluorescent probes 4 + Na+, 8 +
Na+ and 9 + Na+ were calculated from plots of fluorescence
intensities of 2, 8 and 9 versus Na+ ion concentrations.[13] In all
cases, we observed a 1:1 complexation between 4, 8 and 9 with
Na+, indicated by the slopes (~1) of the plots.[13] For 4 + Na+, 8 +
Na+ and 9 + Na+, we found Kd values of 155 mM, 48 mM and
223 mM, respectively (cf. Table 2). Herein, 4 and 2 consisting of
different Na+ ionophores, but they showed the nearest Kd values
(2 + Na+, Kd = 117 mM, 4 + Na+, Kd = 155 mM). For a set of
fluoroionophores, which were all equipped with the N-
methylacetate-aza-15-benzo-crown-5 ionophore (cf. 4), but with
different fluorophore moieties, were found a wide range of Kd
values for Na+, which differed from 89 mM to 226 mM.[5a]
Furthermore, the N-(o-methoxyphenyl)aza-15-crown-5 based
fluoroionophores 2, 7 and 9 showed also different Kd values for
Na+ in the following order 9 > 2 > 8.
Interestingly, we found that with an increased Na+-complex
stability in the following order: 8 (48 mM) > 2 (117 mM) > 9 (223
mM), the Na+ induced FE of 8 (FEF = 10.9) > 2 (FEF = 5.0) > 9
(FEF = 2.0) is also increased. Further, 8 formed the strongest
Na+-complex, but also complexes to K+ (FEF = 2.7). A negligible
K+ influence showed fluorescent probe 9 but also a smaller Na+
induced FE. Thus, the Na+/K+ selectivity is decreased in the
order 9 < 2 < 8.
Again, we tested the suitability of 8 to measure Na+ in the
presence of physiological relevant K+ levels. Thus, we prepared
different Na+ solutions, containing 10 mM KCl or 135 mM KCl to
simulate the extra- or intracellular K+ background, respectively.
As found for 8 + Na+ the Kd value in K+-free solutions is 48 mM.
Herein, the Kd value of 8 + Na+ in the presence of 10 mM K+ (Kd
= 45 mM) or 135 mM K+ (Kd = 42 mM) is slightly influenced by K+
(see Figure S18a and S19a). However, we observed different
Na+-induced FEs, depending on the adjusted K+ concentration.
For 8 + Na+ in the presence of 10 mM K+, we found a FEF of
10.2 and in the presence of 135 mM a FEF of 5.5 (Figure S18b
and S19b). Thus the performance of 8 is only slightly influenced
by extracellular K+ levels.
Figure 7. Fluorescence intensities changes of a) 8 (c = 10-5 M, ex = 440 nm,
10 mM Tris buffer, pH = 7.2) at 517 nm and b) 9 (c = 10-5 M, ex = 420 nm, 10
mM Tris buffer, pH = 7.2) at 613 nm in the presence of 10 mM for K+, 2 mM for
Mg2+, Ca2+, Li+, Rb+, Cs+ and 100 µM for Ni2+, Cu2+, Zn2+ (black bars) and
subsequent addition of 135 mM of Na+ (grey bars).
To verify the pH sensitivity of 8 and 9, we measured the
fluorescence at different pH values.[13] Thus, we found only
minor effects towards the sensitivity of 8 or 9 at various
concentrations of Na+ ions within the pH range from 6.8 to 8.8
(see Figures S21a and S21b).
Table 1. Photophysical properties of 2, 4, 7, 8 and 9 in H2O.[a]
The Na+-selectivity of fluorescent probes 8 and 9 was verified
by titration experiments with other metal ions. Particularly, we
focused on biological cations such as K+, Ca2+, Mg2+, Li+, Ni2+,
Cu2+ and Zn2+ at their physiological concentrations (Figure 7a for
8 and 6b for 9). These cations show only negligible effects on
the fluorescence of 8 and 9. Moreover, 8 and 9 recognized Na+
even in the co-presence of these metal ions, as shown in Figure
7a and 7b. Therefore, 8 and 9 were capable fluorescent probes
to determine extracellular Na+ levels in the presence of other
biological important cation concentrations.
[d]
abs
f
f[b]
FEF[c]
Kd
[nm]
[nm]
[mM]
2[e]
422
422
422
423
423
423
424
440
440
440
500
500
500
500
500
500
501
517
517
517
0.009
0.048
0.017
0.014
0.093
0.048
0.106
0.025
0.275
0.067
-
5.0
1.5
-
-
117
276
-
2 + Na+
2 + K+
4
4 + Na+
4 + K+
7
6.6
3.4
-
155
270
-
8
-
-
8 + Na+
8 + K+
10.9
2.7
48
68
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General synthetic procedures for 11 and 12: To a mixture of the
corresponding aldehyde derivatives (N,N’-bis(o-methoxyphenyl)diaza-15-
9
418
418
418
613
613
613
0.002
0.004
0.002
-
-
crown-5-aldehyde[3b]
or
N-methylacetate-aza-15-benzo-crown-5-
9 + Na+
9 + K+
2.0
1.1
223
aldehyde[5a]) (2.43 mmol) and 673 mg (4.86 mmol) K2CO3 in 35 mL dry
methanol was added 564 mg (2.93 mmol) dimethyl-1-diazo-2-
oxopropylphosphonate[12]. This suspension was stirred for 20 hours at
room temperature. After addition of 60 mL CHCl3, the organic layer was
extracted with water (10 mL), separated, dried with MgSO4 and
concentrated in vacuo. The resulting residue was purified by column
chromatography on silica with CHCl3/CH3OH (v/v, 95/5) as an eluent
mixture to afford 11 or 12 as pale oils.
[f]
-
[a] All data were measured in H2O/DMSO mixtures (v/v, 99/1) in 10
mM Tris buffer (pH = 7.2) and in the absence and presence of 1 M
NaCl or 1 M KCl. [b] Fluorescence quantum yield, (± 15)%. [c]
Fluorescence enhancement factor, [FEF
= I/I0], (± 0.2). [d]
Dissociation constants Kd for Na+- or K+- complexes. [e] See
reference [8] for 2. [f] The fluorescence intensity changes were too
weak to determine the Kd value of 9 + K+ accurately.
N-(4-Ethynyl-2-methoxyphenyl)-N’-(2-
methoxyphenyl)diaza[15]crown-5 ether (11): Yield 37% (408 mg); 1H
NMR (CDCl3, 300 MHz): δ = 7.05-6.81 (m, 7H), 3.81 (s, 3H), 3.79 (s, 3H),
3.63-3.38 (m, 20H), 3.02 ppm (s, 1H); 13C NMR (CDCl3, 75 MHz): δ =
153.19, 151.78, 140.35, 139.05, 125.21, 122.55, 121.51, 120.64, 119.87,
115.28, 114.46, 111.82, 84.14, 75.71, 70.40, 70.34, 69.54, 69.50, 69.45,
69.35, 55.44, 55.33, 52.73, 52.60, 52.45, 52.33 ppm; HRMS (ESI): m/z
calcd for C26H34N2O5+H+: 455.2546 [M+H+]; found: 455.1314.
Conclusions
In summary, we have synthesized a set of Na+-responsive
fluoroionophores 1, 3, 4, 5, 8 and 9. At first, we found that the
flexibility of the aza-15-crown-5 moiety influences obviously the
fluorescence quenching process within 1-4, 8 and 9 in CH3CN.
Particularly, the conjugation of the nitrogen lone pair with the
aromatic ring determines the donor ability of the ionophore
moiety and therefore the PET efficiency within 1-4, 8 and 9.
Herein, 1 exhibited a low f of 0.014 in CH3CN, as required for
an effective Na+ sensing. Thus, 1 showed the highest Na+-
induced FEF of 44.5 and 1 + Na+ exhibited a Kd value of 867 µM
in CH3CN. However, the most stable Na+-complexes formed the
N-(o-methoxyphenyl)aza-15-crown-5 based fluoroionophores 2,
8 and 9 with Kd values of 5 µM, 6 µM and 7 µM in CH3CN.
Herein in CH3CN, the Na+-complex stability is influenced by the
ionophore moiety. Secondly, the N-(o-methoxyphenyl)aza-15-
crown-5 based fluoroionophores 2, 8 and 9 showed different Kd
values and Na+/K+ selectivities in H2O. The most stable Na+-
complex formed 8 + Na+ with a Kd value of 48 mM, but showed a
small K+ induced FE at intracellular K+ concentrations. A higher
Na+/K+ selectivity exhibited fluorescent probe 9, but also a higher
Kd value of 223 mM. Thus, fluorescent probe 8 is a suitable tool
to monitor selective physiological Na+ levels in the presence of
extracellular K+ concentrations. Further, 9 is a highly Na+/K+
selective fluoroionophore capable to visualize extracellular Na+
concentrations. Overall, we have shown that not only the N-(o-
methoxyphenyl)aza-15-crown-5 ionophore specified the Na+-
complex stability and Na+/K+ selectivity, but also the fluorophore
moiety influences the Na+ binding properties.
N-Methylacetate-aza-4-ethynylbenzo[15]crown-5 ether (12): Yield
45% (396 mg); 1H NMR (CDCl3, 300 MHz): δ = 7.02 (dd, J = 8.2, 1.4 Hz,
1H), 6.92 (d, J = 1.4 Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H), 4.15 (s, 2H), 4.13-
4.10 (m, 2H), 3.88-3.86 (m, 2H), 3.78 (t, J = 5.9 Hz, 2H), 3.72-3.60 (m,
11H), 3.50 (t, J = 5.9 Hz, 2H), 2.99 ppm (s, 1H); 13C NMR (CDCl3, 75
MHz): δ = 171.99, 150.61, 140.84, 125.59, 119.40, 116.62, 114.89, 84.05,
75.71, 70.89, 70.62, 70.26, 70.09, 69.54, 69.09, 68.50, 53.21, 52.10,
51.55 ppm; HRMS (EI): m/z calcd for C19H25NO6: 363.1682 [M+]; found:
363.1644.
General CuACC procedure for 1, 3, 4, 5, 8 and 9: A mixture of the
corresponding alkynyl substituted crown compound 10[11a], 11, 12, 13[11b]
or 14[8] (0.415 mmol) and the corresponding azidocoumarin
derivatives[10](0.415 mmol) or the -extended azidocoumarin derivative[13]
(0.415 mmol), CuSO4·5H2O (5.3 mg) and sodium ascorbate (8.2 mg) in 9
ml THF/H2O (v/v, 2/1) was stirred at 60 °C for 48 hours. After that 5 mL
H2O were added to the mixture and then extracted with CHCl3 (30 mL).
The organic layer was dried with MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography on silica using
CHCl3/CH3OH (v/v, 99/5) as an eluent mixture to afford 1, 3-5 as yellow
solids, 8 as an orange solid and 9 as a red solid.
3-(4-(4-(1,4,7,10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl)-1H-
1,2,3-triazol-1-yl)-7-(diethylamino)-2H-chromen-2-one (1): Yield 20%
(48 mg); M.p.: 168°C (decomp.); 1H-NMR (CDCl3, 300 MHz): δ = 8.65 (s,
1H), 8.42 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.9 Hz, 1H), 6.77-
6.64 (m, 3H), 6.56 (d, J = 2.0 Hz, 1H), 3.78 (d, J = 5.5 Hz, 4H), 3.66 (q, J
= 9.4 Hz, 16H), 3.45 (q, J = 7.0 Hz, 4H), 1.24 ppm (t, J = 7.0 Hz, 6H);
13C-NMR (CDCl3, 75 MHz): δ = 156.96, 155.68, 151.39, 148.02, 147.55,
134.16, 129.89, 127.01, 118.71, 118.05, 117.20, 111.59, 109.98, 107.21,
97.02, 71.30, 70.22, 70.17, 68.56, 52.54, 44.95, 12.42 ppm; HRMS (ESI):
m/z calcd for C31H39N5O6+H+: 578.2979 [M+H+]; found: 578.2933.
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 or 500 MHz
instruments, respectively. Data are reported as follows: chemical shifts in
ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of
doublets, m = multiplet), integration, coupling constant (Hz). ESI spectra
were recorded using a Micromass Q-TOF micro mass spectrometer in a
positive electrospray mode. Column chromatography was performed with
silica gel (Merck; silica gel 60 (0.04-0.063 mesh)).
7-(diethylamino)-3-(4-(3-methoxy-4-(13-(2-methoxyphenyl)-1,4,10-
trioxa-7,13-diazacyclopentadecan-7-yl)phenyl)-1H-1,2,3-triazol-1-yl)-
2H-chromen-2-one (3): Yield 75% (221 mg); M.p.: 45°C (decomp.); 1H
NMR (CDCl3, 500 MHz): δ = 8.70 (s, 1H), 8.38 (d, J = 3.1 Hz, 1H), 7.45-
7.30 (m, 3H), 7.08-6.80 (m, 5H), 6.64-6.61 (m, 1H), 6.53-6.50 (m, 1H),
3.90-3.38 (m, 30H), 1.20 ppm (t, J = 6.9 Hz, 6H); 13C NMR (CDCl3, 125
MHz): δ = 156.78, 155.59, 153.07, 152.94, 151.33, 147.50, 134.26,
134.21, 129.85, 121.35, 120.90, 120.58, 119.57, 118.19, 116.83, 111.70,
109.89, 109.86, 109.17, 106.96, 96.81, 75.03, 74.83, 70.43, 70.34, 70.28,
69.42, 55.51, 55.27, 52.64, 52.59, 52.50, 52.44, 44.83, 17.13, 12.29
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ppm; HRMS (ESI): m/z calcd for C39H48N6O7+H+: 713.8560 [M+H+];
found: 713.8563.
[3]
a) H. Szmacinski, J. R. Lakowicz, Anal. Biochem. 1997, 250, 131-138;
b) A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, M.
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2-(16-(1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-triazol-4-
yl)-2,3,5,6,8,9,11,12-octahydro-13H-
benzo[k][1,4,7,10]tetraoxa[13]azacyclopentadecin-13-
yl)methylacetate (4): Yield 38% (98 mg); M.p.: 163°C (decomp.); 1H
NMR (CDCl3, 300 MHz): δ = 8.70 (s, 1H), 8.41 (s, 1H), 7.45-7.33 (m, 3H),
6.98 (d, J = 7.5 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 6.54 (s, 1H), 4.26-3.53
(m, 21H), 3.48-3.39 (m, 4H), 1.22 ppm (t, J = 6.8 Hz, 6H); 13C NMR
(CDCl3, 75 MHz): δ = 172.18, 156.89, 155.67, 151.73, 151.43, 147.47,
139.51, 134.32, 129.93, 124.25, 120.51, 119.63, 118.65, 116.86, 110.54,
110.00, 107.05, 96.91, 70.81, 70.63, 70.24, 70.10, 69.62, 68.99, 68.50,
53.48, 52.26, 51.52, 44.94, 12.38 ppm; HRMS (ESI): m/z calcd for
C32H39N5O8+H+: 622.2877 [M+H+]; found: 622.2842.
[4]
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7-(diethylamino)-3-(4-(2,3,5,6,8,9,11,12-
octahydrobenzo[b][1,4,7,10,13]pentaoxacyclopentadecin-15-yl)-1H-
1,2,3-triazol-1-yl)-2H-chromen-2-one (5): Yield 32% (73 mg); M.p.:
184°C (decomp.); 1H-NMR (CDCl3, 300 MHz): δ = 8.72 (s, 1H), 8.42 (s,
1H), 7.53-7.35 (m, 3H), 6.92 (d, J = 8.3 Hz, 1H), 6.66 (dd, J = 8.9, 2.3 Hz,
1H), 6.54 (d, J = 2.1 Hz, 1H), 4.27-4.14 (m, 4H), 3.99-3.89 (m, 4H), 3.75
(s, 8H), 3.44 (q, J = 7.0 Hz, 4H), 1.22 ppm (t, J = 7.0 Hz, 6H); 13C-NMR
(CDCl3, 75 MHz): δ = 156.91, 155.69, 151.45, 149.32, 149.11, 147.38,
134.35, 129.94, 123.81, 119.68, 118.77, 116.85, 114.05, 111.36, 110.03,
107.07, 96.94, 71.10, 71.07, 70.46, 70.43, 69.55, 69.51, 69.01, 68.93,
44.95, 12.38 ppm; HRMS (ESI): m/z calcd for C29H34N4O7+H+: 551.2506
[M+H+]; found: 551.2507.
[6]
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a) P. Nandhikonda, M. P. Begaye, M. D. Heagy, Tetrahedron Lett. 2009,
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D. Kim, Q. P. Xuan, H. Moon, Y. W. Jun, K. H. Ahn, Asian J. Org.
Chem. 2014, 3, 1089-1096.
10-(4-(4-(1,4,7,10-tetraoxa-13-azacyclopentadecan-13-yl)-3-
methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2,3,6,7-tetrahydro-1H,5H,11H-
pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (8): Yield 20% (52 mg);
M.p.: 126°C (decomp.); 1H NMR (CDCl3, 300 MHz): δ = 8.72 (s, 1H),
8.32 (s, 1H), 7.47 (s, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 7.8 Hz,
1H), 6.99 (s, 1H), 3.94 (s, 3H), 3.80-3.33 (m, 20H), 3.32-3.28 (m, 4H),
2.90 (t, J = 6.4 Hz, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.02-1.92 ppm (m, 4H);
13C NMR (CDCl3, 75 MHz): δ = 157.04, 150.69, 147.19, 146.88, 134.65,
130.80, 125.84, 119.83, 119.72, 118.48, 118.40, 115.80, 115.39, 109.20,
109.10, 106.83, 106.06, 70.39, 70.18, 69.95, 69.88, 69.75, 55.65, 49.95,
49.53, 27.34, 21.02, 20.12, 20.08 ppm; HRMS (ESI): m/z calcd for
C34H41N5O7+H+: 632.3084 [M+H+]; found: 632.3058.
[10] K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill, Q. Wang,
Org. Lett. 2004, 6 (24), 4603-4606.
[11] a) J. D. Lewis, J. N. Moore, Dalton Trans. 2004, 1376-1385; b) L.-H.
Lee, V. Lynch, R. J. Lagow, J. Chem. Soc., Perkin Trans. 1 2000, 2805-
2809.
[12] J. Pietruszka, A. Witt, Synthesis 2006, 24, 4266-4268.
[13] For the synthesis of 3-azido-8-(dimethylamino)-2H-benzo[g]chromen-2-
one and for the determination of the Kd values see supporting
information.
[14] a) V. V. Rostovtsev, L. G. Green, V. V. Folkin, K. B. Sharpless, Angew.
Chem. Int. Ed. 2002, 41, 2596-2599; b) C. W. Tornoe, C. Christensen,
M. Meldal, J. Org. Chem. 2002, 67, 3057-3062.
3-(4-(4-(1,4,7,10-tetraoxa-13-azacyclopentadecan-13-yl)-3-
methoxyphenyl)-1H-1,2,3-triazol-1-yl)-8-(dimethylamino)-2H-
benzo[g]chromen-2-one (9): Yield 33% (86 mg); M.p.: 135°C
(decomp.); 1H-NMR (CDCl3, 300 MHz): δ = 8.78 (s, 1H), 8.58 (s, 1H),
7.94 (s, 1H), 7.83-7.75 (m, 1H), 7.50-7.32 (m, 3H), 7.24-7.21 (m, 1H),
7.13 (d, J = 8.9 Hz, 1H), 6.79 (s, 1H), 4.05-3.38 (m, 23H), 3.11 ppm (s,
6H); 13C-NMR (CDCl3, 125 MHz): δ = 156.57, 150.26, 149.86, 145.55,
137.40, 132.44, 129.85, 129.51, 123.87, 123.85, 120.35, 118.85, 118.62,
116.39, 113.18, 109.92, 109.50, 109.41, 109.30, 106.13, 103.95, 70.70,
70.50, 70.41, 70.31, 56.21, 55.77, 40.21 ppm; HRMS (ESI): m/z calcd for
C34H39N5O7+H+: 630.2928 [M+H+]; found: 630.2917.
[15] D. Rehm, A. Weller, Isr. J. Chem. 1970, 8, 259-271.
[16] For 1, 2 and 4 a reductive PET process from the ionophore to the
fluorophore has different GPET values according to the Rehm-Weller
[15]
equation GPET = Eox - Ered - E00 - Gion pair
.
The oxidation potential
Eox of 1 is 0.75 V, of 2 is 0.77 V, of 4 is 0.80 V, of 5 is 1.30 V and of 7 is
1.37 V, respectively. The reduction potential of 1, 2, 4, 5 and 7 Ered is –
1.90 V (Fc/Fc+ = 0.09 V in MeCN). In MeCN the E00 for 1, 2, 4, 5 and 7
ˆ

2.77 eV). The ion-pairing energy Gion pair is ca.
is found at 448 nm (
0.1 eV under our conditions. Finally: GPET (1) = -0.22 eV, GPET (2) = -
0.20 eV, GPET (4) = -0.17 eV,GPET (5) = +0.33 eV, GPET (7) = +0.39
eV.
Keywords: sodium • potassium • fluorescence • fluorescent
probes • crown compounds
[17] T. Lopez Arbeloa, F. Lopez Arbeloa, M. J. Tapia, I. Lopez Arbeloa, J.
Phys. Chem. 1993, 97, 4704-4707.
[18] G. Jones II, M. A. Rahman, J. Phy. Chem. 1994, 98, 13028-13037.
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Bers, W. H. Barry, S. Despa, Cardiovasc. Res. 2003, 57, 897-912.
A. Minta, R. Y. Tsien, J. Biol. Chem. 1989, 264, 19449-19457.
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10.1002/chem.201605986
Chemistry - A European Journal
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Thomas Schwarze, Holger Müller, Darya
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Design of Na+-Selective Fluorescent
Probes – A Systematic Study of the
Na+-Complex Stability and the Na+/K+
Selectivity in Acetonitrile and Water
Herein, we report on the development of Na+-selective fluorescent probes, which
containing of 15-membered crown ether ionophores and of coumarin or -extended
coumarin derivatives, as fluorophores. These fluoroionophores show different Na+-
complex stabilities and Na+/K+ selectivities in acetonitrile and water.
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