A Ratiometric Fluorescent Probe for K+ in Water Based on a Phenylaza-18-Crown-6 Lariat Ether

Article abstract of DOI:10.1002/chem.201802306

This work presents two molecular fluorescent probes 1 and 2 for the selective determination of physiologically relevant K⁺ levels in water based on a highly K⁺/Na⁺ selective building block, the o-(2-methoxyethoxy)phenylaza-18-crown-6 lariat ether unit. Fluorescent probe 1 showed a high K⁺-induced fluorescence enhancement (FE) by a factor of 7.7 of the anthracenic emission and a dissociation constant (K_d) value of 38 mm in water. Further, for 2+K⁺, we observed a dual emission behavior at 405 and 505 nm. K⁺ increases the fluorescence intensity of 2 at 405 nm by a factor of approximately 4.6 and K⁺ decreases the fluorescence intensity at 505 nm by a factor of about 4.8. Fluorescent probe 2+K⁺ exhibited a K_d value of approximately 8 mm in Na⁺-free solutions and in combined K⁺/Na⁺ solution a similar K_d value of about 9 mm was found, reflecting the high K⁺/Na⁺ selectivity of 2 in water. Therefore, 2 is a promising fluorescent tool to measure ratiometrically and selectively physiologically relevant K⁺ levels.

Full text of DOI:10.1002/chem.201802306

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Accepted Article
Title: A Ratiometric Fluorescent Probe for K+ in Water based on a
Phenylaza-18-Crown-6 Lariat Ether
Authors: Hans-Jürgen Holdt and Thomas Schwarze
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A Ratiometric Fluorescent Probe for K+ in Water based on a
Phenylaza-18-Crown-6 Lariat Ether
Thomas Schwarze,*[a] Janine Riemer[a] and Hans-Jürgen Holdt*[a]
Abstract: Herein, we present two molecular fluorescent probes 1 and
2 (Scheme 1) for the selective determination of physiologically
relevant K+ levels in water based on a highly K+/Na+ building block the
very sensitive to cellular dye concentration changes. Further,
fluoroionophores without a spacer in a donor -conjugated
acceptor arrangement show cation induced spectral shifts of their
wavelengths and small intensity changes, caused by the
modulation of the intramolecular charge transfer (ICT). A typical
representative for an ICT-type fluoroionophore is the ratiometric
fluorescent probe fura-2 introduced by Tsien et al..[3] Fura-2 is the
most powerful tool to measure physiologically relevant Ca2+ levels
in vivo. The determination of intracellular Ca2+ takes place by the
Ca2+-induced change of two wavelengths in the excitation
spectrum of fura-2. This method allows a fast and a more precise
Ca2+ determination in vivo than by the use of single wavelength
fluorescent dyes. A benefit of ratiometric metal ion detection in
vivo is that this procedure reduces or eliminates distortions
caused by photobleaching effects, indicator concentration
changes or illumination stabilities.
o-(2-methoxyethoxy)phenylaza-18-crown-6
lariat
ether
unit.
Fluorescent probe showed
1
a
high K+-induced fluorescence
enhancement (FE) by a factor of 7.7 of the anthracenic emission and
a dissociation constant (Kd) value of 38 mM in water. Further, for 2 +
K+, we observed a dual emission behavior at 405 nm and 505 nm. K+
increases the fluorescence intensity of 2 at 405 nm by a factor of ~4.6
and K+ decreases the fluorescence intensity at 505 nm by a factor of
~4.8. Fluorescent probe 2 + K+ exhibited a Kd value of ~8 mM in Na+
free solutions and in combined K+/Na+ solution a similar Kd value of
~9 mM, reflecting the high K+/Na+ selectivity of 2 in water. Therefore,
2 is a promising fluorescent tool to measure ratiometrically and
selectively physiologically relevant K+ levels.
So far, for the detection of cellular K+ levels, Minta and Tsien
designed in an ICT-type architecture the first fluorescent probe
PBFI (potassium-binding benzofuranisophthalate) with a similar
signal readout as fura-2. However, K+ induces only small changes
in the excitation spectra of PBFI at two wavelengths.[4] In addition
to it, PBFI lacks an approbate K+/Na+ selectivity to track
exclusively K+ levels in vivo. Generally, only a few molecular
fluorescent probes exist, which show a K+-dependent dual
emission behavior.[5a-c] As a rare example, Boens et al. reported
on a highly K+ selective ratiometric fluorescent indicator, which
was only tested in pure acetonitrile.[5a] Teramae et al. showed a
Introduction
Fluorescent probes for biological important cations, such as K+,
are powerful tools to visualize and monitor cations in vitro and in
vivo. Therefore, fluorescence spectroscopy is a very popular and
sensitive method to track K+ concentrations in vivo. Thereby, the
non-fluorescent K+ is detected by fluorescent probes, so called
fluoroionophores, consisting of a signaling element (fluorophore)
and an ion-specific receptor (ionophore). Particularly, the K+/Na+
selectivity of the ionophore is of fundamental importance to
produce precise and true data. In animal cells, often Na+ is the
antagonist to K+ and both show contrary concentrations in the
extra- ([K+] = ~4 mM, [Na+] = ~140 mM) or intracellular ([K+] =
~120 mM, [Na+] = ~10 mM) medium. Further, the design of the
fluoroionophore architecture affects the fluorescence signal.
Fluoroionophores, which are electronically decoupled by a spacer
between the electron donor and acceptor unit often use a K+-
induced off switching of a photoinduced electron transfer (PET),
which leads normally to intensity enhancements at a single
emission wavelength.[1a-d] Most of the developed fluorescent
probes for K+ analysis in vitro and in vivo based on fluorescence
intensity changes at a single emission wavelength.[2a-g] However,
this K+ determination method requires a costly calibration and is
ratiometric fluorescent probe based on
a
K+-controlled
intramolecular exciplex formation in pure acetone.[5b] Further,
Clark et al. developed a ratiometric nanosensor for K+ based on a
Förster resonance energy transfer (FRET) between a quantum
dot and a chromoionophore in water.[5c]
However, it is a challenge to develop ratiometric ICT-type
fluoroionophores for the monovalent K+, because K+ induces only
small spectral wavelength shifts in such a -conjugated donor
acceptor arrangement.[6a,b] So far, to the best of our knowledge, a
ratiometric and K+-selective fluorescent probe, which shows a K+-
induced change at two emission wavelengths in water has not
been reported. Therefore, an improved design concept for
fluoroionophores, which are capable for the ratiometric detection
of K+ ions by a dual emission behavior, is required.
Recently, we developed highly K+ selective PET
fluoroionophores to determine exclusively K+ by fluorescence
intensity enhancements[7a-e], when excited by a one photon[7a-c] or
two photon process[7d] and by fluorescence lifetime changes[7e]. In
a further report, we explored a K+ responsive fluorescent probe,
which consists of a phenylaza-18-crown-6 ionophore and an
anthracene unit as a locally excited (LE) fluorophore, which are
connected by a 1,2,3-triazole moiety in a fully -conjugated
[a]
Dr. T. Schwarze, 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
E-mail: schwarth@uni-potsdam.de
Supporting information for this article is given via a link at the end of
the document.
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fashion (cf. Scheme 1, fluorescent probe 3).[8] Further, 3 exhibits
a weakly emissive charge transfer (CT) state (S1) at 539 nm and
a locally excited (LE) state (S2) at 415 nm in CH3CN. In the
presence of K+, the CT is decreased and energetically shifted
upwards and the LE state becomes the dominating fluorescent
state (S1). The fluorescent probe 3 is a light up probe for K+ with
a K+-induced state reversal in water. Thus, K+ induces a 12-fold
increase of the fluorescence intensity in water, but the Kd value 3
+ K+ (about 300 mM) was too high to measure physiologically
relevant K+ levels. In a further paper, we found that the o-(2-
methoxyethoxy)phenylaza-18-crown-6 lariat ether moiety is a
highly K+-selective building block for the determination of
physiologically relevant K+ levels.[7b] Nowadays, this K+ ionophore
is part of a set of highly K+ selective indicators.[9a-c]
Scheme 1. Studied fluorescent probes 1, 2 and reference compounds 3[8] and
Herein, we synthesized fluorescent probes 1 and 2 on the
basis of this highly K+ selective building block the o-(2-
methoxyethoxy)phenylaza-18-crown-6 lariat ether (ionophore) to
measure physiologically relevant K+ levels. Further, the aniline
moieties of fluorescent probes 1 and 2 were connected to position
1 and the fluorophore moiety to position 4 of the triazole unit (cf.
Scheme 1), owing to the higher K+/Na+ selectivity of this
arrangement.[7b] As a fluorophore moiety, we introduced typically
LE fluorophores such as anthracene (1) or pyrene (2) (cf.
fluorescent probes 1 and 2 in Scheme 1). As is well known, that
these LE fluorophores become a fluorescent CT state in polar
solvents, when they were substituted by an electron rich
dimethylaniline unit in a fully -conjugated arrangement.[10] For
the pyrene aniline derivative an intense fluorescent CT state with
a quantum yield f of 0.95 and a fluorescence emission maximum
em of 532 nm was found in acetonitrile.[10] However, for the
corresponding anthracene aniline analogue a lower f of 0.47 (em
= 582 nm) was observed.[10] Further, we found for the 1,2,3-triazol
-linked fluorescent probe 3 a very low fluorescent CT state in
acetonitrile and water.[8] However, Bag and Kundu showed for the
the 1,2,3-triazol -linked pyrene derivative 4 (cf. Scheme 1) a dual
4[11]
.
The UV/Vis absorption spectra of 1 and 2 in Tris buffered (10
mM, pH of 7.2) H2O/DMSO mixtures 99/1 (v/v) show
superimpositions of charge transfer (CT) bands and the typical
polycyclic aromatic absorption bands (cf. Figures S1a,b). For 1
and 2, we observed two CT bands about 300 nm and 330 nm and
for 2 about 285 nm and 370 nm, respectively. The CT band at 330
nm for 1 and at 370 nm for 2 were mainly superimposed by the
typical anthryl or pyrenyl absorption bands. The short-wavelength
CT absorptions about 300 nm (1) and 285 nm (2) correspond to a
CT from the nitrogen lone pair of the aromatic ring (donor) to the
1,2,3-triazole acceptor unit, which is also found for -conjugated
anilino-1,2,3-triazol-1,4-diyl – fluoroionophores.[7a,b] Further, the
CT absorption band of 1 at 330 nm belongs to a CT from the
anilinotriazole donor moiety to the anthryl acceptor unit as also
shown for probe 3.[8] Herein, we observed for 1 compared to 3 (CT
at 423 nm) a blue-shifted and less intense CT band about 330 nm
(cf. Figure S1a), because 1 possess a weaker electron donor unit
as the reference compound 3, as already discussed for series of
crown compounds with a bulky substituent in ortho position of the
aniline donor moiety.[15] Further, we found for 2 superimposed
absorptions about 370 nm, which partially corresponding to a CT
from the anilinotriazole donor moiety to the pyrenyl acceptor unit.
The fluorescence spectrum of 1 (ex = 365 nm) showed the
typical anthracenic emission peaks (cf. Table 1) in Tris buffered
(10 mM, pH of 7.2) H2O/DMSO mixtures of 99/1 (v/v). Further, we
observed for 2 in the range from 370 nm to 440 nm the typical
pyrene emission from the LE state and a broad emission peaked
at 505 nm, which corresponds to a fluorescent CT state, as also
observed for 4[11], but 1 exhibits no intense fluorescent CT state
in the range from 450 nm to 650 nm in water. Notably, the
fluorescence spectrum of 2 (ex = 345 nm) shows compared to 1
a distinctive dual emission in water, which can be underlined by
excitation spectra of 2 at a em of 390 nm and a em of 505 nm,
respectively (Figure S3). The excitation spectrum of 2 (em = 390
nm) exhibits the typical pyrene fine structure from 300 nm to 370
nm with no superposition of the CT band (cf. Figure S3a) and the
excitation spectrum of 2 (em of 505 nm) shows only a broad and
well-defined CT band in the same region of the spectrum ranging
from 300 nm to 400 nm (cf. Figure S3b). Concluding that 2 has a
emission behavior in dioxane/water mixtures.[11a-c] Even
a
fluorescent CT state in polar solvents is observed, when a
dimethylaniline unit is -linked via a 1,2,3-triazol-1,4-diyl unit with
a benzene moiety.[12] Thus, we favored the pyrene substituted
fluorescent probe 2 to exhibit a fluorescent CT and LE state for a
dual recognition behavior of K+ in water. Further, for fluorescent
probe 1, we expect a high K+-induced fluorescence enhancement
in water as already observed for fluorescent probe 3. Overall, the
new fluorescent probes 1 and 2 (Scheme 1) should operate in the
presence of K+ in a similar mechanistic way as found for 3 + K+.
Results and Discussion
The feasible synthesis of the fluorescent probes 1 and 2 were
realized by Cu(I)-catalyzed 1,3-dipolar azide alkyne cycloaddition
(CuAAC) reactions[13a,b] of the azido functionalized phenylaza-18-
crown-6 lariat ether compound[7b] with 9-ethynylanthracene[14] or
with the commercial available 1-ethynylpyrene, respectively (cf.
Scheme S1).
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fluorescent CT state (S1) and a lower fluorescent LE state (S2) in
a H2O/DMSO mixture of 99/1 (v/v). However, the CT emission of
2 at 505 nm becomes more fluorescent, when the volume fraction
of H2O is increased from 1/1 to 99/1 (H2O/DMSO mixtures, cf.
Figure S8). The excitation spectrum of 1 (em = 424 nm) showed
the typical anthryl bands, respectively (cf. Figure S2), but we
found not such a pronounced CT band as observed for 2, when
excitation spectra of 1 were recorded in a em range from 450 to
550 nm. Further, we observed for 1 a fluorescence quantum yield
f of 0.024 and for 2 a higher f value of 0.092 (cf. Table 1).
After that, we investigated the influence of K+ and Na+ on the
fluorescence of 1 and 2 in the physiological relevant concentration
range of 1 mM to 160 mM K+ and Na+. We observed an
extraordinary K+-induced (FEF = 7.7, see Figure 2a and S4a) and
a slightly Na+-induced (FEF = 1.7 at 160 mM Na+, see Figure S5a)
fluorescence enhancement of 1. About 140 mM K+, we found for
1 + K+ the maximum signal change with a F value of 0.193 (Table
1). The fluorescence signal of 2 + K+ showed remarkable intensity
changes at 405 nm and 505 nm (cf. Figure 1b). K+ reduces the
fluorescene intensity of the CT state at 505 nm by a factor of ~4.8
to a concentration of around 50 mM K+. However, K+ enhances
the fluorescence intensity (FEF = ~4.6, see Figure S7a) of the LE
state at 405 nm. Thus, 2 showed a dual fluorescence emission
behavior in the presence of K+. Overall, we observed a reduced
f of 0.023 for 2 + 160 mM K+ and a slightly Na+-influenced f with
a value of 0.081 (see Table 1).
Table 1. Photophysical data of 1 and 2 in a H2O/DMSO mixture of 99/1 (v/v)
in the absence and presence of 160 mM KCl and 160 mM NaCl.
[a]

abs [nm]

em [nm]
f
KdK+ [mM][b]
-
Figure 1. Fluorescence intensity changes of a) 1 (c = 10-5 M, exc = 365 nm) and
b) 2 (c = 10-5 M,exc = 345 nm) in the presence of various K+ concentrations
under simulated physiological conditions (10 mM Tris-buffer, pH 7.2) with a
constant ionic strength of 160 mM by varying Na+ concentrations equal to [K+]
+ [Na+] = 160 mM.
1
386, 367, 349,
330, ~300
423, 405
423 , 405
423, 405
0.024
1 + K+
1 + Na+
386, 367, 349,
333, ~300
0.193
0.040
38
-
386, 367, 349,
330, ~300
In the next step, the high K+/Na+-selectivity was further verified
by using solutions containing both, K+ and competing Na+ ions.
Figure 1 shows the fluorescence behavior of 1 (cf. Figure 1a) and
2 (cf. Figure 1b) in a buffered H2O/DMSO mixture (99/1, v/v; 10
mM Tris; pH = 7.2) in combined K+/Na+ solutions equal to 160 mM.
The FE factor of 1 + 160 mM K+ (FEF = 4.5) is lower than that in
the experiment without Na+ (FEF = 7.7, cf. Figure 1a and Figure
S4b) and for 2 + K+ in the presence Na+, we found similar
fluorescene intensity changes as in the experiment without Na+
(cf. Figure 1b and Figure S7b). Further, the run of the titration
curves of 1 + K+ and 2 + K+ were only slightly influenced by
competing Na+ ions (cf. Figure 2a and 2b), again demonstrating
the high K+/Na+ selectivity of the fluorescent probes 1 and 2.
Dissociation constants were calculated from plots of fluorescence
intensities versus K+ concentrations.[16] The Kd of 1 in Na+ free
solutions is ∼38 mM and increases to ∼41 mM in combined
K+/Na+ solutions. We also found an slightly increased Kd value for
2 + K+ from ∼8 mM to ∼9 mM in the presence of Na+. Thus, the
presence of Na+ has a negligible effect on the performance of 1
and 2 under simulated physiological conditions.
2
370, 285
350, 282
370, 283
505, 405, 385
495, 405, 385
505, 405, 385
0.092
0.023
0.081
-
2 + K+
2 + Na+
8
-
[a] Fluorescence quantum yield, (±15%). [b] Dissociation constants Kd for K+
complexes.
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Conclusions
Herein, we synthesized by CuAAC reactions two fluorescent
probes 1 and 2, which both consisting of a K+-selective building
block the o-(2-methoxyethoxy)phenylaza-18-crown-6 lariat ether
(ionophore). Fluorescent probes 1 and 2 only differ by the
fluorophore unit, whereby 1 possess an anthracene unit and 2 a
pyrene moiety. In the presence of K+, 1 and 2 showed K+-induced
fluorescence intensity changes. For 1 + K+, we observed a high
FE with a factor of 7.7 and for 2 + K+, we found a dual emission
behavior at 405 nm and 505 nm in water. The CT band at 505 nm
is decreased and the LE emission at 405 nm is increased by K+.
Thus, we have shown, that the fluorescent probe 2 (Kd = 8 mM) is
able to measure extracellular K+ levels (1 mM - 10 mM K+) by
analyzing the intensity ratio at two emission wavelengths in vitro.
This ratiometric behavior of 2 + K+ is caused by a K+-induced state
reversal in water. Overall, we present an improved design
principle for the ratiometric fluorescence analysis of analytes in
water. Currently, we functionalizing fluorescent probe 2 with alkyl
chains to anchor it into cell membranes to analyze extracellular
K+ levels in vivo. Prospectively, fluorescent probe 2 should
embedded in a hydrogel as a part of an optode to measure local
K+ levels.
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 instrument, 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)).
Figure 2. Titration curves of a) 1 (c = 10-5 M) and b) 2 (c = 10-5 M) in the
presence of various K+ concentrations (black circles) and in combined K+/Na+
solutions (red triangles) with a constant ionic strength equal to [K+] + [Na+] = 160
mM for 1 and 2, respectively.
Contrary to 1, 2 exhibits a more pronounced emissive charge
transfer (CT) state (S1) at 505 nm and also a locally excited (LE)
state (S2) about 400 nm in water (cf. Figure 3). Thus, we observed
for 2 in the presence of K+ a decreased and a slightly energetically
upwards shifted CT band and the LE state becomes the
dominating fluorescent state (S1) (cf. Figure 3). We assume, that
the FE for 1 + K+ is also caused by a K+-induced state reversal
with the difference that 1 exhibits only a very weak emissive CT
state in water
General CuACC procedure for 1 and 2: A mixture of N-(4-azido-2-
methoxyethoxyphenyl)aza-18-crown-6 ether[7b] 189 mg (0.415 mmol) and
0.415 mmol of 9-ethynylanthracene[14] or 1-ethynylpyrene, respectively,
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, 9/1)
as an eluent mixture to afford 1 and 2 as pale yellow oils.
Fluorescent probe 1: Yield 20% (55 mg); 1H-NMR ((CD3)2SO, 300 MHz):
δ = 9.22 (s, 1H), 8.83 (s, 1H), 8.37 (s, 2H), 8.24 (d, J = 8.6 Hz, 2H), 7.91
(d, J = 8.6 Hz, 2H), 7.65-7.54 (m, 5H), 4.33-4.26 (m, 2H), 3.82-3.75 (m,
2H), 3.76-3.18 ppm (m, 27H); 13C-NMR ((CDCl3), 75 MHz): δ = 144.49,
131.42, 131.30, 128.68, 128.46, 128.41, 127.09, 126.16, 125.96, 125.55,
125.22, 125.02, 112.84, 112.72, 108.73, 106.71, 70.87, 70.73, 70.59,
70.50, 70.31, 69.42, 68.27, 58.97, 53.12 ppm; HRMS (ESI): m/z calcd for
C37H44N4O7+H+: 657.3288 [M+H+]; found: 657.3237.
Fluorescent probe 2: Yield 35% (99 mg); 1H NMR (CD3CN, 300 MHz): δ
= 8.85 (d, J = 9.3 Hz, 1H), 8.67 (s, 1H), 8.37-8.01 (m, 7H), 7.59 (s, 1H),
7.48-7.37 (m, 2H), 7.19 (d, J = 8.6, 1H), 4.22-4.17 (m, 2H), 3.75-3.71 (m,
2H), 3.61-3.40 (m, 24H), 3.37 ppm (s, 3H); 13C NMR (CD3CN, 75 MHz): δ
Figure 3. K+-induced state reversal of fluorescent probe 2 in water.
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= 153.32, 148.26, 132.36, 132.15, 131.84, 129.20, 129.03, 128.81, 128.33,
128.16, 127.40, 126.88, 126.51, 126.29, 126.20, 126.02, 126.00, 125.76,
125.36, 123.02, 113.72, 106.86, 79.15, 71.59, 71.25, 71.12, 71.02, 70.35,
68.88, 59.06, 53.48 ppm; HRMS (ESI): m/z calcd for C39H44N4O7+H+:
681.3288 [M+H+]; found: 681.3315.
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Keywords: fluorescence • potassium • ratiometric • charge
transfer • crown compounds
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Supporting Information.
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10.1002/chem.201802306
Chemistry - A European Journal
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In this paper, we show a ratiometric
fluorescent probe to measure
physiologically relevant K+ levels by
K+-induced intensity changes at two
emission wavelengths.
Thomas Schwarze*, Janine Riemer
and Hans-Jürgen Holdt*
Page No. – Page No.
A Ratiometric Fluorescent Probe
for K+ in Water based on a
Phenylaza-18-Crown-6 Lariat Ether
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.