High Na+ and K+-induced fluorescence enhancement of a π-conjugated phenylaza-18-crown-6-triazol-substituted coumarin fluoroionophore

Article abstract of DOI:10.1039/c0cc04370b

The new π-conjugated 1,2,3-triazol-1,4-diyl fluoroionophore 1 generated via Cu(i) catalyzed [3 + 2] cycloaddition shows high fluorescence enhancement factors (FEF) in the presence of Na⁺ (FEF = 58) and K⁺ (FEF = 27) in MeCN and high

Full text of DOI:10.1039/c0cc04370b

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COMMUNICATION
High Na+ and K+-induced fluorescence enhancement of a p-conjugated
phenylaza-18-crown-6-triazol-substituted coumarin fluoroionophorew
Sandra Ast,a Holger Mu
Hans-Ju
¨
¨
ller,a Roman Flehr,a Tillmann Klamroth,a Bernd Walzb and
rgen Holdt*a
Received 12th October 2010, Accepted 23rd February 2011
DOI: 10.1039/c0cc04370b
The new p-conjugated 1,2,3-triazol-1,4-diyl fluoroionophore 1
generated via Cu(I) catalyzed [3 + 2] cycloaddition shows high
fluorescence enhancement factors (FEF) in the presence of
Na+ (FEF = 58) and K+ (FEF = 27) in MeCN and high
selectivity towards K+ under simulated physiological conditions
(160 mM K+ or Na+, respectively) with a FEF of 2.5 for K+.
(FEFNa+: 58; FEFK+: 27). The signal transduction in 1 also
works nicely under simulated physiological conditions. In the
presence of 160 mM K+ a FEF of 2.5 was observed, whereas
the same concentration range of Na+ resulted in an almost
negligible fluorescence enhancement. We assume that compound 1
is a PET-fluoroionophore with a virtual spacer between
the anilinotriazole electron donor unit and the coumarin
electron acceptor moiety. The constitutional isomer of 1, the
1,2,3-triazole-fluoroionophore 2 shows smaller cation induced
FEFs than 1, suggesting that in the presented conjugated
sensors the substitution on the 1,2,3-triazole ring has a basic
influence on the quality of the fluorionophore.
Recently 1,2,3-triazol-1,4-diyl fluoroionophores for Zn2+ 1
,
Ni2+,2 Cu2+,3 Hg2+,4 Ag+,4 and Al3+,5 were generated by
Cu(I) catalyzed reaction between an azide and an alkyne
(CuAAC). In these fluoroionophores the 1,2,3-triazol-1,4-diyls
serve, in addition to the conventional function as covalent linkers,
as both chelating ligand of the metal ions and as electronic
transmitter of a coordination event to the fluorophore.1a Only
Zn2+ and Al3+ could be detected by fluorescent enhancement,
whereas the other metal ions were analysed through fluores-
cence quenching. In these known 1,2,3-triazol-1,4-diyl fluoro-
ionophores, no electronically conjugated signal transduction
chain: ionophore-1,2,3-triazole-fluorophore is used. Either
receptor and 1,2,3-triazole are connected through a deconjugated
linker1,2 or the triazole and the fluorophoric group are
deconjugated.3–5
The CuAAC of the ethinyl-functionalized N-phenylaza-
18-crown-6 ether8 with 3-azido-7-diethylaminocoumarin9 afforded
1,2,3-triazole-fluoroionophore 1. The triazole-isomer 2 was
obtained by the reaction of the azido-functionalized N-phenyl-
aza-18-crown-6 ether10 with 3-ethinyl-7-diethylaminocoumarin.11
Dye 3, in which the crown has been replaced by a diethylamino
group was synthesised in order to represent a reference
compound for 1 (Scheme 1). A further reference compound
for 1 is compound 4, in which the p-phenylene linker between
aza-18-crown-6 and the triazole ring is replaced by a deconjugated
methylene group. Compound 4 was obtained by the reaction
of N-propargylaza-18-crown-6 ether12 with 3-azido-7-diethyl-
aminocoumarin. The new 1,4-disubstituted 1,2,3-triazoles
1–4 are stable in solutions of MeCN and DMSO at room
temperature for many weeks. UV-irradiation over a period of
several hours did not show any decomposition of the fluoro-
ionophores 1 and 2.
In a systematic investigation Diederich et al. showed the
capacity of the 1,2,3-triazol-diyls as active p-linkers in Charge
Transfer (CT) chromophores.6 Such push–pull chromophores
are only weakly or even nonfluorescent. 1(4-Butoxyphenyl)-
4-pyridyl-1,2,3-triazole displays turn-on fluorescence upon
addition of metal cations.7
We found that in the simple CuAAC-generated fluoro-
ionophore 1, the electronic conjugation of the N-phenylaza-
18-crown-6 ether and the 7-diethylaminocoumarin fluorophore
through a 1,2,3-triazol-1,4-diyl p-linker results in a perfect
signal transduction chain for the sensing of Na+ and K+.
In MeCN high cation-induced FEFs were obtained for 1
a Universitat Potsdam, Institut fur Chemie,
¨
¨
Karl-Liebknecht-Strasse 24-25, 14467 Potsdam, Germany.
E-mail: holdt@chem.uni-potsdam.de; Fax: +49 331 9775055;
Tel: +49 331 9775180
b Universitat Potsdam, Institut fur Biochemie und Biologie,
¨
Zoophysiologie, Karl-Liebknecht-Strasse 24-25, 14467Potsdam,
¨
Germany
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c0cc04370b
Scheme 1 Conjugated fluoroionophores 1 and 2 with 1,2,3-triazol-
1,4-diyl as the p-linker, and reference compounds 3 and 4.
c
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Chem. Commun., 2011, 47, 4685–4687 4685

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Table 1 Photophysical properties of 1–4 in MeCN
Ligand
1
2
3
4
l
l
em/nma
ex/nmb
484
410
0.008
0.62
0.23
470
412
0.03
0.79
0.40
493
410
0.017
0.018
0.016
480
408
0.56
0.55
0.58
Ff(ligand)c
Ff(ligand + Na+ d
Ff(ligand + K+ d
)
)
a
b
Emission maxima. Excitation maxima. Fluorescence quantum
c
d
yields (Ff). Ff were determined in the presence of 40 mM of NaPF6
or 40 mM KPF6, respectively.
Fig. 1 UV/VIS absorption spectra of 1–4 in MeCN in the presence
of increasing concentrations of NaPF6: (a) 1 +0.00–2.40 mM,
(b) 2 +0.00–2.80 mM, (c) 3 +0.00–2.80 mM, (d) 4 +0.00–4.60 mM.
The UV/Vis absorption spectra of the 3-(anilino-1,2,3-triazolyl)
substituted 7-diethylaminocoumarins 1–4 in MeCN show the
typical long-wavelength coumarin CT absorption band between
lmax = 408 and 412 nm (Fig. 1a–d), which are similar to that
of 3-(2-methylbenzimidazol-1-yl)-7-diethylaminocoumarin at
lmax = 407 nm.13
Fig. 2 Fluorescence spectra of 1 in MeCN (black) upon addition of
0–2.5 mM NaPF6 (blue) and 0–1.16 mM KPF6 (red), respectively.
Na+ and K+ in the cavity of the aza-18-crown-6 ether of 1
and 2 in MeCN.
The 1,2,3-triazol-1,4-diyl constitutional isomerism in the case
of fluoroionophores 1 and 2 has only a small effect on the lmax
of the long-wavelength CT absorption. However, the intensity
of this CT band is higher for 2 (e = 40 050 Mꢀ1 cmꢀ1) than
for 1 (e = 21 250 Mꢀ1 cmꢀ1). This might be caused by the
more efficient conjugation between the triazole and coumarin
unit in 2 through the sp2-hybridized carbon atom C(4) of the
triazole, whereas in 1 the coumarin unit is connected to the
triazole ring at N(1).
The fluorescence quantum yield of 1 in MeCN is extremely
low (Ff(1) = 0.008, Table 1). The fluorescence spectra of 1 in
the presence of Na+ and K+, respectively (Fig. 2) show the
impressive fluorescence enhancement upon coordination of
the alkali metal ions. High FEFs could be determined for
Na+ (FEF = 58) and for K+ (FEF = 27).14
The B3LYP/6-31G*-optimized geometry of 1 (Fig. S10, ESIw)
shows that the p-electron system of 1 is twisted at the coumarin–
triazole link (61) owing to the steric hindrance caused by the
carbonyl unit. The anilino moiety is nearly coplanar with the
triazole ring. As found in many twisted systems this twisting
should result in a virtual spacer.15a Thus, the well-delocalized
planar anilinotriazole unit serves as a good PET donor and the
coumarin serves as a PET acceptor. The planarity of the anilino-
triazole ensures a low oxidation potential. Hence, a very
small fluorescence quantum yield is found. Complexation of
Na+ or K+ by the azacrown increases the oxidation potential
of the anilinotriazole and the fluorescence switch-on happens
by the stoppage of the PET. Also, in the UV/Vis-spectra, the
lowest energy coumarin absorption is essentially unchanged
upon Na+ or K+ addition, which typically characterises fluores-
cent PET systems. Only the higher energy anilinotriazole
absorption is blue-shifted, owing to the removal of the anilino
electron pair by binding to Na+ or K+. Similar blue-shifts of
higher-energy bands combined with unchanged lower-energy
bands are known from the UV/Vis-spectra of PET sensors.15b,c
The fluorescence quantum yield of the constitutional isomer
2 in MeCN is higher than that of 1 (Ff(2) = 0.03, Table 1). The
fluorescence of 2 is also enhanced in the presence of increasing
concentrations of Na+ and K+ (ESIw, Fig. S7 and S8,
respectively). However the observed FEFs for Na+ (FEF = 17)
and K+ (FEF = 13) are significantly lower than for the isomer 1.
The B3LYP/6-31G*-optimized geometry of 2 revealed that in 2
the anilino unit is twisted against the triazole ring with a torsion
In the UV/Vis-spectra of 1 and 2 we observe next to the
long-wavelength CT transition a second short-wavelength CT
transition at lmax = 289 and 294 nm, respectively (Fig. 1a and b).
The CT nature of these absorption bands is confirmed by the
reduction in intensity in the presence of Na+ (Fig. 1a and b)
or K+ (Fig. S1 and S2, ESIw), respectively. The coordination
of Na+ or K+ in the cavity of the azacrown reduces the
electron donor character of the anilino moiety. Thus the CT
towards the conjugated electron poor triazole (acceptor) is
energetically shifted upwards. We observed such a CT absorption
band around 290 nm also in the UV/Vis absorption spectrum
of a 1,2,3-triazol-fluoroionophore in which the conjugated
N-phenylaza-18-crown ether-1,2,3-triazol-1-yl unit is connected
to a 9-anthracenyl moiety by a methylene-spacer. Addition of
Na+ leads to a complete disappearance of the CT absorption
band at lmax = 289 and 294 nm in the spectra of 1 and 2,
respectively (Fig. 1a and b), whereas the presence of K+ reduces
the intensity of this CT absorption band to a lesser extent
(Fig. S1 and S2, ESIw). This can be explained by the larger
charge density of Na+. The complexation of Na+ or K+ by the
azacrown of 1 or 2 has only very little influence on the long-
wavelength CT absorption at lmax = 410 or 412 nm.
The UV/Vis absorption spectrum of the reference compound 3
in MeCN is barely changed in the presence of Na+ (Fig. 1c)
or K+ (Fig. S3, ESIw). This result proves the coordination of
c
4686 Chem. Commun., 2011, 47, 4685–4687
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angle of about 241 (Fig. S10, ESIw). The poorer planarity
of the anlinotriazole unit in 2 results in a higher oxida-
tion potential. Therefore, free 2 shows a stronger fluorescence
than 1 and lower FEF values are found.
Recently He et al.16a and the Verkman group16b designed
fluorescence switch-on PET sensors with high K+/Na+ selectivity
and sensitivity for physiological K+ in the concentration
range of 0–40 mM. In these K+ fluoroionophores a [3.2.2]-
cryptand represents the ionophore. A drawback though, is the
very expensive synthetic procedure.16c
The reference compound 4 consists of a poorer PET donor
due to the aliphatic amine which is electronically separated from
the triazole. Hence 4 has a high quantum yield (Ff(4) = 0.56,
Table 1) and is therefore less affected by the presence of
Na+ and K+.
To investigate the pH-sensitivity of 1, the fluorescence
intensity was measured in water at different pH values. The
resulting pKa of 1 is near 4.5 (Fig. S9, ESIw) meaning that 1 is
less pH-sensitive than the o-methoxyphenylaza-15-crown-5
Na+-fluoroionophore (pKa E 5.5) of He et al.16
We further investigated the influence of Na+ and K+ on the
fluorescence of 1 under simulated physiological conditions.
The ligand was exposed to aqueous solutions in the physio-
logical interesting concentration range of 0–160 mM Na+ or K+,
respectively. Additionally, the solutions contained 2 mM Ca2+
and 2 mM Mg2+. The pH was adjusted to 7.2 with 10 mM Tris
and a constant ionic strength of 180 mM was maintained with
choline chloride. Under these conditions, the fluorescence of 1
(lex = 424 nm, lem = 500 nm, Ff = 0.07) is hardly increased
in the presence of Na+ whereas increasing concentrations of
K+ resulted in a modest fluorescence enhancement (Fig. 3a).
In the presence of 160 mM K+ a FEF of 2.5 is observed
with very little variations in the FEF values (Fig. 3b). It is
noteworthy, that the presence of physiological important
extracellular cations, such as Mg2+ and Ca2+ does not affect
the signal response.
In summary, we have shown for the first time that an
electronically conjugated 1,2,3-triazole-fluoroionophore con-
sisting of the signaling transduction chain: triazole-fluorophore
works as an effective sensor for Na+ and K+ in acetonitrile
with high cation-induced FEFs. Under simulated physio-
logical conditions 1 selectively detects K+ with the modest
FEF being limited by the rather simple receptor unit. The
efficient signaling transduction chain in the fluoroionophore 1
should be a promising design concept for generating novel
highly sensitive fluoroionophores for Na+, K+, Mg+ and Ca+
with interesting potential for biological applications. Further
studies towards the fine tuning of the receptor unit are currently
under investigation in our laboratories.z
Notes and references
z Patent application filed on March 9th 2011, EP11157413.3
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In comparison, the FEF of 1 for K+ in MeCN is clearly
smaller than the FEF in water under simulated physiological
conditions. This can be explained by the significantly smaller
stability constants of the K+ complex with the N-phenylaza-
18-crown-6 in water [lg K (H2O) 4 0.5; lg K (MeCN) =
3.95 ꢁ 0.08]. The fact that the fluorescence of 1 in water is
exclusively enhanced in the presence of K+ and not by Na+
can be rationalized by the stronger hydration enthalpy of
Na+. However, the fluorescence enhancement of 1 in the
presence of K+ under simulated physiological conditions
shows that the signaling transduction chain in this fluoro-
ionophore works well in water.
The dissociation constant (Kd) of 1 + K+ amounts to
B260 mM in solutions which approximates the physiological
ionic strength. To measure intracellular or extracellular con-
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Fig. 3 Fluorescence measurements of 1 (lex = 424 nm) under
simulated physiological conditions (ESIw). (a) Fluorescence spectra
in the presence of 0–160 mM Na+ (blue) and K+(red), respectively
and (b) the corresponding FEF with error bars in the presence of
0–160 mM K+ or Na+
.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4685–4687 4687