Development of a potassium ion-selective fluorescent sensor based on 3-styrylated BODIPY

Article abstract of DOI:10.1016/j.bmcl.2011.08.056

We have developed a red-emitting fluorescent K⁺ probe, B3TAC, which also shows a wavelength shift upon binding to K⁺. The probe was synthesized by conjugating a cryptand-based chelator, 2-triazacryptand [2,2,3-1-(2-methoxyethoxy)benz

Full text of DOI:10.1016/j.bmcl.2011.08.056

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6090–6093
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a potassium ion-selective fluorescent sensor
based on 3-styrylated BODIPY
⇑
Tomoya Hirata, Takuya Terai, Toru Komatsu, Kenjiro Hanaoka, Tetsuo Nagano
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 0033, Japan
a r t i c l e i n f o
a b s t r a c t
We have developed a red-emitting fluorescent K+ probe, B3TAC, which also shows a wavelength shift
upon binding to K+. The probe was synthesized by conjugating a cryptand-based chelator, 2-triazacrypt-
and [2,2,3]-1-(2-methoxyethoxy)benzene (TAC), to position 3 of the BODIPY fluorophore through a styryl
linker. In water–acetonitrile mixed solvent, it responded to K+ in the physiological concentration range
with high selectivity over Na+ and other metal ions. B3TAC is potentially useful for measuring cellular
K+ ion concentration, as well as for simple, naked-eye detection of K+ in solution.
Article history:
Received 6 June 2011
Revised 10 August 2011
Accepted 11 August 2011
Available online 19 August 2011
Keywords:
Potassium ion
BODIPY
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorescent probe
Fluorescent probes are useful tools for analysis of physiological
events, including ion channel activity, localization of metal ions,
and enzyme activity, based on the changes of their optical proper-
ties, such as fluorescence intensity and excitation/emission wave-
length, as a result of specific interactions with target molecules.1,2
For example, the role of Ca2+ ion as a signaling molecule is now
much better understood, following the introduction and applica-
tion of a variety of Ca2+ ion probes with different fluorescence colors
and dissociation constants.2,3 K+ ion, which is the most abundant
metal ion in cells, plays essential roles in cardiac and neuronal
excitability, cellular ionic homeostasis and cell proliferation.4,5
Hence, there is a great need for chemical tools that are capable of
visualizing intracellular and extracellular K+ concentration, [K+].
But, in contrast to the case of Ca2+ probes, there have been a few
biological applications of commercially available K+ ion probes
incorporating an 18-crown-6 based chelator, such as PBFI,6 due to
their relatively low fluorescence and poor selectivity for K+ over
Na+. On the other hand, a K+-selective cryptand-based chelator,
2-triazacryptand [2,2,3]-1-(2-methoxyethoxy)benzene (TAC), was
absorption spectra is insensitive to [K+]. Considering the demand
for simple colorimetric detection of molecules in aqueous sam-
ples,12,13 we believe there is a strong need for selective fluorescent
K+ indicators that meet the following requirements: (i) excitation at
over 550 nm, (ii) significant color change in response to [K+], and
(iii) compatibility with aqueous media (>50% H2O).
Here we report the synthesis and spectroscopic properties of a
novel red-emitting, fluorogenic K+ probe, B3TAC, which is also
applicable for colorimetric detection of K+ ion. As an ionophore,
we selected TAC because of its good selectivity for K+ and fast re-
sponse to changes of ion concentration.9 As a chromophore, we
adopted 8-phenyl BODIPY because of its sharp absorption and
emission spectra, high extinction coefficient and quantum effi-
ciency, and stability to light and chemical reactions.14 Several
BODIPY derivatives bearing ion-recognizing groups at a styryl moi-
ety introduced at position 3 of the chromophore have recently
been synthesized.15–18 In most cases, they have red emission due
to the extension of
p-conjugation, and they show both fluores-
cence intensity change and wavelength shift of the absorption/
emission spectra, presumably derived from the change of elec-
tronic environment as a result of coordination of metal ions to
recently synthesized,7 and several fluorescent K+ probes incorpo-
8–10
rating TAC have been reported by Verkman et al.
However,
long-wavelength-emitting K+ probes that can be excited at over
550 nm, the optimal region for biological studies in terms of auto-
fluorescence and photo-damage to biological samples, are still
rare.8,9 Furthermore, although these TAC-based probes show good
K+ selectivity, their fluorescence properties are controlled by
photoinduced electron transfer (PeT),11 and so the shape of the
the nitrogen atom linked to the
p-system. Those results prompted
us to design B3TAC as a novel fluorescent K+ probe that would sat-
isfy the above requirements. Although sensors for Cu2+ 15 Hg2+ 16
,
,
and FÀ17 have been developed based on the styryl BODIPY scaffold,
K+ has not previously been targeted.
The synthetic route to B3TAC is depicted in Scheme 1. B3TAC
was obtained by Knoevenagel-type condensation of TAC-aldehyde
(1) and 8-phenyl-1,3,5,7-tetramethyl BODIPY (2) in a Dean–Stark
apparatus with 15% yield. The purity of B3TAC was confirmed by
1H-NMR, HRMS and HPLC (Supplementary data).
⇑
Corresponding author. Tel.: +81 3 5841 4850.
E-mail address: tlong@mol.f.u-tokyo.ac.jp (T. Nagano).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.056

T. Hirata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6090–6093
6091
O
O
O
O
N
N
N
N
B
AcOH
piperidine
F
F
O
O
N
+
N
N
O
B
toluene
O
F
F
O
N
O
O
reflux, 6 h
2
O
N
H
O
O
O
N
1
O
O
B3TAC
Scheme 1. Synthesis of B3TAC.
K= 150 mM
K+= 150 mM
0.05
a
b
c
K
= 100 mM
K+= 100 mM
+
K= 80 mM
K = 80 mM
Color
+
Fluorescence
K= 60 mM
K = 60 mM
0.04
0.03
0.02
0.01
+
K= 50 mM
K = 50 mM
+
K= 40 mM
K = 40 mM
+
K= 30 mM
K = 30 mM
+
KK== 2200mmMM
K+= 8 mM
K= 8 mM
K+= 4 mM
K= 4 mM
K+= 2 mM
K= 2 mM
K+= 1 mM
K= 1 mM
K+= 0 mM
K= 0mM
-K+
+K+
-K+
+K+
0
450
500
550
600
650
700
d
e
Wavelength (nm)
600
500
400
300
200
100
0
600
500
400
300
200
100
0
450
500
550
600
650
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Figure 1. Optical properties of B3TAC. Change in (a) color and, (b) fluorescence (excitation at 365 nm) of a MeCN solution containing B3TAC with or without K+, (c)
Absorption, (d) excitation (kem = 571 nm) and, (e) emission (kex = 560 nm) spectra of 1
l
M B3TAC in 5 mM HEPES (pH 7.0)/MeCN = 60:40 containing the indicated
concentration of K+ as the ClO4 salt. Ionic strength was maintained at 150 mM by addition of NaClO4. The mixture contained 2% DMSO as a cosolvent.
À
First, we preliminarily evaluated the response of B3TAC to K+
ion in a glass sample vial, and confirmed that, in acetonitrile,
B3TAC showed almost no fluorescence in the absence of K+ ion,
but emitted strong yellow to orange fluorescence when excess
KClO4 was added (Fig. 1b). As expected, a significant color change
of the sample from blue to pink was also observed (Fig. 1a). We
next examined whether B3TAC worked in aqueous environment
(Fig. S1). As the percentage of 5 mM HEPES buffer (pH 7.0) in

6092
T. Hirata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6090–6093
Table 1
Optical properties of B3TAC in 5 mM HEPES (pH 7.0)/MeCN = 60:40 containing the indicated concentrations of Na+ and K+
K+/Na+ (mM)
Absmax (nm)
Exmax (nm)
Emmax (nm)
e
(MÀ1 cmÀ1
)
uFL
Buffer only
578
581
561
560
559
562
568
569
570
7.3 Â 104
7.4 Â 104
8.9 Â 104
0.022
0.018
0.39
Buffer + 150 mM Na+
Buffer + 150 mM K+
acetonitrile was increased up to 70%, little change of the absorption
spectrum was observed, although the fluorescence intensity was
decreased to some extent. However, when the percentage of HEPES
buffer was 80% or more, the absorption spectrum was broadened
and the fluorescence disappeared completely with a concomitant
increase of scattered light. This is considered to be due to aggrega-
tion of B3TAC, because of its hydrophobic nature and poor solubil-
ity in pure water. Hence, subsequent experiments were performed
in a mixed aqueous media containing 60% HEPES buffer, in which
the probe should behave as individual molecules.
the mechanism remains to be examined, excess Na+ might com-
pete with K+ and fix B3TAC in the K+-free ‘quenched’ conformation.
From a plot of fluorescence intensity versus [K+], the apparent
dissociation constant (KDapp
)
was calculated to be 53 mM22
(Fig. S4). Since extracellular K+ concentration is reported to be
about 5 mM, while intracellular K+ concentration is around
150 mM,23 this value of KDapp means that the probe is suitable
for measuring physiological K+ concentrations.
We also examined the selectivity of B3TAC for K+ (Fig. 2). Com-
pared to the fluorescence intensity in 5 mM HEPES (pH 7.0)/
MeCN = 60:40, B3TAC showed no increase in fluorescence in the
presence of Na+ (at 150 mM), but showed slightly increased fluo-
We next performed a titration experiment with B3TAC and K+ in
aqueous 5 mM HEPES (pH 7.0)/MeCN = 60:40 by increasing the
concentration of KClO4 under the condition that the total ionic
strength was maintained at 150 mM with NaClO4 (Figs. 1c–e and
S2). In the absence of K+, a rather broad and structureless absorp-
tion spectrum was observed with kmax = 581 nm, and the probe
emitted very low fluorescence (kex = 560 nm).19 In the presence
of 150 mM K+, however, the absorption spectrum became sharp,
with a 20 nm blue shift of the peak to 561 nm, and the fluorescence
intensity was elevated dramatically, by approximately 100-fold.20
As shown in Figure 1c, the shape of the absorption spectrum grad-
ually changed in a [K+]-dependent manner with a clear isosbestic
point at 570 nm. The results indicated the conversion of K+-free
B3TAC to a K+-bound form, probably the 1:1 complex.9 Notably,
B3TAC was capable of detecting 1 mM K+ even in the presence of
a high concentration of Na+. The photophysical properties of
B3TAC and its K+ complex are summarized in Table 1. Using cresyl
rescence with other metal ions such as Li+, Mg2+
,
Ca2+ (at
50 mM), Zn2+, Fe3+ and Cu2+ (at 100
lM). Importantly, however,
B3TAC recognized K+ even in the presence of these ions, and the
fluorescence intensity of the K+-added sample was not greatly af-
fected by the co-presence of the other ions. On the other hand,
strong fluorescence was observed in the presence of Rb+ or Cs+
(at 50 mM), which are alkali metal ions with a larger radius than
K+; these ions are known to be chelated by TAC.8 For biological
applications, however, this should not be a problem because the
predominant metal ions in extracellular and intracellular fluids
are Na+ and K+, respectively, and Rb+ and Cs+ are not present at rel-
evant concentrations. The pH dependence of the probe was also
examined, and it was confirmed that the effect of pH is negligible
in the physiological range (Fig. S6).
Although the mechanism of fluorescence quenching in the ab-
sence of K+ remains to be fully investigated, B3TAC is different
from previously reported TAC-based sensors7–10 in that the change
of fluorescence intensity is accompanied with a distinct color
change. This type of blue shift of the absorption spectrum in re-
sponse to the coordination of ions is a general phenomenon of
amine-containing BODIPYs and other fluorophores, and has been
attributed to the suppression of internal charge transfer
(ICT).3,16,24 However, in contrast to these typical ICT-based probes,
the shape of the excitation spectrum of B3TAC was not influenced
by [K+] (Figs. 1d and S2). In other words, the unbound form of
violet (
efficiency (
u
FL = 0.54 in methanol) as a standard,21 the quantum
FL) of B3TAC was determined to be 0.022 in buffer,
u
0.018 at 150 mM Na+ and 0.39 at 150 mM K+, but when the absorp-
tion wavelength change is taken into account, the effective activa-
tion of fluorescence intensity is much larger (Fig. 1e). High
concentrations of Na+ caused a small (ca. 3 nm) red shift of the
absorption peak and a slight decrease of quantum efficiency
compared to the buffer-only condition. Also, the fluorescence
intensity of B3TAC in the presence of 80 mM K+ was decreased to
some extent when >100 mM Na+ was added (Fig. S3). Although
500
450
400
350
300
250
200
150
100
50
0
Li+
Na+
Rb+
Cs+
Mg2+ Ca2+ Zn2+
Fe3+ Cu2+
Buffer
K+ 80 mM -
+
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
+
-
-
Figure 2. Ion selectivity of B3TAC. Emission spectra were measured with 1
l
M B3TAC dissolved in 5 mM HEPES (pH 7.0)/MeCN = 60:40 containing 2% DMSO as a co-solvent
(kex = 560 nm). Concentrations were as follows: Li+, Rb+, Cs+, Mg2+ and Ca2+ 50 mM, Na+ 150 mM, Zn2+, Fe3+ and Cu2+ 100
l
M, each as the ClO4 salt.
À

T. Hirata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6090–6093
6093
B3TAC appears to be quenched very efficiently by charge transfer
References and notes
from the fluorophore-linked amine of TAC, although some contri-
bution of PeT involving the other phenyl rings in the chelator can
not be excluded.
1. Terai, T.; Nagano, T. Curr. Opin. Chem. Biol. 2008, 12, 515.
2. The Molecular Probes Handbook; Johnson, . I., Spence, M. T. Z., Eds., 11th ed.; Life
Technologies: Carlsbad, 2010.
In conclusion, we have developed a novel fluorescent probe for
K+, B3TAC, which possesses many desirable properties for biologi-
cal applications, including high selectivity for K+, suitability for col-
orimetric detection, long excitation wavelength, solubility in
aqueous media, and large fluorescence increase in response to K+.
This probe was able to sense physiological concentrations of K+
with high values of signal-to-noise ratio, enabling quantification
of potassium concentration in blood or cell lysate in the presence
of some organic solvent to improve the solubility. A change of KDapp
value in pure water cannot be ruled out, but we believe that
improving the water solubility of B3TAC by appropriate modifica-
tion of the fluorophore in the future should allow for intracellular
or extracellular [K+] measurement in terms of fluorescence change
in the red wavelength region even in the absence of organic sol-
vent. Furthermore, to our knowledge, B3TAC is the first colorimet-
ric K+ sensor that allows for naked-eye detection of the ion in
aqueous media. With these properties, B3TAC should serve as a
potentially useful tool to measure [K+] in solution.
3. Grzegorz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 3440.
4. Shieh, C. C.; Coghlan, M.; Sullivan, J. P.; Gopalakrishman, M. Pharmacol. Rev.
2000, 52, 557.
5. Bonnet, S.; Archer, S. L.; Allalunis-Turner, J.; Haromy, A.; Beaulieu, C.;
Thompson, R.; Lee, C. T.; Lopaschuk, G. D.; Puttagunta, L.; Bonnet, S.; Harry,
G.; Hashimoto, K.; Porter, C. J.; Andrade, M. A.; Thebaud, B.; Michelakis, E. D.
Cancer Cell 2007, 11, 37.
6. Minta, A.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 19449.
7. He, H.; Mortellaro, M. A.; Leiner, M. J. P.; Fraatz, R. J.; Tusa, J. K. J. Am. Chem. Soc.
2003, 125, 1468.
8. Padmawar, P.; Yao, X.; Bloch, O.; Manley, G. T.; Verkman, A. S. Nat. Methods
2005, 2, 825.
9. Magzoub, M.; Padmawar, P.; Dix, J. A.; Verkman, A. S. J. Phys. Chem. 2006, 110,
21216.
10. Namkung, W.; Padmawar, P.; Mills, A. D.; Verkman, A. S. J. Am. Chem. Soc. 2008,
130, 7794.
11. Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima, H.; Kikuchi, K.;
Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 14079.
12. Zhang, C.; Suslick, K. S. J. Am. Chem. Soc. 2005, 127, 11548.
13. Palacios, M. A.; Nishiyabu, R.; Marquez, M.; Anzenbacher, P., Jr. J. Am. Chem. Soc.
2007, 129, 7538.
14. Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem., Int. Ed. 2008, 47, 1184.
15. Wang, D.; Shiraishi, Y.; Hirai, T. Chem. Commun. 2011, 47, 2673.
16. Atilgan, S.; Ozdemir, T.; Akkaya, E. U. Org. Lett. 2010, 12, 4792.
17. Shiraishi, Y.; Maehara, H.; Sugii, T.; Wang, D.; Hirai, T. Tetrahedron Lett. 2009,
50, 4293.
Acknowledgments
18. Lee, J. S.; Kim, H. K.; Feng, S.; Vendrell, M.; Chang, Y. T. Chem. Commun. 2011, 47,
2339.
This work was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology of Japan (Grant Nos.
22000006 to T.N., 21659024 to K.H. and 21750135 to T.T.), and
by a grant from the New Energy and Industrial Technology Devel-
opment Organization (NEDO) of Japan (to T.T.). T.T. was also sup-
ported by the Cosmetology Research Foundation, Japan.
19. Excitation at 580 nm gave essentially the same results, as shown in Fig. S2.
20. The emission spectrum showed a minor peak at 616 nm, but the shape of the
excitation spectrum with kem = 616 nm was the same as that with
kem = 570 nm (Fig. S2).
21. Magde, D.; Brannon, J. H.; Cremers, T. L.; Olmsted, J. I. I. I. J. Phys. Chem. 1979,
83, 696.
22. Kamoto, M.; Umezawa, N.; Kato, N.; Higuchi, T. Chem. Eur. J. 2008, 14, 8004.
23. Neuroscience: Exploring the Brain; Bear, M. F., Connors, B. W., Paradiso, M. A.,
Eds., 3rd ed.; Lippincott Williams & Wilkins/Wolters Kluwer Health: Chicago,
2007.
24. Baruah, M.; Qin, W.; Vallee, R. A. L.; Beljonne, D.; Rohand, T.; Dehaen, W.;
Boens, N. Org. Lett. 2005, 7, 4377.
Supplementary data
Supplementary data (detailed synthetic procedure and charac-
terization of B3TAC, methods of optical measurements, Figs. S1–
S6) associated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2011.08.056.