Unmodified fluorescein as a fluorescent chemosensor for fluoride ion detection

Article abstract of DOI:10.1016/j.tetlet.2007.10.086

Unmodified fluorescein (1) behaves as a fluorescent chemosensor for F^- detection, where the F^--induced fluorescence enhancement is driven by a transfer of the phenolic OH protons to F^-.

Full text of DOI:10.1016/j.tetlet.2007.10.086

Available online at www.sciencedirect.com
Tetrahedron Letters 48 (2007) 8803–8806
Unmodified fluorescein as a fluorescent chemosensor
for fluoride ion detection
Xuan Zhang, Yasuhiro Shiraishi* and Takayuki Hirai
Research Center for Solar Energy Chemistry, and Division of Chemical Engineering, Graduate School of Engineering Science,
Osaka University, Toyonaka 560-8531, Japan
Received 12 September 2007; revised 12 October 2007; accepted 18 October 2007
Available online 22 October 2007
Abstract—Unmodified fluorescein (1) behaves as a fluorescent chemosensor for Fꢀ detection, where the Fꢀ-induced fluorescence
enhancement is driven by a transfer of the phenolic OH protons to Fꢀ.
Ó 2007 Elsevier Ltd. All rights reserved.
The recognition and sensing of anions have attracted a
great deal of attention because of biological and
environmental importance of anions.1 Fluoride ion
(Fꢀ) is one of the most important anions due to its
pivotal roles in dental care and the treatment of osteo-
porosis.2 Design of fluorescent probe for Fꢀ detection
has therefore attracted much attention due to high
sensitivity and simplicity of the fluorescence analysis.3
a formation of the spirocycle-opened ‘emissive’ fluores-
cein species. Very recently, a fluorescein bearing a thio-
urea group was synthesized by Kim et al.,7c which shows
an anion-induced fluorescence enhancement. In that,
hydrogen-bonding interaction between anion and thio-
urea group leads to a spirocycle opening of the fluores-
cein moiety, resulting in fluorescence enhancement.
Needless to say, practical anion sensing requires inex-
pensive and easily preparable sensors;8 however, all of
these fluorescein-based anion sensors require a synthesis
step for sensor preparation.7
Fluorescein is a dye used extensively as bio-labeling
reagents and fluorescent probes due to its excellent
photophysical properties, such as long-wavelength
absorption and emission, high fluorescence quantum
yield, and high stability against light.4 Considerable
effort has been devoted to the development of fluores-
cent probes for reactive oxygen species (ROS)5 and
metal cations6 based on the fluorescein platform. There
are, however, only three reports of fluorescein-based
fluorescent anion sensor.7 Yoon et al.7a synthesized a
fluorescein derivative conjugated with boronic acid
and aminomethyl groups. This material shows a Fꢀ-
induced emission enhancement due to a suppression of
the photoinduced electron transfer from the amine
nitrogen to the photoexcited fluorescein moiety by a
cooperative coordination of Fꢀ with the ligand groups.
Yang et al.7b synthesized a fluorescein derivative whose
two phenolic OH are protected by tert-butyldimethylsi-
lyl (TBS) groups. This shows a Fꢀ-induced fluorescence
enhancement due to the deprotection of TBS, leading to
Here we report that a commercially-available ‘unmodi-
fied’ fluorescein (1, Fig. 1) behaves as a fluorescent
chemosensor for Fꢀ detection, enabling selective Fꢀ
sensing among the halide anions. We describe here that
the strong fluorescence enhancement of 1 by Fꢀ is sim-
ply triggered by a transfer of the phenolic OH protons to
Fꢀ, leading to a formation of the spirocycle-opened
emissive anionic species.
As shown in Figure 2, 1 (1 lM) dissolved in acetonitrile
(MeCN) is nonfluorescent. Addition of 10 equiv of Fꢀ
(as a n-Bu4N+ salt) to the solution, however, leads to
O
COOEt
COOMe
O
HO
O
OH
O
O
OH
O
O
OMe
2
3
1
*
Corresponding author. Tel.: +81 6 6850 6271; fax: +81 6 6850
6273; e-mail: shiraish@cheng.es.osaka-u.ac.jp
Figure 1. Structures of fluorescein (1) and its derivatives (2, 3).
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.10.086

8804
X. Zhang et al. / Tetrahedron Letters 48 (2007) 8803–8806
F−
600
400
200
0
AcO−
F−
0.8
1 only, Cl−, Br−,
I−, HSO4−
H2PO4−
0.6
AcO−
500
550
600
650
0.4
0.2
0.0
λ / nm
1 only, Cl−, Br−,
Figure 2. Fluorescence spectra of 1 (1 lM) in MeCN measured with
10 equiv of respective anions as n-Bu4N+ salt (kex = 480 nm). (Inset)
change in fluorescence color.
−
I−, HSO4
−
H2PO4
300
350
400
450
500
550
λ / nm
an appearance of a strong green fluorescence at 500–
600 nm. Moderate and weak emission enhancement is
Figure 4. Absorption spectra of 1 (10 lM) measured in MeCN with
4 equiv of respective anions. (Inset) Color change.
observed for AcOꢀ and H2PO ꢀ, respectively, whereas
4
no ꢀ enhancement is observed for other anions
ðCl ; Brꢀ; Iꢀ; and HSO4ꢀÞ. As shown in Figure 3, fluo-
rescence titration with Fꢀ reveals that the fluorescence
enhaꢀncement is saturated upon the addition of 10 equiv
of F , where the fluorescence quantum yield is 0.61.9
The emission enhancement is determined to be 2100-
fold, which is the highest value among the reported
fluorescein-based fluorescent Fꢀ sensors.7 In contrast,
addition of even 100 equiv of other halide anions (Clꢀ,
Brꢀ, and Iꢀ) shows no emission enhancement (Fig. 3).
Notably, as shown in Figure S1,10 the fluorescence
response of 1 toward Fꢀ is unaffected by the presence
of other halide anions (Clꢀ, Brꢀ, and Iꢀ), indicating that
1 is potentially available for selective Fꢀ detection
among the halide anions.
to an appearance of a strong absorption (kmax
=
514 nm), along with a clear color change of the solution
from colorless to yellow-green. Moderate and weak
absorption increase is observed for AcOꢀ and
H2PO4ꢀ, respectively, but almost no change is observed
for other anions ðClꢀ; Brꢀ; Iꢀ; and HSO4ꢀÞ.
As shown in Figure 5, absorption titration of 1 with Fꢀ
reveals that addition of first 2 equiv of Fꢀ leads to an
increase in the entire absorption at 400–550 nm (see blue
line). Further addition of 2 equiv of Fꢀ (see red line)
leads to further increase in 465–550 nm absorbance,
but also leads to a decrease in 400–465 nm absorbance
with an isosbestic point at 465 nm. These findings imply
that two kinds of fluorescein species form in response to
the interaction between 1 and Fꢀ with 1:2 and 1:4 stoi-
chiometry. This is confirmed by the Job’s plots of 1 with
Fꢀ: as shown in Figure 6, 460 nm absorbance shows a
maximum at X (=[Fꢀ]/([Fꢀ] + [1])) = 0.66, whereas
Figure 4 shows absorption spectra of 1 (10 lM) in
MeCN. Without anions, 1 is colorless and exhibits
almost no absorption at 400–550 nm, indicating that 1
exists as a spirocycle-closed form. This is confirmed by
a distinctive spiro-carbon shift at 83.51 ppm in the 13C
NMR spectrum of 1.11 Addition of Fꢀ, however, leads
F−
F
11.0
[[F ]
F−
0.8
0.6
0.4
0.2
0.0
600
00.8
AAcO
O−
AcO−
44.0eq.
33.8eq.
00.6
[F−]
600
400
200
0
400
200
0
A
−
00.4
H2
P
O4
10eq.
9eq.
00.2
H2PO4−
C
l
,
B
r
,
I
,
H
S
O4
−
00.0
0
2
4
6
8
1
0
1
2
22.0eq.
Cl−, Br−, I−, HSO4−
60 80 100
[[Anionn] / [1]
0
20
40
1eq.
0eq.
[Anion] / [1]
00.2eq.
00.0eq.
300
350
400
450
500
550
500
550
600
650
λ / nm
λ / nm
Figure 5. Absorption titration of 1 (10 lM) with Fꢀ. (Inset) Change in
absorbance monitored at 514 nm. The detailed absorption spectrum
change: see Figure S2.10
Figure 3. Fluorescence titration of 1 (1 lM) with Fꢀ (kex = 480 nm).
(Inset) Change in fluorescence intensity (kem = 532 nm).

X. Zhang et al. / Tetrahedron Letters 48 (2007) 8803–8806
8805
ion, in which both phenolic OH protons of 1 are
removed. This means that anionic species of 1 are actu-
ally produced by the Fꢀ-induced proton removal. The
Fꢀ sensing mechanism of 1 can therefore be summarized
as Scheme 1: first 2 equiv of Fꢀ leads to removal of one
phenolic OH proton of 1 via a hydrogen-bonding inter-
action, resulting in a formation of a FHFꢀ dimer and
the emissive monoanion of 1.15 Another phenolic OH
proton of the monoanion is removed continuously with
further addition of 2 equiv of Fꢀ, forming FHFꢀ dimer
and the emissive dianion.
0.08
0.06
0.04
0.02
0.00
0.24
0.18
0.12
0.06
0.00
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
X ( = [F− ] / ([F−] + [1]))
The proposed Fꢀ sensing mechanism of 1 (Scheme 1) is
further confirmed by absorption and fluorescence
behaviors of the control compounds, 2 and 3 (Fig. 1;
see Materials10). Compound 2 has a spirocycle-open
form by the esterification of the carboxylic acid group
of 1; therefore, even without anions, 2 exhibits an
absorption at 400–500 nm (Figure S510). This absorp-
tion spectrum is similar to that of 1 obtained with
<2 equiv of Fꢀ (Fig. 5), meaning that the removal of
the phenolic OH proton of 1 actually leads to the spiro-
cycle opening (Scheme 1). Upon the addition of Fꢀ to 2,
a new absorption appears at 524 nm, along with a de-
crease in 400–475 nm absorbance (isosbestic point:
475 nm), as is also the case for 1 with 2–4 equiv of Fꢀ.
This means that the phenolic OH proton of 2 is also
removed by Fꢀ. Job’s plot of 2 with Fꢀ (Figure S610)
shows a maximum absorption at X = 0.66, indicating
that the proton removal of 2 by Fꢀ occurs in a 1:2 stoi-
chiometry.17 These indicate that 2 equiv of Fꢀ leads to
removal of one phenolic OH proton; this fact supports
the proposed deprotonation mechanism of 1 (Scheme
1). Compound 2 is weakly fluorescent, but addition of
Figure 6. Job’s plot for Fꢀ versus 1 in MeCN measured (closed circle)
at 514 nm and (open circle) at 460 nm. [Fꢀ] + [1] = 20 lM.
514 nm absorbance shows a maximum at X = 0.8. The
respective absorption spectra of 1 obtained with 2 and
4 equiv of Fꢀ (Fig. 5) are similar to those of the mono-
anion and dianion species of 1 observed in water.12 As
reported,12a,13 1 is emissive in the anionic forms, espe-
cially in the dianion form. These findings suggest that,
as proposed in Scheme 1, two phenolic OH protons of
1 are removed via the interaction with Fꢀ in the ground
state, resulting in the formation of ‘emissive’ two anionic
species.
For further confirmation of the proposed Fꢀ sensing
mechanism of 1, H NMR titration of 1 was carried
1
out with Fꢀ in DMSO-d6 (Figure S310). Upon the addi-
tion of 0.2 equiv of Fꢀ, phenolic OH proton of 1
(10.09 ppm) shifts downfield (11.02 ppm) with a signifi-
cant intensity decrease. This indicates the occurrence
of a hydrogen-bonding interaction between the phenolic
OH protons of 1 and Fꢀ.14 Addition of 2 equiv of Fꢀ
leads to a complete disappearance of the OH proton.
In addition, all of the aromatic protons of 1 shift upfield.
This means that net electron density of the aromatic ring
of 1 increases,15 indicative of the removal of phenolic
OH proton from 1. The upfield shift of the aromatic
protons almost stops upon the addition of 4 equiv of
Fꢀ, meaning that the two phenolic OH protons of 1
are completely removed at this stage. This result is con-
sistent with the absorption titration and the Job’s plot
data (Figs. 5 and 6). In addition, upon the addition of
>4 equiv of Fꢀ, a new triplet signal appears at
16.1 ppm (J = 121 Hz), which is ascribed to a FHFꢀ
dimer,1h,3d,g,16 meaning that the phenolic OH protons
of 1 are actually removed by Fꢀ. The removal of the
phenolic OH protons is also confirmed by ESI–MS anal-
ysis (Figure S410): the MS chart obtained with 1 and
5 equiv of Fꢀ in MeCN shows a strong peak at m/z
1056.8, which is ascribed to ([1ꢀ2H] + 3[n-Bu4N])+
Fꢀ leads to
a fluorescence enhancement (Figure
S710).18 This means that the removal of the phenolic
OH proton produces stronger emitting species; this also
supports the proposed mechanism (Scheme 1).
Compound 3 (Fig. 1) has a spirocycle-open form, where
both carboxylic acid and phenolic OH groups of 1 are
methylated. This compound does not show any absorp-
tion or fluorescence response to Fꢀ (Figure S1010). This
is because 3 does not produce anionic species due to the
lack of a phenolic OH proton. This finding again
support the proposed mechanism: the removal of the
phenolic OH proton of 1 by Fꢀ triggers the strong
fluorescence enhancement (Scheme 1).
In conclusion, we found that the unmodified ‘ready-
made’ fluorescein (1) behaves as a fluorescent Fꢀ sensor,
which shows potential for selective Fꢀ detection among
the halide anions. As is usually observed for the fluores-
cent anion sensors,3,7 the present fluorescein system has
difficulty in the application for aqueous samples.19 How-
ever, the fluorescein is photoexcited by visible light and
shows high fluorescence quantum yield and high sensi-
tivity; therefore, the fluorescein may be applicable as a
fluorescent Fꢀ sensor. The sensing mechanism clarified
here, which cleverly detects Fꢀ by simple proton transfer
processes, may contribute to the design of more effective
and more sensitive fluorescent anion sensor based on the
fluorescein platform.
O
FHF−
FHF−
O
O
2F−
OH
2F−
O
O
O
HO
O
O
O
OH
O
O
O
neutral
"nonfluorescent"
monoanion
"fluorescent"
dianion
"highly fluorescent"
Scheme 1. Proposed Fꢀ sensing mechanism of 1.

8806
X. Zhang et al. / Tetrahedron Letters 48 (2007) 8803–8806
5644–5645; (c) Hirano, T.; Kikuchi, K.; Urano, Y.;
Acknowledgment
Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2000, 122,
12399–12400; (d) Komatsu, H.; Iwasawa, N.; Citterio, D.;
Suzuki, Y.; Kubota, T.; Tokuno, K.; Kitamura, Y.; Oka,
K.; Suzuki, K. . J. Am. Chem. Soc. 2004, 126, 16353–
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Sheng, M.; Lippard, S. J. Inorg. Chem. 2006, 45, 9748–
9757.
This work was partly supported by Grants-in-Aid for
Scientific Research (No. 19760536) from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan (MEXT).
7. (a) Swamy, K. M. K.; Lee, Y. J.; Lee, H. N.; Chun, J.;
Kim, Y.; Kim, S.-J.; Yoon, J. J. Org. Chem. 2006, 71,
8626–8628; (b) Yang, X.-F.; Ye, S.-J.; Bai, Q.; Wang,
X.-Q. J. Fluoresc. 2007, 17, 81–87; (c) Wu, J.-S.; Kim, H.
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Tetrahedron Lett. 2007, 48, 3159–3162.
Supplementary data
Supplementary data (Materials and Figures S1–S16).
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2007.10.086.
8. Miyaji, H.; Sessler, J. L. Angew. Chem., Int. Ed. 2001, 40,
154–157.
9. The fluorescence quantum yield of fluorescein (0.85 in
0.1 M NaOH aqueous solution) was used as a standard:
Parker, C. A.; Rees, W. T. Analyst 1960, 85, 587–600, The
measurement was carried out at 300 K in an aerated
condition. The fluorescence quantum yield of fluorescein is
insensitive to oxygen.
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17. The formation of the anion form of compound 2 is
confirmed by 1H NMR titration (Fig. S8) and ESI–MS
(Fig. S9) analyses. Upon the addition of Fꢀ, the phenolic
OH proton of 2 at 11.06 ppm (in DMSO-d6) disappears
and all of the aromatic protons shift upfield, as is also the
case for 1. Triplet FHFꢀ dimer signals also appear at
16.1 ppm (J = 121 Hz). ESI–MS analysis of a MeCN
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