Design and synthesis of a novel fluorescence probe for Zn2+ based on the spirolactam ring-opening process of rhodamine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.05.074

The spirolactam ring-opening process of rhodamine derivative is one of the most useful mechanisms for controlling fluorescence properties. However, the open/closed equilibrium reaction of rhodamine spirolactam has not been well characterized. Therefore, w

Full text of DOI:10.1016/j.bmc.2010.05.074

Bioorganic & Medicinal Chemistry 19 (2011) 1072–1078
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Bioorganic & Medicinal Chemistry
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Design and synthesis of a novel fluorescence probe for Zn2+ based on the
spirolactam ring-opening process of rhodamine derivatives
Hiromi Sasaki a,b, Kenjiro Hanaoka a,b, Yasuteru Urano a, Takuya Terai a,b, Tetsuo Nagano a,b,
*
a Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
b CREST, Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The spirolactam ring-opening process of rhodamine derivative is one of the most useful mechanisms for
controlling fluorescence properties. However, the open/closed equilibrium reaction of rhodamine spiro-
lactam has not been well characterized. Therefore, we examined the relationship between the spirolac-
tam ring-opening process of rhodamine derivatives and the structure of the xanthene moiety. Based on
the results of this investigation, we selected a candidate xanthene moiety for a Zn2+ sensor, and success-
Received 2 March 2010
Revised 24 May 2010
Accepted 26 May 2010
Available online 9 June 2010
fully developed a new fluorescence probe for Zn2+
.
Keywords:
Fluorescence
Zinc ion
Ó 2010 Elsevier Ltd. All rights reserved.
Rhodamine
Sensors
1. Introduction
switching because of its modifiable structure. Moreover, from a
chemical and biological viewpoint, rhodamine is one of the most
attractive fluorescent dyes due to its excellent spectroscopic prop-
erties, such as longer wavelength (>500 nm), large molar extinc-
tion coefficient (>75,000 cmꢀ1 Mꢀ1), high fluorescence quantum
yield, and high photostability. The development of fluorescence
probes based on rhodamine spirolactam was pioneered by Czarnik
et al. in 1997.17 Since that work, many papers on fluorescence
probes utilizing rhodamine spirolactam-opening, mainly for metal
ions, have been reported.18–21
Fluorescence imaging is one of the most powerful techniques
for visualization of the temporal and spatial biological events in
living cells, and is employed in many fields of research. In particu-
lar, fluorescence sensor probes offer ease of use, high sensitivity,
and high selectivity in visualizing biomolecules. To develop novel
fluorescence sensor probes, it is important to find mechanisms
for controlling fluorescence properties. For example, widely used
mechanisms include Förster resonance energy transfer (FRET),1,2
intramolecular charge transfer (ICT),2–4 and photoinduced electron
transfer (PeT).2,5
In the present work, we focused on the spiro-ring-opening
reaction of xanthene fluorophores, such as fluorescein and rhoda-
mine. It is well-known that the C-9 position of xanthenes shows
high electrophilicity, therefore, and is readily attacked by a
nucleophilic substituent on the benzene moiety, resulting in
spiro-ring formation. Xanthene fluorophores in the spiro-ring
configuration are colorless and non-fluorescent. This unique
property can be used for fluorescence on/off switching, and in-
deed, has been employed in many fluorescence sensors for vari-
ous biomolecules.6–14
However, we thought that these probes leave room for
improvement. In general, there are several important requirements
for fluorescence sensor probes. Firstly, fluorescence probes for bioi-
maging are required to work in physiological conditions. Secondly,
it is better for sensors to work reversibly, because then it is possi-
ble to examine more complex behavior of a target biomolecule. Fi-
nally, although there are many reports on fluorescence sensors
utilizing the rhodamine spirolactam ring-opening process, the
mechanism of fluorescence on/off switching has not been well
characterized. Specifically, we investigated the influence of xan-
thene structure on the spirolactam ring-opening process, and uti-
lized the results to develop a fluorescence probe for Zn2+. There
were two reasons for selecting Zn2+ as a target; one is the impor-
tance of Zn2+ in physiology,22–24 and the other is that no probe
based on rhodamine spirolactam has yet been developed for
There are various types of spiro-ring configurations of xanthene
fluorophore, including (thio)spirolactam,15,16 (thio)spirolactone,6
and (thio)spirofuran.7 Among these, we chose the spirolactam con-
figuration of rhodamine as a good candidate for fluorescence on/off
detecting Zn2+ 25–29
Rhodamines in a spirolactam form have nei-
.
ther absorbance nor fluorescence in the visible region, so this kind
of fluorescence probes would potentially offer a better signal-to-
noise ratio in cellular applications.
* Corresponding author. Tel.: +81 3 5841 4850; fax: +81 3 5841 4855.
E-mail address: tlong@mol.f.u-tokyo.ac.jp (T. Nagano).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.074

H. Sasaki et al. / Bioorg. Med. Chem. 19 (2011) 1072–1078
1073
2. Results and discussion
Zn2+ chelating moiety
O
N
HO
O
OH
2.1. Zn2+ sensors based on symmetric rhodamines
4'
H
5'
6'
8
3'
N
2' OH
Based on published reports, we hypothesized that the opening
mechanism of rhodamine spirolactam upon addition of metal ions
is as shown in Scheme 1.17,19,30 Rhodamine B is mainly used as a
scaffold for such fluorescence sensor probes,9,12,18,20,21 but little is
known about the effect of variation of the xanthene moiety. In
other words, the relationship between the rhodamine spirolac-
tam-opening process and the structure of the xanthene moiety of
rhodamine is not been well characterized. Therefore, we designed
and synthesized three candidate Zn2+ sensors (5a–c) with symmet-
ric xanthene moieties (Fig. 1). Rhodamine 101, B, and 6G, which are
commercially available, were used as xanthene moieties, and novel
fluorescence sensors for Zn2+ (5a–c) were synthesized by the com-
bination of the rhodamine derivatives (4a–c) and the chelator moi-
ety, which is a ligand for Zn2+. The photochemical properties in
100 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfo-
nic acid) buffer (pH 7.4) of 4a–c are shown in Table 1. Compounds
5a–c were synthesized by treating rhodamine derivatives (4a–c)
with POCl3, followed by addition of the Zn2+ chelating moiety,
whose carboxylic acids were protected with tert-butyl groups.
After column chromatography using AcOEt/hexane as an eluent,
the tert-butyl groups of products were eliminated with TFA. The
products were purified by preparative HPLC in 30–50% yields. In
addition, the ethyl ester of rhodamine 6G was eliminated to obtain
4c. The synthetic schemes for 4c and 5a–c and details of the chem-
ical characterization of compounds are presented in Supplemen-
tary data. Although the spirolactam configuration of rhodamine
generally shows poor water solubility, the water solubility of 5a–
c was improved by incorporating two carboxylic acid groups into
the Zn2+ chelating moiety. The improved water solubility enables
us to use the probes under physiological conditions.
1'
1
9
O
O
R1
R1
R2
R1
R1
2
7
6
R2
R2
R
2 N
R3
O
N
N
R3
O
N
3
5
4
R4
R4
R4
R4
R3
R3
5a-c
4a-c
N
O
N
N
O
N
N
O
N
H
H
Rhodamine 101 (a)
Rhodamine B (b)
Rhodamine 6G (c)
Figure 1. Structures of rhodamine derivatives 4a–c and 5a–c. Compounds 5a–c
were synthesized by combination of 4a–c and a ligand for Zn2+
.
Table 1
Photochemical properties of 4a–c in 100 mM HEPES buffer (pH 7.4)
kabs (nm)
kem (nm)
ec (cmꢀ1 Mꢀ1
)
UFL
a
b
d
Compounds
4a
4b
4c
576
554
521
598
577
548
116,000
120,000
106,000
0.30
0.18
0.48
a
The maximum absorption wavelengths were measured in 100 mM HEPES
buffer (pH 7.4).
The maximum emission wavelengths were measured in 100 mM HEPES buffer
(pH 7.4).
The molar extinction coefficients were measured in 100 mM HEPES buffer (pH
7.4).
b
c
d
Fluorescence quantum efficiencies were measured in 100 mM HEPES buffer (pH
7.4) by using fluorescein in 0.1 N NaOH aq (UFL = 0.85) as a reference.
We assessed the spectroscopic characteristics of 5a–c under
physiological conditions (100 mM HEPES, pH 7.4; Fig. 2). The
absorbance intensity changes of 5a–c were monitored during
lue of 4a in 100 mM HEPES buffer (pH 7.4), the fraction of open
form of 5a was over 50% at pH 2.0 in 100 mM sodium phosphate
buffer, as was the case in the presence of Zn2+ (1.0 equiv). The acid
dissociation constant (pKa) of 5a, as judged from its pH-profile, was
Zn2+ addition (Fig. 2a). The absorbance of 5a (10
lM) increased sig-
nificantly upon addition of Zn2+ (0ꢀ1.0 equiv). We also measured
6.1. On the other hand, the absorbance of 5b (10
creased under acidic conditions, and its pH-profile was bell-
shaped. From the above value of 4b in 100 mM HEPES buffer
lM) hardly in-
the molar extinction coefficients (e) of rhodamine derivatives in
100 mM HEPES buffer (pH 7.4) (Table 1); the
e value of Rho101
e
(4a) in this buffer was 116,000. This value was used for calcula-
tion of the fraction of open form of 5a (=open form/(open for-
m + closed form)) (Scheme 1), that is, when the spirolactam ring
of 5a (10 lM) is completely open, the absorbance would be 1.16.
e
(pH 7.4), the fraction of open form of 5b was at most 6% at pH
4.5, at which 5b showed its absorbance maximum, in 100 mM so-
dium phosphate buffer. Compound 5b has two acid dissociation
constants, and the pKa1 and pKa2 of 5b obtained from the pH-pro-
From this calculation, the fraction of open form of 5a appeared to
be over 50% under these conditions. On the other hand, the absor-
file were 3.8 and 5.7. Further, the absorbance of 5c (10
creased remarkably under acidic conditions, and its pH-profile
was bell-shaped. As judged from the above value of 4c in
lM) in-
bance of 5b (10 lM) or 5c (10 lM) hardly increased upon addition
e
of Zn2+ (0ꢀ1.0 equiv). The molar extinction coefficients (
e) of 4b
100 mM HEPES buffer (pH 7.4), the fraction of open form of 5c
was nearly 50% at pH 3.0, at which 5c showed its absorbance max-
imum in 100 mM sodium phosphate buffer. The pKa value of 5c ob-
tained from its pH-profile was 4.3.
and 4c in 100 mM HEPES buffer (pH 7.4) were measured by us to
be 120,000 and 106,000, respectively, and the fractions of open
form of 5b and 5c were both less than 5%.
Thus, the absorbance changes in response to Zn2+ addition
(0ꢀ1.0 equiv) and acidic conditions (pH 2.0ꢀ7.0) were completely
different among the three compounds (5a–c). Compound 5b based
on rhodamine B, which has been most widely used as a basic scaf-
Furthermore, we monitored the absorbance intensity changes
of 5a–c in 100 mM sodium phosphate buffer at various pH values
(pH 2.0ꢀ12.0) ( Fig. 2b). The absorbance of 5a (10
lM) also in-
creased significantly under acidic conditions. From the above
e va-
Mn+
Mn+
O
N
O
O
Mn+
chelator
chelator
chelator
N
N
R2N
O
NR2
R2N
O
NR2
R2N
O
NR2
Open Form
Strongly Fluorescent
Closed Form
Closed Form
Non-Fluorescent
Non-Fluorescent
Scheme 1. Proposed mechanism of rhodamine spirolactam-opening process upon addition of a metal ion.

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H. Sasaki et al. / Bioorg. Med. Chem. 19 (2011) 1072–1078
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
a
Zn2+ chelating moiety
HO
O
N
O
OH
H
OH
N
O
O
R1
R2
R5
R1
N
R5
R2
R6
R6
N
O
N
R7
O
N
R7
R4
R8
R4
R8
R3
R3
5d-f
4d-f
N
O
N
N
H
O
N
N
H
O
N
0
2
4
6
8
10
[Zn2+] (µM)
Rhodamine 101/B (d)
Rhodamine B/6G (e)
Rhodamine 101/6G (f)
Figure 3. Structures of asymmetric rhodamine derivatives (4d–f and 5d–f).
1.0
0.8
0.6
0.4
0.2
0
b
erties, that is, lower background and higher spirolactam-opening
fractions in the presence of Zn2+. So, we designed and synthesized
asymmetric rhodamine 101/B (4d), rhodamine B/6G (4e), and rho-
damine 101/6G (4f) ( Fig. 3). The photochemical properties in
100 mM HEPES buffer (pH 7.4) of 4d–f are shown in Table 2. Next,
we synthesized asymmetric rhodamine derivatives (5d–f) based
on 4d–f by using the same synthetic scheme as for 5a–c (Fig. 3).
The same Zn2+ chelating moiety as in 5a–c was also used for 5d–
f. The synthetic schemes for 4d–f and 5d–f and details of their
chemical characterization are provided in the Supplementary data.
We performed spectroscopic analysis of 5d–f under physiolog-
ical conditions (100 mM HEPES, pH 7.4; Fig. 4), in the same way as
for 5a–c. The absorbance intensity changes of 5d–f were moni-
2
4
6
8
10
12
pH
tored during Zn2+ addition. The absorbance of 5d (10
lM) in-
Figure 2. (a) Absorption changes of 10 lM 5a (591 nm; blue, ), 5b (570 nm; red,
), and 5c (539 nm; green, ) in 100 mM HEPES buffer (pH 7.4) containing 0.1%
creased upon addition of Zn2+ (0ꢀ1.0 equiv), and its absorbance
DMSO as a cosolvent in the presence of various amounts of Zn2+ (0ꢀ1.0 equiv). (b)
with 1.0 equiv of Zn2+ was intermediate between those of 5a and
Absorption changes of 10
(530 nm; green,
lM 5a (586 nm; blue, ), 5b (562 nm; red, ), and 5c
)
at various pH values (pH 2.0ꢀ12.0) in 100 mM sodium
5b. The molar extinction coefficient (e) of 4d in 100 mM HEPES
buffer (pH 7.4) was measured to be 125,000 (Table 2), and the frac-
tion of open form of 5d was approximately 20% under these condi-
phosphate buffer containing 0.1% DMSO as a cosolvent. These samples were kept at
room temperature for 2 h before measurements.
tions. On the other hand, the absorbance of 5e (10 lM) hardly
increased upon addition of Zn2+ (0ꢀ1.0 equiv) compared with 5d
fold for fluorescence sensor probes based on rhodamine spirolac-
tam until now, was hardly open in the presence of Zn2+ or under
acidic conditions. On the other hand, 5a based on rhodamine 101
showed large absorption responses to both Zn2+ and pH compared
with 5b. Compound 5c based on rhodamine 6G was unique, being
not quite open upon addition of Zn2+, but considerably open in acid
conditions. As 5a–c have the same Zn2+ chelator, it is considered
that the difference of responses to Zn2+ and pH between 5a–c are
due to the xanthene moieties of 5a–c. We think that the xanthene
substituents in 5a preferentially stabilize the cationic open form
compared with 5b and 5c.
Compounds 5b and 5c decolorized under strong acidic condi-
tions (5b: pKa1 = 3.8, 5c: pKa1 <2). A similar phenomenon was re-
ported by Belov et al.31 This is probably due to protonation of the
N atoms of the xanthene moiety, and it is considered that this pro-
tonation led to the formation of the spirolactam-ring via nucleo-
philic attack of the N atom of the amide moiety on the C-9
position of the xanthene moiety.
or 5f, and its absorbance with 1.0 equiv of Zn2+ was intermediate
between those of 5b and 5c. The molar extinction coefficient (e)
of 4e in 100 mM HEPES buffer (pH 7.4) was measured to be
107,000 (Table 2), and the fraction of open form of 5e was less than
5%. The absorbance of 5f (10 l
M) increased upon addition of Zn2+
(0–1.0 equiv), and its absorbance with 1.0 equiv of Zn2+ was inter-
mediate between those of 5a and 5c. The molar extinction coeffi-
cient (e) of 4f in 100 mM HEPES buffer (pH 7.4) was measured to
be 126,000 (Table 2), and the fraction of open form of 5f was
approximately 20%.
We also monitored the absorbance intensity changes of 5d–f in
100 mM sodium phosphate buffer at various pH values (pH
Table 2
Photochemical properties of 4d–f in 100 mM HEPES buffer (pH 7.4)
a
b
d
Compounds
kabs (nm)
kem (nm)
ec (cmꢀ1 Mꢀ1
)
UFL
4d
4e
4f
566
539
554
587
566
580
125,000
107,000
126,000
0.34
0.12
0.32
2.2. Zn2+ sensors based on asymmetric rhodamines
a
As described above, 5a, 5b, and 5c showed interesting features
in the spirolactam-ring-opening reaction. However, these three
rhodamine derivatives were not suitable for use as Zn2+ sensor, be-
cause 5a had high background absorbance and fluorescence, while
the spirolactam ring of 5b or 5c was hardly opened upon addition
of Zn2+. From these results, we expected that asymmetric rhoda-
mine derivatives, 5d and 5f (Fig. 3), might have intermediate prop-
The maximum absorption wavelengths were measured in 100 mM HEPES
buffer (pH 7.4).
The maximum emission wavelengths were measured in 100 mM HEPES buffer
(pH 7.4).
The molar extinction coefficients were measured in 100 mM HEPES buffer (pH
7.4).
Fluorescence quantum efficiencies were measured in 100 mM HEPES buffer (pH
7.4) by using fluorescein in 0.1 N NaOH aq (UFL = 0.85) as a reference.
b
c
d

H. Sasaki et al. / Bioorg. Med. Chem. 19 (2011) 1072–1078
1075
pected. The pKa values of 5d–f also basically lay in the middle of
those of 5a–c.
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
a
2.3. Fluorescence and chemical properties of 5f
Among 5a–f, 5f is the most suitable compound for use as a Zn2+
sensor because of its approximately 12-fold enhancement in both
absorbance at 571 nm and fluorescence intensity at 595 nm; this
enhancement is the largest among the six compounds (5a–f) (Table
S2 and Fig. S3, Supplementary data). Therefore, we chose 5f as a
fluorescence probe for Zn2+, and its photochemical properties in
the presence of Zn2+ were examined in detail. The UV–vis absorp-
tion and fluorescence emission spectral changes of 5f (10 lM) dur-
0
2
4
6
M)
8
10
ing Zn2+ addition (0ꢀ1.0 equiv) in 100 mM HEPES buffer (pH 7.4)
are shown in Figure 5. Compound 5f displayed a major absorption
band centered at k = 571 nm, and the fluorescence emission spec-
trum of 5f had a major band at 595 nm. The fluorescence emission
intensity of 5f remained almost at a plateau level in the presence of
an excess of Zn2+ (the inset of Fig. 5b). Compound 5f achieved equi-
librium between open and closed forms upon addition of Zn2+
within 30 s (Fig. S7, Supplementary data). Photographs of absor-
bance and fluorescence of a solution of 5f (the inset of Fig. 5a
and b) show the marked color change between without Zn2+ and
with Zn2+ (1.0 equiv). Further, the Job’s plot of the absorbance of
5f at 571 nm indicates that the coordination of 5f with Zn2+ occurs
[Zn2+] (
µ
b 1.0
0.8
0.6
0.4
0.2
0
2
4
6
8
10
12
pH
Figure 4. (a) Absorption changes of 10
pink, ), and 5f (571 nm; light green,
containing 0.1% DMSO as a cosolvent upon addition of various amounts of Zn2+
M 5d (574 nm; light blue, ), 5e
lM 5d (581 nm; light blue, ), 5e (557 nm;
)
in 100 mM HEPES buffer (pH 7.4)
(0ꢀ1.0 equiv). (b) Absorption changes of 10
l
(547 nm; pink, ), and 5f (564 nm; light green, ) at various pH values in 100 mM
sodium phosphate buffer containing 0.1% DMSO as a cosolvent. These samples were
kept at room temperature for 2 h before measurements.
2.0ꢀ12.0) (Fig. 4b). The absorbance of 5d (10
acidic conditions, and its pH-profile was also bell-shaped. From the
above value of 4d in 100 mM HEPES buffer (pH 7.4), the fraction
lM) increased under
e
of open form of 5d was approximately 25% at pH 3.5, at which 5d
showed its absorbance maximum in 100 mM sodium phosphate
buffer, and this fraction was intermediate between those of 5a
and 5b. The pKa value of 5d obtained from its pH-profile was 5.5.
On the other hand, the absorbance of 5e (10
slightly compared with that of 5d or 5f in acidic conditions, and
its pH-profile was bell-shaped. From the above value of 4e in
lM) was increased
e
100 mM HEPES buffer (pH 7.4), the fraction of open form of 5e
was around 12% at pH 3.5, at which 5e showed its maximum
absorbance in 100 mM sodium phosphate buffer. This fraction of
5e was intermediate between those of 5b and 5c. Compound 5e
has two acid dissociation constants, and pKa1 and pKa2 of 5e ob-
tained from its pH-profile were 2.2 and 5.1. Further, the absor-
bance of 5f (10
that of 5e under acidic conditions. From the above
l
M) was increased significantly compared with
value of 4f
e
in 100 mM HEPES buffer (pH 7.4), the fraction of open form of 5f
was over 50% at pH 2.0. This fraction of 5f was intermediate be-
tween those of 5a and 5c, and the pKa value of 5f obtained from
its pH-profile was 5.2.
Figure 5. (a) Absorption spectra of 5f (10 lM) in 100 mM HEPES buffer (pH 7.4) in
the presence of various amounts of Zn2+ (0ꢀ1.0 equiv). Inset: the Job’s plot; the total
The spirolactam-ring-opening processes in response to Zn2+
(0ꢀ1.0 equiv) and pH (2.0ꢀ12.0) of the above three asymmetric
compounds (5d–f) had the intermediate properties between those
of symmetric rhodamine derivatives (5a–c). In particular, 5d based
on 4a and 4b, and 5f based on 4a and 4c, both had lower back-
ground compared with 5a, and their fractions of open form were
larger than that of 5b or 5c upon addition of Zn2+, as we had ex-
concentration of ([Zn2+] + [5f]) was 10
lM. The photograph shows the color change
of 5f (10 l
M) upon addition of Zn2+ (0 equiv or 1.0 equiv) in 100 mM HEPES buffer
(pH 7.4). (b) Fluorescence emission spectra of 5f (10 lM) in 100 mM HEPES buffer
(pH 7.4) with various amounts of Zn2+ (0ꢀ1.0 equiv). The excitation wavelength
was 565 nm. Inset: fluorescence intensities at 595 nm of 5f (10 lM) with various
amounts of Zn2+ (0ꢀ2.0 equiv). The photograph shows the fluorescence color of 5f
(10 l
M) upon addition of Zn2+ (0 equiv or 1.0 equiv) in 100 mM HEPES buffer (pH
7.4) with 365 nm excitation.

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H. Sasaki et al. / Bioorg. Med. Chem. 19 (2011) 1072–1078
to form a 1:1 complex (the inset of Fig. 5a). The apparent dissoci-
On the other hand, fluorescence emission enhancement of 5f
ation constant Kd of 5f for Zn2+ was calculated to be 4.1
lM from
(10 l
M) was observed only upon addition of Zn2+, showing high
the Zn2+ titration curves of fluorescence intensity at 594 nm
(Fig. S4, Supplementary data), and this Kd value is sufficiently small
for biological applications.23 The values of fluorescence quantum
efficiency (UFL) of 5f measured with fluorescein in 0.1 N NaOH aq
selectivity for Zn2+ ( Fig. 6a and Fig. S6c, Supplementary data).
The fluorescence intensity of 5f was weakened by the addition of
several cations (particularly Ni2+, Cu2+, and Co2+) together with
Zn2+ in comparison with that upon addition of Zn2+ alone
,
(
UFL = 0.85) as a reference were 0.13 in the presence of Zn2+
(Fig. 6b). However, Zn2+ can be distinguished from those heavy
metals, since only Zn2+ chelation enhances the fluorescence inten-
sity. In addition, although Zn2+ sensors generally show poor selec-
tivity between Zn2+ and Cd2+, 5f did not show any fluorescence
enhancement upon addition of Cd2+. The equilibrium of 5f between
open and closed forms upon addition of Cd2+ also did not change
(1.0 equiv with respect to 5f) in 100 mM HEPES buffer (pH 7.4)
and 0.28 in 100 mM sodium phosphate buffer (pH 2.0) (Table S3,
Supplementary data). In addition, we added an aqueous solution
of ethylenediamine-N,N,N0,N0-tetraacetic acid, disodium salt (ED-
TAꢁ2Na) to 5f (10
l
M) with 1.0 equiv of Zn2+ in 100 mM HEPES buf-
fer (pH 7.4) to examine the reversibility of complex formation of 5f
and Zn2+ (Table S4 and Fig. S5, Supplementary data). When EDTA
(1.0 equiv with respect to 5f) was added to the solution, the color
of solution changed from purple to colorless. Next, when Zn2+
(1.0 equiv with respect to 5f) was further added to the solution,
the purple color was recovered. This result indicated that 5f could
for at least 10 min (Fig. S7, Supplementary data). Moreover, Ca2+
,
Mg2+, Na+, and K+, which exist at high concentrations in living cells,
also did not enhance the fluorescence intensity of 5f (Fig. 6 and
Fig. S6, Supplementary data). These results indicate that 5f has
high selectivity for Zn2+ over various cations, especially those likely
to be encountered in biological applications.
work as a reversible chemosensor for Zn2+
.
Furthermore, the effect of adding various heavy metal ions
(1.0 equiv with respect to 5f), Ca2+ (5 mM), Mg2+ (5 mM), Na+
(100 mM), and K+ (100 mM) on the absorbance and the fluores-
cence intensity of 5f was examined in 100 mM HEPES buffer (pH
7.4) (Fig. 6 and Fig. S6, Supplementary data). Absorbance change
3. Conclusion
In conclusion, we anticipated that the open/closed equilibrium
reaction of rhodamine spirolactam would be profoundly influenced
by the xanthene structure of rhodamine derivatives. Therefore, we
designed and synthesized a series of Zn2+ sensors based on sym-
metric or asymmetric rhodamine derivatives (5a–f), and examined
the changes of their absorbance and fluorescence spectra in re-
of 5f (10
l
M) was observed upon addition of Zn2+, Ni2+,Co2+, or
Cu2+ (1.0 equiv with respect to 5f) (Fig. S6ab, Supplementary data).
sponse to ring-opening induced by Zn2+
.
We selected the rhodamine derivative 5f as a candidate fluores-
cence probe for Zn2+, on the basis of the above results, and con-
firmed that 5f could selectively recognize Zn2+ in physiological
solution in terms of fluorescence responses. In addition, the Job’s
plot showed that 5f formed a 1:1 complex with Zn2+, and the
reversibility of the reaction of 5f with Zn2+ was confirmed by treat-
ment with EDTA followed by further addition of Zn2+. Compound
5f also showed high selectivity over various cations, including
Cd2+, which most Zn2+ sensor probes are unable to distinguish from
Zn2+. There have been many reports of rhodamine-based on/off
a
1600
1200
800
400
0
Zn2+
5f only, Ni2+,Mn2+,
Co2+, Cd2+, Fe2+, Fe3+,
Ca2+, Mg2+, Na+, K+
Cu2+
fluorescence sensor molecules for various cations, such as Hg2+
,
Cu2+, and Fe3+, by using the spirolactam-ring-opening process.
However, compound 5f is the first Zn2+-selective fluorescence sen-
sor molecule based on the spirolactam-ring-opening process of
rhodamines. We believe our design strategy for the rhodamine spi-
rolactam-ring has great potential for further development of excel-
lent fluorescence probes, and could be applied to develop
fluorescence sensors for other molecules of interest in biological
applications.
550
600
650
700
Wavelength (nm)
b
1600
1200
800
400
0
4. Experimental
4.1. Materials
All reagents and solvents were of the best commercial grade,
supplied by Tokyo Chemical Industry Co. Ltd (Tokyo, Japan), Wako
Pure Chemical Industries, Ltd (Osaka, Japan), or Aldrich Chemical
Co. Inc. (Milwaukee, WI), and were used without further purifica-
tion. Rhodamine 101 was purchased from Acros Organics (Geel, Bel-
gium). Rhodamine B and rhodamine 6G were purchased from Tokyo
Chemical Industry Co, Ltd (Tokyo, Japan). All other rhodamines
were prepared as described in the Supplementary data. Water
(H2O, fluorometric grade), dimethyl sulfoxide (DMSO, fluorometric
grade), dimethyl formamide (DMF, fluorometric grade), methyl
alcohol (MeOH, fluorometric grade), acetonitrile (CH3CN, fluoro-
metric grade), acetone (fluorometric grade), methylene chloride
(CH2Cl2, fluorometric grade), chloroform (CHCl3, fluorometric
Figure 6. (a) Fluorescence emission spectra of 5f (10
l
M) in the presence of various
cations. (b) Bars indicate the fluorescence intensities of 5f (10
at 594 nm in the presence of various cations. Colored bars: each cation was added.
Colorless bars: each cation was added in the presence of Zn2+ (10
M). Heavy metal
ions (1.0 equiv. with respect to 5f) were added as NiSO4, MnSO4, CoSO4, CdSO4,
CuSO4, FeCl2, and FeCl3. Other cations were added as ZnSO4 (10 M), CaCl2 (5 mM),
lM) aqueous solution
l
l
MgSO4 (5 mM), NaNO3 (100 mM), and KNO3 (100 mM). All samples were measured
in 100 mM HEPES buffer (pH 7.4) with 565 nm excitation.

H. Sasaki et al. / Bioorg. Med. Chem. 19 (2011) 1072–1078
1077
grade), tetrahydrofuran (THF, fluorometric grade), toluene
(fluorometric grade), benzene (fluorometric grade), 2-[4-(2-
hydroxymethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), and
ethylenediamine-N,N,N0,N0-tetraacetic acid, disodium salt, dehy-
drate (EDTAꢁ2Na) used for spectrometric measurements were pur-
chased from Dojindo (Kumamoto, Japan). Silica gel column
chromatography was performed using Chromatorex-NH (Fuji Sily-
sia Chemical Ltd, Aichi, Japan), Silica Gel 60 (Kanto Chemical Co.
Inc., Tokyo, Japan), or Silica Gel 60 N (Kanto Chemical Co. Inc., To-
kyo, Japan).
[Zn2+
8.4, 9.0, 9.3, 9.6, 9.7.
[Zn2+
M): 0.040, 0.10, 0.16, 0.40, 0.63, 2.5, 4.0, 6.3, 10, 16,
]total (mM): 0.085, 0.21, 0.33, 0.80, 1.2, 3.5, 4.6, 5.8, 6.8, 7.7,
]
free
(l
25, 40, 63, 100, 160.
4.5. Metal ion selectivity measurements
The absorption and the fluorescence emission enhancement of 5f
were measured in 100 mM HEPES buffer (pH 7.4) (excitation
565 nm). Heavy metals (10
CoSO4, CdSO4, CuSO4, FeCl2, and FeCl3. Other cations were added as
ZnSO4 (10 M), CaCl2 (5 mM), MgSO4 (5 mM), NaNO3 (100 mM),
lM) were added as NiSO4, MnSO4,
4.2. Instruments and general methods
l
and KNO3 (100 mM).
HPLC preparation or analysis was performed on a reverse-phase
column (Inertsil ODS-3 10 mm ꢂ 250 mm for purification and
Inertsil ODS-3 4.6 ꢂ 250 mm for analysis; GL Sciences (Tokyo,
Japan)) using eluent A (H2O containing 0.1% TFA (v/v)) and eluent
B (CH3CN with 20% H2O containing 0.1% TFA (v/v)), fitted on a Jasco
PU-2080 system. NMR spectra were recorded on a JNM-LA300 or
JNM-LA400 (JEOL Ltd, Tokyo, Japan) instrument at 300 MHz or
400 MHz for 1H NMR and at 75 MHz or 100 MHz for 13C NMR,
respectively. Mass spectra (MS) were measured with a JEOL JMS-
T100LC AccuTOF mass spectrometer (ESI+ or ESIꢀ).
Acknowledgments
This work was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology of the Japan (Grant Nos.
20689001, 19890047 and 21659024 to K.H., 19205021 and
20117003 to Y.U., and 21750135 to T.T.), and by a grant from the
Industrial Technology Development Organization (NEDO) of Japan
(to T.T.). K.H. was also supported by The Mochida Memorial Foun-
dation for Medical Pharmaceutical Research, and Sankyo Founda-
tion of Life Science.
4.3. Measurements of photochemical properties
Supplementary data
Absorption spectra were obtained with a UV–vis spectrometer
UV-1650 (Shimadzu Corp., Kyoto, Japan). Fluorescence spectro-
scopic studies were performed with a F-4500 Hitachi fluorescence
spectrophotometer (Hitachi, Ltd, Tokyo, Japan). The slit width was
2.5 nm for both excitation and emission. The photomultiplier volt-
age was 700 V. All experiments were carried out at 25 °C. Photo-
chemical properties of dyes were examined in 100 mM HEPES
buffer (pH 7.4) or 100 mM sodium phosphate buffer (pH
2.0ꢀ12.0) containing less than 1% (v/v) DMSO as a cosolvent. Val-
ues of relative quantum efficiency of fluorescence (UFL) were
determined based on fluorescein in 0.1 N NaOH aq (UFL = 0.85) as
a standard, using the following equation:
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.074.
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pKa1 ¼ 9:33; pKa2 ¼ 2:58; log KðZnLÞ ¼ 7:27
Protonation constants must be corrected upward by 0.11 when
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centration was calculated according to the method described in
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2þ
½Zn
ꢃ
¼ ½Zn2þꢃtotal=KðZnLÞ ꢂ aM ꢂ ½Lꢃ
ð1Þ
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free
a2Þ
aM ¼ 1 þ 10ðpHꢀpK Þ þ 10ð2pHꢀpK
a1
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2þ
½Lꢃfree ¼ ½Lꢃtotal ꢀ ½Zn
ꢃ
total
[L]total was 10 mM, and [Zn2+
]
was varied from 0 to 9.7 mM.
total
The value of [Zn2+
]
was obtained from Eq. 1.
free

1078
H. Sasaki et al. / Bioorg. Med. Chem. 19 (2011) 1072–1078
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