Development of a fluorescein analogue, TokyoMagenta, as a novel scaffold for fluorescence probes in red region

Article abstract of DOI:10.1039/c1cc00078k

We present a design strategy for fluorescence probes with a high off/on activation ratio in the red wavelength region, based on a novel fluorescein analogue in which the O atom at the 10 position of the xanthene chromophore is replaced with a Si atom. To

Full text of DOI:10.1039/c1cc00078k

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Development of a fluorescein analogue, TokyoMagenta, as a novel
scaffold for fluorescence probes in red regionw
Takahiro Egawa,a Yuichiro Koide,ab Kenjiro Hanaoka,ab Toru Komatsu,a Takuya Teraiab
and Tetsuo Nagano*ab
Received 5th January 2011, Accepted 11th February 2011
DOI: 10.1039/c1cc00078k
We present a design strategy for fluorescence probes with a high
off/on activation ratio in the red wavelength region, based on a
novel fluorescein analogue in which the O atom at the 10 position
of the xanthene chromophore is replaced with a Si atom. To
demonstrate the usefulness of this strategy, we designed and
synthesized a red-fluorescent probe for b-galactosidase, and
showed that it works in live HEK293 cells.
since we expected that they would be useful for dual-color
imaging with various kinds of indicators having green
fluorescence, such as GFP, fluorescein derivatives and so on.
Here, we describe the development of a novel fluorescein
analogue, TokyoMagenta (TM) (Fig. 1), in which the O atom
at the 10 position of the xanthene chromophore is replaced
with a Si atom (Scheme 1). Our synthetic strategy was to
synthesize the xanthene moiety from 3-bromoaniline (1).
To synthesize the dimer 3, a N atom is more suitable than
an O atom because of its high electron-donating ability, so
3-bromoaniline was chosen as a starting material instead of
3-bromophenol. As a protecting group, we chose allyl because
of its weak electron-withdrawing character. This results in less
influence on the dimerization reaction, and also permits the
Since the first fluorescence indicators for calcium ions were
reported by Tsien et al.,1 many fluorescence probes have been
developed and have contributed greatly to biological and
medical research.2 In particular, fluorescein (Fig. 1) has many
favorable characteristics, such as high water solubility,
high fluorescence quantum yield and high molar extinction
coefficient, and it has been utilized as the fluorescent core for a
large number of probes, including, for example, fluorescence
probes for metal ions3 and reactive oxygen and nitrogen
species.4 However, fluorescence probes based on fluorescein
and its derivatives, such as TokyoGreen (TG) derivatives
(Fig. 1), have only ‘green’ fluorescence. Therefore, we set out
to develop fluorescence probes in another color region, following
the report that Si-substituted pyronine has 90 nm longer
absorption and fluorescence wavelengths than pyronine Y,5
Fig. 1 Chemical structures of fluorescein, TokyoGreens and Tokyo-
Magentas. R1 = 2-Me: 2-Me TG. R2 = 2-Me: 2-Me TM.
a Graduate School of Pharmaceutical Sciences, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
E-mail: tlong@mol.f.u-tokyo.ac.jp; Fax: +81 3 5841 4855;
Tel: +81 3 5841 4850
b CREST, Japan Science and Technology Agency, Sanbancho-blg,
5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
w Electronic supplementary information (ESI) available: Detailed
descriptions of materials and general methods, and synthetic procedures.
Data on the measurement of the pH profiles and pKa plots of absorbance,
fluorescence and excitation and the photobleaching test of 8, fluorescence
quantum yield of 9, and the time-dependent fluorescence change of 9 in
the presence of b-galactosidase. See DOI: 10.1039/c1cc00078k
Scheme 1 Synthetic scheme of 2-Me TM.
c
4162 Chem. Commun., 2011, 47, 4162–4164
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Fig. 2 Photophysical properties of 2-Me TM. (a) pH-dependency of
absorption spectra of 1 mM 2-Me TG and 2-Me TM in 0.1 M sodium
phosphate buffer containing 1% DMSO. (b) pH-dependency of
fluorescence spectra (Ex = 582 nm) of 2-Me TM under the same
conditions as in (a). (c) Photophysical properties of 2-Me TM,
measured in sodium phosphate buffer at pH 9 for the anion form
and pH 3 for the neutral form. For the determination of fluorescence
quantum yields, Rhodamine B in EtOH (Ffl = 0.65) was used as a
fluorescence standard. (d) The value of the ratio of fluorescence
intensity (anion form (pH 9)/neutral form (pH 3)) with excitation at
the absorption maximum of the respective anion form (492 nm for
2-Me TG and 582 nm for 2-Me TM).
Fig. 3 (a) Reaction scheme of 2-Me TM bgal with b-galactosidase.
(b) Absorption and (c) fluorescence spectra of 1 mM 2-Me TM bgal
before and after reaction with 6 units b-galactosidase at 37 1C in
0.1 M sodium phosphate buffer (pH 7.4) containing 1% DMSO.
(d) Visualizing b-galactosidase activity in live cells using 2-Me TM
bgal. HEK293 cells (lacZ(+) or lacZ(À)) were incubated with 10 mM
2-Me TM bgal in DMEM containing 0.1% DMSO for 30 min. Bright
field images (left) and fluorescence images (right). The excitation and
emission wavelengths were 580 and 600–620 nm, respectively.
use of sec-butyllithium for the synthesis of Si-containing
xanthone (4) by lithiation. Thus, diaminoxanthone (5) was
synthesized, and then transformed into dihydroxyxanthone
(6). Compound 7 was a key intermediate in the synthesis of
TMs with various kinds of benzene moiety in one step. First,
we synthesized 2-Me TM as a prototype.
TM bgal was at a shorter wavelength than that of the neutral
form of 2-Me TM, and 2-Me TM bgal could serve as a
substrate for b-galactosidase. Further, 2-Me TM bgal showed
a large redshift of the absorbance spectrum following the
enzymatic reaction (Fig. 3b), and the fluorescence intensity
upon excitation at 582 nm was also greatly increased (Fig. 3c).
We next applied 2-Me TM bgal to live cells. When cultured
HEK293 cells (lacZ(+) or lacZ(À)) were incubated with
10 mM 2-Me TM bgal for 30 min, a large fluorescence
increment was observed in the intracellular region of lacZ(+)
cells, but not lacZ(À) cells, on excitation at 580 nm (Fig. 3d).
In conclusion, we have developed a novel scaffold for
fluorescence probes operating in the red wavelength region,
i.e., a Si-substituted fluorescein analogue, which we call
TokyoMagenta. TM showed an extremely large change of its
absorption spectrum upon deprotonation, and this phenomenon
can be utilized to obtain red-fluorescent sensor probes with
a high off/on ratio. This design strategy for controlling
fluorescence intensity is noteworthy, because fluorescein-based
probes require control by means of PeT or spiro cyclization
strategies to achieve a high off/on ratio. Furthermore, since
TMs can be highly activated without any additional strategy,
their benzene moieties can be flexibly modified compared to
TGs; i.e., for example, modification of the benzene moiety of
TGs is highly restricted because control of the oxidation
potential is extremely important for the PeT strategy. Thus,
it should be easy to design fluorescence sensor probes based on
TMs for a range of applications by flexibly modifying the
Fig. 2 shows the photophysical properties of 2-Me TM in
aqueous solution. A fascinating feature of 2-Me TM is that
deprotonation of the hydroxyl group of the fluorophore
triggered a 110 nm redshift of the absorbance (Fig. 2a). It is
considered that this redshift is due to s*–p* conjugation,6
similar to that of Si-containing pyronine,5 and the strength of
the s*–p* conjugation may be different between the neutral
form and the anion form of TM. This redshift is very large
compared to that of fluorescein derivatives; for example, 2-Me
TG shows only a 52 nm redshift on deprotonation (Fig. 2a).
Because of this large redshift, the fluorescence intensity of the
anion form of 2-Me TM is much larger than that of the neutral
form when excited at 582 nm (Fig. 2b). Thus, as 78% of 2-Me
TM is present in the anion form at pH 7.4, which corresponds
to the intercellular pH, fluorescence sensor probes with a high
off/on ratio can be obtained simply by utilizing the large
redshift of TM (Fig. 2c and d), without the need for additional
controls, such as the photoinduced electron transfer (PeT)7 or
spiro cyclization strategies,8 which are usually required for
fluorescein-based sensor probes.
To examine whether hydroxyl group substitution alone can
be used to develop fluorescence probes with a large off/on ratio
based on the dramatic redshift of TM, we designed and
synthesized 2-Me TM bgal as a fluorescence probe for
b-galactosidase (Fig. 3a). The absorbance maximum of 2-Me
c
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benzene moiety. Also, the switches of reported fluorescein-
based sensor probes for bioimaging could be easily applied to
TM to obtain equivalent probes operating in the red region,
instead of green. Because fluorescein-based fluorescence
probes have been widely utilized in biological and medical
research, sensor probes based on TMs should also be useful in
those fields as red-colored indicators, e.g., for dual-color
imaging.
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This research was supported in part by the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(Grant Nos. Specially Promoted Research 22000006 to T.N.,
20689001 and 21659204 to K.H.), and by the Industrial
Technology Development Organization (NEDO) of Japan
(to T.T.). K.H. was also supported by the Research Foundation
for Pharmaceutical Sciences.
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4164 Chem. Commun., 2011, 47, 4162–4164
This journal is The Royal Society of Chemistry 2011