Highly activatable and rapidly releasable caged fluorescein derivatives

Article abstract of DOI:10.1021/ja070376d

Caged fluorophores, which recover fluorescence activity upon brief illumination with ultraviolet (UV) light, have played an important role in investigating cell lineage and cellular protein dynamics. In this paper, we report novel nitrobenzyl-caged fluore

Full text of DOI:10.1021/ja070376d

Published on Web 05/03/2007
Highly Activatable and Rapidly Releasable Caged Fluorescein Derivatives
Tomonori Kobayashi,†,‡ Yasuteru Urano,†,§ Mako Kamiya,†,‡ Tasuku Ueno,†,‡ Hirotatsu Kojima,†,‡ and
Tetsuo Nagano*,†,‡
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan, and PRESTO, JST, and CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Received January 18, 2007; E-mail: tlong@mol.f.u-tokyo.ac.jp
The cellular dynamics of living cells can be investigated by using
functional small organic molecules, such as fluorescence probes.1
One approach is to use caged compounds, in which critically
important functional groups for bioactivity are protected with caging
groups (photoremovable protecting groups),2 which can be removed
upon brief illumination with ultraviolet (UV) light to release the
original bioactive molecules, such as neurotransmitters, second
messengers, and drugs. It is then possible to supply these molecules
with high temporal and spatial resolution in a noninvasive manner
by controlling both the time and application site of the excitation
light. Caged fluorophores have proved to be very useful for
investigating cell lineage or for fluorescence labeling of cellular
proteins.3 Desirable properties for a caged fluorophore include fast
release of the fluorophore and a large enhancement of the
fluorescence in response to brief irradiation, in order to minimize
cell damage. Among many caged fluorophores, caged fluorescein
is one of the most popular dyes because fluorescein has many
advantages for bioimaging, including high fluorescence quantum
efficiency, high extinction coefficient around 490 nm, and high
water solubility under physiological conditions. However, traditional
caged fluoresceins have various drawbacks. Since almost all of the
Figure 1. (A) Uncaging scheme of caged fluorescein. (B) Uncaging scheme
and structures of caged TokyoGreen. The pictures were taken before and
after UV irradiation of an aqueous solution of TG-NPE (1 µM).
reported caged fluoresceins are bis-caged lactone forms, full
activation of them requires deprotection of two photoremovable
protecting groups. Thus, full fluorophore release and a large
fluorescence increase require quite prolonged irradiation with high-
intensity UV light. Moreover, photobyproducts derived from caging
groups may show cytotoxicity, as in the case of 2-nitrobenzyl
derivatives. Therefore, for several reasons, it would be preferable
to use a single caging group in a molecule. Krafft et al. reported a
mono-caged fluorescein ether fixed in the lactone form which had
a single caging group, but the photoactivated product was less
fluorescent than fluorescein.4 There is thus a need for a rapidly
releasable and highly activatable caged fluorescein for bioimaging.
In this study, we report novel caged fluorescein derivatives which
showed rapid uncaging and a large fluorescence enhancement. We
first synthesized mono-2-nitrobenzyl-caged fluorescein (MonoNB-
FL) according to Scheme S1. Since MonoNB-FL has a single
protecting group, we expected that it would be more rapidly
activated than commercially available bis(5-carboxymethoxy-2-
nitrobenzyl)-caged fluorescein (BisCMNB-FL, Figure 1A). As
expected, MonoNB-FL showed a faster increase of fluorescence
than BisCMNB-FL upon 20 s irradiation with UV light at around
350 nm in 100 mM sodium phosphate buffer (pH 7.4), as shown
in Figure 2B. However, MonoNB-FL has a high fluorescence
quantum efficiency (Φ ) 0.117) before photoactivation. Generally,
nitro-containing dyes have almost no fluorescence due to quenching
of the singlet excited state by photoinduced electron-transfer (PeT)
Figure 2. (A) Absorption spectra of caged compounds (1 µM) in 100 mM
sodium phosphate buffer, pH 7.4, containing 0.1% DMF as a cosolvent.
(B) Fluorescence increase and fluorescence ratio values at the emission
maximum wavelength (caged fluorescein: 516 nm, caged TokyoGreen: 513
nm) upon UV irradiation at around 350 nm for 20 s. Fluorescence increase
and fluorescence ratio denote the increase and the ratio, respectively, of
the fluorescence intensity after 20 s UV irradiation with respect to the
fluorescence before irradiation at the emission maximum wavelength.
process.5 However, in this case, PeT from the xanthene moiety to
the nitrobenzyl group is less favorable because the electron density
of the xanthene moiety is lowered by the electron-withdrawing
effect of the nitrobenzyl group. Although MonoNB-FL was easily
releasable, its high background fluorescence before photoactivation
would result in a low signal-to-noise ratio, and therefore it is not
expected to be suitable for bioimaging.
Next, we tried lowering the fluorescence quantum efficiency
before photoactivation by using TokyoGreen instead of fluorescein.
We have shown that the fluorescent properties of a wide range of
† The University of Tokyo.
‡ CREST, JST.
§ PRESTO, JST.
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10.1021/ja070376d CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
living cells. HeLa cells were incubated with TG-NPE AM (1 µM),
the membrane-permeable acetoxymethyl ester of TG-NPE, and a
selected cell was irradiated with UV light (330-385 nm) from a
100 W high-pressure mercury lamp through the objective lens of a
fluorescence microscope. After irradiation for 10 s, the irradiated
cell became brightly fluorescent (Figure 3A). In contrast, similar
UV irradiation (10 s) of BisCMNB-FL AM (1 µM) loaded cells
did not result in any detectable fluorescence increase (Figure 3B).
Even after longer irradiation (up to 600 s) or the use of a higher
BisCMNB-FL AM concentration (10 µM), the fluorescence inten-
sity did not increase as much as that of TG-NPE AM. No
cytotoxicity of TG-NPE AM was observed under these experimental
conditions with the use of the Calcein AM/EthD-1 assay. Thus,
caged TokyoGreen was concluded to be superior to traditional caged
fluoresceins. Furthermore, a macromolecular TG-NPE-dextran
conjugate was prepared, and it was confirmed that caged Tokyo-
Green also worked in this context, in both cuvette and cell systems
(Supporting Information).
In conclusion, we have designed and developed novel caged
fluorescein derivatives (caged TokyoGreens) which are rapidly
activatable upon brief irradiation and show a large fluorescence
enhancement. In a biological application, fluorescence labeling of
a single living cell was far more efficient with TG-NPE AM than
with a traditional caged fluorescein. Moreover, in this TokyoGreen-
based molecular design, a wide variety of photoremovable protect-
ing groups susceptible to different wavelengths of uncaging light
could be used to create designed-to-order caged fluorophores for
various experimental conditions. We believe caged TokyoGreen
represents a breakthrough in caged fluorophore technology.
Figure 3. Cell application of (A) TG-NPE AM and (B) BisCMNB-FL
AM. Differential interference contrast (DIC), fluorescence images before
(0 s) and after (10 s) irradiation, and merge.
fluorophores, including fluorescein, rhodamine, and BODIPY, can
be controlled precisely by utilizing the concept of PeT.6 Tokyo-
Greens are fluorescein derivatives in which the carboxylic group
of fluorescein is replaced with a methyl or methoxy group, and
their fluorescence quantum yield can be precisely controlled by
adjusting the oxidation potential of their benzene moiety.7,8 On the
basis of the TokyoGreen platform, we have designed and synthe-
sized three caged TokyoGreens (Figure 1B) according to Scheme
S2. In these derivatives, that is, the 2-nitrobenzyl (TG-NB), 1-(2-
nitrophenyl)ethyl (TG-NPE), and 4,5-dimethoxy-2-nitrobenzyl (TG-
DMNB) derivatives, the oxidation potential of the benzene moiety
is finely tuned to show a drastic change of fluorescence upon
cleavage of the caging group. Before photoactivation, caged
TokyoGreen should be almost nonfluorescent because of quenching
of the singlet excited state via the PeT process, while removal of
the caging group by UV illumination should result in a large
fluorescence enhancement, as the PeT process becomes less
favorable. As expected, the fluorescence quantum efficiency was
decreased to less than 1/100 of that of MonoNB-FL in the cases of
TG-NB and TG-NPE and less than 1/50 in the case of TG-DMNB.
Low background fluorescence was thus achieved by rational
molecular design based on the TokyoGreen platform. All three
derivatives showed almost the same spectral features, except that
TG-DMNB had a higher extinction coefficient at around 300-400
nm, as reported elsewhere (Figure 2A).9 Next, aqueous solutions
of the caged compounds (1 µM in 100 mM sodium phosphate
buffer, pH 7.4) were illuminated with UV light in a cuvette of a
monochromator system. In practical applications, a rapid increase
of fluorescence upon brief irradiation is critical to minimize cell
damage. Figure 2B shows the fluorescence increase and ratio values
at the emission maximum wavelength upon brief irradiation (330-
370 nm, 1.88 mW/cm2 at 350 nm, 20 s). The fluorescence increase
was the largest with TG-NPE, followed by TG-DMNB, TG-NB,
MonoNB-FL, and BisCMNB-FL; indeed, BisCMNB-FL showed
no fluorescence change in this setting. The fluorescence ratio was
the largest for TG-NPE, followed by TG-NB, TG-DMNB, and
MonoNB-FL. That of BisCMNB-FL could not be determined. The
uncaging quantum efficiency of TG-NPE was determined to be 0.03
by means of HPLC analyses, being slightly smaller than that of
BisCMNB-FL (Supporting Information). In cuvette experiments,
the caged TokyoGreens were clearly superior to commercially
available caged fluorescein in terms of fluorescence enhancement.
Among our compounds, TG-NPE appeared to be the best, affording
the greatest fluorescence activation upon brief UV irradiation.
To determine the effectiveness of caged TokyoGreen in a
biological system, we examined the fluorescence labeling of single
Acknowledgment. This study was supported in part by a grant
from Hoansha Fundation to T.N. This study was supported in part
by research grants (Grant Nos. 14103018, 16651106, and 16689002
to Y.U.) from the Ministry of Education, Culture, Sports, Science
and Technology of the Japanese Government, and by a grant from
the Kato Memorial Bioscience Foundation to Y.U.
Supporting Information Available: Synthesis and characterization
of caged TokyoGreens, and experimental detail of photoirradiation
experiments in cuvette and cell assay. This material is available free
of charge via the Internet at http://pubs.acs.org.
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