Development of 2,6-carboxy-substituted boron dipyrromethene (BODIPY) as a novel scaffold of ratiometric fluorescent probes for live cell imaging

Article abstract of DOI:10.1039/b917209b

Ratiometric fluorescent probes based on boron dipyrromethene (BODIPY) were developed based on a novel design strategy, in which a change of the electron-withdrawing character of the 2,6-substituents resulting from reaction with a target molecule generates a fluorescence wavelength change. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b917209b

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Development of 2,6-carboxy-substituted boron dipyrromethene
(BODIPY) as a novel scaffold of ratiometric fluorescent probes
for live cell imagingw
Toru Komatsu,ac Yasuteru Urano,a Yuuta Fujikawa,ac Tomonori Kobayashi,ac
Hirotatsu Kojima,b Takuya Terai,a Kenjiro Hanaokaa and Tetsuo Nagano*abc
Received (in Cambridge, UK) 20th August 2009, Accepted 22nd September 2009
First published as an Advance Article on the web 8th October 2009
DOI: 10.1039/b917209b
Ratiometric fluorescent probes based on boron dipyrromethene
(BODIPY) were developed based on a novel design strategy, in
which a change of the electron-withdrawing character of the
2,6-substituents resulting from reaction with a target molecule
generates a fluorescence wavelength change.
employed photoinduced electron transfer (PeT)10,11 to make
them suitable for single wavelength measurement. Nevertheless,
the sharp absorbance and fluorescence peaks of BODIPY are
desirable features in a scaffold for ratiometric probes, since a
slight wavelength shift can yield a greater ratio change than
would be expected in the case of dyes with relatively flat
or wide peaks. It is known that some substitutions of
BODIPY have marked effects on the absorbance/fluorescence
wavelengths,8,9 and a few ratiometric fluorescent probes have
been developed on this basis,12 but most studies so far have
focused on substitution at the 1,3,5,7-positions. We thought
that an understanding of the substituent effects at the
2,6-positions of the fluorophore might lead to a new rational
means to develop ratiometric probes based on BODIPY, since
a range of substituents can be easily introduced at these
positions.8
Fluorescent probes are molecules whose fluorescence
characteristics, such as fluorescence intensity, wavelength,
and lifetime, change as a result of specific reactions with target
molecules. Many fluorescent probes have been developed to
study biologically important phenomena in living cells.1 The
use of ratiometric measurements, monitoring fluorescence
wavelength change, offers many advantages for imaging living
cells, because this approach minimizes interference with the
signal owing to variations in factors such as cell size, probe
concentration, excitation intensity, and detector sensitivity.2
In combination with recent advances in fluorescence micro-
scope technology that have increased the accuracy in detection
of fluorescence wavelength change, ratiometric measurements
have proved useful for determination of metal ions,2,3 pH,4
reactive oxygen species,5 and various enzyme activities.6,7
However, the theoretical basis to predict fluorescence wave-
length change without alteration of fluorescence intensity is
weak, and rational approaches to the design of functional
ratiometric fluorescent probes are urgently needed.
A consideration of the structure–wavelength relationship of
various BODIPYs indicated that substitution with electron-
withdrawing groups at the 2,6-positions often causes a blue
shift, while electron-donating groups result in a red shift. This
led us to hypothesize that the electron-withdrawing character
of the 2,6-substituents would be the key to controlling the
fluorescence wavelength of BODIPY. In order to confirm and
extend this hypothesis, we focused on the BODIPY scaffold in
which the 2,6-positions are directly substituted with carboxylic
acid derivatives, since various potential target reactions would
produce carboxylate as a product. While ester (CO2R), amide
(CONHR), and carboxylic acid (CO2H) would act as strongly
electron-withdrawing groups, carboxylate (CO2À) would have
a lower electron-withdrawing capacity owing to its negative
charge13 and, therefore, we expected that an ester or amide
cleavage reaction that generates carboxylate at the 2,6-positions
under neutral pH conditions would be suitable as a switch for
ratiometric fluorescent probes.
Here we propose a design strategy for ratiometric fluorescent
probes based on fluorescence wavelength change resulting
from modification of the substituents at the 2,6-positions of
boron dipyrromethene (BODIPY). BODIPY dyes are among
the most useful fluorophores as a platform for fluorescent
probes.8,9 BODIPY shows strong and sharp absorbance and
fluorescence peaks in the visible light region, and these are
little influenced by environmental factors, such as solvent
polarity and pH. In addition, its chemical and photochemical
stability are compatible with many kinds of chemical
modifications. Many fluorescent probes have been developed
based on this fluorophore, though most of them have
Therefore, we synthesized 2,6-diCO2H-BDP (1), whose
2,6-positions were directly substituted with carboxylic acid
(CO2H) groups (Fig. 1). Among these carboxylic acid derivatives,
only 2,6-diCO2Et-BDP, whose 2,6-positions were directly
substituted with CO2Et groups, was a known compound,10
and there has been no report on carboxylic acid, amide, or
other ester derivatives. Basic hydrolysis of 2,6-diCO2Et-BDP
did not give 2,6-diCO2H-BDP, but resulted in destruction
of the fluorophore, presumably via nucleophilic attack on
boron.8 Therefore, we designed another synthetic route
to 2,6-diCO2H-BDP via benzyl-protected carboxy-pyrrole
(8) (Scheme S1w). Although several steps were needed to
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 Chemical Biology Research Initiative, The University of Tokyo,
Japan
c CREST, Japan Science and Technology Agency, Japan
w Electronic supplementary information (ESI) available: Experimental
details, pKa calculation and fluorescence spectra and images. See DOI:
10.1039/b917209b
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Fig. 1 Normalized absorbance (a) and relative fluorescence (b)
spectra of 2,6-diCO2H-BDP (1) and its derivatives (amide (2) and
ester (3)) in phosphate buffer (pH 7.4).
Fig.
2
Ratiometric measurement of esterase activity using
obtain photochemically pure products, the overall yield of
2,6-diCO2H-BDP was high.
2,6-diCO2AM-BDP (4). (a) Structure of 2,6-diCO2AM-BDP (4).
(b) Fluorescence spectral change of 2,6-diCO2AM-BDP (1 mM) after
addition of porcine liver esterase (0.1 units mLÀ1) in phosphate buffer
(100 mM, pH 7.4) containing 20% DMSO at 37 1C. Spectra were
measured every 30 s. Scattered excitation light (lex. = 500 nm) can be
seen in the spectra. (c) Fluorescence ratio (525 nm/505 nm) increase of
2,6-diCO2AM-BDP (1 mM) after addition of different concentrations
of PLE.
2,6-DiCO2H-BDP was sufficiently stable under basic/acidic
conditions, and its derivatization to amides (2) and esters (3)
could be easily achieved through SN2 reaction of the carboxylate
or via formation of the acid chloride. 2,6-DiCO2H-BDP (1)
took the carboxylate (CO2À) form in solutions of neutral pH
(apparent pKa = 4.5–5.0 for the two CO2H groups, Fig. S1w).
Based on the Hammett constants,13 the electron-withdrawing
character of substituents is in the order of ester (sp = 0.37) >
amide (sp = 0.20) > carboxylate (sp = 0.00). Absorbance
and fluorescence spectra of 2,6-diCO2H-BDP (carboxylic acid, 1),
2,6-diCONHR-BDP (amide, 2), and 2,6-diCO2R-BDP (ester, 3)
(R = CH2CO2H) are illustrated (Fig. 1, Table 1). All showed
strong fluorescence (FFL = 0.37–0.66). As we had hoped, the
fluorescence wavelength changed among these derivatives
in the order of electron-withdrawing character of the
2,6-substituents. In particular, the carboxylate (CO2À) form of
2,6-diCO2H-BDP showed a red-shift of the absorbance and
fluorescence peak wavelengths by 20 nm compared to the
ester form.
we synthesized fluorescent probes to visualize activities of
esterases. Esterases are a group of enzymes that mediate
cleavage of various esters in living cells.14,15 Since esterases
have a role in the metabolism of many ester-bearing drugs and
prodrugs, we think that a precise understanding of esterase
activities in living cells of different types and under different
conditions should be helpful to predict and evaluate the
efficacy of various therapeutic drugs. An advantageous feature
of our design is that any protecting group for a carboxylic acid
can be a reaction site, and we selected the acetoxymethyl (AM)
group, which is a readily cleavable protective group for esterases.
The structure of the synthesized probe, 2,6-diCO2AM-BDP (4)
is shown in Fig. 2a and Scheme S2w. It was confirmed that the
probe was stable in phosphate buffer (pH 7.4) for >1 h in the
absence of enzyme, but upon addition of porcine liver esterase
(PLE), a fluorescence wavelength change from 505 nm (ester)
to 525 nm (carboxylate) was observed (Fig. 2b, Scheme S2w).
In in vitro experiments, PLE activity could be determined by
monitoring the increase of fluorescence ratio of the probe
(Fig. 2c).
The finding that the ester and carboxylate forms of
2,6-diCO2H-BDP (1) showed emissions at different wavelengths
allows us to design ratiometric fluorescent probes to visualize
exo-type ester cleavage reactions that leave the carboxylic acid
as a product. In other words, we can use any carboxylic
acid-protective group that is a potential target of a molecule
of interest, and so this platform offers a novel means to
develop fluorescent probes for various ester cleavage reactions.
In order to confirm the feasibility of this design approach,
Since the wavelength change of the probe can detect esterase
activity, we examined whether the probe could be used for
ratiometric imaging of esterase activity in living cells. Since the
water-solubility of compound 4 was not high enough for
cellular experiments, a probe with increased water-solubility
was synthesized by modification of the 8-phenyl group, and
the resulting compound, tetraCO2AM-BDP (5) was used for
further experiments (Fig. 3d, Scheme S3w). To visualize esterase
activity by means of ratiometric imaging, we adjusted the
filters of the epifluorescence microscope so that the fluorescence
ratio increase that occurred when the ester was hydrolyzed to
carboxylate could be observed (Fig. S3w). When the probe was
Table 1 Photochemical characteristics of synthesized BODIPYs
a
sp
b
Substituents
lex/lem (nm)
FFL
À
CO2 (1)
CONHR (2)
CO2R (3)
0.00
0.20
0.37
510/525
500/510
495/505
0.66
0.61
0.37
a
sp represents the Hammett constant of the substituent (taken from
ref. 13). Quantum yield was measured with an absolute photo-
b
luminescence quantum yield measurement system (C9920-02,
Hamamatsu Photonics) under excitation at 475 nm.
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of functional probes to study esterase activities, both in vitro
and in living cells. While further work might be required to
design a probe in which a large emission wavelength change is
induced by cleavage of a single ester moiety, our results
show that the novel scaffold, 2,6-diCO2H-BDP, should be
useful for the development of a range of fluorescent probes
for various exo-type ester cleavage reactions. We believe that
the design strategy of utilizing a chemical reaction at the
2,6-substituents of BODIPY would be applicable to other
substituents, not only to carboxylic acid derivatives, and we
think it will be possible to develop a range of novel ratiometric
fluorescent probes to visualize a variety of reactions in
living cells.
Fig. 3 Bright-field transmission and fluorescence ratiometric images
of tetraCO2AM-BDP (5)-loaded HeLa cells. (a) Bright-field transmission
and ratiometric images after addition of tetraCO2AM-BDP (10 mM) to
extracellular solution. (b) Fluorescence ratiometric change in the cell
areas (1–3) and an extracellular area depicted in (a). (c) Slope of ratio
increase of cellular and extracellular areas (n = 6). Data on control
cells and cells pretreated with esterase inhibitor, AEBSF, are shown.
See ESI Fig. S4 for a details.w (d) Structure of tetraCO2AM-BDP (5).
Notes and references
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added to medium containing HeLa cells, entry of the probe
into the cells (an increase of short-wavelength fluorescence),
and subsequent hydrolysis of the probe inside the cells (an
increase of the ratio) were observed immediately (Fig. 3a and b).
Pre-treatment of cells with 1 mM serine hydrolase inhibitor,
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), which
inhibited ester cleavage reaction to the extent of about 50%
in in vitro lysate experiments (unpublished data), blocked the
increase of the ratio to the same extent, indicating that the
calculated ratio increase represents the activity of esterases in
the living cells (Fig. 3c).
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In conclusion, ratiometric fluorescence imaging is a powerful
technique for investigating cellular processes, and recent
advances in fluorescence microscope technology have increased
the accuracy of detection of fluorescence wavelength change.
The main shortcoming has been the difficulty of developing
a range of fluorescent molecules suitable for ratiometric
measurement. Here we present a rational design strategy for
ratiometric probes based on the BODIPY scaffold. Since
chemical modification of BODIPY is straightforward, our
approach should allow the design and synthesis of a variety
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 7015–7017 | 7017