Enhancing the Stokes' shift of BODIPY dyes via through-bond energy transfer and its application for Fe3+-detection in live cell imaging

Article abstract of DOI:10.1039/c2cc31011b

Two energy transfer cassettes that exhibit a large pseudo Stokes' shift (up to 400 nm) due to efficient through-bond energy transfer (up to 99%) have been constructed. Selective binding of Fe(iii) with the donor entity significantly suppresses the excitation energy transfer resulting in fluorescence quenching in aqueous solution and in living cells. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc31011b

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COMMUNICATION
Enhancing the Stokes’ shift of BODIPY dyes via through-bond energy
transfer and its application for Fe3+-detection in live cell imagingw
Xingyu Qu, Quan Liu, Xiaoning Ji, Huachao Chen, Zhikuan Zhou and Zhen Shen*
Received 11th February 2012, Accepted 8th March 2012
DOI: 10.1039/c2cc31011b
Two energy transfer cassettes that exhibit a large pseudo Stokes’
shift (up to 400 nm) due to efficient through-bond energy transfer
(up to 99%) have been constructed. Selective binding of Fe(III)
with the donor entity significantly suppresses the excitation
energy transfer resulting in fluorescence quenching in aqueous
solution and in living cells.
The following characteristics are highly desirable for intracellular
imaging: (i) large Stokes shifts for easy separation of excitation and
emission; (ii) fluorescence maxima appearing in the NIR region
(650–900 nm).5 In addition to the near-infrared probes,6a two-
photon absorption materials6b and up-conversion nanoparticles,6c
the TBET system is a very promising pathway to achieve the
above criteria.6d Recently several TBET cassettes which
showed large quasi-Stokes shifts have been developed.7 However,
the fluorescent probes based-on TBET and their application in
intracellular imaging are still very rare.8 Herein, we report two
TBET cassettes containing 5-(quinolin-2-yl)thiophen-2-yl (TQ) as
an energy donor linked to a BODIPY or NIR tetrastyryl-
substituted BODIPY acceptor. The two cassettes exhibit excellent
optical performance with high energy transfer efficiency (up to
99%) and large pseudo Stokes’ shift (up to 398 nm). Their sensing
properties have been investigated in solution and living cell imaging.
The synthesis of TBET cassettes 1 and 2 are outlined in
Scheme 1. 5-(Quinolin-2-yl)thiophene-2-carbaldehyde 3 is
prepared by treatment of 2-(thiophen-2-yl)quinoline 4 with
n-butyl lithium in THF solution (yield 59%). The trifluoroacetic
acid catalyzed condensation reaction of 3 with 2,4-dimethyl-
pyrrole gives compound 2 in 65% yield.9a 1 can be obtained in
10% yield by condensation of 2 with 4-methoxybenzaldehyde
using the Knoevenagel condensation method.9b
Excitation energy transfer (EET) plays a key role in nature’s
photosynthetic process which captures solar energy to the reaction
centre to initiate a multistep electron-transfer cascade.1 The
EET process can be classified into two pathways: fluorescence
resonance energy transfer (FRET) and through-bond energy
transfer (TBET). Both systems consist of an energy donor and
an energy acceptor linked through either a nonconjugated
spacer (in the case of FRET) or a conjugated spacer (in the
case of TBET), respectively. The FRET efficiency is controlled
by the spectral overlap between the emission spectrum of the
energy donor and the absorption spectrum of the energy
acceptor. In contrast to the FRET system, the TBET system
is not limited by such a kind of spectral overlap and exhibits
high energy transfer efficiencies, fast energy transfer rates and
large pseudo-Stokes’ shift. The TBET cassettes have wide
applications as optical materials, as photosynthetic models, in
biotechnology and as chemosensors.2 The 4,4-difluoro-4-bora-
3a,4a-diaza-s-indacene (BODIPY) dyes have been investigated
intensively as labelling reagents,3a fluorescent switches,3b
chemosensors3c and laser dyes3d in the recent years owing to
their favorable photophysical properties, such as high extinction
coefficient, high fluorescence quantum yields (>0.8), facile
derivativation, and good photostability.4 However, the Stokes’
shift of BODIPY is about 5–20 nm that limited its application in
living cell imaging due to the backscattering effects from the
excitation source. Since the absorption and emission bands of
the unmodified BODIPY lie at ca. 500 nm, introducing four
styryl substituents at 1-, 3-, 5- and 7-positions significantly red
shifts the main BODIPY absorption band to the NIR region.4f
The structure of cassette 2 has been determined by X-ray
analysis. Single crystal of 2 is obtained by slow evaporation of
hexane/CH2Cl2 solution at ambient temperature. Similar to the
previously reported structures of alkyl substituted BODIPYs,9c the
meso-thiophene is virtually orthogonal to the indacene plane with
the torsion angle being 89.81. Both the 2-(thiophen-2-yl)quinoline
moiety and the indacene plane are nearly planar with the deviations
State Key Laboratory of Coordination Chemistry, Nanjing National
Laboratory of Microstructures, Nanjing 210093, P. R. China.
E-mail: zshen@nju.edu.cn; Fax: +86 (0)25-83314502;
Tel: +86 (0)25-83686679
w Electronic supplementary information (ESI) available: X-Ray
analysis of BODIPY 2, synthesis details, etc. CCDC reference number
856179. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2cc31011b
Scheme 1 Synthetic procedures for cassettes 1 and 2: (i) n-butyl
lithium and DMF in THF, À78 1C, 59%; (ii) 2,4-dimethyl-pyrrole and
trifluoroacetic acid in CH2Cl2, DDQ, triethylamine, BF3ÁOEt2, rt, 65%;
(iii) 4-methoxybenzaldehyde, AcOH/piperidine, toluene, reflux, 10%.
c
4600 Chem. Commun., 2012, 48, 4600–4602
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Fig. 2 The absorption (left) and emission (right) spectra of 1, 2, 4 and
meso-unsubstituted BODIPY10 in CH2Cl2 (10 mM, lex = 334 nm).
Fig. 1 ORTEP views of the molecular structure of 2 with the thermal
ellipsoids set at 30% probability (left) and packing diagram (right).
from the mean plane being 0.0285 A and 0.0598 A, respectively. It
is interesting to note that in the unit cell, the indacene plane and
2-(thiophen-2-yl)quinoline moiety in the neighboring molecules
are near-parallel to each other. The distance between the
closest overlapping p-surfaces is 3.64 A in average, indicating
the existence of weak p–p interactions (Fig. 1).
yields of the BODIPY upon excitation of the TQ moiety are higher
than that obtained by direct excitation of the BODIPY or NIR
tetrastyryl-BODIPY acceptor (Table 1). The fluorescence quantum
yield of 1 in CH2Cl2 is 0.6, which is about 14-fold than that of 2.
Such a remarkable difference might be attributed to the 1,7-
substituted 4-methoxystyryl group that prevents free rotation
of the meso-2-(thiophen-2-yl)quinoline thus reducing loss of
energy from the excited states via non-irradiative molecular
motion.13 Consistently, the fluorescence quantum yield of
2 increased significantly to about 0.7 in paraffin oil which is
14-fold than that measured in non-viscous solvents.
The UV/vis absorption and emission spectra of 1, 2, 4 and
meso-unsubstituted BODIPY are measured in various solvents
of different polarity and the photophysical properties are
summarized in Table 1. As shown in Fig. 2, the absorption
maxima of 1 and 2 are centered at approximately 708 nm and
514 nm, respectively, and they do not show any peak change with
increasing solvent polarity (Table S1, ESIw). The absorption
spectrum of 2 is nearly the sum of the absorption spectra of 4
and meso-unsubstituted BODIPY, indicating that there is almost
no electronic coupling between the donor and acceptor entities in
the ground state. The absorption band at 370 nm for 1 can be
assigned to the intramolecular charge transfer (ICT) band due to
an electron-donating 4-methoxystyryl group.11 The band at 708 nm
attributed to the S0 - S1 transition of BODIPY is significantly
red-shifted due to the extended p-conjugation with the 4-methoxy-
styryl substituent. Exciting the 2-(thiophen-2-yl)quinoline moiety at
334 nm in 4 provides an emission band at 386 nm. Whereas the
emission from the TQ moiety in cassettes 1 and 2 is almost
quenched completely upon excitation at 334 nm, strong emissions
typical for the BODIPY fluorophore at 732 nm for 1 and 524 nm
for 2, respectively, are observed. Whereas the fluorescence spectrum
of a 1 : 1 mixture of meso-unsubstituted BODIPY and 4 exhibits
dual emission bands at 386 nm and 524 nm which resembles
exactly the sum of the emission spectra of 4 and meso-unsubstituted
BODIPY upon excitation at 334 nm (Fig. S11, ESIw). These results
imply that efficient through-bond energy transfer from the donor to
the BODIPY acceptor is the prevalent mechanism in cassettes
1 and 2. The energy transfer efficiency is evaluated according
to the equation: 100 Â [1 À (fluorescence intensity of the donor
in the cassette)/(fluorescence intensity of the free donor)]%.12a
In addition, due to the antenna effect,12b the fluorescence quantum
The chelating property of 2-(thiophen-2-yl)quinoline 4 in
the presence of various metal ions is tested, only Fe3+ exhibits
specific fluorescence quenching property (Fig. S8, ESIw). The
response of Fe3+ in cassettes 1 and 2 is investigated. Upon
excitation of the TQ part at 334 nm, the fluorescence intensities
from the BODIPYs of 2 at 523 nm and 1 at 732 nm decrease
remarkably with a virtually unchanged peak position (Fig. 3),
whereas excitation of the BODIPY part at 490 nm for 2 and
640 nm for 1, the emission band from the BODIPYs at 523 nm
for 2 and 732 nm for 1 still remain. The most obvious
explanation for the fluorescent quenching is that the energy
transfer from 2-(thiophen-2-yl)quinoline to the BODIPY is
inhibited after binding with Fe3+. Almost no change is
observed in the fluorescence intensities of 2 by addition of
120 equiv. of Zn2+, Cu2+, Cd2+, Co2+, Fe2+, Mn2+, Ni2+
Hg2+, Al3+, Pb2+, Cr3+, 400 equiv. of Mg2+, Na+, Ca2+, K+,
Na+, Li+, and also 1 by addition of 40 equiv. of Zn2+, Cu2+
Cd2+, Co2+, Mn2+, Ni2+, Hg2+, Al3+, Pb2+, Ca2+, Cr3+
,
,
,
respectively (Fig. S9, ESIw). To evaluate the utility of 2 as a Fe3+
selective fluorescent sensor, the ion interference experiments are
carried out. As shown in Fig. S9 (ESIw), its Fe3+ response is not
interfered in the background containing appropriate metal ions.
The practical application of 2 as a Fe3+ probe within living
cells has been evaluated.14 As shown in Fig. 4, by incubating
the MCF-7 cells with 10 mM of 2 for 30 min at 37 1C, a
significant green emission from the intracellular region could
Table 1 Optical properties of 1, 2, 4 and meso-unsubstituted BODIPY in CH2Cl2 at 298 K
max
Compounds
l
(nm)/log emax
l
flu/nm Quasi-Stokes’ shift/nm Dl1/2a/nm Ffex334 b Faf cceptor b ETEc(%) td/ns
abs
1
708/5.23
334/4.53
514/4.77
334/4.39
334/4.27
732
524
24
398
10
190
52
8
34
25
0.62
0.36
98
99
4.51
3.47
2
0.066
0.054
4
386
515
50
20
0.09
—
—
0.9
—
—
—
—
meso-Unsubstituted BODIPY 507/4.32
334/3.38
a
b
Half width at maximum height. Fluorescence quantum yield. Energy transfer efficiency. Fluorescence lifetime.
c
d
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4600–4602 4601

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Fig. 4 Confocal fluorescence and bright field images of MCF-7 cells.
(a) Cells incubated with 10 mM of 2 for 30 min at 37 1C upon excitation
at 405 nm and (c) upon excitation at 488 nm. (e) Cells supplemented
with 50 mM of Fe3+ in the growth media for 1 h at 37 1C upon
excitation at 405 nm. (g) Cells supplemented with 50 mM of Fe3+ in
the growth media for 1 h at 37 1C upon excitation at 488 nm. (b), (d),
(f), and (h) Bright field images of cells.
be observed either excited at 405 nm or at 488 nm (Fig. 4a and c).
Upon supplementing cells with 50 mM of Fe3+ in the growth
medium for 1 h at 37 1C, microscope images showed only weak
intracellular fluorescence when excited at 405 nm (Fig. 4e), while
the emission could still be observed when excited at 488 nm
(Fig. 4g). Bright field measurements with both Fe3+ and 2 after
treatment confirmed that the cells are viable throughout the
imaging experiments (Fig. 4b, d, f, h). These results indicate
that 2 can be used as an effective intracellular Fe3+ imaging
agent. Due to the limit of equipment detection wavelength, we
could not obtain cell imaging for 1.
In summary, large Stokes’ shift and strong emission could
be successfully achieved via through-bond energy transfer in
2-(thiophen-2-yl)quinoline appended BODIPY cassettes. That
combined with the selective detection of Fe3+ based-on the
quenching of excitation energy transfer from the donor group
could provide considerable scope for developing chemosensors
based on the TBET cassettes containing BODIPY fluorophores.
In addition, 2 can be used as a fluorescence turn-off chemosensor
for Fe3+ in MCF-7 cells indicating its potential application for
studying the effect of Fe3+ in biological systems.
Financial support was provided by the National Natural
Science Foundation of China (No. 20971066 and 21021062)
and the Major State Basic Research Development Program of
China (No. 2011CB808704).
Notes and references
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2003, 125, 14668; (b) F. Puntoriero, F. Nastasi, S. Campagna,
c
4602 Chem. Commun., 2012, 48, 4600–4602
This journal is The Royal Society of Chemistry 2012