Through-bond energy transfer cassettes with minimal spectral overlap between the donor emission and acceptor absorption: Coumarin-rhodamine dyads with large pseudo-stokes shifts and emission shifts

Article abstract of DOI:10.1002/anie.200904515

Chemical Equation Presented Cassette recording: A new class of coumarin-rhodamine through-bond energy-transfer (TBET) cassettes with minimal spectral overlap between the donor emission and the acceptor absorption (see picture) show large pseudo-Stokes shifts (up to 230 nm) and emission shifts (up to 170 nm). The utility of this TBET platform for TBET-based probe development was demonstrated by a new ratiometric fluorescence pH probe.

Full text of DOI:10.1002/anie.200904515

Angewandte
Chemie
DOI: 10.1002/anie.200904515
Fluorescent Dyes
Through-Bond Energy Transfer Cassettes with Minimal Spectral
Overlap between the Donor Emission and Acceptor Absorption:
Coumarin–Rhodamine Dyads with Large Pseudo-Stokes Shifts and
Emission Shifts**
Weiying Lin,* Lin Yuan, Zengmei Cao, Yanming Feng, and Jizeng Song
Small-molecule organic dyes have been widely used in
fluorescent probes,[1] labels,[2] logic gates,[3] light-emitting
materials,[4] and light-harvesting systems.[5] However, the
undesirable photophysical properties of various fluorophores
still constrain the full potential of their applications. For
instance, many bright organic dyes including rhodamine,
fluorescein, boron dipyrromethane (BODIPY), and cyanine
derivatives have the serious disadvantage of very small Stokes
shifts (typically less than 25 nm), which can lead to serious
self-quenching and fluorescence detection errors because of
excitation backscattering effects.[6] Therefore, there is a need
to develop dyes with improved properties.
Since it is still difficult to judiciously design single organic
dyes with desirable photophysical properties, considerable
attention has recently been paid to the exploration of
multifluorophores with energy-donor–acceptor architec-
tures.[1m,6b,7–9] In this regard, some energy-donor–acceptor
systems based on fluorescence resonance energy transfer
(FRET) have been constructed.[6b,8] FRET dyads are usually
linked by a nonconjugated spacer, and the energy transfer
occurs through space. Although the pseudo-Stokes shifts (the
wavelength discrepancy between the donor absorption and
the acceptor emission in an energy transfer system with
almost 100% energy transfer efficiency[7]) of FRET-based
energy cassettes are larger than the Stokes shifts of either the
donor or acceptor dyes, FRET-based cassettes are still limited
by the requirement that the donor emission must have strong
overlap with the acceptor absorption.[10] This requirement
essentially restricts the pseudo-Stokes shifts as well as the
emission shifts (the emission wavelength shift between the
donor and acceptor) of FRET-based systems. Like the
pseudo-Stokes shift, the emission shift is also an important
parameter in energy-transfer dyads. A large emission shift in
energy transfer systems should result in two well-separated
emission peaks, which is favorable for the precise measure-
ment of the peak intensities and ratios.[6b,11] Thus, energy-
transfer dyads with large pseudo-Stokes shifts and emission
shifts are desirable.
By contrast, through-bond energy transfer (TBET) is
theoretically not subjected to the constraint of intense
spectral overlap between the donor emission and the acceptor
absorption.[9] Thus, TBET cassettes may have large pseudo-
Stokes shifts and emission shifts. Unlike through-space
energy-transfer cassettes, in TBET cassettes, the donor and
the acceptor units are joined by a conjugated spacer. Burgess
and co-workers have developed elegant TBET systems based
on the conjugated fluorescein–rhodamine system.[9a,b] How-
ever, the fluorescein (donor) emission overlaps significantly
with the rhodamine (acceptor) absorption and the advantage
of TBET, that is, no requirement of strong spectral overlap
between the donor emission and the acceptor absorption, was
not really capitalized upon in these conjugated fluorescein–
rhodamine energy transfer cassettes. Not surprisingly, the
pseudo-Stokes shifts (< 120 nm) and emission shifts (20–
90 nm) in these fluorescein–rhodamine TBET systems are
rather restricted.[9a,b]
Although it is believed that TBET systems do not require
a strong spectral overlap between the donor emission and the
acceptor absorption, to the best of our knowledge, this
challenge has not been met in small-molecule dual-fluores-
cent dye systems. Thus, we were interested in creating novel
TBET platforms that only have minimal spectral overlap
between the donor emission and the acceptor absorption. The
merits of such a new class of TBET systems should include
large pseudo-Stokes shifts and emission shifts. These advanta-
geous spectral properties are desirable for the applications of
fluorescent dyes in chemistry, biology, medicine, and materi-
als science. Herein, as a proof-of-concept, we present the
coumarin–rhodamine TBET cassettes 1a–d as a small-mole-
cule dual-fluorescent dye energy-transfer platform with
minimal spectral overlap between the donor emission and
the acceptor absorption (Scheme 1). In addition, this TBET
platform was applied to develop a new TBET-based pH
probe. As expected, the probe exhibited the key features of
our TBET platform, namely a large pseudo-Stokes shift and a
significant ratiometric value that arise from a significant
emission shift.
[*] Prof. W. Lin, L. Yuan, Z. Cao, Y. Feng, J. Song
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering, Hunan University
Changsha 410082 (China)
Fax: (+86)731-8882-1464
E-mail: weiyinglin@hnu.cn
The choice of dyes with a minimal spectral overlap as the
donors and acceptors is straightforward. To exemplify the
general concept of our TBET design, coumarin and rhod-
amine dyes were selected as the energy donors and acceptors,
respectively (Scheme 2), as the coumarin emission has
[**] Funding was partially provided by NSFC (20872032, 20972044),
NCET (08-0175), and the Key Project of the Chinese Ministry of
Education (no. 108167).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200904515.
Angew. Chem. Int. Ed. 2010, 49, 375 –379
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375

Communications
the donor and the acceptor moieties from becoming planar
and facilitate the through-bond energy transfer process.
Furthermore, from a practical point of view, such a construct
should also be synthetically accessible. These considerations
led us to select a phenyl moiety as a rigid and conjugated
linker.
The new class of TBET cassettes 1a–d was synthesized by
condensation of coumarin aldehydes 5a–d with 3-(diethyl-
amino)phenol, followed by oxidation with chloranil
(Scheme 1). However, the purification of the products 1a–d
by column chromatography on silica gel proved to be very
challenging. Alternatively, we employed a reduction–oxida-
tion process to successfully purify these products (see the
Supporting Information). The donors 4a–d were prepared by
following standard procedures (see the Supporting Informa-
tion). The new coumarin–rhodamine compounds were fully
characterized by 1H and 13C NMR spectroscopy, and HRMS.
The absorption spectra of the TBET cassettes 1a–d (5 mm)
displayed the characteristic absorption signal of rhodamine
Scheme 1. Synthesis of the coumarin–rhodamine TBET cassettes 1a–d.
a) CH3CH2COOH, 4-toluenesulfonic acid. b) Chloranil, CH2Cl2, MeOH.
c) NaBH4, MeOH, then chloranil, CH2Cl2, MeOH, standard concen-
trated HCl.
Scheme 2. Structures of the energy donors 4a–d and the acceptor 6.
negligible overlap with the rhodamine absorption (Figure 1).
However, the main challenge in the development of the new
TBET system is to connect the coumarin donor with the
rhodamine acceptor with a suitable linker that may prevent
&
^
Figure 2. a) Absorption spectra of equimolar 1a ( ), 1b ( ), 1c (N),
~
*
1d ( ) and 6 ( ) in pH 7.0 phosphate buffer/MeOH (3:2). b) Fluores-
~
&
^
*
cence spectra of equimolar 1a ( ), 1b ( ), 1c (N), 1d ( ), and 6 ( )
excited at 372 nm in phosphate buffer/MeOH (3:2) (see Figure S2–6
in the Supporting Information for the fluorescence spectra at other
excitation wavelengths in phosphate buffer or other solvent systems.).
The inset shows the visual fluorescence color of compounds 6, 1a, 1b,
1c, 1d excited at 365 nm using a hand-held UV lamp. The concen-
tration of all the compounds is 5 mm. Figure S14 in the Supporting
Information is a color version of Figure 2b.
Figure 1. Normalized emission spectra of donor coumarin dyes 4a
~
&
*
( ), 4b ( ), 4c ( ), and 4d (N) and the normalized absorption
spectra of the acceptor rhodamine 6 (&) in pH 7.0 phosphate buffer/
MeOH (3:2).
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Table 1: Photophysical data of 1a–d and acceptor 6.[a]
pseudo-Stokes shifts of up to
230 nm, which are much larger
than those of fluorescein–
Compound
Absorption
Emission
labs [nm][b] log emax labs [nm][c] Log emax lem [nm][d] FEF[e] Ff[f] Dl[g] Dlem
[h]
rhodamine TBET cassettes
1a
1b
1c
1d
6
353
351
354
372
–
4.29
4.30
4.22
4.28
–
560
560
561
561
559
4.92
4.92
4.98
4.92
4.96
582
582
582
582
576
5.8
6.0
4.3
6.7
1.0
0.27 229
0.26 231
0.25 228
0.27 210
0.25 17
153
150
172
143
–
(<120 nm)[9a,b] and those of the
typical FRET-based rhodamine
systems (< 100 nm).[6b,14c,d] Further-
more, the emission shifts between
the coumarin donor and rhoda-
mine acceptor in our TBET cas-
settes are large (up to 172 nm;
Table 1 and Figure S1 in the Sup-
porting Information), which are
also much larger than those of the
fluorescein–rhodamine TBET cas-
[a] Measurements recorded in 25 mm phosphate buffer/MeOH (3:2). [b] The maximal absorption of the
coumarin component; [c] The maximal absorption of the rhodamine component. [d] The maximal
emission of the cassettes. [e] Fluorescence enhancement relative to the acceptor 6. [f] Fluorescence
quantum yields were determined using rhodamine 6G (Ff =0.95 in water) as a standard.[10, 15]
[g] Pseudo-Stokes shifts of the cassettes 1a–d and the Stokes shift of the acceptor 6. [h] Emission
wavelength shifts between the donor emission and acceptor emission in cassettes 1a–d.
derivatives around 560 nm and the typical absorption signal of
coumarin derivatives around 350 nm (in the cases of 1a–c) or
370 nm (in the case of 1d; Figure 2a and Table 1). This result
reveals that there are very weak electronic interactions
between the donor and the acceptor in the ground state,
and the compounds 1a–d behave as cassettes[12] instead of
single dye molecules.
settes (20–90 nm)[9a,b] and those of the typical FRET-based
rhodamine systems (< 75 nm).[6b,8a–c]
Upon excitation of the cassettes 1a–d (5 mm; phosphate
buffer/MeOH 3:2) in the coumarin absorption band, only the
characteristic emission band of the acceptor rhodamine
(around 582 nm) was observed (Figure 2b and Figure S1 in
the Supporting Information). The characteristic emission of
coumarin (410–439 nm) was not observed, thus indicating that
the energy-transfer efficiencies were nearly perfect
(>99%).[13] For comparison, we also examined the fluores-
cence spectra of an equimolar mixture of the coumarin donor
and the rhodamine acceptor. For instance, in an equimolar
mixture of coumarin 4a and rhodamine 6, no visible quench-
ing of the fluorescence of 4a and no marked enhancement of
the fluorescence of 6 was observed upon excitation of the
coumarin absorption band (Figure S7 in the Supporting
Information), thus suggesting that there is essentially no
intermolecular energy transfer between coumarin 4a and
rhodamine 6 in the mixture. The same conclusion can be
drawn for an equimolar mixture of rhodamine 6 and coumarin
4b, 4c, or 4d, respectively (Figure S8–10 in the Supporting
Information). Thus, the superiority of the TBET cassettes for
energy transfer is evident.
Figure 3. a) Brightfield image of nasopharyngeal carcinoma cells
treated with 1b (15 mm); b) fluorescence image of nasopharyngeal
carcinoma cells treated with 1b (15 mm) excited around 355 nm; c) the
overlay image of (a) and (b).
To examine the potential use of these new coumarin–
rhodamine cassettes for fluorescence imaging in living cells,
nasopharyngeal carcinoma cells were incubated with the
representative cassette 1b or 1d for 30 min at 378C. As shown
in Figure 3 and Figure S12 in the Supporting Information, the
novel cassettes were cell-permeable and can be employed for
cell imaging when the coumarin absorption band is excited.
This characteristic suggests that the coumarin–rhodamine
TBET cassettes are potentially useful
for biological applications in living
systems.
As a preliminary illustration of the
utility of our system, we created the
water-soluble fluorescent probe 2 as a
For an energy-transfer cassette to be useful in practical
applications, the fluorescence intensity of the acceptor
component in the cassettes must be greater than that of the
energy acceptor (without the donor) when it is excited at the
donor absorption wavelength. As is evident from Figure 2b,
the fluorescence enhancement factors (FEFs) are 5.8-, 6.0-,
4.3-, and 6.7-fold for the cassettes 1a–d compared to the
acceptor 6, respectively. Notably, these enhancement factors
are much higher than those of other typical FRET-based
rhodamine systems (< 4.0-fold).[14] Moreover, the cassette
fluorescence is significantly brighter than that of the acceptor
6 (Figure 2b, inset), thus confirming the enhanced fluores-
cence of the cassette.
new candidate for a TBET-based pH
probe. The 7-hydroxycoumarin moiety
not only improves the water solubility
of the probe, but also functions as the
H+ sensing unit.[16] Figure 4 shows the
ratiometric fluorescence response of compound 2 to varia-
tions of the pH value. The increase of pH from 3.0 to 8.6
induced a significant decrease (13-fold overall) in the rho-
damine emission signal around 587 nm and a large increase
(19-fold overall) in the coumarin emission signal around
465 nm. Thus, the ratios of emission intensities at 587 and
465 nm (I587/I465) exhibit a dramatic change from 46.5 at
pH 3.0 to 0.2 at pH 8.6. It should be noted that such a large
change of emission intensity ratios at two wavelengths is
desirable for ratiometric fluorescent probes, as the sensitivity
as well as the dynamic range of ratiometric probes are
The photophysical data of the cassettes 1a–d and acceptor
6 are given in Table 1 and Tables S1 and S2 in the Supporting
Information. Indeed, the TBET cassettes have very large
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377

Communications
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controlled by the emission ratio.[7b] Furthermore, the emission
changes evolved with a well-defined isoemissive point at
555 nm. The difference in emission wavelength between the
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In conclusion, we have described coumarin–rhodamine
TBET cassettes as a novel paradigm of small-molecule dual-
fluorescent dye energy-transfer systems with minimal spectral
overlap between the donor emission and the acceptor
absorption. The key features of the novel class of the TBET
platform include large pseudo-Stokes shifts (up to 230 nm)
and emission shifts (up to 170 nm). These advantageous
spectral properties should allow use of the TBET cassettes in
many areas. In addition, we have demonstrated that the new
TBET cassettes are cell-membrane-permeable and poten-
tially useful for biological applications. For a preliminary
application, we created a ratiometric fluorescent TBET pH
probe. This probe showed a large pseudo-Stokes shift as well
as a dramatic change of the ratios of emission intensities at
587 and 465 nm (I587/I465) from 46.5 at pH 3.0 to 0.2 at pH 8.6
because of a large emission shift. We expect that our general
design concept of the new class of the TBET platform with
minimal spectral overlap between the donor emission and the
acceptor absorption should be applicable to other small-
molecule dual-fluorescent dye energy transfer systems based
on a wide variety of dyes. This work, as well as the application
of our TBET platform to the development of ratiometric
fluorescent probes for various analytes of interest is in
progress.
Received: August 13, 2009
Revised: October 14, 2009
Published online: December 8, 2009
Keywords: bioorganic chemistry · dyes · fluorescent probes ·
rhodamine · sensors
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