Convenient and efficient fret platform featuring a rigid biphenyl spacer between rhodamine and BODIPY: Transformation of 'turn-on' sensors into ratiometric ones with dual emission

Article abstract of DOI:10.1002/chem.201002498

We have connected a borondipyrromethene (BODIPY) donor to the 5′ position of a tetramethylrhodamine (TMR) acceptor to form a high efficiency (over 99%) intramolecular fluorescence resonance energy transfer (FRET) cassette, BODIPY-rhodamine platform (BRP).

Full text of DOI:10.1002/chem.201002498

FULL PAPER
DOI: 10.1002/chem.201002498
Convenient and Efficient FRET Platform Featuring a Rigid Biphenyl Spacer
between Rhodamine and BODIPY: Transformation of ꢀTurn-Onꢁ Sensors
into Ratiometric Ones with Dual Emission**
Haibo Yu,[a] Yi Xiao,*[a] Haiying Guo,[a] and Xuhong Qian*[b]
Abstract: We have connected a boron-
dipyrromethene (BODIPY) donor to
the 5’ position of a tetramethylrhoda-
mine (TMR) acceptor to form a high
efficiency (over 99%) intramolecular
fluorescence resonance energy transfer
(FRET) cassette, BODIPY–rhodamine
platform (BRP). While the good spec-
tral overlap between the emission of
BODIPY and the absorption of TMR
was one favorable factor, another fea-
ture of this FRET system was the rigid
and short biphenyl spacer that favored
efficient through-bond energy transfer.
More importantly, in this system, the
2’-carboxyl group of the rhodamine
unit was preserved for the further
modifications, which was as convenient
as those carbonyl groups on the origi-
nal rhodamines without connection to
donors. For this reason, BRP is clearly
differentiated from the previous ratio-
metric sensors based on donor rhoda-
mine systems. To illustrate its value as
a versatile platform, we introduced typ-
ical Hg2+ receptors into BRP, through
convenient one-pot reactions on the 2’-
carboxyl group, and successfully devel-
oped two ratiometric sensors, BRP-1
and BRP-2, with different spirocyclic
receptors that recognized Hg2+ on dif-
ferent reaction mechanisms. Upon exci-
tation at a single wavelength (488 nm),
at which only BODIPY absorbed, both
of the FRET sensors exhibited clear
Hg2+-induced changes in the intensity
ratio of the two strong emission bands
of BODIPY and rhodamine. It should
be noted that these ratiometric Hg2+
sensors exhibited excellent sensitivity
and selectivity Hg2+, as well as pH in-
sensitivity, which was similar to the cor-
responding ꢀturn-onꢁ rhodamine sen-
sors. While both ratiometric probes
were applicable for Hg2+ imaging in
living cells, BRP-1 exhibited higher
sensitivity and faster responses than
BRP-2. Our investigation indicated
that on a versatile platform, such as
BRP, a large number of highly efficient
ratiometric sensors for transition-metal
ions could be conveniently developed.
Keywords: donor–acceptor
sys-
tems · fluorescence · FRET · mer-
cury · sensors
Introduction
signal outputs, cell penetrability, and noninvasive imaging of
cells in vivo.[4] As transition- and heavy-metal ions often act
as efficient fluorescence quenchers, ꢀturn-offꢁ sensors are
common.[5] However, because of their low signal-to-noise
ratio, turn-off sensors are less sensitive than the ꢀturn-onꢁ
ones.[6] Thus, intensive efforts have been directed toward the
development of turn-on fluorescent sensors, which have
become mainstream.[7] Among various strategies, the chemo-
dosimeter approach[8] provides good opportunities to design
turn-on sensors for transition-metal ions with both high sen-
sitivity and selectivity.
Owing to the important roles of transition- and heavy-metal
cations in the areas of biological, environmental, and chemi-
cal systems,[1,2] the development of new chemosensors for
these cations has been of interest.[3] Particularly, in recent
decades, there has been a considerable increase in fluores-
cent sensors research, because of their advantages in optical
[a] H. Yu, Prof. Dr. Y. Xiao, H. Guo
State Key Laboratory of Fine Chemicals
Dalian University of Technology, Zhongshan Road 158
Dalian, 116012 (P.R. China)
Recently, rhodamine spirolactams, as novel chemodosime-
ters, have attracted much attention and have been widely
utilized in the design of turn-on sensors.[9] The turn-on re-
sponse to certain transition cations have involved ring-open-
ing transformations from colorless and nonfluorescent spiro-
cyclic forms to rhodamine fluorophores with intensive ab-
sorptions and emissions in the visible spectrum range
(Scheme 1). The remarkable fluorescence enhancements
ensure the high signal-to-noise ratios of such sensors. More
importantly, by introducing specially designed receptors to
the spirolactamꢁs imide site, high reactivity and selectivity
toward a specific metal cation can be achieved. So far, effi-
cient rhodamine spirolactam sensors for various transition-
Fax : (+86)411-83673488
E-mail: xiaoyi@dlut.edu.cn
[b] Prof. Dr. X. Qian
Laboratory of Chemical Biology
East China University of Science and Technology
Meilong Road 130, Shanghai, 200237 (P.R. China)
Fax : (+86)21-64252603
E-mail: xhqian@ecust.edu.cn
[**] BODIPY=borondipyrromethene.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002498.
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adopted to develop ratiometric sensors, the emission bands
of which shift upon binding a guest.[20] However, before and
after binding the target ions, the maximum excitation wave-
length changes remarkably, and there are large overlaps be-
tween the two emission bands owing to the broad fluores-
cence spectra of ICT fluorophores. These two aspects make
it difficult for ICT sensors to accurately determine the ratio
of the two fluorescence peaks. Theoretically, the above
problems can be avoided by using a FRET-based sensor for
which the single excitation wavelength of the donor fluoro-
phore releases the acceptor emission at a longer wave-
length.[21]
Scheme 1. Ring-opening reaction of rhodamine fluorophores used in the
design of turn-on sensors.
Since 2008, a novel strategy of connecting a shorter-wave-
length fluorophore to a rhodamine spirolactam has brought
about several ratiometric sensors for metal cations
(Scheme 2).[22–26] The idea is a combination of the chemo-
dosimeter approach and a FRET mechanism. Initially, a
rhodamine spirolactam unit with a nonconjugated structure,
which has no absorption in the visible range and cannot act
as energy acceptor, due to the poor spectral overlap, cannot
undergo a FRET process. A ring-opening reaction induced
by metal ions generates intensively colored rhodamine dye,
which is a good energy acceptor, and thus the FRET process
is switched on. This strategy is the so-called spectral over-
lap-modulated FRET process. The successful examples
given in Scheme 2 reveal that the construction of a FRET
cassette by attaching a donor fluorophore to rhodamine is
highly feasible to transform turn-on sensors into ratiometric
ones. However, unexpectedly, this strategy has not been
widely applied, and rhodamine-derived ratiometric sensors
for metal cations are rare, in contrast to the situation in
which large numbers of turn-on rhodamine sensors have
been reported frequently in recent years.
[10]
[11]
[12]
and heavy-metal cations, such as Cu2+
,
Hg2+
,
Fe3+
,
[13]
[14]
[15]
Cr3+
,
Au3+
,
Pb2+
,
and Ag+,[16] have been successfully
developed. Moreover, such a rhodamine spirolactam chemo-
dosimeter strategy has also been extended to sensing active
oxygen species[17] and amino acids.[18] For convenient chemi-
cal modification on the spirolactam imide (carboxyl group),
conventional rhodamine dyes have become versatile plat-
forms to design turn-on sensors. Their applications are im-
portant in the field of fluorescent sensors, as indicated by
the large amount of recent publications on this topic.
Although the significant fluorescence enhancement con-
tributes to cell identification in tissue imaging, turn-on sen-
sors have an intrinsic limitation. Because, for these single-
emission sensors, the accuracy of quantitative detection
might be limited by factors such as fluctuating intensities of
the excitation light, the concentration of the fluorescent
sensor, inner filter effects, or something as trivial as varia-
tion in the distance between the sample and the excitation
light or detector.[19] To solve this problem, some work has
focused on the design of ratiometric sensors. These sensors
utilized the ratios of emission intensity at two different
wavelengths as signal outputs, which, in principle, or ideally,
should be capable of self-calibration and independent of
both excitation light and sample concentration. An intramo-
lecular charge-transfer (ICT) mechanism is frequently
When we attempted to analyze why the extension of such
a promising strategy has been hampered, we concluded that
the lack of a versatile FRET platform was the major reason.
As can be foreseen from the structures in Scheme 2, to syn-
thesize these compounds, chemists will be forced to use an
Scheme 2. Representative FRET ratiometric sensors for different metal ions based on a rhodamine fluorophore.
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A Convenient and Efficient FRET Platform
FULL PAPER
individual and long synthetic pathway for each compound.
If there is a ready FRET platform on which convenient
modifications result in a large number of ratiometric sensors
specific for various analytes, synthetic procedures will be
simplified to large extent. We decided to take on the chal-
lenging task of constructing an efficient and versatile FRET
platform, BRP, to provide the opportunities to develop
many ratiometric sensors in a simplified method, by borrow-
ing the experiences accumulated in the development of
turn-on rhodamine sensors. With BRP as a precursor, we
successfully developed two ratiometric sensors, BRP-1 and
BRP-2, conveniently by introducing two different and
known Hg2+ receptors, and their excellent Hg2+ ratiometric
responses justified our idea.
ence from/to other parts of the molecule (spacer or donor)
for their separation from each other. For the previous ratio-
metric rhodamine sensors 1–4, the receptors were intro-
duced between the rhodamine and donors, so they were in-
tegral parts of the spacers. The spatial proximity of the reac-
tive recognition site to other bulky groups might lower the
sensitivity or selectivity toward the analytes, relative to
those of the original turn-on rhodamine sensors.
In our design, a short and rigid biphenyl spacer was
adopted to satisfy the requirements for consistent and
highly efficient energy transfer. First, the short and rigid bi-
phenyl group ruled out the inaccuracy caused by changes in
the distance and orientation between the donor and the ac-
ceptor. Because in FRET systems with flexible and relative-
ly long spacers, these changes
would be likely to happen dy-
namically in the inhomogene-
ous biological microenviron-
ment,[27] and then affect the
energy-transfer efficiency.[28,29]
Although sensitivities in dis-
tance/orientation were very
useful design features for sen-
sors monitoring conformational
or functional changes in biolog-
ical macromolecules, such as
DNA beacons and enzyme ac-
tivity sensors,[30] unfortunately
Results and Discussion
they interfere unfavorably for sensing small molecular ana-
lytes in local microenvironments. Second, a short, rigid bi-
phenyl spacer with a partly conjugated nature was good for
high-efficiency FRET through bonds. Excellent work by the
group of Burgess and others suggested superfast and effi-
cient energy-transfer systems through bonds with donor and
acceptor fragments linked with various rigid spacers.[31] The
rigid biphenyl spacer as a twisted, but otherwise conjugated,
p-electron system could keep the energy-transfer time on
the picosecond scale, which is faster than that of through-
space energy transfer.[32]
Considerations in the construction of the BRP platform:
BRP, with a borondipyrromethene (BODIPY) donor con-
nected to a tetramethylrhodamine (TMR) acceptor, is an in-
tramolecular FRET cassette. BRP could be regarded as a
versatile platform because the 2’-carboxyl group of the rhod-
amine unit was preserved for further modifications. This car-
boxyl group in the BRP structure maintained a similar reac-
tivity to those of ordinary rhodamine dyes without connect-
ed donors. As is known, the 2’-carboxyl group of the rhod-
A
We preferred to use BODIPY rather than other fluoro-
phores in the construction of a platform for ratiometric sen-
sors. In our understanding of the spectral overlap-modulated
FRET strategy, whereas the rhodamine unit should be sensi-
tive to cations, the donor fluorophore should have a strong
and stable fluorescence that is insensitive to environmental
factors, such as polarity and pH, since the donor would si-
multaneously act as the internal standard for ratiometric de-
tection. BODIPY fulfills this requirement and should be su-
perior to other donors, such as dansyl amide, naphthalimide,
and fluorescein, adopted in previous ratiometric FRET sen-
sors, for example, sensor 2–4. Moreover, the emission band
of BODIPY is so narrow that it can be easily separated
from the emission band of rhodamine, which is similarly
narrow. The distinct dual nonoverlapping fluorescence sig-
nals of BODIPY-rhodamine systems are highly desirable in
the process of ratio measurement. Although BODIPY is
very popular in FRET investigations,[33] its good match to
receptors to produce spirolactam-type turn-on sensors for
several transition-metal cations. Clearly, all of these known
receptors, and those developed in the future, could be intro-
duced into BRP under similar conditions. The difference is
that spirocyclic sensors built on BRP will be ratiometric
ones based on the FRET mechanism. With BRP as a plat-
form, and by using knowledge of rhodamine turn-on sensors,
the originally difficult task to search for ratiometric sensors
is considerably simplified, only the facile installation of
ready-made components is required. To the best of our
knowledge, there has been no BRP-like versatile platform
for ratiometric sensors of metal cations, although rhodamine
dyes have been adopted as acceptors in some FRET systems
that have been designed for other applications. Incidentally,
another important and unique feature of BRP is that the
spirocyclic receptors induced on the remaining 2’-carboxyl
group can recognize the corresponding cations without influ-
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Y. Xiao, X. Qian et al.
rhodamine has only been re-
vealed recently.[22,34] This excel-
lent FRET pair has not yet
been extensively studied.
Synthesis of the BRP platform
and BRP-1 and BRP-2 sensors:
The synthesis of the BRP plat-
form is shown in Scheme 3. The
compounds 4’-B-BODIPY and
5’-Br-TMR were prepared ac-
cording literature procedures.[35]
BRP was efficiently and
straightforwardly obtained by
Scheme 3. The synthesis of BRP, BRP-1, and BRP-2: a) Pd
b) 1) NH2NH2, CH3OH; 2) PhNCS, DMF; c) 1) POCl3, ClCH2CH2Cl; 2) Na2S, CH3CN.
ACHTUGNRTEN(NNUG OAc)2, PPh3, Na2CO3, n-propanol/methanol;
the Suzuki coupling of 4’-B-BODIPY and 5’-Br-TMR cata-
lyzed by Pd(OAc)2 in the presence of PPh3 and Na2CO3.
mine emission band was not detectable, indicating that
FRET could not occur. The reason was that the rhodamine
ACHTUNGTRENNUNG
The target product BRP was easily obtained and isolated in
78% yield after purification by column chromatography.
The BODIPY moiety was connected to the 5’ position of
TMR, and the 2’-carboxyl group was preserved in BRP. This
rigid connection keeps the orientation of donor and accept-
or constant to favor through-bond FRETs.[36]
To demonstrate BRP as a platform to design ratiometric
sensors, two FRET sensors, BRP-1 and BRP-2, were synthe-
sized. BRP-1 and BRP-2 were conveniently obtained ac-
cording to the synthetic method used in the design of turn-
on rhodamine sensors.[37,38] BRP-1 and BRP-2 were pre-
pared in moderate and good yields, respectively, from plat-
form BRP by using two-step procedures. Details of the syn-
theses are given in the Experimental Section. The conven-
ient synthetic routes of sensors BRP-1 and BRP-2 further
indicated that most receptors reported in turn-on rhodamine
sensors could be easily introduced into the BRP platform to
design ratiometric sensors.
Spectral properties of platform BRP: BRP is not only a pre-
cursor or platform, but may also be a sensor or logic gate
molecule, since the reversible switching of a rhodamine dye
between a zwitterion and a lactone is sensitive to tempera-
ture, pH, polarity, proticity, and so forth. As illustrated in
Figure 1, BRP switches between the two structures as readi-
ly as ordinary rhodamine dyes, which means that, within
BRP, energy-transfer processes can be switched off or on. In
aprotic solvent, such as acetonitrile, BRP emitted a strong
green fluorescence; the fluorescence spectrum showed a
single band (lflu =510 nm) characteristic for BODIPY. The
fluorescence quantum yield of BRP in acetonitrile was 0.43,
which was lower than that of 4’-Br-BODIPY (F=0.61).
There would be photoinduced electron transfers from the
spirolactone of rhodamine moiety to the BODIPY fluoro-
phore in the BRP platform, which would result in partial
quenching of the fluorescence of BODIPY. This is under-
standable because the spirolactone form of the rhodamine
moiety with amino groups is relatively rich in electron densi-
ty. In recent work, we found that electron transfer might be
an important factor that required careful consideration in
the design of FRET systems.[39] A longer-wavelength rhoda-
Figure 1. a) The structural changes of BRP between zwitterion and lac-
tone. b) Normalized absorption and emission spectra of BRP in acetoni-
~
*
trile ( ) and methanol ( ), respectively. c) The overlap between the
emission of 4’-Br-BODIPY (left) and the absorption of 5’-Br-TMR
(right).
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A Convenient and Efficient FRET Platform
FULL PAPER
unit was in the spirolactone form, which, without absorption
in the visible range, could not act as an acceptor. In protic
solvent, such as methanol, BRP emitted a strong orange
fluorescence (F=0.47). The fluorescence spectrum showed
only a band (lflu =568 nm) characteristic for rhodamine,
upon excitation of BODIPY at 488 nm, which indicated that
FRET was switched on. The rhodamine unit existed in the
form of a ring-opened zwitterion, which had a characteristic
absorption peak at 540 nm. It acted as an efficient acceptor,
the absorption of which overlapped well with the emission
of BODIPY. The overlap integral was also assessed as
shown in Figure 1c.
The Forster critical radius, R0 (in ꢃ), was calculated by
using Equation (1):
Z
1
1=6
R0 ¼ 0:211½k2nꢀ4FD
IDðlÞeAðlÞl4dlꢁ
ð1Þ
0
in which k2 is the orientational factor, FD is the quantum
yield of fluorescence emission of the donor, n is the average
refractive index of the medium in the wavelength range
where spectral overlap is significant, ID(l) is the normalized
fluorescence spectrum of the donor, eA(l) is the molar ab-
sorption coefficient of the acceptor (in dm3 molꢀ1 cmꢀ1), and
l is the wavelength in nanometers. As previously reported,
the orientation factor was assumed to be equal to the dy-
namic average that is 2/3. The Forster critical radius of the
BRP platform was 20.3 ꢃ. The efficiency of energy transfer
(EET) was calculated according to Equation (2):[40]
Table 1. Spectral data for 4’-Br-BODIPY, 4’-B-BODIPY, 5’-Br-TMR,
and BRP.
[h]
Compound
Solvent labs
[nm][a]
loge[b] lem
[nm][a]
F[c]
hEET
[%]
4’-Br-
BODIPY
4’-B-
BODIPY
5’-Br-TMR
BRP
MeCN 498
MeOH 499
MeCN 498
MeOH 498
MeOH 541
MeCN 498
4.96
5.00
4.96
4.98
5.00
4.94
5.14
5.02
514
0.61[d]
0.54[d]
0.55[d]
0.53[d]
0.56[e]
0.43[d]
0.47[f]
0.49[g]
–
–
hEET ¼ 1 ꢀ FFðdyadÞ=FFðdonorÞ
ð2Þ
515
513
513
565
510
in which hEET is the efficiency of energy transfer, FF(dyad) is
the fluorescence quantum yields of the dyad (the donor part
in FRET system), and FF(donor) is the fluorescence quantum
yields of the donor when not connected to the acceptor. The
fluorescence quantum yields (FF(donor)) of 4’-Br-BODIPY[41]
in methanol was 0.54, whereas that of the BODIPY part in
BRP (FF(donor)) was greatly reduced to 0.005. Thus, the effi-
ciency of BRP (hEET) in the on state is very high, up to
99%.
–
–
498
MeOH
540
BRP
568
99
[a] labs =absorption maximum; lem =fluorescence emission maximum.
[b] e=molar extinction coefficient. [c] F=fluorescence quantum yield.
[d] The quantum yield was determined by using fluorescein as a standard.
[e] The quantum yields were determined by using rhodamine 6G as a ref-
erence.[42] [f] lex =488 nm. [g] lex =530 nm. [h] hEET =the efficiency of
energy transfer.
The BRP platform exhibited an interesting equilibrium
between lactone and zwitterion, which depended on the sol-
vent hydrogen-bond-donating ability and solvent dielectric/
polarizability characteristics.[43] Although BRP can respond
to many common parameters, such as temperature and pH,
the switch could be controlled easily in some specific condi-
tions. Because the switching of ordinary rhodamine dyes has
been successfully applied in many fields, such as imprint-
ing,[44] indicators,[45] and molecular sensors,[46] we anticipated
that the BRP platform might also be used as a sensor or in-
dicator.
The spectra data of BRP and intermediate compounds
are shown in Table 1. The maximum absorption bands of
BRP in methanol were at 498 and 540 nm, which corre-
sponded to the absorption of 4’-Br-BODIPY and 5’-Br-
TMR, respectively. Upon excitation of BRP at 488 nm, the
emission peak was shown at 568 nm owing to FRET and the
pseudo-Stokes shift was up to 70 nm. During the process of
energy transfer, the spectral properties of the TMR unit
were not changed relative to 5’-Br-TMR. The fluorescence
quantum yields of BRP (F=0.47, 0.49) in methanol were
constant upon excitation at 488 and 540 nm, respectively,
which was attributed to through-bond energy transfer from
BODIPY to the TMR fluorophore. According to results re-
ported by Burgess et al.,[32] the energy-transfer time in the
range of picoseconds and the
Properties of sensor BRP-1: The mechanism of the reaction
between BRP-1 and Hg2+ is shown in Scheme 4. The thiose-
micarbazides section of BRP-1 was transformed into a 1,3,4-
oxadiazide promoted by Hg2+, resulting in the irreversible
rigid connection between donor
and acceptor were significant
characteristics for through-bond
energy transfer. The rigid bi-
phenyl group was
a critical
factor for energy transfer within
the BRP platform, which could
keep the orientation of donor
to acceptor constant to favor
through-bond FRET.
Scheme 4. Hg2+-induced ring opening and cyclization of BRP-1.
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Y. Xiao, X. Qian et al.
ring-opening reaction of the rhodamine section. The product
of the reaction between BRP-1 and Hg2+ was characterized
by ESI-MS analyses (m/z 824.4310 C50H45BF2N7O2+; Fig-
ure S1 in the Supporting Information). A similar Hg2+-pro-
moted reaction of thiosemicarbazides to form 1,3,4-oxadia-
zoles was reported previously for a turn-on rhodamine
sensor.[37] The absorption and fluorescence titration of Hg2+
was conducted by using 0.3 mm BRP-1 in ethanol/water (80/
20 v/v) at pH 7.2. In the absence of Hg2+ the absorption and
emission spectra of BRP-1 only displayed the features of
BODIPY. Apparently, the rhodamine spirolactam did not
absorb in the visible range and could not accept the energy
from BODIPY. Upon the addition of Hg2+, the new absorp-
tion band at 562 nm appeared and increased, which indicat-
ed that the rhodamine chromophore was generated. Conse-
quently, the large spectral overlap between the emission of
BODIPY and the adsorption of rhodamine switched the
FRET on.
was a curvilinear function with the Hg2+ concentration over
the range of 0.03 to 0.3 mm (6–60 ppb). The Hg2+ response
range of one order of magnitude was relatively narrow, re-
flecting the high sensitivity and quantitatively reactive char-
acteristic of the rhodamine spirolactam chemodosimeter.[37]
The detection limit of Hg2+ is at the parts per billion (ppb)
levels. The response to Hg2+ was very fast and all spectra
were recorded within 5 min after addition of Hg2+
.
The selectivity of BRP-1 for different metal ions was in-
vestigated. Various alkali, alkaline earth metal ions, and
transition-metal ions (Na+, Mg2+, Ca2+, Al3+, Pb2+, Cu2+
,
Cd2+, Fe3+, Fe2+, Zn2+, Cr3+, Co2+, and Ni2+ etc.) were
measured. Only the Ag+ ion promoted a small fluorescence
intensity enhancement at 584 nm and a distinct decrease at
510 nm, whereas the other metal ions did not induce any
fluorescence or color changes of BRP-1 upon the addition
of metal ions (10 equiv) (Figure S3 in the Supporting Infor-
mation). The selectivity observed for Hg2+ over other metal
ions was remarkably high. In addition, the ratiometric fluo-
rescence changes resulting from the addition of Hg2+ was
not influenced by the subsequent addition of other metal
ions. Although the interference of Ag+ was not negligible,
the I584/I510 ratio of BRP-1 showed good selectivity toward
Hg2+ ions (Figure 3a). Due to a solvent mixture with 80%
alcohol or acetonitrile content used in the experiment, the
pH values obtained from the pH meter were different from
that obtained in water. Therefore, the acid/base concentra-
tion was used to reveal the interaction between BRP-1-
As shown in Figure 2, with increasing Hg2+ concentra-
tions, the BODIPY emission signal at 510 nm decreased,
and the rhodamine emission signal at 584 nm appeared and
gradually increased in intensity. Upon addition of Hg2+
from 0.03–0.15 mm, the emission intensities at 510 and
584 nm were linearly proportional to the amount of Hg2+
(Figure S2 in the Supporting Information). However, the
ratio of the emission intensities at 584 and 510 nm (I584/I510
)
AHCTUNGTREG(NNNU BRP-2) and acid/base. The acid/base responses of BRP-1
and BRP-1 in the presence of Hg2+ (5.0 equiv) are shown in
Figure 3b. These solutions remained stable over a wide con-
centration range of acid/base (from log
G
[NaOH]=ꢀ5.0), suggesting that the response was insensi-
tive to acid/base and the spirolactam form of BRP-1 was
still preferred in that range in the absence of mercury ion.
Properties of sensor BRP-2: The mechanism of BRP-2 was
different from BRP-1. In the presence of Hg2+, the high thi-
ophilicity of Hg2+ induced the ring opening of thiospirolac-
tone in the rhodamine moiety; this was called a metal-coor-
dination-induced ring-opening reaction. The ESI-MS spectra
showed three kinds of binding ratio between Hg2+ and
BRP-2, as shown in Figure 4. When Hg2+ (1.0 equiv) was
added to BRP-2 in H2O/CH3CN, the signal at m/z 961.2106
given in Figure 4, corresponding to [HgACTHNUTRGNEUNG
(BRP-2)Cl]+ (la-
beled 2 in Figure 4) was clearly observed, which indicated a
1:1 binding ratio between Hg2+ and BRP-2 existed in sol-
vent. The signal at m/z 825.2647 given in Figure 4, corre-
sponding to [HgACTHNUTRGNEUNG
(BRP-2)2]2+ (1:2) (labeled 1 in Figure 4)
was also found in ESI-MS spectra. In the solvent, there was
another complex (labeled 3 in Figure 4) at m/z 1685.9658
given in Figure 4, corresponded to [HgACTHNUTRGNEUNG
(BRP-2)2Cl)]+. When
the mercury salt was changed from chloride to perchloride,
a single binding mode was obtained from ESI-MS measure-
ments, as shown in Figure 4c. The signal at m/z 824.7800
corresponding to [Hg
Due to the different coordinating ability of the counteran-
Figure 2. Changes in the absorption (a) and emission spectra (b) of BRP-
1 (0.3 mm) in C2H5OH/H2O (80:20) at pH 7.2 (0.01m HEPES) upon the
gradual addition of Hg2+ from 0.03 to 0.35 mm. Inset: the ratio intensity
(BRP-2)2]2+ (1:2) was clearly observed.
ACHTUNGTRENNUNG
at 584 and 510 nm upon the gradual addition of Hg2+
.
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A Convenient and Efficient FRET Platform
FULL PAPER
BRP-2 upon the addition of
metal ions (10 equiv) (Fig-
ure 6a). In addition, the en-
hancement in fluorescence in-
tensity at 585 nm resulting from
the addition of Hg2+ was not
influenced by the subsequent
addition of other metal ions.
Thus, BRP-2 displayed better
selectivity toward Hg2+ than
BRP-1, the Hg2+ recognition of
which was disrupted by Ag+.
BRP-2 also exhibited insensitiv-
ity to acid/base over a wide
concentration
range
(Fig-
ure 6b). However, the Hg2+ de-
tection limit of BRP-2 was only
on the ppm scale, thus its sensi-
tivity was much lower than that
of BRP-1, the detection limit of
which was on the ppb scale.
This could be ascribed to their
different recognition mecha-
nisms. The irreversible ring-
opening reaction of BRP-1 in-
duced by Hg2+ might be much
more efficient than the reversi-
Figure 3. a) I584/I510 ratio of BRP-1 (0.3 mm) in C2H5OH/H2O (80:20 v/v, pH 7.2) in the presence of various
metal ions (10 equiv). Black bars represent the addition of an excess of the appropriate metal ion (3 mm) to a ble reaction of BRP-2. Despite
0.3 mm solution of BRP-1. Gray bars represent the subsequent addition of 3 mm Hg2+ to this solution. b) I584/I510
its lower sensitivity relative to
2+
&
*
ratio of BRP-1 ( ) and BRP-1 in present of 5.0 equiv Hg ( ) in C2H5OH/H2O (80:20 v/v) versus different
BRP-1, BRP-2 also had advan-
tages as a potential reusable
sensor because its coordination
concentrations of HCl and NaOH at room temperature (lex =488 nm.)
ions, the binding complex between BRP-2 and Hg2+ would
be different, which was also indicated in Jobꢁs plot (Fig-
ure S4 in the Supporting Information). Although Hg2+ with
different counteranions could switch on the energy transfer
of BRP-2 to similar degrees (Figure S5 in the Supporting In-
formation), small but apparent differences in the extents of
ratiometric changes in the two emissions were observed.
Thus, in the application of BRP-2, the interference of coun-
teranions is not negligible.
with Hg2+ was reversible and it could be recovered from the
complex of Hg2+–BRP-2, if excess competing coordinating
reagents, such as KI (Figure S4 in the Supporting Informa-
tion), were added to attract Hg2+
.
Intracellular imaging for Hg2+ of BRP-1: To further confirm
the applicability of these two sensors in biological imaging,
fluorescence microscopy images in living cells were investi-
gated. Generally, there are two methods used for the imag-
ing of metal ions in living cells during cellular incubation of
sensors and metal ions in literature.[47,48] One method is to
incubate cells first with ions for a certain length of time and
then to incubate the cells with fluorescent sensors. The
second method involves the incubation of first the sensor
and then the addition of ions. Some chemists, such as Tae
and co-workers,[47] often used the former method in the cel-
lular imaging of metal ions. This method is suitable for turn-
on sensors and is usually encountered in analytical chemis-
try. The latter was often used by others in imaging experi-
ments not only with turn-on sensors,[48,49] but also with ratio-
metric sensors.[50] In our experiments, we decided that the
latter method would be more appropriate for ratiometric
sensors. The process of visible fluorescence (from green to
red) responses to metal ions could be demonstrated.
The Hg2+-induced ring-opening reaction of thiospirolac-
tone was similar to previously reported Hg2+-induced ring-
opening reactions.[38] The absorption and fluorescence titra-
tion of Hg2+ was also investigated by using 3.25 mm BRP-2
in acetonitrile/water (80/20 v/v). Upon the addition of Hg2+
,
the trend of variation in absorption and emission spectra
was similar to that of BRP-1. The changes of absorption and
emission spectra are shown in Figure 5. The I584/I510 ratio
was linearly proportional to the Hg2+ concentration over
the range of 6.5 to 62.5 mm (1.3–12.5 ppm). The response to
Hg2+ of BRP-2 was also very fast and spectral data were re-
corded within 5 min after the addition of Hg2+
.
Changes in the I584/I510 ratio of BRP-2 caused by other
metal ions are shown in Figure 6a. BRP-2 showed an excel-
lent selectivity toward Hg2+. The other metal ions did not
cause any apparent changes in the optical properties of
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3185

Y. Xiao, X. Qian et al.
ed with these sensors (2.5 mm)
for 15 min at room temperature
and then the cells were washed
three times with phosphate-buf-
fered saline (PBS; pH 7.4).
Upon excitation with blue light,
a
strong green intracellular
fluorescence was detected,
which indicated that both sen-
sors were cell permeable. Sup-
plementing cells incubated with
sensor BRP-1 with 2.5 mm
HgCl2 in PBS for 10 min at
room temperature resulted in
significant color changes, as
shown in Figure 7a. The re-
sponse of BRP-1 to Hg2+ was
very fast. Hela cells showed
color changes from green to
yellow–orange after incubation
with Hg2+ for 6 min. After
10 min, orange–red cell images
were obtained. The above re-
sults demonstrated that BRP-1
could sense and image intracel-
lular mercury ions sensitively
and quickly. BRP-2 also exhib-
ited good cell permeability and
bright green intracellular fluo-
rescence. However, the re-
sponse of BRP-2 to intracellu-
lar Hg2+ was much slower and
less sensitive than that of BRP-
1. To clearly demonstrate the
color changes, a high concentra-
tion of Hg2+ (10 equiv) was
used in the cellular imaging of
BRP-2. As shown in Figure 7b,
supplementing Hela cells incu-
bated with sensor BRP-2 with
25 mm HgCl2 (10 equiv) in PBS
at room temperature resulted in
gradual color changes from
green to green–yellow after 2 h.
After Hela cells were incubated
with mercury ions for 3 h,
orange cell images were ob-
served. Compared with BRP-1,
even in the presence of Hg2+
(10 equiv), a longer equilibrium
time of BRP-2 to Hg2+ was re-
quired to achieve apparent flu-
orescent color changes. Al-
though BRP-2 responded to
Figure 4. a) Hg2+-coordination-induced ring-opening reaction of BRP-2. b) ESI-MS spectrum of BRP-2 in the
presence of HgCl2 (1 equiv) in H2O/CH3CN. c) ESI-MS of BRP-2 in the presence of Hg
H2O/CH3CN solvent.
ACHTUNGTRENN(NUG ClO4)2 (1 equiv) in
In preliminary experiments, an inverted fluorescence mi-
croscope was used to observe the process of Hg2+ entering
into and diffusing within Hela cells. The cells were incubat-
Hg2+ in aqueous solution as quickly as BRP-1 did, its lower
performance in living cells might be ascribed to the recogni-
tion mechanism on coordination-induced reaction. There
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A Convenient and Efficient FRET Platform
FULL PAPER
I515ꢂ15. When cells stained with BRP-1 were incubated with
HgCl2 (2.5 mm) for 5 min, the color of the MCF-7 cells
showed significant changes from green to yellow–orange. In
the imaging, partial quenching of the green fluorescence in-
tensity and a partial increase in the red fluorescence intensi-
ty was observed (Figure 8b). Because Hg2+ only partially re-
acted to BRP-1 within 5 min, the pseudo-color of the ratio
imaging in Figure 8b showed real-time responses between
Hg2+ and BRP-1 in different cells. When MCF-7 cells
stained with BRP-1 were incubated with HgCl2 for 10 min,
the green fluorescence intensity decreased significantly and
the red fluorescence intensity remarkably increased (Fig-
ure 8c). The pseudo-color of ratio imaging in Figure 8c
showed different distributions of Hg2+ in MCF-7 cell. By
producing ratio images, it is possible to construct a map
showing local ion concentrations throughout the field of
view.
The fast and drastic green-to-red color changes of BRP-1
in intracellular Hg2+ imaging could be clearly distinguished
by using confocal fluorescence microscopy; this satisfied the
requirement for real-time observations. The visible color
changes should indicate to the two narrow emission bands
of BODIPY and TMR fluorophores. These two well-sepa-
rated emissions of BRP-1 by a single excitation wavelength
(488 nm) were highly desirable for fluorescence ratiometric
sensing, which not only ensured the detection accuracy, as
discussed in the Introduction, but also simplified the excita-
tion source to a single one and reduced the expense of the
detection device. The ratio changes of fluorescence intensi-
ties at 584 and 510 nm were converted into a pseudo-color,
which could show the distribution of Hg2+ in a cell.
Figure 5. Changes in the absorption (a) and emission spectra (b) of BRP-
2 (3.25 mm) in CH3CN/H2O (80:20) at pH 7.2 (0.01m HEPES) upon the
gradual addition of Hg2+ from 6.5 to 62.5 mm. Inset: the ratio intensity at
585 and 510 nm upon the gradual addition of Hg2+
.
are many thiol-containing biological molecules that might
compete with BRP-2 in the coordination of mercury ions.
Quantitatively detecting intracellular mercury ions re-
quired ratiometric imaging data that could not be obtained
by an ordinary inverted fluorescence microscope. Thus, con-
focal microscopy experiments were further carried out. Dif-
ferent from an inverted fluorescence microscope, a confocal
microscope has multichannel detectors, which provide good
opportunities to observe the changes of emission intensity at
different wavelengths. During the confocal imaging experi-
ments, an Ar laser provided the single excitation wavelength
(488 nm) that was suitable for the absorption of the
BODIPY fluorophore. Then, in this experiment fluores-
cence images were obtained at (515ꢂ15) (green channel)
and (590ꢂ25) nm (red channel), respectively. Ratiometric
sensor BRP-1 was chosen as an indicator due to its fast re-
sponse to mercury ions. MCF-7 cells incubated with BRP-1
(2.5 mm) for 15 min at room temperature showed a clear
green intracellular fluorescence, as shown in Figure 8a. In
the green channel a strong green fluorescence could be
seen. Meanwhile, there was little red fluorescence in the red
channel, which indicated that BRP-1 mainly existed in the
form of the spirolactam. As shown in the ratio imaging in
Conclusion
An intramolecular BRP FRET cassette with BODIPY and
TMR fluorophores was constructed based on a spectral
overlap-modulated FRET strategy. A rigid biphenyl group,
which favored through-bond energy transfer and kept a
fixed donor–acceptor distance and orientation, was used to
connect donor and acceptor moieties. To realize such a con-
nection, regioisomerically pure 5’-Br-TMR, a useful synthet-
ic building block, was developed according to a stepwise
synthetic strategy. The BODIPY moiety was connected to 5’
position of TMR efficiently through Suzuki coupling. BRP
was the first FRET platform in which the 2’-carboxyl group
of the rhodamine unit was preserved for further modifica-
tions. The spectral changes in different solvents indicated
that the FRET of BRP could be switched off and on under
proper conditions and the energy-transfer efficiency was up
to 99% in the on state. As a versatile FRET platform,
chemical modifications on BRP were convenient and
straightforward. Two Hg2+ sensors, BRP-1 and BRP-2, were
efficiently developed on the BRP platform as representative
ratiometric sensors. The recognition mechanisms of BRP-1
and BRP-2 were different: the mechanism for BRP-1 was
an irreversible ring-opening reaction induced by mercury
Figure 8a, there were no significant ratio changes of I590ꢂ25
/
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3187

Y. Xiao, X. Qian et al.
sensors exhibited clear Hg2+-in-
duced changes in the intensity
ratio of the two strong emission
bands of BODIPY and rhoda-
mine. While both were applica-
ble to the imaging of Hg2+ in
living cells, BRP-1 displayed
higher sensitivity and a faster
response. There have already
been many turn-on rhodamine
spirocyclic sensors, the numbers
of which are still increasing rap-
idly. All experiACTHNUTRGNEUGNences in turn-on
rhodamine spirocyclic sensors
may be used in ratiometric sen-
sors based on a FRET switch-
on strategy. The BRP platform,
as well as its analogues, should
be applicable to a broad range
of ratiometric sensors for vari-
ous analytes.
Experimental Section
General: All chemicals were obtained
from commercial suppliers and used
without further purification. The silica
gel used was 200–300 mesh. 1H and
13C NMR spectra were measured on
Varian 400 and Bruker 400 spectrome-
ters in CDCl3 or [D6]DMSO with tet-
ramethylsilane (TMS) as the internal
reference. Mass spectra were mea-
sured on a UPLC/Q-TOF mass spec-
Figure 6. a) I584/I510 ratios of BRP-2 (3.25 mm) in CH3CN/H2O (80:20 v/v, pH 7.2) in the presence of various
metal ions (10 equiv). Black bars represent the addition of an excess of the appropriate metal ion (32.5 mm) to
a 3.25 mm solution of BRP-2. Gray bars represent the subsequent addition of 32.5 mm Hg2+ to this solution.
2+
&
*
b) I585/I510 ratio of BRP-2 ( ) and BRP-2 in presence of Hg (5 equiv; ) in CH3CN/H2O (80:20 v/v) versus
different concentrations of HCl and NaOH at room temperature (lex =488 nm.)
ions, but the ring-opening reaction of BRP-2 was reversible.
Excited at the single excitation wavelength (488 nm), both
Figure 7. Images of Hela cells under an inverted fluorescent microscope. a) The cells were incubated with BRP-1 (2.5 mm) for 15 min at room tempera-
ture. Fluorescence images of Hela cells then further incubated with HgCl2 (2.5 mm) for 10 min were recorded. b) The cells were incubated with BRP-2
(2.5 mm) for 15 min at room temperature. Fluorescence image of Hela cells incubated with 25 mm HgCl2 for 3 h at room temperature were recorded.
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A Convenient and Efficient FRET Platform
FULL PAPER
Figure 8. Confocal fluorescence imaging of Hg2+ in MCF-7 cell with BRP-1 (2.5 mm) for 15 min. a) cells without Hg2+. b) cells incubated with HgCl2
(2.5 mm) for 5 min. c) cells incubated with HgCl2 (2.5 mm) for 10 min.
trometer. Fluorescence spectra were measured on spectroflurophotome-
ter FP-6500. Absorption spectra were determined on a UV/Vis spectro-
photometer HP-8453.
heated at 608C for 6 h. During the reaction the color of the solution
changed from red to pink. Then, the solvent was removed in vacuo to
give a red solid. This red solid was dissolved in DMF (15 mL) and then a
solution of phenyl isothiocyanate (50 mL, 0.28 mmol) in DMF (1.5 mL)
was added. The reaction mixture was heated at 608C for 4 h. The crude
product was purified by silica-gel column chromatography with a mixture
of CH2Cl2/CH3OH (300:1, v/v) as the eluent. After recrystalization in
mixture of dichloromethane and n-hexane, BRP-1 was obtained as red
crystals (75 mg, 63%). M.p. 242–2458C; 1H NMR (400 MHz, CDCl3,
Platform BRP: A mixture of compound 1 (600 mg, 1.33 mmol), 5’-bro-
motetramethylrhodamine (500 mg, 0.98 mmol), PPh3 (46 mg, 0.18 mmol),
PdACHTUNGTRENNUNG(OAC)2 (6.6 mg, 0.03 mmol), Na2CO3 (415.5 mg, 3.92 mmol), and n-
propanol/methanol (20 mL/20 mL) were placed in a 100 mL round-bot-
tomed flask with a magnetic stirrer bar under a nitrogen atmosphere .
The mixture was heated at 758C for 6 h. Then the crude product was pu-
rified by silica-gel column chromatography with a mixture of CH2Cl2/
258C, TMS): d=8.13 (d, 3J
A
G
4
3
8, J
(H,H)=3.6 Hz, 1H; ArH), 7.70 (d, J
G
CH3OH/NACHTUNGTRENNUNG(C2H5)3 (100:3.5:2.5, v/v) as the eluent. A red solid was ob-
(s, 1H; ArH), 7.51(s, 1H; ArH), 7.33 (d, 3J
N
tained. This solid was dissolved in methanol (5 mL) and added to HBF4
(2m), then the precipitate was collected and recrystalized in mixture of
dichloromethane and n-hexane. BRP was obtained as a bright black crys-
tal (42 mg, 78%). M.p. 274–2768C; 1H NMR (400 MHz, [D6]DMSO,
3
3
7.23–7.19 (t, J
ArH), 7.07
E
(d, 3J
G
R
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
258C, TMS): d=8.28 (d, 3J(H,H)=8.0 Hz, 1H; ArH), 8.23 (d, 3J
ACTHNUGTRENNUGN ACHTUNGTRENNNUG
A
ACHTUNGTRENNUNG
3
AHCTUNGTRENNUNG
7.2 Hz, 1H; ArH), 8.01 (d, JACHTGNUTRENNUNG
1.38 ppm (s, 6H; pyrrole-CH3); 13C NMR (100 MHz, CDCl3, 258C,
TMS): d=182.8, 166.9, 155.9, 154.2, 152.2, 151.2, 146.5, 143.1, 140.9,
140.4, 137.9, 135.5, 131.5, 129.1, 128.7, 128.5, 128.4, 127.7, 126.2, 125.1,
124.7, 123.1, 121.5, 109.4, 105.2, 99.7, 67.5, 40.3, 14.8 ppm; HRMS (ES):
m/z calcd for C50H47BN7O2F2S: 858.3573 [M+]; found: 858.3544.
3
7.50 (d, JACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(s, 6H; pyrrole-CH3); 13C NMR (100 MHz, [D6]DMSO, 258C, TMS): d=
166.1, 156.5, 155.0, 142.6, 141.2, 138.2, 134.5, 131.3, 130.8, 130.5, 129.7,
128.8, 128.2, 127.8, 121.4, 114.3, 112.7, 96.2, 39.9, 14.2 ppm; HRMS (ES):
m/z calcd for C43H40BN4O3F2: 709.3162 [M+]; found: 709.3134.
Sensor BRP-2: A solution of BRP (100 mg, 0.14 mmol) in dry 1,2-di-
chloroethane (20 mL) was stirred until the solid dissolved completely,
and phosphorus oxychloride (18 mL) was added with vigorous stirring at
Sensor BRP-1: Hydrazine monohydrate (0.10 mL (excess)) was added to
BRP (100 mg, 0.14 mmol) in MeOH (15 mL). The reaction solution was
Chem. Eur. J. 2011, 17, 3179 – 3191
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3189

Y. Xiao, X. Qian et al.
room temperature for 5 min. Then, the solution was heated at reflux for
2 h. The reaction mixture was cooled and evaporated in vacuo to give
BRP acid chloride. The crude acid chloride was dissolved in acetonitrile
(15 mL) and added dropwise to a solution of Na2S (22 mg, 0.28 mmol) in
water (5 mL). After stirring overnight, the crude product was purified by
silica-gel column chromatography with a mixture of dichloromethane and
n-hexane (1.5:1, v/v) as the eluent. BRP-2 was obtained as red crystals
[8] a) J. V. Ros-Lis, B. Garcꢄa, D. Jimꢅnez, R. Martꢄnez-MꢆÇez, F. San-
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(74 mg, 73%) M.p. 278–2818C; H NMR (400 MHz, CDCl3, 258C, TMS):
d=7.95 (d, 3J(H,H)=8.0 Hz, 1H; ArH), 7.78 (d, 3J
ACHTUNGTRENNUNG ACHTUNTGREN(NUGN H,H)=8.0 Hz, 1H;
3
3
ArH), 7.62 (d, J
(H,H)=8.0 Hz, 2H; ArH), 6.84 (d, 3J
3J
(H,H)=8.0 Hz, 4H; ArH), 5.96 (s, 2H; pyrrole-CH), 2.97 (s, 12H; N-
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(CH3)2), 2.54 (s, 6H; pyrrole-CH3), 1.37 ppm (s, 6H; pyrrole-CH3);
[10] a) Y. Zhao, X. B. Zhang, Z. X. Han, L. Qiao, C. Y. Li, L. X. Jian,
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13C NMR (100 MHz, CDCl3, 258C, TMS): d=196.9, 158.7, 155.9, 152.2,
151.3, 146.3, 143.2, 141.1, 140.1, 135.4, 131.4, 129.9, 128.8, 128.3, 127.8,
125.4, 123.4, 121.5, 109.7, 109.2, 98.8, 62.7, 40.4, 14.9, 14.7 ppm; HRMS
(ES): m/z calcd for C43H40BN4O2F2S: 725.2933 [M+]; found: 725.2906.
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Acknowledgements
We thank the NSF of China (no. 20876022), the Fundamental Research
Funds for the Central University (DUT10ZD114) and the Program for
Changjiang Scholars and Innovative Research Team in University
(IRT0711) for financial support. We also thank Dr. Liji Jin (Dalian Uni-
versity of Technology) for help with cell culture experiments and Dr.
Xiaolin Yuan (Dalian University) for help with confocal microscopy ex-
periments.
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Received: August 30, 2010
Published online: February 10, 2011
Chem. Eur. J. 2011, 17, 3179 – 3191
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3191