Through-bond energy transfer cassettes for multicolor encoding

Article abstract of DOI:10.1021/jo500653r

Through-bond energy transfer (TBET) has been proposed as a versatile strategy to develop encoded microspheres. Together with the donor molecule, two TBET cassettes with high intramolecular TBET efficiencies (98% and 99%) and pseudo-Stokes shifts about 70 and 160 nm have been codoped into PS microspheres. Upon exclusive excitation at 480 nm, these microspheres emit simultaneously triple peaks at 512, 570, and 656 nm. Further confocal imaging and flow cytometric analysis demonstrates satisfactory performances of the new encoded microspheres.

Full text of DOI:10.1021/jo500653r

Note
pubs.acs.org/joc
Through-Bond Energy Transfer Cassettes for Multicolor Encoding
Xinfu Zhang, Yi Xiao,* Ling He, and Yuhui Zhang
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
S
* Supporting Information
ABSTRACT: Through-bond energy transfer (TBET) has
been proposed as a versatile strategy to develop encoded
microspheres. Together with the donor molecule, two TBET
cassettes with high intramolecular TBET efficiencies (98% and
99%) and pseudo-Stokes shifts about 70 and 160 nm have
been codoped into PS microspheres. Upon exclusive excitation
at 480 nm, these microspheres emit simultaneously triple
peaks at 512, 570, and 656 nm. Further confocal imaging and
flow cytometric analysis demonstrates satisfactory perform-
ances of the new encoded microspheres.
low fluorescence microspheres array is a high-throughput
than 10 nm).7,8 However, within the microspheres, there is no
Ftechnique that can perform multiple discrete assays in a guarantee of the sufficiently short distances. In addition,
because of the randomness in dyes’ distributions, the
intermolecular FRET efficiencies are not stable in different
batches of microspheres and so the fluorescence coding is not
repeatable.
single tube. This detection combines the specificity of
fluorescence-encoded microspheres and the high sensitivity of
flow cytometry.1 It is of great demand in both research and
clinical applications, such as gene profiling, clinical diagnostics,
and environmental analysis.2,3 Although the concept of
multicolor encoding has been proposed for nearly 20 years,
the technical core of how to produce these uniform 5−10 μM
microspheres is highly unpublicized, which is unfavorable for
widespread applications.
It looks easy but is challenging to achieve single-excitation
several-emissions, the essential requirement for multicolor
encoding. Generally, two or more fluorophores with different
and large Stokes shifts are the first choices.4,5 Inorganic
fluorophores, e.g., quantum dots (QDs), have been employed,6
but limiting factors of QD, e.g., “blink” and their compatibility
within biosystems, prevent them from widespread applications.
A few large Stokes shift dyes with ICT (intramolecular charge
transfer) nature had also been attempted, but they suffered
from the broad emission and low excitation efficiency,5 which
decreases the sensitivity and accuracy. In contrast to QD and
ICT dyes, most organic dyes with high fluorescence quantum
yields have small Stokes shifts. Thus, for encoding microspheres
excited at a single wavelength, it is almost impracticable for two
fluorophores to emit in different spectral ranges independently.
The practical way to mediate emission signatures should be
fluorescence resonance energy transfer (FRET).
To overcome the problems of intermolecular FRET encoding
mode, we propose a platform approach to three-color
fluorescence encoded microspheres by codoping two intra-
molecular through-bond energy transfer (TBET) cassettes and
the donor. In a so-called TBET cassette, the donor dye and the
acceptor are connected by a rigid, short, and conjugated linker,
which makes the excitation energy-transfer processes “through
bond”.9−11 First, TBET is not subjected to the constraint of
spectral overlap between the donor emission and the acceptor
absorption, which means TBET cassettes may have very large
pseudo-Stokes shifts.11,12 Second, TBET occurs extremely fast
(in picosecond or even femtosecond range) and highly
efficiently (approaching 100%), which is not as severely
affected by surrounding medium molecules as FRET is. Burgess
et al.9,10,12 and Akkaya et al.13,14 did the pioneering work on
construction of TBET cassettes, and Ziessel et al.,15,16Lin,11 our
group,17,18 and others19,20 adopted this concept to design
ratiometric probes and solar energy harvesters. Very recently,
we and co-workers21,22 had extended this concept to develop
multiply pumped laser dyes with unprecedented laser perform-
ance. Inspired by these previous works, especially by Akkaya et
al., who proposed the strategy for higher TBET effciency,13 we
clearly know TBET cassettes’ photophysical features, such as its
large pseudo-Stokes shift and stable and high energy transfer
efficiency,23 are exactly what we desire for fluorescence coding
microspheres.
However, in practice, intermolecular FRET for fluorescence
encoding is still imperfect. To obtain usable FRET efficiency,
one of the preconditions is a considerable overlap of the
emission band of donor and the absorption band of acceptor.4,5
However, owing to the small Stokes shifts of most excellent
fluorophores, the above-mentioned overlap is also accompanied
by the hardly avoidable crosstalk of two emission bands.
Another precondition for efficient FRET is the spatial distance
between donor and acceptor should be as short as possible (less
Herein, two TBET cassettes, BDP-A1 and BDP-A2, were
prepared efficiently (Figure 1a). Three BODIPY derivatives
Received: March 22, 2014
Published: June 12, 2014
© 2014 American Chemical Society
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Note
Figure 1. (a) Structures of three fluorophores BDP, BDP-A1, and BDP-A2. Normalized (b) absorption and (c) fluorescence spectra of three
fluorophores in THF.
Table 1. Photophysical Properties of BDP, BDP-A1, and BDP-A2 in THF
a
e
f
g
h
solvent
λabs (nm)
λem (nm)
ε (M−1 cm−1
)
Φf
Φd
ETE (%)
R0 (Å)
R
(Å)
ETE (%)
BDP
502
512
69200
0.77
0.77
b
c
BDP-A1
BDP-A2
502, 561
502, 618
512, 570
512, 656
71300, 87600
73200, 92400
0.83 , 0.78
0.015
0.007
98.1
83.52
10.35
10.35
99.9
99.9
b
d
0.35 , 0.29
99.1
70.76
a
b
c
d
e
Rhodamine B is used as standard (Φf 0.69, in methanol). Exciting wavelength, λex = 480 nm. λex = 540 nm. λex = 580 nm. Energy transfer
f
efficiency, ETE =1-Φd/Φd0, Φd is the quantum yield of donor part in BDP-A1 or BDP-A2, Fd0 is the quantum yield of BDP. Forster radius,
̈
g
calculated according to the equation, R0 = 0.211[k2n−4ΦD∫ 0∞ID(λ)εA(λ)λ4dλ]1/6 k2 = 2/3, n (THF) = 1.4050. Radius, distance between the D and A
h
6
6
groups (estimated using standard bond lengths). Calculated according to the equation ETE = R0 /[R0 + R6].
Figure 2. Fluorescence spectra changes (λex = 480 nm) of singly labeled microspheres (in water) against doping amount. Inset: intensity of emission
maxima vs doping amount, unit 10−7 mol/(g of microspheres). (a) For BDP, slit 1 nm, 2.5 nm; (b) for BDP-A1, slit 2.5 nm, 2.5 nm; (c) for BDP-
A2, slit 5 nm, 2.5 nm.
with well-separated emission bands are chosen to construct the
cassettes. A biphenyl group is adopted as a linker for consistent
and highly efficient TBET according to our previous results.22
In the case of poor solubility for BDP-A2 due to its relatively
large conjugation structure, a Newkome dendrimer is
introduced. Figure 1b shows the absorbance spectra of the
cassettes are essentially equal to the sum of donor and acceptor
parts. They both maintain high molar extinction coefficients for
the donor part (∼73200 M−1 cm−1). Upon excitation of three
fluorophores at 480 nm, sharp fluorescence with different
colors (512 nm, 570 and 656 nm) is observed (Figure 1c and
Table 1). These emission spectra show very small overlap with
each other. For BDP-A1 and BDP-A2, they emit predom-
inantly from their acceptor fragments. The emission peaks are
very weak from donor at 512 nm. The quantum yields of BDP-
A1 are measured to be 0.83 and 0.78 for two different excitation
wavelengths (480 and 540 nm). BDP-A2 shows a similar result
with two quantum yields as 0.35 and 0.29 excited at 480 and
580 nm, respectively. That is to say, the fluorescence quantum
yields do not vary significantly with excitation wavelength,
which suggests almost complete energy transfer from donor to
acceptor. Calculated values of energy-transfer efficiency (ETE)
by two methods are listed in Table 1. Both methods provide
high ETE values over 98% for BDP-A1 and 99% for BDP-A2.
In addition, these fluorophores show good solubility in a broad
variety of organic solvents and they are proven as environ-
mental factor-independent fluorophores.
Microspheres labeled by BDP, BDP-A1n or BDP-A2 singly
show linearly increases in emission intensity against the doping
amount: (2−20) × 10−7 mol/(g of microspheres) as recorded
in Figure 2. Good linear correlation is observed in each case.
Importantly, upon excitation of microspheres singly labeled by
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Note
two cassettes at 480 nm (absorption of donor) the emission of
donor at 512 nm is ignorable, compared with the intensive
emissions from the acceptors. Thus, the TBET efficiencies in
microspheres are still very high. Further studies on excitation
spectra give a consistent result (Figure S1, Supporting
Information) that TBET cassettes can be efficiently excited at
the absorption of donor. Thus, properties of singly labeled
microspheres indicate that BDP-A1 and BDP-A2 are ideal
modules for fluorescence encoding microspheres.
Triply doped microspheres exhibit controllable emission
signatures along with the changes in doping amounts of BDP,
BDP-A1, and BDP-A2. Three fluorophores are successfully
codoped into microspheres under a same doping procedure
(for dye loading amounts, see Table S1, Supporting
Information). Non-normalized fluorescent spectra of fluores-
cence coding microspheres dispersed in water are shown in
Figure 3. The spectra show three clearly separated emission
For better demonstrating the advantage of TBET cassette
over conventional intermolecular FRET dye pair, we also labeled
microspheres by the detached donor and acceptor. Here, we
choose the second acceptor A2 promising a larger pseudo-
Stokes shift. No surprise, the result shows quite low FRET
efficiency. In the experiment of codoping a fixed amount of A2
[20 × 10−7 mol/(g of microspheres)] and a varied amount of
donor BDP [0−20 × 10−7 mol/(g of microspheres)] in
microspheres, emission spectra show only a very slight increase
of A2’s emission intensity despite the remarkable and linear
increase of the emission of donor (Figure 4). The inset clearly
shows the different increasing trends of the two emission
maxima. Furthermore, in another experiment of codoping a
fixed amount of donor BDP and a varied amount of A2, spectra
show moderate energy transfer from BDP to A2 when the
amounts of acceptor become large. However, to obtain equal
emission intensity between BDP and A2, a large amount of A2
up to 64 × 10−7 mol/(g of microspheres) is used. That is quite
a high doping concentration and brings about a clear red shift
of emission and a trend of fluorescence self-quenching. Hence,
the low intermolecular FRET efficiency results in unsatisfactory
tunability of donor−acceptor emission ratio, which is
unfavorable for fluorescence encoding.
Confocal microscope images intuitively illustrate the
encoding results. We set three channels to collect three
emissions from BDP, BDP-A1, and BDP-A2, respectively. In
Figure 5, there are six different microspheres that show different
emission intensities in three channels (dye loading amounts,
see Table S2, Supporting Information). For example, micro-
spheres 1 and 5 are marked with their emissions (for others, see
Figure S4, Supporting Information). Microspheres 1 are singly
labeled with BDP, which shows bright fluorescence in green
channel but no fluorescence in the other two channels. It
demonstrates nearly no crosstalk between channels. Micro-
spheres 5 codoped with three fluorophores exhibit fluorescence
signals in all the three channels. Not only can the beads be
easily distinguished by the color difference, but in addition,
their encoding information can be readily demonstrated by
emission fingerprints extracted from the microscopic images. As
discussed previously, this type of simultaneous excitation with
well-separated emissions is hardly possible for microspheres
loaded with conventional organic dyes.
Figure 3. Fluorescence spectra of triple labeled microspheres in water
(λex = 480 nm, λem = 490−750 nm, slit 5 nm, 5 nm).
peaks, which is a result that previous methods cannot achieve.
The doping amount of each fluorophore is adjusted at three
levels (just for illustration; no problem for more levels), and
thus, there are three levels of intensity changes for each
emission maximum. Moreover, under a same labeling
operation, intensity of each emission maximum shows a relative
stable value against doping amount. That is to say, we can
adjust the intensity of each emission maximum simply by
increasing or decreasing the amount of corresponding
fluorophore without affecting the others’ emissions. Thus,
fluorescence coding microspheres based on TBET cassette
exhibit tailored emission fingerprints other than a “this is it”
result.
Flow cytometric analysis also confirms that these fluores-
cence encoded microspheres are suitable for high throughput
assays. In two-color flow cytometric analysis, the fluorescence
intensity ratio from different channel is the basis for the
recognition of different fluorescent microspheres. Thus,
Figure 4. Fluorescence spectra changes of microspheres (in water, λex = 480 nm) labeled by BDP (λem = 512 nm) and A2 (λem = 656 nm). (a)
Fluorescence spectra change vs doping amount of BDP, inset: intensity of emission maxima vs amount of BDP. (b) Fluorescence spectra change vs
doping amount of A2. Inset: intensity of emission maxima vs amount of A2; unit 10−7 mol/(g of microspheres).
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spheres with different I656/I570 ratios can also be discriminated
by a flow cytometer (Figure 6b). Flow cytometric analysis of
five microspheres labeled by different amounts of BDP-A1 and
BDP-A2 is given in the Supporting Information (Figures S7
and S8, dye loading amounts, Table S4).
In conclusion, the TBET cassette strategy has been proposed
for rational development of encoded microspheres for the first
time. By the connections of two different long wavelength
BODIPY dyes to a short wavelength BODIPY dye via a
biphenyl linker, two TBET cassettes have been constructed.
Together with the donor they are loaded into self-produced
polystyrene microspheres which demonstrate tailored emission
fingerprints upon single excitation of the donor. The good
fluorescence encoding performance should be ascribed to the
high efficiency of TBET. In contrast, within those microspheres
loaded with unbound donor and acceptor, the encoding
performance is unsatisfactory, resulted from the poor efficiency
of intermolecular FRET. Confocal imaging and flow cytometric
analysis demonstrates that the TBET-based encoded micro-
spheres are of practical applicability in multiplex and high
throughput assays. Since there are a variety of excellent dyes as
candidates for donors and acceptors, above TBET strategy is a
versatile approach for DIY production of fluorescence encoded
microspheres by researchers.
Figure 5. Confocal microscope images of triply doped fluorescence
microspheres (λex = 488 nm): (a) green channel (λem = 495−555 nm);
(b) orange channel (λem = 560−620 nm); red channel (λem = 655−
755 nm); (d) overlap of a, b, and c.
EXPERIMENTAL SECTION
■
different emissions with small spectral crosstalk makes
quantification of intensity from different channels easy, which
means a precise ratio, namely, a precise decoding. Five
randomly chosen microspheres with different I512/I570 ratios
(0.05, 2, 2.5, 7.5, and 10) adjusted by codoping different
amounts of BDP and BDP-A1 (Figures S5 and S6, dye loading
amounts, Table S3, Supporting Information) are studied as
representatives. The data dots for each group of microspheres
are mostly distributed in linear banding regions that show
similar slopes close to 1, which indicates that in each group all
microspheres present an equable I512/I570, namely, that they are
evenly labeled by the identical ratio of BDP to BDP-A1 (Figure
S6, Supporting Information). Interestingly, as shown in Figure
6a, the mixture of the above microspheres exhibits five clearly
separated banding regions parallel to each other without
overlapping, which indicates that differently encoded beads can
be readily discriminated by a flow cytometer. The same
experiment is also conducted by using microspheres with
different codoping amounts of BDP-A1 and BDP-A2. Micro-
Microsphere Synthesis and Fluorophore Doping. Mono-
disperse polystyrene microspheres, with carboxy group outside, are
prepared by two-stage dispersion polymerization. These microspheres
are monodisperse and uniform in size (∼5.5 μm) (Figure S2,
Supporting Information), which is usually required for a flow
cytometric measurement.24,25 Scanning electron microscope revealed
that the beads had a 5.5 μm diameter in size (diameter). Incorporation
of three fluorophores is achieved by swelling the beads in a solvent
mixture containing 22% (v/v) THF and 78% (v/v) H2O and by
adding a controlled amount of fluorophores to the mixture (FigureS3,
Supporting Information). Fluorophores were prepared as a 1 mM
solution in THF. After 4 min, the embedding process was completed
by adding a large amount of water followed by centrifugation to obtain
the fluorescence microspheres. The beads were washed with ethanol
twice in case unlabeled dyes were present.
Synthesis of Compounds. The BDP,26 DC-SPC,26 4′-B-
BODIPY,27 and Newkome dendrimer28 were prepared following the
reported methods. For others, see below:
A1. BDP (1.11 mmol, 500 mg) and benzaldehyde (1.11 mmol, 118
mg) were added to a 100 mL round bottomed flask containing 50 mL
of toluene, and to this solution were added piperidine (1 mL) and
acetic acid (1 mL). The mixture was heated under reflux by using a
Dean−Stark trap, and reaction was monitored by TLC 1:5 CH2Cl2/
hexanes (Rf 0.3). When all the starting material had been consumed,
the mixture was cooled to room temperature and solvent was
evaporated. Water (300 mL) was added to the residue, and the
product was extracted into the CH2Cl2 (3 × 200 mL). The organic
phase was dried over Mg2SO4 and evaporated, and the residue was
purified by silica gel column chromatography using 1:5 CH2Cl2/
hexanes as the eluent to yield the desired product A1 as a purple
Figure 6. Flow cytometric analysis of a mixture sample of five
microspheres: (a) co-doping by BDP and BDP-A1, FITC-A is the
name for green channel: 530
30 nm; PE-A is the name for red
channel: 585 42 nm; (b) co-doping by BDP-A1 and BDP-A2, PE-A
is the name for red channel: 585 42 nm; PerCP-Cy5.5-A is the name
for NIR channel: 670LP.
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Note
powder (179 mg, 30%): mp 261−263 °C; 1H NMR (400 MHz,
CDCl3) δ 7.86 (d, 2H), 7.67 (d, 1H), 7.59 (d, 2H), 7.37 (t, 2H), 7.30
(t, 1H), 7.25 (d, 1H), 7.08 (d, 1H), 6.62 (s, 1H), 6.02 (s, 1H), 2.59 (s,
3H), 1.47 (s, 3H), 1.44 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
156.0, 152.9, 142.7, 138.9, 138.3, 136.5, 136.3, 134.6, 130.2, 128.9,
128.7, 127.5, 121.6, 119.1, 117.7, 94.7, 14.9, 14.7, 14.7; MS m/z (TOF
MS ES) calcd M+ for C26H22BN2F2I 538.0889, found 538.0851.
3H), 2.14 (t, 6H), 1.91 (t, 6H), 1.65 (s, 3H), 1.45 (s, 3H), 1.41 (s,
27H); 13C NMR (100 MHz, CDCl3) δ 172.9, 163.9, 141.0, 140.6,
138.6, 134.1, 130.1, 128.7, 126.5, 126.0, 123.8, 123.2, 120.8, 120.7,
120.2, 109.3, 109.2, 95.4, 81.0, 58.2, 53.2, 39.0, 30.0, 29.6, 28.1, 12.6,
12.2; MS m/z (TOF MS ES) calcd M+ for C59H67BN7O7F2NaCl2I
1254.3483, found 1254.3463.
BDP-A1. A1 (0.19 mmol, 100 mg), 4′-B-BODIPY (0.19 mmol, 86
mg), PPh3 (4.9 mg, 0.019 mmol), Pd(OAC)2 (6.6 mg, 0.03 mmol),
Na2CO3 (42 mg, 0.4 mmol), n-propanol/THF/water (3 mL/6 mL/0.3
mL), and a magnetic stir bar were placed in a 25 mL round-bottom
flask under a nitrogen atmosphere. The mixture was heated at 75 °C
for 3 h. Then the crude product was purified through silica gel column
chromatography with a mixture of 1:4 CH2Cl2/hexanes as eluent. A
BDP-A2. A2 (0.08 mmol, 100 mg), 4′-B-BODIPY (0.08 mmol, 36
mg), PPh3 (4.9 mg, 0.019 mmol), Pd(OAC)2 (6.6 mg, 0.03 mmol),
Na2CO3 (21 mg, 0.2 mmol), n-propanol/THF/water (3 mL/6 mL/0.2
mL), and a magnetic stir bar were placed in a 25 mL round-bottom
flask under a nitrogen atmosphere. The mixture was heated at 75 °C
for 3 h. Then the crude product was purified through silica gel column
chromatography with a mixture of 1:2 hexanes/ethyl acetate as eluent.
1
brown solid was obtained (128 mg, 92%): mp >300 °C; H NMR
(400 MHz, CDCl3) δ 7.83 (d, 4H), 7.61 (d, 1H), 7.59 (d, 2H), 7.44−
7.37 (m, 6H), 7.30 (t, 1H), 7.26 (d, 1H), 6.64 (s, 1H), 6.04 (s, 1H),
6.01 (s, 2H), 2.62 (s, 3H), 2.58 (s, 6H), 1.54 (s, 3H), 1.50 (s, 3H),1.48
(s, 6H); 13C NMR (100 MHz, CDCl3) δ 155.9, 155.7, 152.7, 151.0,
143.0, 142.9, 142.2, 141.2, 140.7, 140.0, 136.5, 136.1, 134.7, 132.8,
131.9, 131.4, 129.0, 128.8, 128.7, 127.6, 127.5, 14.9, 14.7, 14.7, 14.6;
MS m/z (TOF MS ES) calcd M+ for C45H40B2N4F4 734.3375, found
734.3348.
1
A dark blue solid was obtained (99 mg, 85%): mp 215−216 °C; H
NMR (400 MHz, CDCl3) δ 8.37 (d, 1H), 8.26 (s, 1H), 8.16 (d, 1H),
7.84 (s, 4H), 7.79 (d, 1H), 7.74 (s, 1H), 7.53 (d, 2H), 7.48 (t, 1H),
7.43−7.38 (m, 5H), 7.28 (d, 1H), 6.74 (s, 1H), 6.01 (s, 2H), 5.66 (s,
2H), 4.80 (s, 2H), 2.67 (s, 3H), 2.58 (s, 6H), 2.15 (t, 6H), 1.92 (t,
6H), 1.66 (s, 3H), 1.52 (s, 3H), 1.49 (s, 6H), 1.48 (s, 3H), 1.42 (s,
27H); 13C NMR (100 MHz, CDCl3) δ 172.8, 163.8, 155.7, 151.8,
148.3, 144.3, 143.0, 141.1, 141.0, 140.6, 138.4, 139.8, 139.0, 136.8,
134.7, 134.1, 131.4, 130.7, 128.9, 128.8, 128.7, 127.8, 127.6, 126.5,
125.9, 123.7, 123.1, 121.3, 120.8, 120.6, 120.1, 109.3, 109.2, 80.9, 58.2,
53.1, 38.9, 29.9, 29.7, 14.7, 12.6, 12.2; MS m/z (TOF MS ES) calcd
M+ for C78H85B2N9O7F4NaCl2 1450.5969, found 1450.5920.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, characterization and additional
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: xiaoyi@dlut.edu.cn.
■
Notes
A2. Under a nitrogen atmosphere, CuSO4·5H2O (2.8 mg, 0.012
mmol) in H2O (1 mL) and Na−ascorbate (4.2 mg, 0.022 mmol) in
H2O (1 mL) followed by DIPEA (30 mL, 0.174 mmol) were added to
a solution of Newkome dendrimer (52 mg, 0.106 mmol) in EtOH (8
mL). Then a solution of DC-SPC (63 mg, 0.086 mmol) in toluene (20
mL) was slowly added dropwise to the mixture in the absence of light.
The reaction mixture was stirred in the dark for 12 h at rt. Still in the
absence of light, the solvent was evaporated off under vacuum, and the
product was purified by silica gel column chromatography using 2:1
hexanes/ethyl acetate as the eluent yielded the desired product A2 as
dark blue solid (100 mg, 95%): mp 167−169 °C; 1H NMR (400 MHz,
CDCl3) δ 8.30 (d, 1H), 8.25 (s, 1H), 8.15 (d, 1H), 7.99 (d, 2H),
7.80−7.70 (m, 2H), 7.53 (d, 2H), 7.48 (t, 3H), 7.37 (s, 1H), 7.29 (d,
1H), 7.05 (d, 2H), 6.74 (s, 1H), 5.66 (s, 2H), 4.80 (s, 2H), 2.65 (s,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Y.X. thanks the National Natural Science Foundation of China
(Nos. 21174022 and 21376038), the National Basic Research
Program of China (No. 2013CB733702), and the Specialized
Research Fund for the Doctoral Program of Higher Education
(No. 20110041110009) for financial support.
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