Conjugated polyelectrolytes with aggregation-enhanced emission characteristics: Synthesis and their biological applications

Article abstract of DOI:10.1002/asia.201300501

Conjugated polyelectrolytes are promising candidates for the construction of fluorescent bioprobes. In this study, a series of water-soluble fluorescent polyelectrolytes have been designed and synthesized by means of the quaternization of their tetrapheny

Full text of DOI:10.1002/asia.201300501

FULL PAPER
DOI: 10.1002/asia.201300501
Conjugated Polyelectrolytes with Aggregation-Enhanced Emission
Characteristics: Synthesis and their Biological Applications
Rongrong Hu,[a, b] Ruquan Ye,[b] Jacky W. Y. Lam,[a, b] Min Li,[b] Chris W. T. Leung,[b] and
Ben Zhong Tang*[a, b, c]
Dedicated to Professor Chunli Bai on the occasion of his 60th birthday
Abstract: Conjugated polyelectrolytes
are promising candidates for the con-
struction of fluorescent bioprobes. In
this study, a series of water-soluble flu-
orescent polyelectrolytes have been de-
signed and synthesized by means of the
quaternization of their tetraphenyle-
thene-containing polyyne precursors.
The polyynes can be facilely prepared
through Hay–Glaser polycoupling in
high yields (up to 99%) with high mo-
lecular weights (up to 38900). All the
polymers exhibit a phenomenon of ag-
gregation-induced or -enhanced emis-
sion. The fluorimetric titrations of bio-
molecules such as heparin, calf thymus
DNA, RNA, bovine serum albumin,
and human serum albumin to buffer
solutions of the polyelectrolytes sug-
gest that they are promising fluorescent
bioprobes with high sensitivity and fast
response. The emission intensity of the
polyelectrolytes is enhanced by up to
sevenfold upon binding with biomole-
cules through electrostatic and hydro-
phobic cooperative interactions. The
polyelectrolytes can also serve as fluo-
rescent visualizers for intracellular
imaging with good biocompatibility
and low autofluorescence interference.
Keywords: aggregation
· electro-
lytes · fluorescent probes · imaging
agents · polymers
Introduction
rescent nanoparticles offer improved photostability and high
luminescence quantum efficiency, they introduce new prob-
lems such as difficult synthesis, limited variety, and high cy-
totoxicity.[4] Organic dyes, on the other hand, provide the
opportunity for chemical-structure modification, thus ena-
bling fine-tuning of their photophysical properties by sys-
tematic structural variation.[5] Although much progress has
been made in this field, major challenges that limit the prac-
tical high-tech applications of fluorescent bioprobes, such as
poor solubility in aqueous media, poor stability under ambi-
ent conditions, insufficient biocompatibility, expensive prices
due to painstaking syntheses, and so on, remain to be
solved.[6] The development of readily accessible and environ-
mentally stable bioprobes with high sensitivity and selectivi-
ty is thus in urgent demand.
Water-soluble fluorescent conjugated polyelectrolytes
(CPEs) have long been recognized as an important class of
materials owing to the unique combination of the optoelec-
tronic properties of the conjugated polymers and the poten-
tial electrostatic interactions between the polyelectrolytes
and the analytes. They afford a unique platform for the de-
velopment of highly sensitive fluorescence-based sensors
with fast response for biological targets and are of great aca-
demic and technological importance.[7] Most conjugated
polyelectrolyte-based biosensors rely on the fluorescence-in-
tensity change upon binding with the bioanalyte.[8] The ana-
lyte-induced aggregation of polymer backbones through
electrostatic and hydrophobic cooperative interactions was
the most widely adopted sensing mechanism for CPEs.[9] In
this process, multiple charges and hydrophobic cavities in
The development of new technologies for the detection of
biomolecules has attracted much academic and industrial in-
terest because of the rapidly increasing demand for genetic
analysis, clinical diagnosis, biowarfare, environmental analy-
sis, and homeland defense.[1] Among various methods such
as the traditionally used colorimetric techniques[2] for pro-
tein quantitation, fluorescence-based probes have received
special attention owing to their high sensitivity, selectivity,
and rapidity.[3] Although inorganic quantum dots and fluo-
[a] Dr. R. Hu, Dr. J. W. Y. Lam, Prof. B. Zhong Tang
The Hong Kong University of Science & Technology (HKUST)
Shenzhen Research Institute
No. 9 Yuexing 1st RD, South Area, Hi-tech Park
Nanshan, Shenzhen, 518057 (China)
Fax : (+85)2-23581594
E-mail: tangbenz@ust.hk
[b] Dr. R. Hu, R. Ye, Dr. J. W. Y. Lam, M. Li, C. W. T. Leung,
Prof. B. Zhong Tang
Department of Chemistry
Institute of Molecular Functional Materials
and Division of Biomedical Engineering, HKUST
Clear Water Bay, Kowloon, Hong Kong (P.R. China)
[c] Prof. B. Zhong Tang
Guangdong Innovative Research Team
SCUT-HKUST Joint Research Laboratory
State Key Laboratory of Luminescent Materials and Devices
South China University of Technology (SCUT)
Guang Zhou 51064 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201300501.
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the biomolecules facilitate the formation of aggregates,
thereby resulting in a remarkable fluorescence change of
the aqueous solution and thus realizing analyte detection.
Typical CPE-based fluorescence assays allow detection of
the target analytes in the nanomolar[10] or even picomolar[11]
concentration ranges. This is a major advantage of CPEs,
considering that the detection sensitivity is one of the most
important aspects in biosensor development with the ulti-
mate goal being the trace detection of genes or proteins in
biological fluids.
In this study, we designed and synthesized a series of
TPE-containing conjugated polyelectrolytes and investigated
their utilities as fluorescent bioprobes for the detection of
heparin, nucleic acids (ctDNA and RNA), and proteins
(BSA and HSA). The fluorescence of the TPE-containing
CPEs in aqueous buffer solutions was greatly enhanced
upon binding to the biomacromolecules by means of electro-
static attraction and hydrophobic interaction on account of
their multiple positive charges and the hydrophobic nature
of their polymer backbones.
Poly(p-phenylenevinylene)s, poly(thiophene)s, poly(phen-
ylene ethynylene)s, poly(p-phenylene)s, and polyfluorenes
are widely used as conjugated polyelectrolytes for the con-
struction of bioprobes.[12] However, these CPEs generally do
not exist as isolated chains and form simple solutions in
pure water. Instead, they tend to form aggregates in aque-
ous buffers.[13] The aggregation of the polymer chains favors
the formation of detrimental species such as excimers and
exciplexes and causes nonradiative relaxation of the excited
states. Such an aggregation-caused quenching (ACQ) effect
of light emission in the aggregated state adversely affects
their emission properties and has consequently hampered
their application as bioprobes.[14] Recently, we and other
groups observed a phenomenon of aggregation-induced
emission (AIE) that is the exact opposite to the ACQ
effect: a series of propeller-shaped molecules such as tetra-
phenylethenes (TPEs), siloles,
Results and Discussion
Polymer Synthesis
Polyelectrolytes P1–P4 were synthesized according to the re-
action routes shown in Schemes 1 and 2. The palladium-cat-
alyzed Sonogashira coupling reaction of 4,4’-dibromobenzo-
phenone (10) with trimethylsilylacetylene (TMSA) fur-
nished product 8. Treatment of 4,4’-dihydroxybenzophenone
(11) with an excess amount of 1,2-dibromoethane afforded
9, which then underwent a McMurry coupling reaction with
8 to generate intermediate 7. The trimethylsilyl groups of 7
were cleaved under basic conditions, thereby furnishing the
desirable TPE-containing monomer 5. Hay–Glaser polycou-
pling of 5 at 508C in the presence of CuCl and N,N,N’,N’,-
butadienes, pyrans, and ful-
venes are nonemissive in the
solution state but become
highly luminescent in the ag-
gregated state.[15] Through
a series of designed experi-
ments and theoretical calcula-
tions, we proposed the restric-
tion of intramolecular rotation
as the main cause for the AIE
effect. Decorating AIE-active
compounds with ionic or polar
functional groups renders the
dyes
water-soluble,
which
means they can be utilized as
fluorescent probes for bioanal-
yses.[16] In fact, we and many
research groups have succeed-
ed in utilizing TPE-based AIE
luminogens for nucleic acid de-
tection, enzymatic activity
assay, and metallic ion trac-
ing.[17] Such promising results
prompted us to solve the noto-
rious ACQ problem of current
CPE-based bioprobes by utiliz-
ing AIE-active CPEs and ex-
plore their applications in bio-
analyses further.
Scheme 1. Synthetic routes to polymers P1 and P2.
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Ben Zhong Tang et al.
absorption peaks related to the
stretching of the diyne function-
ality are observed at 2205 and
2143 cmÀ1 (see the Supporting
Information). The IR spectra of
P1–P4 largely resemble those of
P5 and P6, except for the new
peaks associated with aliphatic
À
C H stretching vibrations at
2872–2678 cmÀ1 (see the Sup-
porting Information).
Similar results are obtained
1
from the H NMR spectroscop-
ic analyses. The singlet peak at
d=3.05 ppm in the spectrum of
5 originates from the absorp-
tion of its terminal acetylene
proton, which weakens substan-
tially in the spectrum of P5 (see
the Supporting Information).
The same trend was observed
in the spectra of 6 and P6: the
peak at d=3.06 ppm on ac-
count of the terminal acetylene
proton resonance of 6 disap-
pears in the spectrum of P6,
Scheme 2. Synthetic routes to polymers P3 and P4 (TBAF: tetrabutylammonium fluoride).
tetramethylethenediamine (TMEDA) in 1,2-dichloroben-
zene (o-DCB) for 5 h produced P5 with a weight-average
molecular weight (Mw) of 38900 in nearly quantitative yield.
Treatment of P5 with an excess amount of trimethylamine
or triethylamine furnished polyelectrolytes P1 and P2 in
high yields. Similarly, TPE-containing polyelectrolytes P3
and P4 with one trialkylammonium group on each repeating
unit were successfully obtained by similar synthetic path-
ways by using monohydroxybenzophenone (14) instead of
11 as the starting material.
which is indicative of the complete consumption of the
triple bond by the polymerization. Meanwhile, the absorp-
tion peaks of P6 are significantly broadened relative to
those of 6 owing to its polymeric nature (see the Supporting
Information). Strong peaks due to the absorptions of the
methyl and ethyl groups of the ammonium salt moieties are
observed in the spectra of P1 and P2 at d=3.33–3.27 and
3.46 and 1.26 ppm, respectively. These findings verify their
polyelectrolyte structures and suggest that the quaterniza-
tion reaction proceeds to a great extent.
Structural Characterization
Solubility
All the intermediates and products were characterized by
IR and NMR spectroscopies and gave satisfactory analytical
data that corresponded to their expected molecular struc-
tures (see the Experimental Section for details). Their high-
resolution mass spectra gave M+ peaks at m/z 624.0311
(624.0300, calcd for 5) and 502.0933 (502.0932, calcd for 6),
thereby confirming the formation of the expected monomers
(see the Supporting Information).
CPEs P1–P4 dissolve completely or partially in water,
whereas P5 and P6 possess good solubility in common or-
ganic solvents such as toluene, dichloromethane, chloroform,
THF, and so on. On the other hand, all the polymers are
soluble in DMSO. CPEs with trimethylammonium groups
(P1 and P3) show better water solubility than those with
triethylammonium functionalities (P2 and P4). The magni-
tude of charge in each repeating unit also affects the water
solubility. After taking into the account all these aspects, P1
possesses the best water solubility and can facilely dissolve
in water or aqueous buffer.
The IR spectra of 5 and P5 are given in the Supporting
Information as an example. The strong absorption bands ob-
served at 3290 and 2105 cmÀ1 in 5 are associated with its ꢀ
À
C H and CꢀC stretching vibrations, respectively. These
bands are absent in the spectra of P5, and new absorption
Photophysical Properties
À
peaks related to CꢀC CꢀC stretching vibrations emerge
at 2202 and 2141 cmÀ1, thereby suggesting that all the triple
The absorption spectra of the polymers in dilute solutions
(10 mm) are shown in Figure 1. The post-polymerization
slightly alters the ground-state electronic transition, and the
absorption maximum of P1 and P2 as well as their precursor
bonds of 5 have been consumed by the polycoupling reac-
À
tion. Similarly, the spectrum of P6 shows no ꢀC H and Cꢀ
C stretching vibrations of 6 at 3290 and 2109 cmÀ1, and new
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Figure 1. Normalized absorption spectra of P1 and P2 in DMSO, and P5
and P6 in THF. Concentration: 10 mm.
Figure 3. A) Photographs of P6 in THF/water mixtures with different
water fractions (fw) taken under 365 nm UV irradiation from a hand-held
UV lamp. B) Emission spectra of P6 in THF/water mixtures with differ-
ent fw values. C) Plot of relative PL intensity (I/I0) versus the composition
of the aqueous mixture of P6. Solution concentration: 10 mm; excitation
wavelength: 366 nm.
P5 is located at 372 nm. The absorption of P6 is blueshifted
6 nm from that of P5, thereby revealing that the bromoe-
thoxy group contributes to the conjugation of the polymer.
Studies of the photoluminescence (PL) properties of P5
and P6 reveal that they exhibit the typical phenomenon of
aggregation-enhanced emission (AEE) or AIE. The iconic
impression of the AIE/AEE features of P5 and P6 are given
by their fluorescent photographs in THF and THF/water
mixtures, which are shown in Figures 2A and 3A. Whereas
the solution of P5 in THF emits weak yellow light, addition
of water, a nonsolvent of the polymer, into the solution in-
duces the polymer chains to aggregate and enhance its emis-
sion intensity. The emission remained weak until 30 vol%
water was added, after which it started to increase. The solu-
tion of P6 in THF, however, emitted no light under UV irra-
diation, whereas an intense yellow emission was observed
when its chains aggregated in the presence of a large
amount of water (ꢁ70 vol%).
To have a quantitative picture, the emission behaviors of
P5 and P6 in THF and THF/water mixtures were investigat-
ed with a PL spectrophotometer. Compound P5 emits faint-
ly at 536 nm in THF (Figure 2). Addition of water increases
the emission intensity, progressively accompanied by a blue-
shift in the emission maximum. From the isolated species in
THF to aggregates in a 90% aqueous mixture, the emission
intensity rises 15-fold, whereas the emission maximum blue-
shifts by 11 nm. Unlike P5, no emission is detected from the
solution of P6 in THF. When its chains aggregate in the
presence of water, an emission peak emerges at 536 nm, the
intensity of which increases gradually with increasing water
content in the THF/water mixture. In 90% aqueous mixture,
the intensity is 65-fold higher than that in THF. The steric
effect of the bromoethoxy groups in P5 might inhibit the
free rotation of the TPE units, thus rendering the polymer
emissive in solution. It also makes the polymer adopt
a more twisted structure, and a bluer emission is detected in
the aggregated state than in that of P6.
Fluorimetric Heparin Titration
Heparin is a highly sulfated glycosaminoglycan with an aver-
age molecular weight of approximately 15000 Da; it is
widely used as an injectable anticoagulant.[18] Among all
known biological molecules, heparin has the highest nega-
tive charge density. The anticoagulant activity of heparin is
due to its interaction with the proteins involved in the
blood-clotting cascade. It is thus crucial to monitor the hep-
arin level. With this in mind, fluorescence titrations of hepa-
rin using P1 and P2 in aqueous solutions were carried out.
The polymers were first dissolved in DMSO to give solu-
Figure 2. A) Photographs of P5 in THF/water mixtures with different
water fractions (fw) taken under 365 nm UV irradiation from a hand-held
UV lamp. B) Emission spectra of P5 in THF/water mixtures with differ-
ent fw values. C) Plot of relative PL intensity (I/I0) versus the composition
of the aqueous mixture of P5. Solution concentration: 10 mm; excitation
wavelength: 372 nm.
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Ben Zhong Tang et al.
tions with a concentration of 10À3 m, and then they were di-
luted to 5 mm with water. As shown in Figure 4, the aqueous
solution of P1 shows an emission peak at 520 nm. When
a small amount of heparin solution was added, the emission
Figure 4. A) Fluorimetric titration of heparin to an aqueous solution of
P1 in water. B) Plot of I/I0 at 524 (P1) and 527 nm (P2) versus the hepa-
rin concentration. I0 =emission intensity in the absence of heparin. [P1]=
[P2]=5 mm; excitation wavelength: 330 nm.
was enhanced approximately threefold along with a 4 nm
redshift in the emission maximum. The addition of more
heparin does not cause further enhancement in the emission
intensity, thereby suggesting that the interaction between P1
and heparin is saturated at low heparin concentration. Simi-
larly, the aqueous solution of P2 emits at 527 nm, with inten-
sities increasing by up to 1.6-fold in the presence of heparin
(see the Supporting Information and Figure 4B).
Figure 5. Fluorimetric titration of A) ctDNA and C) RNA to an aqueous
solution of P1 in water. Plot of I/I0 at 524 (P1) and 527 nm (P2) versus
the B) ctDNA and D) RNA concentration. I0 =emission intensity in the
absence of ctDNA or RNA. [P1]=[P2]=5 mm; excitation wavelength=
330 nm.
low ctDNA concentration and gradually became saturated
as the ctDNA concentration increased.
In aqueous media, the cationic polymers spontaneously
bind to the negatively charged heparin, driven by electro-
static forces. When the polymer strands are bound to the
biomolecules, the intramolecular rotation of the TPE units
is restricted, which blocks the nonradiative relaxation chan-
nels and hence renders the polymers emissive. The free rota-
tion of the TPE phenyl rings in P2 is somewhat hindered in
the aqueous solution owing to the relatively poor water sol-
ubility, whereas the free rotation in P1 is still severe. The P2
solution thus shows a higher pristine emission intensity I0 in
the absence of heparin and lower relative fluorescent en-
hancement I/I0 than P1.
The fluorescence intensity of P1 also becomes higher
upon binding to RNA (Figure 5C). The plot of relative PL
intensity (I/I0) versus the RNA concentration shows that the
emission rises initially with increasing RNA concentration
but decreases slightly when more RNA is present in the
aqueous solution. Compared with P1, P2 shows a similar
emission enhancement but with a lower sensitivity (see the
Supporting Information and Figure 5B,D).
Protein Probe
Besides nucleic acid detection, fluorescent biosensors for
protein have also attracted much attention and are of great
clinical value for qualitative and quantitative analysis of pro-
tein. Bovine serum albumin (BSA) and human serum albu-
min (HSA) are the most abundant proteins in bovine and
human blood plasma, respectively. They play a role in multi-
ple biological functions and are widely studied as protein
standards. The detection of BSA and HSA is hence of great
importance.[20]
To explore the utility of these CPEs for protein analysis,
the emission spectra of P1 and P2 in the absence and pres-
ence of BSA were measured (see Figure 6A and the Sup-
porting Information). The PL response of their neutral poly-
mer precursor P5 to the presence of protein was also studied
(see the Supporting Information). In the absence of BSA,
Nucleic Acid Detection
Fluorescent probes, the emission of which is activated upon
complexation with biomacromolecules such as nucleic acids,
are useful biomarkers in genomics.[19] By taking advantage
of the “light-up” property, cationic AIE-active CPEs can be
employed as probes for the detection of nucleic acids. Calf
thymus DNA (ctDNA) and RNA from torula yeast were
chosen as models for spectrometric titrations. The PL spec-
trum of a dilute solution of P1 in water peaked at 524 nm.
The addition of a small amount of ctDNA increased the
fluorescence intensity but caused no change in the spectral
profile (Figure 5A). The peak intensity increased rapidly at
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spectively (see the Supporting Information and Fig-
ures 6C,D). The relative fluorescent enhancement for such
polyelectrolytes after interaction with biomolecules is gener-
ally lower than the water-soluble AIE-active small mole-
cules in the literature.[17c] The TPE units in the polyelectro-
lytes are knitted together by covalent bonds, which practi-
cally restricts their intramolecular rotation and results in an
emissive solution. Furthermore, the AEE-active polyelectro-
lytes with a rigid hydrophobic conjugated polymer backbone
do not have as good solubility as the small cationic AIE
molecules. The pristine emission intensity I0 is hence higher
for polyelectrolytes than for small compounds, and the rela-
tive fluorescent enhancement I/I0 is the opposite.
To serve as fluorescent visualizers for intracellular cell
imaging, it is desirable to prepare AIE materials with longer
wavelength emissions because they suffer little interference
from cell autofluorescence.[22] With their good water solubili-
ty, strong emission efficiency in the aggregated state, and
green emission color, the AIE-active CPEs are promising
fluorescent staining agents for cell imaging. After 8 h, P1 se-
lectively stains the cytoplasm of living HeLa cells but not
their nuclei (Figure 7). The cells display their normal mor-
Figure 6. Fluorimetric titration of A) BSA and C) HSA to an aqueous so-
lution of P1 in water. Plot of I/I0 at 514 (P1), 524 (P2), and 534 nm (P5)
versus the B) BSA or D) HSA concentration. I0 =emission intensity in
the absence of BSA or HSA. [P1]=[P2]=[P5]=5 mm; excitation wave-
length: 330 (P1 and P2) and 367 nm (P5).
the aqueous buffer solution (5 mm) of P1 emitted weakly at
520 nm. The addition of 1 mm BSA aqueous buffer solution
resulted in a steep rise in emission intensity by sevenfold,
thus demonstrating that P1 is a promising candidate for
trace protein analysis.[21] Unlike heparin and nucleic acid de-
tection, the emission maximum of P1 shifts to a shorter
wavelength of 514 nm when interacting with protein, proba-
bly due to the more hydrophobic regions present in the pro-
tein molecules, which might cause the CPE chains to adopt
a more twisted conformation. The plot of I/I0 at 514 nm
versus the BSA concentration shows a steep upward line at
low protein concentration but an almost flat line at high
concentration of BSA (Figure 6B). Compound P2, which
carries triethylammonium groups, shows a merely threefold
increase in PL intensity upon binding with BSA because it
only dissolves partially in water. P5 is not soluble in water
and emits in DMSO at 536 nm. Its emission intensity in-
creases twofold in the presence of BSA, presumably due to
the hydrophobic interaction between the polymer strands
and the protein molecules. Quaternization of P5 furnishes
water-soluble polyelectrolytes, which diminishes the emis-
sion but enables the polymers to interact with the protein
molecules electrostatically. This makes the polymer more
emissive and thus results in higher sensitivity upon protein
analysis. A similar study was conducted for HSA, and the
emission of P1, P2, and P5 was enhanced by approximately
5-, 3.5-, and 1.5-fold upon binding with HSA molecules, re-
Figure 7. A) Fluorescent, B) bright-field, and C) overlapping images of
HeLa cells stained with P1 for 8 h.
phology, which is indicative of the good biocompatibility of
P1. Endocytosis is believed to be the major route for P1 to
enter the living cells, in which the CPE molecules are first
enclosed by the cell membrane to form small vesicles and
be internalized by the cells. They are then processed in the
endosomes and lysosomes and eventually released to the cy-
toplasma.[23]
Conclusion
In this paper, TPE-containing polyynes P5 and P6 were pre-
pared by Hay–Glaser polycoupling and converted into poly-
electrolytes P1–P4 by quaternization with amines. All the
polymers were characterized by standard spectroscopic
methods with satisfactory results. The neutral polyynes P5
and P6 are non- or weakly emissive in THF but become
strong emitters when aggregated in poor solvents, thus dem-
onstrating a phenomenon of AIE or AEE. Compound P1
was found to possess excellent water solubility, whereas P2–
P4 are partially soluble in aqueous media. The applications
of these fluorescent polyelectrolytes as sensitive, rapidly re-
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Sample Preparation
sponsive, and environmentally stable sensors for biomole-
cules such as heparin, ctDNA, RNA, BSA, and HSA were
explored. The charges and hydrophobic aromatic rings of
the polyelectrolytes facilitate their binding with the biomol-
ecules through electrostatic and hydrophobic cooperative in-
teractions, which restrict the intramolecular rotations of
their TPE units and leads to a remarkable fluorescence en-
hancement. They have also proven capable of being used as
fluorescent visualizers for intracellular imaging with good
biocompatibility and low interference from autofluores-
cence. The AIE-active conjugated polyelectrolytes are thus
promising fluorescent materials for bioprobes and bioimag-
ing. Studies of biologically important processes using these
AIE-active fluorescent polyelectrolytes are underway.
Stock solutions of BSA and HSA with a concentration of 100 mm were
prepared by dissolving appropriate amounts of protein in PBS (pH 7.0).
Heparin, ctDNA, and RNA solutions were prepared in PBS
(1.0 mgmLÀ1). Fluorescence measurement was carried out by adding ali-
quots (10–100 mL) of heparin (or ctDNA, RNA, BSA, and HSA) solution
to the polyelectrolyte solution (1 mL, 5 mm). The mixtures were vortexed
prior to the measurements.
Cell Culture and Imaging
HeLa cells were incubated in minimum essential medium (MEM) com-
plemented with 10% FBS, penicillin G (100 UmLÀ1 ), and streptomycin
(100 mgmLÀ1) in a 5% CO2, 90% relative humidity incubator at 378C.
Compound P1 was dissolved in PBS (pH 7.4) at a concentration of
5 mgmLÀ1 as a stock solution. HeLa cells were seeded overnight on
a cover slip mounted onto a 35 mm Petri dish and then stained with P1
(50 mgmLÀ1) for 8 h (by adding stock solution of P1 (10 mL) to fresh cul-
ture medium (990 mL)). After rinsing three times with PBS, the cells
were imaged using confocal laser scanning microscopy (CLSM; Carl
Zeiss, Germany) at an excitation wavelength of 405 nm and a collection
wavelength of 450–600 nm.
Experimental Section
Materials
Synthesis
THF was distilled under normal pressure from sodium benzophenone
ketyl under nitrogen immediately prior to use. Compounds 10, 11, and
14; [PdACHTUNGTRENNUNG(PPh3)2Cl2], CuI, PPh3, zinc powder, TiCl4, tetrabutylammonium
The synthetic routes for polyelectrolytes P1–P4 are shown in Schemes 1
and 2. Detailed experimental procedures are given below.
fluoride (TBAF), K2CO3, trimethylsilylacetylene, copper(I) chloride,
TMEDA, o-dichlorobenzene, 1,2-dibromoethane, triethylamine, aqueous
trimethylamine solution (33%), ethanol, DMSO, and other chemicals
and solvents were all purchased from Aldrich and used as received with-
out further purification. Heparin sodium was purchased from Acros.
BSA, HSA, ctDNA, and RNA from torula yeast were purchased from
Sigma–Aldrich and used as received. Phosphate-buffered saline (PBS)
with pH 7.0 was purchased from Merck. Water was purified with a Milli-
pore filtration system. Human cervical cancer HeLa cells were obtained
from American Type Culture Collection (ATCC; Rockville, USA). Cul-
ture medium and fetal bovine serum (FBS) were purchased from Gibco
Laboratories (Grand Island, USA). All the experiments were performed
at room temperature.
4,4’-Bis(2-trimethylsilylethynyl)benzophenone (8)
ACHTUNGRENN[UG PdCl2ACHTUGNTRENN(GUN PPh3)2] (619 mg, 0.88 mmol), CuI (223 mg, 1.17 mmol), PPh3
(154 mg, 0.59 mmol), 10 (10.0 g, 29.4 mmol), and a solvent mixture of
THF/Et3N (225 mL/75 mL) were added into a 500 mL two-necked round-
bottomed flask under an atmosphere of nitrogen. After all the com-
pounds dissolved, (trimethylsilyl)acetylene (16.6 mL, 117.6 mmol) was in-
jected into the flask, and the mixture was stirred at 708C for 24 h. The re-
action was quenched by adding saturated aqueous NH4Cl aqueous solu-
tion (200 mL). The aqueous phase was extracted with dichloromethane
(200 mL, three times). The solvent was removed, and the crude product
was purified on a silica gel column by using hexane as eluent. A pale
brown solid was obtained in 98% yield. 1H NMR (400 MHz, CDCl3,
TMS): d=7.71 (d, J=8.4 Hz, 4H), 7.56 (d, J=8.4 Hz, 4H), 0.27 ppm (s,
Instruments
18H; Si
1625, 1600, 1552, 1400, 1307, 1288, 1271, 1249, 1219, 1174, 929, 848, 758,
650 cmÀ1
ACHTUGNTRENUN(NG CH3)3); IR (KBr): n˜ =3301, 3062, 2959, 2899, 2157, 1931, 1655,
1H NMR spectra were measured with Bruker ARX 400 NMR spectrome-
ters using CDCl3 as solvent and tetramethylsilane (TMS; d=0 ppm) as
internal standard. UV/Vis absorption spectra were measured with
a Milton Roy Spectronic 3000 array spectrophotometer. Photolumines-
cence spectra were recorded with a Perkin–Elmer LS 55 spectrofluorom-
eter. IR spectra were recorded with a Perkin–Elmer 16 PC FTIR spectro-
photometer. Relative number (Mn) and weight-average (Mw) molecular
weights and polydispersity indices (PDI or Mw/Mn) of the polymer were
.
4,4’-Bis(2-bromoethoxy)benzophenone (9)
1,2-Dibromoethane (35.1 g, 186.7 mmol) was added to a mixture of 4,4’-
dihydroxybenzophenone (10.0 g, 46.7 mmol) and potassium carbonate
(38.7 g, 280.1 mmol) in acetone. The mixture was heated to reflux under
stirring overnight. After filtration and solvent evaporation, the crude
product was purified by a silica gel column using hexane/ethyl acetate
(4:1 v/v) as eluent. Compound 9 was obtained as a white powder in 87%
yield. 1H NMR (400 MHz, [D6]DMSO): d=7.70 (d, J=8.4 Hz, 4H), 7.10
(d, J=8.4 Hz, 4H), 4.43 (t, J=5.2 Hz, 4H), 3.84 ppm (t, J=5.2 Hz, 4H);
IR (KBr): n˜ =3063, 2948, 2893, 1633, 1603, 1579, 1507, 1308, 1295, 1255,
1226, 1171, 1150, 1024, 854, 841, 763 cmÀ1; HRMS (MALDI-TOF): m/z
calcd: 425.9466 [M+]; found: 425.9470.
estimated with
a Waters Associates gel permeation chromatography
(GPC) system equipped with RI and UV detectors. THF was used as
eluent at a flow rate of 1.0 mL. A set of monodispersed linear polystyr-
enes that covered the molecular-weight range of 103–107 was used as
standards for the molecular-weight calibration. High-resolution (HR)
mass spectra were recorded with a GCT premier CAB048 mass spec-
trometer operating in MALDI-TOF mode.
Preparation of Aggregates
2,2-Bis[4-(2-bromoethoxy)phenyl]-1,1-bis[4-(2-
trimethylsilylethynyl)phenyl]ethene (7)
Stock solution of P5 and P6 in THF with a concentration of 0.1 mm was
prepared. An aliquot (1 mL) of this stock solution was transferred to
a 10 mL volumetric flask. After addition of an appropriate amount of
THF, water was dropped slowly under vigorous stirring to furnish a 10 mm
THF/water mixture with a specific water fraction. The water content was
varied in the range of 0–90 vol%. Absorption and emission spectra of
the resulting solutions and aggregates were measured immediately after
the sample preparation.
Compound 8 (2.00 g, 5.34 mmol), 9 (2.28 g, 5.33 mmol), and zinc dust
(0.70 g, 10.7 mmol) were placed into a 500 mL two-necked round-bot-
tomed flask with a reflux condenser. The flask was evacuated under
vacuum and flushed with dry nitrogen three times. Then THF (150 mL)
was added. The mixture was cooled to À788C, and TiCl4 (1.71 mL,
5.33 mmol) was added dropwise using a syringe. The mixture was slowly
warmed to room temperature, stirred for 0.5 h, and then heated to reflux
for 24 h. The reaction was quenched with 10% aqueous K2CO3 solution.
The resulting mixture was filtered, and the filtrate was extracted with di-
Chem. Asian J. 2013, 8, 2436 – 2445
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Ben Zhong Tang et al.
chloromethane three times. The organic layer was washed with water and
dried over Na2SO4. After solvent evaporation, the residue was purified
by silica gel chromatography using petroleum ether/ethyl acetate (4:1 v/
v) as eluent. Compound 7 was obtained as a white solid in 29% yield.
3H), 7.01–6.90 (m, 8H), 6.65 (d, J=8.8 Hz, 2H), 4.23 (t, 2H), 3.61 (t,
2H), 3.06 (s, 1H; HCꢀ), 3.04 ppm (s, 1H; HCꢀ); IR (KBr): n˜ =3290 (ꢀ
À
C H stretching), 3046, 3031, 2960, 2929, 2861, 2107 (CꢀC stretching),
1603, 1506, 1290, 1243, 1174, 836, 823, 760 cmÀ1; HRMS (MALDI-TOF):
m/z calcd: 502.0932 [M+]; found: 502.0933.
2,2-Bis[4-(2-bromoethoxy)phenyl]-1,1-bis(4-ethynylphenyl)ethene (5)
Polymerization
Compound 7 (2.00 g, 2.59 mmol) and K2CO3 (0.36 g, 2.59 mmol) were
placed into a 100 mL round-bottomed flask. The reactants were dissolved
in methanol (24 mL), THF (15 mL), and CHCl3 (5 mL). The mixture was
stirred under nitrogen at room temperature for 3 h, diluted with dichloro-
methane, and washed three times with water (100 mL). The mixture was
extracted three times with dichloromethane (200 mL). The organic layer
was then washed twice with brine. The crude product was condensed and
purified on a silica gel column using hexane as eluent. A light yellow
solid of 5 was obtained in 60% yield. 1H NMR (400 MHz, CD2Cl2,
TMS): d=7.22 (d, J=8 Hz, 2H), 6.94 (d, J=8 Hz, 2H), 6.91 (d, J=
7.2 Hz, 2H), 6.65 (d, J=7.2 Hz, 2H), 4.21 (t, 2H), 3.61 (t, 2H), 3.08 ppm
The polymerization reaction and manipulation were carried out in an
open atmosphere. Typical experimental procedures for the polymeri-
zation are given below.
Polymer P5
CuCl (0.4 mg, 0.004 mmol), TMEDA (1.7 mg, 0.015 mmol), and o-di-
chlorobenzene (2 mL) were placed into a test tube equipped with a mag-
netic stir bar. The catalyst mixture was bubbled with a slow steam of
compressed air and stirred at 508C for 15 min. Monomer 5 (120 mg,
0.19 mmol) was dissolved in o-dichlorobenzene (1 mL) and then added
dropwise into the catalyst mixture. After stirring at room temperature for
5 h, the polymerization was terminated by dropping the reaction mixture
into methanol (300 mL) acidified with HCl solution (1 mL, 37%). The
polymer precipitate was filtered by a Gooch crucible, washed with meth-
anol and hexane, and dried under vacuum overnight at room tempera-
ture. A yellow powder was obtained in 99% yield. Mw: 14900; Mw/Mn:
À
(s, 1H; HCꢀ); IR (KBr): n˜ =3290 (ꢀC H stretching), 3031, 2964, 2925,
2860, 2105 (CꢀC stretching), 1603, 1507, 1242, 1175, 1017, 832 cmÀ1
HRMS (MALDI-TOF): m/z calcd: 624.0300 [M+]; found: 624.0311.
;
4-(2-Bromoethoxy)benzophenone (13)
1,2-Dibromoethane (5.21 g, 27.8 mmol) was added to a mixture of 14
(5.00 g, 25.2 mmol) and potassium carbonate (10.4 g, 75.7 mmol) in ace-
tone. The mixture was heated to reflux under stirring overnight. After fil-
tration and solvent evaporation, the crude product was purified by using
a silica gel column with hexane/ethyl acetate (4:1 v/v) as eluent. Com-
pound 13 was obtained as a white powder in 63% yield. 1H NMR
(400 MHz, [D6]DMSO): d=7.74 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.4 Hz,
2H), 7.63 (m, 1H), 7.52 (m, 2H), 7.10 (m, 2H), 4.42 (t, J=5.2 Hz, 2H),
3.83 ppm (t, J=5.2 Hz, 2H); IR (KBr): n˜ =3284, 3097, 2975, 2933, 2884,
1651, 1599, 1572, 1508, 1446, 1421, 1476, 1389, 1318, 1255, 1279, 1151,
1175, 1082, 1011, 922, 849, 820, 795, 742, 707, 698 cmÀ1; HRMS (MALDI-
TOF): m/z calcd: 304.0099 [M+]; found: 304.0089.
1
1.91; H NMR (400 MHz, CD2Cl2, TMS): d=7.22 (m, 2H), 6.92 (m, 4H),
6.66 (m, 2H), 4.22 (m, 2H), 3.61 ppm (m, 2H); IR (KBr): n˜ =3287, 3030,
À
À
2965, 2926, 2857, 2204 (CꢀC CꢀC stretching), 2141 (CꢀC CꢀC
stretching), 1602, 1574, 1505, 1241, 1173, 1016, 830 cmÀ1
.
Polymer P1
A 250 mL flask with a magnetic stir bar was charged with P5 (50 mg) in
THF (67 mL). Aqueous trimethylamine solution (12 mL, 33%) was
added to this solution. The mixture was heated to reflux and stirred for
3 d. After removal of THF, excess amounts of trimethylamine, and water,
a
yellow powder was obtained in 61% yield. 1H NMR (400 MHz,
[D6]DMSO): d=7.48, 7.37, 7.10–6.92, 4.49, 3.86, 3.27 ppm; IR (KBr): n˜ =
2-[4-(2-Bromoethoxy)phenyl]-2-phenyl-1,1-bis[4-(2-
trimethylsilylethynyl)phenyl]ethene (12)
À
À
3030, 2958, 2872, 2210 (CꢀC CꢀC stretching), 2143 (CꢀC CꢀC
stretching), 1633, 1603, 1508, 1482, 1241, 1177, 952, 833 cmÀ1
.
Compound 8 (4.00 g, 10.7 mmol), 13 (3.34 g, 10.9 mmol), and zinc dust
(1.40 g, 21.4 mmol) were placed into a 500 mL two-necked round-bot-
tomed flask with a reflux condenser. The flask was evacuated under
vacuum and flushed three times with dry nitrogen. THF (200 mL) was
then added to dissolve the reactants. The mixture was cooled to À788C,
and TiCl4 (1.17 mL, 10.7 mmol) was added dropwise with a syringe. The
mixture was slowly warmed to room temperature, stirred for 0.5 h, and
then heated to reflux for 24 h. The reaction was then quenched with
10% aqueous K2CO3 solution. The resulting mixture was filtered, and
the filtrate was extracted with dichloromethane three times. The organic
layer was washed with water and dried over Na2SO4. After solvent evap-
oration, the residue was purified by silica gel chromatography using pe-
troleum ether/ethyl acetate (4:1 v/v) as eluent to give 12 as a white solid
in 26% yield. 1H NMR (400 MHz, CDCl3, TMS): d=7.22 (d, J=8.4 Hz,
2H), 7.18 (d, J=8.4 Hz, 2H), 7.10 (m, 3H), 6.99 (m, 2H), 6.94–6.88 (m,
6H), 6.64 (d, J=8.8 Hz, 2H), 4.22 (t, 2H; CH2), 3.61 (t, 2H; CH2), 0.23
Polymer P2
A 250 mL flask with a magnetic stir bar was charged with P5 (50 mg) in
THF (67 mL). Triethylamine (6 mL) was added to the solution. The mix-
ture was then heated to reflux and stirred for 3 d. During this period,
water (5 mL) was added at regular time intervals. After evaporation of
THF, excess amounts of triethylamine, and water, a yellow powder was
obtained in 90% yield. 1H NMR (400 MHz, [D6]DMSO): d=7.47, 7.37,
7.09, 7.03, 6.97, 6.87, 6.76, 4.32, 3.86, 3.10, 1.26 ppm; IR (KBr): n˜ =3032,
À
À
2935, 2974, 2677, 2209 (CꢀC CꢀC stretching), 2143 (CꢀC CꢀC
stretching), 1603, 1507, 1242, 1174, 1016, 831, 808 cmÀ1
.
Polymer P6
CuCl (0.7 mg, 0.007 mmol), TMEDA (2.8 mg, 0.024 mmol), and o-di-
chlorobenzene (3 mL) were placed into a test tube equipped with a mag-
netic stir bar. The catalyst mixture was bubbled with a slow steam of
compressed air and stirred in an oil bath at 508C for 15 min. Monomer 6
(161 mg, 0.32 mmol) was dissolved in o-dichlorobenzene (2 mL) and then
added dropwise into the catalyst mixture. After stirring at room tempera-
ture for 5 h, the polymerization was terminated by dropping the reaction
mixture into methanol (300 mL) acidified with HCl solution (1 mL,
37%). The polymer precipitate was filtered by a Gooch crucible, washed
with methanol and hexane, and dried under vacuum overnight. A yellow
powder was obtained in 91% yield. Mw: 38900; Mw/Mn: 3.85; 1H NMR
(400 MHz, CDCl3, TMS): d=7.20 (m, 4H), 7.12 (m, 3H), 7.00–6.90 (m,
8H), 6.65 (m, 2H), 4.22 (m, 2H), 3.61 ppm (m, 2H); IR (KBr): n˜ =3030,
(s, 9H; SiACHTUNGTRENNUNG(CH3)3), 0.22 ppm (s, 9H; SiACHUTGNTREN(NNGU CH3)3); IR (KBr): n˜ =3075, 3032,
2959, 2898, 2156 (CꢀC stretching), 1605, 1507, 1249, 1176, 1018, 861,
841, 760, 670 cmÀ1; HRMS (MALDI-TOF): m/z calcd: 646.1723 [M+];
found: 646.1695.
2-[4-(2-Bromoethoxy)phenyl)]-2-phenyl-1,1-bis(4-ethynylphenyl)ethene
(6)
A solution of 12 in THF (40 mL, 1.80 g, 2.78 mmol) was placed into
a 100 mL round-bottomed flask. A solution of tetrabutylammonium fluo-
ride in THF (10 mL, 1m) was then added. After stirring for 3 h, water
(40 mL) was added, and the mixture was extracted with dichloromethane
(200 mL, three times). The organic layer was washed twice with brine.
The crude product was condensed and purified on a silica gel column
using hexane as eluent. A light yellow solid of 6 was obtained in 57%
yield. 1H NMR (400 MHz, CD2Cl2, TMS): d=7.22 (m, 4H), 7.11 (m,
À
À
2962, 2928, 2204 (CꢀC CꢀC stretching), 2142 (CꢀC CꢀC stretching),
1601, 1505, 1241, 1174, 1016, 834, 816, 670 cmÀ1
.
Chem. Asian J. 2013, 8, 2436 – 2445
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Ben Zhong Tang et al.
Polymer P3
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A 250 mL flask with a magnetic stir bar was charged with P6 (60 mg) in
THF (100 mL). Aqueous trimethylamine solution (10 mL, 33%) was
added to this solution. The mixture was heated to reflux and stirred for
3 d. After removal of THF, excess amounts of trimethylamine, and water,
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a
yellow powder was obtained in 65% yield. 1H NMR (400 MHz,
[D6]DMSO): d=7.47, 7.27, 7.07, 6.92, 4.48, 3.95, 3.84 ppm; IR (KBr): n˜ =
À
À
3031, 2957, 2871, 2207 (CꢀC CꢀC stretching), 2143 (CꢀC CꢀC
stretching), 1633, 1601, 1507, 1481, 1466, 1238, 835, 815, 757, 701 cmÀ1
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Polymer P4
A 50 mL flask with a magnetic stir bar was charged with P6 (64 mg) dis-
solved in THF (20 mL). Triethylamine (10 mL) and water (1 mL) were
added to this solution. The mixture was heated to reflux and stirred for
3 d. During this period, water (2 mL) was added at regular time intervals.
After removal of THF, excess amounts of triethylamine, and water,
a
yellow powder was obtained in 84% yield. 1H NMR (400 MHz,
[D6]DMSO): d=7.46, 7.26, 7.08, 6.89, 6.65, 4.35, 3.86, 3.61, 1.34 ppm; IR
À
(KBr): n˜ =3029, 2964, 2933, 2739, 2677, 2201 (CꢀC CꢀC stretching),
À
2141 (CꢀC CꢀC stretching), 1601, 1506, 1261, 1240, 1017, 1033, 834,
806, 700 cmÀ1
.
Acknowledgements
This work was partially supported by the National Basic Research Pro-
gram of China (973 Program; 2013CB834701), the RPC and SRFI Grants
of HKUST (RPC11SC09 and SRFI11SC03PG), the Research Grants
Council of Hong Kong (602212, HKUST2/CRF/10 and N_HKUST620/
11), and the University Grants Committee of HK (AoE/P-03/08). B.Z.T.
is grateful for the support of the Guangdong Innovative Research Team
Program (201101C0105067115).
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Received: April 11, 2013
Published online: July 10, 2013
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