Water-soluble hyperbranched poly(phenyleneethynylene)s: Facile synthesis, characterization, and interactions with dsDNA

Article abstract of DOI:10.1016/j.polymer.2014.12.068

Two novel water-soluble hyperbranched poly(p-phenyleneethynylene)s (HBP1′ and HBP2′) bearing different contents of oligo(ethylene oxide) (OEO) side chains with ammonium end groups were synthesized by the facile "A₂ + B₂ (or A2′) + C<

Full text of DOI:10.1016/j.polymer.2014.12.068

Accepted Manuscript
Water-Soluble Hyperbranched Poly(phenyleneethynylene)s: Facile Synthesis,
Characterization, and Interactions with dsDNA
Yan-Qin Huang, Rui Zhang, Cai-Xia Song, Rong-Cui Jiang, Xing-Fen Liu, Guang-Wei
Zhang, Qu-Li Fan, Lian-Hui Wang, Wei Huang
PII:
S0032-3861(15)00002-6
10.1016/j.polymer.2014.12.068
JPOL 17527
DOI:
Reference:
To appear in: Polymer
Received Date: 11 September 2014
Revised Date: 27 November 2014
Accepted Date: 31 December 2014
Please cite this article as: Huang Y-Q, Zhang R, Song C-X, Jiang R-C, Liu X-F, Zhang G-W, Fan Q-L,
Wang L-H, Huang W, Water-Soluble Hyperbranched Poly(phenyleneethynylene)s: Facile Synthesis,
Characterization, and Interactions with dsDNA, Polymer (2015), doi: 10.1016/j.polymer.2014.12.068.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Water-Soluble Hyperbranched Poly(phenyleneethynylene)s: Facile
Synthesis, Characterization, and Interactions with dsDNA
Yan-Qin Huang,1,* Rui Zhang,3 Cai-Xia Song,1 Rong-Cui Jiang,1 Xing-Fen Liu,1
Guang-Wei Zhang,1 Qu-Li Fan,1 Lian-Hui Wang,1 Wei Huang1, 2,
*
(1. Key Laboratory for Organic Electronics and Information Displays & Institute of
Advanced Materials(IAM), Jiangsu National Synergistic Innovation Center for
Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9
Wenyuan Road, Nanjing 210023, China.
2. Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced
Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced
Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road,
Nanjing 211816, China.
3. Department of Ophthalmology, Zhongda Hospital, Southeast University, Nanjing
211189, China.)
*
To whom correspondence should be addressed: E-mail: iamyqhuang@njupt.edu.cn,
Tel +86 25 8586 6396, Fax +86 25 8586 6396; E-mail: wei-huang@njtech.edu.cn,
Fax: +86 25 5813 9988; Tel: +86 25 5813 9001.
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Abstract
Two novel water-soluble hyperbranched poly(p-phenyleneethynylene)s (HBP1’
and HBP2’) bearing different contents of oligo(ethylene oxide) (OEO) side chains
with ammonium end groups were synthesized by the facile ‘‘A2 + B2 (or A2’) + C3’’
protocol based on Sonogashira polymerization. Their linear analogue (LP2’) was also
synthesized for comparative investigation. The optical properties of the neutral
precursory polymers in THF and final cationic conjugated polyelectrolytes (CCPs) in
aqueous solution were studied. Compared with LP2’, HBP1’ exhibited increased
water solubility and fluorescence quantum yield despite its lower charge density, and
HBP2’, with the similar charge density as LP2’, showed the best water solubility and
the highest fluorescence quantum yield among the three CCPs. This indicated that the
introduction of hyperbranched structure into conjugated polyelectrolytes was an
efficient way to improve water solubility and fluorescence quantum yield because
intermolecular aggregation was remarkably prevented. The interactions among the
three CCPs and double-stranded DNA (dsDNA) were studied using ethidium bromide
(EB) as the fluorescent probe. The electrostatic bindings of the three CCPs with
dsDNA/EB complex resulted in displacement of EB from dsDNA to the solution
accompanied by the quenching of EB fluorescence. Both HBP1’ and HBP2’ bound to
dsDNA more efficiently than LP2’, and HBP2’ formed the most stable complex with
dsDNA, suggesting that dsDNA might enter the cavities of single-molecular globular
architectures of these hyperbranched conjugated polyelectrolytes and induced
additional host-guest spatial interactions. Hence, HBP1’ and HBP2’ may be proved
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very useful in gene delivery or DNA biosensor applications.
Keywords: Hyperbranched conjugated polyelectrolytes; Fluorescence; dsDNA
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1. Introduction
Conjugated polyelectrolytes (CPEs) are synthetic macromolecules with highly
delocalized π-conjugated backbones and hydrophilic polar side chains, which endow
them with optoelectronic advantages of traditional conjugated polymers as well as
water solubility and ionic nature of polyelectrolytes[1-3]. Amphiphilic CPEs can form
complexes with acceptors (e.g., oppositely charged species, biological molecules)
through noncovalent interactions, mainly including electrostatic interactions,
hydrophobic interactions and π-π aromatic interactions. Thus, excitons can efficiently
transfer to lower electron/energy acceptor sites along long conjugated parts to
superquench the fluorescence of CPEs or to amplify the signals of acceptors [1-3].
Over the past decade, these advantages have resulted in the wide exploration of CPEs
as promising biosensor platform [1-10]. Beyond sensing, new functions of CPEs have
also been recently achieved in biological imaging and biomedical applications,
including gene delivery, drug delivery and release, etc. [11-16].
Although CPEs have been proven useful in these applications, most of them
reported in the literature have linear backbone structures and thus are “rigid-rod” like
molecules [1-3]. Such molecular configuration cannot adapt to the range of secondary
structures presented by biological macromolecules [17]. In aqueous media, the
intermolecular interactions of CPEs with linear backbones result in tight aggregates,
which may not only lead to fluorescence quenching due to π-stacking between the
backbones of CPEs, but also reduce spatial interactions with biological
macromolecules [17-19]. Therefore, the efficiencies of linear CPEs in their
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applications above would be weakened. As promising materials, hyperbranched CPEs
have been anticipated to overcome these drawbacks of linear CPEs [20-25].
Hyperbranched polymers are intriguing highly branched macromolecules with
three-dimensional dendritic architectures [26-28]. In contrast with multigenerational
dendritic polymers which require stepwise synthesis with complicated purification
processes, hyperbranched polymers are easier to be synthesized in a one-pot
procedure as well as show comparable properties [25]. We have employed the steric
repulsions induced by highly branched and globular molecular structures of
hyperbranched polymers to prevent the strong aggregation of linear conjugated
polymers (CPs), and thus have developed some stable and strong blue light emitting
CPs [29]. Furthermore, as the water-soluble derivatives of hyperbranched CPs,
amphiphilic hyperbranched CPEs can exhibit single-molecular globular architectures
with many cavities in aqueous media, and the functional groups on hydrophilic side
chains extending into the aqueous solvent can provide binding forces for
bioconjugation [20,24,25]. Such molecular configuration can be expected to improve
contacts and binding stability between the molecules of hyperbranched CPEs and
biological macromolecules as compared with the aggregate structure of linear CPEs.
Double-stranded DNA (dsDNA) with high density of negatively charged
phosphate has been demonstrated in the studies of gene delivery to form rather stable
complexes with polycations through electrostatic interactions [30,31]. Binding
stability of these complexes is one of the chief requirements in the applications of
gene delivery. Based on this point, some dendronized conjugated polyelectrolytes
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with cationic charges have been developed for studying gene delivery [32].
Publications introducing the studies of hyperbranched CPEs in gene delivery have
been very limited to date [33]. Herein, we designed and synthesized two structurally
analogous hyperbranched water-soluble poly(phenyleneethynylene)s (PPEs) with
different cationic charge densities through a simple ‘‘A2 + B2 (or A2’) + C3’’ protocol
based on Sonogashira polymerization. Water-soluble PPEs were used here because
they show good optical responses to environmental variations and are suitable for
studying structure–property relationships [34-37]. We successfully modulated their
charge densities by adjusting the content of hydrophilic oligo(ethylene oxide) (OEO)
side chain with ammonium end group. Meanwhile, the linear analogue of these
hyperbranched CPEs was also synthesized to study the influence of molecular
configuration on their properties, including optical properties and their interactions
with dsDNA. The studies on the complex formations of these cationic conjugated
polyelectrolytes (CCPs) with dsDNA indicated that the hyperbranched CPE with
higher cationic charge density on the side chains formed the most stable complex with
dsDNA.
2. Experimental Section
2.1. Materials
Toluene was purified by distillation from sodium in the presence of benzophenone.
Ethidium bromide (EB) was purchased from Shanghai Genebase Gene-Tech Co., Ltd.
(China). All other chemical reagents were purchased from either J&K Scientific Ltd.
(Shanghai, China) or Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), and
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used without further purification. All oligonucleotides were purchased from Shanghai
Sangon Biological Engineering Technology & Service Co., Ltd. (China). All aqueous
solutions were prepared with MilliQ water (18.2 MQ cm) from a Millipore system. 1,
2-Bis(2-bromoethoxy)ethane [38], 1,4-dibromo-2,5-hydroquinone [39] and
1,4-diethynylbenzene (Monomer B2) [40] were synthesized according to the literature
procedures.
2.2. General Methods
NMR spectra were collected on a Bruker Ultra shield Plus 400 spectrometer with
tetramethylsilane as the internal standard. UV-Vis spectra were recorded on a
Shimadzu 3600 PC spectrophotometer. Photoluminescence (PL) measurement was
carried out on a Shimadzu RF-5301 PC spectrofluorophotometer with a xenon lamp
as a light source. Mass spectra were recorded on a Shimadzu GCMS-QP2010 plus
equipped with DB-5 ms column. Elemental microanalyses were carried out on a Vario
EL III CHNOS Elemental Analyzer. Molecular weight measurements were performed
by Shimadzu Shim-pack GPC-800 gel permeation chromatography with polystyrenes
as the standard and tetrahydrofuran (THF) as the eluant.
The optical properties of polymers were studied in dilute solutions (c = 1 µM,
based on polymer repeat unit). All fluorescence spectra were recorded in a 3 mL
quartz cuvette with an optical path length of 1.0 cm. Milli-Q water used in preparing
the aqueous solutions of the polymers and quenchers was purged with nitrogen for 4 h
before using. DNA concentrations were determined by measuring the UV-vis
absorbance at 260 nm in 3 mL quartz cuvettes. The double-stranded DNA (dsDNA)
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was obtained by annealing the mixtures of complementary strands in a buffer solution
(10 mM tris-HCl, 100 mM NaCl, pH 7.5) at 2°C below the melting temperature Tm
for 20 min and then slowly cooled to room temperature.
2.3. Synthesis of 1,4-Bis(2-(2-(2-bromoethoxy)ethoxy)ethoxy)-2,5-dibromobenzene
(Compound 1)
A 100 mL round-bottom flask with magnetic stirring bar was charged with
anhydrous potassium carbonate (8 g, 57.3 mmol), 1,4-dibromo-2,5-hydroquinone
(2.56 g, 9.52 mmol), and 25 mL of acetonitrile. When the temperature of the mixture
reached 70 °C, 1, 2-bis(2-bromoethoxy)ethane (26.27g, 95.2 mmol) was added into
the flask, then the reaction mixture was stirred at this temperature for 24 h. After it
was cooled to room temperature, the mixture was poured into a large volume of water
and extracted with dichloromethane. The organic phase was dried over anhydrous
sodium sulfate, filtered and stripped of solvent by rotary evaporation. The residue was
purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5:1) to
give a white solid (3.2 g, yield: 50.5 %). Mp: 46-48ꢀ. 1H NMR (400 MHz, CDCl3): δ
(ppm) 7.16 (s, 2H), 4.13 (t, J = 6.5 Hz, 4H), 3.86–3.91 (m, 4H), 3.83 (t, J = 6.3 Hz,
4H), 3.77–3.79 (q, 4H), 3.69–3.72 (q, 4H), 3.48 (t, J = 6.3 Hz, 4H). Anal. Calcd for
C18H26Br4O6: C, 32.86; H, 3.98. Found: C, 32.82; H, 3.96.
2.4.
Synthesis
of
1,4-Bis(2-(2-(2-N,N-diethylaminoethoxy)ethoxy)ethoxy)-2,5-dibromobenzene
(Monomer A2)
Compound 1 (0.8 gꢁ1.2 mmol) was dissolved in 20 mL boiling diethylamine and
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the reaction mixture was refluxed for 10 h. After the removal of excess diethylamine,
water was added to dissolve the precipitate and then extracted with ether for three
times. The combined organic layer was dried over anhydrous sodium sulfate. After
removing the solvent, the residue was purified by silica gel column chromatography
(petroleum ether/ethyl acetate/triethylamine = 75:25:3) to give a yellow oil (0.68 g,
yield: 87.2%). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.14 (s, 2H), 4.13 (t, J = 6.5 Hz,
4H), 3.84 –3.92 (m, 4H), 3.73–3.79 (m, 4H), 3.61–3.67 (m, 4H), 3.57 (t, J = 6.5 Hz,
4H), 2.67 (q, J = 6.7 Hz, 4H), 2.56 (q, J = 7.1 Hz, 8H), 1.02 (t, J = 7.0 Hz, 12H). 13C
NMR (100 MHz, CDCl3): δ (ppm) 147.5, 120.4, 110.8, 70.5, 70.2, 70.0, 69.8, 68.7,
50.9, 49.9, 13.3. Anal. Calcd for C26H46Br2N2O6: C, 48.61; H, 7.22; N, 4.36. Found: C,
48.58; H, 7.20; N, 4.33.
2.5.
Synthesis
of
1,4-
Bis[(trimethylsilyl)ethynyl]-2,5-[bis(2-(2-(2-N,N-diethylaminoethoxy)ethoxy)
ethoxy) benzene (Compound 2)
Under nitrogen protection, Pd(PPh3)2Cl2 (49.2 mgꢁ0.07 mmol), and CuI (26.8
mgꢁ0.14 mmol) were added to a solution of Monomer A2 (1.5 gꢁ2.336 mmol) in 15
mL of diisopropylamine. Trimethylsilyl acetylene (0.46 g, 4.7 mmol) was slowly
added to the mixture at room temperature. The reaction mixture was then refluxed
under nitrogen for 6 h. The solvent was stripped off under reduced pressure. The
residue was passed through a short column of silica gel using petroleum ether/ethyl
acetate/triethylamine (75:25:3) as the eluent. Evaporation of the solvent led to a
1
yellow oil (0.866 g, yield: 61%). H NMR (400 MHz, CDCl3): δ (ppm) 6.90 (s, 2H),
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4.13 (t, J = 6.5 Hz, 4H), 3.85–3.93 (m, 4H), 3.77–3.79 (m, 8H), 3.61–3.63 (m, 4H),
3.55 (t, J = 6.5 Hz, 4H), 2.65 (q, J = 6.7 Hz, 4H), 2.55 (q, J = 7.1 Hz, 8H), 1.01 (t, J =
7.0 Hz, 12H), 0.25 (s, 18H). Anal. Calcd for C36H64N2O6Si2: C, 63.86; H, 9.53; N,
4.14. Found: C, 63.88; H, 9.52; N, 4.16
2.6.
Synthesis
of
1,4-Diethynyl-2,5-[bis(2-(2-(2-N,N-diethylaminoethoxy)ethoxy)ethoxy)benzene
(Monomer A2’)
Methanol (5 mL) and aqueous potassium hydroxide (0.4 g, 20%) were added to a
stirred solution of Compound 2 (0.8 g, 1.32 mmol) in THF (8 mL) at room
temperature. After the reactant was stirred at room temperature for 1 h, a large volume
of water was poured into it, and then the mixture was extracted with dichloromethane.
The extract was washed with water for three times, with brine once, and then dried
over anhydrous sodium sulfate. After the solvent was removed, the residue was
passed through a short column of silica gel using petroleum ether/ethyl
acetate/triethylamine (60:30:2) as the eluent. Evaporation of the solvent led to a
yellow oil (0.51 gꢁyieldꢂ72 %). 1H NMR (400 MHz, CDCl3): δ (ppm) 6.99 (s, 2H),
4.14 (t, J = 6.5 Hz, 4H), 3.85-3.88 (m, 4H), 3.73-3.76 (m, 4H), 3.61-3.63 (m, 4H),
3.54-3.57 (t, J = 6.5 Hz, 4H), 3.34 (s, 2H), 2.65 (q, J = 6.7 Hz, 4H), 2.55 (q, J = 7.1 Hz,
13
8H), 1.01 (t, J = 7.0 Hz, 12H). C NMR (100 MHz, CDCl3): δ (ppm) 151.4, 118.4,
112.3, 82.4, 81.5, 70.5, 70.2, 70.0, 69.9, 68.7, 50.8, 49.9, 13.2. Anal. Calcd for
C30H48N2O6ꢂC, 67.64; H, 9.08; N, 5.26. Found: C, 67.68; H, 9.04; N, 5.23.
2.7. General procedures for the synthesis of linear neutral polymers LP1 and LP2
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Under nitrogen protection, degassed diisopropylamine/toluene (1:2, 12 mL) was
added to a 25 mL round-bottom flask containing 0.128 g (0.2 mmol) of Monomer A2
and 0.22 mmol diethynylbenzene monomer (0.028 g Monomer B2, or 0.117 g of
Monomer A2’), 6 mg (0.005 mmol) of Pd(PPh3)4, and 2 mg (0.01 mmol) of CuI. After
the reaction mixture was stirred at 75 °C for 24 h, bromobenzene (0.1 g, 0.5 mmol)
was added for end-capping the polymer for an additional 2 h. Upon cooling to room
temperature, the reaction mixture was subjected to a CHCl3/H2O workup. The organic
phase was washed with water for three times, and then dried over anhydrous sodium
sulfate. After the solution was concentrated, it was dropped into hexane (200 mL).
The precipitate was filtered, dissolved in chloroform, and reprecipitated in hexane
twice to obtain a yellow fibrous solid.
LP1ꢂ0.085 g (yield 70 %).1H NMR (400 MHz, CDCl3): δ (ppm) 7.56–7.44 (m, ArH),
7.05 (m, 2H), 4.11–4.27 (m, 4H), 3.45–3.90 (m, 16H), 2.55–2.90 (m, 12H), 1.05 (m,
12H). 13C NMR (100 MHz, CDCl3): δ (ppm) 151.3, 118.4, 112.5, 131.9, 122.5, 95.9,
84.3, 70.6, 70.3, 70.0, 69.8, 68.8, 50.9, 49.7, 13.3.
1
LP2ꢂ0.151 g (yield 74 %). H NMR (400 MHz, CDCl3): δ (ppm) 7.03 (m, ArH),
4.11–4.27 (m, 4H), 3.45–3.95 (m, 16H), 2.5–2.90 (m, 12H), 1.06 (m, 12H). 13C NMR
(100 MHz, CDCl3): δ (ppm) 151.6, 118.6, 112.4, 84.4, 70.8, 70.4, 70.1, 69.9, 68.9,
50.8, 49.8, 13.2.
2.8. General procedures for the synthesis of hyperbranched neutral polymers HBP1
and HBP2
Under nitrogen protection, degassed diisopropylamine/toluene (1:2, 12 mL) was
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added to a 25 mL round-bottom flask containing 0.128 g (0.2 mmol) of Monomer A2,
0.22 mmol diethynylbenzene monomer (0.028 g Monomer B2, or 0.117 g of Monomer
A2’), and Monomer C3(4 mgꢁ0.0126 mmol), 6 mg (0.005 mmol) of Pd(PPh3)4, and 2
mg (0.01 mmol) of CuI. After the reaction mixture was stirred at 75 °C for 48 h,
bromobenzene (0.1 g, 0.5 mmol) was added for end-capping the polymer for an
additional 2 h. The following procedures were the same as those of the linear neutral
polymers. The products were both brown fibrous solid.
HBP1ꢂ0.066 g (yield 54%). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.6–7.69 (m, ArH),
7.38–7.58 (m, ArH), 7.0–7.18 (m, ArH), 4.11–4.27 (m, 4H), 3.25–3.90 (m, 16H),
13
2.55–2.90 (m, 12H), 1.08 (m, 12H). C NMR (100 MHz, CDCl3): δ (ppm) 151.7,
135.8, 131.9, 122.4, 121.8, 118.5, 112.3, 95.9, 84.2, 70.7, 70.4, 70.1, 69.9, 68.8, 50.7,
49.9, 13.2.
HBP2ꢂ0.124 g (yield 61%). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.5–7.65 (m, ArH),
6.97–7.15 (m, ArH), 4.11–4.29 (m, 4H), 3.45–3.95 (m, 16H), 2.45–2.90 (m, 12H),
13
1.08 (m, 12H). C NMR (100 MHz, CDCl3): δ (ppm) 151.7, 135.9, 121.9, 118.6,
112.5, 93.4, 84.3, 70.7, 70.3, 70.0, 69.7, 68.8, 50.7, 49.8, 13.3.
2.9. General procedure for the synthesis of cationic conjugated polyelectrolytes (CCPs)
via quaternization of the neutral polymers
Ethyl bromide (6 mL) was added to 0.1 mmol of the neutral polymers dissolved in
10 mL THF at room temperature. After 10 min stirring, some precipitate was
observed, which was redissolved by addition of 1 mL H2O. After 5 days, the reaction
mixture was concentrated and then poured into acetone to form a precipitate. After
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stirring for two hours, the collected precipitate was redissolved with methanol. The
solution was concentrated and poured into acetone again, and the precipitate was
collected and dried at room temperature in vacuo to yield the target polymers.
1
LP2’ꢂ 0.107 g (yield 74%). H NMR (400 MHz, CD3OD): δ (ppm) 7.1 (br, ArH),
13
4.14–4.30 (br, 4H), 3.5–3.95 (m, 16H), 2.55–3.0 (br, 12H), 1.15 (br, 12H). C NMR
(100 MHz, CDCl3): δ (ppm) 151.6, 118.5, 112.4, 84.3, 70.7, 70.3, 70.0, 68.8, 68.2,
60.2, 55.4, 8.3.
1
HBP1’ꢂ0.065 g (yield 79%). H NMR (400 MHz, CD3OD): δ (ppm) 7.1–7.8 (m,
ArH), 3.2–4.5 (m, 20H), 2.6–3.1 (m, 12H), 1.16 (br, 12H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 151.6, 135.8, 132.0, 122.5, 121.7, 118.6, 112.5, 95.8, 84.1, 70.7,
70.5, 70.2, 68.8, 68.2, 60.1, 55.5, 8.4.
1
HBP2’ꢂ0.12 g (yield 82%). H NMR (400 MHz, CD3OD): δ (ppm) 7.15–7.75 (m,
ArH), 4.15–4.32 (br, 4H), 3.5–4.0 (m, 16H), 2.55–3.05 (br, 12H), 1.14 (br, 12H). 13C
NMR (100 MHz, CDCl3): δ (ppm) 151.7, 135.8, 121.9, 118.5, 112.4, 93.3, 84.2, 70.6,
70.4, 70.1, 68.7, 68.1, 60.1, 55.4, 8.3.
3. Results and Discussion
3.1. Synthesis and characterization
The preparation of linear neutral polymers was accomplished via Sonogashira
polymerization of Monomer A2 and Monomer B2 (or A2’) [34]. A simple ‘‘A2 + B2 (or
A2’) + C3’’ protocol based on Sonogashira polymerization was employed to synthesize
the neutral hyperbranched polymers [22,29].
The synthetic routes for monomers A2 and A2’ were shown in Scheme 1. In a basic
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deprotonation condition, 1,4-dibromo-2,5-hydroquinone was alkylated with 1,
2-bis(2-bromoethoxy)ethane to afford Compound 1 [41,42], and then Monomer A2
was synthesized by refluxing the mixture of Compound 1 and excess diethylamine
[42]. Compound 2 was obtained via Sonogashira reaction of Monomer A2 with
trimethylsilyl acetylene, which was then stirred in the mixed solution of methanol and
aqueous potassium hydroxide to produce Monomer A2’. Monomer B2 was prepared
according to literature procedures [40]. The correct structures of Monomer A2 ,
1
Monomer A2’ and Monomer B2 were affirmed by H NMR and 13C NMR
spectroscopy and elemental analysis.
Scheme 1. Synthetic routes for the monomers. Reagents and conditions: (a) 1,
2-bis(2-bromoethoxy)ethane,K2CO3, acetonitrile, 70 °C,24 h; (b) (C2H5)2NH, reflux,
10 h; (c) (CH3)3SiCCH, (PPh3)2PdCl2, CuI, diisopropylamine, 6 h; (d) KOH,
CH3OH-THF, rt, 1 h.
The synthetic routes for the polymers were illustrated in Scheme 2. GPC revealed
that the four neutral polymers had close weight-average molecular weights (Mw)
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ranging from 7250 to 7680 with the polydispersity indices in the range of 1.17–1.42
(Table 1). The solubility of the hyperbranched neutral polymers was dominantly
determined by the molar ratio of the branching unit C3. HBP1 and HBP2 with 3% C3
(produced in the molar ratios of Monomer A2:B2(or A2’):C3) = 48:52:3) were fully
soluble in common organic solvents, e.g., THF, chloroform, and CH2Cl2. Further
increasing the contents of C3 leads to a lower yield of soluble polymer due to
crosslinking [22,29]. It was found that the solubility of the hyperbranched neutral
polymers was obviously better than those of the linear neutral polymers. Moreover,
LP1 showed poor solubility in THF and chloroform while LP2 showed better
solubility than LP1 most likely due to the much higher density of side chains in LP2.
Table 1. Characterization of the Neutral Precursory Polymers and Cationic
Conjugated Polyelectrolytes.
GPC
λabs,max /nm
λem,max /nm
Entry
Mn
Mw
PDI
THF
H2O
THF
H2O
LP1
LP2
5350 7600 1.42
5450 7250 1.33
5870 7680 1.31
6360 7450 1.17
395
417
387
406
451
472
445
467
HBP1
HBP2
LP2’
339
330
333
467
442
463
HBP1’
HBP2’
Scheme 2. Synthetic routes for the neutral and cationic polymers. Reagents and
conditions: (a) Pd(PPh3)4, CuI, diisopropylamine/toluene, 75 °C, 24 h; (b) C2H5Br,
THF-H2O, 5 days; (c) Pd(PPh3)4, CuI, diisopropylamine/toluene, 75 °C, 48 h;
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1
13
The structures of the polymers were characterized by H NMR and C NMR
spectra. As shown in Fig. 1, all protons on the side chains of HBP1 and HBP2 could
be well assigned, and the single peaks at δ =1.08 were assigned to the signals from
–NCH2CH3– protons, evidently indicating the existence of the amine groups [36, 43].
Moreover, the characteristic proton signals of the branching units and phenylene units
were observed at δ = 7.6–7.69, 7.38–7.58 and 7.0–7.18 ppm for HBP1, and at δ =
7.5–7.65 and 6.97–7.15 ppm for HBP2. Thus, the intergral ratios between the
branching unit and phenylene of these two polymers can be approximately estimated
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1
from H NMR spectra, that is about 1:65 for HBP1 and 1:43 for HBP2, respectively,
which can roughly reflect the relative amount of benzene unit [22,29]. The CCPs were
synthesized through the postquaternization treatment of the neutral polymers with
ethyl bromide in THF-H2O solution [36]. However, the quaternization of LP1 was not
1
achieved perhaps due to the poor solubility in THF. In the H NMR spectra of the
CCPs LP2’, HBP1’, and HBP2’, nearly all signals moved to the lower field compared
with those of LP2, HBP1, and HBP2, and there were no split peaks arising from the
quaternized (low field) and unquaternized components, clearly indicating complete
quaternization of these three neutral polymers [36,42,43]. The solubility for LP2’,
HBP1’, and HBP2’ in water (25 °C) was measured to be 8, 11 and 20 mg/mL,
respectively. In view of the slight differences in the molecular weights of these
WSCPs, it was rational to propose that the existence of hyperbranched structure as
well as hydrophilic side chains would improve the water solubility of CPEs.
j
(a)
h
i
g
f
e
d
a
b
c
j
b
g
f
e
i
h
d
c
a
8
7
6
5
4
3
2
1
ppm
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h
i
g
f
(b)
e
d
c
a
b
i
g
f
h
e
d
c
b
a
8
7
6
5
4
3
2
1
ppm
1
Fig. 1. H NMR spectra of the hyperbranched neutral polymers (a) HBP1 and (b)
HBP2.
3.2. Optical properties
The UV–vis absorption and PL spectra of the neutral precursory polymers in
dilute THF solution resembled previously reported spectra of structurally analogous
neutral PPEs (Fig. 2) [36]. The corresponding optical data were summarized in Table
1. The absorption and emission maxima of HBP1 were blueshifted 8 nm and 6 nm,
respectively, as compared with the absorption and emission maximum of LP1. When
comparing the optical data of HBP2 and LP2, we also found these similar phenomena.
This can be attributed to the introduction of benzene branching unit in the structure of
hyperbranched polymers, which interrupted the linear π-system and led to reduction in
the effective conjugation length [22]. On the other hand, the absorption and emission
maxima of HBP2 were redshifted 19 nm and 22 nm, respectively, as compared with
the absorption and emission maximum of HBP1. These similar phenomena were also
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observed while comparing the optical data of LP2 and LP1. In view of the higher
density of side chains in the structure of HBP2 and LP2, these phenomena should be
ascribed to the nonbonding electron pairs on the oxygen atoms of side chains, which
contributed to the conjugation of the main chain and led to increase in the effective
conjugation length [43,44].
(a)
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
HBP1
LP1
0.0
0.0
1.0
(b)
1.0
HBP2
LP2
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
350
400
450
500
550
600
Wavelength (nm)
Fig. 2. UV–vis absorption and PL spectra of the neutral polymers (a) LP1 and HBP1,
and (b) LP2 and HBP2 in THF.
The UV–vis absorption and PL spectra of LP2’, HBP1’ and HBP2’ in water were
shown in Fig. 3. In comparison with those of their neutral precursory polymers, the
absorption and emission peaks of all quaternized polymers presented blue-shifts, and
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the blue-shifts were especially obvious in the cases of absorption. This was likely due
to the mutual repulsion among the positive charges, which resulted in a more twisted
main chain conformation, hence a decreased effective conjugation length [36]. The
fluorescence quantum yields (ΦF) for LP2’, HBP1’ and HBP2’ in water were
measured to be 2.47%ꢃ2.7%ꢃand 6.26%, respectively. HBP2’ exhibited a much
higher ΦF than its linear analogue, LP2’. Moreover, although HBP1’ had a lower
density of hydrophilic side chains than LP2’, it also presented a slightly higher ΦF.
Although the ΦF of these PPEs were lower than those of the linear and hyperbranched
polyfluorenes reported in a previous study, similar relationships between the structure
and ΦF have been found [22]. It was noted that ΦF for the three CCPs followed the
same order as their solubility in water. Therefore, it was rational to propose that the
incorporation of hyperbranched structures into the CPEs can efficiently reduce the
intermolecular aggregation, thus significantly improving the fluorescence efficiency
as well as water solubility [20,22].
LP2'
HBP1'
HBP2'
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
300
350
400
450
500
550
600
Wavelength (nm)
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Fig. 3. UV–vis absorption and PL spectra of the CCPs LP2’, HBP1’, and HBP2’ in
water.
3.3. Interactions of the cationic conjugated polyelectrolytes with DNA
The interactions between dsDNA and the three CCPs were investigated by
probing the PL intensity changes of the CCPs. The oligonucleotide used in these
experiments was 5’-GAA CAT GGC AAG CTG-3’(ssDNA). The dsDNA was
obtained by hybridization of the ssDNA with its complementary strand, 5’-CAG CTT
GCC ATG TTC-3’(ssDNAC). As shown in Fig. 4(a), the maxima and shapes of the PL
spectra of LP2’ did not change and no new peaks appeared, and there was a
substantial decrease in the emission of LP2’ ([LP2’] = 1.0 × 10-6 M) upon adding
dsDNA ([dsDNA] = 0 ~ 1.0 × 10-8 M) in the tris buffer solution (10 mM tris-HCl, 100
mM NaCl, pH = 7.5). The inset of Fig. 4(a) also exhibited that the PL intensity of
LP2’ obviously dropped after the interaction with dsDNA. It can be calculated from
Fig. 4(b) that the PL intensity dropped about 27.5 percent as the concentration of
dsDNA reached 1.0 × 10-8 M. The ammonium groups on the CCPs would be partially
neutralized with the formation of CCP/dsDNA complex, leading to decreased charge
density and thus a decrease in the intermolecular electrostatic repulsion. Therefore,
LP2’, with the planar and linear main chain of PPE [34-37], tended to form π-stacking
aggregates near the negatively charged dsDNA, leading to self-quenching [32, 45, 46].
By contrast, such substantial quenching of emission was not found in the case of
HBP2’. As shown in Fig. 4(c), the PL intensity dropped less than 7 percent as the
concentration of dsDNA reached 1.0 × 10-8 M. Similar result was also obtained for
HBP1’. These results can also be explained by the highly branched and rigid globular
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molecular structures of HBP2’ and HBP1’, which efficiently reduce the intra- and
intermolecular aggregation with the addition of dsDNA, thus significantly inhibiting
self-quenching [22,32].
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Fig. 4. (a) PL spectra of LP2' with the presence of dsDNA at different concentrations,
[dsDNA] = 0 to 1.0 × 10-8 M, [LP2’] = 1.0 × 10-6 M, λex = 339 nm. The inset shows
the fluorescent photos of LP2’/dsDNA solutions when dsDNA concentration was 0
and 1.0 × 10-8 M, respectively. (b) Normalized PL intensity of LP2’ as a function of
dsDNA concentration. (c) PL spectra of HBP2’ with the presence of dsDNA at
different concentrations, [dsDNA] = 0 to 1.0 × 10-8 M, [HBP2’] = 1.0 × 10-6 M, λex =
333 nm. The inset shows the fluorescent photos of HBP2’/dsDNA solutions when
dsDNA concentration was 0 and 1.0 × 10-8 M, respectively. Measurements were
performed in the tris buffer solution (10 mM tris-HCl, 100 mM NaCl, pH = 7.5). The
fluorescent photos were taken under a portable UV lamp with excitation at 365 nm.
The interactions between dsDNA and the three CCPs were also investigated by
using cationic ethidium bromide (EB) as a fluorescent probe. EB is a dsDNA-specific
intercalator which shows an evident increase in fluorescence intensity upon
intercalating into the double helix of dsDNA [47,48]. However, free EB exhibits a
very low fluorescence intensity in buffer solution. Hence, the binding properties of
other cationic species to dsDNA can be studied by using EB as competitor [32,49].
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The electrostatic bindings of the three CCPs with dsDNA/EB complex would lead to
displacement of intercalated EB from dsDNA to the solution due to the electrostatic
repulsion, and this was accompanied by the quenching of EB fluorescence. Thus, the
stability of the CCP/dsDNA complex can be evaluated by studying the fluorescence
changes of EB. First, the dsDNA and EB were premixed in the tris buffer solution (10
mM tris-HCl, 100 mM NaClꢁpH = 7.5) ([dsDNA] = 1.0 × 10-8 M, [EB] = 3.0× 10-7
M), and the CCP was successively added to the solution ([HBP2’] = 0 to 1.0 × 10-7 M,
[HBP1’] = [LP2’] = 0 to 1.0 × 10-6 M) followed by excitation at the absorption
maximum of EB (480 nm). As shown in Fig. 5(a), the PL intensity of dsDNA/EB
decreased gradually as the concentration of HBP2’ increased. The decrease of PL
intensity were also observed for HBP1’ and LP2’. The normalized PL intensity of
dsDNA/EB (I/I0) as functions of the CCP concentrations were illustrated in Fig. 5(b,
c). It was noted that the linear plots were obtained for both HBP2’ and HBP1’,
whereas an upsloping nonlinear line was observed for LP2’. Moreover, the decline of
I/I0 upon adding HBP2’ exhibited nearly ten times the rate of decline upon adding
HBP1’. Interestingly, although the charge density of HBP1’ was much lower than that
of LP2’, the value of I/I0 with the addition of HBP1’ decreased more greatly than with
the addition of LP2’. Most importantly, as the stability of the CCP/dsDNA complex
has close relationship with the ratio of positive charge to negative charge, we also
studied the variation of I/Io with molar ratio of amine (on LP2’, HBP1’ and HBP2’) to
phosphate (on dsDNA). The data in Fig. 5(d) showed similar trends to those in Fig.
5(b, c), namely, the rate of decline of I/I0 also followed the order HBP2’ >HBP1’>
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LP2’. All these results indicated that the hyperbranched CPEs HBP2’ and HBP1’
bound to dsDNA more efficiently than their linear analogue LP2’.
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Fig. 5. (a) PL spectra of dsDNA/EB as a function of HBP2’ concentration. (b)
Normalized PL intensity of dsDNA/EB as a function of HBP2’ concentration. (c)
Normalized PL intensity of dsDNA/EB as functions of HBP1’ and LP2’
concentrations. (d) Normalized PL intensity of dsDNA/EB as functions of molar ratio
of amine (on LP2’, HBP1’ and HBP2’) to phosphate (on dsDNA). [dsDNA] = 1.0 ×
10-8 M, [EB] = 3.0× 10-7 M, [HBP2’] = 0 to 1.0 × 10-7 M, [HBP1’] = [LP2’] = 0 to 1.0
× 10-6 M, λex = 480 nm. Measurements were performed in the tris buffer solution (10
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mM tris-HCl, 100 mM NaCl, pH = 7.5).
We compared these results with those in a previous literature which also studied
the interactions between water-soluble conjugated polyfluorenes with dendritic side
chains and dsDNA using EB as fluorescent probe [32]. Among three polymers with
the same conjugated backbones, the polymer with highest generation of dendritic side
chains, namely, highest charge density, was reported to form most stable complex
with dsDNA [32]. However, in our cases, the charge density of HBP2’ was close to
that of LP2’, and for HBP1’, it was even lower than LP2’. Hence, in addition to the
charge densities of the CCPs, the state of aggregation may be the other crucial factor
that influenced the stability of the CCP/dsDNA complexes. As discussed above,
compared with HBP2’ and HBP1’, LP2’ exhibited a greater tendency to form
π-stacking aggregates, which may make the polymer less accessible to dsDNA [43,
50], subsequently reduce the binding efficiency and lead to the upsloping nonlinear
line in Fig. 5c. However, as for HBP2’ and HBP1’, the single-molecular globular
architectures with many cavities and with cationic ammonium end groups on
extending side chains, can not only effectively prevent the formation of intra- and
intermolecular aggregates, but also may provide the advantages of more chances to
contact dsDNA. According to previous literatures, these molecular configuration
features of hyperbranched CPEs may make them useful host molecular carriers for
organic molecules and biomacromolecules in applications [20,24,25]. Therfore, as
shown in Scheme 3, we suggested that dsDNA might enter the cavities of highly
branched structure as a guest molecule, which resulted in more spatial interactions
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and thereby significantly improved the binding stability of hyperbranched
CPE/dsDNA complex.
4. Conclusion
In summary, two structurally analogous water-soluble hyperbranched PPEs
(HBP1’ and HBP2’) with different cationic charge densities were synthesized by the
simple ‘‘A2 + B2 (or A2’) + C3’’ protocol based on Sonogashira polymerization. Their
linear analogue (LP2’) was also synthesized for comparative investigation. The
optical properties of these CCPs and their corresponding neutral polymers were
studied by UV–vis and PL spectroscopy. All the three CCPs had good water solubility.
In particular, HBP1’ and HBP2’ exhibited obviously increased water solubility and
fluorescence quantum yield compared with that of their linear analogue LP2’,
indicating that the introduction of hyperbranched structures into CPEs is an efficient
way to improve water solubility and fluorescence quantum yield. Moreover, among
these CCPs, HBP2’ with higher content of charge density in the two hyperbranched
CPEs showed the best water solubility and the highest fluorescence quantum yield.
Studies on the interactions between the three CCPs and dsDNA displayed that the
electrostatic bindings of the CCPs with dsDNA/EB complex led to displacement of
EB from dsDNA to the solution. HBP2’ formed the most stable complex with dsDNA,
and interestingly, although the charge density of HBP1’ was much lower than that of
LP2’, HBP1’ also bound to dsDNA more efficiently than LP2’. We suggested that
dsDNA might enter the cavities of hyperbranched structure, and that the additional
host-guest spatial interactions thereby significantly improved the binding stability of
28