Synthesis of cationic diacetylene-co-carbazole-co-fluorene polymers and their sensitive fluorescent quenching properties with DNA

Article abstract of DOI:10.1002/pola.24197

Two neutral precursor conjugated copolymers based 2,7-diethynylfluorene and 3,6-diethynylcarbazole units in the main chain (PFC and PF2C) were prepared by Hay coupling polymerization. Their cationic copolymers (CPFC and CPF2C) were prepared by the methylation of their diethylpropylamino groups with CH ₃I. For comparison, neutral conjugated homopolymers of 2,7-diethynylfluorene (PF), 3,6-diethynylcarbazole units (PC) and their cationic polymers (CPF and CPC) were also prepared with the same method. A comparative study on the optical properties of cationic polymers CPFC and CPF2C in DMF and DMF/H₂O showed that they underwent water-induced aggregation. The spectral behaviors of CPFC and CPF2C with calf thymus DNA showed that a distinct fluorescent quenching took place with minute addition of CT DNA (3.3 × 10^-13 M). The results showed that the polymers would be promising biosensor materials for sensitive detection of DNA.

Full text of DOI:10.1002/pola.24197

Synthesis of Cationic Diacetylene-co-Carbazole-co-Fluorene Polymers
and Their Sensitive Fluorescent Quenching Properties with DNA
WENNAN ZENG,1,2 SHOUPING LIU,3 HANXUN ZOU,1 LINYUN WANG,1 ROGER BEUERMAN,3 DERONG CAO1
1School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China
2School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Hunan, Xiangtan 411201, China
3Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751
Received 6 May 2010; accepted 22 June 2010
DOI: 10.1002/pola.24197
Published online in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: Two neutral precursor conjugated copolymers
based 2,7-diethynylfluorene and 3,6-diethynylcarbazole units in
the main chain (PFC and PF2C) were prepared by Hay coupling
polymerization. Their cationic copolymers (CPFC and CPF2C)
were prepared by the methylation of their diethylpropylamino
groups with CH3I. For comparison, neutral conjugated homo-
polymers of 2,7-diethynylfluorene (PF), 3,6-diethynylcarbazole
units (PC) and their cationic polymers (CPF and CPC) were also
prepared with the same method. A comparative study on the
optical properties of cationic polymers CPFC and CPF2C in
DMF and DMF/H2O showed that they underwent water-induced
aggregation. The spectral behaviors of CPFC and CPF2C with
calf thymus DNA showed that a distinct fluorescent quenching
took place with minute addition of CT DNA (3.3 ꢀ 10ꢁ13 M).
The results showed that the polymers would be promising bio-
C
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sensor materials for sensitive detection of DNA.
2010 Wiley
Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 4168–
4177, 2010
KEYWORDS: aggregation; binding interaction; biological applica-
tions of polymers; cationic conjugated polymers; conducting
polymers; CT DNA; fluorescence; photophysics; UV-Vis spectroscopy
INTRODUCTION Conjugated polymers (CPs) offer several
advantages as the response basis for chemical and biological
detection based on optical methods. CPs may be viewed as a
collection of short, conjugated units kept in close proximity
by virtue of the polymer backbone. Modification of CPs with
anionic or cationic functional groups yields materials that
possess the properties of conjugated polymers and water
solubility which is essential for interaction with biological
substrates1 such as proteins2 and DNA.3 Cationic conjugated
polymers (CCPs), in particular, have been proven to be very
useful for DNA sequence detection based on electrostatic
interaction with the negatively charged phosphate back-
bone.4 CCPs reported previously for DNA detection were ei-
ther polythiophene derivatives5 or poly(fluorene-co-phenyl-
ene) derivatives.6
reported, including diacetylene polycarbonate,9 diacetylene
polyurethanes,10 diacetylene polyamides,11 diacetylene poly-
esters12 and other examples.13 However, to our knowledge,
no efforts have been made to the development of cationic
conjugated polymers based on diacetylene in the main chain
for electronic and optical applications in DNA–biosensors.
In this article, novel conjugated copolymers PFxCy (PF1C
and PF2C) containing diacetylene, carbazole and fluorene
moieties in the main chain with tertiary amines in the side
chains were synthesized as precursor polymers with differ-
ent ratios of fluorene and carbazole units by oxidative
coupling of acetylene groups under oxygen atmosphere with
CuCl as catalyst.14 Addition of CH3I to PFxCy gave the
cationic polymers CPFxCy (Scheme 1). CPFC and CPF2C pro-
vided electrostatic interactions with the negatively charged
analytes,15 such as DNA. The interaction between calf
thymus DNA (CT DNA) and CPFC or CPF2C by means of
absorption and emission spectroscopy was preliminarily
investigated. The disturbance by the buffer solution was
also studied. For comparison, neutral conjugated homopoly-
mer (PF) of 2,7-diethynylfluorene and homopolymer (PC)
of 3,6-diethynylcarbazole, as well as their cationic polymers
(CPF and CPC) were also prepared and characterized
(Scheme 1).
Carbazole is a conjugated unit that has interesting optical
and electronic properties such as photoconductivity and pho-
torefractivity.7 Polymers containing carbazole moieties in the
main chain or side chain have been widely studied because
of their unique properties which allow them to become vari-
ous photoelectronic materials including photoconductive,
electroluminescent, and photorefractive materials.8 Mean-
while, various semicrystalline and amorphous polymers
containing diacetylene moiety in the backbone have been
Correspondence to: D. Cao (E-mail: drcao@scut.edu.cn); or R. Beuerman (E-mail: rwbeuer@mac.com)
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Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 4168–4177 (2010) 2010 Wiley Periodicals, Inc.
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SCHEME 1 Synthetic routine of
polymers PFxCy and CPFxCy.
3,6-Diethynyl-N-[60-(N,N-diethylamino)-hexyl]carbazole (2) was
prepared with carbazole as starting material by using the
same procedure as for the synthesis of 1 (Scheme 3). The IR
spectrum of monomer 2 showed the characteristic ab-
sorption of the acetylene at 3307 cmꢁ1 for CBCAH and at
2107 cmꢁ1 for CBC. Additional structural proof was
obtained from 1H NMR and 13C NMR spectra. 1H NMR
showed ethynyl proton as a singlet at 3.06 ppm. Two charac-
teristic peaks for acetylenic carbons were found at 77.8 and
85.6 ppm in 13C NMR spectrum.
RESULTS AND DISCUSSION
Synthesis and Characterization
2,7-Diethynyl-9,9-bis[60-(N,N-diethylamino)-hexyl]fluorene (1)
was synthesized by a modified routine (Scheme 2). Accord-
ing to the synthetic routine in the literature,16 9,9-bis(6-bro-
mohexyl)-2,7-diethynyl-9H-fluorene was the intermediate
which was prepared from 2,7-diethynyl-9H-fluorene. How-
ever, its yield was low because C-9 position of 2,7-diethynyl-
9H-fluorene was easily oxidized under the strong base condi-
tion and gave 2,7-diethynyl-fluorenone as the main product.
In our synthetic routine, the introduction of bromohexyl at
C-9 position of fluorene was performed from 2,7-diiodo-9H-
fluorene 4 with high yield (71%) (Scheme 2). The classical
Sonogashira coupling reaction between 2,7-diiodo-9,9-bis(60-
Oxidative coupling of acetylene groups of monomer 1 and 2
with two different feed ratios under oxygen atmosphere
using CuCl in 1,2-dichlorsobenzene gave the neutral precur-
sor polymers PFxCy in 51–82% yields. The molar ratios of
2,7-diethynylfluorene unit to 3,6-diethynylcarbazole unit in
two copolymers (PFC and PF2C) were determined by 1H
NMR spectra from the ratio of the protons of PFC at
0.58 ppm corresponding to monomer 1 (0.48–0.56 ppm, 4H,
CH2) and the protons of PFC at 4.27 ppm corresponding to
monomer 2 (4.23 ppm, 2H, N–CH2), as shown in Figure 1.
bromohexyl)-9H-fluorene
5 and 2-methyl-3-butyn-2-ol 6
with Pd(PPh3)2Cl2 and CuI as catalysts gave 2,7-bis(20-
hydroxy-20-methylbutynyl)-9,9-bis(60-bromohexyl)-fluorene 7
in excellent yield. Finally, monomer 1 was obtained by reac-
tion of 7 with diethylamine following by treatment with
KOH in 57% yield for two steps.
SCHEME 2 Synthetic routine of monomer 1.
Reagents and conditions: (i) I2, KIO3, AcOH,
H2SO4, H2O, 80 ꢂC, 10 h, 79%; (ii) BrC6H12Br,
KOH, H2O, Bu4NBr, DMSO, N2, rt, 24 h, 71%;
(iii) (PPh3)2PdCl2, 2-methyl-3-butyn-2-ol 6, CuI,
Et3N, THF, N2, rt, 12 h, 92%; (iv) HNEt2, reflux,
12 h, 73%; (v) KOH, toluene-EtOH, N2, 120 ꢂC,
3 h, 78%.
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SCHEME 3 Synthetic routine of monomer 2.
With the calculation, the average ratios of 2,7-diethynylfluor-
ene to 3,6-diethynylcarbazole group were estimated to be
about 0.99:1 and 2.02:1 for PFC and PF2C, respectively. For
comparison, the homopolymers of 2,7-diethynylfluorene (PF)
and 3,6-diethynylcarbazole (PC) were also prepared with the
same method. Molecular weights and polydispersity indexes
are listed in Table 1. Mn and Mw ranged from 5201 to 26118
(Mn) and from 5331 to 41326 (Mw), respectively, with poly-
dispersity indices (PDI) ranging from 1.03 to 1.64. The neu-
tral precursor polymer PFxCy were isolated as yellow pow-
der and displayed excellent solubility in chlorinated
hydrocarbons, DMF, N-methylpyrrolidinone, N,N-dimethylace-
tamide, and tetrahydrofuran (THF), but insoluble in metha-
nol and hexane.
Figure 1, the quaternization degrees of CPFC were nearly
100%, because after the treatment with CH3I, all signals cor-
responding to ACH2NA moved to the lower field.17
Optical Properties of Polymers
The UV-vis and PL spectra of the neutral precursor polymers
PFxCy in THF are shown in Figure 3 and their optical data
are summarized in Table 1. As compared with absorption
spectra with the maxima peaks at 325 nm for monomer 1
and 297nm for monomer 2, the absorption maxima of PFC,
PF2C, PF, PC were located at 381, 411, 419 and 352 nm,
respectively, and were red-shifted. This fact means the exten-
sion of conjugation length upon transformation from the
monomers to polymers. When PFxCy were excited at the
absorption maxima, they showed strong emission at the blue-
green region and the maximum emission peaks of PFC, PF2C,
PF and PC at about 426, 427, 456 and 429 nm, respectively.
1
The H NMR and IR spectra of the polymers were consistent
with their assigned structures. As shown in Figure 1, no
residual ethynyl protons were observed in 1H NMR of the
polymers. The 1H NMR spectra of polymers displayed the
broader peaks than those of monomers. As shown in Figure
2, the characteristic absorptions bands of CBCAH and CBC
The solvent effect of water on quaternary ammonium poly-
mers CPFxCy measured by UV–vis absorption and PL spectra
in DMF and DMF/H2O (1:50, v/v) was shown in Figure 4. As
shown in Table 1, the absorption peaks of cationic polymers
CPFxCy in DMF and DMF/H2O were observed at 389/412
and 392/423 nm (CPF), 390/414 and 394/424 nm (CPF2C),
420 and 421nm (CPF), 319/363/391 and 327/364/394 nm
(CPC), respectively. The corresponding PL spectrum of
CPFxCy in DMF and DMF/H2O was peaked at 437/460 and
448/473 nm (CPFC), 434/458 and 446/473 nm (CPF2C),
435 and 445nm (CPF), 405 and 468 nm (CPC), respectively.
With increased volume fraction of H2O in the solvent, the
absorption and emission peaks of all quaternized polymers
presented obvious red-shift absorption and broadening. The
absorption and emission data were consistent with a signifi-
cant increase in pAp stacking interactions of the conjugated
polymer chains in water, which suggested that while water
for 1 (3293 and 2105 cmꢁ1) and 2 (3307 and 2107 cmꢁ1
)
were no longer observable in the IR spectra of polymers
PFxCy and two new stretching bands ascribed to CBCACBC
of copolymers appeared at 2208 and 2137 cmꢁ1 for PFC,
2204 and 2135 cmꢁ1 for PF2C, and four new stretching
bands of homopolymers appeared at 2133, 2191 cmꢁ1
(CBCACBC) and 2324, 2358 (CBC) for PF, 2133, 2198
cmꢁ1 (CBCACBC) and 2327, 2354 cmꢁ1 (CBC) for PC,
respectively. This confirmed efficient polymerization of
monomer 1 and 2 to afford PFxCy.
The cationic polymers CPFxCy were obtained by treating of
the neutral precursor polymers PFxCy with CH3I for 5 days.
As compared with 1H NMR spectra of PFC and CPFC in
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FIGURE
1
1H NMR spectra of
PFC in CDCl3, and CPFC in
DMSO-d6.
was added to the polymer solution in DMF, the polymer
chains most likely underwent water-induced aggregation
into a morphology where their planar phenylene rings and
diacetylene in adjacent main chains tightly packed face-to-
face and the hydrophilic side chains extended more fully into
water to optimize water-solubility. This is consistent with
the previously reported water-soluble PPE-SOꢁ3 .18 In that
case, PPE-SOꢁ3 showed significantly decreased PL intensity,
progressively red-shifted and broader absorption with
increase of volume fraction of H2O, which are prominent
characteristics of conjugated polymer aggregation. Another
possible explanation for such red-shift could be explained
by the increased torsion angle of the polymer main chains,
due to the increased repulsion between the positive ammo-
nium groups in the side chains and the adjacent fluorene
and carbazole units in the polar solvents.19
Effect of Buffer Solution and Na3PO4 on UV-Vis
Absorption and Photoluminescence of Cationic
Conjugated Polymers
DNA is more commonly stored in buffered solutions than in
pure water. The buffer ions are expected to weaken the
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TABLE 1 Molecular Weights and Photophysical Properties of Polymers
UV, kmax (nm)
DMF
PL, kmax (nm)
Polymer
MW
5,331
Mn
5,201
PDI
THF
DMF/H2O
THF
DMF
DMF/H2O
PFC
PF2C
PF
1.03
1.10
1.46
1.64
381
411
419
352
426
427
456
429
8,953
38,302
41,326
8,120
26,118
25,155
PC
CPFC
CPF2C
CPF
CPC
389/412
390/414
420
392/423
394/424
421
437/460
434/458
435
448/473
446/473
445
319/363/391
327/264/394
405
468
electrostatic interactions between the positively charged
polyelectrolytes and dsDNA, which is often referred to as
electrostatic screening. Because the external ions decrease or
collapse, the electrostatic interaction of counterions result in
a decrease in the potential between the two oppositely
charged pseudo surfaces.20 With these considerations
in mind, the absorption and PL spectral changes of CPFC
([RU] ¼ 2 ꢀ 10ꢁ5M) were firstly compared in water and in
Tris-HCl (0.1 M, pH ¼ 7.2) buffer solution. The results
showed that both absorption and PL spectra still exhibited
few change in terms of shape, wavelength and intensity
except that the UV-vis absorption was slightly red-shifted
and the PL intensity increased by less than 2% in the buffer.
Then the optical response of CPFC to CTDNA (5 ꢀ 10ꢁ5M)
FIGURE 3 (a) UV-vis absorption and (b) PL emission spectra of
PFxCy in THF. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.].
FIGURE 2 IR spectra of monomers 1, 2 and PFxCy.
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FIGURE 4 (a) and (b) UV-vis absorption spectra; (c) and (d) PL emission spectra of CPFxCy in DMF or DMF/H2O (1:50, v/v). [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
in Tris-HCl buffer (0.1 mol, pH ¼ 7.2) and water was tested,
the fluorescence quenching was about 37% and 38% in
water and buffer, respectively. Thus, the presence of buffer
ions made only a minor modification of the assay response.
The remarkably amplified fluorescence increase upon addi-
tion of Na3PO4 might be a characteristic of CPFC as sensitive
fluorescent sensors for detection of DNA. For comparison,
Na3PO4 was added to the homopolymers solution of CPF
and CPC. It was found that the usual fluorescence quenching
of CPF and CPC when [Na3PO4] ¼ 2 ꢀ 10ꢁ5 M.
More recently, energy transfer between oppositely charged
polyelectrolytes has been used to obtain fluorescence super-
quenching.21 These mixtures offer possibilities of ‘‘charge
reversal’’, where it is possible to encourage interactions
between a given polyelectrolyte and a fluorescence quencher
with similar charge. For example, mixing MPS 6 PPV with a
cationic poly-L-lysine with appended cyanine dyes resulted in
a complex where energy transfer from MPS 6 PPV to the
dye took place followed by quenching with a negatively
charged anthraquinone derivative.21 In our experiments, it
was surprised that when Na3PO4 was added to the aqueous
solution of CPFC and the molar ratio of [Na3PO4]:[CPFC]
went up to 10:1, Na3PO4 caused amplified fluorescence
increase (fivefold) concomitant with almost unchanged shape
of absorption spectra as shown in Figure 5. When Na3PO4
was added to the copolymer solution of CPF2C, the similar
phenomenon was observed. This result was just opposite to
the usually fluorescence quenching of cationic conjugated
polymers with the addition of negatively charged substances.
UV-Vis Absorption and Photoluminescence of
Copolymers CPFC and CPF2C with DNA
The preliminary study of the interaction between copolymers
and CT DNA was monitored by UV-Vis absorption spectra by
fixing polymer concentration ([RU]0 ¼ 2.0 ꢀ 10ꢁ5 M) upon
addition of CT DNA (4.3 ꢀ 10ꢁ9 M to 8.3 ꢀ 10ꢁ5 M). The
representative absorption spectra of CPFC in the absence
and presence of CT DNA were shown in Figure 6. When DNA
concentration was lower than that of polymer repeat units,
an absorption increase of polymers was observed with no
significant changes in the spectral shapes. When DNA con-
centration was near or above the polymer concentration, a
dramatic decrease of absorbance was observed, accompanied
by slight red shift and broadening. The maximum decrease
of the peak in the absorbance at 396 nm was found to be
37.4% (CPFC), 53.1% (CPF2C), with the CT DNA concentra-
tions of 2.7 ꢀ 10ꢁ5 M and 2.0 ꢀ 10ꢁ5 M, respectively.
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tion. From the previous studies, the interaction of opposite
charged analytes influences chain conformation, aggregation,
and thereby fluorescence of CPE. Herein, a possible mecha-
nism is that the multidimensions from the interchain aggre-
gation of polymers may touch DNA in different directions
and conduce to enhancement of the quenching ability of
tertiary amino group on conjugated backbone, resulting in
more fluorescence quenching.23 Another reason is that
interchain aggregation could lead to further amplification of
fluorescence quenching through the occurrence of interchain
exciton migration.24
The Stern-Volmer quenching constant (KSV) was obtained
from the initial linear part of a plot of the ratio of fluores-
cence intensity in the presence and the absence of quencher
as a function of quencher concentration.25 The Stern-Volmer
plot in Figure 7(b) is typical for those obtained with the
mixtures of CPFC and CPF2C and CT DNA. A linear region
was observed at low quencher concentrations, followed by
FIGURE 5 (a) UV–vis absorption and (b) PL spectra of CPFC
([RU] ¼ 2 ꢀ 10ꢁ5 M) in DMF/H2O with addition of PO43ꢁ ([PO34ꢁ
]
¼ 0, 4.5 ꢀ 10ꢁ8 ꢃ 2 ꢀ 10ꢁ4 M, from bottom to top), kexc
¼
390 nm.
From the quenching experiments of CPFC a decrease of fluo-
rescence upon the addition of CT DNA ([DNA] ¼ 3.0 ꢀ
10ꢁ13 to 8.3 ꢀ 10ꢁ5 M) was monitored [Fig. 7(a)]. Because
the maxima and shapes of the fluorescence spectra did not
change and no new peaks appeared, possible mechanism for
the quenching of CPFC was attributed to its aggregation
near the negatively charged dsDNA, leading to self-quench-
ing.22 Similar results were obtained for CPF2C.
CPFC and CPF2C exhibited high sensitivity with CT DNA.
Addition of a very small amount of DNA ([CT DNA] ¼ 3.3 ꢀ
10ꢁ13 M) could induce distinct fluorescent quenching (ca.
2.7% and 1.6% quenching respectively for CPFC and CPF2C,
kmax ¼ 449 nm). Electrostatic attractions between positively
charged conjugated polymer and negatively charged phos-
phate groups in DNA play an important role in their close
proximity. Therefore, the interaction between DNA and cati-
onic conjugated polyelectrolyte (CPE) is important for detec-
FIGURE 6 Absorption spectra of CPFC in the absence and
presence of CT DNA ([RU]0 ¼ 2 ꢀ 10ꢁ5 M): (a) [CT DNA] ¼
6.5 ꢀ 10ꢁ7 to 3.2 ꢀ 10ꢁ6 M; (b) [CT DNA] ¼ 1.3 ꢀ 10ꢁ5 to 8.3 ꢀ
10ꢁ5 M.
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decreased at a slower rate with higher DNA concentrations.
Thus, the fluorescence could not be completely quenched
even at high DNA concentrations. Similar behavior was
observed with other CCPs.27 This phenomenon was consist-
ent with the changes of UV-vis spectra.
EXPERIMENTAL
Materials
CT DNA was purchased from Sigma-American Biotechnology
Company. Double distilled water was used to prepare the
buffer solutions. Tris(hydroxymethyl)-aminomethane buffer
(Tris) was prepared with Tris (0.1 M) and adjusted to pH of
7.2 with hydrochloric acid. All other reagents and chemicals,
unless otherwise stated, were purchased from commercial
sources and used without further purification. Solvents were
dried and distilled. 2,7-Bis(20-hydroxy-20-methylbutynyl)-9,9-
bis(60-bromohexyl)-fluorene (7) and 3,6-bis(20-hydroxy-20-
methylbutynyl)-N-(60-(N,N-diethylamino)-hexyl]-carbazole (12)
were prepared by a typical procedure of Sonogashira coupling
reaction.28
Measurements
1H NMR and 13C NMR spectra were recorded on a Bruker-
400 MHz instrument using tetramethylsilane as an internal
reference. Infrared (IR) spectra were obtained with a Nicolet
380 FT-IR spectrometer. Elemental microanalyses were
carried out on a Vario EL III CHNS Elemental Analyzer. The
molecular weights of the polymers were determined by gel
permeation chromatography (GPC) relative to polystyrene
standards, using THF at room temperature at a flow rate of
1.0 mL/min. UV-vis absorption and PL spectra were recorded
on an UV-2450 and F-4500 spectrometer respectively.
Synthesis of 2,7-Diethynyl-9,9-bis[60-(N,N-diethylamino)-
hexyl]fluorene (1)
Compound 7 (1.65 g, 2.5 mmol) was dissolved in 20 mL of
diethylamine by heating and the solution was refluxed for
12 h. After the excess diethylamine was removed, KOH
(0.84 g, 15 mmol), toluene (10 mL) and methanol (5 mL)
were added. The mixture was refluxed under N2 at 120 ꢂC
for 3 h, extracted with CH2Cl2 (3 ꢀ 30 mL). The combined
organic layer was washed with water by three times, dried
over anhydrous Na2SO4. After removing solvent, the residue
was purified by silica gel column chromatography (EtOAc/
petroleum ether/Et3N ¼ 1:1:0.01, v/v/v) to give 1 as yellow
oil (0.69 g, 57%).
FIGURE 7 (a) Emission of CPFC decreased in intensity upon
complexation with additions of CT DNA ([CT DNA] ¼ 0 to 3 ꢀ
10ꢁ5 M); (b) Stern-Volmer plot of CPFC and CPF2C quenched
by CT DNA (1 ꢀ 10ꢁ9 to 1 ꢀ 10ꢁ8 M) in Tris-HCl buffer at pH ¼
7.2. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
an up-sloping nonlinear ‘‘superlinear’’ region where the
energy transfer may be best described by a ‘‘sphere-of-
action’’ mechanism.26 The non-linear parts were originated
from the coexistence of static and dynamic quenching
processes for aggregation of DNA with chromophore.
1H NMR (400 MHz, CDCl3, d, ppm): 0.48–0.56 (m, 4H), 0.93
(t, J ¼ 7.2 Hz, 12H), 1.00–1.03 (m, 8H), 1.20–1.22 (m, 4H),
1.88–1.92 (m, 4H), 2.24 (m, 4H), 2.42 (q, J ¼ 7.2 Hz, 8H),
3.12 (s, 2H), 7.41–7.45 (m, 4H), 7.59 (d, J ¼ 7.6 Hz, 2H). IR
(cmꢁ1): 3293 (CBCAH), 2105 (ACBCA).
The Stern-Volmer quenching constants calculated for CPFC
and CPF2C by CT DNA in DMF/Tris-HCl (1:600, v/v, pH ¼
7.2) were 1.9 ꢀ 108, 5.2 ꢀ 107 Mꢁ1 respectively, indicating
CPFC has better sensitivity of CCP fluorescence toward
quenching by DNA than that of CPFC. Such high Ksv values
indicated an efficient fluorescence quenching of CPFC and
CPF2C when they were bound to CT DNA through electro-
static interaction. Therefore, it is possible to detect DNA
easily at very low concentrations with CPFC and CPF2C.
However, the maximum fluorescence quenching was only
59% (CPFC) and 56% (CPF2C). The fluorescence intensity
Synthesis of 3,6-Diethynyl-N-[60-(N,N-diethylamino)-
hexyl]carbazole (2)
To a stirred solution of 13 (2.71 g, 5.6 mmol) in toluene
(20 mL) and methanol (10 mL) was added KOH (1. 91 g,
34.1 mmol). The mixture was refluxed under N2 at 120 ꢂC
for 12 h, extracted with CH2Cl2 (3 ꢀ 60 mL). The combined
organic layer was washed with water three times, dried over
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anhydrous Na2SO4, and evaporated to dryness. The crude
product was purified by silica gel column chromatography
using EtOAc/petroleum ether/Et3N (1:1:0.01, v/v/v) as
eluent to afford 2 as yellow viscous liquid (1.86 g, 90%).
and dried overnight under vacuum at room temperature to
give PC (51% yield).
1H NMR (400 MHz, CDCl3, d, ppm): 1.01 (m, 6H), 1.34–1.42
(m, 6H), 1.84 (m, 2H), 2.39 (m, 2H), 2.51 (m, 4H), 4.12 (m,
2H), 7.22–7.29 (m, 2H), 7.61–7.63 (m, 2H), 8.11–8.19 (m,
2H). IR (cmꢁ1): 2327, 2354 (CBC), 2133, 2198 (CBCACBC).
1H NMR (400 MHz, CDCl3, d, ppm): 0.96 (t, J ¼ 7.2 Hz, 6H),
1.27–1.38 (m, 6H), 1.80–1.84 (m, 2H), 2.33 (t, J ¼ 7.2 Hz,
2H), 2.46 (q, J ¼ 7.2 Hz, 4H), 3.06 (s, 2H), 4.23 (t, J ¼ 7.2
3
4
Hz, 2H), 7.29 (d, J ¼ 8.4 Hz, 2H), 7.57 (dd, J ¼ 8.4 Hz, J ¼
General Procedure of Synthesis of Cationic Quaternary
Ammonium Polymers CPFxCy
4
1.2 Hz, 2H), 8.18 (d, J ¼ 1.2 Hz, 2H); 13C NMR (C5D5N, 100
MHz, d, ppm): 12.2, 27.3, 27.4, 27.6, 29.3, 43.3, 47.1, 53.0,
77.8, 85.6, 110.1, 113.6, 122.7, 125.3, 130.6, 141.1. ACPI:
371 [Mþ]. IR (cmꢁ1): 3307 (CBCAH), 2107 (ACBCA).
CH3I (0.5 mL) was added to a solution of neutral precusor
polymers PFxCy in 10 mL of THF at room temperature. After
5 min stirring, some precipitate was observed, which was
dissolved by addition of 2 mL of DMF. Additional CH3I
(1 mL) was added and the mixture was stirred for 5 d at
room temperature. The precipitated solid was filtered and
washed with THF several times to afford cationic polymers
CPFxCy as yellow solids.
General Procedure of Synthesis of Neutral Precusor
Polymers (PFxCy)
To a dried three-neck round-bottomed flask attached an
inlet and outlet for oxygen were added CuCl (10 mol %),
N,N,N0,N0-tetramethylethylenediamine (TMEDA) 0.1mL and
6 mL of anhydrous 1,2-dichlorobenzene. The mixture was
warmed to 70ꢂC while oxygen was bubbled through. After
20 min, a solution of 3,6-diethynylcarbazole, 1, 2,7-diethynyl-
fluorene, 2 in 10 mL of 1,2-dichlorobenzene was added to
the mixture. After 3 h, the solid was precipitated in 10 mL
of methanol. The precipitate was filtered, washed several
times with methanol, and dried under vacuum at room
temperature to afford PFC and PF2C as yellow solids.
CPFC: yield 90%. 1H NMR (400 MHz, DMSO-d6, d, ppm):
0.49 (br, 4H), 1.08–1.16 (m, 26H), 1.30–1.79 (m, 12H), 2.49–
3.24 (m, 33H), 4.47 (br, 2H), 7.64 (m, 2H), 7.79–7.76
(m, 6H), 7.99 (m, 2H), 8.58 (m, 2H).
CPF2C: yield 94%. 1H NMR (400 MHz, DMSO-d6, d, ppm):
0.49 (br, 8H), 1.08–3.35 (m, 102H), 4.49 (br, 2H), 7.65
(m, 4H), 7.80 (m, 8H), 7.99 (m, 4H), 8.59–8.60 (m, 2H).
CPF: yield 85%. 1H NMR (400 MHz, DMSO-d6, d, ppm): 0.46
(br, 4H), 1.06–1.14 (m, 20H), 1.41 (m, 4H), 2.07 (m, 2H),
2.83–2.85 (m, 6H), 3.04 (m, 8H), 3.21–3.22 (m, 8H), 7.60–
7.64 (m, 2H), 7.80 (m, 2H), 7.99–8.00 (m, 2H).
1
PFC: yield 78%; H NMR (400 MHz, CDCl3, d, ppm): 0.58 (m,
4H), 0.97–1.05 (m, 26H), 1.27–1.37 (m, 12H), 1.86–1.94 (m,
6H), 2.30–2.35 (m, 6H), 2.48 (m, 12H), 4.27 (s, 2H), 7.34–
7.36 (m, 2H), 7.48–7.50 (m, 4H), 7.65–7.63 (m, 4H), 8.26
(m, 2H). Mw: 5331; Mw/Mn: 1.03 (GPC). IR (cmꢁ1): 2208,
2137(CBCACBC).
PF2C: yield 82%. 1H NMR (400 MHz, CDCl3, d, ppm): 0.58
(m, 8H), 1.01–1.04 (m, 46H), 1.32–1.43 (m, 16H), 1.83–1.94
(m, 10H), 2.39 (m, 10H), 2.55–2.55 (sm, 20H), 4.29 (m, 2H),
7.35–7.37 (m, 2H), 7.48–7.52 (m, 8H), 7.64–7.66 (m, 6H),
8.26–8.27 (m, 2H). Mw: 8953; Mw/Mn: 1.10 (GPC). IR (cmꢁ1):
2204, 2135(CBCACBC).
1
CPC: yield 88%. H NMR (400 MHz, DMSO-d6, d, ppm): 1.15
(m, 6H), 1.30 (m, 4H), 1.52 (m, 2H), 1.80 (m, 2H), 2.86–2.88
(m, 2H), 3.09 (m, 3H), 3.21–3.23 (m, 4H), 4.48 (m, 2H), 7.75
(m, 4H), 8.57 (m, 2H).
DNA Binding Study
All experiments involving the interaction of copolymers
CPFC and CPF2C with CT DNA were performed in Tris
buffer. The experiment was performed by fixing the con-
centrations of the polymer (the concentration of CPFC and
CPF2C were calculated in terms of repeat units (RU), [RU] ¼
2.0 ꢀ 10ꢁ5 M) while increasing amount of DNA. DNA con-
Synthesis of Polymer PF
The above procedure was followed using CuCl (2 mg, 0.01
mmol), TMEDA 0.1mL, monomer 1 (55 mg, 0.1 mmol), and
4 mL of 1,2-dichlorobenzene. The polymerization was carried
centrations ranged from 0, 3.0 ꢀ 10ꢁ13 M to 8.3 ꢀ 10ꢁ5
M
in base pairs. DNA concentration was determined by absorp-
tion spectrometry at 260 nm using a molar extinction coeffi-
cient 6600 Mꢁ1ꢄcmꢁ1 (A260/A280 > 1.80).29
ꢂ
out at 70 C. The light yellow fibrous was filtered and dried
overnight under vacuum at room temperature to give PF
(65% yield).
CPFC and CPF2C can be dissolved in DMF/H2O mixtures
well, but they have a low solubility in water. Stock polymer
solutions with concentrations of around 4 ꢀ 10ꢁ3 M in
repeat units were prepared in 10 mL of DMF/H2O (1:3, v/v).
Aliquots of this solution were diluted to give solutions with
polymer concentrations of 8 ꢀ 10ꢁ7 to 4 ꢀ 10ꢁ4 M in terms
of repeat units that were used for the photometric measure-
ments. In DNA binding experiments, stock polymer solutions
were diluted with Tris-HCl (0.1 M, pH ¼ 7.2) to give the test
polymer concentrations of 2 ꢀ 10ꢁ5 M in DMF/Tris-HCl
(1:600, v/v).
1H NMR (400 MHz, CDCl3, d, ppm): 0.57 (m, 4H), 0.95 (m,
12H), 1.04 (m, 8H), 1.23 (m, 4H), 1.92 (m, 4H), 2.27 (m, 4H),
2.43–2.45 (m, 8H), 7.48 (m, 4H), 7.63–7.65 (m, 2H). IR
(cmꢁ1): 2324, 2358 (CBC), 2133, 2191 (CBCACBC).
Synthesis of Polymer PC
The above procedure was followed using CuCl (2 mg, 0.01
mmol), TMEDA (0.1 mL), monomer 2 (46 mg, 0.1 mmol),
and 4 mL of 1,2-dichlorobenzene. The polymerization was
carried out at 70 ꢂC. The light yellow fibrous was filtered
4176
WILEYONLINELIBRARY.COM/JOURNAL/JPOLA

ARTICLE
CONCLUSIONS
7 Brizius, G.; Kroth, B.; Macromolecules 2002, 35, 5317; (b)
Roh, Y.; Bauld, N. L. Adv Synth Catal 2002, 344, 192; (c) Gao,
D.; Roh, Y.; Bauld, N. L. Adv Synth Catal 2001, 343, 269.
In conclusion, two novel cationic quaternary ammonium con-
jugated copolymers CPFC and CPF2C containing fluorenyl,
diacetylene and carbazolyl units were successfully synthe-
sized via an oxidative coupling approach. The polymers were
characterized by IR, 1H NMR, UV-vis, and fluorescence. The
water-induced aggregation of the polymers in DMF/H2O was
confirmed by spectroscopy. The polymers emitted intense
blue fluorescence and exhibited high quenching efficiency
with DNA, which indicated that the polymers might be
applied as sensitive fluorescent materials for detection of
DNA.
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This work was supported by the National Natural Science Foun-
dation of China (20872038, 20904010) and the Fundamental
Research Funds for the Central Universities (2009ZM0170).
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SYNTHESIS OF CATIONIC DIACETYLENE-co-CARBAZOLE-co-FLUORENE POLYMERS, ZENG ET AL.
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