Optimization of the molecular orbital energies of conjugated polymers for optical amplification of fluorescent sensors

Article abstract of DOI:10.1021/ja055382t

Cationic water-soluble poly(fluorene-co-phenylene)s with electron withdrawing or donating substituents on the conjugated backbone were designed and synthesized. Fluorescence resonance energy transfer (FRET) experiments between these conjugated polymers and dye-labeled single-stranded DNA (ssDNA-C*) reveal the importance of matching donor and acceptor orbital energy levels to improve the sensitization of C* emission. Quenching of polymer fluorescence with ssDNA-C* and differences in C* emission suggest involvement of photoinduced charge transfer (PCT) as an energy wasting mechanism. The HOMO and LUMO energy levels of the conjugated polymers and C* serve as a preliminary basis to understand the competition between FRET and PCT. Dilution of C* in polymer/ssDNA-C* complexes by addition of ssDNA yields insight into C*...C* self-quenching. Under optimized conditions, where there is no probe self-quenching and minimum PCT, efficient signal amplification is demonstrated despite poor spectral overlap between polymer and C*.

Full text of DOI:10.1021/ja055382t

Published on Web 01/06/2006
Optimization of the Molecular Orbital Energies of Conjugated
Polymers for Optical Amplification of Fluorescent Sensors
Bin Liu and Guillermo C. Bazan*
Contribution from the Department of Materials and Chemistry and Biochemistry,
Institute for Polymers and Organic Solids, UniVersity of California at Santa Barbara,
Santa Barbara, California 93106
Received August 6, 2005; E-mail: bazan@chem.ucsb.edu
Abstract: Cationic water-soluble poly(fluorene-co-phenylene)s with electron withdrawing or donating
substituents on the conjugated backbone were designed and synthesized. Fluorescence resonance energy
transfer (FRET) experiments between these conjugated polymers and dye-labeled single-stranded DNA
(ssDNA-C*) reveal the importance of matching donor and acceptor orbital energy levels to improve the
sensitization of C* emission. Quenching of polymer fluorescence with ssDNA-C* and differences in C*
emission suggest involvement of photoinduced charge transfer (PCT) as an energy wasting mechanism.
The HOMO and LUMO energy levels of the conjugated polymers and C* serve as a preliminary basis to
understand the competition between FRET and PCT. Dilution of C* in polymer/ssDNA-C* complexes by
addition of ssDNA yields insight into C*‚‚‚C* self-quenching. Under optimized conditions, where there is
no probe self-quenching and minimum PCT, efficient signal amplification is demonstrated despite poor
spectral overlap between polymer and C*.
Introduction
biosensor applications, one relies on CPs with charged pendant
2,7
groups (i.e., conjugated polyelectrolytes). These water soluble
CPs have been used as light harvesting molecules that deliver
excitations to signaling fluorescent dyes attached to biomolecular
probes, thereby providing increased signal intensities and sensi-
Conjugated polymers (CPs) form the basis of new methods
for the trace detection of analytes in a variety of environments.
Their delocalized electronic structure allows for electronic
coupling between optoelectronic segments and efficient intra-
and interchain energy transfer. Important properties, such as
charge transport, conductivity, emission intensity,
exciton migration are easily perturbed by external agents,
leading to substantial changes in measurable signals. For inter-
1
,2
6,8
tivities above those of single molecule reporters. Nonspecific
contacts between nontarget species and the hydrophobic CP
backbone need special attention for attaining selectivity, as these
interactions can lead to misleading interpretations of results.9
3
4
5
2a,2f,2g
and
4
6
rogation in aqueous media, conditions typically required for
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1188
9
J. AM. CHEM. SOC. 2006, 128, 1188-1196
10.1021/ja055382t CCC: $33.50 © 2006 American Chemical Society

Molecular Orbital Energies of Conjugated Polymers
A R T I C L E S
Scheme 2. Major Interactions within CCP/DNA-C* Complexes
Scheme 1. Effect of Relative Orbital Energy Levels on FRET
versus PCT Preferences
that Influence Optical Performance
One specific approach that improves the sensitivity of
fluorescent DNA assays involves using a cationic conjugated
polymer (CCP) and a chromophore labeled DNA (DNA-C*),
which are selected to favor fluorescence resonance energy
transfer (FRET) from the CCP to the DNA-C*.2c,8d Amplifica-
tion of C* emission by excitation of CCPs, relative to direct
excitation of C*, originates from the polymer’s large absorption
cross-section. Efficient CCP to C* FRET is a required additional
condition to increase sensitivity.10 Eq 1 describes how the FRET
rate (kt) changes as a function of the donor-acceptor distance
in one of the optical partners, as in situation B, donor excitation
1
4
may lead to photoinduced charge transfer. As shown in
Scheme 1, donor excitation would lead to photoinduced electron
transfer to the acceptor. Excitation of the acceptor would lead
to a similar charge separated state via hole transfer to the
1
3
donor. While Scheme 1 is widely used for choosing suitable
optical partners for a specific application, it fails to be accurate
for intermediate cases since it neglects contributions from the
exciton binding energy, the intermolecular charge transfer state
energy, and the stabilization of the charged species by the
(r), the orientation factor (κ), and the overlap integral (J), where
FD(λ) is the corrected fluorescence intensity of the donor in the
range of λ to λ + ∆λ and ꢀA(λ) is the extinction coefficient of
the acceptor at λ.11
14d
medium. The mechanism by which FRET or PCT is preferred
is complex for conjugated polymer blends and may involve
geminate electron-hole pairs that convert to exciplexes and
1
2
kt ∝ κ J(λ)
(1)
6
r
1
5
ultimately excitons. Despite these uncertainties, Scheme 1B
∞
4
J(λ) )
∫
F (λ)ꢀ (λ)λ dλ
D A
provides for a necessary, but not sufficient, condition for PCT.
0
Electrostatic interactions between CCPs and phosphate groups
in DNA bring CCPs and DNA-C* into close proximity to satisfy
the FRET distance requirement. Hydrophobic interactions are
also operative; however, these are less well understood and may
According to eq 1, maximized overlap (J(λ)) between the
emission of the CCP and the absorption of C* should provide
for optimum FRET conditions.
1
6
Recent studies of conjugated phenylenevinylene-based den-
drimers and binary conjugated polymer blends highlight the
competition between energy transfer and photoinduced charge
transfer (PCT) events as a function of the HOMO and LUMO
be controlled to some extent by solvent choice. CCP/DNA-
C* complexes are dynamic structures of varying sizes. Three
major types of interactions that influence optical performance
within these structures can be readily identified (shown in
Scheme 2): CCP‚‚‚C* (A), CCP‚‚‚CCP (B), and C*‚‚‚C* (C).
Close association between CCP and DNA-C* will favor FRET,
while close contact among C* units may cause self-quenching,
ultimately leading to decreased signal amplification. Interchain
contacts (CCP‚‚‚CCP) can also lead to donor self-quenching
and a reduction of fluorescence amplification. Despite the
general success of using CCPs in biosensor schemes,3
12
energy levels in the donor and acceptor pairs. Scheme 1
provides a simplified illustration of two situations that may occur
upon donor excitation.13 Situation A corresponds to the ideal
situation for FRET, where the HOMO and LUMO energy levels
of the acceptor are located within the orbital energy levels of
the donor. Upon excitation of the donor, energy transfer from
the donor to the acceptor takes place, leading to an emissive
process, provided that the emission quantum yield of the
acceptor is sufficiently large. There is no quenching of the
acceptor under Situation A.
i,3j,8d,16b,17
there is little information available on how the geometry, size,
and orientation between optical partners in these kinetically
formed CCP/DNA-C* structures influence optical output.18
When the energy levels of the acceptor are not contained
within the orbital energies of the donor, in other words, when
both the electron affinity and the ionization potential are higher
(14) (a) Sariciftci, N. S.; Smilowitz, L.; Heeger, A. J.; Wudl, F. Science 1992,
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Bardeen, A. J.; Thayumanavan, S. J. Am. Chem. Soc. 2005, 127, 373.
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Phys. ReV. Lett. 2004, 92, 247402.
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Chem. Soc. 2003, 125, 6705.
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(b) Gaylord, B. S.; Massie, M. R.; Feinstein, S. C. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 34. (c) Xu, H.; Wu, H. P.; Huang, F.; Song, S. P.; Li,
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(
10) F o¨ rster, T. Ann. Phys. 1948, 2, 55.
(
11) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/
Plenum Publisher: New York, 1999.
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Holmes, A. B.; Br e´ das, J. L.; Friend, R. H. Phys. ReV. B 1999, 60, 5721.
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M. Chem. Commun. 2001, 8, 707. (c) Guldi, D. M.; Swartz, A.; Luo, C.;
G o´ mez, R.; Segura, J.; Mart ´ı n, N. J. Am. Chem. Soc. 2002, 124, 10875.
13) Cornil, J.; Lemaur, V.; Steel, M. C.; Dupin, H.; Burquel, A.; Beljonne, D.;
Bredas, J. L. In Organic PhotoVoltaics; Sun, S. J., Sariciftci, N. S., Eds.;
Taylor and Francis: Boca Raton, FL, 2005; p 161.
(
J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006 1189

A R T I C L E S
Liu and Bazan
Scheme 3. Synthesis of CCPs 1, 2, and 3a
Table 1. Summary of Absorption and PL Spectra in 25 mM
Phosphate Buffer (pH ) 7.4)
(cm- L mol-1)
1
×
104
Φa (%)
compounds
λmax abs. (nm)
λmax em. (nm)
ꢀ
1
2
3
Fl
TR
385
363
369
495
590
415
410
414
515
615
4.54
4.05
4.18
7.50
8.30
24
32
29
85
b
90
a
Using quinine sulfate as the standard, (5% error.22 b Molecular Probes
(
http://probes.invitrogen.com).
Scheme 4. Chemical Structures of Fl and TR with Linkers
(8-Amino-3,6-dioxaoctanoic Acid) Prior to ssDNA Attachment
a
Reaction conditions: (i) t-BuLi/pentane, 2-isopropoxy-4,4,5,5-tetram-
ethyl-1,3,2-dioxaborolane, THF, -78 °C, 3 h; (ii) Pd(PPh3)4, 2 M Na2CO3,
toluene; and (iii) NMe3, THF/H2O.
In this contribution, we probe the importance of matching
the energy levels between CCPs and C* for determining C*
emission and FRET efficiency. We begin by examining the
optical and electronic properties of three specifically designed
CCP structures and the FRET from the CCPs to fluorescein-
Poly[9,9′-bis(6′′-(N,N,N-trimethylammonium)-hexyl)fluorene-
3i
co-alt-1,4-phenylene dibromide] (1), poly[9,9′-bis(6′′-(N,N,N-
trimethylammonium)-hexyl)fluorene-co-alt-2,5-dimethoxy-1,4-
phenylene dibromide] (2), and poly[9,9′-bis(6′′-(N,N,N-trimethyl-
ammonium)-hexyl)fluorene-co-alt-2,5-difluoro-1,4-phenylene di-
bromide] (3) were synthesized via Suzuki coupling under
refluxing conditions in a mixture of 2 M Na2CO3/toluene with
(Fl) or Texas Red- (TR) labeled ssDNA. We then report studies
on CCP self-quenching with unlabeled ssDNA and on C*
emission quenching upon CCP/DNA-C* complex formation.
The HOMO and LUMO energy levels of the CCPs and C* serve
as a basis for understanding the competition between energy
transfer and PCT processes from a thermodynamics perspective.
Finally, we show how this information is useful to control
ssDNA-TR/polymer interactions and to improve signal ampli-
fication.
2
0
Pd(PPh3)4 as the catalyst, followed by trimethylamine treat-
ment (Scheme 3). Suzuki copolymerization of 2,7-bis[9,9′-bis-
6′′-bromohexyl)-fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]dioxaboro-
lane and 1,4-dibromobenzene derivatives provides the neutral
polymer precursors 1a, 2a, and 3a in 73-78% yields. Forma-
tion of the ionic water-soluble polymers 1-3 was achieved in
(
8
3-89% yields via quaternization of the neutral polymers with
Results
1
13
trimethylamine in a THF/water mixture. H and C NMR
spectroscopies confirm the molecular structures in Scheme 3.
The degree of quaternization is greater than 90%, as calculated
from the ratio of the integrated areas for CH2CH2X (X ) Br,
N(CH3)3) and CH2CH2N(CH3)3 in the H NMR spectra. GPC
determined molecular weights (Mn) are 27 000 (PDI ) 2.2), 35
Synthesis. Scheme 3 shows the synthetic route to the CCP
structures used in this study. The sequence of steps circumvents
time-consuming protection-deprotection steps or tedious pu-
rification procedures associated with amine-containing inter-
1
19
mediates. The key intermediate, 2,7-bis[9,9′-bis(6′′-bromohexyl)-
fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]dioxaborolane was synthe-
sized in one step, starting from 2,7-dibromo-9,9′-bis(6′′-bro-
mohexyl)fluorene.1 After the addition of t-BuLi, excess 2-iso-
propoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was added in
a single portion to the reaction mixture, and the reaction was
continued for another 2 h. It is necessary to keep the reaction
temperature at -78 °C to minimize nucleophilic reaction
between the aliphatic bromide at the fluorene 9-position and
the lithium salt byproduct. The boronic ester was purified by
chromatography using silica support, and the molecular structure
was confirmed by MS and NMR spectroscopies. The availability
of the alkylbromide-containing arylboronates greatly simplifies
the synthesis of water-soluble conjugated polymer precursors
that rely on Suzuki cross-coupling protocols.
000 (PDI ) 2.0), and 28 000 (PDI ) 1.5) for 1-3, respectively.
Optical and Electronic Properties. Table 1 summarizes of
6b
the absorption and photoluminescence (PL) spectra of 1-3, Fl,
and TR (structures shown in Scheme 4). Figure 1 shows the
PL spectra of 1-3 and the absorption of Fl and TR attached to
the 5′-position of ssDNA-C* (sequence: 5′-C*-ATC TTG ACT
ATG TGG GTG CT-3′, C* ) Fl or TR). Measurements were
made in 25 mmol phosphate buffer at pH ) 7.4, conditions
typically used in DNA detection schemes. For 1, the absorption
maximum (λabs) appears at 385 nm and the emission maximum
(λem) at 415 nm. The absorption maxima of 2 and 3 are slightly
blue shifted relative to that of 1, probably as a result of steric
interference, which prevents an equally coplanar arrangement
between aromatic units and thus yields less effective π-conjuga-
(
18) (a) Wang, S.; Bazan, G. C. Chem. Commun. 2004, 21, 2508. (b) Wang, S.;
Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446. (c) Tan,
C. Y.; Pinto, M. R.; Kose, M. E.; Ghiviga, I.; Schanze, K. S. AdV. Mater.
(20) (a) Suzuki, A. J. Organomet. Chem. 1999, 576, 147. (b) Liu, B.; Bazan,
G. C. In Organic Electroluminescence; Kafafi, Z. H., Ed.; Taylor and
Francis: Boca Raton, FL, 2005; p 179. (c) Liu, B.; Yu, W. L.; Lai, Y. H.;
Huang, W. Chem. Commun. 2000, 7, 551. (d) Liu, B.; Yu, W. L.; Lai, Y.
H.; Huang, W. Chem. Mater. 2001, 13, 1984. (e) Liu, B.; Yu, W. L.; Lai,
Y. H.; Huang, W. Macromolecules 2002, 35, 4975.
2
004, 16, 1208.
(
19) (a) Stork, M.; Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. AdV. Mater.
002, 14, 361. (b) Wang, S.; Liu, B.; Gaylord, B. S.; Bazan, G. C. AdV.
Funct. Mater. 2003, 13, 463.
2
1190 J. AM. CHEM. SOC.
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VOL. 128, NO. 4, 2006

Molecular Orbital Energies of Conjugated Polymers
A R T I C L E S
FRET Measurements. We now examine how the CCP
substituents influence the FRET to ssDNA-Fl by monitoring
the dye emission intensity upon CCP excitation. Measurements
were carried out in buffer (25 mM phosphate buffer, pH ) 7.4),
-
8
at a fixed ssDNA-Fl concentration (2 × 10 M based on
-7
strands), with [RU] varying from 0 to 4 × 10 M (RU ) poly-
-
8
mer repeat unit), with an increase of 5 × 10 M upon each
polymer addition. Figure 2a shows a direct comparison of Fl
-7
emission upon CCP excitation ([RU] ) 4 × 10 M). The most
intense Fl emission was observed for 3/ssDNA-Fl, which is ap-
proximately 2-fold more intense than that observed for 1/ss-
DNA-Fl and is over an order of magnitude larger than that for
2
/ssDNA-Fl. For 3/ssDNA-Fl, the integrated Fl emission is
approximately 5-fold greater than that obtained by direct
excitation of Fl at its absorption maximum (495 nm) in the ab-
sence of the CCPs, while over 20-fold enhancement is observed
relative to direct Fl excitation in the presence of 3. This enhance-
ment is indicative of the signal amplification provided by the
light harvesting capabilities of the CCPs. Additionally, the CCP
sensitized Fl emission is ∼10 nm red-shifted, relative to the
emission upon direct Fl excitation in the absence of the CCP.
FRET experiments using ssDNA-TR as the acceptor (with
an identical base sequence to ssDNA-Fl) were also performed
Figure 1. Photoluminescence (PL) spectra of 1 (a), 2 (b), and 3 (c) and
absorbance of ssDNA-C* (Fl, d and TR, e) in 25 mM phosphate buffer
(
pH ) 7.4). Excitation wavelength is 385 nm for 1 and 365 nm for 2
and 3.
Table 2. Optical Bandgap and HOMO and LUMO Energies of the
a
Neutral Precursors of 1, 2, 3, Fl, and TR
compound
band gap (eV)
HOMO (eV)
LUMO (eV)
1
2
3
FL
TR
a
a
a
2.80
2.80
3.05
2.30
2.08
-5.6
-5.4
-5.8
-5.9
-5.4
-2.8
-2.6
-2.7
-3.6
-3.3
-
8
-7
(Figure 2b, [ssDNA-TR] ) 2 × 10 M and [RU] ) 4 × 10
M). The TR emission intensities are similar for 1/ssDNA-TR
and 3/ssDNA-TR, which are approximately twice more intense
than that observed with 2/ssDNA-TR. For 3/ssDNA-TR, the
integrated TR emission through FRET is approximately twice
greater than that obtained by direct excitation of TR at its
absorption maximum (590 nm) in the absence of CCPs, while
there is a 10-fold enhancement relative to direct excitation of
TR in the presence of 3. Of particular significance is that the
TR emission with 2/ssDNA-TR is more intense than the Fl
emission with 2/ssDNA-Fl, despite the less effective spectral
overlap (J(λ) in eq 1).
2
6
a
Cyclic voltammetry data were collected in THF or DMF using 0.1 M
Bu4NPF6 as the electrolyte at a scan rate of 100 mV/s with a glass carbon
electrode (1.6 mm diameter) as the working electrode, Pt wire as the
auxiliary electrode, and Ag/AgNO3 as the reference electrode.
tion.21 However, the three CCPs show similar λem, PL band
shape (Figure 1), and PL quantum yields (1, 24%; 2, 32%; and
3
, 29% within (5% error). The spectral overlap (J(λ)) between
the three CCPs and the fluorescent dyes is thus similar.
The HOMO energy levels of the CCPs were estimated from
the oxidation potentials of the neutral precursors determined
Polymer Quenching with ssDNA and ssDNA-Fl. Figure 3
shows the decrease of CCP emission intensity ([RU] ) 1 ×
2
3
by cyclic voltammetry (CV) in THF (Table 2). Direct
measurement of the oxidation and reduction potentials of CCPs
in water was not successful. The corresponding LUMO levels
were obtained by taking into consideration the optical bandgap
from the absorption spectra. The oxidation potential onset for
-6
1
0
M) when [ssDNA] or [ssDNA-Fl] (identical base se-
-8
quences) is varied from 0 to 3.5 × 10 M. For the three CCPs,
there is a decrease in PL intensity upon the addition of ssDNA,
which we attribute to chain aggregation induced by the
negatively charged ssDNA and the resulting interchain quench-
1
a is 1.2 V versus SCE.24 Introduction of the fluorine atoms
ing process. At similar CCP concentrations, the fluorescence
19b
on the central core increases the oxidation potential by ap-
proximately 0.2 V, while the methoxy groups lower the
oxidation potential by approximately 0.2 V. In DMF, Fl displays
a reversible reduction peak, with an onset reduction potential
at -0.84 V versus SCE. This result is close to previous
work with 5-carboxyfluorescein (-0.47 V vs NHE; NHE is
quenching of 3 is less effective than that observed with 1 and
2. Within the conditions in Figure 3, the loss of polymer
emission upon complexation/aggregation with ssDNA varies
from 0 to 20% for 3 and from 0 to 40% for 1 and 2.
When ssDNA-Fl is added to solutions containing the CCPs,
one observes a more pronounced decrease in emission, relative
to the measurements with ssDNA (Figure 3). Polymer absorption
was also monitored to show that there is less than 10%
absorbance loss upon ssDNA-Fl addition.
2
5 26
-
0.2412 V vs SCE ). The first reduction potential of TR
2
7
was previously reported as -0.88 V versus NHE. Taking
into account the optical bandgap of 2.08 eV, one obtains TR
HOMO and LUMO energies of -5.36 and -3.28 eV, respec-
tively.
Acceptor Quenching Study. Changes in the intrinsic emis-
sion properties of the reporter dyes upon CCP/ssDNA-C*
(
26) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.; Kamagata, Y.;
(
21) Berggren, M.; Ingan a¨ s, O.; Gustafsson, G.; Rasmusson, J.; Anderson, M.
R.; Hjertberg, T.; Wennerstr o¨ m, O. Nature 1994, 372, 444.
22) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991.
23) Gomer, R.; Tryson, G. J. Chem. Phys. 1977, 66, 4413.
24) Mallavia, R.; Montilla, F.; Pastor, I.; Velasquez, P.; Arredondo, V.; Alvarez,
A. L.; Mateo, C. R. Macromolecules 2005, 38, 3185.
Kanagawa, T.; Kurane, R. Anal. Sci. 2001, 17, 155.
(27) (a) Rompaey, E. V.; Sanders, N.; Smedt, S. C.; Demeester, J.; Craenen-
broeck, E. V.; Engelborghs, Y. Macromolecules 2000, 33, 8280. (b)
Rompaey, E. V.; Engelborghs, Y.; Sanders, N.; Smedt, S. C.; Demeester,
J. Pharm. Res. 2001, 18, 928. (c) Lucas, B.; Rompaey, E. V.; Smedt, S.
C.; Demeester, J.; Oostveldt, P. V. Macromolecules 2002, 35, 8152. (d)
Lucas, B.; Remaut, K.; Braeckmans, K.; Haustraete, J.; Smedt, S. C.;
Demeester, J. Macromolecules 2004, 37, 3832.
(
(
(
(
25) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; John Wiley &
Sons: New York, 1980.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006 1191

A R T I C L E S
Liu and Bazan
Figure 2. Fluorescence spectra of ssDNA-Fl (a) and ssDNA-TR (b) in the presence of 1 (black), 2 (blue), and 3 (red) in 25 mmol phosphate buffer at
-
8
-7
[
ssDNA-Fl or ssDNA-TR] ) 2 × 10 M and [RU] ) 4 × 10 M. The excitation wavelengths are 385 nm for 1 and 365 nm for 2 and 3. Direct excitation
of ssDNA-Fl and ssDNA-TR prior to polymer addition is also shown in green and orange, respectively.
Figure 3. Normalized integrated PL intensity changes in the 390-520 nm
Figure 4. PL spectra of CCP/ssDNA-Fl complexes upon excitation at 495
-
6
range for 1-3 ([RU] ) 1 × 10 M) in the presence of ssDNA or ssDNA-
-8
-7
nm with [ssDNA-Fl] ) 2 × 10 M and [RU] ) 4 × 10 M, at æ ) 2:
Fl in 25 mM phosphate buffer with the concentration of DNA varying from
(a) prior to CCP addition and in the presence of (b) 3, (c) 1, and (d) 2.
-
8
0
to 3.5 × 10 M (based on strands). F0 and F are defined as the integrated
polymer emission in the absence and in the presence of ssDNA (or ssDNA-
Fl), respectively. Excitation wavelength is 385 nm for 1 and 365 nm for 2
and 3.
complexation were monitored by direct excitation of the dyes
at their absorption maxima. These experiments were conducted
-
8
at [ssDNA-C*] ) 2 × 10 M, with polymer [RU] varying
from 0 to 4 × 10 M. Figure 4 compares the Fl PL for
/ssDNA-Fl, 2/ssDNA-Fl, and 3/ssDNA-Fl at æ ) 2 (æ is the
-
7
1
ratio of CCP positive charges relative to ssDNA negative
charges), upon direct excitation at 495 nm. An 80% decrease
in Fl emission intensity is observed for 1/ssDNA-Fl and a 90%
decrease is observed for 2/ssDNA-Fl, relative to ssDNA-Fl.
Similarly, a 65% decrease takes place with 3/ssDNA-Fl.
Monitoring Fl absorption reveals less than 15% loss upon CCP
addition, indicating that the complexes remain in solution and
that there is little precipitation or adsorption to surfaces.
Similar experiments to those in Figure 4 were performed
using ssDNA-TR instead of ssDNA-Fl. Upon interaction with
CCPs, the TR emission is red shifted to 620 nm, relative to
free ssDNA-TR (615 nm). TR emission upon direct excitation
at its absorption maximum (590 nm) also decreases with
increasing [CCP]. Figure 5 compares the TR emission for CCP/
ssDNA-TR at æ ) 2, upon direct excitation at 590 nm. Relative
to free ssDNA-TR emission, the decreases in emission intensities
are 70, 80, and 60% for 1-3, respectively. When one compares
Figure 5. PL spectra of CCP/ssDNA-TR complexes upon excitation at
-
8
-7
590 nm with [ssDNA-TR] ) 2 × 10 M and [RU] ) 4 × 10 M, at
æ ) 2: (a) prior to CCP addition and in the presence of (b) 3, (c) 1, and
(d) 2.
the spectra in Figure 5 to those in Figure 4, the intensity loss
for TR is only slightly lower than that for Fl.
To probe the possibility of self-quenching, we monitored the
Fl emission upon complexation with 1 in ssDNA-Fl/ssDNA
mixtures. In these experiments, the ssDNA sequences were
1192 J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006

Molecular Orbital Energies of Conjugated Polymers
A R T I C L E S
Figure 6. F/F0 vs æ for 1/(ssDNA-Fl+ssDNA) complexes with [ssDNA]T
-
7
Figure 7. F/F0 vs æ for CCP/(ssDNA-Fl+ssDNA) solutions prepared by
the addition of the CCPs (a for 3, b for 1, and c for 2) to ssDNA-Fl (2 ×
)
2 × 10 M, prepared by adding 1 to ssDNA-Fl + ssDNA mixtures.
Excitation wavelength is 495 nm.
-
8
-7
10
M) + ssDNA (1.8 × 10 M) mixtures. Excitation wavelength is
495 nm.
identical and differed only by the labeling with Fl. Measure-
ments were carried out by adding 1 to solutions with different
ratios of ssDNA-Fl and ssDNA, while keeping a constant total
[ssDNA]T ) [ssDNA-Fl] + [ssDNA]. F0 and F values cor-
respond to the emission intensities of Fl in the absence and
presence of the CCP, respectively, upon direct excitation, and
the ratio F/F0 provides a measure of the Fl quenching. In the
four traces of Figure 6, the ratio of [ssDNA] to [ssDNA-Fl] is
varied. The x-axis (æ) corresponds to the ( charge ratio. Three
important observations can be drawn from Figure 6. First, at
all [ssDNA] to [ssDNA-Fl] ratios, the addition of 1 induces a
decrease of Fl emission intensity. Second, a higher fraction of
ssDNA-Fl in the mixture leads to more pronounced quenching.
This observation suggests Fl‚‚‚Fl self-quenching, which is more
pronounced when the local concentration of ssDNA-Fl is
highest. Third, the extent of quenching, as indicated by the
decrease in F/F0, reaches its maximum in the æ )1 f 2 range.
Upon further polymer addition, the Fl emission increases. For
æ > 2, one observes a more effective emission recovery when
the ssDNA-Fl content in the mixture is lowest. However, even
at æ ) 7, the maximum recovery of Fl emission corresponds
to approximately 60% of the original PL emission intensity.
Figure 8. F/F0 vs æ for CCP/(ssDNA-Fl + ssDNA) solutions prepared
-8
by addition CCP (a for 3, b for 1, and c for 2) to ssDNA-TR (2 × 10 M)
-
7
+ ssDNA (1.8 × 10 M) mixtures. Excitation wavelength is 590 nm.
differences as a function of CCP structures at high æ values
are not related to differences in the ratio of free to complexed
ssDNA-Fl.
The effect of polymer structure on Fl emission quenching
was examined as shown in Figure 7. In these experiments,
A comparison of TR emission of different CCP/ssDNA-TR
-
7
mixtures with [ssDNA]T ) 2 × 10 M and [ssDNA-TR] ) 2
-
7
-8
[
ssDNA]T ) 2.0 × 10 M and [ssDNA-Fl] ) 2 × 10 M,
-
8
×
10 M upon addition of different quantities of 1-3 is shown
conditions under which self-quenching is reduced. Different
quantities of the CCPs were then added to these solutions, and
the decrease in Fl emission, upon direct excitation, was
measured. Figure 7 shows that interaction with 2 gives rise to
the most efficient quenching with little or no recovery as more
polymer is added. More recovery occurs with the addition of 1
or 3, with 1 being intermediate in terms of net quenching and
in Figure 8. There is virtually no quenching observed for TR
upon interaction with polymer 3 under these conditions, and
only ∼15% loss of TR emission is observed upon complexation
with polymer 1. When compared to the data in Figure 7, the
quenching of acceptor dye emission induced by complexation
with the CCPs is less pronounced for TR relative to Fl.
Improved Energy Transfer Conditions. Results from FRET
studies between 3 and ssDNA-TR with the TR diluted within
3
providing the least perturbation. At æ ) 7, ∼25% loss in Fl
emission takes place for 3/ssDNA-Fl + ssDNA, while there is
more than 80% loss for 2/ssDNA-Fl + ssDNA under similar
conditions. Examination of the Fl emission in
the presence of CCPs reveals a similar shape upon direct
excitation or sensitization by the CCPs. Therefore, over 90%
of ssDNA-Fl is complexed with the CCPs, and the F/F0
-8
the total DNA content ([ssDNA-TR] ) 2 ×10 M + [ssDNA]
-7
)
1.8 ×10 M) are shown in Figure 9. Under these conditions,
self-quenching of TR is nearly nonexistent. An 18-fold enhance-
ment of the TR emission intensity is observed with [3] ) 4
-
6
×10 M, relative to direct excitation in the absence of 3.
Comparison of these results against the spectra in Figure 2,
J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006 1193

A R T I C L E S
Liu and Bazan
the rigid CCPs and the ssDNA. Additionally, from Figure 3,
the three CCPs are quenched to the same extent by ssDNA-Fl.
In other words, the chemical nature of the dyes at the terminus
of the ssDNA does not appear to influence the general
arrangement of the components so that vastly different optical
coupling occurs. While there are gaps in our knowledge of the
precise arrangement of optical partners in these aggregates,
differences in the sensitization of Fl or TR cannot be attributed
to different abilities of 1-3 to serve as FRET donors.
Direct excitation of the dyes within the CCP and ssDNA-C*
mixtures (where C* is either Fl or TR) shows that there is a
considerable decrease in the emission efficiency of the dyes,
relative to unbound ssDNA-C*. Figures 4 and 5 show the
magnitude of this quenching and that the CCP structure plays
a role in determining the extent of quenching. From these data,
one observes that the degree of quenching matches the dye
emission intensity upon CCP excitation, as shown in Figure 2.
That is, with 3, one observes best sensitization of Fl or TR and
also the least degree of dye quenching. Similarly, 1 is the
intermediate case, in both sensitization and quenching, and 2
gives least energy transfer and most effective quenching. By
monitoring the Fl intensity in CCP/ssDNA-Fl + ssDNA
mixtures at different æ values and [ssDNA-Fl]/[ssDNA] ratios,
as in Figure 6, one observes that the Fl emission recovers at
higher CCP/ssDNA ratios (æ > 2). This recovery is due to
diminished dye self-quenching as a result of the dilution within
the polyplex structures. Similar quenching behavior of dyes
attached to DNA upon condensation with polycations has been
observed by using single and dual color fluorescence fluctuation
Figure 9. Fluorescence spectra of 3/ssDNA-TR upon (a) excitation of 3
at 365 nm, (b) direct excitation of TR at 590 nm. Conditions: [ssDNA-
-
8
-7
-6
TR] ) 2 × 10 M, [ssDNA] ) 1.8 × 10 M, [3] ) 4 × 10 M.
where [ssDNA-TR] is similar to that in Figure 9, but only a
2-fold increase in emission is observed, highlights the improved
TR emission efficiency in the complex and the impact of TR
self-quenching on signal amplification. The net effect of dilution
is thus a 9-fold increase in TR emission intensity (upon
excitation of 3).
Discussion
Scheme 3 provides a straightforward access to water-soluble
cationic conjugated polymers (CCPs) with fluorene-co-phen-
ylene repeat units. Suzuki cross-coupling polymerization of the
key intermediate, 2,7-bis[9,9′-(6′′-bromohexyl)-fluorenyl]-
2
7
spectroscopies. However, Figure 6 shows that the recovery is
not complete, even at high levels of dilution. This latter
observation indicates that a fraction of dye quenching by
interaction with the CCP is also taking place.
4,4,5,5-tetramethyl-[1.3.2]dioxaborolane, with 1,4-dibromo-2,5-
substituted phenylene derivatives, affords the neutral CCP
precursors. The ionic versions 1-3 are produced in 83-89%
yields by treatment of the neutral polymers with excess
trimethylamine.
Examination of Fl or TR emission changes (F/F0) in ssDNA-
C*+ssDNA/CCP mixtures, upon direct excitation, as in Figures
7
and 8, leads to the following comments. First, the data further
While slight differences exist in the absorption maxima of
confirm that Fl is more effectively quenched than TR. Second,
the quenching of the dye by the CCP under conditions where
the dye is diluted with unlabeled ssDNA is dependent on CCP
structure. Finding the conditions where dye self-quenching is
eliminated is essential for elucidating this CCP/dye quenching
phenomenon. Polymer 3 is the least effective quencher. By using
1-3, their PL spectra, absorption coefficients, and PL quantum
yields are very close to each other. Furthermore, the three CCPs
have similar rigid backbone structures and molecular weights/
polydispersities, and similar transition moment orientations upon
interactions with a given acceptor site are expected. Therefore,
by taking into account eq 1, one would estimate nearly identical
donor capabilities for the three CCPs. However, Figure 2 shows
that upon excitation of 1 the emission intensities from Fl and
TR are similar, despite the considerably red-shifted absorption
band of TR. More striking is that excitation of 2 leads to more
intense emission by TR, relative to Fl. Therefore, consideration
of eq 1 does not provide a complete picture of the energy transfer
processes. Understanding the failure of eq 1 to predict FRET
efficiencies and the influence of polymer structure on the
emission output of the two acceptors provides the main
motivation for subsequent studies.
From the decrease in the CCPs’ PL intensities as a function
of [ssDNA] provided in Figure 3, one concludes that the three
structures show similar levels of self-quenching upon complex-
ation (or aggregation) with ssDNA. The exact sizes, shapes,
and orientation of the components in these polyplexes are
unknown at this stage but are likely to be dominated by the
structural attributes of the polyelectrolyte components, namely,
3
and taking into account that dye self-quenching can be reduced
by “diluting” ssDNA-TR with unlabeled DNA, it is possible to
find conditions where there is no quenching of TR emission,
relative to free ssDNA-TR. A similar success could not be
achieved with ssDNA-Fl.
Our efforts to understand how the polymer molecular structure
influences the optical performance of the dyes within the
polyplex structures begin by examination of the molecular
orbital energy levels of the optical partners and the background
provided in Scheme 1. The HOMO and LUMO energy levels
of the different molecular components were estimated using
cyclic voltammetry and absorption spectroscopy and are listed
in Table 2. Although we do not have the cyclic voltammetry
data of CCPs in water, the neutral precursors allow a relative
comparison of donor/acceptor orbital energy levels. Additionally
recent studies have shown that there is negligible difference in
the HOMO and LUMO levels of a neutral precursor and a
1194 J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006

Molecular Orbital Energies of Conjugated Polymers
A R T I C L E S
charged counterpart in the solid state.28 The difference in
oxidation and reduction potentials between the neutral polymers
in THF and the CCPs in water is thus estimated to be (0.1 V,
assuming that there is no coupled electron and proton transfer
between the CCPs and water.29
TR---TR self-quenching is operative, it is possible to unambigu-
ously rule out PCT.
Mechanistic insight can be applied to optimize solutions of
3 with ssDNA-TR so that an 18-fold signal amplification in
the TR emission can be obtained by FRET from 3, despite poor
spectral overlap. In the absence of TR dilution, only a 2-fold
enhancement is observed. The degree of enhancement is
considerably lower with 1 or 2, primarily because of the
underlying quenching of the dye emission by the polymers.
From Table 2 we note that CCP HOMO and LUMO energies
track the expected trend based on the electron releasing/
withdrawing properties of the substituents on the phenylene
comonomer.2 Fluorine substitution lowers the energy levels,
while the electron donating methoxy group raises the levels,
relative to the unsubstituted parent structure 1. The Fl LUMO
energy (-3.6 eV) is contained within the HOMO-LUMO gap
of the three polymers. However, the Fl HOMO energy (-5.9
eV) is lower than those of polymers 1 (-5.6 eV) and 2 (-5.4
eV). For these two structures it is reasonable to expect that
situation B in Scheme 1, i.e. PCT to the LUMO of Fl, is taking
place. For polymer 3, with a HOMO energy of -5.8 eV, the
situation is less clear and given the limitations of Scheme 1
both processes may be taking place to some extent. We point
out that there is ( 0.3 eV uncertainty on the average energy
level values and that a range of energy levels can be expected
in a sample, given the molecular weight distributions and ranges
of environment within the polyplex structures, i.e., interchain
delocalization.30 Despite these uncertainties, it is evident that
electron withdrawing substituents allow the Fl HOMO to be
better contained within the HOMO-LUMO gap of the π-con-
jugated system and give rise to a reduced driving force for
charge transfer. Indeed, this is the situation observed experi-
mentally. Excitation of polymer 3 provides the highest Fl
emission intensity, while polymer 2 is the least effective in this
0d
Conclusion
An improved access to cationic conjugated polymers (CCPs)
with fluorene-phenylene repeat units containing substituents that
modulate the electron density in the polymer backbone has been
developed. These materials were used to dissect the efficiency
of energy transfer to dye labeled DNA. We found that the three
CCPs under study are quenched to similar extent by Fl or TR.
For both dyes, we found that there is considerable self-quenching
upon complexation with the CCPs. This self-quenching can
be minimized upon “dilution” of ssDNA-C* with unlabeled
ss-DNA. The efficiencies of dye emission are more significantly
affected by the interactions with the CCPs, with Fl being more
effectively quenched than TR. Reduction of electron density
on the polymer backbone through substitution with electron
withdrawing fluorine leads to a reduction of the quenching
process. Examination of the molecular orbital energies deter-
mined by electrochemical and optical techniques and correlation
against the dependence of FRET and PCT on the relative
ordering of donor and acceptor orbital energies provides for a
possible rationale of the results. Taking into account these mech-
anistic considerations, it is possible to optimize the emission
of the reporter dye by reducing the total fraction of DNA that
is labeled with the reporter dye. Furthermore, it is interesting
to note that much of the previous sensitivities of CCP sensors
that operate by energy transfer to Fl-labeled bimolecular probes
can be considerably improved by utilizing less electronegative
dyes.
3
1
respect.
The TR HOMO level (-5.4 eV) is higher than those of 1
and 3 and is close in energy to the level of 2 (Table 1). In fact,
the HOMO-LUMO levels of TR are well contained between
the levels of 3, as in situation A in Scheme 1 which should
favor FRET over PCT. Additionally, that the energy gap
between TR and the three CPs is greater than that observed
with Fl should provide additional driving force for energy
transfer, relative to charge transfer.1
4c
Experimental Procedures
Direct dye excitation experiments provide complementary
information to the FRET experiments. That the Fl PL quantum
efficiency is considerably reduced (Figures 4 and 7), is consistent
with the analysis of orbital energies provided above and with
the possibility of PCT quenching, by hole transfer to the CCP
HOMO.13 Direct excitation of TR in CCP/ssDNA-TR poly-
plexes shows that the quenching is much less severe than for
Fl (Figures 5 and 8). When one examines the PL output of TR
when in the presence of polymer 3 under conditions where no
General Details. H and 13C NMR spectra were collected on a Varian
ASM-100 200 MHz spectrometer. The UV-Vis absorption spectra were
recorded on a Shimadzu UV-2401 PC diode array spectrometer.
Fluorescence was measured using a PTI (Lawrenceville, NJ) Quantum
Master fluorometer equipped with a xenon lamp excitation source and
a Hamamatsu (Japan) 928 PMT, using 90 degree angle detection for
solution samples. HPLC purified DNA oligonucleotides were obtained
from Sigma Genosys and the concentrations were determined using
1
2
(
60 nm absorbance measurements in 200 µL quartz cells in a Beckman
Fullerton, CA) DU800 spectrophotometer. All the reagents and
chemicals were obtained from Aldrich Co. and were used as received.
,7-dibromo-9,9-bis(6′-bromohexyl)fluorene was synthesized according
to the reported procedure.
(
28) Huang, F.; Wu, H.; Wang, D.; Yang, W.; Cao, Y. Chem. Mater. 2004, 16,
7
08.
2
(
29) Seidel, C. A.; Schulz, M.; Sauer, H. J. Phys. Chem. 1996, 100, 5541.
30) (a) Gebhardt, V.; Bacher, A.; Thelakkat, M.; Stalmach, U.; Meier, H.;
Schmidt, H. W.; Haarer, D. AdV. Mater. 1999, 11, 119. (b) Chang, R.;
Hsu, J. H.; Fann, W. S.; Liang, K. K.; Chang, C. H.; Hayashi, M.; Yu, J.;
Lin, S. H.; Chang, E. C.; Chuang, K. R.; Chen, S. A. Chem. Phys. Lett.
16b
(
Synthesis. 2,7-bis[9,9′-bis(6′′-bromohexyl)-fluorenyl]-4,4,5,5-tetra-
methyl-[1.3.2]dioxaborolan e. A 50 mL round-bottom flask was charged
with 9,9′-bis(6′-bromohexyl)-2,7-dibromofluorene (0.65 g, 1 mmol) in
2
000, 317, 142.
(
31) We reemphasize here that the framework we provide for understanding
the level of quenching of the dye by the different CCP structures is based
on the thermodynamic driving force for PCT. Possible kinetic differences
based on the acceleration of charge transfer with increasing driving force
within the normal region, as described by Marcus, are not included and
may bear an important contribution to the different levels of quenching.
See: (a) Creutz, C.; Keller, A. D.; Sutin, N.; Zipp, A. P. J. Am. Chem.
Soc. 1982, 104, 3618. (b) Dossot, M.; Allonas, X.; Jacques, P. Phys. Chem.
Chem. Phys. 2002, 4, 2989. (c) Sim, E. J. Phys. Chem. B 2005, 109, 11829
and ref 14b.
2
0 mL of dry THF and cooled to -78 °C with a dry ice/acetone bath.
At -78 °C, 2.5 eq. of t-BuLi in pentane (1.8 mL, 1.7 M) was added
drop by drop and the mixture was stirred for an hour. It was followed
immediately by adding 2-isopropoxy-4,4,5,5-tetramethyl-[1.3.2]-di-
oxaborolane (1.3 mL, 6 mol) in one shot. The resulting solution was
kept at -78 °C for additional 2 h. The resulting mixture was quenched
by water, and the solution was concentrated by rotary evap-
J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006 1195

A R T I C L E S
Liu and Bazan
oration and extracted with chloroform. The organic phase was separated
and dried over magnesium sulfate. After evaporation, the reside was
(50 MHz, CDCl
120.5, 118.1, 55.2, 40.3, 34.2, 32.8, 29.2, 27.9, 23.8.
3
): δ 158.4, 153.6, 151.3, 140.7, 133.8, 128.1, 123.6,
purified with silica gel column chromatography (CH
to yield 0.32 g (42%) of the product as white crystals. H NMR (400
MHz, CDCl ): δ 7.83-7.72 (m, 6H), 3.27-3.24 (t, J ) 6.8 Hz, 4H),
.03-1.99 (q, J ) 4.0 Hz, 4H), 1.64-1.57 (q, J ) 7.2 Hz, 4H), 1.39
s, 24 H), 1.17-1.13 (q, 4H), 1.06-1.02 (q, 4H), 0.55 (m, 4H). 13
NMR (100 MHz, CDCl ): δ 150.27, 144.09, 133.99, 128.93, 119.67,
2 2
Cl /hexane 1:3)
Poly[(9,9′-bis(6′′-(N,N,N,-trimethylammonium)-hexyl)fluorene-co-
alt-1,4-phenylene) dibromide] (1). Condensed trimethylamine (2 mL)
was added dropwise to a solution of the neutral polymer 1a (60 mg) in
THF (10 mL) at -78 °C. The mixture was allowed to warm to room
temperature. The precipitate was redissolved by the addition of water
1
3
2
(
C
3
(10 mL). After the mixture was cooled to -78 °C, extra trimethylamine
8
3.97, 55.24, 40.12, 34.19, 32.84, 29.15, 27.93, 23.55. MS(EI) m/z
(2 mL) was added and the mixture was stirred for 24 h at room
+
7
44 (M ).
temperature. After removing most of the solvent, acetone was added
Poly(9,9′-bis(6′′-bromohexyl)fluorene-co-alt-1,4-phenylene) (1a).
,7-Dibromo-9,9′-bis(6′′-bromohexyl)fluorene (325 mg, 0.5 mmol), 1,4-
1
to precipitate 1 (72 mg, 89%) as an off-white powder. H NMR (200
2
MHz, CD
3
OD): δ 8.0-7.8 (m, 10H), 3.3-3.2 (t, 4H), 3.1 (s, 18H),
phenyldiboronic acid (82.9 mg, 0.5 mmol), Pd(dppf)Cl
2
(7 mg) and
13
2
.3 (br, 4H), 1.6 (br, 4H), 1.3 (br, 8H), 0.8 (br, 4H). C NMR (50
potassium carbonate (830 mg, 6 mmol) were placed in a 25 mL round-
bottom flask. A mixture of water (3 mL) and THF (6 mL) was added
to the flask and the reaction vessel was degassed. The mixture was
refluxed at 85 °C for 24 h, and then precipitated into methanol. The
polymer was filtered and washed with methanol and acetone, and then
MHz, CD OD): δ 151.8, 140.9, 140.4, 140.0, 127.6, 126.1, 121.2,
3
1
20.5, 66.7, 55.7, 52.5, 40.2, 29.2, 25.8, 23.7, 22.5.
Poly[9,9′-bis(6′′-N,N,N-trimethylammonium)-hexyl)fluorene-co-alt-
,5-dimethoxy-1,4 phenylene) dibromide] (2). Condensed trimethyl-
2
amine (2 mL) was added dropwise to a solution of the neutral polymer
2a (70 mg) in THF (10 mL) at -78 °C. The mixture was allowed to
warm to room temperature. The precipitate was redissolved by the
addition of water (10 mL). After the mixture was cooled to -78 °C,
extra trimethylamine (2 mL) was added and the mixture was stirred
for 24 h at room temperature. After removing most of the solvent,
dried under vacuum for 24 h to afford 1a (220 mg, 78%), as an off-
1
white solid. H NMR (200 MHz, CDCl
4
3
): δ 7.8-7.5 (m, 10H), 3.3 (t,
H), 2.1 (m, 4H), 1.7 (m, 4H), 1.3-1.2 (m, 8H), 0.8 (m, 4H). 13C NMR
50 MHz, CDCl ): δ 151.9, 140.9, 140.7, 140.2, 128.1, 126.6, 121.8,
(
3
1
20.8, 55.7, 40.9, 34.5, 33.2, 29.6, 28.3, 24.2.
Poly(9,9′-bis(6′′-bromohexyl)fluorene-co-alt-2,5-dimethoxy-
,4-phenylene) (2a). 2,7-Bis[9,9′-bis(6′′-bromohexyl)-fluorenyl]-4,4,5,5-
acetone was added to precipitate 2 (80 mg, 83%) as an off-white
1
1
powder. H NMR (200 MHz, CD
3
OD): δ 7.8-7.6 (m, 8H), 3.9 (s,
tetramethyl-[1.3.2]dioxabor olane (372 mg, 0.5 mmol), 1,4-dibromo-
,5-dimethoxybenzene (148 mg, 0.5 mmol), Pd(PPh (5 mg) and
2
8
1
3
H), 3.3-3.2 (m, 4H), 3.1 (s, 18H), 2.2 (br, 4H), 1.6 (br, 4H), 1.3 (br,
2
3 4
)
H), 0.8 (br, 4H). 13C NMR (50 MHz, CD
3
OD): δ 152.5, 151.7, 141.6,
potassium carbonate (830 mg, 6 mmol) were placed in a 25 mL round-
bottom flask. A mixture of water (3 mL) and toluene (5 mL) was added
to the flask and the reaction vessel was degassed. The mixture was
vigorously stirred at 85 °C for 24 h and then precipitated into methanol.
The polymer was filtered and washed with methanol and acetone, and
38.7, 132.3, 129.6, 126.3, 120.8, 116.5, 67.8, 57.6, 56.4, 53.6, 31.0,
0.5, 27.0, 25.0, 23.7.
Poly[9,9′-bis(6′′-N,N,N-trimethylammonium)-hexyl)fluorene-co-alt-
2,5-difluoro-1,4-phenylene) dibromide] (3). Condensed trimethylamine
(2 mL) was added dropwise to a solution of the neutral polymer 3a
(60 mg) in THF (10 mL) at -78 °C. The mixture was allowed to warm
to room temperature. The precipitate was redissolved by the addition
of water (10 mL). After the mixture was cooled to -78 °C, extra
trimethylamine (2 mL) was added and the mixture was stirred for 24
h at room temperature. After removing most of the solvent, acetone
then dried under vacuum for 24 h to afford 2a (240 mg, 76%), as white
1
fibers. H NMR (200 MHz, CDCl
3
): δ 7.8-7.1(m, 8H), 3.8 (s, 6H),
3
4
1
.3-3.2 (t, 4H), 2.1 (m, 4H), 1.7 (m, 4H), 1.3-1.2 (m, 8H), 0.9 (m,
13
H). C NMR (50 MHz, CDCl ): δ 151.3, 150.7, 140.2, 137.2, 132.4,
3
28.3, 124.7, 119.7, 115.7, 57.2, 55.2, 40.4, 34.2, 33.0, 29.5, 28.1, 24.2.
Poly(9,9′-bis(6′′-bromohexyl)fluorene-co-alt-2,5-difluoro-1,4-
phenylene) (3a). 2,7-Bis[9,9′-bis(6′′-bromohexyl)-fluorenyl]-4,4,5,5-
tetramethyl-[1.3.2]dioxabor olane (372 mg, 0.5 mmol), 1,4-dibromo-
1
was added to precipitate 3 (74 mg, 84%) as a white powder. H NMR
(
1
200 MHz, CD
3
OD): δ 7.8-7.5 (m, 8H), 3.3-3.2 (m, 4H), 3.1 (s,
2
,5-difluorobenzene (136 mg, 0.5 mmol), Pd(PPh
)
3 4
(5 mg) and
13
8H), 2.2 (br, 4H), 1.6 (br, 4H), 1.3 (br, 8H), 0.8 (br, 4H). C NMR
potassium carbonate (830 mg, 6 mmol) were placed in a 25 mL round-
bottom flask. A mixture of water (3 mL) and toluene (5 mL) was added
to the flask and the reaction vessel was degassed. The mixture was
vigorously stirred at 85 °C for 24 h, and then precipitated into methanol.
The polymer was filtered and washed with methanol and acetone, and
(
1
3
50 MHz, CD OD): δ 159.8, 151.7, 154.9, 152.6, 142.4, 135.1, 129.4,
24.8, 121.6, 119.2, 67.8, 53.5, 50.4, 41.2, 31.0, 30.5, 27.1, 23.7.
Acknowledgment. This work was supported by a National
Institute of Health Grant (GM 62958-01), the NSF (DMR-
097611), and the Institute for Collaborative Biotechnologies.
then dried under vacuum for 24 h to afford 3a (221 mg, 73%), as white
0
1
fibers. H NMR (200 MHz, CDCl
4
3
): δ 7.8-7.2 (m, 8H), 3.3-3.2 (t,
H), 2.1 (m, 4H), 1.7 (m, 4H), 1.3-1.2 (m, 8H), 0.9 (m, 4H). 13C NMR
JA055382T
1196 J. AM. CHEM. SOC.
9
VOL. 128, NO. 4, 2006