Design and synthesis of new cationic water-soluble pyrene containing dendrons for DNA sensory applications

Article abstract of DOI:10.1002/pola.25031

A series of new water-soluble cationic pyrene-dendron derivatives, G1, G2, and G3, was successfully synthesized and characterized. These new dendrons were designed with the quaternized amino moieties at the periphery of the dendrons for DNA detection and functionalized with pyrene as a fluorescent probe. The electrostatic interactions between the plasmid DNA (pDNA) and cationic charged dendrons in an aqueous solution resulted in a change in the photophysical properties of pyrene, which could be shown in the UV-vis and fluorescence spectra. Pyrene dendrons showed a high and rapid fluorescence response upon the addition of pDNA, which was strongly dependent on the size and hydrophobicity of the dendrons.

Full text of DOI:10.1002/pola.25031

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Design and Synthesis of New Cationic Water-Soluble Pyrene Containing
Dendrons for DNA Sensory Applications
Ching-Yi Chen,1y Yumiko Ito,2y Yu-Cheng Chiu,3 Wen-Chung Wu,4 Tomoya Higashihara,2
Mitsuru Ueda,2 Wen-Chang Chen1,3
1Institute of Polymer Science and Engineering, National Taiwan University, Taipei 10617, Taiwan
2Department of Organic and Polymeric Materials, Graduate School of Science and Engineering, Tokyo Institute of Technology,
Tokyo 152-8552, Japan
3Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan
4Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
Correspondence to: M. Ueda (E-mail: ueda.m.ad@m.titech.ac.jp) or W.-C. Chen (E-mail: chenwc@ntu.edu.tw)
Received 10 August 2011; accepted 29 September 2011; published online 22 October 2011
DOI: 10.1002/pola.25031
ABSTRACT: A series of new water-soluble cationic pyrene-den-
dron derivatives, G1, G2, and G3, was successfully synthesized
and characterized. These new dendrons were designed with the
quaternized amino moieties at the periphery of the dendrons for
DNA detection and functionalized with pyrene as a fluorescent
probe. The electrostatic interactions between the plasmid DNA
(pDNA) and cationic charged dendrons in an aqueous solution
resulted in a change in the photophysical properties of pyrene,
which could be shown in the UV-vis and fluorescence spectra. Py-
rene dendrons showed a high and rapid fluorescence response
upon the addition of pDNA, which was strongly dependent on the
C
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size and hydrophobicity of the dendrons. 2011 Wiley Periodi-
cals, Inc. J Polym Sci Part A: Polym Chem 50: 297–305, 2012
KEYWORDS: dendrons; DNA; polyamide; pyrene; sensor; water-
soluble polymers
INTRODUCTION Dendrimers are well-defined, three-dimen-
sional hyperbranched macromolecules with a large number of
terminal groups that can be easily utilized for introducing
functionalities at the periphery of the dendrimers. Such a
structural specificity has received great interests as new poly-
meric materials for applications in the fields of molecular light
harvesting,1 catalysts,2 liquid crystals,3,4 molecular encapsula-
tion,5,6 and drug delivery systems.7–10 Dendrimers containing
fluorescent components not only facilitate the detailed investi-
gation of the self-assembly process and molecular interaction
in the dendrimers, but also extend their applications in
fluorescence-based sensing materials.11–15 Pyrene is a good
candidate as a fluorescent probe because its fluorescence
properties are well known and highly sensitive to the micro-
environment.16,17 In addition, it has a strong tendency to form
excimers via intermolecular p–p stacking, which exhibit a
broad and structure-less fluorescent emission red-shifted with
respect to that of monomeric pyrene. In the past decades, sev-
eral pyrene-labeled dendrimers have been developed by a
noncovalent incorporation of pyrene into the cavities of the
dendrimers, or covalent attachment of pyrene to the core or
periphery of the dendrimers.18–26 However, few of them have
been reported for use in sensory applications.21
For sensory applications, electrostatic interactions between
cationic moieties and negatively charged analyte targets (e.g.,
DNA or RNA) are commonly coordinated when designing flu-
orescence-based sensors. Various cationic fluorescence-based
sensors, such as cationic amphiphilic molecules,27,28 cationic
conjugated polymers,29–31 or cationic charged dendrimers,12
have been designed and synthesized for DNA or RNA detec-
tion. Recently, we also reported the aligned electrospun
nanofibers formed from cationic polyfluorene that showed
an enhanced sensitivity to plasmid DNA (pDNA).32 Although
cationic fluorescent conjugated polymers have been proven
to have a high sensitivity due to the excitation energy trans-
fer mechanism resulting in amplification of the fluorescent
signal, their rigid conformation led to its poor response to
various secondary structures naturally present in biological
macromolecules, such as the double helices of DNA.33 In
addition, the uncontrolled molecular weight variation of con-
jugated polymers caused reproduction of their sensitivity dif-
ficult with different batches of conjugated polymers. Den-
drimers have more conformational freedom and precise
molecular structures that allows them to be more suitable
for DNA detection. However, the synthesis of cationic
†Ching-Yi Chen and Yumiko Ito contributed equally to this work.
Additional Supporting Information may be found in the online version of this article.
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2011 Wiley Periodicals, Inc.
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SCHEME 1 Chemical structures of G1, G2, and G3 dendrons.
dendrimers might require a tedious multistep procedure
with a repetitive protection–deprotection and purification
process for each generation. Recently, we demonstrated the
simple and rapid synthesis of dendritic polyamides and suc-
cessfully obtained third generation dendrimers.34–37
Instrumentation
1H NMR spectra were recorded in deuterated dimethylsulfox-
ide (DMSO-d6) or chloroform (CDCl3) on a BRUKER DPX-300
spectrometer at 300 MHz. Infrared spectra were recorded on a
Horiba FT-720 spectrophotometer. Matrix-assisted laser
desorption ionization with time of flight (MALDI-TOF) mass
spectra were recorded on a Kratos Kompact MALDI instru-
ment operated in linear detection mode to generate positive
ion spectra using dithranol as a matrix, Tetrahydrofuran (THF)
as a solvent, sodium trifluoroacetate as an additive agent. Size
exclusion chromatography (SEC) was performed on a Jasco
GULLIVER 1500 system equipped with two polystyrene gel
columns (Plgel 5-mm MIXED-C) eluted with CHCl3 at a flow
rate of 1.0 mL minꢁ1 calibrated by standard polystyrene sam-
ples. Absorption and photoluminescence spectra were meas-
ured with a Jasco V-550 spectrophotometer. Transmission
electron microscopy (TEM) images were obtained with a Phi-
lips TEM (CM 100) instrument operating at a voltage of 80 kV
with a Morada CCD camera. The samples of G1–G3 dendrons
were prepared with concentration of 100 lM.
Hence, we now report the synthesis and investigation of a
different generation of new cationic charged dendrons with
functional pyrene as a fluorescent probe (G1, G2, and G3
dendrons) for sensing DNA molecules (Scheme 1).
The quaternized amino moieties at the periphery of den-
drons were designed for favorable electrostatic attraction
with negatively charged nucleic acids. The number of
charged moieties, the distance between the cationic groups
and fluorescent units, as well as the hydrophobicity of the
dendron generation were also examined to reveal their sensi-
tivity to a pDNA, since it has been reported that the binding
affinity of the cationic molecules to pDNA was strongly de-
pendent on the structures of the cationic molecules.38,39
EXPERIMENTAL
Synthesis
Materials
The pyrene-dendrons (G1, G2, and G3) were synthesized by
a divergent method as shown in Schemes 2 and 3.
N-Methyl-2-pyrrolidinone (NMP) was distilled under reduced
pressure from calcium hydride, and then stored under nitro-
gen. Triethylamine (TEA) was distilled from calcium hydride
under nitrogen, and then stored under nitrogen. Diphenyl(2,3-
dihydro-2-thioxo-3-benzoxazolyl)phosphonate (DBOP) was
supplied from KYOCERA Chemical Corporation and recrystal-
lized from hexane, then stored under nitrogen in a refrigera-
tor. The pDNA molecules (base pair: 700) used for studying
the responsive properties of the pyrene-dendrons were pur-
chased from Sigma (Milwaukee). The stock solutions of pDNA
were prepared by dissolving certain amount of solid pDNA in
double distilled water and stored at 4 ꢀC in the dark. Other
reagents and solvents were obtained commercially and used
as received unless otherwise noted.
Preparation of 3-Bromo-propyl-1-NHBoc (2)40
1M NaOH aq. (10 mL) was added to a solution of 3-bromo-
propylamine (1.00 g, 4.56 mmol) and di-tert-butyl dicarbon-
ate (0.945 g, 4.10 mmol) in dioxane/water (20 mL/10 mL).
The mixture was stirred at room temperature for 1 h, and
then diluted with ethyl acetate and water. The organic layer
was washed successively with 1N HCl aq., 5 wt % of NaHCO3
aq., and brine, and then dried with MgSO4. After filtration,
the solvent was removed under reduced pressure. The prod-
uct was obtained as colorless oil (0.898 g, 92% yield). 1H
NMR (CDCl3, d, ppm): 1.44 (s, 9H), 2.05 (triplet–triplet
appearing as a quintet, 2H, J ¼ 6.6 Hz), 3.27 (triplet–doublet
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appearing as a quartet, 2H, J ¼ 6.6 Hz), 3.45 (t, 2H, J ¼ 6.6
as a quintet, 4H, J ¼ 6.9 Hz), 3.20 (t, 4H, J ¼ 6.9 Hz), 4.19 (t,
4H, J ¼ 6.9 Hz), 6.79 (s, 1H), 7.38 (s, 2H), 8.04–8.40 (m, 9H),
Hz), 4.67 (s, 1H).
10.68 (s, -CONH-, 1H). Anal. calcd for
C33H31F6N3O7
Preparation of Protected-AB2 Building Block (3)
1.08H2O: C, 55.43; H, 4.67; N, 5.88. Found: C, 55.20; H, 4.54;
N, 6.11.
To a mixture of compound 2 (3.63 g, 15.0 mmol) and K2CO3
(2.07 g, 15.0 mmol) in DMF (10 mL) was added methyl 3, 5-
dihydorxybenzoate (0.84 g, 5.00 mmol) at room temperature
under nitrogen. The reaction mixture was stirred at 60 ꢀC
overnight, and then poured into water. The precipitate was
filtered and dried under reduced pressure to give a white
solid (2.24 g, 93% yield). M.p. 123–124 ꢀC. 1H NMR (CDCl3,
d, ppm ): 1.44 (s, 18H), 1.99 (triplet–triplet appearing as a
quintet, 4H, J ¼ 6.6 Hz), 3.32 (triplet–doublet appearing as a
quartet, 4H, J ¼ 6.6 Hz), 3.90 (s, 3H), 4.04 (t, 4H, J ¼ 6.6 Hz)
4.73 (s, 2H), 6.64 (t, 1H), 7.17 (d, 2H).
Synthesis of Protected-Second Generation Dendrons
(protected-G2)
To a solution of 4 (0.539 g, 1.15 mmol) in NMP (5 mL) were
added DBOP (0.422 g, 1.10 mmol) and TEA (0.154 mL, 1.10
mmol) under nitrogen. The reaction solution was stirred at
room temperature for 1 h. Then, G1 (0.347 g, 0.500 mmol)
and TEA (0.280 mL, 2.00 mmol) were added to the solution
and the solution was stirred at room temperature for 6 h.
The reaction solution was poured into water, and the precip-
itate was collected and dried. The crude product was dis-
solved in methanol (100 mL) and water (25 mL) was added
to this solution. The precipitate was collected and dried in
vacuo at 80 ꢀC to give a gray powder (0.636 g, 93% yield).
M.p. 118–121 ꢀC. IR (KBr, cmꢁ1): 1164 (Ar-O-alkyl), 1689
(C¼¼O, amide, and carbamate), 2935, 2973 (Ar-H), 3313,
3355 (NAH, amide, and carbamate). 1H NMR (DMSO-d6, d,
Preparation of AB2 Building Block (4)41
A mixture of compound 3 (3.38 g, 7.00 mmol) and KOH
(0.550 g, 9.80 mmol) in methanol/water (42 mL/14 mL)
was refluxed for 2 h. The reaction solution was cooled to
room temperature and acidified with acetic acid. Then, the
organic layer was diluted with ethyl acetate and washed
with water three times, and dried over MgSO4. After filtra-
tion, the solvent was removed under reduced pressure to
ꢀ
ppm, 40 C): 1.36 (s, 36H), 1.81 (triplet–triplet appearing as
1
a quintet, 8H, J ¼ 6.8 Hz), 2.03 (triplet–triplet appearing as a
quintet, 4H, J ¼ 6.8 Hz), 3.07 (triplet–doublet appearing as a
quartet, 8H, J ¼ 6.8 Hz), 3.45 (triplet–doublet appearing as a
quartet, 4H, J ¼ 6.8 Hz), 3.99 (t, 8H, J ¼ 6.8 Hz), 4.16 (t, 4H,
J ¼ 6.8 Hz), 6.59 (s, 2H), 6.69–6.80 (m, 5H), 7.00 (s, 4H),
7.35 (s, 2H), 8.03–8.37 (m, 9H), 8.43 (t, 2H, J ¼ 5.0 Hz), 10.7
(s, 1H). Anal. calcd for C75H97N7O17: C,65.82; H, 7.14; N,
7.16. Found: C, 65.38; H,6.93; N,7.05. MALDI-TOF MS: Calcd.:
[M]þ ¼ 1367.7, Found: [MþNa]þ ¼ 1389.5.
ꢀ
afford a white solid (2.85 g, 87% yield). M.p. 128–129 C. H
ꢀ
NMR (DMSO-d6, d, ppm, 40 C): 1.37 (s, 18H), 1.82 (triplet–
triplet appearing as a quintet, 4H, J ¼ 6.5 Hz), 3.08 (triplet–
doublet appearing as a quartet, 4H, J ¼ 6.5 Hz), 4.00 (t, 4H,
J ¼ 6.5 Hz), 6.69 (t, 1H, J ¼ 2.2 Hz), 6.77 (s, 2H), 7.04
(d, 2H, J ¼ 2.2 Hz).
Synthesis of Protected-First Generation Dendron
(protected-G1)
To a solution of 4 (1.48 g, 3.15 mmol) and 1-aminopyrene
(0.652 g, 3.00 mmol ) in NMP (3 mL) were added DBOP
(1.21 g, 3.15 mmol) and TEA (0.440 mL, 3.15 mmol) under
nitrogen. The reaction solution was stirred at room tempera-
ture for 3 h, and then poured into water. The precipitate was
collected, washed with methanol, and dried in vacuo at 80
ꢀC to give a gray powder (1.60 g, 80% yield). M.p. 161–162
ꢀC. IR (KBr, cmꢁ1): 1180 (Ar-O-alkyl), 1585 (C¼¼O, amide
and carbamate), 3039, 3058 (Ar-H), 3421, 3532 (NAH, am-
Synthesis of Second Generation Dendron (G2)
Protected-G2 (0.550 g, 0.402 mmol) was dissolved in 5 mL
of TFA and stirred for 1.5 h. The solvent was evaporated to
dryness and ether was added. The precipitate was collected
and dried in vacuo at 80ꢀC to give a gray powder (0.544 g,
95% yield). IR (KBr, cmꢁ1): 1172 (Ar-O-Alkyl), 1203 (C-F),
1592 (C¼¼O, carboxylate), 1677 (C¼¼O, amide), 2700–3200
(NAH, ammonium), 3370, 3432 (NAH, amide). 1H NMR
ꢀ
1
ꢀ
(DMSO-d6, d, ppm, 40 C): 1.94–2.12 (m, 12H), 2.96 (t, 8H, J
ide, and carbamate). H NMR (DMSO-d6, d, ppm, 40 C): 1.38
(s, 18H), 1.89 (triplet-triplet appearing as a quintet, 4H, J ¼
6.5 Hz), 3.13 (triplet–doublet appearing as a quartet, 4H, J ¼
6.5 Hz), 3.22 (s, 3H), 4.10 (t, 4H, J ¼ 6.5 Hz), 6.74 (t, 1H, J ¼
2.0 Hz), 6.80 (s, 2H), 7.32 (d, 2H, J ¼ 2.0 Hz), 8.05- 8.35 (m,
9H), 10.65 (s, ACONHA, 1H). Anal. calcd for C39H45N3O7: C,
70.14; H, 6.79; N, 6.29. Found: C, 70.04; H, 6.68; N, 6.37.
¼ 6.6 Hz), 3.46 (triplet–triplet appearing as a quintet, 4H, J
¼ 6.8 Hz), 4.08 (t, 8H, J ¼ 6.6 Hz), 4.16 (t, 4H, J ¼ 6.8 Hz),
6.65 (s, 2H), 6.77 (s, 1H), 7.07 (s, 4H), 7.36 (s, 2H) 8.04-
8.36 (m, 9H), 8.48 (t, 2H, J ¼ 4.9 Hz) 10.70 (s, ACONHA,
1H). Anal. calcd for C63H69F12N7O17 1.18H2O: C, 52.35;
H,4.98; N, 6.78. Found: C, 51.89; H, 4.90; N, 7.24.
Synthesis of First Generation Dendron (G1)
Synthesis of Protected-Third Generation Dendron
(protected-G3)
Protected-G1 (0.500 g, 0.749 mmol) was dissolved in tri-
fluoroacetic acid (TFA) (5 mL) and stirred at room tempera-
ture for 1.5 h. The solvent was evaporated to dryness and
ether was added. The precipitate was collected and dried in
vacuo at 80 ꢀC to give a gray powder (0.485 g, 93% yield).
M.p. 176–185 ꢀC. IR (KBr, cmꢁ1): 1176 (Ar-O-Alkyl), 1203
(C-F), 1592 (C¼¼O, carboxylate), 1677 (C¼¼O, amide), 2700–
3200 (NAH, ammonium), 3404, 3432 (NAH, amide). 1H
DBOP (0.248 g, 0.644 mmol) and TEA (90.0 lL, 0.644 mmol)
were added to a solution of 4 (0.316 g, 0.672 mmol) in NMP
(1 mL) under nitrogen and the solution was stirred at room
temperature for 1 h. To this solution were added G2 (0.200
g, 0.140 mmol) and TEA (0.240 mL, 1.68 mmol) and the so-
lution was stirred at room temperature overnight. The reac-
tion solution was poured into water, and the precipitate was
collected and dried. The crude product was dispersed in
ꢀ
NMR (DMSO-d6, d, ppm, 40 C): 2.07(triplet–triplet appering
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SCHEME 2 Synthesis of AB2 building block (4).
methanol (20 mL), and water (5 mL) was added to this mix-
ꢀture. The precipitate was collected and dried in vacuo at 80
C to give a gray powder (0.345 g, 89% yield). IR (KBr,
cmꢁ1): 1164 (Ar-O-alkyl), 1693 (C¼¼O, amide and carba-
mate), 2935, 2973 (Ar-H), 3343 (NAH, amide and carba-
mate). H NMR (DMSO-d6, d, ppm, 40 C): 1.25 (s, 72H), 1.71
(triplet–triplet appearing as a quintet, 16H, J ¼ 6.6 Hz),
1.79–1.99 (m, 12H), 2.97 (triplet–doublet appearing as a
quartet, 16H, J ¼ 6.6 Hz), 3.21–3.40 (m, 12H), 3.87 (t, 16H, J
¼ 6.6 Hz), 3.94 (t, 8H, J ¼ 6.3 Hz), 4.06 (t, 4H, J ¼ 6.6 Hz),
6.48 (s, 4H), 6.52 (s, 2H), 6.56–6.71 (m, 7H), 6.88 (s, 8H),
6.94 (s, 4H) 7.92–8.24 (m, 9H), 8.24–8.39 (m, 6H), 10.6 (s,
1H). Anal. calcd for C147H261N15O37: C, 63.73; H,7.31; N,7.58.
Found: C,63.40; H,7.25; N,7.59. MALDI-TOF MS: Calcd.: [M]þ
¼ 2768.4, Found: [MþNa]þ ¼ 2790.5.
monitored. The fluorescence spectra were excited at 340 nm
for G1 and G2 and 347 nm for G3. No obvious influence on
both UV-vis and fluorescence spectra was observed due to
the limited water volume added.
1
ꢀ
RESULTS AND DISCUSSION
Synthesis of AB2 Building Block and Dendrons
Scheme 2 shows the synthetic route for an AB2 building
block. Compound 2 was prepared according to a previous
paper.40 Methyl 3,5-dihydroxybenzoate reacted with 2 in the
presence of K2CO3 to yield a protected AB2 building block
precursor (3), which was converted into an AB2 building
block (4) by the hydrolysis of the methyl ester group of 3.
1
The H NMR spectrum of the AB2 building block 4 is shown
in the supporting information (Fig. S1). All peaks are well
assigned to the corresponding structure.
Synthesis of Third Generation Dendron (G3)
Protected-G3 (0.260 g, 0. 0939 mmol) was dissolved in TFA
(5 mL) and stirred at room temperature for 1.5 h. The sol-
vent was evaporated to dryness and ether was added. The
The dendrons were grown from the 1-aminopyrene core by a
divergent approach using DBOP as the condensing agent.42
Coupling reactions for the synthesis of the protected-G1, G2,
and G3 were conducted by a two-step method36 consisting of
(i) the activation of a carboxylic acid moiety of the AB2 build-
ing block by DBOP to generate an active amide moiety and
(ii) the condensation of the active amide with 1-aminopyrene,
G1 or G2. The tert-butyl carbamate group of the protected-G1,
G2, and G3 was deprotected with TFA to afford the cationic
charged dendrons (G1, G2, and G3) (Scheme 3). It should be
noted that this new synthetic method is the first example of
the synthesis of hemi-aliphatic polyamide dendrons by two-
step method using DBOP. All products were characterized by
ꢀ
precipitate was collected and dried in vacuo at 80 C to give
a gray powder (0.254 g, 94% yield). IR (KBr, cmꢁ1): 1172
(Ar-O-Alkyl), 1203 (C-F), 1592 (C¼¼O, carboxylate), 1681
(C¼¼O, amide), 2700–3200 (NAH, ammonium), 3440 (NAH,
amide). 1H NMR (DMSO-d6, d, ppm, 40 ꢀC): 1.88–2.12 (m,
28H), 2.96 (t, 16H, J ¼ 6.7 Hz), 3.36–3.52 (m, 12H), 4.00–
4.20 (m, 28H), 6.60–6.66 (m, 6H), 6.78 (s, 1H), 7.04 (s, 12H),
8.03–8.34 (m, 9H), 8.42–8.55 (m, 6H), 10.70 (s, -CONH-, 1H).
Anal. calcd for C123H145F24N15O37 2.39 H2O: C,50.51; H, 5.16;
N,7.18. Found: C,50.07; H, 5.09; N, 7.63.
1
Preparation of G1–G3 Solutions and pDNA Sensing
Studies
IR, H NMR spectroscopy, and elemental analysis. The IR spec-
trum of G3 showed strong absorptions at 3440 and 1681
cmꢁ1 due to the characteristic NAH and C¼¼O stretchings of
the amide groups, respectively. Furthermore, the characteristic
carboxylate and ether stretching were observed at 1592 and
1172 cmꢁ1, respectively (Fig. S2). The 1H NMR spectrum of
G3 showed signals corresponding to the amide protons (f, l,
and r) at 8.42–8.55 and 10.69 ppm, pyrene protons (s) at
8.04–8.40 ppm, and alkyl protons of the end unit (a) at 2.96
ppm, respectively (Fig. 1).
The sample solutions were prepared by dissolving certain
amount of pyrene-dendrons in double distilled water with
ꢀ
gentle shaking and stored at 4 C in the dark before titration.
For the pDNA titration, the sample solutions were prepared
to 5 lM. To 2 mL of sample solution was added 0.5- to 5-lL
aliquots of pDNA stock solutions. Upon each addition, the
solution was stirred for 2 min to reach equilibrium. UV-vis
spectrum and fluorescence spectrum were subsequently
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SCHEME 3 Synthesis of first–third generation water-soluble polyamide dendrons (G1, G2, and G3).
1
The H NMR spectra of G1 and G2 are shown in the support-
ing information (Fig. S3). Moreover, Figure 2 shows the
MALDI-TOF MS spectra of the protected-G2 and G3, which
exhibited peaks observed at M/Z ([MþNa]þ) ¼ 1389.5 and
2790.5 and well agreed with the calculated mass (1389.7 and
2790.4), together with minor peaks, which could be assignable
to partially deprotected dendrons. The deprotection probably
occurred during the measurement. Moreover, no peaks derived
from dendrons having defect structures were observed in Fig-
ure 2. The SEC curves for protected-G1, G2, and G3 showed
quite narrow polydispersities (Fig. S4). These findings clearly
indicated the formation of the target dendrons.
mation of the dendron/pDNA complex. Although the pyrene
absorption peak of G2 kept increasing during the addition of
pDNA, the pyrene absorption peak of G3 increased up to a
15 mM pDNA solution addition and then became saturated.
This is probably because the larger molecular size and more
charged terminal groups of G3 compared with the G2 ones
induced a higher affinity with pDNA and formed a stiffer
aggregation. The formation of the G3/pDNA complex aggre-
gation did not change with the further addition of pDNA.
Photophysical Properties of pDNA Sensibility
The sensing ability of this new series of cationic charged
dendrons as a function of the pDNA molar concentration
was investigated in an aqueous solution. Figure 3 shows the
UV-vis spectra of G1, G2, and G3 with various concentrations
of pDNA in an aqueous solution. The dendron concentration
was fixed at 5.0 lM. The absorption peak attributed to py-
rene at 340 nm was observed in the UV-vis spectra of G1,
but it gradually decreased and red-shifted when pDNA was
added to the G1 aqueous solution [Fig. 3(A)]. The absorption
spectra of G2 and G3 showed the absorption peak of pyrene
at 347 nm and slightly red-shifted to 350 nm with the
increasing pDNA concentration [Fig. 3(B,C)]. The red shift
due to pyrene groups absorption may be caused by the for-
FIGURE 1 1H NMR spectrum of third generation dendron (G3).
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on the emission of the pDNA/dendrons in detail, the fluores-
cence intensities for the G1–G3 dendrons are plotted versus
the various ratios of the pDNA/dendrons (Fig. 5).
As shown in the figure, the G1–G3 dendrons exhibited a dif-
ferent fluorescence tendency and maximum emission inten-
sities at the different pDNA/dendrons charge ratios. Initially,
FIGURE 2 MALDI-TOF-MS spectrum of (A) protected-second
generation dendron (protected-G2), and (B) protected-third
generation dendron (protected-G3).
The fluorescence emission spectra of the G1, G2, and G3 den-
drons with the increasing pDNA concentration are shown in
Figure 4. The emission at 385 nm corresponds to the mono-
meric pyrene emission. Without pDNA, G1 showed a strong
excimer fluorescence at 442 nm due to intermolecular p–p
stacking, however, no excimer emission was observed for G2
and G3. This is attributed to the large dendron size and
more highly charged groups of G2 and G3 that resulted in
the increased steric hindrance and charge repulsion to form
the pyrene excimer. When the pDNA concentration increases,
G1 gradually showed a decrease in the fluorescent emission
from the pyrene excimer. However, G2 and G3 initially
showed an enhancement of the fluorescent emission, and
then a decrease in its fluorescence intensity with an increase
in the pDNA concentration, which is different from the
behavior of the G1/pDNA complexes. This result indicates
that the excimer formation of pyrene might be strongly de-
pendent on the size and hydrophobicity of the dendrons
because G2 and G3 have a larger size and more hydrophobic
units than G1.27 In addition, the formation of the G2/pDNA
and G3/pDNA complexes reduces the electrostatic repulsion
between the cationic charges of G2 and G3, which gives rise
to the formation of more pyrene excimers. To illustrate the
effect of the ratios of the positive charge to negative charge
FIGURE 3 UV-vis spectra of (A) G1, (B) G2 and (C) G3 as a
function of added pDNA.
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decreasing after the addition of pDNA, suggesting that the
pyrene excimer formation was disrupted by the electrostatic
interactions between the negatively charged pDNA and posi-
tively charged amino groups in the dendrons. When excess
pDNA was added (charge ratio > 1) to the G2 aqueous solu-
tion, the fluorescence intensities decreased and reached satu-
ration. This might be because an excessive amount of pDNA
reduces the number of dendrons attached to each pDNA
strand and thus prevents formation of the pyrene excimers.
On the other hand, G3 showed only a slight decrease in the
fluorescence intensities after the charge ratio of 1. G3, which
possesses a greater number of charged terminal groups
probably has a stronger interaction with pDNA and formed
aggregates during the addition. The aggregates might not col-
lapse with the further addition of pDNA. These results agree
with the results of UV-VIS spectra [Fig. 3(B,C)].
Morphologies of Self-assembled Pyrene Dendrons and
pDNA Complexes
The morphologies of G1–G3 dendrons and dendron/pDNA
complexes were also investigated by TEM with a fixed con-
centration of dendrons and the pDNA/dendrons ratio of 3.0.
(Fig. 6). Without addition of pDNA, interesting morphologies
of G1–G3 dendrons are observed. G1 dendron showed the
aggregation of the sphere-like nanostructures with diameter
around 20–60 nm [Fig. 6(A)]. G2 and G3 dendrons exhibit
entangled fibrous network with widths around 20–100 nm
for G2 and 15–80 nm for G3, respectively [Fig. 6(B,C)]. On
the other hand, when addition of the pDNA into the dendron
solutions, the large lamellar-like sheet of G1/pDNA com-
plexes is observed in Figure 6(D), however, the small lamel-
lar aggregations of G2/pDNA and G3/pDNA complexes are
shown in Figure 6(E,F), respectively. The morphologies of
the dendron/pDNA complexes are significantly different from
FIGURE 4 Fluorescence spectra of (A) G1, (B) G2, and (C) G3
as a function of added pDNA.
with the increasing pDNA/dendrons ratios, pDNA plays a
role in either the disruption or helps with the formation of
the pyrene excimers, which gradually decrease or increase
the emission intensities of dendrons. This result also indi-
cates the interactions between the pDNA and cationic den-
drons are significantly dependent on the cationic charged
moieties and hydrophobicity of the dendron generation.
Actually, the fluorescent emission intensities of G1 kept
FIGURE 5 Fluorescence emissions at 442 nm for the G1 den-
dron and 472 nm for G2 and G3 dendrons with concentration
of 5 lM and various ratios of the pDNA base pairs (negative
charge)/dendron cations in aqueous solution.
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FIGURE 6 TEM images of self-assembled (A–C) G1–G3 dendrons, and (D–F) G1–G3 dendrons/pDNA complexes, respectively.
those of G1–G3 dendrons, which indicated the existence of
electrostatic interactions between the negatively charged
pDNA and positively charged amino groups in the dendrons.
The larger lamellar aggregation of G1/pDNA complexes com-
pared to those of G2/pDNA and G3/pDNA complexes might
be attributed to the number of the cationic charged moieties
in the dendron. Since G1 has the less cationic charges, the
more G1 may interact on each pDNA strand. In addition, G2
and G3 with large size may reduce the number of dendrons
attached to each pDNA strand due to steric hindrance effect
and prevent the formation of large aggregates.
The fluorescence responses of the pyrene-dendrons upon
addition of pDNA were strongly dependent on the size,
hydrophobicity and number of cationic charges of the den-
drons. The TEM images of dendron/pDNA complexes also
demonstrated the existence of electrostatic interactions
between the negatively charged pDNA and positively charged
amino groups in the dendrons.
The financial supports of this work from National Science
Council, The Ministry of Economics Affairs, and the Excellence
Research Program of National Taiwan University are highly
appreciated. Y. I. thanks Japan Society of the promotion of Science
(JSPS, #2208196) for financial supports. Supporting Information
is available online from Wiley InterScience or from the author.
CONCLUSIONS
We have designed and successfully synthesized a new series
of water-soluble cationic pyrene-dendrons, G1, G2, and G3.
The UV-vis and fluorescence spectral investigation demon-
strated that the electrostatic interactions between the pDNA
and cationic charged dendrons in an aqueous solution
caused a change in the photophysical properties of pyrene
due to the formation or disassembly of the pyrene excimers.
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