Detection of CAG repeat DNA sequences by pyrene-functionalized pyrrole-imidazole polyamides

Article abstract of DOI:10.1016/j.bmc.2007.07.055

Five N-methylpyrrole-N-methylimidazole (Py-Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of conjugates 1-5 was examined in the presence and absence of (CAG)₁₂-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the conjugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at 475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concentration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon binding in the minor groove of DNA.

Full text of DOI:10.1016/j.bmc.2007.07.055

Bioorganic & Medicinal Chemistry 15 (2007) 6937–6942
Detection of CAG repeat DNA sequences by
pyrene-functionalized pyrrole-imidazole polyamides
Toshikazu Bando,* Jun Fujimoto, Masafumi Minoshima, Ken-ichi Shinohara,
Shunta Sasaki, Gengo Kashiwazaki, Masatoshi Mizumura and Hiroshi Sugiyama*
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan
Received 26 July 2007; accepted 30 July 2007
Available online 24 August 2007
Abstract—Five N-methylpyrrole-N-methylimidazole (Py–Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc
solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of con-
jugates 1–5 was examined in the presence and absence of (CAG)12-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the con-
jugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at
475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concen-
tration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon bind-
ing in the minor groove of DNA.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
specific sequences of duplex DNA for potential diag-
nostic applications.8–11
A number of chemical and biological studies have dem-
onstrated that N-methylpyrrole-N-methylimidazole
(Py–Im) polyamides precisely recognize each of the
four Watson–Crick base pairs.1,2 Sequence-specific
DNA recognition by Py–Im polyamides bound in the
minor groove depends on the sequence of side-by-side
aromatic amino acid pairings oriented in the amino-
carboxyl (N–C) direction with respect to the 50–30
direction of the DNA helix. An antiparallel pairing
of N-methylimidazole (Im) opposite N-methylpyrrole
(Py) (Im/Py) recognizes a GÆC base pair, whereas a
Py/Py pair recognizes TÆA and AÆT base pairs. A b-ala-
nine/b-alanine (b/b) pairing reads TÆA and AÆT pairs in
the same way as do Py/Py pairs. Thus, Py–Im polya-
mides can sequence-specifically recruit various func-
tional groups in DNA.3,4 We have developed various
types of sequence-specific alkylating Py–Im polyamides
and have investigated their chemical and biological
properties.5–7 In addition to such basic applications,
the sequence-specific binding properties of Py–Im
polyamides have been explored in the detection of
Fluorescent biosensors are powerful tools for probing
the sequence and structures of biomolecules. A typical
example of such probe systems is the molecular bea-
cons, which have proven to be of significant utility
in the detection of DNA sequences. With respect to
fluorescent biosensors, we are especially interested in
pyrene, which has been used for detecting DNA
sequences.12 Pyrene-based fluorescence exhibits large
extinction coefficients, excellent quantum yields, and
good stability in aqueous solution. Pyrene also forms
an excited state dimer, termed an excimer, with readily
detected emission that is red-shifted by about 100 nm
relative to the monomer. In spite of these useful prop-
erties, pyrene excimer emission has only been used in
applications for the detecting of single-strand DNA
sequences.13–18 Because formation of an excimer is
strongly distance- and geometry-dependent, specific
DNA binding of Py–Im polyamides could precisely
control excimer-based fluorescence. To develop a fluo-
rescence-based probe for the detection of repeated
DNA sequences in duplex form, we have designed a
novel Py–Im conjugate, which changes fluorescence
when bound in the minor groove of duplex DNA.
Here we report the molecular design of a pyrene exci-
mer-based Py–Im conjugate 1, synthesized by Fmoc
Keywords: Pyrene; Excimer; Py–Im polyamide; CAG repeat.
*
Corresponding authors. Tel.: +81 75 753 4002; fax: +81 75 753
3670; e-mail: hs@kuchem.kyoto-u.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.055

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T. Bando et al. / Bioorg. Med. Chem. 15 (2007) 6937–6942
solid-phase methods, which has potential in the field
of chemical biosensors for double-stranded DNA.
2. Results and discussion
2.1. Molecular design and synthesis
It has been clearly demonstrated that an expansion of
triplet repeat DNA sequences is associated with various
diseases. As a model, we examined techniques to detect
the CAG repeat sequence, which is known to be ex-
panded in Huntington disease.19,20 Py–Im polyamides
possessing more than five contiguous Py–Im rings show
poor binding to the minor groove of B-DNA because of
over-curvature compared with the minor groove of
DNA. By introducing b-alanine (b) as an aliphatic sub-
stitute for a Py ring in the hairpin polyamides, we take
advantage of the better flexibility of the b residue to
allow the crescent-shaped polyamide to match the
curvature of B-DNA.21 Accordingly, we have designed
and synthesized Py–Im polyamides targeting a seven-
base pair sequence, 50-AGCAGCA-30 (Fig. 1).
Scheme 1. Preparation of Fmoc-protected pyrenylbutyl-pyrrole mono-
mer for solid-phase synthesis.
The polyamides 1–5 were synthesized by Fmoc solid-
phase synthesis, starting with Fmoc-b-Ala-CLEAR-acid
resin and using Fmoc protected Py/Im monomers and
compound 9.22–24 After completion of the solid-phase
synthesis, the polyamides were cleaved from the resin
by aminolysis with 3-(dimethylamino)propylamine (Dp),
to produce the desired polyamides 1–5, in which 4-pyre-
nylbutyl groups were incorporated at the corresponding
N1-position of Py in the hairpin Py–Im polyamide.
After purification by HPLC, Py–Im polyamides 1–5
were obtained as a yellow powder. The purity of polya-
mides was >95% as judged by HPLC analysis. The
bis-pyrenyl conjugates 1–5 adopt a hairpin conforma-
tion in the DNA minor groove when complexed with
their target DNA duplex (Fig. 2).
We prepared 4-(9-fluorenylmethoxy carbonyl) amino-1-
pyrenylbutylpyrrole-2-carboxylic acid (compound 9) as
a monomeric unit to introduce a pyrene functionalized
Py residue to Py–Im polyamides. Compound 9 was syn-
thesized according to the five-step procedure shown in
Scheme 1. Methyl 4-nitro-1-(4-pyrenylbutyl)pyrrole-2-
carboxylate (compound 7) was prepared from the corre-
sponding starting material 6 in a mixture of 1-(4-bromo-
butyl)pyrene and potassium carbonate in the presence of
a small amount of tetrabutylammonium iodide. The
resultant compound 7 was converted to Boc-protected
pyrrole carboxylic acid (compound 8) by subsequent
reduction and protection using palladium-carbon and
(Boc)2O under hydrogen gas pressure, then hydrolysis
by LiOH. Under these conditions, a relatively high yield
of compound 8 was obtained without isolation of an O2-
sensitive amino pyrrole intermediate. The removal of the
t-Boc protecting group, followed by Fmoc protection of
compound 8, produced compound 9.
2.2. Emission of conjugates 1–5 in the presence of CAG
repeats
To evaluate the ability of conjugates to detect CAG re-
peat sequences, the fluorescence of conjugates 1–5 was
examined in the presence and absence of (CAG)12-con-
taining ODNs 1 and 2. In the presence of ODNs 1
and 2, conjugate 1 showed an increasing emission cen-
tered at 475 nm with increasing concentrations of ODNs
1 and 2 (Fig. 3). The dramatic increase of emission can
be explained by the formation of a pyrene excimer on
Figure 1. Molecular interaction model of Py–Im polyamides with
double-stranded DNA. The excimer-based probe for the detection of
the CAG repeat sequences is composed of two N-(1-pyrenyl) butyl
groups conjugated to a Py–Im polyamide. The pyrene moieties are
segregated in the absence of target (left). Upon target binding, excimer
formation occurs with consequent longer wavelength excimer emission
(475 nm) (right).
Figure 2. The molecular design of the DNA minor groove binder for
the CAG repeat sequences. Chemical structures of bis-pyrenyl Py–Im
polyamides 1–5.

T. Bando et al. / Bioorg. Med. Chem. 15 (2007) 6937–6942
6939
Figure 3. Fluorescence titration spectra of the conjugates 1–5 (10 lM) in a 5 mM Na phosphate buffer (pH 7 containing 7% v/v DMF) in the presence
of a target repeated ODN-1: 50-TA(CAG)12TTA-30/ODN-2: 50-AA(CTG)12TAA-30 (0–5 lM); kex = 345 nm.
binding of conjugate 1 to the minor groove of DNA.
Importantly, the pyrene excimer of conjugate 1 did not
form in the absence of DNA. In contrast, conjugates 2
and 4 showed excimer emission centered at 462 nm in
the absence of ODNs 1 and 2, and the intensity of exci-
mer fluorescence increased in a nonlinear fashion with
the amount of target ODNs. In the case of conjugates
3 and 5, fluorescence of the monomeric pyrene was
observed even in the presence of ODNs 1 and 2. The
results imply that, on binding to DNA, conjugate 1
changes its conformation to one in which two pyrene
units can form an excimer. Accordingly, conjugate 1
represents an ‘excimer-based’ bis-pyrene Py–Im polyam-
ide probe with fluorescence at 380 and 398 nm that shifts
to 475 nm after the conjugate binds to target oligonucle-
otides in the minor groove of double-stranded DNA.
contrast, excimer emission was weak using ODNs 7
and 8, without CAG repeat, and emission did not in-
crease with the amount of ODNs 7 and 8 used (Fig. 4c).
3. Conclusions
We have demonstrated that the excimer emission of Py–
Im polyamides is dramatically changed by the substitu-
tion of a pyrenylbutyl group in the hairpin. The emis-
sions of conjugate 1 excimer increased linearly with
the number of CAG repeats and the concentration of
ODNs containing CAG repeats, indicating the possible
application of conjugate 1 for the quantification of
CAG repeat DNA sequences.
Because conjugate 1, containing two pyrene units,
showed optimal excimer fluorescence on DNA binding,
we examined whether this excimer emission could be
used to quantify the number of CAG repeat sequences
(Fig. 4). Strong excimer emission of conjugate 1 was
observed with 4–12 CAG repeat sequences, as shown
in Figure 4b. The emission of conjugate 1 increased
linearly with the concentration of target ODNs. In
4. Experimental
4.1. General
Reagents and solvents were purchased from standard
suppliers and used without further purification.
Abbreviations of some reagents: HCTU, 1-[bis(dimethyl-
amino)methylene]-5-chloro-1H-benzotriazolium 3-oxide

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T. Bando et al. / Bioorg. Med. Chem. 15 (2007) 6937–6942
(ESI-TOF-MS) was produced on a BioTOF II (Bruker
Daltonics) mass spectrometer. DNA oligonucleotides
were purchased from Sigma–Aldrich Co. Fluorescence
measurements were performed on Spectrofluorometer
FP-6300 (JASCO). All machine-assisted polyamide syn-
theses were performed on Liberty Microwave Peptide
Synthesizer (CEM Co.) with a computer-assisted opera-
tion system at a 0.10 mmol scale (200 mg of CLEAR
resin, 0.52 meq/g) by using Fmoc solid-phase chemistry.
HPLC purification was performed with a Chemcobond
5-ODS-H reversed phase column (10 · 150 mm) in
0.1% AcOH with acetonitrile as eluent at a flow rate
of 6.0 mL/min, appropriate gradient elution conditions,
and detection at 254 or 345 nm. Methyl 4-nitropyrrole-
2-carboxylate 6 was prepared according to the published
procedure.25,26
4.2. General preparation of the Py–Im polyamides 1–5 by
Fmoc solid-phase synthesis
Typically, Fmoc-b-Ala-CLEAR-acid resin (200 mg,
0.10 mmol) was swollen in 3 mL of DMF in a 50-mL
plastic centrifuge tube for 30 min and placed on the
board of microwave synthesizer. DMF, DCM, 20%
piperidine in DMF, 20% acetic anhydride in DMF,
i
0.45 M HCTU in DMF, 2 M Pr2NEt in DMF were
placed in the external bottle, respectively. The amino
acids were weighted out in 4 molar excess amounts
and added DMF to get standard 0.2 M amino acid solu-
tion used on the instrument. Each monomer in DMF
solution was placed on the loading position of micro-
wave synthesizer, respectively. The synthesis was then
started, controlled by the computer using the established
program. The reactions were performed using deprotec-
tion steps of 180 s at 30 W (Tmax = 75 ꢁC) and coupling
steps of 300 s at 25 W (Tmax = 75 ꢁC) and a final capping
step of 120 s at 50 W (Tmax = 75 ꢁC). All couplings were
carried out with single-couple cycles. After the comple-
tion of the synthesis on the peptide synthesizer, the resin
was washed with four times DMF, methanol, and
DCM, respectively, and dried in a desiccator at room
temperature in vacuo.
Figure 4. (a) Sequences of ODNs 1–8. (b) Fluorescence titration
spectra of conjugate 1 (10 lM) in a 5 mM Na phosphate buffer (pH 7
containing 7% v/v DMF) in the presence of each ODNs (5 lM),
respectively. Black line shows spectra in the absence of ODNs. (c)
Fluorescence titration graph of conjugate1 ([1], 10 lM; [Na phos-
phate], 5 mm, 7% v/v DMF, at pH 7.0, kem = 475 nm) in the presence
of each ODN (0–5lM). The relative intensity was obtained by
subtracting the background level of emission of 1 in the absence of
ODNs at 475 nm. Error bars represent standard deviation of the
means of triplicate samples. kex = 345 nm.
4.2.1. AcImPy-b-ImPy*-c-ImPy*-b-ImPy-b-Dp (1). The
resin was then placed in a 10-mL glass scintillation vial,
3 mL of (dimethylamino)propylamine was added, and
the solution stirred at 55 ꢁC overnight. Resin was re-
moved by filtration through a pad of Celite and washed
thoroughly with DCM. The resultant filtrates were re-
moved in vacuo. The residue was dissolved in approxi-
mately 0.5 mL of DMF. The polyamide solution was
analyzed and purified by preparatory HPLC at
345 nm. Appropriate fractions were lyophilized to give
the final desired polyamide 1 (3.5 mg, 4% for 12 steps)
hexafluorophosphate; (Boc)2O, di-tert-butyl dicarbon-
ate; Fmoc-OSu, 9-fluorenylmethyl succinimidyl carbonate;
DMF, N,N-dimethylformamide; DCM, dichloromethane;
AcOEt, ethyl acetate; THF, tetrahydrofuran. NMR
spectra were recorded with a JEOL JNM-FX 400 nucle-
ar magnetic resonance spectrometer, and tetramethylsil-
ane was used as the internal standard. Proton NMR
spectra were recorded in parts per million (ppm)
downfield relative to tetramethylsilane. The following
abbreviations apply to spin multiplicity: s (singlet), d
(doublet), t (triplet), m (multiplet), br (broad). Electro-
spray ionization time-of-flight mass spectrometry
1
as light-yellow powder. H NMR (400 MHz, DMSO-
d6) d 10.30 (s, 2H, NH), 10.25 (s, 2H, NH), 9.88 (s,
4H, NH), 8.29–7.85 (m, 14H, NH+PyreneCH), 7.44 (s,
4H, CH), 7.30 (s, 2H, CH), 7.19 (s, 2H, CH), 6.92 (s,
2H, CH), 6.90 (s, 2H, CH), 4.36 (br s, 4H, CH2), 3.93
(s, 18H, NCH3), 3.92 (s, 6H, NCH3), 3.40 (m, 12H,
CH2), 3.03 (br s, 2H, CH2), 2.52 (br s, 2H, CH2), 2.32
(m, 6H, CH2), 2.14 (br s, 2H, CH2), 2.05 (s, 6H,
NCH3), 2.00 (s, 3H, COCH3), 1.85 (m, 4H, CH2), 1.70

T. Bando et al. / Bioorg. Med. Chem. 15 (2007) 6937–6942
6941
(m, 4H, CH2), 1.48 (m, 4H, CH2); ESI-TOF-MS m/e
Calcd for C102H111N26O13: [M+H]+ 1907.9. Found:
1908.0.
compound 8 (1.46 g, 88% yield for 2 steps) as a yellow
powder. 1H NMR (400 MHz, CDCl3) d = 8.13 (d,
J = 8.8 Hz, 1H), 8.06 (d, J = 7.6 Hz, 2H), 8.01–7.98
(m, 3H), 7.92 (s, 2H), 7.88 (t, J = 7.6 Hz, 1H), 7.74 (d,
J = 7.6 Hz, 1H), 7.16 (s, 1H), 6.63 (s, 1H), 6.17 (s,
1H), 4.22 (br t, 2H), 3.24 (t, J = 7.2 Hz, 2H), 1.88 (m,
2H), 1.77 (m, 2H), 1.42 (s, 9H); ESI-TOF-MS m/e Calcd
for C30H31N2O4 [M++H] 483.23. Found: 483.27.
A synthetic procedure similar to that used for the
synthesis of polyamide 1 was followed to prepare
polyamides 2–5, with yields of 2–4% for 12 steps as
light-yellow powder.
4.2.2. AcImPy*-b-ImPy-c-ImPy*-b-ImPy-b-Dp (2). ESI-
TOF-MS m/e Calcd for C102H111N26O13: [M+H]+
1907.9. Found: 1908.1.
4.2.8. 4-[(9-Fluorenylmethoxycarbonyl)amino]-1-(4-pyre-
nylbutyl)pyrrole-2-carboxylic acid (9). The reaction mix-
ture of compound 8 (1.46 g, 3.03 mmol) in 12 M HCl–
AcOEt (1:3, 60 mL) was stirred for 5 h at room temper-
ature. The precipitate was collected by filtration, washed
with diethyl ether, and dried to produce crude amine
(1.16 g) as a gray powder, which was used in the
next step without further purification. To a solution
of crude amine in water–DMF (1:1, 60 mL) was
added NaHCO3 (927 mg, 11.04 mmol) and Fmoc-OSu
(1.02 g, 3.04 mmol). The reaction mixture was stirred
for 5 h at rt under Ar gas. After the quenching by 5%
aq HCl, the precipitate was removed by filtration. The
solution was partitioned between AcOEt (200 mL) and
water (20 mL). The organic phase was separated,
washed with water (3· 20 mL), saturated NH4Cl
(20 mL), saturated NaCl (20 mL), dried over anhydrous
Na2SO4. After the filtration, the solution was concen-
trated to a residue, which was subjected to column chro-
matography (silica gel, 25% AcOEt in hexane) to
produce compound 9 (1.23 g, 65% yield for 2 steps) as
4.2.3. AcImPy*-b-ImPy-c-ImPy-b-ImPy*-b-Dp (3). ESI-
TOF-MS m/e Calcd for C102H111N26O13: [M+H]+
1907.9. Found: 1908.4.
4.2.4. AcImPy*-b-ImPy*-c-ImPy-b-ImPy-b-Dp (4). ESI-
TOF-MS m/e Calcd for C102H111N26O13: [M+H]+
1907.9. Found: 1908.4.
4.2.5. AcImPy-b-ImPy*-c-ImPy-b-ImPy*-b-Dp (5). ESI-
TOF-MS m/e Calcd for C102H111N26O13 [M+H]+
1907.9. Found: 1908.2.
4.2.6. Methyl 4-nitro-1-(4-pyrenylbutyl)pyrrole-2-carbox-
ylate (7). To a solution of 1-(4-bromobutyl)pyrene
(1.23 g, 3.64 mmol) in acetone (20 mL) was added
K2CO3 (0.75 g, 5.46 mmol) and Bu4NI (0.27 g,
0.72 mmol). After compound 6 (0.62 g, 3.64 mmol)
was added, the reaction mixture was refluxed for 11 h.
Evaporation of the solvent gave a brown residue, which
was collected by filtration, washed with water, 5% aq
HCl, then dried to produce 7 (1.46 g, 94% yield) as a
a
yellow powder. 1H NMR (400 MHz, CDCl3)
d = 8.12 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 7.6 Hz, 2H),
8.00–7.95 (m, 3H), 7.90 (s, 2H), 7.87 (t, J = 7.6 Hz,
1H), 7.71 (m, 3H), 7.53 (d, J = 7.2 Hz, 2H), 7.33 (br t,
2H), 7.25 (br t, 2H), 7.13 (s, 1H), 6.66 (s, 1H), 6.34 (s,
1H), 4.42 (d, J = 6.4 Hz, 2H), 4.23 (m, 3H), 3.24 (br t,
2H), 1.87 (m, 2H), 1.76 (m, 2H); ESI-TOF-MS m/e
Calcd for C40H33N2O4: [M++H] 605.24. Found: 605.30.
1
brown powder. H NMR (400 MHz, CDCl3) d = 8.16–
8.09 (m, 3H), 8.04 (d, J = 8.8 Hz, 2H), 7.96 (s, 2H),
7.93 (t, J = 7.6 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.49
(d, J = 2.0 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H), 4.30 (t,
J = 7.2 Hz, 2H), 3.75 (s, 3H), 3.32 (t, J = 7.2 Hz, 2H),
1.89 (m, 2H), 1.84 (m, 2H); ESI-TOF-MS m/e Calcd
for C26H23N2O4: [M++H] 427.17. Found: 427.19.
4.3. Steady state fluorescence measurements of DNA
fragments containing CAG/CTG repeats sequences
4.2.7. 4-[(tert-Butoxycarbonyl)amino]-1-(4-pyrenylbutyl)pyr-
role-2-carboxylic acid (8). To a solution of compound
7 (1.46 g, 3.43 mmol) in DMF (25 mL) was slowly
Fluorescence spectra were obtained on a Spectrofluo-
rometer FP-6300 (JASCO) with an excitation
wavelength of 345 nm in hybridization buffer, and oligo-
nucleotides at corresponding concentrations of
conjugates 1–5. No special efforts were made to remove
oxygen from the reaction solution at room temperature.
i
added 10% Pd–C (440 mg). After Pr2NEt (1.84 mL,
10.30 mmol) and (Boc)2O (1.18 mL, 5.14 mmol) were
added dropwise, the reaction mixture was stirred for
5 h at room temperature under hydrogen gas pressure.
The catalyst was removed by filtration through celite
using AcOEt (200 mL). The organic phase was sepa-
rated, washed with water (3· 20 mL), saturated NaCl
(20 mL), dried over anhydrous Na2SO4. After the filtra-
tion, the solvent was evaporated in vacuo to produce
crude amine, which was used in the next step without
further purification. To a solution of crude amine in
water–THF (1:2, 60 mL) was added LiOH monohydrate
(2.9 g, 68.60 mmol). The reaction mixture was refluxed
for 28 h and quenched by acetic acid (4.5 mL), then
AcOEt (200 mL) was added. The organic phase was sep-
arated, washed with water (3· 20 mL), saturated NaCl
(20 mL), dried over anhydrous Na2SO4. After the filtra-
tion, the solvent was evaporated in vacuo to produce
Acknowledgment
This work was supported by a Grant-in-Aid for Priority
Research from the Ministry of Education, Culture,
Sports, Science, and Technology, Japan.
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