DNA Alkylation by Pyrrole−Imidazole seco-CBI Conjugates
A R T I C L E S
SCIEX) and BioTOF II (Bruker Daltonics) mass spectrometer. Poly-
acrylamide gel electrophoresis was performed on a HITACHI SQ5500-S
and HITACHI SQ5500-E DNA sequencer. PCR amplification was
carried out with an iCycler (BIO-RAD). Ex Taq DNA polymerase was
purchased from Takara Co.; the Thermo Sequenase core sequencing
kit and loading dye (formamide with fuschin red) were from Amersham
Co. Ltd.; 5′-Texas Red-modified DNA oligomer (20-mer) was from
Proligo Co. Ltd.; and 50% Long Ranger gel solution was from FMC
bioproducts. Surface plasmon resonance (SPR) assays were performed
on a BIACORE X system (Sweden), and processing of data was carried
out using BIAevalution version 4.1. Biotinylated hairpin DNAs were
obtained from Proligo and used without further purification. HBS-EP
buffer (0.01 M HEPES, pH 7.4, containing 0.15 M NaCl, 3 mM EDTA,
and 0.005% Surfactant P20) and sensor chip SA were purchased from
BIAcore AB (Sweden).
(s, 1H; NH), 10.46 (s, 1H; OH), 8.12 (d, J ) 7.2 Hz, 1H; CH), 7.96 (s,
1H; NH), 7.85 (d, J ) 8.0 Hz, 1H; CH), 7.71 (s, 1H; CH), 7.59 (d, J
) 8.0 Hz, 1H; CH), 7.53 (t, J ) 7.2 Hz, 1H; CH), 7.37 (t, J ) 8.0 Hz,
1H; CH), 7.29 (s, 1H; NH), 7.22 (d, J ) 8.0 Hz, 1H; CH), 4.80 (t, J
) 10.0 Hz, 1H; CH), 4.54 (d, J ) 12.0 Hz, 1H; CH), 4.24 (brt, 1H;
CH), 4.01 (dd, J ) 8.0 Hz, 3.2 Hz, 1H; CH), 3.87 (dd, J ) 7.6 Hz, 3.6
Hz, 1H; CH), 3.33 (s, 2H; NH
2
). ESI-TOF-MS m/z calcd for C22
H
18
-
+
ClN O
3 2
[M + H] 392.11, found 392.15.
AcImImPy-â-ImPy-Indole-seco-CBI (4). To a solution of com-
i
pound 1 (5.0 mg, 6.7 µmol) in DMF (150 µL) were added Pr NEt
2
(2.3 µL, 1.3 µmol) and HATU (2.6 mg, 6.7 µmol). The reaction mixture
was stirred for 2 h at room temperature. After the conversion from 1
i
to activated ester was confirmed by HPLC and ESIMS analysis, Pr -
2
NEt (2.3 µL, 13 µmol) and 3′ (5.2 mg, 13 µmol) were added to the
reaction vessel. The reaction mixture was stirred overnight at room
Solid-Phase Synthesis of Py-Im Polyamides. AcImImPy-â-
2
temperature under N atmosphere. Evaporation of the solvent gave a
ImPyCO
2
H (1). AcImImPy-â-ImPyCO
2
-oxime resin was synthesized
yellow residue by filtration, which was washed with chloroform (2 µL
× 2) and water (2 µL × 2) and was subjected to column chromatog-
in a stepwise reaction by Fmoc solid-phase protocol. A sample of resin
was cleaved with 6 mL of 1 N aqueous NaOH/DMF (1/1, v/v) for 1 h
at 55 °C, and purified by HPLC using a Chemcobond 5-ODS-H column
2 2
raphy (silica gel 5-10% MeOH in CH Cl , gradient elution) to produce
compound (3.4 mg, 3.0 µmol, 45%) as a yellow powder. 4 was used
1
(
0.1% AcOH/CH
3
CN 0-100% linear gradient, 0-30 min, 254 nm),
6
in the DNA alkylation reaction. H NMR (400 MHz, DMSO-d ): δ
1
to produce 1 (20.0 mg, 26.8 µmol, 27%) as a yellow powder. H NMR
11.67 (s, 1H; NH), 10.43 (s, 1H; OH), 10.29 (s, 2H; NH), 10.26 (s,
1H; NH), 9.96 (s, 1H; NH), 9.82 (s, 1H; NH), 9.32 (s, 1H; NH), 8.11
(d, J ) 8.0 Hz, 1H; CH), 8.07 (brs, 1H; NH), 7.96 (s, 1H; CH), 7.85
(d, J ) 8.0 Hz, 1H; CH), 7.55 (s, 1H; CH), 7.52 (m, 1H; CH), 7.50 (s,
2H; CH), 7.47 (s, 1H; CH), 7.43 (d, J ) 9.6 Hz, 1H; CH), 7.36 (t, J
) 8.0 Hz, 1H; CH), 7.32 (s, 1H; CH), 7.20 (s, 1H; CH), 7.17 (s, 1H;
CH), 6.97 (s, 1H; CH), 4.80 (t, J ) 10.0 Hz, 1H; CH), 4.54 (d J )
(
(
1
400 MHz, DMSO-d
s, 1H; NH), 10.01 (s, 1H; NH), 9.32 (s, 1H; NH), 8.08 (t, J ) 5.2 Hz,
H; NH), 7.55 (s, 1H; Im-H), 7.50 (s, 1H; Im-H), 7.45 (s, 1H; Im-
H), 7.44 (s, 1H; Py-H), 7.22 (s, 1H; Py-H), 6.96 (s, 1H; Py-H),
6
): δ 10.30 (s, 1H; NH), 10.27 (s, 1H; NH), 10.26
6
3
.92 (s, 1H; Py-H), 3.99 (s, 3H; NCH
H; NCH ), 3.81 (s, 3H; NCH ), 3.80 (s, 3H; NCH
), 2.58 (t, J ) 7.2 Hz, 2H; CH ), 2.03 (s, 3H; COCH
ESI-TOFMS m/z calcd for C32
[M + H]+ 745.28, found
3
), 3.97 (s, 3H; NCH
), 3.44 (q, J ) 6.4
).
3
), 3.93 (s,
3
3
3
Hz, 2H; CH
2
2
3
3
12.0 Hz, 1H; CH), 4.22 (brt, 1H; CH), 3.99 (s, 3H; CH ), 3.97 (s, 6H;
H
36
N O
14 8
CH
CH
3
), 3.87 (s, 3H; CH
), 2.58 (t, J ) 7.2 Hz, 2H; CH
3
), 3.81 (s, 3H; CH ), 3.45 (q, J ) 6.4 Hz, 2H;
3
7
45.27.
2
2
), 2.03 (s, 3H; COCH
3
). ESI-TOF-
+
MS m/z calcd for C54
H52ClN17O
9
[M + H] 1118.38, found 1118.65.
2
AcImPyPy-â-ImPyCO H (2). A synthetic protocol similar to that
used for the preparation of compound 1 was followed to prepare
compound 2 (26.3 mg, 35.4 µmol, 35%), which was confirmed by
A synthetic procedure similar to that used for the preparation of
compound 4 was followed to prepare compound 5.
HPLC and ESI-TOFMS analysis. ESI-TOFMS m/z calcd for C33
H
37
N
13
O
8
AcImPyPy-â-ImPy-Indole-seco-CBI (5). A synthetic procedure
similar to that used for the preparation of compound 4 was followed
to prepare compound 7, with a yield of 53% for one step from 2. After
purification by column chromatography (silica gel 5-10% MeOH in
+
[M + H] 744.29, found 744.32.
BocHN-Indole-seco-CBI. To a solution of H
2
N-indole-CO
(1/1, v/v) was
O (553 mg, 2.54 mmol), and the reaction mixture was stirred
2
H (157
mg, 0.892 mmol) in 1.6 mL of DME/aqueous NaHCO
added Boc
3
CH
2
Cl
2
, gradient elution), 5 was used in the DNA alkylation reaction.
2
+
for 10 h at room temperature. After completion of the reaction
confirmed by HPLC analysis, the solution was concentrated and washed
ESI-TOFMS m/z calcd for C55
H53ClN16
O
9
[M + H] 1117.39, found
1117.57.
by 10% aqueous hydrochloric acid. Boc-NH-indole-CO
as a purple powder (271 mg, 0.98 mmol, quant). Next, to a solution of
Boc-NH-indole-CO H in DMF was added seco-CBI (245 mg, 1.05
mmol) with EDCI (764 mg, 4.00 mmol) and NaHCO (336 mg, 4.00
mmol). The reaction mixture was stirred overnight at room temperature
under N atmosphere. After the completion of the reaction was
2
H was obtained
AcImImPyPy-â-PyPyPy-â-Dp (6). AcImImPyPy-â-PyPyPy-â-
CLEAR resin was synthesized in a stepwise reaction by Fmoc solid-
phase protocol. A sample of resin was cleaved with 3-(dimethylamino)-
1-propylamine for 10 h at 55 °C, and purified by HPLC using a
2
3
Chemcobond 5-ODS-H column (0.1% AcOH/CH CN 0-100% linear
3
2
gradient, 0-30 min, 254 nm), to produce 6 (2.9 mg, 2.5 µmol, 2.5%)
confirmed by HPLC analysis, the solution was concentrated and washed
with 1% aqueous hydrochloric acid. The product was obtained as a
brown solid (493 mg, 1.00 mmol, quant). H NMR (400 MHz, DMSO-
as a yellow powder.
After further purification by HPLC, 6 was used in the DNA
1
1
alkylation reaction. H NMR (400 MHz, DMSO-d
6
): δ 10.32 (s, 1H;
d
6
): δ 11.63 (s, 1H; NH), 10.42 (s, 1H; OH), 9.16 (s, 1H; NH), 8.13
NH), 10.29 (s, 1H; NH), 9.92 (s, 1H; NH), 9.89 (s, 1H; NH), 9.88 (s,
1H; NH), 9.87 (s, 1H; NH), 9.32 (s, 1H; NH), 8.05 (t, J ) 5.2 Hz, 1H;
NH), 7.97 (t, J ) 5.2 Hz, 1H; NH), 7.86 (t, J ) 5.2 Hz, 1H; NH), 7.59
(s, 1H; Im-H), 7.50 (s, 1H; Im-H), 7.28 (s, 2H; Py-H), 7.22 (s, 1H;
Py-H), 7.19 (s, 2H; Py-H), 7.14 (s, 1H; Py-H), 7.02 (s, 1H; Py-H),
6.87 (s, 1H; Py-H), 6.85 (s, 1H; Py-H), 6.82 (s, 1H; Py-H), 4.00 (s,
(d, J ) 8.0 Hz, 1H; CH), 7.95 (s, 1H; CH), 7.84 (d, J ) 8.0 Hz, 1H,
CH), 7.79 (s, 1H; CH), 7.51 (t, J ) 8.0 Hz, 1H; CH), 7.37 (s, 1H;
CH), 7.34 (d, J ) 8.0 Hz, 1H; CH), 7.31 (t, J ) 9.2 Hz, 1H; CH), 7.10
(s, 1H; CH), 4.77 (t, J ) 10.0 Hz, 1H; CH), 4.30 (d, J ) 12.0 Hz, 1H;
CH), 4.21 (brt, 1H; CH), 4.00 (dd, J ) 8.0 Hz, 3.2 Hz, 1H; CH), 3.85
(dd, J ) 7.6 Hz, 3.6 Hz, 1H; CH), 1.48 (s, 9H; CH
3
). ESI-TOF-MS
[M + H] 492.16, found 492.31
3H; NCH
NCH ), 3.81 (s, 3H; NCH
3.04 (q, J ) 6.4 Hz, 2H; CH
J ) 7.2 Hz, 2H; CH ), 2.19 (t, J ) 7.2 Hz, 2H; CH
NCH ), 2.04 (s, 3H; COCH ) 1.51 (t, J ) 7.2 Hz, 2H; CH
TOFMS m/z calcd for C53
3
), 3.97 (s, 3H; NCH
), 3.79 (s, 3H; NCH
), 2.66 (t, J ) 7.2 Hz, 2H; CH
), 2.10 (s, 6H;
). ESI-
10 [M + H] 1143.53, found 1143.75.
3
), 3.84 (s, 3H; NCH
), 3.46 (m, 4H; CH
), 2.31 (t,
3
), 3.83 (s, 6H;
+
m/z calcd for C27
H26ClN O
3 4
3
3
3
2
),
H
2
N-Indole-seco-CBI (3). To a solution of BocHN-indole-CO
115 mg, 0.35 mmol) in 1 mL of CH Cl was added 1 mL of TFA/
O (10/1, v/v), and the reaction mixture was stirred for 1 h at room
2
H
2
2
(
H
2
2
2
2
2
3
3
2
+
temperature. After completion of the reaction was confirmed by HPLC
analysis, the solution was concentrated and washed with 10% aqueous
hydrochloric acid. Compound 3 was obtained as a black powder (82
mg, 0.35 mmol, quant), which was used in the next coupling step
without further purification. H NMR (400 MHz, DMSO-d
66 20
H N O
AcImImPy-â-ImPyPyPy-â-Dp (7). AcImImPy-â-ImPyPyPy-â-
CLEAR resin was synthesized in a stepwise reaction by Fmoc solid-
phase protocol. A sample of resin was cleaved with 3-(dimethylamino)-
1-propylamine for 10 h at 55 °C, and purified by HPLC using a
1
6
): δ 12.00
J. AM. CHEM. SOC.
9
VOL. 129, NO. 17, 2007 5389