Sequence-Specific Alkylation of Human Sequence
A R T I C L E S
(
MSNT) were dissolved in 2 mL of DCM followed by 217 µL of
cleanup kit (Sigma Aldrich). The 5′-Texas Red labeled DNA fragments
were alkylated by various concentrations of alkylation polyamides in
10 µL of 5 mM sodium phosphate buffer (pH 7.0) containing 10%
DMF at 25 °C for 20 h. The reaction was quenched by the addition of
calf thymus DNA (1 mM, 1 µL) and by heating the mixture for 30
min at 95 °C. The DNA was recovered by vacuum centrifugation. The
pellet was dissolved in 5 µL of loading dye (formamide with fuschsin
red), heated at 95 °C for 30 min, and then immediately placed on ice.
The 2 µL aliquot was subjected to electrophoresis on a 6% denaturing
polyacrylamide gel using a Hitachi SQ5500-E DNA Sequencer.
Analysis of Growth Inhibition against 39 Human Cancer Cell
Lines. The human cancer cell lines that were composed of five breast
cancer (HBC-4, BSY-1, HBC-5, MCF-7, MDA-MB-231), six brain
cancer (U251, SF-268, SF-295, SF-539, SNB-75, SNB-78), colon
cancer (HCC2998, KM-12, HT-29, HCT-15, HCT-116), seven lung
cancer (NCI-H23, NCl-H226, NCI-H522, NCI-H460, A5449,
DMS273, DMS114), one melanoma (LOX-IMVI), five ovarian cancer
(OVCAR-3, OVCAR-4, OVCAR-6, OVCAR-8, SK-OV-3), two renal
cancer (RXF-631L, ACHN), six stomach cancer (St-4, MKN1, MKN7,
MKN28, MKN45, MKN74), and two prostate cancer (DU-145, PC-3)
were all plated at an appropriate density in 96-well plates in RPMI1640
(sigma) containing 10% heat-inactivated fetal bovine serum (Life
Technologies), penicillin (100 U/mL), and streptomycin (100 µg/mL).
The cells were maintained at 37 °C in humidified atmosphere of 95%
N-methylimidazole (NMI) and added to 400 mg of CLEAR-acid resin
and 2 mL of DMF. The coupling was allowed to proceed for 10 h at
room temperature. The resin was then acethylated with 166 µL of acetic
anhydride (Ac
AcPyPyPy-γ-ImIm-CO
Fmoc solid-phase methods on the Im-coupled CLEAR-acid resin with
Fmoc-Py-CO H and Fmoc-Im-CO H monomers. A peptide can be
cleaved from CLEAR-acid resin to yield primary carboxylic acid with
acidic condition (TFA/dimethyl sulfide/triisopropylsilane/H O ) 91:
:3:3, 30 min, room temperature), and purified by HPLC using a
2
O), 4.3 mg of DMAP, and 3 mL of DMF for 1 h.
2
H was synthesized in a stepwise reaction by
2
2
2
3
Chemcobond 5-ODS-H column (0.1% AcOH/CH CN 0-100% linear
gradient, 0-30 min, 254 nm), to produce 7 (2.4 mg, 7%) as a yellow
powder. H NMR (400 MHz, DMSO-d
(
3
1
6
): δ 10.38 (s, 1H; NH), 9.88
s, 1H; NH), 9.87 (s, 1H; NH), 9.80 (s, 1H; NH), 9.60 (s, 1H; NH),
.01 (s, 1H; NH), 7.62 (s, 1H; CH), 7.51 (s, 1H; CH), 7.22 (s, 1H;
8
CH), 7.16 (s, 1H; CH), 7.13 (s, 1H; CH), 7.03 (s, 1H; CH), 6.87 (s,
1
3
2
H; CH), 6.85 (s, 1H; CH), 3.95 (s, 3H, NCH
.84 (s, 3H; NCH ), 3.82 (s, 3H; NCH ), 3.79 (s, 3H; NCH
H, J ) 8.0 Hz; CH ), 2.35 (t, 2H, J ) 7.2 Hz; CH ), 1.96 (s, 3H;
). ESI-TOF-MS m/e: calcd for
3
), 3.92 (s, 3H; NCH
3
),
3
3
3
), 3.19 (t,
2
2
COCH
C
3
) 1.78 (qu, 2H, J ) 9.2 Hz; CH
2
+
34
H
40
N O
13 8
[M + H] , 758.3; found, 758.4.
NH
carboxylate (870 mg, 4.2 mmol) in CH
commercial available 1-methyl-2-trichloroacetyl-4-nitroimidazole (1.2
2
-Im-Indole-CO
2
H (8). To a solution of 5-aminoindole-2-
2
Cl (50 mL) was added
2
1
4
2
air and 5% CO . The cells were exposed to drugs for 48 h. The cell
i
15
g, 4.2 mmol) and Pr
2
NEt (1.5 mL, 8.4 mmol), and the reaction mixture
atmosphere to
produced compound 8 (210 mg, 0.64 mmol). H NMR (400 MHz,
DMSO-d ): δ 11.68 (s, 1H; NH), 9.75 (s, 1H; NH), 8.10 (s, 1H; CH),
.48 (dd, 1H, J ) 2.1, 10.0 Hz; CH), 7.36 (d, 1H, J ) 8.2 Hz; CH),
.03 (s, 1H; CH), 6.51 (s, 1H; CH), 3.89 (s, 3H; CH ), 2.88 (s, 2H;
).
growth was determined according to the sulforhodamine B assay.
was stirred for 18 h at room temperature under an H
2
Absorbance for the control well (C), the treated well (T) and the treated
1
well at time 0 (T
0
) were measured at 525 nm. The 50% growth inhibition
)/(C - T )] ) 50. The mean
(IC50) was calculated as 100 × [(T - T
0
0
6
7
7
NH
graphs, which show the differential growth inhibition of the drugs in
the cell line panel, were drawn on the basis of a calculation using a set
3
16
of IC50
. Pearson correlation coefficients (r) were calculated using the
2
following formula:
Preparation of DNA Fragments Containing Four Repeats of the
Human Telomere Sequence. All DNA fragments and primers for
cloning or DNA amplification were purchased from Proligo. The DNA
fragments were annealed in a final volume of 20 µL containing 50 µM
of fragment set (5′-GCAGAGTGAGGGTTAGGGTTAGGGTTAGG-
GTTACAAGCCCTCA-3′, 3′-ACGTCTCACTCCCAATCCCAATCC-
CAATCCCAATGTTCGGGAG-5′). Products were identified by sepa-
ration in TBE (10% native polyacrylamide gel with 0.5 µg/mL ethidium
bromide using low weight DNA marker [New England BioLabs]) and
visualization under UV illumination. The annealed fragments were
ligated into pGEM-T easy vectors (Promega). Escherichia coli DH5R
competent cells (Toyobo) were transformed and cultured on LB plates
with 100 µg/mL ampicillin and 32 µg X-gal /400 µg IPTG overnight
at 37 °C. White colonies were identified by colony direct PCR in 20
µL of the reaction mixtures containing 250 nM of primer set (T7: 5′-
TAATACGACTCACTATAGGG-3′, sp6: 5′-GATTTAGGTGACAC-
TATAG-3′), 200 µM of deoxynucleotide triphosphates (Sigma Aldrich),
2
2 1/2
r ) (S(x - x )(y - y ))/(S(x - x ) S(y - y ) )
i
m
i
m
i
m
i
m
i i
where x and y are log IC50 of drug A and drug B, respectively, against
each cell line, and x
respectively.
Effects for Normal Human Cell Lines. The human normal cell
lines used in this study were MRC-5, NHSF46, and WI-38, which are
derived from male embryonic lung fibroblast, skin fibroblast, and female
embryonic lung fibroblast, respectively. All normal cell lines were
obtained from the RIKEN Cell Bank. MRC-5 and NHSF46 cells were
maintained in MEM-alpha (Gibco), and WI-38 cells were grown in
Dulbecco’s modified Eagle’s medium (Sigma) at 37 °C in a humidified
m m i i
and y are the mean values of x and y ,
2
atmosphere of 5% CO in 95% air, and both of the media were
supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/
mL penicillin, and 100 mg/mL streptomycin. The growth inhibiting
effects against these normal cell lines were assessed as above.
2
units taq DNA polymerase, and 1x ThermoPol reaction buffer (New
England Bio Labs). Amplification cycles were carried out with an
iCycler (BIO-RAD). The reaction mix was incubated at 95 °C for 5
min then followed by 30 incubation cycles of 95 °C for 30 s, 55 °C for
Acknowledgment. This work was supported by a Grant-in-
Aid for Priority Research from the Ministry of Education,
Culture, Sports, Science, and Technology, Japan.
3
0 s, and 72 °C for 30 s, with a final extension step of 72 °C for 7
min. The appropriate colony was selected for transfer to 5 mL of LB
medium with 100 µg/mL ampicillin and cultured overnight at 37 °C.
The plasmids with inserts were extracted using GenElutePlasmid
miniprep kit (Sigma Aldrich) and identified by PCR (program and
reaction mixtures same as above).
(
14) (a) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica,
D.; Hose, C.; Langley, J.; Cronise, P.; Vai-gro-Wolff, A.; Gray-Goodrichi,
M.; Campbell, H.; Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
(
b) Yamori, T.; Sato, S.; Chikazawa, H.; Kadota, T. Jpn. J. Cancer Res.
1997, 88, 11205.
(
15) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica,
D.; Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R. J. Natl. Cancer
Inst. 1990, 82, 1107.
Preparation of 5′-Texas Red-Modified DNA Fragment and High-
Resolution Gel Electrophoresis. The 5′-Texas Red-modified DNA
fragments containing telomere sequence was prepared by PCR using a
primer set of 5′-Texas Red labeled T7 and sp6 promoter primer or that
of T7 and 5′-Texas Red labeled sp6 promoter primer; 1 ng of the
telomere fragment inserted pGEM-T easy vector (program and other
reagents same as above). The fragment was purified by GenElute PCR
(
16) (a) Boyd, M. R. Status of the National Cancer Institute Preclinical Antitumor
Drug Discovery Screen: Implications For Clinical Trial. In Cancer:
Principles and Practice of Oncology; Devista, V. T., Jr., Hellman, S.,
Rosenberg, S. A., Eds.; Lippincott: Philadelphia, PA, 1989; Vol. 3, pp
1
-12. (b) Paull, K. D.; Schoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,
D.; Rubinstein, L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer Inst. 1989,
81, 1088.
J. AM. CHEM. SOC.
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