Mimicry of Damaged DNA Bases
A R T I C L E S
Hz). 13C NMR (CDCl3, 100 MHz) δ 158.66, 144.56, 141.31, 138.75,
135.68, 135.58, 132.64, 130.25, 129.54, 128.29, 128.02, 127.06, 123.59,
123.33, 113.30, 109.86, 86.85, 85.12, 85.08, 71.88, 63.15, 55.31, 38.94,
16.61. HRMS (ESI+) m/z [M + Na]+ calcd for C34H33N2O5NaCl
607.1975, found 607.1974.
21.13, 20.46, 20.39, 20.25, 20.19, 14.27. 19F NMR (CDCl3, 376 MHz)
δ -123.02 (dd, J ) 9.8, 4.9 Hz), -123.08 (dd, J ) 9.8, 4.9 Hz). 31P
NMR (CDCl3, 161 MHz) δ 149.27, 148.60. HRMS (ESI+) m/z [M +
Na]+ calcd for C43H48N3O6FNaPCl 810.2851, found 810.2875.
5′-Triphosphate Synthesis. The nucleoside (0.1 mmol) was stirred
in trimethylphosphate (0.25 mL) with proton sponge (32 mg, 0.15
mmol) at 0 °C. POCl3 (10 mL, 0.1 mmol) was added and the mixture
was stirred for 2 h at 0 °C. A solution of tributylammonium
pyrophosphate (240 mg) in anhydrous N,N-dimethylformamide (DMF)
(1.0 mL) and n-tributylamine (0.1 mL) were added to the reaction
mixture under vigorous stirring at 0 °C. After 5 min, 0.25 M aqueous
triethylammonium bicarbonate (TEAB) buffer (10 mL) was poured into
the solution. The aqueous layer was washed with dichloromethane (10
mL × 2) and concentrated under reduced pressure. The residue was
purified by reverse-phase HPLC (Zorbox ODS 9.4 mm × 25 cm, SB-
C18 column) with a linear gradient of CH3CN/50 mM TEAA buffer
(pH 7.5). The fractions containing triphosphate were collected and
concentrated in vacuo. The residue of triethylammonium nucleoside
triphosphate was dissolved in HPLC-grade methanol (0.2 mL) and
precipitated by adding 0.6 M NaClO4 solution in HPLC-grade acetone
(1.2 mL). The precipitated sodium salt was collected by centrifugation,
washed with acetone (1.0 mL × 4) and dried under vacuum.
2-Chloro-1-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-â-D-ribofurano-
syl]-4-fluoroindole (6). The compound was isolated as a colorless
foam: 1H NMR (CDCl3/TMS, 400 MHz) δ 7.48-7.43 (m, 3H), 7.38-
7.34 (m, 4H), 7.30-7.21 (m, 3H), 6.82 (dd, 4H, J ) 8.8, 1.7 Hz),
6.76-6.71 (m, 2H), 6.55 (s, 1H), 6.49 (dd, 1H, J ) 8.2, 6.7 Hz), 4.72-
4.68 (m, 1H), 4.01-3.98 (m, 1H), 3.78 (s, 6H), 3.56-3.48 (m, 2H),
2.82 (ddd, 1H, J ) 13.9, 8.2, 5.6 Hz), 2.21 (ddd, 1H, J ) 13.9, 6.7,
3.2 Hz), 1.97 (d, 1H, J ) 3.5 Hz). 13C NMR (CDCl3, 100 MHz) δ
158.66, 156.61, 154.16, 144.64, 136.41, 135.74, 135.65, 130.25, 128.30,
128.02, 127.05, 124.74, 122.65, 122.59, 117.17, 113.30, 108.50, 105.99,
105.80, 97.96, 94.51, 86.82, 84.54, 84.50, 72.09, 63.19, 55.31, 38.89.
19F NMR (CDCl3, 376 MHz) δ -122.87 (dd, J ) 8.5, 7.3 Hz). HRMS
(ESI+) m/z [M + Na]+ calcd for C34H31NO5FNaCl 610.1772, found
610.1795.
General Procedure for Preparation of Phosphoramidite Deriva-
tives. To a solution of the DMTr derivative (∼0.3 mmol) and iPr2NEt
(3 equiv) in anhydrous CH2Cl2 was added iPr2NP(Cl)OC2H4CN (6
equiv) at 0 °C. After being stirred for 1 h at the same temperature, the
reaction mixture was quenched with saturated NaHCO3 solution and
extracted with EtOAc. The organic layer was separated, dried over Na2-
SO4, and concentrated in vacuo. The crude compound was purified by
column chromatography (silica gel, hexane/AcOEt) to give the purified
material, which was precipitated from hexane at -78 °C. The hexane
was removed by decantation and the solid material was dried in vacuum.
2-Chloro-1-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-O-(N,N-diiso-
propyl-â-cyanoethylphosphoramidyl)-â-D-ribofuranosyl]-4-methyl-
1H-benzimidazole (11). The compound was isolated as a white
powder: 1H NMR (CDCl3/TMS, 400 MHz) δ 7.58-7.52 (m, 1H),
7.48-7.41 (m, 2H), 7.37-7.15 (m, 7H), 6.99 (d, 1H, J ) 7.5 Hz),
6.85-6.75 (m, 4H), 6.73-6.70 (m, 1H), 6.45-6.40 (m, 1H), 4.88-
4.77 (m, 1H), 4.23-4.18 (m, 1H), 3.87-3.80 (m, 1H), 3.78 (s, 3H),
3.77 (s, 3H), 3.76-3.66 (m, 1H), 3.64-3.39 (m, 4H), 2.87-2.74 (m,
1H), 2.66-2.60 (m, 1H), 2.59 (s, 3H), 2.57-2.40 (m, 1.5H), 2.34 (ddd,
0.5H, J ) 13.6, 5.3, 2.3 Hz), 1.25-1.12 (m, 9H), 1.06-1.04 (m, 2H),
0.88-0.83 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ 158.62, 158.60,
144.51, 141.35, 138.65, 135.68, 135.65, 135.56, 135.51, 132.67, 130.37,
130.35, 130.32, 129.47, 129.44, 129.20, 128.45, 128.40, 127.94, 127.96,
127.15, 127.02, 126.98, 123.51, 123.27, 117.44, 113.21, 110.19, 86.71,
85.38, 85.32, 85.05, 84.75, 73.28, 73.10, 72.85, 72.66, 62.78, 62.59,
58.42, 58.26, 55.31, 55.28, 43.43, 43.35, 43.30, 43.22, 38.24, 24.67,
24.63, 24.55, 20.48, 20.41, 20.27, 20.19, 16.59. 31P NMR (CDCl3, 161
MHz) δ 149.39, 148.78. HRMS (ESI+) m/z [M + Na]+ calcd for
C43H50N4O6NaPCl 807.3054, found 807.3041.
2-Chloro-1-[2′-deoxy-â-D-ribofuranosyl]-4-methyl-1H-benzimi-
dazole 5′-Triphosphate (4). 1H NMR (D2O, 400 MHz) δ 7.63 (d, 1H,
J ) 7.6 Hz), 7.29 (t, 1H, J ) 7.6 Hz), 7.16 (d, 1H, J ) 7.6 Hz), 6.55
(t, 1H, J ) 7.4 Hz), 4.81-4.77 (m, 1H), 4.31-4.23 (m, 2H), 4.22-
4.19 (m, 1H), 2.89-2.85 (m, 1H), 2.47 (s, 3H), 2.37-2.30 (m, 1H).
31P NMR (D2O, 161 MHz) δ -8.4, -13.4, -24.0. LRMS (ESI-) m/z
[M - H]- calcd for C13H17N2O12P3Cl 520.0, found 520.6.
2-Chloro-1-[2′-deoxy-â-D-ribofuranosyl]-4-fluoroindole 5′-Triph-
1
osphate (3). H NMR (D2O, 400 MHz) δ 7.57-7.49 (m, 1H), 7.24-
7.19 (m, 1H), 6.91-6.86 (m, 1H), 6.66 (s, 1H), 6.57 (dd, 1H, J ) 8.4,
7.0 Hz), 4.81-4.77 (m, 1H), 4.29-4.22 (m, 2H), 4.15-4.14 (m, 1H),
2.96-2.89 (m, 1H), 2.28-2.22 (m, 1H). 19F NMR (D2O, 376 MHz) δ
-123.5 (dd, J ) 11.0, 4.9 Hz). 31P NMR (D2O, 161 MHz) δ -6.2,
-11.0, -22.0. LRMS (ESI-) m/z [M - H]- calcd for C13H15NO12P3-
ClF 524.6, found 524.3.
Oligodeoxyribonucleotide Synthesis. Oligonucleotides were syn-
thesized by the standard DNA synthesis procedures (trityl-on mode)
on an ABI 394 DNA/RNA synthesizer. Rapid-deprotecting chemistry
was used [with PAC-A, Ac-C, and iPr-PAC-G phosphoramidites (Glen
Research)]. Deprotection of synthesized oligonucleotides was done with
50 mM potassium carbonate in methanol (3 mL) at room temperature
for 18 h. 5′-DMT oligonucleotides were purified by reverse-phase
HPLC. The DMTr protecting group was cleaved in 10% aqueous acetic
acid at room temperature for 30 min, the resulting DMTr-OH was
removed by washing with ether, and the solvents were neutralized and
concentrated under reduced pressure. Structures of the synthetic
oligonucleotides were confirmed by 1H NMR and matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) MS measurements
(see Supporting Information for data).
Thermal Denaturation Experiments. Duplex DNA solutions were
heated to 95 °C for 5 min and annealed by slow cooling (over 2 h to
5 °C). The melting studies were carried out in 1-cm path length quartz
cells on a Varian Cary 100 Bio UV-vis spectrophotometer equipped
with thermoprogrammer. The samples were warmed at a rate of 0.5
°C/min from 5 to 80 °C. Absorbance was monitored at 260 nm. The
data were analyzed by the program MeltWin v. 3.0. Melting temper-
atures (Tm values) were determined by computer fit of the first derivative
of absorbance with respect to 1/T. van’t Hoff methods were used to
calculate thermodynamic parameters.
Steady-State Kinetics Methods: 1. Steady-State Kinetics. Poly-
merase-catalyzed nucleotide single insertion reactions were carried out
in 10 mL volumes in the reaction buffer [50 mM Tris-HCl (pH 7.5),
10 mM MgCl2, 1 mM dithiothreitol, and 50 mg/mL BSA for Klenow
(exo-) DNA polymerase I (Amersham Bioscience)]. As polymerase
2-Chloro-1-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-O-(N,N-diiso-
propyl-â-cyanoethylphosphoramidyl)-â-D-ribofuranosyl]-4-fluoroin-
dole (7). The compound was isolated as a white powder: 1H NMR
(CDCl3/TMS, 400 MHz) δ 7.60-7.54 (m, 1H), 7.49-7.44 (m, 2H),
7.38-7.30 (m, 4H), 7.28-7.16 (m, 3H), 6.85-6.78 (m, 4H), 6.73-
6.68 (m, 1H), 6.61-6.56 (m, 1H), 6.55 (s, 1H), 6.46-6.41 (m, 1H),
4.87-4.79 (m, 2H), 4.16 (t, 1H, J ) 3.7 Hz), 3.78 (s, 3H), 3.77 (s,
3H), 3.63-3.50 (m, 4H), 3.47-3.43 (m, 1H), 2.89-2.80 (m, 1H), 2.60
(t, 1H, J ) 6.6 Hz), 2.42 (t, 1H, J ) 6.4 Hz), 2.37-2.29 (m, 0.5H),
2.28-2.24 (m, 0.5H), 1.25-1.12 (m, 9H), 1.06-1.04 (m, 2H), 0.88-
0.83 (m, 1H). 13C NMR (CDCl3, 100 MHz) δ 158.65, 158.60, 158.59,
156.55, 154.11, 144.65, 144.61, 136.40, 135.77, 135.73, 135.66, 135.61,
135.56, 130.40, 130.37, 130.35, 129.20, 128.46, 128.43, 127.94, 126.98,
126.94, 126.94, 124.69, 124.66, 122.65, 122.57, 117.50, 117.46, 116.91,
113.25, 113.20, 108.97, 108.93, 105.94, 105.76, 98.05, 97.85, 94.50,
86.65, 84.90, 84.83, 84.62, 84.59, 84.36, 84.30, 73.27, 73.10, 72.84,
72.65, 62.79, 62.57, 60.47, 58.45, 58.26, 55.31, 55.28, 43.40, 43.32,
43.28, 43.20, 38.21, 24.71, 24.67, 24.62, 24.55, 22.71, 22.45, 22.37,
9
J. AM. CHEM. SOC. VOL. 129, NO. 28, 2007 8843