DNA Polymerase I Klenow Fragment, Inhibitors of
1761
equivalent) suspended in dry methylene chloride (2 mL). After stirring at room tem-
perature for 5 hr under nitrogen, the reaction mixture was diluted with ethyl ether
(50 mL) The black solid was removed by suction filtration and washed with ethyl
ether (50 mL). The combined ether layer was extracted with sodium bicarbonate (10%,
50 mL). The organic layer was evaporated to remove solvent. The product was then
purified by silica gel chromatography, eluting with hexane/ethyl acetate (9:1). The
appropriate fractions were pooled and roto-evaporated, yielding 0.4 g (80%) of crude
1
product as a yellowish oil. H-NMR (CDCl3) d 3.67 (d, J = 2.1, 2H, C2H), 7.07 (d,
J = 6.6, 2H, PhH), 7.18 (d, J = 6.6, 2H, PhH), 9.72 (t, J = 2.1, 1H, C1H).
2-Chloro-2-(4-azidophenyl)acetaldehyde (6): Freshly prepared 4-azidophenylac-
etaldehyde (4, 0.2 g, 1.24 mmol) was dissolved in acetic acid (2 mL), and t-butyl
hypochlorite (170 mL, 1.1 equivalent) in acetic acid (1 mL) was added dropwise. After
stirring 1 hr at room temperature, the reaction mixture was diluted with water (5 mL)
and extracted with three volumes of ethyl ether. The combined ether extracts were
washed with water until the aqueous phase was neutral, dried, and filtered (do not
extract with base as the product decomposes). The final crude product contained 70%
1
aldeyhyde product and 30% acid by NMR: H-NMR (CDCl3) d 5.19 (d, J = 2.7 Hz,
1H, C2H), 6.95–7.21 (m, 4H, PhH), 9.49 (d, J = 2.7 Hz, 1H, C1H).
2-Bromo-2-(4-azidophenyl)acetaldehyde (7). Freshly-prepared 2-(4-azidophenyl)-
acetaldehyde (4, 0.1g, 0.62 mmol) and p-toluenesulfonic acid (about 0.1 equivalent)
were dissolved in dry THF (2 mL), and NBS (0.11 g, 1 equivalent) was added in small
portions. The reaction was stirred at room temperature until the brown color
disappeared (about 30 min). The reaction mixture was diluted with methylene chloride
(20 mL) and extracted with three volumes of water. Evaporation of the organic layer
1
yielded the crude product as a yellowish oil. H-NMR (CDCl3) d 5.19 (d, J = 2.7 Hz,
1H, C2H), 6.95–7.21 (m, 4H, PhH), 9.49 (d, J = 2.7 Hz, 1H, C1H).
a-(4-Azidophenyl)-1,N6-etheno-2’-deoxyadenosine 5’-monophosphate (3m). A
solution of dAMP (50 mg, 0.14 mmol) and sodium acetate (0.24 g, 1.76 mmol ) in
water (1 mL) was added to a 10-fold molar excess of freshly prepared a-halo-2-(4-
azidophenyl)acetaldehyde (6 or 7) in acetonitrile (1 mL), and the heterogeneous
solution was stirred at 40°C for 24 hr. The reaction was then diluted with water
(10 mL), neutralized with 0.1N NaOH and extracted with three volumes of ethyl ether.
The aqueous phase was lyophilized, and the product was purified by ion-exchange
chromatography on a DEAE-sephadex column with an average purification yield of
20%: IR (KBr) 2120 (N3); UV lMax (e  10À4): 0.05N NaOH, 275 (1.46), 298 (1.96);
0.05N HCl, 275 (1.72), 298 (1.93); 0.01M ammonium acetate, 276 (1.38), 296 (1.83);
1H-NMR d 2.58 (m, 1H, C2’H1), 2.88 (m, 1H, C2’H2), 3.97 (t, J = 3.9 Hz, 2H, C5’H),
4.21 (m, 1H, C4’H), 4.68 (br s, 1H, C3’H), 6.55 (t, J = 6.9 Hz, 1H, C1’H), 7.01 (d,
J = 7.8 Hz, 2H, PhH), 7.46 (d, J = 7.8 Hz, 2H, PhH), 7.50 (s, 1H, C10H), 8.52 (s, 1H,
C2H), 8.92 (s, 1H, C8H); high resolution FAB-MS m/e 471.0953; calcd. for
C18H16N8O6P1 (M–H+), 471.0930.
a-(4-Azidophenyl)-1,N6-etheno-2’-deoxyadenosine 5’-triphosphate (3) was syn-
thesized from dATP and a 10-fold molar excess of 6 (or 7) as described in the synthesis
of 3 with an average yield of 20%, or by the pyrophosphorylation of 4m as described