Hydrolytic Reactions of Nucleoside Phosphoramidates
A R T I C L E S
Scheme 8 a
4.12 (m, 1H), 3.94 (m, 1H), 3.86 (m, 1H), 3.84 (m, 1H), 3.77 (m, 1H),
3.72 (m, 1H), 3.70 (m, 1H), 3.45 (m, 1H), 0.87 (s, 9H), 0.87 (s, 9H),
0.84 (s, 9H), 0.81 (s, 9H), 0.06 (s, 6H), 0.05 (s, 6H), 0.04 (s, 6H), 0.01
(s, 6H). ESI--MS: m/z 1004.7 [M - H]-.
3′-Amino-3′-deoxyuridylyl-3′,5′-uridine (2a). The tert-butyldi-
methylsilyl-protected phosphoramidate 15 was dissolved in 1 mol L-1
tetrabutylammonium fluoride (0.265 g, 1.02 mmol) in tetrahydrofuran
(1 mL), and the solution was stirred for 16 h at room temperature. The
mixture was evaporated to dryness, and the product was purified by
reversed-phase chromatography on a Lobar RP-18 column (37 × 440
mm, 40-63 µm) eluting with a mixture of 0.1 mol L-1 aqueous
ammonium acetate and acetonitrile, the acetonitrile content of which
was linearly increased from 0% to 20%. The buffer salts were removed
on the same column by eluting with a mixture of water and acetonitrile.
Finally, the product was passed through a Na+-form Dowex 50-W
(100-200 mesh) cation exchange column. 31P NMR (δP) (202 MHz,
1
D2O): 8.85. H NMR (δH) (500 MHz, D2O): 7.93 (d, 1H, J ) 8.1
Hz), 7.90 (d, 1H, J ) 8.1 Hz), 5.80 (d, 1H, J ) 3.4 Hz), 5.71 (d, 1H,
J ) 8.1 Hz), 5.65 (d, 1H, J ) 8.1 Hz), 5.59 (s, 1H), 4.22 (dd, 1H, J )
5.5 Hz), 4.17 (m, 1H), 4.14-4.09 (m, 2H), 3.95-3.86 (m, 4H), (m,
1H), 3.78 (m, 1H). ESI--MS: m/z 321.9 [M - H]-.
3′-Amino-3′-deoxyuridine 2′-Phosphate (3). 5′-O-(4,4′-dimethoxy-
trityl)-3′-deoxy-3′-azidouridine was reduced to the corresponding 5′-
O-(4,4′-dimethoxytrityl)-3′-amino-3′-deoxyuridine as described above
for the 2′,5′-bis-O-TBDMS-protected analogue 10. The 3′-amino group
was then acylated43 with S-ethyl trifluorothioacetate, and the 2′-hydroxyl
group was phosphorylated44 with phosphoryltris(triazole). The 5′-O-
(4,4′-dimethoxytrityl) group was removed with a mixture of trifluoro-
acetic acid, dichloromethane, and methanol (3:1:3, v/v/v, 15 min at 22
°C). The volatile components were evaporated. The residue was
dissolved in water and washed with dichloromethane, and the product
was purified on a Lobar RP-18 column (37 × 440 mm, 40-63 µm)
using 2% aqueous acetonitrile as an eluent. The 3′-N-trifluoroacetyl
group was removed with methanolic ammonia within 5 h at 22 °C.
The mixture was concentrated, and the product was purified on the
Lobar RP-18 column using water as an eluent. 31P NMR (δP) (162
a Reagents and conditions: (a) t-BuMe2SiCl, (b) (1) (Ph)3P, (2) aqueous
NH3, (c) CNCH2CH2OH, PivCl, (d) 11, CCl4, Et3N, (e) NH3/MeOH, (f)
TBAF.
the solution was stirred for 6 h at 50 °C, heating was stopped, and a
mixture of aqueous NH3 (33%, 6 mL) and pyridine (10 mL) was added.
After being stirred for 16 h, the solution was concentrated in vacuo to
a syrup, which was dissolved in 100 mL of dichloromethane, washed
with water, and concentrated in vacuo. The product was purified on a
silica column eluted with a mixture of ethyl acetate and hexane (70:
30, v/v). 1H NMR (δH) (400 MHz, CDCl3): 8.15 (d, 1H, J ) 8.1 Hz),
5.68 (s, 1H), 5.57 (d, 1H), 4.02 (dd, 1H, J ) 5.3 Hz), 4.00 (m, 1H),
3.82 (s, 1H), 3.79 (s, 1H), 3.31 (m, 1H), 1.35 (m, 2H), 0.86 (s, 18H),
0.03 (s, 12H). ESI--MS: m/z 470.4 [M - H]-.
1
MHz, D2O): 3.47. H NMR (δH) (400 MHz, D2O): 7.59 (d, 1H, J )
3′-Amino-2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-deoxyuridylyl-
3′,5′-[2′,5′-bis-O-(tert-butyldimethylsilyl)uridine] 2-Cyanoethyl Ester
(14). The triethylammonium salt of 2′,5′-bis-O-(tert-butyldimethylsilyl)-
uridine 5′-hydrogenphosphonate (12) was prepared as described
earlier,45 and 0.26 g (0.49 mmol) of the product was dissolved in a
mixture of anhydrous pyridine (2.0 mL) and acetonitrile (2.0 mL).
2-Cyanoethanol (24 µL, 0.34 mmol) and pivaloyl chloride (63 µL, 0.51
mmol) were added. The cyanoethyl ester 13 was not isolated, but after
the solution was stirred for 2 h at room temperature, 3′-amino-3′-
deoxyuridine 3 (0.185 g, 0.393 mmol), carbon tetrachloride (2.5 mL),
and triethylamine (0.17 mL) were added, and stirring was continued
for 75 min. The mixture was then poured into 35 mL of dichloro-
methane and washed with saturated aqueous NaCl (3 × 30 mL). The
organic layer was dried with Na2SO4 and concentrated. The product
was purified on a silica gel column using a mixture of dichloromethane
and methanol as eluent (70:30, v/v). ESI+-MS: m/z 1081.9 [M + Na]+.
3′-Amino-2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-deoxyuridylyl-
3′,5′-[2′,3′-bis-O-(tert-butyldimethylsilyl)uridine] (15). Compound 14
was dissolved in saturated methanolic ammonia (3 mL). After being
stirred for 2 h, the solution was evaporated to dryness, and the product
was purified on a silica gel column eluting with a mixture of
dichloromethane and methanol, the methanol content of which was
increased stepwise from 0% to 60%. 31P NMR (δP) (202 MHz, DMSO-
d6): 8.23. 1H NMR (δH) (500 MHz, DMSO-d6): 11.37 (s, 1H), 11.30
(s, 1H), 8.21 (d, 1H, J ) 7.9 Hz), 7.79 (d, 1H, J ) 8.1 Hz), 5.85 (d,
1H, J ) 4.9 Hz), 5.79 (d, 1H, J ) 6.0 Hz), 5.62 (d, 1H, J ) 8.1 Hz),
5.58 (d, 1H, J ) 8.1 Hz), 4.32 (dd, 1H, J ) 4.9 Hz), 4.17 (dd, 1H),
8.1 Hz), 5.76 (d, 1H, J ) 3.36 Hz), 5.73 (d, 1H, J ) 8.1 Hz), 4.90 (dd,
1H, J ) 5.50 Hz), 4.19 (m, 1H), 3.99 (m, 1H), 3.78 (dd, 1H), 3.68
(dd, 1H). ESI--MS: m/z 321.9 [M - H]-.
Kinetic Measurements. The reactions were carried out in sealed
tubes immersed in a thermostated water bath, the temperature of which
was adjusted within (0.1 °C. The hydronium ion concentration of the
reaction solutions was adjusted with hydrogen chloride, sodium
hydroxide and formate, acetate, triethanolamine, 4-(2-hydroxyethyl)-
piperazine-1-ethanesulfonic acid (HEPES), and glycine buffers. The
pH values of the buffer solutions were calculated from the literature
data of the pKa values of the buffer acids under the experimental
conditions.46 A low buffer concentration was used (<0.06 mol L-1) to
minimize the effects of possible buffer catalysis on the reactions. At
pH 3 and 8 the effect of the buffer concentration was determined by
carrying out two runs at different buffer concentrations. The effect on
the reaction rate was in both cases less than 30% when the total buffer
concentration was 0.2 mol L-1
.
The initial substrate concentration in the kinetic runs was ca. 10-4
mol L-1. The composition of the samples withdrawn at appropriate
intervals was analyzed by HPLC on a Hypersil ODS 5 column (4 ×
250 mm, 5 µm) using acetic acid/sodium acetate buffer (0.045/0.015
mol L-1) containing 0.1 mol L-1 ammonium chloride and 1.0%
acetonitrile as an eluent. The observed retention times (tR, min) for the
hydrolytic products of 2a (the flow rate was 1 mL min-1) were as
follows: 18.0 (2a), 5.4 (4), 4.2 (5), 4.2 (7), 3.1 (3), and 2.5 (6). 5 and
its 3′-N-phosphoramidate (7) could be separated from each other by
(46) Serjeant, E. P.; Dempsey, B. Ionization Constants of Organic Acids in
Aqueous Solution; IUPAC Chemical Data Series No. 23; Pergamon:
Oxford, 1979.
(45) Almer, H.; Stawinski, J.; Stro¨mberg, R.; Thelin, M. J. Org. Chem. 1992,
57, 6163.
9
J. AM. CHEM. SOC. VOL. 124, NO. 48, 2002 14371