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Helvetica Chimica Acta Vol. 86 (2003)
CHCl3/MeOH 10 :1) to yield an off-white crystalline product in 85% yield. X-Ray-quality crystals were
obtained from MeOH. UV: lmax 293 (3290), 242 (7330); lmin 268, 223; pH 1: lmax 307 (1800), 250 (6790); lmin 283,
236; pH 12: lmax 278 (10370); lmin 240; e 260 (mm) 3.0, e 280 (mm) 2.6. 1H-NMR: 1.80 (m, 1 HÀC(2')); 2.14
(m, 1 HÀC(2')); 2.49 (s, Me); 3.49 (m, CH2(5')); 3.62 (m, HÀC(4')); 4.16 (m, HÀC(3')); 4.40 (s, CH2(5)); 4.79
(t, 5'ÀOH); 5.13 (d, 3'ÀOH); 6.24 (t, HÀC(1')); 7.34 (s, HÀC(4)); 10.29 (br. s, HÀN(8)). 13C-NMR: 21.1 (Me);
35.1 (C(2')); 47.8 (C(5)); 61.8 (C(5')); 70.6 (C(3')); 83.1 (C(1')); 86.0 (C(4')); 120.8 (C(4a)); 124.8 (C(4)); 151.8
(C(7)); 153.2 (C(3)); 155.2 (C(8a)). HR-MS: 303.10790 ([M Na] , C12H16N4NaO4 ; deviation 3.12 ppm).
2'-Deoxy-N4-hydroxy-5-(2-hydroxyiminopropyl)cytidine (15). Compound 7 (0.85 g, 3.1 mmol) was dis-
solved in 5 cm3 of MeOH, and a 50% soln. of NH2OH in H2O (20 cm3) was added. The mixture was stirred at r.t.
for overnight, and the solvent was concentrated. The compound was purified by silica-gel chromatography with
CHCl3/MeOH mixture. To completely remove NH2OH from the desired compound, either HPLC or a further
1
column was required. Yield: 0.65 g (66%). UV: lmax 232, 275. H-NMR: 1.72 (s, Me); 2.00 (m, CH2(2')); 3.72
(m, CH2(5')); 3.75 (m, HÀC(4')); 4.05 (s, CH2); 4.20 (m, HÀC(3')); 4.88 (t, 5'ÀOH); 5.20 (m, 3'ÀOH); 6.16
(t, HÀC(1')); 6.89 (s, HÀC(6)); 10.36 (s, NH). 13C-NMR: 13.27 (Me); 32.80 (C(2')); 38.62 (CH2); 61.77 (C(5'));
70.79 (C(3')); 83.27 (C(1')); 86.86 (C(4')); 106.81 (C(5)); 127.81 (C(6)); 128.05, 143.12, 149.12 (C(2) or
CNOH), 153.00 (C(2) or CNOH). ES-TOF-MS: 313.37 (C12H17N4O6; calc. 313.29). HR-MS: 337.11220
([M Na] , C12H18N4NaO6 ; deviation À 0.70 ppm).
2'-Deoxy-5-(2-oxopropyl)uridine (16). Compound 7 (1 g, 3.7 mmol) was dissolved in 25 cm3 of THF, and
5 cm3 of 2n NaOH soln. was added. The mixture was stirred at r.t. for 4 h. After concentration, the compound
was purified by silica-gel chromatography with 0 20% MeOH/CHCl3. Yield: 0.23 g (22%), UV: lmax 266.
1H-NMR: 2.08 (m, 1 HÀC(2')); 2.12 (s, Me); 3.40 (m, 1 HÀC(2')); 3.54 (m, CH2(5')); 3.76 (m, HÀC(4')); 4.07
(s, CH2COMe); 4.22 (m, HÀC(3')); 5.01 (t, 5'ÀOH); 5.27 (d, 3'ÀOH); 6.16 (t, HÀC(1')); 7.71(s, HÀC(6));
11.37 (br. s, NH). 13C-NMR: 29.22 (Me); 38.66 (C(2')); 40.53 (CH2COMe); 61.31 (C(5')); 70.31 (C(3')); 84.04
(C(1')); 87.31 (C(4')); 108.34 (C(5)); 138.24 (C(6)); 150.24 (C(2)); 162.98 (C(4)); 204.90 (COMe). ES-TOF-MS:
283.33 (C12H15N2O6; calc. 283.26). HR-MS: 307.09110 ([M Na] , C12H18N2NaO6 ; deviation 1.51 ppm).
6-(b-d-2-Deoxyribofuranosyl)-5,6,7,8-tetrahydro-3-methylpyrimido[4,5-c]pyridazin-7-one 5'-Triphosphate
(dHTP). To 140 mg (0.5 mmol) of dried 11 in a 50-cm3 round-bottom flask under Ar was added 5 cm3 of
trimethyl phosphate. The homogenous soln. was cooled via an ice-bath, and redistilled phosphorus oxychloride
(70 ml, 0.75 mmol) was added. The mixture was stirred with cooling for 1 h. TLC Analysis indicated the reaction
to be ca. 70% complete. Therefore, 25 ml (0.27 mmol, 0.51 equiv.) more phosphorus oxychloride was added, and
the mixture was allowed to stir for an additional hour. Both 1m tributylammonium pyrophosphate in anh. DMF
(2.5 cm3, 2.5 mmol) and Bu3N (0.6 cm3, 2.5 mmol) were then simultaneously added slowly to the cooled soln.
After addition, the mixture was stirred for 30 min, then warmed to r.t., and stirred for 1 h. Cooled 1m TEAB
buffer (pH 7.0) was added to the mixture until the soln. became neutral as observed by pH paper. The buffer was
then added to a final volume of 40 cm3, and the mixture was stirred overnight at r.t. The soln. was evaporated
under high vacuum to a viscous soln., and the crude mixture was then applied to a 50 Â 300 mm DeltaPak (15 m
C18 HPLC column), which was eluted with a linear gradient over 25 min with 0.1m TEAB (pH 7.0) to 0.1m
TEAB in 25% MeCN at a flow rate of 130 ml per min. A peak containing the product was collected at 10.5 min.
After evaporation, the crude triphosphate was finally purified on a 21 Â 250 mm Synchropak Ax100 HPLC
column with a linear gradient for 30 min at 15 ml per min of 0.1m TEAB in 40% MeCN to 1m TEAB in 40%
MeCN. 31P-NMR (D2O): À 9.26 (d); À 10.20 (d); À 22.43 (t). HPLC (DeltaPak C18, 3.9 Â 30 cm, 0.1m TEAB
(pH 7.0) to 0.1m TEAB in 25% MeCN in 30 min at 1 ml per min) 12.7 min. UV: lmax 237, 292.
6-[5-O-Dimethoxytrityl-3-O-(2-cyanoethyl N,N-Diisopropylphosphoramidite)-b-d-2-deoxyribofuranosyl]-
5,6,7,8-tetrahydro-3-methylpyrimido[4,5-c]pyridazin-7-one. Compound 11 (1.16 g, 4.1 mmol) was co-evaporated
with anhydrous pyridine and redissolved in 30 cm3 of anh. pyridine. Dimethoxytrityl chloride (4.15 g,
12.24 mmol) was added to the stirred soln. at r.t. under Ar. After 3.5 h, the mixture was evaporated under
reduced pressure, and residue was dissolved in CHCl3. The org. layer was washed with sat. NaHCO3 soln.,
dried (Na2SO4) and evaporated under reduced pressure. The residue obtained was purified by CC (silica gel;
CHCl3 ! CHCl3/MeOH 95:5) to afford the 6-(5-Dimethoxytrityl-b-d-2-deoxyribofuranosyl)-5,6,7,8-tetrahydro-
3-methylpyrimido[4,5-c]pyridazin-7-one (2.35 g, 99%). ES-TOF-MS (cone 100v, MeCN/0.1m TEAB): 581.24
([MH À 1]).
5'-DMT-Protected 11 (583 mg, 1.0 mmol) was co-evaporated with anh. pyridine, followed by toluene, and
dissolved in anh. CH2Cl2 (5 cm3). To the stirred soln. under a slow stream of Ar, at r.t., Et(i-Pr)2N (0.7 cm3,
4.0 mmol) was added, followed by dropwise addition of 2-cyanoethoxy N,N-diisopropylaminochlorophosphine
(0.28 cm3, 1.25 mmol). After 30 min, TLC in 10% MeOH/CHCl3 indicated completion of the reaction. The
mixture was diluted with CH2Cl2, washed with 10% Na2CO3, and the org. layer after drying (Na2SO4) was