7176
4. Zhang, H.-Z.; Rao, K.; Carr, S. F.; Straub, K.; Wu, C. J.; Fried, J. J. Med. Chem. 1997, 40, 4±8.
5. Winte, H.; Maeda, J.; Ucheda, H.; Mitsuya, H.; Zemlicka, J. J. Med. Chem. 1997, 40, 2191±2195.
6. van der Marel, G. A.; van Boeckel, C. A. A.; Wille, G.; van Boom, J. H. Nucleic Acids Res. 1982, 10, 2337±2351.
7. Michelson, A. M.; Todd, A. R. J. Chem. Soc. 1949, 2476±2486.
8. Takaku, H.; Shimanda, Y.; Arai, K. Bull. Chem. Soc. Japan 1974, 47, 779±780.
9. Claesen, C. A.; Segers, R. P.; Tesser, G. I. Rec. Trav. Chim. Pays-Bas 1985, 104, 119±122.
10. Horn, T.; Urdea, M. S. Tetrahedron Lett. 1986, 27, 4705±4708.
11. Tanaka, T.; Yamada, Y.; Ikehara, M. Tetrahedron Lett. 1986, 27, 3267±3270, 5641±5644.
12. Uhlman, E.; Engels, J. Tetrahedron Lett. 1986, 27, 1023±1026.
13. Ferris, J. P.; Rayser, J. R. Nucleosides & Nucleotides 1994, 13, 1087±1111.
14. Shaller, H.; Khorana, H. G. J. Am. Chem. Soc. 1963, 85, 3828±3835.
15. Nadeau, J. G.; Singleton, C. K.; Kelly, G. B.; Weith, H. L.; Gough, G. R. Biochemistry, 1984, 23, 6153±6159.
16. Rommler, D. H.; Lapidot, Y.; Khorana, H. G. J. Am. Chem. Soc. 1963, 85, 1989±1997.
17. Shaller, H.; Weimann, G.; Lerch, B.; Khorana, H. G. J. Am. Chem. Soc. 1963, 85, 3821±3828.
18. Albert, A. In Synthetic Procedures in Nucleic Acid Chemistry; Zorbach, W. W.; Tipson, R. S., Eds.; Wiley: New
York, 1973; Vol. 2, pp. 47±123.
19. General procedure for phosphorylation: The new phosphorylating reagent, 20-O-(4,40-dimethoxytrityl)ethyl-
sulfonylethan-2-yl-phosphate (1) was synthesized from sulfonyldiethanol (Aldrich) as described previously by
us.1c Phosphorylation was carried out on a 0.2 mmolar scale as follows: 0.2 mmol of starting nucleoside derivative
to be phosphorylated, containing free 50 or 30 hydroxyl groups, was dried by repeated evaporation with anhydrous
pyridine. Compound 1 (1 mmol, 600 mg), TPS-TAZ or TPS-NT (2 mmol) and 5 mL of pyridine were added under
nitrogen. The reaction mixture was refrigerated for 1 h, then kept for an additional 2 h at 25 ꢁC, partitioned
between methylene chloride (100 mL) and saturated NaHCO3 (10 mL) and the organic layer was dried and
evaporated. In the case of compounds 2±6, the residue was dissolved in pyridine (5 mL) and treated with 2 M
NaOH in ethanol (5 mL). The reaction mixture was stirred at 25ꢁC for 20 min. Deprotection of the DMTr-group
on the phosphate was monitored by TLC. The reaction mixture was partitioned between methylene chloride (100
mL) and water (10 mL). The organic layer was evaporated, co-evaporated with toluene (basic pH has to be
maintained; it is adjusted by adding triethylamine). The residue was puri®ed on a short silica gel column. The
target phosphorylated compounds were eluted with 10±15% MeOH in methylene chloride. Further puri®cations
were carried out by reversed phase HPLC on a C-18 column with ethanol±water as the eluting solvent. For
compounds 12±16, the residue of the phosphorylation reaction was stirred with conc. NH4OH (10 mL) at 25ꢁC for
1 h. The reaction mixture was evaporated and puri®ed by reversed-phase HPLC. Final deprotections for both
series were carried out as indicated in Schemes 1 and 2. The products were characterized by spectral data. For
example, for compound 2 (triethylammonium salt): UV (H2O) 233 nm (14000), 264 nm (8000). 1H NMR (MeOH-d4),
7.82 (d, J=7.8 Hz, 1H, H-6), 7.41 (d, J=7.6 Hz, 2H), 7.30±7.22 (m, 7H) and 7.86 (d, J=7.6 Hz, 4H) (DMTr), 6.30
(t, J=6.4 Hz, 1H, 10), 5.21 (d, J=7.8 Hz, 1H, H-5), 5.04 (s, 1H, 30), 4.29 (s, 1H 40), 3.78 (s, 6H, CH3O), 3.53±3.50
(m, 1H, 50a), 3.38±3.36 (m, 1H, 50b), 3.11 (s, 6H, CH2N), 2.56±2.52 (m, 1H, 20a), 2.39 (qw, J=7.6 Hz, 1H, 20b), 1.27
(s, 9H, CH3CH2N); (in CDCl3), 8.69 (s. 1H, NH), 7.34±7.21 (m, 9H) and 6.83 (d, J=11.2 Hz, 4H) (DMTr), 6.35 (t,
J=8.8 Hz, 1H, 10), 5.38 (d, J=10.8 Hz, 1H, H-5), 5.14 (br s, 1H, 30), 4.16 (s, 1H, 40), 3.77 (s, 6H, CH3O), 3.30 (s,
2H, 50), 2.98 (q, J=9.8 Hz, 6H, CH2N), 2.53±2.49 (m, 1H, 20a), 2.30±2.25 (m, 1H, 20b), 1.23 (t, J=9.8 Hz, 9H,
CH3CH2N). 13C NMR (MeOH-d4), 166.1 (C-4), 160.3 (unidenti®ed), 152.0 (C-2), 142.5 (C-6), 146.0, 131.5, 131.4,
130.5, 129.5, 129.4, 128.9, 128.0, 114.2 and 113.9 (DMTr), 102.4 (C-5), 88.3 (Ph3C), 87.1 and 87.0 (10), 86.6 (40),
76.4 (30), 64.9 (50), 55.7 (CH3O), 47.6 (CH2N), 40.7 (20), 9.2 (CH3CH2N). 31P NMR: (MeOH-d4), 3.74 (s). FAB
HRMS: (M^H)^ calcd for C30H30N2O10P: 609.1638; found: 609.1631.