Experimental
Materials
The NMR spectra were recorded on a Bruker AM 200 or a Jeol
400 spectrometer. The 1H NMR chemical shifts (at 300 K) were
referred to internal TMS, and the 31P NMR shifts (202 MHz,
300 K) to external orthophosphoric acid. The mass spectra
were acquired using a Perkin Elmer Sciex API 365 triple
quadrupole LC/MS/MS spectrometer.
The organic solvents were dried by refluxing over calcium
hydride and distilled. The solid starting materials were dried by
coevaporation with the anhydrous solvent. Thymidine, thymine
and thymidine 5Ј-phosphate were products of Sigma. 3Ј-Azido-
3Ј-deoxythymidine was prepared as described previously23 and
converted to 3Ј-amino-3Ј-deoxythymidine by treatment with
triphenylphosphine in anhydrous pyridine followed by addition
of aqueous ammonia.24 Dinucleoside thiophosphoramidate
analogs of thymidylyl-3Ј,5Ј-thymidine (2) were prepared using
H-phosphonate methodology, in which nucleoside H-phos-
phonothioate 17 was converted into a pyridine adduct of
a nucleoside thiometaphosphate followed by reaction with
5Ј-protected 3Ј-amino-3Ј-deoxythymidine 18 (Scheme 6), as
described previously by Stawinski et al.4 Dinucleoside phos-
phoramidate analog of thymidylyl-3Ј,5Ј-thymidine (3) was
obtained by oxidative amination of the appropriately protected
thymidine 5Ј-(H-phosphonate) 2-cyanoethyl ester 21 in the
presence of 3Ј-amino-3Ј-deoxythymidine (Scheme 7). The same
reaction sequence has previously been used for the synthesis of
oligoribonucleotide phosphoramidates on a solid support.6,7
Scheme
7
Reagents and conditions: (i) CNCH2CH2OH, DPCP,
CH3CN–pyridine, (ii) 4, CCl4, Et3N, (iii) NH3–MeOH, (iv) TBAF–
THF.
pyridine (2 ml) were added (Scheme 6). The crude product was
isolated by a conventional aqueous work up, and purified on a
silica gel column eluted with a mixture of dichloromethane and
methanol (80 : 20%, v/v). The tert-butyldimethylsilyl protected
thiophosphoramidate (19) was dissolved in 1 mol LϪ1 solution
of tetrabutylammonium fluoride (0.265 g, 1.02 mmol) in tetra-
hydrofuran (1 mL), and the solution was stirred for 16 h at
room temperature. The mixture was evaporated to dryness and
the diastereomers were purified and separated from each other
by reversed phase chromatography on a Lobar RP-18 column
(37 × 440 mm, 40–63 µm) eluting with a mixture of water and
acetonitrile (90 : 10%, v/v). Finally, the product was passed
through a Naϩ-form Dowex 50-W (100–200 mesh) cation
exchange column. 2 (the faster eluted diastereomer): 31P NMR:
1
δP (202 MHz, D2O) = 53.63. H NMR: δH (400 MHz, D2O) =
7.71 (s, 1H, H6), 7.70 (s, 1H, H6), 6.15 (t, 1H, H1Ј, J = 6.62),
5,80 (dd, 1H, H1Ј, J = 7.05, J = 2.14), 4.43 (m, 1H, H3Ј), 4.00
(m, 2H, H4Ј), 3.93 (m, 2H, H5Ј, H5Љ), 3.83–3.79 (m, 1H, H5Ј),
3.71 (m, 1H, H5Љ), 3.70 (m, 1H, H4Ј), 3.63–3.54 (m, 1H, H3Ј),
2.37–2.13 (m, 4H, H2Ј, H2Љ), 1.73 (s, 3H, CH3), 1.70 (s,
3H,CH3). ESIϪ-MS: m/z 560.3 [M Ϫ H]Ϫ. 2 (The slower eluted
diastereomer): 31P NMR: δP (202 MHz, D2O) = 53.59. 1H
NMR: δH (400 MHz, D2O) = 7.62 (s, 2H, 2 × H6), 6.16 (t, 1H,
H1Ј, J = 6.62), 5.90 (dd, 1H, H1Ј, J = 6.83, J = 3.63), 4.42 (m,
1H, H3Ј), 4.00 (m, 1H, H4Ј), 4.00–3.95 (m, 1H, H5Ј), 3.91–3.86
(m, 1H, H5Љ), 3.81 (m, 1H, H5Љ), 3.72 (s, 2H, H3Ј, H4Ј), 3.76–
3.68 (m, 1H, H5Љ), 2.33–2.18 (m, 4H, H2Ј, H2Љ), 1.80 (s, 3H,
CH3), 1.72 (s, 3H, CH3). ESIϪ-MS: m/z 560.3 [M Ϫ H]Ϫ.
(3Ј-Amino-3Ј-deoxythymidylyl)-(3Ј 5Ј)-thymidine (3)
Compound 3 was prepared, analogously with the previously
described method,6,7 by reacting the triethylammonium salt
of 5Ј-O-(tert-butyldimethylsilyl)thymidine 5Ј-hydrogenphos-
phonate 20 (0.52 g, 1.24 mmol) with cyanoethanol (76 µL,
1.11 mmol) and DPCP (304 µL, 1.49 mmol) in a mixture of
anhydrous pyridine (4.0 mL) and acetonitrile (6.0 mL) (Scheme
7). The formed cyanoethyl ester 21 was not isolated. After 2 h
of stirring at room temperature, 3Ј-amino-3Ј-deoxythymidine 4
(0.47 g, 1.34 mmol), carbon tetrachloride (5.0 mL) and triethyl-
amine (0.22 mL) were added and the mixture was left to stand
overnight. The mixture was then poured into 90 mL of di-
chloromethane and washed with saturated aqueous NaCl
(3 × 50 mL). The organic layer was dried with Na2SO4 and
concentrated. The residue was purified on a silica gel column
using a mixture of dichloromethane and methanol as eluent
(92 : 8%, v/v) to give the fully protected product 22. The
cyanoethyl group was removed by treatment with saturated
Scheme 6 Reagents and conditions: (i) TMSCl, py, (ii) I2, (iii) Et3N, py,
(iv) TBAF–THF.
The (RP)- and (SP)-thiophosphoramidate analogs of (3Ј-amino-
3Ј-deoxythymidylyl)-3Ј,5Ј-thymidine (2)
Nucleoside H-phosphonothioate25 17 (0.25 g, 0.57 mmol) and
trimethylsilyl chloride (0.19 g, 1.72 mmol) were dissolved in
10 mL of anhydrous pyridine. After stirring for 5 min at RT,
iodine (0.22 g, 2.87 mmol) was added. After an additional 7 min
of stirring, 5Ј-O-(tert-butyldimethylsilyl)-3Ј-amino-3Ј-deoxy-
thymidine 18 (0.20 g, 0.57 mmol) and triethylamine (0.40 ml) in
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
598