The Synthesis of Base-Sensitive Methyl-SATE Oligonucleotide Prodrugs
FULL PAPER
prooligonucleotides, the solid materials (0.2 mg) were suspended in
5Ϫ10 µL of a saturated solution of the matrix 2,4,6-(trihydroxy)-
acetophenone (THAP, 45 mg, ammonium citrate, 4 mg) in 500 µL
of acetonitrile/water (1:1, v/v). The mixture (1 µL) was spotted onto
the stainless steel probe plate and allowed to air-dry before analysis.
aromatic), 6.76 (d, 4 H, Hα of OCH3), 6.17 (t, 1 H, 1Ј-H), 4.99 (s,
2 H, CH2OSi), 4.59 (m, 1 H, 3Ј-H), 4.11 (d, 1 H, 4Ј-H), 3.70 (s, 6
H, 2 ϫ OCH3), 3.55 (m, 4 H, 2 ϫ CH2), 3.34 (m, 1 H, CH), 3.23
(m, 1 H, CH), 3.02 (m, 1 H, 5-HЈ), 2.92 (m, 1 H, 5ЈЈ-H), 2.81 (m,
1 H, 2Ј-H), 2.41 (m, 1 H, 2ЈЈ-H), 2.19 (s, 3 H, SCOCH3), 1.12
For analysis of prooligonucleotides released from the solid support, [s, 12 H, N{CH(CH3)2}2), 0.92 [s, 9 H, C(CH3)3] ppm. 31P NMR
each of the samples [1 µL, 0.1 OD260nm in 100 µL water/acetonitrile
(1:1, v/v)] was exchanged on DOWEX 50 W X8 resin (ammonium
form) prior to the addition of the THAP matrix solution (5Ϫ10
µL). The resulting solution (1 µL) was then spotted onto the stain-
less steel probe plate and allowed to air-dry before analysis.
(CD3CN): δ ϭ 148.91 and 148.86 ppm.
General Procedure for the Trimethylsilylethyl Phosphitylation of N-
SiOMB-5Ј-O (4,4Ј-Dimethoxytrityl)-2Ј-deoxynucleosides (3a؊d): A
solution of bis(N,N-diisopropylamino)chlorophosphane (320 mg,
1.2 mmol) in dry CH2Cl2 (1.2 mL) was added dropwise over 3 min
to a cooled (Ϫ20 °C) and magnetically stirred solution of N-Si-
OMB-5Ј-O-Dmtr-2Ј-deoxynucleoside 1aϪd (1.0 mmol) and (diiso-
propyl)ethylamine (0.28 mL, 1.3 mmol) in dry CH2Cl2 (6.5 mL).
The reaction mixture was warmed to room temperature while stir-
ring was maintained (2 h). The 2-trimethylsilyl alcohol (0.17 mL,
1.16 mmol) was added followed by diisopropylammonium tetrazol-
ide (86 mg, 0.5 mmol), and the reaction mixture was stirred over-
night. Then CH2Cl2 (15 mL) was added and the reaction mixture
was washed with saturated aqueous sodium hydrogen carbonate
(20 mL) and brine (2 ϫ 20 mL) and dried with anhydrous sodium
sulfate. The residue obtained after evaporation of the organic layer
under reduced pressure was purified on a silica-gel column pre-
pared with 1% triethylamine in cyclohexane and washed with cyclo-
hexane (1-bed volume) before the products were deposited. Elution
was performed with a stepwise gradient of ethyl acetate (0Ϫ50%)
in cyclohexane, fractions containing the desired product were com-
bined and the solvents evaporated to dryness. The residue was ly-
ophilized from dioxane affording a colorless powder: 3a (59%), 3b
(38%), 3c (57%) and 3d (70%). 31P NMR showed also the presence
of few percent of hydrolyzed chlorophosphane (ഠ 7 ppm) and
phosphite triester (ഠ140 ppm). As these side compounds will not
react, the mixture was used without a second purification. Owing
to their high molecular weight FAB MS showed only characteristic
fragments of the molecules.
General Procedure for the Me-SATE Phosphitylation of N-SiOMB-
5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-deoxynucleosides (2a؊c): Prior to
use, the N-SiOMB-5Ј-O-Dmtr-2Ј-deoxynucleosides 1aϪc (1 mmol)
and diisopropylammonium tetrazolide (85.5 mg, 0.5 mmol) were
separately dried three times by coevaporation with anhydrous
acetonitrile, and then mixed and dissolved in anhydrous dichloro-
methane (7 mL). A solution of [2-(acetylthio)ethyloxy]bis(diisopro-
pylamino)phosphane (421 mg, 1.2 mmol) in dichloromethane
(3 mL) was added under argon. The resulting mixture was stirred
at room temperature overnight, then diluted with ethyl acetate
(50 mL) and washed with saturated aqueous NaHCO3 (25 mL) and
then with brine (50 mL). The organic layer was dried (Na2SO4),
filtered, and the solvents were evaporated to dryness. The residues
were purified by flash column chromatography (silica gel; gradient
30Ϫ100% ethyl acetate/1% Et3N/cyclohexane). The appropriate
fractions were combined, concentrated to dryness, dissolved in a
few mL of toluene, and then precipitated in cold hexane (Ϫ78 °C).
The resulting precipitate was dried under vacuum to afford the re-
sulting phosphoramidites 2a (70%), 2b (82%), or 2c (84%), as color-
less powders.
N6-{2-[(tert-Butyl)(diphenyl)silyloxymethyl]benzoyl}-5Ј-O-(4,4Ј-
dimethoxytrityl)-2Ј-deoxyadenosine 3Ј-O-[2-(acetylthio)ethyl] Diiso-
propylphosphoramidite (2a): 0.77 mmol, 710 mg of 1a gave 631 mg,
70% yield, TLC (Cyclohexane/EtOAc/Et3N, 20:70:10, v/v/v), Rf ϭ
0.55. 1H NMR (CDCl3): δ ϭ 8.37 (s, 1 H, 8-H), 8.09 (s, 1 H, 2-H),
7.63Ϫ7.01 (m, 23 H, H aromatic), 6.68 (d, 4 H, Hα of OCH3), 6.32
(q, 1 H, 1Ј-H), 4.92 (s, 2 H, CH2OSi), 4.81Ϫ4.68 (m, 1 H, 3Ј-H),
4.17Ϫ4.09 (m, 1 H, 4Ј-H), 3.62 (s, 6 H, 2 ϫ OCH3), 3.30Ϫ3.15 (m,
4 H, 2 ϫ CH2), 3.01Ϫ2.86 (m, 4 H, 5Ј-H 5ЈЈ-H 2 ϫ CH), 2.58Ϫ2.41
(m, 1 H, 2Ј-H), 2.17 (s, 3 H, SCOCH3), 2.13Ϫ2.01 (m, 1 H, 2ЈЈ-H),
1.04 [s, 12 H, N[CH(CH3)2]2), 0.92 [s, 9 H, C(CH3)3] ppm. 31P
NMR (CD3CN): δ ϭ 148.9 and 148.7 ppm.
N6-{2-[(tert-Butyl)(diphenyl)silyloxymethyl]benzoyl}-5Ј-O-(4,4Ј-
dimethoxytrityl)-2Ј-deoxyadenosine
3Ј-O-[(2-trimethylsilyl) ethyl]
Diisopropylphosphoramidite (3a): 1a (552 mg, 0.60 mmol) gave 3a
in 59% yield (413 mg). TLC (EtOAc/cyclohexane/Et3N, 60:30:10,
v/v/v). Rf ϭ 0.57. 31P NMR (CD3CN): δ ϭ 147.13 and 146.89 ppm,
plus 1.3% of hydrolyzed chlorophosphane (δ ϭ 7.1 ppm).
N4-{2-[(tert-Butyl)(diphenyl)silyloxymethyl]benzoyl}-5Ј-O-(4,4Ј-
dimethoxytrityl)-2Ј-deoxycytidine 3Ј-O-[(2-trimethylsilyl) ethyl] Di-
isopropylphosphoramidite (3b): 1b (724 mg, 0.80 mmol) gave 3b in
38% yield (351 mg). TLC (EtOAc/cyclohexane/Et3N, 50:40:10, v/v/
v). Rf ϭ 0.62. 31P NMR (CD3CN): δ ϭ 147.31 and 147.14 ppm
plus 7% of hydrolyzed chlorophosphane (δ ϭ 7.2 ppm) and 10%
phosphite triester (δ ϭ 140.5 ppm).
N4-{2-[(tert-Butyl)(diphenyl)silyloxymethyl]benzoyl}-5Ј-O-(4,4Ј-
dimethoxytrityl)-2Ј-deoxycytidine 3Ј-O-[2-(acetylthio)ethyl] Diiso-
propylphosphoramidite (2b): 3.0 mmol, 2.8 g of 1b gave 2.8 g, 82%
yield, TLC (Cyclohexane/EtOAc/Et3N, 40:60:10, v/v/v): Rf ϭ 0.41
and 0.45. 1H NMR (CD3CN): δ ϭ 9.15 (br. s, 1 H, NH), 8.16 (dd,
1 H, 6-H), 6.99Ϫ7.61 (m, 24 H, H aromatic), 6.78 (dd, 4 H, H
aromatic meta of OCH3), 6.04 (q, 1 H, 1Ј-H), 4.89 (s, 2 H, CH2Si),
4.51 (d, 1 H, 3Ј-H), 4.02 (q, 1 H, 4Ј-H), 3.65 (s, 6 H, 2 ϫ OCH3),
3.51 (m, 3 H, CH2 ϩ2 ϫ CH), 3.32 (m, 2 H, CH2), 3.01 (m, 1 H,
5ЈЈ-H), 2.89 (m, 1 H, 5Ј-H), 2.50 (m, 1 H, 2Ј-H), 2.22 (m, 1 H, 2ЈЈ-
H), 2.13 (s, 3 H, SCOCH3), 1.09 [d, 12 H, N{CH(CH3)2}2), 0.90
[s, 9 H, C(CH3)3] ppm. 31P NMR (CD3CN): δ ϭ 149.16 and 148.90
ppm. ϩFAB (thioglycerol): m/z ϭ 1151 [M ϩ H]ϩ.
N2-{2-[(tert-Butyl)(diphenyl)silyloxymethyl]benzoyl}-5Ј-O-(4,4Ј-
dimethoxytrityl)-2Ј-deoxyguanosine
3Ј-O-[(2-trimethylsilyl) ethyl]
Diisopropylphosphoramidite (3c): 1c (0.94 mmol, 889 mg) gave 3c in
57% yield (678 mg). TLC (CH2Cl2/EtOAc/Et3N, 50:40:10, v/v/v).
Rf ϭ 0.41. 31P NMR (CD3CN): δ ϭ 147.17 and 147.09 ppm, plus
5% of hydrolyzed chlorophosphane (δ ϭ 7.0 ppm) and 18% of
phosphite triester (δ ϭ 140.0 ppm).
5Ј-O-(4,4Ј-Dimethoxytrityl)thymidine 3Ј-O-[2-(Trimethylsilyl)ethyl]
N2-{2-[(tert-Butyl)(diphenyl)silyloxymethyl]benzoyl}-5Ј-O-(4,4Ј- Diisopropylphosphoramidite (3d): 1d (3.3 mmol, 1.14 g) gave 3d in
dimethoxytrityl)-2Ј-deoxyguanosine 3Ј-O-[2-(acetylthio)ethyl] Diiso-
propylphosphoramidite (2c): 1c (365.mg, 0.39 mmol) gave 389 mg, v). Rf ϭ 0.48. 31P NMR (CD3CN): δ ϭ 147.0 and 146.7 ppm,
84%, TLC (EtOAc/CH3CN/Et3N, 60:30:10, v/v/v). Rf ϭ 0.46. 1H
plus 7% of hydrolyzed chlorophosphane (δ ϭ 7.7 ppm) and 9% of
NMR (CDCl3): δ ϭ 7.89 (s, 1 H, 8-H), 7.75Ϫ7.12 (m, 23 H, H phosphite triester (δ ϭ 140.1 ppm).
70% yield (1.81 g). TLC (EtOAc/cyclohexane/Et3N, 50:40:10, v/v/
Eur. J. Org. Chem. 2003, 2327Ϫ2335
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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