Nucleic Acid Containing 3′-C-P-N-5′ Ethyl Phosphonamidate Ester and 2′-Methoxy Modifications in Combination; Synthesis and Hybridisation
Properties

Abstract

The preparation of thymidine-thymidine and thymidine-5-methylcytidine dinucleosides containing a 3′-C-P-N-5′ ethyl phosphonamidate ester linkage, with defined phosphorus stereochemistry, in combination with a 2′-methoxy substituent in the lower sugar residue, is described. Incorporation of these dinucleosides into DNA oligonucleotides and the effect upon duplex stability with complimentary RNA is reported.

antisense - modified oligonucleotides - nucleosides - phosphorus - stereoselective

References

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New address: Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex, RH12 5AB, UK.

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Reaction conditions were as described in ref. [5] Flash column chromatography was performed using Merck Silica Gel 60 (0.040-0.063 mm). NMR spectra were recorded with a Brucker AC400 instrument. Key distinguishing ¹H resonances for each diastereoisomer are assigned. ³¹P NMR shifts are given as ppm values relative to phosphoric acid. Mass spectroscopy was carried out using a Fisons Instruments VG Platform II spectrometer. A reaction carried out on a 3.11 mmol scale gave;
5: White amorphous foam; 1.07 g; ³¹P NMR (CDCl₃, 162 MHz): δ = 32.52 ppm; ¹H NMR (CDCl₃, 400 MHz): δ = 9.67 (br s, 1 H), 9.52 (s, br, 1 H), 7.69-7.61 (m, 4 H), 7.45-7.34 (m, 7 H), 7.13 (s, 1 H), 6.10-6.02 (m, 1 H, H1′ upper sugar), 5.50 (d, J = 2 Hz, 1 H, H1′ lower sugar), 4.26-3.64 (m, 8 H), 3.50 (s, 3 H), 3.36-3.17 (m, 3 H), 2.74-2.61 (m, 1 H), 2.51-2.42 (m, 1 H), 2.32-2.20 (m, 1 H), 2.04-1.82 (m, 2 H), 1.84 (s, 3 H), 1.74-1.59 (m, 1 H), 1.58 (s, 3 H), 1.27 (t, J = 7 Hz, 3 H), 1.04 (s, 9 H). MS (ES+): m/z (%) = 840(27) [M + H], 862(100) [M + Na].
6: White amorphous foam; 0.94 g; ³¹P NMR (CDCl₃, 162 MHz): δ = 32.81 ppm; ¹H NMR (CDCl₃, 400 MHz): δ = 9.38 (br s, 1 H), 9.17 (s, br, 1 H), 7.70-7.62 (m, 4 H), 7.44-7.34 (m, 7 H), 7.13 (s, 1 H), 6.14-6.06 (m, 1 H, H1′ upper sugar), 5.55 (d, J = 2 Hz, 1 H, H1′ lower sugar), 4.24-3.64 (m, 8 H), 3.51 (s, 3 H), 3.50-3.18 (m, 3 H), 2.80-2.68 (m, 1 H), 2.50-2.39 (m, 1 H), 2.34-2.23 (m, 1 H), 2.01-1.84 (m, 1 H), 1.84 (s, 3 H), 1.77-1.58 (m, 2 H), 1.61 (s, 3 H), 1.20-1.10 (m, 3 H), 1.04 (s, 9 H). MS (ES+): m/z (%) = 840(8) [M + H], 862(100) [M + Na].

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Reaction conditions were as described in ref. [5] A reaction carried out on a 1.46 mmol scale gave;
13: White amorphous foam; 1.54 g; ³¹P NMR (CDCl₃, 162 MHz): δ = 32.70 ppm; ¹H NMR (CDCl₃, 400 MHz): δ = 8.62 (s, 1 H), 8.15 (d, 2 H, J = 7 Hz), 7.58-7.44 (m, 8 H), 7.35-7.31 (m, 1 H), 7.30-7.17 (m, 16 H), 7.08 (s, 1 H), 5.96-5.90 (m, 1 H, H1′ upper sugar), 5.42 (d, 1 H, J = 2 Hz, H1′ lower sugar), 3.97-3.89 (m, 1 H), 3.86-3.77 (m, 2 H), 3.69-3.53 (m, 4 H), 3.10 (s, 3 H), 2.98-2.81 (m, 2 H), 2.76-2.63 (m, 1 H), 2.59-2.48 (m, 1 H), 2.29-2.20 (m, 1 H), 2.03-1.94 (m, 1 H), 1.91 (s, 3 H), 1.76-1.60 (m, 1 H), 1.44 (s, 3 H), 1.38-1.14 (m, 1 H), 1.07-0.99 (m, 3 H), 0.94 (s, 9 H), 0.91 (s, 9 H). Minor (S _P)-diastereoisomer; ³¹P NMR (CDCl₃, 162 MHz): δ = 31.71 ppm; ¹H NMR (CDCl₃, 400 MHz): key distinguishing resonances δ = 5.90-5.83 (m, 1 H, H1′ upper sugar), 5.29 (d, 1 H, J = 2 Hz, H1′ lower sugar).

<A NAME="RD01702ST-14A">14a</A> Oligonucleotides were prepared using an ABI 390 DNA synthesiser following standard phosphoramidite chemistry, according to: Gait: MJ. Oligonucleotide Synthesis: A Practical Approach   IRL Press; Oxford: 1984. 

<A NAME="RD01702ST-14B">14b</A>

For the steps involving incorporation of modified dimers, double couplings with double reaction times were employed.

<A NAME="RD01702ST-15A">15a</A> The thermal denaturation of DNA/RNA hybrids was performed at 260 nm using a Gifford Response II spectrophotometer (Ciba-Corning Diagnostics Corp., Oberlin, OH) absorbance vs. temperature profiles were measured at 4 µm of each strand in 10 mM phosphate pH 7.0 (Na salts), 100 mM total (Na⁺) 0.1 mM EDTA. T_m’s were obtained from fits of absorbance vs. temperature curves to a two-state model with linear slope baselines: Freier SM. Albergo TD. Turner DH. Biopolymers  1982,  22:  1107 

<A NAME="RD01702ST-15B">15b</A>

All values are averages of at least three experiments. The absolute error of the T_m values is ±0.5 °C.

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