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.

Key words

antisense - modified oligonucleotides - nucleosides - phosphorus - stereoselective

References

• 2a Wahlestedt C. Good L. Curr. Opin. Drug Discov. Dev.  1999,  2:  142 
• 2b Bennett CF. Exp. Opin. Invest. Drugs  1999,  8:  237 
• 2c Ma DDF. Rede T. Naqvi NA. Cook PD. Biotechnol. Annu. Rev.  2000,  5:  155 
• 2d Bennett CF. Butler M. Cook PD. Geary RS. Levin AA. Mehta R. Teng C.-L. Deshmukh H. Tillman L. Hardee G. Gene Ther.   Marcel Dekker Inc.; New York: 2000.  p.305 
• 2e Uhlmann E. Curr. Opin. Drug Discovery Dev.  2000,  3:  203 
• 2f Galderisi U. Cipollaro M. Cascino A. Emerging Drugs  2001,  6:  69 
• 3a Freier SM. Altmann K.-H. Nucleic Acids Res.  1997,  25:  4429 
• 3b Altmann K.-H. Cuenoud B. Von Matt P. In Applied Antisense Oligonucleotide Technology   Krieg AM. Stein CA. Wiley-Liss Inc.; New York: 1998. 
• 3c Egli M. Gryaznov SM. Cell. Mol. Life Sci.  2000,  57:  1440 
• 3d Engels JW. Uhlmann E. Pharm. Aspects Oligonucleotides  2000,  35 
• 3e An H. Wang T. Maier MA. Manoharan M. Ross BS. Cook PD. J. Org. Chem.  2001,  66:  2789 
• 4a Crooke ST. Lemonidis KM. Neilson L. Griffey R. Lesnik EA. Monia BP. Biochem.  1995,  312:  599 
• 4b McKay RA. Miraglia LJ. Cummins LL. Owens SR. Sasmor H. Dean NM. J. Biol. Chem.  1999,  274:  1715 
• 4c Cramer H. Pfleiderer W. Nuleosides, Nucleotides, Nucleic Acids  2000,  19:  1765 
• 4d Malchere C. Verheijien J. Van Der Laan S. Bastide L. Van Boom J. Lebleu B. Robbins I. Antisense Nucleic Acid Drug Dev.  2000,  10:  463 
• 4e Kværnø L. Wengel J. Chem. Commun.  2001,  1419 
• 5 Fairhurst RA. Collingwood SP. Lambert D. Taylor RJ. Synlett  2001,  467 
• 6 Fairhurst RA. Collingwood SP. Lambert D. Synlett  2001,  473 
• 7a Griffey RH. Lesnik E. Freier S. Sanghvi YS. Teng K. Kawasaki A. Guinosso C. Wheeler P. Mohan V. Cook PD. ACS Symp. Ser.  1994,  580:  212 
• 7b Manoharan M. Biochim. Biophys. Acta  1999,  1489:  117 
• 8a Inoue H. Hayase Y. Imura A. Iwai S. Miura K. Ohtsuka E. Nucleic Acids Res.  1987,  15:  6131 
• 8b Lamond AI. Sproat BS. FEBS Lett.  1993,  325:  123 
• 9a De Mesmaeker A. Lesueur C. Bévièrre M.-O. Waldner A. Fritsch V. Wolf VF. Angew. Chem., Int. Ed. Engl.  1996,  35:  2790 
• 9b De Mesmaeker A. Lebreton J. Jouanno C. Fritsch V. Wolf RM. Wendeborn S. Synlett  1997,  1287 
• 9c Pfundheller HM. Wengel J. Bioorg. Med. Chem. Lett.  1999,  9:  2667 
• 10 Huang J. McElroy EB. Widlanski TS. J. Org. Chem.  1994,  59:  3520 
• 12a Kim CH. Marquez VE. Broder S. Mitsuya H. Driscoll JS. J. Med. Chem.  1987,  30:  862 
• 12b Ross BS. Springer RH. Vasquez G. Andrews RS. Cook PD. Acevedo OL. J. Heterocycl. Chem.  1994,  31:  765 
• 14a 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. 
• 14b

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

• 15a 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 
• 15b

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

• 16 Martin P. Helv. Chim. Acta  1995,  78:  486 
• 17a Pless RC. Ts’o POP. Biochemistry  1977,  16:  1239 
• 17b Summers MF. Powell C. Egan W. Byrd RA. Wilson WD. Zon G. Nucleic Acids Res.  1986,  14:  7421 
• 17c Ferguson DM. Kollman PA. Antisense Research and Development  1991,  1:  243 
• 18 Sanghvi YS. Hoke GD. Freier SM. Zounes MC. Gonzalez C. Cummins L. Sasmor H. Cook PD. Nucleic Acids Res.  1993,  21:  3197 

1

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

11

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].

13

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).