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. 1996 May 15;24(10):1841–1848. doi: 10.1093/nar/24.10.1841

Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

S Peyrottes 1, J J Vasseur 1, J L Imbach 1, B Rayner 1
PMCID: PMC145873  PMID: 8657564

Abstract

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.

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Selected References

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  1. Alul R. H., Singman C. N., Zhang G. R., Letsinger R. L. Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives. Nucleic Acids Res. 1991 Apr 11;19(7):1527–1532. doi: 10.1093/nar/19.7.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloch E., Lavignon M., Bertrand J. R., Pognan F., Morvan F., Malvy C., Rayner B., Imbach J. L., Paoletti C. Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase. Gene. 1988 Dec 10;72(1-2):349–360. doi: 10.1016/0378-1119(88)90162-x. [DOI] [PubMed] [Google Scholar]
  3. Dagle J. M., Walder J. A., Weeks D. L. Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis. Nucleic Acids Res. 1990 Aug 25;18(16):4751–4757. doi: 10.1093/nar/18.16.4751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Durand M., Maurizot J. C., Asseline U., Barbier C., Thuong N. T., Hélène C. Oligothymidylates covalently linked to an acridine derivative and with modified phosphodiester backbone: circular dichroism studies of their interactions with complementary sequences. Nucleic Acids Res. 1989 Mar 11;17(5):1823–1837. doi: 10.1093/nar/17.5.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fathi R., Huang Q., Syi J. L., Delaney W., Cook A. F. (Aminomethyl)phosphonate derivatives of oligonucleotides. Bioconjug Chem. 1994 Jan-Feb;5(1):47–57. doi: 10.1021/bc00025a007. [DOI] [PubMed] [Google Scholar]
  6. Froehler B., Ng P., Matteucci M. Phosphoramidate analogues of DNA: synthesis and thermal stability of heteroduplexes. Nucleic Acids Res. 1988 Jun 10;16(11):4831–4839. doi: 10.1093/nar/16.11.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giles R. V., Tidd D. M. Enhanced RNase H activity with methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides. Anticancer Drug Des. 1992 Feb;7(1):37–48. [PubMed] [Google Scholar]
  8. Giles R. V., Tidd D. M. Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures. Nucleic Acids Res. 1992 Feb 25;20(4):763–770. doi: 10.1093/nar/20.4.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goodchild J. Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjug Chem. 1990 May-Jun;1(3):165–187. doi: 10.1021/bc00003a001. [DOI] [PubMed] [Google Scholar]
  10. Kean J. M., Cushman C. D., Kang H., Leonard T. E., Miller P. S. Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides. Nucleic Acids Res. 1994 Oct 25;22(21):4497–4503. doi: 10.1093/nar/22.21.4497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Letsinger R. L., Bach S. A., Eadie J. S. Effects of pendant groups at phosphorus on binding properties of d-ApA analogues. Nucleic Acids Res. 1986 Apr 25;14(8):3487–3499. doi: 10.1093/nar/14.8.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Milligan J. F., Matteucci M. D., Martin J. C. Current concepts in antisense drug design. J Med Chem. 1993 Jul 9;36(14):1923–1937. doi: 10.1021/jm00066a001. [DOI] [PubMed] [Google Scholar]
  13. Milligan J. F., Matteucci M. D., Martin J. C. Current concepts in antisense drug design. J Med Chem. 1993 Jul 9;36(14):1923–1937. doi: 10.1021/jm00066a001. [DOI] [PubMed] [Google Scholar]
  14. Morvan F., Porumb H., Degols G., Lefebvre I., Pompon A., Sproat B. S., Rayner B., Malvy C., Lebleu B., Imbach J. L. Comparative evaluation of seven oligonucleotide analogues as potential antisense agents. J Med Chem. 1993 Jan 22;36(2):280–287. doi: 10.1021/jm00054a013. [DOI] [PubMed] [Google Scholar]
  15. Nielsen P., Kirpekar F., Wengel J. Incorporation of (R)- and (S)-3',4'-seco-thymidine into oligodeoxynucleotides: hybridization properties and enzymatic stability. Nucleic Acids Res. 1994 Mar 11;22(5):703–710. doi: 10.1093/nar/22.5.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Polushin N. N., Morocho A. M., Chen B. C., Cohen J. S. On the rapid deprotection of synthetic oligonucleotides and analogs. Nucleic Acids Res. 1994 Feb 25;22(4):639–645. doi: 10.1093/nar/22.4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Potts J. D., Dagle J. M., Walder J. A., Weeks D. L., Runyan R. B. Epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor beta 3. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1516–1520. doi: 10.1073/pnas.88.4.1516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sinha N. D., Davis P., Usman N., Pérez J., Hodge R., Kremsky J., Casale R. Labile exocyclic amine protection of nucleosides in DNA, RNA and oligonucleotide analog synthesis facilitating N-deacylation, minimizing depurination and chain degradation. Biochimie. 1993;75(1-2):13–23. doi: 10.1016/0300-9084(93)90019-o. [DOI] [PubMed] [Google Scholar]
  19. Stein C. A., Subasinghe C., Shinozuka K., Cohen J. S. Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 1988 Apr 25;16(8):3209–3221. doi: 10.1093/nar/16.8.3209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stein C. A., Tonkinson J. L., Yakubov L. Phosphorothioate oligodeoxynucleotides--anti-sense inhibitors of gene expression? Pharmacol Ther. 1991 Dec;52(3):365–384. doi: 10.1016/0163-7258(91)90032-h. [DOI] [PubMed] [Google Scholar]
  21. Vyazovkina E. V., Savchenko E. V., Lokhov S. G., Engels J. W., Wickstrom E., Lebedev A. V. Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC). Nucleic Acids Res. 1994 Jun 25;22(12):2404–2409. doi: 10.1093/nar/22.12.2404. [DOI] [PMC free article] [PubMed] [Google Scholar]

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