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. 1990 Sep 11;18(17):5197–5205. doi: 10.1093/nar/18.17.5197

Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

W H Kuijpers 1, J Huskens 1, L H Koole 1, C A van Boeckel 1
PMCID: PMC332142  PMID: 2402444

Abstract

A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included.

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Selected References

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  1. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  2. Gallo K. A., Shao K. L., Phillips L. R., Regan J. B., Koziolkiewicz M., Uznanski B., Stec W. J., Zon G. Alkyl phosphotriester modified oligodeoxyribonucleotides. V. Synthesis and absolute configuration of Rp and Sp diastereomers of an ethyl phosphotriester (Et) modified EcoRI recognition sequence, d[GGAA(Et)TTCC]. A synthetic approach to regio- and stereospecific ethylation-interference studies. Nucleic Acids Res. 1986 Sep 25;14(18):7405–7420. doi: 10.1093/nar/14.18.7405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hassner A., Strand G., Rubenstein M., Patchornik A. Letter: Levulinic esters. An alcohol protecting group applicable to some nucleosides. J Am Chem Soc. 1975 Mar 19;97(6):1614–1615. doi: 10.1021/ja00839a077. [DOI] [PubMed] [Google Scholar]
  4. Ma Y., Sonveaux E. The 9-fluorenylmethyloxycarbonyl group as a 5'-OH protection in oligonucleotide synthesis. Biopolymers. 1989 May;28(5):965–973. doi: 10.1002/bip.360280505. [DOI] [PubMed] [Google Scholar]
  5. Marcus-Sekura C. J. Techniques for using antisense oligodeoxyribonucleotides to study gene expression. Anal Biochem. 1988 Aug 1;172(2):289–295. doi: 10.1016/0003-2697(88)90447-2. [DOI] [PubMed] [Google Scholar]
  6. Matsukura M., Zon G., Shinozuka K., Stein C. A., Mitsuya H., Cohen J. S., Broder S. Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV). Gene. 1988 Dec 10;72(1-2):343–347. doi: 10.1016/0378-1119(88)90161-8. [DOI] [PubMed] [Google Scholar]
  7. Miller P. S., Barrett J. C., Ts'o P. O. Synthesis of oligodeoxyribonucleotide ethyl phosphotriesters and their specific complex formation with transfer ribonucleic acid. Biochemistry. 1974 Nov 19;13(24):4887–4896. doi: 10.1021/bi00721a003. [DOI] [PubMed] [Google Scholar]
  8. Miller P. S., Fang K. N., Kondo N. S., Ts'o P. O. Syntheses and properties of adenine and thymine nucleoside alkyl phosphotriesters, the neutral analogs of dinucleoside monophosphates. J Am Chem Soc. 1971 Dec;93(24):6657–6665. doi: 10.1021/ja00753a054. [DOI] [PubMed] [Google Scholar]
  9. Miller P. S., McParland K. B., Jayaraman K., Ts'o P. O. Biochemical and biological effects of nonionic nucleic acid methylphosphonates. Biochemistry. 1981 Mar 31;20(7):1874–1880. doi: 10.1021/bi00510a024. [DOI] [PubMed] [Google Scholar]
  10. Miller P. S., Reddy M. P., Murakami A., Blake K. R., Lin S. B., Agris C. H. Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates. Biochemistry. 1986 Sep 9;25(18):5092–5097. doi: 10.1021/bi00366a017. [DOI] [PubMed] [Google Scholar]
  11. Miller P. S., Ts'o P. O. A new approach to chemotherapy based on molecular biology and nucleic acid chemistry: Matagen (masking tape for gene expression). Anticancer Drug Des. 1987 Oct;2(2):117–128. [PubMed] [Google Scholar]
  12. Moody H. M., van Genderen M. H., Koole L. H., Kocken H. J., Meijer E. M., Buck H. M. Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides. Nucleic Acids Res. 1989 Jun 26;17(12):4769–4782. doi: 10.1093/nar/17.12.4769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Scanlon D., Haralambidis J., Southwell C., Turton J., Tregear G. Purification of synthetic oligodeoxyribonucleotides by ion-exchange high-performance liquid chromatography. J Chromatogr. 1984 Dec 7;336(1):189–198. doi: 10.1016/s0378-4347(00)85141-5. [DOI] [PubMed] [Google Scholar]
  14. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Stein C. A., Cohen J. S. Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer Res. 1988 May 15;48(10):2659–2668. [PubMed] [Google Scholar]
  16. Summers M. F., Powell C., Egan W., Byrd R. A., Wilson W. D., Zon G. Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2. Nucleic Acids Res. 1986 Sep 25;14(18):7421–7436. doi: 10.1093/nar/14.18.7421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Toulmé J. J., Hélène C. Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review. Gene. 1988 Dec 10;72(1-2):51–58. doi: 10.1016/0378-1119(88)90127-8. [DOI] [PubMed] [Google Scholar]
  18. Zon G. Oligonucleotide analogues as potential chemotherapeutic agents. Pharm Res. 1988 Sep;5(9):539–549. doi: 10.1023/a:1015985728434. [DOI] [PubMed] [Google Scholar]

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