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Synthesis of backbone-modified DNA analogues for biological applications

Abstract

The phosphite triester method was adapted for automated synthesis of phosphate (“backbone”)-modified analogues of DNA. The use of phosphoramidite reagents to achieve substitution of the nonequivalent (diastereotopic) oxygens of an internucleotide phosphate linkage is nonselective, and leads to nonequivalent (diastereomeric) strands of DNA with R p and S p absolute configurations at the chiral, modified phosphorus position. Indications of the scope and limitations of the use of reversed-phase HPLC to separate these diastereomers have been obtained through studies of backbone-modified DNA analogues having phosphorothioate (P-S), phosphotriester (P-OR), and alkanephosphonate (P-R) linkages. Incorporation of these modifications in the self-complementary octanucleotide d(GGAATTCC) led to separable diastereomers, which form nonequivalent R p · R p and S p · S p duplexes. A combination of chemical and nuclear Overhauser effect NMR spectroscopic methods was developed to assign unambiguously, for the first time, the absolute configurations at phosphorus in these prototypal cases. The effects of backbone ethylation on DNA structure and dynamics were evaluated by NMR methods. Modified duplexes were used to probe for proposed phosphate contacts for EcoRI endonuclease, and to define, in concert with base-modified analogues, a recognition site for monoclonal anti-native DNA autoantibody.

References

  1. Andrews, B. S., Eisenberg, R. S., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. J., Murphy, E. D., and Roths, J. B. (1978). J. Exp. Med. 148, 1198–1215.

  2. Broido, M. S., James, T. L., Zon, G., and Keepers, J. (1985). Eur. J. Biochem. 150, 117–128.

  3. Bryant, R. F., and Benkovic, S. J. (1979). Biochemistry 18, 2825–2828.

  4. Burgers, P. M. J., Eckstein, F., and Hunneman, D. H. (1979). J. Biol. Chem. 254, 7476–7478.

  5. Caruthers, M. H. (1985). Science 230, 281–285.

  6. Chacko, K. K., Lindner, K., Saenger, W., and Miller, P. S. (1983). Nucleic Acids Res. 11, 2801–2814.

  7. Connolly, B. A., Potter, B. V. L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984a). Biochemistry 23, 3443–3453.

  8. Connolly, B. A., Eckstein, F., and Pingoud, A. (1984b). J. Biol. Chem. 259, 10760–10763.

  9. Eckstein, F. (1983). Angew. Chem. Int. Ed. Engl. 22, 423–439.

  10. Frederick, C. A., Grable, J., Melia, M., Samudzi, C., Jen-Jacobson, L., Wang, B.-C., Greene, P., Boyer, H. W., and Rosenberg, J. M. (1984). Nature 309, 327–331.

  11. Jäger, A., and Engels, J. (1984). Tetrahedron Lett. 25, 1437–1440.

  12. Koziołkiewicz, M., Uznański, B., Stec, W. J., and Zon, G. (1986). Chem. Scr., 26, 251–260.

  13. Lu, A.-L., Jack, W. E., and Modrich, P. (1981). J. Biol. Chem. 256, 13200–13206.

  14. Miller, P. S., Chandrasegaran, S., Dow, D. L., Pulford, S. M., and Kan, L. S. (1982). Biochemistry 21, 5468–5474.

  15. Noble, S. A., Fisher, E. F., and Caruthers, M. H. (1984). Nucleic Acids Res. 12, 3387–3404.

  16. Phillips, L. R., Gallo, K. A., Zon, G., Stec, W. J., and Uznański, B. (1985). Org. Mass. Spectrom. 20, 781–783.

  17. Potter, B. V. L., Connolly, B. A., and Eckstein, F. (1983). Biochemistry 22, 1369–1377.

  18. Stec, W. J., and Zon, G. (1984a). Tetrahedron Lett. 25, 5279–5282.

  19. Stec, W. J., and Zon, G. (1984b). Tetrahedron Lett. 25, 5275–5278.

  20. Stec, W. J., and Zon, G. (1985). In Natural Products Chemistry 1984 (Zalewski, R. I., and Skolik, J. J., eds.), Elsevier, Amsterdam, pp. 495–510.

  21. Stec, W. J., Zon, G., Egan, W., and Stec, B. (1984). J. Am. Chem. Soc. 106, 6077–6079.

  22. Stec, W. J., Zon, G., and Uznański, B. (1985a). J. Chromatogr. 326, 263–280.

  23. Stec, W. J., Zon, G., Gallo, K. A., Byrd, R. A., Uznański, B., and Guga, P. (1985b). Tetrahedron Lett. 26, 2191–2194.

  24. Stec, W. J., Zon, G., Egan, W., Byrd, R. A., Phillips, L. R., and Gallo, K. A. (1985c) J. Org. Chem. 50, 3908–3913.

  25. Stollar, B. D. (1981). Clin. Immunol. Allergy 1, 243–260.

  26. Stollar, B. D. (1985). CRC Crit. Rev. Biochem., in press.

  27. Stollar, B. D., and Papalian, M. (1980). J. Clin. Invest. 66, 210–219.

  28. Stollar, B. D., Huang, B. S., and Blumenstein, M. (1986). In Fourth Conversation in Biomolecular Stereodynamics (Sarma, R. H., ed.), Academic Press, New York, in press.

  29. Uznański, B., Koziołkiewicz, M., Stec, W. J., Zon, G., Shinozuka, K., and Marzilli, L. G. (1986). Chem. Scr., 26, 221–224.

  30. Yamana, K., and Letsinger, R. L. (1985). Nucleic Acids Res. Symp. Ser. 16, 169–172.

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This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.

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Zon, G. Synthesis of backbone-modified DNA analogues for biological applications. J Protein Chem 6, 131–145 (1987). https://doi.org/10.1007/BF00247762

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Key words

  • synthesis using phosphoramidites
  • absolute stereochemistry of phosphorothioate
  • phosphotriester
  • methane-phosphonate internucleotide linkages