Application of Reverse (5’ to 3’) Oligonucleotides

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The chemical synthesis of DNA using the phosphoramidite method proceeds in a 3’
to 5’ direction principally as a consequence of the use of building blocks activated as 3’-O-phosphoramidites. The primary 5’-OH group is significantly more reactive than the secondary 3’-OH (or 2’-OH) group, making it straightforward to protect with the DMT group leaving the 3’-OH available to form the phosphoramidite. In contrast, ‘reverse’ oligonucleotide synthesis (i.e. in a 5’ to 3’ direction) has not been utilised to nearly the same extent.

Nevertheless, there are several applications of this chemistry, most notably in nuclease resistance. An interesting addition to the protection of antisense oligonucleotides is to modify the terminal linkages from the natural 3’-5’ to 3’-3’ and/or 5’-5’ linkages. In this way, the oligonucleotides are protected against exonuclease activity, especially 3’-exonuclease activity which is by far the most significant enzymatic degradation route, resulting in nucleosides with no toxicity concerns. This strategy has been applied by Beaucage and co-workers who have
used 5’-O-phosphoramidites in the formation of oligonucleotides having alternating 3’-3’ and 5’-5’ linkages to maintain effective hybridisation.1 A simpler approach is in fact to modify only the linkage at the 3’ terminus.2 This is conveniently carried out and results in effective resistance with minimal disruption to hybridisation.

In addition to the established applications in nuclease resistance and hairpin loops, other technologies exploit the flexibility of reverse oligo synthesis. For example, with the increasing use of DNA chip technology, interest has focused upon the synthesis of support-bound, fully deprotected oligonucleotides.3 Such molecules are accessible through the use of 2-(4-nitrophenyl)-ethyl/[2-(4-nitrophenyl)ethoxy]carbonyl (npe/npeoc) protecting groups4 on the nucleobase.

The use of 5’-O-phosphoramidites has not generally been used for the elaboration
of oligonucleotides, even though this approach offers a facile route to 3’-modified
oligodeoxynucleotides. The potential for this approach has recently been demonstrated by Hecht and co-workers using a phosphoramidite derived from tyrosine.5 The derived oligonucleotide was shown to have chromatographic and electrophoretic properties identical with the modified oligo resulting from the proteinase K digestion of a topoisomerase-DNA complex.

We offer reverse phosphoramidites (2020 - 2023, 2093) and solid supports (2294, 2298, 2355, 2356), with classical heterocyclic base protection groups.

Applicable products: 2020, 2021, 2022, 2023, 2093, 2294, 2298, 2355, 2356.

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References

  1. (a) Alternating α,β-oligothymidylates with alternating (3’-3’)- and (5’-5’)-internucleotidic phosphodiester linkages as models for antisense oligodeoxyribonucleotides, M. Koga, M.F. Moore and S.L. Beaucage, J. Org. Chem., 56, 3757-3759, 1991; Abstract (b) Synthesis and physicochemical properties of alternating α,β-oligodeoxyribonucleotides with alternating (3’-3’)- and (5’-5’)-internucleotidic phosphodiester linkages, M. Koga, A. Wilk, M.F. Moore, C.L. Scremin, L. Zhou and S.L. Beaucage, J. Org. Chem., 60, 1520-1530, 1995.
  2. (a) Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation, J.F.R. Ortigao, H. Rosch, H. Selter, A. Frohlich, A. Lorenz, M. Montenarh and H. Seliger, Antisense Res. & Dev., 2, 129-146, 1992; (b) Oligonucleotide analogs with terminal 3’-3’- internucleotidic and 5’-5’-internucleotidic linkages as antisense inhibitors of viral gene-expression, H. Seliger, A. Frohlich, M. Montenarh, J.F.R. Ortigao and H. Rosch, Nucleosides & Nucleotides, 10, 469-477, 1991.
  3. Synthesis of 2’-deoxyribonucleoside 5’-phosphoramidites: New building blocks for the inverse (5’-3’)-oligonucleotide approach, T. Wagner and W. Pfleiderer, Helv. Chim. Acta., 83, 2023-2035, 2000.
  4. Improved synthesis of oligodeoxyribonucleotides, K.P. Stengele and W. Pfleiderer, Tetrahedron Lett., 31, 2549-2552, 1990.
  5. 3’-Modified oligonucleotides by reverse DNA synthesis, C.D. Claeboe, R. Gao and S. M. Hecht, Nucleic Acids Research, 31, 5685-5691, 2003. Full Text PDF
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