Key featuresShow Hide
- CPG has aminopropyl-succinyl linker
- Standard luer or Supercolumn pipette column types available in different synthesis scales
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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. We provide a range of 5'-3’ “reverse” (or “inverse”) phosphoramidites and CPGs, with a variety of pore sizes and linkers consistent with our unmodified DNA and RNA CPG products. The protecting group strategies are compatible with the usual DNA and RNA chemistries. Depending on the product, various column types are available.
- (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; (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.
- (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.
- Extinction Coefficient at 260 nm: 8400
Spectral properties measured in water, for the cleaved and deprotected nucleoside.
- Protecting group deblock conditions: 3'DMT groups detritylate slowly. Increase detritylation time by 50%.
- Cleavage conditions: Use concentrated ammonia for 90 minutes at 25 °C when using standard amidites or 1:1 ammonia:methylamine (AMA) for 25 minutes at 25 °C when using fast deprotecting amidites.
- Deprotection conditions: When using fast deprotecting amidites (eg. C-Ac, G-DMF, G-PAC) use concentrated ammonia for 1 hour or AMA for 30 minutes at 60 °C. When using standard amidites (eg. C-Bz, G-iBu) use concentrated ammonia for 5 hours at 60 °C. Image of cleaved and deprotected structure:
Storage and handling:
- Storage conditions: +5 °C in sealed bag or container