Technical resources: 2'-CEM™-amidites
A novel RNA synthetic method with a 2-cyanoethoxymethyl (CEM™)
protecting group at the 2'-hydroxyl position of ribose
- Introduction
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The discovery of RNA interference has had a great impact on the life sciences, and there has been an increasing demand for synthetic RNA in all areas from basic research to the development of new medicines. Throughout the history of the chemical synthesis of RNA, the most important point has been the selection of a suitable 2'-hydroxyl protecting group. Several good 2'-hydroxyl protecting groups have been developed, including triisopropylsilyloxymethyl (TOM), bis[2-acetoxyethyloxy] methyl (ACE), and the widely used t-butyldimethylsilyl (TBDMS), but there is still a lot of room for improvement in efficiency, cost, scale-up, and the synthesis of long RNA. Recently, Nippon Shinyaku Co. developed a completely new 2'-hydroxyl protecting group, 2-cyanoethoxymethyl (CEM™), that is superior in all of these respects and is particularly suitable for the synthesis of long RNA oligomers (Refs 1 and 2).
- Synthesis of RNA oligomers
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"The 2'-hydroxyl protecting group that we have developed, 2-cyanoethoxymethyl (CEM™), is achiral, has low steric hindrance, and can be removed under mild conditions (Ref. 1). The CEM™ method is compatible with standard, unmodified DNA synthesizer equipment. To verify the potential and practicality of the CEM™ method, we undertook the total chemical synthesis of a 110-nucleotide RNA oligomer (Ref. 2). As far as we know, this was the first chemical synthesis of an RNA oligomer longer than 100 nucleotides."
Fig. 1. Chemical structure of CEM™-phosphoramidite. - Synthesis of RNA oligomers
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- The CEM™ method can give the desired RNA oligomer in high yield, with a 99% coupling efficiency at each step.
- The CEM™ method allows the synthesis of RNA oligomers of more than 100 nucleotides.
- The CEM™ method is comparable with DNA synthesis in its efficiency and the ease of purification of the final oligonucleotide.
Fig. 2. HPLC of 40mer RNA oligomers (crude reaction mixture). Fig. 3. Capillary gel electrophoresis of a 110mer RNA oligomer (crude reaction mixture).
[References]
1) T. Ohgi et al., Org. Lett. 7, 3477-3480 (2005).
2) Y. Shiba et al., Nucleic Acids Res. 35, 3287-3296 (2007).