The dinucleotide pdCpA consists of a DNA/RNA hybrid that’s useful for preparing suppressor t-RNACUAs, enabling site-specific incorporation of unnatural amino acids into proteins. Synthesis of fully mature t-RNAs, t-RNACUA-aa, is accomplished via ligation of the aminoacylated dinucleotide, pdCpA-aa, to the abbreviated suppressor t-RNACUA-COH, lacking the two terminal 3′-nucleotides (CA). Preparation of pdCpA-aa first requires synthesis of the dinucleotide followed by coupling of the N-protected and activated amino acid. This methodology is cumbersome, requiring several synthetic steps prior to ligating to the suppressor t-RNA. Moreover, this route yields a semi-synthetic tRNA that contains a deoxyribonucleotide that could affect its binding to the ribosomal A site and participation in peptide elongation. Outlined herein is a strategy employing a solid phase synthesis for the preparation of aminoacylated nucleotide, A-aa and dinucleotide CpA-aa. Solid phase synthesis of oligonucleotides have high coupling efficiencies, are easy to purify and can be carried out autonomously. Unfortunately, traditional phosphoramidite oligonucleotide synthesis proceeds in the 3′ to 5′-direction making it impossible to attach the amino acid following dinucleotide synthesis. Recent developments, however, have led to new molecules enabling the synthesis of the same oligonucleotides in a 5′ to 3′-direction. The reverse RNA amidites and their corresponding solid supports can be used for the preparation of CpA-aa and the resulting aminoacylated dinucleotide can be ligated to the corresponding truncated tRNA (t-RNACUA-COH).

New strategy for the synthesis of aminoacylated dinucleotide CpA-aa

256th National Meeting (2018)

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