Synthesis of oligodeoxynucleotides using fully protected deoxynucleoside 3'-phosphoramidite building blocks and base recognition of oligodeoxynucleotides incorporating N3-cyano-ethylthymine

Abstract

Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side products, is of great importance to biochemistry-based technology development. One side reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this reaction by synthesizing ODNs using fully protected deoxynucleoside 3'-phosphoramidite building blocks, where the remaining reactive nucleobase residues were completely protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of cyanoethylation at the nucleobase site showed that N3-protection of the thymine base efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating N3-cyanoethylthymine was synthesized using the phosphoramidite method, and primer extension reactions involving this ODN template were examined. As a result, the modified thymine produced has been proven to serve as a chain terminator.

Figure 1

Fully protected monomers.

Scheme 1

Synthesis of fully protected 2′-deoxythymidine derivative 4.

Scheme 2

Synthesis of fully protected 2′-deoxycytidine derivative 6 and 2′-deoxyadenosine derivative 8.

Scheme 3

Synthesis of fully protected 2′-deoxyguanosine derivative 13.

Figure 2

The remaining amount (%) of T or T^Bz in its reaction with acrylonitrile. Open and filled circles refer to the remaining amounts (%) of T and T^Bz in use of T and T^Bz, respectively. Conditions: 100 equiv of acrylonitrile in DBU/CH₃CN.

Figure 3

Analysis of cyanoethylation of (i) the fully protected monomers (C^phth, A^phth, and G^iBu·dibe) and (ii) the common protected monomers (C^ac, A^pac, and G^iPrpac) by reverse-phase HPLC; (iii) Analysis of deprotection of the fully protected monomers by concentrated NH₃ at 55 °C for 6 h.

Figure 4

Anion-exchange HPLC profiles of ODNs obtained by using (i) the fully protected monomers (T^Bz, C^phth, A^phth, and G^iBu·dibe); (ii) only T^Bz monomer and (iii) T^Bz, C^phth, A^phth, and G^iPrpac monomers after removal of the cyaonoethyl groups of the internucleotidic phosphate groups by treatment with DBU-CH₃CN (1:9, v/v); (iv) Anion-exchange HPLC profile of deprotection of ODN having the fully protected monomers (T^Bz, C^phth, A^phth, and G^iBu·dibe) by concentrated NH₃.

Figure 5

Analysis of depurination using ODNs containing (A) A^phth or (B) G^iBu·dibe by reverse-phase HPLC.

Scheme 4

Synthesis of T^CE phosphoramidite unit 23.

Figure 6

Single dNTP insertion reactions using Taq DNA polymerases. (A) Sequences of 5′-FAM labeled 18-nt primer and 25-nt templates; (B) PAGE analysis of single-insertion reactions.