A comparative study of the thermal stability of oligodeoxyribonucleotides containing 5-substituted 2'-deoxyuridines

Two series of modified oligonucleotides based on the self-complementary dodecamer d(CGCTAATTAGCG) were synthesized. The first contained the -C ≡ CCH₂R linker at C5 of deoxyuridine at position 4 (T*) of d(CGCT*AATTAGCG) and the second contained the -SR linker. The goal of the study was to evaluate and compare these two types of side chains for suitability as tethers for linking reporter groups to oligonucleotides. Our primary concern was how these tethers would effect duplex stability. The modified nucleosides were synthesized by palladium-mediated coupling reactions between the substituted alkyne and 5'-(4,4'-dimethoxytrityl)-5-iodo-2'-deoxyuridine and between a disulfide and 5-chloromercurio-2'-deoxyuridine. The C5 deoxyuridine side chains evaluated included C ≡ CCH₃, C ≡ CCH₂NHC(O)CH₃, C ≡ CCH₂N(CH₃)₂, C ≡ CCH₂NHC(O)C₅H₄N, C ≡ CCH₂NHC(O)C₁₀H₁₅, SCH₃, SC₆H₅ and SCH₂CH₂NHC(O)CH₃. The nucleosides containing these substituents were incorporated into oligodeoxyribonucleotides by standard phosphoramidite methodology. Melting studies demonstrated that the sequence containing the C ≡ CCH₃ side chain had the highest T(m) value (59.1°C) in comparison with the control sequence (T(m) = 55.2°C) and that any additional substituent on C3 of the propynyl group lowered the T(m) value relative to propynyl. Nevertheless, even the most destabilizing substituent, adamantylcarbamoyl, yielded an oligodeoxyribonucleotide that dissociated with a T(m) of 54°C, which is only 1.2°C less than the control sequence. In contrast, the thioether substituents led to lower T(m) values, ranging from as low as 45.1°C for SPh up to 52.2°C for SMe. Replacing the methyl of the SMe substituent with a CH₂CH₂NHC(O)CH₃ tether led to no further reduction in melting temperature. The T(m) value of the CH₂CH₂NHC(O)CH₃-containing oligonucleotide was less than the natural sequence by 1.6°C/substituent. This is sufficiently small that it is anticipated that the C5 thioether linkage may be as useful as the acetylenic linkage for tethering reporter groups to oligonucleotides. More importantly, the thioether linkage provides a means to position functional groups to interact specifically with opposing complementary (target) sequences.