A comparative study of the base pairing properties of oligodeoxynucleotides containing 5-substituted-2\u27-deoxyuridines

Two series of modified dodecamer oligonucleotides were synthesized. The first contained the -C=CCH$\sb2$R linker at C5 of deoxyuridine at position 4 (T*) of 5$\sp\prime$-d(CGCT*AATTAGCG)-3$\sp\prime$ and the second contained the -SR linker. The goal of the study was to evaluate and compare these two types of side-chains for suitablity as tethers for linking reporter groups to oligonucleotides. Our primary concern was how these tethers would affect duplex stability. The modified nucleosides were synthesized by palladium mediated coupling reactions between the substituted alkyne and 5$\sp\prime$-(4,4$\sp\prime$-dimethoxytrityl)-5-iodo-2$ \sp\prime$-deoxyuridine and between a disulfide and 5-chloromercurio-2$\sp\prime$-deoxyuridine. The C5 deoxyuridine side chains evaluated included C$\equiv$CCH$\sb3,$ C$\equiv$CCH$\sb2$NHC(O)CH$\sb3,$ C$\equiv$CCH$\sb2$N(CH$\sb3)\sb2,$ C$\equiv$CCH$\sb2$NHC(O)C$\sb5$H$\sb4$N, C$\equiv$CCH$\sb2$NHC(O)C$\sb{10}$H$\sb{15}$, SCH$\sb3,$ SC$\sb6$H$\sb5,$ and SCH$\sb2$CH$\sb2$NHC(O)CH$\sb3.$ The nucleosides containing these substituents were incorporated into oligodeoxyribonucleosides by standard phosphoramidite methodology. Melting studies demonstrated that the sequence containing the C$\equiv$CCH$\sb3$ side chain had the highest $T\sb{m}$ (60.0$\sp\circ$C) in comparison to the control sequence $(T\sb{m}$ 55.9$\sp\circ$C), and that any additional substituent on C3 of the propynyl group lowered the $T\sb{m}$ relative to propynyl. Nevertheless, even the most destabilizing substituent, adamantylcarbamoyl, yielded an oligodeoxyribonucleotide that melted at a $T\sb{m}$ (55.4$\sp\circ$C) within 0.5$\sp\circ$C of the control sequence. In contrast the thioether substituents led to lower $T\sb{m}$\u27s ranging from 46.4$\sp\circ$C for SPh to 53.3$\sp\circ$C for SCH$\sb3.$ However, the oligonucleotide containing the thioether substituent, SCH$\sb2$CH$\sb2$NHC(O)CH$\sb3$ $(T\sb{m}$ = 52.5$\sp\circ$C) melted with higher cooperativity than any of the sequences tested and was shown to have the largest negative free energy of formation $\rm (G\sb{25\sp\circ} = {-}14.1).