5-Substituted 2-Aminopyridine C-Nucleosides as Protonated Cytidine Equivalents:  Increasing Efficiency and Selectivity in DNA Triple-Helix Formation

The easily accessible C-nucleoside 2-amino-5-(2‘-deoxy-β-d-ribofuranosyl)pyridine (P) and its 3-methyl ( Me P) and 2‘-O-methyl (P OMe ) derivatives were synthesized and incorporated as protonated cytidine equivalents in homopyrimidine oligodeoxynucleotides. T m measurements indicate that oligonucleotides containing P or Me P have a higher affinity to double-stranded DNA over the pH range of 6−8 than 5-methylcytidine (MeC) containing oligonucleotides. This increase in stability is most pronounced above pH 7.0. The average increase in T m/modification for the dissociation of oligonucleotide d(TTTTTMePTMePTMePTMePTMePT) from a 21-mer target duplex at pH 7.5 is 2.3 °C relative to oligonucleotide d(TTTTTMeCTMeCTMeCTMeCTMeCT). The pH dependence and sequence composition effects are much less pronounced for Me P (and also P) containing oligonucleotides than for MeC containing ones. While oligonucleotide d(TTTMeCMeCMeCMeCTTTTMeCTTT) shows no longer any affinity to the target duplex above pH 6.5, oligonucleotide d(TTTMePMePMePMePTTTTMePTTT) displays preserved binding with a T m of 32.5 °C at pH 7.0 and even binds with a T m of 23.3 °C at pH 8.0. Oligonucleotides containing P OMe show distinctly less stable triple helices. The average decrease in T m/modification for oligonucleotide d(TTTTTPOMeTPOMeTPOMeTPOMeTPOMeT) at pH 6.5 is 6.7 °C relative to the MeC containing oligonucleotide. DNase I footprint titration experiments indicate that d(TTTTTMePTMePTMePTMePTMePT) binds not only five times stronger to a 229 base pair DNA fragment than d(TTTTTMeCTMeCTMeCTMeCTMeCT) but also with higher selectivity. UV-melting experiments show that duplexes of d(TTTTTCTXTCTCTCT) (where X = P, Me P, or P OMe ) with their antiparallel Watson−Crick complement are dramatically less stable (ΔT m < −12 °C) at pH 8.0 than the corresponding natural duplex. Thus the new bases P and Me P show Hoogsteen specific pairing behavior.