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 ( Me C) 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(TTTTT Me PT Me PT Me PT Me PT Me PT) from a 21-mer target duplex at pH 7.5 is 2.3 °C relative to oligonucleotide d(TTTTT Me CT Me CT Me CT Me CT Me CT). The pH dependence and sequence composition effects are much less pronounced for Me P (and also P ) containing oligonucleotides than for Me C containing ones. While oligonucleotide d(TTT Me C Me C Me C Me CTTTT Me CTTT) shows no longer any affinity to the target duplex above pH 6.5, oligonucleotide d(TTT Me P Me P Me P Me PTTTT Me PTTT) 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(TTTTTP OMe TP OMe TP OMe TP OMe TP OMe T) at pH 6.5 is 6.7 °C relative to the Me C containing oligonucleotide. DNase I footprint titration experiments indicate that d(TTTTT Me PT Me PT Me PT Me PT Me PT) binds not only five times stronger to a 229 base pair DNA fragment than d(TTTTT Me CT Me CT Me CT Me CT Me CT) 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 (Δ T 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.
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Hildbrand et al. (1997) studied this question.