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-beta-D-ribofura nosyl)py (P) and its 3-methyl (P-Me) and 2'-O-methyl (P-OMe) derivativ es were synthesized and incorporated as protonated cytidine equivalent s in homopyrimidine oligodeoxynucleotides. T-m measurements indicate t hat oligonucleotides containing P or P-Me have a higher affinity to do uble-stranded DNA over the pH range of 6-8 than, 5-methylcytidine (C-M e) containing oligonucleotides. This increase in stability is most pro nounced above pH 7.0. The average increase in T-m/modification for the dissociation of oligonucleotide d((TTTTTPTPTPTPTPT)-P-Me-P-Me-P-Me-P- Me-P-Me) from a 21-mer target duplex at pH 7.5 is 2.3 degrees C relati ve to oligonucleotide. d((TTTTTCTCTCTCTCT)-C-Me-C-Me-C-Me-C-Me-C-Me). The pH dependence and sequence composition effects are much less prono unced for P-Me (and also P) containing oligonucleotides than for C-Me containing ones. While oligonucleotide d((TTTCCCCTTTTCTTT)-C-Me-C-Me-C -Me-C-Me-C-Me) shows no longer any affinity to the target duplex above pH 6.5, oligonucleotide d((TTTPPPPTTTTPTTT)-P-Me-P-Me-P-Me-P-Me-P-Me) displays preserved binding with a T-m of 32.5 degrees C at pH 7.0 and even binds with a T-m of 23.3 degrees C at pH 8.0. Oligonucleotides c ontaining P-OMe show distinctly less stable triple helices. The averag e decrease in T-m/modification for oligonucleotide d(TTTPTPOMeTPOMeTPO MeTPOMeTPOMeT) at pH 6.5 is 6.7 degrees C relative to the C-Me contain ing oligonucleotide. DNase I footprint titration experiments indicate that d((TTTTTPTPTPTPTPT)-P-Me-P-Me-P-Me-P-Me-P-Me) binds not only five times stronger to a 229 base pair DNA fragment than d((TTTTTCTCTCTCTC T)-C-Me-C-Me-C-Me-C-Me-C-Me) but also with higher selectivity; UV-melt ing experiments show that duplexes of d(TTTTTCTXTCTCTCT) (where X = P, P-Me, or P-OMe) With their antiparallel Watson-Crick complement are d ramatically less stable (Delta T-m < -12 degrees C) at pH 8.0 than the corresponding natural duplex. Thus the new bases P and P-Me show Hoog steen specific pairing behavior.