2′-O-aminoethyl oligoribonucleotides containing novel base analogues:: Synthesis and triple-helix formation at pyrimidine/purine inversion sites

The synthesis of a common sugar intermediate for the 2'aminoethyl-ribonucleoside synthesis in 9 steps and an overall yield of 33% starting from D-ribose is described. This intermediate was successfully used for the synthesis of the 2'-aminoethyl-ribonucleosides containing the bases thymine (t), 5-methylcytosine (c), 5-methyl-2-pyrimidinone (x), 2-aminopurine (ap) and guanine (g). These were subsequently transformed into the corresponding cyanoethyl phosphoramidite building blocks for oligonucleotide synthesis. 2'-Aminoethyl oligonucleotide 15-mers were prepared with a DNA synthesizer, and an optimized post-synthetic deprotection protocol has been developed which prevents cyanoethylation of the 2'-amino side chains during conventional ammonia deprotection. A series of fully modified, triplex forming 2'-aminoethyl oligoribonucleotides (2'AE-TFOs) were prepared in which x was designed to bind to CG inversion sites and ap as well as g to TA inversion sites on a double-helical DNA target. The affinity of x-CG base-triple formation in different sequence contexts was assessed by UV- and CD melting analysis. It was found that TFO 15-mers containing up to 5 x residues still form stable triplexes even in the case where all x residues are consecutively arranged in the TFO. The nearest neighbor properties of x have been probed and it was found that triplex stability decreases in the local sequence order Axt- > -txc- >> -cxc-. TFOs containing ap and g were found to bind to their DNA targets with TA inversion sites with less affinity and less selectivity compared to TFOs containing the corresponding deoxyribonucleosides, irrespective whether they were incorporated in TFOs with a DNA or a 2'-AE-RNA backbone. The obtained data suggest that guanine-TA or aminopurine-TA base-triple formation is strongly sensitive to TFO conformation and more efficient in TFOs with a DNA than an RNA backbone. (c) Wiley-VCH Verlag GmbH & Co.