Synthesis and Triplex‐Forming Ability of 2′,4′‐BNAs Bearing Imidazoles as a Nucleobase.

Abstract

Triplex-forming oligonucleotide (TFO) is able to hybridize with a double-stranded DNA (dsDNA) target in a sequence-specific manner to form a triplex DNA, and it has attracted great interest because of its applications to gene therapy and diagnosis. 2-4) There are two fashions in triplex formation. One is a parallel motif triplex, where a TFO consisting of a homopyrimidine sequence hybridizes with a dsDNA target in parallel with a purine strand of the target duplex via Hoog-steen hydrogen bond. The other is an antiparallel motif triplex, in which the TFO and the purine strand of the duplex are in an antiparallel orientation. In both triplex motifs, the TFOs are able to hybridize only with a homopurine-homopy-rimidine tract in the dsDNA target. This is a severe limitation of the practical use of TFOs. To overcome this problem, extensive research on nucleic acid analogues has so far been carried out. 5-8) We have focused on the parallel type triplex, and synthesized 2-O,4-C-methylene-bridged nucleic acid (2,4-BNA 5,9-13) /locked nucleic acid (LNA) 14)) bearing unnatural nucleobases to recognize pyrimidine-purine interruption in the target dsDNA. 15-19) Recently, it was found that the 2,4-BNA bearing oxazole 15,16) (O B : Fig. 1) or 2-pyridone 17-19) as a nucleobase effectively recognized a CG interruption in homopurine-homopyrimidine dsDNA. Here, we have selected imidazoles as unnatural nucle-obases for recognition of pyrimidine-purine interruption. Synthesis of the 2,4-BNA monomers bearing imidazoles (I B and aI B : Fig. 1) and triplex-forming ability of the oligonu-cleotide derivatives are described. Results and Discussion Synthesis of 2,4-BNA Monomers and Their TFO Derivatives As shown in Chart 1, 2,4-BNA amidite units bearing imidazole and 2-aminoimidazole were synthesized by using 1 20) as the starting material. Coupling reaction of 1 with 2-nitroimidazoles gave 2b, whereas the reaction of un-substituted imidazole gave the desired compound 2a along with an imidazolium salt 2a. 21) Next, 2a and 2b were reacted with K 2 CO 3 in MeOH to give 3a and 3b. Reduction of 3a and 3b smoothly proceeded to afford 2,4-BNA monomers 4a and 4b, respectively. Protection of an amino group in 4b gave the corresponding N,N-dimethylformamidine derivative 4b. The 5-hydroxy groups of 4a and 4b were then protected with a dimethoxytrityl (DMTr) group to afford 5a and 5b, respectively. The preparation of phosphoramidites 6a and 6b was carried out by phosphitilation of 5a and 5b, respectively. Incorporation of the obtained amidites 6a and 6b into oligonucleotides was successfully achieved by using a standard phosphoramidite method on an automated DNA synthesizer. After purification by reverse-phase HPLC, composition of the oligonucleotides was confirmed by MALDI-TOF-MS. To expand the sequence of double-stranded DNA (dsDNA) targets in a triplex formation, 2,4-BNAs (2-O,4-C-methylene bridged nucleic acids) having imidazoles as a nucleobase were synthesized and incorporated into oligonucleotides. Triplex-forming ability of the modified olig...