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 sequencespecific manner to form a triplex DNA, and it has attracted great interest because of its applications to gene therapy and diagnosis. [2][3][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 Hoogsteen 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-homopyrimidine 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][6][7][8] We have focused on the parallel type triplex, and synthesized 2Ј-O,4Ј-C-methylene-bridged nucleic acid (2Ј, [9][10][11][12][13] /locked nucleic acid (LNA) 14) ) bearing unnatural nucleobases to recognize pyrimidine-purine interruption in the target dsDNA. [15][16][17][18][19] Recently, it was found that the 2Ј,4Ј-BNA bearing oxazole 15,16) : Fig. 1) and triplex-forming ability of the oligonucleotide derivatives are described. Results and DiscussionSynthesis of 2,4-BNA Monomers and Their TFO Derivatives As shown in Chart 1, 2Ј,4Ј-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 unsubstituted 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 2Ј,4Ј-BNA monomers 4a and 4b, respectively. Protection of an amino group in 4b gave the corresponding N,N-dimethylformamidine derivative 4b. The 5Ј-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 oligonucleotides was evaluated by using melting temperature (T m ) measurements.Key words methylene bridged nucleic acid (BNA); locked nucleic acid (LNA); triplex; imidazole; oligonucleotide Reagents: a) 1-trimethylsilylimidazole, trimethylsilyl trifluoromethanesulfonate,...

Towards the Sequence‐Selective Recognition of Double‐Stranded DNA Containing Pyrimidine‐Purine Interruptions by Triplex‐Forming Oligonucleotides

“…[30] We thus evaluated a combination of 2Ј,4Ј-BNA/LNA and five-membered heteroaro- matic nucleobases such as oxazoles and imidazoles in the recognition of pyrimidine-purine interruption in dsDNA. [31,32] TFOs incorporating 2Ј,4Ј-BNA/LNAs with 2phenyloxazol-5-yl (pO B ) or imidazol-1-yl (I B ) nucleobases showed no stabilization of the triplexes formed with dsDNA containing a CG base pair whereas a 2Ј,4Ј-BNA/ LNA with the oxazol-5-yl nucleobase (O B ) did show an apparent interaction with a CG base pair (Figure 7, a). In general, a heteroatom at the α-position -but not the βposition -of the glycosyl bond in a five-membered nucleobase might play a role in the recognition of the CG base pair ( Figure 8).…”