Synthesis, RNA selective hybridization and high nuclease resistance of an oligonucleotide containing novel bridged nucleic acid with cyclic urea structurew

Since the Human Genome Project was completed, much attention has been given to genome technologies to regulate target gene expression or function. The most simple and promising approach to down-regulate target gene expression in a living cell is antisense strategy, which prevents translation by hybridization of an oligonucleotide with its complementary RNA strand. For practical use in antisense strategy, chemical modification of oligonulcleotides is essential to achieve high resistance towards nuclease degradation, high affinity to target mRNA with sequence specificity and RNA selectivity. 20,40-BNA (20,40-Bridged Nucleic Acid)/LNA whereby the sugar moiety is restricted to North-type (N-type) conformation was developed by our group and Wengel’s group independently (Fig. 1). Oligonucleotides (ONs) containing 20,40-BNA confer moderate resistance against enzymatic degradation and strong affinity with their RNA complements, but they still showed high affinity with their DNA complements. On the other hand, other bridged nucleic acids bearing a different type of bridge structure between 20and 40-positions have been developed to date (Fig. 1). Depending on the ring size and/or the elements which compose the bridged structure, the modified ONs varied among properties such as enzymatic stability and RNA selectivity. In general, ONs containing bridged nucleosides with a large bridged ring size revealed higher resistance against enzymatic degradation (i.e., 20,40-BNA/LNA vs. ENA, aza-ENA, 20,40-BNA, 20,40-BNA, PrNA, etc.). In addition, incorporation of heteroatoms in an appropriate position of the bridged moiety showed a tendency to enhance hybridization ability (i.e., PrNA vs. 20,40-BNA, or carbocyclic ENA vs. 20,40-BNA ) and in some cases RNA selectivity (i.e., aza-ENA, 20,40-BNA, 20,40-BNA ). However, it is still unclear how the bridged moiety itself affects the hybridization properties of ONs, and further evaluation of the relationship between the bridged structure and properties of the modified ONs is ongoing. Here, we focus on a urea structure, containing both N–H and CQO groups as a proton donor and acceptor, respectively, and introduced it into the bridged moiety connecting the 20and 40-positions of nucleoside to evaluate hybridization properties and enzymatic stability. As shown in Scheme 1, the phosphoramidite derivative of a novel bridged nucleic acid bearing a cyclic urea moiety was synthesized from known 40-hydroxymethyl nucleoside derivative 1. At first, 1 was treated with trifluoromethanesulfonyl chloride in the presence of DMAP to afford a 2,20-anhydro intermediate, which was then converted to an arabino-type nucleoside under alkaline conditions. Subsequent triflation at the 20-hydroxy group afforded ditriflate 2. Replacement of the two triflate groups by azide groups successfully proceeded to give diazide 3. Reduction under Staudinger conditions, followed by a ring-closure reaction with p-nitrophenyl chloroformate gave desired compound 4.