Yoshiyuki Hari

Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2'-O,4'- C-methyleneribonucleosides

Bicyclic nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneuridine and -cytidine, were incorporated into oligonucleotides, and the binding efficiency of the modified oligonucleotides to the complementary DNA and RNA as well as the CD spectra of the modified DNA-DNA and modified DNA-RNA duplexes were studied.

Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering

2′-O,4′-C-Methyleneuridine and -cytidine, novel bicyclic nucleoside analogs having a typical C3′-endo sugar puckering, were synthesized starting from uridine via a several-step sequence.

Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addition of an unnatural base pair, promise to expand the biotechnological applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogues, we performed a detailed structure-activity relationship analysis, which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.

Triplex-forming enhancement with high sequence selectivity by single 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA) modification

Triplex-forming ability of the oligonucleotides containing one 2′-O,4′-C-methyleneribonucleic acid (2′,4′-BNA) unit was investigated by measurement of the melting temperature (Tm), and the 2′,4′-BNA modification promoted the marked triplex stabilization in a highly sequence-selective manner.

Synthesis and conformation of 3′,4′-BNA monomers, 3′-O,4′-C-methyleneribonucleosides

Abstract In order to develop novel 2′,5′-linked oligonucleotide analogues aimed for antivirus reagents and antisense/antigene oligonucleotides, novel nucleoside analogues, 3′-O,4′-C-methyleneribonucleosides (3′,4′-BNA monomers) were synthesized via two synthetic routes. The first route starting from uridine utilized a regioselective ring-closure reaction of the 4′-C-(p-toluenesulfonyl)oxymethyluridine derivative. The second route involved a coupling reaction of 1,2,3-tri-O-acetyl-4-C-(p-toluenesulfonyl)oxymethylribofuranose derivative with nucleobases followed by oxetan-ring formation to afford the 3′,4′-BNA monomers bearing all four nucleobases. By means of 1H NMR, X-ray crystallography and computational analysis, the sugar puckering of the 3′,4′-BNA monomers was found to be restricted in S-conformation (C1′-exo–C2′-endo puckering mode).

A bridged nucleic acid, 2′,4′-BNACOC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2′,4′-BNACOC monomers and RNA-selective nucleic-acid recognition

Recently, we synthesized pyrimidine derivatives of the 2′-O,4′-C-methylenoxymethylene-bridged nucleic-acid (2′,4′-BNACOC) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNACOC) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNACOC consisting of 2′,4′-BNACOC monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2′,4′-BNACOC monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNACOC/BNACOC duplex possessed excellent thermal stability and that the BNACOC increased thermal stability with a complementary RNA strand. On the other hand, BNACOC/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNACOC generally improved the sequence selectivity with Watson–Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNACOC formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.

Triplex formation by an oligonucleotide containing conformationally locked C-nucleoside, 5-(2-O,4-C-methylene-β-d-ribofuranosyl)oxazole

The triplex-forming ability of oligonucleotide analogues containing conformationally locked C-nucleosides, 5-(2-O,4-C-methylene-β-d-ribofuranosyl)oxazole or its 2-phenyl congener, towards a purine sequence of duplex DNA with a single C·G base pair interruption is studied.

Amido-Bridged Nucleic Acids (AmNAs): Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro Antisense Potency

Since their discovery, 2’,4’-bridged nucleic acids (2’,4’-BNAs) [or locked nucleic acids (LNAs)] have been considered promising candidates for antisense technology because of their unprecedented high binding affinities toward complementary strands, 2] sequence selectivities, and potential for in vivo applications. 5] Our group and others are continuing efforts to develop other bridged nucleic acids with improved properties based on the LNA structural concept. Recently, our group has focused on developing new bridged nucleic acids with carbonyl functionality in the bridge. The introduction of a carbonyl group into the bridge would be expected, as a consequence of its trigonal planarity, to restrict the flexibility of the sugar moiety. We have so far reported two analogues of bridged nucleic acids with carbonyl functionality in their bridges; both exhibited interesting properties. In one report we described a seven-membered bridged nucleic acid with a cyclic urea structure, which showed high nuclease resistance along with high RNA selectivity. We recently introduced another interesting class of LNA analogues: hydroxamate-bridged nucleic acids (HxNAs), which possessed unique nuclease resistance. Among the challenges to the development of a potent antisense oligonucleotide, in vivo enzymatic digestion of the oligonucleotide remains an important factor. Previous studies have shown that the nuclease resistance of a bridged nucleic acid can be enhanced by increasing the size of the bridge, but this reduces binding affinity. 7] Optimization of the bridged nucleic acids to improve nuclease resistance without losing binding affinity is therefore needed. To accomplish this, the relationship between the size of the bridge and the properties of the bridged nucleic acid were investigated. Here we report the synthesis and properties of a new set of analogues of LNAs with cyclic amide structures, named amido-bridged nucleic acid (AmNAs). This is the first report of an amide linkage in a bridged nucleic acid, although amide functionality has found other uses in nucleic acid chemistry, for example, in modification of the phosphate linkage 13] and in peptide nucleic acids (PNAs). Structurally, an AmNA has a five-membered bridged structure similar to those of an LNA or an 2’-amino-LNA. Previous reports on 2’-amino-LNAs demonstrated that their binding affinities toward complementary strands are comparable to those of LNAs. Introduction of a carbonyl group into the LNA structure, as in an AmNA, should enhance the rigidity and polarity of the structure. These structural characteristics of an AmNA would be expected to impart properties appropriate for antisense oligonucleotides. Compound 1 (Scheme 1), synthesized by known procedures, was used as the starting material for the synthesis of AmNAs. Triflylation of 1 followed by an SN2 reaction with NaN3 yielded 3 in good yield. The silyl protecting group of 3 was removed (tributylammonium fluoride, TBAF) to produce 4 with a free hydroxy moiety that was oxidized with pyridinium dichromate (PDC) in DMF to afford the carboxylic acid 5. By means of a Staudinger reaction (i.e. , treatment of 5 with PBu3 in THF), the azido group at the 2’-position was converted into an amino group to yield 6. The carboxylic acid function at the 4’-position and the amino function at the 2’-position of 6 were linked together by a condensation reaction, activated by N’-(3dimethylaminopropyl)-N-ethylcarbodiimide (EDCI) to afford the desired cyclic product 7. Debenzylation of 7 by catalytic hydrogenolysis yielded monomer 8 in excellent yield. The desired monomer containing a carbonyl function in a five-membered bridge had thus been successfully synthesized. The functionalizable nitrogen at the 2’-position allowed the synthesis of various N-substituted derivatives of the monomer. The first attempt to methylate this functionalizable nitrogen also caused N-methylation at the thymine moiety, so the thymine moiety was first protected with a benzyloxymethyl (BOM) protecting group, followed by N-methylation of the bridge nitrogen and then by debenzylation by catalytic hydrogenolysis to afford the desired N-methylated monomer 10. To incorporate the synthesized monomers 8 and 10 into oligonucleotides, the primary hydroxy groups of 8 and 10 were selectively tritylated with 4,4’-dimethoxytrityl chloride (DMTrCl), followed by phosphitylation of secondary hydroxy groups with 2-cyanoethyl-N,N,N’,N’-tetraisopropylphosphorodiamidite to afford the desired thymine phosphoramidites 9 and 12. For interconversion of the nucleobase (i.e. , thymine into cytosine), the secondary hydroxy group of tritylated nucleoside 11 was protected with a triethylsilyl (TES) group, and subsequent treatment with 2,4,6-triisopropylbenzenesulfonyl chloride (iPr3ArSO2Cl) in the presence of 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) sulfonated the 4-oxo group of the thymine moiety. The sulfonylated nucleoside was subjected to substitution with ammonia to afford a 5-methylcytidine derivative. The exocyclic amino moiety of the 5-methylcytidine was protected with an acetyl group, followed by desilylation and phosphitylation of the free secondary hydroxy group by 2cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite to afford the desired N-acetyl-protected 5-methylcytidine phosphordiamidite 13 (Scheme 1). [a] A. Yahara, Dr. A. R. Shrestha, Dr. T. Yamamoto, Dr. Y. Hari, T. Osawa, M. Yamaguchi, Dr. M. Nishida, Dr. T. Kodama, Prof. Dr. S. Obika Graduate School of Pharmaceutical Sciences, Osaka University 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan) E-mail : obika@phs.osaka-u.ac.jp Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200506.

Effective synthesis of C-nucleosides with 2′,4′-BNA modification

Abstract The effective synthesis of some C -nucleosides with 2′- O ,4′- C -methylene bridged nucleic acid (2′,4′-BNA) modification was accomplished by using the coupling reaction of a tetrahydrofurancarbaldehyde 1 with the magnesium or lithium derivatives of aromatic heterocycles followed by the Mitsunobu cyclization. Moreover, it was clearly shown by 1 H NMR spectra and X-ray crystallography that the sugar conformation in the synthesized C -nucleosides, independent of the nucleobases, was fixed in N-form.

Synthesis of conformationally locked C-nucleosides having a 2,5-dioxabicyclo[2.2.1]heptane ring system

Abstract Some novel C-nucleosides having a 2,5-dioxabicyclo[2.2.1]heptane ring system were successfully synthesized via the coupling reaction of tetrahydrofurancarbaldehyde 1 with the lithium and the magnesium derivatives of aromatic heterocycles.