Synthesis of fluorescent 5-heteroarylpyrimidine-containing oligonucleotides post-synthetic trifluoromethyl conversion.

Post-synthetic conversion of the trifluoromethyl group to a heteroaryl group at the C5 position of the pyrimidine base in DNA oligonucleotides was achieved. Specifically, the oligonucleotides containing 5-trifluoromethylpyrimidine bases were treated with -phenylenediamines and -aminothiophenols as nucleophiles to afford the corresponding 5-(benzimidazol-2-yl)- and 5-(benzothiazol-2-yl)-pyrimidine-modified bases. Furthermore, evaluation of the fluorescence properties of the obtained oligonucleotides revealed that among them the oligonucleotide containing 5-(5-methylbenzimidazol-2-yl)cytosine exhibited the highest fluorescence intensity.

Synthesis of a Hydrophobic Phenanthrene-Containing Universal Support for Solid-Phase Oligonucleotide Synthesis.

Universal solid supports are widely used in solid-phase oligonucleotide (ON) synthesis based on phosphoramidite chemistry. Herein, we describe the synthesis of hydrophobic universal linkers, namely phenanthrene ring-fused 7-oxabicyclo[2.2.

Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA.

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design.

Chemical Conversion of 5-Fluoromethyl- and 5-Difluoromethyl-Uracil Bases in Oligonucleotides Using Postsynthetic Modification Strategy.

This article describes the postsynthetic modification of oligonucleotides (ONs) containing 2'-deoxy-5-fluoromethyluridine (dU ) and 2'-deoxy-5-difluoromethyluridine (dU ). Reactions of fully protected and controlled pore glass (CPG)-attached ONs containing dU and dU in basic solutions result in deprotection of all protecting groups except for the 4,4'-dimethoxytrityl group, cleavage from CPG, and conversion of the fluoromethyl or difluoromethyl groups to afford the corresponding ONs containing 5-substituted 2'-deoxyuridines. Moreover, the difluoromethyl group can be converted to formyl, oxime, or hydrazone via the postsynthetic conversion of protection- and CPG-free ON containing dU .

Synthesis of purine derivatives of Me-TaNA and properties of Me-TaNA-modified oligonucleotides.

We previously reported that pyrimidine derivatives of methylated 2'-,4'--methyleneoxy-bridged nucleic acid (Me-TaNA), a unique consecutive three-acetal-containing nucleic acid, are promising building blocks for chemically modified oligonucleotides. Herein, purine derivatives of Me-TaNA (Me-TaNA-A and -G) were synthesized and introduced into oligonucleotides. During the synthesis, we found stereoselective introduction of a substituent on the 4' carbons by using 2',3'-carbonate compounds as substrates.

Expansion of Phosphoramidite Chemistry in Solid-Phase Oligonucleotide Synthesis: Rapid 3'-Dephosphorylation and Strand Cleavage.

In solid-phase oligonucleotide synthesis, a solid support modified with a universal linker is frequently used to prepare oligonucleotides bearing non-natural- or non-nucleosides at the 3'-end. Generally, harsh basic conditions such as hot aqueous ammonia or methylamine are required to release oligonucleotides by 3'-dephosphorylation via the formation of cyclic phosphate with the universal linker. To achieve 3'-dephosphorylation under milder conditions, we used -alkyl phosphoramidites instead of the commonly used -cyanoethyl phosphoramidites at the 3'-end of oligonucleotides.

Generation of 4'-Carbon Radicals via 1,5-Hydrogen Atom Transfer for the Synthesis of Bridged Nucleosides.

The rapid and facile generation of 4'-carbon radicals from oxime imidates of nucleosides via 1,5-hydrogen atom transfer induced by iminyl radicals was developed. The cyclization of 4'-carbon radicals with olefins, followed by the hydrolysis of imidate residues, provided various 2'-,4'-- and 3'-,4'--bridged nucleosides. This operationally simple approach can be applied to the few-step syntheses of 6'-methyl-2'-,4'--ethylene-bridged 5-methyluridine (6'-Me-ENA-T) and -constrained ethyl-bridged 5-methyluridine (-cEt-T).

Synthesis and Properties of Oligonucleotides Containing 2'-,4'--Methyleneoxy-Bridged Pyrimidine Derivatives.

In this study, 2'-,4'--methyleneoxy-bridged nucleic acid, a unique consecutive three-acetal-containing nucleic acid (TaNA), was designed. Pyrimidine derivatives of methylated TaNA (Me-TaNA) were also synthesized and introduced into oligonucleotides via solid-phase synthesis. The Me-TaNA-modified oligonucleotides exhibited higher stabilities when forming duplexes with single-stranded RNA or triplexes with double-stranded DNA, relative to the natural oligonucleotides and modified oligonucleotides containing another 2',4'-bridged 5-methyluridine, such as 2',4'-BNA/LNA and 2',4'-ENA.

Synthesis of Nucleobase-Modified Oligonucleotides by Post-Synthetic Modification in Solution.

Oligonucleotides containing modified nucleobases have applications in various technologies. In general, to synthesize oligonucleotides with different nucleobase structures, each modified phosphoramidite monomer needs to be prepared over multiple steps and then introduced onto the oligonucleotides, which is time-consuming and inefficient. Post-synthetic modification is a powerful strategy for preparing many types of modified oligonucleotides, especially nucleobase-modified ones.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes.

2',4'-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance*.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex-forming abilities and enhanced nuclease resistance. 2'-O,4'-C-Methylene-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) with 1,3-diaza-2-oxophenoxazine (BNAP (B )) was previously reported. Herein, a novel B analogue, 2',4'-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-oxophenoxazine (G-clamp), named BNAP-AEO (B ), was designed.

Synthesis and hybridization properties of iridium(III) polypyridyl complex-conjugated oligonucleotide.

An Ir(III) polypyridyl complex-conjugated 14-mer oligonucleotide (Ir-DNA) was synthesized and its hybridization properties with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) were evaluated by UV-melting experiments. The stabilities of the duplexes of Ir-DNA with 14-, 20-, and 26-mer ssDNAs were higher than those of the unconjugated oligonucleotides. The triplex of Ir-DNA with 14-mer dsDNA was also stabilized.

Synthesis of Oligonucleotides Containing 2'--alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) Moieties Using Post-Synthetic Modification Strategy.

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2'--pentafluorophenoxycarbonyl-2'-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2'--pentafluorophenoxycarbonyl-2'-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM KCO in MeOH at room temperature for 4 h).

Synthesis of 2'-,4'--Methyleneoxy-Bridged Thymidine Derivatives and Properties of Modified Oligonucleotides.

2',4'-Bridged nucleic acid (2',4'-BNA) analogues are used for therapeutic oligonucleotides, owing to their excellent hybridizing ability with complementary RNA and high resistance toward enzymatic degradation. We developed 2',4'-BNA analogues with oxygen atoms at 6'-positions (e.g.

Construction of Pyrimidine Bases Bearing Carboxylic Acid Equivalents at the C5 Position by Postsynthetic Modification of Oligonucleotides.

This unit describes postsynthetic modification of oligonucleotides (ONs) containing 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine. In ONs, the trifluoromethyl group at the C5 position of pyrimidine bases is converted into a variety of carboxylic acid equivalents using alkaline and amine solutions. In addition, treating fully protected and controlled pore glass (CPG)-attached ONs with methylamine and sodium hydroxide aqueous solution results in deprotection of all protecting groups (except the 4,4'-dimethoxytrityl group), cleavage from CPG, and simultaneous conversion of the trifluoromethyl group to afford the corresponding ONs containing 5-substituted pyrimidine bases.

Synthesis of the Methyl Analog of 2'-,4'--Ethylene-Bridged 5-Methyluridine via Intramolecular Radical Cyclization and Properties of Modified Oligonucleotides.

The synthesis of 6'-Me-2'-,4'--ethylene-bridged 5-methyluridine (6'-Me-ENA-T) was achieved using visible light-mediated stereoselective radical cyclization as a key step. This is the first example of a method for constructing a 2',4'-bridged structure from a 4'-carbon radical intermediate. The 6'-Me-ENA-T monomer was successfully incorporated into oligonucleotides, and their properties were examined.

2'-C,4'-C-Ethyleneoxy-Bridged 2'-Deoxyribonucleic Acids (EoDNAs) with Thymine Nucleobases: Synthesis, Duplex-Forming Ability, and Enzymatic Stability.

This chapter describes procedures for (1) the synthesis of six 2'-C,4'-C-ethyleneoxy-bridged thymidine phosphoramidites, i.e., methylene-EoDNA-T, (R)-Me-methylene-EoDNA-T, (S)-Me-methylene-EoDNA-T, EoDNA-T, (R)-Me-EoDNA-T, and (S)-Me-EoDNA-T phosphoramidites, (2) the introduction of the phosphoramidites into oligonucleotides, (3) UV-melting experiments of the duplexes of the modified oligonucleotides and complementary RNA, and (4) nuclease degradation experiments of the modified oligonucleotides.

Effective syntheses of 2',4'-BNA monomers bearing adenine, guanine, thymine, and 5-methylcytosine, and the properties of oligonucleotides fully modified with 2',4'-BNA.

We efficiently synthesized 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2'-O-substituted 5-methyluridines to evaluate the effects of 2'-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2',4'-BNA monomers bearing adenine or guanine were obtained over several steps.

Photoredox-Catalyzed Deformylative 1,4-Addition of 2'-Deoxy-5'- O-phthalimidonucleosides for Synthesis of 5'-Carba Analogs of Nucleoside 5'-Phosphates.

A concise approach for the synthesis of the 5'-carba analogs of nucleoside 5'-phosphates from 2'-deoxy-5'- O-phthalimidonucleosides by a visible-light-mediated deformylative 1,4-addition was developed. This method enabled rapid and facile generation of 4'-carbon radicals of nucleosides. Moreover, this synthetic strategy was applicable to the 5'-carba analogs of nucleoside 5'-phosphates as well as other 5'-carba nucleosides bearing methoxycarbonyl, cyano, and N-methylsulfamoyl groups.

Synthesis and hybridizing properties of isoDNAs including 3'-O,4'-C-ethyleneoxy-bridged 5-methyluridine derivatives.

3',4'-Ethyleneoxy-bridged 5-methyluridine derivatives with methyl groups in the bridge, (R)-Me-3',4'-EoNA-T and (S)-Me-3',4'-EoNA-T, were synthesized, and these two analogs and unsubstituted 3',4'-EoNA-T were successfully incorporated into a 2',5'-linked oligonucleotide (isoDNA). Their duplex-forming ability with complementary DNA and complementary RNA, and triplex-forming ability with double-stranded DNA, were evaluated by UV-melting experiments. The results indicated that isoDNAs, including these 3',4'-EoNA analogs, could hybridize exclusively with complementary RNA.

Synthesis and thermal stabilities of oligonucleotides containing 2'-O,4'-C-methylene bridged nucleic acid with a phenoxazine base.

We designed and synthesized a novel artificial 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2',4'-BNA/LNA with 5-methylcytosine or 2'-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits properties associated with the phenoxazine moiety in DNA/DNA duplexes and characteristics associated with the 2',4'-BNA/LNA moiety in DNA/RNA duplexes.

Synthesis, duplex-forming ability, enzymatic stability, and in vitro antisense potency of oligonucleotides including 2'-C,4'-C-ethyleneoxy-bridged thymidine derivatives.

We synthesized thymidine derivatives of 2'-C,4'-C-ethyleneoxy-bridged 2'-deoxyribonucleic acids with an 8'-methyl group ((R)-Me-EoDNA and (S)-Me-EoDNA) and without any substituent (EoDNA). Oligonucleotides including these EoDNAs showed high hybridization abilities with complementary RNA and excellent enzymatic stabilities compared with natural DNA. Moreover, the in vitro antisense potency of oligonucleotides with these EoDNAs and our recently reported methylene-EoDNAs was investigated and compared with that of LNA, which is a practical chemical modification for oligonucleotide-therapeutic agents.

Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures.

Synthesis and properties of novel 2'-C,4'-C-ethyleneoxy-bridged 2'-deoxyribonucleic acids with exocyclic methylene groups.

Three 2'-C,4'-C-ethyleneoxy-bridged 2'-deoxyribonucleic acids possessing six-membered bridges with 6'-oxygen and 8'-exocyclic methylene groups (methylene-EoDNAs) were designed and synthesized in nine to ten steps from 5-methyluridine. The methylene-EoDNA-modified oligonucleotides showed excellent binding affinity with target ssRNA and extremely high nuclease resistance compared with natural oligonucleotides. These results proved the potential of methylene-EoDNAs for nucleic acid based technology.