Synthesis of fluorescent 5-heteroarylpyrimidine-containing oligonucleotides via 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 o-phenylenediamines and o-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. These results indicated that post-synthetic trifluoromethyl conversion, which is practical and operationally simple, is a powerful tool for exploring functional oligonucleotides.

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[2.2.1]heptane-2,3-diol derivatives (PT linkers), their coupling to solid supports [e.g., controlled pore glass (CPG) and polystyrene (PS)], and the use of the resulting PT-linker-modified solid supports in ON synthesis. PT linkers were synthesized in four steps from commercial materials and subsequently attached to CPG and PS resins through succinyl and diethylene glycol-containing spacers, respectively. Cleavage of the desired ON from the resins was accomplished under standard basic conditions, indicating that the reactivity of the PT linkers was comparable to that of conventional universal linkers. Furthermore, owing to their high hydrophobicity, the desired ON could be readily separated from impurities originating from the PT linker by reversed phase HPLC. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phenanthrene ring-fused 7-oxabicyclo[2.2.1]heptane-2,3-diol (PT linker) derivatives Basic Protocol 2: Preparation of PT-linker-modified CPG and PS resins Basic Protocol 3: Solid-phase ON synthesis using PT-linker-modified solid supports and cleavage of ONs from resins.

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. We developed novel Ps-TFOs containing 2'-O,4'-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2 , 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.

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 (dUCH2F ) and 2'-deoxy-5-difluoromethyluridine (dUCHF2 ). Reactions of fully protected and controlled pore glass (CPG)-attached ONs containing dUCH2F and dUCHF2 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 dUCHF2 . © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of fully protected and CPG-attached oligonucleotides containing 2'-deoxy-5-fluoromethyluridine and 2'-deoxy-5-difluoromethyluridine Basic Protocol 2: Postsynthetic modification of fully protected and CPG-attached oligonucleotides containing 2'-deoxy-5-fluoromethyluridine Basic Protocol 3: Postsynthetic modification of fully protected and CPG-attached oligonucleotide containing 2'-deoxy-5-difluoromethyluridine Basic Protocol 4: Postsynthetic modification of protection- and CPG-free oligonucleotide containing 2'-deoxy-5-difluoromethyluridine Support Protocol: Synthesis of 2'-deoxy-5-fluoromethyluridine and 2'-deoxy-5-difluoromethyluridine phosphoramidites.

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

We previously reported that pyrimidine derivatives of methylated 2'-O,4'-C-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. When forming duplexes with single-stranded RNA, the modified oligonucleotides, including purine derivatives of Me-TaNA, showed higher duplex stability than the natural oligonucleotide. This study enabled the use of Me-TaNA for the chemical modification of various oligonucleotide sequences because synthesis of Me-TaNAs with all four nucleobases was achieved.

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 O-alkyl phosphoramidites instead of the commonly used O-cyanoethyl phosphoramidites at the 3'-end of oligonucleotides. Alkylated phosphotriesters are more alkali-tolerant than their cyanoethyl counterparts because the latter generates phosphodiesters via E2 elimination under basic conditions. Among the designed phosphoramidites, alkyl-extended analogs exhibited rapid and efficient 3'-dephosphorylation compared to conventional cyanoethyl and methyl analogs under mild basic conditions such as aqueous ammonia at room temperature for 2 h. Moreover, nucleoside phosphoramidites bearing 1,2-diols were synthesized and incorporated into oligonucleotides. 1,2,3,4-Tetrahydro-1,4-epoxynaphthalene-2,3-diol-bearing phosphoramidite behaved like a universal linker at the 3'-terminus, allowing dephosphorylation and strand cleavage of the oligonucleotide chain to occur efficiently. Our strategy using this new phosphoramidite chemistry is promising for the tandem solid-phase synthesis of diverse 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'-O,4'-C- and 3'-O,4'-C-bridged nucleosides. This operationally simple approach can be applied to the few-step syntheses of 6'S-methyl-2'-O,4'-C-ethylene-bridged 5-methyluridine (6'S-Me-ENA-T) and S-constrained ethyl-bridged 5-methyluridine (S-cEt-T).

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

In this study, 2'-O,4'-C-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. Furthermore, Me-TaNA within oligonucleotides significantly enhanced nuclease resistance.

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. Depending on the stage of modification, post-synthetic modification can be divided into two stages: "solid-phase modification," wherein an oligonucleotide attaches to the resin, and "solution-phase modification," wherein an oligonucleotide detaches itself from the resin. In this review, we focus on post-synthetic modification in solution for the synthesis of nucleobase-modified oligonucleotides, except the modifications to linkers for conjugation. Moreover, the reactions are summarized for each modified position of the nucleobases.

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. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

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 (BH )) was previously reported. Herein, a novel BH analogue, 2',4'-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-oxophenoxazine (G-clamp), named BNAP-AEO (BAEO ), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104 -, 152-, and 11-fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2'-deoxycytidine (C), 2',4'-BNA/LNA with 5-methylcytosine (L), or 2'-deoxyribonucleoside with G-clamp (PAEO ), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3'-exonuclease than ODNs with phosphorothioate linkages.

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

An Ir(III) polypyridyl complex-conjugated 14-mer oligonucleotide (IrIII-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 IrIII-DNA with 14-, 20-, and 26-mer ssDNAs were higher than those of the unconjugated oligonucleotides. The triplex of IrIII-DNA with 14-mer dsDNA was also stabilized. However, the triplexes of IrIII-DNA with 20- and 26-mer dsDNAs, flanked by 3 and 6 base pairs at the both ends of 14-mer dsDNA target, were destabilized. This is presumably because of steric repulsion between the Ir(III) complex and the protruding 3- and 6-mer dsDNA moieties which are inflexible compared to ssDNA.

Synthesis of Oligonucleotides Containing 2'-N-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'-N-pentafluorophenoxycarbonyl-2'-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2'-urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2'-amino-LNA. Thus, 2'-urea-LNAs could be promising units for the modification of oligonucleotides; the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.

Synthesis of 2'-C,4'-C-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., EoNA and EoDNAs) and demonstrated that the presence of 6'-oxygen atoms in the bridge structure could show positive effect on the properties of the modified oligonucleotides. Herein, we designed and synthesized 7'-methyl derivatives of methyleneoxy-bridged 2'-deoxyribonucleic acid (MoDNA), possessing a five-membered bridge with 6'-oxygen atom via radical cyclization for the bridge construction. The synthesized monomers were incorporated into the oligonucleotides by solid-phase oligonucleotide synthesis. The MoDNA-modified oligonucleotides showed high affinity toward single-stranded RNA and double-stranded DNA, as well as excellent resistance toward nuclease compared with the corresponding natural oligonucleotide.

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. © 2019 by John Wiley & Sons, Inc.

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

The synthesis of 6'S-Me-2'-O,4'-C-ethylene-bridged 5-methyluridine (6'S-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'S-Me-ENA-T monomer was successfully incorporated into oligonucleotides, and their properties were examined. The oligonucleotides containing 6'S-Me-ENA-T exhibited a highly selective hybridization affinity toward single-stranded RNA and an excellent enzymatic stability, compared to the corresponding LNA- and ENA-modified oligonucleotides.

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

We efficiently synthesized 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNANC) 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'-BNANC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2',4'-BNANC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2',4'-BNANC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2',4'-BNANC monomers in both or either strands were found to possess excellent thermal stabilities.

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. In particular, 3',4'-EoNA-T and (R)-Me-3',4'-EoNA-T modifications within isoDNA could stabilize the duplexes with complementary RNA compared with unmodified or 3',4'-BNA-modified isoDNAs.