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.

Photoisomerization of "Partially Embedded Dihydropyridazine" with a Helical Structure.

Herein, we report the synthesis of two "partially embedded fused-dihydropyridazine N-aryl aza[5]helicene derivatives" (PDHs) and the demonstration of their intrinsic photo-triggered multi-functional properties based on a Kekulé biradical structure. Introducing bulky electron-withdrawing trifluoromethyl or pentafluoroethyl groups into the aza[5]helicene framework (PDH-CF3 and -C2 F5 ) gives PDH axial chirality based on the helicity of the P and M forms, even at room temperature. Upon photo-irradiation of PDH-CF3 in a frozen solution, an ESR signal from the triplet biradical with zero-field splitting values, generated by N-N bond dissociation, was observed. However, when the irradiation was turned off, the ESR signal became silent, thus indicating the existence of two equilibria: between the biradical and quinoidal forms based on the Kekulé structure, and between N-N bond cleavage and recombination. The observed photo- and thermally induced behaviors indicate that T-type photochromic molecules are involved in the photoisomerization mechanism involving the two equilibria. Inspired by the photoisomerization, chirality control of PDH by photoracemization was achieved. Multiple functionalities, such as T-type photochromism, photo-excitation-mediated triplet biradical formation, and photoracemization, which are attributed to the "partially embedded dihydropyridazine" structure, are demonstrated.

Photoisomerization of "Partially Embedded Dihydropyridazine" with a Helical Structure.

Invited for the cover of this issue are the groups of Kazuteru Usui and Satoru Karasawa at Showa Pharmaceutical University and Yasuhiro Kobori of Kobe University. The image depicts chirality control of helical compounds through cycles of photocleavage and recombination under sunlight with a "Jack and the Beanstalk" motif. Read the full text of the article at 10.1002/chem.202302413.

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.

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.

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.

Artificial Host Molecules to Covalently Capture 8-Nitro-cGMP in Neutral Aqueous Solutions and in Cells.

New 1,3-diazaphenoxazine derivatives (nitroG-Grasp-Guanidine, NGG) have been developed to covalently capture 8-nitro-cGMP in neutral aqueous solutions, which furnish a thiol reactive group to displace the 8-nitro group and a guanidine unit for interaction with the cyclic phosphate. The thiol group was introduced to the 1,3-diazaphenoxazine skeleton through a 2-aminobenzylthiol group (NGG-H) and its 4-methyl (NGG-pMe) and 6-methyl (NGG-oMe) substituted derivatives. The covalent adducts were formed between the NGG derivatives and 8-nitro-cGMP in neutral aqueous solutions. Among the NGG derivatives, the one with the 6-methyl group (NGG-oMe) exhibited the most efficient capture reaction. Furthermore, NGG-H showed a cell permeability into HEK-293 and RAW 264.7 cells and reduced the intracellular 8-nitro-cGMP level. The NGG derivatives developed in this study would become a valuable tool to study the intracellular role of 8-nitro-cGMP.

Push-Pull Bisnaphthyridylamine Supramolecular Nanoparticles: Polarity-Induced Aggregation and Crystallization-Induced Emission Enhancement and Fluorescence Resonance Energy Transfer.

Emissive push-pull-type bisnaphthyridylamine derivatives (BNA-X: X=Me, Et, Bzl, Ph, BuBr, and BuTEMPO) aggregate in aqueous methanol. Furthermore, a two-step emission and aggregation process is controllable by varying the methanol-to-water ratio. At 2:3 MeOH/H2 O, crystallization-induced emission enhancement (CIEE) occurs via formation of an emissive crystal phase, whereas, at 1:9 MeOH/H2 O, aggregation-induced emission enhancement (AIEE) occurs, induced by emissive supramolecular nanoparticles (NPs). For BNA-Ph, the emission quantum yield was 25 times higher in aqueous methanol than that in pure methanol. Despite the high hydrophobicity of BNA-X (C log P=6.1-8.0), the spherical NPs were monodisperse (polydispersity indices <0.2). Moreover, the emissive NPs exhibited fluorescence resonance energy transfer (FRET) with pyrene; however, for BNA-X bearing the TEMPO radical (BNA-BuTEMPO), no FRET was observed because of quenching. In particular, the BNA-BuTEMPO NPs have a slow rotational correlation time (1.3 ns), suggesting applications as magnetic resonance imaging contrast agents with large relaxivity.

Characterization and Water-Proton Longitudinal Relaxivities of Liposome-Type Radical Nanoparticles Prepared via a Supramolecular Approach.

For the construction of metal-free magnetic resonance imaging (MRI) contrast agents, radical-based nanoparticles (RNPs) are promising materials because they allow the water-proton longitudinal relaxivity (r1) to be enhanced not only by paramagnetic resonance effects but also by prolonging the rotational correlation times (τR). However, the τR effect is limited because the radical units are often located within the central hydrophobic core of oil-in-water (o/w) emulsions, resulting in a lack of water molecules surrounding the radical units. In this study, to construct supramolecular RNPs that have high r1 values, we designed a liposome-type RNP in which the radical units are located at positions with sufficient surrounding water molecules. Using this strategy, PRO1 with a PROXYL framework was prepared by introducing hydrophilic groups on both sides of the radical unit. The RNP composed of PRO1 formed spherical nanoparticles approximately 100 nm in size and yielded a higher r1 value (0.26 mM-1 s-1) compared to those of small radical species and similar supramolecular o/w emulsion-type nanoparticles (0.17 mM-1 s-1 in PRO2).

Characterization of Push-Pull-Type Benzo[X]quinoline Derivatives (X = g or f): Environmentally Responsive Fluorescent Dyes with Multiple Functions.

Benzo[X]quinoline (X = g or f: BQX) derivatives bearing bis-trifluoromethyl and amine groups have been designed as push-pull-type fluorescent dyes. Through the synthesis of BQX derivatives from 2,7-diaminonaphthalene, linear-type (BQL) and angular-type (BQA) structural isomers were obtained. X-ray structures of single crystals from six given BQX derivatives revealed that the BQL and BQA series adopt planar- and bowl-shaped structures. In the fluorescence spectra, interestingly, the BQL series emitted in the near-infrared region over 700 nm in polar solvents. Based on the visible absorptions and base properties related to the amine moiety, the ammonia responsiveness was investigated using an ion-exchange reaction by the BQX-HCl salt. By exploiting the environmentally responsive fluorescence probe, cell imaging through confocal laser microscopy was conducted using HeLa and 3T3-L1 cells, emitting specific lipid droplets. The results indicate that BQX derivatives have multiple functions and may be applied in materials chemistry and biochemistry.

A fully synthetic 6-aza-artemisinin bearing an amphiphilic chain generates aggregates and exhibits anti-cancer activities.

Installation of a nitrogen at the C6 position of artemisinin facilitates the addition of a functional unit on the cyclohexane moiety (C-ring). In this study, conjugation of an amphiphilic chain, composed of sequentially connected hydrophilic oligoethylene glycol, hydrophobic alkyl chain, urea, and 4,4'-disubstituted biphenyl linker, imparted self-assembling properties. The fully synthetic mid-molecular weight 6-aza-artemisinin 6 bearing the amphiphilic moiety formed aggregates (approx. 200 nm) at ambient temperature and exhibited increased in vitro anti-cancer activities compared to the N-benzylated aza-artemisinin 5.

Fluorescence Properties and Exciplex Formation of Emissive Naphthyridine Derivatives: Application as Sensors for Amines.

Water-soluble donor-acceptor-type fluorophore 15Nap-Cl having two trifluoromethyl groups and a Cl group on a 1,5-aminonaphthyridine framework was prepared. Fluorophore 15Nap-Cl showed strong solvatochromic fluorescence, and, as the solvent polarity increased, a bathochromic shift was observed accompanied by an increase in the fluorescence quantum yield. In addition, in the presence of amines such as ethylamine, diethylamine, and aniline, further considerable bathochromic shifts in the fluorescence were observed. Density functional calculations identified the source of the fluorescence behavior as exciplex formation between 15-Nap-Cl and the corresponding amine. The fluorescence behavior was exploited to fabricate a sensor that can identify various primary, secondary, and tertiary amines.

Development of Turn-On Probes for Acids Triggered by Aromaticity Enhancement Using Tricyclic Amidine Derivatives.

Two fluorophores consisting of tricyclic amidine derivatives (DHIm and DHPy) were prepared as selective turn-on probes for acids, which were triggered by an aromaticity enhancement. Both amidine derivatives were expanded rings prepared by condensed reactions between the corresponding dibromoalkanes and an aminonaphthyridine analogue. In X-ray analyses, DHIm, in which the dihydroimidazole ring was condensed into aminonaphthyridine, showed high planarity, compared to DHPy, with condensed dihydropyrimidine. The fluorescence properties of DHIm exhibited a higher quantum yield than DHPy due to the difference in planarity. Under acidic conditions, such as in the presence of H+ and M(II), protonations and complexations occurred, exhibiting a higher quantum yield than the neutral DHX (X = Im or Py). The nucleus-independent chemical shift values from the density functional theory calculations suggested that the protonations and complexations caused an enhancement of the aromaticity within the frameworks. These aromaticity changes led to intense fluorescence, and DHX behaved as a selective turn-on probe for acids and metal ions. Interestingly, this fluorescence turn-on system triggered by the aromaticity-based enhancement is not a typical system, such as the photoinduced electron transfer, aggregation-induced enhanced emission, and twisted intramolecular charge transfer systems, but is classified as a novel turn-on system.

Luminescent europium sensors for specific detection of 8-oxo-dGTP by time-gated fluorescence.

The 9-hydroxy-1,3-diazaphenoxazine-2-one unit was conjugated with the Eu3+-cyclen complex through a linker. This diazaphenoxazine group was expected as an antenna unit for the excitation of europium ion, and a selective recognition site for 8-oxo-dGTP base. Among the synthesized three derivatives, the highest fluorescence emission was obtained by the complex constructed of an ethylene linker and the cyclen unit with three N,N-dimethylacetamide groups. The Eu3+-cyclen complex exhibited a selective response to the 8-oxo-dGTP in aqueous media by a time-resolved fluorescence assay.

Synthetic receptor molecules for selective fluorescence detection of 8-oxo-dGTP in aqueous media.

A series of 9-hydroxy-1,3-diazaphenoxazine-2-one derivatives were synthesized as fluorescent receptor molecules for 8-oxo-dGTP, which attach the cyclen-zinc complex at the 3-N position as the binding site for the triphosphate and the (2-aryloxycarbonylamino)ethyl group at the 9-O position as the hydrogen bonding site for 8-oxoguanine. Among these molecules, the receptor molecule 5a-Zn constructed of the ethyl linker at 3-N and the (2-benzyloxycarbonyl amino)ethyl group at 9-O displayed the best recognition ability for 8-oxoguanosine triphosphate (8-oxo-dGTP) in aqueous media. The receptor 5a-Zn was also shown to selectively detect 8-oxo-dGTP in a cell lysate solution.

New NitroG-Grasp Molecules with Enhanced Capture Reactivity for 8-Nitroguanosine in the Aqueous Media.

8-Nitroguanosine is formed by the nitration of guanosine, and has been conventionally classified as a genotoxic material. Recently, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) has become of great interest due to its biological role as an intracellular signaling molecule. In an attempt to develop recognition molecules for 8-nitroguanosine, we have determined a 1,3-diazaphenoxazine nucleoside derivative (nitroG-grasp) bearing a thiol group with a urea linker, which efficiently displaces the nitro group through the formation of multiple hydrogen-bonded complexes with 8-nitroguanosine. However, a drawback of this capture molecule was that the displacement efficiency was not sufficient to capture 8-nitroguanosine in water. Taking into account that both the flexibility of the linker and the pKa value of the thiol group are determinants of the reactivity, new nitroG-grasp molecules were designed to have a fixed linker structure with different pKa values. Compared to the previous nitroG-grasp, the new compounds with the (2-aminophenyl)methanethiol or the propylene acetal of 3-amino-1-mercaptopropan-2-one unit have exhibited more than 10-100 times faster reactivity in the aqueous media. These compounds may be potential components to construct new nitroG-grasp molecules to capture 8-nitro-cGMP in the biological systems.

Development of new 1,3-diazaphenoxazine derivatives (thioG-grasp) to covalently capture 8-thioguanosine.

The derivatives of 8-thioguanosine are thought to be included in the signal transduction system related to 8-nitroguanosine. In this study, we attempted to develop new 1,3-diazaphenoxazine (G-clamp) derivatives to covalently capture 8-thioguanosine (thioG-grasp). It was expected that the chlorine atom at the end of the linker would be displaced by the nucleophilic attack by the sulfur atom of 8-thioguanosine via multiple hydrogen-bonded complexes. The thioG-grasp derivative with a propyl linker reacted efficiently with 8-thioguanosine to form the corresponding adduct.

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.

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).

Photophysical Properties of Emissive Pyrido[3,2-c]carbazole Derivatives and Apoptosis Induction: Development towards Theranostic Agents in Response to Light Stimulus.

Pyridocarbazole moieties are present in many natural products, such as olivacine and ellipticine, and their derivatives are well-known anticancer agents. To develop functional therapeutic and diagnostic compounds, three emissive pyrido[3,2-c]carbazole derivatives, PC-X, containing secondary or tertiary amine groups, were synthesized from an aminoquinoline derivative using a palladium complex as the catalyst. X-ray diffraction analyses revealed that PC-X showed highly planar structures between the pyridine ring and the carbazole framework, exhibiting high fluorescence intensities along with solvatochromic behavior. Imaging of HeLa cells treated with PC-X showed no specific accumulation into the organelles; however, a comparative examination showed that the accumulation in mitochondria was the highest as compared to nuclei and lysosomes. Cytotoxicity analysis using HeLa cells showed that PC-H, containing a secondary amine group showed the highest cytotoxicity (IC50 ≈20 µm) as compared to another PC-X having a tertiary amine group. Colocalization with MitoTracker, a typical mitochondrial staining dye, showed apoptosis-like behavior with remarkable appearance of blebbing during irradiation with near UV light (403 nm), suggesting that the PC-H may not only behave as a fluorescence probe for the imaging organelles, but also as a therapeutic agent for inducing apoptosis in HeLa cells, thereby functioning as a theranostic agent.