Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Modified (2'-deoxy)adenosines activate autophagy primarily through AMPK/ULK1-dependent pathway.

Autophagy is a conserved self-digestion process, which governs regulated degradation of cellular components. Autophagy is upregulated upon energy shortage sensed by AMP-dependent protein kinase (AMPK). Autophagy activators might be contemplated as therapies for metabolic neurodegenerative diseases and obesity, as well as cancer, considering tumor-suppressive functions of autophagy. Among them, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), a nucleoside precursor of the active phosphorylated AMP analog, is the most commonly used pharmacological modulator of AMPK activity, despite its multiple reported "off-target" effects. Here, we assessed the autophagy/mitophagy activation ability of a small set of (2'-deoxy)adenosine derivatives and analogs using a fluorescent reporter assay and immunoblotting analysis. The first two leader compounds, 7,8-dihydro-8-oxo-2'-deoxyadenosine and -adenosine, are nucleoside forms of major oxidative DNA and RNA lesions. The third, a derivative of inactive N6-methyladenosine with a metabolizable phosphate-masking group, exhibited the highest activity in the series. These compounds primarily contributed to the activation of AMPK and outperformed AICAr; however, retaining the activity in knockout cell lines for AMPK (ΔAMPK) and its upstream regulator SIRT1 (ΔSIRT1) suggests that AMPK is not a main cellular target. Overall, we confirmed the prospects of searching for autophagy activators among (2'-deoxy)adenosine derivatives and demonstrated the applicability of the phosphate-masking strategy for increasing their efficacy.

Direct C-H borylation of vinylporphyrins via copper catalysis.

A method of direct borylation of vinyl-substituted porphyrinoids (porphyrins and chlorins) has been developed based on the copper catalyzed vinylic C-H activation. Ni(II) complexes of meso- and ß-vinylporphyrinoids have been transformed to the corresponding pinacolboronated derivatives with good yields and high (E)-stereoselectivity. The method provides an easy and direct access to the valuable synthons which were shown to act as nucleophylic partners in the Suzuki cross-coupling building tetrapyrrole derivatives with π-conjugation through the carbon-carbon double bond.

Carbene functionalization of porphyrinoids through tosylhydrazones.

Here, we investigated methods for carbene functionalization of porphyrinoids through metal catalyst-free thermal decomposition of their tosylhydrazones. For the first time, tetrapyrrolyl substituted carbenes were obtained via thermolysis of tosylhydrazones of the corresponding tetrapyrrolyl aldehydes and ketones in the presence of a base. The carbenes formed reacted thermally with substrates without a metal catalyst or light irradiation. Carbenes at the ß-pyrrolic position of porphyrinoids reacted with styrene leading to cyclopropane derivatives of tetrapyrroles. Carbenes also reacted with 1,4-dioxane with their insertion into the C-H bond yielding a tetrapyrrole 1,4-dioxane conjugate. Thermolysis of tosylhydrazones of meso-formyl-ß-octaalkylporphyrinoids led exclusively to the corresponding cyclopentane fused porphyrinoids via intramolecular carbene C-H insertion. A plausible reaction mechanism was discussed based on DFT calculations of the intermediates. The tetrapyrrolyl carbenes were found to be considerably more stable than other carbenes. The products of the functionalization of porphyrinoids via hydrazone formation and subsequent carbene reactions exhibited modified optical spectra. The method for carbene functionalization of porphyrinoids through thermal decomposition of their tosylhydrazones created a new synthetic pathway for tailoring the perimeter of tetrapyrrolic macrocycles. Moreover, this method allows the obtainment of dyes with controllable spectral optical properties. In particular, new tetrapyrrole derivatives possessing phytoporphyrin carbon skeletons which have not been accessible were obtained using a convenient straightforward procedure.

Phenoxazine-based scaffold for designing G4-interacting agents.

G-quadruplexes (G4) represent one class of non-canonical secondary nucleic acid structures that are currently regarded as promising and attractive targets for anti-cancer, anti-viral and antibacterial therapy. Herein, we probe a new i-clamp-inspired phenoxazine scaffold for designing G4-stabilizing ligands. The length of the protonated aminoalkyl tethers ('arms') of the phenoxazine-based ligand was optimized in silico. Two double-armed ligands differing in the relative orientation of their arms and one single-armed ligand were synthesized. The two-armed ligands significantly enhanced the thermal stability of the G-quadruplex structures (increasing the melting temperature by up to 20 °C) and displayed G4 selectivity over duplex DNA. The ligands look promising for biological studies and the phenoxazine scaffold could be a starting point for designing new G4-interacting compounds.

i-Clamp phenoxazine for the fine tuning of DNA i-motif stability.

Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements. However, iM stability is moderate, which in turn limits its in vivo applications. Here, we report the rational design of a new substituted phenoxazine 2'-deoxynucleotide (i-clamp) for iM stabilization. This residue contains a C8-aminopropyl tether that interacts with the phosphate group within the neighboring chain without compromising base pairing. We studied the influence of i-clamp on pH-dependent stability for intra- and intermolecular iM structures and found the optimal positions for modification. Two i-clamps on opposite strands provide thermal stabilization up to 10-11°C at a pH of 5.8. Thus, we developed a new modification that shows significant iM-stabilizing effect both at strongly and mildly acidic pH and increases iM transition pH values. i-Clamp can be used for tuning iM-based pH probes or assembling extra stable iM structures for various applications.

Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA.

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.

Synergism of primary and secondary interactions in a crystalline hydrogen peroxide complex with tin.

Despite the significance of H2O2-metal adducts in catalysis, materials science and biotechnology, the nature of the interactions between H2O2 and metal cations remains elusive and debatable. This is primarily due to the extremely weak coordinating ability of H2O2, which poses challenges in characterizing and understanding the specific nature of these interactions. Herein, we present an approach to obtain H2O2-metal complexes that employs neat H2O2 as both solvent and ligand. SnCl4 effectively binds H2O2, forming a SnCl4(H2O2)2 complex, as confirmed by 119Sn and 17O NMR spectroscopy. Crystalline adducts, SnCl4(H2O2)2·H2O2·18-crown-6 and 2[SnCl4(H2O2)(H2O)]·18-crown-6, are isolated and characterized by X-ray diffraction, providing the complete characterization of the hydrogen bonding of H2O2 ligands including geometric parameters and energy values. DFT analysis reveals the synergy between a coordinative bond of H2O2 with metal cation and its hydrogen bonding with a second coordination sphere. This synergism of primary and secondary interactions might be a key to understanding H2O2 reactivity in biological systems.

Cationic Perylene Antivirals with Aqueous Solubility for Studies In Vivo.

Perylene-based compounds are attracting significant attention due to their high broad-spectrum antiviral activity against enveloped viruses. Despite unambiguous results of in vitro studies and high selectivity index, the poor water solubility of these compounds prevented in vivo evaluation of their antiviral properties. In this work, we synthesized a series of compounds with a perylene pharmacophore bearing positively charged substituents to improve the aqueous solubility of this unique type of antivirals. Three types of charged groups were introduced: (1) quaternary morpholinium salts (3a-b); (2) a 2'-O-l-valinyl-uridine hydrochloride residue (8), and (3) a 3-methylbenzothiazolium cation (10). The synthesized compounds were evaluated based both on antiviral properties in vitro (CHIKV, SARS-CoV-2, and IAV) and on solubility in aqueous media. Compound 10 has the greatest aqueous solubility, making it preferable for pre-evaluation by intragastrical administration in a mouse model of lethal influenza pneumonia. The results indicate that the introduction of a positively charged group is a viable strategy for the design of drug candidates with a perylene scaffold for in vivo studies.

Anticancer activity of G4-targeting phenoxazine derivatives in vitro.

G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC50 in the micromolar range). Importantly, the CC50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action.