1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties.

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.

PAR-CLIP Assay in Ferroptosis.

Ferroptosis is a regulatory cell death process that is accompanied by large amounts of iron ion accumulation and lipid peroxidation. Photoactivated ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) is a method used to identify the binding sites of RNA-binding proteins (RBPs) on target RNAs with high resolution at the nucleotide level. By inserting photosensitive ribonucleoside analogs into new RNA transcripts of living cells, characteristic mutations can be generated during reverse transcription and be used to accurately locate the crosslinking position of RNAs and RBPs. The use of PAR-CLIP to detect interactions and determine precise crosslinking sites between RNAs and RBPs, or to search for RNAs upstream or downstream of ferroptosis pathways genes through known proteins, can help to clarify and verify the occurrence and regulation mechanisms of the various signaling pathways of ferroptosis. Furthermore, it may reveal new targets for ferroptosis detection and improve the treatment efficiency of ferroptosis-related diseases such as cancer and neurodegenerative diseases. Here, we introduce a specific PAR-CLIP protocol for monitoring the ferroptosis process.

Optimized infrared photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (IR-PAR-CLIP) protocol identifies novel IGF2BP3-interacting RNAs in colon cancer cells.

The conserved family of RNA-binding proteins (RBPs), IGF2BPs, plays an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs: IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells, and its expression correlates with a poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we modified the protocol to use highly sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified method "IR-PAR-CLIP." Third, we compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.

Bioactive Furanyl- or Thienyl-Substituted Nucleobases, Nucleosides and Their Analogues.

Five-membered heterocycles, including furan and thiophene, play a prominent role in drug design as structural units of bioactive molecules. This review is intended to demonstrate the importance of the furan-2-yl, furan-3-yl, thien-2-yl and thien-3-yl substituents in the medicinal chemistry of purine and pyrimidine nucleobases, nucleosides and selected analogues. Data presented in the article are limited to compounds containing heteroaromatic ring connected through a bond and not fused to other systems. The impact of bioisosteric replacement of aryl substituents with heteroaryl ones on activities was assessed by comparison of the title compounds with their aryl counterparts. A total of 135 heteroaryl-substituted and 35 aryl-substituted derivatives are mentioned in the text and shown in the figures. The following classes of compounds are included in the article: (i) 5-heteroaryl-2'-deoxyuridines and related compounds; (ii) 8-heteroaryl- 2,9-disubstituted adenine derivatives; (iii) O6-(heteroarylmethyl)guanines; (iv) 6-heteroaryl tricyclic guanine analogues; (v) 6-heteroaryl-9-benzylpurines and analogous compounds; (vi) N4- furfurylcytosine, N6-furfuryladenine, their derivatives and analogues; (vii) 6-heteroaryl purine and 7- deazapurine ribonucleosides; (viii) 7-heteroaryl-7-deazaadenosines, their derivatives and analogues; (ix) 4-heteroaryl fused 7-deazapurine nucleosides. In most cases various modifications of the lead compound structure performed in order to obtain the most favorable activity and selectivity are briefly discussed. The reviewed structure-activity relationship studies exemplify the search for compounds with optimized antiviral, antitumor, antimycobacterial or antiparkinsonian action.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

Aqueous-Microdroplet-Driven Abiotic Synthesis of Ribonucleotides.

Phosphorylation for ribonucleotide formation is a critical step in the origin of life but has had limited success due to the thermodynamic and kinetic constraints in aqueous media. Here, we report that the production of ribonucleotides from ribonucleosides in the presence of monopotassium phosphate (KH2PO4) spontaneously proceeded in aqueous microdroplets under ambient conditions and without using a catalyst. A full set of ribonucleotides including adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), and cytidine monophosphate (CMP) were generated on the scale of a few milliseconds. The aqueous microdroplets could transfer the ribonucleotides to oligoribonucleotides and showed mutual compatibility for individual phosphorylation. Conditions established the dependence of the conversion ratio on the droplet size and suggested that the condensation reactions occurred at or near the microdroplets' surface. This aqueous microdroplet approach also provides a route for elucidating phosphorylation chemistry in the prebiotic era.

Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.

We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent Km values for α-R and ATP were 358 and 297 µM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and ß-ribazole (ß-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate ß-N-linked glycosides like ß-adenosine or ß-R. Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (S. enterica) strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated α-Ado in vitro. When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica α-Ado transport may be inefficient. We conclude that GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target.

The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.

7-Iodo-5-aza-7-deazaguanine ribonucleoside: crystal structure, physical properties, base-pair stability and functionalization.

The positional change of nitrogen-7 of the RNA constituent guanosine to the bridgehead position-5 leads to the base-modified nucleoside 5-aza-7-deazaguanosine. Contrary to guanosine, this molecule cannot form Hoogsteen base pairs and the Watson-Crick proton donor site N3-H becomes a proton-acceptor site. This causes changes in nucleobase recognition in nucleic acids and has been used to construct stable `all-purine' DNA and DNA with silver-mediated base pairs. The present work reports the single-crystal X-ray structure of 7-iodo-5-aza-7-deazaguanosine, C10H12IN5O5 (1). The iodinated nucleoside shows an anti conformation at the glycosylic bond and an N conformation (O4'-endo) for the ribose moiety, with an antiperiplanar orientation of the 5'-hydroxy group. Crystal packing is controlled by interactions between nucleobase and sugar moieties. The 7-iodo substituent forms a contact to oxygen-2' of the ribose moiety. Self-pairing of the nucleobases does not take place. A Hirshfeld surface analysis of 1 highlights the contacts of the nucleobase and sugar moiety (O-H...O and N-H...O). The concept of pK-value differences to evaluate base-pair stability was applied to purine-purine base pairing and stable base pairs were predicted for the construction of `all-purine' RNA. Furthermore, the 7-iodo substituent of 1 was functionalized with benzofuran to detect motional constraints by fluorescence spectroscopy.

Naturally occurring modified ribonucleosides.

The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as the "fifth" ribonucleotide in 1951. Since then, the ever-increasing number and complexity of modified ribonucleosides have been found in viruses and throughout all three domains of life. Such modifications can be as simple as methylations, hydroxylations, or thiolations, complex as ring closures, glycosylations, acylations, or aminoacylations, or unusual as the incorporation of selenium. While initially found in transfer and ribosomal RNAs, modifications also exist in messenger RNAs and noncoding RNAs. Modifications have profound cellular outcomes at various levels, such as altering RNA structure or being essential for cell survival or organism viability. The aberrant presence or absence of RNA modifications can lead to human disease, ranging from cancer to various metabolic and developmental illnesses such as Hoyeraal-Hreidarsson syndrome, Bowen-Conradi syndrome, or Williams-Beuren syndrome. In this review article, we summarize the characterization of all 143 currently known modified ribonucleosides by describing their taxonomic distributions, the enzymes that generate the modifications, and any implications in cellular processes, RNA structure, and disease. We also highlight areas of active research, such as specific RNAs that contain a particular type of modification as well as methodologies used to identify novel RNA modifications. This article is categorized under: RNA Processing > RNA Editing and Modification.

New N-ribosides and N-mannosides of rhodanine derivatives with anticancer activity on leukemia cell line: Design, synthesis, DFT and molecular modelling studies.

N-ribosylation and N-mannosylation compounds have a great role in compounds activity as anticancer. The reaction of 2-thioxo-4-thiazolidinone (rhodanine) derivatives, as aglycon part, was done with ribofuranose and mannopyranose sugars (glycone part) followed by deacetylation without cleavage of the rhodanine under acidic medium. Conformational analysis has been studied using NMR methods (2D, DQF-COSY, HMQC and HMBC). All final the new deprotected nucleosides were screened against leukemia 1210, and were found to be considerably less potent (Ic50% 1.4-10.6 µM) than doxorubicin (Ic50% 0.02 µM). Compounds 10d and 10e which contain ribose moiety have better activity than those with mannose sugar. DFT calculations with B3LYP/6-31 + G (d) level were used to analyze the electronic and geometric characteristics deduced from the stable structure of the compounds. The principal quantum chemical descriptors showed a good correlation with the experimental observations. Rapid Overlay Comparison Similarity (ROCS) study was operated to explain the compounds similarity and to figure out the most important pharmacophoric features.

Discovery and applications of nucleoside antibiotics beyond polyoxin.

Nucleoside antibiotics possess various biological activities such as antibacterial, antifungal, anticancer, and herbicidal activities. RIKEN scientists contributed to this area of research with two representative antifungal nucleoside antibiotics, blasticidin S and polyoxin. Blasticidin S was the first antibiotic exploited in agriculture worldwide. Meanwhile, the polyoxins discovered by Isono and Suzuki are still used globally as an agricultural antibiotic. In this review article, the research on nucleoside antibiotics mainly done by Isono and his collaborators is summarized from the discovery of polyoxin to subsequent investigations.

Gallic acid functionalized UiO-66 for the recovery of ribosylated metabolites from human urine samples.

Early diagnosis of disease biomarkers has been focused in recent years through Omics sciences. Nucleosides are the biomarkers of cancers including lung cancer, colorectal cancer, thyroid cancer, bladder cancer, cervical cancer and breast cancer. Nucleosides are directly excreted in the urine of diseased states whereas they are decomposed into other forms as modified nucleosides in healthy conditions. A dual affinity probe (gallic acid modified UiO-66) is prepared and reported for the first time in selectively enriching the ribosylated metabolites and modified nucleosides. Material is characterized by SEM, EDX, FTIR and Nitrogen adsorption porosimetry. The enrichment is benefitted from the interaction ability of zirconium towards glycosylated molecules, rich surface chemistry (3 terminal hydroxyl groups) on gallic acid and high surface area (384 m2/g) of 3-dimensional porous structure of metal organic frameworks (MOFs). Material shows selectivity of 1:500, recovery up to 137.1% and binding capacity of 2340.9 µg/g. Forty-three (43) nucleosides are enriched from human urine samples and 12 potential nucleoside biomarkers from colorectal cancer samples are quantified and their concentration is found higher than in the healthy controls.

Novel Synthesis and Biological Evaluation of the First Pyrazole Thioglycosides as Pyrazofurin Analogues.

This study reports a novel and efficient method for the synthesis of the first reported novel class of pyrazole thioglycosides 6a-h. These series of compounds were designed through the reaction of sodium 2-cyano-3-oxo-3-(4-substitutedphenylamino)prop-1-ene-1,1-bis(thiolate) salts 2 with hydrazine hydrate in ethanol at room temperature to give the corresponding sodium 5-amino-4-(substitutedphenylcarbamoyl)-1H-pyrazole-3-thiolates 3a-d. The latter compounds were treated with protected α-D-gluco- and galacto-pyranosyl bromides 4a,b in DMF at ambient temperature to give in a high yields the corresponding pyrazole thioglycosides 6a-h. Treatment of pyrazole salts 3a-d with hydrochloric acid at amobient temperature afforded the corresponding 3-mercaptopyrazole derivatives 5. The latter compounds were treated with peracetylated sugars 4 in sodium hydride in ethanol at ambient temperature to tolerate the S-glycosyl 6a-h compounds. Ammonolysis of the pyrazole thioglycosides 6a-h afforded the corresponding free thioglycosides 7a-h. The toxicity and antitumor activities of the synthesized compounds were studied.

Design, synthesis, and biological evaluation of novel 2'-deoxy-2'-fluoro-2'-C-methyl 8-azanebularine derivatives as potent anti-HBV agents.

Hepatitis B virus (HBV) is a global health problem requiring more efficient and better tolerated anti-HBV agent. In this paper, a series of novel 2'-deoxy-2'-fluoro-2'-C-methyl-ß-d-arabinofuranosyl 8-azanebularine analogues (1 and 2a) and N4-substituted 8-azaadenosine derivatives (2b-g) were designed, synthesized and screened for in vitro anti-HBV activity. Two concise and practical synthetic routes were developed toward the structural motif construction of 2'-deoxy-2'-fluoro-2'-C-methyl-ß-d-arabinofuranosyl 8-azainosine from the ribonolactone 3 under mild conditions. The in vitro anti-HBV screening results showed that these 8-azanebularine analogues had a significant inhibitory effect on the expression of HBV antigens and HBV DNA at a concentration of 20 µM. Among them, halogen-substituted 8-azaadenosine derivative 2g displayed activities comparable to that of 3TC. In particular, 2g retained excellent activity against lamivudine-resistant HBV mutants.

Regioselective 5'-position phosphorylation of ribose and ribonucleosides: phosphate transfer in the activated pyrophosphate complex in the gas phase.

Herein, we present a rapid, efficient and regioselective phosphorylation method at the 5'-position of unprotected ribose and ribonucleosides with pyrophosphate in the gas phase, which involves the formation of anionic complexes via electrospray ionization and collisional activation to induce phosphorylation within the complexes.

Synthesis and Cytotoxic and Antiviral Profiling of Pyrrolo- and Furo-Fused 7-Deazapurine Ribonucleosides.

Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

Combinatorial Synthesis of New Pyrimidine- and Purine-ß-d-Ribonucleoside Nucleolipids: Their Distribution Between Aqueous and Organic Phases and Their In Vitro Activity Against Human- and Rat Glioblastoma Cells In Vitro.

Two series of nucleolipids, O-2',3'-heptanylidene- as well as O-2',3'-undecanylidene ketals of six ß-d-ribonucleosides (type A) and partly N-farnesyl derivatives thereof (type B) were prepared in a combinatorial manner. All novel compounds were characterized by elemental analysis and/or ESI mass spectrometry and by UV-, 1 H-, and 13 C-NMR spectroscopy. Conformational parameters of the nucleosides and nucleolipids were calculated from various 3 J(H,H), 3 J(1 H,13 C), and 5 J(F,H) coupling constants. For a drug profiling, the parent nucleosides and their lipophilic derivatives were studied with respect to their distribution (log P) between water and n-octanol as well as water and cyclohexane. From these data, qualitative conclusions were drawn concerning their possible blood-brain barrier passage efficiency. Moreover, nucleolipids were characterized by their molecular descriptor amphiphilic ratio (a.r.), which describes the balance between the hydrophilicity of the nucleoside headgroup and the lipophilicity of the lipid tail. All compounds were investigated in vitro with respect to their cytostatic/cytotoxic activity toward human glioblastoma (GOS 3) as well as rat malignant neuroectodermal BT4Ca cell lines in vitro. In order to differentiate between anticancer and side-effects of the novel nucleolipids, they were also studied on their activity on differentiated human THP-1 macrophages.