Synthesis Of Nucleosides Research Articles

Synthesis and Biological Evaluation of Glycosides and Acyclic Nucleosides Derived 2‐Oxonicotinonitriles

Synthesis and biological evaluations of a series of 2‐oxonicotinonitriles (2‐ONNs) derivatives are described. Incorporation of branching and solubilizing groups to the 2‐ONN derivative 7 was attained by coupling with several organo‐halides/alkylating agents including three glycosyl bromides under basic conditions. Coupling of 2‐ONNs occurred mainly at the ring nitrogen position and to a lesser extent at the 2‐oxo position giving the O‐alkylated products. Alkylated ONNs and free nucleosides/glycosides derived from the 2‐ONN derivative 7 were tested for their antibacterial, antifungal, and antiviral activities. N‐propargyl and N‐allyl derivatives 23 and 25 showed significant anti‐SARS‐CoV. The N‐butylacetate and O‐allyl derivatives 18 and 26 showed potent antibacterial activities against both Gram‐positive (Staphylococcus aureus, Micrococci luteus) and Gram‐negative (Pseudomonas aeruginosa, Escherichia coli) bacterial strains. The N‐glucoside derivative 8 showed significant antifungal activity.

An Eco-Friendly Technique: Solvent-Free Microwave Synthesis and Docking Studies of Some New Pyridine Nucleosides and Their Pharmacological Significance.

Two series of novel 5-arylazo-3-cyano-2-(2″,3″,4″,6″-tetra-O-acetyl-β-d-galacto pyranosyloxy) pyridines and 3-cyano-2-(2″,3″,4″,6″-tetra-O-acetyl-β-d-galactopyranosyloxy) pyridines were synthesized in high yields utilizing a microwave-assisted synthesis tool guided by the principles of green chemistry. The chemical structures of the new substances were confirmed on the basis of their elemental analysis and spectroscopic data (FT-IR, 1D, 2D-NMR). Activity against different bacterial strains was studied. The anticancer potential of the new compounds is also discussed. Molecular docking was used as a tool in this research work to get better insight into the possible interactions, affinities, and expected modes of binding of the most promising derivatives of the potential chemotherapeutic target (DHFR).

Leflunomide Synergizes with Gemcitabine in Growth Inhibition of PC Cells and Impairs c-Myc Signaling through PIM Kinase Targeting

The immunosuppressive agent leflunomide has been used in the treatment of over 300,000 patients with rheumatoid arthritis. Its active metabolite, teriflunomide (Ter), directly inhibits dihydroorotate dehydrogenase (DHODH), an enzyme involved in nucleoside synthesis. We report that Ter not only shows in vitro anti-proliferative activity in pancreatic cancer (PC) cells as a single agent but also synergizes with the chemotherapeutic gemcitabine (Gem) in growth inhibition of PC cells. The growth-inhibitory effects of Ter are not solely caused by inhibition of DHODH. Through a kinase screening approach, we identified the PIM-3 serine-threonine kinase as a novel direct target. Subsequent dose-response kinase assays showed that Ter directly inhibited all three PIM family members, with the highest activities against PIM-3 and -1. The PIM-3 kinase was the PIM family member most often associated with PC oncogenesis and was also the kinase inhibited the most by Ter among more than 600 kinases investigated. Ter in PC cells induced changes in phosphorylation and expression of PIM downstream targets, consistent with the effects achieved by overexpression or downregulation of PIM-3. Finally, pharmacological inhibition of PIM proteins not only diminished PC cell proliferation, but also small-molecule pan-PIM and PIM-3 inhibitors synergized with Gem in growth inhibition of PC cells.

Synthesis and Conformation of Pentopyranoside Nucleoside Phosphonates.

In contrast to natural nucleosides, where the nucleobase is positioned at the anomeric center, we report the synthesis of pentopyranoside nucleosides with a phosphonate functionality at the 1'-anomeric oxygen. Starting from l-arabinose, key functionalized l- glycero- and l- erythro-pentopyranose carbohydrate synthons were prepared and further elaborated into the final six-membered ring nucleosides via nucleobase incorporation and phosphonomethylation reactions. NMR analysis demonstrated that these nucleoside phosphonates exist in solution as conformers predominantly adopting a chair structure in which the base moiety is equatorially positioned. Such conformation prevents unfavorable 1,3-diaxial steric and electronic interactions. Notably, the stereochemical outcome of the Vorbrüggen glycosylation step utilized en route to the thymine analogue clearly suggests the absence of anchimeric assistance, as opposed to what is usually observed during nucleoside synthesis using protected furanose precursors. The finding that the diphosphates of the compounds developed in this study are recognized by DNA polymerases is important in view of the future selection of artificial genetic systems and dedicated polymerases as well as applications in therapy.

Synthesis and biological evaluation of novel imidazole nucleosides as potential anti-dengue virus agents.

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-β-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4'-thio and 4'-seleno derivatives of EICAR and EICNR 3-6 were prepared. The resulting 4'-thioEICAR and 4'-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.

Editorial

Editorial

Re-Engineering RNA Molecules into Therapeutic Agents.

Efforts to chemically modify nucleic acids got underway merely a decade after the discovery of the DNA double helix and initially targeted nucleosides and nucleotides. The origins of three analogues that remain staples of modification strategies and figure prominently in FDA-approved nucleic acid therapeutics can be traced to the 1960s: 2'-deoxy-2'-fluoro-RNA (2'-F RNA), 2'- O-methyl-RNA (2'- OMe RNA), and the phosphorothioates (PS-DNA/RNA). Progress in nucleoside phosphoramidite-based solid phase oligonucleotide synthesis has gone hand in hand with the creation of second-generation (e.g., 2'- O-(2-methoxyethyl)-RNA, MOE-RNA) and third-generation (e.g., bicyclic nucleic acids, BNAs) analogues, giving rise to an expanding universe of modified nucleic acids. Thus, beyond site-specifically altered DNAs and RNAs with a modified base, sugar, and/or phosphate backbone moieties, nucleic acid chemists have created a host of conjugated oligonucleotides and artificial genetic polymers (XNAs). The search for oligonucleotides with therapeutic efficacy constitutes a significant driving force for these investigations. However, nanotechnology, diagnostics, synthetic biology and genetics, nucleic acid etiology, and basic research directed at the properties of native and artificial pairing systems have all stimulated the design of ever more diverse modifications. Modification of nucleic acids can affect pairing and chemical stability, conformation and interactions with a flurry of proteins and enzymes that play important roles in uptake, transport or processing of targets. Enhancement of metabolic stability is a central concern in the design of antisense, siRNA and aptamer oligonucleotides for therapeutic applications. In the antisense approach, uniformly modified oligonucleotides or so-called gapmers are used to target a specific RNA. The former may sterically block transcription or direct alternative splicing, whereas the latter feature a central PS window that elicits RNase H-mediated cleavage of the target. The key enzyme in RNA interference (RNAi) is Argonaute 2 (Ago2), a dynamic multidomain enzyme that binds multiple regions of the guide (antisense) and passenger (sense) siRNAs. The complexity of the individual interactions between Ago2 and the siRNA duplex provides significant challenges for chemical modification. Therefore, a uniform (the same modification throughout, e.g., antisense) or nearly uniform (e.g., aptamer) modification strategy is less useful in the pursuit of siRNA therapeutic leads. Instead, unique structural features and protein interactions of 5'-end (guide/Ago2MID domain), seed region, central region (cleavage site/Ago2 PIWI domain), and 3'-terminal nucleotides (guide/Ago2 PAZ domain) demand a more nuanced approach in the design of chemically modified siRNAs for therapeutic use. This Account summarizes current siRNA modification strategies with an emphasis on the regio-specific interactions between oligonucleotide and Ago2 and how these affect the choice of modification and optimization of siRNA efficacy. In addition to standard assays applied to measure the effects of modification on the stability of pairing and resistance against nuclease degradation, structural insights based on crystallographic data for modified RNAs alone and in complex with Ago2 from molecular modeling studies are a valuable guide in the design of siRNA therapeutics. Thus, this comprehensive approach is expected to result in accelerated generation of new siRNA-based therapies against various diseases, now that the first siRNA has obtained approval by the US FDA for treatment of hereditary hATTR amyloidosis.

Integrated synthesis of nucleotide and nucleosides influenced by amino acids

Research on prebiotic chemistry and the origins of nucleic acids and proteins has traditionally been focussed on only one or the other. However, if nucleotides and amino acids co-existed on the early Earth, their mutual interactions and reactivity should be considered explicitly. Here we set out to investigate nucleotide/nucleoside formation by simple dehydration reactions of constituent building blocks (sugar, phosphate, and nucleobase) in the presence of different amino acids. We demonstrate the simultaneous formation of glycosidic bonds between ribose, purines, and pyrimidines under mild conditions without catalysts or activated reagents, as well as nucleobase exchange, in addition to the simultaneous formation of nucleotide and nucleoside isomers from several nucleobases. Clear differences in the distribution of glycosylation products are observed when glycine is present. This work demonstrates that reaction networks of nucleotides and amino acids should be considered when exploring the emergence of catalytic networks in the context of molecular evolution.

Efficient synthesis and antifungal investigation of nucleosides’ quaternaryammonium salt derivatives

Quaternary ammonium salts are a group of compounds with diverse biological properties, the most important of which are their antiviral, antibacterial, and antifungal activities. The quaternization reactions of 5'-O-tosyl derivatives of uridine and thymidine with triethylamine, trimethylamine, 4-(N ,N -dimethylamino)pyridine, 2-methylpyridine, and pyridine are described in this article. Two of the synthesized compounds are exceptional because they are first of this type that demonstrate concentration-dependent antifungal in vitro activity against two species of the genus Candida in minimal YNB-SG medium. The experimental results have been extended by adding full atom molecular dynamics simulations and substrates and products energies evaluation.

Enzymatic synthesis of novel purine nucleosides bearing a chiral benzoxazine fragment.

A series of ribo- and deoxyribonucleosides bearing 2-aminopurine as a nucleobase with 7,8-difluoro- 3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (conjugated directly or through an aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction. Nucleosides 3-6 were resistant to deamination under action of adenosine deaminase (ADA) Escherichia coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was evaluated against herpes simplex virus type 1 (HSV-1, strain L2). It has been shown that at sub-toxic concentrations, nucleoside (S)-4-[2-amino-9-(β-D-ribofuranosyl)-purin-6-yl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine exhibit significant antiviral activity (SI>32) on the model of HSV-1 in vitro, including an acyclovir-resistant virus strain (HSV-1, strain L2/R).

Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5′-monophospate analogues

The present work describes the functional and structural characterization of adenine phosphoribosyltransferase 2 from Thermus thermophilus HB8 (TtAPRT2). The combination of structural and substrate specificity data provided valuable information for immobilization studies. Dimeric TtAPRT2 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles by two different strategies: a) an enzyme immobilization at pH 8.5 to encourage the immobilization process by N-termini (MTtAPRT2A, MTtAPRT2B, MTtAPRT2C) or b) an enzyme immobilization at pH 10.0 to encourage the immobilization process through surface exposed lysine residues (MTtAPRT2D, MTtAPRT2E, MTtAPRT2F). According to catalyst load experiments, MTtAPRT2B (activity: 480 IU g−1biocatalyst, activity recovery: 52%) and MTtAPRT2F (activity: 507 IU g−1biocatalyst, activity recovery: 44%) were chosen as optimal derivatives. The biochemical characterization studies demonstrated that immobilization process improved the thermostability of TtAPRT2. Moreover, the potential reusability of MTtAPRT2B and MTtAPRT2F was also tested. Finally, MTtAPRT2F was employed in the synthesis of nucleoside-5′-monophosphate analogues.

Efficient Synthesis of Spirocyclic Nucleosides via Michael Addition-Initiated Intermolecular Cyclopropanation Reaction

Efficient Synthesis of Spirocyclic Nucleosides via Michael Addition-Initiated Intermolecular Cyclopropanation Reaction

Bio-catalytic synthesis of unnatural nucleosides possessing a large functional group such as a fluorescent molecule by purine nucleoside phosphorylase

Unnatural nucleosides are attracting interest as potential diagnostic tools, medicines, and functional molecules.

Synthesis and Characterization of New Isatin Nucleosides

In this research, a series of new nucleoside analogues 25–36 of substituted isatin as nucleobase were synthesized in three steps. The reaction sequence is started from the condensation of free sugar (D-glucose and L-rhamnose) with different anilines to afford the N-arylglycosylamines 1–12 which subsequently O-acetylated in the presence of acetic anhydride/pyridine. The resulting acetylated N-arylglycosylamines 13–24 were then subjected to an intramolecular cyclization with oxalyl chloride in the presence of anhydrous aluminum chloride to yield the target isatin nucleosides. The synthesized compounds were characterized by the FTIR, 1H-NMR and13 C-NMR spectroscopy. The stereochemistry was established based on the coupling constants observed between vicinal hydrogens of the glycon that confirmed the β-configuration of the synthesized nucleosides with a 4C1 conformation of the glucosides and a 1C4 conformation of the rhamnosides.

Biocatalytic synthesis of seleno-, thio- and chloro-nucleobase modified nucleosides by thermostable nucleoside phosphorylases

Selenium-containing nucleosides are the building blocks of the Se-nucleic acids useful for structure-and-function study, drug discovery and for targeting nucleic acids and their protein complexes. However, chemical synthesis of these Se-containing nucleosides is labor-intensive and provides low overall yields. Thus, we decided to explore biocatalytic synthesis of the modified nucleosides (such as Se-nucleosides) by employing thermostable nucleoside phosphorylases from thermophilic microorganisms. We were surprised to discover that these enzymes can still recognize the nucleobases containing rather larger atoms, Se vs O atom. These enzymes also showed excellent adaptability to some modified purines with high substrate conversion (up to 97%).

Synthesis of Chiral Acyclic Nucleosides by Sharpless Asymmetric Dihydroxylation: Access to Cidofovir and Buciclovir.

An efficient method to construct chiral acyclic nucleosides via Sharpless asymmetric dihydroxylation of N-allylpyrimidines or N-alkenylpurines is reported. A range of chiral acyclic nucleosides with two adjacent hydroxyl groups present on the side chains could be produced in good yields (up to 97% yield) and excellent enantioselectivities (90-99% ee). The synthetic utility of the reaction was demonstrated by the catalytic asymmetric synthesis of ( S)-Cidofovir and ( R)-Buciclovir.

Daniel McGillivray Brown. 3 February 1923—24 April 2012

Dan Brown was a nucleic acids chemist of the highest order, beginning with pioneering work under Lord Alexander Todd in the 1950s at University of Cambridge on chemical methods for synthesis of nucleosides and nucleotides. This work helped to confirm the furanose chemical structure of the sugar in nucleosides as well as the 3′-5′ phosphodiester linkage in DNA and RNA, perhaps the most well thought of achievement of his career. Later, as a chemistry department lecturer, he established the chemical structures of glycerol monophosphoinositides as well as triphosphoinositides. Turning back to the nucleic acids in 1961, he became fascinated by the effect of mutagens on DNA. He elucidated the mechanism for the reaction of hydroxylamine on cytidine to form an initial ‘bis-adduct’ and thereafter N 6 -hydroxycytidine. Moving in 1982 to the MRC Laboratory of Molecular Biology, he developed a method to prepare single-stranded DNA probes for detection of RNA sequences and in addition worked on a novel automated device for oligonucleotide synthesis. Reverting to his interest in mutagens, he then designed and synthesized hydrogen bonding degenerate bases and developed novel P and K modified pyrimidine and purine bases respectively as transition mutagens. Finally, he synthesized the base analogue 5-nitroindole as a potential universal base, which became useful in cycle DNA sequencing, and in addition developed the concept of ‘error catastrophe’ for the ribonucleoside of the P base as an antiviral agent. The P, K and 5-nitroindole bases became the most valued chemical entities of his career to molecular biologists. His legacy to the nucleic acids includes both his significant contributions to studies of the chemical nature of DNA and RNA and their constituents as well as a variety of enabling nucleic acids chemistry methods and mechanisms of DNA mutagenicity.

Synthesis of Nucleosides and Non-nucleosides Based 4,6-disubstituted-2-oxo-dihydropyridine-3-carbonitriles as Antiviral Agents.

Viral diseases are considered main threats that face the humanity worldwide. The emergence of new viruses like influenza viruses emphasizes the significance of designing novel antiviral drugs. The aim of this work is to synthesize a new set of nucleoside and non-nucleoside cyanopyridine, characterized and evaluated for their in vitro antiviral properties against various strains. More of the compounds showed variable antiviral potential against a panel of eighteen DNA and RNA viruses. The screening data suggested that the order of activity of the active compounds are in the order of O-glycosyl > O-alkyl > N-alkyl > S-alkyl derivatives. In addition, the 4-fluoro substituted compounds are more effective among the O- and N-alkyl analogs, whereas remarkable antiviral activity was ascribed to the methoxylated O-glycosyl derivatives. Most of the active compounds proved to be more selective towards the inhibition of the replication of DNA rather than the RNA-viruses. The analogs 1a, 2a, 12b, 14b and 16b possessed broad spectrum and noticeable antiviral potential against most of the tested DNA- and RNA-viruses (EC50 ≈ 0.8-20 µM), accompanied with considerably low cytotoxic margin (MCC ≈ 4-20 µM), and comparable with reference standard antiviral agents.

Taxonomic and metabolic shifts in the Coorong bacterial metagenome driven by salinity and external inputs

The Coorong estuary lies at the terminus of Australia’s largest river system, the Murray-Darling; both are strongly influenced by human activities; including farming and extensive flow modification. Metagenomic approaches were used to determine the planktonic bacterial community composition and potential metabolic function at two extremes in the Coorong, the river mouth which exhibits marine-like salinity, and the hypersaline upper-reaches of the estuary. Significant shifts in taxa and metabolic function were seen between the two sites. The river mouth exhibited an increase in abundance of Rhodobacteriaceae and Alteromonadaceae; families readily able to adapt to change in nutrient conditions; and the potentially pathogenic families Brucellaceae, Enterobacteriaceae and Vibrionaceae. Metabolisms over-represented include motility and chemotaxis, RNA metabolism and membrane transport, all of which are involved in actively searching for and obtaining nutrients. Also over-represented were metabolisms involved in population succession and stress response. An over-representation of taxa and metabolisms indicative of environmental change is reflective of anthropogenically affected riverine input. In the hypersaline upper reaches of the estuary, the halophilic family Ectothiorhodospiraceae was over-represented, as were the families Flavobacteriaceae, Cytophagaceae and Nocardioidaceae, members of which are able to survive over a wide salinity range. Metabolisms over-represented here were reflective of increased bacterial growth, characteristic of hypersaline environments, and included DNA metabolism, nucleotide and nucleoside synthesis and cell cycle. Coorong metagenomes clustered taxonomically and metabolically with other planktonic metagenomes, but remained an outlier of this group with only 71% and 84% similarity, respectively. This indicates that the Coorong exhibits a unique planktonic bacterial community that is influenced by riverine input at the river mouth and salinity in the upper-reaches.

Conception and Synthesis of Oxabicyclic Nucleoside Phosphonates as Internucleotidic Phosphate Surrogates in Antisense Oligonucleotide Constructs.

The stereocontrolled synthesis of a novel oxabicyclic nucleoside phosphonate comprising a perhydrofuropyran core unit was achieved. It was incorporated in an oligonucleotide sequence as a 5'-3' phosphonate-phosphate insert, and the stability properties of the resulting duplex were measured. The oxabicyclic nucleoside framework was designed so as to restrict rotation around angles γ, δ, and ε of a natural nucleoside.