Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19 F NMR Spectroscopy.

Development of versatile probes that can enable the study of different conformations and recognition properties of therapeutic nucleic acid motifs by complementing biophysical techniques can greatly aid nucleic acid analysis and therapy. Here, we report the design, synthesis and incorporation of an environment-sensitive ribonucleoside analogue, which serves as a two-channel biophysical platform to investigate RNA structure and recognition by fluorescence and 19 F NMR spectroscopy techniques. The nucleoside analogue is based on a 5-fluorobenzofuran-uracil core and its fluorescence and 19 F NMR chemical shifts are highly sensitive to changes in solvent polarity and viscosity. Notably, the modified ribonucleotide and phosphoramidite substrates can be efficiently incorporated into RNA oligonucleotides (ONs) by in vitro transcription and standard solid-phase ON synthesis protocol, respectively. Fluorescence and 19 F readouts of the nucleoside incorporated into model RNA ONs are sensitive to the neighbouring base environment. The responsiveness of the probe was aptly utilized in detecting and quantifying the metal ion-induced conformational change in an internal ribosome entry site RNA motif of hepatitis C virus, which is an important therapeutic target. Taken together, our probe is a good addition to the nucleic acid analysis toolbox with the advantage that it can be used to study nucleic acid conformation and recognition simultaneously by two biophysical techniques.

Synthesis of xanthosine 2-phosphate diesters via phosphitylation of the carbonyl group.

O2-Phosphodiesterification of xanthosine has been achieved by a one-pot procedure consisting of the phosphitylation of the 2-carbonyl group of appropriately protected xanthosine derivatives using phosphoramidites and N-(cyanomethyl)dimethylammonium triflate (CMMT), oxidation of the resulting xanthosine 2-phosphite triesters, and deprotection. In addition, a study on the hydrolytic stability of a fully deprotected xanthosine 2-phosphate diester has revealed that it is more stable at higher pH.

Prebiotic Photochemical Coproduction of Purine Ribo- and Deoxyribonucleosides.

The hypothesis that life on Earth may have started with a heterogeneous nucleic acid genetic system including both RNA and DNA has attracted broad interest. The recent finding that two RNA subunits (cytidine, C, and uridine, U) and two DNA subunits (deoxyadenosine, dA, and deoxyinosine, dI) can be coproduced in the same reaction network, compatible with a consistent geological scenario, supports this theory. However, a prebiotically plausible synthesis of the missing units (purine ribonucleosides and pyrimidine deoxyribonucleosides) in a unified reaction network remains elusive. Herein, we disclose a strictly stereoselective and furanosyl-selective synthesis of purine ribonucleosides (adenosine, A, and inosine, I) and purine deoxynucleosides (dA and dI), alongside one another, via a key photochemical reaction of thioanhydroadenosine with sulfite in alkaline solution (pH 8-10). Mechanistic studies suggest an unexpected recombination of sulfite and nucleoside alkyl radicals underpins the formation of the ribo C2'-O bond. The coproduction of A, I, dA, and dI from a common intermediate, and under conditions likely to have prevailed in at least some primordial locales, is suggestive of the potential coexistence of RNA and DNA building blocks at the dawn of life.

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 anti-trypanosomal activity of 3'-fluororibonucleosides derived from 7-deazapurine nucleosides.

Trypanosoma brucei parasites cause Human African Trypanosomiasis and the current drugs for its treatment are often inefficient and toxic. This urges the need to development of new antitrypanosomal agents. We report the synthesis and biological profiling of 3'-deoxy-3'-fluororibonucleosides derived from 7-deazaadenine nucleosides bearing diverse substituents at position 7. They were synthesized through glycosylation of 6-chloro-7-bromo- or -7-iodo-7-deazapurine with protected 3'-fluororibose followed by cross-coupling reactions at position 7 and/or deprotection. Most of the title nucleosides displayed micromolar or submicromolar activity against Trypanosoma brucei brucei. The most active were the 7-bromo- and 7-iododerivatives which exerted double-digit nanomolar activity against T. b. brucei and T. b. gambiense and no cytotoxicity and thus represent promising candidates for further development.

Synthesis and Cytotoxic and Antiviral Activity Profiling of All-Four Isomeric Series of Pyrido-Fused 7-Deazapurine Ribonucleosides.

All four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total synthesis of each isomeric fused heterocycle through multistep heterocyclization was followed by glycosylation and derivatization at position 4 by cross-coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and antiviral activity. The most active were pyrido[4',3':4,5]pyrimidine nucleosides bearing MeO, NH2 , MeS, or CH3 groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double-strand breaks and apoptosis.

Design, synthesis, in vitro and in silico studies of novel 4-oxoquinoline ribonucleoside derivatives as HIV-1 reverse transcriptase inhibitors.

Human immunodeficiency virus type 1 (HIV-1) is a public health problem that affects over 38 million people worldwide. Although there are highly active antiretroviral therapies, emergence of antiviral resistant strains is a problem which leads to almost a million death annually. Thus, the development of new drugs is necessary. The viral enzyme reverse transcriptase (RT) represents a validated therapeutic target. Because the oxoquinolinic scaffold has substantial biological activities, including antiretroviral, a new series of 4-oxoquinoline ribonucleoside derivatives obtained by molecular hybridization were studied here. All synthesized compounds were tested against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and 9a and 9d displayed the highest antiviral activities, with IC50 values of 1.4 and 1.6 µM, respectively. These compounds were less cytotoxic than AZT and showed CC50 values of 1486 and 1394 µM, respectively. Molecular docking studies showed that the most active compounds bound to the allosteric site of the enzyme, suggesting a low susceptibility to the development of antiviral resistance. In silico pharmacokinetic and toxicological evaluations reinforced the potential of the active compounds as anti-HIV candidates for further exploration. Overall, this work showed that compounds 9a and 9d are promising scaffold for future anti-HIV-1 RT drug design.

Synthesis of 7-trifluoromethyl-7-deazapurine ribonucleoside analogs and their monophosphate prodrugs.

Novel 7-trifluoromethyl-7-deazapurine ribonucleoside analogs (13a-c) and their Protides (15a-c) were successfully synthesized from ribolactol or 1-α-bromo-ribose derivatives using Silyl-Hilbert-Johnson or nucleobase-anion substitution reactions followed by key aromatic trifluoromethyl substitution. Newly prepared compounds were evaluated against a panel of RNA viruses, including HCV, Ebola or Zika viruses.

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-ß-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.

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.

An Improved Strategy for the Chemical Synthesis of 3',5'-Cyclic Diguanylic Acid.

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.

Oxanosine Monophosphate Is a Covalent Inhibitor of Inosine 5'-Monophosphate Dehydrogenase.

Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-ß-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.

Novel synthesis of new pyrazole thioglycosides as pyrazomycin analogues.

This study reports a novel method for the synthesis of a new class of pyrazole thioglycosides 7a-h as pyrazomycin analogues. 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 phenyl hydrazine in ethanol at room temperature to give the corresponding sodium 5-amino-4-(substitutedphenylcarbamoyl)-1-phenyl-1H-pyrazole-3-thiolates 3a-d. The latter compounds were treated with tetra-acetylated glycosyl bromides 4a,b in DMF at ambient temperature to give the corresponding pyrazole thioglycosides 6a-h. Treatment of pyrazole salts 3a-d with hydrochloric acid at room temperature afforded the corresponding 3-mercaptopyrazole derivatives 5. The latter compounds were treated with tetra-acetylated glycosyl bromides 4 in sodium hydride-DMF to tolerate the S-glycosyl 6a-h compounds. Ammonolysis of the latters afforded the corresponding free thioglycosides 7a-h. The structures of the reaction products were elucidated based on spectral data and elemental analysis.

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.

Synthesis and characterization of various 5'-dye-labeled ribonucleosides.

Hitherto unknown chromophoric nucleosides are reported. This novel set of visibly coloured dye-labeled 5'-nucleosides, including 1,2,4,5-tetrazine, dicyanomethylene-4H-pyran, benzophenoxazinone, 9,10-anthraquinone and azobenzene chromophores, were prepared mainly under Cu-catalyzed azide-alkyne cycloaddition (CuAAC). The design criteria are outlined. Several derivatives possess in supplement a fluorescence property. The absorption and fluorescence spectra of all coloured nucleosides were recorded to study their potential as visible-range probes. Such nucleodyes are of great interest for future competitive lateral flow test MIP-based strips.

6-Chloropurine Ribonucleosides from Chloropyrimidines: One-Pot Synthesis.

A one-pot glycosylation and cyclization procedure is described for the synthesis of 6-chloropurine ribonucleosides from chloropyrimidines. From such a procedure and modification of the obtained chloropurine ribonucleosides, many drug candidates or molecular tools for biological study designed from their similarity to naturally occurring nucleosides could be obtained. The synthesis begins by preparation of several amidinoaminochloropyrimidines as precursors for the one-pot procedure. Then, by adding trimethylsilyl trifluoromethanesulfonate (TMSOTf) to a mixture of a pyrimidine and 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribose, different 6-chloropurine ribonucleosides are obtained. This methodology allows the straightforward introduction of an alkyl substituent at position 8 of purine ribonucleosides, which then can be functionalized at positions 2 and 6. © 2018 by John Wiley & Sons, Inc.

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.

Practical Synthesis of 4'-Thioribonucleosides from L-Arabinose via Novel Reductive Ring-Contraction Reaction and Pummerer-Type Thioglycosylation.

The detailed practical synthesis of 4'-thionucleosides starting from L-arabinose is described here. 1,4-Anhydro-2,3-O-isopropylidene-4-thioribitol, which is the key intermediate for the synthesis of 4'-thionucleosides, is obtained from L-arabinose in several steps, including a novel reductive ring-contraction reaction. After oxidation of the key intermediate, the sulfoxide is subjected to Pummerer-type thioglycosylation in the presence of persilylated nucleobases to obtain the 4'-thioribonucleosides in good yield and ß-selectively. © 2017 by John Wiley & Sons, Inc.

Synthesis of 5'-Thio-3'-O-ribonucleoside Phosphoramidites.

The chemical synthesis of phosphoramidite derivatives of all four 5'-deoxy-5'-thioribonucleosides is described. These phosphoramidites contained trityl (A, G, C, and U), dimethoxytrityl (A and G), or tert-butyldisulfanyl (G) as the 5'-S-protecting group. The application of several of these phosphoramidites for solid-phase synthesis of oligoribonucleotides containing a 2'-O-photocaged 5'-S-phosphorothiolate linkage or 5'-thiol-labeled RNAs is also further investigated.