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.

Synthesis of 4'-Substituted-2'-Deoxy-2'-α-Fluoro Nucleoside Analogs as Potential Antiviral Agents.

Nucleoside analogs are widely used for the treatment of viral diseases (Hepatitis B/C, herpes and human immunodeficiency virus, HIV) and various malignancies. ALS-8176, a prodrug of the 4'-chloromethyl-2'-deoxy-2'-fluoro nucleoside ALS-8112, was evaluated in hospitalized infants for the treatment of respiratory syncytial virus (RSV), but was abandoned for unclear reasons. Based on the structure of ALS-8112, a series of novel 4'-modified-2'-deoxy-2'-fluoro nucleosides were synthesized. Newly prepared compounds were evaluated against RSV, but also against a panel of RNA viruses, including Dengue, West Nile, Chikungunya, and Zika viruses. Unfortunately, none of the compounds showed marked antiviral activity against these viruses.

2'-Deoxyribonucleoside 5'-triphosphates bearing 4-phenyl and 4-pyrimidinyl imidazoles as DNA polymerase substrates.

We developed a versatile access to a series of 4-substituted imidazole 2'-deoxynucleoside triphosphate bearing functionalized phenyl or pyrimidinyl rings. 4-Iodo-1H-imidazole was enzymatically converted into the corresponding 2'-deoxynucleoside, which was then chemically derived into its 5'-triphosphate, followed by 4-arylation via Suzuki-Miyaura coupling using (hetero)arylboronic acids. Both KF (exo-) and Deep Vent (exo-) DNA polymerases incorporated these modified nucleotides in primer-extension assays, adenine being the preferred pairing partner in the template. The 4-(3-aminophenyl)imidazole derivative (3APh) was the most efficiently inserted opposite A by KF (exo-) with only a 37-fold lower efficiency (Vmax/KM) than that of the correct dTTP. No further extension occurred after the incorporation of a single aryl-imidazole nucleotide. Interestingly, the aryl-imidazole dNTPs were found to undergo successive incorporation by calf thymus terminal deoxynucleotidyl transferase with different tailing efficiencies among this series and with a marked preference for 2APyr polymerization.

Synthesis and anti-HCV activity of ß-d-2'-deoxy-2'-α-chloro-2'-ß-fluoro and ß-d-2'-deoxy-2'-α-bromo-2'-ß-fluoro nucleosides and their phosphoramidate prodrugs.

We report herein the synthesis and evaluation of a series of ß-d-2'-deoxy-2'-α-chloro-2'-ß-fluoro and ß-d-2'-deoxy-2'-α-bromo-2'-ß-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.

Synthesis of l-Deoxyribonucleosides from d-Ribose.

The preparation of 2-deoxy-l-ribose derivatives or mirror image deoxyribonucleosides (l-deoxyribonucleosides) from d-ribose is reported. Starting from inexpensive d-ribose, an acyclic d-form carbohydrate precursor was synthesized to study a unique carbonyl translocation process. In this novel radical reaction, not only was the configuration of the sugar transformed from the d-form to the l-form, but also deoxygenation at the C(2) position of the sugar was successfully achieved. This is one of the most practical methods for converting a d-sugar to a 2-deoxy-l-sugar in a one-step reaction. To further identify the reaction product, radical reactions followed by treatment with 1,3-propanedithiol and then benzoylation were performed to afford a dithioacetal derivative. The stereochemistry and configuration of the 2-deoxy-l-ribose dithioacetal derivative were confirmed by its X-ray crystal structure. To further apply this methodology, a diethyl thioacetal derivative was formed, followed by selective benzoyl protection, and an NIS-initiated cyclization reaction to give the desired ethyl S-l-2-deoxyriboside, which can be used as a 2-deoxy-l-ribosyl synthon in the formal total synthesis of various l-deoxyribonucleosides, such as l-dT.

Protected 2'-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA.

2'-Deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)cytosine (5hmC) protected with photocleavable groups (2-nitrobenzyl or 6-nitropiperonyl) were prepared and studied as substrates for the enzymatic synthesis of oligonucleotides and DNA containing a photocaged epigenetic 5hmC base. DNA probes containing photocaged or free 5hmC in the recognition sequence of restriction endonucleases were prepared and used for the study of the photorelease of caged DNA by UV or visible light at different wavelengths. The nitrobenzyl-protected dNTP was a slightly better substrate for DNA polymerases in primer extension or PCR, whereas the nitropiperonyl-protected nucleotide underwent slightly faster photorelease at 400 nm. However, both photocaged building blocks can be used in polymerase synthesis and the photorelease of 5hmC in DNA.

6-Aryl-4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (benzo-fused 7-deaza-dATP analogues): Synthesis, fluorescent properties, enzymatic incorporation into DNA and DNA-protein binding study.

Four 6-substituted 4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (dA(BX)TPs) were prepared by glycosylation of 4,6-dichloropyrimidoindole followed by ammonolysis, cross-coupling and triphosphorylation. They were found to be moderate to good substrates for DNA polymerases in primer extension. They also exerted fluorescence with emission maxima 335-378nm. When incorporated to oligonucleotide probes, they did not show significant mismatch discrimination but the 6-benzofuryl 4-amino-pyrimido[4,5-b]indole nucleotide displayed a useful sensitivity to protein binding in experiment with SSB protein.

Synthesis and nonenzymatic template-directed polymerization of 2'-amino-2'-deoxythreose nucleotides.

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2'-amino modified TNA nucleosides (2'-NH2-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2'-NH2-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3'-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C4 RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h(-1) on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM N-(hydroxylethyl)imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h(-1), causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2'-NH2-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems.

Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

5-Nitroindole oligonucleotides with alkynyl side chains: universal base pairing, triple bond hydration and properties of pyrene "click" adducts.

Oligonucleotides with 3-ethynyl-5-nitroindole and 3-octadiynyl-5-nitroindole 2'-deoxyribonucleosides were prepared by solid-phase synthesis. To this end, nucleoside phosphoramidites with clickable side chains were synthesized. The 3-ethynylated 5-nitroindole nucleoside was hydrated during automatized DNA synthesis to 3-acetyl-5-nitroindole 2'-deoxyribonucleoside. Side product formation was circumvented by triisopropylsilyl protection of the ethynyl side chain and was removed with TBAF after oligonucleotide synthesis. All compounds with a clickable 5-nitroindole skeleton show universal base pairing and can be functionalized with almost any azide in any position of the DNA chain. Functionalization of the side chain with 1-azidomethylpyrene afforded click adducts in which the fluorescence was quenched by the 5-nitroindole moieties. However, fluorescence was slightly recovered during duplex formation. Oligonucleotides with a pyrene residue and a long linker arm are stabilized over those with non-functionalized side chains. From the UV red shift of the pyrene residue in oligonucleotides and modelling studies, pyrene intercalation was established for the long linker adduct showing increased duplex stability over those with a short side chain.

Synthesis of 3',4'-difluoro-3'-deoxyribonucleosides and its evaluation of the biological activities: discovery of a novel type of anti-HCV agent 3',4'-difluorocordycepin.

Upon reacting 3',4'-unsaturated cytosine (8 and 9) and adenine nucleosides (13 and 14) with XeF(2)/BF3 · OEt(2), the respective novel 3',4'-difluoro-3'-deoxyribofuranosyl nucleosides (10-12 and 15-18) could be obtained. Formation of anti-adducts (11, 16 and 18) revealed that the fluorination involved oxonium ions as incipient intermediates. TBDMS-protected 3',4'-unsaturated adenosine provided the ß-face adducts as sole stereoisomers whereas α-face-selectivity was observed with the TBDPS-protected adenosine 14. The evaluation of the novel 3'-deoxy-3',4'-difluororibofuranosylcytosine-(19-21) and adenine nucleosides (22-25) against antitumor and antiviral activities revealed that 3',4'-difluorocordycepin (24) was found to possess anti-HCV activity. The SI of 24 was comparable to that of the anti-HCV drug ribavirin. However, sofosbuvir, FDA-approved novel anti-HCV drug, showed better SI value. Our finding revealed that the introduction of the fluoro-substituent into the 4'-position of cordycepin derivatives decreased the cytotoxicity to the host cell with retention of the antiviral activity.

A tractable and efficient one-pot synthesis of 5'-Azido-5'-deoxyribonucleosides.

Synthetic routes to 5'-azidoribonucleosides are reported for adenosine, cytidine, guanosine, and uridine, resulting in a widely applicable one-pot methodology for the synthesis of these and related compounds. The target compounds are appropriate as precursors in a variety of purposive syntheses, as the synthetic and therapeutic relevance of azido- and amino-modified nucleosides is expansive. Furthermore, in the conversion of alcohols to azides, these methods offer a tractable alternative to the Mitsunobu and other more difficult reactions.

Synthesis of 2'-deoxy-9-deaza nucleosides using Heck methodology.

During the synthesis of a series of 2'-deoxy-9-deaza nucleosides using Heck methodology, the necessity for a pyrrole protecting group was discovered. The results of this brief study revealed that the benzyloxymethyl (BOM) group proved optimal, and Heck coupling using Jeffery conditions increased the coupling yield significantly. The results are reported herein.

Synthesis of 2,6-disubstituted pyridin-3-yl C-2'-deoxyribonucleosides through chemoselective transformations of bromo-chloropyridine C-nucleosides.

2-Bromo-6-chloro- and 6-bromo-2-chloropyridin-3-yl deoxyribonucleosides were prepared by the Heck coupling of bromo-chloro-iodopyridines with TBS-protected deoxyribose glycal. Some of their Pd-catalyzed cross-coupling reactions proceeded chemoselectively at the position of the bromine, whereas nucleophilic substitutions were unselective and gave mixtures of products. The mono-substituted intermediates were used for another coupling or nucleophilic substitution giving rise to a small library of title 2,6-disubstituted pyridine C-deoxyribonucleosides. The title nucleosides did not exert antiviral or cytostatic effects.

Reactivity of nucleosides with a hydroxyl radical in non-aqueous medium.

DNA damage: The reactivity of HO(.) with silylated 2'-deoxyribonucleosides was investigated in acetonitrile by means of a time-resolved technique. The obtained rate constants were in general slightly lower than those reported for the natural nucleosides in water. Analysis of the reaction mixture by UPLC-MS revealed that HO(.) attack occurred at the nucleobase (see scheme).

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides.

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.

Investigation of excess-electron transfer in DNA double-duplex systems allows estimation of absolute excess-electron transfer and CPD cleavage rates.

To investigate the parameters and rates that determine excess-electron transfer processes in DNA duplexes, we developed a DNA double-duplex system containing a reduced and deprotonated flavin donor at the junction of two duplexes with either the same or different electron acceptors in the individual duplex substructures. This model system allows us to bring the two electron acceptors in the duplex substructures into direct competition for injected electrons and this enables us to decipher how the kind of acceptor influences the transfer data. Measurements with the electron acceptors 8-bromo-dA (BrdA), 8-bromo-dG (BrdG), 5-bromo-dU (BrdU), and a cyclobutane pyrimidine dimer, which is a UV-induced DNA lesion, allowed us to obtain directly the maximum overall reaction rates of these acceptors and especially of the T=T dimer with the injected electrons in the duplex. In line with previous observations, we detected that the overall dimer cleavage rate is about one order of magnitude slower than the debromination of BrdU. Furthermore, we present a more detailed explanation of why sequence dependence cannot be observed when a T=T dimer is used as the acceptor and we estimate the absolute excess-electron hopping rates.

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.

Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase-DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA-protein interaction than do tethered fluorophores. Here we report the incorporation of the 5'-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tC(O)), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.

Synthesis and anti-HIV activity of 2'-deoxy-2'-fluoro-4'-C-ethynyl nucleoside analogs.

Based on the favorable antiviral profiles of 4'-substituted nucleosides, novel 1-(2'-deoxy-2'-fluoro-4'-C-ethynyl-beta-D-arabinofuranosyl)-uracil (1a), -thymine (1b), and -cytosine (2) analogs were synthesized. Compounds 1b and 2 exhibited potent anti-HIV-1 activity with IC(50) values of 86 and 1.34 nM, respectively, without significant cytotoxicity. Compound 2 was 35-fold more potent than AZT against wild-type virus, and also retained nanomolar antiviral activity against resistant strains, NL4-3 (K101E) and RTMDR. Thus, 2 merits further development as a novel NRTI drug.

DNA as a supramolecular framework for the helical arrangements of chromophores: towards photoactive DNA-based nanomaterials.

Nucleic acids have been emerging as supramolecular structural scaffolds for the helical organization of chromophores in the creation of functional nanomaterials mainly because of the their unique structural features and synthetic accessibility. A large number of chromophores have been successfully incorporated into DNA or RNA as C-nucleosides, as base surrogates or as modified sugars using solid phase phosphoramidite chemistry. Moreover, multiple incorporations yield the helical organization of the chromophores inside or outside the DNA or RNA double helix depending upon the conjugation of the chromophores. Significant photophysical interactions are observed in the chromophore stacks resulting in unique optical properties that are significantly different from the monomer properties. In this feature article, multichromophore labelled nucleic acids are reviewed with special emphasis on the self-assembly induced modulation of the optical properties.