Synthesis and photophysical properties of novel push-pull-type solvatochromic 7-deaza-2′-deoxypurine nucleosides

We have synthesized two novel push-pull-type fluorescent 7-deazapurine nucleosides, ^CNZA and ^CNZG, and investigated their photophysical properties. In particular, ^CNZA was found to exhibit a remarkable solvatofluorochromicity (Δλ_fl.max = 60 nm). We incorporated ^CNZA into oligonucleotides and found that ^CNZA can form a stable base pair with both thymine and cytosine. Such environmentally sensitive fluorescent nucleosides have a potential as a fluorescence sensor for structural studies of nucleic acids.     

Template-directed ligation: from DNA towards different versatile templates

A systematic approach evaluating template-directed ligation reactions has now resulted in a simple outline for a two-stage replication cycle. This cycle builds on an efficient method for reading the information encoded in DNA into an amplified translation product. It is further demonstrated that the translation product strand is capable of catalyzing the synthesis of the original DNA strand. We propose that this cycle represents just one of many possible solutions; other chemical ligation or polymerization reactions could be accommodated with different templates. In that context, a new template, derived by modest changes to the DNA backbone, has been developed and has been shown to hybridize under reaction conditions different than those accessible to DNA. Therefore, the conceptual groundwork has been laid for extending this approach to encoding and reading stored information in molecules other than the natural biopolymers at the densities found in biology.     

Reactivity between acetone and single-stranded DNA containing a 5′-capped 2′-fluoro-N7-methyl guanine

DNA-mediated catalysis is an emerging field in bioorganic chemistry and chemical biology. However, the functional group diversity and known reactivity of DNA (A, T, C, and G) is relatively limited in scope. This relatively defined reactivity can limit the utility of DNA as a catalyst. In an effort to expand the functional group diversity and chemical reactivity of DNA, we sought to explore reactions involving single-stranded DNA equipped with a stabilized variant of N7-methyl guanine (2′-fluoro-5′-N7-methyl guanine). Here, we show that 5′-capped 2′-fluoro-N7-methyl guanine-labeled single-stranded DNA reacts with a ketone to afford a ketone-labeled DNA. This reaction likely proceeds through a reactive ylide or N-heterocyclic carbene. Taken together, our findings suggest that 2′-fluoro-5′-N7-methyl guanine is a stable adduct that can be selectively incorporated into ssDNA and functionalized with a ketone moiety by reaction with a simple ketone. Incorporation of this nucleoside into ssDNA may be useful for the evolution of novel deoxyribozymes that catalyze new reactions, including those which proceed via a reactive ylide or N-heterocyclic carbene-mediated chemistry.     

Hoogsteen-Duplex DNA: Synthesis and base pairing of oligonucleotides containing 1-deaza-2′-deoxyadenosine

Oligodeoxyribonucleotides containing 1-deaza-2′-deoxyadenosine ( = 7-amino-3-(2-deoxy-β-D -erythro-pentofuranosyl)-3H-imidazo[4, 5-b]pyridine; 1b ) form Hoogsteen duplexes. Watson-Crick base pairs cannot be built up due to the absence of N(1). For these studies, oligonucleotide building blocks – the phosphonate 3a and the phosphoramidite 3b – were prepared from 1b via 4a and 5 , as well as the Fractosil-linked 6b , and used in solid-phase synthesis. The applicability of various N-protecting groups (see 4a – c ) was also studied. The Hoogsteen duplex d[(c¹A)₂₀] · d(T₂₀) ( 11 · 13 ; T_m 15°) is less stable than d(A₂₀) · d(T₂₀) ( 12 · 13 ; T_m 60°). The block oligomers d([c¹A)₁₀–;T₁₀] ( 14 ) and d[T₁₀–(c¹A)₁₀] ( 15 ) containing purine and pyrimidine bases in the same strand are also able to form duplexes with each other. The chain polarity was found to be parallel.     

Concise and efficient synthesis of 3′-O-triphosphates of 2′-deoxyadenosine and 2′-deoxycytidine

We describe concise and efficient synthesis of 2′-deoxyadenosine-3′-O-triphosphate (2′-d-3′-ATP) and 2′-deoxycytidine-3′-O-triphosphate (2′-d-3′-CTP) which are well known for their various biological applications. One-pot synthetic methodology was used to convert N⁶-Benzoyl-5′-O-levulinoyl-2′-deoxyadenosine into N⁶-Benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-triphosphate in 72% yield. One-step concurrent deprotection of N⁶-Benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted in 2′-d-3′-ATP in 75% yield. The same synthetic strategy was successfully employed to convert N⁴-Benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-CTP in 66% yield.     

Synthesis of 8-halogenated-7-deaza-2′-deoxyguanosine as an 8-oxo-2′-deoxyguanosine analogue and evaluation of its base pairing properties

8-Halogenated-7-deaza-2′-deoxyguanosines (8-halo-7-deaza-dG) were designed to structurally mimic 8-oxo-2′-deoxyguanosine (8-oxo-dG), which is representative of an oxidized nucleoside. It has been shown by NMR that the conformation around the N-glycosidic bond of (8-halo-7-deaza-dG) is preferably syn, similar to 8-oxo-dG. The base pairing properties of 8-halo-7-deaza-dG were studied by measuring the thermal denaturation temperature of the duplexes, showing that their base pair with dC is destabilized compared with natural dG. These results also support their preference for syn conformation. Unlike 8-oxo-dG, 8-halo-7-deaza-dG did not form a stable base pair with dA, most likely due to the lack of N7-H hydrogen bonding with dA. In conclusion, the newly-designed 8-halo-7-deaza-dG analogs resemble 8-oxo-dG in its shape and preference for syn conformation, but they do not form Hoogsteen base pair with the opposing dA.     

A convenient approach towards boron cluster modifications with adenosine and 2′-deoxyadenosine

New 2′-deoxyadenosine and adenosoine modifications: 8-[(2-dimethylaminoethyl)amino]-2′-deoxyadenosine and 8-[(2-dimethylaminoethyl)amino]adenosine were prepared and their reactivity towards cyclic oxonium adducts of closo-dodecaborate and cobalt-bis-dicarbollide was studied. The cleavage reactions of clusters oxonium rings by N,N-dimethylanio group of modified nucleosides led to the first [B₁₂H₁₂]²⁻ and new [Co(C₂B₉H₁₁)₂]⁻ conjugates with adenosine and 2′-deoxyadenosine respectively. The proposed methodology provides a convenient route for the synthesis of libraries of boron cluster modified adenosine and 2′-deoxyadenosine derivatives for biological screening.     

Synthesis of 2′,3′-dideoxy-6′-fluorocarbocyclic nucleosides via Reformatskii-Claisen rearrangement

2′,3′-Dideoxy-6′-fluorocarbocyclic nucleosides, analogues of highly bioactive carbovir and abacavir were synthesized. The notable steps were the incorporation of fluoromethylene group by way of silicon-induced Reformatskii-Claisen rearrangement of allyl bromofluoroacetate, the construction of the carbocyclic ring via ring-closing metathesis (RCM) and the introduction of base by Mitsunobu reaction.     

The first Cu- and amine-free Sonogashira-type cross-coupling in the C-6-alkynylation of protected 2′-deoxyadenosine

The Sonogashira cross-coupling reaction offers a convenient route to C(sp)-C(sp²) bond formation. Although the Sonogashira reaction has traditionally been carried out in the presence of Pd catalyst and a co-catalyst of Cu(I) salt, the use of Cu(I) salt is often not efficient because it leads to the formation of unwanted side-products. This has prompted interest in recent years in the development of Cu-free Sonogashira cross-coupling reaction conditions. In addition, the development of Cu-free Sonogashira cross-coupling conditions for the alkynylation of nucleoside derivatives remains largely unexplored. Herein, we demonstrate that Cu- and amine-free Sonogashira-type cross-coupling lead to successful alkynylation of aryl bromides and heteroaryl bromides. For the first time, we have extended this method for the alkynylation of protected 2′-deoxyadenosine at the C-6 position.     

Synthesis of 4′-substituted 2,2′:6′,2″-terpyridines via a Mitsunobu reaction

Treatment of 2,6-di(pyridin-2-yl)pyridin-4(1H)-one with various appropriately protected ω-substituted primary alcohols or a nucleoside (3,3′-O-diBz-dUrd) in dry THF in the presence of triphenylphosphine and diisopropylazodicarboxylate gives the corresponding 4′-substituted terpyridines in high yield.     

A label-free ultrasensitive assay of 8-hydroxy-2′-deoxyguanosine in human serum and urine samples via polyaniline deposition and tetrahedral DNA nanostructure

Oxidative damage is an important factor in causing various human disease and injury. As an oxidative DNA damage product, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a key marker, which is widely used to study oxidative damage mechanism in diseases. Most reported electrochemical methods were based on oxidation current of 8-OHdG. In this work, a simple electrochemical biosensor for ultrasensitive detection of 8-OHdG was proposed based on it triggered polyaniline (PANI) deposition on tetrahedral DNA nanostructure (TDN). TDN was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences. 8-OHdG-aptamer on the top of TDN formed a hemin/G-quadruplex structure in the presence of 8-OHdG and hemin, which have high catalytic activity to trigger PANI deposition. Numerous negative charges on the duplex DNAs contained in hemin/G-quadruplex and TDN supplied exquisite environment for PANI deposition, which improved the detection sensitivity greatly by increasing the DPV current to10-fold (∼3 μA) compared to our previously reported method without TDN. The response signals correlated linearly with the concentration of 8-OHdG ranging from 10 pM to 2 nM, with a detection limit of 1 pM (S/N = 3). The sensitivity was improved to almost 300-fold when compared with most of previously reported electrochemical methods. The method was also simple and reliable, avoiding complex, expensive label procedures and nanomaterial synthesized procedures. The method had been successfully applied to quantify 8-OHdG in urine and human serum samples with satisfactory results.     

A new, facile, and protection-free one-pot chemical synthesis of 2′-deoxynucleoside-5′-tetraphosphates

A new straight forward, reliable, and an efficient method for the chemical synthesis of 2′-deoxynucleoside-5′-tetraphosphate is reported. The present ‘one-pot, three step’ synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide the corresponding 2′-deoxynucleoside-5′-tetraphosphate in moderate yield with high purity.     

Straightforward synthesis of 3′-deoxy-3′,4′-didehydronucleoside-5′-aldehydes via 2′,3′-O-orthoester group elimination: a simple route to 3′,4′-didehydronucleosides

Straightforward, high-yielding syntheses of 3′-deoxy-3′,4′-didehydronucleoside-5′-aldehydes and 3′-deoxy-3′,4′-didehydronucleosides starting from 2′,3′-O-orthoester derivatives of ribonucleosides are described.     

Development of a novel nucleoside analogue with S-type sugar conformation: 2′-deoxy-trans-3′,4′-bridged nucleic acids

Two novel trans-3′,4′-bridged nucleic acid (trans-3′,4′-BNA) monomers, one with a 3,5,8-trioxabicyclo[5.3.0]decane structure and the other with a 4,7-dioxabicyclo[4.3.0]nonane structure, were successfully synthesized from thymidine. The locked trans-fused ring structures of the nucleoside analogues were confirmed by X-ray crystallography, which also indicated that their furanose rings had a typical S-type conformation involving C_2′-endo or C_3′-exo sugar puckering, respectively, and the same ring conformation as that observed in the B-type helical structure of the DNA duplex.     

2′-Lipid-modified oligonucleotides via a ‘Staudinger-Vilarrasa’ reaction

We report a new access to 2′-amido-2′-deoxyuridine via a Staudinger-Vilarrasa coupling reaction for the preparation of lipid-modified oligonucleotides. One or two lipidic moieties were inserted within the oligonucleotidic sequence (LONs) leading to a repertoire of original antagomir-like molecules targeting micro RNA (miRNA or miR). Melting temperature (Tm) experiments revealed that the stability of the duplexes depends on the lipid position and the number of lipid moieties inserted within the oligonucleotide sequence. Single lipid conjugations of positions 11 and 19 of LONs targeting miR-122 do not destabilize the duplexes.     

A concise synthesis of 3′-α-fluoro-2′,3′-dideoxyguanosine (FddG) via 3′-α-selective fluorination of 8,2′-thioanhydronucleoside

The antiviral nucleoside 3′-α-fluoro-2′,3′-dideoxyguanosine (FddG) was synthesized via 3′-α-selective fluorination of 8,2′-thioanhydronucleoside as the key step. Desulfurization of 3′-α-fluoro-3′-deoxy-8,2′-thioanhydronucleoside could be achieved by the treatment with Raney Ni in toluene. This method provides a concise route to 3′-α-fluoro-2′,3′-dideoxynucleosides that avoids the use of explosive and expensive SF₄-related fluorinating reagents.     

Radical-based transformation of vicinal diols to olefins via thioxocarbamate derivatives: a simple approach to 2′,3′-didehydro-2′,3′-dideoxynucleosides

The bis-O-thioxocarbamate derivatives obtained from the reaction of vicinal diols with phenyl isothiocyanate are shown to be reduced with tris(trimethylsilyl)silane in the presence of azobisisobutyronitrile to afford the corresponding olefins in good yields. In this way, 2′,3′-didehydro-2′,3′-dideoxy analogs of adenosine, guanosine, inosine, cytidine and uridine were prepared by the radical-based deoxygenation of the corresponding ribonucleosides via the bis-O-thioxocarbamate derivatives.     

Syntheses of 5′-amino-2′,5′-dideoxy-2′,2′-difluorocytidine derivatives as novel anticancer nucleoside analogs

A novel class of 5′-amino-2′,5′-dideoxy-2′,2′-difluorocytidine derivatives has been synthesized in order to identify anticancer nucleoside analogs. Several synthetic routes were devised and implemented which relied upon either S_N2 displacement or reductive amination to provide the desired derivatives.     

Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA

Several genomic disorders are caused by an excessive number of DNA triplet repeats. We developed a DNA-templated reaction in which product formation occurs only when the number of repeats exceeds a threshold indicative for the outbreak of Chorea Huntington. The combined use of native chemical PNA ligation and auxiliary DNA probes enabled reactions on templates obtained from human genomic DNA.