An Isocytidine Derivative with a 2‐Amino‐6‐methylpyridine Unit for Selective Recognition of the CG Interrupting Site in an Antiparallel Triplex DNA

Sequence‐specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non‐natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N²‐arylmethyl isodC derivatives and incorporated them into triplex‐forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2‐amino‐6‐methylpyridine moiety (AP‐isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.     

Base‐Pairing Behavior of a Carbocyclic Janus‐AT Nucleoside Analogue Capable of Recognizing A and T within a DNA Duplex

Janus‐type nucleosides are heterocycles with two faces, each of which is designed to complement the H‐bonding interactions of natural nucleosides comprising a canonical Watson–Crick base pair. By intercepting all of the hydrogen bonds contained within the base pair, oligomeric Janus nucleosides are expected to achieve sequence‐specific DNA recognition through the formation of J‐loops that will be more stable than D‐loops, which simply replaces one base‐pair with another. Herein, we report the synthesis of a novel Janus‐AT nucleoside analogue, J_AT, affixed on a carbocyclic analogue of deoxyribose that was converted to the corresponding phosphoramidite. A single J_AT was successfully incorporated into a DNA strand by solid phase for targeting both A and T bases, and characterized through biophysical and computational methods. Experimental UV‐melting and circular dichroism data demonstrated that within the context of a standard duplex, J_AT associates preferentially with T over A, and much more poorly with C and G. Density functional theory calculations confirm that the J_AT structure is well suited to associate only with A and T thereby highlighting the importance of the electronic structure in terms of H‐bonding. Finally, molecular dynamics simulations validated the observation that J_AT can substitute more effectively as an A‐analogue than as a T‐analogue without substantial distortion of the B‐helix. Overall, this new Janus nucleotide is a promising tool for the targeting of A–T base pairs in DNA, and will lead to the development of oligo‐Janus‐nucleotide strands for sequence‐specific DNA recognition.     

Synthesis and Biological Evaluation of Pyrrolo[2,1‐f][1,2,4]triazine C‐Nucleosides with a Ribose, 2′‐Deoxyribose,and 2′,3′‐Dideoxyribose Sugar Moiety

The synthesis of hitherto unknown pyrrolo[2,1‐f][1,2,4]triazine C‐nucleosides is described. Structural variations (chlorine, bromine, iodine, and cyano groups) were introduced at position 7 of 4‐aza‐7,9‐dideazaadenine. In addition, pyrrolo[2,1‐f][1,2,4]triazine C‐nucleosides bearing a 2′‐deoxy‐, 2′,3′‐dideoxy‐, and 2′,3′‐dehydrodideoxyribose moiety were also prepared. Among these analogues, the pyrrolo[2,1‐f][1,2,4]triazine C‐ribonucleosides with either a hydrogen atom or cyano group at position 7 of the nucleobase displayed potent cytotoxic activity in a panel of various cancer cell lines.     

Gold electrodes modified with poly(4‐aminophenol): incorporation of nitrogenated bases and an oligonucleotide

BACKGROUND: Investigations of chemical modification of electrode surfaces and immobilization of nitrogenated bases and oligonucleotides are considered essential for the construction of DNA electrochemical nanodevices. Modification of gold electrode surfaces with poly(4‐aminophenol) was carried out in order to produce polymers capable of immobilizing purine bases and oligonucleotides. RESULTS: Gold electrodes coated with poly(4‐aminophenol) showed improved analytical characteristics and considerably enhanced the electrochemical signals associated with the detection of adenine and guanine by factors of ca 3 and ca 6, respectively, when compared with non‐coated gold surfaces. Impedance studies indicated higher charge transfer impedance to modified electrodes containing adenosine monophosphate. Atomic force microscopy images showed that nitrogenated bases have a strong influence over the morphology of the modified electrode surface. It was observed that the modified electrode containing guanine presents globular morphology. CONCLUSION: The modified electrodes increased the amplitude of the current signal of nitrogenated bases when compared to non‐coated gold surfaces and produced good response and peaks to the detection of an oligonucleotide. This work presents, for the first time, the electropolymerization of 4‐aminophenol on gold electrodes, as well as the detection of nitrogenated bases and an oligonucleotide incorporated on these modified electrodes. Copyright © 2007 Society of Chemical Industry     

Proteins as Possible Targets for Cytotoxic trans‐Platinum(II) Complexes with Aliphatic Amine Ligands: Further Exceptions to the DNA Paradigm

The reactivity of three cytotoxic trans‐Pt^II complexes bearing aliphatic amine ligands, with transferrin and single‐stranded oligonucleotides as DNA models, was investigated by ESI‐MS and the results obtained are discussed in comparison with cisplatin. Tandem MS studies provided additional information on the preferential Pt binding sites. To determine whether trans‐Pt^II complexes can migrate from a peptide to an oligonucleotide, transfer experiments were also performed using ESI‐MS, and competitive binding of the trans‐Pt^II complexes toward a model peptide and different oligonucleotides was also investigated. Significant differences in the reactivity of the trans complexes with respect to cisplatin were observed. In general, adduct formation with the selected peptide is favored for the trans compounds, whereas cisplatin shows a preference for oligonucleotides, especially if adjacent G–G residues are present. The results are discussed in relation to the possible mechanism of action of the trans‐Pt^II complexes.     

Inhibition of xanthine oxidase by thiosemicarbazones, hydrazones and dithiocarbazates derived from hydroxy-substituted benzaldehydes

Nonpurine xanthine oxidoreductase (XOR) inhibitors represent important alternatives to the purine analogue allopurinol, which is still the most widely used drug in the treatment of conditions associated with elevated uric acid levels in the blood. By condensing mono‐, di‐ and trihydroxybenzaldehydes with aromatic thiosemicarbazides, aryl hydrazides and dithiocarbazates, three series of structurally related Schiff bases were synthesised, characterised and tested for XOR inhibitory activity. Hydroxy substitution in the para‐position of the benzaldehyde component was found to confer high inhibitory activities. Acyl hydrazones were generally less potent than thiocarbonyl‐containing Schiff bases. Within the thiosemicarbazone series, chloro and cyano substituents in the para‐position of the thiosemicarbazide unit increased activities further, up to potencies approximately four‐times higher than that of the benchmark allopurinol, as measured under the same assay conditions. In order to illustrate the potential of the Schiff bases to bind directly to the molybdenum centre in the active site of the enzyme, a representative example (H₂L) of each inhibitor series was co‐ordinated to a cis‐dioxomolybdenum(VI) unit, and the resulting complexes, [MoO₂(L)MeOH], were structurally characterised. Subsequent steady‐state kinetic investigations, however, indicated mixed‐type inhibition, similar to that observed for inhibitors known to bind within the substrate access channel of the enzyme, remote from the Mo centre. Enzyme co‐crystallisation studies are thus required to determine the exact binding mode. Finally, the coordination of representative inhibitors to copper(II) gave rise to significantly decreased IC₅₀ values, revealing an additive effect that merits further investigation.     

Dendritic Chiral Phosphine Lewis Bases‐Catalyzed Asymmetric Aza‐Morita–Baylis–Hillman Reaction of N‐Sulfonated Imines with Activated Olefins

A series of polyether dendritic chiral phosphine Lewis bases was synthesized, and successfully applied to the asymmetric aza‐Morita–Baylis–Hillman reaction of N‐sulfonated imines (N‐arylmethylidene‐4‐methylbenzenesulfonamides) with methyl vinyl ketone (MVK), ethyl vinyl ketone (EVK), and acrolein to give the adducts in good to excellent yields along with up to 97 % ee, which are more effective than our previously reported original chiral phosphine Lewis bases. In addition, the dendrimer‐supported chiral phosphine Lewis bases can be easily recovered and reused.     

Novel Chiral Biheteroaromatic Diphosphine Oxides for Lewis Base Activation of Lewis Acids in Enantioselective Allylation and Epoxide Opening

Enantiomerically pure biheteroaromatic diphosphine oxides were synthesised and tested as organocatalysts in two different reactions involving trichlorosilyl compounds. The allylation of aldehydes with allyl(trichloro)silane afforded homoallylic alcohols in fair to good yields and up to 95% ee. Preliminary experiments showed that this new class of metal‐free catalysts was able also to promote the stilbene oxide opening by addition of tetrachlorosilane with enantioselectivity higher than 80%. The interesting results in terms of chemical and stereochemical efficiency open the way to further studies towards the development of new chiral heteroaromatic Lewis bases as efficient metal‐free catalysts.     

Production,Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila

Purine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over‐expressed in Escherichia coli, purified, characterized about its substrate specificity and used for the preparative synthesis of some 6‐substituted purine‐9‐ribosides. Substrate specificity towards natural nucleosides showed that this PNP catalyzes the phosphorolysis of both 6‐oxo‐ and 6‐aminopurine (deoxy)ribonucleosides. A library of nucleoside analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1‐, 2‐, 6‐ and 7‐modified nucleosides are accepted as substrates, whereas 8‐substituted nucleosides are not. A few transglycosylation reactions were carried out using 7‐methylguanosine iodide ( 4 ) as a D ‐ribose donor and 6‐substituted purines as acceptor. In particular, following this approach, 2‐amino‐6‐chloropurine‐9‐riboside ( 2c ), 6‐methoxypurine‐9‐riboside ( 2d ) and 2‐amino‐6‐(methylthio)purine‐9‐riboside ( 2g ) were synthesized in very high yield and purity.     

Synthesis and Antiviral Evaluation of TriPPPro‐AbacavirTP,TriPPPro‐CarbovirTP,and Their 1′,2′‐cis‐Disubstituted Analogues

Herein we describe the synthesis of lipophilic triphosphate prodrugs of abacavir, carbovir, and their 1′,2′‐cis‐substituted carbocyclic analogues. The 1′,2′‐cis‐carbocyclic nucleosides were prepared by starting from enantiomerically pure (1R,2S)‐2‐((benzyloxy)methyl)cyclopent‐3‐en‐1‐ol by a microwave‐assisted Mitsunobu‐type reaction with 2‐amino‐6‐chloropurine. All four nucleoside analogues were prepared from their 2‐amino‐6‐chloropurine precursors. The nucleosides were converted into their corresponding nucleoside triphosphate prodrugs (TriPPPro approach) by application of the H‐phosphonate route. The TriPPPro compounds were hydrolyzed in different media, in which the formation of nucleoside triphosphates was proven. While the TriPPPro compounds of abacavir and carbovir showed increased antiviral activity over their parent nucleoside, the TriPPPro compounds of the 1′,2′‐cis‐substituted analogues as well as their parent nucleosides proved to be inactive against HIV.     

Improvement of SNAr Reaction Rate by an Electron‐Withdrawing Group in the Crosslinking of DNA Cytosine‐5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor

DNA cytosine 5‐methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron‐deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism‐based modified nucleosides, 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) containing other nucleoside analogues, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐3‐cyano‐4‐halopyridine‐C‐nucleosides (d^XP^CN) and ODNs containing d^XP^CN, as more reactive inhibitors of DNMTs. Nucleophilic aromatic substitution (S_NAr) of the designed nucleosides, d^XP^CN, was faster than that of d^XP, and the ODN containing d^XP^CN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron‐withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.     

Fiber‐reinforced nanopigmented poly(methyl methacrylate) as improved denture base

Nanopigmented and fiber‐reinforced poly (methyl methacrylate) (PMMA) were synthesized for denture bases, by incorporating E‐glass fibers, flock fibers, or polyethylene fibers into the PMMA powder formulation to improve the flexural behavior and porosity; decreasing the Candida albicans adherence and being noncytotoxic. The commercial acrylic resin, Lucitone 199 was used as a control group. Scanning electron microscopy analysis was performed to the PMMA particles and the reinforcing fibers. Flexural strength increased by adding E‐glass fibers in the PMMA powder as compared to flock and polyethylene fibers. The reinforced PMMA with flock fibers showed the lower porosity even smaller than Lucitone 199. The synthesized PMMA and the fiber reinforced nanopigmented PMMA groups reduced significantly the C. albicans adherence when compared to the commercial acrylic resin. All the tested groups were found to be nontoxic materials after being in contact with mouse fibroblast culture during 24 h, showing that these novel nanostructured composites are suitable for producing adequate and nontoxic reinforced materials with antimicrobial properties for dentistry applications. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Quenching of fluorescent nucleobases by neighboring DNA: the "insulator" concept

Fluorescent nucleosides are widely used as probes of biomolecular structure and mechanism in the context of DNA, but they often exhibit low quantum yields because of quenching by neighboring DNA bases. Here we characterize the quenching by DNA of fluorescent nucleosides that have pyrene (Y), perylene (E), benzopyrene (B), or 2-aminopurine (2AP) as nucleobase replacements, and we investigate the effect of inserting varied nucleosides as potential "insulators" between the fluorescent nucleosides and other nearby DNA bases as a strategy for increasing quantum yields. The data show that the hydrocarbons are quenched by adjacent pyrimidines, with thymine being the strongest quencher. The quantum yield of pyrene is quenched 120-fold by a single adjacent T, that of benzopyrene tenfold, and that of perylene by a factor of 2.5. Quenching of excimer and exciplex dinucleoside labels (Y-Y, Y-E, E-E, etc.) was considerably lessened, but was strongest with neighboring thymine. 2-Aminopurine (2AP) is most strongly quenched (15-fold) by neighboring G. We tested four different insulator candidates for reducing this quenching by measuring the fluorescence of short oligonucleotides containing insulators placed between a fluorescent base and a quenching base. The insulators tested were a C(3) abasic spacer (S), dihydrothymidine nucleoside (DHT), terphenyl nucleoside (TP), and adenine deoxynucleoside (dA). Results showed that the abasic spacer had little effect on quenching, while the other three had substantial effects. DHT and terphenyl enhanced fluorescence of the fluorophores by factors of 5 to 70. Adenine base reduced the quenching of pyrene 40-fold. The results underscore the importance of the nearest neighbors in DNA-quenching mechanisms, and establish simple strategies for enhancing fluorescence in labeled DNAs.     

Diastereoselective Synthesis of N‐Substituted Planar Chiral Ferrocenes Using a Proline Hydantoin‐Derived Auxiliary

An N‐substituted ferrocene bearing a proline‐derived chiral directing group undergoes diastereoselective lithiation–electrophile quench to give planar chiral products in >95:5 dr. The starting material is synthesized by imidation of iodoferrocene with L ‐proline hydantoin, followed by hydrosilylation. Although the auxiliary is stable to lithium bases of the types RLi and R₂NLi, the ortho‐substituted products may be converted to solely planar chiral derivatives with simple reagents thanks to a labile triethylsilyloxy moiety.     

Flexibility of C3h‐Symmetrical Linkers in Tris‐oligonucleotide‐Based Tetrahedral Scaffolds

Flexibility of tris‐oligonucleotides is determined by the length of their connecting hydrocarbon chains. Tris‐oligonucleotides are branched DNA building blocks with three oligonucleotide arms attached to a C_3h‐symmetrical linker core at these chains. Four tris‐oligonucleotides hybridise into a tetrahedral nanocage by sequence‐determined self‐assembly. The influence of methylene, ethylene and propylene chains was studied by synthesising sets of tris‐oligonucleotides and analysing the relative stability of the hybridisation products against digestion by mung bean nuclease by using gel electrophoresis. Linkers with ethylene chains showed sufficient flexibility, whereas methylene‐chain linkers were too rigid. Tris‐oligonucleotides based on the latter still formed tetrahedral scaffolds in intermixing experiments with linkers of higher flexibility. Thus, a new generation of versatile isocyanurate‐based linkers was established.     

Novel gel‐entrapped base catalysts for the Claisen–Schmidt reaction

Novel gel‐entrapped base catalysts (GEBCs) were prepared by entrapping aqueous solutions of bases in a gel matrix of agar agar. The bases used were NaOH, KOH, morpholine and piperidine. Ternary phase diagrams were constructed for the water–base–agar agar system to identify the various phases and especially the solid phase, useful as a solid base catalyst. The 10% NaOH solid gel was used to effect the Claisen–Schmidt reaction between benzaldehydes and acetophenones in ethanol under heterogeneous conditions to obtain 70–100% yield of the products. The solid GEBCs obtained using other bases were also used for the same reaction; however, the yields were lower. The catalyst needed no activation prior to use and could be recycled. Copyright © 2004 Society of Chemical Industry     

Thioamide-Bridged Nucleic Acid (thioAmNA) Containing Thymine or 2-Thiothymine: Duplex-Forming Ability,Base Discrimination,and Enzymatic Stability

Oligonucleotides containing bridged nucleic acids (BNAs) show high duplex-forming ability towards target single-stranded RNA, so many BNAs have been developed for antisense applications. Amide-bridged nucleic acids (AmNAs), which are BNA analogues bearing an amide bond at the bridge, exhibit high duplex-forming ability, enzymatic stability, and antisense activity; thus, the AmNA motif represents a promising BNA scaffold. The high enzymatic stability of the AmNA motif is presumably attributable to the bulky amide structure, because it inhibits the access of nucleases to the phosphodiester linkage. Here, to improve enzymatic stability further, we designed thioAmNAs: thioamide-bridged nucleotides that have a bulkier bridge structure than AmNA. The synthesis of thioAmNAs bearing either thymine (thioAmNA-T) or 2-thiothymine (thioAmNA-S²T) bases was successful, and the obtained monomers were introduced into designed oligonucleotides without noticeable by-product generation. The thioAmNA-T- and thioAmNA-S²T-modified oligonucleotides showed strong binding affinity toward complementary single-stranded RNA, with the thioAmNA-S²T-modified oligonucleotide displaying excellent base-discrimination capability. Moreover, both thioAmNA-T and thioAmNA-S²T endowed oligonucleotides with higher resistance to enzymatic degradation than AmNA-T. These results indicate that thioAmNAs are potentially useful chemical modifications for oligonucleotide-based therapeutics.     

A Conformational Mimetic Approach for the Synthesis of Carbocyclic Nucleosides as Anti‐HCV Leads

Computer‐aided approaches coupled with medicinal chemistry were used to explore novel carbocyclic nucleosides as potential anti‐hepatitis C virus (HCV) agents. Conformational analyses were carried out on 6‐amino‐1H‐pyrazolo[3,4‐d]pyrimidine (6‐APP)‐based carbocyclic nucleoside analogues, which were considered as nucleoside mimetics to act as HCV RNA‐dependent RNA polymerase (RdRp) inhibitors. Structural insight gained from the modeling studies revealed the molecular basis behind these nucleoside mimetics. The rationally chosen 6‐APP analogues were prepared and evaluated for anti‐HCV activity. RdRp SiteMap analysis revealed the presence of a hydrophobic cavity near C7 of the nucleosides; introduction of bulkier substituents at this position enhanced their activity. Herein we report the identification of an iodinated compound with an EC₅₀ value of 6.6 μM as a preliminary anti‐HCV lead.     

Hepatocyte‐Specific Delivery of siRNAs Conjugated to Novel Non‐nucleosidic Trivalent N‐Acetylgalactosamine Elicits Robust Gene Silencing in Vivo

We recently demonstrated that siRNAs conjugated to triantennary N‐acetylgalactosamine (GalNAc) induce robust RNAi‐mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non‐nucleosidic linker, were developed to yield simplified trivalent GalNAc‐conjugated oligonucleotides under solid‐phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3′‐end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.     

Effect of Schiff Bases Containing Pyridyl Group as Corrosion Inhibitors for Low Carbon Steel in 0.1 M HCl

The inhibiting effect of four newly synthesised Schiff bases containing pyridyl group was investigated on the corrosion of low carbon steel in 0.1 M hydrochloric acid solution under various conditions by potentiodynamic polarisation method and impedance measurements. The Schiff bases used were 2-((1Z)-1-aza-2-(2-pyridyl)vinyl)benzene-1-thiol, (1Z)-1-aza-1,2- di(2-pyridyl)ethene, [((1Z)-1-aza-2-(2-pyridyl)vinyl)amino]benzene-1-thione and 2-((1Z)-1-aza-2-(2-pyridyl)vinyl)benzothiazole. All the Schiff bases inhibit corrosion of low carbon steel and their inhibition efficiencies increase with decrease in temperature and increase in concentration. The difference in protection actions of the inhibitors can be attributed to the presence of substituents in the structures that increase or decrease the electron density on the azomethine (–C=N–) group. Polarisation curves indicate that the Schiff bases act as anodic inhibitors. AC impedance and potentiodynamic polarisation measurements reveal that the compounds are adsorbed on the steel surface and the adsorption obeys the Temkin isotherm. Activation parameters (E_a, ΔH*, ΔS*) for the corrosion of low carbon steel in 0.1 M HCl were calculated and showed that corrosion was much reduced in the presence of inhibitors.