Molecular Design of an Environmentally Sensitive Fluorescent Nucleoside, 3‐Deaza‐2′‐Deoxyadenosine Derivative: Distinguishing Thymine by Probing the DNA Minor Groove

An environmentally sensitive fluorescent nucleoside containing a 3‐deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3‐naphthylethynylated 3‐deaza‐2′‐deoxyadenosine (^3nzA, 1 ) exhibited dual fluorescence emission from an intramolecular charge‐transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength.     

Potent Triple Helix Stabilization by 5′,3′‐Modified Triplex‐Forming Oligonucleotides

Anthraquinone and pyrene analogues attached to the 3′ and/or 5′ termini of triplex‐forming oligonucleotides (TFOs) by various linkers increased the stability of parallel triple helices. The modifications are simple to synthesize and can be introduced during standard solid‐phase oligonucleotide synthesis. Potent triplex stability was achieved by using doubly modified TFOs, which in the most favourable cases gave an increase in melting temperature of 30 °C over the unmodified counterparts and maintained their selectivity for the correct target duplex. Such TFOs can produce triplexes with melting temperatures of 40 °C at pH 7 even though they do not contain any triplexstabilizing base analogues. These studies have implications for the design of triplex‐forming oligonucleotides for use in biology and nanotechnology.     

O4‐Alkylated‐2‐Deoxyuridine Repair by O6‐Alkylguanine DNA Alkyltransferase is Augmented by a C5‐Fluorine Modification

Oligonucleotides containing various adducts, including ethyl, benzyl, 4‐hydroxybutyl and 7‐hydroxyheptyl groups, at the O⁴ atom of 5‐fluoro‐O⁴‐alkyl‐2′‐deoxyuridine were prepared by solid‐phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5‐fluoro‐2′‐deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B‐form DNA structure. O⁶‐Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5‐fluoro‐O⁴‐benzyl‐2′‐deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O⁴‐ethyl and benzyl adducts of 5‐fluoro‐2‐deoxyuridine. Computational assessment of N1‐methyl analogues of the O⁴‐alkylated nucleobases revealed that the C5‐fluorine modification had an influence on reducing the electron density of the O⁴?C_α bond, relative to thymine (C5‐methyl) and uracil (C5‐hydrogen). These results reveal the positive influence of the C5‐fluorine atom on the repair of larger O⁴‐alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT.     

Highly Efficient Fluorescent Interstrand Photo‐crosslinking of DNA Duplexes Labeled with 5‐Fluoro‐4‐thio‐2′‐O‐methyluridine

The formation of a fluorescent photoadduct between 5‐fluoro‐4‐thiouridine ( ^FS U ), in the sequence context 5′‐A ^FS U A‐3′ and incorporated into a synthetic oligonucleotide either at its 3′‐ or 5′‐end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo‐crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo‐crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo‐crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations.     

O6‐Alkylguanine‐DNA Alkyltransferase‐Mediated Repair of O4‐Alkylated 2′‐Deoxyuridines

O⁶‐Alkylguanine‐DNA alkyltransferases (AGTs) are responsible for the removal of O⁶‐alkyl 2′‐deoxyguanosine (dG) and O⁴‐alkyl thymidine (dT) adducts from the genome. Unlike the E. coli OGT (O⁶‐alkylguanine‐DNA‐alkyltransferase) protein, which can repair a range of O⁴‐alkyl dT lesions, human AGT (hAGT) only removes methyl groups poorly. To uncover the influence of the C5 methyl group of dT on AGT repair, oligonucleotides containing O⁴‐alkyl 2′‐deoxyuridines (dU) were prepared. The ability of E. coli AGTs (Ada‐C and OGT), human AGT, and an OGT/hAGT chimera to remove O⁴‐methyl and larger adducts (4‐hydroxybutyl and 7‐hydroxyheptyl) from dU were examined and compared to those relating to the corresponding dT species. The absence of the C5 methyl group resulted in an increase in repair observed for the O⁴‐methyl adducts by hAGT and the chimera. The chimera was proficient at repairing larger adducts at the O⁴ atom of dU. There was no observed correlation between the binding affinities of the AGT homologues to adduct‐containing oligonucleotides and the amounts of repair measured.     

NBD‐Based Green Fluorescent Ligands for Typing of Thymine‐Related SNPs by Using an Abasic Site‐Containing Probe DNA

The binding behavior of green fluorescent ligands, derivatives of 7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N¹‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)propane‐1,3‐diamine (NBD‐NH₂) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52×10⁶ M ^?1. From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD‐NH₂ is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2‐amino‐6,7‐dimethyl‐4‐hydroxypteridine (diMe‐pteridine).     

Recognition and Excision Properties of 8‐Halogenated‐7‐Deaza‐2′‐Deoxyguanosine as 8‐Oxo‐2′‐Deoxyguanosine Analogues and Fpg and hOGG1 Inhibitors

Cellular DNA continuously suffers various types of damage, and unrepaired damage increases disease progression risk. 8‐Oxo‐2′‐deoxyguanine (8‐oxo‐dG) is excised by repair enzymes, and their analogues are of interest as inhibitors and as bioprobes for study of these enzymes. We have developed 8‐halogenated‐7‐deaza‐2′‐deoxyguanosine derivatives that resemble 8‐oxo‐dG in that they adopt the syn conformation. In this study, we investigated their effects on Fpg (formamidopyrimidine DNA glycosylase) and hOGG1 (human 8‐oxoguanine DNA N‐glycosylase 1). Relative to 8‐oxo‐dG, Cl‐ and Br‐deaza‐dG were good substrates for Fpg, whereas they were less efficient substrates for hOGG1. Kinetics and binding experiments indicated that, although hOGG1 effectively binds Cl‐ and Br‐deaza‐dG analogues with low K_m values, their lower k_cat values result in low glycosylase activities. The benefits of the high binding affinities and low reactivities of 8‐oxo‐dG analogues with hOGG1 have been successfully applied to the competitive inhibition of the excision of 8‐oxoguanine from duplex DNA by hOGG1.     

Probing Local Folding Allows Robust Metal Sensing Based on a Na+-Specific DNAzyme

Fluorescent metal sensors based on DNA often rely on changes in end-to-end distance or local environmental near fluorophore labels. Because metal ions can also nonspecifically interact with DNA through various mechanisms, such as charge screening, base binding, and increase or decrease in duplex stability, robust and specific sensing of metal ions has been quite challenging. In this work, a side-by-side comparison of two signaling strategies on a Na⁺-specific DNAzyme that contained a Na⁺-binding aptamer was performed. The duplex regions of the DNAzyme was systematically shortened and its effect was studied by using a 2-aminopurine (2AP)-labeled substrate strand. Na⁺ binding affected the local environmental of the 2AP label and increased its fluorescence. A synergistic process of Na⁺ binding and forming the duplex on the 5′-end of the enzyme strand was observed, and this end was close to the aptamer loop. Effective Na⁺ binding was achieved with a five base-pair stem. The effect on the 3′-end is more continuous, and the stem needs to form first before Na⁺ can bind. With an optimized substrate binding arm, a FRET-based sensor has been designed by labeling the two ends of a cis form of the DNAzyme with two fluorophores. In this case, Na⁺ failed to show a distinct difference from that of Li⁺ or K⁺; thus indicating that probing changes to the local environment allows more robust sensing of metal ions.     

Quick Click: The DNA‐Templated Ligation of 3′‐O‐Propargyl‐ and 5′‐Azide‐Modified Strands Is as Rapid as and More Selective than Ligase

The copper(I)‐mediated azide–alkyne cycloaddition (CuAAC) of 3′‐propargyl ether and 5′‐azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate–sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA‐templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k_obs value of 1.1 min^?1, comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3′‐propargyl ether and 5′‐azide‐terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.     

6‐Substituted 2‐Aminopurine‐2′‐deoxyribonucleoside 5′‐Triphosphates that Trace Cytosine Methylation

Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2′‐deoxy 5‐methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O⁶‐alkylated 2′‐deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6‐substituted 2‐aminopurine‐2′‐deoxyribonucleoside 5′‐triphosphates modified at position 6 with various functionalities. We found that sensing of 5‐methylation by this class of nucleotides is more general, not being restricted to O⁶‐alkyl modification of dGTP but also applying to other functionalities.     

A fluorescent chemosensor based on optically active 2,2′‐binaphtho‐20‐crown‐6 for metal ions

A chiral conjugated polymer can be obtained by the polymerization of (S)‐6,6′‐dibromo‐2,2′‐binaphtho‐20‐crown‐6 and 1,4‐divinyl‐2,5‐dibutoxybenzene via a palladium‐catalyzed Heck cross‐coupling reaction. The chiral conjugated polymer shows strong green‐blue fluorescence. The responsive properties of the chiral polymer to metal ions were investigated using fluorescence and UV‐visible absorption spectra. K⁺, Pb²⁺, Cd²⁺ and Ba²⁺ enhance the fluorescence of the polymer; in contrast, Hg²⁺ causes effective quenching of the fluorescence of the polymer. The obvious influences on the fluorescence indicate that the 2,2′‐binaphtho‐20‐crown‐6 moiety plays an important role in fluorescence recognition for Hg²⁺ due to the effective photo‐induced electron transfer or charge transfer between the conjugated polymer backbone and the receptor ions. The responsive properties of the polymer to metal ions show that the chiral conjugated polymer incorporating 2,2′‐binaphtho‐20‐crown‐6 moieties in the main‐chain backbone as recognition sites can act as an excellent fluorescent probe for the sensitive detection of Hg²⁺. Copyright © 2010 Society of Chemical Industry     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

Tropolone-Conjugated DNA: Fluorescence Enhancement in the Duplex

Tropolone (2-hydroxycyclohepta-2,4,6-triene-1-one and tautomer) is a non-benzenoid bioactive natural chromophore with pH-dependent fluorescence character and extraordinary metal binding affinities, especially with transition-metal ions Cu²⁺/Zn²⁺/Ni²⁺. This report describes the syntheses and biophysical studies of a new tropolonyl thymidine [(4(5)-hydroxy-5(4)-oxo-5(4)H-cyclohepta-1,3,6-trienyl)thymidine] (tr-T) nucleoside and of corresponding tropolone-conjugated DNA oligonucleotides that form B-form DNA duplex structures with a complementary DNA strand, although their duplex structures are less stable than that of the control. Furthermore, the stabilities of those DNA duplex structures are lowered by the presence of increasing numbers of tr-T residue or by decreasing pH of their environments. Most importantly, these duplex structures are made fluorescent because of the presence of the tropolone moieties conjugated to the thymidine residues. The fluorescence behavior of those duplex structures exhibits pH dependence, with stronger fluorescence at lower pH and weaker fluorescence at high pH. Importantly, the fluorescence characters of tr-DNA oligonucleotides are significantly enhanced by nearly threefold after duplex structure formation with their complementary control DNA oligonucleotide. Further, the fluorescence behavior of these tr-DNA duplex structures is also dependent on the pH conditions. Hence, tropolonyl-conjugated DNA represents a class of new fluorescent analogues that might be be employed for sensing DNA duplex formation and provide opportunities to improve fluorescence properties further.     

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.     

Neglected Aspects of Anthracenide (Anthracenidyl) Chemistry — Reactions with two N‐Benzoylaziridines

Reaction of anthracenide A ^·‐ with N‐benzoylaziridines 1a,b forms charged radicals 3a,b by single electron transfer and homolytic ring opening. Reactions follow that are known or expected as e.g. coupling with position 9 of A ^·‐ forming dihydroanthracene anions 9a,b that yield amidoethylated dihydroanthracenes 10a,b , or react with 1a,b giving finally 9,10‐bis‐amidoethylated dihydroanthracenes 11a,b . Results depend on experimental conditions and on the counter ions Na⁺ or Li⁺. Coupling is not regiospecific: contributions by positions 2 and 1 reach 29% or 4%, respectively, of total coupling with the primary radical 3a ; much higher contributions are possible with Li. Product 21s (probably 3,3′‐disubstituted tetrahydrobianthryl) may arise by hydrogen detachment from the first intermediate ( 29 ) of coupling with position 2 and dimerization of the formed 2‐substituted A ^·‐ ( 30 ). Coupling products may be fully aromatized or may be hydroxylated in one of the benzylic positions. With counter ion Li⁺ a non‐SET reaction of 1a with the dimer of A ^·‐ is indicated by the isolation of 9‐benzoyl‐dihydroanthracene 15 and by 19% yield of 16a (aromatized 10a ). Reaction of 3b with anthracene is indicated by 10,10′‐disubstituted tetrahydrobianthryl 37 .     

Structural Insights into the Recognition of N2‐Aryl‐ and C8‐Aryl DNA Lesions by the Repair Protein XPA/Rad14

Aromatic amines are strongly carcinogenic. They are activated in the liver to give reactive nitrenium ions that react with nucleobases within the DNA duplex. The reaction occurs predominantly at the C8 position of the dG base, thereby giving C8‐acetyl‐aryl‐ or C8‐aryl‐dG adducts in an electrophilic aromatic substitution reaction. Alternatively, reaction with the exocyclic 2‐NH₂ group is observed. Although the C8 adducts retain base‐pairing properties, base pairing is strongly compromised in the case of the N² adducts. Here we show crystal structures of two DNA lesions, N²‐acetylnaphthyl‐dG and C8‐fluorenyl‐dG, within a DNA duplex recognized by the repair protein Rad14. The structures confirm that two molecules of the repair protein recognize the lesion and induce a 72 or 78° kink at the site of the damage. Importantly, the same overall kinked structure is induced by binding of the repair proteins, although the structurally different lesions result in distinct stacking interactions of the lesions within the duplex. The results suggest that the repair protein XPA/Rad14 is a sensor that recognizes flexibility. The protein converts the information that structurally different lesions are present in the duplex into a unifying sharply kinked recognition motif.     

Polymerization of 4‐(4′‐N‐1,8‐naphthalimidophenyl)‐1,2,4‐triazolidine‐3,5‐dione with diisocyanates

4‐(4′‐Aminophenyl)‐1,2,4‐triazolidine‐3,5‐dione ( 1 ) was reacted with 1,8‐naphthalic anhydride ( 2 ) in a mixture of acetic acid and pyridine (3 : 2) under refluxing temperature and gave 4‐(4′‐N‐1,8‐naphthalimidophenyl)‐1,2,4‐triazolidine‐3,5‐dione ( NIPTD ) ( 3 ) in high yield and purity. The compound NIPTD was reacted with excess n‐propylisocyanate in N,N‐dimethylacetamide solution and gave 1‐(n‐propylamidocarbonyl)‐4‐[4′‐(1,8‐naphthalimidophenyl)]‐1,2,4‐triazolidine‐3,5‐dione ( 4 ) and 1,2‐bis(n‐propylamidocarbonyl)‐4‐[4′‐(1,8‐naphthalimidophenyl)]‐1,2,4‐ triazolidine‐3,5‐dione ( 5 ) as model compounds. Solution polycondensation reactions of monomer 3 with hexamethylene diisocyanate ( HMDI ), isophorone diisocyanate ( IPDI ), and tolylene‐2,4‐diisocyanate ( TDI ) were performed under microwave irradiation and conventional solution polymerization techniques in different solvents and in the presence of different catalysts, which led to the formation of novel aliphatic‐aromatic polyureas. The polycondensation proceeded rapidly, compared with conventional solution polycondensation, and was almost completed within 8 min. These novel polyureas have inherent viscosities in a range of 0.06–0.20 dL g^?1 in conc. H₂SO₄ or DMF at 25°C. Some structural characterization and physical properties of these novel polymers are reported. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 2861–2869, 2003     

Chemistry,Properties, and in Vitro and in Vivo Applications of 2′‐O‐Methoxyethyl‐4′‐thioRNA,a Novel Hybrid Type of Chemically Modified RNA

We report the synthesis, properties, and in vitro and in vivo applications of 2′‐O‐methoxyethyl‐4′‐thioRNA (MOE‐SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE‐SRNA showed a moderate improvement of T_m value (+3.4 °C relative to an RNA:RNA duplex). However, the results of a comprehensive comparison of the nuclease stability of MOE‐SRNA relative to 2′‐O‐methoxyethylRNA (MOERNA), 2′‐O‐methyl‐4′‐thioRNA (Me‐SRNA), 2′‐O‐methylRNA (MeRNA), 4′‐thioRNA (SRNA), and natural RNA revealed that MOE‐SRNA had the highest stability (t_1/2>48 h in human plasma). Because of the favorable properties of MOE‐SRNA, we evaluated its in vitro and in vivo potencies as an anti‐microRNA oligonucleotide against miR‐21. Although the in vitro potency of MOE‐SRNA was moderate, its in vivo potency was significant for the suppression of tumor growth (similar to that of MOERNA).     

Studies on Electrophilic Interaction of 2,3‐Allenols with Electrophilic Halogen Reagents: Selective Synthesis of 2,5‐Dihydrofurans, 3‐Halo‐3‐alkenals,or 2‐Halo‐2‐alkenyl Ketones

The reaction of primary 2,3‐allenols with iodine (I₂) afforded 2,5‐dihydrofurans while that of readily available 1‐aryl or 1‐methyl substituted 2,3‐allenols with bromine (Br₂), N‐bromosuccinimide (NBS), I₂ or N‐iodosuccinimide (NIS) formed the not easily available but synthetically useful 3‐halo‐3‐alkenals and 2‐halo‐2‐alkenyl ketones with good selectivity and yields via a sequential electrophilic interaction of X⁺ with the allene moiety, 1,2‐aryl or 1,2‐proton shift, and H⁺ elimination process.