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.     

Inhibitory Effect of 8‐Halogenated 7‐Deaza‐2′‐deoxyguanosine Triphosphates on Human 8‐Oxo‐2′‐deoxyguanosine Triphosphatase,hMTH1, Activities

hMTH1 (8‐oxo‐2′‐deoxyguanine triphosphatase) hydrolyzes oxidized nucleoside triphosphates; its presence is non‐essential for survival of normal cells but is required for survival of cancer cells. In this study, 8‐halogenated‐7‐deaza‐2′‐deoxyguanosine triphosphate (8‐halogenated‐7‐deazadGTP) derivatives were synthesized. Interestingly, these triphosphates were poor substrates for hMTH1, but exhibited strong competitive inhibition against hMTH1 at nanomolar levels. This inhibitory effect is attributed to slower rate of hydrolysis, possibly arising from enzyme structural changes, specifically different stacking interactions with 8‐halogenated‐7‐deazadGTP. This is the first example of using nucleotide derivatives to inhibit hMTH1, thus demonstrating their potential as antitumor agents.     

Synthesis and Cytostatic and Antiviral Activities of 2′‐Deoxy‐2′,2′‐difluororibo‐ and 2′‐Deoxy‐2′‐fluororibonucleosides Derived from 7‐(Het)aryl‐7‐deazaadenines

A series of sugar‐modified derivatives of cytostatic 7‐heteroaryl‐7‐deazaadenosines (2′‐deoxy‐2′‐fluororibo‐ and 2′‐deoxy‐2′,2′‐difluororibonucleosides) bearing an aryl or heteroaryl group at position 7 was prepared and screened for biological activity. The difluororibonucleosides were prepared by non‐ stereoselective glycosidation of 6‐chloro‐7‐deazapurine with benzoyl‐protected 2‐deoxy‐2,2‐difluoro‐D ‐erythro‐pentofuranosyl‐1‐mesylate, followed by amination and aqueous Suzuki cross‐couplings with (het)arylboronic acids. The fluororibo derivatives were prepared by aqueous palladium‐catalyzed cross‐coupling reactions of the corresponding 7‐iodo‐7‐deazaadenine 2′‐deoxy‐2′‐fluororibonucleoside 20 with (het)arylboronic acids. The key intermediate 20 was prepared by a six‐step sequence from the corresponding arabinonucleoside by selective protection of 3′‐ and 5′‐hydroxy groups with acid‐labile groups, followed by stereoselective S_N2 fluorination and deprotection. Some of the title nucleosides and 7‐iodo‐7‐deazaadenine intermediates showed micromolar cytostatic or anti‐HCV activity. The most active were 7‐iodo and 7‐ethynyl derivatives. The corresponding 2′‐deoxy‐2′,2′‐difluororibonucleoside 5′‐O‐triphosphates were found to be good substrates for bacterial DNA polymerases, but are inhibitors of human polymerase α.     

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.     

Triplexes with 8‐Aza‐2′‐Deoxyisoguanosine Replacing Protonated dC: Probing Third Strand Stability with a Fluorescent Nucleobase Targeting Duplex DNA

The fluorescent 8‐aza‐2′‐deoxyisoguanosine ( 4 ) as well as the parent 2′‐deoxyisoguanosine ( 1 ) were used as protonated dCH⁺ surrogates in the third strand of oligonucleotide triplexes. Stable triplexes were formed by Hoogsteen base pairing. In contrast to dC, triplexes containing nucleoside 1 or 4 in place of dCH⁺ are already formed under neutral conditions or even at alkaline pH values. Triplex melting can be monitored separately from duplex dissociation in cases in which the third strand contains the fluorescent nucleoside 4 . Third‐strand binding of oligonucleotides with 4 , opposite to dG, was selective as demonstrated by hybridisation experiments studying mismatch discrimination. Third‐strand binding is more efficient when the stability of the DNA duplex is reduced by mismatches, giving third‐strand binding more flexibility.     

Structure‐Based Design,Synthesis, and Evaluation of 2′‐(2‐Hydroxyethyl)‐2′‐deoxyadenosine and the 5′‐Diphosphate Derivative as Ribonucleotide Reductase Inhibitors

Analysis of the recently solved X‐ray crystal structures of Saccharomyces cerevisiae ribonucleotide reductase I (ScRnr1) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2′‐hydroxy group of the nucleoside ribose. In this study 2′‐(2‐hydroxyethyl)‐2′‐deoxyadenosine 1 and the 5′‐diphosphate derivative 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxymethylene group, to generate novel RNR inhibitors as potential antitumor agents. Herein we report the synthesis of analogues 1 and 2 , and the co‐crystal structure of adenosine diphosphate analogue 2 bound to ScRnr1, which shows the conserved water molecule is displaced as hypothesized.     

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.     

8‐Cyclopropyl‐2′‐Deoxyguanosine: A Hole Trap for DNA‐Mediated Charge Transport

DNA duplexes containing 8‐cyclopropyl‐2′‐deoxyguanosine (^8CPG) were synthesized to investigate the effect of the C8‐modified deoxyguanosine as a kinetic trap for transient hole occupancy on guanines during DNA‐mediated hole transport (HT). Thermal denaturation and CD spectra show that DNA duplexes containing ^8CPG are able to form stable B‐form duplexes. Photoirradiation of terminal tethered anthraquinone can induce oxidative decomposition of ^8CPG through DNA HT along adenine tracts with lengths of up to 4.8 nm. Shallow and periodic distance dependence was observed in a long adenine tract with intervening guanines. The efficient charge transport indicates that ^8CPG can electronically couple well with a DNA bridge and form HT‐active conformational domains to facilitate transient hole delocalization over an adenine tract.     

In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage

The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8‐oxoguanine, are a main source of these mutations, and the enzyme 8‐oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8‐oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel‐based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8‐oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8‐oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60‐fold light‐up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.     

Synthesis and Biological Evaluation of Purine 2′‐Fluoro‐2′‐deoxyriboside ProTides as Anti‐influenza Virus Agents

2′‐Fluoro‐2′‐deoxyguanosine has been reported to have potent anti‐influenza virus activity in vitro and in vivo. Herein we describe the synthesis and biological evaluation of 6‐modified 2′‐fluoro‐2′‐deoxyguanosine analogues and their corresponding phosphoramidate ProTides as potential anti‐influenza virus agents. Whereas the parent nucleosides were devoid of antiviral activity in two different cellular assays, the 5′‐O‐naphthyl(methoxy‐L ‐alaninyl) ProTide derivatives of 6‐O‐methyl‐2′‐fluoro‐2′‐deoxyguanosine, 6‐O‐ethyl‐2′‐fluoro‐2′‐deoxyguanosine, and 2′‐deoxy‐2′‐fluoro‐6‐chloroguanosine, and the 5′‐O‐naphthyl(ethoxy‐L ‐alaninyl) ProTide of 6‐O‐ethyl‐2′‐fluoro‐2′‐deoxyguanosine displayed antiviral EC₉₉ values of ～12 μM . The antiviral results are supported by metabolism studies. Rapid conversion into the L ‐alaninyl metabolite and then 6‐modified 2′‐fluoro‐2′‐deoxyguanosine 5′‐monophosphate was observed in enzymatic assays with yeast carboxypeptidase Y or crude cell lysate. Evidence for efficient removal of the 6‐substituent on the guanine part was provided by enzymatic studies with adenosine deaminase, and by molecular modeling of the nucleoside 5′‐monophosphates in the catalytic site of a model of ADAL1, thus indicating the utility of the double prodrug concept.     

siRNA Modified with 2′‐Deoxy‐2′‐C‐methylpyrimidine Nucleosides

(2′S)‐2′‐Deoxy‐2′‐C‐methyluridine and (2′R)‐2′‐deoxy‐2′‐C‐methyluridine were incorporated in the 3′‐overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2′S)‐2′‐deoxy‐2′‐C‐methylcytidine was incorporated in the 6‐position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC₅₀ values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3′‐overhang region by (2′S)‐ or (2′R)‐2′‐deoxy‐2′‐C‐methyluridine, reducing IC₅₀ to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.     

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     

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.     

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.     

N,N,N′,N′‐Tetramethylchloroformamidinium Chloride‐Mediated Cyclizations of β‐Oxo Amides: Facile and Divergent One‐Pot Synthesis of Substituted 2H‐Pyrans, 4H‐Pyrans and Pyridin‐2(1H)‐ones

An efficient and divergent one‐pot synthesis of substituted 2H‐pyrans, 4H‐pyrans and pyridin‐2(1H)‐ones from β‐oxo amides based on the selection of the reaction conditions is reported. Mediated by N,N,N′,N′‐tetramethylchloroformamidinium chloride, β‐oxo amides underwent intermolecular cyclizations in the presence of triethylamine at room temperature to give substituted 2H‐pyrans in high yields, which could be converted into substituted 4H‐pyrans in the presence of sodium hydroxide in ethanol at room temperature, or into substituted pyridin‐2(1 H)‐ones under reflux.     

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.     

Cover Picture: Chemical Restriction: Strand Cleavage by Ammonia Treatment at 8‐Oxoguanine Yields Biologically Active DNA (ChemBioChem 7/2003)

The cover picture shows a chemical restriction method in which DNA double strands are cleaved at the position of the artificial base 8‐oxoguanine (G^oxo). As as result of the mild cleavage conditions, the DNA fragments remain biologically active and can be used in gene cloning experiments. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine‐modified primers, restricted by treatment with ammonia, ligated into a plasmid vector, transformed in Escherichia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure, and allows the design of any 3′ overhang, independent of the sequence of the cloned DNA. Further information can be found in the article by Giese et. al. on pp. 610–614     

A New Class of 3′‐Sulfonyl BINAPHOS Ligands: Modulation of Activity and Selectivity in Asymmetric Palladium‐Catalysed Hydrophosphorylation of Styrene

Palladium‐catalysed monophosphorylation of (R)‐2,2′‐bisperfluoroalkanesulfonates of BINOL (R^F=CF₃ or C₄F₉) by a diaryl phosphinate [Ar₂P(O)H] followed by phosphine oxide reduction (Cl₃SiH) then lithium diisopropylamide‐mediated anionic thia‐Fries rearrangement furnishes enantiomerically‐pure (R)‐2′‐diarylphosphino‐2′‐hydroxy‐3′‐perfluoralkanesulfonyl‐1,1′‐binaphthalenes [(R)‐ 8ab and (R)‐ 8g–j ], which can be further diversified by Grignard reagent (RMgX)‐mediated CF₃‐displacement [→(R)‐ 8c–f ]. Coupling of (R)‐ 8a–j with (S)‐1,1′‐binaphthalene‐2,2′‐dioxychlorophosphine (S)‐ 9 generates 3′‐sulfonyl BINAPHOS ligands (R,S)‐ 10a–j in good yields (43–82%). These new ligands are of utlility in the asymmetric hydrophosphonylation of styrene ( 1 ) by 4,4,5,5‐tetramethyl‐1,3,2‐dioxaphospholane 2‐oxide ( 2 ), for which a combination of the chiral ligands with either [Pd(Cp)(allyl)] or [Pd(allyl)(MeCN)₂]⁺/NaCH(CO₂Me)₂ proves to be a convenient and active pre‐catalyst system. A combination of an electron‐rich phosphine moiety and an electron‐deficient 3′‐sulfone moiety provides the best enantioselectivity to date for this process, affording the branched 2‐phenethenephosphonate, (−)‐iso‐ 3 , in up to 74% ee with ligand (R,S)‐ 10i , where Ar=p‐anisyl and the 3′‐SO₂R group is triflone.