Thymine Analogues Research Articles

Asymmetric synthesis of L-cyclopentyl carbocyclic nucleosides

Asymmetric synthesis of L-carbocyclic nucleosides, (+)-β-L-aristeromycin ( 11) and its thymine analog ( 12) was accomplished. The key intermediate 3 was synthesized by a regioselective conjugate addition to the enone 1 followed by DIBAL-H reduction. Coupling of 4 with heterocycle or construction of heterocyle by a linear approach gave 11 and 12. This is the first asymmetric synthesis of L-cyclopentyl carbocyclic nucleosides.

Model studies towards the synthesis of 4′-azaerythrofuranosyladenines as analogues of the antiviral drug 2′,3′-dideoxyadenosine (ddA) 1

The [3 + 2] dipolar cycloaddition of nitrones to N-9-vinyladenine provides a regioselective and stereoselective method for the obtainment of 4′-aza-analogues of 2′,3′-dideoxyadenosine. The latter may act as antiviral agents by analogy with the behaviour in vitro of the corresponding thymine analogue. The formation of the unsubstituted isoxazolidino derivative requires the protection of the 6-NH2 function of the dipolarophile, which is not necessary when preformed nitrones are used. The obtainment of a specific enantiomer has been exploited by enzyme-catalysed hydrolysis of an ester function introduced into the isoxazolidino nucleus.

BMS-200475, a novel carbocyclic 2′-deoxyguanosine analog with potent and selective anti-hepatitis B virus activity in vitro

BMS-200475, a novel carbocyclic analog of 2′-deoxyguanosine, is a potent inhibitor of hepatitis B virus in vitro (ED 50 = 3 nM) with relatively low cytotoxicity (CC 50 = 21–120 μM). A practical 10-step asymmetric synthesis was developed affording BMS-200475 in 18% overall chemical yield and >99% optical purity. The enantiomer of BMS-200475 as well as the adenine, thymine, and iodouracil analogs are much less active.

ChemInform Abstract: N,N′‐Cyclization of Carbodiimides with 2‐(Bromomethyl)acrylic Acid. A Direct Entry to the System 5‐Methylene‐6H‐pyrimidine‐2,4‐dione, a New Class of Thymine Analogues.

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N,N′‐Cyclization of carbodiimides with 2‐(bromomethyl)acrylic acid. A direct entry to the system 5‐methylene‐6H‐pyrimidine‐2,4‐dione, a new class of thymine analogues

AbstractCarbodiimides react under very mild conditions with 2‐(bromomethyl)acrylic acid at both N atoms to give 1,3‐disubstituted‐5‐methylene‐6H‐pyrimidine 2,4‐dione derivatives in moderate to good yields. These products behaved as good Michael acceptors towards a variety of nucleophiles such as bromine, hydrochloric acid, hydrides, etc. A plausible mechanism is proposed based on theoretical approaches and experimental results.

Synthesis and oligomerization of Nδ-Boc- Nα-(thymin-1-ylacetyl)ornithine

The synthesis of a new DNA analogue based on ornithine is described. Only the thymine analogue was synthesised. A 10 mer entirely made up of the thymine monomer forms a triple helix with poly RNA A. The T m of the triplex was 21°C.

Identification of a Tus protein segment that photo-cross-links with TerB DNA and elucidation of the role of certain thymine methyl groups in the Tus-TerB complex using halogenated uracil analogues.

Six potential hydrophobic sites of the Tus-TerB complex were analyzed using bromodeoxy-uridine and iododeoxyuridine as isosteric analogues of thymine. Analogues were incorporated at individual sites, and dissociation rates were measured in 150 mM potassium glutamate, pH 8.0, using a nitrocellulose filter assay. These halogenated analogues serve as a probe of the environment in which they reside. Our measurement revealed at least two types of responses. Three sites showed increases in stability with the introduction of the bromo and iodo derivatives. The enhanced stability is proposed to result from polar or charged molecules in the vicinity of the halogenated analogues through dipole-dipole, dipole-ion, or dipole-induced dipole interactions. The other three sites exhibited the opposite trend, being destabilized by the introduction of these analogues. The destabilizing effects are attributed to a hydrophobic environment which cannot accommodate polar molecules. The photoreactivity of these analogues was utilized to specifically cross-link the Tus protein and TerB DNA. Using the substitution of bromodeoxyuridine at position 8 in the TerB DNA, Tus protein was covalently attached to the DNA, and by trypsin digestion a fragment of Tus was isolated. Sequencing of the peptide fragment revealed a segment that matched the amino acid composition from 122-139 of the Tus protein.

The paradoxical influence of thymine analogues on restriction endonuclease cleavage of oligodeoxynucleotides.

Thymine residues in the DNA of eucaryotes may be replaced occasionally by uracil (U) or 5-(hydroxymethyl)uracil (H) as consequences of dUMP misincorporation or thymine oxidation, respectively. In this study, we constructed a series of 44-base oligonucleotides containing site-specific U or H residues and 5'-fluorescein labels in order to probe the influence of such modifications on sequence-specific DNA-protein interactions using several type II restriction endonucleases. We find that substitution within the recognition sites of several restriction endonucleases increases initial cleavage velocity by up to an order of magnitude. These results contrast dramatically with several previous studies which demonstrated that U substitution in short oligonucleotides inhibits or prevents nuclease cleavage. We propose that this apparent paradox results because the rate-limiting step in the cleavage of longer oligonucleotides is product release whereas for shorter oligonucleotides substrate binding is most probably rate-limiting. For longer oligonucleotides and DNA, more rapid release of the cleaved, substituted oligonucleotides results in more rapid turnover and a faster apparent cleavage rate. The sequence length at which the transition in rate-limiting step occurs likely corresponds to the size of the enzyme footprint on its DNA recognition site. We conclude that both U and H do perturb sequence-specific DNA-protein interactions, and the magnitude of this effect is site-dependent.

Synthesis and reactions of [formula omitted] derivatives of 5-alkyl- and 5-halo-pyrimidines

Chemical syntheses of 1-(2-O- acetyl-5-azido-5-deoxy-3-O-p- toluenesulfonyl-β- d-xylofuranosyl)-5-iodo -, -5-fluoro-, and -5-trifluoromethyl-uracil nucleosides ( 11–13) as well as the thymine analogue 10 were performed from a sugar precursor and the corresponding uracil bases. Partial deblocking of 10–13 gave the 5′-azido-5′-deoxynucleosides 14–17. The 3′,5′-diazido-3′,5′-dideoxyribonucleosides were obtained in the same way. The 2′,3′-anhydro analogue 20 was prepared by treatment of 10 with potassium carbonate in methanol or a basic ion-exchange resin. Reaction of 10 with azide or methanethiolate ions gave 2′-azido- and 2′-thiomethyl-ribonucleosides, respectively. Similarly, 13 gave a 2′-thiomethylribonucleoside on treatment with methanethiolate ion. Treatment of 16 with phenoxythiocarbonyl chloride in basic medium afforded a 2′,3′-anhydro derivative and not the expected ester.

Generation of Thymidine Kinase-Deficient Mutants of Infectious Laryngotracheitis Virus

Current vaccines for the avian respiratory disease infectious laryngotracheitis consist of naturally attenuated strains of the causative agent—the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3′-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce β-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-β-d-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.

Comparative stability study of thymidine and (dideoxy- d- erythro-hexopyranosyl)thymine analogues monitored by capillary electrophoresis

A capillary zone electrophoretic method was developed for monitoring the stability of hexopyranosyi analogues of thymidine. The final analytical conditions adopted were as follows: capillary, fused silica, 70 cm (62 cm to detector) × 75 μm I.D.; background electrolyte, 20 m M sodium tetraborate (pH 10.5); voltage, 30 kV; detection, UV at 262 nm; and temperature. 15°C. The performance of this analytical system is discussed. The stability study at three pH values allows to compare the stability of the analogues with that of thymidine and to investigate the influence of the configuration of the N-glycosidic bond on the rate constant of degradation.

Binding of T and T analogs to CG base pairs in antiparallel triplexes.

The goal of this study was to address antiparallel triplex formation at duplex targets that do not conform to a strict oligopurine.oligopyrimidine motif. We focused on the ability of natural bases and base analogs incorporated into oligonucleotide third strands to bind to so-called CG inversions. These are sites where a cytosine base is present in an otherwise purine-rich strand of a duplex target. Using a 26-base-triplet test system, we found that of the standard bases, only thymine (T) shows substantial binding to CG inversions. This is quantitatively similar to the report of Beal and Dervan [Science (1991), 251, 1360-1363]. Binding to CG inversions was only slightly weaker than binding to AT base pairs. Binding of T to CG inversions was also evaluated in two other sequences, with qualitatively similar results. Six different analogs of thymine were also tested for binding to CG inversions and AT base pairs. Significant changes in affinity were observed. In particular, 5-fluoro-2'-deoxyuridine was found to increase affinity for CG inversions as well as for AT base pairs. Studies with oligonucleotides containing pyridin-2-one or pyridin-4-one suggest that thymine O4 plays a critical role in the T.CG interaction. Possible models to account for these observations are discussed.

Synthesis of 1-(3-Azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)-, 1-(2,3-Dideoxy-β-D-erythro-hexofuranosyl)- and 1-(2,3-Dideoxy-β-D-erythro-hex-2-enofuranosyl)pyrimidine Nucleosides

1-(3-Azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)uracil (XXII) and 1-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)uracil (VIII) were prepared starting from 1-(2-O-acetyl-3,5,6-tri-O-benzoyl-β-D-glucofuranosyl)uracil (I) by a procedure described previously for thymine analogs. 1-(2,3-Dideoxy-β-D-erythro-hexofuranosyl)uracil (XIII) was obtained by catalytic hydrogenation of 1-(5,6-di-O-benzoyl-2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)uracil (VII) and subsequent methanolysis. Reaction of dibenzoyl derivative VII, 1-(5,6-di-O-benzoyl-2,3-dideoxy-β-D-erythro-hexofuranosyl)uracil (XII) and the diacetate prepared by acetylation of azido derivative XXII with Lawesson's reagent, followed by methanolysis, afforded 1-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)-4-thiouracil (X), 1-(2,3-dideoxy-β-D-erythro-hexofuranosyl)-4-thiouracil (XV) and 1-(3-azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)-4-thiouracil (XXIII), respectively. Heating of thio derivatives X, XV and XXIII with methanolic ammonia at 100 °C gave 1-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)cytosine (XI), 1-(2,3-dideoxy-β-D-erythro-hexofuranosyl)cytosine (XVI) and 1-(3-azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)cytosine (XXIV).

Reactions of Some 2,3-Anhydro Pyrimidine Nucleosides with Dilithium Tetrahalocuprates

Abstract The reaction of 1-(2,3-anhydro-5-0-trityl-β-D-lyxofuranosyl)-2-0-methyluracil (2a) and its thymine analogue (2b) with dilithium tetrahalocuprates (Li2CuX4) revealed an excellent to perfect regioselectivity, yielding 2,2′-anhydro-3′-halonucleosides (3a-d), while the same reactions with 2,3-anhdro uracil and thymine nucleosides (5a,b) gave arabinosyl (6a-d) and xylosyl halohydrins (7a-d) with respective product ratios of 7:3 to 8:2 which were estimated after mesylation to 8a-d and 9a-d.

Structure-activity relationships of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine analogs: effect of substitutions at the C-6 phenyl ring and at the C-5 position on anti-HIV-1 activity

The effect of substitution on the pyrimidine moiety of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine (HEPT-S) on anti-HIV-1 activity was investigated by synthesizing a series of 5-methyl-6-(arylthio) and 5-substituted-6-(phenylthio) derivatives. Preparation of the 5-methyl-6-(arylthio) derivatives was carried out based on either LDA lithiation of 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]thymine (3) and 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2-thiothymine (4) followed by reaction with diaryl disulfides or an addition-elimination reaction of 1-[[2-(tert-butyldimethylsiloxy)ethoxy]-methyl]-6- (phenylsulfinyl)thymine (31) with aromatic thiols. Preparation of the 5-substituted-6-(phenylthio) derivatives was carried out based on either C-5 lithiation of the 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-6-(phenylthio)uraci l (41) with LTMP or the LDA lithiation of 5-alkyl-1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2-thiouraci l derivatives 45-47. Substitution at the meta position of the C-6-(phenylthio) ring by the methyl group improved the original anti-HIV-1 activity of HEPT, and introduction of two m-methyl groups to the phenylthio ring further potentiated the activity [EC50: 6-[(3,5-dimethylphenyl)thio]-1-[(2-hydroxyethoxy)methyl]thymine (28), 0.26 microM; 6-[(3,5-dimethylphenyl)thio]-1-[(2-hydroxyethoxy)methyl]-2-thiothymin e (30), 0.22 microM]. When the 5-methyl group was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity of HEPT was also improved remarkably [EC50: 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (48), 0.11 microM; 5-isopropyl-1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)-2-thiouracil (50), 0.059 microM; 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (54), 0.12 microM; 5-isopropyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (56), 0.063 microM]. 6-[(3,5-Dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]thymine derivatives 51 and 57 and 6-[(3,5-dimethylphenyl)thio]-5-isopropyl-1-[(2- hydroxyethoxy)methyl]thymine derivatives 52 and 58 inhibited the replication of HIV-1 in the nanomolar concentration range.

Interaction of TFIID in the minor groove of the TATA element

TFIID binding in the minor groove of DNA at the TATA element was demonstrated by methylation interference and hydroxyl radical footprinting assays, and by binding studies with thymine analog substituted oligonucleotides. These results provide an explanation for TFIID-dependent DNA bending at the TATA element. TFIID binding shows phosphate contacts with the same residues that were found to be essential for TFIID interactions by methylation and thymine-specific modification interference assays. Based on previous studies implicating residues conserved between the direct repeats in DNA binding, as well as models of prokaryotic DNA binding proteins, these results also suggest a model in which the direct repeats of TFIID form two basic antiparallel β ribbon arms that could contact DNA through the minor groove.

Microfluorimetric analysis of spatial and temporal patterns of immobilized cell growth

Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high-resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse-labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass-transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr-alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.

Phosphate Modified Analogues of 5'-O-Phosphorylated 2',3'-Dideoxynucleosides: Synthesis and Anti-HIV Activity

Abstract Phosphonate analogues of 5′-O-phosphoryl-2′,3′-dideoxyribofuranosyl adenine, cytosine, hypoxanthine and thymine were synthesized. The hypoxanthine and thymine analogues were inactive against HIV induced cyopathy in the CEM-4 T-cell lines. The 2′,3′-ddC and 2′,3′-ddA analogues were marginally active.

Research Note: 5-Azacytidine-Induced Increase in Plasma Prolactin Levels of Immature Turkeys

Immature female turkeys (7 wk old) were treated with 5-azacytidine, a cytidine analog that has been shown to inhibit cytosine methylation in eukaryotic DNA. Such methylation has been repeatedly implicated in the control of specific gene expression in higher vertebrates. Turkeys administered 5-azacytidine (5 mg/kg) by intravenous injection daily for 5 days exhibited elevated (P<.05) circulating prolactin (Prl) levels, whereas hens treated with saline, cytidine, or 6-azathymine did not show altered circulating Prl levels. Absence of response upon treatment with the normal nucleoside cytidine or the 6-aza analog of thymine reasonably eliminates nonspecific drug-induced stress as the cause of Prl level elevation. Based on these findings, DNA methylation is implicated in the control of circulating Prl levels in the young turkey.

Kinetic mechanisms and interaction of rat liver DNA methyltransferase with defined DNA substrates.

DNA substrate analogs were constructed from poly(dC-dG), M13, and XP12 DNA which do not contain a mixture of types of methylation sites. These were used to distinguish different kinetic mechanisms for maintenance and de novo methylation using a highly purified rat liver DNA (cytosine-5) -methyltransferase (DMase+) preparation. De novo methylation on single (ss) and double-stranded (ds) DNA was found to obey Michaelis-Menten kinetics while methylation of hemimethylated sites showed differences depending on size of the hemimethylated region. On long stretches analogous to maintenance methylation of newly replicated DNA, saturation could not be achieved and the kinetics showed non-ideal positive cooperative kinetics, while short stretches showed non-Michaelis-Menten kinetics and rapid saturation. Two types of DMase-DNA complexes could be distinguished by means of affinity chromatography on DNA -agarose matrices and in preincubation assays. The later complex, which is engaged in methyl group turnover, exhibited enhanced stability. The competitiveness of variously configured DNAs was found to parallel the stability of complex formation, e.g., ss, hemi- and ds DNA, respectively. In studies utilizing 5-bromodeoxyuridine, the thymine analog left the basic reaction mechanisms unchanged but increased the km and S0.5 while reducing the velocity of these reactions.