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Biotechnology Letters Jan 20123'-O-stearoylation of 6-azauridine was achieved enzymatically for the first time. Among eight commercially available lipases, that from Burkholderia cepacia displayed a...
3'-O-stearoylation of 6-azauridine was achieved enzymatically for the first time. Among eight commercially available lipases, that from Burkholderia cepacia displayed a 3'-regioselectivity of 80% towards the acylation of 3-hydroxyl of 6-azauridine. Using an immobilized lipase from Burkholderia cepacia, the 3'-regioselectivities of the acylations could be reversed by lengthening the aliphatic chain of the acyl donors (C2-C18). The possible reason might be the presence of the interaction between the base moiety and the acyl group.
Topics: Acylation; Azauridine; Burkholderia cepacia; Enzymes, Immobilized; Lipase; Substrate Specificity
PubMed: 21898129
DOI: 10.1007/s10529-011-0737-y -
Physical Chemistry Chemical Physics :... May 2010Excited state characteristics of 6-azauridine (6AUd), which is known as a medicine against psoriasis and neoplastic, were investigated with laser plash photolysis,...
Excited state characteristics of 6-azauridine (6AUd), which is known as a medicine against psoriasis and neoplastic, were investigated with laser plash photolysis, time-resolved thermal lensing, and near IR single photon counting method. The triplet-triplet absorption spectrum of 6AUd was observed for the first time. The formation quantum yield of excited triplet 6AUd (Phi(ISC)) was estimated by acetone triplet sensitization and actinometry with benzophenone to be 1.00 +/- 0.07 (248 nm excitation) and 0.78 +/- 0.05 (308 nm excitation). This excitation wavelength effect could be explained by intersystem crossing (ISC) to the excited triplet manifolds occurring during the relaxation on the potential energy surface (PES) of the S(1)(npi*) state and be in competition with internal conversion to the S(0) state after the relaxation to the minimum of the S(1)(npi*) state. 6AUd had a lower Phi(ISC) value than 6-azauracil (6AU) with the 308 nm excitation (Phi(ISC) = 0.93 +/- 0.04 for 6AU). The nucleoside has more vibrational modes than 6AU, and therefore the ribose would accelerate intramolecular vibrational energy redistribution and the relaxation to the minimum of the PES of the S(1)(npi*) state. Sensitized singlet oxygen formation of 6AUd was also detected in the O(2)-saturated condition with quantum yields of 0.49 +/- 0.01 with the 248 nm excitation, indicating the high phototoxicity of 6AUd.
Topics: Azauridine; Molecular Conformation; Quantum Theory; Singlet Oxygen; Thermodynamics; Ultraviolet Rays
PubMed: 20445916
DOI: 10.1039/b921568a -
Biotechnology Progress 2009CAL-B-catalyzed synthesis of different 5'-O-monoester derivatives of 6-azauridine via a one-step highly regioselective enzymatic acylation route was successfully...
CAL-B-catalyzed synthesis of different 5'-O-monoester derivatives of 6-azauridine via a one-step highly regioselective enzymatic acylation route was successfully performed for the first time. The effects of some crucial factors on the enzymatic undec-10-enoylation of 6-azauridine were examined. The optimal reaction medium, molar ratio of 6-azauridine to vinyl undec-10-enoate and reaction temperature were found to be anhydrous acetone, 1:3 and 50 degrees C, under which the reaction rate, the substrate conversion and the regioselectivity were 22.3 mM/h, 99.0% and 99.0%, respectively. In addition, the enzyme recognition of acyl donors was investigated. The results showed that the enzyme activity varied widely with different acyl donors owing to the specific structure of the lipase active site and the acyl donors. 5'-O-Monoesters of 6-azauridine were achieved exclusively with all the acyl donors tested.
Topics: Acylation; Azauridine; Biocatalysis; Fungal Proteins; Kinetics; Lipase; Stereoisomerism; Substrate Specificity
PubMed: 19452525
DOI: 10.1002/btpr.237 -
Journal of Molecular Biology Feb 2009Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells...
Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS(-/-) plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.
Topics: Amino Acid Sequence; Arabidopsis; Cell Nucleus; Cell Survival; DNA, Bacterial; Gene Deletion; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene Knockout Techniques; Genes, Reporter; Genetic Complementation Test; Green Fluorescent Proteins; Homozygote; Molecular Sequence Data; Mutagenesis, Insertional; Phylogeny; Protein Structure, Tertiary; Recombinant Fusion Proteins; Recombination, Genetic; Saccharomyces cerevisiae; Seeds; Sequence Alignment; Sequence Homology, Amino Acid; Transcriptional Elongation Factors
PubMed: 19150360
DOI: 10.1016/j.jmb.2008.12.066 -
Biochemistry Mar 2008Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical...
Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis-Menten kinetics with K m = 350 +/- 60 nM and V max = 2.70 +/- 0.10 micromol/min/mg protein. Inhibition patterns for nucleoside 5'-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5'-monophosphate ( K i = 3.6 +/- 0.7 nM) > xanthosine 5'-monophosphate (XMP, K i = 4.4 +/- 0.7 nM) > 6-azauridine 5'-monophosphate (AzaUMP, K i = 12 +/- 3 nM) > allopurinol-3-riboside 5'-monophosphate ( K i = 240 +/- 20 nM). XMP is an approximately 150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of the enzyme. Both the phosphate-binding loop and the betaalpha5-loop have conformational flexibility, which may be associated with substrate capture and product release along the reaction pathway.
Topics: Animals; Binding Sites; Crystallization; Crystallography, X-Ray; Dimerization; Escherichia coli; Humans; Kinetics; Models, Molecular; Molecular Weight; Orotidine-5'-Phosphate Decarboxylase; Plasmodium falciparum; Recombinant Proteins; Ribonucleotides; Species Specificity; Uridine Monophosphate
PubMed: 18303855
DOI: 10.1021/bi702390k -
Organic & Biomolecular Chemistry Feb 2008The stereoselective syntheses of 6-azauracil- and 8-aza-7-deazaadenine 2'-deoxy-2'-fluoro-beta-d-arabinofuranosides and employing nucleobase anion glycosylation with...
The stereoselective syntheses of 6-azauracil- and 8-aza-7-deazaadenine 2'-deoxy-2'-fluoro-beta-d-arabinofuranosides and employing nucleobase anion glycosylation with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-d-arabinofuranosyl bromide as the sugar component are described; the 6-azauracil 2'-deoxy-2'-fluoro-beta-d-ribofuranoside was prepared from 6-azauridine via the 2,2'-anhydro intermediate and transformation of the sugar with DAST. Compounds show a preferred N-conformer population (100% N for , and 78% N for ) being rather different from nucleosides not containing the combination of a fluorine atom at the 2'-position and a nitrogen next to the glycosylation site. Oligonucleotides incorporating and were synthesized using the phosphoramidites and . Although the N-conformation is favoured in the series of 6-azauracil- and 8-aza-7-deazaadenine 2'-deoxy-2'-fluoroarabinonucleosides only the pyrimidine compound shows an unfavourable effect on duplex stability, while oligonucleotide duplexes containing the 8-aza-7-deazaadenine-2'-deoxy-2'-fluoroarabinonucleoside were as stable as those incorporating dA or 8-aza-7-deaza-2'-deoxyadenosine .
Topics: Adenine; Base Pairing; Base Sequence; Crystallography, X-Ray; DNA; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Nitrogen; Nucleic Acid Denaturation; Nucleosides; Oligonucleotides; Organophosphorus Compounds; RNA; Stereoisomerism; Transition Temperature; Uracil
PubMed: 18219432
DOI: 10.1039/b715512c -
The Journal of Nutrition Jun 2007The urinary excretion of orotic acid, an intermediate in the pyrimidine biosynthetic pathway, is markedly increased in many inborn errors of the urea cycle and in a... (Review)
Review
The urinary excretion of orotic acid, an intermediate in the pyrimidine biosynthetic pathway, is markedly increased in many inborn errors of the urea cycle and in a number of other disorders involving arginine metabolism. Carbamoyl phosphate, which accumulates within hepatic mitochondria in patients with ornithine transcarbamoylase deficiency, can diffuse to the cytosol and enter the pyrimidine pathway, resulting in greatly increased orotic acid production and excretion. This orotic aciduria also occurs in inborn errors of the mitochondrial ornithine/citrulline transporter, arginase, argininosuccinate synthetase, and argininosuccinate lyase. Increased orotic acid excretion is also found in a number of hypoargininemic states, such as lysinuric protein intolerance. However, orotic aciduria should not be used uncritically as an index of arginine deficiency because it is found in patients with arginase deficiency who exhibit hyperargininemia. Increased orotic acid excretion can also arise as a result of impairments of pyrimidine synthesis, whether brought about by a genetic defect (e.g., in UMP synthase) or by drugs that inhibit the terminal part of the pathway (e.g., allopurinol or 6-azauridine). When used appropriately, measurement of urinary orotic acid is a valuable tool for the study of many derangements of arginine metabolism, including arginine depletion, and to assess the efficacy of therapies used to replete this amino acid.
Topics: Arginine; Humans; Hyperargininemia; Orotic Acid
PubMed: 17513443
DOI: 10.1093/jn/137.6.1656S -
Journal of Medicinal Chemistry Aug 2006Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the...
Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.
Topics: Calorimetry; Computer Simulation; Drug Design; Kinetics; Models, Molecular; Orotidine-5'-Phosphate Decarboxylase; Thermodynamics; Uridine Monophosphate
PubMed: 16884305
DOI: 10.1021/jm060202r -
Antimicrobial Agents and Chemotherapy Jun 2006Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in... (Comparative Study)
Comparative Study
Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, beta-D-N(4)-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 microg/ml, 2 microM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 microM, >200 nM, >200 nM, >100 microM, and 80 microM, respectively). These agents may be investigated further for the treatment of coronavirus infections.
Topics: Animals; Antiviral Agents; Azauridine; Base Sequence; Cell Line; Cell Survival; Coronavirus; Coronavirus Infections; Cytidine; Cytopathogenic Effect, Viral; Humans; Inhibitory Concentration 50; Macaca mulatta; Molecular Structure; Neutralization Tests; Nucleosides; RNA Interference; RNA, Small Interfering; RNA, Viral; Receptors, Virus; Time Factors; Virus Replication
PubMed: 16723558
DOI: 10.1128/AAC.01598-05 -
Chemical & Pharmaceutical Bulletin Mar 2005Seventy eight N(3)-substituted derivatives of uridine (1), thymidine (2), 2'-deoxyuridine (3), 6-azauridine (4), 2',3'-O-isopropylideneuridine (5), and...
Seventy eight N(3)-substituted derivatives of uridine (1), thymidine (2), 2'-deoxyuridine (3), 6-azauridine (4), 2',3'-O-isopropylideneuridine (5), and arabinofuranosyluracil (6) were synthesized and their antinociceptive effects were evaluated. N(3)-(2',4'-Dimethoxyphenacyl)uridine (1l), N(3)-(2',4'-dimethoxyphenacyl)2'-deoxyuridine (3l), and N(3)-(2',5'-dimethoxyphenacyl)arabinofuranosyluracil (6m) possessed 93, 86, and 82% of the antinociceptive effects tested by hot plate, respectively. The antinociceptive effects of three derivatives were 5.8, 5.4, and 5.1-folds of the effect of N(3)-phenacyluridine (1h) (16%), respectively. The structure-activity relationship of N(3)-substituted pyrimidine nucleosides was also discussed.
Topics: Analgesics; Animals; Male; Mice; Pyrimidine Nucleosides; Structure-Activity Relationship; Uridine
PubMed: 15744105
DOI: 10.1248/cpb.53.313