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To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.

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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.

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The purpose was to study in vitro metabolism of 2',3',5'-triacetyl-6-azauridine (1) by porcine liver esterase (PLE) and in human plasma using an automated analytical system developed previously. A gradient-LC method was developed to study the concentration-time course of 1 and its metabolites. A fast-LC assay was used to study the temperature effect on the metabolism of 1 by the PLE. 1 and all of its proposed possible metabolites were separated by the gradient-LC method in less than 10 min. Two simplified kinetic schemes were developed to describe the time course of 1, the intermediates and final metabolites with only five rate constants for the metabolisms of 1 by PLE and four rate constants in human plasma. Both enthalpy and entropy of activation in the in vitro metabolism of 1 by PLE were obtained.

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Two similar automated analytical systems using liquid chromatography (LC) and microdialysis as an on-line sampling technique were applied to studies of enzyme kinetics. 2',3',5'-Triacetyl-6-azauridine (azaribine) with porcine liver esterase (PLE) and N-acetylphenylalanyl-3,5-diiodotyrosine (AcFY') with pepsin were used as model compounds. The microdialysis sampling technique permitted the rapid separation of low molecular weight analytes from macromolecules, thus simultaneously achieving clean-up of the samples and quenching of the reaction. The combination of rapid LC analysis and microdialysis sampling provided selectivity and automation. The systems are rugged and give reproducible results in agreement with those from manual sampling methods.

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3-Phenylpyrimido-[3,4-a]-s-triazines exhibit antiparasitic, antibacterial and antifungal activity. In order to study the metabolism of these heterocycles, 9,9-diethyl-3-phenyl-6,8-dioxo-2,3,4,5,6,7,8,9-octahydropyrimido[3 ,4-a]-s- triazine (TZ) was administered to dogs. Three potential metabolites were synthesized, and these models were identified and quantified with gas chromatography-mass spectrometry. The heterobicyclic compounds, TZ and its hydroxy derivative, underwent thermal degradation under chromatographic conditions. Dog urine spiked with the model metabolites was extracted, and the substances were quantified. The urine of dogs treated with TZ was studied, and two of the potential metabolites were recovered, identified and quantified.

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Triazine nucleosides represent highly active compounds affecting different cellular processes. While 6-azauridine displays a rather selective inhibitory effect, biological action of 5-azacytidine reflects the polyvalent inhibitory mechanism of the drug (interaction with pyrimidine synthesis de novo, incorporation into RNA and DNA, depressed maturation of ribosomal RNA, inhibition of RNA and DNA methylation, etc.) and the analog displays pronounced cytostatic and immunosuppressive activity. 5-Aza-2'-deoxycytidine action is directed against DNA synthesis similar to that of 5-azacytosine arabinoside. N4-Substituted derivatives of 5-azacytidine affect gastric secretion and together with 5-azacytosine and 5-azacytidine represent a new type of drugs with antiulcer activity. 6-Amino-5-azacytosine nucleosides interfere with the metabolism of purines rather than pyrimidines as evidenced by the character of their inhibitory mechanism and measurement of conformation. 6-Azauridine (as 2',3',5'-triacetate) and 5-azacytidine were used with certain success in human chemotherapy, the first one as a drug affecting recalcitrant psoriasis, the second one for the treatment of different forms of leukemia. The inhibitory mechanisms of individual azapyrimidine nucleosides are discussed in relation to their known biological effects.

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The transport and metabolic conversion of 6-azauridine differed when compared in HeLa and chick embryo (CE) cells. The values of 9-(S)-(2, 3-dihydroxypropyl) adenine transported into the cells were found different for ZP cells (rabbit lung cell line), HeLa and CE cells. These differences were less expressed if relating the values of cellular uptake and metabolic conversion to the cell volume of the respective cell type. The differences seem to play a role in quantitation of the antiviral potency of the compounds in different host cells.

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A relatively rapid, high-resolution chromatographic procedure, using mini-columns of DEAE cellulose equilibrated with 10 mM sodium phosphate, pH 6.0, is described in sufficient detail to permit ready replication. This initial paper demonstrates the quantitative separation, using suction, of the acidic nucleosides, 3-deazauridine and 6-azauridine, from their phosphorylated derivatives. The chemically stable, tritium-labeled nucleosides are eluted from the mini-columns (capacity approximately equal to 1.8 ml) with 10 mM sodium phosphate, pH 6.0; subsequently, the nucleotides are eluted completely with 0.5 M HCl/0.5 M NaCl. Quantitation is based on liquid scintillation counting of aliquots of the eluates.

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A new uridine analog, 4-hydroxy-1-(beta-D-ribonfuranosyl)-pyridazin-6-one (3-deaza-6-azaUrd), inhibited the growth of L1210 cells in culture, with a concentration to reduce growth rate to 50% of control of 7 X 10(-5) M. After treatment for 24 or 48 h with 5 X 10(-4) M 3-deaza-6-azaUrd, 80% of the cells were unable to resume growth when the analog was removed from the cultures; also, 99% of the cells were killed, as determined by colony formation in soft agar. Studies on the prevention of the cytotoxic effects of 5 X 10(-4) M 3-deaza-6-azaUrd showed that uridine or cytidine gave complete protection. 2'-Deoxycytidine also gave partial protection, but orotic acid or thymidine had no effect on the growth inhibition by 3-deaza-6-azaUrd. These results suggested that growth inhibition by 3-deaza-6-azaUrd might be due to interference in pyrimidine biosynthesis. Activation of 3-deaza-6-azaUrd to its 5'-phosphate derivative appeared to be catalyzed by uridine kinase. 3-Deaza-6-azaUrd was shown to complete with uridine for phosphorylation (Ki = 4.7 mM) and, therefore, to be a possible alternative substrate for uridine kinase from mouse kidney (Km for uridine = 82 microM). The enzyme was partially purified by streptomycin sulfate precipitation, ammonium sulfate fractionation, and gel filtration. This preparation was found to be free of pyrimidine nucleoside phosphorylase and uridine monophosphate kinase.

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The cytostatic effect of 6-azauridine on cell growth is generally regarded to be a consequence of the inhibition of de novo pyrimidine biosynthesis by the metabolite, 6-azauridine 5'-monophosphate. We show here that wheat embryonic axes further metabolize 6-azauridine to the 5'-triphosphate and incorporate the analogue into RNA, thus offering an alternative mechanism for growth inhibition. At a level of 6-azauridine required to maximally inhibit UTP biosynthesis, the ratio of 6-azaUTP to UTP is about 2:1 and substitution of 6-azauridine for uridine in new RNA is on the order of 1 in 18. The new metabolites of 6-azauridine are identified by high pressure and thin layer chromatography coupled with enzyme treatments.

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Cytidine deaminase, an enzyme found in the supernatant fluid of hepatocytes, granulocytes and tumor cells, and in plasma, degrades the antitumor agents cytosine arabinoside and 5-azacytidine. Uridine and its analogs, 3-deazauridine, 5-bromodeoxyuridine, 5-fluorodeoxyuridine and 6-azauridine, were found to competitively inhibit cytidine deaminase; the most potent inhibitor was 3-deazauridine (K(i) = 1.9 x 10(-5) M). In addition, deoxycytidylate deaminase, which degrades cytosine arabinoside monophosphate to the inactive uracil arabinoside monophosphate (K(m) = 9 x 10(-4) M), was competitively inhibited by 3-deazauridine monophosphate, as well as by the nucleotides of other uridine analogs. These results suggest that uridine analogs such as 3-deazauridine may have value in protecting cytosine arabinoside, 5-azacytidine and their monophosphate nucleotides from degration by neucleoside and nucleotide deaminases.

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Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.

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