thiouridine and 4-thio-2--deoxyuridine

In the search for effective radiosensitizers for tumor cells, halogenated uracils have attracted more attention due to their large cross section for dissociation upon the attachment of low-energy electrons. In this study, we investigated dissociative electron attachment (DEA) to 5-iodo-4-thio-2'-deoxyuridine, a potential radiosensitizer using a crossed electron-molecule beam experiment coupled with quadrupole mass spectrometry. The experimental results were supported by calculations on the threshold energies of formed anions and transition state calculations. We show that low-energy electrons with kinetic energies near 0 eV may effectively decompose the molecule upon DEA. The by far most abundant anion observed corresponds to the iodine anion (I

Topics: Anions; Electrons; Radiation-Sensitizing Agents; Thiouridine

Functionalized oligonucleotides (ONs) are widely applied as target recognition molecules for biosensing and gene regulation. Herein, we describe a general method for post-synthetic modification of ONs based on the oxidative amination of 4-thio-2'-deoxyuridine (4SdU) with sodium periodate and several amines. Alkyne-/azide-, biotin-, and fluorophore-modified ONs were prepared by modifying 4SdU-containing ONs with the corresponding amines and characterized for their bioorthogonal reactivity, streptavidin-binding affinity, and fluorescence properties, respectively. We synthesized three fluorophore-modified ONs with and without the aromatic fluorophores conjugated to modified nucleobases and investigated their emission properties. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Post-synthetic modification of ONs Supporting Protocol: Preparation of dansyl ethylenediamine Basic Protocol 2: Quantification of ON reaction yields Basic Protocol 3: Characterization of modified ONs.

Topics: Amination; Oligonucleotides; Oxidative Stress; Thiouridine

Functionalized oligonucleotides (ONs) are widely applied as target binding molecules for biosensing and regulators for gene expression. Numerous efforts have been focused on developing facile methods for preparing these useful ONs carrying diverse modifications. Herein, we present a general method for postsynthetic modification of ONs via oxidative amination of 4-thio-2'-deoxyuridine (4SdU). 4SdU-containing ON can be derived by both alkyl and aromatic amines. Using this approach, ONs are successfully attached with alkyne/azide, biotin and dansylamide moieties, and these as-prepared ONs possess the expected biorthogonal reactivity, streptavidin affinity and fluorescent property, respectively. Furthermore, we also directly install fluorophores to the ON nucleobase based on oxidative amination of 4SdU, and these fluorophores exhibit distinct luminescence behaviors before and after conjugation. We believe our method will be a versatile strategy for constructing various functionalized ONs used in a wide range of nucleic acid applications.

Topics: Amination; Oligonucleotides; Oxidation-Reduction; Thiouridine

Nucleosides, especially pyrimidines modified in the C5-position, can act as radiosensitizers via a mechanism that involves their enzymatic triphosphorylation, incorporation into DNA, and a subsequent dissociative electron attachment (DEA) process. In this paper, we report 5-iodo-4-thio-2'-deoxyuridine (ISdU) as a compound that can effectively lead to ionizing radiation (IR)-induced cellular death, which is proven by a clonogenic assay. The test revealed that the survival of cells, pre-treated with 10 or 100 µM solution of ISdU and exposed to 0.5 Gy of IR, was reduced from 78.4% (for non-treated culture) to 67.7% and to 59.8%, respectively. For a somewhat higher dose of 1 Gy, the surviving fraction was reduced from 68.2% to 54.9% and to 40.8% for incubation with 10 or 100 µM ISdU, respectively. The cytometric analysis of histone H2A.X phosphorylation showed that the radiosensitizing effect of ISdU was associated, at least in part, with the formation of double-strand breaks. Moreover, the cytotoxic test against the MCF-7 breast cancer cell line and human dermal fibroblasts (HDFa line) confirmed low cytotoxic activity of ISdU. Based on the results of steady state radiolysis of ISdU with a dose of 140 Gy and quantum chemical calculations explaining the origin of the MS detected radioproducts, the molecular mechanism of sensitization by ISdU was proposed. In conclusion, we found ISdU to be a potential radiosensitizer that could improve anticancer radiotherapy.

Topics: Breast Neoplasms; Cell Survival; DNA Breaks, Double-Stranded; Drug Screening Assays, Antitumor; Female; Histones; Humans; MCF-7 Cells; Phosphorylation; Quantum Theory; Radiation-Sensitizing Agents; Thiouridine; X-Ray Therapy

Radiosensitizing properties of substituted uridines are of great importance for radiotherapy. Very recently, we confirmed 5-iodo-4-thio-2'-deoxyuridine (ISdU) as an efficient agent, increasing the extent of tumor cell killing with ionizing radiation. To our surprise, a similar derivative of 4-thio-2'-deoxyuridine, 5-bromo-4-thio-2'-deoxyuridine (BrSdU), does not show radiosensitizing properties at all. In order to explain this remarkable difference, we carried out a radiolytic (stationary and pulse) and quantum chemical studies, which allowed the pathways to all radioproducts to be rationalized. In contrast to ISdU solutions, where radiolysis leads to 4-thio-2'-deoxyuridine and its dimer, no dissociative electron attachment (DEA) products were observed for BrSdU. This observation seems to explain the lack of radiosensitizing properties of BrSdU since the efficient formation of the uridine-5-yl radical, induced by electron attachment to the modified nucleoside, is suggested to be an indispensable attribute of radiosensitizing uridines. A larger activation barrier for DEA in BrSdU, as compared to ISdU, is probably responsible for the closure of DEA channel in the former system. Indeed, besides DEA, the XSdU anions may undergo competitive protonation, which makes the release of X

Topics: Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Chromatography, Liquid; Halogens; Histones; Humans; Models, Molecular; Molecular Conformation; Molecular Structure; Radiation-Sensitizing Agents; Tandem Mass Spectrometry; Thiouridine

The interaction between 5-Bromo-4-thio-2'-deoxyuridine (4-SBrdU) and human serum albumin (HSA) was investigated by the methods of UV-vis absorbance, fluorescence and circular dichroism (CD) spectroscopy and molecular docking under simulative physiological conditions. The results showed that the quenching mechanism of HAS by 4-SBrdU was dynamic fluorescence quenching, hydrophobic interaction was the main intermolecular force based on thermodynamic data, the fluorescence experimental results were in agreement with results obtained by the molecular docking study.

Topics: Binding Sites; Bromodeoxyuridine; Circular Dichroism; Humans; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Protein Binding; Protein Conformation; Serum Albumin, Human; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thiouridine

5-Substituted-4-thio-2'-deoxyuridine nucleosides have been chemically synthesized and studied by NMR and UV spectroscopy. The results have been analyzed and discussed in connection with the previous data. The imino proton signal and the carbon signal of the thiocarbonyl group in the 5-substituted-4-thio-2'-deoxyuridines were found to be at much lower field, offering a potential for monitoring these modified bases at the DNA level. All 4-thionucleosides have strong absorptions at around 340 nm and consequently would be useful as potential UVA-induced anticancer agents.

Topics: Magnetic Resonance Spectroscopy; Molecular Structure; Thiouridine; X-Ray Diffraction

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.

Topics: Animals; Binding Sites; Catalysis; Cross-Linking Reagents; Deoxyguanosine; Guanine; Guanosine; Nucleic Acid Conformation; Organophosphorus Compounds; Photochemistry; Pyrimidines; RNA, Catalytic; Schistosoma mansoni; Sequence Analysis, RNA; Substrate Specificity; Thionucleosides; Thiouridine

The modified nucleosides 4-thio-2'-deoxyuridine (s4dU) and 4-thio-2'-deoxythymidine (s4dT) are incorporated into dinucleosides, and s4dT is incorporated into a DNA hairpin loop to provide divalent metal ion binding sites. Binding of two different metal ions to these sites is studied, including Cd(II) as an NMR spectroscopy probe and Cu(II) as a reactive metal ion for DNA cleavage. Binding of Cd(II) to 4-thiouridine (s4U) and s4dT nucleosides, s4dU- and s4dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s4dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 degrees C in solutions containing 20.0-40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl2. Cd(II) binds to the N3 deprotonated form of s4dT with a binding constant (K = 1.1 x 10(4) M(-1)) that is similar to that for Cd(II) binding to d(Tps4T) (K = 9.2 x 10(3) M(-1)). Apparent binding constants (Kapp) at pH 7.7 of Cd(II) to dinucleosides d(Gps4T), d(s4TpG), and d(Gps4U) are similar to those of their respective nucleosides s4U and s4dT, suggesting that neither the phosphate diester nor the second nucleoside has a major effect on Cd(II) binding. Binding of Cd(II) to s4U and d(Gps4U) is studied by use of 113Cd NMR and 1H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps4U) as studied by 1H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s4dT in the loop portion of a DNA hairpin loop (Kapp = 2.7 x 10(3) M(-1) at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in T(m) of 14 degrees C in the presence of 10.0 mM Cd(II) as determined by optical melting experiments. Cu(II) oxidizes s4dT to form the disulfide of s4dT, limiting the usefulness of further studies with Cu(II).

Topics: Cadmium; Cations, Divalent; DNA; Metals, Heavy; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligonucleotides; Temperature; Thionucleosides; Thiouridine; Thymidine

The thiopurines, 6-thioguanine and 6-mercaptopurine, are antileukemic agents that are incorporated into DNA following retrieval by the purine salvage pathway (see [1] for a review). Their toxicity requires active DNA mismatch repair (MMR), and thiopurine resistance is an acknowledged phenotype of MMR-defective cells [2, 3]. In addition to these direct cytotoxic effects, DNA thiobases have distinctive photochemical properties [4], the therapeutic potential of which has not been extensively evaluated. We report here that the thiopyrimidine nucleoside 4-thiothymidine is incorporated into DNA. It does not induce MMR-related toxicity, but it interacts synergistically with UVA light and dramatically sensitizes cultured human cells to very low, nonlethal UVA doses. 4-thiothymidine induced UVA dose enhancements of around 100-fold in DNA repair-proficient cells. Nucleotide excision repair-defective xeroderma pigmentosum cells were sensitized up to 1000-fold, implicating bulky DNA photoproducts in the lethal effect. The synergistic action of thiothymidine plus UVA required thymidine kinase, indicating a selective toxicity toward rapidly proliferating cells. Cooperative UVA cytotoxicity is a general property of DNA thiobases, and 6-thioguanine and 4-thiodeoxyuridine were also UVA sensitizers. Thiobase/UVA treatment may offer a novel therapeutic approach for the clinical management of nonmalignant conditions like psoriasis or for superficial tumors that are accessible to phototherapy.

Topics: Cell Line; DNA; DNA Repair; Humans; Mercaptopurine; Phototherapy; Thioguanine; Thiouridine; Ultraviolet Therapy; Xeroderma Pigmentosum

The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4'-thio-2'-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20-600 times) than that shown by the corresponding 4'-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4'-thio-2'-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4'-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4'-thio-2'-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4'-oxy- and 4'-thio-2'-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4'-thio-2'-deoxyuridines are better inhibitors than their 4'-oxy counterparts of [(3)H]thymidine incorporation in HSV-1-infected cells; (vi) 4'-thio-2'-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4'-thio-2'-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4'-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.

Topics: Antiviral Agents; Blood Platelets; Cell Line; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA; DNA-Directed DNA Polymerase; DNA, Complementary; HeLa Cells; Humans; Kinetics; Nucleosides; Phosphorylation; Simplexvirus; Thiouridine; Thymidine Kinase; Thymidine Phosphorylase

We report a facile, quantitative regeneration of the thiocarbonyl group from S-alkylated 4-thio-2'-deoxyuridine residues by treatment with NaSH within oligodeoxynucleotides.

Topics: Base Sequence; Chromatography, High Pressure Liquid; DNA; Spectrophotometry, Ultraviolet; Thiouridine

A method for the introduction of depurinated lesions in DNA is described, and is based on the formation of a covalent cross-link between an antisense oligonucleotide probe and the target DNA sequence followed by an unexpectedly mild thermal depurination.

Topics: Acetamides; Antisense Elements (Genetics); Cross-Linking Reagents; Deoxyguanosine; DNA; DNA Damage; Heating; Oligonucleotides, Antisense; Thiouridine

Full details of the template-directed covalent cross-linking of duplex oligodeoxynucleotides are presented. 4-Thio-2'-deoxyuridine was incorporated synthetically into a 17mer oligodeoxynucleotide, and the thiocarbonyl group of the modified base was alkylated with a variety of alpha-bromoacetyl-derivatized diamines. Covalent cross-linking was initiated by annealing the electrophilic probe oligomers with their complementary sequences, where a dG base was targeted at the position complementary to the modified 4-thio-2'-deoxyuridine. The sequence selectivity of cross-link formation as a function of tether topology and rigidity was examined, and the thermal stability of the modified duplexes was measured by UV melting experiments.

Topics: Base Sequence; Chromatography, High Pressure Liquid; Cross-Linking Reagents; DNA; DNA Replication; Kinetics; Models, Chemical; Nucleic Acid Conformation; Oligonucleotides; Thiouridine

Topics: Animals; Bacillus subtilis; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry; Circular Dichroism; Deoxyribonucleases; Deoxyribonucleotides; Deoxyuridine; DNA Nucleotidyltransferases; Drug Stability; Methylation; Micrococcal Nuclease; Phosphoric Diester Hydrolases; Phosphorus Isotopes; Poly A-U; Polynucleotides; Snakes; Spectrophotometry, Ultraviolet; Spleen; Sulfonic Acids; Temperature; Thiouridine; Ultracentrifugation; Venoms