- Synthesis and stability of nucleoside 3a?2,5a?2-cyclic phosphate triesters masked with enzymatically and thermally labile phosphate protecting groups
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Appropriately protected structurally modified nucleoside 3a?2,5a?2-cyclic monophosphates are known to show antiviral activity. For this reason, a straightforward synthesis of nucleoside 3a?2,5a?2-cyclic phosphates protected with three different enzymatically removable groups, viz. 3-acetyloxy-2,2-bis-(ethoxycarbonyl)propyl (in 1 and 4), 4-acetylthio-2,2-di-methyl-3-oxobutyl (in 2), and 4-(tert-butyldisulfanyl)-2,2-di-methyl-3-oxobutyl (in 3) groups, is described. Removal of these protecting groups at pH 7.5 and 37?°C was monitored by reverse-phase HPLC.
- Sontakke, Vyankat A.,Shinde, Vaishali S.,L??nnberg, Harri,Ora, Mikko
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- A solid-state 17O nuclear magnetic resonance study of nucleic acid bases
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We report a systematic solid-state 17O NMR study of free nucleic acid bases: thymine (T), uracil (U), cytosine (C), and guanine (G). Site-specifically 17O-enriched samples were synthesized: [2-17O]thymine (1), [4-17O]thymine (2), [2-17O]uracil (3), [4-17O]uracil (4), [2-17O]cytosine (5), and [6-17O]guanine monohydrate (6). Magic-angle-spinning (MAS) and static 17O NMR spectra were acquired at 11.75 T for compounds 1-6, from which information about the 170 chemical shift and electric field gradient tensors was obtained. Extensive quantum chemical calculations were performed at the B3LYP/6-311++G(d,p) level of theory for 17O NMR properties in various molecular models. The calculated 17O NMR tensors are highly sensitive to the description of intermolecular hydrogen-bonding interactions at the target oxygen atom. A reasonably good agreement between experimental solid-state 17O NMR data and B3LYP/6-311++G(d,p) calculations is achievable only in molecular cluster models where a complete hydrogen-bond network is considered. Using this theoretical approach, we also investigated the 17O NMR tensors in two unusual structures: guanine- and uracil-quartets.
- Wu, Gang,Dong, Shuan,Ida, Ramsey,Reen, Nitin
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- Thermal decomposition of the tert-butyl perester of thymidine-5′- carboxylic acid. Formation and fate of the pseudo-C4′ radical
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Thermal decomposition of the tert-butyl perester of thymidine-5′- carboxylic acid 1 carried out at 85°C in different solvents affords the tert-butylacetal 4a, deriving from in cage decomposition, and pseudo C4′ radicals 2. Radicals 2 can be reduced to 5 by hydrogen atom abstraction from thiol (thiophenol or glutathione) or THF, or can be oxidized to cations 8 by dioxygen or perester 1 itself. Cations 8 are stereoselectively trapped by the nucleophilic solvent (tert-butanol, methanol, water) to give acetals 4a-c.
- Montevecchi, Pier Carlo,Manetto, Antonio,Navacchia, Maria Luisa,Chatgilialoglu, Chryssostomos
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- Sources of 2,5-diaminoimidazolone lesions in DNA damage initiated by hydroxyl radical attack
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The present study reports radiation-chemical yields of 2.5-diaminoimidazolone (Iz) derivatives in X-irradiated phosphate-buffered solutions of guanosine and double-stranded DNA. Various gassing conditions (air, N20/O2 (4:1), N2O, vacuum) were employed to elucidate the contribution of several alternative pathways leading to Iz in reactions initiated by hydroxyl radical attack on guanine. In all systems, Iz was identified as the second by abundance guanine degradation product after 8-oxoguanine, formed in 1:5 (guanosine) and 1:3.3 (DNA) ratio to the latter in air-saturated solutions. Experimental data strongly suggest that the addition of molecular oxygen to the neutral guanine radical G(-H)? plays a major in Iz production in oxygenated solutions of double-stranded DNA while in other systems it may compete with recombination of G(-H)? with superoxide and/or alkyl peroxyl radicals. The production of Iz through hydroxyl radical attack on 8-oxoguanine was also shown to take place although the chemical yield of Iz (ca 6%) in this process is too low to compete with the other pathways. The linearity of Iz accumulation with dose also indicates a negligible contribution of this channel to its yield in all systems.
- Thomas, Caroline Suzanne,Pollard, Hannah Catherine,Razskazovskiy, Yuriy,Roginskaya, Marina
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- The kinetic mechanism of Human Thymidine Phosphorylase-a molecular target for cancer drug development
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Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1- phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding.
- Deves, Candida,Rostirolla, Diana Carolina,Martinelli, Leonardo Kras Borges,Bizarro, Cristiano Valim,Santos, Diogenes Santiago,Basso, Luiz Augusto
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- Synthesis, stability, and biological evaluation of 1,3-dihydrobenzo[c] furan analogue of d4T and its SATE pronucleotide
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The anti-HIV activity and stability studies of 1,3-dihydrobenzo[c]furan analogue of d4T are reported. The corresponding mononucleoside phosphotriester derivative bearing a S-pivaloyl-2-thioethyl (tBuSATE) group, as biolabile phosphate protection, is also studied.
- Len, Christophe,Selouane, Abdelmajid,Postel, Denis,Villa, Pierre,Aubertin, Anne-Marie,Egron, David,Gosselin, Gilles,Perigaud, Christian
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- Mass spectrometry and theoretical studies on N-C bond cleavages in the N-sulfonylamidino thymine derivatives
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Abstract The reactivity of new biologically active thymine derivatives substituted with 2-(arylsulfonamidino)ethyl group at N1 and N3 position was investigated in the gas phase using CID experiments (ESI-MS/MS) and by density functional theory (DFT) calculations. Both derivatives show similar chemistry in the negative mode with a retro-Michael addition (Path A-) being the most abundant reaction channel, which correlate well with the fluoride induced retro-Michael addition observed in solution. The difference in the fragmentation of N-3 substituted thymine 5 and N-1 substituted thymine 1 in the positive mode relates to the preferred cleavage of the sulfonyl group (m/z 155, Path B) in N-3 isomer and the formation of the acryl sulfonamidine 3 (m/z 309) via Path A in N-1 isomer. Mechanistic studies of the cleavage reaction conducted by DFT calculations give the trend of the calculated activation energies that agree well with the experimental observations. A mechanism of the retro-Michael reaction was interpreted as a McLafferty type of fragmentation, which includes Hβ proton shift to one of the neighboring oxygen atoms in a 1,5-fashion inducing N1(N3)-Cα bond scission. This mechanism was found to be kinetically favorable over other tested mechanisms. Significant difference in the observed fragmentation pattern of N-1 and N-3 isomers proves the ESI-MS/MS technique as an excellent method for tracking the fate of similar sulfonamidine drugs. Also, the observed N-1 and/or N-3 thymine alkylation with in situ formed reactive acryl sulfonamidine 3 as a Michael acceptor may open interesting possibilities for the preparation of other N-3 substituted pyrimidines.
- Kobeti?, Renata,Kazazi?, Snje?ana,Kova?evi?, Borislav,Glasovac, Zoran,Krstulovi?, Luka,Baji?, Miroslav,?ini?, Biserka
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- A METHOD FOR THE FLASH VACUUM THERMOLYSIS OF NON-VOLATILE COMPOUNDS
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A method for evaporation of non-volatile compounds is described.Examples are given of the application of this method to flash vacuum thermolysis of organic compounds.
- Magrath, Joe,Fowler, Frank W.
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- Apoprotein-Assisted Unusual cyclization of Neocarzinostatin Chromophore
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The reaction of neocarzinostatin chromophore (1) with 2-mercaptoethanol in the presence and absence of apoprotein was investigated.When complexed with apoprotein, 1 was found to react with 2-mercaptoethanol to provide unprecedented cyclization product 5 as a major product together with a minor amount of known cyclization product 4c.The structure of 5 was elucidated by spectroscopic data including 1H- and 13C-NMR, FABMS, and FTIR.Addition of isopropanol to the reaction system dramatically changed the ratio of 5 to 4c.In a Tris-HCl buffer containing 80percent isopropanol, 4c was formed as a major product together with a minor amount of 5.The reaction of isolated pure 1 with 2-mercaptoethanol in Tris-HCl buffer containing isopropanol gave 4c as a major product together with trace of 5.These results indicate that 1 shows a quite different reactivity toward thiols when complexed with apoprotein: nucleophilic attack of the thiol at C12 of 1 complexed with apoprotein gives a different type of enynecumulene 7 which undergoes cycloaromatization followed by hydration to provide 5.The fact that the binding constant of 5 toward apoprotein is one order of magnitude larger than that of 4c suggests that the stability of the apoprotein-5 complex may be closely related to the preferential formation of 5 in an apoprotein complex.
- Sugiyama, Hiroshi,Yamashita, Katsuhiro,Fujiwara, Tsuyoshi,Saito, Isao
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- Disposition of [1′-14C]stavudine after oral administration to humans
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The disposition of stavudine, a potent and orally active nucleoside reverse transcriptase inhibitor, was investigated in six healthy human subjects. Before dosing humans with [1′-14C]stavudine, a tissue distribution study was performed in Long-Evans rats. Results from this study showed no accumulation of radioactivity in any of the tissues studied, indicating that the position of the 14Clabel on the molecule was appropriate for the human study. After a single 80-mg (100 μCi) oral dose of [1′- 14C]stavudine, approximately 95% of the radioactive dose was excreted in urine with an elimination half-life of 2.35 h. Fecal excretion was limited, accounting for only 3% of the dose. Unchanged stavudine was the major drug-related component in plasma (61% of area under the plasma concentration-time curve from time zero extrapolated to infinite time of the total plasma radioactivity) and urine (67% of dose). The remaining radioactivity was associated with minor metabolites, including mono- and bis-oxidized stavudine, glucuronide conjugates of stavudine and its oxidized metabolite, and an N-acetylcysteine (NAC) conjugate of the ribose (M4) after glycosidic cleavage. Formation of metabolite M4 was shown in human liver microsomes incubated with 2′,3′-didehydrodideoxyribose, the sugar base of stavudine, in the presence of NAC. In addition, after similar microsomal incubations fortified with GSH, two GSH conjugates, 3′-GS-deoxyribose and 1′-keto-2′,3′-dideoxy-3′-GS-ribose, were observed. This suggests that 2′,3′-didehydrodideoxyribose underwent cytochrome P450-mediated oxidation leading to an epoxide intermediate, 2′,3′- ribose epoxide, followed by GSH addition. In conclusion, absorption and elimination of stavudine were rapid and complete after oral dosing, with urinary excretion of unchanged drug as the predominant route of elimination in humans. Copyright
- Zhou, Lian,Kaul, Sanjeev,Liu-Kreyche, Peggy,Tran, Scott B.,Espina, Robert R.,Warrack, Bethanne M.,Roongta, Vikram A.,Iyer, Ramaswamy A.
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- Hydrolysis of the cis-Phenyl Ester of Thymidine 3′,5′-Cyclic Monophosphate: PH-Dependent Competition between Depyrimidination and Phosphotriester Hydrolysis via CO and PO Bond Ruptures
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Hydrolytic reactions of the cis-phenyl ester of thymidine 3′,5′-cyclic monophosphate (1a) have been followed by HPLC over a wide pH range. Under acidic conditions (pH 80%) at pH 10, the hydrolysis is hydroxide-ion-catalyzed, yielding the three phosphodiester products in a 42:42:16 ratio ([2]:[3]:[4]). From pH 7 to 10, the pH-rate profile is nonlinear, possibly due to N3H deprotonation of the thymine moiety. In the same pH range, the site of bond cleavage appears to be changed. The product analyses of the corresponding methanolysis reactions suggest that the pH-independent reaction predominantly takes place via cleavage of the C5′O bond, while the alkaline reaction proceeds by rupture of one of the PO bonds. Consistent with this proposal, the pH-independent hydrolysis yields at high concentrations of sodium chloride 5′-chloro-5′-deoxythymidine 3′-(phenyl phosphate) and in concentrated acetate buffers 5′-O-acetylthymidine 3′-(phenyl phosphate). Accordingly, the hydrolytic reactions of 1a markedly differ from those of more simple 2-aryloxy-2-oxo-1,3,2-dioxaphosphorinanes.
- Varila, Jaana,Hankam?ki, Teemu,Oivanen, Mikko,Koole, Leo H.,L?nnberg, Harri
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- Selective hydrolysis of nucleotides to nucleosides and free bases
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The kinetics of the hydrolysis of 2'-deoxyadenosine-5'-monophosphoric acid (dAMP), 2'-deoxycytidine-5'-monophosphoric acid (dCMP), 2'-deoxyguanosine-5'-monophosphoric acid (dGMP) and tymidine-5'-monophosphoric acid (dTMP) was studied in the presence of Xanthomonas maltophilia [1]. The reaction products are nucleosides: 2'-deoxyadenosine (dA), 2'-deoxycytidine (dC), 2'-deoxyguanosine (dG) and tymidine (dT), respectively, or the respective free bases. Hydrolysis of dTMP and dGMP proceeded stepwise according to the sequence: nucleotide→nucleoside→free base, whereas no accumulation of the free base was observed during the hydrolysis of dAMP and dCMP. Copyright (C) 1999 Elsevier Science S.A.
- Chmielowiec, Urszula,Kruszewska, Hanna,Cybulski, Jacek
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- Nucleophilic Participation in the Transition State for Human Thymidine Phosphorylase
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Recombinant human thymidine phosphorylase catalyzes the reaction of arsenate with thymidine to form thymine and 2-deoxyribose 1-arsenate, which rapidly decomposes to 2-deoxyribose and inorganic arsenate. The transition-state structure of this reaction was determined using kinetic isotope effect analysis followed by computer modeling. Experimental kinetic isotope effects were determined at physiological pH and 37 °C. The extent of forward commitment to catalysis was determined by pulse-chase experiments to be 0.70%. The intrinsic kinetic isotope effects for [1′-3H]-, [2′R-3H]-, [2′S-3H]-, [4′- 3H]-, [5′-3H]-, [1′-14C]-, and [1-15N]-thymidines were determined to be 0.989 ± 0.002, 0.974 ± 0.002, 1.036 ± 0.002, 1.020 ± 0.003, 1.061 ± 0.003, 1.139 ± 0.005, and 1.022 ± 0.005, respectively. A computer-generated model, based on density functional electronic structure calculations, was fit to the experimental isotope effect. The structure of the transition state confirms that human thymidine phosphorylase proceeds through an SN2-like transition state with bond orders of 0.50 to the thymine leaving group and 0.33 to the attacking oxygen nucleophile. The reaction differs from the dissociative transition states previously reported for N-ribosyl transferases and is the first demonstration of a nucleophilic transition state for an N-ribosyl transferase. The large primary 14C isotope effect of 1.139 can occur only in nucleophilic displacements and is the largest 14C primary isotope effect reported for an enzymatic reaction. A transition state structure with substantial bond order to the attacking nucleophile and leaving group is confirmed by the slightly inverse 1′-3H isotope effect, demonstrating that the transition state is compressed by the impinging steric bulk of the nucleophile and leaving group.
- Birck, Matthew R.,Schramm, Vern L.
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- N2-amination of guanine to 2-hydrazinohypoxanthine, a novel in vivo nucleic acid modification produced by the hepatocarcinogen 2-nitropropane
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2-Nitropropane, an industrial chemical and a hepatocarcinogen in rats, induces aryl sulfotransferase-mediated liver DNA and RNA base modifications [Sodum, R. S., Sohn, O. S., Nie, G., and Fiala, E. S. (1994) Chem. Res. Toxicol. 7, 344-351]. Two of these modifications were previously identified as 8-aminoguanine and 8-oxoguanine. We now report that the base moiety of the so far unidentified third nucleic acid modification, namely RX1 in RNA and DX1 in DNA, is 2-hydrazinohypoxanthine (N2-aminoguanine). 2-Hydrazinoinosine and 2-hydrazinodeoxyinosine, synthesized by adapting published procedures, cochromatographed with RX1 and DX1 of liver RNA and DNA, respectively, from 2-nitropropane-treated rats. 2-Hydrazinoinosine and 2-hydrazinodeoxyinosine are unstable in solution like the in vivo products RX1 and DX1. At neutral pH, hypoxanthine nucleoside is the major product of decomposition, while at pH 10 or above, xanthine nucleoside is also formed. RX1 and DX1 could be generated in the anaerobic reactions of hydroxylamine-O-sulfonic acid, an intermediate in the proposed activation pathway of 2-nitropropane, with guanine nucleosides. These results provide further evidence for the activation of 2-nitropropane and other carcinogenic secondary nitroalkanes to a reactive species capable of aminating nucleic acids and proteins.
- Sodum, Rama S.,Fiala, Emerich S.
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- Michael versus retro-Michael reaction in the regioselective synthesis of N-1 and N-3 uracil adducts
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By controlling the temperature or reaction time in the base-catalysed Michael-type addition of 5-substituted uracil derivatives we were able to synthesise N-1 or N-3 uracil adducts using methyl acrylate and acrylonitrile as acceptors. The mechanism of this chemical inequivalence was established using 1H NMR spectroscopic studies. The investigations revealed that formation of the N-1 adduct was achievable under kinetically controlled conditions irrespective to the type of the base used (TEA, DBU). In turn, synthesis of the N-3 adducts proceeded from the initially formed N-1,N-3 diadduct via a retro-Michael reaction which dominates at elevated temperature or prolonged reaction time.
- Boncel, S?awomir,McZka, MacIej,Walczak, Krzysztof Z.
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- Lipase catalyzed diastereoselective deacetylations of anomeric mixtures of peracetylated 2'-deoxynucleosides
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Lipase catalyzed deacetylations of anomeric mixtures of peracetylated 2'- deoxyribofuranosyl- and 2'-deoxyribopyranosyl thymine nucleosides 1 and 5 have been investigated. Generally, the diastereoselectivity was more pronounced in pure phosphate buffer than in phosphate buffer containing 10% DMF. Wheat Germ Lipase and Porcine Liver Esterase catalyzed diastereoselective deacetylation of 1 affording the pure β-anomer thymidine (4β) as the only completely deprotected nucleoside product.
- Damkjaer,Petersen,Wengel
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- Kinetics and mechanism of the oxidation of sugars and nucleotides by oxoruthenium(IV): Model studies for predicting cleavage patterns in polymeric DNA and RNA
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Kinetic parameters for the oxidation of D-ribose, 2-deoxy-D-ribose, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), 2'-deoxyadenosine 5'-monophosphate (dAMP), cytidine 5'-monophosphate (CMP), 2'-deoxycytidine 5'-monophosphate (dCMP), and thymidine 5'-monophosphate (TMP) by Ru(tpy)(bpy)O2+ were determined in pH 7 phosphate buffer (tpy = 2,2',2''-terpyridine, bpy = 2,2'-bipyridine). Plots of k(obs) vs [substrate] were linear for the oxidation of ribose, 2-deoxyribose, TMP, and dCMP, yielding second-order rate constants of 0.029, 0.082, 0.38, and 0.46 M-1 s-1, respectively. From the temperature dependence of the rate constant, activation parameters consistent with the oxidation of other organic molecules by hydride transfer were found. For GMP, AMP, and dAMP, k(obs) vs [substrate] plots were curved due to electrostatic binding of Ru(tpy)(bpy)O2+ to the dianionic nucleotides, and the data were treated using double-reciprocal plots, yielding effective second-order rate constants of 0.10, 0.39, and 2.5 M-1 s-1, respectively. Product analysis by HPLC shows that a quantitative yield of free cytosine is obtained upon oxidation of dCMP based on nucleotide consumed. In TMP oxidations, an 80% yield of free thymine is observed based on Ru(tpy)(bpy)O2+ consumed. The kinetics and product analyses are consistent with sugar oxidation at the 1' position, and the increased reactivity of DNA compared to RNA can be understood on the basis of deactivation of the sugar oxidation product by the polar effect of the 2'-hydroxyl. The oxidation of the guanine base in GMP by Ru(tpy)(bpy)O2+ proceeds via an oxo transfer mechanism where the initial step is formation of a bound Ru(III)OR2+ intermediate. The ratio of the rate-determining rate constant for oxidation of guanine nucleotides to the average rate constant of sugar oxidation predicts the relative yields of base and sugar oxidation on sequencing gels.
- Neyhart,Cheng,Thorp
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- Phosphodiester and N-glycosidic bond cleavage in DNA induced by 4-15 eV electrons
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Thin molecular films of the short single strand of DNA, GCAT, were bombarded under vacuum by electrons with energies between 4 and 15 eV. Ex vacuo analysis by high-pressure liquid chromatography of the samples exposed to the electron beam revealed the formation of a multitude of products. Among these, 12 fragments of GCAT were identified by comparison with reference compounds and their yields were measured as a function of electron energy. For all energies, scission of the backbone gave nonmodified fragments containing a terminal phosphate, with negligible amounts of fragments without the phosphate group. This indicates that phosphodiester bond cleavage by 4-15 eV electrons involves cleavage of the C-O bond rather than the P-O bond. The yield functions exhibit maxima at 6 and 10-12 eV, which are interpreted as due to the formation of transient anions leading to fragmentation. Below 15 eV, these resonances dominate bond dissociation processes. All four nonmodified bases are released from the tetramer, by cleavage of the N -glycosidic bond, which occurs principally via the formation of core-excited resonances located around 6 and 10 eV. The formation of the other nonmodified products leading to cleavage of the phosphodiester bond is suggested to occur principally via two different mechanisms: (1) the formation of a core-excited resonance on the phosphate unit followed by dissociation of the transient anion and (2) dissociation of the CO bond of the phosphate group formed by resonance electron transfer from the bases. In each case, phosphodiester bond cleavage leads chiefly to the formation of stable phosphate anions and sugar radicals with minimal amounts of alkoxyl anions and phosphoryl radicals.
- Zheng, Yi,Cloutier, Pierre,Hunting, Darel J.,Wagner, J. Richard,Sanche, Leon
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- Acyl Migration in the Production of Thymine Propenal from 3'-O-Benzoyl-5'-deoxy-4'-hydroperoxythymidine: A Reinterpretation of a Putative Model for Bleomycin-Mediated DNA Degradation
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Studies of Saito et al. (Saito, I.; Morii, T.; Matsuura, T.J.Org.Chem. 1987, 52, 1008) analyzing the decomposition of 3'-O-benzoyl-5'-deoxy-4'-hydroperoxythymidine (7) claimed to model the decomposition of the putative 4'-hydroxyperoxynucleotide intermediate in the bleomycin (BLM) mediated production of base propenal, 3'-phosphoglycolate, and 5'-phosphate termini from double-stranded DNA.A number of puzzling observations reported in this paper prompted a reinvestigation of this model system in detail. 18O2>-7 and its 4'-epimer 8 were prepared and their fate in aqueous solution as a function of pH was examined.Compound 7 decompos ed in aqueous solution to produce thymine propenal accompanied by stoichiometric formation of benzoate containing 1 atom of 18O.In addition, thymine accompanied by stoichiometric amounts of malondialdehyde and 18O>benzoate was also observed.Acetate containing 1 atom of 18O accompanied production of both thymine and thymine propenal.The ratio of thymine propenal to thymine varied as a function of pH and temperature.Production of 18O>benzoate and a detailed kinetic analysis of the decomposition of 7 unequivocally demonstrated that conversion of 7 to thymine propenal required the intermediacy of a 4'-perbenzoate ester.This perester produced by migration of the 3'-benzoyl blocking group of 7 to the terminal oxygen of its 4'-hydroperoxy moiety would then greatly facilitate heterolytic cleavage of the oxygen-oxygen bond.For stereochemical reasons a similar intramolecular benzoyl migration cannot occur with 8, explaining its lack of reactivity.These results call into question the relevance of the Model proposed by Saito et al. to understanding the base propenal pathway in the BLM-catalyzed degradation of DNA.In addition, preparation of a second model of a putative intermediate in the base propenal pathway, oxy>-3-oxopropyl>thymine (12) is reported.The detailed kinetics of its decomposition as well as identification of the products accompanying ist decomposition are reported.The relevance of these two model systems to the mechanism of degradation of DNA by BLM is discussed.
- McGall, Glenn H.,Stubbe, JoAnne,Kozarich, John W.
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- The photochemistry of thymine in frozen aqueous solution: Trimeric and minor dimeric products
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Early work identified three compounds, namely the c,s cyclobutane dimer, the so-called (6-4) photoproduct (5-hydroxy-6-4′-(5-methylpyrimidin- 2′-one)-5,6-dihydrothymine) and a trimer hydrate, as products formed upon UV irradiation of thymine in frozen aqueous solution. More recent work has shown that an (α-4) product, namely α-4′-(5′- methylpyrimidine-2′-one)-thymine, is a likely product formed under these reaction conditions. During a thorough reinvestigation of the photochemistry of Thy in ice at -78.5°C, we found that a variety of other products could be detected. In addition to the c,s dimer, the other three known cyclobutane dimers, namely the c,a, t,s and t,a forms, are produced, although in considerably smaller amounts. The so-called spore product of thymine (5,6-dihydro-5-(α-thyminyl)thymine) is likewise formed. Two other dimers have been identified as minor products; one of these has been determined to be 5-(thymin-3-yl)-5,6-dihydrothymine and the other has been tentatively assigned to be a (5-4) adduct (6-hydroxy-5-4′-(5-methylpyrimidin-2′-one)-5,6- dihydrothymine). Compounds with the behavior expected of true trimeric compounds have been isolated via HPLC and characterized by mass spectrometry and photochemical behavior. One of these materials, putatively containing an oxetane ring, decomposes thermally to a secondary trimeric product that is then converted into the known trimer hydrate.
- Shetlar, Martin D.,Basus, Vladimir J.
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- Identification of the oxidized products formed upon reaction of Chromium(V) with thymidine nucleotides
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Two pathways have been observed for Cr(V)-mediated nucleotide oxidation in reactions of bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) [CrO(ehba)2]- with thymidine nucleotides dTMP, dTDP, and dTTP. The extent of Cr(V)-induced nucleotide oxidation was greatest for thymidine diphosphate (dTDP), as measured by the production of thiobarbituric acid reactive species (TBARS; indicative of pathway 1) and thymine release (indicative of pathway 2). The nucleoside thymidine showed no reaction, suggesting a phosphate-dependent oxidation. Amounts of TBARS and thymine were maximal at a pH range of 6.0-6.5, and both TBARS formation and thymine release correlated with decay of the Cr(V) EPR signal. Formation of TBARS was maximal in 100% O2 but decreased markedly under argon, whereas thymine release was maximal under argon, but remained the major product observed under aerobic conditions. Pathway 1 for the reaction of Cr(V) with dTDP led to formation of glycolic acid and trans-thymine propenal at approximately equimolar amounts, consistent with a mechanism involving oxygen-dependent sugar oxidation following hydrogen atom abstraction at the C-4' carbon of the deoxyribose sugar. Pathway 2 led to release of free thymine, but much less (barely detectable) 2-deoxy-D-pentitol was formed from postreduction of the reactive aldehydic sugar fragment. Thus, the oxygen-independent release of thymine does not appear to result from reaction at the C-4' hydrogen unless decomposition of the aldehydic intermediate occurred. Determination of the oxidation state of chromium responsible for the observed oxidative damage was carried out using Mn(II), a Cr(IV)-specific reductant. Mn(II) essentially abolished all activity for both TBARS formation (pathway 1) and thymine release (pathway 2). These results suggest that Cr(IV), formed upon disproportionation of Cr(V), oxidizes the nucleotide deoxyribose sugar moiety via a phosphate-bound intermediate. pathway 1 involves oxygen-dependent oxidation at the C-4' position; however, the mechanism for oxygen-independent thymine release (pathway 2) is still unclear.
- Sugden, Kent D.,Wetterhahn, Karen E.
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- Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: A backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography
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Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5′-deoxythymidine-5′-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 to-3.1 °C per phosphoroselenolate) when introduced at the 5′-Termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 ?. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.
- Conlon, Patrick F.,Eguaogie, Olga,Wilson, Jordan J.,Sweet, Jamie S. T.,Steinhoegl, Julian,Englert, Klaudia,Hancox, Oliver G. A.,Law, Christopher J.,Allman, Sarah A.,Tucker, James H. R.,Hall, James P.,Vyle, Joseph S.
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- The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**
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The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4–10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 106 for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.
- Kaspar, Felix,Neubauer, Peter,Kurreck, Anke
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p. 1385 - 1390
(2021/01/29)
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- Efficient biocatalytic synthesis of dihalogenated purine nucleoside analogues applying thermodynamic calculations
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The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.
- Giessmann, Robert T.,Kaspar, Felix,Klare, Hendrik F. T.,Kurreck, Jens,Neubauer, Peter,Paulick, Katharina,R?hrs, Viola,Wagner, Anke,Westarp, Sarah,Yehia, Heba
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- Anion exchange resins in phosphate form as versatile carriers for the reactions catalyzed by nucleoside phosphorylases
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In the present work, we suggested anion exchange resins in the phosphate form as a source of phosphate, one of the substrates of the phosphorolysis of uridine, thymidine, and 1-(β-D-arabinofuranosyl)uracil (Ara-U) catalyzed by recombinant E. coli uridine (UP) and thymidine (TP) phosphorylases. α-D-Pentofuranose-1-phosphates (PF-1Pis) obtained by phosphorolysis were used in the enzymatic synthesis of nucleosides. It was found that phosphorolysis of uridine, thymidine, and Ara-U in the presence of Dowex 1X8 (phosphate; Dowex-nPi) proceeded smoothly in the presence of magnesium cations in water at 20-50 °C for 54-96 h giving rise to quantitative formation of the corresponding pyrimidine bases and PF-1Pis. The resulting PF-1Pis can be used in three routes: (1) preparation of barium salts of PF-1Pis, (2) synthesis of nucleosides by reacting the crude PF-1Pi with an heterocyclic base, and (3) synthesis of nucleosides by reacting the ionically bound PF-1Pi to the resin with an heterocyclic base. These three approaches were tested in the synthesis of nelarabine, kinetin riboside, and cladribine with good to excellent yields (52-93%).
- Artsemyeva, Julia N.,Buravskaya, Tatiana N.,Esipov, Roman S.,Konstantinova, Irina D.,Litvinko, Natalia M.,Mikhailopulo, Igor A.,Miroshnikov, Anatoly I.,Remeeva, Ekaterina A.
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p. 2607 - 2622
(2020/11/26)
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- Thermodynamic Reaction Control of Nucleoside Phosphorolysis
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Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose-1-phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase-catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate-specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature-dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis. (Figure presented.).
- Kaspar, Felix,Giessmann, Robert T.,Neubauer, Peter,Wagner, Anke,Gimpel, Matthias
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supporting information
p. 867 - 876
(2020/01/24)
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- Biomimetic Oxidative Deamination Catalysis via ortho-Naphthoquinone-Catalyzed Aerobic Oxidation Strategy
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An ortho-naphthoquinone-catalyzed oxidative deamination reaction has been developed where the molecular oxygen and water serve as the sole oxidant and nucleophile. The current aerobic deamination reaction proceeds via the ketimine formation between ortho-naphthoquinones and amines followed by the prototropic rearrangement and hydrolysis by water, representing a biomimetic oxidative deamination of amine species in the human body by the liver and kidneys. The compatibility of ortho-naphthoquinone organocatalysts with molecular oxygen and water opens up a new biomimetic catalyst system that can function as versatile deaminases for a variety of amine-containing molecules such as amino acids and DNA nuclear bases.
- Golime, Gangadhararao,Bogonda, Ganganna,Kim, Hun Young,Oh, Kyungsoo
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p. 4986 - 4990
(2018/05/14)
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- Methyl ketone derivative, and preparation method and applications thereof
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The invention discloses a methyl ketone derivative, and a preparation method and applications thereof. The preparation method comprises following steps: a ketone derivative and an organic peroxide are dissolved in a solvent, and reaction is carried out at 80 to 130 DEG C so as to obtain methyl pyrimidone and a methyl pyrimidone derivative. According to the preparation method, the ketone derivative is taken as a starting material; the raw materials are easily and widely available; products of different kinds can be obtained via the preparation method, and can be used directly or used in other further reaction. No metal is involved, so that the preparation method is suitable to be applied in pharmaceutical preparation technology. The preparation method is short in route, mild in reaction conditions, simple in reaction operation and postprocessing process, and high in yield, and is suitable for large-scale production.
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Paragraph 0039
(2017/08/28)
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- Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis
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Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.
- Alexeev,Sivets,Safonova,Mikhailov
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p. 107 - 121
(2017/02/05)
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- Biotransformation of halogenated nucleosides by immobilized Lactobacillus animalis 2′-N-deoxyribosyltransferase
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An immobilized biocatalyst with 2′-N-deoxyribosyltransferase (NDT) activity, Lactobacillus animalis NDT (LaNDT), was developed from cell free extracts. LaNDT was purified, characterized and then immobilized by ionic interaction. Different process parameters were optimized, resulting in an active derivative (2.6 U/g) able to obtain 1.75 mg/g of 5-fluorouracil-2′-deoxyriboside, an antimetabolite known as floxuridine, used in gastrointestinal cancer treatment. Furthermore, immobilized LaNDT was satisfactorily used to obtain at short reaction times other halogenated pyrimidine and purine 2′-deoxynucleosides such as 6-chloropurine-2′-deoxyriboside (4.9 U/g), 6-bromopurine-2′-deoxyriboside (4.3 U/g), 6-chloro-2-fluoropurine-2′-deoxyriboside (5.4 U/g), 5-bromo-2′-deoxyuridine (2.8 U/g) and 5-chloro-2′-deoxyuridine (1.8 U/g) compounds of pharmaceutical interest in antiviral or antitumor treatments. Besides, increasing the biocatalyst amount 8 times per volume unit allowed obtaining a 5-fold improvement in floxuridine biotransformation. The developed biocatalyst proved to be effective for the biosynthesis of a wide spectrum of nucleoside analogues by employing an economical, simple and environmentally friendly methodology.
- Britos, Claudia N.,Lapponi, María José,Cappa, Valeria A.,Rivero, Cintia W.,Trelles, Jorge A.
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- Sonochemical transformation of thymidine: A mass spectrometric study
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Abstract Ultrasound is extensively used in medical field for a number of applications including targeted killing of cancer cells. DNA is one of the most susceptible entities in any kind of free radical induced reactions in living systems. In the present work, the transformation of thymidine (dT) induced by ultrasound (US) was investigated using high resolution mass spectrometry (LC-Q-ToF-MS). dT was subjected to sonolysis under four different frequencies (200, 350, 620 and 1000 kHz) and at three power densities (10.5, 24.5 and 42 W/mL) in aerated as well as argon saturated conditions. A total of twenty modified nucleosides including non-fully characterized dT dimeric compounds were detected by LC-Q-ToF-MS. Out of these products, seven were obtained only in the argon atmosphere and two only in the aerated conditions. Among the identified products, there were base modified products and sugar modified products. The products were formed by the reaction of hydroxyl radical and hydrogen atom. Under aerated conditions, the reactions proceed via the formation of hydroperoxides, while in argon atmosphere disproportionation and radical recombinations predominate. The study provides a complete picture of sonochemical transformation pathways of dT which has relevance in DNA damage under ultrasound exposure.
- Chandran, Jisha,Aravind, Usha K.,Aravindakumar
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p. 178 - 186
(2015/06/02)
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- Thymidylate synthase inspired biomodel reagent for the conversion of uracil to thymine
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Inspired by TSase catalysis for dUMP conversion to dTMP, a biomodel reagent is developed. The presence of NH2, Gly-(S)-Cys and (S)-oxiran methyl, at C5, C4 and N-10 of acridine, respectively, in addition to the pH of the reaction mixture, allows for good complementary inter- and intra-molecular interactions and chiral discrimination for the reagent to achieve conversion of uracil to thymine.
- Singh, Palwinder,Kumar, Arun,Kaur, Sukhmeet,Singh, Amrinder
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supporting information
p. 9961 - 9964
(2015/06/22)
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- Iodine Monochloride Facilitated Deglycosylation, Anomerization, and Isomerization of 3-Substituted Thymidine Analogues
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The reaction of thymidine, 3-mono-, and 3,3′,5′-trialkylsubstitued thymidine analogues with iodine monochloride (ICl) was investigated. Treatment with ICl resulted in rapid deglycosylation, anomerization, and isomerization of thymidine and 3-substituted thymidine analogues under various reaction conditions leading to the formation of the nucleobases as the major products accompanied by minor formation of α-furanosidic-, α-pyranosidic-, and β-pyranosidic nucleosides. On the other hand, 3,3′,5′-trisubstitued thymidine analogues were only deglycosylated and anomerized. These results are similar to those observed for the acidic hydrolysis of the glycoside bond in nucleosides, but were presumably caused by the Lewis acid character of an iodine electrophile.
- Khalil, Ahmed,Ishita, Keisuke,Ali, Tehane,Tiwari, Rohit,Riachy, Ramy,Toppino, Antonio,Hasabelnaby, Sherifa,Sayfullin, Naum,Oliver, Allen G.,Gallucci, Judith,Huang, Zhenguo,Tjarks, Werner
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p. 786 - 799
(2015/10/19)
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- Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli
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A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.
- Arivett, Brock,Farone, Mary,Masiragani, Ranjith,Burden, Andrew,Judge, Shelby,Osinloye, Adedoyin,Minici, Claudia,Degano, Massimo,Robinson, Matthew,Kline, Paul
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p. 656 - 662
(2014/03/21)
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- Discovery of a bacterial 5-methylcytosine deaminase
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5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 105, 2.9 × 104, and 1.1 × 103 M-1 s-1, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 105, 6.8 × 104, and 2.0 × 102 M-1 s-1, respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85), 5-fluorocytosine (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.
- Hitchcock, Daniel S.,Fedorov, Alexander A.,Fedorov, Elena V.,Almo, Steven C.,Raushel, Frank M.
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p. 7426 - 7435
(2015/04/16)
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- The muraminomicin biosynthetic gene cluster and enzymatic formation of the 2-deoxyaminoribosyl appendage
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Muraminomicin is a lipopeptidyl nucleoside antibiotic produced by Streptosporangium amethystogenes SANK 60709. Similar to several members of this antibiotic family such as A-90289 and muraymycin, the structure of muraminomicin consists of a disaccharide comprised of two modified ribofuranose units linked by an O-β(1 → 5) glycosidic bond; however, muraminomicin holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Fives enzymes are likely involved in the assembly and attachment of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-d-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis, and the specificity for this appendage is dictated primarily by the two characterized enzymes. The Royal Society of Chemistry 2013.
- Chi, Xiuling,Baba, Satoshi,Tibrewal, Nidhi,Funabashi, Masanori,Nonaka, Koichi,Van Lanen, Steven G.
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p. 239 - 243
(2013/03/13)
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- A comparison between immobilized pyrimidine nucleoside phosphorylase from Bacillus subtilis and thymidine phosphorylase from Escherichia coli in the synthesis of 5-substituted pyrimidine 2′-deoxyribonucleosides
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Pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli (EcTP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2′-deoxyribonucleosides (14-18) by transglycosylation in fully aqueous medium. From the comparative study of the two biocatalysts, no remarkable differences emerged about their substrate specificity, bioconversion yield, stability in organic cosolvents (DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2′,3′- dideoxynucleosides as substrates, whereas they catalyze bioconversions involving 5′-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14-18 proceeded at a similar conversion (33-68% for BsPyNP and 25-62% for EcTP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2′-deoxyuridine (14) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% (EcTP) or 76% (BsPyNP) conversion (about 9 g/L).
- Serra, Immacolata,Bavaro, Teodora,Cecchini, Davide A.,Daly, Simona,Albertini, Alessandra M.,Terreni, Marco,Ubiali, Daniela
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- Enzymatic synthesis of 2-deoxy-β-d-ribonucleosides of 8-azapurines and 8-aza-7-deazapurines
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The enzymatic synthesis of 8-azapurine and 8-aza-7-deazapurine 2-deoxyribonucleosides has been studied. Two methods have been used: (i) transglycosylation employing 2-deoxyguanosine, 2-deoxycytidine, 2-deoxyuridine, and 2-deoxythymidine as 2-deoxy-d-ribofuranose donors and recombinant E. coli purine nucleoside phosphorylase (PNP) as biocatalyst, and (ii) one-pot synthesis from 2-deoxy-d-ribose and nucleobases employing recombinant E. coli ribokinase (RK), phosphopentomutase (PPM) and PNP as biocatalysts. Good substrate activity was observed for all bases studied except 2-amino-8-aza-6-chloro-7-deazapurine, which afforded the desired N9-nucleoside in moderate yield due to very low solubility of the base and partial replacement of C6-chloro atom of the base and formed nucleoside with a hydroxy group. The participation of Ser90 Oγ of E. coli PNP in the binding of 8-aza-7-deazapurines in the catalytic center of PNP followed by the formation of a productive complex and glycosidic bond is suggested. Georg Thieme Verlag Stuttgart · New York.
- Stepchenko, Vladimir A.,Seela, Frank,Esipov, Roman S.,Miroshnikov, Anatoly I.,Sokolov, Yuri A.,Mikhailopulo, Igor A.
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experimental part
p. 1541 - 1545
(2012/09/08)
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- Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: Implications for the clinical efficacy of nucleoside analogues
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In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymesmay compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km = 473 μM) and deoxyuridine (Km = 578 μM), it prefers uridine (K m =92 μM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NPI class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses. The Authors Journal compilation
- Vande Voorde, Johan,Gago, Federico,Vrancken, Kristof,Liekens, Sandra,Balzarini, Jan
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experimental part
p. 113 - 123
(2012/10/23)
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- PHOSPHORAMIDATE DERIVATIVES OF 5 - FLUORO - 2 ' - DEOXYURIDINE FOR USE IN THE TREATMENT OF CANCER
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Phosphoramidate derivatives of 5-fluoro-2'-deoxyuridine are disclosed for use in the treatment of cancer, especially in the treatment of cancer where the patient shows resistance, for example, in a patient with cells with a lowered level of nucleoside transporter proteins and/or with nucleoside kinase-deficient cells and/or with mycoplasma-infected cells and/or with cells with a raised level of thymidylate synthase.
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Page/Page column 66-67
(2012/09/21)
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- Investigation of reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides
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Model 3′-azido-3′-deoxynucleosides with thiol or vicinal dithiol substituents at C2′ or C5′ were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides. Esterification of 5′-(tert-
- Dang, Thao P.,Sobczak, Adam J.,Mebel, Alexander M.,Chatgilialoglu, Chryssostomos,Wnuk, Stanislaw F.
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experimental part
p. 5655 - 5667
(2012/09/25)
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- Photorepair of cyclobutane pyrimidine dimers by 8-oxopurine nucleosides
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The 8-oxopurine nucleosides 2′,3′,5′-tri-O-acetyl-8-oxo- 7,8-dihydroguanosine (OG) and 2′,3′,5′-tri-O-acetyl- ribosyluric acid (RU) were studied for their ability to mediate the photochemical (λ>300nm) reversion of cyclobutane pyrimidine dimers to their parent pyrimidines thymine and uracil. The bimolecular reactions of these monomers proceeded at very slow rates compared with recently published work using oligonucleotide contexts; nevertheless, it was possible to make comparisons between the efficacy of OG and RU as photocatalysts as a function of pH. Although RU has a lower redox potential and anionic character, it was only equivalent to OG in facilitating thymine dimer photorepair over a broad pH range. Only OG showed pH-dependent behavior with higher activity at pH 8-9 where the base becomes deprotonated. Despite the overall low activity of OG and RU, the results are instructive with respect to a comparison of the two 8-oxopurines, and support the hypothesis that 8-oxopurine nucleosides may have played primordial roles as precursors to modern-day flavins in redox reactions of the RNA world. Copyright
- Nguyen, Khiem Van,Burrows, Cynthia J.
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experimental part
p. 574 - 577
(2012/08/28)
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- Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics
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Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an l-methionine:uridine-5′-aldehyde aminotransferase; (ii) LipP, a 5′-amino-5′-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-d-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.
- Chi, Xiuling,Pahari, Pallab,Nonaka, Koichi,Van Lanen, Steven G.
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supporting information; experimental part
p. 14452 - 14459
(2011/11/04)
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- The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2′-deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes
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A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2′-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue.
- Vande Voorde, Johan,Liekens, Sandra,McGuigan, Christopher,Murziani, Paola G.S.,Slusarczyk, Magdalena,Balzarini, Jan
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experimental part
p. 441 - 452
(2012/01/14)
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- Opened-ring adducts of 5-methylcytosine and 1,5-dimethylcytosine with amines and water and evidence for an opened-ring hydrate of 2′- deoxycytidine
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A variety of nucleic acid components and related compounds undergo photoreaction with water to form so-called photohydrates (e.g. uracil forms 6-hydroxy-5,6-dihydrouracil). However, the corresponding hydrates of 5-methylcytosine (a minor nucleobase in eukaryotic DNA) and related compounds have not been characterized. We report the preparation of opened-ring forms of such products for 5-methylcytosine (m5C) and 1,5-dimethylcytosine (DMC). This was accomplished via thermal reaction of ring-opened amine adducts (e.g. N-carbamoyl-3-amino-2-methylacrylamidine (IVa) or N-(N′-methylcarbamoyl)- 3-amino-2-methylacrylamidine (IVb)) produced by photo-induced reactions of m5C with ammonia or methylamine. When these adducts were treated with dilute trifluoroacetic acid, the amino group at the 3-position was replaced with a hydroxyl group; with IVa, N-carbamoyl-3-hydroxy-2-methylacrylamidine (Va) was formed, while reaction of IVb led to N-(N′-methylcarbamoyl)-3-hydroxy-2- methylacrylamidine (Vb). These compounds are ring-opened isomers of 5,6-dihydro-6-hydroxy-5-methylcytosine (Ia and IIa) and 5,6-dihydro-6-hydroxy-1, 5-dimethylcytosine (Ib and IIb). Compounds Va and Vb each undergo thermal ring closure reactions to form two unstable compounds with chemical and UV spectral properties expected for Ia and IIa (or Ib and IIb). The latter compounds have been identified as minor products in UV-irradiated aqueous solutions of m5C and DMC. Evidence is also presented that the 2′-deoxycytidine photohydrates coexist with an opened-ring form, possibly similar in nature to Vb. The nucleobase 5-methylcytosine (m5C) reacts photochemically with ammonia to form an opened-ring adduct (IVa); similar reactions occur with 1,5-dimethylcytosine and with methylamine. Subjection of such adducts to hydrolysis in dilute acid (e.g. 0.1% trifluoracetic acid) produces opened-ring hydrates of m5C (Va) and DMC. Photoreaction of the nucleoside 2′-deoxycytidine in water results in formation of closed-ring hydrates that equilibrate thermally with a compound that has the UV spectral properties expected for an adduct similar in nature to Va.
- Shetlar, Martin D.,Chung, Janet
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experimental part
p. 818 - 832
(2012/07/27)
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- Serendipitous discovery of a zidovudine guanidine complex: A superior process for the production of zidovudine
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A superior process for the commercial production of zidovudine (AZT) has been developed. It was discovered that an AZT-guanidine complex formed when a crude zidovudine solution was treated with guanidine. This readily precipitated from protic solvents resulting in the exclusion of impurities and permitted the development of a superior isolation and purification of AZT.
- Radatus, Bruno K.
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experimental part
p. 1281 - 1286
(2012/01/13)
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- PROCESS FOR STRAIGHTENING KERATIN FIBRES WITH A HEATING MEANS AND DENATURING AGENTS
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The invention relates to a process for straightening keratin fibres, comprising: (i) a step in which a straightening composition containing at least two denaturing agents is applied to the keratin fibres, (ii) a step in which the temperature of the keratin fibres is raised, using a heating means, to a temperature of between 110 and 250° C.
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- HUMANIZED ANTI-EGFL7 ANTIBODIES AND METHODS USING SAME
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The present invention concerns antibodies to EGFL7 and the uses of same.
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- Synthesis of 3-fluoro-6-S-(2-S-pyridyl) nucleosides as potential lead cytostatic agents
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The 3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-β-d-glucopyranosyl nucleoside analogs 7 were prepared via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a-e, were obtained by the sequence of deacetylation of 3-deoxy-3-fluoro-β-d-glucopyranosyl nucleosides 2a-e, selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired thiolpyridine protected analogs 7a-c,f,g were obtained by the sequence of deacetylation of 5a-c followed by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly synthesized peracetylated 6-S-(2-S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluorouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and selective cytostatic action against a variety of murine and human tumor cell cultures.
- Tsoukala, Evangelia,Tzioumaki, Niki,Manta, Stella,Riga, Alexandra,Balzarini, Jan,Komiotis, Dimitri
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experimental part
p. 285 - 293
(2011/02/23)
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- Studies on the synthesis of N′-acetyl AZA-analogues of Ganciclovir - Unexpected liability of N′-(2-hydroxyethyl)-azanucleosides under basic conditions
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The O′-pivaloyl diesters of N′-acetyl-azanucleosides were obtained from N-[1,3-di(pivaloyloxy)prop-2-yl]-N-(pivaloyloxymethyl)acetamide and a silylated nucleobase under Vorbruggen′s conditions. Unexpectedly, de-pivaloylation of the diesters under basic conditions afforded the corresponding nucleobase and N-acetylserinol. Mechanistic investigations showed that these products result from the following cascade of spontaneous transformations initiated by the mono de-pivaloylation of the starting diesters. N′-Deacetylation of the resultant mono-esters via the intramolecular N-O acetyl migration is the key step of the cascade; the corresponding NH-azanucleosides in the form of O-acetyl-O′-pivaloyl diesters are formed. Fragmentation of these diester intermediates gives the nucleobase and O-acetyl-O'-pivaloylserinol. Conversion of the latter to N-acetylserinol involves the selective O-N acetyl migration followed by de-pivaloylation of the resulting N-acetyl-O-pivaloylserinol. Copyright Taylor and Francis Group, LLC.
- Koszytkowska-Stawinska, Mariola
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experimental part
p. 768 - 785
(2011/05/04)
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- Transition state analysis of thymidine hydrolysis by human thymidine phosphorylase
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Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched thymidines were synthesized enzymatically from glucose or (deoxy)ribose, and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1′- 14C]-, [1-15N]-, [1′-3H]-, [2′R-3H]-, [2′S-3H]-, [4′- 3H]-, and [5′-3H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to the irreversible nucleophilic attack by water. The results establish activation of the thymine leaving group without requirement for phosphate. A transition state constrained to match the intrinsic KIEs was found using density functional theory. An active site histidine (His116) is implicated as the catalytic base for activation of the water nucleophile at the rate-limiting transition state. The distance between the water nucleophile and the anomeric carbon (rC-O) is predicted to be 2.3 A at the transition state. The transition state model predicts that deoxyribose adopts a mild 3′-endo conformation during nucleophilic capture. These results differ from the concerted bimolecular mechanism reported for the arsenolytic reaction (Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 2447-2453).
- Schwartz, Phillip A.,Vetticatt, Mathew J.,Schramm, Vern L.
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supporting information; experimental part
p. 13425 - 13433
(2010/12/19)
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- Self-sacrificing acetylation observed during attempted desilylation of 1-[4-benzenesulfonyl-3-O-(tert-butyldimethylsilyl)-2-deoxy-5-O-methanesulfonyl- α-l-threo-pentofuranosyl]thymine
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Desilylation of 1-[4-benzenesulfonyl-3-O-(tert-butyldimethylsilyl)-2-deoxy- 5-O-methanesulfonyl-α-l-threo-pentofuranosyl]thymine (4) with Bu 4NF/THF, when carried out at room temperature, gave four products. Among these, there were 1-[3-O-acetyl-4-benzenesulfonyl-2-deoxy-5-O- methanesulfonyl-α-l-threo-pentofuranosyl]thymine (7) and thymine. A possible reaction mechanism is proposed, which suggests the origin of 3′-O-acetyl group of 7 and thymine as well as structures of the other two products (9a and 9b).
- Shimada, Hisashi,Kubota, Yutaka,Tanaka, Hiromichi
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body text
p. 4253 - 4255
(2010/09/07)
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- Novel acyclic amide-conjugated nucleosides and their analogues
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An effective one-step synthesis of new amide-conjugated nucleosides and their analogues, in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMT-MM) as the condensing agent, is presented. Substrate subunits carrying carboxylic group were obtained by acidic hydrolysis of Michael-type adducts of various 5-substituted uracil derivatives to methyl acrylate. Amine substrate was synthesized by reduction of 1-(2′- cyanoethyl)thymine with sodium borohydride in the presence of nickel (II) chloride as catalyst. Other applied amine substrates were 5′-amino- 5′-deoxythymidine and 5-aminouracil. Copyright Taylor & Francis Group, LLC.
- Boncel, Slawomir,Walczak, Krzysztof
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experimental part
p. 103 - 117
(2009/06/18)
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- ANTIBODIES TO INSULIN-LIKE GROWTH FACTOR RECEPTOR
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The invention provides various antibodies that bind to insulin-like growth factor-I receptor (IGF-1R), methods for making such antibodies, compositions and articles incorporating such antibodies, and their uses in treating, for example, cancer or aging. The antibodies include murine, chimeric, and humanized antibodies.
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- ANTIBODIES TO LYMPHOTOXIN-ALPHA
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The invention provides various antibodies that bind to lymphotoxin-α, methods for making such antibodies, compositions and articles incorporating such antibodies, and their uses in treating, for example, an autoimmune disorder. The antibodies include murine, chimeric, and humanized antibodies.
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- Antibodies and immunoconjugates and uses therefor
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Anti-STEAP-1 antibodies and immunoconjugates thereof are provided. Methods of using anti-STEAP-1 antibodies and immunoconjugates thereof are provided.
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- New [11C]phosgene based synthesis of [11C]pyrimidines for positron emission tomography
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Thymine, 5-FU, and uracil were successfully synthesized through a procedure involving a cyclocondensation of triphosgene with newly developed (α-substituted β-aminoacrylamides intermediates (1a, X= Me; 1b, X= F; 1c, X= H). The radioligands [2-11C]thymine and [2- 11C]5-fluorouracil were synthesized in high radiochemical yields in 16-17 minutes from the end of bombardment by applying the cyclocondensation method with [11C]COCl2.
- Seki, Koh-Ichi,Nishijima, Ken-Ichi,Sanoki, Kimihito,Kuge, Yuji,Takahashi, Masayuki,Akizawa, Hiromichi,Tamaki, Nagara,Wiebe, Leonard I.,Ohkura, Kazue
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experimental part
p. 1307 - 1321
(2010/10/03)
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- Protecting groups transfer: Unusual method of removal of tr and TBDMS groups by transetherification
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The triphenylmethyl (Tr) group undergoes a transfer (transetherification or disproportionation) between the molecules of 5'-O-Tr-2'-deoxynucleosides in a process mediated by anhydrous sulfates of Cu+2, Fe+2, or Ni+2 to yield mixtures of 3',5'-bis-O-Tr and 3'-O-Tr products. If phenylmethanol is present in a reaction medium, detritylation results with concomitant formation of phenylmethyl triphenylmethyl ether. The behavior of t-butyldimethylsilyl (TBDMS) group in 5'-O-TBDMS-2'-deoxynucleosides is exactly the same. Such type of transetherifications was not observed before for the O-Tr and O-TBDMS groups. Copyright Taylor & Francis Group, LLC.
- Cabral, Nadia L. D.,Thiessen, Luciano Hoeltgebaum,Doboszewski, Bogdan
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p. 931 - 948
(2008/12/21)
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- Kinetic parameters and recognition of thymidine analogues with varying functional groups by thymidine phosphorylase
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Thymidine phosphorylase (TP, EC 2.4.2.4) recognized the structure of the substrate with high specificity, via both the base and the ribosyl moieties. The replacement of 3′-OH of thymidine markedly influenced its catalytic activity with TP. The conversion
- Hatano, Akihiko,Harano, Aiko,Takigawa, Yoshikatsu,Naramoto, Yasuhiro,Toda, Keisuke,Nakagomi, Yuuichi,Yamada, Hideyuki
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p. 3866 - 3870
(2008/12/20)
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- α,β-Methylene-2′-deoxynucleoside 5′-triphosphates as noncleavable substrates for DNA polymerases: Isolation, characterization, and stability studies of novel 2′-deoxycyclonucleosides, 3,5′-cyclo-dG, and 2,5′-cyclo-dT
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We report synthesis and characterization of a complete set of α,β-methylene-2′-dNTPs (α,β-m-dNTP; N = A, C, T, G, 12-15) in which the α,β-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2′-deoxynucleoside 5′-diphosphate precursors, followed by an enzymatic γ-phosphorylation. All four synthesized α,β-m-dNTPs were found to be potent inhibitors of polymerase β, with Ki values ranging 1-5 μM. During preparation of the dG and dT derivatives of α,β-methylene diphosphate, we also isolated significant amounts of 3,5′-cyclo-dG (16) and 2,5′-cyclo-dT (17), respectively. These novel 2′-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 → C5′ and O2 → C5′, respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5′-cyclo-2′-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.
- Liang, Fengting,Jain, Nidhi,Hutchens, Troy,Shock, David D.,Beard, William A.,Wilson, Samuel H.,Chiarelli, M. Paul,Cho, Bongsup P.
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experimental part
p. 6460 - 6470
(2009/10/23)
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- Fluorinase-coupled base swaps: Synthesis of [18F]-5′- deoxy-5′-fluorouridines
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(Chemical Equation Presented) Making F-ases: One-pot fluorination/base-swap biotransformations were developed by coupling the fluorinase enzyme to nucleoside phosphorylases to generate 5′-deoxy-5′-fluoronucleosides (FDAs). These biotransformations are amenable to radiolabeling syntheses starting from [18F]fluoride ion, as exemplified by the synthesis of [18F]-5′-deoxy-5′-fluorouridines (see scheme), and demonstrate a new application of fluorinase as a catalyst for 18F-C bond formation.
- Winkler, Margit,Domarkas, Juozas,Schweiger, Lutz F.,O'Hagan, David
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supporting information; experimental part
p. 10141 - 10143
(2009/05/30)
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- Substrate specificity of thymidine phosphorylase of E. coli: Role of hydroxyl groups
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Substrate specificity of E. coli thymidine phosphorylase to pyrimidine nucleoside modified at 5′-, 3′-, and 2′-positions of sugar moiety has been studied. Equilibrium (Keq) and kinetics constants of phosphorolysis reaction of nucleosides were measured. Th
- Panova, Natalya G.,Alexeev, Cyril S.,Polyakov, Konstantin M.,Gavryushov, Sergei A.,Kritzyn, Anatoliy M.,Mikhailov, Sergey N.
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scheme or table
p. 1211 - 1214
(2009/04/06)
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- Photochemical generation of oligodeoxynucleotide containing a C4′-oxidized abasie site and its efficient amine modification: Dependence on structure and microenvironment
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(Chemical Equation Presented) Bleomycin-induced oxidative DNA damage under limited oxygen conditions results in the formation of the C4′-oxidized abasic site (1). We synthesized the oligodeoxynucleotides (ODN) 5, which contains 4′-o-nitrobenzyloxythymidine (3), and 6, which contains 2-nitrobenzyloxy-4′-methoxy-2′-deoxy-D-ribofuranoside (4) as the caged precursors of 7, an ODN containing 1, to study its reactivity with amines Photoirradiation of the single- and double-stranded 5 led to the formation of 7. Uncaging of the duplex was faster and the yield of 7 was higher with the double-stranded than with the single-stranded ODN. It was suggested that a low dielectric environment of the o-nitrobenzyloxy group in the minor groove of the duplex might accelerate the uncaging rate. Similarly, 6 and its duplex yielded 7 by photoirradiation However, the yields of 7 were lower than those of 5, and duplex formation slowed the uncaging rate. Reaction of the obtained 7 with an amine resulted in the formation of the lactam 2b in good yield in both single- and double-stranded forms, showing that amine modification of biomolecules by an ODN containing 1 is possible under physiologic conditions.
- Usui, Kazuteru,Aso, Mariko,Fukuda, Mitsuhiro,Suemune, Hiroshi
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p. 241 - 248
(2008/09/19)
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- BINDING POLYPEPTIDES WITH OPTIMIZED SCAFFOLDS
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The invention provides variant heavy chain variable domains (VH) with increased folding stability. Libraries comprising a plurality of these polypeptides are also provided. In addition, compositions and methods of generating and using these polypeptides and libraries are provided.
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