Repair effect of phenylpropanoid glycosides on thymine radical anion induced by pulse radiolysis

Repair effects on thymine radical anion by six phenylpropanoid glycosides (PPGs), isolated from Pedicularis species, were studied using pulse radiolysis method. The thymine radical anion was produced by the reaction of hydrated electron with thymine. PPGs were added into the thymine solution saturated with N(2). Kinetic analysis showed that transient absorption spectrum of thymine radical anion formed at first, and then after several microseconds of pulse radiolysis changed to that of PPG radical anion. The evidence indicated that thymine radical anion was repaired through one-electron transfer between the radical anion and PPG. Electrophilic phenyl-substituted unsaturated carboxylic group containing in PPGs' structure was able to capture electron from thymine radical anion before it undergo reversible protonation. The reaction rate constants of electron transfer from thymine radical anion to PPGs were within 1.16-2.29 x 10(9) dm(3) mol(-1) s(-1).     

Perturbation of hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations

Perturbation of the hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine...thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at O6 leads to destabilization of the adenine...thymine pair; divalent cations (Mg2+, Ca2+) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

A kinetic study of thymine 7-hydroxylase from neurospora crassa.

The steady-state kinetics of thymine 7-hydroxylase (thymine, 2-oxoglutarate dioxygenase, EC 1.14.11.6) has been investigated. Initial velocity plots were all found to be linear and intersecting. Variation in concentration of two of the substrates, when the third substrate was at a constant high or low concentration, gave initial velocity plots that conform to an ordered sequential mechanism, where thymine is the second substrate to add. With 5-carboxyuracil, which is the end product in the sequential oxygenation of thymine, a competitive inhibition pattern was observed when 2-ketoglutarate was the variable substrate. When either thymine or oxygen was the variable substrate a noncompetitive inhibition pattern was obtained. When either 2-ketoglutarate or thymine was the variable substrate the inhibition patterns observed with bicarbonate were noncompetitive. With succinate noncompetitive inhibition patterns with hyperbolic intercept replots were obtained. These results are consistent with an ordered sequential kinetic mechanism, where 2-ketoglutarate is added first, followed by thymine and oxygen, and the products are released in the order: bicarbonate, succinate, and 5-hydroxymethyluracil. The order of the two last mentioned products, however, is changed in the presence of succinate.     

Formation of Thymine from Thymine Glycol by Gamma-irradiation in an Aqueous System

When an aqueous solution of thymine glycol, a main radiolysis product from thymine, was gamma-irradiated in the absence of oxygen, a characteristic UV absorption which was absent before irradiation was found to be developed. From the chromatographical and spectrophotornetrical investigations and microbiological assay, one of the main UV positive products was identified to be thymine. The initial G-values with 2 mm thymine glycol solution were 1.1 and 0.4 for thymine glycol decomposition and thymine formation, respectively. The formation was suppressed by the presence of oxygen or nitrous oxide, while it was enhanced by potassium thiocyanate or sodium formate. Thus, the presence of a new reversible reaction between thymine and thymine glycol through radiolysis in an aqueous system was proposed.     

Exomethylene pyranonucleosides: efficient synthesis and biological evaluation of 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine

A new series of unsaturated pyranonucleosides with an exocyclic methylene group and thymine as heterocyclic base have been designed and synthesized. d-Galactose (1) was readily transformed in three steps into the corresponding 1-(beta-d-galactopyranosyl)thymine (2). Selective protection of the primary hydroxyl group of 2 with a t-butyldimethylsilyl (TBDMS) group, followed by specific acetalation, and oxidation gave 1-(6-O-t-butyldimethylsilyl-3,4-O-isopropylidene-beta-d-lyxo-hexopyranosyl-2-ulose)thymine (5). Wittig reaction of the ketonucleoside 5, deprotection and tritylation of the 6'-hydroxyl group gave 1-(2-deoxy-2-methylene-6-O-trityl-beta-d-lyxo-hexopyranosyl)thymine (9). Exomethylene pyranonucleoside 9 was converted to the olefinic derivative 10, which after detritylation afforded the title compound 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine (11). These novel synthesized compounds were evaluated for antiviral activity against rotaviral infection and cytotoxicity in colon cancer. As compared to AZT, compounds 1-(2-deoxy-2-methylene-beta-d-lyxo-hexopyranosyl)thymine (7) and 1-(beta-d-lyxo-hexopyranosyl-2-ulose)thymine (8) showed to be more efficient, in rotavirus infections and in treatment of colon cancer.     

The role of specific DNA base damages in the X-ray-induced inactivation of bacteriophage PM2

Two types of X-ray-induced base damages, alkali-labile sites and thymine ring saturation products, were quantitated in PM2 DNA irradiated in the phage capsid under oxic and anoxic conditions. The extent of formation of these base damages was compared with the number of single- and double-strand breaks and lethal hits produced under the same conditions. The individual inactivation efficiencies of alkali-labile sites and thymine ring saturation products were determined by selectively inducing each of these damages in isolated PM2 DNA by chemical means in vitro and determining the rate of biological inactivation of the treated DNA by transfection. For each lethal X-ray hit induced in oxic conditions there were 1.06 alkali-labile sites, 0.40 thymine ring saturation products, 2.09 singe-strand breaks and 0.11 double-strand breaks in the PM2 genome. In anoxic conditions, the respective number of lesions was 1.00, 0.19, 1.73 and 0.09. The individual inactivation efficiencies of thymine ring saturation products and alkali-labile sites were found to be essentially equal, 7-8 lesions per lethal event in the PM2 genome. Alkali-labile sites and thymine ring saturation products together accounted for 15-20% of the biological inactivation of X-irradiated bacteriophage PM2. The presence or absence of oxygen during irradiation did not affect the contribution to inactivation made by alkali-labile sites, but the contribution by thymine ring saturation products to inactivation was about 2-fold higher in oxic compared with anoxic conditions. With the 4 lesions measured, we have accounted for some 28-34% of the lethal events in X-irradiated PM2 phage, most of the remaining events being caused by as yet unidentified base damages.     

1,2-DISUBSTITUTED CYCLOHEXANE CARBOCYCLIC ANALOGUES OF NUCLEOSIDES

Several compounds of a new series of cyclohexane-based 1,2-disubstituted carbonucleoside analogues, were synthesized. The adenine and uridine derivatives, were prepared by construction of the heterocyclic base on the primary amino group of 2-aminocyclohexylmethanol, and the thymine derivative by condensation of 2-hydroxycyclohexylmethanol with thymine using the Mitsunobu reaction.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

Preferential uptake of thymidine by thymineless enterobacteria: its significance in DNA labelling.

Minimum satisfactory concentrations of thymine and thymidine were determined for the growth of a high thymine-requirng (thy) mutant to Escherichia coli strain J5-3. Cultures were then grown in the presence of these concentrations of non-radioactive ('cold') pyrimidine together with 5 microCi/ml [methyl-3H)thymine, or [methyl-3H)thymidine (specific activities 5 Ci/m mole), and the uptake of radioactivity into ice cold trichloroacetic acid insoluble material determined. By far the most efficient labelling system was obtained if the label was supplied as radioactive thymidine and growth requirements satisfied by thymine alone. The addition of deoxyadenosine to the labelled thymidine/unlabelled thymine system dramatically reduced uptake of label. The addition of radioactive thymine with either thymine or thymidine to ensure satisfactory growth gave poor labelling. Using the [methyl-3H] thymidine/thymine system it was possible to increase the concentration of thymine from 8 to 64 microgram/ml with only a 25% reduction in label uptake after a 2 h period. The same system was also shown to be most efficient for labelling a thy derivative of another K12 strain, a thymine low-requiring (tir) K12 strain, a thy mutant of Klebsiella aerogenes 418 and a tir derivative of Salmonella typhimurium LT2.     

Repair effect of thymine radical anion by echinocoside using pulse radiolysis

Repair activities of thymine radical anion by echinocoside, isolated from Pedicularis plicata. were studied using pulse radiolysis technique. The thymine radical anion was produced by the reaction of hydrated electron with thymine. Echinocoside. one of the polyphenols of phenylpropanoid glycoside, was added to the thymine aqueous solution saturated with N2. Kinetic analysis by transient absorption spectrum showed that thymine radical anion was formed at first, and then after several decades of microseconds of pulse radiolysis. the spectrum of thymine radical anion was changed to that of echinocoside radical anion. The evidence indicated that thymine radical anion was repaired through one-electron-transfer between the DNA base radical anion and echinocoside. The rate constant of electron transfer by echinocoside was 1.45× 109 dm3 · mol1 · s 1.     

Synthesis of C-1′-Branched Acyclic Nucleosides

Abstract

Two C-1′-branched acyclic thymine derivatives, 1-[2-hydroxy-1-(2-hydroxyethoxy)ethyl]thymine and 1-[3-hydroxy-1-(2-hydroxyethoxy)-propyl]thymine were synthesized by a novel iodine-activated reaction of a tolylthio derivative with ethylene glycol. This synthetic method provides a potentially versatile synthetic entry to C-1′-branched acyclic nucleosides.     

Photochemical reactions on the synthetic polymers containing thymine bases

Photodimerizations of N-2-isobutyloxyethyl thymine (T-M), bis[2-(5-methyl-1-pyrimidinyl)ethyl]glutarate (T-T), poly-N-2-methacryloyloxethyl thymine (P-MAOT) and poly-N-2-acryloyloxyethyl thymine (P-AOT) were studied in dimethylformamide solution. Quantum efficiencies of intramolecular photodimerizations were determined to be 0.0012 for T-T, 0.0084 for P-MAOT and 0.010 for P-AOT. In the case of T-M, however, intermolecular photodimerization did not occur under the reaction condition used. Quenching studies by using isoprene suggest that the photodimerization of T-T occurs predominantly through an excited triplet state, while that of P-MAOT and P-AOT occur through both singlet and triplet states. For the effect of adding model compounds containing adenine bases on this reaction, adenine derivatives acts as an inhibitor against this reaction by quenching the excited singlet state of thymine. The photodimers of T-T, P-MAOT and P-AOT were concluded to be two syn-fused cyclobutane- type dimers (cis-syn and trans-syn).     

Synthesis of 4'-C-ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta- D-ribo-pentofuranosyl pyrimidines, and their biological evaluation.

4'-C-Ethynyl-beta-D-arabino-pentofuranosyl thymine (14) and cytosine (16), and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosyl thymine (25) and cytosine (27) were synthesized by properly protected 4'-C-hydroxy-methyl-3,5-di-O-benzyl-alpha-D-ribo-pentofuranose (1) from D-glucose. Among them, 2'-deoxy derivatives 25 and 27 exhibited antiviral activity, while cytidine derivatives 16 and 27 inhibited the growth of neoplastic cells.     

Synthesis and antiviral evaluation of the beta-L-enantiomers of some thymine 3'-deoxypentofuranonucleoside derivatives

3'-Deoxy-beta-L-erythro- (3), 3'-deoxy-beta-L-threo- (6), 2'-fluoro- (7) and 2'-azido-2',3'-dideoxy-beta-L-erythro- (10) pentofuranonucleoside derivatives of thymine have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of thymine with a suitable peracylated 3-deoxy-L-erythro-pentofuranose sugar (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.     

Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA.

Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded.     

The synthesis of novel 6,5- and 6,6-membered fused heterocyclic compounds derived from thymine

Novel pyrimido[1,2-a]pyrimidinones 14, 15 and 16 and imidazo[1,2-a] pyrimidinones 19 and 20, designed as conformationally constrained analogues of 1-(3-amino-2-hydroxypropyl)thymine and 1-(2-amino-3-hydroxypropyl)thymine, respective ly, were synthesized by the ring-opening/ ring-closure rearrangement of the corresponding byciclic oxygen-containing amino compounds 12 and 17.     

Determination of intra- and intermolecular tritium isotope effects in the reaction of thymine 7-hydroxylase

Different [7-3H]thymine preparations have been used to determine the inter- and intramolecular isotope effects of the 2-oxoglutarate-dependent thymine hydroxylation, catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.6). Specific activity ratios of products, viz., 3H2O and 5-hydroxymethyluracil, and remaining substrate to initial substrate have been determined. The influence on these ratios of intra- and intermolecular isotope effects at different degrees of tritium substitution has been analyzed. An intramolecular isotope effect with a kH/kT of about 6.5 has been found. No intermolecular isotope effect of TV/K could be detected when oxygen concentration was varied from 0.4 to 0.01 mM. This agrees with a mechanism in which 2-oxoglutarate is irreversibly changed before the bond-breaking in thymine takes place.     

Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

Synthesis of Cyclobutane Nucleosides 2-Preparation of Thymine and Uracil Analogues

1-(2-Oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione and 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-5-methyl-2,4(1H,3H)-pyrimidinedione can be prepared by reaction of uracil and thymine, respectively, with 3-benzoyloxymethyl-2-bromocyclobutanone. The N-alkylation gave both cis and trans isomers with the trans isomer predominating for uracil whereas the trans isomer was the only product which could be isolated for thymine. Both series were subjected to borohydride reduction followed by transesterification with methoxide giving the corresponding uracil and thymine nucleoside analogues. The uracil derivative 1-(2-oxocyclobutyl-4-benzoyloxymethyl)-2,4(1H,3H)-pyrimidinedione was irradiated in aqueous acetonitrile to generate isonucleoside analogues.     

Incorporation of hexose nucleoside analogues into oligonucleotides: synthesis, base-pairing properties and enzymatic stability.

Oligonucleotides containing 1-(2,4-dideoxy-beta-D-erythro-hexopyranosyl)thymine (2) and 1-(3,4-dideoxy-beta-D-erythro-hexopyranosyl)thymine (3) were synthesized on a solid support using the phosphoramidite approach. The properties of these oligonucleotides were compared with the earlier reported characteristics of oligonucleotides containing 1-(2,3-dideoxy-beta-D-erythro-hexopyranosyl) thymine (1). The order in enzymatic stability of end-substituted oligonucleotides is 3 greater than 1 much greater than 2. The hybridization properties of the modified oligonucleotides are in reverse order: 2 much greater than 1 greater than 3.