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PubMed1997

Effect of N9-methylation and bridge atom variation on the activity of 5-substituted 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii.

The effect of N9-methylation and bridge atom variation on inhibitory potency and selectivity of 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases (DHFR) was studied. Specifically three nonclassical 2,4-diamino-5-((N-methylanilino)methyl)pyrrolo[2,3-d]pyrimidines with 2',5'-dimethoxyphenyl (2), 3',4'-dichlorophenyl (3), 1'-naphthyl (4), one classical analogue with a 4'-L-glutamate substituent (10), and four nonclassical 2,4-diamino-5-((phenylthio)methyl)pyrrolo[2,3-d]pyrimidines with 3',4'-dimethoxyphenyl (5), 3',4'-dichlorophenyl (6), 1'-naphthyl (7), and 2'-naphthyl (8) substituents were synthesized. The classical and nonclassical analogues were obtained by displacement of the intermediate 2,4-diamino-5-bromomethylpyrrolo[2,3-d]pyrimidine, 14, with appropriately substituted N-methylaniline, thiophenols, or 4-(N-methylamino)benzoyl-L-glutamate. Compounds 2-8 and 10 were evaluated against Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) DHFRs. The N-methyl and thiomethyl analogues were more inhibitory than their corresponding anilinomethyl analogues (previously reported) against all three DHFRs. The inhibitory potency of these analogues was greater against rlDHFR than against tgDHFR which resulted in a loss of selectivity for tgDHFR compared to the N9-H analogues. The classical N9-methyl analogue 10 was more potent and about 2-fold more selective against tgDHFR than its corresponding desmethyl analogue. All of the analogues, 2-8 and 10, were more selective than trimetrexate (TMQ) against pcDHFR (except 4) and significantly more selective than TMQ against tgDHFR.

Animals↗

PubMed1997

Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines.

In the course of the random screening of a pool of CIBA chemicals, the two pyrazolopyrimidines 1 and 2 have been identified as fairly potent inhibitors of the EGF-R tyrosine kinase. Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGF-R protein tyrosine kinase (PTK), the class of the pyrazolo[3,4-d]pyrimidines was then optimized in an interactive process leading to a series of 4-(phenylamino)-1H-pyrazolo[3,4-d]-pyrimidines as highly potent inhibitors of the EGF-R tyrosine kinase. The most potent compounds 13, 14, 15, 17, 19, 22, 26, 28, and 30 of this series inhibited the EGF-R PTK with IC50 values below 10 nM. High selectivity toward a panel of nonreceptor tyrosine kinases (c-Src, v-Abl and serine/threonine kinases (PKC alpha, CDK1) was observed. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by compounds 13, 15, 19, 22, and 23 at IC50 values below 50 nM, whereas PDGF-induced tyrosine phosphorylation was not affected by concentrations up to 10 microM, thus indicating high selectivity for the inhibition of the ligand-activated EGF-R signal transduction pathway. Compounds 15 and 19 inhibited proliferation of the EGF-dependent MK cell line with IC50 values below 0.5 microM. In addition, two compounds, 9 and 11, showing satisfactory oral bioavailability in mice after oral administration, exhibited good in vivo efficacy at doses of 12.5 and 50 mg/kg in a nude mouse tumor model using xenografts of the EGF-R overexpressing A431 cell line. From SAR studies, a binding mode for 4-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidines, especially for compound 15, at the ATP-binding site of the EGF-R tyrosine kinase is proposed. 4-(Phenylamino)-1H-pyrazolo[3,4-d]pyrimidines represent a new class of highly potent tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the potential for further evaluation as anticancer agents.

3T3 Cells↗

PubMed1997

2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimid ine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]- 6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 microM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.

Animals↗

PubMed2003

3-Bromopyrazolo[3,4-d]pyrimidine 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides: modified DNA constituents with an unusually rigid sugar N-conformation.

The nucleobase anion glycosylation of 3-bromo-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-6-amine (6) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-d-arabinofuranosyl bromide (5) furnished the protected N(1)-beta-d-nucleosides 7 (60%) and 8 (ca. 2%) along with the N(2)-beta-d-regioisomer 9 (9%). Debenzoylation of compounds 7 and 9 yielded the nucleosides 10 (81%) and 11 (76%). Compound 10 was transformed to the 2'-deoxyguanosine derivative 1 [6-amino-3-bromo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-one] (85% yield) and the purine-2,6-diamine analogue 2 [3-bromo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4, 6-diamine] (78%). Both nucleosides form more than 98% N-conformer population (P(N) ca. 358 degrees and psi(m) ca. 37 degrees ) in aqueous solution. Single-crystal X-ray analysis of 1 showed that the sugar moiety displays also the N-conformation [P = 347.3 degrees and psi(m) = 34.4 degrees ] in the solid state. The remarkable rigid N-conformation of the pyrazolo[3,4-d]pyrimidine 2'-deoxy-2'-fluoro-beta-d-arabinonucleosides 1 and 2 observed in solution is different from that of the parent purine 2'-deoxy-2'-fluoro-beta-d-arabinonucleosides 3 and 4, which are in equilibrium showing almost equal distribution of the N/S-conformers.

Arabinonucleosides↗

PubMed2004

Stereoselectivity and regioselectivity in nucleophilic ring opening in derivatives of 3-phenylisoxazolo[2,3-a]pyrimidine. Unpredicted dimerization and ring transformation. Syntheses of derivatives of pyrimidinylmethylamine, pyrimidinylmethylamino acid amides, and alpha-amino-2-pyrimidinylacetamides.

The nucleophilic ring opening of the isoxazolone ring in 2-oxo-3-phenylisoxazolo[2,3-a]pyrimidine derivatives by optically active amino acid amides and ephedrine led to pyrimidinylmethylamino acid amides. Using amides of different L-amino acids and (-)-ephedrine resulted in different degrees of stereoselectivity. The degree of streoselectivity depended mostly on the nucleophile used. When applying hydroxy amines such as ephedrine, the attack via the secondary amino group was found as the favored regioselectivity. Upon replacement of the oxo group in position 2 in the phenylisoxazolo[2,3-a]pyrimidine system by an imino group, it was expected that the spontaneous decarboxylation that follows the ring opening would not take place, thus achieving amino acid amide derivatives of 2-pyrimidinylacetamide, which are closely related to pyrimidoblamic acid, an important constituent of Bleomycins, used in cancer therapy. However, by heating 5,7-dimethyl-2-imino-3-phenylisoxazolo[2,3-a]pyrimidine in solution, it underwent an unprecedented dimerization process that involved both the phenyl and the imino group. After protecting the imino group by acetylation, the ring opening by nucleophiles was possible, resulting in the formation of derivatives of 2-pyrimidinylacetamide. 2-Acetylimino-5,7-dimethyl-3-phenylisoxazolo[2,3-a]pyrimidine also underwent a ring transformation, yielding an interesting indolone derivative. Selectivity in ring opening and mechanisms of dimerization and ring transformation are discussed.

Amides↗

PubMed2006

Efficient synthesis of highly functionalized dihydropyrido[2,3-d]pyrimidines by a double annulation strategy from alpha-alkenoyl-alpha-carbamoyl ketene-(S,S)-acetals.

A convenient and efficient synthesis of highly functionalized dihydropyrido[2,3-d]pyrimidines via a double [5 + 1] annulation strategy starting from easily available alpha-alkenoyl-alpha-carbamoyl ketene-(S,S)-acetals 1 and cheap reagents (NH4OAc, DMF, and POCl3) has been developed. In the first step of the double annulation route, 2-amino-3-carbamoyl-5,6-dihydro-4-pyridones 2 were created in high to excellent yields by a formal [5C + 1N] annulation reaction of ketene-(S,S)-acetals 1 with ammonia (from ammonium acetate). In the second step of the double annulation strategy, the highly functionalized dihydropyrido[2,3-d]pyrimidine derivatives, 7,8-dihydropyrido[2,3-d]pyrimidin-4(3H)-ones 3 (when R1 = aryl) and 7,8-dihydropyrido[2,3-d]pyrimidines 4 (when R1 = H), were constructed, respectively, in fair to good yields by reacting 2 with excessive Vilsmeier reagent (DMF/POCl3). A mechanism involved in the second [5 + 1] annulation step, including a formal [5 + 1] annulation and accompanied chlorovinylation, chloroformylation, amination, and aromatization reactions, is proposed.

Acetals↗

PubMed2004

Identification of O2-substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)- 1-(3-pyridyl)-1-butanone and 4-(acetoxymethylnitros- amino)-1-(3-pyridyl)-1-butanol with DNA.

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2'-position of the tobacco specific carcinogen N'-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH(2)OAc) with DNA, dCyd, and dThd. NNKCH(2)OAc and NNALCH(2)OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH(2)OAc and NNALCH(2)OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 degrees C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH(2)OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH(2)OAc were similarly characterized. The dThd adduct of NNKCH(2)OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH(4) gave material, which was identical to that produced upon reaction of NNALCH(2)OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH(2)OAc with DNA are O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH(2)OAc are O(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) andO(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH(2)OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH(2)OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH(2)OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.

Alkylating Agents↗

PubMed1997

Pyrimidine biosynthesis in Pseudomonas stutzeri ATCC 17588.

The de novo pyrimidine biosynthetic enzymes in the denitrifying bacterium Pseudomonas stutzeri ATCC 17588 were assayed and their activities were lower in glucose-grown cells than in succinate-grown cells. When P. stutzeri was grown in the presence of uracil, the de novo enzyme activities in succinate-grown cells were lowered while they remained largely unchanged in glucose-grown cells. A uracil auxotroph of P. stutzeri, deficient for aspartate transcarbamoylase activity, was isolated and its auxotrophic requirement was met by only uracil and cytosine. The inability of pyrimidine ribonucleosides to meet the auxotrophic requirement was related to the limited ability of P. stutzeri to transport uridine and cytidine. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be repressible by a uracil-related compound in succinate-grown P. stutzeri cells. Regulation of pyrimidine synthesis in P. stutzeri was similar to that observed for other pseudomonads classified within rRNA homology group I.

Aspartate Carbamoyltransferase↗

PubMed1982

Ligation of oligonucleotides by pyrimidine dimers--a missing 'link' in the origin of life?

One of the principal photochemical reactions of DNA on exposure to UV is the formation of intrastrand cyclobutane-type pyrimidine dimers. The efficiency of this reaction depends on both the wavelength of the UV2 and the specific nucleotide sequence in the DNA. The formation of the pyrimidine dimer and its repair in living cells have been studied extensively. We have examined the possibility that pyrimidines at the ends of DNA strands may be adequately juxtaposed for dimer formation by the presence of a complementary strand, even when no phosphodiester linkage joins their sugars. In these conditions the formation of a dimer will 'ligate' two DNA strands end-to-end. We report here that thymidine oligonucleotides annealed to polydeoxyadenylate can be ligated end-to-end by UV irradiation, via thymine dimerization of the terminal nucleotides in adjacent oligonucleotides. The linkages are susceptible to direct photoreversal by 254 nm UV, as expected for cyclobutane-type thymine dimers, but they are not cleaved by the bacteriophage T4 endonuclease V, a dimer-specific DNA repair enzyme. We demonstrate that the ligating dimers are also resistant to photolyase from Escherichia coli. Although the phosphodiester backbone is not required for dimer formation, it is required for recognition of dimers by these DNA repair enzymes. We discuss the possibility that high molecular weight polynucleotides in primordial seas might have been generated from oligonucleotides by pyrimidine dimerization under the intense solar UV flux unattenuated by an ozone layer.

Endonucleases↗

PubMed2005

Genetic factors influencing pyrimidine-antagonist chemotherapy.

Pyrimidine antagonists, for example, 5-fluorouracil (5-FU), cytarabine (ara-C) and gemcitabine (dFdC), are widely used in chemotherapy regimes for colorectal, breast, head and neck, non-small-cell lung cancer, pancreatic cancer and leukaemias. Extensive metabolism is a prerequisite for conversion of these pyrimidine prodrugs into active compounds. Interindividual variation in the activity of metabolising enzymes can affect the extent of prodrug activation and, as a result, act on the efficacy of chemotherapy treatment. Genetic factors at least partly explain interindividual variation in antitumour efficacy and toxicity of pyrimidine antagonists. In this review, proteins relevant for the efficacy and toxicity of pyrimidine antagonists will be summarised. In addition, the role of germline polymorphisms, tumour-specific somatic mutations and protein expression levels in the metabolic pathways and clinical pharmacology of these drugs are described. Germline polymorphisms of uridine monophosphate kinase (UMPK), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and methylene tetrahydrofolate reductase (MTHFR) and gene expression levels of OPRT, UMPK, TS, DPD, uridine phosphorylase, uridine kinase, thymidine phosphorylase, thymidine kinase, deoxyuridine triphosphate nucleotide hydrolase are discussed in relation to 5-FU efficacy. Cytidine deaminase (CDD) and 5'-nucleotidase (5NT) gene polymorphisms and CDD, 5NT, deoxycytidine kinase and MRP5 gene expression levels and their potential relation to dFdC and ara-C cytotoxicity are reviewed.

Antineoplastic Agents↗

PubMed1968

Precursors of the pyrimidine moiety of thiamine.

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-(14)C]methionine or [G-(14)C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-(14)C]glycine and [2-(14)C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.

Amino Acids↗

PubMed1975

Free radicals formed by exposure of pyrimidine solids to sodium atoms: an electron spin resonance study.

Sodium atoms have been deposited on various pyrimidine powders under high vacuum at 77 degrees K and the electron spin resonance spectra of the resulting free radicals have been observed. Generally, the spectra show that the electron of the Na goes into a molecular orbital of the pyrimidine ring and the Na+ ions become attached to a carbonyl oxygen of the resulting pyrimidine anion. In 5-fluorouracil and 5-chlorouracil, however, the halogen is evidently abstracted by the Na to form NaF or NaC1 and the neutral uracil radical. Thymine shows evidence for H-addition radicals as well as the Na+-[thymine]-complex. The H source for the addition radicals may be an H-2-0 impurity in the sample, with which the Na atoms combine to release the H atoms. In addition to a resonance with g equals 2.00 from the pyrimidine anion radical, broad resonances with g greater than 2 were observed for 5-bromouracil, 5-chlorouracil and 5-iodouracil, as well as for alloxan and cytosine. These resonances, generally unstable at room temperature, are believed to arise from electrons trapped in interstitial sties or vacancies in the lattice.

Alloxan↗

PubMed1997

Investigation of the pyrimidine preference by the c-Myb DNA-binding domain at the initial base of the consensus sequence.

The principal determinant of the pyrimidine preference by the c-Myb DNA-binding domain at the initial base of the consensus sequence was investigated by mutation of both the protein and the DNA base pairs, with analysis by a filter binding assay. Amino acid residue 187 was revealed to interact with the pyrimidine base position, as estimated from our previous complex structure. Unexpectedly, since the pyrimidine preference is retained even in the Gly187 mutant, the principal origin of the base specificity should not occur via the direct-readout mechanism, but by an indirect-readout mechanism, namely in the intrinsic "bendability" of the pyrimidine-purine step of the DNA duplex. A significant but rather small positive base pair roll is detectable in the conformation of DNA in complex with the c-Myb DNA-binding domain. Following the conventional chemical rules of the direct-readout mechanism, amino acid mutagenesis at position 187 yielded several new base preferences for the protein.

Amino Acid Sequence↗

PubMed1998

Leflunomide inhibits pyrimidine de novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans.

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic-acid dehydrogenase. Leflunomide (25 and 50 microM), like Brequinar (0.5 and 1 microM), a demonstrated dihydroorotic-acid dehydrogenase inhibitor, was cytostatic, not cytotoxic, with proliferation being halted in the G1 phase. Both drugs restricted the normal 4-8-fold mitogen-induced expansion of pyrimidine pools over 72 h to concentrations found in nonstimulated T-cells and [14C]bicarbonate incorporation into UTP, ATP, and GTP. Uridine (50 microM) restored expansion of all pools, but [14C]bicarbonate incorporation into ATP and GTP only, not UTP. [14C]Hypoxanthine salvage was also restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors. [14C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 microM Leflunomide. 100 microM Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidine-related effects reported for Leflunomide at 100 microM and above.

Adenosine Triphosphate↗

PubMed1999

Poly(L-lysine)-graft-dextran copolymer promotes pyrimidine motif triplex DNA formation at physiological pH. Thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗

PubMed1999

The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, Chlorella virus-pdg.

The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Site-directed mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed.

Bacteriophage T4↗

PubMed2000

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗

PubMed1987

Radioprotection of pyrimidines by oxygen and sensitization by phosphate: a feature of their electron adducts.

The reactions of the electron adducts of thymine, uracil and 1,3-dimethylthymine in the presence of phosphate buffer and low oxygen concentrations have been investigated. Oxygen reacts with the pyrimidine electron adducts and their O(4)-protonated forms to restore the pyrimidine and produce O2-./HO2.. Thus oxygen acts as a radiation protector. In the presence of high buffer concentrations the electron adducts are irreversibly protonated at C(6). On reaction of this radical with oxygen no restitution of the original pyrimidine occurs and the pyrimidine is destroyed. Thus phosphate buffer acts as a radiation sensitizer. Some speculations are made as to the possible relevance of these reactions to biological systems.

Oxygen↗