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D Shugar

Publications and source records attributed to D Shugar.

At least 91 records · Page 5Linked to original sources
PubMed1984

Hydroxylamine and methoxyamine mutagenesis: displacement of the tautomeric equilibrium of the promutagen N6-methoxyadenosine by complementary base pairing.

The imino-amino tautomeric equilibrium of the promutagenic adenosine analogue N6-methoxy-2',3',5'-tri-O-methyladenosine [OMe6A(Me)3], in solvents of various polarities, has been studied with the aid of 1H and 13C NMR spectroscopy. The high energy barrier (free enthalpy delta G = 80 +/- 5 kJ X mol-1) between the two tautomeric species renders possible direct observation of the independent sets of all 1H and 13C signals from each of them. The equilibrium ranges from 10% imino in CCl4 to 90% in aqueous medium. Thermodynamic parameters, including energy barriers and lifetimes, were calculated from the temperature dependence of the equilibrium. Essentially similar results prevail for the promutagenic N6-hydroxy analogue. The conformations of the sugar moieties, and of the base about the glycosidic bond, for both tautomers are similar to those for adenosine. The conformation of the exocyclic N6-OCH3 group, which determines the ability of each species to form planar associates (hydrogen-bonded base pairs), has also been evaluated. Formation of autoassociates of OMe6A(Me)3 and of heteroassociates with the potentially complementary 2',3',5'-tri-O-methyluridine and -cytidine, in chloroform solution, was also investigated. The amino form base pairs with uridine and the imino form with cytidine. Formation of a complementary base pair by a given tautomeric species was accompanied by an increase of up to 10% in the population of this species and a concomitant decrease in population of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1984

Tautomerism and conformation of the promutagenic analogue N6-methoxy-2',3',5'-tri-O-methyladenosine.

N6-Methoxy-2',3',5'-tri-O-methyladenosine crystallizes in space group P2(1)2(1)2(1) with cell dimensions a = 4.693, b = 11.412, c = 31.741 A. Least-squares refinement of diffractometer data converged at R = 0.038. The location of a hydrogen atom at N1 and the observed bond lengths and bond angles indicate unequivocally the imino tautomer of the adenine moiety. The N6-methoxy group is oriented syn to N1 and the glycosidic torsion angle XCN is -3.6 degrees, i.e. in the anti range. The furanose ring has a C2'-exo/C3'- endo pucker (P = 0.9 degrees) and is unusually flattened (tau m = 30.0 degrees). The conformations of the O-methyl groups of the ribose ring are compared with those of monomethylated nucleosides, including the biologically important 2'-O-methyl nucleosides. Evidence is presented for the existence of C-H ... N intermolecular hydrogen bonds between adenine moieties. Bearing in mind that N6-methoxyadenosine is a promutagenic analogue, the results are compared with those for the corresponding promutagenic N4-methoxycytidine. They are also discussed in relation to the tautomerism, the conformation of the N6-methoxy group, and the associated base-pairing abilities in the absence and presence of polymerases.

Adenosine↗
PubMed1984

Studies on the dynamic syn-anti equilibrium in purine nucleosides and nucleotides with the aid of 1H and 13C NMR spectroscopy.

Analyses of 1H and 13C NMR spectra have been utilized to extend studies on the dynamic equilibrium syn-anti about the glycosidic bond of purine nucleosides and nucleotides. With the aid of chemically synthesized model analogous in fixed syn and anti conformations, and the introduction of appropriate corrections for the conformation of the exocyclic chain of the sugar moiety, it is possible to evaluate quantitatively the relative populations of the syn and anti conformers from the experimentally observed chemical shifts of H(2') and C(2'). The resulting agreement between the data based on H(2') chemical shifts with those deduced from C(2') chemical shifts extends the validity of this procedure, and furnishes more accurate results than those previously based on uncorrected H(2') chemical shifts alone. The overall findings are briefly compared with those derived from measurements of proton relaxation times and the Overhauser effect, as well as by X-ray diffraction in the solid state. Attention is drawn to the potential utility of the results, including chemical shift data, in studies on interactions of nucleosides and nucleotides with appropriate enzyme systems.

Carbon Isotopes↗
PubMed1984

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis.

Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage phi W-14 DNA (in which half the thymine residues are replaced by alpha-putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37 degrees C after DNA uptake at 30 degrees C in the presence of low concentrations of phi W-14 DNA (0.1 microgram/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of phi W-14 DNA was enhanced by an increase in its concentration (to 0.5-1 microgram/ml), or by increasing the temperature of uptake (to 37 degrees C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 microgram/ml) or untreated with phi W-14 DNA, and incubated for 20 min at 37 degrees C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with phi W-14 DNA. The influence of a high concentration of phi W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗
PubMed1984

Acyclonucleosides: acyclobenzimidazole nucleoside and nucleotide analogues and conformations of the acyclic chains by means of NMR spectroscopy.

A number of acyclo nucleosides of benzimidazole derivatives has been synthesized, in which the benzimidazole ring includes substituents at C(5), C(6) and C(2). The acyclic chains which replace the sugar moiety are 2',3'-dihydroxypropyl, 2'-hydroxyethoxymethyl and 1',5'-dihydroxy-4'-hydroxymethyl-3'- oxypentyl -2' (R), each of which corresponds to some fragment of the ribose ring. 1H NMR spectroscopy has been employed to determine the conformations of these acyclic chains in solutions of fully deuterated dimethylsulfoxide and methanol, utilizing for this purpose vicinal proton-proton coupling constants, and the new Karplus relation developed by Haasnoot , de Leeuw & Altona ( Tetrahedron , 36, 2783-2792, 1980). The data thus obtained are compared with those available for the solid state from X-ray diffraction data, and should be applicable to other classes of acyclonucleosides . Nucleotides of the three types of acyclo benzimidazole nucleosides have also been prepared, and their susceptibilities to snake venom 5'-nucleotidase examined. In contrast to acycloG , the nucleoside analogues did not exhibit significant in vitro activity against herpes simplex virus type 1 or influenza virus.

Benzimidazoles↗
PubMed1984

Mutagenesis induced by 5-bromouracil and methyl methane sulfonate: role of DNA polymerase I.

A polA1 mutation in the DNA polymerase I gene of E. coli results in a drastic reduction of the frequency of mutagenesis induced by 5-bromo-2'-deoxyuridine (BUdR). Comparisons of the effect of a polA1 mutation on mutagenesis induced by methyl methane sulfonate (MMS), ultraviolet irradiation (UV) and 2-aminopurine (2-AP) demonstrated that a similar effect of a polA1 mutation is observed with MMS. This effect is much less marked with UV-and-2-AP-induced mutagenesis. It follows that DNA polymerase I plays a key role in the process of mutagenesis induced by BU and MMS. Bearing in mind that mutagenesis provoked by UV, MMS and BU involves participation of the accompanying induced error-prone system, the sources of the differences in requirement for DNA polymerase I are critically examined.

Bromodeoxyuridine↗
PubMed1984

5-substituted arabinofuranosyluracil nucleosides: synthesis and antiviral properties.

A number of 5-alkyl (ethyl, propyl, isopropyl, butyl) analogues of araU, their alpha-anomers and N3-isomers have been synthesized by a number of different procedures, based on the catalytic condensation of the appropriate 5-alkyl-2,4-bis-(trimethylsilyloxy)-pyrimidine with 2,3,5-tri-O-benzyl-alpha-D-arabinofuranosyl chloride. The resulting protected nucleosides were deblocked by a new procedure based on the use of BF3 X Et2O in C2H5SH. The chloromercuri derivative of araU, on reaction with allyl chloride in the presence of Li2PdCl4, gave the 5-allyl derivative, which was catalytically reduced to the corresponding 5-propyl analogue. The antiviral activities of these compounds have been evaluated. 5-Allyl-araU showed moderate specific activity (MIC 20 micrograms/ml) against herpes simplex type 1 virus in PRK cell cultures. Structure-activity relationships are discussed for the 5-alkyl deoxy- and arabino- uracil nucleoside series.

Antiviral Agents↗
PubMed1984

Hydroxylamine and methoxyamine mutagenesis: tautomeric equilibrium of the promutagenic, N6-methoxyadenosine in solvents of different polarities.

Ultraviolet (UV) and Infrared (IR) spectroscopy have been applied to a study of the tautomeric equilibrium, in solvents of varying polarities and differing hydrogen bond donor-acceptor properties, of the promutagenic analogue N6- methoxyadenosine , the product of the reaction of the mutagen methoxyamine with adenosine. In the non-polar solvent CCl4, the analogue is predominantly in the amino form, with KT approximately 10. On transfer from CCl4 to chloroform, dimethyl sulfoxide, and water, the equilibrium is shifted stepwise towards the imino form, attaining a KT approximately 10 in favour of the imino species in aqueous medium. Both the UV and, particularly, the IR spectra exhibit two sets of absorption bands which were assigned to the respective tautomers , in dynamic equilibrium with each other. The significance of the foregoing results in the mechanism of hydroxylamine (and methoxyamine) mutagenesis is considered. It is also shown that base pairing of each tautomeric species is significantly dependent on the conformation of the exocyclic N6-methoxy group. It is further demonstrated that infrared spectroscopy provides data which both supplement and extend those obtained by NMR spectroscopy, under conditions where application of the latter is technically limited.

Adenosine↗
PubMed1983

Hydroxylamine mutagenesis: observation of inverted Watson-Crick base-pairing between N4-methoxycytosine and adenine with the aid of natural-abundance high-resolution 15N NMR spectroscopy.

Natural-abundance high-resolution 15N NMR spectra have been recorded for 1-methyl-N4-methoxycytosine (the product of reaction of 1-methylcytosine with the mutagen methoxyamine), 2',3',5'-tri-O-methyladenosine, and a 1:1 mixture of the two, all in chloroform solution. The spectra were obtained by application of a sequence of impulses of the type referred to as insensitive nuclear enhancement by polarization transfer (INEPT). Assignments of all the nitrogen resonances were based on nitrogen-proton coupling constants and literature data. Analysis of the 15N chemical shifts demonstrated the formation of base pairs with hydrogen bonds involving the ring N(1) and an N6-H of adenine, and the N(3)-H and C(2) = O of N4-methoxycytosine, viz. N(1) . H-N(3) and N6-H . O2 = C. The hydrogen-bond-stabilized complex formed thus involves an inverse type of Watson-Crick base-pairing. The data excluded formation of Hoogsteen-type base-pairing. The results are considered in relation to hydroxylamine and methoxyamine mutagenesis, to types of base-pairing at the monomer and polymer levels, and to interpretations of 15N NMR spectra of heterocyclic rings in general.

Adenine↗
PubMed1983

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA.

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.

Bacteriophage lambda↗
PubMed1983

Identification of uracil as a major lesion in E. coli DNA following the incorporation of 5-bromouracil, and some of the accompanying effects.

Cultivation of E. coli cells in the presence of 5-bromodeoxyuridine (BUdR) leads to formation of lesions in the cellular DNA which affect its secondary structure, as reflected by changes in temperature profiles. Such DNA contains single-stranded regions susceptible to endonuclease S1. One of the major sources of the BU-induced lesions appears to be dehalogenation of incorporated 5-bromouracil (BU) residues, with accompanying formation of uracil. The presence of uracil residues in such DNA was demonstrated directly by chromatography of hydrolyzates, and by the susceptibility of such residues to uracil-DNA glycosylase. The number of uracil residues was dependent on the extent of damage in the DNA, and decreased during the DNA repair that accompanied reactivation of bromouracil-inactivated cells. Dehalogenation of incorporated BU presumably results in formation of apyrimidinic sites by uracil-DNA glycosylase, and then single-strand nicks either by AP-endonuclease and/or dehalogenation. The findings are relevant to the mechanism of BU-induced mutagenesis.

Bromodeoxyuridine↗
PubMed1983

Structure of the planar complex of N4-methoxycytosine with adenine, and its relevance to the mechanism of hydroxylamine mutagenesis.

Infrared, and 1H- and 13C-NMR spectroscopy has been applied to a study of the planar interaction in apolar media between 1-substituted N4-methoxycytosine (and the corresponding 5-methyl analogue) and 9-substituted adenines. In both chloroform and carbon tetrachloride solutions, the exocyclic N4-methoxy group of N4-methoxycytosine, and of 5-methyl N4-methoxycytosine (which is in the oxime form under these conditions), is so oriented that it is predominantly syn to the ring N(3), and neither compound forms planar auto-associates. In chloroform solution, both the N(3)-H and the C(2) = O interact weakly with the solvent, the interaction being of the nature of non-typical hydrogen bonding. The 13C-NMR chemical shift of the C(2) of N4-methoxycytosine is modified during formation of hetero-associates with 9-substituted adenine, in accordance with the C(2) = O of the former being the acceptor of an adenine amino proton. The resulting planar hetero-associate is a non-Watson-Crick type of base-pair. This was further substantiated by infrared absorption studies of the carbonyl frequency during complex formation. The results are examined in the light of the mechanism of hydroxylamine (and methoxyamine) mutagenesis.

Adenine↗
PubMed1982

5-Substituted 2'-deoxyuridines: correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase.

A large variety of 5-substituted 2'deoxyuridines (dUrds) and 2'-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their Ki/Km values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP greater than 5-formyl-dUMP greater than 5-fluoro-dUMP greater than 5-oxime of 5-formyl-dUMP greater than 5-azidomethyl-dUMP greater than (E)-5-(2-bromovinyl)-dUMP. The ki/Km values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37 degrees). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5'-monophosphates followed by an inhibition of dTMP synthetase.

Animals↗
PubMed1982

Specific inhibition of hnRNA synthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Requirement of a free 3'-hydroxyl group, but not 2'- or 5'-hydroxyls.

Five structural analogues of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), all with modified sugar moieties, have been examined for their inhibitory activities on RNA transcription in salivary glands of Chironomus tentans. The well-known ability of the parent DRB at 65 microM concentration to selectively inhibit hnRNA/mRNA synthesis by approx. 90% was essentially abolished on methylation of the 3'-OH; but, at an overdose the analogue suppressed labeling of all RNA classes examined (hnRNA/mRNA, rRNA, 4-5 S RNA) by 70-80%. By contrast, the 2'-O-methyl derivative of DRB was almost as effective as DRB itself in blocking transcription of hnRNA/mRNA genes. Blocking of both the 2' and 3' hydroxyls (2',3'-O-isopropylidene-DRB) completely abolished inhibitory activity, irrespective of the concentration employed. The 5'-deoxy-5'-chloro derivative of DRB was only slightly less effective than the parent DRB. An unusual aspect of the activities of 2'-O-methyl-DRB and 5'-deoxy-5'-chloro-DRB was their ability to stimulate synthesis of 4-5 S RNA by 25-45%. Also investigated was the influence of the various analogues on the rate of formation of [3H]UTP from [3H]uridine used as an RNA precursor. The rate of such formation of [3H]UTP was suppressed 2-6-fold by treatment with 2'-O-methyl or 3'-O-methyl-DRB, but was unaffected by 5'-deoxy-5'-chloro-DRB or 5,6-dichloro-1-alpha-D-arabinofuranosylbenzimidazole. The overall data point to the importance of a free 3'-OH in the ribose moiety of DRB for selective inhibitory activity. The alpha-D-arabinofuranosyl analogue, although less selective in inhibition of RNA transcription, still exhibits about 50% of the activity of DRB.

Amanitins↗
PubMed1982

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines.

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, RAji and TK- (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK-. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log ID50 (median inhibitory dose) for L1210 cell growth, on the one hand, and log ID50 for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log ID50 values for the two human lymphoblast cell lines. However, there was no significant correlation (r less than 0.40) either between the log ID50 for the TK- Raji cells and the parental TK+ Raji cells, or between the log ID50 for the TK- L1210/BdUrd cells and the parental TK+ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.

Animals↗
PubMed1982

Pyrimidine ribonucleoside phosphorylase activity vs 5- and/or 6-substituted uracil and uridine analogues, including conformational aspects.

The pyrimidine ribonucleoside phosphorylase from Salmonella typhimurium phosphorylyses 6-methyluridine, a uridine analogue sterically constrained to the syn conformation about the glycosylic bond, as effectively as uridine itself. In conjunction with the observation that 3-methyluridine is a very poor substrate compared to 5-methyluridine and 5,6-dimethyluridine, it follows that the phosphorolysis reaction involves the initial conversion of uridine, and other 5-substituted uridines (including 5-fluorouridine), to the syn conformation during interaction with the enzyme. Furthermore, and consistent with the foregoing, the enzyme recognizes as substrates, to varying degrees, the N(3)-ribosides of xanthine and uric acid, and will also catalyze the formation of these ribosides from the corresponding purines, which may be considered formally as 5,6-disubstituted uracils. Similar observations are reported for the synthetic 5,6-trimethyleneuridine. The enzyme does not, however, recognize 6-methyluracil and 5,6-tetramethyleneuridine in the reverse, synthetic, reaction. The conformational aspects of these reactions are discussed. Since it was previously shown that 6-methyluridine is an equally effective substrate for the pyrimidine phosphorylase of primary rabbit kidney cells, at least some of these conformational requirements apply to the enzyme from mammalian sources, and are consequently of relevance in the design of chemotherapeutic agents, for which some examples are cited.

Molecular Conformation↗
PubMed1982

Uptake and fate of bacteriophage phi W-14 DNA in competent Bacillus subtilis.

Phage phi W-14 DNA (in which one-half of the thymine residues are replaced by alpha-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of phi W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of phi W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that phi W-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of phi W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of phi W-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of phi W-14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded phi W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in phi W-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.

Azides↗