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Biomedical subjects

D Shugar

Publications and source records attributed to D Shugar.

At least 145 records · Page 8Linked to original sources
PubMed1977

Synthesis and determination of antiviral activity of the 2'(3')-O-methyl derivatives of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide).

Diazomethane treatment of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) in the presence of SnCl2 as catalyst led to quantitative formation of the 2'-O-methyl and 3'-O-methyl derivatives of the parent compound. The products were successfully fractionated on a basic ion-exchange column and isolated in crystalline form. Indentification was based on the elution sequence from the column and on 1H NMR spectroscopy. Both derivatives were found to be inactive, relative to the parent compound, against several virus types in cell culture. Unlike ribavirin itself, the 2'(3')-O-methyl derivatives did not suppress cellular DNA synthesis. NMR data showed that the loss of biologic activity upon 2'(3')-O-methylation was not due to a change of conformation of the nucleoside sugar moiety.

Animals↗
PubMed1977

Kinetics of deamination of cytosine nucleosides with etherified sugar hydroxyls.

The kinetics of deamination of derivatives of the therapeutically important 1-beta-D-arabinofuranosylcytosine with etherified (methylated) sugar hydroxyls has provided additional direct evidence for involvement of the 2'-hydroxyl in intramolecular catalysed deamination. In the case of 1-beta-D-lyxofuranosylcytosine, the kinetics of deamination of its 2'-O-methyl and 3'-O-methyl derivatives pointed to similar involvement of the "up" 2'-OH in intramolecular catalysed deamination. Participation by the 3'-OH, which is also in the "up" position, was excluded. A qualitative correlation was shown to exist between the electron density distributions on C(4) and C(6) of the cytosine rings in cytosine, 1-methylcytosine and cytidine, and their relative susceptibilities to deamination.

Cytidine↗
PubMed1977

Preparation of O'-METHYL derivatives of 9-beta-D-xylofuranosyladenine.

Treatment of the therapeutically important 9-beta-D-xylofuranosyladine in strongly alkaline medium with dimethyl sulphate led principally to etherification of sugar hydroxyls and, to a minor extent, to formation of products with a methylated exocyclic amino group. The various O'-methyl derivatives of xylofuranosyladenine were fractionated on a strongly basic ion exchange column, and isolated in pure form. Also isolated was 9-beta-D-xylofuranosyl-N6-methyladenine and its 2'-O-methyl derivative. The products were identified from their 1H NMR spectra, for which extensive data are tabulated. The susceptibilities of the various derivatives to calf intestinal adenosine deaminase were examined in relation to those of other adenine nucleosides; in particular, 5'-O-methylation led to total loss of substrate properties for the riboside, arabinoside and xyloside of adenine.

Adenosine↗
PubMed1977

Preparation and properties of formycin analogues methylated on the pyrazolo ring nitrogens and/or the ribose cis-hydroxyls.

1. Diazomethane treatment of formycin A in the presence or absence of SnCl2 as catalyst, was used for the preparation of the 2'-O-methyl, 3'-O-methyl, N1-methyl and N2-methyl derivatives. The four possible dimethylated derivatives, 2'(3")-O,N1(N2)-dimethylformycins, were obtained by controlled treatment of formycin with diazomethane in the presence of SnCl2, and subsequent column chromatography for product isolation. 2. All the foregoing products were characterized and identified by chromatography, ultraviolet absorption spectra, and proton magnetic resonance spectroscopy. Extensive u.v. spectral data, and spectrally determined pK values, for the various derivatives are presented. 3. N2-Methylformycin B was also prepared by enzymatic deamination of the parent N2-methylformycin A. 4. The sequence of elution of N1-methylformycin and N2-methylformycin on a strongly basic ion exchange column suggested that the latter is in the syn conformation. The susceptibility of N2-methylformycin to adenosine deaminase shows that this analogue may adopt the anti conformation on reaction with the enzyme. 5. The active species in the SnCl2-catalysed monomethylation of the 2'(3') cishydroxyls of ribonucleosides by diazomethane was shown to be an organo-tin product of the reaction of SnC2 with diazomethane. This product, not identified, contained no nitrogen or chlorine. 6. A simple column chromatographic procedure is described for the desalting of heterocyclic bases and their nucleosides with pK values for ring protonation down to about 0.

Antibiotics, Antineoplastic↗
PubMed1976

Discrimination of competent Bacillus subtilis with respect to ribonucleic acids.

A study has been made of the affinity of double-stranded helical RNA for DNA receptors in competent Bacillus subtilis. In competition experiments, using homologous and heterologous DNA samples which had been sheared to molecular weights comparable to that of the RNA (about 2 x 10(6)), and which still exhibited appreciable competition in DNA uptake experiments, the replicative form of phage f2 RNA showed no evidence of affinity for receptor sites. A second double-stranded RNA preperation from a widely different source, a mycophage of Penicillium chrysogenum, behaved similarly to the f2 RNA. Transfer RNA and 23S ribosomal RNA, which reduce transformation frequencies in pneumococcus, did not compete for B. subtilis receptors. Lack of competition was not due to enzymatic degradation of the RNA, since the latter was recovered intact following exposure to competent cells. Under conditions where homologous native DNA undergoes normal uptake, there was virtually no uptake of native double-stranded RNA. The results are examined in the light of reports on transformation by RNA and DNA-RNA hybrids, and also in relation to the characterization of the specificity of cell-nucleic acid interactions.

Bacillus subtilis↗
PubMed1976

Mechanism of hydroxylamine mutagenesis. Crystal structure and conformation of 1,5-dimethyl-N4-hydroxycytosine.

The crystal structure of the title compound, which is a formal analogue of 5-methyl-N4-hydroxycytosine nucleosides, has been determined by X-ray diffraction. The space group is P2(1)/c with a = 7.368 (2), b = 12.096 (3), c = 9.192 (4) A, beta = 113.94 (3) degrees. Three-dimensional intensity data were collected with a four-circle diffractometer, and the structure was refined by block-diagonal least-squares to R = 0.053. The compound is in the imino form, and the exocyclic N4-OH is located essentially in the plane of the pyrimidine ring, and syn to the ring (N(3). There is an intramolecular hydrogen bond involving the N(3)-H as donor and O(4) as acceptor, viz. N(3)-H(31)----O(4)-H. With this conformation, which probably prevails also in solution, the compound would be unable to participate in normal Watson-Crick base pairing. It is shown that a similar situation may prevail for N4-hydroxycytosine nucleosides. The implications with regard to the molecular mechanism of hydroxylamine mutagenesis, with particular reference to the T-even bacteriophages, are discussed. Analogous considerations are applied to an examination of the possible behaviour of hydroxylamine-modified adenine nucleosides.

Cytosine↗
PubMed1976

Conformation in aqueous medium of the neutral, protonated and anionic forms of 9-beta-D-arabinofuranosyladenine.

Proton magnetic resonance spectroscopy was employed to study the solution conformations of the neutral, protonated and dissociated forms of the therapeutically active 9-beta-D-arabinofuranosyladenine (araA). In particular, in strongly basic medium, increasing alkalinity led to pronounced changes in chemical shifts and coupling constants of some pentose protons, due to ionization of the pentose hydroxyls, especially the 2'-OH. The neutral form of araA may be characterized as approx. 25% C(2')endo and approx. 60% gauche-gauche, hence somewhat different from that of the therapeutically active 1-beta-D-arabinofuranosylcytosine (araC). By contrast, the conformations of the anionic forms of both of these are identical, predominantly (greater than 80%) C(2')endo and gauche-gauche. With the aid of the 3'-O-methyl derivatives of araA and araC, where only the 2'-OH ionizes, and the accompanying conformational changes are similar, it follows that the conformation C(2')endo and gauche-gauche for all the foregoing is constrained to this form via a strong intramolecular hydrogen bond, viz. O(5')H...O(2')(-). The influence of the foregoing hydrogen bond on the chemical shifts of the adenine H(8) in the araA anion points to the existence of the latter in the form anti. A similar effect of the doubly ionized phosphate group on H(8) in 5'-araAMP shows the nucleotide to also prefer the form anti, as previously demonstrated for 5'-AMP. The conformations of the sugar rings of the neutral forms of araA and adenosine in aqueous medium differ appreciably, whereas in the solid state they are very similar. PMR spectroscopy is shown to be an effective method for following sugar hydroxyl dissociation. The extent of ionization of a given hydroxyl is provided by the resulting chemical shifts of neighbouring (geminal and vicinal) protons. When ionization is accompanied by a change in conformation, the process may be followed also by changes in proton-proton vicinal coupling constants.

Adenine Nucleotides↗
PubMed1976

Human pancreatic-type ribonucleases with activity against double-stranded ribonucleic acids.

Purified acid-thermostable ribonuclease (Ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) from human pancreas degrades double-stranded RNA at 2% the rate for single-stranded RNA. The activities against single-stranded RNA and double-stranded RNA were shown to be due to a single enzyme with properties similar to bovine pancreatic RNAase A. For purposes of comparison the activities against double-stranded RNA of crystalline ribonucleases of the whale, rat and cow were assayed and found to be 0.4%, 0.03% and 0.003%, respectively, of their activities against single-stranded RNA. Both human serum and urine contain RNAse components of pancreatic origin which hydrolyze double-stranded RNA at 2% and 0.4%, respectively, of the rates against single-stranded RNA. By contrast, purified acid-thermostable RNAases from human spleen and liver hydrlyze double-stranded RNA at least 20-fold more slowly than human pancreatic RNAase, relative to the corresponding rates against single-stranded RNA. The human pancreatic and serum enzymes exhibit appreciable activity against the poly(C) component of the double-stranded poly(I)-poly(C); they also attack poly(C) itself at approximately 25 times the rate for poly(U) and at more than 50 times the rate for single-stranded RNA.

Animals↗
PubMed1976

Novel activity of potato nucleotide pyrophosphatase.

The classical Kornberger-Pricer procedure for purification of potato nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to yield a preparation purified 2500-fold. In addition to the known activity against pyrophosphate linkages in pyrophosphates located at the 5'-OH of nucleosides, and phosphodiester linkages in aryl esters of nucleoside-5'-phosphates, the enzyme has now been shown to catalyze the cleavage of: (a) aryl esters of nucleoside-3'-phosphates and orthophosphates, (b) nucleotide pyrophosphate linkages of the type (3')-pp-(3'), and (c) pm7G from m7GpppGm-terminated fragments of viral mRNA. Activities against aryl esters of nucleoside-3'- and 5'-phosphates, and NAD, were shown to be due to the same protein by three criteria: (a) constant ratio of activities during purification and gel electrophoresis, (b) identical chromatographic properties in various systems, and (c) similarities in pH-dependence, heat inactivation, and the effects of cations and other substances. Since potato nucleotide pyrophosphatase does not exhibit exonuclease or phosphatase activities against natural substrates for the latter enzymes, but does cleave synthetic aryl esters of nucleotide-3'- and 5'-phosphates and of orthophosphate, it follows that these substrates are not suitable for detection of such activities in higher plants.

Hydrogen-Ion Concentration↗
PubMed1976

Methylation of adenosine in strongly alkaline medium: Preparation and Properties of o'-methyl derivatives of adenosine and N6-methyladenosine.

In strongly alkaline aqueous medium, 9-substituted adenines, including adenine nucleosides, are relatively resistant to alkylation of the ring nitrogens and the exocyclic amino group. This fact was utilized to obtain the various possible Omicron'-methyl derivatives of adenosine by dimethyl sulfate treatment of the latter in alkaline medium, followed by separation of the products on a Dowex OH-column. In strongly alkaline aqueous dimethyl sulfoxide, several derivatives additionally methylated on the amino group were obtained, including N6,O2'-dimethyladenosine, a component located recently at the 5' terminus of animal cell and viral mRNAs. The latter was also prepared by diazomethane methylation of N6-methyladenosine in the presence of SnCl2. Alkylation in alkaline medium possesses the advantage that the products are not limited to those involving etherification of cis hydroxyls and is applicable to adenine nucleosides with sugar components other than ribose. The properties of the various O'-methylderivatives, including proton magnetic resonance data, are presented in detail.

Adenosine↗
PubMed1976

Preparation and properties of an analogue of poly(A) and poly(G): poly(isoguanylic acid).

Isoguanosine-5'-pyrosphosphate, in the presence of an oligonucleotide primer, was polymerized by Escherichia coli polynucleotide phosphorylase under conditions analogous to those required for polymerization of 5'-GMP. The resulting poly(isoguanylic acid), poly(isoG), was a multistranded helix with a stability considerably higher than that of poly(G), and fully resistant to various nucleolytic enzymes. The polymer exhibited a two-step temperature transition profile in moderately alkaline propylene glycol. Alkaline titration in aqueous medium, by ultraviolet and circular dichroism spectroscopy, showed two clearly defined transitions, the second of which was fully cooperative. The accompanying changes in sedimentation constants were consistent with a structure for poly(isoG) of a fourstranded helix, like neutral poly(G). In acid medium, spectral and potentiometric titrations demonstrated the existence of more than one transition in the pH range 6-12, with accompanying protonation of the isoguanosine residues. In neutral medium the polymer formed no complexes with other potentially complementary homopolymers. In acid medium, on the other hand, the protonated form of poly(isoG) did form a triple-stranded complex with poly(I), viz. 2poly(I) . poly(isoG)+. Possible structures are formulated for the neural and protonated forms of poly(isoG) which account for the two-step thermal transition in alkaline propylene glycol and on alkaline titration in aqueous medium. The nature of the protonated form, and its complex with poly(I) is also discussed.

Binding Sites↗
PubMed1976

Purification and properties of human acid-thermostable ribonucleases, and diagnosis of childhood pancreatic fibrosis.

Acid-thermostable ribonucleases were isolated from human pancreas, duodenal contents, liver, spleen, serum and urine, and purified 15--1000-fold. The pH optima, ionic requirements, and some of the specificity requirements, of these enzymes were investigated. The isolated enzymes formed two distinct groups: (a) The ribonucleases of the pancreas, duodenal contents and fraction A of serum and urine exhibit a pH optimum of 8.5, are inhibited by An2+ and Cu2+, and relatively rapidly hydrolyze the synthetic substrate uridine 3'-(alpha-naphthylphosphate); (b) the ribonucleases of the liver and spleen, and of fractions B of the serum and urine, with a pH optimum of 7, are less sensitive to An2+ and Cu2+, and exhibit negligible activity versus uridine 3'-(alpha-naphthylphosphate). Determination of the serum level of pancreatic-type ribonuclease activity, with the use of uridine 3'-(alpha-naphthylphosphate) or RNA as substrates, appears to be a valid diagnostic tool for pancreatic fibrosis in children.

Child↗
PubMed1976

Preparation and properties of poly 2'-O-ethylcytidylic acid.

Poly 2'0-ethylcytidylic acid (poly (Ce)) was prepared by polymerization of 2'-0-ethylcytidine-5'-pyrophosphate with Escherichia coli polynucleotide phosphorylase in the presence of Mn++, and its properties compared with those of poly (rC), poly (Cm) and poly (dC). The neutral form of pOLY (Ce) exhibits properties similar to those of poly (rC) and poly (Cm). It also forms an acid twin-stranded helix with a transition pH of 5.9 in 0.1 M NaCl. The neutral form readily forms a double-stranded helical complex with poly (rI). Relative to poly (Cm), replacement of the 2'-0-methyl by 2-0-ethyl leads to increased enhancement of the thermal stabilities of both the acid helical form of poly (Ce) and its complex with poly (rI).

Enzyme Activation↗
PubMed1976

Poly 2'-O-ethylcytidylate, an inhibitor and poor template for AMV reverse transcriptase.

Poly(2'-O-ethylcytidylate) is a poor template-primer for purified avian myeloblastosis virus reverse transcriptase; the relative activities of the template-primers poly(C)-oligo(dG), poly(Cm)-oligo(dG) and poly(Ce)-oligo(dG) are 23:16:1. A mixture of poly(Ce) and poly(dI) is inactive as template-primer, in agreement with the observed inability of these to form a helical complex. By contrast the inactivity of poly(Ce)-poly(I) is shown to be due to the influence of the 2'-O-ethyl residue. Poly(Ce) inhibits poly(A)-oligo(dT)-directed polymerase activity, with Ki = 3 muM, but marked inhibition with poly(A)-poly(dT) occurs only at low concentrations of the latter. Poly(Ce) did not inhibit template-primer activity of poly(C)-poly(dI) and poly(dC)-poly(dI). Qualitative physico-chemical studies show only partial complex formation between oligo(dG) and poly(C) and its 2'-O-alkyl analogues. This is discussed in relation to the widespread use of poly(C)-oligo(dG) as the template-primer for reverse transcriptase.

Avian Leukosis Virus↗
PubMed1976

Effect of cytosine, arabinoside, iododeoxyuridine, ethyldeoxyuridine, thiocyanatodeoxyuridine, and ribavirin on tail lesion formation in mice infected with vaccinia virus.

Mice infected intravenously with vaccinia virus develop characteristic lesions over the entire tail surface. This experimental virus infection presents a highly sensitive and reliable model for evaluating the antivaccinia activity of antiviral compounds. Ara-C (1-beta-D-arabinofuranosylcytosine), ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), IUdR (5-iodo-2'-deoxyuridine) as well as two novel analogs of IUdR, EtUdR (5-ethyl-2'-deoxyuridine), and NCSUdR (5-thiocyanato-2'-deoxyuridine), were found to inhibit the formation of vaccinia tail lesions, when administered intraperitoneally once daily for 7 days starting immediately after virus infection. The order of (decreasing) activity was: ara-C greater than IUdR greater than NCSUdR greater than ribavirin greater than EtUdR. Various drug combinations, involving IUdR + ara-C, NCSUdR + ara-C, NCSUdR + IUdR, NSCUdR + ribavirin, etc., were evaluated but none proved more efficacious than either compound administered alone.

Animals↗
PubMed1976

Preparation and properties of the O-methyl derivatives of 9-beta-D-arabinofuranosyladenine.

The previously reported resistance of 9-substituted adenines to ring alkylation in alkaline medium was profited from to prepare all seven possible O' methyl derivatives of the therapeutically important 9-beta-D-arabinofuranosyladenine (araA) by mild methylation of the latter with dimethylsulfate in aqueous alkaline medium. All the products were fractionated and isolated in a single step on a Dowex OH- column. The sequence of elution of the various derivatives was strikingly similar to that for the O' methyl derivatives of 1-beta-D-arabinofuranosylcytosine, previously reported, suggestive of a similar sequence of acidities of the sugar hydroxyls. The products were identified by various criteria, including PMR spectroscopy, extensive data for which are supplied. The 2'-O-methyl and 3'-O-methyl derivatives of araA exhibited appreciably lower susceptibility to calf intestinal adenosine deaminase than the parent araA. The 5'-O-methyl analogue was fully resistant to enzymatic deamination.

Chromatography, Ion Exchange↗