Mutagenicity of hydroxylamine: reaction with analogues of cytosine, 5(6)-substituted cytosines and some 2-keto-4-ethoxypyrimidines.
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Chemical Phenomena↗
Biomedical subjects
D Shugar
Publications and source records attributed to D Shugar.
Synthesis of uridine-3'-naphthylphosphate and cytochemical localization of ribonuclease by the azo-dye coupling technique.
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Azo Compounds↗
Reversible photochemical transformation of 5-fluorouracil analogues and poly-5-fluorouridylic acid.
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Chemical Phenomena↗
Viral and host-cell protein kinases: enticing antiviral targets and relevance of nucleoside, and viral thymidine, kinases.
Numerous targets are known for development of antiviral agents, and some significant successes have been achieved with nucleoside analogues. These are "activated" by phosphorylation by viral and/or host-cell nucleoside kinases, the final target being principally the viral polymerase. With latency of herpes viruses, the viral thymidine kinase may be the ultimate target. Less attention has been devoted to viral protein kinases as antiviral targets, largely because 5 years ago, these the study of such enzymes was considered "still in its infancy." In the interim, identification of viral and host-cell protein kinases involved in viral gene expression, and viral replication, has made impressive advances. In conjunction with current progress in development of specific inhibitors of cellular protein kinases, and the differences in sequence motifs between these and the viral enzymes, the latter are indeed attractive targets, as are also some host-cell protein kinases. Examples include, amongst others, the essential protein kinases of vaccinia virus; the nonsegmented negative-strand RNA viruses, all essentially dependent on host-cell kinases, e.g., protein kinase CK-II (casein kinase-II), for which good inhibitors, such as halogenated benzimidazoles and benzotriazoles, are known; herpes viruses, with emphasis on human cytomegalovirus, the UL97 gene of which codes for a protein kinase that, like viral thymidine kinases, "activates," by phosphorylation, a nonpeptide antiviral acyclonucleoside ganciclovir, an analogue of the antiherpes aciclovir. The latter, in turn, is active against animal cytomegaloviruses following phosphorylation by the products of their UL97 gene homologues. Attention is also directed to the antiviral activity of the cyclic phosphate of ganciclovir, a structural analogue of the second messenger cyclic GMP.
Animals↗
Interactions of calf spleen purine nucleoside phosphorylase with antiviral acyclic nucleoside phosphonate inhibitors: kinetics and emission studies.
Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.
Animals↗
Studies on purine-pyrimidine hydrogen bonded base pairing by means of absorption and emission sectroscopy.
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Hydrogen↗
Spectrophotometric determination of the pK values for dissociation of the sugar hydroxyls in pyrimidine arabinonucleosides.
Spectrophotometric titration in the ultraviolet has been employed to determine the pK values for dissociation of the sugar hydroxyls in pyrimidine arabinonucleosides and some of their O'-methyl and O'-ethyl derivatives. The order of dissociation of the sugar hydroxyls in the arabinofuranose ring was 2'-OH greater than 3'-OH greater than 5'-OH. The higher acidity (lower pK) of the 2'-OH was interpreted in terms of formation of an intramolecular hydrogen bond of the form 5'-OH....2'-O(--) and the accompanying changes in conformation of the arabinose ring. The various factors affecting the dissociation of specific hydroxyls in some of the O'-alkyl derivatives are discussed in relation to steric, conformational and other effects.
Arabinofuranosyluracil↗
Tautomerism of isoguanosine and solvent-induced keto-enol equilibrium.
Ultraviolet and infrared absorption spectroscopy, in aqueous and non-aqueous media, have been employed to study the tautomerism of 9-substituted isoguanines, including the nucleoside isoguanosine. With the aid of a series of model compounds, it was shown that 9-substituted isoguanines, and isoguanosine, in aqueous medium are predominantly in the form N (1) H, 2-keto-6-amino. In dioxane solution the tautomeric equilibrium is shifted in the direction of the enol form. The shift towards this form is accentuated for those analogues in which the exocyclic amino group is methylated. With the aid of N6,N6, 9-trimethylisoguanine, and 9-octyl analogue, the tautomeric constant was studied as a function of concentration, temperature, and solvent polarity, and the results applied to evaluate the tautomeric equilibria of 9-methylisoguanine and isoguanosine as a function of these variables. In general the enol form is favoured by a decrease in solvent polarity, by a decrease in concentration in dioxane, or an increase in temperature in chloroform solution. Syntheses are described for several N6 amino and methylamino derivatives of 2-methoxy-9-methylpurine, and 3-methyl-5-oxo-7,8-dihydroimidazo (2,1-i) purine, which served as an analogue of the unavailable 1,9-dimethylisoguanine.
Chemical Phenomena↗
Unusual photochemical transformation of 5-ethylorotate to 5-ethylidenehydroorotate.
Ultraviolet irradiation of 5-ethylorotate in aqueous medium led to formation of 5-ethylidenehydroorotate via a 1,3-hydrogen rearrangement from the ethylene group of the 5-substituent to the ring C6. The same product was also obtained by an alternate pathway via a hitherto unidentified intermediate. The structure of this unusual pyrimidine analogue, established in the solid state by X-ray diffraction, was shown with the aid of spectroscopic methods to be the same in solution, and some of its properties are described. Heating of 5-ethylidenehydroorotate in the solid state led by direct conversion to a mixture of the parent 5-ethylorotate and 5-ethyluracil. By contrast, ultraviolet irradiation in aqueous medium resulted in quantitative photochemical conversion of the photoproduct to 5-ethyluracil.
Hot Temperature↗
The crystal structure of the pyrimidine analogue calcium 5-ethylidenehydroorotate-1.5 hydrate, a photoproduct of ethylorotate.
X-ray diffraction methods have been employed to establish the crystal structure of a new, unusual pyrimidine analogue, 5-ethylidenehydroorotate, obtained by a photochemical rearrangement of 5-ethylorotate. Crystals of the calcium salt of the title compound are monoclinic, space group Pc, cell constants a=14.631, b=10.038, c=19.168 A, beta=137.7 degrees, and contain four molecules, two cations and three water molecules per asymmetric unit. The structure was solved by direct methods and refined to R=5.2% on the basis of 2653 diffractometer measured data. The four independent molecules represent two pairs of enantiomers with slightly differing conformations linked together by an intricate system of hydrogen bonding and Ca2+-coordination (pentagonal bipyrimidal). The structure of the compound in aqueous medium, established by spectral methods, is the same as that in the crystal.
Models, Molecular↗
A 1H NMR study of the syn-anti dynamic equilibrium in adenine nucleosides and nucleotides with the aid of some synthetic model analogues with fixed conformations.
The syn-anti equilibrium about the glycosidic bond in adenosine and some related analogues was studied by means of 1H NMR spectroscopy, with the aid of several model analogues fixed in given conformations either by intramolecular bonding, or by introduction of a bulky substituent. A model unambiguously and exclusively in the syn conformation is 8- (alpha-hydroxyisopropyl) adenosine; while one fixed in the anti conformation is 8,5'-anhydro-8-oxoadenosine. A new analogue, fixed in the high anti conformation, is 8,2-'O-isopropylidenearabinofuranosyladenine. Several additional new model compounds were synthesized and their properties are described. With the aid of these models, the syn-anti dynamic equilibrium was examined for adenosine and some related compounds in different solvent systems, and the conformer populations evaluated quantitatively. The validity of the procedure applied, and the accuracy of the results are critically examined, and compared with findings obtained by other procedures. Available literature data on the syn-anti equilibrium in other 8-substituted adenosines are re-analyzed in the light of the present results. An analysis is also presented of the interdependence of the various conformational parameter, i. e. conformation about the glycosidic bond and those of the sugar ring and exocyclic carbinol group, in adenosine and 2',3'-O-isopropylidenadenosine.
Adenine Nucleotides↗
Sensitive fluorimetric assay for adenosine deaminase with formycin as substrate; and substrate and inhibitor properties of some pyrazolopyrimidine and related analogues.
The nucleoside antibiotic formycin, 7-amino-3-(beta-D-ribofuranosyl)pyrazolo(4,3-d)pyrimidine, a structural analogue of adenosine, is deaminated about 10-fold faster by adenosine deaminase than adenosine itself, and is therefore a superior substrate for both routine assays and kinetic studies with the purified enzyme. The luminescence properties of formycin have been profited from to develop a fluorimetric assay for adenosine deaminase which is considerably more sensitive than the spectrophotometric procedure widely employed with adenosine as substrate. Examples are presented of its application to routine assays of adenosine deaminase levels in cellular extracts, as well as to kinetic studies with the purified enzyme, including the properties of some pyrazolopyrimidine and purine substrates and inhibitors.
Adenosine Deaminase↗
Conformation about the glycosidic bond and susceptibility to 5'-nucleotidase of 8-substituted analogues of 5'-GMP.
Susceptibilities to snake venom 5'-nucleotidase (EC 3.1.3.5) have been evaluated for several 8-substituted analogues of 5'-GMP with varying populations of syn/anti conformations about the glycosidic bond. Improved syntheses of some of these are described, including direct chlorination of 5'-GMP to give 8-chloro-5'-GMP, a procedure which should be applicable to other purine nucleotides. The conformations of the various analogues were determined by means of 1H NMR spectroscopy, with particular emphasis on the glycosidic bond conformations. All the 8-substituted derivatives of 5'-GMP were relatively poor substrates of 5'-nucleotidase. This was shown to result largely from steric effects and the nature of the 8-substituent, and consistent with a requirement for the anti conformation. Although ribose-5-phosphate was not a substrate, it was a weak inhibitor, and its inhibitory properties account in part for the weak inhibitory properties of the 8-substituted 5'-GMP, and other, analogues. Attention is drawn to the hitherto largely neglected differences in properties of 5'-nucleotidases from different sources and their relevance to the present findings.
5'-Nucleotidase↗
Purification and properties of adenosine kinase from rat liver: separation from deoxyadenosine kinase activity.
Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS-polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 mumol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources. Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2'-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (approximately 1.5%) under optimal conditions (pH 7.5) vs 2'-deoxy-adenosine, the lowest yet reported for such a preparation, with a Km of 670 microM, as compared to 0.3 microM for adenosine. The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a Ki of 70 microM. Competitive inhibition was also exhibited by cordycepin (3'-deoxyadenosine) with a Ki of 150 microM. A more potent competitive inhibitor was tubercidin, the Ki for which was 1.9 microM.
Adenosine Kinase↗
Phosphorylation of coformycin and 2'-deoxycoformycin, and substrate and inhibitor properties of the nucleosides and nucleotides in several enzyme systems.
Under conditions where 2'-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5'-phosphate in 15-20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of less than or equal to 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5'-phosphate in 20% overall yield. Coformycin-5'-phosphate was characterized by various criteria, including 1H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2'-deoxycoformycin-5'-phosphate, coformycin-5'-phosphate was a feeble substrate of snake venom 5'-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5'-AMP. With 5'-AMP analogues as substrate, the 5'-phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with Ki values greater than 0.3 mM. The 5'-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (Ki greater than 0.2 mM), but are potent inhibitors of adenylate deaminase (Ki less than or equal to 10(-9) M). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5'-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented.
5'-Nucleotidase↗
Stereospecific synthesis by the sodium salt glycosylation method of halogeno benzimidazole 2'-deoxyribose analogues of the inhibitor of hnRNA synthesis, 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB).
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the beta-D-2'-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
Animals↗
Solution conformations of some acyclo nucleoside and nucleotide analogues of antiviral acyclonucleosides, and their substrate/inhibitor properties in several enzyme systems.
Chemical and enzymatic procedures have been employed for the preparation of various phosphorylated derivatives of the acyclonucleoside 9-(1,3-dihydroxy-2-propoxymethyl)adenine, an analogue of the active antiviral agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG). In combination with the previously reported 2',3'-seco nucleosides and their phosphates and cyclic phosphates (Stolarski et al., Z. Naturforsch. 41c, 758-770, 1986), this made available a broad class of acyclonucleosides and nucleotides, the acyclic moieties of which are capable of mimicking the ribose and 2'-deoxyribose rings. The solution conformations of the foregoing were determined with the aid of 1H, 13C and 31P NMR, and compared with those of DHPG and 9-(hydroxyethoxymethyl)guanine (Acyclovir, ACV). Particular attention was devoted to conformations about C-O bonds in different acyclic fragments, which demonstrated well-defined differences between 2',3'-seco derivatives on the one hand (conformational "rigidity") and derivatives with DHP and AC acyclic chains on the other (rotation about the C(1')-O(4') bond). The overall results are in good general agreement with reported crystal structures, and are compared with those obtained by quantum mechanical calculations. The conformational features of the various compounds are also discussed in relation to their substrate and/or inhibitor properties in a number of enzyme systems, including adenosine deaminase, phosphodiesterases, nuclease P1,3'-nucleotidase and herpes virus type 1 thymidine kinase.
Antiviral Agents↗
Properties of 5'-AMP deaminase and its inhibitors with the aid of a continuous fluorimetric assay with formycin-5'-phosphate as substrate.
A new continuous fluorimetric assay for AMP deaminase activity is described. The method makes use of a fluorescent analog of 5'-AMP, formycin-5'-phosphate (5'-FMP), which undergoes deamination to formycin B-5'-phosphate, not fluorescent at neutral pH. The pH-dependence for deamination of 5'-FMP is similar to that for 5'-AMP, but shifted about 0.2 units to more acidic pH. Deamination of 5'-FMP may also be followed spectrophotometrically at 306 nm, permitting better assays of crude extracts. Some kinetic results obtained by means of the new method for AMP deaminase from chick and rabbit skeletal muscle are presented. In particular it was found that the natural product of deamination, 5'-IMP exhibited allosteric inhibition of the chick enzyme with Ki values 1.6 mM, 1.2 mM and 1.0 mM at pH 5.8, 6.5 and 7.3, respectively. Activation by diadenosine tetraphosphate, Ap4A, reported for mouse muscle AMP deaminase, has not been noted for the chick enzyme. Inhibition by the transition state analogs, coformycin and 2'-deoxycoformycin, was observed for both rabbit and chick deaminases with Ki values approximately 1 microM and approximately 1.6 microM respectively. Kinetic data for coformycin-5'-phosphate show it to be a tight-binding inhibitor with Ki less than 0.6 x 10(-9) M as compared to 1 x 10(-9) M for 2'-deoxycoformycin-5'-phosphate.
AMP Deaminase↗