guanosine-monophosphate and guanosine-5--phosphoimidazolide

For deeper understanding the roles of the mRNA cap structure in cellular processes isotopically labeled dinucleotide cap analogues have been synthesized as tools for NMR and in vivo studies. Tritium or carbon C-13 labeled methyl iodide was used as a source of the isotope material. In order to minimize the number of steps during the radioisotopic synthesis the methylation with tritium labeled methyl iodide was performed with Gp(3)G as a substrate. The C-13 isotope was introduced into the cap dinucleotide by methylation of GDP with C-13 Methyl iodide, followed by coupling the product with guanosine 5'-phosphorimidazolide in DMF with zinc chloride as a catalyst.

Topics: Carbon Isotopes; Catalysis; Dinucleoside Phosphates; Guanosine Monophosphate; Hydrocarbons, Iodinated; Isotope Labeling; Methylation; Nuclear Magnetic Resonance, Biomolecular; RNA Caps; RNA, Messenger; Tritium

Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

Topics: Catalysis; DNA; Exobiology; Guanosine Monophosphate; Lead; Molecular Structure; Oligonucleotides; RNA; Sugar Alcohols; Templates, Genetic

Aliphatic amines react with phosphoimidazolide-activated derivatives of guanosine and cytidine (ImpN) by replacing the imidazole group. The kinetics of reaction of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) with glycine ethyl ester, glycinamide, 2-methoxyethylamine, n-butylamine, morpholine, dimethylamine (Me2NH), ethylmethylamine (EtNHMe), diethylamine (Et2NH), pyrrolidine, and piperidine were determined in water at 37 degrees C. With primary amines, a plot of the logarithm of the rate constant for attack by the amine on the protonated substrate, log kSH(A), versus the pKa of the amine exhibits a good linear correlation with a Bronsted slope, beta nuc = 0.48. Most of the secondary amines tested react with slightly higher reactivity than primary amines of similar pKa. Interestingly, some secondary amines show substantially lower reactivity than might be expected: EtNHMe reacts about eight times, and Et2NH at least 100 times, more slowly than Me2NH although all three amines are of similar basicity. For comparison, the kinetics of reaction of guanosine 5'-phosphoimidazolide (ImpG) and cytidine 5'-phosphoimidazolide (ImpC) were determined with Me2NH, EtNHMe, and Et2NH, and similar results were obtained. These results establish that the increased steric hindrance observed with the successive addition of ethyl groups are not due to any special steric requirements imposed by the guanosine or the methyl on the 2-methylimidazole leaving group of 2-MeImpG. It is concluded that addition of ethyl and, perhaps, groups larger than ethyl dramatically increases the kinetic barrier for addition of aliphatic secondary amines to the P-N bond of ImpN. This study supports the observation that the primary amino groups on the natural polyamines are at least 2 orders of magnitude more reactive than the secondary amino groups in the reaction with ImpN.

Topics: Amines; Chemical Phenomena; Chemistry; Guanosine Monophosphate; Kinetics; Nucleosides

We have prepared hairpin oligonucleotides in which a 5'-terminal single-stranded segment contains cytidylate (C) and guanylate (G) residues. When these hairpin substrates are incubated with a mixture of cytidine 5'-phosphoro(2-methly)imidazolide (2-MeImpC) and guanosine 5'-phosphoro(2-methyl)imidazolide (2-MeImpG), the 5'-terminal segment acts as a template to facilitate sequence-specific addition of G and C residues to the 3'-terminus of the hairpin. If an isolated G residue is present at the 3'-end of the template strand, it is copied regiospecifically in the presence of 2-MeImpC and 2-MeImpG to give a product containing an isolated C residue linked to its G neighbors by 3'-5'-internucleotide bonds. However, if only 2-MeImpC is present in the reaction mixture, very little reaction occurs. Thus, the presence of 2-MeImpG catalyzes the incorporation of C. If the template strand contains a short sequence of G residues, it is copied in the presence of a mixture of 2-MeImpC and 2-MeImpG. If only 2-MeImpC is present in the reaction mixture, efficient synthesis occurs to give a final product containing one fewer C residue than the number of G residues in the template.

Topics: Base Sequence; Cytidine; Cytidine Monophosphate; Directed Molecular Evolution; Guanosine; Guanosine Monophosphate; Molecular Sequence Data; Oligonucleotides; Templates, Genetic

Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 < or = pH < or = 8.6. This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phosphate. The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate. Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities. The results show that both H2PO4- and HPO4(2-) participate in both hydrolysis and nucleophilic substitution. Statistically corrected biomolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile. The low Bronsted value beta = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before. The beta nuc = 0.35 calculated for water, H2PO4-, HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR- with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (beta nuc = 0.25). With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phosphate, assuming the concentrations of phosphate, Pi, on prebiotic Earth were similar to those in the present oceans ([Pi] approximately 2.25 micromoles).

Topics: Catalysis; Guanosine Monophosphate; Hydrolysis; Nitrogen; Nucleotides; Phosphates; Phosphorus; Polynucleotides; Templates, Genetic

Nucleoside-5'-phosphorimidazolides react readily with acylating agents to give N-substituted products that are highly activated. In most cases these acylated derivatives undergo rapid hydrolysis to give nucleoside 5'-phosphates, whether or not a complementary template is present. However, guanosine 5'-phosphorimidazolide reacts with diethyl pyrocarbonate to give a derivative that oligomerizes rapidly and efficiently in the presence of polycytidylic acid and Pb2+. The reaction is complete in about 1 h, whereas the corresponding reaction in the absence of an acylating agent takes several days. However, the final yield of long oligomers is lower when diethyl pyrocarbonate is present.

Topics: Acylation; Adenosine Monophosphate; Diethyl Pyrocarbonate; Guanosine Monophosphate; Hydrolysis; Kinetics; Poly C

Selected imidazolide-activated nucleotides have been subjected to hydrolysis under conditions similar to those that favor their template-directed oligomerization. Rate constants of hydrolysis of the P-N bond in guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5'-monophosphate imidazolide (ImpG), kh, have been determined in the presence/absence of magnesium ion as a function of temperature and polycytidylate [poly(C)] concentration. Using the rate constant of hydrolysis of 2-MeImpG and the rate constant of elongation, i.e., the reaction of an oligoguanylate with 2-MeImpG in the presence of poly(C) acting as template, the limiting concentration of 2-MeImpG necessary for oligonucleotide elongation to compete with hydrolysis can be calculated. The limiting concentration is defined as the initial concentration of monomer that results in its equal consumption by hydrolysis and by elongation. These limiting concentrations of 2-MeImpG are found to be 1.7 mM at 37 degrees C and 0.36 mM at 1 degrees C. Boundary conditions in the form of limiting concentration of activated nucleotide may be used to evaluate a prebiotic model for chemical synthesis of biopolymers. For instance, the limiting concentration of monomer can be used as a basis of comparison among catalytic, but nonenzymatic, RNA-type systems. We also determined the rate constant of dimerization of 2-MeImpG, k2 = 0.45 +/- 0.06 M-1 h-1 in the absence of poly(C), and 0.45 +/- 0.06 less than or equal to k2 less than or equal to 0.97 +/- 0.13 M-1 h-1 in its presence at 37 degrees C and pH 7.95.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Guanosine Monophosphate; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Magnesium; Nucleotides; Poly C; Temperature; Templates, Genetic

Magnesium, an ion necessary in enzymatic as well as in nonenzymatic template-directed polynucleotide-synthesizing reactions, has been found to catalyze the hydroxide ion attack on the P-N bond of selected 5'-monophosphate imidazolide derivatives of nucleotides, such as guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG), guanosine 5'-monophosphate imidazolide (ImpG), and adenosine 5-monophosphate 2-methylimidazolide (2-MeImpA). Calcium ion behaves similarly, but quantitatively the effects are smaller. Pseudo-first-order rate constants of 2-MeImpG and ImpG hydrolysis as a function of Mg2+ concentration have been obtained in the range 6 < or = pH < or = 10 at 37 degrees C. Mg2+ catalysis is particularly effective around pH 10 where a 0.02 M concentration leads to 15-fold acceleration and a 0.2 M concentration to a 115-fold acceleration of the rate. At other pH values Mg2+ catalysis is less dramatic, mainly because the noncatalyzed reaction is faster. Mg2+ catalysis is attributed to the reaction of the zwitterionic form of the substrate (SH+/-, imidazolide moiety protonated) with OH- rather than reaction of the anionic form (S-, imidazolide moiety deprotonated) with water. This conclusion is based on a study of the N-methylated substrates N-MeImpG and 1,2-diMeImpg, respectively, which were generated in situ by the equilibrium reaction of ImpG with N-methylimidazole and 2-MeImpG with 1,2-dimethylimidazole, respectively. In contrast, the absence of Mg2+ the reaction of S- with water competes with the reaction of SH+/- with OH-. The present study bears on the mechanism of the Mg2(+)-catalyzed template-directed synthesis of oligo-and polynucleotides derived from 2-MeImpG and on the competition between oligonucleotide synthesis and hydrolysis of 2-MeImpG.

Topics: Calcium; Catalysis; Evolution, Molecular; Guanosine Monophosphate; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Magnesium; Nitrogen; Nucleotides; Phosphorus; Polynucleotides; Templates, Genetic

Topics: Chromatography, High Pressure Liquid; Guanine Nucleotides; Guanosine Monophosphate

Oligodeoxycytidylic acids and polydeoxycytidylic acid are effective templates for the polymerization of guanosine 5'-(phospho-2-methylimidazolide). They may be substituted for the corresponding ribo-oligomers without greatly changing the course of the reactions. Since oligomers of deoxynucleotides are much more easily synthesized than the ribo-oligomers, this finding, if it proves general, should greatly facilitate the study of the template properties of oligomers containing two or more bases. Oligodeoxycytidylates facilitate the synthesis of oligoguanylates up to one residue longer than the template in high yield, and oligoguanylates up to twice the length of the template in significant yield. The time-course and regiospecificity of these reactions suggest that "sliding" and "double-templating" are important factors in determining the pattern of reaction products.

Topics: Chromatography, High Pressure Liquid; Guanosine Monophosphate; Hydrogen-Ion Concentration; Oligodeoxyribonucleotides; Oligonucleotides; Poly C; Polynucleotides; Polyribonucleotides; Templates, Genetic

The Pb2+ and Zn2+ ions are efficient catalysts for the polycytidylic acid-directed polymerization of an activated guanylic acid derivative, guanosine 5'-phosphorimidazolide. The products include oligomers of 30 to 40 units in length. The nucleotide residues are predominantly 2'-5' linked when Pb2+ is the catalyst, and predominantly 3'-5' linked in the presence of Zn2+. The significance of these results in the context of the prebiotic evolution of RNA polymerase is discussed.

Topics: DNA-Directed RNA Polymerases; Guanine Nucleotides; Guanosine Monophosphate; Lead; Pancreas; Poly C; Polyribonucleotides; Ribonucleases; Templates, Genetic; Zinc

Topics: Biopolymers; Catalysis; Guanine Nucleotides; Guanosine Monophosphate; Lead; Magnesium; Poly C; Polyribonucleotides; Templates, Genetic; Time Factors

Topics: Catalysis; Chromatography, High Pressure Liquid; Guanine Nucleotides; Guanosine Monophosphate; Kinetics; Magnesium; Oligonucleotides; Poly C; Polyribonucleotides; Zinc

Topics: Bismuth; Catalysis; Chromatography, High Pressure Liquid; Guanine Nucleotides; Guanosine; Guanosine Monophosphate; Lead; Magnesium; Metals; Oligonucleotides; Poly C; Polyribonucleotides; Tin; Zinc