10 The montmorillonite-catalyzed synthesis of RNA dimer

A synthesis has been developed, providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5′-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately, 30% of the imidazolide is converted into each 2′-5′ dimer and 3′-5′ dimer with the rest hydrolyzed to the 5′-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5′-monophosphates. Both homo- and heterochiral combinations of reagents have been tried. The montmorillonite-catalyzed oligomerization of 5′-activated nucleotides leads to oligomers up to 50 residues in length (Huang & Ferris, 2007) using the excellent catalyst Volclay®. However, all oligomers must necessarily begin as dimers, so we considered it important to study in detail the formation of these products under prebiotic conditions. Then, a meaningful comparison could be drawn between our syntheses and the formation of long oligomers that is part of our studies of the origins of life. In the synthesis of trimers from these dimers, we looked for alternative synthetic methods via a 5′-phosphate dimer with activated nucleotides as well as 5′-hydroxy nucleotide dimers with the same reactant. The method has shown promise in targeting trimer synthesis and the procedure lends itself to the development of combinatorial libraries. The use of enzymatic hydrolysis has played a crucial role in this work, facilitating product identity across the spectrum of products prepared. The yields of the corresponding homochiral and heterochiral dimers from A and U will require careful modeling of the reactants in their interactions with both the clay and one another to locate the source of the similarities and differences. The lack of reactivity of arabino- and xylo-nucleosides also poses interesting structural, modeling, and origins of life issues. Results with clays that catalyze long oligomer formation only poorly reveal that they too catalyze these dimer syntheses, albeit less well than Volclay.^®      

A Nucleotide Dimer Synthesis Without Protecting Groups Using Montmorillonite as Catalyst

A synthesis has been developed providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5′-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately 30% of the imidazolide is converted into each 2′-5′ dimer and 3′-5′ dimer with the rest hydrolyzed to the 5′-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5′-monophosphates.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental files.]     

Clay Catalysis of Oligonucleotide Formation: Kinetics of the Reaction of the 5'-Phosphorimidazolides of Nucleotides with the Non-Basic Heterocycles Uracil and Hypoxanthine

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine if substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.     

11 Findings and hurdles on the path leading from formamide to the spontaneous generation of RNA

Our laboratories analyze the synthetic reactions leading from formamide, NH₂COH to prebiotically relevant compounds in the presence of catalysts. We have described the formation of all the biological nucleic bases of carboxylic acids of two aminoacids, and of condensing agents in the presence of catalysts of terrestrial origin (Saladino et al., 2012) and of one meteorite. Heat-dependent synthetic reactions from NH₂COH lead to the synthesis of acyclonucleosides, not (yet?) to that of nucleosides [hurdle # 1]. Nucleosides are phosphorylated in the presence of NH₂COH and a phosphate source yielding cyclic nucleotides as well. (Costanzo et al., 2007). 3′,5′-cyclic GMP nonenzymatically polymerizes up to at least 25mers, as shown by PAGE, MALDI ToF, ³¹P-NMR, specific RNAse and inhibitors analyses (Costanzo et al., 2012).The reaction is stimulated by 1,8-diazabicycloundec-7-ene and dimethylformamide. 3′,5′-cUMP does not polymerize spontaneously [hurdle # 2], 3′,5′-cAMP polymerizes very poorly [hurdle # 3]. We will discuss data on the polymerization of 3′,5′-cCMP and on a ribozyme activity exerted by oligomers neosynthesized from cyclic nucleotides. This approach finds its larger perspective in the evolutionary scenario depicted by Trifonov (2009).     

Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C): An RNA polymerase model

(Guanosine 5′-phosphor)-2-methylimidazolide (2-MeImpG), unlike guanosine 5′-phosphorimidazolide (ImpG), undergoes an efficient, buffer-independent, template-directed oligomerization in the presence of poly(C) at pH values above 7.6. The reaction occurs in a Watson-Crick double helix and yields predominantly 3′-5′-linked oligomers up to the 50-mer in above 80% yield. Synthesis proceeds in the 5′ → 3′ direction and has high fidelity in the sense that nucleotides other than G are not incorporated significantly into oligomers. Under some conditions, oligomers corresponding to approximately one and two turns of the helix are obtained in higher yield than somewhat longer or somewhat shorter oligomers.In the protonated triple-helical structure formed below pH 7, the efficiency of the oligomerization is much lower. Oligomers up to about the 10-mer are obtained. The most abundant products are “capped” at the 5′ terminus with a GppG pyrophosphate group.     

Enzymatic synthesis of perfluoroalkylated DNA

Thymidine analogues 5-trifluoromethyl-, 5-pentafluoroethyl- and 5-(heptafluoro-n-propyl)-2′-deoxyuridines were synthesised and converted into the corresponding 5′-triphosphates 1a–c. Performing DNA polymerase-catalyzed primer extension reactions these modified nucleotides were incorporated into DNA to create perfluoroalkylated nucleic acids. Although single modified nucleotides were enzymatically incorporated and further elongated quite similar to the natural TTP, the enzymatic synthesis of multi-modified nucleic acids was initial only feasible with modifications at every fourth base. Nevertheless, as the effects of the modified dUTPs on DNA polymerases varied significantly with the used enzyme, Therminator DNA polymerase was proficient in incorporating 11 adjacent 5-trifluoromethyl-2′-deoxyuridine moieties.     

Purine and Pyrimidine 5′-Nucleotide Catabolism in Tetrahymena pyriformis W1

SYNOPSIS Deamination at pH 7.5 of adenosine, deoxyadenosine, cytidine and deoxycytidine by cell-free preparations of Tetrahymena pyriformis W was observed both in the presence and absence of fluoride. Deamination of 5′-AMP, 5′-dAMP, 5′-CMP, and 5′-dCMP was found only in the absence of fluoride. Dephosphorylation of the above nucleotides by acid phosphatases occurred at pH 4.5; reduced activity was noted at pH 7.5. Fluoride effectively blocked acid phosphatase activity at both pH values. This correlation of phosphatase and deaminase activities suggests a catabolic pathway for 5′-AMP and 5′-CMP whereby dephosphorylation precedes deamination. Radiolabelled substrates were used to test this hypothesis. The experiments were designed so that conversion of as little at 1.0% of the radiolabelled substrate to the deaminated product could be detected. No 5′-IMP or 5′-UMP, the expected deamination products of 5′-AMP and 5′-CMP, respectively, was recovered after incubation of the radiolabelled substrates with cell-free enzyme preparations. Thus, it appears that Tetrahymena has no 5′-AMP or 5′-CMP deaminases and that these compounds are deaminated only after conversion to nucleosides. Acid phosphatase activity toward 5′-GMP, 5′-dGMP, 5′-TMP, 5′-UMP, and 5′-XMP was also found.     

Prebiotic nucleotide oligomerization in a fluctuating environment: Effects of kaolinite and cyanamide

Summary The clay kaolinite was tested for its ability to promote nucleotide oligomerization in model prebiotic systems. Heterogeneous mixtures of clay, water and nucleotide were repeatedly evaporated to dryness at 60°C and redissolved in water in cyclic fashion in the presence or absence of cyanamide and/ or ammonium chloride. With or without cycling, kaolinite alone did not promote the oligomerization of nucleotides at detectable levels. Cycling of clay in combination with cyanamide, however, promoted high levels of condensation to a mixture of oligonucleotides and dinucleotide pyrophosphate without requiring ammonium chloride. Although cycling with clay favored synthesis of dinucleotide pyrophosphate, cycling without clay enhanced formation of oligonucleotides. These results support the hypothesis that the presence of clays in fluctuating environments would have influenced the course of prebiotic condensation reactions.     

Catalysis of accurate poly(C)-directed synthesis of 3'-5'-linked oligoguanylates by Zn2+

Zn²⁺ is an efficient catalyst for the oligomerization of guanosine 5′-phosphorimidazolide on a polycytidylic acid template. Up to 75% of the input ImpG² is converted to oligomers with a mean chain length up to 10. Material longer than (pG)₃₀ can be detected. The oligomeric products are predominantly 3′-5′-linked.If poly(C) is incubated with ImpG and an equimolar quantity of the 5′-phosphorimidazolides of adenosine, uridine or cytidine, in the presence of Zn²⁺, ImpG is incorporated at least 200 times more efficiently than “incorrect” nucleotides.     

Significance of Mineral Salts in Prebiotic RNA Synthesis Catalyzed by Montmorillonite

The montmorillonite-catalyzed reactions of the 5′-phosphorimidazolide of adenosine used as a model generated RNA type oligomers. These reactions were found to be dependent on the presence of mineral salts. Whereas montmorillonite (pH 7) produced only dimers and traces of trimer in water, addition of sodium chloride (0.1–2.0 M) enhanced the chain length of oligomers to 10-mers as detected by HPLC. Maximum catalytic activity was observed with sodium chloride at a concentration between 0.8 and 1.2 M. This concentration of sodium chloride resembled its abundance in the ancient oceans (0.9–1.2 M). Magnesium chloride produced a similar effect but its joint action with sodium chloride did not produce any difference in the oligomer chain length. Therefore, Mg²⁺ was not deemed necessary for generating longer oligomers. The effect of monovalent cations upon RNA chain length was: Li⁺ > Na⁺ > K⁺. A similar effect was observed with the anions with enhanced oligomer length in the following order: Cl^? > Br^? > I^?. Thus, the smaller ions facilitated the formation of the longest oligomers. Inorganic salts that tend to salt out organic compounds from water and salts which show salt-in effects had no influence on the oligomerization process indicating that the montmorillonite-catalyzed RNA synthesis is not affected by either of these hydrophobic or hydrophilic interactions. A 2.3-fold decrease in the yield of cyclic dimer was observed upon increasing the sodium chloride concentration from 0.2 to 2.0 M. Inhibition of cyclic dimer formation is vital for increasing the yield of linear dimers and longer oligomers. In summary, sodium chloride is likely to have played an essential role in any clay mineral-catalyzed prebiotic RNA synthesis.     

Nucleoside Azide–Alkyne Cycloaddition Reactions Under Solvothermal Conditions or Using Copper Vials in a Ball Mill

Novel nucleoside analogues containing photoswitchable moieties were prepared using ‘click’ cycloaddition reactions between 5′-azido-5′-deoxythymidine and mono- or bis-N-propargylamide-substituted azobenzenes. In solution, high to quantitative yields were achieved using 5 mol% Cu(I) in the presence of a stabilizing ligand. ‘Click’ reactions using the monopropargylamides were also effected in the absence of added cuprous salts by the application of liquid assisted grinding (LAG) in metallic copper reaction vials. Specifically, high speed vibration ball milling (HSVBM) using a 3/32^″ (2.38 mm) diameter copper ball (62 mg) at 60 Hz overnight in the presence of ethyl acetate lead to complete consumption of the 5′-azido nucleoside with clean conversion to the corresponding 1,3-triazole.     

Chemical Synthesis of Cap Analogues: P1,P2-Dinucleoside-5′-diphosphates

Abstract

A procedure was developed for the chemical synthesis of P¹,P²-dinucleoside-5′-diphosphates (N₁(5′)pp(5′)N₂) on a nanomolar scale Reaction conditions for activating purine-5′-monophosphates (pA, pG, and pm⁷G) by 1,1′-carbonyldiimidazole were studied and optimized in respect to solvents and amount of activating reagent used. Various dinucleoside-5′-diphosphates were synthesized in 62-98% yield by incubating activated and non-activated purine-5′-monophosphates. Two unexpected by-products were formed by competition reactions: the imidazolidate of the non-activated nucleotide and the corresponding symmetrically substituted dinucleoside-5′-diphosphate. A mechanism is proposed to explain the observed side reactions.     

Non-enzymatic oligomerization of racemic adenosine 5'-phosphorimidazolide on Na(+)-montmorillonite.

In this study, we have investigated non-enzymatic oligomerization of an activated racemic mononucleotide in the presence of Na(+)-montmorillonite. Oligomers up to the decamer in length were formed by oligomerization reactions of activated D- and L-mononucleotides. Similarly, oligomerization of an activated racemic mononucleotide results in the formation of oligomers up to the octamer. These results suggest that montmorillonite catalysis is quite efficient for the oligomerization of racemic monomers, though it is somewhat less efficient than that of D- and L-monomers.     

Regio- and Diastereo-Selectivity of Montmorillonite-Catalyzed Oligomerization of Racemic Adenosine 5′-Phosphorimidazolide

Clay is a possible candidate for an effective catalyst in prebiotic chemical evolution of biomolecules. Montmorillonite was reported to effectively catalyze oligomerization of racemic adenosine 5′-phosphorimidazolide (DL-ImpA). In the oligomerization reaction, considerable amounts of cyclic dimers as well as linear dimers were produced in the oligomerization reactions. To assess the regio- and diastereo-selectivities of the oligomerization reaction, the dimer products including cyclic dimers were completely identified by means of enzymatic degradation reactions of the products.     

Template-directed oligomerization of 5′-deoxy-5′-nucleosideacetic acid derivatives

5-Deoxy-5-nucleosideacetic acids II–V are isostructural analogues of nucleotides with a carboxylate group in the place of the 5-phosphate group. We have studied their oligomerization in aqueous solution using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogues IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.     

Reconstitution of a hormone-sensitive adenylate cyclase with membrane extracts from Neurospora and avian erythrocytes

Adenylate cyclase activity associated to wild type Neurospora membranes is highly dependent on Mn2+ and insensitive to fluoride, guanyl nucleotides, and cholera toxin. These membranes are able to interact with components of detergent extracts from turkey erythrocyte ghosts. The reconstituted cyclase system is catalytically active in the presence of Mg2+ and it is activated by guanyl-5'-yl imidodiphosphate plus isoproterenol and fluoride. When detergent extracts were prepared from avian erythrocyte membranes treated with cholera toxin, the reconstituted system was stimulated by guanyl-5'-yl imidodiphosphate in the absence of isoproterenol and cyclase activities were higher than those observed with extracts from membranes not treated with the toxin. Dose-response curves for isoproterenol and fluoride in the reconstituted system were similar to those reported for avian erythrocyte and liver membranes, respectively.     

Adenine nucleotide metabolism in relation to purine enzymes in liver,erythrocytes and cultured fibroblasts

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hypoxanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5′-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5′-nucleotidase present in this tissue interferred with the formation of LaCl₃ salts. The lanthanum and the thin-layer chromatographic methods agreed with-in 10%.Liver cell sap had the highest activities of all purine enzymes except for 5′-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erythrocytes were devoid of 5′-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue.Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5′-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control of adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte > liver > fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Stimulation of polypeptide synthesis by cyclic 3'-5'-guanosine monophosphate

3′-5′-Cyclic GMP was found to stimulate polyphenylalanine synthesis in a cell-free system derived from rat liver. The effect of cyclic GMP is detected either in the presence or absence of GTP and appears quite specific since it is not produced by other cyclic nucleotides.