Phosphorimidazolide Of Adenosine Research Articles

The role of fluoride in montmorillonite-catalyzed RNA synthesis.

The montmorillonite-catalyzed reactions of the 5'-phosphorimidazolide of adenosine in the presence of fluoride were investigated to complete our study on the effect of salts on this type of reaction. Both anions and cations have been found to influence the oligomerization reactions of the activated nucleotides, being used here as a model system for pre-biotic RNA synthesis. However, in total contrast to the behavior of the activated nucleotides in the presence of montmorillonite and other salts, alkali metal fluorides did not yield any detectable oligomerization products except in very dilute (<0.005 M) solutions of fluoride. Instead, 5'-phosphorofluoridates were formed. Their identity was confirmed by a combination of HPLC, mass spectrometry, synthesis, and NMR.

Significance of Mineral Salts in Prebiotic RNA Synthesis Catalyzed by Montmorillonite

The montmorillonite-catalyzed reactions of the5'-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 (0.1-2.0M) 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 1.2M. This concentration of sodium chloride resembled its abundance in the ancient oceans (0.9-1.2M). Magnesium chloride produced a similar effect but its joint action with sodium chloride did not produce any difference in the oligomer chain length. Therefore, Mg(2+) 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 2.0M. 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.

9 Prebiotic RNA synthesis: significance of mineral salts in montmorillonite-catalyzed reactions

The dual properties of RNA as an enzyme catalyst and its ability to store genetic information suggest that early life was based on RNA, and DNA and protein evolved from it. Our lab has demonstrated synthesis of long RNA oligomers by Na+-montmorillonite-catalyzed reactions of 5′-end-activated mononucleotides (Joshi et al., 2009). The Na+-montmorillonite not only catalyzes the prebiotic synthesis of RNA but also facilitates homochiral selection (Joshi et al., 2011, 2013). The montmorillonite-catalyzed reactions of 5′-phosphorimidazolide of adenosine were further investigated to study the effect of salts. These reactions were found to be dependent on the nature of mineral salts present. While montmorillonite (pH 7) produced only dimers in water, addition of sodium chloride (1 M) enhanced the chain length of oligomers to 10-mers as detected by HPLC. Magnesium chloride produced a similar effect but the presence of both sodium chloride and magnesium chloride did not produce any difference in the oligomer chain length. The effect of monovalant cations in RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl− > Br− > I−. Inorganic salts that tend to salt out organic compounds from water and salts which show salt-in effects had no effect in the oligomerization process, indicating that the montmorillonite-catalyzed RNA synthesis is not affected by 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 M to 2.0 M. Inhibition of cyclic dimer formation is essential for increasing the yield of linear dimers as well as the overall chain length. The results of this study show that the presence of salts is essential in prebiotic RNA synthesis catalyzed by clay minerals.

Montmorillonite catalysis of 30-50 mer oligonucleotides: laboratory demonstration of potential steps in the origin of the RNA world.

Elongation of the primer 32pdA(pdA)8pA proceeds by the reaction of the 5'-phosphorimidazolides of adenosine and uridine in the presence of montmorillonite clay. Daily addition of the activated nucleotides for up to 14 days results in the formation of 40-50 mers using the 5'-phosphorimidazolide of adenosine (ImpA) and 25-30 mers using the 5'-phosphorimidazolide of uridine (ImpU). The limitation on the lengths of the chains formed is not due to the inhibitors formed since the same chain lengths were formed using 2-3 times the amount of montmorillonite catalyst. The shorter oligomers formed by the addition of U monomers is not due to its greater rate of decomposition since it was found that both the A and the U adducts decompose at about the same rates. Alkaline phosphatase hydrolysis studies revealed that some of the oligomers are capped at the 5'-end to form, with ImpA, Ap32pdA(pdA)8pA(pA)n. The extent of capping depends on the reaction time and the purine or pyrimidine base in the activated mononucleotide. Hydrolysis with ribonuclease T2 followed by alkaline phosphatase determined the sites of the 3', 5'- and 2', 5'-phosphodiester bonding to the primer. The potential significance of the mineral catalyzed formation of 50 mer oligonucleotides to the origin of life based on RNA (the RNA world scenario) is discussed.

Studies on the reaction of the 5′-phosphorimidazolide of adenosine with Cu(II)-exchanged hectorite

The role of clay minerals in the prebiotic synthesis of nucleotide oligomers has received considerable attention in recent years. Scanning force microscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are used to identify oligomers of adenylic acid formed on the clay mineral Cu(II)-exchanged hectorite in simulated prebiotic cycling experiments. Electron-spin resonance and x-ray diffraction data indicate that the monomer (5′-phosphorimidazolide of adenosine, or ImpA) penetrates into the intergallery regions of the mineral substrate, and complexes the gallery Cu(II) cations. It is postulated that polymerization of the monomer is initiated in the clay intergallery regions, producing oligomers of adenylic acid up to 8 units in length or more.

Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5'-phosphorimidazolide of adenosine on Na(+)-montmorillonite.

The rate constants for the condensation reaction of the 5'-phosphorimidazolide of adenosine (ImpA) to form dinucleotides and oligonucleotides have been measured in the presence of Na(+)-volclay (a Na(+)-montmorillonite) in pH 8 aqueous solution at 25 degrees C. The rates of the reaction of ImpA with an excess of adenosine 5'-monophosphoramidate (NH2pA), P1,P2-diadenosine 5',5'-pyrophosphate (A5'ppA), or adenosine 5'-monophosphate (5'-AMP or pA) in the presence of the montmorillonite to form NH2pA3'pA, A5'ppA3'pA, and pA3'pA, respectively, were measured. Only 3',5'-linked products were observed. The magnitude of the rate constants decrease in the order NH2pA3'pA > A5'-ppA3'pA > pA3'pA. The binding of ImpA to montmorillonite was measured, and the adsorption isotherm was determined. The binding of ImpA to montmorillonite and the formation of higher oligonucleotides is not observed in the absence of salts. Mg2+ enhances binding and oligonucleotide formation more than Ca2+ and Na+. The rate constants for the oligonucleotide formation were determined from the reaction products formed from 10 to 40 mM ImpA in the presence of Na(+)-montmorillonite using the computer program SIMFIT. The magnitudes of the rate constants for the formation of oligonucleotides increased in the order 2-mer < 3-mer < 4-mer ... 7-mer. The rate constants for dinucleotide and trinucleotide formation are more than 1000 times larger than those measured in the absence of montmorillonite. The rate constants for the formation of dinucleotide, trinucleotide, and tetranucleotide are 41,2.6, and 3.7 times larger than those for the formation of oligo(G)s with a poly(C) template. The hydrolysis of ImpA was accelerated 35 times in the presence of the montmorillonite. The catalytic ability of montmorillonite to form dinucleotides and oligonucleotides is quantitatively evaluated and possible pathways for oligo(A) formation are proposed.

Oligomerization of mononucleotides on montmorillonite: A potential approach to the prebiotic synthesis of RNA

The condensation of the 5'-phosphorimidazolide of adenosine (ImpA) on montmorillonite in a pH 8 aqueous solution yields oligomers containing up to 10 monomer units. The regiospecificity of 3',5'-phosphodiester bond formation is enhanced by addition of 10% diadenosine pyrophosphate (AppA) to the reaction mixture. A series of activated derivatives of 5'-AMP was prepared to investigate the effect of the leaving group on oligomer formation. The benzimidazole and p-dimethylamino-pyridine derivatives gave the best yields of oligomers. Factors important for oligomer formation is discussed.

Oligomerization reactions of ribonucleotides: The reaction of the 5′ -phosphorimidazolide of adenosine with diadenosine pyrophosphate on montmorillonite and other minerals

The reaction of the 5′ -phosphorimidazolide of adenosine (5′-ImpA) with diadenosine pyrophosphate (A5′ppA) in the presence of Na+-montmorillonite in aqueous, pH 8 solution results in the regiospecific formation of A5′ppA3′pA and A5ppA3′pA3′ pA. The formation of oligomers of general structure (pA)n decreases in the presence of A5′ppA. A5′ppA3′pA is the principal reaction product when a 1:1 ratio of ImpA and A5′ppA is used. The yield of A5′ppA3′pA3′pA is optimal when 9:1 or 4:1 ratios of ImpA: A5′ppA are used. The overall regiospecificity of formation of 3′,5′-links is about 80%. The reaction between ImpA and A5′ppA on montmorillonite differs from the self-condensation of ImpA in that it proceeds in the absence of Mg2+ and there are only small differences in oligomer yields when Na+, Li+ Ca2+, and NH4+ are the exchangeable cations on the montmorillonite. The reaction is inhibited by 0.4 M imidazole but the inhibition is suppressed with 0.4 M Mg2+. Little or no phosphodiester bond formation was observed with Mg2+- or Al3+-montmorillonite. Montmorillonites other than 22A and Volclay exhibited no catalysis for the formation of adducts between ImpA and A5′ppA and no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, or sepiolite.

ChemInform Abstract: Oligomerization of Ribonucleotides on Montmorillonite: Reaction of the 5′‐Phosphorimidazolide of Adenosine.

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Oligomerization of ribonucleotides on montmorillonite: reaction of the 5'-phosphorimidazolide of adenosine.

The regiospecific formation of oligomers from unblocked monomers in aqueous solution is one of the central tenets in research on the origins of life on earth. Direct experimental support for this hypothesis has been obtained in studies of the condensation of the 5'-phosphorimidazolide of adenosine (ImpA) with itself and with P1,P2-diadenosine-5',5'-pyrophosphate (AppA) in water in the presence of a montmorillonite clay. Oligomers of up to ten nucleotides in length are formed. Analysis of the trimers, tetramers, and pentamers formed from a 9:1 ImpA:AppA mixture has shown that 85% of the bonds formed are 3',5'-linked and that any 2',5'-linkages present are at the phosphodiester bond next to the 3'-terminus of the oligomers.