Michael F. Aldersley

Mechanism of montmorillonite catalysis in the formation of RNA oligomers.

The montmorillonite clay-catalyzed reactions of nucleotides generate oligomers as long as 50-mers. The extent of catalysis depends on the magnitude of the negative charge on the montmorillonite lattice and the number of cations associated with it. When cations in raw montmorillonites are replaced by sodium ions, the resulting Na(+)-montmorillonite does not catalyze oligomer formation because they saturate the interlayers between the platelets of montmorillonites, which blocks the binding of the activated monomers. Treating the montmorillonite with dilute hydrochloric acid replaces the cations on the raw montmorillonite with protons. The protonated montmorillonite, titrated to pH 6-7, serves as a catalyst for the formation of RNA oligomers. The titration does not add sufficient sodium ions to the interlayers of the montmorillonite platelets to prevent the activated monomer from entering. It was noted that noncatalytic montmorillonites have a higher negative charge on their platelets that is due mainly to the natural substitution of the tetravalent and trivalent elements in the montmorillonite lattice with trivalent and divalent metal ions, respectively. The larger negative charge on these montmorillonites was demonstrated by the almost 2-fold greater amounts of sodium hydroxide needed to titrate noncatalytic montmorillonites as compared to the catalytic montmorillonites. Adsorption isotherms established that the equilibrium binding is strongest for ImpA and weakest for ImpU. Of the 22 montmorillonites investigated, 12 were catalysts. This research provides insight into the mechanism of the catalytic process.

Homochiral Selectivity in RNA Synthesis: Montmorillonite-catalyzed Quaternary Reactions of D, L-Purine with D, L- Pyrimidine Nucleotides

Selective adsorption of D, L-ImpA with D, L-ImpU on the platelets of montmorillonite demonstrates an important reaction pathway for the origin of homochirality in RNA synthesis. Our earlier studies have shown that the individual reactions of D, L-ImpA or D, L-ImpU on montmorillonite catalyst produced oligomers which were only partially inhibited by the incorporation of both D- and L-enantiomers. Homochirality in these reactions was largely due to the formation of cyclic dimers that cannot elongate. We investigated the quaternary reactions of D, L-ImpA with D, L-ImpU on montmorillonite. The chain length of these oligomers increased from 9-mer to 11-mer as observed by HPLC, with a concominant increase in the yield of linear dimers and higher oligomers in the reactions involving D, L-ImpA with D, L-ImpU as compared to the similar reactions carried out with D-enantiomers only. The formation of cyclic dimers of U was completely inhibited in the quaternary reactions. The yield of cyclic dimers of A was reduced from 60% to 10% within the dimer fraction. 12 linear dimers and 3 cyclic dimers were isolated and characterized from the quaternary reaction. The homochirality and regioselectivity of dimers were 64.1% and 71.7%, respectively. Their sequence selectivity was shown by the formation of purine-pyrimidine (54–59%) linkages, followed by purine-purine (29–32%) linkages and pyrimidine-pyrimidine (9–13%) linkages. Of the 16 trimers detected, 10 were homochiral with an overall homochirality of 73–76%. In view of the greater homochirality, sequence- and regio- selectivity, the quaternary reactions on montmorillonite demonstrate an unexpectedly favorable route for the prebiotic synthesis of homochiral RNA compared with the separate reactions of enantiomeric activated mononucleotides.

Derivatives of naphtho[2,3-c]pyran-5,10-dione ; a simple synthesis and a note of their chromogenic properties.

Abstract Appropriate N -ylides convert 2-methyl-1,4-naphthoquinone into 3-(acylmethyl) derivatives which can be cyclised to naphtho[2,3-c]pyran-5,10-diones by treatment with bromine and dehydrobromination with triethylamine; these diones give a variety of striking colours in acid media.

Intramolecular catalysis of amide hydrolysis by the carboxy-group. Rate determining proton transfer from external general acids in the hydrolysis of substituted maleamic acids

The highly efficient intramolecular catalysis by the carboxy-group of the hydrolysis of simple dialkylmaleamic acids is itself subject to external general acid catalysis. The kinetic characteristics of the general acid catalysed reaction are those expected for a diffusion-controlled proton transfer. At high concentrations of general acid, external catalysis disappears. This is shown to result from a change in rate-determining step, and is thus evidence for an intermediate on the reaction pathway. The intermediate can only reasonably be a tetrahedral addition intermediate. Kinetic evidence is now available for all the major steps on the reaction pathway, and the requirements for an enzyme catalyst carrying out the reaction can be specified in detail. The full mechanism specifically implicates the O-protonated amide as the reactive species in dilute acid.

Intramolecular displacement of alkoxide ions by the ionised carboxy-group: hydrolysis of alkyl hydrogen dialkylmaleates

The pH-independent hydrolysis reactions of the title ester acids and their anions, and the acid catalysed hydrolysis of the ester acids, are all subject to highly efficient intramolecular nucleophilic catalysis. The carboxylate group of the ester anions can displace leaving groups as poor as isopropoxide, 1013 times more basic, in an unassisted reaction which shows the highest sensitivity to leaving group ever measured for ester hydrolysis. Intramolecular catalysis by the carboxy-group is more efficient for esters derived from alcohols with pKa > 13·6, and is itself subject to external general base catalysis.

Progress in Studies on the RNA World

The montmorillonite-catalyzed reactions of D, L-ImpA with D, L-ImpU generates RNA-like oligomers. The structures of the dimers to pentamers were investigated and homochiral products were identified in greater amounts than would be expected if theoretical amounts of each were formed. The homochirality increased from 64% to 97% as the chain length increased from dimers to pentamers. Investigation of the effect of pH, occupancy of the interlayer space and the influence of various cations in the reaction provided further insight into physical process in the mechanism of the catalysis. A detailed analysis of dimers was carried out in view of there being key intermediates towards formation of higher oligomers. The study was extended to the synthesis of non-standard dimers including those formed with deoxy-ribonucleotides.

Pyridinium ylides in syntheses of naphthopyrandiones and in regioselective syntheses of acylated anthraquinones related to fungal and bacterial metabolites

Improvements have been made in the use of acylated pyridinium ylides for the transformation of 2-methyl-1,4-naphthoquinone into derivatives (15) and (16) of naphtho[2,3-c]pyran-5,10-dione, containing furan and thiophene groups. The substitution and cyclisation steps can be combined effectively by using 2-phenoxymethyl- instead of 2-methyl-naphthoquinone. The use of better leaving groups than phenoxy (especially 4-nitrophenoxy) allows the quinone to react with two proportions of ylide and leads regiospecifically to 1-aroyl-2-arylanthracene-9,10-diones such as (20a). If the leaving group is nuclear bromine as in 2-bromo-3-methyl-1,4-naphthoquinone, another reaction with 2 mol equiv. of ylide leads to complex red intermediates of type (31) which in contact with alumina are quantitatively converted into the regioisomeric 2-aroyl-3-aryianthracene-9,10-diones such as (22a).The structures have been determined by standard methods but special features of the NMR spectra are reported including a case of extreme line broadening by traces of iron. Mechanisms are suggested for the diverse reactions between the quinones and the ylides.

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.

Signal Enhancement of Abiotically-Synthesized RNA Oligonucleotides and other Biopolymers using Unmodified Fused Silica in MALDI-MS

Metal is the standard desorption platform for MALDI-MS but other surfaces have been shown to offer advantages for particular types of analytes or applications. One such substrate is fused silica, which has been employed for matrix-free detection of low mass analytes and for affinity MALDI-MS in which binding ligands are immobilized at the fused silica surface. The present work reports improved MALDI-MS detection of RNA oligonucleotides, including polyA, polyU, and polyA/U, at the high end of the mass range when unmodified fused silica is used instead of stainless steel as the MALDI target. The RNA oligonucleotides were abiotically synthesized from activated monomers on catalytic clay surfaces. Further investigation found enhanced signals as well for other anionic biopolymers, including DNA oligonucleotides and heparin. Enhancement also was observed for dextran, which is neutral, indicating that the effect is not restricted to anionic biopolymers. Among more general analytical applications, the results are particularly relevant to rapid screening of abiotic RNA polymerization toward elucidating pathways to life on Earth.

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.®