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.].

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.

Progress in demonstrating total homochiral selection in montmorillonite-catalyzed RNA synthesis.

The Na(+)-montmorillonite-catalyzed reactions of 5'-phosphorimidazolides of nucleosides generates RNA oligomers. The question arises as to how chiral selectivity was introduced into this biopolymer from a simple chemical system. We have demonstrated homochiral selection in quaternary reactions of a racemic mixture of D,L-ImpA and D,L-ImpU on Na(+)-montmorillonite. The dimer, trimer, tetramer and pentamer fractions were investigated for homochiral selection. The products were collected via ion exchange HPLC and their terminal 5'-phosphate was cleaved by alkaline phosphatase. These fractions were analyzed by reverse phase HPLC for the identification of homochiral and heterochiral isomers. Encouraged by favorable homochiral excesses of dimer (63.5 ± 0.8%) and trimer (74.3 ± 1.7%), the study was extended to the analysis of higher oligomers. The tetramer and pentamer of the quaternary reaction were separated into 26 and 22 isomers, respectively, on a reverse phase column. Their co-elution with those formed in the binary reactions of d-ImpA and D-ImpU on Na(+)-montmorillonite revealed 92.7 ± 2.0% and 97.2 ± 0.5% homochirality of the tetramer and pentamer, respectively. These results suggest that Na(+)-montmorillonite not only catalyzes the prebiotic synthesis of RNA but it also facilitates homochiral selection.

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.

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 concomitant 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.

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.

Selectivity of montmorillonite catalyzed prebiotic reactions of D, L-nucleotides.

The montmorillonite-catalyzed reactions of the 5'-phosphorimidazolides of D, L-adenosine (D, L-ImpA) (Figure 1a. N = A, R = H) and D, L-uridine (Figure 1a., N = U, R = H) yields oligomers that were as long as 7 mers and 6 mers, respectively. The reactions of dilute solutions of D-ImpA and D-ImpU under the same conditions gave oligomers as long as 9 and 8 mers respectively. This demonstrated that oligomer formation is only partially inhibited by incorporation of both the D- and L-enantiomers. The structures of the dimers, trimers and tetramer fractions formed from D, L-ImpA was investigated by selective enzymatic hydrolysis, comparison with authentic samples and mass spectrometry. Homochiral products were present in greater amounts than would be expected if theoretical amounts of each were formed. The ratio of the proportion of homochiral products to that of the amount of each expected for the dimers (cyclic and linear), trimers and tetramers, was 1.3, 1.6, and 2.1, respectively. In the D, L-ImpU reaction homochiral products did not predominate with ratios of dimers (cyclic and linear), trimers and tetramers 0.8, 0.44, and 1.4, respectively. The proportions of cyclic dimers in the dimer fraction were 52-66% with D, L-ImpA and 44-69% with D, L-ImpU. No cyclic dimers were formed in the absence of montmorillonite. The differences in the reaction products of D, L-ImpA and D, L-ImpU are likely to be due to the difference in the orientations of the activated monomers when bound to the catalytic sites on montmorillonite. The consequences of the selectivity of montmorillonite as a prebiotic catalyst are discussed.

Montmorillonite-catalysed formation of RNA oligomers: the possible role of catalysis in the origins of life.

Large deposits of montmorillonite are present on the Earth today and it is believed to have been present at the time of the origin of life and has recently been detected on Mars. It is formed by aqueous weathering of volcanic ash. It catalyses the formation of oligomers of RNA that contain monomer units from 2 to 30-50. Oligomers of this length are formed because this catalyst controls the structure of the oligomers formed and does not generate all possible isomers. Evidence of sequence-, regio- and homochiral selectivity in these oligomers has been obtained. Postulates on the role of selective versus specific catalysts on the origins of life are discussed. An introduction to the origin of life is given with an emphasis on reaction conditions based on the recent data obtained from zircons 4.0-4.5Ga.

Studies in the mineral and salt-catalyzed formation of RNA oligomers.

Activated mononucleotides oligomerize in the presence of montmorillonite clay to form RNA oligomers. In the present study, effects of salts, temperature and pH on the clay-catalyzed synthesis of RNA oligomers were investigated. This reaction is favored by relatively high concentration of salts, such as 1 M NaCl. It was shown that the presence of divalent cations was not required for this reaction. High concentrations of NH4+ and HCO3- and 0.01 M HPO4(2-) inhibit the reaction. The yields of RNA oligomers decreased as the temperature was raised from 4 degrees C to 50 degrees C. A5' ppA was the major product at pH's below 6. The catalytic activity of a variety of minerals and three meteorites were investigated but none of them except galena catalyzed the oligomerization. ATP was generated from ADP but it was due to the presence of HEPES buffer and not due to the minerals. Meteorites catalyzed the hydrolysis of the pyrophosphate bonds of ATP. The results suggest that oligomers of RNA could have formed in pH 7-9 solutions of alkali metal salts in the presence of montmorillonite clay.

One-step, regioselective synthesis of up to 50-mers of RNA oligomers by montmorillonite catalysis.

5'-Nucleotides of A and U with the phosphate activated with 1-methyladenine generate RNA oligomers containing 40-50 monomers in 1 day in reactions catalyzed by montmorillonite. The corresponding monomers of C give oligomers that are 20-25-mers in length after a 9-day reaction. It was not possible to determine the chain lengths of the oligomers of G since they did not give well-defined bands on gel electrophoresis. Co-oligomers of A and U as well as A, U, G, and C were also prepared. The oligo(A)s formed were separated by gel electrophoresis, and the bands of the 7-39-mers were isolated, the 3',5'-phosphodiester bonds were cleaved by RNase T(2), and the terminal phosphate groups were cleaved with alkaline phosphatase. HPLC analysis revealed that the proportions of A(5)'pp(5)'A, A, A(2)'pA, and A(2)'pA(2)'pA formed were almost the same for the long and shorter oligomers. A similar structure analysis performed on the oligo(U)s established that the proportions of U(5)'pp(5)'U, U, U(2)'pU, U(2)'pU(2)'pU, U(2)'pU(2)'pU(2)'pU, and U(2)'pU(2)'pU(2)'pU(2)'pU did not vary with chain length. The structural analysis of the oligomers of A revealed that 74% of the phosphodiester bonds were 3',5'-linked a value slightly greater than 67% observed when imidazole was the activating group. 61% of the bonds in the U oligomers were 3',5'-linked, which is almost 3 times greater than the 20% measured when imidazole was the activating group. The potential significance of these data to the origin and early evolution of life is discussed.

MALDI analysis of oligonucleotides directly from montmorillonite.

Oligonucleotides synthesized on a montmorillonite catalyst were analyzed directly. By mixing the catalyst with a matrix (2,4,6-trihydroxyacetophenone or 6-aza-2-thiothymine) and dibasic ammonium citrate, higher molecular weight products were detected compared with "classical" methods such as gel electrophoresis and HPLC with UV as a detector. The oligomers (30-mers and higher) were detected by mass spectrometry even though their concentration was less than 10(-4)% of the total content of the RNA. This method is different from the (MALDI) analysis of the eluates from montmorillonite, which otherwise requires desalting. Placing reaction mixtures with a high concentration of buffers on homoionic, preferably Li-containing, montmorillonite does not require desalting.

Catalysis and selectivity in prebiotic synthesis: initiation of the formation of oligo(U)s on montmorillonite clay by adenosine-5'-methylphosphate.

Adenosine-5'-methylphosphate (MepA) initiates the oligomerization of the 5'-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5'-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69-84% of the 5-9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA --> MepA3'pU --> MepA3'pU2'pU --> MepA3'pU2'pU3'pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA3'pU and MepA2'pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA3'pA and MepA2'pA the ImpA reacts 5.2 times faster with MepA3'pA. In the competitive reaction of ImpU and ImpA with MepA3'pA and MepA3'pU the elongation proceeds on MepA3'pA 5.6 times more rapidly than with MepA3'pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2',5'-linkages in the oligomer chain proceeds more slowly than the addition to a single 2',5'-link or a 3',5'-link and either chain termination or elongation by a 3',5'-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed.

Cations as mediators of the adsorption of nucleic acids on clay surfaces in prebiotic environments.

Monovalent ([Na+] > 10 mM) and divalent ([Ca2+], [Mg2+] > 1.0 mM) cations induced the precipitation of nucleic acid molecules. In the presence of clay minerals (montmorillonite and kaolinite), there was adsorption instead of precipitation. The cation concentration needed for adsorption depended on both the valence of the cations and the chemical nature of the nucleic acid molecules. Double-stranded nucleic acids needed higher cation concentrations than single-stranded ones to be adsorbed to the same extent on clay. Divalent cations were more efficient than monovalent ones in mediating adsorption. Adsorption to the clay occurred only when both nucleic acids and cations were present. However, once the complexes were formed, the cations could not be removed from the system by washing, indicating that they are directly involved in the association between nucleic acids and mineral surfaces. These observations indicate that cations take part directly in the formation of nucleic acid-clay complexes, acting as a 'bridge' between the negative charges on the mineral surface and those of the phosphate groups of the genetic polymer. The relatively low cation concentrations needed for adsorption and the ubiquitous presence of clay minerals in the environment suggest that the adsorption of nucleic acids on mineral surfaces could have taken place in prebiotic habitats. This may have played an important role in the formation and preservation of nucleic acids and/or their precursor polymers.

Synthesis of 35-40 mers of RNA oligomers from unblocked monomers. A simple approach to the RNA world.

RNA oligomers greater than 35-40 mers in length form in one day in the montmorillonite clay-catalyzed reaction of unblocked RNA monomers at 25 degrees C in aqueous solution.

Sequence- and regioselectivity in the montmorillonite-catalyzed synthesis of RNA.

The possible role of catalysis in forming a limited number of RNAs from activated monomers is investigated by examining the sequence- and regioselectivity in the montmorillonite-catalyzed formation of RNA dimers and trimers. The reactivity of A was similar to that of G, and C was comparable in reactivity to U. Yet the reactivity of the purine nucleotides differed from that of the pyrimidines. In the reaction of nucleotides (pN) with activated monomers (ImpN), the sequence- and regioselectivity was Pu(3')Py > Pu(3')Pu = Pu(2')Py > Pu(2')Pu. The 5'-pyrimidine initiated dimers formed less efficiently than the 5'-purine initiated dimers. Trimer formation was investigated by the synthesis of 8 dimers (pNpN) and measuring the yields of trimers formed in the reaction of each dimer with a mixture of equal molar amounts of four activated monomers. The reactivity of the dimers depended on the nucleotide attached to the 3'-end of the RNA and the regiochemistry of the phosphodiester bond. Rules based on these studies are proposed to predict the sequence- and regioselectivity of the RNAs formed in montmorillonite-catalyzed reactions. These rules are consistent with the structures of the 2-5-mers formed in the reaction of equimolar amounts of ImpA and ImpC. This research establishes that the montmorillonite catalyst limits the number of RNA oligomer isomers formed. The potential significance of these findings to the origins of life is discussed.

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.