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To identify key parameters which influence the efficiency of nonenzymatic template-directed oligonucleotide reactions, a kinetic study of oligoguanylate synthesis on a polycytidylate (poly(C)) template has been performed. This oligomerization is satisfactorily described by three kinetic processes: (i) dimerization to form the first primer (k2), (ii) elongation of a preformed primer (ki, i ≥ 3), and (iii) hydrolysis of the monomer to form deactivated material (kh), with kh < k2 < ki. This is the first reported study that includes rate determinations of ki as a function of the concentration of both poly(C) template and the activated monomer, guanosine 5‘-monophosphate-2-methylimidazolide (2-MeImpG), in the range 2 mM ≤ [poly(C)] ≤ 50 mM and 5 mM ≤ [2-MeImpG] ≤ 50 mM. ki values determined under conditions where the template is fully saturated with monomer are practically independent of both monomer and polymer concentration and thus strongly support a template-directed elongation model. Values of ki determi...

The title reaction leads to the formation of the zwitterionic Michael adduct T/sup +/-/ (PhCH(R/sub 2/NH/sup +/)C(COCH/sub 3/)/sub 2//sup -/) which is in rapid acid-base equilibrium with its anionic form T/sup -/ (PhCH(R/sub 2/N)C(COCH/sub 3/)/sub 2//sup -/). Rate (K/sub 1/, k/sub -1/) and equilibrium constants (K/sub 1/) for nucleophilic addition and the pK/sub a/ of the T/sup +/-/-adducts were determined in 50% Me/sub 2/SO-50% water at 20/sup 0/C. From an interpolation of the rate constants to K/sub 1/ = 1 an intrinsic rate constant, log k/sub 0/ = 0.3, was determined. This value deviates negatively by approximately 2.5 log units from a correlation of log k/sub 0/ for amine addition to five olefins of the type PhCH=CXY, with log k/sub 0/ for the deprotonation of the corresponding carbon acids CH/sub 2/XY. Two major factors are believed to contribute to this depressed intrinsic rate constant or enhanced intrinsic barrier: (1) steric inhibition of resonance in T/sup +/-/ with the steric effect developing ahead of C-N bond formation (this conclusion is supported by an X-ray crystallographic study of p-methoxybenzylideneacetylacetone which shows that steric hindrance to optimal ..pi..-overlap in the adduct T/sup+/-/ is already present in the substrate); (2) intramolecular hydrogen bonding inmore » T/sup +/-/, which is inferred from abnormally high pK/sub a/ values and whose development lags behind C-N bond formation. These effects are shown to be manifestations of the Principle of Nonperfect Synchronization.« less

The RNA world hypothesis requires a plausible mechanism by which RNA itself (or precursor RNA-like polymers) can be synthesized nonenzymatically from the corresponding building blocks. Simulation experiments have exploited chemically reactive mononucleotides as monomers. Solutions of such monomers in the prebiotic environment were likely to be very dilute, but in experimental simulations of polymerization reactions dilute solutions of activated mononucleotides in the millimolar range hydrolyze extensively, and only trace amounts of dimers and trimers are formed. We report here that random medium-size RNA analogues with mixed sequences (5- to 17-mers with traces of longer products) can be synthesized in ice eutectic phases that are produced when dilute solutions of activated monomers and catalysts (Mg(II) and Pb(II)) are frozen and maintained at -18 degrees C for periods up to 38 days. Under these conditions, the monomers are concentrated as eutectics in an ice matrix. Hydrolysis of the activated mononucleotides was suppressed at low-temperature ranges, and polymerization was enhanced with yields up to 90%. Analysis of the mixed oligomers established that incorporation of both purine and pyrimidine bases proceeded at comparable rates and yields. These results suggest that ice deposits on the early Earth could have facilitated the synthesis of short- and medium-size random sequence RNA analogues and thereby provided a microenvironment suitable for the formation of biopolymers or their precursors.

A kinetic study of oligoguanylate synthesis on a polycytidylate template, poly(C), as a function of the concentration of the activated monomer, guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, is reported. Reactions were run with 0.005-0.045 M 2-MeImpG in the presence of 0.05 M poly(C) at 23 degrees C. The kinetic results are consistent with a reaction scheme (eq 1) that consists of a series of consecutive steps, each step representing the addition of one molecule of 2-MeImpG to the growing oligomer. This scheme allows the calculation of second-order rate constants for every step by analyzing the time-dependent growth of each oligomer. Computer simulations of the course of reaction based on the determined rate constants and eq 1 are in excellent agreement with the product distributions seen in the HPLC profiles. In accord with an earlier study (Fakhrai, H.; Inoue, T.; Orgel, L. E. Tetrahedron 1984, 40, 39), rate constants, ki, for the formation of the tetramer and longer oligomers up to the 16-mer were found to be independent of length and somewhat higher than k3 (formation of trimer), which in turn is much higher than k2 (formation of dimer). The ki (i > or = 4), k3, and k2 values are not true second-order rate constants but vary with monomer concentration. Mechanistic models for the dimerization (Scheme I) and elongation reactions (Scheme II) are proposed that are consistent with our results. These models take into account that the monomer associates with the template in a cooperative manner. Our kinetic analysis allowed the determination of rate constants for the elementary processes of covalent bond formation between two monomers (dimerization) and between an oligomer and a monomer (elongation) on the template. A major conclusion from our study is that bond formation between two monomer units or between a primer and a monomer is assisted by the presence of additional next-neighbor monomer units. This is consistent with recent findings with hairpin oligonucleotides (Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 317). Our study is the first of its kind that shows the feasibility of a thorough kinetic analysis of a template-directed oligomerization and provides a detailed mechanistic model of these reactions.

Results are presented from a detailed study of the P-N bond hydrolysis in guanosine 5-prime-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-prime-imidazolide (ImpG) in the presence of 0-0.50 M Mg(2+). Pseudo-first-order rate constants of these compounds were obtained as a function of Mg(2+) concentration, for pH values between 6 and 10 and 37 C. It was found that Mg(2+) catalysis was most effective at pH 10, where a 15-fold increase in hydrolysis was achieved in 0.02 M Mg; at 0.2 M, a 115-fold increase was observed. Implication of these results for the mechanism of template-directed oligomerization is discussed.

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 6or = pHor = 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.